Myocardin reverses insulin resistance and ameliorates cardiomyopathy by increasing IRS-1 expression in a murine model of lipodystrophy caused by adipose deficiency of vacuolar H+-ATPase V0d1 subunit.

Aim: Adipose tissue (AT) dysfunction that occurs in both obesity and lipodystrophy is associated with the development of cardiomyopathy. However, it is unclear how dysfunctional AT induces cardiomyopathy due to limited animal models available. We have identified vacuolar H+-ATPase subunit Vod1, encoded by Atp6v0d1, as a master regulator of adipogenesis, and adipose-specific deletion of Atp6v0d1 (Atp6v0d1AKO) in mice caused generalized lipodystrophy and spontaneous cardiomyopathy. Using this unique animal model, we explore the mechanism(s) underlying lipodystrophy-related cardiomyopathy. Methods and Results: Atp6v0d1AKO mice developed cardiac hypertrophy at 12 weeks, and progressed to heart failure at 28 weeks. The Atp6v0d1AKO mouse hearts exhibited excessive lipid accumulation and altered lipid and glucose metabolism, which are typical for obesity- and diabetes-related cardiomyopathy. The Atp6v0d1AKO mice developed cardiac insulin resistance evidenced by decreased IRS-1/2 expression in hearts. Meanwhile, the expression of forkhead box O1 (FoxO1), a transcription factor which plays critical roles in regulating cardiac lipid and glucose metabolism, was increased. RNA-seq data and molecular biological assays demonstrated reduced expression of myocardin, a transcription coactivator, in Atp6v0d1AKO mouse hearts. RNA interference (RNAi), luciferase reporter and ChIP-qPCR assays revealed the critical role of myocardin in regulating IRS-1 transcription through the CArG-like element in IRS-1 promoter. Reducing IRS-1 expression with RNAi increased FoxO1 expression, while increasing IRS-1 expression reversed myocardin downregulation-induced FoxO1 upregulation in cardiomyocytes. In vivo, restoring myocardin expression specifically in Atp6v0d1AKO cardiomyocytes increased IRS-1, but decreased FoxO1 expression. As a result, the abnormal expressions of metabolic genes in Atp6v0d1AKO hearts were reversed, and cardiac dysfunctions were ameliorated. Myocardin expression was also reduced in high fat diet-induced diabetic cardiomyopathy and palmitic acid-treated cardiomyocytes. Moreover, increasing systemic insulin resistance with rosiglitazone restored cardiac myocardin expression and improved cardiac functions in Atp6v0d1AKO mice. Conclusion: Atp6v0d1AKO mice are a novel animal model for studying lipodystrophy- or metabolic dysfunction-related cardiomyopathy. Moreover, myocardin serves as a key regulator of cardiac insulin sensitivity and metabolic homeostasis, highlighting myocardin as a potential therapeutic target for treating lipodystrophy- and diabetes-related cardiomyopathy.

Grape Polyphenols Attenuate Diet-Induced Obesity and Hepatic Steatosis in Mice in Association With Reduced Butyrate and Increased Markers of Intestinal Carbohydrate Oxidation.

A Western Diet (WD) low in fiber but high in fats and sugars contributes to obesity and non-alcoholic fatty liver disease (NAFLD). Supplementation with grape polyphenols (GPs) rich in B-type proanthocyanidins (PACs) can attenuate symptoms of cardiometabolic disease and alter the gut microbiota and its metabolites. We hypothesized that GP-mediated metabolic improvements would correlate with altered microbial metabolites such as short chain fatty acids (SCFAs). To more closely mimic a WD, C57BL/6J male mice were fed a low-fiber diet high in sucrose and butterfat along with 20% sucrose water to represent sugary beverages. This WD was supplemented with 1% GPs (WD-GP) to investigate the impact of GPs on energy balance, SCFA profile, and intestinal metabolism. Compared to WD-fed mice, the WD-GP group had higher lean mass along with lower fat mass, body weight, and hepatic steatosis despite consuming more calories from sucrose water. Indirect and direct calorimetry revealed that reduced adiposity in GP-supplemented mice was likely due to their greater energy expenditure, which resulted in lower energy efficiency compared to WD-fed mice. GP-supplemented mice had higher abundance of Akkermansia muciniphila, a gut microbe reported to increase energy expenditure. Short chain fatty acid measurements in colon content revealed that GP-supplemented mice had lower concentrations of butyrate, a major energy substrate of the distal intestine, and reduced valerate, a putrefactive SCFA. GP-supplementation also resulted in a lower acetate:propionate ratio suggesting reduced hepatic lipogenesis. Considering the higher sucrose consumption and reduced butyrate levels in GP-supplemented mice, we hypothesized that enterocytes would metabolize glucose and fructose as a replacement energy source. Ileal mRNA levels of glucose transporter-2 (GLUT2, SLC2A2) were increased indicating higher glucose and fructose uptake. Expression of ketohexokinase (KHK) was increased in ileum tissue suggesting increased fructolysis. A GP-induced increase in intestinal carbohydrate oxidation was supported by: (1) increased gene expression of duodenal pyruvate dehydrogenase (PDH), (2) a decreased ratio of lactate dehydrogenase a (LDHa): LDHb in jejunum and colon tissues, and (3) decreased duodenal and colonic lactate concentrations. These data indicate that GPs protect against WD-induced obesity and hepatic steatosis by diminishing portal delivery of lipogenic butyrate and sugars due to their increased intestinal utilization.

Effect of miR-132 on bupivacaine-induced neurotoxicity in human neuroblastoma cell line.

BACKGROUND: Local anesthetics (LAs) may generate neurotoxicity in neurons. In the current study, we explored the mechanisms by which microRNA-132 (miR-132) regulated the neurotoxicity of human neuroblastoma cells (SH-SY5Y) induced by bupivacaine (BUP). METHODS: CCK-8, flow cytometry, EdU detection, qRT-PCR and western blotting were used to explore the cell viability, apoptosis and gene expression, respectively. RESULTS: In this study, we found that 600 µM BUP dramatically inhibited SH-SY5Y cells viability. In addition, BUP induced cell apoptosis and neurotoxicity via increasing active caspase-3 and cleaved PARP1 levels. More importantly, the level of miR-132 was significantly up-regulated in BUP-treated cells, which was significantly reversed by miR-132 inhibitor. In addition, dual-luciferase assay indicated IGF1R was the directly binding target of miR-132 in cells. Our study further indicated that the level of IGF1R was markedly decreased by BUP interference, while miR-132 inhibitor exerted the opposite effect. Furthermore, BUP induced apoptosis and neurotoxicity in SH-SY5Y cells were attenuated by IGF1, which further confirmed IGF1R was the downstream target of BUP in SH-SY5Y cells. CONCLUSION: In the present study, miR-132 played important roles in regulating BUP-induced neurotoxicity through IGF1R and may act as a promising molecular target for the treatment of human neurotoxicity induced by BUP.