Improving erectile function in diabetic male mice by rescuing depalmitoylated FBP1 to reduce cavernosal lactate

Domain: chemistry
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
The high prevalence of diabetes mellitus-induced erectile dysfunction (DMED) in males and the limited efficacy of existing therapies necessitate deeper mechanistic insights. We identify pathological lactate accumulation in the corpus cavernosum as a major driver of DMED in male mice, promoting structural degeneration and smooth muscle cell loss. Mechanistically, transcriptional repression and functional impairment of fructose-1,6-bisphosphatase 1 (Fbp1), a rate-limiting gluconeogenic enzyme, underlie this lactate accumulation. Epigenetic profiling revealed suppression of Fbp1 transcription associated with histone H3 lysine 9 trimethylation and histone H3 lysine 27 trimethylation. Palmitoylation at the FBP1 Cys282 residue, catalyzed by zinc finger DHHC-type containing 13, further disrupted its gluconeogenic function, exacerbating lactate accumulation. To rescue these defects, we engineered lipid nanoparticles loaded with the Fbp1-C282S mutant mRNA, restoring FBP1 expression, blocking pathological palmitoylation, and ultimately improving erectile function in diabetic male mice. Here, our study in male mice identifies cavernosal lactate accumulation as a central pathological factor, elucidates its molecular regulation, and provide a targeted therapeutic strategy for DMED.

Introduction
Erectile dysfunction (ED), defined as the persistent inability to attain or sustain an erection sufficient for satisfactory sexual performance, represents a common clinical condition that significantly diminishes quality of life and psychological well-being1. Diabetes mellitus is a major risk factor for ED, with diabetes mellitus-induced erectile dysfunction (DMED) exhibiting a prevalence ranging from 35.8 to 86.1% across diverse populations2,3. Compared to ED in non-diabetic individuals, DMED is characterized by earlier onset, more severe symptoms, and markedly reduced responsiveness to conventional therapies, presenting a substantial clinical management challenge4,5. While the pathogenesis of DMED involves a variety of cellular alterations and mediating factors, the precise mechanisms remain unclear.

Within the penile microenvironment, corpus cavernosum smooth muscle cells (CCSMCs) serve as the terminal effector regulating vascular tone and hemodynamics during erection6. Under diabetic conditions, CCSMCs sustain structural and functional damage through mitochondrial impairment, oxidative stress, and metabolic dysregulation7, while concurrently undergoing a contractile-to-synthetic phenotypic transition that drives excessive extracellular matrix deposition and cavernosal fibrosis8. Consequently, preserving CCSMC integrity and function has emerged as a plausible therapeutic strategy for DMED, although the precise regulatory mechanisms governing these cells demand further investigation.

Lactate, once considered merely a metabolic waste product, is now recognized as a key signaling molecule with pleiotropic roles in cardiovascular, neurodegenerative, inflammatory, and oncological pathologies9. Elevated circulating lactate levels in diabetic patients—detectable even during prediabetic stages—correlate with early diabetes-associated complications10,11. Interestingly, sildenafil citrate, a first-line ED pharmacotherapy, was recently shown to reduce lactate accumulation in muscle following strenuous exercise and improve exercise performance in rats12. Gluconeogenesis maintains a critical homeostatic relationship with lactate metabolism. Under physiological conditions, lactate generated via glycolysis during hypoxia or high metabolic demand is reconverted to glucose through gluconeogenesis in the Cori cycle. Conversely, impaired gluconeogenesis disrupts this recycling axis, driving pathological lactate accumulation in susceptible tissues. Within this gluconeogenic pathway, fructose-1,6-bisphosphatase 1 (FBP1) serves as a critical regulatory node governing lactate-to-glucose conversion. In cancers, FBP1 downregulation drives lactate-driven Warburg effect amplification, accelerating tumor invasion and metastasis13. In non-alcoholic fatty liver disease, suppressed FBP1 induces hepatic lactate buildup, exacerbating inflammation and steatosis14.

Here, we show that pathological cavernosal lactate accumulation drives DMED through structural degeneration and CCSMC loss. Transcriptional repression and functional impairment of Fbp1 constitute the core mechanism underlying lactate dysregulation, mediated by histone methylation and palmitoylation modifications. Critically, nanoparticle delivery of a palmitoylation-resistant Fbp1 mutant mRNA restores gluconeogenic flux, normalizes lactate levels, and rescues erectile function in diabetic mice. This work establishes aberrant lactate metabolism as a pathogenic cornerstone of DMED and proposes a metabolic reprogramming therapy.

Results
Supplemental lactate impairs the CCSMC function and leads to DMED
After obtaining clinical ethics approval (LYEC2025-0188), we conducted an observational case-control study and recruited 60 patients with DMED—33 with mild, 19 with moderate, and 8 with severe ED—and 60 age-matched controls (Fig. 1a, b and Supplementary Fig. 1a). DMED patients exhibited significantly higher serum lactate and blood glucose levels compared to controls (Fig. 1c and Supplementary Fig. 1b). When all 120 participants were stratified into tertiles based on serum lactate levels, the overall incidence of ED increased significantly with ascending lactate tertiles (Fig. 1d). Moreover, within the DMED subgroup, the incidence of moderate-to-severe ED rose in a lactate-dependent manner (Fig. 1e).

To further investigate the correlation between lactate and DMED, we established a mouse model of DMED using a combination of low-dose streptozotocin and a high-fat diet. Cavernosal lactate concentrations and intracavernous pressure (ICP) were measured at 0, 4, 8, and 12 weeks post-modeling (Supplementary Fig. 1c). We observed a time-dependent increase in cavernosal lactate levels, accompanied by a progressive decline in ICP (Supplementary Fig. 1d–f). DMED mice also exhibited significantly elevated plasma lactate and blood glucose levels compared to controls (Supplementary Fig. 1g, h). Furthermore, cavernosal tissue in DMED mice showed pronounced lactate accumulation, with its levels positively correlating with plasma lactate concentrations (Fig. 1f–h).

To evaluate the effect of lactate on erectile function, mice were randomly assigned to receive saline or different doses of lactate (Fig. 1i). Serum lactate kinetics analysis showed that the 0.5 g/kg dose significantly elevated the concentration-time curve compared to the 0.25 g/kg group, whereas the 0.75 g/kg dose did not further increase lactate levels within the short term (Supplementary Fig. 1i). Therefore, the 0.25 and 0.5 g/kg doses were selected for subsequent experiments. Exogenous lactate administration elevated cavernosal lactate levels and reduced ICP in a concentration-dependent manner compared with the saline group (Fig. 1j–l). Histological analysis further revealed that lactate treatment significantly reduced the lumen-to-parenchyma ratio, smooth muscle-to-collagen ratio, and α-SMA fluorescence intensity, while increasing Collagen I fluorescence intensity (Fig. 1m–r and Supplementary Fig. 1j, k). These results demonstrate that elevated lactate levels disrupt erectile function by reducing smooth muscle content.

Based on the above findings, we further investigated the toxic effects of lactate accumulation on CCSMCs. Primary CCSMCs were treated with lactate at different concentrations (0, 2, 4, 6, 8, 10, 12 mmol/L). Cell viability was not significantly affected at lactate concentrations up to and including 4 mmol/L (Supplementary Fig. 1l). Thus, 5 and 10 mmol/L were selected as low and high lactate stimulation concentrations, respectively, for subsequent experiments (Fig. 1s). Lactate treatment impaired CCSMCs’ activity and motility in a concentration-dependent manner (Fig. 1t–v). Western blot analysis showed that exposure to high lactate levels downregulated contractile markers (SM22α and CALPONIN) and upregulated synthetic markers (VIMENTIN and OSTEOPONTIN) in CCSMCs (Fig. 1w, x). Transcriptome sequencing further suggested that excessive autophagy activation may underlie the lactate-induced impairment of CCSMCs (Supplementary Fig. 2a–c). In vitro experiments were conducted by pretreating CCSMCs with the autophagy inhibitor 3-methyladenine (3-MA). The results demonstrated that 3-MA significantly abrogates lactate-induced excessive autophagy activation, impairment of cell viability, and phenotypic switching toward the synthetic smooth muscle phenotype. (Supplementary Fig. 2d–l). In animal models, we further analyzed autophagic activity in the corpus cavernosum of lactate-challenged mice and DMED mice. Intriguingly, progressive elevation of the LC3-II/LC3-I ratio was observed in both models during disease progression, whereas P62 protein expression exhibited a dynamic change characterized by an initial decrease followed by a subsequent increase (Supplementary Fig. 3). Building upon these observations, we performed interventions with 3-MA and SMER28, respectively. Our data showed that 3-MA effectively suppressed excessive autophagy and remarkably improved erectile function as well as tissue architecture in DMED mice. In contrast, although SMER28 promoted P62 degradation, it failed to exert significant therapeutic efficacy (Supplementary Fig. 4). Collectively, these results indicate that lactate-induced excessive autophagy is a key mechanism contributing to DMED.

FBP1 is a key factor for cavernosal lactate accumulation in DMED
Lactate serves as a key intermediate in glucose metabolism. Under anaerobic conditions, glycolysis rapidly converts glucose to lactate, while gluconeogenesis reversibly converts lactate back to glucose to maintain systemic glucose homeostasis (Fig. 2a). Consequently, tissue lactate accumulation can arise from either glycolytic overactivation, which leads to excessive lactate production, or impaired gluconeogenesis, which results in inadequate lactate clearance15. We have validated that CCSMC also possesses gluconeogenic function, consistent with what has been observed in smooth muscle cells from other sites16,17 (Supplementary Fig. 5). Using RNA-seq data from our previously established DMED mouse and rat models18, we analyzed the transcription profiles of key genes involved in glycolysis and gluconeogenesis. The results revealed no upregulation of glycolytic pathway genes. Furthermore, a brief exploratory treatment of DMED mice with sodium oxamate, a glycolysis inhibitor, did not significantly reduce cavernosal lactate levels (Supplementary Fig. 6a–c), suggesting that lactate accumulation in the corpus cavernosum primarily results from defective gluconeogenesis rather than enhanced glycolysis. Among gluconeogenic enzymes, Fbp1 showed consistent transcriptional downregulation (Fig. 2b, c). Analysis of public single-cell sequencing data from the Human Male Health Atlas (http://malehealthatlas.cn/)19 also indicated reduced FBP1 expression in the corpus cavernosum of DMED patients (Fig. 2d). Consistent with this, validation in DMED mouse models confirmed significant downregulation of FBP1, whereas the expression of G6PC3 and PCK1 remained unchanged (Fig. 2e–g and Supplementary Fig. 6d–f).

FBP1 catalyzes the hydrolysis of fructose-1,6-bisphosphate into fructose 6-phosphate and inorganic phosphate, playing a critical role in lactate metabolism20. Its deficiency is associated with metabolic disturbances, including hypoglycemia and lactic acidosis21. We therefore asked whether FBP1 downregulation contributes to the development of ED. Our results demonstrated that Fbp1 knockdown significantly increased cavernosal lactate levels and reduced ICP, lumen-to-parenchyma ratio, and α-SMA fluorescence intensity compared to normal mice (Fig. 2h–n). Under low-lactate conditions in vitro, Fbp1 knockout further impaired CCSMC viability, motility, and contractile marker expression (Fig. 2o–q). Conversely, Fbp1 overexpression partially counteracted the detrimental effects of high lactate concentrations on CCSMCs (Fig. 2r–t). Notably, the G260R variant (the glycine at position 260 of the FBP1 is substituted with arginine), a well-characterized enzymatically inactive form of FBP1 linked to metabolic disorders22,23, was found in our study to fail in alleviating the harmful effects of high lactate concentrations on CCSMCs (Fig. 2r–t). These findings indicate that FBP1 modulates lactate-induced effects on CCSMCs in an enzyme activity-dependent manner.

Aberrant accumulation of H3K9me3 or H3K27me3 in the Fbp1 promoter epigenetically represses its transcription in DMED
Transcriptional regulation represents a fundamental mechanism controlling gene expression. As previously described, the transcriptional downregulation of Fbp1 was observed across species in penile cavernosal tissues. Given that histone methylation modifications can induce epigenetic repression of gene expression by altering chromatin structure, and have been implicated in various diabetic complications24–26, we hypothesized that these marks might contribute to Fbp1 silencing. To identify potential repressive histone modifications on the Fbp1 promoter, we performed an initial screening using the ChIP-Atlas database (https://chip-atlas.org/). The analysis revealed significant enrichment of the repressive marks histone H3 lysine 27 trimethylation (H3K27me3)27 and histone H3 lysine 9 trimethylation (H3K9me3)28 within the Fbp1 promoter region (Fig. 3a). We further validated these findings through ChIP-qPCR assays, which confirmed strong enrichment of both H3K9me3 and H3K27me3 at the Fbp1 promoter in MOVAS cells and primary mouse CCSMCs, and also characterized additional epigenetic features of the Fbp1 promoter (Fig. 3b, c and Supplementary Fig. 7a–g). Furthermore, we demonstrated that the enrichment of H3K9me3 or H3K27me3 were significantly elevated in the corpus cavernosum of DMED mice compared to normal mice (Fig. 3d, e). Since histone methylation is catalyzed by specific methyltransferases, we screened for enzymes associated with H3K9me3 and H3K27me3. This revealed substantial upregulation of suppressor of variegation 3-9 homolog 1 (SUV39H1) and enhancer of zeste homolog 2 (EZH2) in DMED mice (Fig. 3f, g). Overexpression of Suv39h1 increased H3K9me3 levels and inhibited the FBP1 expression in both MOVAS cells (Fig. 3h, i) and CCSMCs (Supplementary Fig. 7h–i). Conversely, Suv39h1 knockdown using siRNA or inhibition with its specific inhibitor chaetocin29 led to the upregulation of FBP1 (Fig. 3j–m and Supplementary Fig. 7j–m). Similarly, Ezh2 overexpression elevated H3K27me3 and repressed FBP1, whereas Ezh2 knockdown or inhibition with EPZ643830 increased FBP1 expression (Supplementary Figs. 7n–s and 8). Given that the EZH2–H3K27me3 axis has been previously implicated in FBP1 regulation31–33, we focused on identifying readers of H3K9me3 at the Fbp1 promoter. Integrated analysis using ChIP-Atlas and ChIP-qPCR identified chromobox protein homolog 3 (CBX3), a known reader of H3K9me334, binding to the Fbp1 promoter (Fig. 3n, o and Supplementary Fig. 7t). CBX3 enrichment was significantly higher in DMED mice compared to normal mice (Fig. 3p, q). We further demonstrated that CBX3 negatively regulates FBP1 expression in vitro (Fig. 3r–y and Supplementary Fig. 7u–z), and to mechanistically target this regulation, we employed PROTAC technology35 to develop a selective CBX3 degrader that induces its ubiquitination-mediated degradation (Supplementary Fig. 9). Collectively, these findings indicate that aberrant enrichment of H3K9me3 and H3K27me3 at the Fbp1 promoter epigenetically represses its transcription in DMED.

The effects of H3K9me3- and H3K27me3-related inhibitors on DMED mice
Next, we evaluated the effects of the three inhibitors on Fbp1 function by measuring glucose consumption and lactate production. Treatment with inhibitors or siRNA targeting Cbx3, Suv39h1, or Ezh2 significantly reduced both glucose consumption and lactate production in cultured cells. However, with the exception of siCbx3 (Fig. 4b), most inhibitors failed to reverse the metabolic alterations caused by Fbp1 knockout, as reflected in lactate production and glucose uptake (Fig. 4a–l). Moreover, inhibition of Cbx3, Suv39h1, or Ezh2 did not restore Fbp1 expression in Fbp1-knockout cells (Fig. 4m–o and Supplementary Fig. 10a–c). These results indicate that Fbp1 acts as a key downstream effector of Cbx3, Suv39h1, and Ezh2, although Cbx3 and Suv39h1 may also modulate lactate and glucose metabolism through additional pathways. Subsequently, we administered H-PROTAC, chaetocin, and EPZ6438 to DMED mice and Fbp1−/+ mice, respectively. Although the three inhibitors elicited statistically significant effects in DMED mice, the improvements were modest: they led to only a slight reduction in cavernosal lactate levels and partial recovery of ICP, tissue morphology, and smooth muscle content. Notably, none of the inhibitors produced any beneficial effect in Fbp1−/+ mice (Fig. 4p–u). Although combined inhibition of both marks led to stronger Fbp1 upregulation in vitro (Supplementary Fig. 10d, e), the combined in vivo administration of the three agents—chaetocin (intraperitoneally), EPZ6438 (orally), and H-PROTAC (intracavernously)—was poorly tolerated in DMED mice. Therefore, rather than pursuing complex combination regimens, it is more compelling to explore alternative mechanisms governing FBP1 expression and function.

ZDHHC13 mediated S-palmitoylation of FBP1 at C282 site
The above findings indicate that although histone methylation modifications strongly suppress Fbp1 transcription in DMED, interventions targeting these epigenetic regulators failed to robustly restore erectile function in vivo. This discrepancy led us to reevaluate the regulatory landscape of FBP1 and hypothesize that its activity might be further modulated by post-translational modifications (PTMs). Given the critical role of the G260 residue in FBP1 enzymatic function and gluconeogenesis, we focused on identifying potential PTMs near this key site. Analysis of all PTMs within ±25 amino acids around the G260 of FBP1 has shown that, apart from the previously reported acetylation at K26936 and phosphorylation at Y26537, a palmitoylation site at C282 has been identified by using the CSS-palm 4.0 tool (Fig. 5a). Palmitoylation of FBP1 was confirmed via acyl-biotinyl exchange (ABE) assay in cells expressing HA-tagged Fbp1, with streptavidin blotting revealing a clear palmitoylation signal (Fig. 5b and Supplementary Fig. 11a). This finding was further validated using biotin-alkyne labeling coupled with click chemistry, which showed increased palmitoylated proteins in Fbp1-transfected cells; hydroxylamine treatment abolished this signal, confirming its specificity (Fig. 5c and Supplementary Fig. 11b, c). We also demonstrated that endogenous FBP1 undergoes palmitoylation (Supplementary Fig. 11d). Sequence alignment revealed that C282, located adjacent to the functional G260 residue, is evolutionarily conserved across species (Fig. 5d). To identify the enzyme responsible for FBP1 palmitoylation, we generated a series of Zdhhc knockout cell lines using CRISPR technology. Among these, knockout of zinc finger DHHC-type containing 13 (Zdhhc13) markedly reduced FBP1 palmitoylation (Fig. 5e), suggesting it acts as a key palmitoyltransferase for FBP1. The direct physical interaction between FBP1 and ZDHHC13 was further confirmed by molecular docking, co-immunoprecipitation, confocal imaging, and GST pull-down assays (Fig. 5f–j and Supplementary Fig. 11e). Transcriptome analysis and immunohistochemistry of the corpus cavernosum showed that ZDHHC13 expression was elevated in DMED mice (Fig. 5k, l), suggesting its involvement in FBP1 palmitoylation in vivo. In cellular models, knockdown of Zdhhc13 decreased FBP1 palmitoylation, whereas its overexpression increased palmitoylation levels (Fig. 5m, n and Supplementary Fig. 11f, g). Mutation of the cysteine at position 282 to serine (FBP1-C282S) abolished FBP1 palmitoylation (Fig. 5o and Supplementary Fig. 11h). To clarify the enzyme dependency, we reintroduced either wild-type ZDHHC13 (ZDHHC13-WT) or an enzymatically inactive mutant (ZDHHC13-C456S)38 into Zdhhc13-knockout cells. Only ZDHHC13-WT restored FBP1 palmitoylation, confirming the requirement for ZDHHC13 enzymatic activity (Fig. 5p and Supplementary Fig. 11i, j). To rule out interference from endogenous FBP1, we expressed FBP1‑WT or FBP1‑C282S in Fbp1-knockout cells. Exogenous FBP1‑WT underwent palmitoylation, whereas the FBP1‑C282S mutant did not (Fig. 5q and Supplementary Fig. 11k, l). Finally, the palmitoylation assay confirmed that ZDHHC13‑WT mediated palmitoylation of FBP1‑WT but not FBP1‑C282S, and ZDHHC13‑C456S failed to catalyze the modification (Fig. 5r). Together, these data demonstrate that ZDHHC13 catalyzes S-palmitoylation of FBP1 at cysteine 282.

ZDHHC13-mediated S-palmitoylation of FBP1 impairs its gluconeogenic function
S-palmitoylation is known to regulate protein stability, localization, and function39,40. To investigate how ZDHHC13-mediated S-palmitoylation influences FBP1, we initially assessed its effects on protein degradation and subcellular localization, but observed no significant changes in either (Fig. 6a, b). We then employed PyMOL to assess whether palmitoylation at C282 induces conformational changes in FBP1. Palmitoylation reduced the solvent-accessible surface area (SASA) of the critical G260 residue by ~20% (from 3.296 to 2.657 Å²), while the total SASA of chain A decreased by ~388 Å² (Supplementary Fig. 12a). Together, these data suggest that palmitoylation at C282 promotes a more compact conformational state of FBP1, characterized by reduced solvent exposure of G260 and enhanced local structural folding. Fbp1 knockout significantly elevated glucose consumption, lactate production, and extracellular acidification rate (ECAR), while suppressing the oxygen consumption rate (OCR). Reintroduction of the Fbp1 C282S mutant into Fbp1‑knockout cells produced a greater reduction in glucose consumption, lactate production, and ECAR, alongside a more substantial increase in OCR (Fig. 6c–f and Supplementary Fig. 12b–g). Knockdown of Zdhhc13 significantly decreased glucose consumption, lactate production, and ECAR, and increased OCR. However, these metabolic changes were abolished in Fbp1-knockout cells (Fig. 6g–j and Supplementary Fig. 12h–k). Conversely, overexpression of Zdhhc13 increased glucose consumption, lactate production, and ECAR, while decreasing OCR; again, these effects were absent in Fbp1-knockout cells (Fig. 6k–n and Supplementary Fig. 12l–o). The enzymatically inactive mutant Zdhhc13-C456S had no significant effect on glycolytic activity or lactate production (Supplementary Fig. 12p–w). Furthermore, in Fbp1-knockout cells reconstituted with the Fbp1C282S mutant, Zdhhc13 overexpression failed to alter glucose consumption or lactate production (Supplementary Fig. 12x, y). Finally, we injected an adeno-associated virus (AAV) encoding Zdhhc13-targeting shRNA into the corpus cavernosum of DMED mice and Fbp1−/+ mice, respectively (Supplementary Fig. 12z). Knockdown of Zdhhc13 partially reduced cavernosal lactate levels and moderately improved ICP in DMED mice; however, it produced no effect in Fbp1−/+ mice (Fig. 6o–t). These results underscore that targeting Zdhhc13 alone provides limited therapeutic benefit in DMED. Building on these limitations, we next sought to elucidate the mechanism underlying Zdhhc13 upregulation in DMED. We analyzed differentially expressed genes using KEGG pathway enrichment, which revealed significant activation of the PI3K-AKT pathway—the most upregulated signaling pathway in DMED (Supplementary Fig. 13a). Given established knowledge of PI3K-AKT signaling in regulating SRY-related HMG-box gene 9 (SOX9) activity41,42, we investigated this potential connection. Interestingly, ChIP-Atlas data indicated SOX9 binding to the promoters of both Zdhhc13 and Cbx3 (Supplementary Fig. 13b). Consistent with these findings, we confirmed elevated phosphorylated AKT and SOX9 levels in the cavernosum of DMED mice (Supplementary Fig. 13c). Sox9 knockdown downregulated CBX3 and ZDHHC13 expression, while its overexpression increased them (Supplementary Fig. 13d–i). However, when we administered AAV-shSox9 to the cavernosum of DMED mice, we observed no significant improvement in cavernosal lactate levels or ICP (Supplementary Fig. 13j–m). This unexpected outcome prompted us to explore alternative strategies capable of concurrently addressing both Fbp1 transcriptional repression and palmitoylative inactivation.

The effect of lipid nanoparticles loaded with Fbp1-C282S mRNA on DMED mice
Currently, single-site inhibitors demonstrate only modest efficacy in vivo, failing to substantially ameliorate elevated lactate levels or restore erectile function in DMED mice. Furthermore, combination therapy with multiple agents may provoke serious adverse reactions. To address this challenge, we synthesized the Fbp1-C282S mRNA using site-directed mutagenesis, which was subsequently encapsulated in lipid nanoparticles (Fbp1-C282S-LNP) via the Ignite Nano Assay system (Fig. 7a). The Fbp1-C282S-LNPs exhibited an average particle size of 99.3 nm, polydispersity index (PDI) of 0.083, encapsulation efficiency of 90.8%, and zeta potential of −3.44 mV (Fig. 7b). Scanning electron microscopy was used to further characterize the nanoparticles (Fig. 7c). The Fbp1-C282S-LNPs significantly upregulated FBP1 level without exhibiting obvious cytotoxicity in primary mouse CCSMCs (Fig. 7d–f). Functional assays confirmed that the Fbp1-C282S-LNPs also markedly downregulated glucose consumption, lactate production and ECAR, while upregulating cellular OCR (Fig. 7g–n). Subsequently, we encapsulated Flag-tagged Fbp1-C282S mRNA into LNPs and evaluated cellular uptake within the corpus cavernosum using multiplex immunofluorescence staining. Robust LNPs uptake was observed in CCSMCs, with detectable internalization also occurring in endothelial cells and fibroblasts (Supplementary Fig. 14a). Additionally, in vivo assessments confirmed this formulation induced no significant hepatic or renal toxicity in mice (Supplementary Fig. 14b–e). Concurrently, we completed pharmacokinetic and tissue distribution studies of Fbp1-C282S-LNPs to define their in vivo profiles (Supplementary Fig. 14f, g). To assess dose-dependent therapeutic efficacy, DMED mice received intracavernous injections of Fbp1-C282S-LNPs at 0.1, 0.25, or 0.5 mg/kg. The 0.1 mg/kg group showed no significant improvement in cavernosal lactate levels or erectile function relative to controls. In contrast, the 0.25 mg/kg group significantly reduced lactate levels, increased FBP1 expression, improved ICP, enhanced smooth muscle content, ameliorated histological structure, and reduced cavernous fibrosis. However, the 0.5 mg/kg group provided no additional therapeutic benefit over the 0.25 mg/kg dose (Fig. 7o–y). We validated that Fbp1 C282S-LNP treatment can also efficiently reverse autophagic abnormalities in the corpus cavernosum of DMED mice (Supplementary Fig. 14h-j). Results of subsequent multi-tissue validation demonstrated that, in contrast to the significant increase in FBP1 protein expression observed in the corpus cavernosum following treatment, FBP1 protein expression in the liver and skeletal muscle exhibited an upward trend but failed to reach statistical significance (Supplementary Fig. 15). Furthermore, Fbp1-C282S-LNPs demonstrated sustained efficacy in long-term studies of DMED mice (Supplementary Fig. 16) and consistently improved outcomes across multiple diabetic models, including type 1 diabetes (Supplementary Fig. 17), db/db mice (Supplementary Fig. 18), and Fbp1−/+ mice (Supplementary Fig. 19). These results firmly establish Fbp1-C282S-LNP as a highly promising therapeutic strategy for DMED.

Methods
Clinical specimen collection
The study protocol was approved by the Institutional Review Board (IRB) of the Second Xiangya Hospital of Central South University (Approval No. LYEC2025-0188). The collection of clinical patient information and specimens was registered with the Chinese Clinical Trial Registry (ChiCTR; registration number: ChiCTR2500107001) as an observational case-control study. Written informed consent was obtained from all participants prior to sample collection. All participants were assessed using the IIEF-5 questionnaire; those with a score below 22 were defined as having DMED. Venous blood samples were collected for subsequent lactate measurement. No therapeutic or experimental interventions were administered to the participants during the study period.

Reagents
Apomorphine, streptozotocin, and lactate were purchased from Sigma-Aldrich (USA). The lactic acid assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute (China). Glucose test kit and Nuclear and cytoplasmic protein extraction kit were obtained from Beyotime Biotechnology (China). The primary antibodies used were as follows: α-SMA (#GB111364, Servicebio, 1:1000), SM22α (#60213, Proteintech, 1:5000), CALPONIN (#13938, Proteintech, 1:5000), VIMENTIN (#60330, Proteintech, 1:20,000), OSTEOPONIN (#83341, Proteintech, 1:1000), FBP1 (#12842, Proteintech, 1:500), H3K9me3 (#ab8898, Abcam, 1:1000), H3K27me3 (#ab6002, Abcam, 1:1000), SUV39H1 (#10574, Proteintech, 1:2000), SUV39H2 (#11338, Proteintech, 1:1000), SETDB1 (#66293, Proteintech, 1:20,000), SETDB2 (#ab13712, Abcam, 1:500), EZH1 (#ab263961, Abcam, 1:1000), EZH2 (#66476, Proteintech, 1:5000), β-ACTIN (#60008, Proteintech, 1:5000), CBX3 (#66446, Proteintech, 1:2000), CBX5 (#11831, Proteintech, 1:500), CBX1 (#10241, Proteintech, 1:1000), ZDHHC13 (#24759, Proteintech, 1:500), FLAG (#66008, Proteintech, 1:5000), HA-tag (#51064, Proteintech, 1:5000), HIS-tag (#66005, Proteintech, 1:5000), AGK (#ab137616, Abcam, 1:1000), GST (#2622, Cell Signaling Technology, 1:500), Histone H3 (#17168, Proteintech, 1:1000), LC3 (#14600, Proteintech, 1:1000), P62 (#18420, Proteintech, 1:1000), G6PC (#A21168, ABclonal, 1:2000), PCK1 (#GB11966, Servicebio, 1:500), Phospho-RPB1(Ser2) (#13499, Cell Signaling Technology, 1:500), H3K4me3 (#39915, Proteintech, 1:1000), H3K27ac (#A22264, ABclonal, 1:5000), H2AK119ub (#8240, Cell Signaling Technology, 1:500), CRBN (#28494, Proteintech, 1:2000), Phospho-AKT (#66444, Proteintech, 1:1000), AKT (#10176, Proteintech, 1:1000), SOX9 (#67439, Proteintech, 1:5000), vWF (#GB120019, Servicebio, 1:500), and Collagen I (#GB11022, Servicebio, 1:1000). 13C-labeled sodium lactate (#HY-B0389S14), 3-MA (#HY-19312), SMER28 (#HY-100200), lactate Chaetocin (#HY-N2019), EPZ6438 (#HY-13803), and sodium oxamate (#HY-W013032A) were purchased from the MedChemExpress. H-PROTAC was developed by our research group. AAV was provided by Ji-kai Biotechnology (Shanghai, China). As previously mentioned64, Flag-Suv39h1, Flag-Cbx3, Flag-Ezh2, Flag-Zdhhc13, and HA-Fbp1 were constructed by cloning the corresponding cDNA sequences into the OmicLinkTM Expression Clone (CMV Promoter; GeneCopoeia, EX-V0006-M14). The point mutations of Zdhhc13 and Fbp1 were synthesized by using the Hieff Mut Multi Site-Directed Mutagenesis Kit (#11004ES10, Yeasen Biotechnology). The siRNAs were obtained from RiboBio. The sequences of the siRNAs and single-guide RNAs (sgRNAs) are provided in Supplementary Table S1. Cells were transfected with the plasmids or siRNAs using Lipofectamine 2000 (Thermo Fisher Scientific). Other reagents used in this study are detailed in the respective sections.

Cell culture
The MOVAS cell line (CRL-2797), derived from mouse aortic smooth muscle, was obtained from Xiamen Immocell Biotechnology (China). Primary mouse CCSMCs were isolated using the tissue block method. Briefly, the penis of an 8-week-old C57BL/6 male mouse was excised, and the glans penis, urethra, dorsal blood vessels, and nerves were removed. The remaining tissue was thoroughly washed before being finely minced into ~1 mm³ fragments. These tissue pieces were carefully placed at the base of a culture flask. The flask was then inverted and incubated for 4 h to facilitate initial adhesion. Subsequently, Dulbecco’s Modified Eagle’s Medium (C11885500BT, Gibco) supplemented with 20% bovine calf serum was gently added to the culture to support continuous cell growth62.

Cell viability
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8, DOJINDO) assay. Briefly, cells were treated with lactate at various concentrations for different durations. After the treatment periods, 100 μL of the CCK-8 working solution was added to each well according to the manufacturer’s instructions. The optical density values were measured, which reflect viable cell numbers since the assay quantifies dehydrogenase activity present only in live cells.

Transwell assay
Serum-free cell suspension was added to the upper chamber, while the lower chamber was supplemented with serum-containing medium. Following a 24-h incubation, cells adhering to the underside of the membrane were fixed and stained. The cells in the upper chamber were gently removed, and the migrated cells in the lower chamber were visualized under a microscope to evaluate migration capacity.

qRT-PCR
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and subsequently reverse-transcribed with the PrimeScript RT Kit (Takara). Complementary DNA (cDNA) was amplified using TB Green Fast qPCR Mix (Takara). Relative gene expression levels were quantified using the 2−ΔCt method, with β-actin serving as the internal reference gene. Primer sequences are listed in Supplementary Table S2.

Transfection of siRNA and plasmids
The designated siRNA, plasmids, and Lipofectamine 2000 (Thermo Fisher Scientific) were combined in 1 mL of serum-free Opti-MEM and incubated for 20 min at room temperature. The transfection mixture was then added to cells that had been serum-starved for 12 h. After a 6-h transfection period, the culture medium was replaced with complete DMEM, and the cells were maintained under standard culture conditions for an additional 72 h.

Co-immunoprecipitation and Western blot
Cells were harvested and lysed using RIPA lysis buffer (#P0013, Beyotime, China). The lysates were then incubated with 30 µL of protein A/G beads (#P2029, Beyotime, China), along with either IgG (#A7007, Beyotime, China) or a specific primary antibody, for over 24 h at 4°C. Following incubation, the beads were extensively washed with RIPA buffer. For Western blot analysis, protein lysates were denatured by boiling, separated via SDS-PAGE, and transferred onto a nitrocellulose membrane. Protein bands were detected using an Amersham Imager 600 (General Electric, USA).

Chromatin immunoprecipitation (ChIP) and ChIP-qPCR
The ChIP assay was performed following the protocols provided with Abcam’s chromatin extraction kit and magnetic one-step ChIP kit, briefly outlined below: Cells or tissues were fixed with 1% formaldehyde and then quenched with glycine. Nuclei were isolated via stepwise lysis using buffers, and chromatin was fragmented to 200–1000 bp by sonication (as verified by electrophoresis). The chromatin was then diluted, supplemented with protease inhibitors, and incubated overnight at 4°C with either specific antibodies or IgG (as a negative control). Pre-blocked Protein A magnetic beads were added for capture at room temperature for 1 hour. After sequential washing with low-salt buffer, high-salt buffer, and a final rinse with TE buffer, complexes were eluted using lysis buffer containing 1% SDS. Incubation at 65°C facilitated elution and subsequent NaCl-mediated reversal of cross-links (overnight, or accelerated via a 2-h incubation at 95°C). Finally, DNA was purified following Proteinase K digestion; the analysis of this purified DNA was largely comparable to that of cDNA in RT-qPCR. Primer sequences for ChIP-qPCR are provided in Supplementary Table S3.

Glutathione S-transferase (GST) pull-down assay
Cells were lysed thoroughly using RIPA buffer. GST fusion proteins were immobilized on BeyoMag Anti-GST Magnetic Beads (P2138, Beyotime). After washing with RIPA buffer, the beads were incubated with cell lysates for 4 h at 4°C. The beads were then washed again with the same buffer and resuspended in loading buffer. The proteins bound to the beads were analyzed via Western blotting64.

Acyl-biotinyl exchange assay
The cell pellets were lysed in buffer containing 50 mM Tris (pH 7.2), 150 mM NaCl, 1% NP-40, and 1 mM EDTA, followed by the addition of 20 mM methyl methanethiosulfonate (Sigma-Aldrich) and 1 mM PMSF (Beyotime). After incubation for 1 h at room temperature, excess reagents were removed by chloroform–methanol precipitation (one volume of chloroform, four volumes of methanol, and three volumes of ddH₂O). The pre-blocked samples were then treated with hydroxylamine (Sigma-Aldrich) at pH 7.0 to reach a final concentration of 0.5 M and incubated for 2 h at room temperature to cleave thioester bonds. Subsequently, 0.2 mM biotin-HPDP (TopScience) was added, and samples were incubated in the dark for 2 h to ensure efficient biotinylation. Excess biotin was again removed by chloroform–methanol precipitation. The resulting proteins were purified with Streptavidin beads (Yeasen) and analyzed by SDS-PAGE and Western blotting.

Click-iT pull down
Cells were transfected with plasmids for 48 h, and then labeled with 100 μmol/L palmitic acid-azide (Thermo Fisher Scientific) for 6 h. After that, the cells were lysed to extract proteins. The Click-iT kit (Thermo Fisher Scientific) was used to catalyze the reaction between biotin-alkyne and the azide group, forming a biotinylated palmitoylated protein complex. The complex was captured by streptavidin magnetic beads. After washing with gradient salt buffers, the proteins were eluted with SDS-PAGE buffer. Finally, the palmitoylation modification was analyzed by Western blotting65.

Semi-in vitro palmitoylation assay
Cells were transfected with GST-ZDHHC13 (WT) or the C456S mutant. After 24 h, cells were lysed with NP-40 lysis buffer, and GST fusion proteins were purified with glutathione agarose beads. The semi-purified GST-ZDHHC13 was incubated with purified HIS-FBP1 or HIS-FBP1 (C282S) in the presence of palmitoyl-alkyne-CoA at 25°C for 1 h (with or without 2-bromopalmitate as a control). Reaction products were then subjected to a Click-iT reaction, followed by streptavidin pull-down and Western blot analysis of FBP1 palmitoylation.

CRISPR technique
Multiple single-guide RNAs (sgRNAs) targeting Zdhhcs and Fbp1 were designed using the platform available at https://www.synthego.com. Subsequently, these sgRNAs were cloned into the lentiCRISPR v2 vector (Addgene)64. The sequences of the sgRNAs are presented in Supplementary Table S1.

Nuclear-cytoplasmic protein separation
Cytoplasmic and nuclear proteins were extracted following the manufacturer’s protocol (#P0028, Beyotime). Briefly, Cytoplasmic Protein Extraction Reagent A was mixed with a protease inhibitor at a 100:1 ratio and added to the cell pellet. The mixture was vortexed for 5 s and incubated on ice for 10–15 min. Subsequently, Reagent B was added, and the mixture was vortexed again for 5 s. The sample was centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant containing cytoplasmic proteins was collected. The remaining pellet was resuspended in Nuclear Protein Extraction Reagent mixed with a protease inhibitor at a 100:1 ratio. The suspension was vortexed for 10 s and incubated on ice with intermittent vortexing (10–15 s every 2 min) for 30 min. Finally, the sample was centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant containing nuclear proteins was collected.

Glucose consumption and lactate production experiment
Cells in the logarithmic growth phase were seeded into 96-well plates and cultured in complete medium supplemented with glucose for 24 h. After washing with basal medium, the medium was replaced with one containing a known concentration of glucose. Following an additional 48-h incubation, the supernatant was collected, and glucose and lactate levels were quantified using specific detection kits. Glucose consumption and lactate production were calculated based on the concentration differences before and after incubation and the total medium volume.

Seahorse extracellular flux assays
The Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). For the OCR analysis, the Seahorse XF Cell Mito Stress Test Kit (Agilent) was used. According to the manufacturer’s protocol, oligomycin, p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP), and a mix of rotenone/antimycin A were injected sequentially into the microplate wells. For the ECAR analysis, the Seahorse XF Glycolysis Stress Test Kit was employed, and glucose, oligomycin, and 2-deoxy-D-glucose (2-DG) were sequentially injected into the wells.

Identification of gluconeogenic function in CCSMCs
Primary CCSMCs were individually seeded into three 75-cm² culture flasks and maintained in a humidified incubator with 5% CO₂ at 37°C until confluence was achieved. Subsequently, the culture medium was aspirated and replaced with serum-free medium for a 24-h starvation period, followed by the addition of 13C-labeled sodium lactate to a final concentration of 2 mM to continue cultivation. Upon completion of incubation, the culture supernatants were collected, lyophilized using a vacuum centrifugal concentrator, and resuspended in 2 mL of deuterium oxide. ¹³C nuclear magnetic resonance (NMR) spectral acquisition and data analysis were performed on a Bruker AVANCE NEO 600M NMR spectrometer, following protocols described in previously published relevant literature16,17. The obtained spectra were subjected to phase and baseline correction using TopSpin software, and qualitative analysis was performed based on the typical ¹³C chemical shift range of glucose.

PROTAC
The CBX3-targeting ligand was designed based on the H3K9me3-derived QTARM3L peptide motif ((Q/N)(T/V)A(R/I/F/W/V)Kme3(S/T)). Meanwhile, the immunomodulatory drug Pomalidomide was incorporated on the opposite terminus as the E3 ubiquitin ligase recruiter, specifically engaging the CRL4^(CRBN) complex (Cereblon E3 ubiquitin ligase), which is widely employed in PROTAC design for its potent protein degradation properties. A critical aspect of the design involved the synthesis and optimization of a bridging linker that covalently connects these two ligands; its length, composition, and rigidity were meticulously engineered based on structural insights to ensure proper spatial alignment and facilitate efficient formation of a productive ternary complex, thereby enabling targeted ubiquitination and subsequent proteasomal degradation of CBX3. The degradation efficiency and specificity of the synthesized CBX3-PROTAC were validated by Western blotting to quantify the reduction in CBX3 protein levels35.

LNP
A cysteine-to-serine substitution at position 282 of the mouse Fbp1 gene was introduced by site-directed mutagenesis. The mutated Fbp1 gene was amplified by standard PCR, which included initial denaturation, followed by cycles of denaturation, annealing, and extension steps. The PCR product was excised from an agarose gel and purified using a commercial DNA gel extraction kit. The purified DNA was then double-digested, along with a T7 promoter-driven vector, using appropriate restriction enzymes. After recovery, the digested fragments were ligated using T4 DNA ligase. The ligation product was transformed into competent E. coli cells, and positive colonies were selected on antibiotic-containing plates. Single colonies were cultured, and the plasmid DNA was extracted and verified by Sanger sequencing. The confirmed recombinant plasmid was linearized and used as a template for in vitro transcription. The resulting mRNA was purified using an mRNA purification kit.
For LNP formulation, the aqueous phase was prepared by diluting mRNA to a concentration of 0.35 mg/mL in 100 mM sodium acetate buffer (pH 4.0). The organic phase was prepared by dissolving the lipid mixture in ethanol to a final concentration of 25 mM. The lipid composition was as follows: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate: distearoylphosphatidylcholine: cholesterol: [1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine]-N-[methoxy(polyethyleneglycol)-2000] (50:10:38.5:1.5, mol%). LNPs were assembled using an Ignite microfluidic chip (Precision Nanosystems) with a PNI Ignite instrument at a flow rate ratio of 3:1 (aqueous to organic phase) and a total flow rate of 12 mL/min.

Animal
All experimental procedures were approved by the Animal Use and Care Committee of the Second Xiangya Hospital, Central South University (Approval No. 20250042) and were conducted in accordance with the ARRIVE Guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The following mice were used in this study: C57BL/6, C57BLKS/J-leprdb/leprdb (db/db), Fbp1−/+ (heterozygous Fbp1 knockout, C57BL/6JGpt background, GemPharmatech Co., Ltd; derived from C57BL/6JGpt-Fbp1em1Gpt). The animals were generated, housed, and bred under a 12 h light/dark cycle at a temperature of 22±2°C and 55±5% relative humidity, with free access to food and water throughout the study. The standard chow diet used was Catalog No. YNDWSL01-TJ, and the high-fat diet for DMED model induction was Catalog No. D12492 (Medison).
A mouse model of DMED was induced by a high-fat diet combined with streptozotocin administration66. Male C57BL/6 mice were acclimatized for 2 weeks prior to random assignment into control or experimental groups. All groups were rigorously age-matched to minimize confounding effects. Mice in the experimental group received a high-fat diet for four weeks, followed by intraperitoneal injections of streptozotocin (50 mg/kg/day) for 5 consecutive days. Diabetes was confirmed when blood glucose levels exceeded 16.7 mmol/L on three consecutive measurements. Diabetic mice were maintained on a high-fat diet for an additional 8 weeks to promote the development of ED. Apomorphine was dissolved in 0.9% sodium chloride solution containing 0.2 mg/mL ascorbic acid and administered subcutaneously at 100 μg/kg. Erectile responses were recorded within 30 min post-injection. Mice that did not exhibit penile erection were classified as having DMED. Of the 220 C57BL/6 mice initially enrolled, 24 died due to severe hyperglycemia or procedural complications, yielding 196 surviving diabetic mice. Among these, 181 met the DMED criteria (absence of erectile response to apomorphine) and were randomly assigned to treatment subgroups: control (normal saline, i.p.), 3-MA (10 mg/kg/day, p.o.), SMER28 (50 μg/kg/day, i.p.), Chaetocin (0.2 mg/kg every three days, i.p.), EPZ6438 (250 mg/kg/day, p.o.), H-PROTAC (5 mg/kg/week, intracavernosal), sodium oxamate (0.5 g/kg/day, i.p.), AAV9 empty vector, AAV9-Zdhhhc13KD (3×10¹³ vg/mL, intracavernosal), AAV9-Sox9KD (3×10¹³ vg/mL, intracavernosal), LNP control, and Fbp1 C282S-LNP (1 mg/mL/week, intracavernosal). Four weeks post-treatment, we evaluated cavernosal lactate levels, ICP, and histopathological changes.
A type 1 diabetes model was induced as previously described67. Eight-week-old male C57BL/6 mice were fasted for 12 h and then given a single intraperitoneal injection of streptozotocin (150 mg/kg). Diabetes was confirmed 72 h post-injection by fasting blood glucose >16.7 mmol/L. Mice were maintained for eight additional weeks before erectile function was assessed via the apomorphine test.
Additionally, 20-week-old male db/db mice (C57BLKS/J-leprdb/leprdb) were obtained from Shulaibao Biotechnology (Wuhan, China). Fbp1−/+ mice were generated by Shulaibao Biotechnology (Wuhan, China). For lactate exposure experiments, 8-week-old male C57BL/6 mice were randomly divided into three groups receiving intraperitoneal injections of normal saline, lactate (0.25 g/kg), or lactate (0.5 g/kg) for 8 weeks. Control mice were strictly age-matched and underwent the same standardized procedures.

ICP
The ICP was measured using the MP160 system and BL-420F Functional Biological Experiment System (China). After anesthesia induction with isoflurane, the penile crura and cavernous nerves were bilaterally exposed. A 24-gauge needle was inserted into one crus, and the ipsilateral cavernous nerve was stimulated using a bipolar hook-shape electrode. ICP recordings were obtained via a pressure transducer connected to the needle63.

Hematoxylin-Eosin (H&E) and Masson staining
For H&E staining, dewaxed and rehydrated tissue sections were stained with hematoxylin for 5 min, differentiated in 1% hydrochloric acid-ethanol for 1 min, blued in ammonia water for 1 min, and counterstained with eosin for 1 min. The stained samples were then dehydrated, mounted, and examined under a microscope. For Masson staining, sections were sequentially treated with hematoxylin for 5 minutes, Ponceau acid-fuchsin for 6 min, 1% phosphomolybdic acid for 1 min, and 2.5% aniline blue for 2 min, followed by rinsing and differentiation with 1% glacial acetic acid. Morphometric analysis was conducted using ImageJ software on three randomly selected non-overlapping fields from each section. In H&E-stained sections, well-defined structural regions were imported into ImageJ, and the threshold function was applied with manual adjustment to distinguish luminal spaces from parenchymal areas based on grayscale intensity. In Masson-stained sections, red-stained areas were identified as muscle fibers, while dark blue regions indicated collagen fibers; both were quantified sequentially using threshold-based segmentation. All analyses were performed by two independent investigators blinded to group assignments, and result were cross-validated with mean values used for statistical analysis.

Immunofluorescence (IF) and immunohistochemistry (IHC)
For IF, samples were permeabilized with Triton X-100, blocked with goat serum, and incubated with primary antibodies at 4°C overnight, followed by incubation with fluorophore-conjugated secondary antibodies and DAPI at room temperature. For IHC, paraffin-embedded sections were dewaxed in xylene, rehydrated in a graded ethanol series, and subjected to heat-induced antigen retrieval. After blocking with 5% goat serum, sections were incubated with primary antibodies at 4°C overnight, followed by incubation with biotinylated secondary antibodies, ABC reagent, and DAB chromogenic detection. All stained sections were visualized using a fluorescence or bright-field microscope, respectively. For quantitative analysis, three non-overlapping fields per sample were randomly selected and examined using ImageJ software by two independent investigators blinded to the experimental conditions.

Pharmacokinetic studies and tissue distribution
The intracavernosal injections of the test agent (0.25 mg/kg) were administered to C57BL/6 mice, with blood samples gathered at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h post-injection. These blood samples were then centrifuged at 11,000 × g for 10 min at 4°C. A 50 μL portion of serum was combined with 150 μL methanol, followed by 10 min of sonication on ice. The resulting supernatant underwent high-performance liquid chromatography analysis, with pharmacokinetic parameters derived from these analytical results. For characterizing the tissue distribution of LNPs, healthy C57BL/6 mice received intracavernosal injections of DiD-labeled LNPs that encapsulated Fbp1 C282S mRNA. At 0.25, 1, 4, and 12 h after injection, the mice were euthanized, and tissues, including the corpus cavernosum, liver, skeletal muscle, spleen, and brain, were collected. Fluorescence intensity in the supernatants of these tissues was measured using a multimode microplate reader68.

RNA sequencing and bioinformatic analysis
Total RNA was isolated from primary mouse CCSMCs under control and high-lactate conditions (10 mM) using TRIzol Reagent (Thermo Fisher Scientific). RNA concentration and purity were measured on a Nanodrop spectrophotometer, with A260/A280 ratios between 1.8 and 2.0 deemed acceptable. RNA integrity was assessed using an Agilent Bioanalyzer 2100 system, and samples with an RNA integrity number (RIN) ≥ 7.0 were subjected to sequencing. Sequencing was performed by Shanghai Jiayin Biotechnology Co., Ltd. on the Illumina NovaSeq platform under paired-end 150 bp mode. After quality control (including adapter trimming and low-quality read filtering via FastQC), differential gene expression analysis was conducted using DESeq2 in RStudio (v1.4). KEGG pathway enrichment analysis was performed using clusterProfiler, and results were visualized using barcharts.

Statistical analysis
All experimental data were analyzed using GraphPad Prism 7.0 software. Comparisons between two groups were conducted using an unpaired two-sided Student’s t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript explores the relationship between lactate metabolism and diabetes-related erectile dysfuncton(DMED), especially the role of lactate in the pathogenesis of DMED. The study found that blood glucose, serum lactate and penile lactate levels were significantly increased in diabetic mice, and after lactate supplementation, the function of penile smooth muscle cells (CCSMC) was significantly impaired, showing a shift from a contractile phenotype to a synthetic phenotype. Further studies found that downregulation of Fbp1 (gluconeogenic enzyme) led to blockage of the gluconeogenic pathway and accumulation of lactate in penile tissue, thereby affecting erectile function. The study also revealed the role of histone methylation and fatty acylation (S-palmitoylation) in the regulation of Fbp1 function, especially Zdhhc13 promoted palmitoylation of Fbp1 through its acyltransferase activity, thereby impairing cell function. Although inhibition of Fbp1 acylation or histone methylation failed to completely restore erectile function in DMED mice, the development of a Fbp1-C282S mRNA-encapsulated lipid nanoparticle (LNP) treatment regimen successfully reduced lactate levels and significantly improved erectile function in the DMED mouse model, showing new therapeutic prospects. Finally, the study pointed out that there is still room for optimization of this treatment strategy, and the possibility of more drug combination therapy should be explored in the future. It has a scientific value in this field. But there are still some issues and questions that need to be addressed. Lacks of study in human erectile tissues (this is very important because, as far as I know, there is no study showing the relationship between lactate and ED in human erectile tissue) and absence of data related to other important cellular components of erectile tissue, such as endothelial cells, pericytes, and neuronal cells, are a major limitation of this study.\n1. Was there a specific time-point of lactate accumulation in the corpus cavernosum during the progression of DMED?\n2. Lactate supplementation: How do the doses of lactate given (0.25 g/kg/d and 0.5 g/kg/d) relate to physiological levels? Are these doses intended to mimic pathological conditions? Are there data on how lactate affects healthy tissues? How should the dose be chosen?\n3. Clinical relevance of lactate measurements: While the study focuses on lactate accumulation in the corpus cavernosum, how directly do these findings correlate to human DMED? Are plasma lactate levels in DMED patients also elevated to the same extent as observed in the mice?\n4. Role of Zdhhc13-mediated palmitoylation: The role of Zdhhc13 in Fbp1 palmitoylation and its subsequent impact on gluconeogenesis is intriguing. Could the manuscript expand on the mechanism by which palmitoylation at C282 disrupts Fbp1 function? Is there a pathway implicated in the degradation or inactivation of Fbp1 upon palmitoylation?\n5. Therapeutic potential of Fbp1 rescue: Fbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNP) are proposed as a potential therapeutic strategy. Can the manuscript include more detailed pharmacokinetic or pharmacodynamic data on the efficacy of these nanoparticles in vivo, including potential off-target effects?\n6. Histological analysis: More specific histological analysis (e.g., immunohistochemistry or immunofluorescence) could help validate the effects of lactate on smooth muscle function and fibrosis. Were markers of fibrosis (e.g., collagen type I) assessed in the cavernosal tissue?\n7. Comparative analysis of other gluconeogenic enzymes: Given the role of Fbp1 in gluconeogenesis, it might be beneficial to compare the expression of other key gluconeogenic enzymes (such as G6PC, PCK1) in the corpus cavernosum of DMED mice to ensure that the observed effects are Fbp1-specific and not part of a broader metabolic dysfunction.\n8. The introduction could benefit from a clearer explanation of the significance of Fbp1 in diabetic erectile dysfuncton(DMED). Why is Fbp1 a key player in the pathophysiology of DMED? Why other targets, such as Hk2, Pklr, Pck1 were not select? These targets expression pattern looks similar with Fbp1.\n9. While Cbx3 is shown to interact with H3K9me3, what are its specific effects on Fbp1 function in terms of cellular metabolism or other pathways? Could metabolic assays help elucidate its role further?\n10. Although Fbp1-C282S-LNP improves various functional parameters in DMED mice, the manuscript could benefit from a more detailed discussion of how this strategy compares to traditional therapeutic interventions for DMED. Could this approach be combined with other treatment modalities?\n11. Fbp1 knockout mice are used in several experiments; however, the role of Fbp1 in other tissues or systemic effects (e.g., liver or muscle) should be considered. Could the observed effects be specific to the corpus cavernosum or systemic in nature?\n12. Lactate Accumulation and Erectile Dysfunction: The link between lactate accumulation and erectile dysfunction is mentioned, but the underlying cellular mechanisms are not fully explained. Could the authors elaborate on how lactate directly impairs CCSMC and erectile function at the molecular or cellular level?\n13. LNP-Based Therapy: The use of LNPs for delivering the Fbp1-C282S mRNA is promising, but the manuscript would benefit from a deeper discussion of how LNPs are taken up by specific cell types, particularly in cavernosal tissue. What are the advantages of LNP delivery over other methods?\n14. Limitations in Cell-Type Specificity: The study focuses on the effect of Fbp1 on CCSMCs, but the corpus cavernosum contains various cell types. How might Fbp1-C282S affect other cells like endothelial cells, fibroblasts, pericytes, or macrophages? Would the delivery system need to target these cells specifically?\n15. In Vivo Validation of mRNA Strategy: While the Fbp1-C282S mRNA strategy shows promising outcomes, did the authors observe any long-term effects or changes in other metabolic pathways? How sustained are the improvements in erectile function over time?", "reviewer_2": "This investigation is concerned with the impact of diabetes on erectile dysfunction. The authors specifically investigate lactate accumulation, the causes of this accumulation, and its impact on the function of the corpus cavernosum. They propose that transcriptional and functional repression of Fbp1 is the cause of the lactate accumulation, and investigate strategies to correct Fbp1 activity and erectile function.\nMajor concerns – models and approaches\n1. The experimental model of diabetes is extreme. A blood glucose concentration of 30mM is exceptionally high and would be fatal in humans. It is important to establish whether the findings are consistent in a model system that achieves a clinically relevant blood glucose concentration.\n2. The in vitro models (e.g. Figure 1O) use extracellular lactate concentrations of 5mM and 10mM which massively exceed the plasma lactate concentrations observed in the animal model (~3mM). Why?\n3. Glucose concentrations in standard culture media are often supra-physiological. Please explain which concentrations of glucose were used for the in vitro experiments.\n4. The DMED mice are a highly selected group. Mice are first treated to induce hyperglycaemia, and then erectile function is tested. Only those mice that show ED are investigated in the DMED group. What fraction of the hyperglycaemic group is this?\n5. All diabetic mice receive injections of streptozotocin (daily for 5 days) and apomorphine. Were control mice injected with vehicle? There are established impacts of stress (e.g. by daily injections) on plasma lactate that must be controlled for in these experiments.\n6. The description of the acyl biotin exchange assay (line 615) is not adequate. What buffer was used for lysis? What cleanup steps were done to eliminate ‘surplus reagents’ (e.g after biotinylation)? What was the final concentration of hydroxylamine in the assay? Why was DTT absent from SDS PAGE buffer?\nMajor concerns – figures\n1. Statistical analysis. Throughout – the legends should indicate the names of the statistical tests used. When reporting values in the main text either state ‘significantly different / not different’ OR state values, measure of variation, or units (for example – lines 107 currently simply says ‘0.47 VS. 0.52 VS. 0.76’, line 108 currently says 116.3 VS. 103.7 VS. 56.3 – needs units and SD / SEM). In the methods section, please state whether one-tailed or two-tailed statistical tests were performed. Where ANOVA results are reported, please provide the F values and degrees of freedom. Where t-test results are reported, please provide the t-values and degrees of freedom.\n2. Fig 2A-2D. The lack of detail in the legend makes it very difficult to understand what this represents. What are N1, T1, etc? Same problem in Fig 5K.\n3. Fig 3V appears to show that a ‘PROTAC’ has been developed that induces degradation of Cbx3. No experimental evidence is presented that this molecule binds to Cbx3. The characterisation in Fig S3 is inadequate. What is the in vivo bioavailability / half life of this reagent (Fig 4)?\n4. Palmitoylation assays should be presented with a palmitoylated housekeeping protein as a control for the assay (e.g. throughout Fig 5).\n5. No quantification of palmitoylation assays is presented making it impossible to draw conclusions about whether any changes in palmitoylation described are statistically significant (e.g. throughout Fig 5).\n6. The molecular model presented in Fig 5F is meaningless. No information is provided about how this was generated. It is impossible to distinguish which protein is which, how they are interacting, etc.\n7. zDHHC13 is a polytopic membrane protein, which are notoriously difficult to purify in an intact, functional state. Only zDHHC20 and zDHHC9 from this family have been purified enzymatically competent (PMIDs: 29326245, 38182928). It is highly unlikely that GST-zDHHC13 (purified from what expression system?) retains catalytic activity during purification in RIPA buffer (Fig 5R).\n8. Fig 6B – palmitoylation anchors proteins to membranes. Why does the amount of Fbp1 in the cytosolic fraction not change when zDHHC13 is silenced? Fig 5 indicated reduced palmitoylation of Fbp1 in the absence of zDHHC13, which would reduce its membrane association.\nMinor concerns – figures\n1. Fig 1D – colour code the individual points on this graph red and blue to indicate which group of mice each data point belongs to.\n2. Fig 1E – 1N: it is not clear from the text or the legend accompanying the figure whether these experiments were conducted in healthy mice or DMED mice.\n3. Fig 1G – what does the x axis in these traces represent? Please add a title (in addition to units) to the y axis.\n4. The description of the analysis of images such as Fig 1I, 1K, 2K, 7T (and others) is not adequate. How were these images analysed? Were investigators blinded during analysis?\n5. Fig 1P – how was viability assessed? This appears to show cell growth not viability.\n6. Fig 1Q, 2P, 2S (and other figures) – the scale bar is incorrectly labelled 200x.\n7. Fig 3I, 3K, 3M – these graphs are incorrectly labelled mRNA, they appear to show quantification of the western blots.\n8. Quality of some western blots is poor. For example – Fig 3L H3K9, Fig 4N Fbp1, Fig 4O H3K27: the intensity of the bands changes from left to right, indicative of poor / inconsistent transfer.\n9. Fig 4P – 4U: in the description of these data the authors should state the normal range values for lactate and ICP to emphasise that the correction they have achieved is modest at best.\n10. Fig 5 – change the title of this figure. There is no data about Fbp1 function in this figure.\n11. Fig 5R – why is there beta actin in these samples? They are purified recombinant proteins.\n12. Fig 6C – 6N: the authors should show that the effects of expressing zDHHC13 on glucose consumption & lactate production occur in the presence of Fbp1 but not in the presence of C282S Fbp1.\n13. Fig 7H – quantification of Fbp1 expression is missing.\n14. Figure S3C – please include a description of what the numbers on the right of the graph refer to.\nOther comments:\n1. In the abstract the authors state that the “Suv39h1-mediated H3K9 trimethylation and Ezh2-dependent H3K27 trimethylation synergistically suppressed Fbp1 transcription”. In the manuscript, evidence is provided for each of these repressing transcription individually, however no evidence is presented to support “synergistic effects”. Does simultaneously increasing (or decreasing) Suv39h1-mediated H3K9 trimethylation and Ezh2-dependent H3K27 trimethylation cause additional modulation of Fbp1 transcription compared to increasing (or decreasing) only one of these?\n2. How realistic is Fbp1 or lactate accumulation as a therapeutic target? Is modulating processes involved in cellular metabolism likely to be associated with undesired effects? Data presented from MOVAS cells (derived from mouse aortic smooth muscle) indicates that the effects of Fbp1 modulation are very similar to those in the CCSMCs. Very similar effects in both cell types would presumably mean that interventions targeting or involving Fbp1 would risk off target effects in vascular smooth muscle cells, and that therapeutic drugs would have to be very selectively targeted to CCSMCs.\n3. At various points throughout the results section, reference is made to ‘increases’ or ‘decreases’ in various measures in some groups compared to other groups. ‘Increases’ or ‘decreases’ implies that measurements have been made from the same group before and after an intervention. When comparing values between different groups, please use ‘higher’ or ‘greater’ and ‘lower’ instead to describe differences. Please adjust this in lines 107, 100, 108, 113, 162, 163, 212, 217, 232 and any other instances.\n4. zDHHC13 is a Golgi-localised enzyme (PMIDs: 16647879, 19299482), yet Fbp1 appears to be present in the plasma membrane and the nuclear membrane. How is it possible that Fbp1 interacts with and is a substrate for zDHHC13 when they are expressed in different cellular compartments?\n5. Fbp1 palmitoylation has been reported before by others: https://swisspalm.org/proteins/Q9QXD6 . Other palmitoylation sites have been described in this protein (C39, C93). How do you account for mutation of only C282 abolishing palmitoylation?\n6. What was the dosing frequency of the LNP (line 728)?\n7. Were control and DMED mice age-matched? Please include the ages of mice used for these experiments.", "reviewer_3": "In the present study the authors suggest that excessive lactate accumulation in the corpus cavernosum is a key pathological driver of DMED, promoting structural degeneration, smooth muscle cell loss, and erectile dysfunction in diabetic mice. Mechanistically, they show that transcriptional repression and functional impairment of Fbp1 (fructose-1,6-bisphosphatase 1, Fbp1), a rate-limiting gluconeogenic enzyme, underlined corpus cavernosum lactate pathological accumulation. Epigenetic profiling revealed that Suv39h1-mediated H3K9 trimethylation and Ezh2-dependent H3K27 trimethylation synergistically suppressed Fbp1 transcription. In addition, Zdhhc13-mediated palmitoylation at the Fbp1 C282 site further disrupted its gluconeogenesis related function, aggravating lactate accumulation. To rescue these defects, the authors engineered Fbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNP), which restored Fbp1 expression and abrogated pathological palmitoylation in the corpus cavernosum, improving erectile function in DMED mice.\nMain points\n1-The authors suggested that, compared with the normal mice, the Fbp1 knockout significantly increased the cavernosal lactate and decreased the ICP, the lumen--to--parenchyma ratio, and the α--Sma fluorescence intensity (Figure 2H -N). Would the administration of LDH inhibitors be beneficial in this condition (Fbp1 knockout mice with increased Lactate production)?\n2- The authors show by ChIP qPCR assay that H3K9me3 or H3K27me3 were enriched on the promoter of Fbp1 in the MOVAS cells and primary mouse CCSMCs (Figure 3B, C, Supplementary Figure 1A, B). The promoter should be better epigenetically characterized by verifying H3K4me3, H3K27ac, DNA methylation and members of the PRC1 and 2 complexes as well as the active pol 2.\n3- In Fig 3 D-E, the authors showed that the enrichment of H3K9me3 or H3K27me3 were significantly increased in the corpus cavernosum of DMED mice compared to normal mice (Figure 3D 3E). Is this regulation specific of the corpus cavernosum? Is it also reported in patients? Is it also at the RNA level? Many more epi-regulators target H3K9me3 or H3K27me3 in respect of the onec shown. How is their expression?\n4- The authors employed PROTAC technology to synthesize a Cbx3 specific inhibitor by spatially bridging E3 ligase and the target protein, thereby inducing ubiquitination mediated degradation of Cbx3 (Supplementary Figure 3A, K). In the methods the authors give a very short description: “PROTAC In designing a Cbx3 targeting PROTAC, we used a molecular bridging strategy [31]. One ligand, the H3K9me3 peptide, binds Cbx3 due to its specific recognition ability. The other, Pomalidomide, attaches to the E3 ubiquitin ligase CRL4CRBN, widely used in PROTACs for promoting target protein degradation. The bridge molecule, with optimized length and rigidity, links these two ligands to ensure effective binding and maintain activity\". From this description, the strategy might also degrade other binders. The specificity and selectivity of the characterization should be shown in depth. Please also include dose curves and IC50.\n5- The authors applied H PROTAC, Chaetocin, and EPZ6438 to the mice with DMED or Fbp1 knockout. The 3 inhibitors decreased the cavernosal lactate, partially restored the ICP, the structure and CCSMC content of corpus cavernosum in DMED mice but had no effect on the Fbp1-knockout mice (Figure 4P, U). Would a combinatory use of the inhibitors display a better therapeutic value for DMED mice?\n6- The authors report CBX3 upregulation in the DMED mice (Fig.3P). Which is the mechanism(s) behind this deregulation?\n7- The transcriptome and immunohistochemistry of the corpus cavernosum showed that the expression of Zdhhc13 was increased in DMED mice (Figure 5K, L), suggesting that Zdhhc13 may be involved in the palmitoylation of Fbp1 in vivo. Why is Zdhhc13 overexpressed? Is it tissue specific or?\n8- The Fbp1 C282S LNP reduced the cavernosal lactate, improved the ICP and the structure and CCSMC content, both in DMED and Fbp1 knockout mice (Figure 7Q, V) supporting that the Fbp1 C282S LNP is a promising therapeutic strategy for DMED. Were there detected collateral effects for Fbp1 C282S LNP? Was a Pharmacokinetic and a pharmacodynamic approach be performed?\n9- The methods section should be deeply revised, implementing all details. If there is space limitation, likely it might go into the additional materials.\n10- All raw data (images, uncropped blots etc) should be uploaded as additional material.\n11-English grammar and typos need deep revision.\nMinor points\nsome examples....\n• Lane 25 please correct underlied\n• Lane 28 please correct in addation…\n• Lane 204 please check… histone methyltrantion…\n• Lane 380 arase one of the 2 significantly….", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The study is interesting and consistent with recent data showing high lactate level in patients with diabetes. The authors have performed an extensive series of studies to demonstrate that fructose-1,6-bisphosphatase 1 (Fbp1) was reduced in a model of diabetes induced by high fat diet and STZ. The reduction of Fbp1 was associated with palmitoylation via Zdhhc13. Restoration of Fbp1 via a lipid nanovesicle injection directly into the corpus cavernosum restored erectile dysfunction.\nMajor comments\n1. Additional models of diabetes such as db/db mice and type 1 models should be evaluated as well as patient samples\n2. No discussion of endothelial dysfunction was described which has been previously found to play a key role in human erectile dysfunction.\n3. The results suggest that Zdhhc13 mediated S-palmitoylation on Fbp1 decreases its gluconeogenesis function, but the therapeutic potential of Zdhhc13 inhibition in DMED mice remains limited. It is possible that Fbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNP) can restore Fbp1 expression and abrogated pathological palmitoylation in the corpus cavernosum and restore erectile dysfunction. However priapism should be avoided and different models should be tested."}, "author_response": "Response to Reviewers\nWe sincerely thank the Editors and Reviewers for their thoughtful comments and\nconstructive suggestions on our manuscript entitled “Rescuing the depalmitoylated\nFBP1 ameliorates diabetes mellitus-induced erectile dysfunction through decreasing\nthe cavernosal lactate.” We have carefully revised the manuscript according to all the\ncomments, and we believe that the revisions have substantially improved the clarity,\nrigor, and impact of our work. Below, we provide a detailed, point-by-point response\nto each comment. All changes in the revised manuscript are highlighted.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript explores the relationship between lactate metabolism and\ndiabetes-related erectile dysfuncton(DMED), especially the role of lactate in the\npathogenesis of DMED. The study found that blood glucose, serum lactate and penile\nlactate levels were significantly increased in diabetic mice, and after lactate\nsupplementation, the function of penile smooth muscle cells (CCSMC) was\nsignificantly impaired, showing a shift from a contractile phenotype to a synthetic\nphenotype. Further studies found that downregulation of Fbp1 (gluconeogenic\nenzyme) led to blockage of the gluconeogenic pathway and accumulation of lactate in\npenile tissue, thereby affecting erectile function. The study also revealed the role of\nhistone methylation and fatty acylation (S-palmitoylation) in the regulation of Fbp1\nfunction, especially Zdhhc13 promoted palmitoylation of Fbp1 through its\nacyltransferase activity, thereby impairing cell function. Although inhibition of Fbp1\nacylation or histone methylation failed to completely restore erectile function in\nDMED mice, the development of a Fbp1-C282S mRNA-encapsulated lipid\nnanoparticle (LNP) treatment regimen successfully reduced lactate levels and\nsignificantly improved erectile function in the DMED mouse model, showing new\ntherapeutic prospects. Finally, the study pointed out that there is still room for\noptimization of this treatment strategy, and the possibility of more drug combination\ntherapy should be explored in the future. It has a scientific value in this field. But\nthere are still some issues and questions that need to be addressed. Lacks of study in\nhuman erectile tissues (this is very important because, as far as I know, there is no\nstudy showing the relationship between lactate and ED in human erectile tissue) and\nabsence of data related to other important cellular components of erectile tissue, such\nas endothelial cells, pericytes, and neuronal cells, are a major limitation of this study.\n1. Was there a specific time-point of lactate accumulation in the corpus\ncavernosum during the progression of DMED?\nResponse: We sincerely appreciate the reviewer for raising this important question\nregarding the temporal dynamics of lactate accumulation in corpus cavernosum—a\ncore aspect our study aimed to elucidate. Your insightful comment has provided\nvaluable guidance for improving the presentation of our results.\nIn direct response to your inquiry, our experimental design specifically incorporated\nlongitudinal assessments to capture the dynamic process of cavernosal lactate\naccumulation. Using an established Type 2 DMED mouse model validated in previous\nstudies (e.g., PMID: 37217862; PMID: 37445656), we systematically measured\ncavernosal lactate concentrations at four time points: baseline (Week 0), Week 4,\nWeek 8, and Week 12 post-modeling. Our results demonstrated a progressive,\ntime-dependent increase in cavernosal lactate accumulation, which correlated\ninversely with declining intracavernosal pressure. These data are now clearly\npresented in Supplementary Figure 1 and described in the revised Results section 1.\nWe have revised the manuscript to more explicitly highlight the temporal relationship\nbetween metabolic changes and functional impairment in DMED progression. Thank\nyou again for this constructive suggestion, which has significantly strengthened our\ndata presentation.\n2. Lactate supplementation: How do the doses of lactate given (0.25 g/kg/d and\n0.5 g/kg/d) relate to physiological levels? Are these doses intended to mimic\npathological conditions? Are there data on how lactate affects healthy tissues?\nHow should the dose be chosen?\nResponse: We sincerely thank the reviewer for raising these critical questions\nregarding our lactate supplementation approach. Their comments have helped us\nclarify the rationale behind our dose selection and improve the interpretation of our\nresults.\nIn response, we would like to explain that the lactate doses used (0.25 and 0.5 g/kg/d)\nwere specifically chosen to replicate pathological lactate levels observed in our\ndiabetic mouse model, rather than physiological conditions. Our data demonstrated\nsignificantly elevated lactate concentrations in both serum and corpus cavernosum\ntissue in diabetic mice, with a positive correlation between these compartments\n(Figure1).\nTo determine the appropriate dosing regimen, we first consulted previous studies\ninvolving lactate supplementation(PMID:38925041). We then conducted pilot\nexperiments testing three doses (0.25, 0.5, and 0.75 g/kg/d). Results revealed that 0.5\ng/kg/d significantly increased the serum lactate exposure compared to the lower dose,\nwhile the highest dose provided no further increase. Using our established correlation\nbetween serum and cavernosal lactate levels, we found that 0.5 g/kg/d effectively\nelevated cavernosal lactate to the pathological range observed in DMED mice. These\npreliminary data supported the selection of the two doses(0.25 and 0.5 g/kg/d) for\nformal experiments, as they allowed us to mimic pathological conditions while\navoiding potential non-specific effects at higher doses. These data are now clearly\npresented in Supplementary Figure 1 and described in the revised Results section 1.\nSubsequent experiments confirmed that exogenous lactate administration resulted in\ndose-dependent impairments in erectile function in mice and corpus cavernosum\nsmooth muscle cells (Figure1).\nWe thank the reviewer again for these insightful questions, which have significantly\nimproved the rigor and clarity of our experimental design.\n3. Clinical relevance of lactate measurements: While the study focuses on lactate\naccumulation in the corpus cavernosum, how directly do these findings correlate\nto human DMED? Are plasma lactate levels in DMED patients also elevated to\nthe same extent as observed in the mice?\nResponse: We sincerely appreciate your thoughtful questions regarding the clinical\nrelevance of lactate measurements. The issues you raised—specifically, the\nassociation between corpus cavernosum lactate accumulation in mice and human\nDMED, as well as the characteristics of lactate changes in DMED patients—are\ncentral to validating the clinical significance of our study and crucial for strengthening\nthe translational value of our findings. Below, we provide a point-by-point response\nintegrating both clinical data and experimental details.\nTo directly address the relationship between corpus cavernosum lactate accumulation\nin mice and human DMED, we first obtained approval from the Clinical Ethics\nCommittee (LYEC2025-0188). We then recruited 60 DMED patients—classified as\n33 mild, 19 moderate, and 8 severe cases—along with 60 healthy controls, carefully\nmatching for confounders such as age. Serum lactate levels were systematically\nmeasured in all participants. The results revealed significantly higher serum lactate\nlevels in DMED patients (2.4 mmol/L) compared to healthy controls (1.7 mmol/L; p =\n0.0002). This observation of elevated lactate aligns with the increased lactate levels\nobserved both in serum and corpus cavernosum tissue in our DMED mouse model,\nthereby offering clinical support for the role of lactate in the pathophysiology of\nDMED.\nIt should be noted that due to ethical constraints and practical challenges in procuring\nhuman corpus cavernosum tissue, direct measurement of local lactate concentrations\nin DMED patients was not feasible. However, in our mouse model of DMED, we\nidentified a significant positive linear correlation between plasma lactate and corpus\ncavernosum lactate levels. This correlation provides an important basis for inferring\nthat local lactate concentrations within the corpus cavernosum of DMED patients may\nalso be elevated, further supporting the translational relevance of our animal findings\nto human disease.\nTo further assess the clinical association between lactate levels and DMED, we\nstratified all 120 participants into three tertiles based on serum lactate levels: lower\nthird (<1/3), middle third (1/3–2/3), and upper third (>2/3). In the overall cohort, the\nincidence of erectile dysfunction (ED) exhibited a significant increasing trend across\nrising lactate tertiles (p=0.002). The incidence of ED in the upper third was\nsignificantly higher than that in the lower third (p=0.001; Cramer’s V = 0.376).\nPost-hoc pairwise comparisons were performed using the chi-square partition method\nwith a corrected significance level of α' = 0.017.\nAmong DMED patients specifically, the incidence of moderate-to-severe ED also rose\nwith increasing lactate levels (p=0.014). Notably, the rate of severe ED was\nsignificantly higher in patients within the upper lactate tertile compared to those in the\nlower tertile (p=0.004; Cramer’s V=0.376). These results not only reinforce the\nassociation between elevated lactate and DMED but also demonstrate a dose-response\nrelationship wherein higher lactate levels correlate with greater disease severity,\nthereby enhancing the clinical applicability of our conclusions.\nIn summary, our clinical data support the involvement of lactate in DMED\npathophysiology through several lines of evidence: consistency between animal and\nhuman lactate trends, stratification analysis confirming a dose-dependent relationship,\nand extrapolation from plasma-cavernosal lactate correlation in mice—compensating\nfor the inability to directly measure human tissue lactate. These aspects have been\nincorporated into Figure 1 and Supplementary Figure 1 of the revised manuscript.\nWe are truly grateful for your insightful comments, which have prompted us to more\nclearly articulate the clinical implications of our work and further improve the rigor\nand persuasiveness of our conclusions.\n4. Role of Zdhhc13-mediated palmitoylation: The role of Zdhhc13 in Fbp1\npalmitoylation and its subsequent impact on gluconeogenesis is intriguing. Could\nthe manuscript expand on the mechanism by which palmitoylation at C282\ndisrupts Fbp1 function? Is there a pathway implicated in the degradation or\ninactivation of Fbp1 upon palmitoylation?\nResponse:\nWe sincerely appreciate your valuable comments regarding the mechanism by which\nC282 palmitoylation disrupts Fbp1 function, as well as the potential involvement of\ndegradation or inactivation pathways. Your questions are essential for further\nelucidating the molecular mechanism of Zdhhc13-mediated palmitoylation. Based on\nour experimental data, we have organized the following response:\nFbp1 is a rate-limiting enzyme in lactate metabolism. Previous studies have\nestablished that residue G260 within its catalytic site is critical for enzymatic\nactivity—any disruption at this site impairs Fbp1 function and leads to lactate\naccumulation (PMID: 20151204, 28720574). In this study, multiple experimental\napproaches, including glucose consumption, lactate production, and Seahorse\nextracellular flux analyses, consistently demonstrated that Zdhhc13-mediated\npalmitoylation at C282 significantly impairs the gluconeogenic function of Fbp1\n(Figures 6). To investigate the mechanism, we first excluded potential effects on\nprotein stability and subcellular localization, as palmitoylation at C282 did not alter\nFbp1 degradation or localization (Figures 6A–6B). We therefore proposed that\nstructural perturbation may underlie the loss of function.\nSimulation of palmitoylated Fbp1 using PyMOL revealed that modification at C282\nreduces the solvent-accessible surface area (SASA) of the catalytic residue G260 by\napproximately 20% (from 3.296 Å² to 2.657 Å²), while the total SASA of chain A\ndecreased by approximately 388 Å² (Supplementary Figure 8A). These results suggest\nthat palmitoylation induces a more compact conformation in Fbp1, which reduces the\nsolvent exposure of G260 and enhances local structural folding. Since G260 is\nessential for catalytic activity, its impaired accessibility likely hinders substrate or\ncofactor binding, thereby disrupting enzymatic function and gluconeogenesis.\nIn summary, our findings indicate that Zdhhc13-mediated palmitoylation of Fbp1 at\nC282 induces conformational compaction, reduces solvent accessibility of the\ncatalytic residue G260, and thereby inhibits Fbp1 enzymatic activity and\ngluconeogenic function—independent of changes in protein degradation or\nlocalization. We thank you once again for your thorough and insightful questions,\nwhich have helped us better articulate the mechanistic details of this regulatory\npathway.\n5. Therapeutic potential of Fbp1 rescue: Fbp1 C282S-loaded lipid nanoparticles\n(Fbp1 C282S-LNP) are proposed as a potential therapeutic strategy. Can the\nmanuscript include more detailed pharmacokinetic or pharmacodynamic data\non the efficacy of these nanoparticles in vivo, including potential off-target\neffects?\nResponse: We sincerely appreciate your critical questions concerning the therapeutic\npotential of Fbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNPs). In response\nto your inquiries regarding in vivo pharmacokinetics, pharmacodynamics, tissue\ndistribution, cellular uptake, and potential off-target effects, we have summarized the\nkey experimental findings below:\nTo evaluate the therapeutic efficacy of Fbp1 C282S-LNPs, we administered\nintracavernous injections to DMED mice at three dose levels (0.1 mg/kg, 0.25 mg/kg,\nand 0.5 mg/kg), along with a control group. The results indicated that the 0.1 mg/kg\ndose did not effectively reduce lactate levels in the corpus cavernosum or improve\nerectile function. In contrast, the 0.25 mg/kg dose significantly decreased lactate\naccumulation and enhanced erectile performance. The 0.5 mg/kg dose did not yield\nstatistically superior efficacy compared to the 0.25 mg/kg group, suggesting that 0.25\nmg/kg is an effective dose approaching pharmacodynamic saturation.\nMultiplex immunofluorescence staining (using Flag tag for Fbp1 C282S-LNPs, and\nmarkers for endothelial cells, smooth muscle cells, and fibroblasts: vWF, α-SMA, and\nvimentin, respectively) revealed strong LNP uptake in smooth muscle cells, with\ndetectable internalization in endothelial cells and fibroblasts. This indicates that the\nLNPs can target multiple relevant cell types within the penile tissue.\nFurther pharmacokinetic analysis via LC-MS/MS of orbital blood samples showed\nthat Fbp1 C282S-LNPs have an elimination half-life of 12.57±1.51 hours. To assess\ntissue distribution and potential off-target effects, DiD-labeled LNPs were used: the\nhighest fluorescence intensity was observed in the corpus cavernosum at 0.25 hours\npost-injection, followed by a gradual decline, confirming rapid target tissue\naccumulation and sustained local concentration. In the liver—the primary clearance\norgan—fluorescence intensity initially increased and then decreased without abnormal\nretention. Only minimal fluorescence was detected in skeletal muscle and the spleen,\nand due to the blood-brain barrier, brain fluorescence remained negligible throughout,\neffectively ruling out central nervous system exposure. Additionally, measurements of\nliver and kidney function markers (ALT, AST, serum creatinine, and blood urea\nnitrogen) showed no significant differences between treated and control groups,\nsupporting the absence of hepatorenal toxicity.\nIt is worth emphasizing that although LNPs were taken up by both penile and\nnon-penile tissues (such as liver and skeletal muscle), we believe this “non-strict\ntargeting” profile enhances rather than diminishes therapeutic efficacy through two\ncomplementary mechanisms: uptake in liver and muscle enhances systemic lactate\nmetabolism, thereby reducing lactate delivery to the corpus cavernosum; meanwhile,\nuptake within penile endothelial cells and fibroblasts contributes to local lactate\nconsumption. Thus, the broad targeting capability of LNPs supports a synergistic\ntherapeutic strategy combining local and systemic effects, with no significant adverse\neffects observed.\nAll relevant data—including dose-response outcomes, pharmacokinetic parameters,\ntissue distribution, cellular uptake, and safety assessments—have been added as\nFigure 7 and Supplementary Figure 10 in the revised manuscript. These results\ncollectively support the therapeutic potential and safety of Fbp1 C282S-LNPs. We are\ntruly grateful for your thorough and insightful comments, which have helped us\npresent these findings more comprehensively and strengthen the overall rigor of our\nstudy.\n6. Histological analysis: More specific histological analysis (e.g.,\nimmunohistochemistry or immunofluorescence) could help validate the effects of\nlactate on smooth muscle function and fibrosis. Were markers of fibrosis (e.g.,\ncollagen type I) assessed in the cavernosal tissue?\nResponse: We sincerely appreciate your valuable suggestions regarding the\nhistological analysis. Your focus on validating the effects of lactate on cavernous\nsmooth muscle function and fibrosis—particularly through immunohistochemistry or\nimmunofluorescence targeting markers such as collagen type I—has provided\nessential guidance for strengthening the evidence linking lactate to pathological\nchanges in the corpus cavernosum. Based on your comments, we have conducted\nadditional experiments and integrated the findings as follows:\nTo further investigate the role of lactate in cavernous fibrosis, we performed\nimmunofluorescence staining for collagen type I. The results demonstrated a positive\ncorrelation between the concentration of intraperitoneally injected sodium lactate and\nthe fluorescence intensity of collagen type I within cavernous tissue. This outcome\naligns with the fibrotic features previously observed via hematoxylin-eosin (HE)\nstaining and Masson’s trichrome staining, together providing compelling evidence\nthat elevated lactate levels promote cavernous fibrosis.\nFurthermore, we expanded the scope of our investigation to include type 1 diabetes,\ntype 2 diabetes, and db/db mouse models (a spontaneous type 2 diabetes model).\nIn these models, we supplemented the evaluation with assessments of collagen type I\nexpression in cavernous tissue following treatment with Fbp1 C282S-LNPs. This\nadditional analysis aims to more comprehensively evaluate the therapeutic potential of\nthis strategy in alleviating lactate-mediated cavernous fibrosis.\nWe are truly grateful for your thorough and insightful suggestions, which have guided\nus to deepen our investigation into the relationship between lactate and cavernous\ntissue damage at the level of fibrosis markers. Your comments have significantly\nenhanced the scientific soundness and rigor of our conclusions. All results from these\nsupplementary experiments have been included in the revised manuscript.\n7. Comparative analysis of other gluconeogenic enzymes: Given the role of Fbp1\nin gluconeogenesis, it might be beneficial to compare the expression of other key\ngluconeogenic enzymes (such as G6PC, PCK1) in the corpus cavernosum of\nDMED mice to ensure that the observed effects are Fbp1-specific and not part of\na broader metabolic dysfunction.\nResponse: Thank you for suggesting the comparison with other key gluconeogenic\nenzymes (e.g., G6PC and PCK1), which helps confirm the specific role of Fbp1—\nrather than general metabolic dysfunction—in lactate accumulation in the corpus\ncavernosum of DMED mice. We have conducted further investigations using\ntranscriptomic data and validation experiments, and provide the following response:\nGlucose can be rapidly metabolized to lactate via glycolysis, while lactate can be\nreconverted to glucose through gluconeogenesis to help maintain systemic glucose\nhomeostasis. Tissue lactate accumulation typically arises from one of two scenarios:\nexcessive glycolytic activation leading to increased lactate production, or impaired\ngluconeogenesis resulting in reduced lactate consumption. To identify the primary\ncause of lactate accumulation in our DMED model, we analyzed RNA-seq data from\nboth mouse and rat DMED models, focusing on key enzymes in glycolysis and\ngluconeogenesis. The results indicated that glycolytic enzymes were broadly\nsuppressed, suggesting that lactate accumulation in the corpus cavernosum is more\nlikely due to impaired lactate consumption—specifically, deficient gluconeogenic\nfunction.\nSupporting this hypothesis, transcriptomic analysis in both species revealed\nsignificant downregulation of Fbp1 at the mRNA level. Subsequent validation in\nDMED mice confirmed that Fbp1 expression was markedly reduced at both the\ntranscriptional and protein levels. In contrast, the other two key gluconeogenic\nenzymes, G6pc3 and Pck1, showed inconsistent transcriptional changes across species\n(e.g., G6pc3 was unchanged in mice but slightly upregulated in rats). Furthermore,\nqPCR and protein-level assays in DMED mice confirmed that neither G6pc3 nor Pck1\nexhibited significant alterations, effectively excluding their involvement in lactate\naccumulation.\nIn summary, our findings indicate that lactate accumulation in the corpus cavernosum\nof DMED mice results primarily from the specific downregulation and functional\nimpairment of Fbp1, rather than from a general disruption of gluconeogenic enzymes.\nWe thank you once again for your insightful suggestions, which have helped us more\nprecisely delineate the central role of Fbp1 in DMED pathology and strengthened the\nspecificity and reliability of our conclusions. The relevant data have been added to\nFigure 2 and Supplementary Figure 2 in the revised manuscript.\n8. The introduction could benefit from a clearer explanation of the significance of\nFbp1 in diabetic erectile dysfuncton(DMED). Why is Fbp1 a key player in the\npathophysiology of DMED? Why other targets, such as Hk2, Pklr, Pck1 were not\nselect? These targets expression pattern looks similar with Fbp1.\nResponse: We sincerely thank you for your insightful comments, which are\ninvaluable for refining the introduction and clarifying the central role of Fbp1 in\nDMED. In response, we have provided a concise explanation based on our research\nrationale and available evidence:\nAlthough no studies have directly linked Fbp1 to ED, we have highlighted its\npotential role in DMED in the revised introduction through two key aspects: (1) the\nmechanistic connection between impaired gluconeogenesis and lactate accumulation,\nand (2) the pathological relevance of Fbp1 deficiency-mediated lactate accumulation\nin various diseases. As the rate-limiting enzyme of gluconeogenesis, dysregulation of\nFbp1 directly impedes lactate clearance via gluconeogenesis, thereby contributing to\nrelated pathological damage.\nRegarding the selection of targets such as Hk2, Pklr, and Pck1, we previously\nprovided a detailed response (Question 6), which can be summarized as follows: Hk2\nand Pklr, both glycolytic enzymes that promote lactate production, were found to be\ndownregulated in transcriptomic data from mouse and rat models, suggesting they do\nnot drive lactate accumulation in DMED. Although Pck1 is involved in\ngluconeogenesis, its expression remained unchanged in DMED mouse tissues. Thus,\nthese targets were not selected for further investigation.\nWe are truly grateful for your suggestions, which have greatly improved the focus and\nlogical soundness of our introduction and strengthened the foundation for presenting\nour subsequent findings.\n9. While Cbx3 is shown to interact with H3K9me3, what are its specific effects on\nFbp1 function in terms of cellular metabolism or other pathways? Could\nmetabolic assays help elucidate its role further?\nResponse: We sincerely appreciate your question, which insightfully addresses the\ncentral relationship between Cbx3 and Fbp in metabolic regulation. Our experimental\nresults have clearly elucidated the underlying mechanisms and functional\nconsequences:\nSpecifically, we have demonstrated that Cbx3 transcriptionally regulates Fbp1.\nFunctional assays, including glucose consumption and lactate production\nmeasurements, further confirm that Cbx3 modulates the gluconeogenic activity of\nFbp1 through this regulatory mechanism. The process can be summarized as follows:\nupregulation of Cbx3 suppresses Fbp1 expression. Since Fbp1 serves as a key\nrate-limiting enzyme in gluconeogenesis, its downregulation directly impairs\ngluconeogenic function, leading to inadequate lactate consumption through this\nmetabolic pathway and consequent lactate accumulation.\nThank you once again for your valuable input. These validated findings provide\ncritical evidence clarifying the role of the “Cbx3–Fbp1–lactate metabolism” axis in\nthe pathological process.\n10. Although Fbp1-C282S-LNP improves various functional parameters in\nDMED mice, the manuscript could benefit from a more detailed discussion of\nhow this strategy compares to traditional therapeutic interventions for DMED.\nCould this approach be combined with other treatment modalities?\nResponse: We sincerely thank you for your insightful comments, which have greatly\ninspired our research. In response, we have added the following content to the\nDiscussion section to clarify the differences between Fbp1-C282S-LNP and\nconventional DMED treatments, as well as their potential for combination therapy:\nPhosphodiesterase 5 (PDE5) inhibitors, as first-line therapeutics for ED, act by\nmaintaining intracellular cGMP levels to promote smooth muscle relaxation and\nenhance penile blood flow [PMID: 34183196]. However, it should be noted that\nPDE5 inhibitors target only vasodilatory signaling pathways and do not address the\nunderlying structural damage to smooth muscle in DMED. Consequently, their\nefficacy in DMED patients remains limited [PMID: 32257866; PMID: 22282735].\nIn contrast, Fbp1-C282S-LNP delivers therapeutic mRNA directly to the damaged\ntissue, reducing local lactate accumulation, mitigating smooth muscle impairment,\nand promoting functional recovery. This approach targets the “structural damage”\nitself—a dimension beyond the reach of PDE5 inhibitors. The complementary\nmechanisms of these two strategies suggest strong potential for combination therapy:\nFbp1-C282S-LNP restores tissue integrity, establishing a structural foundation for\nfunctional recovery, while PDE5 inhibitors enhance vasodilatory signaling. Such\nsynergy may significantly improve treatment outcomes, particularly in patients with\nresistance to PDE5 inhibitors.\nWe are truly grateful for your valuable suggestions, which have helped us more\nclearly articulate the unique advantages of the Fbp1-C282S-LNP strategy, broaden our\nperspective on combination therapy, and enhance the depth and clinical relevance of\nthe Discussion. Your feedback provides important guidance for refining our future\nresearch directions.\n11. Fbp1 knockout mice are used in several experiments; however, the role of\nFbp1 in other tissues or systemic effects (e.g., liver or muscle) should be\nconsidered. Could the observed effects be specific to the corpus cavernosum or\nsystemic in nature?\nResponse: We sincerely appreciate your insightful feedback, which has prompted us\nto more thoroughly consider potential systemic effects within our study design. Based\non our current findings and limitations, we provide the following response:\nOur study demonstrated that blood lactate levels were significantly elevated in DMED\npatients compared to healthy controls. Preclinical data also showed increased lactate\nconcentrations in the corpus cavernosum of DMED mice, which correlated positively\nwith systemic lactate levels. The primary focus of our research was to elucidate the\neffect of lactate on penile function and the mechanism underlying its local\naccumulation in the corpus cavernosum. That said, we fully acknowledge the\nlimitations of our current work. First, The Fbp1-/+ mice used in this study are not\npenile tissue-specific knockdown models. It has been reported in the literature that\nFbp1-/+ mice exhibit inhibited Fbp1 gene expression in multiple tissues/organs\n[38834617]. Second, although intracavernous injection of Fbp1-C282S-LNP\nimproved local retention, some uptake was still detected in the liver and skeletal\nmuscle. Therefore, we cannot entirely exclude the possibility that lactate derived from\nsystemic sources (e.g., liver or muscle) may contribute to pathological changes in the\ncorpus cavernosum.\nWe are truly grateful for your valuable suggestions, which have encouraged deeper\nreflection on the tissue specificity and systemic implications of our results. Your\ncomments not only help us present the limitations of this study more objectively but\nalso provide important guidance for future research—such as developing\ntissue-specific knockdown models and improving the targeting efficiency of LNPs.\n12. Lactate Accumulation and Erectile Dysfunction: The link between lactate\naccumulation and erectile dysfunction is mentioned, but the underlying cellular\nmechanisms are not fully explained. Could the authors elaborate on how lactate\ndirectly impairs CCSMC and erectile function at the molecular or cellular level?\nResponse: We sincerely thank you for your valuable suggestion, which accurately\nidentifies a central gap in understanding the molecular link between lactate and\nDMED. Your comment has provided essential guidance for further mechanistic\ninvestigation.\nTo clarify the specific molecular mechanism by which lactate impairs CCSMCs, we\nperformed transcriptome sequencing on primary CCSMCs following lactate treatment.\nBioinformatics analysis revealed a marked activation of autophagy-related pathways\nin lactate-exposed cells. Supported by subsequent in vitro validation and existing\nliterature [PMID:24307209], we propose that lactate may induce persistent\noveractivation of autophagy, thereby compromising the structural and functional\nintegrity of CCSMCs. This ultimately leads to reduced cell viability and impaired\ncontractile–relaxation capacity. These findings provide clear molecular evidence\nsupporting a direct role of lactate in CCSMC damage, and the relevant data have been\nincluded in Supplementary Figure 1.\nWe are truly grateful for your insightful input, which encouraged us to pursue\ntranscriptome-level mechanistic evidence. Your suggestion has not only strengthened\nthe logical chain connecting lactate, excessive autophagy, and CCSMC damage, but\nalso laid a foundation for further clinical validation of this pathway.\n13. LNP-Based Therapy: The use of LNPs for delivering the Fbp1-C282S mRNA\nis promising, but the manuscript would benefit from a deeper discussion of how\nLNPs are taken up by specific cell types, particularly in cavernosal tissue. What\nare the advantages of LNP delivery over other methods?\nResponse: We greatly appreciate your insightful question and valuable input.\nIn response, we have provided detailed explanations in Questions 5 and 10, and would\nlike to briefly summarize the key findings here: During in vivo distribution studies of\nthe LNPs, significant uptake was observed not only in cavernous tissue but also in\nother organs. To further investigate the cellular specificity of LNP uptake within\npenile tissue, we encapsulated Flag-tagged Fbp1-C282S mRNA into LNPs and\nperformed multiplex immunofluorescence staining. The results demonstrated LNP\nuptake across major penile cell types—endothelial cells, smooth muscle cells, and\nfibroblasts—with the highest efficiency observed in smooth muscle cells, followed by\ndetectable uptake in endothelial cells and fibroblasts.\nBased on these findings, we propose that although smooth muscle cells display high\nmetabolic activity, lactate-induced pathological damage within the corpus cavernosum\nis not confined to a single cell type. Moreover, enhanced gluconeogenic function in\nendothelial cells and fibroblasts following LNP uptake may help reduce local lactate\nlevels, thereby mitigating lactate-mediated tissue injury.\nWe have also expanded the Discussion section to include a comparison between the\nLNP delivery system and other treatment strategies (e.g., PDE5 inhibitors), along with\na detailed explanation of the potential for combination therapies.\nOnce again, we sincerely thank you for your thoughtful comments, which have helped\nus present our experimental findings and therapeutic concepts more systematically\nand convincingly.\n14. Limitations in Cell-Type Specificity: The study focuses on the effect of Fbp1\non CCSMCs, but the corpus cavernosum contains various cell types. How might\nFbp1-C282S affect other cells like endothelial cells, fibroblasts, pericytes, or\nmacrophages? Would the delivery system need to target these cells specifically?\nResponse: We sincerely thank you for your insightful question regarding the\nmulti-cellular role within the corpus cavernosum and the delivery strategy of LNPs.\nYour feedback has been invaluable in refining our interpretation. Our key findings are\nsummarized below:\nExperimental results indicate that LNPs are taken up not only by CCSMCs, but also\nby other penile cell types, including endothelial cells and fibroblasts, as well as by\nsystemic tissues such as the liver and skeletal muscle. As a rate-limiting enzyme in\ngluconeogenesis, Fbp1-C282S delivered via LNPs enhances lactate metabolism both\nwithin and outside the penile tissue. This action promotes the conversion of lactate to\nglucose, thereby directly and indirectly modulating cavernous lactate levels.\nSpecifically, within non-smooth muscle cells of the penis, Fbp1-C282S may help\nreduce lactate-induced cytotoxicity, improving the local pathological\nmicroenvironment and supporting functional recovery. Systemically, enhanced lactate\nmetabolism in organs such as the liver and skeletal muscle may further contribute to\nlowering lactate levels in the corpus cavernosum. The broad biodistribution of\nnon-targeted LNPs allows them to simultaneously engage multiple cell types and\ntissues involved in lactate metabolism, resulting in a synergistic therapeutic effect.\nThus, rather than being a limitation, this non-specificity offers a therapeutic advantage\nby avoiding metabolic “blind spots” that could arise from overly restrictive targeting.\nWe are truly grateful for your suggestions, which have helped us present a more\nrigorous and coherent mechanism of action, emphasizing the benefits of multi-target\nmetabolic regulation.\n15. In Vivo Validation of mRNA Strategy: While the Fbp1-C282S mRNA strategy\nshows promising outcomes, did the authors observe any long-term effects or\nchanges in other metabolic pathways? How sustained are the improvements in\nerectile function over time?\nResponse: We sincerely appreciate your valuable suggestions. This question\naccurately points to the long-term efficacy and metabolic safety of the mRNA\ntherapeutic strategy, providing a key direction for us to improve the demonstration of\nthe clinical translational value of our research.\nIn previous experiments, we found that the 0.25 mg/kg dose significantly reduced\ncavernous lactate levels in DMED mice, alleviated tissue fibrosis. To further evaluate\nits long-term efficacy, we additionally set a 1-month drug withdrawal observation\nperiod after the completion of the aforementioned initial treatment. Notably, all the\ntherapeutic effects observed in the early stage (including improved lactate metabolism,\ntissue structure repair, and enhanced indicators related to erectile function) maintained\ngood sustainability during this period. the relevant data have been included in\nSupplementary Figure 11.\nWe hypothesize that the core reason for this durability of therapeutic effects lies in the\nfact that Fbp1-C282S-LNP not only exerts its effects through short-term regulation of\nmetabolic pathways, but more importantly, it can promote fundamental improvements\nin the structural integrity of the corpus cavernosum (such as smooth muscle\nregeneration and fibrosis reversal)—functional recovery driven by structural repair\noften exhibits a more stable long-term maintenance effect.\nAt present, we have not yet observed any abnormal effects of this therapeutic strategy\non other metabolic pathways. However, in subsequent studies, we will further deepen\nrelevant research through metabolomic analysis to more comprehensively evaluate its\nmetabolic safety.\nOnce again, we sincerely thank you for your profound inspiration. Your attention has\nprompted us to expand our research perspective from \"short-term efficacy\" to\n\"long-term benefits\", which not only provides more sufficient evidence for the clinical\napplication potential of this mRNA therapeutic strategy, but also points out a refined\ndirection for safety assessment in subsequent studies.\nReviewer #2 (Remarks to the Author):\nThis investigation is concerned with the impact of diabetes on erectile dysfunction.\nThe authors specifically investigate lactate accumulation, the causes of this\naccumulation, and its impact on the function of the corpus cavernosum. They propose\nthat transcriptional and functional repression of Fbp1 is the cause of the lactate\naccumulation, and investigate strategies to correct Fbp1 activity and erectile function.\nMajor concerns – models and approaches\n1. The experimental model of diabetes is extreme. A blood glucose concentration\nof 30mM is exceptionally high and would be fatal in humans. It is important to\nestablish whether the findings are consistent in a model system that achieves a\nclinically relevant blood glucose concentration.\nResponse: We sincerely appreciate your insightful comments regarding the clinical\nrelevance of the diabetic model used in our study. Your feedback is invaluable for\nclarifying the rationale behind our model selection and strengthening the translational\nvalue of our findings.\nRegarding the blood glucose levels in DMED mouse model, we would like to address\nthree key points: the rationale for model establishment, its standardization, and its\nclinical relevance.\nFirst, the DMED model was established using a well-validated and widely adopted\nprotocol in the field (PMID: 37217862; PMID: 37445656; PMID: 39536767; PMID:\n40041301). The modeling process consisted of two phases: induction of metabolic\ndisorder via a 4-week high-fat diet, followed by pancreatic β-cell impairment through\nfive consecutive low-dose streptozotocin injections. Diabetes was confirmed only\nwhen blood glucose levels consistently exceeded 16.7 mmol/L—a threshold aligned\nwith the clinical definition of hyperglycemia in human diabetes. The elevated glucose\nlevel of 30 mmol/L observed during ED assessment reflects the natural progression of\nuntreated diabetes, rather than an initial modeling target. This approach is supported\nby multiple studies (PMID: 35068334; PMID: 37445656), which have demonstrated\nthat such glucose levels are well-tolerated in mice and effectively mimic the\npathological state of long-term uncontrolled diabetes, resembling the clinical course\nof poorly managed diabetic patients.\nMost importantly, the central conclusion of our study does not rely on the absolute\nvalue of hyperglycemia, but rather on the association between lactate accumulation\nand DMED. Through clinical sample analysis, we confirmed that blood lactate levels\nare significantly elevated in human DMED patients compared to healthy controls and\nare positively correlated with ED severity. Although the absolute blood glucose value\nin mice is higher than typical clinical readings, the core mechanism of\nlactate-mediated ED remains consistent across species. This cross-species mechanistic\nalignment underscores the clinical translatability of our findings.\nWe thank you once again for raising this important point. Your comments have\nenabled us to better articulate the scientific basis of model selection, emphasizing that\nhyperglycemia is a result of disease progression rather than a model limitation, and\nhighlighting the critical role of mechanism-backed clinical relevance—significantly\nenhancing the credibility and impact of our conclusions.\n2. The in vitro models (e.g. Figure 1O) use extracellular lactate concentrations of\n5mM and 10mM which massively exceed the plasma lactate concentrations\nobserved in the animal model (~3mM). Why?\nResponse: We sincerely appreciate your insightful observation regarding the\ndifference between the lactate concentrations used in our in vitro experiments (5 mM\nand 10 mM) and the plasma lactate levels measured in our animal model (~3 mM).\nThis question is crucial for evaluating the scientific soundness of our experimental\ndesign, and we are pleased to provide a systematic explanation covering the following\nthree aspects: clinical and preclinical evidence, rationale for in vitro concentration\ngradients, and the scientific basis for the in vitro–in vivo concentration difference.\nFirstly, both clinical and animal studies support the presence of elevated lactate levels\nin DMED and their correlation with disease severity. With approval from the clinical\nethics committee, we recruited 60 DMED patients and age-matched healthy controls.\nSerum lactate was significantly higher in DMED patients, with some exceeding 5\nmmol/L, and a clear positive correlation was observed between lactate levels and ED\nseverity.\nIn animal models, we confirmed that both serum and corpus cavernosum lactate levels\nwere markedly increased in DMED mice, and a linear correlation was found between\nlocal and systemic lactate concentrations (Figs. 1F-1H). These results confirm that\nhigh lactate levels do occur in vivo, particularly within diseased tissues, thereby\njustifying the use of elevated lactate concentrations in vitro.\nSecondly, the lactate concentration gradients used in vitro were carefully designed\nbased on existing literature and preliminary experiments. In vivo, we tested three\nintraperitoneal lactate injection doses (0.25, 0.5, and 0.75 g/kg) referencing\nestablished methods (PMID: 38925041). The 0.5 g/kg dose produced a significant\nincrease in serum lactate, while 0.75 g/kg did not yield further short-term elevation;\nhence, the former was selected for subsequent in vivo studies.\nFor in vitro experiments, we treated CCSMCs with seven lactate concentrations (0–\n12 mmol/L). Pre-experimental data indicated that concentrations ≤4 mmol/L had no\nsignificant effect on cell viability—consistent with reports in the literature (PMID:\n38855868; PMID: 36302626)—while concentrations ≥5 mmol/L induced clear\nfunctional alterations. We therefore selected 5 mM and 10 mM as representative low\nand high stimulatory concentrations for further study (Figs. 1S–1X).\nLastly, the use of higher lactate concentrations in vitro than those observed\nsystemically in vivo is scientifically justified and aligns with common practices in\npathological simulation. Isolated cells in vitro are removed from the complex\nmetabolic and regulatory microenvironment of the body, often requiring higher\nstimulus concentrations to elicit responses comparable to those in living tissues.\nWe thank you once again for raising this important question. Your comments have\nallowed us to better articulate the pathological relevance of elevated lactate, the\nexperimental rationale behind our concentration selection, and the scientific principles\nunderlying in vitro–in vivo concentration differences—thereby strengthening the\nvalidity and impact of our experimental design. Relevant results supporting the above\npoints can be found in Figure 1 and Supplementary Figure 1.\n3. Glucose concentrations in standard culture media are often\nsupra-physiological. Please explain which concentrations of glucose were used\nfor the in vitro experiments.\nResponse: We sincerely appreciate your careful question regarding the glucose\nconcentration in the cell culture medium, which is essential for standardizing\nexperimental conditions and ensuring the reproducibility of our results.\nIn all in vitro experiments in this study—including cultures of corpus cavernosum\nsmooth muscle cells and lactate intervention assays—we consistently used standard\nDMEM medium from GIBCO (Cat No: C11885500BT), which contains 1 g/L glucose,\nequivalent to 5.56 mM. This helps avoid artificial metabolic perturbations caused by\nhigh glucose media, allowing a more accurate assessment of the specific effects of\nlactate on smooth muscle cell function.\nThank you once again for your attention to this important methodological detail. Your\nfeedback enhances the rigor and transparency of our experimental approach and\nprovides a clear reference for reproducibility in future studies. We have supplemented\nthis information in the Methods section.\n4. The DMED mice are a highly selected group. Mice are first treated to induce\nhyperglycaemia, and then erectile function is tested. Only those mice that show\nED are investigated in the DMED group. What fraction of the hyperglycaemic\ngroup is this?\nResponse: We sincerely appreciate your attention to the screening criteria for DMED\nmice, which relates directly to the stability of our model and the representativeness of\nexperimental samples. Your question also offers an opportunity to clarify key\nmethodological details and improve the transparency of our approach.\nAll animal procedures were approved by the Animal Use and Care Committee of The\nSecond Xiangya Hospital of Central South University (Approval No. 20250042) and\nconducted in accordance with the principles of the Declaration of Helsinki. Male\nC57BL/6 mice were acclimatized for two weeks and then randomly divided into\ncontrol and experimental groups, with strict age-matching to minimize confounders.\nMice in the experimental group received a high-fat diet for 4 weeks, followed by\nintraperitoneal streptozotocin injections (50 mg/kg/day) for 5 consecutive days.\nDiabetes was confirmed only when blood glucose levels exceeded 16.7 mmol/L on\nthree consecutive measurements. These diabetic mice then continued on a high-fat\ndiet for another 8 weeks to induce DMED. Erectile function was assessed using\napomorphine induction. Mice that showed no penile erection within 30 minutes were\nclassified as successful DMED models.\nOut of 200 initially enrolled mice, 23 died due to severe hyperglycemia or procedural\ncomplications, leaving 177 diabetic survivors. Among these, 163 met the DMED\ncriteria (no erectile response) and were randomly assigned to treatment subgroups.\nThe success rate of DMED modeling was 92.1% (163/177), demonstrating the\nefficiency and reliability of this method.\nWe thank you again for raising this important point. Your feedback allows us to better\nhighlight the rigor of our model establishment process and further supports the\nvalidity of our experimental results. We have supplemented this information in the\nMethods section.\n5. All diabetic mice receive injections of streptozotocin (daily for 5 days) and\napomorphine. Were control mice injected with vehicle? There are established\nimpacts of stress (e.g. by daily injections) on plasma lactate that must be\ncontrolled for in these experiments.\nResponse: We sincerely thank you for raising this important concern. We fully agree\nthat stress-induced lactate elevation could confound the results, and we have therefore\ntaken careful measures to control for this variable.\nThroughout the experiment, control mice received the same vehicle treatments as the\nDMED group: during the streptozotocin phase, they were injected with citrate buffer\nalone; during apomorphine testing, they received the ascorbic acid-saline vehicle\nwithout apomorphine. All injections were matched in volume, route, and timing to\nthose administered to the DMED group, ensuring that injection-related stress or\nvehicle effects did not affect lactate level comparisons.\nAs emphasized in the Methods section, control mice were strictly age-matched and\nunderwent the same standardized procedures, further minimizing potential\nconfounders such as age or treatment inconsistency.\nWe are grateful for your valuable reminder, which has further improved the rigor of\nour experimental controls and strengthened the validity of our results.\n6. The description of the acyl biotin exchange assay (line 615) is not adequate.\nWhat buffer was used for lysis? What cleanup steps were done to eliminate\n‘surplus reagents’ (e.g after biotinylation)? What was the final concentration of\nhydroxylamine in the assay? Why was DTT absent from SDS PAGE buffer?\nResponse: Thank you for these valuable comments. We have revised and expanded\nthe Methods section to provide the requested details:\nThe cell pellets were lysed in buffer containing 50 mM Tris (pH 7.2), 150 mM NaCl,\n1% NP-40, and 1 mM EDTA, followed by the addition of 20 mM methyl\nmethanethiosulfonate (Sigma-Aldrich) and 1 mM PMSF (Beyotime). After incubation\nfor 1 h at room temperature, excess reagents were removed by chloroform–methanol\nprecipitation (one volume of chloroform, four volumes of methanol, and three\nvolumes of ddH₂O). The pre-blocked samples were then treated with hydroxylamine\n(Sigma-Aldrich) at pH 7.0 to a final concentration of 0.5 M and incubated for 2 h at\nroom temperature to cleave thioester bonds. Subsequently, 0.2 mM biotin-HPDP (Top\nScience) was added, and samples were incubated in the dark for 2 h to ensure efficient\nbiotinylation. Excess biotin was again removed by chloroform–methanol precipitation.\nThe resulting proteins were purified with Streptavidin beads (Yeasen) and analyzed by\nSDS-PAGE and Western blotting.\nRegarding electrophoresis, we used SDS-PAGE loading buffer from YOCHE\n(#PYT009), which employs TCEP instead of DTT. TCEP provides stronger and more\nstable reducing activity with lower toxicity, and we have clarified this point in the\nrevised text to avoid any misunderstanding.\nWe sincerely appreciate the reviewer’s insightful questions, which helped us improve\nthe clarity and completeness of the Methods description.\nMajor concerns – figures\n1. Statistical analysis. Throughout – the legends should indicate the names of the\nstatistical tests used. When reporting values in the main text either state\n‘significantly different / not different’ OR state values, measure of variation, or\nunits (for example – lines 107 currently simply says ‘0.47 VS. 0.52 VS. 0.76’, line\n108 currently says 116.3 VS. 103.7 VS. 56.3 – needs units and SD / SEM). In the\nmethods section, please state whether one-tailed or two-tailed statistical tests\nwere performed. Where ANOVA results are reported, please provide the F values\nand degrees of freedom. Where t-test results are reported, please provide the\nt-values and degrees of freedom.\nResponse: We sincerely appreciate your valuable suggestions regarding the\nstandardization of statistical reporting. Your feedback has been instrumental in\nenhancing the clarity and traceability of our data presentation. In accordance with\nyour comments, we have implemented several revisions to improve the presentation\nof statistical analyses.\nIn the main text, results are now consistently described using clear statements such as\n\"significantly different\" or \"not significantly different,\" avoiding standalone\nnumerical values without contextual interpretation. The \"Statistical Analysis\"\nsubsection in the Methods chapter now explicitly states the primary statistical\nmethods used: the unpaired two-tailed t-test and one-way ANOVA followed by Tukey’\ns post hoc test. For specialized experimental designs involving repeated measures,\nadditional explanations have been included in the corresponding figure legends to\nensure comprehensive and context-appropriate documentation. Furthermore, all raw\nnumerical data have been made fully accessible, including means, variances (or\nstandard deviations/standard errors), t-values, F-values, and degrees of freedom, to\nfacilitate reproducibility and further analysis.\nWe are truly grateful for your guidance, which has significantly improved the\nstandardization and transparency of our statistical reporting, thereby strengthening the\ncredibility of our findings.\n2. Fig 2A-2D. The lack of detail in the legend makes it very difficult to\nunderstand what this represents. What are N1, T1, etc? Same problem in Fig 5K.\nResponse: We sincerely apologize for the insufficient details in the figure legends,\nwhich may have led to difficulties in interpreting the results. We have thoroughly\nrevised all figure legends to enhance clarity, with specific additions made to Figs. 2A–\n2D and 5K as follows:\nIn Fig. 2B, labels \"n1, n2, n3\" denote three independent samples from control mice,\nwhile \"t1, t2, t3\" represent three independent samples from DMED mice. Similarly, in\nFig. 2C, \"N1, N2, N3\" indicate three independent samples from control rats, and \"T1,\nT2, T3\" correspond to three independent samples from DMED rats. In Fig. 5K,\n\"DMED-1, -2, -3\" refer to three independent samples from DMED mice, and \"NC-1,\n-2, -3\" represent three independent samples from control mice.\nAll revised figure legends now clearly define abbreviations, sample sources, and\ngroup assignments, enabling readers to readily grasp the experimental context of each\nfigure. We truly appreciate your feedback on this matter—your comments have\ngreatly improved the clarity and interpretability of the figures in our study.\n3. Fig 3V appears to show that a ‘PROTAC’ has been developed that induces\nd that induces\ndegradation of Cbx3. No experimental evidence is presented that this molecule\nbinds to Cbx3. The characterisation in Fig S3 is inadequate. What is the in vivo\nbioavailability / half life of this reagent (Fig 4)?\nResponse: We sincerely appreciate your careful attention to the experimental\nevidence regarding the Cbx3-targeting PROTAC molecule. Your suggestions have\nprovided essential guidance for systematically clarifying the design rationale,\nmechanism of action, and in vivo properties of this compound. Based on experimental\ndata, we provide the following explanations in logical sequence:\nThe structural design of the PROTAC molecule, as shown in the schematic diagram,\nconsists of a Cbx3-binding ligand, a CRBN-recruiting ligand, and an H-shaped linker.\nExperimental results confirm that H-PROTAC does not regulate Cbx3 at the\ntranscriptional level, supporting its action through a post-translational degradation\nmechanism. To evaluate the concentration-dependent effect on protein degradation,\nCCSMCs were treated with H-PROTAC at six concentrations (0, 0.2, 0.5, 1, 2, and 5\nμM). Cbx3 protein levels were most effectively reduced at 1 μM, which informed the\nselection of these concentrations for subsequent experiments.\nThe specificity of H-PROTAC was validated through multiple experimental\napproaches: compared to an A-linker control PROTAC, H-PROTAC exhibited\nsignificantly stronger degradation activity against Cbx3, highlighting the influence of\nlinker structure on efficacy. Streptavidin pulldown assays further confirmed direct\nbinding of H-PROTAC to both Cbx3 and CRBN, illustrating its role as a molecular\nglue that facilitates target ubiquitination and degradation.\nKey pharmacokinetic parameters, including bioavailability and half-life, have been\ncharacterized for H-PROTAC in vivo. Additionally, safety assessments following\nintracavernous administration in mice showed no significant abnormalities in liver\nand kidney function markers (ALT, AST, BUN, Scr), supporting its preliminary\nbiosafety and potential for further translational study.\nAll relevant data have been incorporated into the revised main text and\nSupplementary Figure 5 to ensure a comprehensive and logically coherent\npresentation. We are grateful for your insightful questions, which have greatly\nenhanced the mechanistic clarity and experimental rigor of the PROTAC-related\nfindings.\n4. Palmitoylation assays should be presented with a palmitoylated housekeeping\nprotein as a control for the assay (e.g. throughout Fig 5).\nResponse: We sincerely appreciate your thoughtful questions regarding the internal\ncontrol design for the palmitoylation assays. Your comments have helped us better\norganize our rationale and clarify potential ambiguities in our previous explanations.\nBased on a review of the literature and our preliminary empirical data, we would like\nto reaffirm the suitability of the selected internal control as follows:\nWe conducted a systematic search of recent studies on protein palmitoylation\ndetection and found that very few publications employ a dedicated \"palmitoylation\ninternal control protein\"—this reflects the current common practice in the field. In\nline with previous work from our group (PMID: 37078777), we selected AGK, a\nprotein known to be stably palmitoylated, as the internal control. In cells, both\noverexpression and knockdown of ZDHHC13—the core regulatory factor of this\nstudy—were performed, and acyl-biotin exchange assays confirmed that the\npalmitoylation level of AGK remained consistent and unaffected by ZDHHC13\nmanipulation. Therefore, AGK not only satisfies the requirement of being\npalmitoylated (making it a valid internal control for palmitoylation assays) but also is\nnot influenced by ZDHHC13, thereby providing a specific and reliable reference for\nevaluating ZDHHC13-mediated palmitoylation of target proteins.\nWe thank you once again for your valuable suggestions, which have improved the\ntraceability of our experimental design and enhanced the methodological rigor of our\nstudy.\n5. No quantification of palmitoylation assays is presented making it impossible to\ndraw conclusions about whether any changes in palmitoylation described are\nstatistically significant (e.g. throughout Fig 5).\nResponse: We sincerely thank you for raising this important point regarding the lack\nof quantitative analysis in our palmitoylation assays. Your comment rightly\nhighlighted the need for statistical validation of the observed changes, and we have\nfully addressed this issue by supplementing the required quantitative evaluations.\nIn addition to using AGK palmitoylation as an internal reference, we have now\nperformed grayscale-based quantification for all palmitoylation assays presented in\nFig. 5. Comparisons between two groups were conducted using unpaired two-tailed\nt-tests, while multi-group comparisons were analyzed by one-way ANOVA followed\nby Tukey’s post hoc test. The full quantitative results are provided in Supplementary\nFig. 5.\nThis statistical validation offers robust support for the reported palmitoylation\nchanges and directly responds to your central concern. We are grateful for your\ninsightful feedback, which has substantially strengthened the rigor and persuasiveness\nof our palmitoylation-related conclusions.\n6. The molecular model presented in Fig 5F is meaningless. No information is\nprovided about how this was generated. It is impossible to distinguish which\nprotein is which, how they are interacting, etc.\nResponse: We sincerely apologize for the lack of key information in the molecular\nmodel presented in Fig. 5, which hindered its interpretability and scientific value. We\nhave now thoroughly revised this section and supplemented essential details to\nimprove clarity and relevance. The specific updates are as follows:\nMolecular docking between ZDHHC13 and FBP1 was visualized using AlphaFold\nand PyMOL. The original model has been replaced with an optimized diagram in\nwhich FBP1 is shown in green and the catalytic domain of ZDHHC13 in blue. Key\ninteraction sites are indicated with red dashed lines, and the binding energy between\nthe two proteins is clearly labeled. These modifications allow readers to easily\ndistinguish the two proteins, identify their binding interface, and evaluate the\nstructural basis of their interaction.\nWe thank you once again for highlighting this issue. The updated molecular model\nand detailed annotations significantly improve both the clarity and scientific validity\nof this part of our study.\n7. zDHHC13 is a polytopic membrane protein, which are notoriously difficult to\npurify in an intact, functional state. Only zDHHC20 and zDHHC9 from this\nfamily have been purified enzymatically competent (PMIDs: 29326245,\n38182928). It is highly unlikely that GST-zDHHC13 (purified from what\nexpression system?) retains catalytic activity during purification in RIPA buffer\n(Fig 5R).\nResponse: We sincerely thank the reviewer for their insightful comment and\napologize for any confusion caused by our initial wording. As rightly noted,\nZDHHC13 is a polytopic membrane protein, and its purification in a fully intact and\ncatalytically active form remains a significant technical challenge in the field.\nIn this study, we did not attempt to purify ZDHHC13 as a complete and functionally\nactive transmembrane enzyme—a task that remains technically demanding. Instead,\nwe utilized a semi‑in vitro palmitoylation assay: GST-tagged ZDHHC13 (wild-type or\nC456S mutant) was transiently expressed in cells and captured using glutathione\nmagnetic agarose under mild, non-denaturing NP-40 lysis conditions (not RIPA\nbuffer). The bead-immobilized GST-zDHHC13 was then directly incubated with\npurified HIS-FBP1, palmitoyl-alkyne CoA, and reaction buffer, followed by Click-iT\nlabeling and streptavidin pulldown. This approach is more accurately characterized as\na cell lysate-based semi-in vitro assay rather than the use of fully purified ZDHHC13.\nWe have revised the Methods section to clarify this point and prevent any potential\nmisunderstanding. Thank you once again for your valuable feedback..\n8. Fig 6B – palmitoylation anchors proteins to membranes. Why does the amount\nof Fbp1 in the cytosolic fraction not change when zDHHC13 is silenced? Fig 5\nindicated reduced palmitoylation of Fbp1 in the absence of zDHHC13, which\nwould reduce its membrane association.\nResponse: We sincerely thank the reviewer for their attentive reading and valuable\nfeedback. Regarding the observation that “Fbp1 palmitoylation decreases upon\nZDHHC13 silencing (Fig. 5), yet its abundance in the cytosolic fraction remains\nunchanged,” we would like to offer the following clarification based on our\nexperimental methodology and the principles of cellular fractionation:\nCytoplasmic and nuclear proteins were separated using a commercial extraction kit\n(#P0028, Beyotime), which employs a stepwise lysis protocol. The cytosolic fraction\nis obtained under hypotonic conditions that lyse the plasma membrane, releasing\nsoluble cytoplasmic components. Importantly, this process also generates membrane\nfragments—including those from organelles such as mitochondria and the\nendoplasmic reticulum—which are inherently present in the cytosolic fraction. As a\nresult, this fraction contains not only truly soluble proteins but also\nmembrane-associated proteins (including palmitoylated proteins) that remain attached\nto these membrane structures.\nIn the case of Fbp1, although ZDHHC13 knockdown reduces its palmitoylation level\n(which would be expected to weaken membrane association), the cytosolic fraction\nstill contains both membrane-attached and freely soluble pools of Fbp1. The\ncombined signal from these two sources may therefore mask a potential change in the\nsoluble fraction alone. Thus, the unchanged abundance of Fbp1 in the cytosolic\nfraction does not contradict the palmitoylation results but rather reflects the\nmethodological particularity of the fractionation process.\nWe are grateful to the reviewer for raising this subtle yet critical point. Your comment\nhas prompted us to revisit and more thoroughly explain the technical and biological\ncontext of these observations, significantly improving the rigor and clarity of our data\ninterpretation. Thank you for your valuable contribution to enhancing the quality of\nthis study.\nMinor concerns – figures\n1. Fig 1D – colour code the individual points on this graph red and blue to\nindicate which group of mice each data point belongs to.\nResponse: We sincerely appreciate this constructive suggestion, which has greatly\nimproved the clarity of our data presentation. As recommended, we have updated this\nfigure by color-coding individual data points: values from the DMED are now shown\nin red, and those from the control group in blue. This adjustment allows immediate\nvisual distinction between the two groups and better highlights the distribution and\nvariation within each dataset.\nThank you once again for your valuable input, which has enhanced both the\nreadability and interpretability of the figure.\n2. Fig 1E – 1N: it is not clear from the text or the legend accompanying the figure\nwhether these experiments were conducted in healthy mice or DMED mice.\nResponse: Thank you for your question. We apologize for the lack of clarity\nregarding the mouse model in the figure and its accompanying legend. All\nexperiments presented you mentioned were conducted in normal (healthy) mice, not\nDMED mice. To eliminate ambiguity, we have revised the figure to explicitly state\nthis information.\nThis revision ensures readers can immediately identify the mouse model used,\naddressing the confusion you pointed out. Thank you again for highlighting this\noversight, which has helped improve the clarity and transparency of our figure\npresentation.\n3. Fig 1G – what does the x axis in these traces represent? Please add a title\n(in addition to units) to the y axis.\nResponse: Thank you very much for your comment. We have supplemented clear\ntitles for both the x-axis and y-axis of the related figure to address your concern, while\nretaining the original units for consistency.\nThe x-axis, which reflects the timeline of the experimental intervention, is now\nlabeled “Stimulate for 1 minute”—this clarifies that the trace records changes over the\nentire 1-minute stimulation period. The y-axis, which denotes the measured\nphysiological indicator, is labeled \"Intracavernous pressure\" (with units, mmHg).\nThis revision ensures the axes convey both the parameter and timeline of the data,\nmaking the traces more interpretable. Thank you again for highlighting this detail,\nwhich has enhanced the clarity of the figure.\n4. The description of the analysis of images such as Fig 1I, 1K, 2K, 7T (and\nothers) is not adequate. How were these images analysed? Were investigators\nblinded during analysis?\nResponse: We sincerely appreciate the reviewer’s attention to the staining and image\nanalysis methodologies. Below we provide a detailed description of the specific\nprocedures used for H&E staining, Masson staining, and morphometric analysis in\nthis study.\nFor H&E staining, dewaxed and rehydrated tissue sections were stained with\nhematoxylin for 5 minutes, differentiated in 1% hydrochloric acid–ethanol for 1\nminute, blued in ammonia water for 1 minute, and counterstained with eosin for 1\nminute. Stained samples were then dehydrated, mounted, and examined under a\nmicroscope. For Masson staining, tissue sections were treated sequentially with\nhematoxylin for 5 minutes, Ponceau acid-fuchsin for 6 minutes, 1% phosphomolybdic\nacid for 1 minute, and 2.5% aniline blue for 2 minutes, followed by rinsing and\ndifferentiation with 1% glacial acetic acid.\nMorphometric analysis was performed using ImageJ software. Three non-overlapping\nfields were randomly selected from each section. In H&E-stained sections,\nwell-defined structural regions were imported into ImageJ, and the “Threshold”\nfunction was applied with manual adjustment to distinguish luminal spaces from\nparenchymal areas based on grayscale intensity. In Masson-stained sections,\nred-stained areas were identified as muscle fibers and dark blue regions as collagen\nfibers; both were quantified using threshold-based segmentation. All analyses were\nperformed by two independent investigators blinded to group assignments. These\nexperimental and analytical details have been added to the Methods section of the\nmanuscript to ensure reproducibility and reliability.\nWe thank the reviewer again for their thorough evaluation, which has greatly\nenhanced the methodological rigor of our study.\n5. Fig 1P – how was viability assessed? This appears to show cell growth not\nviability.\nResponse: We sincerely appreciate your insightful feedback and apologize for any\nambiguity in describing the cell viability assessment method. We are grateful for the\nopportunity to provide further clarification.\nThe cell viability results presented in our article were obtained using the Cell\nCounting Kit-8 (CCK-8) assay. This method is based on the metabolic activity of\nviable cells: functional dehydrogenases in living cells reduce the CCK-8 reagent to a\ncolored formazan product, whereas dead or impaired cells do not produce this signal.\nThe measured optical density (OD) values are directly proportional to the number of\nmetabolically active cells, thereby reflecting true cell viability rather than merely\nquantifying cell proliferation.\nIn our experimental design, we first conducted a preliminary screening in which\nprimary corpus cavernosum smooth muscle cells were treated with a range of lactate\nconcentrations for 48 hours. Cell viability was not significantly affected at lactate\nconcentrations up to and including 4 mmol/L (Sub-Fig. 1K). Thus, 5 mmol/L and 10\nmmol/L were selected as low and high lactate stimulation concentrations, respectively,\nfor subsequent experiments. The CCK-8 assay was used to identify two\nconcentrations—5 mM and 10 mM lactate—that significantly affected cell viability\n(Figure 1T). It is important to emphasize that the experimental objective was\nspecifically to evaluate the impact of lactate on cell viability, rather than on\nproliferative activity alone.\nWe thank you again for your thorough review, which has helped us describe our\nmethodology with greater precision and avoid potential misunderstandings.\n6. Fig 1Q, 2P, 2S (and other figures) – the scale bar is incorrectly labelled 200x.\nResponse: Thank you very much for bringing this issue to our attention. We sincerely\napologize for the incorrect use of “200x” to label the scale bars in the related figures.\nWe have corrected these figures by replacing the erroneous labels with accurate scale\nbars that include explicit length units , ensuring that the magnification is clearly and\ncorrectly represented.\nWe greatly appreciate your careful review, which has significantly improved the\naccuracy and professionalism of our figures. Thank you once again for your valuable\nfeedback.\n7. Fig 3I, 3K, 3M – these graphs are incorrectly labelled mRNA, they appear to\nshow quantification of the western blots.\nResponse: We greatly appreciate your careful review and valuable feedback\nregarding the labeling in the related Figure. We confirm that these figures present\nmRNA expression levels, as originally indicated, with all data derived from RT‑qPCR\nexperiments rather than Western blot analyses. To prevent any potential\nmisunderstanding, we have updated the figure legends to clearly state “Results are\nfrom mRNA analysis,” thereby explicitly distinguishing these findings from the\nWestern blot data presented elsewhere in the manuscript. Your attention to detail has\nsignificantly improved the clarity and accuracy of our data presentation. Thank you\nonce again for your professional and insightful guidance.\n8. Quality of some western blots is poor. For example – Fig 3L H3K9, Fig 4N\nFbp1, Fig 4O H3K27: the intensity of the bands changes from left to right,\nindicative of poor / inconsistent transfer.\nResponse: We sincerely appreciate your valuable feedback regarding the quality of\nthe Western blot images. We fully acknowledge the issue you identified—namely, the\nleft-to-right variation in band intensity, which suggests suboptimal or inconsistent\nprotein transfer during the experimental process.\nIn response, we have repeated the corresponding Western blot experiments and\nreplaced the original images with newly obtained data. The updated images exhibit\nenhanced clarity and more consistent band patterns, with protein transfer now meeting\nreliable technical standards.\nWe are truly grateful for your meticulous attention to experimental details and your\nprofessional critique. Your rigorous review enabled us to promptly identify and\naddress this issue, thereby significantly improving the accuracy and robustness of our\ndata presentation. Your input has been instrumental in enhancing the overall quality of\nour study.\n9. Fig 4P – 4U: in the description of these data the authors should state the\nnormal range values for lactate and ICP to emphasise that the correction they\nhave achieved is modest at best.\nResponse: We greatly appreciate your valuable suggestion, which has helped us more\nclearly present the actual therapeutic effects of the inhibitors. Below, we provide the\nspecific data regarding cavernosal lactate concentration and ICP from age-matched\nhealthy control mice, DMED mice, and the three inhibitor-treated groups included in\nthis study:\nIn healthy control mice, the lactate concentration was 0.4747 ± 0.04735 mmol/gprot,\nand the ICP value was 57.33 ± 5.033 mmHg. In DMED mice, the lactate\nconcentration was 0.7513 ± 0.01620 mmol/gprot, and the ICP value was 17.00 ±\n2.646 mmHg. For the three inhibitor-treated groups, lactate concentrations were\n0.6653 ± 0.01026 mmol/gprot, 0.6760 ± 0.01308 mmol/gprot, and 0.6783 ± 0.01501\nmmol/gprot, respectively, while ICP values were 27.67 ± 3.512 mmHg, 27.33 ± 2.309,\nmmHg and 27.00 ± 3.606,mmHg respectively. As you rightly noted, although the\nthree inhibitor-treated groups showed statistically significant improvements compared\nto the DMED group, their lactate levels remained elevated relative to those of healthy\ncontrols, and their ICP values were still substantially lower. This indicates that the\noverall therapeutic benefit achieved with the inhibitors was limited.\nAll original data and statistical parameters, including sample sizes, analytical methods,\nand exact P-values, have been compiled in the supplementary source files for full\ntransparency. We thank you once again for your meticulous attention to these\nexperimental details. Your feedback has been invaluable in helping us present the\nresearch findings more objectively and comprehensively.\n10. Fig 5 – change the title of this figure. There is no data about Fbp1 function in\nthis figure.\nResponse: We sincerely thank you for your valuable feedback on Figure 5. We fully\nacknowledge that the original title inaccurately referenced \"Fbp1 function,\" which\nwas not substantiated by the actual content of the figure.\nAs suggested, we have revised the title to more accurately reflect the data presented:\n\"Zdhhc13-mediated S-palmitoylation of Fbp1 at the C282 site.\" This change ensures\nthe title aligns precisely with the experimental evidence shown in the figure and\neliminates any potential misinterpretation.\nWe appreciate your careful reading and insightful suggestion, which has helped\nimprove the clarity and accuracy of our manuscript.\n11. Fig 5R – why is there beta actin in these samples? They are purified\nrecombinant proteins.\nResponse: Thank you for your insightful comment. We would like to clarify that the\nGST-Zdhhc13 used in this assay was not a fully purified recombinant protein, but was\ncaptured from lysates of transfected cells using glutathione beads. The presence of\nendogenous β-actin in the input samples reflects the nature of this semi-purified\npreparation, which was confirmed via Coomassie staining and Western blot under\nnon-denaturing conditions. We appreciate the opportunity to clarify this\nmethodological detail.\n12. Fig 6C – 6N: the authors should show that the effects of expressing zDHHC13\non glucose consumption & lactate production occur in the presence of Fbp1 but\nnot in the presence of C282S Fbp1.\nResponse: We sincerely appreciate your valuable suggestion, which has helped us\nmore rigorously verify whether the effect of ZDHHC13 depends on the C282 site of\nFbp1.\nAs recommended, we conducted a key supplementary experiment: in Fbp1-knockout\ncells reconstituted with the Fbp1 C282S mutant, overexpression of Zdhhc13 did not\nalter glucose consumption or lactate production (Supplementary Fig. 8X–8Y). This\nresult stands in clear contrast to the findings presented in Figs. 6, where Zdhhc13\noverexpression significantly influenced these metabolic indicators in the presence of\nwild-type Fbp1. These data confirm that the regulatory effect of ZDHHC13 on\nglucose consumption and lactate production is strictly dependent on the C282 site of\nFbp1. Specifically, the effect occurs only in the presence of functional wild-type Fbp1\nand is abolished when the C282S mutant is expressed.\nThe supplementary data have been included in Supplementary Fig. 8, and\ncorresponding descriptions have been updated in both the figure legends and the main\ntext. We thank you again for your expert guidance, which has greatly strengthened the\nexperimental logic and overall reliability of our conclusions.\n13. Fig 7H – quantification of Fbp1 expression is missing.\nResponse: We sincerely appreciate you highlighting the absence of Fbp1 expression\nquantification in the related figure. Thank you for your thorough and attentive review.\nIn response to your suggestion, we have now supplemented it with quantitative data\nand statistical analysis for Fbp1 expression, clearly illustrating the differences\nbetween experimental groups. These additions have been incorporated into the revised\nfigure and its corresponding legend to improve data completeness and support the\nconclusions drawn.\nThank you once again for your valuable input, which has significantly enhanced the\nrigor and clarity of our results.\n14. Figure S3C – please include a description of what the numbers on the right of\nthe graph refer to.\nResponse: We greatly appreciate your reminder regarding Figure S3C. The numbers\non the right side of the graph refer to the concentrations of the CBX3-PROTAC drug\nused in the experiment. As suggested, we have supplemented the unit \"μM\" for these\nconcentration values in the revised figure to ensure clarity. We appreciate your careful\nreview, which helps improve the accuracy of our data presentation.\nOther comments:\n1. In the abstract the authors state that the “Suv39h1-mediated H3K9\ntrimethylation and Ezh2-dependent H3K27 trimethylation synergistically\nsuppressed Fbp1 transcription”. In the manuscript, evidence is provided for each\nof these repressing transcription individually, however no evidence is presented\nto support “synergistic effects”. Does simultaneously increasing (or decreasing)\nSuv39h1-mediated H3K9 trimethylation and Ezh2-dependent H3K27\ntrimethylation cause additional modulation of Fbp1 transcription compared to\nincreasing (or decreasing) only one of these?\nResponse: We sincerely appreciate your critical observation and valuable question\nregarding the potential synergistic effect between Suv39h1-mediated H3K9\ntrimethylation and Ezh2-dependent H3K27 trimethylation in the transcriptional\nregulation of Fbp1.\nWe acknowledge that our initial data primarily demonstrated the individual repressive\neffects of these two histone modifications on Fbp1 transcription, without directly\nillustrating their synergy. In response to your suggestion, we have conducted\nadditional in vitro experiments in which we simultaneously modulated both pathways.\nSpecifically, by co-overexpressing Suv39h1 and Ezh2 to elevate both H3K9me3 and\nH3K27me3 levels, we observed significantly stronger suppression of Fbp1 expression\ncompared to overexpression of either enzyme alone. These results confirm that the\ntwo modifications act synergistically to regulate Fbp1 transcription.\nWe thank you again for your expert guidance, which has greatly strengthened the\nreliability and persuasiveness of our conclusions.\n2. How realistic is Fbp1 or lactate accumulation as a therapeutic target? Is\nmodulating processes involved in cellular metabolism likely to be associated with\nundesired effects? Data presented from MOVAS cells (derived from mouse aortic\nsmooth muscle) indicates that the effects of Fbp1 modulation are very similar to\nthose in the CCSMCs. Very similar effects in both cell types would presumably\nmean that interventions targeting or involving Fbp1 would risk off target effects\nin vascular smooth muscle cells, and that therapeutic drugs would have to be\nvery selectively targeted to CCSMCs.\nResponse: We sincerely thank the reviewer for their insightful comments regarding\nthe feasibility, potential adverse effects, and off-target risks of targeting Fbp1—all of\nwhich are crucial for evaluating the translational potential of our research. Based on\nsupplementary experimental data, we provide the following clarification:\nRegarding the therapeutic relevance of targeting Fbp1 and lactate accumulation, our\ndose-response study of intracavernously administered Fbp1-LNP showed that 0.25\nmg/kg significantly reduced lactate levels in the corpus cavernosum of diabetic\nerectile dysfunction (DMED) mice and robustly improved erectile function, while 0.1\nmg/kg showed no discernible effect, confirming a clear dose-dependent response.\nImportantly, these therapeutic benefits remained stable for one month after treatment\ncessation, demonstrating that modulating cavernous lactate metabolism via Fbp1\noffers significant and sustained efficacy, supporting its viability as a therapeutic\ntarget.\nConcerning potential adverse effects and off-target risks, pharmacokinetic and tissue\ndistribution analyses revealed that although LNPs were efficiently taken up by the\ncorpus cavernosum, they were also detected in other tissues such as the liver and\nskeletal muscle. However, no overt adverse effects were observed throughout the\nstudy. Furthermore, multiplex immunofluorescence showed that LNPs were\ninternalized not only by smooth muscle cells but also by endothelial cells and\nfibroblasts. While this indicates a degree of broad biodistribution, it may help reduce\nsystemic lactate accumulation in the diabetic state, thereby synergistically\ncontributing to metabolic homeostasis.\nIn summary, our data demonstrate that Fbp1-LNP achieves notable therapeutic effects\nwith a favorable safety profile, even without exclusive targeting of cavernous smooth\nmuscle cells. Therefore, highly specific cellular targeting may not be necessary at this\nstage. The revised manuscript now includes these supplementary data and figures to\naddress these points more comprehensively.\nWe are grateful for the reviewer’s rigorous and constructive feedback, which has\ngreatly helped us strengthen the translational relevance and validity of our study.\n3. At various points throughout the results section, reference is made to\n‘increases’ or ‘decreases’ in various measures in some groups compared to other\ngroups. ‘Increases’ or ‘decreases’ implies that measurements have been made\nfrom the same group before and after an intervention. When comparing values\nbetween different groups, please use ‘higher’ or ‘greater’ and ‘lower’ instead to\ndescribe differences. Please adjust this in lines 107, 100, 108, 113, 162, 163, 212,\n217, 232 and any other instances.\nResponse: We sincerely appreciate you highlighting this important terminological\ndistinction, which is essential for precise scientific communication. We fully agree\nthat terms such as \"increases\" or \"decreases\" should be reserved for within-group\ncomparisons over time (e.g., pre- vs. post-intervention), while \"higher/lower\" or\n\"greater/smaller\" are more appropriate when describing differences between groups.\nWe have carefully revised the relevant descriptions throughout the Results section to\nadhere to this convention. These adjustments improve the accuracy of our results\nreporting and prevent potential misinterpretation.\nThank you once again for your valuable suggestion, which has significantly enhanced\nthe clarity and rigor of our manuscript.\n4. zDHHC13 is a Golgi-localised enzyme (PMIDs: 16647879, 19299482), yet Fbp1\nappears to be present in the plasma membrane and the nuclear membrane. How\nis it possible that Fbp1 interacts with and is a substrate for zDHHC13 when they\nare expressed in different cellular compartments?\nResponse: Thank you very much for your insightful comment. Although ZDHHC13\nis primarily localized to the Golgi apparatus (PMID: 16647879, 19299482), many\nsubstrates of Golgi-resident DHHC enzymes are modified by these enzymes before\nbeing transported via the endoplasmic reticulum–Golgi network to their final\ndestinations. As reported by Shen et al. (PMID: 28526873), ZDHHC13 is capable of\npalmitoylating non-Golgi-resident proteins, supporting the idea that transient\ninteractions between proteins and ZDHHC13 during intracellular trafficking are\nsufficient for palmitoylation to occur. In this context, it is plausible that FBP1 is\nmodified by ZDHHC13 during its transit through the secretory pathway. We\nappreciate your thoughtful question, which has helped clarify this important\nmechanistic detail.\n5. Fbp1 palmitoylation has been reported before by others:\nhttps://swisspalm.org/proteins/Q9QXD6 . Other palmitoylation sites have been\ndescribed in this protein (C39, C93). How do you account for mutation of only\nC282 abolishing palmitoylation?\nResponse: We sincerely appreciate your attention to prior studies on Fbp1\npalmitoylation, including the modification sites (e.g., C39, C93) documented in the\nSwissPalm database (https://swisspalm.org/proteins/Q9QXD6). Your observation\noffers valuable context that helps refine the interpretation of our results regarding the\nC282 mutation.\nOur experimental data indicate that mutating the C282 site to serine (C282S) does not\ncompletely abolish Fbp1 palmitoylation but leads to a substantial reduction in its\nmodification level. In our initial analysis, the imaging parameters were optimized to\ncapture the strong palmitoylation signal in wild-type Fbp1; however, under these\nstandard conditions, the weaker residual signal in the C282S mutant was obscured—\ncreating the misleading impression that palmitoylation was entirely absent.\nTo resolve this ambiguity, we have included an overexposed Western blot image that\nclearly confirms the presence of residual palmitoylation in the C282S mutant. We\nthank you again for prompting this clarification, which further strengthens the\nmechanistic rigor of our study.\n6. What was the dosing frequency of the LNP (line 728)?\nResponse: We greatly appreciate your attention to this information oversight. We\nhave supplemented the specific dosing regimen for LNP in the revised manuscript: the\nstudy employed an intracavernous injection approach, with a dosing frequency of\nonce weekly and continuous intervention for 4 weeks. This information has been\nexplicitly detailed in the \"Methods\" section to ensure the completeness and\nreproducibility of the experimental protocol. Thank you for your meticulous guidance,\nwhich has facilitated the refinement of our study details.\n7. Were control and DMED mice age-matched? Please include the ages of mice\nused for these experiments.\nResponse: We sincerely appreciate your question regarding the age matching between\nthe control and DMED groups. In response, we have added a clarification in the\n“Animal Experiments” section of the revised manuscript stating that all male\nC57BL/6 mice in both the control and DMED groups were strictly age-matched to\nminimize potential confounding effects of age on the experimental outcomes. We\nplaced high priority on ensuring consistency in the baseline characteristics of the\nexperimental animals. All group assignments, including the subgroup allocations\nfollowing successful induction of the DMED model, guaranteed age comparability\nacross all experimental groups. We thank you once again for your meticulous\nattention to this detail, which has helped us enhance the clarity and rigor of our\nmethodological description.\nReviewer #3 (Remarks to the Author):\nIn the present study the authors suggest that excessive lactate accumulation in the\ncorpus cavernosum is a key pathological driver of DMED, promoting structural\ndegeneration, smooth muscle cell loss, and erectile dysfunction in diabetic mice.\nMechanistically, they show that transcriptional repression and functional impairment\nof Fbp1 (fructose-1,6-bisphosphatase 1, Fbp1), a rate-limiting gluconeogenic enzyme,\nunderlined corpus cavernosum lactate pathological accumulation. Epigenetic profiling\nrevealed that Suv39h1-mediated H3K9 trimethylation and Ezh2-dependent H3K27\ntrimethylation synergistically suppressed Fbp1 transcription. In addition,\nZdhhc13-mediated palmitoylation at the Fbp1 C282 site further disrupted its\ngluconeogenesis related function, aggravating lactate accumulation. To rescue these\ndefects, the authors engineered Fbp1 C282S-loaded lipid nanoparticles (Fbp1\nC282S-LNP), which restored Fbp1 expression and abrogated pathological\npalmitoylation in the corpus cavernosum, improving erectile function in DMED mice.\nMain points\n1-The authors suggested that, compared with the normal mice, the Fbp1\nknockout significantly increased the cavernosal lactate and decreased the ICP,\nthe lumen--to--parenchyma ratio, and the α--Sma fluorescence intensity (Figure\n2H -N). Would the administration of LDH inhibitors be beneficial in this\ncondition (Fbp1 knockout mice with increased Lactate production)?\nResponse: We sincerely appreciate your insightful question regarding whether lactate\ndehydrogenase (LDH) inhibitors could confer benefits in Fbp1-/+ mice. This is indeed\na valuable direction for further investigation.\nTheoretically, since LDH is a key enzyme catalyzing the conversion of pyruvate to\nlactate, LDH inhibitors could potentially reduce lactate production and thereby\nalleviate cavernous lactate accumulation resulting from Fbp1 knockout. This\nmechanism has been supported in several metabolic disease models (PMID:\n38852200; PMID: 38925041). To test this hypothesis, we treated DMED mice with\nthe LDH inhibitor sodium oxamate (0.5 g/kg, administered intraperitoneally once\ndaily for two weeks). However, no significant changes in cavernous lactate levels\nwere observed.\nWe propose the following explanation: glucose can be rapidly converted to lactate via\nglycolysis, while lactate can be reconverted to glucose through gluconeogenesis to\nmaintain glucose homeostasis (Fig. 2A). Tissue lactate accumulation typically results\nfrom either enhanced glycolysis or impaired gluconeogenesis. Using RNA-seq data\nfrom our established DMED mouse (Fig. 2B) and rat (Fig. 2C) models, we analyzed\nthe expression of key enzymes in both pathways. The results revealed significant\ndownregulation of lactate dehydrogenase A (LDHA) at the transcriptional level in\nboth species. Further analysis of LDH isozymes (Ldha, Ldhb, Ldhc, Ldhd) indicated\nwidespread transcriptional suppression, suggesting overall inhibition of glycolysis and\nreduced lactate production. Therefore, cavernous lactate accumulation in DMED is\nmore likely attributable to impaired lactate consumption—specifically, deficient\ngluconeogenesis—which may explain why LDH inhibition did not significantly alter\nlactate levels.\nWe thank you once again for raising this compelling question, which has deepened\nour understanding of the metabolic dysregulation caused by Fbp1 deficiency. The\nrelevant supplementary figures have been included in the Supplementary Materials.\n2- The authors show by ChIP qPCR assay that H3K9me3 or H3K27me3 were\nenriched on the promoter of Fbp1 in the MOVAS cells and primary mouse\nCCSMCs (Figure 3B, C, Supplementary Figure 1A, B). The promoter should be\nbetter epigenetically characterized by verifying H3K4me3, H3K27ac, DNA\nmethylation and members of the PRC1 and 2 complexes as well as the active pol\n2.\nResponse: We sincerely thank the reviewer for this valuable suggestion. To address\nthis point, we further investigated the potential roles of H3K4me3, H3K27ac, active\nRNA polymerase II, and the PRC1 complex-associated mark H2AK119ub in the\nregulation of FBP1. Using the ChIP-Atlas database, we detected binding signals for\nH3K4me3, H3K27ac, RNA polymerase II, and H2AK119ub at the FBP1 promoter\nregion. These findings were further validated by our ChIP-qPCR experiments.\nAdditionally, we examined the expression levels of these epigenetic regulators in the\ncorpus cavernosum tissue of diabetic mice via Western blotting. The results indicated\nthat the protein levels of most factors remained largely unaltered, with the notable\nexception of H3K27ac, which was significantly reduced. We hypothesize that this\ndecrease may be attributable to competitive occupancy resulting from the\nupregulation of H3K27me3, which suppresses H3K27ac enrichment and thereby\nimpairs its transcriptional activation function.\nRegarding the PRC2 complex, we would like to emphasize that EZH2—a core\ncatalytic subunit of PRC2—was the focus of our previous experiments. We have\nclearly demonstrated that EZH2 modulates FBP1 expression through\nH3K27me3-mediated repression.\nWe are truly grateful for the reviewer’s insightful comment, which has greatly\nstrengthened the mechanistic depth and rigor of our study. All relevant data have been\nincluded in Supplementary Figure 3.\n3- In Fig 3 D-E, the authors showed that the enrichment of H3K9me3 or\nH3K27me3 were significantly increased in the corpus cavernosum of DMED\nmice compared to normal mice (Figure 3D 3E). Is this regulation specific of the\ncorpus cavernosum? Is it also reported in patients? Is it also at the RNA level?\nMany more epi-regulators target H3K9me3 or H3K27me3 in respect of the onec\nshown. How is their expression?\nResponse: We sincerely appreciate the reviewer’s insightful comments. Diabetes is\na systemic metabolic disorder, and aberrant epigenetic modifications have been\nreported across various tissues under diabetic conditions. For instance, H3K9me3\nlevels are significantly elevated in diabetic retinopathy (PMID: 34238980) and in\nskeletal muscle in the context of diabetes (PMID: 30528377). Similarly, H3K27me3\nis markedly upregulated in endothelial cells from diabetic patients (PMID: 38580969).\nMoreover, EZH2-mediated H3K27me3 has been demonstrated to play a critical role\nin the pathogenesis and progression of diabetes (PMID: 38161208). These findings\nsuggest that the abnormal increases in H3K9me3 and H3K27me3 are not confined to\nthe corpus cavernosum but occur across multiple diabetic tissues.\nIt is important to emphasize that H3K9me3 and H3K27me3 are post-translational\nhistone modifications and are not regulated at the RNA level. In our preliminary\nexperiments, we examined the expression of several key histone methyltransferases\nassociated with these modifications—including SUV39H1, SUV39H2, SETDB1,\nSETDB2, EZH1, and EZH2—in the corpus cavernosum of diabetic mice. The results\nindicated that among these, the H3K9me3 methyltransferase SUV39H1 and the\nH3K27me3 methyltransferase EZH2 showed particularly notable dysregulation.\nWe thank the reviewer again for their valuable feedback, which has helped us deepen\nthe mechanistic insight into epigenetic regulation in diabetic conditions.\n4- The authors employed PROTAC technology to synthesize a Cbx3 specific\ninhibitor by spatially bridging E3 ligase and the target protein, thereby inducing\nubiquitination mediated degradation of Cbx3 (Supplementary Figure 3A, K). In\nthe methods the authors give a very short description: “PROTAC In designing a\nCbx3 targeting PROTAC, we used a molecular bridging strategy [31]. One\nligand, the H3K9me3 peptide, binds Cbx3 due to its specific recognition ability.\nThe other, Pomalidomide, attaches to the E3 ubiquitin ligase CRL4CRBN,\nwidely used in PROTACs for promoting target protein degradation. The bridge\nmolecule, with optimized length and rigidity, links these two ligands to ensure\neffective binding and maintain activity\".\nFrom this description, the strategy might also degrade other binders. The\nspecificity and selectivity of the characterization should be shown in depth.\nPlease also include dose curves and IC50.\nResponse:\nResponse: We sincerely appreciate your careful attention to the experimental evidence\nregarding the Cbx3-targeting PROTAC molecule. Your suggestions have provided\nessential guidance for systematically clarifying the design rationale, mechanism of\naction, and in vivo properties of this compound. Based on experimental data, we\nprovide the following explanations in logical sequence:\nThe structural design of the PROTAC molecule, as shown in the schematic diagram,\nconsists of a Cbx3-binding ligand, a CRBN-recruiting ligand, and an H-shaped linker.\nExperimental results confirm that H-PROTAC does not regulate Cbx3 at the\ntranscriptional level, supporting its action through a post-translational degradation\nmechanism. To evaluate the concentration-dependent effect on protein degradation,\nCCSMCs were treated with H-PROTAC at six concentrations (0, 0.2, 0.5, 1, 2, and 5\nμM). Cbx3 protein levels were most effectively reduced at 1 μM, which informed the\nselection of these concentrations for subsequent experiments.\nThe specificity of H-PROTAC was validated through multiple experimental\napproaches: compared to an A-linker control PROTAC, H-PROTAC exhibited\nsignificantly stronger degradation activity against Cbx3, highlighting the influence of\nlinker structure on efficacy. Streptavidin pulldown assays further confirmed direct\nbinding of H-PROTAC to both Cbx3 and CRBN, illustrating its role as a molecular\nglue that facilitates target ubiquitination and degradation.\nKey pharmacokinetic parameters, including bioavailability and half-life, have been\ncharacterized for H-PROTAC in vivo. Additionally, safety assessments following\nintracavernous administration in mice showed no significant abnormalities in liver\nand kidney function markers (ALT, AST, BUN, Scr), supporting its preliminary\nbiosafety and potential for further translational study.\nAll relevant data have been incorporated into the revised main text and\nSupplementary Figure 5 to ensure a comprehensive and logically coherent\npresentation. We are grateful for your insightful questions, which have greatly\nenhanced the mechanistic clarity and experimental rigor of the PROTAC-related\nfindings.\n5- The authors applied H PROTAC, Chaetocin, and EPZ6438 to the mice with\nDMED or Fbp1 knockout. The 3 inhibitors decreased the cavernosal lactate,\npartially restored the ICP, the structure and CCSMC content of corpus\ncavernosum in DMED mice but had no effect on the Fbp1-knockout mice (Figure\n4P, U). Would a combinatory use of the inhibitors display a better therapeutic\nvalue for DMED mice?\nResponse: We sincerely appreciate your insightful question regarding the potential\ntherapeutic value of combining the three inhibitors—Chaetocin, EPZ6438, and\nH-PROTAC. In response, we conducted targeted experiments, and the results are\nsummarized below:\nAlthough in vitro experiments indicated that simultaneous induction of H3K9me3 and\nH3K27me3 led to stronger suppression of Fbp1 expression compared to activating\neither pathway alone, suggesting possible molecular synergy, in vivo combination\ntherapy proved challenging.\nSpecifically, DMED mice were treated with Chaetocin (intraperitoneal injection),\nEPZ6438 (oral gavage), and H-PROTAC (intracavernous injection) using their\nrespective administration routes. However, after two weeks of combined treatment,\nthe mice exhibited a significant decline in overall health. Due to these pronounced\nadverse effects, which would confound the interpretation of therapeutic outcomes,\nthe combination regimen was discontinued.\nMechanistically, Fbp1 is regulated through a multi-level network: Suv39h1-mediated\nH3K9me3 and EZH2-mediated H3K27me3 synergistically suppress Fbp1\ntranscription, while ZDHHC13-mediated palmitoylation at C282 impairs Fbp1\nenzymatic function, further promoting lactate accumulation. To precisely target these\ndefects, we developed lipid nanoparticles carrying the Fbp1 C282S mutant (Fbp1\nC282S-LNP). This approach not only restores Fbp1 expression in cavernous tissue but\nalso prevents pathological palmitoylation via the C282S mutation, thereby\nconcurrently correcting both transcriptional and post-translational deficiencies of\nFbp1 and ultimately improving erectile function in DMED mice.\nAlthough further exploration of optimized drug combinations may be considered,\nFbp1 C282S-LNP currently represents a more targeted and potentially safer\ntherapeutic strategy, as it specifically addresses the dual dysregulation of Fbp1\nwithout the systemic toxicity associated with multi-drug regimens.\nWe are grateful for your valuable suggestion, which provides important guidance for\nour subsequent research optimization.\n6- The authors report CBX3 upregulation in the DMED mice (Fig.3P). Which is\nthe mechanism(s) behind this deregulation?\nResponse: This question will be answered together with Question 7.\n7- The transcriptome and immunohistochemistry of the corpus cavernosum\nshowed that the expression of Zdhhc13 was increased in DMED mice (Figure 5K,\nL), suggesting that Zdhhc13 may be involved in the palmitoylation of Fbp1 in\nvivo. Why is Zdhhc13 overexpressed? Is it tissue specific or?\nResponse: We sincerely thank the reviewers for raising these two critical questions.\nYou have precisely captured the core mechanistic link between the aberrant\nexpression of CBX3 and Zdhhc13 in the pathological context of DMED. These\ninsights are of critical importance for our comprehensive understanding of the\nmetabolic dysregulation mechanisms in DMED.\nGiven the limitations of single-target intervention and the toxicity of combined drug\ntherapy, we next conducted an in-depth investigation into the underlying mechanisms\nof Zdhhc13 and Cbx3 upregulation in DMED mice. We performed KEGG pathway\nenrichment analysis on differentially expressed genes, which revealed significant\nactivation of the PI3K-AKT pathway—the most prominently upregulated signaling\npathway in the DMED state.\nIt is well established that the PI3K-AKT signaling pathway regulates the activity of\nSRY-related HMG-box protein 9 (Sox9) [PMID: 33985544, 19711351]. Based on this\nresearch background, we further explored the regulatory association between this\npathway and Zdhhc13/Cbx3. Interestingly, prediction results from the ChIP-Atlas\ndatabase showed that Sox9 can bind to the promoter regions of both Zdhhc13 and\nCbx3. Experimental validation confirmed this prediction: we found that the protein\nlevels of phosphorylated AKT (p-AKT, a marker of PI3K-AKT pathway activation)\nand Sox9 were significantly increased in the corpus cavernosum tissue of DMED\nmice.\nTo clarify the regulatory role of Sox9, we conducted functional validation\nexperiments: in primary mouse CCSMCs, siRNA-mediated knockdown of Sox9\nsignificantly downregulated the expression of Cbx3 and Zdhhc13; conversely,\nplasmid-mediated overexpression of Sox9 markedly upregulated their expression.\nHowever, when we delivered adeno-associated virus expressing Sox9-shRNA\n(AAV-shSox9) into the corpus cavernosum of DMED mice to inhibit Sox9 activity in\nvivo, no significant improvements were observed in cavernosal lactate levels or\nintracavernous pressure (ICP).\nTaken together, all findings indicate that the Fbp1-C282S-LNP strategy exhibits more\nsignificant advantages: it can directly target the dual defects of Fbp1 (impaired\ntranscription and pathological palmitoylation), avoiding both the limitations of\nsingle-target intervention and the toxicity risks associated with complex multi-drug\ncombinations. We sincerely thank the reviewers for their insightful questions, which\nhave greatly facilitated the refinement of our research mechanisms and therapeutic\nstrategies. The relevant results have been included in Supplementary Figure 9 and the\nrevised manuscript.\n8- The Fbp1 C282S LNP reduced the cavernosal lactate, improved the ICP and\nthe structure and CCSMC content, both in DMED and Fbp1 knockout mice\n(Figure 7Q, V) supporting that the Fbp1 C282S LNP is a promising therapeutic\nstrategy for DMED. Were there detected collateral effects for Fbp1 C282S LNP?\nWas a Pharmacokinetic and a pharmacodynamic approach be performed?\nResponse: We greatly appreciate your attention to the characteristics of Fbp1\nC282S-LNP across four core dimensions: dose-effect relationship, drug distribution,\npharmacokinetics, and toxicity—these are key indicators for evaluating the safety,\nefficacy, and clinical translation potential of the formulation. Based on targeted\nexperimental designs and data results, we explain each dimension in detail as follows:\nTo evaluate the therapeutic efficacy of Fbp1 C282S-LNPs, we administered\nintracavernous injections to DMED mice at three dose levels (0.1 mg/kg, 0.25 mg/kg,\nand 0.5 mg/kg), along with a control group. The results indicated that the 0.1 mg/kg\ndose did not effectively reduce lactate levels in the corpus cavernosum or improve\nerectile function. In contrast, the 0.25 mg/kg dose significantly decreased lactate\naccumulation and enhanced erectile performance. The 0.5 mg/kg dose did not yield\nstatistically superior efficacy compared to the 0.25 mg/kg group, suggesting that 0.25\nmg/kg is an effective dose approaching pharmacodynamic saturation.\nMultiplex immunofluorescence staining (using Flag tag for Fbp1 C282S-LNPs, and\nmarkers for endothelial cells, smooth muscle cells, and fibroblasts: vWF, α-SMA, and\nvimentin, respectively) revealed strong LNP uptake in smooth muscle cells, with\ndetectable internalization in endothelial cells and fibroblasts. This indicates that the\nLNPs can target multiple relevant cell types within the penile tissue.\nFurther pharmacokinetic analysis via LC-MS/MS of orbital blood samples showed\nthat Fbp1 C282S-LNPs have an elimination half-life of 12.57±1.51 hours. To assess\ntissue distribution and potential off-target effects, DiD-labeled LNPs were used: the\nhighest fluorescence intensity was observed in the corpus cavernosum at 0.25 hours\npost-injection, followed by a gradual decline, confirming rapid target tissue\naccumulation and sustained local concentration. In the liver—the primary clearance\norgan—fluorescence intensity initially increased and then decreased without abnormal\nretention. Only minimal fluorescence was detected in skeletal muscle and the spleen,\nand due to the blood-brain barrier, brain fluorescence remained negligible throughout,\neffectively ruling out central nervous system exposure. Additionally, measurements of\nliver and kidney function markers (ALT, AST, serum creatinine, and blood urea\nnitrogen) showed no significant differences between treated and control groups,\nsupporting the absence of hepatorenal toxicity.\nIt is worth emphasizing that although LNPs were taken up by both penile and\nnon-penile tissues (such as liver and skeletal muscle), we believe this “non-strict\ntargeting” profile enhances rather than diminishes therapeutic efficacy through two\ncomplementary mechanisms: uptake in liver and muscle enhances systemic lactate\nmetabolism, thereby reducing lactate delivery to the corpus cavernosum; meanwhile,\nuptake within penile endothelial cells and fibroblasts contributes to local lactate\nconsumption. Thus, the broad targeting capability of LNPs supports a synergistic\ntherapeutic strategy combining local and systemic effects, with no significant adverse\neffects observed.\nAll relevant data—including dose-response outcomes, pharmacokinetic parameters,\ntissue distribution, cellular uptake, and safety assessments—have been added as\nFigure 7 and Supplementary Figure 10 in the revised manuscript. These results\ncollectively support the therapeutic potential and safety of Fbp1 C282S-LNPs. We are\ntruly grateful for your thorough and insightful comments, which have helped us\npresent these findings more comprehensively and strengthen the overall rigor of our\nstudy.\n9- The methods section should be deeply revised, implementing all details. If\nthere is space limitation, likely it might go into the additional materials.\nResponse: We greatly appreciate your feedback. We fully recognize the critical\nimportance of providing comprehensive methodological details. We sincerely\napologize for our previous omission of sufficient experimental descriptions due to\nconcerns regarding manuscript length.\nIn the revised manuscript, we have now expanded the “Methods” section in full,\nincluding detailed specifications of all experimental procedures—such as reagent\nmodels, instrument parameters, and key steps in operational protocols. For certain\nspecialized protocols and reagent preparation procedures that exceed the space\nlimitations of the main text, we have provided extended descriptions in the\nSupplementary Materials to ensure full transparency, traceability, and to facilitate\nreplication by other researchers.\nWe are truly grateful for your valuable suggestion, which has significantly improved\nthe rigor and reproducibility of our methodological reporting.\n10- All raw data (images, uncropped blots etc) should be uploaded as additional\nmaterial.\nResponse: We sincerely appreciate your reminder. All raw data, including original\nexperimental images, uncropped blot results, and raw quantitative datasets, have been\nsystematically organized and uniformly uploaded as supplementary materials to\nensure full data accessibility and traceability. We are grateful for your rigorous and\nvaluable suggestions, which greatly enhance the transparency and reproducibility of\nour research data.\n11-English grammar and typos need deep revision.\nMinor points\nsome examples....\n• Lane 25 please correct underlied\n• Lane 28 please correct in addation…\n• Lane 204 please check… histone methyltrantion…\n• Lane 380 arase one of the 2 significantly….\nResponse: Thank you very much for your careful proofreading and for pointing out\nissues with the English expression. We take this matter very seriously and have\nundertaken a systematic revision of the manuscript.\nWe have conducted a comprehensive, sentence-by-sentence review of the entire text,\nfocusing on correcting grammatical errors, spelling mistakes, and inaccuracies in\nword usage. Specific issues such as inconsistent verb tenses, incorrect articles, and\ntypographical errors (for example, “underlied” has been changed to “underlined”, “in\naddation” to “in addition”, and “methyltrantion” to “methylation”) have been\nthoroughly addressed.\nAll the specific errors you clearly identified have been checked and corrected one by\none to ensure the accuracy and standardization of the language throughout the\nmanuscript.\nWe sincerely appreciate your meticulous review. Your feedback has been essential in\nimproving the overall quality of our manuscript and ensuring that it meets high\nstandards of academic writing.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nReviewer #5 (Remarks to the Author):\nThe study is interesting and consistent with recent data showing high lactate level in\npatients with diabetes. The authors have performed an extensive series of studies to\ndemonstrate that fructose-1,6-bisphosphatase 1 (Fbp1) was reduced in a model of\ndiabetes induced by high fat diet and STZ. The reduction of Fbp1 was associated with\npalmitoylation via Zdhhc13. Restoration of Fbp1 via a lipid nanovesicle injection\ndirectly into the corpus cavernosum restored erectile dysfunction.\nMajor comments\n1. Additional models of diabetes such as db/db mice and type 1 models should be\nevaluated as well as patient samples\nResponse:\nWe sincerely appreciate your valuable comments, which have provided significant\nsupport for our research. The specific supplementary research contents are as follows:\nFirst, we obtained approval from the Clinical Ethics Committee ( LYEC2025-0188).\nWe then enrolled 60 DMED patients (including 33 mild, 19 moderate, and 8 severe\ncases) along with 60 age-matched healthy controls. Systematic measurement of serum\nlactate levels revealed significantly higher lactate in DMED patients (2.4 mmol/L)\ncompared to healthy controls (1.7 mmol/L; p= 0.0002). This elevation aligns with the\nincreased lactate levels observed in both serum and corpus cavernosum tissue in our\nDMED mouse model, providing clinical evidence supporting the role of lactate in the\npathophysiology of DMED.\nIt should be noted that, due to ethical and practical constraints, we were unable to\ndirectly measure local lactate concentrations in the corpus cavernosum of DMED\npatients. However, in our DMED mouse model, we confirmed a significant positive\ncorrelation between plasma and corpus cavernosum lactate levels. This supports the\ninference that local lactate concentrations may also be elevated in human DMED\npatients, strengthening the translational relevance of our animal data.\nTo further evaluate the clinical association between lactate and DMED, we divided all\n120 participants into three tertiles based on serum lactate levels: lower (<1/3), middle\n(1/3–2/3), and upper (>2/3). The results showed: In the overall cohort, the incidence\nof erectile dysfunction (ED) increased significantly across lactate tertiles (p= 0.002),\nwith the upper tertile showing a significantly higher incidence than the lower tertile\n(p= 0.001; Cramer’s V = 0.376). Post-hoc pairwise comparisons were performed\nusing the chi-square partition method with a corrected significance level of α' = 0.017.\nAmong DMED patients, the incidence of moderate-to-severe ED also rose with\nincreasing lactate levels (p= 0.014). The incidence of severe ED was significantly\nhigher in the upper lactate tertile than in the lower tertile (p= 0.004; Cramer’s V =\n0.376). These results not only confirm the association between elevated lactate and\nDMED but also reveal a dose-response relationship, thereby enhancing the clinical\napplicability of our conclusions.\nA type 1 diabetes mellitus (T1DM) mouse model was established using a published\nprotocol [PMID: 40196453]: 8-week-old male C57BL/6 mice were fasted for 12\nhours and injected intraperitoneally with streptozotocin (STZ; 150 mg/kg). Diabetes\nwas confirmed if fasting blood glucose exceeded 16.7 mmol/L at 72 hours\npost-injection. Mice were maintained for 8 weeks before erectile function was\nassessed via apomorphine test. Additionally, 20-week-old male db/db mice\n(C57BLKS/J-leprdb/leprdb) were obtained from Shulaibao Biotechnology Co., Ltd.\n(Wuhan, China).\nFbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNP) were evaluated in both\nT1DM and db/db models. The formulation demonstrated significant therapeutic\neffects in both models: it effectively reduced cavernous lactate levels, improved\nfibrosis, increased smooth muscle content, and enhanced intracavernous pressure .\nThe efficacy trends were consistent with those observed in the DMED mouse model.\nWe sincerely thank you for your insightful suggestions. These supplementary studies\nstrengthen both the clinical and preclinical evidence linking lactate to DMED and\nunderscore the therapeutic potential of Fbp1 C282S-LNP across multiple models,\nsignificantly enhancing the comprehensiveness and reliability of our research.\n2. No discussion of endothelial dysfunction was described which has been\npreviously found to play a key role in human erectile dysfunction.\nResponse: We sincerely appreciate your valuable feedback. We fully agree that\nendothelial dysfunction is a well-established key mechanism underlying DMED. As\nextensively documented, endothelial cells regulate blood flow within the penile\ncorpus cavernosum through the release of vasoactive substances such as nitric oxide\n(NO), and their impairment directly compromises the vasodilatory response essential\nfor erection [PMID: 26878803].\nIn recent years, alongside the classical focus on endothelial dysfunction, smooth\nmuscle cell dysfunction has gradually emerged as another critical research focus in\nerectile dysfunction, with several important advances reported [PMID: 37353497;\n38874549]. The primary objective of this study is to elucidate how diabetes-induced\nlactate accumulation impairs erectile function. Therefore, our in vitro experiments\nspecifically center on the damaging effects of lactate on smooth muscle cells and the\nunderlying mechanisms—though this focus does not diminish the central role of\nendothelial cells in DMED pathogenesis.\nNotably, in our experimental system, Fbp1 C282S-LNP is taken up not only by\nsmooth muscle cells but also by endothelial cells and other cell types. This\nmulti-cellular uptake likely contributes synergistically to reducing lactate levels in the\ncavernous tissue of DMED mice. The resulting improvement in the metabolic\nmicroenvironment should help support the functional integrity of various penile cell\ntypes—including the vasodilatory capacity of endothelial cells—thereby collectively\ncontributing to the restoration of normal erectile function.\nWe acknowledge a limitation of our current study: we did not systematically analyze\nspecific functional changes in endothelial cells following lactate reduction, nor did we\nfully elucidate the synergistic interactions between endothelial and smooth muscle\ncells during functional recovery. Accordingly, we have further supplemented the\ndiscussion on endothelial cells in the Discussion section. Future studies will focus on\nclarifying how “metabolic repair” coordinately improves DMED across smooth\nmuscle cells, endothelial cells, and other relevant cell types, thereby providing a more\ncomprehensive understanding of the pathophysiology of this condition.\nWe are truly grateful for your insightful comments, which have helped us better\nrecognize the limitations of our current work and will significantly contribute to\nimproving the depth and completeness of our research.\n3. The results suggest that Zdhhc13 mediated S-palmitoylation on Fbp1\ndecreases its gluconeogenesis function, but the therapeutic potential of Zdhhc13\ninhibition in DMED mice remains limited. It is possible that Fbp1 C282S-loaded\nlipid nanoparticles (Fbp1 C282S-LNP) can restore Fbp1 expression and\nabrogated pathological palmitoylation in the corpus cavernosum and restore\nerectile dysfunction. However priapism should be avoided and different models\nshould be tested.\nResponse: We sincerely appreciate your insightful comment. We have evaluated the\ntherapeutic potential of Fbp1 C282S-loaded lipid nanoparticles (Fbp1 C282S-LNP) in\nmultiple diabetic models, including type 1 diabetes mellitus (T1DM) mice, type 2\ndiabetes mellitus (T2DM) models, and db/db mice. In these studies, Fbp1 C282S-LNP\nconsistently restored Fbp1 expression in the corpus cavernosum, eliminated\npathological palmitoylation, and improved erectile function. Importantly, no priapism\n(persistent abnormal erection) was observed during treatment in any of these models,\nnor in the pharmacokinetic, pharmacodynamic, or safety studies conducted in normal\nmice.\nWe are grateful for your suggestion, which prompted us to further validate both the\nsafety and efficacy of Fbp1 C282S-LNP across different models, thereby significantly\nenhancing the reliability of our conclusions.\nSummary\nIn summary, we have fully addressed all reviewers’ comments. The revised\nmanuscript now includes the following key improvements:\n1. Added clinical data linking lactate accumulation to erectile dysfunction in DMED\npatients, clarified the physiological relevance of lactate levels, and strengthened the\nclinical relevance of the study.\n2. Provided mechanistic validations: transcriptomic analysis of lactate-induced\nsmooth muscle cell injury, demonstration of ZDHHC13-downregulation-mediated\nFBP1 conformational change (exposure of G260 impairing gluconeogenesis), and\ninvestigation into the upregulation mechanisms of ZDHHC13/CBX3.\n3. Supplemented with Fbp1-C282S-LNPs data (dose response, pharmacokinetics,\ntissue distribution, hepatorenal toxicity) and extended validation to additional diabetic\nmouse models (T1DM, db/db), enhancing translational value.\n4. Ensured full methodological transparencyand improved figure clarity and\npresentation.\n5. Standardized language, terminology, and figure formatting throughout.\nWe sincerely thank the reviewers for their insightful comments, which have greatly\nimproved the quality of our manuscript.\nResponse to Reviewers\nWe sincerely thank the Editors and Reviewers once again for their valuable time,\nmeticulous re-evaluation, and insightful supplementary feedback. We have carefully\nconsidered every additional comment and have revised the manuscript accordingly.\nWe believe these revisions have further strengthened the rigor, completeness, and\nclarity of our work. Below, we provide a detailed point-by-point response to each\ncomment. All changes and supplementary content in the revised manuscript are\nhighlighted for easy reference.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have substantially and thoughtfully addressed nearly all 15 of the raised\nconcerns. Their rebuttal includes new experimental data (e.g., longitudinal lactate\nmeasurements, collagen immunofluorescence, PK/PD profiles), mechanistic\nclarifications (Zdhhc13-mediated palmitoylation, Cbx3 transcriptional repression),\nand translational considerations (clinical serum lactate data, safety/tissue distribution\nof LNPs).\nWhile the responses are comprehensive, a few areas could still be strengthened,\nincluding:\n1. More detailed quantitative analyses (effect sizes, fibrosis morphometry,\nlongitudinal statistics).\nResponse: We sincerely appreciate your positive recognition of our comprehensive\nrevisions and constructive feedback on further strengthening the manuscript’s\nquantitative rigor. We fully agree that detailed quantitative analyses are essential to\nreinforcing the reliability and biological relevance of our findings.\nAs requested, we have supplemented detailed longitudinal statistical analyses for\ncavernosal lactate and intracavernous pressure. The results confirm a statistically\nsignificant increase over time in the DMED group. These new analyses are presented\nin Supplementary Figures 1d-1f, and described in the main text.\nIn direct response to your suggestion, we have now calculated and reported effect\nsizes for key quantitative comparisons. For comparisons between two groups using\nthe unpaired t-test, we calculated Cohen's d value, as it is the most appropriate and\nwidely accepted measure of standardized effect size for this statistical test. It provides\na clear interpretation of the magnitude of the difference between groups, independent\nof sample size. For one-way ANOVA analyses involving multiple groups, we first\ncalculated the overall effect size using eta-squared (η²) and reported the Cohen's d\nvalue for the relevant pairwise post-hoc comparisons. The values are now reported in\nthe respective figure legends. Additionally, we have supplemented sample sizes (n\nvalues) and detailed statistical methods in the panel of each figure legend, ensuring\nfull transparency for reproducibility.\nWe sincerely thank you for your valuable suggestion regarding the need for detailed\nfibrosis morphometric analysis. In response, we conducted a systematic and in-depth\nre-analysis of our existing histology and immunofluorescence images. Upon careful\nexamination, we found that our results are consistent with the majority of existing\nreports in this research field [PMID: 36064453;32693824；38874549；39536767；\n37157936]. In the DMED model, the core characteristic of fibrotic deposition is a\nsignificant quantitative increase in collagen content, whereas under the resolution of\nour current methodologies (conventional histological staining), no consistent or\nsignificant differences in the higher-order architecture (i.e., morphology) were\ndetected. We fully understand and agree with your insightful perspective that more\nsubtle morphological differences may require more advanced techniques for detection.\nThe absence of observed significant morphological changes in this study may indeed\nreflect the detection limits of the conventional methods we employed.\nWe sincerely thank you for your insightful guidance, which has helped us refine the\nquantitative presentation of our findings, standardize statistical reporting, and clarify\nthe nature of fibrosis-related changes in our model—further enhancing the rigor and\ncomprehensiveness of our work.\n2. Additional causal validation (e.g., autophagy inhibition rescue, metabolic flux\ntracing).\nResponse: We sincerely appreciate the valuable suggestion regarding the necessity of\nsupplementing causal validation experiments. We fully concur that in-depth\nmechanistic verification is pivotal for establishing the pathophysiological association\nbetween lactate and DMED.\nIn the previous revision round, transcriptome sequencing revealed that lactate\ntreatment activates the autophagic pathway in CCSMCs. In response to your\nrecommendation, we have performed more rigorous causal validation in the current\nrevision.\nIn vitro experiments were conducted by pretreating CCSMCs with the autophagy\ninhibitor 3-methyladenine (3-MA). The results demonstrated that 3-MA significantly\nabrogates lactate-induced excessive autophagy activation, impairment of cell viability,\nand phenotypic switching toward the synthetic smooth muscle phenotype.\nIn animal models, we further analyzed autophagic activity in the corpus cavernosum\nof lactate-challenged mice and DMED mice. Intriguingly, progressive elevation of the\nLC3-II/LC3-I ratio was observed in both models during disease progression, whereas\nP62 protein expression exhibited a dynamic change characterized by an initial\ndecrease followed by a subsequent increase. This finding suggests that autophagic\nflux may become dysregulated in the late stage of the disease.\nBuilding upon these observations, we performed interventions with 3-MA and\nSMER28, respectively. Our data showed that 3-MA effectively suppressed excessive\nautophagy and remarkably improved erectile function as well as tissue architecture in\nDMED mice. In contrast, although SMER28 promoted P62 degradation, it failed to\nexert significant therapeutic efficacy.\nFinally, we validated that Fbp1 C282S-LNP treatment can also efficiently reverse\nautophagic abnormalities in the corpus cavernosum of DMED mice.\nCollectively, these results indicate that lactate-induced excessive autophagy is a key\nmechanism contributing to corpus cavernosum smooth muscle cell damage.\nFurthermore, CCSMCs were cultured with 2 mM ¹³C-labeled lactate under starvation\nconditions, and the extracellular medium was analyzed by nuclear magnetic resonance\n(NMR) spectroscopy referring to relevant literature [PMID：12944325, PMID：\n10751328, PMID：10600777]. We successfully detected the production of ¹³C-labeled\nglucose, which directly confirms that CCSMCs possess gluconeogenic capacity to\nconvert lactate into glucose. This finding provides direct experimental evidence\nsupporting the central role of lactate metabolism in CCSMCs.\n3. Longer-term follow-up and repeat-dose safety data to enhance translational\nrelevance.\nResponse:We are grateful for this insightful comment regarding the long-term safety\nprofile and therapeutic sustainability of the Fbp1 C282S-LNP strategy, which\nrepresent key considerations for its clinical translation. In our previous revision, a\npreliminary assessment of treatment durability was conducted, incorporating a\none-month treatment-free observation period following the initial 4-week therapy. It is\nnoteworthy that the principal therapeutic benefits—including improved lactate\nmetabolism, tissue repair, and erectile function—were consistently maintained\nthroughout this follow-up interval (Supplementary Figure 16). These observations\nsuggest that the intervention may promote sustained and restorative outcomes beyond\ntransient symptomatic amelioration.\nWe fully concur that a more systematic evaluation of long-term safety and repeated\ndosing regimens is essential to further de-risk this therapeutic strategy. A study\ndesigned to comprehensively address these aspects would require an extended\ntimeline with multiple treatment cycles and detailed systemic toxicity monitoring,\nwhich lies beyond the scope of the present discovery-phase investigation.\nIn response to your suggestion, we have added a dedicated paragraph in the\nDiscussion section to acknowledge this direction and outline future work: \"Although\nsustained treatment effects were observed following short-term intervention in\nthis study, further investigation is warranted to elucidate the long-term safety\nprofile and optimal dosing frequency of Fbp1 C282S-LNP. Subsequent studies\nemploying extended multi-dose regimens in larger animal models will be\nvaluable to comprehensively assess chronic toxicity and maximize the\ntranslational potential of this strategy.\"\nWe thank you again for this suggestion, which has enhanced the clinical relevance and\nscholarly rigor of our work.\n4. Schematic figures in the introduction or discussion to better communicate the\nproposed mechanism.\nOverall, the revisions substantially improve the manuscript, and the study now\nprovides a clearer mechanistic insight and stronger therapeutic rationale for targeting\nFbp1/lactate metabolism in DMED. I believe the paper is much closer to being\nsuitable for publication after minor additional clarifications.\nResponse: We sincerely appreciate the suggestion to add a schematic diagram in the\nmain text for a clearer presentation of the mechanism pathway. In response to your\nrecommendation, we have inserted a fully annotated mechanism schematic (Figure 8)\nalong with relevant textual explanations in the Discussion section of the paper:\nTranscriptional repression and functional dysfunction of Fbp1 are key causes of\npenile cavernosal lactate accumulation in DMED. Mechanistically, H3K9me3\nmediated by the Suv39h1/Cbx3 complex and H3K27me3 mediated by Ezh2\ncooperatively suppress Fbp1 transcription; meanwhile, Zdhhc13-mediated\npalmitoylation of the FBP1 protein at the C282 site directly impairs its\ngluconeogenic function, exacerbating lactate accumulation. To target the\naforementioned aberrant mechanisms, we developed Fbp1 C282S-LNP, which\nrestores Fbp1 expression and blocks pathological palmitoylation via\nintracavernosal injection, thereby effectively reducing local lactate levels.\nWe believe that the inclusion of this schematic diagram significantly enhances the\nvisual presentation of the mechanism pathway in the paper, enabling readers to more\nintuitively comprehend the logical connection between the molecular mechanisms and\nthe therapeutic strategy. We sincerely thank you again for this valuable suggestion,\nwhich has played a crucial role in improving the quality of the paper.\nReviewer #6 (Remarks to the Author):\nThe manuscript by Xiao et al identifies fructose 1,6 bisphosphatase 1 (FBP1) activity\nin corpus cavernosum smooth muscle cells (CCSMCs) as a potentially important\ncomponent to erectile function in mice and the overall health of CCSMCs. This is by\nvirtue of FBP1's role in the gluconeogenic pathway, which the authors claim is\nnecessary to prevent lactate accumulation that may otherwise impair CCSMC\nfunction. The implications are significant because this study provides a potentially\nnovel target for ED treatment that could in theory be used alone or in conjunction\nwith traditional therapies such as phosphodiesterase inhibitors.\nThe authors report that FBP1 expression and function in CCSMCs is reduced in a\nmodel of diabetes mellitus-induced erectile dysfuncton(DMED). This happens as a\nresult of epigenetic and post-translational events, leading ultimately to elevated lactate\nconcentrations in the corpus cavernosum. The authors go to great lengths to identify\nthe mechanistic basis for changes in FBP1 expression (via histone methylation\npathways) and functon(via palmitoylation), using in vitro methods as well as their\nmouse model of DMED. The molecular biology experiments are fairly thorough by\ndesign (although underpowered), and complimentary approaches are used. In\nparticular, the use of lipid nanoparticles to deliver palmitoylation-resistant FBP1 is a\ncompelling approach to correct aberrant metabolic processes.\nWhile concerns raised by Reviewer 2 on palmitoylation were adequately addressed by\nauthors, there are several major issues, described below, which diminish the overall\nimpact of the manuscript. In general, while many of the experiments shed light on the\nregulatory mechanisms controlling the functional expression of FBP1 in various\nsystems, extrapolation of these findings to the author's central hypothesis is limited by\nseveral concerns.\nMajor concerns:\n1 - The central hypothesis of the manuscript rests on the idea that dysregulation\nof FBP1 in CCSMCs leads to lactate accumulation via suppression of\ngluconeogenesis. However, the authors do not provide evidence, nor do they cite\nprevious work showing that CCSMCs actually carry out gluconeogenesis to a\nsignificant degree. Aside from transcript-level data and a western blot showing\nthat a few other gluconeogenic enzymes were not altered the corpus cavernosum\nof DMED mice (Sup Fig 2), there is no supporting evidence that lactate\naccumulation in the corpus cavernosum happens as a result impaired flux\nthrough the gluconeogenic pathway within CCSMCs. Relying on the\nquantification of enzyme abundance, rather than measuring glucose or\nmetabolite production directly, is not a rigorous way to test the extent to which\nglycolytic or gluconeogenic pathways proceed in CCSMCs under the\nexperimental conditions. Without a clear demonstration that CCSMCs carry out\ngluconeogenesis and that inhibition of this pathway specifically leads to lactate\naccumulation, it is impossible to interpret the study's findings in the context of\nthe authors' proposed model. The authors must definitively show that\nCCSMC-localized FB1 participates in \"lactate clearance via gluconeogenesis,\" so\nto speak, otherwise the premise of the study is not sound.\nThe authors do mention that conversion of lactate into glucose via the Cori Cycle\nis a key part of lactate metabolism. However, in this pathway, lactate generated\nby muscle under anaerobic conditions is converted to glucose in the liver. If a\nsimilar pathway exists within smooth muscle cells, the authors should include a\nreference which describes it. Likewise, many of the references to FBP1 function\nin lactate metabolism and/or diabetic models involve studies using liver cells or\nliver tissue. It is not appropriate to draw parallels between the findings of this\nstudy and others that deal with these pathways specifically in the liver.\nResponse: We sincerely appreciate your insightful comments. You correctly noted\nthat establishing the functional gluconeogenic capacity of corpus cavernosum smooth\nmuscle cells (CCSMCs) is a core premise of this study. To address this, we have\nreviewed foundational work in the field and integrated our experimental data to\nprovide the following systematic evidence:\nSmooth muscle cells have been shown to possess complete gluconeogenic capacity\nand independent gluconeogenic metabolic compartments [PMID: 8789645]. Fbp1, a\nkey functional enzyme in this pathway, catalyzes the critical conversion of exogenous\nfructose-1,6-bisphosphate to glucose [PMID: 10600777] and exhibits unique\nsubcellular localization in smooth muscle cells [PMID: 16200456]. Pathological\nstudies further underscore Fbp1’s functional importance—for instance,\ndownregulation of gluconeogenic enzymes (including Fbp1) during vascular\ncalcification correlates closely with metabolic imbalance [PMID: 21757657].\nCollectively, these findings confirm Fbp1 as a functional, indispensable key enzyme\nfor gluconeogenesis in smooth muscle cells. Unlike hepatic gluconeogenesis (which\nmaintains systemic glucose homeostasis), CCSMC gluconeogenesis may support\nlocal energy demands (e.g., during erection) or clear lactate (preventing local\nacidosis-induced impairment of smooth muscle contraction).\nSecond, building on this theoretical framework, we provide direct experimental\nevidence via metabolic flux analysis. Primary mouse CCSMCs were cultured with 2\nmM ¹³C-labeled lactate under starvation conditions; nuclear magnetic resonance\n(NMR) detected ¹³C-labeled glucose production, directly confirming CCSMCs’\ngluconeogenic capacity.\nMore importantly, we established a complete causal evidence chain in animal models:\n(1) DMED models showed reduced Fbp1 expression and lactate accumulation in\ncavernous tissue; (2) Fbp1+/- mice exhibited significantly elevated cavernous lactate\nlevels; (3) Fbp1 C282S-LNP was extensively taken up by CCSMCs, restoring\nfunctional Fbp1 expression in cavernous tissue, reducing lactate levels, and improving\nerectile function.\nAdditionally, analyses of oxygen consumption rate (OCR), extracellular acidification\nrate (ECAR), and glucose/lactate production flux in the MOVAS cell line further\nsupport Fbp1’s critical role in regulating smooth muscle cell metabolic homeostasis.\nHowever, we acknowledge that limitations in primary cell acquisition prevented\nreplication of some energy metabolism experiments in primary cells—an inherent\nconstraint of primary cell research.\nIn summary, through systematic literature review, direct metabolic flux validation,\nand in vitro/in vivo functional experiments, we provide multi-level evidence for our\ncore hypothesis: \"Impaired FBP1 function in CCSMCs causes lactate accumulation\nby inhibiting gluconeogenesis.\" Thank you for your valuable input, which has\nstrengthened the rigor of our argument.\n2 - Throughout the study, elevations in lactate concentration, either systemically\nor locally, are reported in association with functional/structural/biochemical\nchanges in the corpus cavernosum. However, these observations are correlative\nand there is a lack of evidence suggesting the downstream effects are a direct\nresult of lactate accumulation consequent to suppressed gluconeogenesis/FBP1\nactivity within CCSMCs. In addition, there should be more discussion of how\nexcessive lactate might cause the phenotypic effects that are described. What is\nthe underlying pathological mechanism by which lactate accumulation causes\ndysfunction?\nResponse: We sincerely appreciate this important comment of yours. You correctly\npointed out the need to clarify the causal relationship between lactate accumulation\nand the observed functional dysfunction, as well as to further elaborate on its\nunderlying pathological mechanism. We have addressed this issue through\nsupplementary experiments and in-depth discussions.\nTo confirm that the observed phenotypic changes are a direct consequence of lactate\naccumulation, we conducted gain-of-function and rescue experiments both in vitro\nand in vivo:In in vitro experiments, we treated CCSMCs with exogenous sodium\nlactate (5 mM and 10 mM). The results showed that key pathological\nchanges—including decreased cell viability and phenotypic switching—exhibited a\ndose-dependent pattern, demonstrating lactate’s direct damaging effect. In animal\nexperiments, intraperitoneal injection of sodium lactate into healthy mice successfully\nincreased lactate levels in the corpus cavernosum, accompanied by significant\nimpairment of erectile function and histological changes that recapitulated the core\npathological features of DMED. Most compellingly, targeted restoration of Fbp1\nfunction in the corpus cavernosum using Fbp1 C282S-LNPs effectively reduced\nlactate accumulation and reversed both erectile dysfunction and tissue damage in\nDMED mice. This rescue experiment confirms that correcting lactate metabolic\ndysregulation is sufficient to improve the disease phenotype.\nWe fully understand and highly agree with your insightful suggestion regarding the\nneed to further strengthen the evidence chain for lactate’s downstream mechanism of\naction—this profound perspective helps establish a causal link confirming that lactate\naccumulation is a direct cause of cavernous dysfunction.\nIn the previous revision, transcriptome sequencing revealed that lactate treatment\nactivates the autophagic pathway in CCSMCs.\nFollowing your recommendation, we performed more rigorous mechanistic validation\nin the current revision:\nIn vitro experiments demonstrated that the autophagy inhibitor 3-methyladenine\n(3-MA) significantly suppressed lactate-induced excessive autophagy, impaired cell\nviability, and phenotypic switching.\nAnimal experiments further showed that in both lactate-challenged mice and DMED\nmice, the ratio of the autophagy marker LC3-II/LC3-I was elevated, while P62 protein\nexpression exhibited dynamic changes—suggesting dysregulated autophagic flux.\nIntervention experiments revealed that the autophagy inhibitor 3-MA effectively\nimproved erectile function and tissue structure in DMED mice, whereas the\nautophagy enhancer SMER28, despite promoting P62 degradation, exerted no\nsignificant therapeutic effect.\nFinally, we validated that Fbp1 C282S-LNP treatment can also efficiently reverse\nautophagic abnormalities in the corpus cavernosum of DMED mice.\nThese results indicate that lactate-induced excessive autophagy is a key mechanism\nunderlying corpus cavernosum smooth muscle cell damage.\nWe believe that these newly supplemented experimental data, together with our\nexisting evidence, form a complete causal evidence chain, clearly defining the\npathological mechanism: \"lactate accumulation → excessive autophagy → cell\ndamage → functional dysfunction.\" We again thank you for your valuable comments,\nwhich have played a pivotal role in enhancing the mechanistic depth of our study.\n3 - Do the authors suggest that lactate is produced in CCSMCs under aerobic\nconditions? Does oxygen availability in corpus cavernosum have an impact on\nlactate generation? Is it possible that aerobic metabolism is somehow impaired in\nCCSMCs in the DMED model, and that this leads to lactate accumulation\nindependently of FBP1? If there are other metabolic disturbances in CCSMCs in\nthe DMED model, the mechanism(s) leading to lactate accumulation may or may\nnot be connected to the functional impairment of the corpus cavernosum. As\nmentioned above, it would be helpful to discuss a clear mechanistic link between\nexcessive lactate and impaired CCSMC function.\nResponse:\nWe sincerely appreciate the series of profound questions you raised, which have been\ncrucial for refining the demonstration of metabolic mechanisms in this study.\nFirst, regarding lactate production under aerobic conditions, previous studies have\nindeed shown that even under aerobic conditions, normal metabolic activity generates\nsmall amounts of lactate due to the metabolic compartmentalization of glycolysis and\ngluconeogenesis in cells—though lactate production increases significantly under\nhypoxic conditions. We also fully acknowledge that oxygen supply to the corpus\ncavernosum is a important factor influencing lactate levels, and several studies have\nconfirmed that the pathological process of DMED is closely associated with local\nhypoxia.\nYour question regarding \"whether other metabolic disorders contribute to lactate\naccumulation in the DMED model\" is highly insightful. We honestly acknowledge\nthat this study focuses on elucidating the mechanism of reduced lactate clearance\nmediated by FBP1 dysfunction. Our data—such as the downregulated transcriptional\nlevels of key glycolytic enzymes—indicate that in the DMED model used in this\nstudy, lactate accumulation is more likely driven by impaired clearance. Additionally,\nwe attempted to inhibit glycolysis using an LDHA inhibitor, but this did not\nsignificantly reduce cavernous lactate levels. Of course, we cannot completely rule\nout the possibility of other pathways (e.g., impaired aerobic metabolism), and these\ncomplex metabolic interactions undoubtedly represent important directions for future\nresearch.\nNevertheless, our series of functional experiments strongly confirm that the\nFBP1-mediated lactate metabolic pathway plays an indispensable core role in DMED:\nmanipulating FBP1 function directly affects lactate levels, and restoring FBP1\nfunction via Fbp1 C282S-LNPs specifically improves local lactate metabolism and\nrescues erectile function. This demonstrates that this pathway is a sufficient and\nimportant cause of functional dysfunction.\nFinally, we fully agree with your insight that a clear mechanistic link between\nexcessive lactate and impaired CCSMC function must be established. This was one of\nthe core focuses of our current revision. As addressed in the previous response,\nfollowing your suggestion, we confirmed through experiments that lactate-induced\nexcessive autophagy is the key link in the causal chain connecting these two processes,\nand we have included an in-depth discussion of this mechanism.\nWe again thank you for your insightful questions, which have greatly enhanced the\nlogical rigor and mechanistic depth of our study.\n4 - In the model of the heterozygous Fbp1-/+ mice, genetic disruption is global,\nmaking it difficult to parse out local effects in the corpus cavernosum from\neffects elsewhere in the body (i.e., the liver). Is it possible that metabolic\ndisruption from global Fbp1-/+ produces the functional changes in CCSMCs as\npart of a broader systemic phenotype? Is it possible that CCSMC dysfunction\nhappens coincidentally with an elevation in circulating lactate, but not\nnecessarily because of it? This does not rule out the possibility that elevated\ncirculating lactate in Fbp1-/+ mice has a detrimental effect on CCSMCs, but it is\ndifficult to justify the conclusion that CCSMC-localized FBP1 dysregulation is\ndriving the observed effects.\nResponse:\nWe sincerely appreciate this profound, critical question. Your insight into the\nlimitations of the Fbp1+/- heterozygous mouse model—and concern about potential\nconfounding between systemic metabolic disorders and local effects—are entirely\nreasonable and well-founded. This has prompted us to interpret our conclusions more\ncautiously and strengthen support for the local mechanism with supplementary\nexperiments.\nYou correctly noted that in the systemic Fbp1+/- model, distinguishing between\ncavernous local effects and systemic impacts (e.g., hyperlactatemia from impaired\nhepatic gluconeogenesis) is challenging. Systemically elevated lactate could\nindependently exert direct toxic effects on corpus cavernosum smooth muscle cells\n(CCSMCs), causing dysfunction. We fully agree that phenotypic data from Fbp1+/-\nmice alone would be insufficient to conclude \"local FBP1 dysfunction in CCSMCs\ndrives the phenotype.\"\nIt is noteworthy that we observed elevated plasma lactate levels in clinical patients\nwith DMED, and lactate concentration was significantly correlated with both the\noccurrence and severity of the condition. In a DMED mouse model, we similarly\nfound increased lactate levels in both plasma and penile tissues. Through in vivo and\nin vitro experiments, we further validated the dose-dependent detrimental effect of\nlactate on CCSMCs and erectile function. More importantly, after administration of\nFbp1-C282S-LNP, tissue distribution analysis revealed that the nanoparticles\npreferentially accumulated in the corpus cavernosum, where they were efficiently\ntaken up by CCSMCs. This led to a significant reduction in intracavernosal lactate\nlevels and a marked improvement in the erectile function phenotype of DMED mice.\nIn the current round of experiments, we further added immunofluorescence\nstaining and quantitative tissue analysis of the liver, skeletal muscle, and penile\ncorpus cavernosum. Crucially, pharmacokinetic data first showed Fbp1 C282S-LNPs\n(administered via local cavernous injection) were primarily retained in the injected\npenis. While minor accumulation was detected in the liver and skeletal muscle over\ntime, the combined results of immunofluorescence staining and quantitative tissue\nanalysis confirmed that only the penile corpus cavernosum exhibited a statistically\nsignificant increase in Fbp1 protein expression—no significant changes were\nobserved in the liver or skeletal muscle.\nThese results strongly suggest that elevated lactate levels localized in the corpus\ncavernosum impair CCSMC function and erectile function, and that the corresponding\ntherapeutic effect is primarily driven by the specific upregulation of FBP1 within the\ncavernous tissue, rather than indirect systemic metabolic modulation. We fully agree\nwith your emphasis on methodological rigor, which has helped us sharpen the focus\nand strengthen the core argument of our study. Relevant clarifications have been\nincorporated into the revised manuscript, and we thank you again for your insightful\nfeedback.\n5 - Apomorphine, the drug used to induce erection in mice, is a centrally acting\ndopamine receptor agonist targeting dopamine signaling pathways in the brain.\nIt is thought to be most effective for psychogenic erectile dysfuncton(in humans)\nand works via a mechanism separate from PDE inhibitors, which act locally.\nHow do the authors rule out that experimental interventions (DMED model,\nlactate injection, metabolic changes from Fbp1-/+, etc.) do not interfere with\nneural control of erectile function?\nResponse: We sincerely appreciate the important question you raised. You correctly\npointed out that apomorphine, as a central dopamine receptor agonist, induces\nerections primarily by activating dopamine signaling pathways in the central nervous\nsystem—this mechanism differs from the local action of PDE5 inhibitors. We fully\nagree that excluding potential interference of experimental interventions on the neural\ncontrol pathway of erections is crucial for ensuring the reliability of our study’s\nconclusions.\nWe can clearly rule out such interference through the following experimental design\nand results:\nFirst, in the different animal models involved in this study, the use of apomorphine\nwas strictly limited to baseline screening of erectile function in DMED model mice\n(i.e., administered only once after model establishment and before grouping for\nintervention). This step was used to verify whether the animals had successfully\ndeveloped an ED model and possessed inducible erectile potential, which is a\ncommonly used method in DMED model establishment [PMID: 41090037; 40676888;\n39654344]. In contrast, in the Fbp1+/- mouse model and the lactate injection model,\napomorphine was not used at any stage from the start to the end of the experiment.\nInstead, changes in erectile function were evaluated using intracavernous pressure\n(ICP) measurements and histological examinations. Therefore, the changes in erectile\nfunction observed in these latter two model groups can completely exclude\ninterference from the central effects of apomorphine, and their results directly reflect\nthe independent impact of metabolic alterations (Fbp1 deficiency or exogenous lactate)\non erectile function.\nSecond, during the screening phase of the DMED model, we ensured strict\nconsistency in experimental procedures. After receiving apomorphine (100 μg/kg,\nsubcutaneous injection). The positive erectile rate in the normal control group was\nnearly 100%, which strongly confirms that the central neural pathway dependent on\napomorphine was functionally intact under our experimental conditions. If DMED or\nits associated metabolic interventions (e.g., lactate accumulation) had impaired this\nneural pathway, delayed or absent erectile responses should have been observed first\nin the control group or during the early screening phase—but this was not the case.\nThis indicates that DMED-related erectile dysfunction does not originate from\nprimary damage to the neural control pathway.\nFinally, regarding concerns about drug residue effects, apomorphine has an extremely\nshort elimination half-life in mice. After a single screening injection, the drug was\ncompletely cleared long before the start of the 4-week intervention treatment with\nFbp1 C282S-LNP and other agents. Therefore, all subsequent therapeutic effects were\nsignificantly associated with the restoration of local FBP1 activity and the reduction\nof lactate levels in the corpus cavernosum. This series of results clearly indicates that\nthe mechanism of action stems from the correction of the local metabolic environment,\nrather than sustained effects on the neural pathway revealed by the early apomorphine\ntest.\nWe again thank you for your profound insight, which has made our argument more\nrigorous.\nMinor concerns\n1 - The authors describe H-PROTAC as a CBX3-sepcific degrader. However,\nSupp Fig 5 does not demonstrate specificity. The authors did not test whether\ndegradation of other proteins, aside from CBX3, were altered by H-PROTAC\ntreatment.\nResponse: We sincerely appreciate the important question you raised regarding the\nspecificity verification of H-PROTAC. You correctly pointed out that our description\nof H-PROTAC as a \"CBX3-specific degrader\" in the original manuscript was\ninsufficiently rigorous, and Supplementary Figure 9 indeed failed to fully demonstrate\nits selectivity for other proteins.\nAs stated in our Methods section, we screened protein databases and identified that\nthe QTAR M3L peptide of H3K9me3 can specifically bind to CBX3—based on this\nfinding, we developed a degrader primarily targeting CBX3. However, we fully agree\nthat since CBX1, CBX3, and CBX5 all belong to the HP1 family and are all\nH3K9me3 readers, this degrader may indeed exhibit a certain degree of\ncross-reactivity.\nIn accordance with your suggestion, we have supplemented experiments to verify the\ndegradation effect of H-PROTAC on different CBX protein family members. The\nresults showed that at a concentration of 1 μM, H-PROTAC significantly degraded\nCBX3, while its degradation effect on CBX1 and CBX5 was notably weaker. This\nconcentration gradient experiment not only provided a basis for us to select the\noptimal working concentration but also confirmed that H-PROTAC exhibits relative\nselectivity for CBX3 at specific concentrations.\nWe have revised the relevant description in the original manuscript, and supplemented\nthe corresponding experimental data. These revisions have made our descriptions\nmore accurate and our experimental design more comprehensive. We once again\nthank you for your rigorous review comments, which are of great value for improving\nthe scientific rigor and accuracy of our research.\n2 - Experiments assessing the palmitoylation status of FBP1 should include both\npositive and negative controls (proteins known to be palmitoylated and known to\nnot be palmitoylated) to demonstrate that the assay was successful. In addition,\nthere should be quantitation for these experiments, and not just a representative\nimmunoblot.\nResponse: We sincerely appreciate the critical comment emphasizing the necessity of\nincluding positive/negative controls and performing quantitative analysis in\nexperiments investigating FBP1 palmitoylation. We fully concur that these\nrecommendations are essential for ensuring the reliability of experimental results, and\nsystematic refinements have been implemented in the previous round of revisions.\nSpecifically, in the acyl-biotin exchange (ABE) assay—a gold-standard method for\ndetecting protein palmitoylation—we have established a comprehensive control\nsystem: acylglycerol kinase (AGK), a well-characterized stably palmitoylated protein,\nwas employed as the positive control, while β-actin (for which no evidence of\npalmitoylation has been reported to date) served as the negative control. This control\nstrategy robustly validates the efficacy of our experimental protocol.\nConcurrently, rigorous quantitative analysis of palmitoylation levels was performed\nacross all cell line-based experiments. The full set of quantitative results has been\ncompiled in Supplementary Figure S11, thereby providing reliable data support for\nour experimental findings. It should be noted that in primary cell experiments,\nquantitative analysis is subject to certain limitations due to practical constraints,\nincluding the difficulty in isolating primary cells and the restricted number of viable\ncell passages.\nThrough these supplementary experiments and methodological refinements, we\nbelieve the current experimental data substantially enhance the reliability and\npersuasiveness of our FBP1 palmitoylation detection results. We would like to express our\ngratitude again to the reviewer for this valuable guidance, which plays a pivotal role\nin improving the methodological rigor of the present study.\n3 - Typically, palmitoylation of proteins is thought to promote an interaction with\nlipid membranes. Are there other examples of palmitoylation causing\nstructural/functional changes in cytosolic enzymes? If so, this should be\ndiscussed.\nResponse: We sincerely appreciate the profound question you raised. You correctly\npointed out that palmitoylation modification is generally considered to promote the\ninteraction between proteins and lipid membranes. The recent studies have indeed\nrevealed that palmitoylation can also directly induce conformational changes in\ncytoplasmic enzymes, thereby regulating their activity—and this is highly\nconsistent with the phenomenon we observed in FBP1 [PMID: 16945566; PMID:\n16128592; PMID: 25576832].\nIn accordance with your suggestion, we have supplemented the relevant literature and\ndiscussions in the Discussion section of the paper. These precedents strongly support\nour finding that Zdhhc13-mediated palmitoylation at the C282 site can induce\nconformational changes in FBP1, thereby affecting the accessibility of its active\ncenter and ultimately inhibiting the gluconeogenic function of this enzyme. We\nsincerely thank you for this directional suggestion, as it has placed our research in a\nbroader theoretical context and significantly enhanced the depth of our discussion.\n4 - In general, the figure legends and in-text figure descriptions require much\nmore detailed information. Figure legends in particular should have details of\nthe experimental conditions, spelled out acronyms for the text in the figure\npanels, description of the assays used for generation of each panel within the\nfigure, description of the statistical tests used, etc. Also many of the experiments\nhave an n=3. A larger sample size would increase the rigor of the study.\nResponse:We sincerely appreciate the valuable suggestions regarding the\nimprovement of figure information and the increase in sample size. We fully agree\nthat detailed figure legends and an adequate sample size are crucial for ensuring the\nreproducibility and rigor of the study, and comprehensive revisions have been made in\naccordance with the reviewer’s comments.\nTo address the issue of insufficiently detailed figure information, we have\nsystematically refined all figure legends: each legend now explicitly supplements key\nexperimental methods corresponding to individual subfigures, experimental\nconditions, statistical methods, sample size, and the full names of all abbreviations\nused in the figures. This ensures that readers can accurately understand the details of\neach experimental design and analytical approach.\nRegarding the sample size concern, we sincerely accept your comment. In all new\nexperiments conducted during this round of revisions, the sample size has been\nincreased to n=5. Meanwhile, it should be noted that due to constraints in\nexperimental timeline and resources, we were unable to supplement the sample size\nfor experiments already completed in the manuscript. We acknowledge that this may\naffect the statistical power of some study results, for which we sincerely apologize,\nand this limitation has been described in the \" Limitations\" section of the paper.\nThese improvements have significantly enhanced the transparency, reproducibility,\nand credibility of our study results. We would like to thank the reviewer again for the\ninsightful suggestions, which have played an important role in improving the quality\nof our research work.\n5 - Line 75-80: The previous observation that sildenafil reduced lactate\naccumulation in exercising rats does not necessarily imply a connection between\nlactate metabolism and erectile function. It only suggests that PDE5 inhibition\nreduces lactate accumulation caused be strenuous exercise.\nResponse: We sincerely appreciate the important insight. You are absolutely correct\nthat the previously cited observation regarding sildenafil reducing lactate\naccumulation in exercised rats cannot directly establish a mechanistic link between\nlactate metabolism and erectile function—it merely demonstrates that PDE5\ninhibition can alleviate lactate accumulation induced by strenuous exercise. In\naccordance with your suggestion, we have deleted the potentially misleading\nstatements in the original manuscript. We sincerely thank you for this highly\ninsightful correction, as this revision has significantly enhanced the logical rigor of\nour discussion.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nOur concerns have been addressed in the revisions.so now we have no additional question.\nResponse: We are very grateful for your time and careful review of our manuscript. Your guidance\nhas helped improve our manuscript. Thank you again for your support and comments.\nReviewer #6 (Remarks to the Author):\nThe authors have satisfactorily addressed the concerns raised from the previous review. They have\nadded citations describing the capacity of smooth muscle cells to participate in gluconeogenesis,\nas well as provided experimental evidence of glucose production in CCSM cells. They have\nestablished a more clear mechanistic link between lactate accumulation and CCSM cells\ndysfuncton(via autophagy). They have appropriately acknowledged the limitations of using a\nglobal Fbp1-/+ mouse model in terms of parsing out local versus systemic effects in the corpus\ncavernosum. They included more controls in their CBX3 degradation assays, and added\ninformation to all figure legends to improve clarity. I have no further suggestions for improving\nthe manuscript.\nResponse: We sincerely appreciate your valuable time and meticulous review of our revised\nmanuscript. Thank you again for your support and comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Our concerns have been addressed in the revisions.so now we have no additional question.", "reviewer_6": "The authors have satisfactorily addressed the concerns raised from the previous review. They have added citations describing the capacity of smooth muscle cells to participate in gluconeogenesis, as well as provided experimental evidence of glucose production in CCSM cells. They have established a more clear mechanistic link between lactate accumulation and CCSM cells dysfuncton(via autophagy). They have appropriately acknowledged the limitations of using a global Fbp1-/+ mouse model in terms of parsing out local versus systemic effects in the corpus cavernosum. They included more controls in their CBX3 degradation assays, and added information to all figure legends to improve clarity. I have no further suggestions for improving the manuscript."}, "author_response": "Response to Reviewers\nWe sincerely thank the Editors and Reviewers once again for their valuable time, meticulous re-evaluation, and insightful supplementary feedback. We have carefully considered every additional comment and have revised the manuscript accordingly. We believe these revisions have further strengthened the rigor, completeness, and clarity of our work. Below, we provide a detailed point-by-point response to each comment. All changes and supplementary content in the revised manuscript are highlighted for easy reference.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have substantially and thoughtfully addressed nearly all 15 of the raised concerns. Their rebuttal includes new experimental data (e.g., longitudinal lactate measurements, collagen immunofluorescence, PK/PD profiles), mechanistic clarifications (Zdhhc13-mediated palmitoylation, Cbx3 transcriptional repression), and translational considerations (clinical serum lactate data, safety/tissue distribution of LNPs).\nWhile the responses are comprehensive, a few areas could still be strengthened, including:\n1. More detailed quantitative analyses (effect sizes, fibrosis morphometry, longitudinal statistics).\nResponse: We sincerely appreciate your positive recognition of our comprehensive revisions and constructive feedback on further strengthening the manuscript’s quantitative rigor. We fully agree that detailed quantitative analyses are essential to reinforcing the reliability and biological relevance of our findings.\nAs requested, we have supplemented detailed longitudinal statistical analyses for cavernosal lactate and intracavernous pressure. The results confirm a statistically significant increase over time in the DMED group. These new analyses are presented in Supplementary Figures 1d-1f, and described in the main text.\nIn direct response to your suggestion, we have now calculated and reported effect sizes for key quantitative comparisons. For comparisons between two groups using the unpaired t-test, we calculated Cohen's d value, as it is the most appropriate and widely accepted measure of standardized effect size for this statistical test. It provides a clear interpretation of the magnitude of the difference between groups, independent of sample size. For one-way ANOVA analyses involving multiple groups, we first calculated the overall effect size using eta-squared (η²) and reported the Cohen's d value for the relevant pairwise post-hoc comparisons. The values are now reported in the respective figure legends. Additionally, we have supplemented sample sizes (n values) and detailed statistical methods in the panel of each figure legend, ensuring full transparency for reproducibility.\nWe sincerely thank you for your valuable suggestion regarding the need for detailed fibrosis morphometric analysis. In response, we conducted a systematic and in-depth re-analysis of our existing histology and immunofluorescence images. Upon careful examination, we found that our results are consistent with the majority of existing reports in this research field [PMID: 36064453;32693824；38874549；39536767； 37157936]. In the DMED model, the core characteristic of fibrotic deposition is a significant quantitative increase in collagen content, whereas under the resolution of our current methodologies (conventional histological staining), no consistent or significant differences in the higher-order architecture (i.e., morphology) were detected. We fully understand and agree with your insightful perspective that more subtle morphological differences may require more advanced techniques for detection. The absence of observed significant morphological changes in this study may indeed reflect the detection limits of the conventional methods we employed.\nWe sincerely thank you for your insightful guidance, which has helped us refine the quantitative presentation of our findings, standardize statistical reporting, and clarify the nature of fibrosis-related changes in our model—further enhancing the rigor and comprehensiveness of our work.\n2. Additional causal validation (e.g., autophagy inhibition rescue, metabolic flux tracing).\nResponse: We sincerely appreciate the valuable suggestion regarding the necessity of supplementing causal validation experiments. We fully concur that in-depth mechanistic verification is pivotal for establishing the pathophysiological association between lactate and DMED.\nIn the previous revision round, transcriptome sequencing revealed that lactate treatment activates the autophagic pathway in CCSMCs. In response to your recommendation, we have performed more rigorous causal validation in the current revision.\nIn vitro experiments were conducted by pretreating CCSMCs with the autophagy inhibitor 3-methyladenine (3-MA). The results demonstrated that 3-MA significantly abrogates lactate-induced excessive autophagy activation, impairment of cell viability, and phenotypic switching toward the synthetic smooth muscle phenotype.\nIn animal models, we further analyzed autophagic activity in the corpus cavernosum of lactate-challenged mice and DMED mice. Intriguingly, progressive elevation of the LC3-II/LC3-I ratio was observed in both models during disease progression, whereas P62 protein expression exhibited a dynamic change characterized by an initial decrease followed by a subsequent increase. This finding suggests that autophagic flux may become dysregulated in the late stage of the disease.\nBuilding upon these observations, we performed interventions with 3-MA and SMER28, respectively. Our data showed that 3-MA effectively suppressed excessive autophagy and remarkably improved erectile function as well as tissue architecture in DMED mice. In contrast, although SMER28 promoted P62 degradation, it failed to exert significant therapeutic efficacy.\nFinally, we validated that Fbp1 C282S-LNP treatment can also efficiently reverse autophagic abnormalities in the corpus cavernosum of DMED mice.\nCollectively, these results indicate that lactate-induced excessive autophagy is a key mechanism contributing to corpus cavernosum smooth muscle cell damage.\nFurthermore, CCSMCs were cultured with 2 mM ¹³C-labeled lactate under starvation conditions, and the extracellular medium was analyzed by nuclear magnetic resonance (NMR) spectroscopy referring to relevant literature [PMID：12944325, PMID： 10751328, PMID：10600777]. We successfully detected the production of ¹³C-labeled glucose, which directly confirms that CCSMCs possess gluconeogenic capacity to convert lactate into glucose. This finding provides direct experimental evidence supporting the central role of lactate metabolism in CCSMCs.\n3. Longer-term follow-up and repeat-dose safety data to enhance translational relevance.\nResponse:We are grateful for this insightful comment regarding the long-term safety profile and therapeutic sustainability of the Fbp1 C282S-LNP strategy, which represent key considerations for its clinical translation. In our previous revision, a preliminary assessment of treatment durability was conducted, incorporating a one-month treatment-free observation period following the initial 4-week therapy. It is noteworthy that the principal therapeutic benefits—including improved lactate metabolism, tissue repair, and erectile function—were consistently maintained throughout this follow-up interval (Supplementary Figure 16). These observations suggest that the intervention may promote sustained and restorative outcomes beyond transient symptomatic amelioration.\nWe fully concur that a more systematic evaluation of long-term safety and repeated dosing regimens is essential to further de-risk this therapeutic strategy. A study designed to comprehensively address these aspects would require an extended timeline with multiple treatment cycles and detailed systemic toxicity monitoring, which lies beyond the scope of the present discovery-phase investigation.\nIn response to your suggestion, we have added a dedicated paragraph in the Discussion section to acknowledge this direction and outline future work: \"Although sustained treatment effects were observed following short-term intervention in this study, further investigation is warranted to elucidate the long-term safety profile and optimal dosing frequency of Fbp1 C282S-LNP. Subsequent studies employing extended multi-dose regimens in larger animal models will be valuable to comprehensively assess chronic toxicity and maximize the translational potential of this strategy.\"\nWe thank you again for this suggestion, which has enhanced the clinical relevance and scholarly rigor of our work.\n4. Schematic figures in the introduction or discussion to better communicate the proposed mechanism.\nOverall, the revisions substantially improve the manuscript, and the study now provides a clearer mechanistic insight and stronger therapeutic rationale for targeting Fbp1/lactate metabolism in DMED. I believe the paper is much closer to being suitable for publication after minor additional clarifications.\nResponse: We sincerely appreciate the suggestion to add a schematic diagram in the main text for a clearer presentation of the mechanism pathway. In response to your recommendation, we have inserted a fully annotated mechanism schematic (Figure 8) along with relevant textual explanations in the Discussion section of the paper:\nTranscriptional repression and functional dysfunction of Fbp1 are key causes of penile cavernosal lactate accumulation in DMED. Mechanistically, H3K9me3 mediated by the Suv39h1/Cbx3 complex and H3K27me3 mediated by Ezh2 cooperatively suppress Fbp1 transcription; meanwhile, Zdhhc13-mediated palmitoylation of the FBP1 protein at the C282 site directly impairs its gluconeogenic function, exacerbating lactate accumulation. To target the aforementioned aberrant mechanisms, we developed Fbp1 C282S-LNP, which restores Fbp1 expression and blocks pathological palmitoylation via intracavernosal injection, thereby effectively reducing local lactate levels.\nWe believe that the inclusion of this schematic diagram significantly enhances the visual presentation of the mechanism pathway in the paper, enabling readers to more intuitively comprehend the logical connection between the molecular mechanisms and the therapeutic strategy. We sincerely thank you again for this valuable suggestion, which has played a crucial role in improving the quality of the paper.\nReviewer #6 (Remarks to the Author):\nThe manuscript by Xiao et al identifies fructose 1,6 bisphosphatase 1 (FBP1) activity in corpus cavernosum smooth muscle cells (CCSMCs) as a potentially important component to erectile function in mice and the overall health of CCSMCs. This is by virtue of FBP1's role in the gluconeogenic pathway, which the authors claim is necessary to prevent lactate accumulation that may otherwise impair CCSMC function. The implications are significant because this study provides a potentially novel target for ED treatment that could in theory be used alone or in conjunction with traditional therapies such as phosphodiesterase inhibitors.\nThe authors report that FBP1 expression and function in CCSMCs is reduced in a model of diabetes mellitus-induced erectile dysfuncton(DMED). This happens as a result of epigenetic and post-translational events, leading ultimately to elevated lactate concentrations in the corpus cavernosum. The authors go to great lengths to identify the mechanistic basis for changes in FBP1 expression (via histone methylation pathways) and functon(via palmitoylation), using in vitro methods as well as their mouse model of DMED. The molecular biology experiments are fairly thorough by design (although underpowered), and complimentary approaches are used. In particular, the use of lipid nanoparticles to deliver palmitoylation-resistant FBP1 is a compelling approach to correct aberrant metabolic processes.\nWhile concerns raised by Reviewer 2 on palmitoylation were adequately addressed by authors, there are several major issues, described below, which diminish the overall impact of the manuscript. In general, while many of the experiments shed light on the regulatory mechanisms controlling the functional expression of FBP1 in various systems, extrapolation of these findings to the author's central hypothesis is limited by several concerns.\nMajor concerns:\n1 - The central hypothesis of the manuscript rests on the idea that dysregulation of FBP1 in CCSMCs leads to lactate accumulation via suppression of gluconeogenesis. However, the authors do not provide evidence, nor do they cite previous work showing that CCSMCs actually carry out gluconeogenesis to a significant degree. Aside from transcript-level data and a western blot showing that a few other gluconeogenic enzymes were not altered the corpus cavernosum of DMED mice (Sup Fig 2), there is no supporting evidence that lactate accumulation in the corpus cavernosum happens as a result impaired flux through the gluconeogenic pathway within CCSMCs. Relying on the quantification of enzyme abundance, rather than measuring glucose or metabolite production directly, is not a rigorous way to test the extent to which glycolytic or gluconeogenic pathways proceed in CCSMCs under the experimental conditions. Without a clear demonstration that CCSMCs carry out gluconeogenesis and that inhibition of this pathway specifically leads to lactate accumulation, it is impossible to interpret the study's findings in the context of the authors' proposed model. The authors must definitively show that CCSMC-localized FB1 participates in \"lactate clearance via gluconeogenesis,\" so to speak, otherwise the premise of the study is not sound.\nThe authors do mention that conversion of lactate into glucose via the Cori Cycle is a key part of lactate metabolism. However, in this pathway, lactate generated by muscle under anaerobic conditions is converted to glucose in the liver. If a similar pathway exists within smooth muscle cells, the authors should include a reference which describes it. Likewise, many of the references to FBP1 function in lactate metabolism and/or diabetic models involve studies using liver cells or liver tissue. It is not appropriate to draw parallels between the findings of this study and others that deal with these pathways specifically in the liver.\nResponse: We sincerely appreciate your insightful comments. You correctly noted that establishing the functional gluconeogenic capacity of corpus cavernosum smooth muscle cells (CCSMCs) is a core premise of this study. To address this, we have reviewed foundational work in the field and integrated our experimental data to provide the following systematic evidence:\nSmooth muscle cells have been shown to possess complete gluconeogenic capacity and independent gluconeogenic metabolic compartments [PMID: 8789645]. Fbp1, a key functional enzyme in this pathway, catalyzes the critical conversion of exogenous fructose-1,6-bisphosphate to glucose [PMID: 10600777] and exhibits unique subcellular localization in smooth muscle cells [PMID: 16200456]. Pathological studies further underscore Fbp1’s functional importance—for instance, downregulation of gluconeogenic enzymes (including Fbp1) during vascular calcification correlates closely with metabolic imbalance [PMID: 21757657].\nCollectively, these findings confirm Fbp1 as a functional, indispensable key enzyme for gluconeogenesis in smooth muscle cells. Unlike hepatic gluconeogenesis (which maintains systemic glucose homeostasis), CCSMC gluconeogenesis may support local energy demands (e.g., during erection) or clear lactate (preventing local acidosis-induced impairment of smooth muscle contraction).\nSecond, building on this theoretical framework, we provide direct experimental evidence via metabolic flux analysis. Primary mouse CCSMCs were cultured with 2 mM ¹³C-labeled lactate under starvation conditions; nuclear magnetic resonance (NMR) detected ¹³C-labeled glucose production, directly confirming CCSMCs’ gluconeogenic capacity.\nMore importantly, we established a complete causal evidence chain in animal models: (1) DMED models showed reduced Fbp1 expression and lactate accumulation in cavernous tissue; (2) Fbp1+/- mice exhibited significantly elevated cavernous lactate levels; (3) Fbp1 C282S-LNP was extensively taken up by CCSMCs, restoring functional Fbp1 expression in cavernous tissue, reducing lactate levels, and improving erectile function.\nAdditionally, analyses of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and glucose/lactate production flux in the MOVAS cell line further support Fbp1’s critical role in regulating smooth muscle cell metabolic homeostasis. However, we acknowledge that limitations in primary cell acquisition prevented replication of some energy metabolism experiments in primary cells—an inherent constraint of primary cell research.\nIn summary, through systematic literature review, direct metabolic flux validation, and in vitro/in vivo functional experiments, we provide multi-level evidence for our core hypothesis: \"Impaired FBP1 function in CCSMCs causes lactate accumulation by inhibiting gluconeogenesis.\" Thank you for your valuable input, which has strengthened the rigor of our argument.\n2 - Throughout the study, elevations in lactate concentration, either systemically or locally, are reported in association with functional/structural/biochemical changes in the corpus cavernosum. However, these observations are correlative and there is a lack of evidence suggesting the downstream effects are a direct result of lactate accumulation consequent to suppressed gluconeogenesis/FBP1 activity within CCSMCs. In addition, there should be more discussion of how excessive lactate might cause the phenotypic effects that are described. What is the underlying pathological mechanism by which lactate accumulation causes dysfunction?\nResponse: We sincerely appreciate this important comment of yours. You correctly pointed out the need to clarify the causal relationship between lactate accumulation and the observed functional dysfunction, as well as to further elaborate on its underlying pathological mechanism. We have addressed this issue through supplementary experiments and in-depth discussions.\nTo confirm that the observed phenotypic changes are a direct consequence of lactate accumulation, we conducted gain-of-function and rescue experiments both in vitro and in vivo:In in vitro experiments, we treated CCSMCs with exogenous sodium lactate (5 mM and 10 mM). The results showed that key pathological changes—including decreased cell viability and phenotypic switching—exhibited a dose-dependent pattern, demonstrating lactate’s direct damaging effect. In animal experiments, intraperitoneal injection of sodium lactate into healthy mice successfully increased lactate levels in the corpus cavernosum, accompanied by significant impairment of erectile function and histological changes that recapitulated the core pathological features of DMED. Most compellingly, targeted restoration of Fbp1 function in the corpus cavernosum using Fbp1 C282S-LNPs effectively reduced lactate accumulation and reversed both erectile dysfunction and tissue damage in DMED mice. This rescue experiment confirms that correcting lactate metabolic dysregulation is sufficient to improve the disease phenotype.\nWe fully understand and highly agree with your insightful suggestion regarding the need to further strengthen the evidence chain for lactate’s downstream mechanism of action—this profound perspective helps establish a causal link confirming that lactate accumulation is a direct cause of cavernous dysfunction.\nIn the previous revision, transcriptome sequencing revealed that lactate treatment activates the autophagic pathway in CCSMCs.\nFollowing your recommendation, we performed more rigorous mechanistic validation in the current revision:\nIn vitro experiments demonstrated that the autophagy inhibitor 3-methyladenine (3-MA) significantly suppressed lactate-induced excessive autophagy, impaired cell viability, and phenotypic switching.\nAnimal experiments further showed that in both lactate-challenged mice and DMED mice, the ratio of the autophagy marker LC3-II/LC3-I was elevated, while P62 protein expression exhibited dynamic changes—suggesting dysregulated autophagic flux.\nIntervention experiments revealed that the autophagy inhibitor 3-MA effectively improved erectile function and tissue structure in DMED mice, whereas the autophagy enhancer SMER28, despite promoting P62 degradation, exerted no significant therapeutic effect.\nFinally, we validated that Fbp1 C282S-LNP treatment can also efficiently reverse autophagic abnormalities in the corpus cavernosum of DMED mice.\nThese results indicate that lactate-induced excessive autophagy is a key mechanism underlying corpus cavernosum smooth muscle cell damage.\nWe believe that these newly supplemented experimental data, together with our existing evidence, form a complete causal evidence chain, clearly defining the pathological mechanism: \"lactate accumulation → excessive autophagy → cell damage → functional dysfunction.\" We again thank you for your valuable comments, which have played a pivotal role in enhancing the mechanistic depth of our study.\n3 - Do the authors suggest that lactate is produced in CCSMCs under aerobic conditions? Does oxygen availability in corpus cavernosum have an impact on lactate generation? Is it possible that aerobic metabolism is somehow impaired in CCSMCs in the DMED model, and that this leads to lactate accumulation independently of FBP1? If there are other metabolic disturbances in CCSMCs in the DMED model, the mechanism(s) leading to lactate accumulation may or may not be connected to the functional impairment of the corpus cavernosum. As mentioned above, it would be helpful to discuss a clear mechanistic link between excessive lactate and impaired CCSMC function.\nResponse:\nWe sincerely appreciate the series of profound questions you raised, which have been crucial for refining the demonstration of metabolic mechanisms in this study.\nFirst, regarding lactate production under aerobic conditions, previous studies have indeed shown that even under aerobic conditions, normal metabolic activity generates small amounts of lactate due to the metabolic compartmentalization of glycolysis and gluconeogenesis in cells—though lactate production increases significantly under hypoxic conditions. We also fully acknowledge that oxygen supply to the corpus cavernosum is a important factor influencing lactate levels, and several studies have confirmed that the pathological process of DMED is closely associated with local hypoxia.\nYour question regarding \"whether other metabolic disorders contribute to lactate accumulation in the DMED model\" is highly insightful. We honestly acknowledge that this study focuses on elucidating the mechanism of reduced lactate clearance mediated by FBP1 dysfunction. Our data—such as the downregulated transcriptional levels of key glycolytic enzymes—indicate that in the DMED model used in this study, lactate accumulation is more likely driven by impaired clearance. Additionally, we attempted to inhibit glycolysis using an LDHA inhibitor, but this did not significantly reduce cavernous lactate levels. Of course, we cannot completely rule out the possibility of other pathways (e.g., impaired aerobic metabolism), and these complex metabolic interactions undoubtedly represent important directions for future research.\nNevertheless, our series of functional experiments strongly confirm that the FBP1-mediated lactate metabolic pathway plays an indispensable core role in DMED: manipulating FBP1 function directly affects lactate levels, and restoring FBP1 function via Fbp1 C282S-LNPs specifically improves local lactate metabolism and rescues erectile function. This demonstrates that this pathway is a sufficient and important cause of functional dysfunction.\nFinally, we fully agree with your insight that a clear mechanistic link between excessive lactate and impaired CCSMC function must be established. This was one of the core focuses of our current revision. As addressed in the previous response, following your suggestion, we confirmed through experiments that lactate-induced excessive autophagy is the key link in the causal chain connecting these two processes, and we have included an in-depth discussion of this mechanism.\nWe again thank you for your insightful questions, which have greatly enhanced the logical rigor and mechanistic depth of our study.\n4 - In the model of the heterozygous Fbp1-/+ mice, genetic disruption is global, making it difficult to parse out local effects in the corpus cavernosum from effects elsewhere in the body (i.e., the liver). Is it possible that metabolic disruption from global Fbp1-/+ produces the functional changes in CCSMCs as part of a broader systemic phenotype? Is it possible that CCSMC dysfunction happens coincidentally with an elevation in circulating lactate, but not necessarily because of it? This does not rule out the possibility that elevated circulating lactate in Fbp1-/+ mice has a detrimental effect on CCSMCs, but it is difficult to justify the conclusion that CCSMC-localized FBP1 dysregulation is driving the observed effects.\nResponse:\nWe sincerely appreciate this profound, critical question. Your insight into the limitations of the Fbp1+/- heterozygous mouse model—and concern about potential confounding between systemic metabolic disorders and local effects—are entirely reasonable and well-founded. This has prompted us to interpret our conclusions more cautiously and strengthen support for the local mechanism with supplementary experiments.\nYou correctly noted that in the systemic Fbp1+/- model, distinguishing between cavernous local effects and systemic impacts (e.g., hyperlactatemia from impaired hepatic gluconeogenesis) is challenging. Systemically elevated lactate could independently exert direct toxic effects on corpus cavernosum smooth muscle cells (CCSMCs), causing dysfunction. We fully agree that phenotypic data from Fbp1+/- mice alone would be insufficient to conclude \"local FBP1 dysfunction in CCSMCs drives the phenotype.\"\nIt is noteworthy that we observed elevated plasma lactate levels in clinical patients with DMED, and lactate concentration was significantly correlated with both the occurrence and severity of the condition. In a DMED mouse model, we similarly found increased lactate levels in both plasma and penile tissues. Through in vivo and in vitro experiments, we further validated the dose-dependent detrimental effect of lactate on CCSMCs and erectile function. More importantly, after administration of Fbp1-C282S-LNP, tissue distribution analysis revealed that the nanoparticles preferentially accumulated in the corpus cavernosum, where they were efficiently taken up by CCSMCs. This led to a significant reduction in intracavernosal lactate levels and a marked improvement in the erectile function phenotype of DMED mice.\nIn the current round of experiments, we further added immunofluorescence staining and quantitative tissue analysis of the liver, skeletal muscle, and penile corpus cavernosum. Crucially, pharmacokinetic data first showed Fbp1 C282S-LNPs (administered via local cavernous injection) were primarily retained in the injected penis. While minor accumulation was detected in the liver and skeletal muscle over time, the combined results of immunofluorescence staining and quantitative tissue analysis confirmed that only the penile corpus cavernosum exhibited a statistically significant increase in Fbp1 protein expression—no significant changes were observed in the liver or skeletal muscle.\nThese results strongly suggest that elevated lactate levels localized in the corpus cavernosum impair CCSMC function and erectile function, and that the corresponding therapeutic effect is primarily driven by the specific upregulation of FBP1 within the cavernous tissue, rather than indirect systemic metabolic modulation. We fully agree with your emphasis on methodological rigor, which has helped us sharpen the focus and strengthen the core argument of our study. Relevant clarifications have been incorporated into the revised manuscript, and we thank you again for your insightful feedback.\n5 - Apomorphine, the drug used to induce erection in mice, is a centrally acting dopamine receptor agonist targeting dopamine signaling pathways in the brain. It is thought to be most effective for psychogenic erectile dysfuncton(in humans) and works via a mechanism separate from PDE inhibitors, which act locally. How do the authors rule out that experimental interventions (DMED model, lactate injection, metabolic changes from Fbp1-/+, etc.) do not interfere with neural control of erectile function?\nResponse: We sincerely appreciate the important question you raised. You correctly pointed out that apomorphine, as a central dopamine receptor agonist, induces erections primarily by activating dopamine signaling pathways in the central nervous system—this mechanism differs from the local action of PDE5 inhibitors. We fully agree that excluding potential interference of experimental interventions on the neural control pathway of erections is crucial for ensuring the reliability of our study’s conclusions.\nWe can clearly rule out such interference through the following experimental design and results:\nFirst, in the different animal models involved in this study, the use of apomorphine was strictly limited to baseline screening of erectile function in DMED model mice (i.e., administered only once after model establishment and before grouping for intervention). This step was used to verify whether the animals had successfully developed an ED model and possessed inducible erectile potential, which is a commonly used method in DMED model establishment [PMID: 41090037; 40676888; 39654344]. In contrast, in the Fbp1+/- mouse model and the lactate injection model, apomorphine was not used at any stage from the start to the end of the experiment. Instead, changes in erectile function were evaluated using intracavernous pressure (ICP) measurements and histological examinations. Therefore, the changes in erectile function observed in these latter two model groups can completely exclude interference from the central effects of apomorphine, and their results directly reflect the independent impact of metabolic alterations (Fbp1 deficiency or exogenous lactate) on erectile function.\nSecond, during the screening phase of the DMED model, we ensured strict consistency in experimental procedures. After receiving apomorphine (100 μg/kg, subcutaneous injection). The positive erectile rate in the normal control group was nearly 100%, which strongly confirms that the central neural pathway dependent on apomorphine was functionally intact under our experimental conditions. If DMED or its associated metabolic interventions (e.g., lactate accumulation) had impaired this neural pathway, delayed or absent erectile responses should have been observed first in the control group or during the early screening phase—but this was not the case. This indicates that DMED-related erectile dysfunction does not originate from primary damage to the neural control pathway.\nFinally, regarding concerns about drug residue effects, apomorphine has an extremely short elimination half-life in mice. After a single screening injection, the drug was completely cleared long before the start of the 4-week intervention treatment with Fbp1 C282S-LNP and other agents. Therefore, all subsequent therapeutic effects were significantly associated with the restoration of local FBP1 activity and the reduction of lactate levels in the corpus cavernosum. This series of results clearly indicates that the mechanism of action stems from the correction of the local metabolic environment, rather than sustained effects on the neural pathway revealed by the early apomorphine test.\nWe again thank you for your profound insight, which has made our argument more rigorous.\nMinor concerns\n1 - The authors describe H-PROTAC as a CBX3-sepcific degrader. However, Supp Fig 5 does not demonstrate specificity. The authors did not test whether degradation of other proteins, aside from CBX3, were altered by H-PROTAC treatment.\nResponse: We sincerely appreciate the important question you raised regarding the specificity verification of H-PROTAC. You correctly pointed out that our description of H-PROTAC as a \"CBX3-specific degrader\" in the original manuscript was insufficiently rigorous, and Supplementary Figure 9 indeed failed to fully demonstrate its selectivity for other proteins.\nAs stated in our Methods section, we screened protein databases and identified that the QTAR M3L peptide of H3K9me3 can specifically bind to CBX3—based on this finding, we developed a degrader primarily targeting CBX3. However, we fully agree that since CBX1, CBX3, and CBX5 all belong to the HP1 family and are all H3K9me3 readers, this degrader may indeed exhibit a certain degree of cross-reactivity.\nIn accordance with your suggestion, we have supplemented experiments to verify the degradation effect of H-PROTAC on different CBX protein family members. The results showed that at a concentration of 1 μM, H-PROTAC significantly degraded CBX3, while its degradation effect on CBX1 and CBX5 was notably weaker. This concentration gradient experiment not only provided a basis for us to select the optimal working concentration but also confirmed that H-PROTAC exhibits relative selectivity for CBX3 at specific concentrations.\nWe have revised the relevant description in the original manuscript, and supplemented the corresponding experimental data. These revisions have made our descriptions more accurate and our experimental design more comprehensive. We once again thank you for your rigorous review comments, which are of great value for improving the scientific rigor and accuracy of our research.\n2 - Experiments assessing the palmitoylation status of FBP1 should include both positive and negative controls (proteins known to be palmitoylated and known to not be palmitoylated) to demonstrate that the assay was successful. In addition, there should be quantitation for these experiments, and not just a representative immunoblot.\nResponse: We sincerely appreciate the critical comment emphasizing the necessity of including positive/negative controls and performing quantitative analysis in experiments investigating FBP1 palmitoylation. We fully concur that these recommendations are essential for ensuring the reliability of experimental results, and systematic refinements have been implemented in the previous round of revisions.\nSpecifically, in the acyl-biotin exchange (ABE) assay—a gold-standard method for detecting protein palmitoylation—we have established a comprehensive control system: acylglycerol kinase (AGK), a well-characterized stably palmitoylated protein, was employed as the positive control, while β-actin (for which no evidence of palmitoylation has been reported to date) served as the negative control. This control strategy robustly validates the efficacy of our experimental protocol.\nConcurrently, rigorous quantitative analysis of palmitoylation levels was performed across all cell line-based experiments. The full set of quantitative results has been compiled in Supplementary Figure S11, thereby providing reliable data support for our experimental findings. It should be noted that in primary cell experiments, quantitative analysis is subject to certain limitations due to practical constraints, including the difficulty in isolating primary cells and the restricted number of viable cell passages.\nThrough these supplementary experiments and methodological refinements, we believe the current experimental data substantially enhance the reliability and persuasiveness of our FBP1 palmitoylation detection results. We would like to express our gratitude again to the reviewer for this valuable guidance, which plays a pivotal role in improving the methodological rigor of the present study.\n3 - Typically, palmitoylation of proteins is thought to promote an interaction with lipid membranes. Are there other examples of palmitoylation causing structural/functional changes in cytosolic enzymes? If so, this should be discussed.\nResponse: We sincerely appreciate the profound question you raised. You correctly pointed out that palmitoylation modification is generally considered to promote the interaction between proteins and lipid membranes. The recent studies have indeed revealed that palmitoylation can also directly induce conformational changes in cytoplasmic enzymes, thereby regulating their activity—and this is highly consistent with the phenomenon we observed in FBP1 [PMID: 16945566; PMID: 16128592; PMID: 25576832].\nIn accordance with your suggestion, we have supplemented the relevant literature and discussions in the Discussion section of the paper. These precedents strongly support our finding that Zdhhc13-mediated palmitoylation at the C282 site can induce conformational changes in FBP1, thereby affecting the accessibility of its active center and ultimately inhibiting the gluconeogenic function of this enzyme. We sincerely thank you for this directional suggestion, as it has placed our research in a broader theoretical context and significantly enhanced the depth of our discussion.\n4 - In general, the figure legends and in-text figure descriptions require much more detailed information. Figure legends in particular should have details of the experimental conditions, spelled out acronyms for the text in the figure panels, description of the assays used for generation of each panel within the figure, description of the statistical tests used, etc. Also many of the experiments have an n=3. A larger sample size would increase the rigor of the study.\nResponse:We sincerely appreciate the valuable suggestions regarding the improvement of figure information and the increase in sample size. We fully agree that detailed figure legends and an adequate sample size are crucial for ensuring the reproducibility and rigor of the study, and comprehensive revisions have been made in accordance with the reviewer’s comments.\nTo address the issue of insufficiently detailed figure information, we have systematically refined all figure legends: each legend now explicitly supplements key experimental methods corresponding to individual subfigures, experimental conditions, statistical methods, sample size, and the full names of all abbreviations used in the figures. This ensures that readers can accurately understand the details of each experimental design and analytical approach.\nRegarding the sample size concern, we sincerely accept your comment. In all new experiments conducted during this round of revisions, the sample size has been increased to n=5. Meanwhile, it should be noted that due to constraints in experimental timeline and resources, we were unable to supplement the sample size for experiments already completed in the manuscript. We acknowledge that this may affect the statistical power of some study results, for which we sincerely apologize, and this limitation has been described in the \" Limitations\" section of the paper.\nThese improvements have significantly enhanced the transparency, reproducibility, and credibility of our study results. We would like to thank the reviewer again for the insightful suggestions, which have played an important role in improving the quality of our research work.\n5 - Line 75-80: The previous observation that sildenafil reduced lactate accumulation in exercising rats does not necessarily imply a connection between lactate metabolism and erectile function. It only suggests that PDE5 inhibition reduces lactate accumulation caused be strenuous exercise.\nResponse: We sincerely appreciate the important insight. You are absolutely correct that the previously cited observation regarding sildenafil reducing lactate accumulation in exercised rats cannot directly establish a mechanistic link between lactate metabolism and erectile function—it merely demonstrates that PDE5 inhibition can alleviate lactate accumulation induced by strenuous exercise. In accordance with your suggestion, we have deleted the potentially misleading statements in the original manuscript. We sincerely thank you for this highly insightful correction, as this revision has significantly enhanced the logical rigor of our discussion.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nOur concerns have been addressed in the revisions.so now we have no additional question.\nResponse: We are very grateful for your time and careful review of our manuscript. Your guidance has helped improve our manuscript. Thank you again for your support and comments.\nReviewer #6 (Remarks to the Author):\nThe authors have satisfactorily addressed the concerns raised from the previous review. They have added citations describing the capacity of smooth muscle cells to participate in gluconeogenesis, as well as provided experimental evidence of glucose production in CCSM cells. They have established a more clear mechanistic link between lactate accumulation and CCSM cells dysfuncton(via autophagy). They have appropriately acknowledged the limitations of using a global Fbp1-/+ mouse model in terms of parsing out local versus systemic effects in the corpus cavernosum. They included more controls in their CBX3 degradation assays, and added information to all figure legends to improve clarity. I have no further suggestions for improving the manuscript.\nResponse: We sincerely appreciate your valuable time and meticulous review of our revised manuscript. Thank you again for your support and comments."}]

Decision letter