Branched-chain α-keto acids impair glucose-stimulated insulin secretion in pancreatic β-cells under diabetes by reactivating the LDHA-lactate axis

Domain: chemistry
Published: 2026-03-02
Editorial outcome: major_revision

Abstract
Dysmetabolism of branched-chain amino acid (BCAA) causes insulin resistance in type 2 diabetes, yet its effect on insulin-producing β-cells remains unclear. Here, we demonstrate that branched-chain α-ketoacids (BCKAs), derived from BCAAs, inhibited glucose-stimulated insulin secretion (GSIS) and glucose fluxes across human islets, mouse islets, and mouse β-cells. In diabetic humans, elevated circulating BCKAs negatively correlated with insulin secretory ability. Treatment with BCKA or its impaired catabolism suppressed GSIS in human islets and male mice, while reducing BCKA improved glucose tolerance and GSIS in male and female diabetic mice. Mechanistically, BCKA redirected glucose metabolism from the TCA cycle to the “β-cell disallowed” lactate dehydrogenase A (LDHA)-lactate axis. BCKA directly bound to LDHA, promoting its dimerization and enhancing enzymatic activity. β-cell-specific LDHA ablation restored GSIS and glucose tolerance in BCKA-fed male mice. Our findings demonstrate that BCKA disrupts insulin secretion through LDHA reactivation, linking aberrant BCAA metabolism to β-cell dysfunction in diabetes.

Introduction
Insulin secretion from pancreatic β-cells is tightly and dynamically regulated by multiple nutrients, hormones, and neural inputs. Glucose, the primary stimulator of biphasic insulin secretion1, is taken up and metabolized via glycolysis to produce pyruvate, which enters the mitochondrial tricarboxylic acid (TCA) cycle to generate ATP. The resulting increase in ATP/ADP ratio closes the ATP-sensitive potassium (K ) channel, inducing membrane depolarization and subsequent calcium influx, which drives a rapid but transient first-phase insulin secretion. Glucose also elicits a gradual but sustained second-phase insulin secretion through the K channel-independent amplifying signals and F-actin remodelling for exocytosis of insulin granules2, 3. This coupling of glucose metabolism to insulin secretion is essential for maintaining systemic glucose homeostasis.

Defective glucose-stimulated insulin secretion (GSIS) in β-cells is a major contributor to type 2 diabetes (T2D). Dysmetabolism of glucose through glycolysis and/or the mitochondrial TCA cycle impairs GSIS. For instance, disruption in pyruvate carboxylase-mediated anaplerotic flux of pyruvate to oxaloacetate, inhibition of pyruvate dehydrogenase-mediated pyruvate entry into the TCA cycle, or impaired TCA flux of α-ketoglutarate to isocitrate via isocitrate dehydrogenase 2 (IDH2), have all been shown to compromise GSIS4, 5, 6, 7. Additionally, altered expression or activity of glycolytic enzymes, such as lactate dehydrogenase A (LDHA; which converts pyruvate into lactate), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphofructose-1-kinase (PFK1), also impairs GSIS5, 8, 9.

Branched-chain amino acids (BCAAs), including leucine, isoleucine and valine, are essential amino acids. Beyond their role in protein synthesis, they also regulate energy metabolism and lipid handling in adipose tissues, as well as insulin sensitivity in the liver and skeletal muscle10, 11, 12. BCAAs are catabolized through transamination into α-ketoacids (BCKAs) via mitochondrial branched-chain aminotransferase (BCAT2), followed by decarboxylation via α-ketoacid dehydrogenase (BCKDH) complex and further oxidation, yielding C3- and C5-acylcarnitines for lipogenesis and acetyl-CoA and succinyl-CoA as TCA cycle substrates. The BCKDH complex is positively and negatively controlled by protein phosphatase 1K (PPM1K) and branched-chain α-ketoacid dehydrogenase kinase (BDK) respectively, both targeting serine 293 of the E1α subunit (p-BCKDHA-Ser293)13. Under diabetic conditions, the activity and expression of these catabolic enzymes are altered in the insulin-responsive tissues14, 15, 16, and enhancing BCAA catabolism improves insulin sensitivity in humans and rodents10, 11, 17.

Interestingly, an in vivo isotope tracing study demonstrated that BCAAs are also catabolized in pancreas to supply approximately 20% of its TCA carbons, a rate much higher than in other tissues in rodents18. Although leucine and its direct catabolite α-ketoisocaproate (KIC) trigger β-cell acute insulin secretion19, 20, 21, genetic alleles associated with high leucine levels correlate negatively with the homeostasis model assessment of β-cell function (HOMA-β) in diabetic patients22. Yet, whether β-cell BCAA metabolism is altered under the diabetic conditions and its contribution to β-cell dysfunction remain poorly understood.

In this study, we utilized various animal models and human samples to demonstrate that disrupted BCAA catabolism in diabetic β-cells drives accumulation of BCKAs, which act as protein linkers to induce dimerization of LDHA, a conventional “disallowed protein” in β-cells23. This LDHA activation redirects glucose metabolism from the TCA cycle to lactate production, reducing ATP production and ultimately impairing GSIS and glucose tolerance. While role of BCAA in insulin-responsive tissues is well recognized, our study demonstrated a novel mechanism linking BCAA dysmetabolism to β-cell dysfunction in T2D, highlighting the therapeutic potential of targeting β-cell metabolic dysregulation to restore glucose homeostasis in diabetes.

Results
Aberrant BCAA catabolism in diabetic islets and β-cells
It remains unknown whether BCAA catabolic machinery and their associated metabolites are altered and contribute to pancreatic β-cell dysfunctions in diabetes. Immunohistochemical staining revealed that total BCKDH-A, PPM1K, and BDK were abundantly expressed in the pancreatic islets of 20-week-old male BKS (db/+) lean mice, whereas BCAT2 was expressed similarly in both islets and exocrine and islets and exocrine cells. Notably, the expression of these proteins was significantly reduced in the pancreatic islets of BKS (db/db) diabetic mice (Fig. 1a-b), which displayed severe obesity, hyperglycemia, reduced insulin secretion, high serum levels of BCAAs and their downstream catabolites BCKAs, including [α-ketoisocaproate (KIC), α-keto-β-methylvalerate (KMV), and α-ketoisovalerate (KIV)] (Supplementary Fig. 1). Despite comparable phosphorylation levels of BCKDH-A—the key enzyme for BCAA catabolism, the p-BCKDH-A/total BCKDH-A ratio was markedly elevated in diabetic islets, suggesting reduced enzymatic activity and impaired BCAA metabolism (Fig. 1a-b). Ratio of p-BCKDH-A/total BCKDH-A was calculated individually for each mouse, rather than being normalized across the entire group, ensuring a more precise and individualized assessment. BCAAs and their metabolites were detected in isolated islets from the lean mice but were significantly higher in those from the db/db mice (Fig. 1c), which might be due to increased uptake of BCAAs from the circulation and/or defective BCAA catabolism in the islets.

We re-analyzed transcriptome and proteome datasets of human islets from HumanIslets online portal.24 There were no significant changes in mRNA expression of the enzymes (BCAT2, BCKDH complex, PPM1K and BDK) responsible for the first two BCAA catabolic processes between normal and T2D subjects (Fig. 1d). By contrast, protein expressions of BCAT2, BCKDH-A, DLD and PPM1K were significantly diminished in islets of T2D subjects compared with those in non-T2D subjects (Fig. 1d). Notably, a previous study of gene co-expression and protein-protein interaction networks highlighted PPM1K as a prominent T2D candidate gene in human islets,25 but its physiological relevance is unexplored. To confirm that PPM1K expression is reduced in pancreatic β-cells, we performed co-immunofluorescence staining with insulin (as a β-cell marker) using the pancreatic sections from human donors with or without T2D (Supplementary Table 1). PPM1K was abundantly expressed in β-cells in non-diabetic (ND) human subjects, but its expression was markedly reduced in T2D subjects (Fig. 1e, g). Aligned with the change in PPM1K, phosphorylation of BCKDH-A-Ser293 (p-BCKDH-A-Ser293) was increased in β-cells of diabetic subjects (Fig. 1f, g), while total BCKDH-A expression remained unchanged. The increased pBCKDH-A/total BCKDH-A ratio, along with reduced PPM1K expression in pancreatic β-cells were consistent with observations from db/db mice (Fig. 1a, b).

Circulating levels of BCKA negatively associate with β-cell function
Although circulating levels of BCAA and BCKA are known to be positively associated with peripheral insulin resistance in humans26, 27, their association with β-cell function is unclear. In line with previous reports, we found that hyperglycemic individuals exhibited significantly higher circulating levels of BCAA and BCKA, along with a slight increase in C3- and C5-acylcarnitine levels (Supplementary Table 2). It is noteworthy that among these metabolites, only α-keto acids KIC and KMV displayed an inverse association with HOMA-β, an index for β-cell function (Supplementary Fig. 2a-h). Additionally, total circulating levels of BCKA negatively correlated fasting blood glucose (Supplementary Fig. 2i).

Next, we examined a direct relationship between insulin secretion and BCAA dysmetabolism in diabetic humans. We measured C-peptide levels during arginine stimulation test and fasting BCAA and their downstream metabolites in human diabetic patients. C-peptide but not insulin was used as an endogenous β-cell secretory index because this can avoid potential confounding effects from exogenous insulin treatment and the variability in hepatic insulin removal28. In T2D subjects, significantly negative associations between C3- and C5-acylcarnitines and arginine-stimulated C-peptide secretion (ArgIC%) were observed, while such associations were not noted for BCAAs or BCKAs (Supplementary Fig. 3a-h). We then categorized the diabetic patients into two groups with a cutoff threshold response of C-peptide secretion at 2.1 ng/mL as previously reported29. The subjects with a level higher than 2.1 ng/mL were classified as “T2D with normal insulin secretion”, while those with a level lower than 2.1 ng/mL were classified as “T2D with defective insulin secretion” (Supplementary Table 3). We found that C3- and C5-acylcarnitines remained significantly negatively associated with ArgIC% in both subgroups, while circulating leucine, valine, KIC, KMV and KIV were negatively correlated with ArgIC% only in the “T2D with defective insulin secretion” group (Supplementary Fig. 3i-o).

Excessive BCKA causes defective GSIS and glucose intolerance
We next examined the capacity of β-cells to utilize BCAA. Both MIN6 and INS-1E cells, the well-established insulinoma β-cell lines, express the BCAA catabolic machinery, including enzymes BCAT2, PPM1K, BDK and BCKDH-A. Acute treatment with mixtures of BCAAs or BCKAs reduced phosphorylation of BCKDAH-A-Ser293, accompanied with accumulation of BCAAs, BCKAs, and C3- and C5-acylcarnitines in β-cells and mouse islets (Supplementary Fig. 4).

Our findings from both humans and animal studies indicate that elevated circulating BCAA or their derived metabolites and/or aberrant β-cell BCAA metabolism may causatively induce β-cell dysfunction in T2D. To test the first possibility, we treated MIN6 β-cells with individual BCAA (leucine, isoleucine, valine), their corresponding BCKA (KIC, KMV, KIV), or acylcarnitines (C3- or C5-) at the concentrations matching their circulating levels in humans and mice with diabetes for 48 hours (Supplementary Fig. 5a-c). Only single BCKA at high concentrations (100 μM of KIC or KIV, or 200 μM of KMV) suppressed GSIS, (Supplementary Fig. 5b). We next evaluated whether combined exposure to BCAA or BCKA metabolites would exert a synergetic effect. A cocktail of BCAAs (leucine, isoleucine, and valine, each at 0.8 mM) or BCKAs (KIC, KMV and KIV each at 10 μM; non-diabetic level) did not influence GSIS, whereas a BCKA cocktail (each at 30 or 100 μM, diabetic concentrations) significantly inhibited GSIS in mouse islets (Fig. 2a and Supplementary Fig. 5d). We replicated the inhibitory effect of BCKA cocktail (each at 30 μM) on GSIS in MIN6 β-cells and human islets (Fig. 2b, c). Importantly, treatment with 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2)—a well-established inhibitor of BDK to facilitate BCKA catabolism11, 30—increased BCKDH-A activity and hence reduced BCKA accumulation in MIN6 cells and mouse pancreatic islets exposed to high BCKAs (Supplementary Fig. 6a-c), accompanied with restoration of GSIS (Fig. 2b). These results indicate that the concurrent elevation of all three BCKAs act synergistically to impair β-cell function. Of note, we used a BCKA cocktail containing KIC, KMV, and KIV (each at 30 μM) to treat the β-cells or islets in the subsequent experiments, unless otherwise specified.

We explored why chronic treatment with BCKA, but not BCAA, impairs GSIS, although BCAAs can also be catabolized into BCKAs. Chronic BCKA treatment for 48 hours dramatically increased intracellular KIC, KMV and KIV by 10-30-fold, without significantly affecting BCAA levels in both MIN6 and INS-1E β-cells (Supplementary Fig. 7). By contrast, BCAA treatment only modestly increased intracellular BCKA levels (Supplementary Fig. 7d-f, j-l). These data indicate that conversion the majority of BCAAs is not used for catabolism in pancreatic β-cells, at least, under normal culture conditions.

We next examined the second possibility of whether blocking β-cell BCKA catabolism impairs GSIS. siRNA-mediated Bckdha or Ppm1k silencing impaired GSIS in INS-1E β-cells, and such impairments were rescued in BCAA-free medium (Fig. 2d-g and Supplementary Fig. 6d-e). Likewise, pancreatic islets from PPM1K global knockout mice displayed a marked reduction of GSIS compared with those from their WT littermates (Fig. 2h). On the other hand, Bcat2 silencing had no impact on GSIS (Supplementary Fig. 6f, g), which is similar to the observation in BCAT2 knockout mouse model31.

Chronic BCKA supplementation causes defective GSIS and glucose intolerance in mice
We then investigated whether manipulation of circulating BCKA affects β-cell functions using two different animal models. In the first model, C57BL/6J mice received either BCKA mixture (KIC, KMV and KIV each at 5 mg/ml) or saline (vehicle control) via drinking water for 6 weeks. Circulating BCKA gradually increased in the BCKA-fed mice to approximately 75 μM, a level comparable to those observed in db/db diabetic mice or T2D subjects (Fig. 3a)17. BCKA administration had no obvious impact on circulating BCAA levels, food intake, body weight, short-term fasting blood glucose and insulin level as well as insulin sensitivity (Supplementary Table 4 and Fig. 3b), but induced progressive glucose intolerance and defective GSIS (Fig. 3c-f). On the other hand, arginine-induced insulin secretion was comparable between BCKA-fed and control mice (Fig. 3g). BCKA feeding upregulated protein expressions of BCKDH-A, PPM1K, and BDK in pancreatic islets, suggesting active transport of dietary BCKA into islets and induction of their catabolic machinery (Supplementary Fig. 8a). Meanwhile, BCKA feeding did not alter insulin content, islet size, or α-/ β-cell distribution (Supplementary Fig. 8b-e). These data suggest that BCKA induces glucose intolerance mainly by disrupting GSIS.

In the second animal model, we tested whether reducing circulating BCAA/BCKA levels improves β-cell function in db/db diabetic mice. After a week of BT2 treatment, circulating BCAAs and BCKAs levels were reduced by approximately 30-40% and 50-60%, respectively (Supplementary Fig. 9a, b, Supplementary Table 5). BT2 treatment alleviated glucose intolerance and improved GSIS (Supplementary Fig. 9c, d), but had minimal effect on insulin sensitivity, arginine-induced insulin secretion (Supplementary Fig. 9e, f) or pancreatic islet size (Supplementary Fig. 9g). The improvement in GSIS might be due to reduced systemic BCKA and/or upregulated β-cell BCKA catabolism, as indicated by decreased BCKDH-A-Ser293 phosphorylation in the diabetic islets (Supplementary Fig. 9h).

To validate the latter speculation, we isolated islets from diabetic and lean mice and treated them with BT2 or vehicle for 4 hours, followed by BCKA supplementation. Diabetic islets accumulated more intracellular BCKA than control islets (Supplementary Fig. 10a-c). As expected, BT2 treatment reversed BCKA accumulation in diabetic islets and restored GSIS (Supplementary Fig. 10a-d). Taken together, our in vivo and ex vivo findings suggest that BT2 effectively improves GSIS by reducing circulating BCKA and/or directly promoting BCKA catabolism in β-cells.

β-cell specific blockage of BCKA catabolism induces glucose intolerance and defective GSIS in mice
To study β-cell-specific BCKA dysmetabolism, we generated β-cell specific PPM1K knockout mice (PPM1K β-KO mice) by crossing PPM1Kflox/flox mice with Ins2-Cre/ERT mice (Supplementary Fig. 11a). We targeted PPM1K but not BDK or BCKDH complex for the below reasons: (1) SNPs and mRNA expression of PPM1K are closely associated with insulin secretion, HbA1c and HOMA-β in humans22, 25, 32; (2) we observed a dramatic reduction of PPM1K protein in the diabetic islets from rodents and humans (Fig. 1a, e); (3) we and others showed that Ppm1k silencing or deletion disrupted BCKA catabolism and impaired GSIS in β-cells25 (Fig. 2g, h). Ppm1k mRNA was decreased by ~80% in pancreatic islets isolated from PPM1K β-KO mice compared to WT controls (Fig. 4a), accompanied by a sharp increase in BCKA levels in the PPM1K-deficient islets (Fig. 4b). On the other hand, Ppm1k mRNA expression in skeletal muscle, subcutaneous white adipose tissue (sWAT) and liver as well as circulating BCAAs, KIC and KMV remained unchanged, while circulating KIV was modestly increased in the PPM1K β-KO mice (Supplementary Fig. 11b-d).

We and others have demonstrated that Ins2-Cre and Ins2-Cre/ERT mice have normal glucose tolerance and GSIS, therefore we only included PPM1Kflox/flox littermates as WT controls3, 33, 34, 35, 36. PPM1K β-KO mice exhibited delayed glucose clearance and lower insulin secretion during GTT (Fig. 4c, d), but normal insulin sensitivity and arginine-induced insulin secretion (Fig. 4e, f). β-cell-specific PPM1K deletion did not significantly affect islet size or β-/α-cell proportions (Supplementary Fig. 12). These findings suggest that selectively abrogating β-cell BCKA catabolism via PPM1K deletion induces defective GSIS and glucose intolerance in mouse model.

BCKA disrupts glucose metabolism in β-cells
In the subsequent mechanistic investigation, we mainly employed BCKA treatment or PPM1K inhibition to mimic β-cell BCKA dysmetabolism, unless otherwise specified. Consistent with the animal studies, ex vivo study of isolated pancreatic islets showed that BCKA treatment or β-cell-selective PPM1K deletion only abolished insulin secretion induced by glucose, but not potassium chloride (KCl; which directly induces insulin secretion via membrane depolarization) (Fig. 3h and 4g). Furthermore, dynamic insulin secretion assay showed that only first- but not second-phase GSIS was blocked by BCKA treatment or β-cell PPM1K deletion (Fig. 3i and 4h). GSIS involves a cascade of events starting with glucose uptake, followed by glycolysis and mitochondrial TCA cycle (Fig. 5a). To pinpoint the defective step in GSIS following chronic BCKA treatment, we assessed glucose uptake, ATP production, total intracellular insulin content, mitochondrial membrane potential, cell viability, caspase 3 activity and apoptotic gene expressions (Fig. 5b and Supplementary Fig. 13). Only glucose-stimulated ATP production was impaired (Fig. 5c), indicating a disruption in glucose metabolism.

Seahorse extracellular flux analysis was performed to monitor glucose metabolism, revealing that chronic BCKA treatment significantly reduced both glucose-stimulated extracellular acidification rate (ECAR; normally indicative for glycolytic flux) and oxygen consumption rate (OCR; reflecting mitochondrial respiration) (Fig. 5d, e). Notably, the interpretation of these results in β-cells is complicated by their unique metabolic characteristics. Unlike most other cell types, β-cells express minimal levels of monocarboxylate transporter 1 (MCT; mediates lactate efflux), and lactate dehydrogenase A (LDHA) under normal conditions37, 38, 39, 40. Indeed, the near absence of MCT1 in normal β-cells makes it unlikely that the observed ECAR changes resulted from lactate-coupled proton efflux in anaerobic glycolysis, as typically assumed in conventional interpretations. Second, both our findings and previous reports showed that oligomycin (mitochondrial ATP synthase inhibitor) reduces ECAR in different β-cells and islets33, 41, 42, 43, 44, 45, suggesting that a substantial portion of ECAR originates from mitochondrial CO -dependent acidification rather than anaerobic glycolysis. Therefore, the ECAR reduction in BCKA-treated β-cells likely reflected diminished respiratory CO from mitochondrial oxidation, typically in β-cell models as previously reported46. The inhibitory effect of BCKA on ECRAR and OCR were also reproduced in EndoC-βH1 cells, a well-characterized human β-cell line that closely mimics primary β-cells in GSIS (Supplementary Fig. 14)47. Furthermore, concomitant treatment with BT2 reversed the effects of BCKA on glucose-stimulated ECAR and OCR (Fig. 5d, e). Importantly, similar defects in glucose-stimulated ATP production, ECAR and OCR responses were observed in Ppm1k-silenced INS-1E cells and β-cell-specific PPM1K-deficient islets, with unchanged glucose uptake (Supplementary Fig. 15).

Metabolomics analysis revealed that BCKA treatment had minimal effect on most glycolytic metabolites, including glucose-6-phosphate (G6P), fructose 6-phosphate (Fru-6-P), fructose-1,6-bisphosphate (FBP), 3-phosphoglycerate (3PG) and phosphoenolpyruvate (PEP) (Supplementary Fig. 16a, b). However, BCKA significantly reduced glucose-stimulated pyruvate and its direct catabolite acetyl-CoA but increased lactate in MIN6 cells (Supplementary Fig. 16b, c). TCA cycle intermediates such as alpha-ketoglutarate (α-KG) and succinate showed a trend of reduction in BCKA-treated β-cells (Supplementary Fig. 16c).

BCKA reconnects glucose metabolism to the inactivated LDHA-lactate axis
We next directly examined whether BCKA affects glucose flux using a stable isotope-labeled glucose tracer and metabolomics analysis. After BCKA treatment, mouse islets and MIN6 cells were incubated with low (2 mM) or high (20 mM) concentrations of 13C -labeled glucose for 30 minutes (mimicking GSIS). 13C -glucose-derived M+3 pyruvate enters TCA cycle via the anaplerotic action of pyruvate carboxylase (PC) to generate M+3 oxaloacetic acid (OAA) and M+3 citrate, or via pyruvate dehydrogenase (PDH) to generate M+2 citrate (Fig. 5f). High-glucose stimulation enhanced 13C incorporation into glucose-derived TCA cycle intermediates (Supplementary Fig. 17c-f). BCKA treatment dramatically diminished 13C - glucose-stimulated productions of pyruvate and downstream TCA metabolites (citrate, α-KG, succinate and malate) but provoked 13C incorporation into lactate in both mouse islets and MIN6 cells (Fig. 5g-l and Supplementary Fig. 17). Mass isotopomer distribution analysis revealed that BCKA reduced citrate production (M+3 and M+2) through both PC- and PDH-mediated pyruvate metabolism, thereby limiting pyruvate entry into the TCA cycle (Fig. 5i and Supplementary Fig. 17c). Importantly, the effects of BCKA on labeled glucose flux into lactate and TCA cycle were consistently observed in pancreatic islets from human donors (Supplementary Fig. 18).

Next, we validated whether the reduced pyruvate entry into the TCA cycle contributes to the suppressive effect of BCKA on GSIS. To this end, we used KCl and cell-permeable TCA metabolites, including pyruvate, α-KG and succinate, which bypass pyruvate metabolism and directly stimulate insulin secretion48, 49, 50. BCKA blocked insulin secretion induced by pyruvate but not by succinate, α-KG or KCl, further supporting an impaired pyruvate metabolism in BCKA-treated β-cells (Supplementary Fig. 19a). To test whether promoting pyruvate entry into the TCA cycle reversed BCKA-induced GSIS defect, we inhibited PDH kinase using dichloroacetate (DCA), an activator of PDH that enhances pyruvate flux51, 52. DCA reduced PDHα1 phosphorylation, restored pyruvate entry into the TCA cycle, and largely reversed BCKA-induced GSIS defect (Supplementary Fig. 19b-i). These findings suggest that BCKA restricts pyruvate entry into the TCA cycle, leading to reduced production of ATP and other coupling factors for insulin secretion.

Inhibition of PC or PDH impairs GSIS by impeding pyruvate entry to the TCA cycle33, 53, 54, whereas LDHA overexpression blocks GSIS by disrupting mitochondrial function via an unknown mechanism9, 55. LDHA expression is suppressed in pancreatic β-cells38, 39, 40, preventing pyruvate-to-lactate conversion to maximize the insulinotropic function of glucose. In BCKA-treated β-cells, the reduced pyruvate flux into TCA cycle could result from (1) impaired PC and/or PDH function, (2) LDHA activation or (3) both. BCKA treatment did not change expression of PC, PDH, phosphorylated PDH at serine 293, Hif1α (Hypoxia-inducible factor 1-alpha; a transcription factor for glycolytic pathway) or LDHA in β-cells (LDHA in Fig. 6a; PC, PDH and Hif1α in Supplementary Fig. 20a, b). However, in vitro enzymatic assays showed that BCKA upregulated LDHA activity in the isolated islets and MIN6 cells (Fig. 6b, c), whereas PDH and PC activities remained unchanged (Supplementary Fig. 20c, d). Likewise, BCKA raised the ratio of lactate to pyruvate stimulated by 13C -glucose in islets from human donors (Fig. 6d). BCKA also directly enhanced the enzymatic activity of purified human LDHA protein to produce lactate (Fig. 6e). Together, these results demonstrate that BCKA activates the “disallowed” LDHA-lactate axis in β-cells at a post-translational level.

β-cell LDHA deficiency restores glucose tolerance and GSIS in BCKA-fed mice
We next questioned whether LDHA inactivation could block the detrimental influences of BCKA on β-cells. Inhibition of LDHA by siRNA-mediated gene silencing or pharmacological inhibitor NHI-1 reversed BCKA-induced LDHA reactivation and defective GSIS (Supplementary Fig. 21a-e). Moreover, NHI-1 treatment directed glucose flux back to the TCA cycle in the BCKA-treated β-cells, as corroborated by increased 13C incorporation into citrate, α-KG and OAA (Supplementary Fig. 21f-i).

To validate the role of LDHA in mediating BCKA effects in vivo, we generated tamoxifen-induced LDHA β-KO (Ldhaflox/flox Ins-Cre+ve) mice. Immunofluorescence staining confirmed a significantly reduced LDHA protein expression in pancreatic islets of LDHA β-KO mice compared to their WT littermates (Fig. 6f). β-cell specific LDHA deletion ameliorated the glucose intolerance and defective GSIS induced by BCKA feeding (Fig. 6g-j), while insulin sensitivity was not affected (Fig. 6k). To confirm these ameliorations are β-cell autonomous, we isolated pancreatic islets and consistently observed that BCKA diminished GSIS only in the islets from WT controls, but not those from LDHA β-KO mice (Fig. 6l).

Conversely, LDHA overexpression had opposite effects, increasing glucose-derived lactate while reducing citrate (Supplementary Fig. 22a-d), leading to impaired GSIS and ATP production in β-cells (Supplementary Fig. 22e-g). These effects were neutralized by BCAA deprivation (Supplementary Fig. 22b-f). Additionally, the inhibited GSIS by LDHA overexpression was counteracted by promoting pyruvate entry to the TCA cycle using DCA (Supplementary Fig. 22g).

Like the observations in the BCKA-treated β-cells, PPM1K or BCKDH-A inactivation enhanced LDHA enzymatic activity, and such increment was abolished by the LDHA inhibitor NHI-1 treatment (Supplementary Fig. 23a). The inhibitory effect of PPM1K and BCKDH-A inactivation or deletion on GSIS was abrogated by LDHA inactivation or PDH activation (Supplementary Fig. 23b-e). Glucose flux analysis showed that Ppm1k silencing upregulated 13C-glucose-derived M+3 lactate and reduced M+2 citrate. Such defects were counteracted by PDH activator DCA (Supplementary Fig. 24a, b). Noticeably, DCA effectively enhanced 13C-glucose incorporation into the TCA cycle metabolites, especially M+2 isotopologues, indicating restored glucose metabolism (Supplementary Fig. 24b-f). Unlike the observation in BCKA-treated β-cells, Ppm1k silencing did not affect M+3 isotopologues of citrate and OAA. The discrepancy might be due to the use of different β-cell lines and different intracellular BCKA levels induced by PPM1K downregulation versus BCKA treatment. These data illustrate that intracellular BCKA dysmetabolism reconnects glucose flux to LDHA-lactate arm, reducing TCA cycle and mitochondrial coupling factor production, and hence impairing insulin secretion.

BCKA bridges LDHA monomer to enhance dimerization and enzymatic activity
A recent protein-metabolite interactomics study showed that LDHA binds multiple regulatory metabolites.56 We hypothesized that BCKA can also bind to LDHA and activate its enzymatic function. To explore this, we performed Computational Molecular Docking Study using MOE (v2022.02) to predict the binding configuration of individual BCKA with LDHA. As shown in the 3D and 2D interaction diagrams (Fig. 7a and Supplementary Fig. 25 a), KIC and KMV bound at the interface of the LDHA dimer, forming a molecular bridge through hydrogen bonds with Ser210 on Chain A and Ser202 and Thr307 on Chain B. Similarly, KIV interacts with Gly203, Asn205, and Ser210 on Chain A, as well as Gly208 on Chain B. This binding configuration securely anchors each BCKA molecule within the binding pocket, as evidenced by the calculated binding free energy, which indicates a favorable interaction between the individual BCKA and the LDHA dimer. This bridging role of BCKA in linking Chain A and Chain B may potentially strengthen the association between the two chains, promoting LDHA dimerization or tetramerization and thereby enhancing its catalytic activity.

We validated the direct BCKA binding to purified human LDHA protein using Surface plasmon resonance (SPR) assays, giving dissociation constants (K ) of 17.2 nM for KIC, 6.8 nM for KMV, and 3.6 nM for KIV, significantly lower than that of pyruvate (132 nM) (Fig. 7b). We further confirmed BCKA-LDHA interaction in β-cells using drug affinity-responsive target stability (DARTS) analysis57. This assay demonstrated that BCKA protected LDHA from proteinase K-induced degradation in a dose-dependent manner, comparable to the effect of pyruvate, the canonical binding substrate for LDHA (Supplementary Fig. 25 b).

LDHA functions as a homotetrameric enzyme, with dimerization as an essential intermediate step for forming a tetramer58. Both tetrameric and dimeric forms of LDHA are more catalytically active than the monomeric form in converting pyruvate to lactate59, 60, 61, 62. We found in mouse islets, MIN6 and INS1-E β-cells, LDHA predominantly exists as monomers and dimers (Fig. 7c and Supplementary Fig. 26a-c). This might also explain LDHA inactivation in healthy β-cells. Treatment with BCKA increased dimeric LDHA in all three β-cell models, with tetramerization observed only in mouse islets (Fig. 7c and Supplementary Fig. 26a). To determine whether all three BCKAs are required for LDHA dimerization, we examined their effects individually and in combination. At 30 μM, no single BCKA induced LDHA dimerization, whereas combinations of two or three BCKAs effectively promoted dimer formation (Supplementary Fig. 26a). However, consistent to our GSIS results, higher concentration of individual BCKA, e.g., 100 μM KIC, 200 μM KMLV or 100μM KIV, was able to enhance LDHA dimerization (Supplementary Fig. 26b). Additionally, DCA treatment did not affect BCKA-induced LDHA dimerization, suggesting the effect was independent of PDH activity (Supplementary Fig. 26c). In addition, BCKA, but not BCAA, dose-dependently enhanced dimerization of recombinant human LDHA protein without promoting tetramerization (Fig. 7d, Supplementary Fig. 26d).

Together, these results demonstrate that BCKA enhances LDHA activity by stabilizing its dimeric form to redirect glucose flux toward lactate production and away from the TCA cycle, thereby impairing insulin secretion.

To experimentally validate the predicted binding sites in LDHA with BCKA, we generated plasmids expressing wild-type (WT) LDHA with Flag epitope and its mutants: 1MUT (Ser210 mutated to alanine [Ser210A]; conserved across all BCKAs), 3MUT (Ser202A/Ser210A/Thr307A; KIC and KMV binding sites), and 4MUT (Gly203A/Gly208A/Asn205A/Ser210A; KIV binding sites). The empty vector or the LDHA plasmids were transfected into HEK293T cells, followed by DARTS assay. BCKA treatment prevented proteinase-induced degradation of WT-LDHA but not the three LDHA mutants (Fig. 7e). Consistently, BCKA treatment enhanced dimerization of WT LDHA but not the three LDHA mutants (Supplementary Fig. 26e). These results confirm that Ser210 is essential to mediate LDHA-BCKA interaction and dimer stabilization.

Methods
Isolation and collection of human pancreatic islets and sections
To obtain human islets, pancreatic specimens were collected from non-diabetic humans by the Department of Liver Surgery and Transplantation in Zhongshan Hospital, Fudan University. The human pancreatic tissues were immediately stored in cold University of Wisconsin (UW) solution until weighing and pancreatic duct perfusion with cold collagenase P (0.2 mg/mL, Roche, Cat# 11213865001) prepared in Hanks' Balanced Salt Solution (HBSS, Gibco™, Cat# 14025076). The perfused pancreas was further digested at 37°C for 20 minutes and subjected to gradient centrifugation using density gradient reagents histopaque 1119 (Sigma-Aldrich, Cat# 1119-1) and 1077 (Sigma-Aldrich, Cat# 1077-1) at 4°C, 339 g for 30 minutes. The purified human islets were collected between the 2nd and 3rd layers of the gradient and then picked manually under a microscope. The isolated islets with similar sizes were subjected to treatment with BCKA and assessments of GSIS (5 size-matched islets in each sample) and isotope glucose flux analysis (500 size-matched islets in each sample). The subjects were diagnosed with or without T2D according to the ADA standards.93 Participants were sex- and age-matched. Sex was determined by self-report (male or female). Written informed consent was obtained from the participants. All procedures were carried out in compliance with the Helsinki Declaration and approved by the Ethical Review Committee of Zhongshan Hospital, Fudan University (No. B2021-853R).

Human pancreatic sections from non-T2D and T2D cadaveric donors were procured from the Department of Hepatology of Zhongshan Hospital, Fudan University. The subjects were diagnosed with or without T2D according to the ADA standards93. Participants were sex- and age-matched. Sex was determined by self-report (male or female). The procedures were approved by the Ethical Review Committee of Zhongshan Hospital, Fudan University (No. BR2021-853R).

Association analysis between BCAA metabolites and pancreatic β-cell function in humans
For the acute arginine-induced C-peptide (ArgIC) assay, 136 patients diagnosed with T2D according to the ADA standards [haemoglobin (HbA1c) over 6.5%] were recruited in the Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University. Patients receiving treatment with insulin secretagogues, such as DPP4i and SGLT2i, or having severe hepatic dysfunction, renal dysfunction, malignant tumors, pregnancy, or mental disorders were excluded. Circulating biomarkers related to glucose metabolism (HbA1c, glucose, insulin and C-peptide) were measured during routine blood tests. ArgIC assay was performed on overnight-fasted patients. A baseline blood sample was collected before arginine injection (0 min). A clamp tube was connected with normal saline to ensure no liquid extravasation, followed by intravenous injection of 5 g of arginine hydrochloride (Shanghai Xinyi Jinzhu Pharmaceutical Co. Ltd., 20 mL per tube) for 30 seconds. After injection, blood samples were obtained at 2, 4 and 6 minutes for measurements of blood glucose, C-peptide and insulin. The response of C-peptide secretion was assessed as the level at first 4 minutes minus the basal level as previously reported29. Subjects with a secretory level higher than 2.1 ng/mL were grouped into “T2D with normal insulin secretory ability”, whereas those with a level lower than 2.1 ng/mL were grouped as “T2D with defective insulin secretory ability” (n=18 and 43, respectively). C-peptide level of each individual is represented as a percentage over its basal level before arginine stimulation and the incremental area under the curve (AUC) was calculated. Clinical and metabolic characteristics of T2D subjects are listed in Supplementary Table 3. All procedures were carried out in compliance with the Helsinki Declaration and approved by the Ethical Review Committee of Zhongshan Hospital, Fudan University (No. B2020-180R).

To assess the associations of circulating BCAA, BCKA and C3/C5-acylcarnitines with fasting blood glucose and HOMA-β (%), human plasma samples were collected from 68- to 74-year-old males and females in the Hong Kong community as we previously described94. Subjects were assigned to the groups of normoglycemia with fasting blood glucose at 4.3-5.2 mM (n=28) or hyperglycemia with fasting blood glucose ≥ 5.6 mM (n=25). Clinical and metabolic characteristics of human subjects are listed in Supplementary Table 2. Participants were sex- and age-matched. Sex was determined by self-report (male or female). The experiment was approved by Human Ethics Committee of the Hong Kong Polytechnic University (approval number: HSEARS20180924002).

Transcriptomes and proteomics analysis of human islets
To compare the mRNA and protein expression of genes involved in BCAAs metabolism in pancreatic islets from non-diabetic (ND) and T2D individuals, we utilized processed transcriptome and proteome datasets from Kolic et al (PMID 38959864)24. Briefly, pancreatic islets were isolated from cadaveric donors, achieving >95% purity, with T2D diagnosis confirmed via HbA1c levels or family declaration. For the transcriptome analysis, RNA was extracted from 50 islets per donor, with quality control requiring an RIN score >5. The dataset comprised samples from 82 ND and 8 T2D age-matched donors. For the proteome analysis, approximately 300 islets per donor were examined from 118 ND and 15 T2D age-matched donors.

Animal models
Ppm1kflox/flox mice (C57BL/6N) were generated by Nanjing Biomedical Research Institute of Nanjing University, China. Briefly, a targeting vector containing homologous arms, a loxP site inserted between exons 2 and 3 of the Ppm1k gene, and a neomycin resistant cassette followed by another loxP site inserted between exons 3 and 4 of the Ppm1k gene were electroporated into ES cells to generate the Flox allele termed Ppm1kflox/flox mice. The full generation report, including floxed site sequencing and genotyping strategy, can be provided upon reasonable request. Ldhaflox/flox mice were well-established, and provided by Prof. Aimin Xu from the University of Hong Kong95. Tamoxifen-induced β-cell-specific Ppm1k and Ldha knockout mice were created by crossing Ppm1kflox/flox and Ldhaflox/flox mice with Ins2-Cre/ERT mice, resulting in PPM1K β-KO (Ppm1kflox/flox Ins-Cre+ve) and LDHA β-KO (Ldhaflox/flox Ins-Cre+ve) mice, respectively. We and others have demonstrated that Ins2-Cre and Ins2-Cre/ERT mice have normal glucose tolerance and GSIS3, 33, 34, 35, 36, therefore only WT littermates Ppm1kflox/flox and Ldhaflox/flox were used as controls in this study. To induce disruption of the Ppm1k and Ldha gene, 5-week-old male PPM1K β-KO, LDHA β-KO and their WT littermates were intraperitoneally injected with tamoxifen (MedChemExpress, Cat# HY-13757A) at a dose of 4 mg per mouse for 4 times over a 7-day period. Three weeks later, the induced knockout efficiency was confirmed by gene or protein expression of Ppm1k and Ldha in the islets. Generation of PPM1K global knockout mice was reported in our previous study96.

For BCKA feeding study, 16-week-old male C57BL/6J mice were obtained from the Hong Kong Polytechnic University. The C57BL/6J mice, LDHA β-KO mice and their WT littermates were given free access to drinking water supplemented with NaCl as vehicle control or BCKA [5 mg/mL of KIC (Cayman Chemical, Cat# 21052), KMV (Sigma-Aldrich, Cat# K7125) and KIV (Sigma-Aldrich, Cat# 198994) each] for 6 weeks.

db/db diabetic mice and their lean littermates (db/+) were obtained from the Chinese University of Hong Kong. BT2 (Santa Cruz Biotechnology, Cat# sc-276559) was freshly dissolved in DMSO at a concentration of 150 mM and further diluted into 5% DMSO with 0.1 M sodium bicarbonate, pH 9.0. 20-24-week-old db/db mice were randomly assigned into two groups and intraperitoneally injected with vehicle (5% DMSO in 0.1 M sodium bicarbonate) or BT2 at 20 mg/kg body weight 5-6 times per week for 4 weeks.

Sex- and age-matched animals were used in each experiment. Animal numbers are specified in the respective figure legends. All the mice were housed in the Centralized Animal Facilities of the Hong Kong Polytechnic University with a 12-hour light/dark cycle, humidity (60-70%), and temperature (23 ± 1 ℃) control. All mice had free access to a standard chow (24.7 kcal% protein, 13.2 kcal% fat, and 62.1 kcal% carbohydrates; Cat# 5053, PicoLab Rodent Diet 2.0 ) and water during the experiments. All animal experiments were approved by the Department of Health, HKSAR Government (Ordinance Cap. 340), and the Animal Subjects Ethics Sub-Committee of the Hong Kong Polytechnic University (22-23/333-HTI-R-GRF and 17-18/87-HTI-R-HMRF).

Glucose tolerance test (GTT), insulin tolerance test (ITT), glucose- and arginine-stimulated insulin secretion in mice
For the GTT, PPM1K β-KO, LDHA β-KO mice and the BCKA-fed mice were fasted for 6 hours before intraperitoneal injection with glucose (2 g/kg body weight). Blood glucose was measured every 15 or 20 minutes via tail vein with a glucose meter (Roche Accu-Chek, USA). db/db diabetic mice and their littermates were fasted for 16 hours, followed by intraperitoneal injection of glucose (1 g/kg body weight). Serum was collected from tail vein for measuring glucose levels using a glucose assay kit (Nanjing Jiancheng Bioengineering Institute, Cat# F006-1-1) following manufacturer’s instructions.

For the ITT, mice were fasted for 6 hours before intraperitoneal injection with insulin (1.25 U/kg for db/db mice and 0.5 U/kg for the other mice; Sigma-Aldrich, Cat# 91077C). Blood glucose was measured every 15 minutes via tail veil using a glucose meter.

For the GSIS, blood samples were collected during GTT at 0, 10 and 30 minutes via tail vein. For arginine-stimulated insulin secretion, mice were fasted overnight before intraperitoneal injection of arginine (1 g/kg body weight), followed by blood collection at 0, 4 and 10 minutes after injection. Serum was separated for measurement of insulin concentration using a mouse insulin ELISA kit (Mercodia, Cat# 10-1247-01) according to manufacturer’s instructions.

Isolation of primary pancreatic islets from mice
Mice were fasted for 4 hours and anesthetized. The common bile duct was clamped off at the duodenum, and the pancreas was perfused with 3 mL of collagenase P (0.6 mg/mL, Roche, Cat# 11213865001) in Hanks' Balanced Salt Solution (HBSS, Gibco™, Cat# 14025076) via common bile duct. The pancreas was then separated and incubated with 5 mL of collagenase P at 37℃ for 20 minutes, followed by vigorous hand shaking for 30 seconds and incubation at 37°C for an additional 5 minutes. The digestion process was halted by adding 15 mL of solution G (HBSS with 0.1% BSA). The digested pancreas was filtered with a 500-μm fine test sieve, and the filtrate was subsequently filtered again with a 70-μm cell strainer. Islets captured by the cell strainer were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Cat# 10270), 1% penicillin-streptomycin (Thermo Fisher Scientific, Cat# 15140122) and 50 μM beta-mercaptoethanol (Bio-Rad Laboratories, Cat# 1610710) overnight at 37°C in an incubator with humidified atmosphere and 5% CO . The islets were manually picked under a microscope for insulin secretion assay or LC-MS/MS targeted metabolomic study.

Cell culture, transfection, and insulin secretion assay
The rat insulinoma cell line INS-1E (AddexBio Technologies, Cat# C0018009) and the mouse pancreatic β-cell line MIN6 (AddexBio Technologies, Cat# C0018008) were cultured in RPMI 1640 (Gibco, Cat# 31800105) or DMEM (Gibco, Cat# 12800082) supplemented with 15% FBS, 1% penicillin-streptomycin and 50 μM beta-mercaptoethanol. Human EndoC-βH1 cells (Human Cell Design)47 were cultured in OPTIβ1® medium (Human Cell Design) according to manufacturer’s instructions. 293T cells (Procell, Cat# CM-0005) were cultured in DMEM (Gibco, Cat# 12800082) supplemented with 10% FBS, 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO incubator. The β-cells or isolated islets were treated with different concentrations of L-leucine (Sigma-Aldrich, Cat# L8000), L-isoleucine (Sigma-Aldrich, Cat# I2752), L-valine (Sigma-Aldrich, Cat# V0500), KIC, KMV, KIV, C2- (Sigma-Aldrich, Cat# A6706), C3-acylcarnitine (Sigma-Aldrich, Cat# 426S02), C5-acylcarnitine (Sigma-Aldrich, Cat# 51371) or BT2 for 48 hours, or treated with NHI-1 (Merck Millipore, Cat# 533966) or sodium dichloroacetate (DCA; Tokyo Chemical Industry Co. Ltd., Cat# D1719) for 2 hours as indicated in each figure legend, followed by an insulin secretion assay. For the BCAA deprivation assay, the siRNA-transfected or adenovirus-infected β-cells were cultured in BCAA-free RPMI 1640 medium (USBiological Life Sciences, custom medium) for 24 hours. The control medium contained normal concentrations of BCAA by supplementing the BCAA-free medium with 0.381 mM of leucine, isoleucine and valine, respectively. INS-1E cells were transfected with either siRNA against Ppm1k (siPpm1k), Bcat2 (siBcat2), Bckdha (siBckdha), Ldha (siLdha) or Scramble controls (GenePharma) using Lipofectamine™ RNAiMAX reagent (Invitrogen, Cat# 13778150) for 48 hours according to the manufacturer’s manual, or infected with adenovirus overexpressing human FLAG-tagged LDHA (WZ Biosciences Inc.) for 48 hours. The sequences of siRNA are listed in Supplementary Table 7. To validate the binding sites of BCKA on LDHA, pcDNA3.1 plasmids encoding LDHA WT, Ser210A (1MUT), Ser202A/Ser210A/Thr307A (3MUT), or Gly203A/Gly208A/Asn205A/Ser210A (4MUT) were constructed by YuYang Biosciences Inc. and further confirmed by DNA sequencing. These plasmids, along with empty vectors, were transfected into 293T cells using PEI (Polysciences, Cat# 23966-100) following the manufacturer’s protocol. The transfected cells underwent binding analysis 48 hours post-transfection or LDHA protein cross-linking assays after 48 hours of BCKA treatment.

For the insulin secretion assay, the cells or isolated islets were first cultured in Krebs buffer containing low glucose (2 mM) and 0.1% fatty acid-free BSA (Sigma-Aldrich, Cat# A8806) for 120 or 30 minutes, respectively. For static insulin secretion analysis, the cells were then stimulated for 30 minutes with low glucose (2 mM) as basal, high glucose (20 mM), KCl (40 mM), or cell-permeable form of methyl pyruvate (10 mM; Sigma-Aldrich, Cat# 371173), dimethyl α-ketoglutaric acid (α-KG; 30 mM; Sigma-Aldrich, Cat# 349631) or dimethyl succinate (30 mM; Sigma-Aldrich, Cat# 73605). For dynamic GSIS assay, conditional medium was collected every 3 minutes from the islets before and after stimulation with high glucose (20 mM). 0-9 min and 9-42 min were defined as the first- and second-phases of insulin secretion, respectively. Insulin concentration in the collected conditional medium was measured using an insulin ELISA kit (Antibody and Immunoassay Services, Cat# 32100) and normalized with protein concentration or islet size.

Targeted measurement of BCAA, BCKA and acylcarnitine by liquid chromatography-mass spectrometry (LC-MS)
Intracellular metabolites were measured by targeted metabolomics using the Agilent 6460 Liquid Chromatography-Electrospray Ionization Triple Quadrupole Mass Spectrometer (University Life Science, The Hong Kong Polytechnic University) connecting with different separating columns. For the measurements of BCAA-related catabolites, calibration curves were prepared using standard chemicals L-leucine, L-isoleucine, L-valine, KIC, KMV, KIV, C2-, C3- and C5-acylcarnitines. 10 μL of standard mixture, human plasma or mouse serum was extracted using 100 μL of chilled absolute methanol containing 0.5 µM D -valine (Cambridge Isotope Laboratories, Cat# DLM-311-PK) and 0.5 µM D -KIV (Cambridge Isotope Laboratories, Cat# DLM-4646-PK) as internal control standards. MIN6 or INS-1E β-cells were seeded onto 60-mm dishes. After the indicated experimental treatments, cells were washed twice with cold PBS, extracted with 400 μL of cold 80% methanol containing internal standards and scraped into Eppendorf tubes. The metabolite extracts were vortexed or sonicated and centrifuged at 12,000 x g for 15 minutes under 4°C. To quantify BCAA and acylcarnitines, 2 μL of supernatant was injected into an Acquity UPLC BEH Amide Column (1.7 μm, 2.1×100 mm, Waters, Cat# 186004801) connected to the mass spectrometer. The target analytes were monitored by multiple reaction monitoring (MRM) in positive electrospray ionization (ESI) mode. The gradient mobile phase consisted of 5 mM ammonium formate (Sigma-Aldrich, Cat# 09735) and 0.1% formic acid (Sigma-Aldrich, Cat# 695076) in mobile phase A (water: acetonitrile, 90:10, v/v) and mobile phase B (water: acetonitrile, 10:90, v/v) at a flow rate of 0.3 mL/min.

Derivatization and measurement of BCKA were performed as previously described with some modifications97. Methanol-extracted cell samples were dried by the Refrigerated CentriVap Centrifugal Concentrator (Labconco) and then reconstituted with 40 μL of 80% methanol, followed by adding 500 μL of 12.5 mM o-phenylenediamine (OPD, Sigma-Aldrich, Cat# P9029) in 2 M HCl and incubation at 80°C for 20 minutes. The derivatized samples were cooled on ice and extracted with 500 μL of ethyl acetate containing 80 mg of Na SO . After centrifugation at 500 g for 5 minutes, the organic phase was transferred, and the aqueous phase was extracted again with 500 μL of ethyl acetate. The two organic phases were mixed with 80 mg of Na SO After vortexing and centrifugation, the organic phase was dried and reconstituted with 200 mM ammonium acetate (Sigma-Aldrich, Cat# A1542). 2 μL of derivatized sample was injected into an Acquity UPLC BEH C18 Column, (130Å, 1.7 µm, 2.1 mm×50 mm, Waters, Cat# 186002350) for LC-MS analysis in positive ESI mode. The gradient mobile phase consisted of 0.1% formic acid in mobile phase A (water) and mobile phase B (acetonitrile) at a flow rate of 0.3 mL/min.

In all LC-MS analyses, the mass spectrometer was operated with the voltage set to 3.5 kV and the source temperature held at 300°C. The fragmentor voltage (70-110 V) and collision energy (5-25 psi) were also optimized for each metabolite. Blank controls were injected every 6 sample injections. The number of samples in each group is specified in the respective figure legends. The target analytes were monitored by MRM with transition (precursor ion → product ion) determined for each metabolite as bellows: leucine (132.1→86.1), isoleucine (132.1→69.2), valine (118.1→72.2), KIC (203.2→161.2), KMV (203.2→174.2), KIV (189.1→119.3), C2-acylcarnitine (204.0→85.0), C3-acylcarnitine (218.0→85.0), C5-acylcarnitine (246.0→85.0), D8-valine (126.0→80.0), D7-KIV (196.0→120.0). The spectra were acquired by Agilent MassHunter Workstation Data Acquisition (VB.06.00) and were then analyzed using Agilent MassHunter Quantitative Analysis Software (V.B.06.00). Enrichment of each metabolite was quantified from calibration curves and corrected by internal control standards and protein concentration or islet size.

13C 6 -labeled glucose tracing study
Stocks of standard chemicals pyruvate (Sigma-Aldrich, Cat# P2256), lactate (BDH, Cat# 101384Q), citrate (Sigma-Aldrich, Cat# 251275), α-KG (Cayman Chemical, Cat# 33693), succinate (BDH, Cat# 102734W), malate (Sigma-Aldrich, Cat# 02288) and oxaloacetic acid (OAA; Cayman Chemical, Cat# 30280) were dissolved in water. Calibration curves were prepared by serial dilution of standard mixture in 80% methanol. MIN6 or INS-1E β-cells were seeded onto 100-mm dishes. After the indicated experimental treatments, MIN6 or INS-1E cells were subjected to serum and glucose starvation in Krebs buffer for 2 hours, while the isolated human and mouse islets were starved for 30 minutes. Subsequently, the cells or islets were stimulated with [U-13C ]-glucose (2 mM or 20 mM, or 10 mM for human islets; Cambridge Isotope Laboratories, Cat# CLM-1396-5) for 30 minutes. Next, the cells were harvested and extracted with chilled 80% methanol containing internal isotope standards. The metabolite extracts and standards were evaporated to dryness in a concentrator. Sample processing and metabolomic analysis were performed and MRM transition of TCA metabolites were acquired as previously described with minor modifications98. Briefly, the metabolite extract was reconstituted in 50 μL of water. Derivatization was performed by adding 50 μL of 500 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC; Sigma-Aldrich, Cat# 341006) and 100 μL of 250 mM O-benzylhydroxylamine hydrochloride (OBHA; Sigma-Aldrich, Cat# B22984) prepared in pyridine buffer (pH 5.0), followed by slow shaking at ambient temperature for 30 minutes. The derivatized metabolites were extracted twice using 400 μL of ethyl acetate and the organic phase was transferred for drying. The residues were resuspended in 30 μL of 50% methanol and injected into an Acquity UPLC BEH C18 column for LC-MS analysis in positive ESI mode. The gradient mobile phase consisted of 0.1% formic acid in mobile phase A (water) and mobile phase B (methanol) at a flow rate of 0.3 mL/min. The acquired spectra for isotopologues were quantified using Agilent MassHunter Quantitative Analysis Software. Signals of the labeled TCA cycle metabolites were corrected with parallel unlabeled control samples.

Targeted measurement of unlabeled glycolysis and TCA cycle metabolites
Following the specified experimental treatments, MIN6 cells were washed twice with cold PBS and flash-frozen in liquid nitrogen. Measurements of unlabeled glycolysis and TCA metabolites were performed by Hangzhou Lianchuan Biotechnology Co., Ltd. In addition, a second batch of samples was analyzed using capillary electrophoresis-mass spectrometry (CE-MS) with assistance from Prof. Hailong Piao at the Dalian Institute of Chemical Physics, Chinese Academy of Sciences99. Briefly, the treated cells were washed with 5% mannitol (Sigma-Aldrich, Cat# M4125), flash-frozen in liquid nitrogen, and extracted with 1 mL cold methanol containing 50 μM internal standards (methionine sulfone, Sigma-Aldrich, Cat# M0876; D-Campher-10-sulfonic acid sodium salt, Sigma-Aldrich, Cat# C2107). The samples were vortexed for 20 seconds, followed by adding 1 mL of chloroform and 400 μL of deionized water, with vortexing for 20 seconds after each addition. The mixtures were centrifuged at 14,000 g for 15 minutes at 4℃. The upper layer was filtered using an ultra-free MC centrifugal filter device (Millipore, Cat# UFC3LCC00) through centrifugation. The filtrate was evaporated to dryness using a concentrator, and then dissolved in water containing Internal Standards 3 (Human Metabolome Technologies, Cat# H3304-1104, 1:200). The internal standard was used to standardize the metabolite intensity and adjust the migration time. The analysis of metabolites was performed using CE-QTOF MS (Agilent Technologies 7100A, connected with G6224A mass spectrometer). All samples were separated by the fused silica capillary [50 μm i.d. and total length of 80 cm; Human Metabolome Technologies (HMT)]. Data were processed and analyzed using Quantitative Analysis Software 10.2 (Agilent).

Intracellular lactate assay
Intracellular lactate was quantified by derivatization method and LC-MS as described above or a lactate assay kit (Sigma-Aldrich, Cat# MAK064) following the manufacturer’s instructions. Briefly, cell lysates with 200 μg of protein were deproteinized with a 10 kDa MWCO spin filter (Millipore, Cat# UFC501096), and reacted with the reaction mixture and probe provided in the kit. Finally, absorbance was recorded at 570 nm by a microplate reader (Bio-rad Benchmark Plus MPM 5.1).

LDHA activity assay
LDHA enzymatic activity was analyzed following an online Worthington protocol (http://www.worthington-biochem.com/ldh/assay.html). Briefly, MIN6 and INS-1E β-cells were homogenized with 0.2 M Tris-HCl buffer (pH 7.3), sonicated and centrifuged. 20 μg of total protein from cell lysates was thoroughly mixed with pre-warmed reaction buffer to make final concentrations of 200 μM NADH (Cayman Chemical, Cat# 16078) and 1 mM sodium pyruvate (Sigma-Aldrich, Cat# P5280). Absorbance was immediately recorded at 340 nm per minute for 10 minutes using a microplate reader at 25°C. The LDHA activity was determined by a decrease in absorbance at 340 nm resulting from NADH oxidation (Bio-rad Benchmark Plus MPM 5.1).

PC activity assay
PC activity was measured using the PC activity kit (Solarbio, Cat# BC0735) according to manufacturer’s instructions. Briefly, 5×106 cells were collected with 1 mL of extraction buffer. The cell lysates were treated with vehicle or 30 μM BCKA for 3 hours, and then mixed with reaction buffer. PC catalyzed pyruvate to oxaloacetic acid, which was then further converted to malate with an oxidation of NADH to NAD+. PC activity was determined by the rate of NADH oxidation, which was monitored by absorbance at 340 nm and normalized against protein concentration.

PDH activity assay
PDH activity was assessed using the PDH activity kit (Solarbio, Cat# BC0385) following manufacturer’s instructions. In brief, 5×106 cells were lysed with extraction buffer. Cell lysates were treated with vehicle or 30 μM BCKA for 3 hours, and then mixed with reaction buffer. PDH catalyzed pyruvate to produce hydroxyethyl thiamine pyrophosphate, which further reduced 2,6-dichlorophenolindophenol to result in a decrease in absorbance at 605 nm. The change in absorbance was normalized against protein concentration.

Molecular docking analysis for metabolite-protein binding
3D molecular docking model and 2D diagram of the binding modes between human dimeric LDHA and KIC, KMV, and KIV were analyzed using Molecular Operating Environment (MOE) software 2022.02. The crystal protein structure of human LDHA was retrieved from the Protein Data Bank (PDB code: 8FW6). The parameters and subsequent minimization were consigned to the MMFF94x force field to optimize the molecular structure by minimizing the energy of the system. The prepared LDHA was defined as the receptor, and the binding site was defined by generating the α-site spheres in the site finder. The docking BCKA (KIC, KMV, and KIV) attack the protein surfaces in their interior grooves for 30 trials until the most stable docking complexes are formed.

Drug affinity responsive target stability (DARTS)
To examine the binding between BCKA and LDHA protein, DARTS was performed as previously described with some modifications100. MIN6 β-cells were washed with PBS and solubilized in lysis buffer (0.2% Triton X-100, 200 mM NaCl, 50 mM Tris-HCl, pH 7.4; protease inhibitor cocktail After sonication and centrifugation, protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific, Cat# 23225). Cell lysates were diluted to the same protein concentration in chilled TNC reaction buffer (50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, pH 8.0), followed by incubation with different concentrations of BCKA and pyruvate for 1 hour on ice. After warming the reaction mixtures to room temperature, limited proteolysis was performed by adding 1 μg of proteinase K (Sigma-Aldrich, Cat# SAE0151) for every 700 μg protein and incubating at room temperature for 5 minutes. Digestion was then quenched by immediately adding SDS loading buffer and heating the mixture at 95°C for 5 minutes. The effect of BCKA or pyruvate to prevent proteolysis was analyzed by immunoblotting.

Surface plasmon resonance (SPR) assay
SPR assay was performed to investigate the interactions between individual BCKA (KIC, KMV and KIV), pyruvate and human recombinant LDHA protein (Abcam, Cat# AB93699) using a Sierra SPR®-32 Pro Analyzer with a high-capacity amine sensor chip (Bruker, Cat# 1862614) at room temperature. Protein immobilization was accomplished by an injection of 25 μg/mL LDHA protein in 10 mM sodium acetate (pH 5.5) and a running buffer containing 30 mM potassium phosphate, 150 mM NaCl, and 0.05% Tween 20 (pH 8.0). Immobilization levels typically reached 15,000 RU. BCKA were injected in a series of concentrations from 4.57 nM to 6.67 μM over the sensor chip with a running buffer containing 10 mM Hepes, 200 mM NaCl, 1% DMSO, and 0.05% Tween 20 (pH 8.5). The binding kinetics (K ) values were calculated based on the fitting curves using Sierra SPR control software R3.

Protein cross-linking assay
To examine the dimer and tetramer formation of LDHA in β-cells, a protein crosslinking assay was performed as previously described.60 Isolated mouse pancreatic islets (470 islets/sample), MIN6 and INS-1E β-cells with or without chronic BCKA treatment were lysed by lysis buffer (40 mM HEPES, 150 mM NaCl, 0.1% NP-40, pH 7.5; protease inhibitor) and centrifuged. The protein crosslinking reaction was initiated by incubating cell lysates with 0.02% glutaraldehyde (Sigma-Aldrich, Cat# G6257) for 30 minutes in a 37°C water bath. The reaction was terminated by a final concentration of 50 mM glycine and SDS loading buffer. Protein samples were then analyzed by immunoblotting using LDHA, β-actin and HSP90 antibodies.

To examine the direct effect of BCKA and BCAA on dimerization and tetramerization of LDHA, 1 μg of human recombinant LDHA protein (Abcam, Cat# AB93699) was incubated with different concentrations of BCKA for 60 minutes on ice. Monomeric LDHA was obtained via heating at 99°C for 5 minutes. Different isoforms of LDHA were separated by SDS-PAGE and visualized by silver staining using a commercially available silver staining kit (Bio-rad, Cat# 1610449).

Immunoblotting analysis
Following the experimental treatments as specified in each figure legend, the cells were solubilized in a lysis buffer composed of 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1% NP40 and proteinase inhibitor (MedChemExpress, Cat# HY-K0010). Protein samples were then resolved by SDS-PAGE and electro-transferred onto PVDF membranes. Proteins were subsequently analyzed with primary antibodies against BCAT2 (Abcam, Cat# ab95976), BCKDH-A (total and phospho-S293; Abcam, Cat# ab90691 and Cat# ab200577), PPM1K (Proteintech, Cat# 14573-AP), HSP90 (Cell Signaling Technology, Cat# 4877), BCKDK (Sigma-Aldrich, Cat# HPA017995), β-actin (Santa Cruz, Cat# sc-8432), LDHA (Cell Signaling Technology, Cat# 2012) FLAG (Sigma-Aldrich, Cat# F7425), PC (Proteintech, Cat# 16588-1-AP), and PDHα1 (total and phosphor-S293; Cell Signaling Technology, Cat# 3205 and Cat# 37115). HSP90 or β-actin was used as a loading control. HRP-conjugated secondary antibodies, anti-rabbit (Cell Signaling Technology, Cat# 7074S) or anti-mouse (Cell Signaling Technology, Cat# 7076S) were used. EPSON Scan 3.10E was used to scan blots from X-ray films. Uncropped and unprocessed scans are provided in the Source Data file.

Histological, immunohistochemical (IHC) and immunofluorescence (IF) analyses
To calculate islet size, pancreas isolated from mice were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned into 5-μm-thick slices, deparaffinized and rehydrated. the pancreatic sections were stained with hematoxylin for 10 minutes and eosin for 3 minutes (H&E staining), followed by dehydration with 100% ethanol twice, clearing in xylene for 10 minutes and mounting with DPX mounting medium. Images were captured using microscopy (BX35-DP80, Olympus) and analyzed using ImageJ V1.50 for quantification of islet area. A minimum of 5 parts of the pancreas and 2 tissue slides for each part were examined.

For IF, human or mouse pancreatic sections were heat retrieved at 98℃ for 15 minutes in citrate buffer (10 mM sodium citrate, 0.05 % Tween 20, pH 6.0) and blocked in PBS with 3% BSA and 10% FBS at ambient temperature for 1 hour. The slides were co-stained with mouse anti-insulin (HyTest, Cat# 2IP10cc-D6C4cc) and rabbit anti- BCKDH-A (total and phospho-S293; Abcam, Cat# ab90691 and Cat# ab200577), PPM1K (Proteintech, Cat# 14573-1-AP) or glucagon (Sigma-Aldrich, Cat# SAB4501137) antibodies overnight at 4℃. The slides were then incubated with Alexa Fluor 594-anti rabbit IgG- (Invitrogen, Cat# A11012) and FITC-anti-mouse IgG-conjugated antibodies (Invitrogen, Cat# A11001) for 90 minutes at ambient temperature in the dark. Finally, slides were mounted with Prolong glass antifade mountant containing DAPI (Invitrogen, Cat# P36984). The fluorescent images were captured using a Nikon Eclipse Ni-U Fluorescent Microscope and analyzed using ImageJ V1.50, normalized to islet size and analyzed consistently across samples. At least ten islets from different pancreatic regions were examined per human donor.

For IHC, mouse pancreatic sections were processed with antigen retrieval buffer and incubated with 3% hydrogen peroxide (H2O2) for 10 minutes at room temperature in the dark to quench endogenous peroxidases. After blocking, the slides were incubated overnight at 4℃ with the primary antibodies against BCAT2 (Abcam, Cat# ab95976), BCKDH-A (total and phospho-S293), PPM1K, BCKDK (Sigma-Aldrich, Cat# HPA017995), LDHA (Cell Signaling Technology, Cat# 2012) or insulin. Following washing with PBST for three times, the slides were incubated with HRP-conjugated anti-rabbit (Cell Signaling Technology, Cat# 7074S) and anti-mouse (Cell Signaling Technology, Cat# 7076S) secondary antibodies for 90 minutes at room temperature. The chromogenic reaction was carried out with a DAB substrate kit (Abcam, Cat# ab64238) before counterstaining with hematoxylin and mounting. Images were acquired using a Nikon Y-THPL microscope and analyzed using ImageJ V1.50, normalized to islet size and analyzed consistently across samples. At least six islets from different regions of the pancreas were examined per mouse, using two levels of tissue sections per animal. Samples from two independent batches of mice were analyzed by two independent investigators.

Measurement of ATP production
MIN6 cells and INS-1E cells were seeded in a white 96-well plate with clear bottom and subjected to treatments as specified in each figure legend. Cells were starved in Krebs buffer supplemented with 2 mM glucose for 90 minutes, followed by stimulation with Krebs buffer supplemented with 2 mM or 20 mM glucose for 10 minutes. ATP level was measured using a luminescence ATP detection kit (PerkinElmer, Cat# 6016943) according to manufacturer’s instructions and the luminescence intensity was acquired using a Varioskan LUX Multimode Microplate Reader 7.0.2 (Thermo Fisher).

2-deoxy-D-glucose (2-DG) uptake assay
Glucose uptake was determined using the Glucose Uptake-Glo™ assay kit (Promega, Cat# J1341) according to the manufacturer’s instructions. BCKA-treated MIN6 cells or siRNA-transfected INS1-E cells were subjected to glucose starvation for 2 hours, followed by incubation with a stable glucose analogue 2-Deoxy-d-glucose (2-DG; Cayman Chemical, Cat# 14S325), which is transported across the membrane and rapidly phosphorylated in the same manner as glucose. The direct product of 2-DG, 2-deoxyglucose-6-phosphate (2DG6P), cannot be further metabolized by glycolysis and therefore reflects glucose uptake by cells. Stop buffer was added to lyse the cells and terminate the uptake of 2-DG. Intracellular production of 2DG6P was determined by sequential additions of neutralization buffer and 2DG6P detection reagent provided in the kit. Finally, luminescence intensity was recorded using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher).

Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was examined using TMRE mitochondrial membrane potential assay kit (Cayman Chemical, Cat# 701310). MIN6 cells were seeded in a black 96-well plate with clear bottom. Following treatment with BCKA for 48 hours, each well was added with 100 μL of 200 nM TMRE in 1 X assay buffer and incubated for 30 minutes at 37°C. The medium was subsequently removed, and the cells were gently rinsed twice with 1 X assay buffer. This was followed by addition of another 100 μL of 1 X assay buffer and the measurement of fluorescence intensity at excitation/emission 530/580 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher).

Assessment of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)
MIN6 cells and INS-1E cells were cultured in XF24 V7 PS tissue culture microplates (Agilent Technologies, Cat# 100777-004) at a confluence of 50-60%. The cells were subjected to treatment as specified in each figure legend. Prior to the analysis, XFe24 sensor cartridge (Agilent Technologies, Cat# 102340-100) was hydrated with Seahorse XF calibrant solution overnight at 37°C in a non-CO incubator. The treated cells were washed twice and incubated with 500 μL of XF Base medium (pH 7.4; Agilent Technologies, Cat# 103335-100) for 1 hour in the non-CO incubator. Following loading of the sensor cartridge and calibration of the Agilent Seahorse XFe24 analyzer, the cell culture plate was loaded into the analyzer. For the glycolysis stress test, ECAR and OCR were recorded during basal condition (2 mM glucose) and sequential injections of 20 mM glucose, 5 μM oligomycin (Cayman Chemical, Cat# 11341) and 50 μM 2-deoxy-D-glucose (2-DG; Cayman Chemical, Cat# 14325). The results were analyzed using Seahorse Wave Desktop 2.4.1 Software (Agilent Technologies) and normalized with protein concentration.

Real-time quantitative PCR analysis
Total RNA was isolated using the TRIzol reagent (Takara, Cat# 9109). cDNA was synthesized with 1 μg of RNA using the GoScriptTM Reverse Transcription Kit (Promega, Cat #A2801). The real-time PCR reaction was conducted using the QuantiNova SYBR Green PCR Kit (Qiagen, Cat# 208056) and analyzed by the ViiA 7 Real-Time PCR System (Applied Biosystems). The mRNA expression level was analyzed using the ∆∆Ct threshold cycle method and normalized against β-actin. The primers used were listed in Supplementary Table 6.

Cell viability assay
To assess cell viability, BCKA-treated β-cells were incubated with medium containing 0.25 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Solarbio Life Sciences, Cat# M1025) at 37°C in a CO incubator for four hours. The culture medium was subsequently removed, and the purple crystal products formed by the cells were solubilized with 200 μL of DMSO, followed by shaking for 15 minutes and measurement of optical density at 570 nm.

Measurement of caspase-3 activity
Cell apoptosis was evaluated by quantifying caspase-3 enzymatic activity using the EnzChek Caspase-3 Assay Kit with the Z-DEVD-AMC substrate (Invitrogen, Cat# E1S3183). In brief, cells were rinsed with PBS, disrupted in the lysis buffer, and the resulting lysates were centrifuged at 5,000 x g for 5 minutes. An aliquot of lysate with 50 µg total protein was reacted with 2× substrate working solution for 30 minutes at room temperature, followed recording of fluorescent intensity at 342 nm excitation and 441 nm emission using a Thermo Scientific Varioskan LUX multimode reader. Caspase-3 activity was expressed relative to the protein content of each sample.

Statistical analysis
The data are expressed as mean ± standard error of mean (SEM). All statistical evaluations were conducted using Prism 10.1.2 (GraphPad Software). The sample size (n) of each experiment is specified in the figure legends. Prior to the application of parametric tests, all the data were pre-evaluated for normality and equal variance among the groups using D’Agostino-Pearson omnibus and Shapiro-Wilk normality test and Brown-Forsythe test, respectively. Statistical significance of normally distributed data with equal variance was evaluated using two-tailed Student’s t-test for comparison between two groups, or one-way ANOVA with Tukey correction for multiple comparisons as indicated in the figure legends. The associations of circulating BCAA, BCKA, C3/C5 acylcarnitine with HOMA-beta, fasting blood glucose and arginine-induced C-peptide secretion were examined using Spearman correlation analysis. A p-value <0.05 was deemed statistical significance. The representation of the p-value was *p<0.05, **p<0.01, or as specified in the figure legend. Animal experiments were conducted at least twice with each animal serving as a biologically independent sample. All cell experiments were replicated a minimum of three times.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript entitled “Branched-chain α-keto acids impair glucose-stimulated Insulin Secretion in Pancreatic β-Cells under diabetes by reactivating the LDHA-lactate axis”, the authors investigated the role of branched-chain α-keto acids (BCKAs) in impairing glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells under diabetic conditions. They concluded that BCKAs bind LDHA to promote its dimerization, thus enhancing enzymatic activity. The work combines animal models, human islet studies, and multi-omics approaches to support the proposed mechanism. I have some questions. Please see below for the detailed comments.\n1. The concentrations of BCKA used in cell culture and mouse models are 30 μM and 5 mg/mL in drinking water, respectively. However, they should be justified against physiological/pathological levels in humans.\n2. The direct target exploration of BCKA was focused on LDHA. Would the authors discuss the possibility of the participation of other protein targets?\n3. Due to the complexity of the proposed mechanism including the role of PPM1K-BCKDH-A signal axis in glucose metabolism and insulin secretion, it’s suggested that a schematic summarizing the mechanism be supplemented in the discussion section. Extended Data Fig. 25 could be modified by the addition of the relationship between the BCKDH-A signal axis and glucose metabolism.\n4. Please discuss the clinical significance of this investigation.", "reviewer_2": "Lin et al., conducted a thorough study to elucidate a mechanism by which branched-chain keto acids contribute to the development of diabetes by examining their role in pancreatic beta cells. While the study is comprehensive, there are a few concerns that require attention.\nMajor Concerns\nFirst the authors show that supplementation of individual BCAAs or BCKAs do not alter GSIS. However, a mix of all three BCKAs are able to illicit an impairment in GSIS. Why does only a combination of all three BCKAs impair GSIS? Are all three needed to bind to LDHA for dimerization? Does increasing the concentration of a singular BCKA to 100 uM impair GSIS?\nSecond, the authors use computational modeling to suggest that it is favorable for all three BCKAs to bind to LDHA which may strengthen LDHA dimerization. The computational modeling suggests several residues of interest for BCKA binding with Ser210 being a conserved site across all three BCKAs. While intriguing, the authors do not validate these binding sites. The manuscript would be significantly strengthened by confirming binding sites through mutagenesis. Are there human mutations on these identified binding sites that correspond to GSIS phenotypes? Further, the authors fail to use a metabolite as a negative control in their modeling and binding assays. Do BCAAs fail to bind to LDHA and induce dimerization?\nThird, the authors systematically measure glucose uptake, insulin content, mitochondrial membrane potential, cell viability, and ATP production to determine where the defect in beta cell GSIS could be occurring. Authors perform seahorse experiment and observe a decrease in glycolysis in BCKA treated cells and speculate that it is driven by a reduction in CO2 production (page 12, line 12-13). Authors should perform radiolabeled glucose oxidation assay to confirm statement.\nMinor Concerns\n• The authors use tracing studies to show that carbons from glucose and pyruvate are redirected from TCA cycle to lactate and this can be rescued by activation of PDH. Does this model (DCA), impair BCKA action on LDHA activity or LDHA dimerization?\n• Supplementation studies with mixture of BCKAs and BCAAs it is unclear whether the concentration reported is total amount of metabolites or individual metabolite.\n• Please confirm figure panel reported in the text matches that in the figures (Page 7 line 15 – refers to Extended Data Fig. 3 not Extended Data Fig. 4).\n• The addition of oligomycin should represent maximal glycolytic capacity in Seahorse assays. However, ECAR and OCR both decrease with the addition of oligomycin. Is this an expected outcome for beta cells?", "reviewer_3": "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_4": "Branched-chain a-keto acids impair glucose-stimulated Insulin secretion in pancreatic b-cells under diabetes by reactivating the LDHA-lactate axis.\nIn this manuscript, Lin and colleagues describe a metabolic rewiring process occurring in pancreatic beta cells under BCKA chronic exposure/BCKA catabolism disruption which promoted impaired glucose-stimulated insulin secretion. In T2D higher levels of circulating BCAA and of their catabolites BCKA have been reported and the authors demonstrated a compromised BCAA catabolic machinery in diabetic pancreatic islets and consequent BCKA accumulation in beta cells. In vivo models demonstrated that high circulating levels of BCKA promoted glucose intolerance whereas reducing circulating BCAA/BCKA levels in diabetic animals improved beta cell function.\nThis “overloading” of BCKA on pancreatic beta cells exert a GSIS-inhibiting effect by decoupling glucose metabolism and TCA/oxidative phosphorylation and promoting an abnormal activation of LDHA. This is mediated by the binding of BCKAs to the monomer of the enzyme, which is normally inactivated in beta-cells and this binding promotes the dimerization of LDHA, thus inducing an abnormal activation of the enzyme and deviating pyruvate catabolism toward lactate synthesis. This would affect ATP synthesis and reduce the first phase of glucose-stimulated insulin secretion. The authors propose that this metabolic rewiring would contribute to glucose intolerance in diabetes.\nThe manuscript is well written, clearly organized and results are convincing.\nThe main aim of this reviewer’s comments will be to improve the manuscript to give the highest translational value possible.\nMajor revisions:\n• The authors used several in vitro (murine and rat beta cells/mouse islets) and in vivo models (db/db, KO models). Unfortunately, not all data were reproduced on human beta cells and islets, but the authors declare these missing data in study limitations.\nAs proof of concept, even if it was not possible to perform all the experiments on human islets, I suggest repeating at least the key experiments on a commercial human beta cells line, such bLox5 cells.\n• The authors already assed cell viability by MTT and mRNA expression of 3 apoptosis-related genes in MIN6 cells treated with BCKA without observing significant differences. However, it would be also interesting to run other in vitro experiments to effectively assess whether BCKA overloading may compromise beta cells survival (like caspase cleavage analysis, cell death assays). Since in the title the authors write about general diabetes and not only T2D, this may give to the proposed mechanism a significant value also in the pathogenesis of T1D.\n• In this sense, please add a section in the discussion paragraph dedicated to the potential relevance of BCAA/BCKA in T1D, since in the last years novel non-immune mediated mechanisms are emerging in promoting beta cells destruction in T1D. In this section, please consider adding the references PMID 35115561 and PMID 38318298.\nMinor revisions:\n• All the figures: where not already specified, please add a note if data were obtained in humans or mice. Ex. In fig 1 e-f add “human islets” to make it clearer for the readers.\n• Page 6 line 14: please add in the title that the analyses were conducted on humans. I would add the most significant data in the main text to give more relevance to the relationship between T2D and BCAA altered catabolism in humans.\n• Page 7 line 14: Probably is extended figure 3?\n• Page 8 line 21: were these results not reproduced in MIN6 cells?\n• Fig 2 d-g add “MIN6 cells”\n• Pag 10 line 11: please add “in vivo” in the title of the paragraph.\n• Pag 13 line 19: I suggest adding significant metabolic data on human islets in the main text."}, "author_response": "NCOMMS-25-27543A Point-by-point response\nPoint-by-point replies to editor and reviewers\nBranched-chain α-keto acids impair glucose-stimulated insulin secretion in pancreatic\nβ-cells under diabetes by reactivating the LDHA-lactate axis (NCOMMS-25-27543A)\nDear Editor and Reviewers,\nThank you for your valuable suggestions and comments, as well as the time you dedicated to\nreviewing our manuscript. The revisions in the manuscript text file are highlighted with yellow.\nPlease find below our point-by-point responses for your further evaluation.\nWe have carefully addressed all reviewer’s comments, including clarifying the details of BCKA\ntreatment concentrations, validating the BCKA binding sites on LDHA, and further elucidating\nthe mechanism by which BCKA regulates LDHA activity. Please find our detailed responses\nas below:\nReviewer #1\nIn this manuscript entitled “Branched-chain α-keto acids impair glucose-stimulated Insulin\nSecretion in Pancreatic β-Cells under diabetes by reactivating the LDHA-lactate axis”, the\nauthors investigated the role of branched-chain α-keto acids (BCKAs) in impairing glucose-\nstimulated insulin secretion (GSIS) in pancreatic β-cells under diabetic conditions. They\nconcluded that BCKAs bind LDHA to promote its dimerization, thus enhancing enzymatic\nactivity. The work combines animal models, human islet studies, and multi-omics approaches\nto support the proposed mechanism. I have some questions. Please see below for the detailed\ncomments.\nComment 1. The concentrations of BCKA used in cell culture and mouse models are 30 μM\nand 5 mg/mL in drinking water, respectively. However, they should be justified against\nphysiological/pathological levels in humans.\nResponse: Thank you for your thoughtful review of our manuscript. The concentrations of\nBCKAs used in cell culture and mouse models were based on their concentration in diabetic\nhumans and animal model. Previous studies have reported that in humans with T2D, plasma\nKIC+KMV levels reach ~60 μM, KIV ~15 μM, and total BCKAs ~75 μM1. These values are\nconsistent with our own clinical measurements in cohorts with hyperglycemia and T2D\n(Supplementary Tables 2 & 3). The circulating levels of total BCKA are around 60-90 μM in\ndb/db mice, and around 30 μM in lean littermates.\nBased on the above data, we treated isolated mouse islets (Fig. 2a) with BCKA cocktails at a\nrange of concentrations from 10 μM (non-diabetic condition) to 100 μM (diabetic conditon).\nWe prepared equal mixtures of KIC, KMV, and KIV (e.g., 30 μM BCKA corresponds to 30\nμM KIC + 30 μM KMV + 30 μM KIV). BCKA at 30 μM and 100 μM produced a clear dose-\ndependent inhibition of GSIS, whereas 10 μM had no obvious effect. The inhibitory effect at\n30 μM was reproduced in both MIN6 β-cells and human islets (Fig. 2b, c). However, 30 μM\nof any individual BCKA did not significantly impair GSIS (Supplementary Fig. 5b; refer to\nresponse to # Review 2, Comment 1). These results indicate that the concurrent elevation of all\nthree BCKAs, the metabolic environment in diabetes, acts synergistically to impair β-cell\nfunction. Therefore, in subsequent cell experiments, we used a BCKA cocktail containing KIC,\nKMV, and KIV (each at 30 μM, a pathophysiological concentration mimicking diabetic\ncondition) to treat the β-cells or islets. The relevant information is now included in the revised\nmanuscript (page 9, line 168-185).\nFor the in vivo studies, we attempted to increase circulating BCKA levels to that in humans or\nrodent with type 2 diabetes. In our pilot study, we fed the mice with two different\nconcentrations of BCKA in drinking water. After 15 days, only 5 mg/ml but not 2.5 mg/ml\nmodestly increased circulating BCKA compared to vehicle controls (see below figure). In\nFigure 3a, the C57BL/6 mice were fed with 5 mg/ml BCKA for 4 weeks and resulted in a\nsignificant increase of circulating BCKA to ~75 μM, levels comparable to those observed in\nT2D humans1. These findings demonstrate that the feeding with 5 mg/mL BCKA recapitulate\nthe diabetic BCKA concentration. The justification is included in the revised manuscript (page\n10, lines 202-206).\nIn summary, both our human plasma data and dose–response experiments confirm that the\nBCKA concentrations applied in vitro (30 μM) and in vivo (5 mg/mL) reliably recapitulate\npathological BCKA levels in diabetic humans and mice.\nPilot data: 12-week-old male C57BL/6J mice\nwere provided drinking water containing\nBCKAs (KIC, KMV, and KIV, each at 2.5 or\n5 mg/mL) or vehicle control (n = 3 per group).\nData are shown as mean ± SEM, and statistical\nsignificance was assessed by one-way\nANOVA with Tukey’s post hoc test.\nComment 2. The direct target exploration of BCKA was focused on LDHA. Would the\nauthors discuss the possibility of the participation of other protein targets?\nResponse: Thanks for the suggestion. We prioritized LDHA because (i) isotope-glucose flux\nanalysis revealed increased lactate and decreased pyruvate production in primary mouse and\nhuman islets (Fig. 5 and Supplementary Fig. 18), and (ii) β-cell–specific LDHA deletion\nreversed BCKA-induced defects in GSIS and glucose tolerance in mice (Fig. 6f-i). We have\ndiscussed the possibility of other protein targets as below, and the relevant discussions are\nincluded in the revised manuscript (page 23, lines 489-504).\nDiscussion: Metabolite-protein interaction has been shown to control cellular response and\nmetabolism. Our current study showed that BCKAs but not BCAAs directly bind to LDHA\nand hence enhance its enzymatic activity toward lactate production. In addition, mutagenesis\nexperiment identified Ser210 of LDHA as a critical residue for mediating the LDHA–BCKA\ninteraction and dimer stabilization. Apart from LDHA-lactate axis, BCKAs have been shown\nto bind mitochondrial pyruvate carrier (MPC) and block pyruvate uptake in hepatocytes2.\nAlthough we did not directly examine the effect of BCKA on pyruvate uptake in mitochondria,\nits involvement in defective GSIS is likely negligible. Because inactivation of LDHA or\ninhibition of PDH kinase by DCA is sufficient to restore glucose flux to the TCA cycle and\nGSIS, arguing that pyruvate flux but not its uptake in mitochondria is impaired. However, a\ndirect labeled pyruvate uptake assay is required to further clarify MPC involvement. In vascular\ncells, BCKAs inhibit prolyl hydroxylase domain (PHD)2 enzyme activity and activate HIF1α,\nthereby inducing LDHA expression and lactate production3. In contrast, our study identifies a\ndistinct mechanism in which BCKAs activate LDHA through post-translational modification\nand dimerization without affecting its expression. Of note, BCKA treatment did not affect Hif-\n1α mRNA or LDHA protein expression in β-cells (Fig. 6a, Supplementary Fig. 20a), thereby\nexcluding the potential effect of BCKAs on the PHD2-HIFα-LDHA axis. Taken together, our\nfindings suggest that BCKAs impair glucose flux and GSIS by binding to LDHA and\npromoting its enzymatic activity toward lactate production.\nComment 3. Due to the complexity of the proposed mechanism including the role of PPM1K-\nBCKDH-A signal axis in glucose metabolism and insulin secretion, it’s suggested that a\nschematic summarizing the mechanism be supplemented in the discussion section.\nSupplementary Fig. 25 could be modified by the addition of the relationship between the\nBCKDH-A signal axis and glucose metabolism.\nResponse: Thank you for the suggestion. The Graphical abstract is now shown in Fig. 8 and\ndiscussed in page 20, lines 428-437. It shows relationship between the PPM1K–BCKDH-A–\nBCKA axis and glucose metabolism. The updated schematic highlights that, under normal\nconditions, BCKAs are catabolized in mitochondria via the BCKDH complex to generate CoA\nderivatives, which enter the TCA cycle. In diabetic β-cells, reduced PPM1K activity leads to\ndeactivation of BCKDH-A and subsequent BCKA accumulation. Elevated BCKA promotes\nthe conversion of LDHA from its inactive monomeric form to the active dimer, diverting\nglucose metabolism away from the TCA cycle toward lactate production. This metabolic shift\ndecreases ATP generation and impairs insulin secretion.\nFig. 8: Graphical abstract. BCKA impairs glucose-stimulated insulin secretion by redirection of\nglucose metabolism to the LDHA-lactate axis.\nComment 4. Please discuss the clinical significance of this investigation.\nResponse: We thank the reviewer for the suggestion, and have now included the below\ndiscussion to highlight the clinical relevance of our findings (page 25-26, lines 542-557).\nDiscussion: Circulating BCKAs are consistently elevated in patients with obesity, insulin\nresistance, and T2D, yet their role in β-cell pathology has remained unclear. Our work provides\ndirect mechanistic evidence that BCKAs are not only as BCAA byproducts but active drivers\nof β-cell dysfunction. Our findings are clinically relevant and important. First, our data suggest\nthat BCKA concentrations could serve as biomarkers for detecting defective insulin secretion\nin β-cells. Second, our results support that dietary restriction of BCAAs or promoting BCKA\ncatabolism might be a feasible approach for treatment and management of diabetes. Indeed,\npharmacological inactivation of BDK using NaPB and hence promoting BCAA/BCKA\ncatabolism has recently demonstrated to improve hyperglycaemia and insulin sensitivity in\nhuman with diabetes 1, despite no assessment on β-cell function. Consistently, a short-term low\nBCAA diet significantly improved insulin sensitivity and hence reduced insulin secretion as\nwell improved mitochondrial efficiency in adipose tissues of humans with T2D.4 Our findings\nfurther support the modulations of β-cell insulin secretion via targeting BCAA dysmetabolism,\nsuch as adjusting BCAA intake, enhancing BCKDH activity, or pharmacologically activating\nPPM1K. Together, our study indicates that the proper BCAA metabolism not only in peripheral\ntissues but also in β-cells is important for glucose homeostasis.\nReviewer #2\nLin et al., conducted a thorough study to elucidate a mechanism by which branched-chain keto\nacids contribute to the development of diabetes by examining their role in pancreatic beta cells.\nWhile the study is comprehensive, there are a few concerns that require attention.\nMajor Concerns\nComment 1. First the authors show that supplementation of individual BCAAs or BCKAs do\nnot alter GSIS. However, a mix of all three BCKAs are able to illicit an impairment in GSIS.\nWhy does only a combination of all three BCKAs impair GSIS? Are all three needed to bind\nto LDHA for dimerization? Does increasing the concentration of a singular BCKA to 100 uM\nimpair GSIS?\nResponse: We appreciate the reviewer’s insightful comment. To clarify the functional\nrelevance of BCKAs, we tested impacts of increasing concentrations of each individual BCKA\non GSIS. Treatment with any single BCKA at 30 μM had no significant impact on GSIS,\nwhereas higher concentrations (100 μM of KIC or KIV, or 200 μM KMV) led to a dose-\ndependent suppression of insulin secretion. This new data is presented in Supplementary Fig.\n5b.\nWe previously demonstrated that each individual BCKA (KIC, KMV, and KIV) can directly\nbind to LDHA, as confirmed by molecular docking and surface plasmon resonance (SPR)\nassays (Fig. 7a, b). To further determine whether all three BCKAs are required for LDHA\ndimerization, we examined their effects individually and in combination. At 30 μM, none of\nthe single BCKA induced LDHA dimerization in INS1E or MIN6 β-cells, whereas\ncombinations of two or all three BCKAs effectively promoted LDHA dimer formation. We\nnext tested whether higher concentration of individual BCKA was able to trigger LDHA\ndimerization. Consistent to our new GSIS data showing that 100μM KIC, 200 μM KMV or\n100μM KIV impaired GSIS, these three treatments also enhanced dimeric form of LDHA.\nThe two new data are now presented in Supplementary Fig. 26a and Supplementary Fig. 26b.\nCollectively, these results indicate that pathophysiological low concentration of individual\nBCKA is insufficient to activate LDHA or impair GSIS. Instead, simultaneous elevation of\nmultiple BCKAs act synergistically to promote LDHA dimerization and β-cell dysfunction.\nThese results reinforce our conclusion that the combined accumulation of BCKAs, rather than\na single metabolite, drives the impairment of insulin secretion in diabetes.\n500\n400\n300\n200\n100\nKIC, KMV or KIV as indicated. n=3-4. All results are presented as mean ± SEM. Statistical\nsignificance was determined using one-way ANOVA with Tukey correction.\nnoiterces\nnilusnI\n)nim\n03/nietorp\ngm/gn(\n0.070\n0.024\n<0.001 0.020\n0.014\n0.011\n(μM) 0 30 30 30 0 30 100 200 30 100 200 30 100 200\nvehicle KIC KMV KIV Vehicle KIC KMV KIV\nBasal Glucose\nSupplementary Fig. 26.\nProtein crosslinking assay\nfor tetrameric, dimeric, and\nmonomeric forms of LDHA\nin INS1E and MIN6 β-cells\nafter a 48-hour culture with\na vehicle, individual or\ncombined BCKA (each at\n30μM) or b individual\nBCKA as indicated.\nNCOMMS-25-27543A Point-by-point response\nComment 2. The authors use computational modeling to suggest that it is favorable for all\nthree BCKAs to bind to LDHA which may strengthen LDHA dimerization. The computational\nmodeling suggests several residues of interest for BCKA binding with Ser210 being a\nconserved site across all three BCKAs. While intriguing, the authors do not validate these\nbinding sites. The manuscript would be significantly strengthened by confirming binding sites\nthrough mutagenesis. Are there human mutations on these identified binding sites that\ncorrespond to GSIS phenotypes? Further, the authors fail to use a metabolite as a negative\ncontrol in their modeling and binding assays. Do BCAAs fail to bind to LDHA and induce\ndimerization?\nResponse: We acknowledge this thoughtful comment and have revised the manuscript\naccordingly. Our molecular docking analysis revealed that KIC and KMV bind at the LDHA\ndimer interface, forming molecular bridges through hydrogen bonds with Ser210 on Chain A\nand Ser202 and Thr307 on Chain B. Similarly, KIV interacts with Gly203, Asn205, and Ser210\non Chain A, and Gly208 on Chain B (Fig. 7a). Among these residues, Ser210 was identified as\na conserved contact site shared across all three BCKAs.\nTo experimentally validate the above prediction, we generated plasmids expressing wild-type\n(WT) LDHA with Flag epitope and its mutants: 1MUT (Ser210 mutated to alanine [Ser210A];\nconserved across all BCKAs), 3MUT (Ser202A/Ser210A/Thr307A; KIC and KMV binding\nsites), and 4MUT (Gly203A/Gly208A/Asn205A/Ser210A; KIV binding sites). The empty\nvector or the LDHA plasmids were transfected into HEK293T cells, followed by DARTS assay.\nThis assay showed that BCKA treatment prevented proteinase-induced degradation of WT-\nLDHA but not the three LDHA mutants (Fig. 7e). Consistently, BCKA treatment enhanced\ndimerization of WT LDHA but not the three LDHA mutants in 293T cells (Supplementary Fig.\n26e). These results confirm that Ser210 is essential to mediate LDHA-BCKA interaction and\ndimer stabilization.\nTo address the reviewer’s question on negative controls, we tested whether BCAA could\npromote LDHA dimerization. To avoid conversion of BCAA to BCKA in cells, purified\nrecombinant human LDHA protein was incubated directly with increasing concentrations of\nBCAA mixture, using BCKA as a positive control. While BCKA strongly induced LDHA\ndimerization, BCAA had no observable effect, confirming the specificity of BCKA–LDHA\nbinding. The result is shown in Supplementary Fig. 26d.\nFig. 7e. 293T cells were transfected with\nempty vector, or plasmids overexpressing\nLDHA WT, Ser210A (1MUT), Ser202A/\nSer210A/Thr307A(3MUT) or Gly203A/\nGly208A/Asn205A/Ser210A(4MUT). The\ncell lysates were incubated with 0, 0.2 mM\nor 2 mM BCKA, followed by DARTS\nanalysis of proteinase K (PK)-mediated\nLDHA degradation. Protein abundance in\ntotal cell lysates (TCL) was visualized\nusing silver staining.\nNCOMMS-25-27543A Point-by-point response\n(left) Supplementary Fig. 26e. 293T cells were transfected with empty vector, or plasmids\noverexpressing LDHA WT or Ser210A (1MUT), Ser202A/Ser210A/Thr307A (3MUT) or Gly203A\n/Gly208A/ Asn205A/Ser 210A (4MUT), followed by culture with vehicle or BCKA (30 μM each of\nKIC, KMV and KIV) for 48 hours. The cells were subjected to protein crosslinking assay for tetrameric,\ndimeric, and monomeric forms of LDHA.\n(right) Supplementary Fig. 26d. Effect of BCAA on the dimerization and tetramerization of purified\nrecombinant human LDHA protein. The upper and lower panels are results of immunoblotting\nanalysis against LDHA and silver staining of total protein, respectively.\nTo explore whether human mutations at the identified LDHA binding residues are associated\nwith GSIS phenotypes, we examined major population and clinical variant databases, including\ngnomAD, dbSNP, and ClinVar. Variants were identified at Ser210 (p.Ser210Pro;\nrs1267663373), Thr307 (p.Thr307Pro; rs368105451), Gly203 (p.Gly203Arg; rs1287059546\nand p.Gly203Ala; rs34305721), and Asn205 (p.Asn205Ile; rs1219647514), while no reported\nvariants were detected at Ser202. In gnomAD, all of these variants are extremely rare, with\nallele frequencies around 10⁻⁴ for Gly203Ala and 10⁻⁶ to 10⁻⁷ for the others (results shown\nbelow). In ClinVar, none of these variants are associated with GSIS or traits related to β-cells.\nThe only clinical annotation (Gly203Ala) is linked to glycogen storage disease. Therefore,\ncurrent human genetic data do not indicate a direct relationship between natural variation at\nthese LDHA residues and insulin secretion, likely due to their ultrarare occurrence and\ninsufficient population-level power to detect functional associations. In addition, the effects of\nLDHA variants on GSIS and β-cell function might be dependent on BCKA levels in the\ncirculation and β-cells.\nNevertheless, LDHA mRNA level in pancreatic β-cells was reported to positively correlated\nwith HbA1c in humans.5 Transcriptomic and proteomic studies have consistently shown\nLDHA dysregulation in β-cells from individuals with T2D. Marselli et al. reported elevated\nLDHA mRNA levels in β-cells isolated from diabetic donors compared with non-diabetic\ncontrols6, while Kolic et al7. identified significant LDHA upregulation in islet proteomes from\n133 human donors in diabetic condition. These findings collectively support a broader\nNCOMMS-25-27543A Point-by-point response\npathogenic role of LDHA activation in T2D, consistent with our mechanistic observations. The\ncorresponding references have been added to the Discussion (page 22, lines 470-472).\nSupporting Data. Allele frequencies of LDHA variants at identified BCKA-binding\nresidues in humans, as reported in the gnomAD database.\nComment 3, the authors systematically measure glucose uptake, insulin content, mitochondrial\nmembrane potential, cell viability, and ATP production to determine where the defect in beta\ncell GSIS could be occurring. Authors perform seahorse experiment and observe a decrease in\nglycolysis in BCKA treated cells and speculate that it is driven by a reduction in CO2\nproduction (page 12, line 12-13). Authors should perform radiolabeled glucose oxidation assay\nto confirm statement.\nResponse: We thank the reviewer for raising this important point. We observed that BCKA\ntreatment markedly reduced glucose-stimulated ECAR in β-cells (Fig. 5d). However, we\ninterpret this reduction as a result of decreased mitochondrial respiratory, rather than decreased\nglycolytic acidification based on several lines of evidence. First, we demonstrated that high\nBCKA exposure increased glucose-derived lactate production, as evidenced by isotope tracing\n(Fig. 5f-l and Supplementary Fig. 17 & 18), and concurrently enhanced LDHA activity in β-\ncells, mouse and human islets, as well as purified recombinant human LDHA proteins (Fig.\n6b-e). These results indicate that glycolysis was not suppressed but redirected toward lactate\nformation. Second, both our findings (Fig. 5d-e and Supplementary Fig. 14, 15d-e) and\nprevious reports showed that oligomycin, which inhibits mitochondrial ATP synthase, reduces\nECAR in β-cells and islets8, 9, 10, 11, 12, 13, suggesting that a substantial portion of ECAR\noriginates from mitochondrial CO₂ hydration rather than anaerobic glycolysis. Third, the near\nNCOMMS-25-27543A Point-by-point response\nabsence of the monocarboxylate transporter MCT1 in normal β-cells14 makes it unlikely that\nECAR reflects lactate-coupled proton efflux, as typically assumed in conventional Seahorse\ninterpretations.\nPrevious studies have demonstrated that extracellular acidification can arise from CO₂\nhydration to H₂CO₃, which dissociates into HCO₃⁻ and H⁺. At present, we are unable to provide\ndirect evidence of CO₂ release, as we currently lack access to a licensed facility to perform\nradiolabeled glucose oxidation assays for its quantification at PolyU. Nevertheless, the study\nby Mookerjee et al. provides important quantitative context15. They measured CO₂-dependent\nacidification in Seahorse assays by calculating the Respiratory Proton Production Rate\n(PPR)—the proportion of total extracellular acidification attributable to CO₂-derived protons.\nBoth INS1E β-cells and dispersed rat islets exhibited nearly fully respiratory acidification\nprofiles, with Respiratory PPR values of approximately 93–96%, markedly higher than those\nobserved in other cell types. These findings indicate that, in β-cell models, extracellular\nacidification predominantly reflects respiratory CO₂ generation rather than glycolytic proton\nrelease.\nCollectively, our data and prior evidence support that the reduced ECAR observed in BCKA-\ntreated β-cells primarily results from diminished mitochondrial respiration and impaired\noxidative metabolism within the TCA cycle. We have now cited the work by Mookerjee et al.\nand further clarify the rationale underlying our glycolytic stress test interpretation (page 13-14,\nlines 269-283)\nMinor Concerns\n• The authors use tracing studies to show that carbons from glucose and pyruvate are\nredirected from TCA cycle to lactate and this can be rescued by activation of PDH. Does this\nmodel (DCA), impair BCKA action on LDHA activity or LDHA dimerization?\nResponse: Thank you for the comment. Our new data indicate that DCA, a PDH activator,\ndoes not affect BCKA-induced LDHA dimerization in β-cells. This suggests that the BCKA–\nLDHA interaction and subsequent dimer formation occur independently of PDH activation.\nThese results are now included in Supplementary Fig. 26c.\nSupplementary Fig. 26c. Protein\ncrosslinking assay for LDHA in whole-\ncell lysates of INS1E β-cells after a 48-\nhour culture with vehicle or BCKA (30\nμM each of KIC, KMV and KIV) in the\npresence or absence of DCA (5 mM) for\n2 hours.\nNCOMMS-25-27543A Point-by-point response\n• Supplementation studies with mixture of BCKAs and BCAAs it is unclear whether the\nconcentration reported is total amount of metabolites or individual metabolite.\nResponse: We thank the reviewer for the comment and have clarified this point throughout the\nmanuscript and figure legends. The text in the Results, Methods, and corresponding legends\nhas been revised to state the composition of each mixture, for example: “BCKA cocktail\ncontaining KIC, KMV, and KIV (each at 30 μM)”, “BCKA (30 μM each of KIC, KMV, and\nKIV)” and “BCKA, with each of KIC, KMV, and KIV at a concentration of 5 mg/mL.”\n• Please confirm figure panel reported in the text matches that in the figures (Page 7 line 15 –\nrefers to Supplementary Fig. 3 not Supplementary Fig. 4).\nResponse: Thank you for pointing this out. We have corrected the figure reference in the text\nto ensure consistency between the text and the corresponding figures.\n• The addition of oligomycin should represent maximal glycolytic capacity in Seahorse assays.\nHowever, ECAR and OCR both decrease with the addition of oligomycin. Is this an expected\noutcome for beta cells?\nResponse: Thank you for the comment. As explained in our response to Major Comment 3,\nthis outcome is expected for β-cells. In our lab, we consistently observed that oligomycin\ntreatment decreased both ECAR and OCR in rodent MIN6 and INS1E β-cell lines, human\nEndoC-βH1 cells (Fig 5d-e, Supplementary Fig. 14 and 15d-e), and primary mouse islets.\nThese results are consistent with previous studies reporting similar findings in β-cell lines and\nprimary rodent and human islets8, 9, 10, 11, 12, 13. This pattern reflects the unique metabolic\ncharacteristics of β-cells, which exhibit low expression of monocarboxylate transporters\n(MCTs, limiting lactate efflux) and LDHA (restricting lactate production). Therefore,\ninhibition of mitochondrial ATP synthase by oligomycin results in untypically concurrent\nreductions in ECAR and OCR.\nReviewer #3\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you very much for reviewing our work.\nNCOMMS-25-27543A Point-by-point response\nReviewer #4\nBranched-chain a-keto acids impair glucose-stimulated Insulin secretion in pancreatic b-cells\nunder diabetes by reactivating the LDHA-lactate axis.\nIn this manuscript, Lin and colleagues describe a metabolic rewiring process occurring in\npancreatic beta cells under BCKA chronic exposure/BCKA catabolism disruption which\npromoted impaired glucose-stimulated insulin secretion. In T2D higher levels of circulating\nBCAA and of their catabolites BCKA have been reported and the authors demonstrated a\ncompromised BCAA catabolic machinery in diabetic pancreatic islets and consequent BCKA\naccumulation in beta cells. In vivo models demonstrated that high circulating levels of BCKA\npromoted glucose intolerance whereas reducing circulating BCAA/BCKA levels in diabetic\nanimals improved beta cell function.\nThis “overloading” of BCKA on pancreatic beta cells exert a GSIS-inhibiting effect by\ndecoupling glucose metabolism and TCA/oxidative phosphorylation and promoting an\nabnormal activation of LDHA. This is mediated by the binding of BCKAs to the monomer of\nthe enzyme, which is normally inactivated in beta-cells and this binding promotes the\ndimerization of LDHA, thus inducing an abnormal activation of the enzyme and deviating\npyruvate catabolism toward lactate synthesis. This would affect ATP synthesis and reduce the\nfirst phase of glucose-stimulated insulin secretion. The authors propose that this metabolic\nrewiring would contribute to glucose intolerance in diabetes.\nThe manuscript is well written, clearly organized and results are convincing.\nThe main aim of this reviewer’s comments will be to improve the manuscript to give the highest\ntranslational value possible.\nMajor revisions:\nComment 1: The authors used several in vitro (murine and rat beta cells/mouse islets) and in\nvivo models (db/db, KO models). Unfortunately, not all data were reproduced on human beta\ncells and islets, but the authors declare these missing data in study limitations.\nAs proof of concept, even if it was not possible to perform all the experiments on human islets,\nI suggest repeating at least the key experiments on a commercial human beta cells line, such\nbLox5 cells.\nResponse: We thank the reviewer for the suggestion. Several key findings have already been\nvalidated in human samples. Specifically, we demonstrated the inhibitory effect of BCKA on\nGSIS in human islets (Fig. 2c) and on isotopic glucose flux (Supplementary Fig. 18);\nanalyzed\nthe expression of BCKA catabolic enzymes in human pancreatic islets under healthy and\ndiabetic conditions (Fig. 1d–g); and identified negative associations between circulating\nBCKA levels and β-cell function indices (Supplementary Fig. 3–4). In addition, we showed\nthat BCKA enhances the enzymatic activity and dimerization of purified human LDHA protein\n(Figs. 6e and 7d).\nTo further strengthen the human relevance, we have now replicated the effect of BCKA on\nglycolysis and mitochondrial respiration in EndoC-βH1 cells—a well-characterized human β-\ncell line that closely mimics primary islet β-cells in glucose-stimulated insulin secretion16.\nConsistent with our findings in rodent cell models, BCKA treatment reduced glucose-\nstimulated ECAR and OCR in EndoC-βH1 cells (Supplementary Fig. 14). We therefore\nvalidated most of the BCKA effects in human β-cells or islets.\nNCOMMS-25-27543A Point-by-point response\nSupplementary Fig 14. Glycolytic stress test in human EndoC-βH1 cells treated with vehicle\nor BCKA (30 μM each of KIC, KMV and KIV) for 48 hours. a ECAR and b OCR in response\nto stimulations with glucose, oligomycin, and 2-DG, n=5.\nComment 2: The authors already assed cell viability by MTT and mRNA expression of 3\napoptosis-related genes in MIN6 cells treated with BCKA without observing significant\ndifferences. However, it would be also interesting to run other in vitro experiments to\neffectively assess whether BCKA overloading may compromise beta cells survival (like\ncaspase cleavage analysis, cell death assays). Since in the title the authors write about general\ndiabetes and not only T2D, this may give to the proposed mechanism a significant value also\nin the pathogenesis of T1D.\nResponse: Thank you for the thoughtful comment. We have now measured caspase-3 activity\nin MIN6 cells following BCKA treatment using the EnzChek Caspase‐3 Enzymatic Assay Kit\n(Catalog # E13183, Invitrogen). We included doxorubicin as a positive control for induction\nof apoptosis. Caspase-3 activities were similar between the cells treated with vehicle or BCKA\ntreated cells. This new result is included in Supplementary Fig. 13d.\nIn addition, we assessed the potential cytotoxic effects of BCKA overload on β-cell survival\nusing multiple complementary approaches. In vivo BCKA supplementation did not reduce β-\ncell mass or β/α-cell ratio in mice (Supplementary Fig. 8). Consistently, BCKA exposure did\nnot affect cell viability (MTT assay; Supplementary Fig. 13c), the mRNA expression of\napoptosis-related genes in β-cell lines (Supplementary Fig. 13e), or total insulin content in\nisolated islets (Supplementary Fig. 13a). Together, these findings indicate that BCKAs do not\ncompromise β-cell survival or apoptosis.\nNCOMMS-25-27543A Point-by-point response\n3\n2\n1\nComment 3: In this sense, please add a section in the discussion paragraph dedicated to the\npotential relevance of BCAA/BCKA in T1D, since in the last years novel non-immune\nmediated mechanisms are emerging in promoting beta cells destruction in T1D. In this section,\nplease consider adding the references PMID 35115561 and PMID 38318298.\nResponse: Thank you for the suggestion. We have added a new paragraph in the Discussion\nfor the potential relevance of BCAA/BCKA metabolism in T1D (page 25, lines 529-541).\nDiscussion: Apart from T2D, aberrant BCAA/BCKA metabolism have also been reported in\nT1D. An inverse association between circulating BCAAs and endogenous insulin secretion\nwas observed in T1D patients17. Longitudinal metabolomics in children progressing to T1D\nalso revealed early metabolic dysregulation preceding autoimmunity, characterized by elevated\nBCAAs and reduced keto-leucine levels before seroconversion18. In addition, high levels of\nBCKA or PPM1K deficiency triggers apoptosis in multiple cell types19, 20, 21. Recent studies\nsuggest that aberrant cell death receptor pathway via TMEM219 also contributes to β-cell\ndestruction in T1D22, 23. In our animal studies, pancreatic β-cell mass was not altered by chronic\ntreatment with BCKA, BT2 or β-cell specific PPM1K deletion. In vitro, treatment with BCKAs\ndid not exert obvious effect on survival or apoptosis in β-cells. Therefore, the direct\ninvolvement of BCKA metabolism in T1D development appears unlikely. Nevertheless,\nfurther validation of the role of PPM1K or BCKA metabolism on β-cell function and mass\nusing appropriate T1D animal models (such as non-obese diabetic mice and streptozotocin\ntreatment) is warranted.\nMinor revisions:\n• All the figures: where not already specified, please add a note if data were obtained in\nhumans or mice. Ex. In fig 1 e-f add “human islets” to make it clearer for the readers.\n• Page 7 line 14: Probably is extended figure 3?\n• Fig 2 d-g add “MIN6 cells”\n• Pag 10 line 11: please add “in vivo” in the title of the paragraph.\nResponse: Thank you for these suggestions. We have proofread the entire manuscript to ensure\nclarity and consistency. All sample types (human, mouse, or cell lines) are now clearly\nytivitca\n3-esapsac\nevitaleR\n)elcihev\nfo\nsdlof(\n< 0.0001\nSupplementary Fig. 13d: Relative caspase 3\nactivity in MIN6 cells treated with vehicle,\nN.S.\nBCKA (30 μM) or doxorubin (30 nM) for 48\nhours. n=5. All results are presented as mean ±\nSEM. Statistical significance was determined\nusing one-way ANOVA with Tukey correction.\nN.S., not significant.\nNCOMMS-25-27543A Point-by-point response\nspecified in the corresponding figure legends, and additional labels have been added within\nfigures where space allows.\n• Page 6 line 14: please add in the title that the analyses were conducted on humans. I would\nadd the most significant data in the main text to give more relevance to the relationship\nbetween T2D and BCAA altered catabolism in humans.\n• Pag 13 line 19: I suggest adding significant metabolic data on human islets in the main text.\nResponse: We appreciate the reviewer’s suggestion to further highlight the human data. In\nthe revised manuscript, we have clarified the type of human samples analyzed in all relevant\nfigures, including the subtitles of Supplementary Fig. 3, 4, 14, and 18. Several key human\nresults are already presented in the main figures (Fig. 1d–g, 2c, 6d–e, and 7d), representing\nthe most central clinical evidence supporting the link between altered BCKA metabolism and\nβ-cell dysfunction. The remaining human datasets are also clinically relevant; however, they\nserve primarily as supportive or confirmatory results and were therefore retained in the\nSupplementary Figures to maintain a clear narrative flow and avoid overcrowding the main\nfigures.\n• Page 8 line 21: were these results not reproduced in MIN6 cells?\nResponse: As indicated in the legend of Fig. 2d–g, the transfection experiments were\nconducted in INS1E β-cells. This decision was based on our pilot data showing that MIN6 β-\ncells exhibited a stronger GSIS response (also see in Fig. 2) but significantly lower transfection\nefficiency compared with INS1E cells. To ensure robustness, we reproduced key findings\nobtained from MIN6 cells in INS1E cells, as well as in primary mouse and human islets,\nconfirming consistency and relevance across multiple β-cell models. These additional results\nin INS2E cells were not included in the manuscript to maintain clarity and avoid redundancy.\nReferences:\n1.Vanweert F, et al. A randomized placebo-controlled clinical trial for pharmacological\nactivation of BCAA catabolism in patients with type 2 diabetes. Nat Commun 13, 3508\n(2022).\n2.Nishi K, et al. Branched-chain keto acids inhibit mitochondrial pyruvate carrier and suppress\ngluconeogenesis in hepatocytes. Cell Rep 42, 112641 (2023).\n3.Xiao W, et al. Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular\ncells. Nat Metab 6, 2138-2156 (2024).\n4.Karusheva Y, et al. Short-term dietary reduction of branched-chain amino acids reduces\nmeal-induced insulin secretion and modifies microbiome composition in type 2 diabetes: a\nrandomized controlled crossover trial. Am J Clin Nutr 110, 1098-1107 (2019).\n5.Malmgren S, et al. Tight coupling between glucose and mitochondrial metabolism in clonal\nbeta-cells is required for robust insulin secretion. J Biol Chem 284, 32395-32404 (2009).\n6.Marselli L, et al. Gene expression profiles of Beta-cell enriched tissue obtained by laser\ncapture microdissection from subjects with type 2 diabetes. PLoS One 5, e11499 (2010).\n7.Kolic J, et al. Proteomic predictors of individualized nutrient-specific insulin secretion in\nhealth and disease. Cell metabolism 36, 1619-1633.e1615 (2024).\nNCOMMS-25-27543A Point-by-point response\n8.Li X, et al. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial\nmetabolism to glucose-stimulated insulin secretion in pancreatic β-cells. Nat Commun 7,\n11740 (2016).\n9.Montemurro C, et al. IAPP toxicity activates HIF1α/PFKFB3 signaling delaying β-cell loss\nat the expense of β-cell function. Nat Commun 10, 2679 (2019).\n10.Cornell D, et al. Pseudoislet Aggregation of Pancreatic β-Cells Improves Glucose\nStimulated Insulin Secretion by Altering Glucose Metabolism and Increasing ATP\nProduction. Cells 11, (2022).\n11.Hals I, et al. Time dynamics of elevated glucose and beta-hydroxybutyrate on beta cell\nmitochondrial metabolism. Islets 17, 2503515 (2025).\n12.Sentjens K, Pillai R, Joseph JW. The effects of free fatty acid-free bovine serum albumin\nand palmitate on pancreatic β-cell function. Islets 17, 2479911 (2025).\n13.Xiong X, et al. Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic\nbeta cells. Diabetologia 59, 151-160 (2016).\n14.Zhao C, Wilson MC, Schuit F, Halestrap AP, Rutter GA. Expression and distribution of\nlactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.\nDiabetes 50, 361-366 (2001).\n15.Mookerjee SA, Goncalves RLS, Gerencser AA, Nicholls DG, Brand MD. The contributions\nof respiration and glycolysis to extracellular acid production. Biochim Biophys Acta 1847,\n171-181 (2015).\n16.Tsonkova VG, et al. The EndoC-βH1 cell line is a valid model of human beta cells and\napplicable for screenings to identify novel drug target candidates. Mol Metab 8, 144-157\n(2018).\n17.Karusheva Y, et al. Branched-Chain Amino Acids Associate Negatively With Postprandial\nInsulin Secretion in Recent-Onset Diabetes. J Endocr Soc 5, bvab067 (2021).\n18.Oresic M, et al. Dysregulation of lipid and amino acid metabolism precedes islet\nautoimmunity in children who later progress to type 1 diabetes. J Exp Med 205, 2975-2984\n(2008).\n19.Funchal C, Gottfried C, De Almeida LM, Wajner M, Pessoa-Pureur R. Evidence that the\nbranched-chain alpha-keto acids accumulating in maple syrup urine disease induce\nmorphological alterations and death in cultured astrocytes from rat cerebral cortex. Glia 48,\n230-240 (2004).\n20.Guo X, et al. BCKA down-regulates mTORC2-Akt signal and enhances apoptosis\nsusceptibility in cardiomyocytes. Biochem Biophys Res Commun 480, 106-113 (2016).\n21.Lu G, et al. A novel mitochondrial matrix serine/threonine protein phosphatase regulates\nthe mitochondria permeability transition pore and is essential for cellular survival and\ndevelopment. Genes Dev 21, 784-796 (2007).\n22.D'Addio F, et al. The IGFBP3/TMEM219 pathway regulates beta cell homeostasis. Nat\nCommun 13, 684 (2022).\n23.D'Addio F, et al. TMEM219 regulates the transcription factor expression and proliferation\nof beta cells. Front Endocrinol (Lausanne) 15, 1306127 (2024).\nNCOMMS-25-27543A Point-by-point reply\nPoint-by-point replies to reviewers\nBranched-chain α-keto acids impair glucose-stimulated insulin secretion in pancreatic\nβ-cells under diabetes by reactivating the LDHA-lactate axis (NCOMMS-25-27543A)\nReviewer #1:\nThe authors have responded to all the comments and made the corresponding revisions. I\nhave no more comments.\nReviewer #4:\nThe authors carefully and deeply addressed all the suggestions of the reviewers, both in terms\nof main text and experiments implementation. The manuscript is of high quality and is ready\nfor publication.\nResponses: We thank the reviewers for their positive assessment of our revisions and for\nrecommending the manuscript for publication.\nReviewer #2:\nThe authors provided new data that addresses most of the reviewers’ comments. One\nremaining issue related to comment #2 and Rev 1’s comment is the model shown in Fig.8.\nBCAA and BCKA require mitochondrial carrier proteins to enter the matrix, and conversely,\nBCKAs would require an exporter to meet LDH in the cytoplasm. BCAA mitochondrial\ncarrier is known, but the authors should examine and discuss the current understanding,\nespecially in beta-cells, and incorporate that in the summary cartoon.\nResponse: We thank the reviewer for this constructive suggestion. We have updated our\nsummary model (Figure 8) to illustrate the mitochondrial transport of BCAAs and BCKAs.\nAdditionally, we have expanded the Discussion section (Page 22, lines 462–477) to address\nthe current understanding of mitochondrial carriers in β-cells.\nDiscussion: Intracellular BCAA and BCKA levels are controlled by their extracellular uptake\nand intracellular catabolism. We showed that BCKAs are accumulated in diabetic islets, which\nare associated with PPM1K reduction and p-BCKDHA induction. However, it is currently\nunclear whether β-cell BCAA/BCKA transportation is altered under the diabetic condition.\nWhile SLC25A44 has been identified as the primary carrier for BCAAs into the mitochondrial\nmatrix1, evidence suggests it may not serve as a universal carrier for their keto acid derivatives.\nNCOMMS-25-27543A Point-by-point reply\nSpecifically, studies have shown that overexpression of SLC25A44 failed to promote the\nmitochondrial uptake of KIV2. Although MCT1 can mediate bidirectional BCKA transport\nacross the plasma membrane3 and is reported to also localize to mitochondrial membrane in\nmost tissues4, it is strictly disallowed within the β-cell plasma membrane to prevent\ninappropriate insulin secretion5, 6. Furthermore, neither MCT1 nor other MCT isoforms can be\ndetected in islet β-cell mitochondria under normal physiological conditions7. Consequently, a\nsignificant knowledge gap remains regarding how BCKAs generated within the matrix are\nexported to the cytoplasm to bind LDHA and activate its activity toward lactate production.\nElucidating these mitochondrial BCKA transport mechanisms is critical to understanding their\npotential role in driving β-cell dysfunction during the pathogenesis of diabetes.\nReference\n1.Yoneshiro T, et al. BCAA catabolism in brown fat controls energy homeostasis through\nSLC25A44. Nature 572, 614-619 (2019).\n2.Walejko JM, et al. Branched-chain α-ketoacids are preferentially reaminated and activate\nprotein synthesis in the heart. Nat Commun 12, 1680 (2021).\n3.Silva LS, et al. Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate\nmacrophage phenotype. EMBO Rep 18, 2172-2185 (2017).\n4.Chen L, et al. MCT1-mediated Lactate Shuttle to Mitochondria Governs Macrophage\nPolarization and Modulates Glucose Homeostasis by Affecting β Cells. Adv Sci (Weinh) 12,\ne14760 (2025).\n5.Pullen TJ, Sylow L, Sun G, Halestrap AP, Richter EA, Rutter GA. Overexpression of\nmonocarboxylate transporter-1 (SLC16A1) in mouse pancreatic β-cells leads to relative\nhyperinsulinism during exercise. Diabetes 61, 1719-1725 (2012).\n6.Schuit F, et al. β-cell-specific gene repression: a mechanism to protect against inappropriate\nor maladjusted insulin secretion? Diabetes 61, 969-975 (2012).\n7.Zhao C, Wilson MC, Schuit F, Halestrap AP, Rutter GA. Expression and distribution of\nlactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.\nDiabetes 50, 361-366 (2001)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded to all the comments and made the corresponding revisions. I have no more comments.", "reviewer_2": "The authors provided new data that addresses most of the reviewers’ comments. One remaining issue related to comment #2 and Rev 1’s comment is the model shown in Fig.8.\nBCAA and BCKA require mitochondrial carrier proteins to enter the matrix, and conversely, BCKAs would require an exporter to meet LDH in the cytoplasm. BCAA mitochondrial carrier is known, but the authors should examine and discuss the current understanding, especially in beta-cells, and incorporate that in the summary cartoon.", "reviewer_4": "The authors carefully and deeply addressed all the suggestions of the reviewers, both in terms of main text and experiments implementation. The manuscript is of high quality and is ready for publication."}, "author_response": "NCOMMS-25-27543A Point-by-point reply\nPoint-by-point replies to reviewers\nBranched-chain α-keto acids impair glucose-stimulated insulin secretion in pancreatic\nβ-cells under diabetes by reactivating the LDHA-lactate axis (NCOMMS-25-27543A)\nReviewer #1:\nThe authors have responded to all the comments and made the corresponding revisions. I\nhave no more comments.\nReviewer #4:\nThe authors carefully and deeply addressed all the suggestions of the reviewers, both in terms\nof main text and experiments implementation. The manuscript is of high quality and is ready\nfor publication.\nResponses: We thank the reviewers for their positive assessment of our revisions and for\nrecommending the manuscript for publication.\nReviewer #2:\nThe authors provided new data that addresses most of the reviewers’ comments. One\nremaining issue related to comment #2 and Rev 1’s comment is the model shown in Fig.8.\nBCAA and BCKA require mitochondrial carrier proteins to enter the matrix, and conversely,\nBCKAs would require an exporter to meet LDH in the cytoplasm. BCAA mitochondrial\ncarrier is known, but the authors should examine and discuss the current understanding,\nespecially in beta-cells, and incorporate that in the summary cartoon.\nResponse: We thank the reviewer for this constructive suggestion. We have updated our\nsummary model (Figure 8) to illustrate the mitochondrial transport of BCAAs and BCKAs.\nAdditionally, we have expanded the Discussion section (Page 22, lines 462–477) to address\nthe current understanding of mitochondrial carriers in β-cells.\nDiscussion: Intracellular BCAA and BCKA levels are controlled by their extracellular uptake\nand intracellular catabolism. We showed that BCKAs are accumulated in diabetic islets, which\nare associated with PPM1K reduction and p-BCKDHA induction. However, it is currently\nunclear whether β-cell BCAA/BCKA transportation is altered under the diabetic condition.\nWhile SLC25A44 has been identified as the primary carrier for BCAAs into the mitochondrial\nmatrix1, evidence suggests it may not serve as a universal carrier for their keto acid derivatives.\nNCOMMS-25-27543A Point-by-point reply\nSpecifically, studies have shown that overexpression of SLC25A44 failed to promote the\nmitochondrial uptake of KIV2. Although MCT1 can mediate bidirectional BCKA transport\nacross the plasma membrane3 and is reported to also localize to mitochondrial membrane in\nmost tissues4, it is strictly disallowed within the β-cell plasma membrane to prevent\ninappropriate insulin secretion5, 6. Furthermore, neither MCT1 nor other MCT isoforms can be\ndetected in islet β-cell mitochondria under normal physiological conditions7. Consequently, a\nsignificant knowledge gap remains regarding how BCKAs generated within the matrix are\nexported to the cytoplasm to bind LDHA and activate its activity toward lactate production.\nElucidating these mitochondrial BCKA transport mechanisms is critical to understanding their\npotential role in driving β-cell dysfunction during the pathogenesis of diabetes.\nReference\n1.Yoneshiro T, et al. BCAA catabolism in brown fat controls energy homeostasis through\nSLC25A44. Nature 572, 614-619 (2019).\n2.Walejko JM, et al. Branched-chain α-ketoacids are preferentially reaminated and activate\nprotein synthesis in the heart. Nat Commun 12, 1680 (2021).\n3.Silva LS, et al. Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate\nmacrophage phenotype. EMBO Rep 18, 2172-2185 (2017).\n4.Chen L, et al. MCT1-mediated Lactate Shuttle to Mitochondria Governs Macrophage\nPolarization and Modulates Glucose Homeostasis by Affecting β Cells. Adv Sci (Weinh) 12,\ne14760 (2025).\n5.Pullen TJ, Sylow L, Sun G, Halestrap AP, Richter EA, Rutter GA. Overexpression of\nmonocarboxylate transporter-1 (SLC16A1) in mouse pancreatic β-cells leads to relative\nhyperinsulinism during exercise. Diabetes 61, 1719-1725 (2012).\n6.Schuit F, et al. β-cell-specific gene repression: a mechanism to protect against inappropriate\nor maladjusted insulin secretion? Diabetes 61, 969-975 (2012).\n7.Zhao C, Wilson MC, Schuit F, Halestrap AP, Rutter GA. Expression and distribution of\nlactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.\nDiabetes 50, 361-366 (2001)."}]

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