Adipocyte small extracellular vesicle-derived microRNA-30a-3p exacerbates hepatic steatosis in high fat diet-fed male mice

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
Adipose tissue dysfunction drives hepatic lipid overload in metabolic dysfunction-associated steatotic liver disease (MASLD), yet the involvement of adipose tissue-derived small extracellular vesicles (sEVs) remains unclear. Herein, we showed that transplanting adipose tissue from high‑fat diet (HFD)-fed male mice exacerbated insulin resistance and hepatic steatosis in lean recipients. Adipose‑specific Sirt3 overexpression (Sirt3AKI) alleviated insulin resistance and liver steatosis in HFD-fed male mice, whereas adipose‑specific Sirt3 knockdown aggravated these phenotypes. Moreover, adipose sEV miRNAs regulated hepatic lipid metabolism in Sirt3AKI male mice. MicroRNA sequencing identified miR-30a-3p was increased in the circulating sEVs from HFD-fed male mice, while decreased in sEVs from Sirt3OE adipocytes and Sirt3AKI male mice. Mechanistically, miR‑30a‑3p promoted hepatic steatosis by targeting Becn1; this process was suppressed when Sirt3 downregulated miR‑30a‑3p transcription via deacetylation of H3K56. These findings highlight the critical role of adipose sEV microRNAs in driving hepatocyte lipotoxicity, and suggest miR-30a-3p inhibition as a promising MASLD therapy.

Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD), currently proliferating globally at an alarming rate, is closely correlated with the progression to end-stage liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma1, 2. MASLD is fundamentally characterized by adipose tissue dysfunction and ectopic accumulation of triglycerides (TG) in the liver3, 4. Adipose tissue secretes free fatty acids (FFAs), adipokines, and cytokines, making the liver more vulnerable to lipotoxicity and subsequent metabolic abnormalities5-8. Thus, gaining a better understanding of adipose tissue-liver communication will be helpful in the development of promising strategies for managing MASLD.

Small extracellular vesicles (sEVs) are cell-derived membrane-surrounded vesicles, which transport specific bioactive cargo, including various proteins, small molecules and RNAs, to recipient cells9, 10. Adipose tissue is the most important source of sEVs, and its dysfunction alters the contents of sEVs qualitatively, thereby leading to the disruption of homeostasis in target organs11, 12. MicroRNAs (miRNAs) are small noncoding RNAs that modulate mRNA degradation and translational efficacy in most cell types13. Adipose tissue-derived sEVs miRNAs serve as key mediators of hepatic gene expression modulation14. Adipose tissue macrophage-derived sEVs miRNAs were reported to improve insulin sensitivity in vivo and in vitro15, indicating their importance in energy metabolism. Accordingly, we hypothesized that adipose tissue-derived sEVs miRNAs, key mediators in adipose-liver inter-organ communication, are critical for MASLD onset and progression.

Sirtuin 3 (Sirt3) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase which deacetylates key substrates involved in energy homeostasis in hepatocytes16. Sirt3 exhibits dual localization in both nucleus and mitochondria17. Some researches suggest that Sirt3 can translocate from the mitochondria to the nucleus under stress conditions18, 19. Additionally, Sirt3 affects gene transcription by regulating the acetylation levels of histone in the nucleus20. Specifically, Sirt3 overexpression reduces acetylated histone H3 lysine 56 (H3K56ac) levels, which enhances electrostatic interactions between positively charged histones and negatively charged DNA, driving chromatin condensation21. These compact nucleosomes obscure the DNA recognition motifs of transcription factors, thereby hindering miR synthesis22. Our previous studies confirmed that Sirt3 activates autophagy to promote lipid mobilization in adipocytes and protects hepatocytes against lipotoxicity23, 24. However, the role of Sirt3 in manipulating adipocyte-hepatocyte crosstalk remains incompletely characterized.

Here, we elucidated an unrecognized adipose-liver communication axis, in which adipose-derived sEVs and their cargo miRNAs can be transferred to the liver, modulating hepatic cellular behavior. Moreover, we demonstrated that reduced Sirt3 expression in the adipose tissue is an important mediator of high-fat diet (HFD)-induced hepatic steatosis. Adipose tissue Sirt3 suppresses the transcription of miR-30a-3p through an epigenetic modification mechanism, resulting in the activation of hepatic autophagy in the liver. It is notable that overexpression of Sirt3 in the adipose tissue, or engineered sEVs loaded with miR-30a-3p antagomir effectively relieved the hepatic steatosis in HFD-fed mice. Taken together, our findings suggest a dual-organ systematic interplay in the progression of MASLD and uncover sEVs miR-30a-3p as a potential non-invasive biomarker and therapeutic target for MASLD.

Results
Transplantation of adipose tissue from HFD-fed mice causes hepatic lipidosis in lean mice, and Sirt3 is downregulated in adipose tissue of MASLD patients

To ascertain the involvement of adipose tissue in hepatic lipidosis, chow-fed mice were received transplantation of visceral adipose tissue (VAT) from HFD-fed mice (Chow TransHFD), chow-fed mice (Chow TransChow), or sham-operated mice (Chow sham) (Fig. 1A). Two weeks post-transplantation, Chow TransHFD mice showed higher liver weight (Fig. 1B), serum TG level (Fig. 1C) and hepatic TG level (Fig. 1D) than those of Chow TransChow mice and Chow Sham mice. Histological examination and Oil red O staining results revealed that Chow TransHFD mice exerted obvious lipid accumulation in the liver compared with Chow TransChow mice and Chow sham mice (Fig. 1E). Furthermore, Chow TransHFD mice exhibited damaged glucose tolerance (Fig. 1F) and insulin sensitivity (Fig. 1G). These results collectively support that adipose tissues from high fat diet-fed male mice release circulating factors that impair insulin sensitivity and promote the development of hepatic steatosis.

Given the reported positive association between Sirt3 deletion and ectopic lipid deposition25. Herein, we confirmed that a decrease of Sirt3 expression was observed in the adipose tissue of MASLD patients, compared to healthy human participants (Fig. 1H). Similarly, the protein and mRNA levels of Sirt3 were lower in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) of HFD-fed mice compared with those of chow-fed mice (Fig. 1I and Supplementary Fig. 1A), which was further corroborated by immunohistochemistry staining (Fig. 1J). In consistent, Sirt3 expression was reduced in mature adipocytes compared to pre-adipocytes (Fig. 1K). Taken together, these findings imply that Sirt3 might be involved in the metabolic crosstalk from adipose tissue to the liver.

Adipose-specific Sirt3 overexpression improves insulin sensitivity and mitigates hepatic steatosis in HFD-fed mice

To explore the function of Sirt3 in adipose tissue-liver crosstalk, we generated adipose-specific Sirt3-overexpressed mice using the Cre-LoxP recombination system. Briefly, Sirt3flox/flox (fl/fl) mice were mated with Adipoq-Cre mice to obtain adipose-specific Sirt3 knock-in mice (ROSA26-Sirt3; Adipoq-Cre, abbreviated as Sirt3AKI mice), which was confirmed by genotyping (Fig. 2A and Supplementary Fig. 1B). As expected, Sirt3 was highly expressed in eWAT and iWAT of Sirt3AKI mice (Fig. 2B and Supplementary Fig. 1C-F). After being fed with an HFD for a period of 10 weeks, HFD-fed Sirt3AKI mice exhibited improved glucose tolerance and enhanced insulin sensitivity (Fig. 2C, D). The histological examination and Oil Red O staining results demonstrated the attenuated lipid accumulation, smaller lipid area and diameter in the liver of Sirt3AKI mice (Fig. 2E). Meanwhile, HFD-fed Sirt3AKI mice exhibited reduced liver index, lower alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and ameliorated lipid profiles of serum and the liver (Fig. 2F-J and Supplementary Fig. 1G, H), indicating that adipose-specific overexpression of Sirt3 confers a significant protection against HFD-provoked hepatic lipid accumulation. To verify the observations, we established an adipose-specific Sirt3 loss-of-function model by administrating AAV carrying Sirt3 shRNA with the adipose-specific promoter adiponectin or control (scram) in mice. AAV-Adipoq-shSirt3 delivery achieved specific Sirt3 knockdown in adipose tissue (Supplementary Fig. 2A-D). As expected, adipose-specific Sirt3 knockdown impaired insulin sensitivity and glucose tolerance in mice under HFD feeding (Supplementary Fig. 2E, F), along with aggravated hepatic lipid accumulation and lipid profiles of serum and the liver (Supplementary Fig. 2G-K), indicating that Sirt3 loss-of-function in adipose tissue exacerbates hepatic steatosis under HFD feeding.

To clarify the impact of Sirt3 in adipocyte-hepatocyte communication, we established Sirt3-overexpressed (Sirt3OE) 3T3-L1 adipocytes (Supplementary Fig. 2L-N). Fully differentiated adipocytes are considered as the pathological state of lipid overload. P/O-stimulated hepatocytes were then co-cultured with conditional medium (CM) from fully differentiated adipocytes (Fig. 2K). Treatment of CM from mature vector adipocytes (vector-CM) significantly aggravated TG level and lipid accumulation in AML12 hepatocytes; while treatment of CM from Sirt3OE adipocytes (Sirt3OE-CM) attenuated P/O-provoked TG level and lipid accumulation in hepatocytes (Fig. 2L, M and Supplementary Fig. 2O), indicating that adipocytes release circulating factors to modulate hepatic lipid metabolism. Taken together, these results indicate that adipocytes might release circulating factors to modulate hepatic lipid metabolism, while Sirt3 acting as a critical regulator in this crosstalk.

Adipose tissue-derived sEVs are responsible for the regulation of hepatic lipid metabolism in HFD-fed Sirt3AKI mice

Adipose tissue is a major source of circulating sEVs that regulate intercellular communication and exchange cellular information26. To evaluate whether sEVs participate in adipocyte-hepatocyte crosstalk, adipocyte-derived sEVs were purified from the culture medium of fully differentiated mature adipocytes, and characterized by morphology and surface marker expression. Transmission electron microscopy (TEM) observation and nanoparticle tracking analyses (NTA) showed that both vector and Sirt3OE adipocyte-derived sEVs possessed high purity with a typical vesicle-shaped morphology (Fig. 3A and Supplementary Fig. 3A-C). The expression of sEVs-specific markers Alix, Syntenin1, TSG101, and HSP70 were readily detectable in these adipocyte-derived sEVs pellets, but not the non-sEVs and lipoprotein markers (Fig. 3B and Supplementary Fig. 3D), indicating that sEVs were well purified and no differences in the sEVs from vector and Sirt3OE adipocytes. PKH67-labeled adipocyte-derived sEVs were efficiently internalized by AML12 hepatocytes within 1-12 hours, as visualized by fluorescence microscopy (Fig. 3C and Supplementary Fig. 3E), which was further confirmed by immunofluorescence detection of sEV markers Alix and TSG101 (Supplementary Fig. 3F, G). The delivery efficiency of sEVs from vector or Sirt3OE adipocytes into hepatocytes showed no obvious difference. Subsequently, P/O-stimulated AML12 cells incubated with Sirt3OE adipocytes-derived sEVs showed decreased TG level and lipid accumulation in hepatocytes, compared with those of hepatocytes treated with vector adipocytes-derived sEVs (Fig. 3D and Supplementary Fig. 3H), suggesting that Sirt3OE adipocyte-derived sEVs attenuated lipid accumulation in hepatocytes. Conversely, Sirt3 knockdown adipocytes-derived sEVs exhibited the opposite results in hepatocytes (Supplementary Fig. 3I, J).

To verify the role of sEVs in the regulating hepatic lipid accumulation in vivo, we depleted sEVs secretion with intraperitoneal injections of GW4869 (a ceramide-induced sEVs inhibitor, 2 mg/kg, 3 times/week) in HFD-fed fl/fl mice and Sirt3AKI mice for two weeks (Fig. 3E). As expected, the administration of GW4869 recovered impairments in insulin sensitivity and glucose tolerance (Fig. 3F and Supplementary Fig. 4A-C), and alleviated hepatic lipidosis (Fig. 3G and Supplementary Fig. 4D, E) under HFD condition. In addition, GW4869 administration enhanced the liver-protective effects of adipose-specific Sirt3 overexpression, as measured by serum TG level (Fig. 3H) and hepatic TG, Total cholesterol (TC), AST and ALT levels (Fig. 3I and Supplementary Fig. 4F-H). Subsequently, sEVs-free medium from both vector and Sirt3OE adipocytes (treated with GW4869) was collected and co-cultured with P/O-stimulated hepatocytes (Fig. 3J). sEVs-free medium from vector adipocytes (VAD) exhibited a relieved lipid accumulation in hepatocytes compared with vector adipocyte-derived sEVs treated group (Fig. 3K, L and Supplementary Fig. 4I, J). Furthermore, sEVs-free medium from Sirt3OE adipocytes (S3AD) caused less lipid accumulation compared with Sirt3OE adipocytes-derived sEVs treated hepatocytes (Fig. 3K, L and Supplementary Fig. 4I, J). Besides, sEVs depletion also decreased MDA and intracellular ROS levels in P/O stimulated hepatocytes (Supplementary Fig. 4K, L). Taken together, these results suggest that adipose tissue from HFD-fed mice sends harmful sEVs to the liver, causing hepatic lipidosis, which is attenuated by Sirt3 overexpression in adipocytes.

sEVs derived from MASLD patients aggravate hepatic steatosis in HFD-fed Sirt3AKI mice

We further collected the serum sEVs from MASLD patients (MASLD-sEVs) with abnormal serum lipid profiles of the serum (Supplementary Fig. 5A-C) and healthy individuals (HI-sEVs). Characterization by TEM and NTA confirmed that both HI-sEVs and MASLD-sEVs were tiny particles (Fig. 4A) with high purity (Supplementary Fig. 5D-F). Additionally, serum-derived sEVs were enriched in markers including Syntenin-1, TSG101, and Alix (Fig. 4B), but lacked of non-sEVs and lipoprotein markers (Supplementary Fig. 5G). Furthermore, in vivo tracking analysis demonstrated efficient hepatic uptake of human-derived sEVs, further confirmed by detection of human-specific CD63 antigen in hepatocytes in vitro (Supplementary Fig. 5H-J). Incubation of MASLD-sEVs significantly increased cellular TG and TC content (Supplementary Fig. 5K, L) and exacerbated lipid accumulation (Fig. 4C) in P/O-stimulated AML12 hepatocytes. To determine whether MASLD-sEVs exacerbates hepatic lipid accumulation in vivo, equal amounts of HI-sEVs or MASLD-sEVs were labeled with PKH67 and intravenously administered to HFD-treated Sirt3AKI mice and fl/fl mice for 2 weeks, respectively (Fig. 4D). Ex vivo organ epifluorescence demonstrated a substantial concentration of HI-sEVs or MASLD-sEVs in the liver with minimal signal in heart, spleen, lungs, kidneys, or eWAT, demonstrating that both groups of human-derived sEVs possess similar biodistribution as well as retention in the liver (Fig. 4E). Oil Red O and H&E staining depicted that MASLD-sEVs treatment intensified hepatic steatosis in the HFD-induced Sirt3AKI mice (Fig. 4F). Additionally, MASLD patient-derived sEVs worsened HFD-induced glucose intolerance, systemic insulin resistance, increased liver index, compared to mice treated with HI-sEVs (Fig. 4G-I and Supplementary Fig. 5M, N). Similarly, compared with HI-sEVs treated Sirt3AKI mice, there existed higher levels of TG, TC, ALT and AST in MASLD-sEVs treated Sirt3AKI mice (Fig. 4J-M and Supplementary Fig. 5O). Collectively, these results suggest that sEVs derived from MASLD patients aggravate insulin resistance and hepatic steatosis in HFD-fed Sirt3AKI mice.

Identification of miRNAs associated with mice circulating sEVs and adipocyte-derived sEVs-guided hepatocyte reprogramming

To identify sEVs cargo mediating hepatocyte reprogramming, the miRNA expression profiles of circulating sEVs from chow (Chow-sEVs), HFD-fed mice (HFD-sEVs) and HFD-fed Sirt3AKI mice (HFD-Sirt3AKI-sEVs) were determined using Illumina HiSeq 2500 high-throughput sequencing. Using a 2-fold change and a P-value threshold of < 0.05, miRNAs were identified as significantly different. Here, 16 upregulated miRNAs were identified in HFD-sEVs compared with those in Chow-sEVs; 11 upregulated miRNAs were identified in HFD-sEVs compared with those in HFD-Sirt3AKI-sEVs (Fig. 5A-D and Supplementary Data 1-4). In addition, the miRNA expression profiles of Sirt3OE and vector adipocytes-derived sEVs were also determined. 69 miRNAs were identified as significantly different between the Sirt3OE and vector adipocyte-derived sEVs. Of these, 35 miRNAs were upregulated, and 34 miRNAs were downregulated in vector adipocyte-derived sEVs compared to Sirt3OE adipocyte-derived sEVs (Fig. 5E and Supplementary Data 5-7). Then the miRNA-sequencing results of adipocyte-derived sEVs were further validated by qPCR (Supplementary Fig. 6A). In contrast to normal mice, the abundance of miR-30a-3p in sEVs from the adipose tissue of HFD mice was significantly higher (Supplementary Fig. 6B). Importantly, miR-196a-5p, miR-30a-3p and miR-511-5p were significantly increased both in mice circulating sEVs and adipocyte-derived sEVs. We further validated these three miRNAs in MASLD patient derived-sEVs, and only miR-30a-3p showed a coherent upregulation trend (Fig. 5F, G), indicating that miR-30a-3p may function as potent modulators of inter-organ crosstalk.

To investigate the potential biological roles of the differentially expressed miRNAs, we conducted Gene Ontology (GO) annotation and enrichment analysis of miRNA profiles in circulating sEVs from chow, HFD-fed mice and HFD-fed Sirt3AKI mice. Among "biological process" (BP) terms, the target genes of differentially expressed miRNAs displayed a main enrichment in regulating autophagy and metabolic process (Supplementary Fig. 6C). This finding aligns closely with GO analysis results based on target genes of miRNAs derived from differentiated Sirt3OE and vector adipocyte-derived sEVs (Supplementary Fig. 6D, E).

Adipose-specific miR-30a-3p overexpression aggravates hepatic steatosis in HFD-fed Sirt3AKI mice

To prove that miR-30a-3p or miR-30a-3p containing sEVs are mainly produced by adipose tissue. Adiponectin, an adipokine predominantly secreted by white adipose tissue, was detected in circulating sEVs from chow, HFD and HFD-fed Sirt3AKI mice (Supplementary Fig. 6F, G). Intriguingly, the adipose-specific Dicer (miRNA-processing enzyme) knockdown mice showed significant decrease of circulating sEVs miR-30a-3p compared with AAV-scram group (Supplementary Fig. 6H, I). Consistently, adipose-specific miR-30a-3p knockdown mice exhibited a dramatic decrease in circulating sEVs miR-30a-3p (Supplementary Fig. 6J). In addition, we transplanted adipose tissue from WPT mice into adipose-specific miR-30a-3p knockdown mice (Supplementary Fig. 6K). As expected, adipose tissue transplantation restored circulating miR-30a-3p content (Supplementary Fig. 6L), which further confirmed that miR-30a-3p containing sEVs are mainly secreted by adipose tissue.

To determine whether miR-30a-3p exerts a harmful effect on lipid accumulation in the liver, AAV specifically expressing miR-30a-3p in adipose tissue (AAV-Adipoq-miR-30a-3p) or its vector control (AAV-GFP) was injected through a tail vein to HFD-fed fl/fl mice and Sirt3AKI mice, respectively (Fig. 6A). Immunofluorescence staining and qPCR revealed a higher expression of miR-30a-3p in adipose tissue (Supplementary Fig. 7A, B). Overexpression of miR-30a-3p in adipose tissue impaired insulin sensitivity in HFD-induced Sirt3AKI and fl/fl mice, as measured by GTT and ITT (Fig. 6B, C and Supplementary Fig. 7C, D). Oil Red O and H&E staining demonstrated that miR-30a-3p overexpression in adipose tissue markedly aggravated hepatic steatosis in HFD-Sirt3AKI mice (Fig. 6D and Supplementary Fig. 7E). Furthermore, AAV-Adipoq-miR-30a-3p-treated mice showed higher liver index, as well as elevated serum TC level, hepatic TG, TC, AST and ALT levels in HFD-fed fl/fl and Sirt3AKI mice compared to the corresponding AAV-GFP treated-mice (Fig. 6E-J). These results indicate that miR-30a-3p mimics aggravates hepatic lipidosis in HFD-fed mice, which partially abolished the hepato-protective role of Sirt3.

In vitro, P/O stimulated AML12 cells were treated with sEVs derived from Sirt3OE adipocytes (Sirt3OE-sEVs) or vector adipocytes (vector-sEVs) co-infected with control (NC) or miR-30a-3p mimics lentivirus (Fig. 6K). TG content and Nile red staining results depicted that lipid accumulation was increased significantly in hepatocytes treated with sEVs from vector adipocytes infected with miR-30a-3p mimics lentivirus (vector-miR30a-3p mimics-sEVs), compared with its control group (vector-NC-sEVs) (Fig. 6L, M). More importantly, overexpression of Sirt3 counteracted the impact of miR-30a-3p on lipid accumulation (Fig. 6L, M). These results elucidated that the role of Sirt3 on hepatocyte-adipocyte crosstalk is facilitated via miR-30a-3p. To validate the function of exogenous miR-30a-3p from adipocyte-derived sEVs, we knocked down endogenous miR-30a-3p in AML12 hepatocytes and treated hepatocytes with adipocyte-derived sEVs (Supplementary Fig. 7F). As expected, exogenous miR-30a-3p from adipocyte-derived sEVs led to severe lipid accumulation (Supplementary Fig. 7G, H). Collectively, these results indicated that miR-30a-3p mimics aggravates hepatic lipidosis in vivo and in vitro, and Sirt3 suppresses the release of sEVs miR-30a-3p in adipocytes to attenuate lipid accumulation in hepatocytes.

Engineered sEVs for targeted delivery of miR-30a-3p antisense oligonucleotide (ASO) attenuate hepatic steatosis in HFD-fed mice

To explore the potential clinical application of miR-30a-3p, we conducted electroporation to load HI-sEVs with miR-30a-3p ASO and functionalized with an N-acetylgalactosamine (GalNAc) ligand to enhance hepatocyte-specific delivery27, 28. TEM analysis confirmed preserved morphology and size distribution with a peak diameter of 50 nm in miR-30a-3p ASO-loaded Engineered sEVs (Engineered-sEVs) and unloaded sEVs (Unloaded-sEVs) (Supplementary Fig. 8A). Ex vivo organ epifluorescence demonstrated a substantial concentration of engineered sEVs in the liver but not in the heart, spleen, lungs, kidneys, or eWAT (Supplementary Fig. 8B, C). Subsequently, equal amounts of Engineered-sEVs and Unloaded-sEVs were intravenously administered to HFD-fed Sirt3AKI and fl/fl mice, respectively, for 4 weeks (Fig. 7A). As shown in Fig. 7B and C, the liver index and serum TG level were remarkably decreased after Engineered-sEVs treatment. It is also straightforward to observe that Engineered-sEVs treatment attenuated HFD-induced glucose intolerance and insulin resistance (Fig. 7D, E). Additionally, Engineered-sEVs treatment ameliorated lipid deposition in the liver to a certain extent (Fig. 7F). Similarly, the hepatic TG, TC, AST and ALT levels were reduced after Engineered-sEVs treatment (Fig. 7G-J), which exhibited more pronounced hepatoprotective effect in Sirt3AKI mice.

Next, the direct implication of miR-30a-3p in hepatocytes was evaluated. The inhibition of miR-30a-3p significantly decreased lipid accumulation in hepatocytes compared with the scramble group (Supplementary Fig. 8D, E). The above data suggest that engineered sEVs loaded with miR-30a-3p ASO or miR-30a-3p inhibition attenuates hepatic steatosis.

Sirt3 suppresses the transcription of miR-30a-3p through H3K56 deacetylation

We further investigated the regulation of Sirt3 in the transcription level of miR-30a-3p. Notably, miR-30a-3p transcript level was significantly decreased in Sirt3OE adipocytes and their derived-sEVs (Fig. 8A), and elevated in Sirt3 knockdown adipocytes (shSirt3) and their derived-sEVs, as well as fat-specific Sirt3 knockdown mice-derived sEVs (Fig. 8B, C and Supplementary Fig. 9A). Sirt3 functions as a transcriptional co-suppressor by facilitating the deacetylation of acetylated lysine residues at positions 9 and 56 on histone H3 (H3K9ac and H3K56ac)21. Notably, overexpression of Sirt3 obviously increased H3K56 deacetylation (Fig. 8D). On the contrary, Sirt3 deficiency obviously reduced H3K56 deacetylation (Fig. 8E), which mainly located in the nuclear (Fig. 8F). Then we found that Sirt3 exhibited dual localization in both nucleus and mitochondria (Supplementary Fig. 9B, C). And the nuclear Sirt3 could directly regulate H3K56 deacetylation (Supplementary Fig. 9D). Thus, histone modifications on the miR-30a-3p promoter in adipocytes were analyzed to elucidate the mechanism by which Sirt3 suppresses miR-30a-3p expression. Based on the deepCAGE database, we obtained the location of the transcription start site (TSS) region of pri-miR-30a-3p and then performed promoter truncation. Five primer pairs targeting the miR-30a-3p promoter were employed (Fig. 8G), and then we used ChIP-qPCR to verify the binding of acetylated H3K56 on the promoter region of mmu-miR-30a-3p (Fig. 8H). The results indicate that acetylated H3K56 physically bind at the upstream promoter region of mmu-miR-30a-3p, promoting the transcription of miR-30a-3p. Conversely, Sirt3 overexpression inhibits the binding between acetylated H3K56 and promoter region of miR-30a-3p, resulting in the suppression of miR-30a-3p transcription.

Then, ChIP-sequencing, a genome-wide immune sequencing assay, revealed slightly elevated H3K56 deacetylation peaks near the TSS regions in Sirt3OE adipocytes compared to vector adipocytes (Fig. 8I and Supplementary Data 8-12). In GEO and KEGG analyses, genes marked by acetylated H3K56 were found to be involved in regulating gene expression, RNA and protein metabolic processes, protein modification, and positive regulation of autophagy (Supplementary Fig. 9E, F and Supplementary Data 13-14). To illustrate the coverage of ChIP-sequencing signal profiles of the different histone modifications across the gene body, we found that acetylated H3K56 peaks were validated at the miR-30a promoters of the selected genes, and slight increases in acetylated H3K56 peaks were observed in Sirt3OE adipocytes (Fig. 8J). Collectively, these results demonstrate that Sirt3 overexpression inhibits the transcription of miR-30a-3p in adipocytes through H3K56 deacetylation.

miR-30a-3p represses autophagy through translational inhibition of Becn1 mRNA

The predicted target genes of differentially expressed miRNAs (from miRNA sequencing) were significantly enriched in Gene Ontology terms or pathways related to autophagy (Supplementary Fig. 6C). Thus, we performed bioinformatics analysis (TargetScan algorithm) and RNA sequencing following Biotin-miR-30a-3p pull-down to screen the autophagy-related target genes by miR-30a-3p. Venn diagram indicated that 42 overlapping genes were identified between autophagy-related pathway genes from RNA sequencing and predicted miR-30a-3p targets (Supplementary Fig. 10A, B and Supplementary Data 15-16). Among the top five genes (Pik3c3, Ctsl, Tax1bp1, Hmgb1, Becn1) (Supplementary Fig. 10C), Beclin1 is a signaling hub in the context of autophagy29 and was chosen for subsequent study. Specifically, miR-30a was found to target the 3'-UTR of Becn1 mRNA at one binding site of base pair 201th to 232th (Fig. 9A). Next, the intact 3'-UTR of Becn1 mRNA was cloned into a luciferase reporter plasmid, and transfected into the AML12 cell lines. miR-30a-3p mimics greatly reduced the luciferase activity of the 3'-UTR reporter of Becn1, while the activity of Becn1-3'-UTR-mutated luciferase reporter was not affected by the miRNA-30a-3p overexpression, demonstrating that miRNA-30a-3p could directly interact with 3'-UTR of Becn1 (Fig. 9B). This direct binding was further verified by Biotin-miR-30a-3p pull-down assay followed by qRT-PCR (Fig. 9C).

Furthermore, we evaluated the effects of Beclin1 in lipid accumulation and autophagy flux. The direct manipulation of Beclin1 in AML12 cells was conducted. As expected, gain of Becn1 expression alleviated lipid accumulation and TG content in P/O-treated hepatocytes, whereas Becn1 knockdown increased lipid accumulation and TG content (Supplementary Fig. 10D, E). To further confirm the effect of Beclin1 on autophagic flux, Beclin1OE and vector cells underwent transfection with an mRFP-GFP-LC3 (microtubule-associated protein 1 light chain 3) tandem construct. More red puncta were detected in Beclin1OE cells, indicating autophagic flux was activated in Beclin1OE cells without disrupting the fusion of autophagosome and lysosome (Supplementary Fig. 10F). To explore whether autophagy was involved in the effects of adipocytes-derived sEVs on the recipient hepatocytes, we detected autophagic level in hepatocytes following treatment with adipocytes-derived sEVs. Expectedly, LC3-II/ LC3-I and Beclin-1 expression was heightened, and p62 expression was suppressed on Sirt3OE adipocytes-derived sEVs treated hepatocytes, compared with those of vector adipocytes-derived sEVs treated cells (Fig. 9D), suggesting the activation of autophagy in Sirt3OE adipocytes-derived sEVs-treated hepatocytes, which was further confirmed in mCherry-GFP-LC3 fluorescence results (Fig. 9E, F). Intriguingly, sEVs inhibition by GW4869 treatment in adipocytes further enhance autophagy in hepatocytes, manifesting as increased ATG5, LC3-II/LC3-I, and decreased p62 expression, and more red puncta (Fig. 9G, H). miR-30a-3p mimics blocked Sirt3-induced autophagic flux with inhibited autophagosome-lysosome fusion (Fig. 9I, J), consistent with observations in vivo (Supplementary Fig. 11A) and MASLD-sEVs treatment (Supplementary Fig. 11B). Conversely, knockdown of miR-30a-3p reversed the P/O-induced blockade of autophagic flux. (Supplementary Fig. 11C, D). As expected, when knocking down endogenous miR-30a-3p in AML12 hepatocytes and treated hepatocytes with adipocyte-derived sEVs, the exogenous miR-30a-3p from adipocyte-derived sEVs led to blocked autophagic flux (Supplementary Fig. 11E). These data suggest that Sirt3OE adipocyte-secreted sEVs alleviate lipotoxicity in hepatocytes by suppressing the translation of Beclin1 mRNA through miR-30a-3p. Additionally, RT2 PCR Profiler arrays were used to assess the involvement of miR-30a-3p in metabolic regulation. The results indicated that miR-30a-3p might also play a role in hepatic lipid metabolism through some lipid metabolism-related genes (Supplementary Fig. 11F-I and Supplementary Data 17).

Methods
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Rabbit monoclonal anti-Sirt3 Cell Signaling Cat# 5490
Technology
Rabbit monoclonal anti-p62 Cell Signaling Cat# #23214
Technology
Mouse monoclonal anti-ATG5 Proteintech Cat#66744-1-Ig
Rabbit monoclonal anti-Beclin1 Cell Signaling Technology Cat#3495
Rabbit monoclonal anti-LC3A/B Cell Signaling Technology Cat#12741
Mouse monoclonal anti-HSP70 Santa Cruz Cat#sc-32239
Mouse monoclonal anti-syntenin-1 Santa Cruz Cat#sc-515538
Mouse monoclonal anti-Alix Santa Cruz Cat#sc-53540
Mouse monoclonal anti-tsg101 Santa Cruz Cat#sc-7964
Rabbit polyclonal anti-acetyl Epigentek Cat#A-4026
H3K56
Rabbit polyclonal anti-Calnexin Proteintech Cat# 10427-2-AP
Rabbit Recombinant anti-Apoe Proteintech Cat# 83728-4-RR
Rabbit polyclonal anti-ARF6 Proteintech Cat# 20225-1-AP
Rabbit monoclonal anti-CD63 Proteintech Cat# 25682-1-AP
Mouse monoclonal anti-CD63 Santa Cruz Cat#sc-5275
Rabbit polyclonal anti-adiponectin Proteintech Cat# 21613-1-AP
Biological samples
Healthy adult and MASLD patient Affiliated Hospital of N/A
serum Guizhou Medical
University
Chemicals, peptides, and recombinant proteins
GW4869 MedChemExpress Cat#HY-19363/CS-6865
Recombinant RNase Inhibitor Takara 2313A
Critical commercial assays
Hieff® Quick exosome isolation YEASEN 41202ES30/41201ES50
kit
RT2 Profiler PCR Array Qiagen PAMM-007ZA
TG, T-CHO, LDL Commercial Nanjing Jiancheng G8090
assay kits
Haematoxylin Eosin Phygene PH0516
Oil red O staining kit Nanjing Jiancheng #D027
Experimental models: Cell lines
3T3-L1 preadipocytes Cells ATCC CRL-11268
AML12 Cells ATCC N/A
Experimental models: Organisms/strains
Mouse: Sirt3AKI Cyagen

Establishment of adipose-specific Sirt3 overexpression mice
To develop conditional targeted overexpression offspring, fertilized mouse eggs were co-injected with the gRNA sequence (CTCCAGTCTTTCTAGAAGATGGG) targeting the mouse ROSA26 gene, the donor vector carrying the CAG promoter-loxP-PGK-Neo-6*SV40 pA-loxP-Kozak-Mouse Sirt3 CDS-rBG pA cassette, and Cas9 mRNA. For adipose tissue-specific Sirt3 overexpression mice, tissue-specific Adipoq-Cre mice were crossed with Sirt3flox/- mice (Cyagen, Suzhou, China) to generate Sirt3flox/flox-Adipoq-Cre mice (denoted Sirt3AKI) and Sirt3flox/flox littermate control mice (denoted fl/fl). Mice were exposed to light-dark cycles for 12 h at 22 ± 2 ˚C with ad libitum access to food and water. Genotypes were performed by PCR followed by sequence analysis. All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Guizhou Medical University (Document No. 2100060) and conducted in compliance with the authorized guidelines and regulations.

Animal experimental procedures
Male Adipoq-Cre+/Sirt3flox/flox mice (Sirt3AKI mice) and Adipoq-Cre-/Sirt3flox/flox mice (fl/fl mice) aged 8 weeks were fed with 60% HFD for 10 weeks, monitoring their body weight once a week. The MASLD-sEVs were adoptively injected into recipient mice via the tail vein (15 μg per injection, twice a week) for 2 weeks, starting at 18 weeks old, and phosphate-buffered saline (PBS, Solarbio) was injected as a control. To inhibit sEVs biogenesis, mice were administered the neutral sphingomyelinase-2 (nSMase2) inhibitor GW4869 (2 mg/kg) intraperitoneally three times a week during weeks 10-12. For AAV transduction, AAV-Adipoq-miR-30a-3p (AAV2/8, 1.5 × 1012 vg/mL) was purified by OBiO Biotechnology (Shanghai, China) and utilized to overexpress miR-30a-3p in adipose tissue. Tissues were harvested 4 weeks post-transduction for immunofluorescence staining, RNA isolation, or protein extraction. For Engineered-sEVs treatment, electroporation was conducted to load HI-sEVs with miR-30a-3p ASO and functionalized with GalNAc ligand to enhance hepatocyte-specific delivery. Then mice were injected with Engineered-sEVs through tail vein for 4 weeks (15 μg per injection, twice a week). Engineered sEV biodistribution was tracked via tail vein injection using an in vivo imaging system. Tissues were harvested 4 weeks post-transduction for Nile red, H&E and Oil red O staining or lipid profile index.

Animal treatment
Adeno-associated virus (AAV) vector with the adipose-specific promoter Adipoq-mediated knockdown of mouse Sirt3 (AAV-Adipoq-shSirt3) , mouse Dicer (AAV-Adipoq-shDicer), mouse miR-30a-3p (AAV-Adipoq-miR-30a-3p KDI), and scrambled control (AAV-Scram) were constructed, amplified, and purified by OBiO biotechnology (Shanghai, China). A total of 50 μL of 3×1011 Vg/ml of each AAV diluted in PBS was injected by tail vein injection. Mice were monitored for changes in glucose tolerance and insulin resistance, then mice were sacrificed and tissues were dissected for further analysis.

Cell culture and differentiation
AML12 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified eagle medium (DMEM; Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher) and ITS-G (5 mg/mL insulin, 5 mg/mL transferrin, and 5 μg/mL selenous acid, Peiyuan Biotechnology, Shanghai, China) at 37 °C in 5% CO2. Both oleic acid (OA) and palmitic acid (PA) were entirely solubilized in 75% (v/v) ethanol at 55 °C, subsequently diluted with 1% (w/v) FFA-free bovine serum albumin (Sigma-Aldrich, A8806, St. Louis, MO, USA) in DMEM to final concentrations of 500 μM OA and 250 μM PA., respectively. The solutions were incubated in a shaker for 2 h and sterilized with 0.22 μm filters to produce a P/O solution. 3T3-L1 preadipocytes (ATCC) were expanded in DMEM containing 10% calf serum (CS, Thermo-Fisher) and 1% penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin, Thermo-Fisher). 3T3-L1 cells were differentiated into adipocytes, as documented previously23. Briefly, 3T3-L1 preadipocytes differentiation was induced with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, and 5 μg/mL insulin (Sigma-Aldrich) in DMEM containing 10% FBS for 48 h. Cells were subsequently treated with DMEM containing 10% FBS and 5 μg/mL insulin for 6 days. Media were changed daily. 3T3-L1 cell differentiation was validated by Oil-Red O staining, as previously outlined. Mature adipocytes were further rinsed twice with DMEM and cultured in serum-free DMEM with 0.2% endotoxin-free and FA-free BSA for 48 h to collect sEVs.

Generation of Sirt3 overexpression cell line
The pcDNA3.1-Sirt3-Flag plasmid was generated as described previously24. Briefly, 3T3-L1 cells (4 × 105) were seeded in 35 mm plates. After 24 h, cells were transfected with 10 μg plasmids (pcDNA3.1 or pcDNA3.1-Sirt3-Flag) using Lipofectamine 2000. Following 24 h transfection, 800 μg/ml G418 (Sigma-Aldrich) was added to select positive cells for 12 days with medium replacement every 24 h.

sEVs isolation and characterization
The 3T3-L1 adipocyte culture media were subjected to centrifugation at 2,000 g for 30 min to remove debris and dead cells, then filtered through a 0.22 μm filter. The cell-free culture media were incubated with 0.5 volumes of Total Exosome Isolation Reagent (Invitrogen) overnight at 4 °C. After centrifugation at 10,000 g at 4 °C for 1 h, the sEVs-containing pellet was collected and resuspended in PBS. sEVs particle size and concentration were measured using NanoSight analysis (Malvern Instruments). sEVs characterization was performed by transmission electron microscopy (TEM), and sEVs-specific markers (HSP70, TSG101, syntenin-1), non-sEVs (Calnexin, ARF6) and lipoprotein (ApoE) markers expression were determined using Western blotting.

Transmission electron microscopy
sEVs were preserved using 2% paraformaldehyde and deposited onto Formvar and carbon-coated copper grids, which were subsequently placed on 2 % gelatin for 20 min at 37 °C. After washing with 0.15 M glycine/PBS and blocking with 1% cold-water fish-skin gelatin, the grids were examined with a JEOL 1200EX II TEM (JEOL, Tokyo, Japan), and images were captured using a Gatan digital camera (Gatan, Pleasanton, CA, USA).

Treatment of hepatocytes with adipocyte-derived sEVs
sEVs intensity was quantified using a BCA protein assay kit (Thermo Fisher). AML12 hepatocytes were treated with adipocyte-derived sEVs (20 μg/mL) for 24 h. sEVs were then fluorescently labeled using the PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich) to assess their uptake by hepatocytes. After incubation for 1 or 12 h, fluorescence signals were measured using confocal microscopy (Olympus, Tokyo, Japan).

miRNA mimic or shRNA transfection
The recombinant lentivirus expressing miR-30a-3p was obtained from Igebiotech (Guangzhou, China). The miRNA precursor sequence is as follows: GCGACTGTAAACATCCTCGACTGGAAGCTGTGAAGCCACAAATGGGCTTTCAGTCGGATGTTTGCAGCTGC. miR-30a-3p shRNA, Beclin1 shRNA, and pcDNA3.1-Beclin1-Flag (Igebiotech, Guangzhou, China) were transfected into AML12 hepatocytes using Lipofectamine 2000 reagent (Thermo Fisher). Furthermore, Sirt3 shRNA, Sirt3 M1-M3 construct were transfected into 3T3-L1 adipocytes using Lipofectamine 2000 reagent. Oligonucleotide sequences of the plasmid were provided in Table S1.

miRNA target prediction and 3′-UTR luciferase reporter assay
Putative targets of miR-30a-3p were predicted using the TargetScan algorithm. The Beclin1 3′-UTR luciferase reporter plasmid (pRL-Beclin1) was provided by Creative Biogene (Shirley, NY, USA). AML12 hepatocytes were co-transfected with the Beclin1 3′-UTR reporter plasmid and recombinant lentivirus encoding miR-30a-3p or miR-146-5p using Lipofectamine 2000, respectively. At 48 h post-transfection, cells were collected and analyzed via a dual-luciferase reporter assay (Promega, Madison, WI, USA) according to the protocols.

RNA extraction and quantitative reverse transcription PCR (qRT-PCR)
RNA was quantified with a Nanodrop (Thermo Fisher) and reverse transcribed to cDNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The cDNA was subsequently subjected to TaqMan® quantitative PCR on a Stratagene 3005P Real-Time PCR system, employing miRNA-specific primers from Applied Biosystems. The Ct values for miR expression were normalized using U6 snRNA. The miR expression was determined utilizing MxPro software (Version 4.00, Stratagene). All reactions were conducted in triplicate. The primers for qRT-PCR were listed in Table S2.

Human adipose tissue samples
Human adipose tissue samples were collected as described in Table S3. The investigations were conducted in accordance with the principles of the Declaration of Helsinki and were approved by the Research Ethics Committee of Guizhou Medical University (Document No. 2023049).

Biotin-labeled miR30a-3p pull-down assay
P/O-treated AML12 cells were transfected with biotin-labeled miR30a-3p (50 nmol/L), and then cells were harvested 48 h later with lysis buffer containing RNase inhibitor. Simultaneously, streptavidin-Dyna beads (Invitrogen) were incubated with rotation at 4 °C for 2 h. Then the beads were washed with splitting buffer and resuspended with lysis buffer. Sample lysates were mixed with precoated beads and incubated overnight at 4 °C on a rotator. Beads were then pelleted to remove unbound materials and washed six times with ice-cold lysis buffer. The bound beads were collected for qRT-PCR and RNA sequencing. The probe sequences were shown as below: miR-30a-3p junction probe: 5'-CUUUCAGUCGGAUGUUUGCAGC-3'; NC probe (brought from Igebiotech, Guangzhou, China) 5'-GACUUGAAGCCUAGCCCGAUCG-3'.

RT2 Profiler PCR Array
AM12 cells treated with P/O were then underwent biotin-miRNA pull-down assay. Total RNA was extracted with Trizol and was quantified by NanoDrop Spectrophotometer (NanoDrop Technologies). 1μg total RNA was reverse transcribed using RT2 First Strand Kit (Qiagen, USA) and analyzed with mouse RT2 profiler PCR array for fatty acid metabolism (PAMM-007Z, Qiagen, USA) following the manufacturer’s instructions. The relative gene expression was calculated using Qiagen web-based PCR Array Data Analysis Software and normalized with the average of 5 housekeeping genes contained in the array.

miRNA library construction and sequencing
sEVs-extracted total RNAs were utilized for miRNAs library construction and sequencing (Majorbio Group, Shanghai, China). The separation of total RNA samples was performed on a 15% Tris-borate-EDTA (TBE) polyacrylamide gel (Invitrogen), and short RNAs measuring 18–30 nt were utilized for library creation. miRNAs were subjected to reverse transcription and subsequent amplification via PCR. Employing the Illumina HiSeq 2500 platform, the PCR products were sequenced.

Gene ontology (GO) analyses of differentially expressed genes
The GO and pathway enrichment analyses were performed on miR sequencing data using the online DAVID 6.8 bioinformatics resources.

Preparation of miR-30a-3p ASO-loaded engineered sEVs
To load miR-30a-3p antisense oligonucleotide (ASO), 1000 μg of sEVs isolated from the serum of healthy individuals were mixed with 1 nM ASO in 200 μL sEVs-Transit Peptide buffer and then added 100 μL reaction buffer according to the manufacturer’s protocol for the ExoLoad® assay kit (ECHO BIOTECH). The mixture was then incubated at 37 °C in dark conditions for 2 h with continuous shaking at 150 rpm. Afterward, free ASO and reaction reagents were washed twice followed by centrifugation (4,000 g, 20 min). ASO-loaded sEVs were conjugated with 50 μM DSPE-PEG-Galnac at room temperature for 2 h with gentle agitation.

3T3-L1 adipocytes and AML12 hepatocytes co-culture model
The co-culture of 3T3-L1 adipocytes and AML12 hepatocytes was performed in accordance with established protocol53. The conditioned medium (CM) of fully differentiated 3T3-L1 adipocytes were collected for 24 h. Then the CM was cleared by centrifugation at 400 g for 10 min. AML12 cells pretreated with P/O solution for a duration of 24 h were incubated with the adipocyte CM or the DMEM for 24 h. Culture supernatants were assayed for metabolic parameters using commercial kits.

Subcellular fractionation
Following treatment, cells were washed with cold PBS and harvested. Cytoplasmic and mitochondrial fractions were isolated using a Cell Mitochondria Isolation Kit (Beyotime, #C3601, Shanghai, China). Subsequently, cytoplasmic and nuclear fractions were separated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, #P0028, Shanghai, China) in accordance with the manufacturer’s instructions.

Immunoblotting
AML12 cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a 1% protease inhibitor mixture and 1% phenylmethanesulfonyl fluoride (Sigma-Aldrich). Protein concentration was quantitated with a BCA protein assay kit (Thermo Fisher). An equivalent amount of protein (20‒30 μg) was fractionated via 5%–12% SDS-PAGE and transferred to PVDF membranes. After a 2-h blocking period with 5% non-fat milk at room temperature, the membranes were subjected to overnight incubation with specified primary antibodies at 4 ºC followed by 1 h incubation with corresponding secondary antibodies. Protein bands were performed with a SuperSignal West Femto Maximum Sensitivity Substrate kit，imaged with a ChemiDoc MP Imaging System (Bio-Rad) and quantified via Image Lab 5.1 (Bio-Rad) .

Immunofluorescence staining and Ad-mCherry-GFP-LC3 transient infection
AML12 cells cultured on collagen-coated glass coverslips were exposed to infection with a lentivirus encoding GFP-LC3 for 72 h prior to different treatment protocols. After 20 min fixation in 4% paraformaldehyde, cells were rinsed with PBS (pH 7.4) and subsequently permeabilized by 0.5% Triton X-100 for 10 min at RT. Samples were incubated overnight at 4 ºC with corresponding primary antibodies. After PBS washes, Texas Red-conjugated anti-rabbit IgG secondary antibody (1:1000) was applied for 1 h at room temperature. Changes in autophagic flux and lipid droplet morphology were evaluated using confocal microscopy (Olympus, Tokyo, Japan).

Nile red, BODIPY and Oil red O staining
Nile red and BODIPY staining was performed according to previously established protocols54 by fixing and staining AML12 cells with Nile red (1 μg/mL) and BODIPY (5 μM), respectively. Intracellular Nile red or BODIPY-stained lipid droplets were observed by fluorescence microscopy and quantitated by Image J software. For Oil Red O staining, the samples were rinsed once with PBS, fixed with 4% paraformaldehyde for 20 min, and stained using the Oil Red O staining kit (#D027; Jiancheng Biotech, China) following the manufacturer’s protocol. The samples were then rinsed with PBS, followed by 60% isopropanol for 30 s, and observed under an optical microscope (Olympus, Japan).

Histological staining
The specimens were preserved in 4% paraformaldehyde for 48 h, followed by sequential dehydration in a series of ethanol solutions. Dehydrated tissues were embedded in paraffin. Sections of 3 μm thickness were obtained using a microtome and subsequently stained with hematoxylin for 3 min and eosin for 1 min at RT. Histological analysis was performed under an optical microscope (Olympus, Japan).

Immunohistological chemistry
3 μm-sectioned paraffin-embedded liver tissues were deparaffinised and rehydrated. Tissue sections were washed with PBS and subsequently boiled in 10 mM citrate buffer (pH 6.0) for 8 min for antigen retrieval. Sections were blocked with 10% goat serum at RT for 1 h, then incubated at 4 °C with primary antibodies overnight. The samples were treated with HRP-conjugated secondary antibodies and then incubated with DAB for color development.

Glucose tolerance tests (GTT), insulin tolerance tests (ITT), and lipid profile determination
Mice were subjected to overnight fasting to conduct GTT. After assessing basal blood glucose levels, mice were intraperitoneally injected with 1.5 g/kg glucose, followed by measuring the blood glucose levels at 30, 60, 90, and 120 min using blood collected from the tail vein. Mice were subjected to a 4-h fasting period to conduct ITT. After measuring the basal blood glucose levels, mice were intraperitoneally injected with insulin (0.75 U/kg, Sigma-Aldrich), followed by measuring the blood glucose levels at 30, 60, 90, and 120 min utilizing blood collected from the tail vein. TG, T-CHO, Low-density lipoprotein (LDL), and MDA levels were measured via commercial assay kits (Nanjing Jiancheng, Nanjing, China).

Co-immunoprecipitation experiment (Co-IP)
RIPA lysis buffer was used to lyse cells on ice for 10 min. After incubating the supernatants with protein A/G agarose beads for 4 h, the supernatants were then incubated overnight at 4 °C with Sirt3 antibody. Following PBS washes, the bound proteins were eluted and heated in 2× loading buffer before Western blotting analysis.

Chromatin immunoprecipitation (ChIP) assay and ChIP sequencing
A ChIP assay was conducted utilizing a kit from Cell Signaling Technology, USA (catalog number 91820S). 3T3-L1 adipocytes were fixed using 4% formaldehyde and subsequently harvested by scraping for lysis. Cross-linked chromatin samples were extracted using nuclease digestion (37 °C for 20 min) and sonication (20% power for 5 s, followed by 10 s, repeated 30 times in total). Soluble fragmented chromatin was immunoprecipitated using H3K56ac antibody as well as Protein A/G. The purified DNA was obtained through incubation with proteinase K and RNase A. Using the qRT-PCR method, ChIP DNA was amplified using miR-30a-3p promoter primers (Table S1). Values represent the percentage of input using the threshold cycle method (2−ΔΔCT).

For ChIP-sequencing, fragmented soluble chromatin was immunoprecipitated for 48 h at 4 °C with H3K56ac antibody and incubated with Protein A/G for 2 h. The beads were then resuspended in ChIP elution buffer, and cross-link reversal was performed at 65 °C for 30 min. The enriched DNA was isolated by incubation with proteinase K and RNase A. After end-repair and A-tailing, the DNA was treated with sequencing adapters and amplified by PCR to obtain the sequencing library. The library was quantified with the help of Qubit2.0, and the MGISEQ-T7 sequencer was used to sequence the various libraries in accordance with the effective concentration as well as the target data volume. After aligning the reads to the reference mouse genome, the ChIP-seq peaks were identified using MACS version 1.4.2.

Statistical analysis
The data are reported as the mean ± standard error of the mean (SEM) based on a minimum of three separate experiments. The analysis was performed through GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) for the purpose of determining the significance of differences between groups. A one-way ANOVA was carried out, after which Tukey's post hoc test was performed through SPSS software version 16.0. P < 0.05 was considered to indicate a significant difference.

Statistics and Reproducibility
No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment unless stated otherwise below. Reproducibility: All experiments were performed at least three independent times. For all representative images (micrographs, Western blots, gels), similar results were obtained in each independent experiment. For quantitative assays (e.g., qPCR), the specific number of biological replicates (n) and independent experiments are indicated in the corresponding Fig. legend. Sample sizes were chosen based on standard practices in the field to ensure adequate statistical power.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Study summary:\nThis manuscript focuses on an adipose-liver crosstalk via exosome containing microRNA miR-30a. Authors first showed that\novernutrition impaired SIRT3 while overexpressing SIRT3 ameliorates adipose and liver metabolic dysfunction. Authors\nthen examined exosome composition regulated by SIRT3 from cultured adipocytes and identified miR-30a as a critical factor\nto induce metabolic dysregulation of liver, supported by efficacy study of miR-30a-ASO. Mechanistically, SIRT3 suppresses\nmiR-30a expression through H3K56ac deacetylation in adipocytes. MiR-30a also impairs autophagy by targeting beclin-1.\nGeneral comments:\nThe manuscript is well-written, logically structured, and builds coherently on the authors’ prior work related to SIRT3. The\nhypothesis and major objectives of the manuscript is a logical extension of authors’ previous work on SIRT3. However, while\nthe data are comprehensive, the study’s novelty and mechanistic depth require strengthening to fully support its central\nclaims.\nThe roles of SIRT3 in metabolic regulation, H3K56ac in epigenetic control, and miR-30a/Beclin-1 in autophagy are well-\ndocumented. The study should clarify how it advances beyond existing literature, particularly in linking these pathways via\nexosome-mediated crosstalk, using more robust methods to examine a tissue-specific SIRT3 role and the selectivity of\nexosome targeting liver.\nKey conclusions rely on SIRT3 overexpression without complementary loss-of-function data (e.g., adipose-selective Sirt3\nKO/KD/mutant). Overexpression may induce non-physiological artifacts, especially for subcellular localization (nuclear vs.\nmitochondrial SIRT3). This is also documented in previous literature.\nSpecific comments:\nIn Fig 1 authors showed decrease of SIRT3 in visceral fat. There is no data on SIRT3 expression in other fat depots and\ncomparison between chow and HFD. There is no clear link between Fig 1 and Fig 2. In Fig 2 there is no SIRT3 mRNA or\nprotein expression in fat depots either, along with adipose tissue morphology. Authors also need to measure circulating free\nfatty acid which could be causing the liver phenotype.\nThe miR profile results were based on OE in adipocytes. Authors also need to show miR in circulating exosome from mice\n(chow, HFD, HFD tg mice), because there are changes of several miRNA not just miR-30a. It is also difficult to prove that\nmiR-30a or miR-30a containing exosome is not produced by other cells including hepatocytes.\nIn Fig 7, for the exosome-miR-30a-ASO delivery, authors did not show the in vivo bio-distribution/uptake of exosome in\nvarious tissues.\nIn Fig 8, the reduction of H3K56ac was not clear in the OE group.\nIn Fig 8, the nuclear vs mitochondrial SIRT3 is still controversial, authors did not include any data on the sub-cellular\nexpression of SIRT3, or any difference in size. Using an overexpression system may induce artifact in expression and may\nnot reveal the physiological changes in vivo. The H3K56ac change could be indirect and not due to a nuclear SIRT3. Loss\nof function approach in vitro would be very useful to support the role of SIRT3 especially to support that nuclear SIRT3\ndirectly regulates H3K56ac.", "reviewer_2": "In this study, the authors investigate the effects of adipose tissue transplantation from high-fat diet (HFD)-fed mice into lean mice on hepatic steatosis. They show that overexpression of SIRT3 in adipose tissue alleviates steatosis through a mechanism mediated by exosomes. These findings are validated in vitro, and through a series of experiments, the authors demonstrate that miR-30a-3p—a microRNA found to be downregulated in exosomes derived from SIRT3-overexpressing adipocytes—is a key mediator of hepatic steatosis. By introducing an antagomir against miR-30a-3p, they show improvements in both insulin sensitivity and hepatic steatosis. Additionally, using a dual-luciferase reporter assay, they confirm Beclin1 as a direct target of miR-30a-3p.\nOverall comments:\nThe authors provide compelling evidence that exosomes from HFD-fed rodents or humans with NAFLD can promote hepatic lipid accumulation. A combination of in vitro and in vivo experiments supports this conclusion, including clear evidence of increased lipid accumulation in hepatocytes treated with exosomes from NAFLD patients. AAV-mediated delivery of miR-30a-3p to the liver promotes hepatic lipid accumulation, while liver-targeted delivery of an antisense oligonucleotide (ASO) against miR-30a-3p inhibits this process.\nThe mechanistic link is well supported, with clear demonstration that miR-30a-3p targets Beclin1 and thereby inhibits autophagy. Overall, the study is rigorous; however, several aspects require clarification or further investigation to fully interpret the findings. Notably, the delivery of miR-30a-3p mimics or inhibitors has a more modest effect than exosomes derived from HFD or SIRT3-overexpressing mice, suggesting that miR-30a-3p does not act alone. Indeed, the small RNA-seq data reveal other downregulated miRNAs that may also contribute to hepatic steatosis. Furthermore, miR-30a-3p likely targets additional genes involved in autophagy or lipid metabolism.\nSpecific comments:\n1. The most recent MISEV guidelines recommend against using the term \"exosomes\" as a synonym for extracellular vesicles, even when classical exosomal markers are present. Please revise the manuscript to use terminology consistent with current guidelines (e.g., \"small extracellular vesicles\" or \"sEVs\").\n2. Please include negative markers (e.g., non-exosomal or lipoprotein markers) to assess the purity of exosome preparations, especially since the exosomes used in this study are derived from blood/plasma, which contains abundant lipoproteins.\n3. Hepatic uptake of exosomes may be confounded by PKH dye degradation and its non-specific incorporation into recipient cell membranes. Please provide clearer confocal images showing subcellular localization of the exosomes, and quantify the delivery of specific cargo (e.g., miR-30a), which is currently lacking in the manuscript. An alternative approach is to use human-derived exosomes (as in Figure 4) labeled with human-specific antigens (e.g., CD63), which can be distinguished from mouse CD63 using specific antibodies.\n4. Please report the number of exosomes per μg of protein to enable comparison with other studies.\n5. Figure 3E–K: GW4869 is a sphingomyelinase inhibitor and can independently alter hepatic lipid metabolism, beyond its effects on exosome secretion. Please include a GW4869-only control group in the in vitro experiments (e.g., AML12 cells treated with lipids) to determine the extent to which GW4869 itself contributes to changes in lipid accumulation.\n6. Figure 6G: Please use a color scheme consistent with that used in Figure 6D (SIRT3-OE data), as it was difficult to interpret whether the miRNAs were upregulated by HFD or SIRT3 overexpression.\n7. In addition to miR-30a-3p, miR-146b-5p was also found to be downregulated by SIRT3OE. Please assess whether miR-146b-5p targets Beclin1 using a dual-luciferase reporter assay, as was done for miR-30a-3p. Additionally, perform lipid accumulation assays in AML12 cells transfected with a miR-146b-5p mimic, as well as miR-214-3p, to determine whether these miRNAs exert similar or opposing effects on hepatic lipid accumulation.\n8. While there is strong evidence that miR-30a-3p targets Beclin1, other genes involved in autophagy or lipid accumulation are also likely targets. An unbiased approach—such as bio-miR-30a-3p co-immunoprecipitation with target mRNAs followed by RNA-seq—would help identify additional mRNA targets in the context of hepatic lipid accumulation (e.g., in AML12 cells treated with palmitate/oleate).", "reviewer_3": "The authors present a rich body of work connecting adipocyte extracellular vesicles to pathological and protective processes in liver steatosis. The experimental design often displays outstanding innovation (e.g. adipose transplantation, making liver-targeting Exos, systemic exo inhibition, cross-species exo administration) to provide evidence to support a multi-step mechanism involving Exos, microRNAs and histone modifications across two tissues. Combination of human, mouse and cell-culture experiments is also a strength of the study, with strong agreement in results between systems.\nThe overall hypothesis appears to be well supported by the experiments presented, with the caveat that a couple of major omissions or errors in the description of some key methods brings some doubt on some conclusions.\nMajor points:\n1. There is no evidence presented that the SIRT3Adipo-Tg mice overexpress Sirt3 in their adipose tissue. It is essential to present an adipose-tissue Sirt3 western, immunostaining and/or RT-qPCR.\n2. It is puzzling how deletion of exons 3 and 4 could lead to overexpression (Figure 2A). I suspect that the loxP sites in figure 2A are wrong and the actual mode of overexpression involves bringing the ROSA26 promoter in close proximity to the Sirt3 CDS in the transgene cassette as described in the methods -CAG promoter-loxP852 PGK-Neo-6*SV40 pA-loxP-Kozak-Mouse Sirt3 CDS-rBG pA cassette. Is that the case?\nThe figure 2 diagram is especially confusing as the ROSA26 locus is the target site, this is therefore a transgenic knock-in of Sirt3 so I would expect the SIRT3Adipo-Tg mice to have less Sirt3 (due to the deletion of exons 3 and 4) than the SIRT3NTg mice which don’t have Cre so have an intact Sirt3 transgene. However, this is the opposite to what is described in the manuscript – i.e. that SIRT3Adipo-Tg mice have higher expression of Sirt3. This crucial point needs clarification.\n3. The nomenclature for the transgenic Sirt3 overexpression mice needs changing throughout the manuscript. The current naming indicates that the endogenous Sirt3 locus is edited when in fact it is a Sirt3 transgene that is altered by the loxP deletion. Please consult https://www.informatics.jax.org/mgihome/nomen/index.shtml\n4. I cannot find any description in the methods for how the SIRT3OE adipocytes were generated. Are they derived from the SIRT3Adipo-Tg mice or made some other way? This lack of explanation makes critique of the manuscript very difficult as these cells, cultured medium and Exos from these cells are central to so many of the experiments.\n5. As with the transgenic mouse, the evidence for Sirt3 expression changes in SIRT3OE adipocytes is not presented. Or is it presented in Figure 8A? But the origins of these ChIP-seq cells is not described either.\n6. For the DAVID analysis in Figure 5G please state exactly what was input into DAVID. Was it all differentially expressed miRs between SIRT3OE and vector, or a subset? This is important for evaluating whether an unbiased investigation into the main biological effects of the SIRT3OE EVs was performed. That in turn is needed for justification of the subsequent focus on autophagy in the study.\n7. I cannot see any evidence that Sirt3 overexpression decreases miR-30a transcript levels (Figure 8 ChIP experiments). There is evidence that there is less miR-30a transcript in Exos from these cells (Figure 5E) but as figure 8 is about transcription of the microRNA, there needs to be evidence that transcription itself is affected in the cells.\n8. It is unclear what Figure 8C displays. Can you please expand on what is being presented?\n9. The importance of Beclin in the proposed mechanism (Figure 9) would be strengthened with direct manipulation of the gene/transcript in AML12 cells, and comparing those effects to the effects of SIRTOE Exos and CM on these cells. As the Exos and CM will likely be affecting many genes it would be reassuring to see that modulation of Beclin itself has the expected effects on autophagosomes, autolysosomes etc in that cell-type.\nMinor points:\n1. 60% HFD. Is this 60% lipid? What is the composition of HFD?\n2. Please present total liver weight for the post-transplant effects (Figure 1B) as the total body weight will have presumably been affected by the difference in the weight of the adipose tissue that was transplanted (i.e. small tissue amount from chow mice donors, large tissue amount from HFD mice donors).\n3. Please correct mentions of ‘quantitative real-time PCR’ to Quantitative reverse transcription PCR’. Also the PCR for the genotyping of SIRT3Adipo-Tg mice is likely to have been PCR, and not qPCR as is stated in the manuscript.\n4. Small RNA-seq data and ChIP-seq data need to be made available either as raw data or the processed list of all transcripts and ChIP peaks, respectively.\n5. Figure 9B change ‘while’ to ‘whole’."}, "author_response": "Response: We thank the reviewer for the kind comments and critical suggestions. In the revised manuscript, we have further demonstrated the role of Sirt3 by performing adipose tissue-specific Sirt3 knockdown in mice. As expected, the newly obtained results are highly consistent with our previous insights, showing that adipose-expressed Sirt3 regulates insulin sensitivity and liver steatosis under HFD feeding. Besides, Sirt3-silenced adipocyte-derived small extracellular vesicles (sEVs) significantly exacerbated lipid accumulation in hepatocytes and exhibited increased miR-30a-3p level. These additional experimental data strongly reinforce our conclusions.\n\n[... Author response continues for all reviewers ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am happy with the response from the authors and the effort to improve the manuscript.\nMy major concerns are the discrepancies between the first version and the revised version, which the authors did not mention in the Response to reviewer’s comments file or elsewhere in the manuscript.\n1. Figure 1C and 2J, the values (or the scale?) of TG contents are different. Authors did not explain.\n2. Figure 2M, authors changed the data from Nile Red to BODIPY, and did not explain why, seems not from reviewer’s request.\n3. Figure 3G, authors used a different set of H&E images.\n4. Figure 3H-I, authors added one group and revised the graphs. However, from the HFD group, the data points are the same on 3I (AST), but different on 3H (TG content), why?\n5. Figure 6G-H (TG and TC content), same issue as Figure 1C, the individual values showed the same variation as the first version, yet the Y-axis scale is different.\n6. Figure S4A-H (S2 in the first version), data looks different, yet the reviewer cannot find relevant information in the response/rebuttal file.", "reviewer_2": "Comments to authors:\nOverall, the revised manuscript is greatly improved and presents robust data supporting a role for miR-30a-3p in the regulation of insulin sensitivity and hepatic steatosis, and validate Beclin1 as a target for miR-30a-3p. However, there are still two unresolved points (comment 3 and 8, outlined below) that this reviewer feels need to be addressed.\nComment 3. Although the additional immunostains provide more evidence of sEV uptake, there is an absence of appropriate controls that cloud the interpretation of the results. Specifically, in Figures S3F-G, TSG101 and Alix are also expressed in AML12 hepatocytes so the inclusion of a control without sEV treatment would help show if there is a relative increase in those markers. In Figures S7F-H, the miR-30 qPCR data shows clear upregulation of miR-30 after sEV treatment, and a strong association with lipid accumulation. However, this data does not demonstrate miR-30 or sEV accumulation into hepatocytes. This could be demonstrated using human sEVs which express human CD63 and there are good antibodies with minimal cross-reactivity between human and mouse isoforms. The authors have attempted this in Figure S5I-J but with the notable omission of a negative control (ie AML12 cells without sEVs). Furthermore, the authors should also stain with antibodies against mouse CD63 and clearly indicate where they are measuring human CD63 (hCD63) vs mouse CD63 (mCD63). This would provide clear evidence of sEV accumulation into hepatocytes.\nComment 8. The authors performed biotin-miR-30a-3p pulldown to identify mRNA targets, confirming Beclin1 as a direct miR-30a-3p target, as well as other genes involved in lipid metabolism such as Ppara, Acox3 and Acca. However, due to insufficient RNA the authors indicate that they were unable to prepare libraries for sequencing. This is somewhat surprising since modern RNA-seq is quite sensitive and requires minimal RNA inputs (often as low as 10ng) and would likely not require deep reads owing to low library complexity. If RNA-seq is not feasible, then the authors should consider a more focused PCR array. For instance, the RT2 PCR Profiler Arrays (Qiagen) require only 25ng of input RNA and can detect 80-300 genes in specific metabolic pathways. The inclusion of this analysis would strengthen the significance of Beclin1 targeting and the involvement of miR-30a-3p in metabolic regulation", "reviewer_4": "These concerns raised originally by #reviewer 3 have been appropriately addressed in the revised manuscript.", "reviewer_5": "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."}, "author_response": "Response: We thank the reviewer for the positive feedback and this important reminder. We sincerely apologize for the oversight in not explicitly listing and discussing the specific discrepancies and changes between the initial and revised manuscripts in our \"Response to Reviewers\" file. We understand that providing a clear summary of revisions is crucial for transparency and for facilitating your final assessment. We are deeply grateful to the reviewer for the opportunity to address the points raised. The key modifications in the revised manuscript were made based on other reviewers’ comments.\n\n[... Author response continues ...]"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed all my comments in the second round of revision, mostly on the discrepancies of data in several sub-panels. Authors have provided reasonable responses. The quality of the data and the manuscript has been greatly improved. I have no further comment.", "reviewer_2": "I am satisfied with the response to comments 3 and 8, specifically the inclusion of appropriate controls to demonstrate sEV uptake by AML12 cells and the RNA-seq of miR-30a-3p target genes after biotin-miR-30a-3p pulldown. These experiments have greatly improved the manuscript and provide strong evidence for miR-30a-3p in the regulation of hepatic steatosis in addition to mechanistic evidence supporting miR-30a-3p targeting of Beclin1 and other key target genes. In summary, after carefully reviewing the revised manuscript, the authors have addressed all major and minor concerns and thus, I recommend this manuscript for publication in Nature Communications.", "reviewer_5": "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."}, "author_response": ""}]

Decision letter