Rengasamy Palanivel

Regulation of fatty acid uptake and metabolism in L6 skeletal muscle cells by resistin

Resistin has been proposed as a potential link between obesity and insulin resistance. It is also well established that altered metabolism of fatty acids by skeletal muscle can lead to insulin resistance and lipotoxicity. However, little is known about the effect of resistin on long chain fatty acid uptake and metabolism in skeletal muscle. Here we show that treating rat skeletal muscle cells with recombinant resistin (50 nM, 24 h) decreased uptake of palmitate. This correlated with reduced cell surface CD36 content and lower expression of FATP1, but no change in FATP4 or CD36 expression. We also found that resistin decreased fatty acid oxidation by measuring 14CO2 production from [1‐14C] oleate and an increase in intracellular lipid accumulation was detected in response to resistin. Decreased AMPK and ACC phosphorylation were observed in response to resistin while expression of ACC and AMPK isoforms was unaltered. Resistin mediated these effects without altering cell viability. In summary, our results demonstrate that chronic incubation of skeletal muscle cells with resistin decreased fatty acid uptake and metabolism via a mechanism involving decreased cell surface CD36 content, FATP1 expression and a decrease in phosphorylation of AMPK and ACC.

An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart

Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1.

Adiponectin stimulates autophagy and reduces oxidative stress to enhance insulin sensitivity during high fat diet feeding in mice

Numerous studies have characterized the antidiabetic effects of adiponectin, yet the precise cellular mechanisms in skeletal muscle, in particular, changes in autophagy, require further clarification. In the current study, we used a high-fat diet (HFD) to induce obesity and insulin resistance in wild-type (WT) or adiponectin knockout (Ad-KO) mice with and without adiponectin replenishment. Temporal analysis of glucose tolerance and insulin sensitivity using hyperinsulinemic-euglycemic clamp and muscle insulin receptor substrate and Akt phosphorylation demonstrated exaggerated and more rapid HFD-induced insulin resistance in skeletal muscle of Ad-KO mice. Superoxide dismutase activity, the reduced glutathione-to-glutathione disulfide ratio, and lipid peroxidation indicated that HFD-induced oxidative stress was corrected by adiponectin. Gene array analysis implicated several antioxidant enzymes, including Gpxs, Prdx, Sod, and Nox4, in mediating this effect. Adiponectin also attenuated palmitate-induced reactive oxygen species production in cultured myotubes and improved insulin-stimulated glucose uptake in primary muscle cells. Increased LC3-II and decreased p62 expression suggested that HFD induced autophagy in muscle of WT mice; however, these changes were not observed in Ad-KO mice. Replenishing adiponectin in Ad-KO mice increased LC3-II and Beclin1 and decreased p62 protein levels, induced fibroblast growth factor-21 expression, and corrected HFD-induced decreases in LC3, Beclin1, and ULK1 gene expression. In vitro studies examining changes in phospho-ULK1 (Ser555), LC3-II, and lysosomal enzyme activity confirmed that adiponectin directly induced autophagic flux in cultured muscle cells in an AMPK-dependent manner. We overexpressed an inactive mutant of Atg5 to create an autophagy-deficient cell model, and together with pharmacological inhibition of autophagy, demonstrated reduced insulin sensitivity under these conditions. In summary, adiponectin stimulated skeletal muscle autophagy and antioxidant potential to reduce insulin resistance caused by HFD.

Increased expression and cell surface localization of MT1-MMP plays a role in stimulation of MMP-2 activity by leptin in neonatal rat cardiac myofibroblasts.

Myocardial matrix remodeling is a well-recognized disease modifier in the pathogenesis of heart failure, although the precise underlying molecular mechanisms remain to be elucidated. Here we investigated the effects of leptin, circulating levels of which are typically increased in obese individuals, on MMP and collagen expression and MMP activity in isolated cardiac myofibroblasts. Neonatal rat myofibroblasts were treated with 6 nM recombinant leptin and the collected supernatant analyzed for MMP-2 activity via gelatin zymography. MMP-2, MT1-MMP and procollagen-I and -III protein expression were determined by western blotting and MMP-2 and MT1-MMP mRNA expression were examined utilizing real-time PCR. Procollagen-I levels were analyzed by confocal microscopy and collagen synthesis was determined through [(3)H]-proline incorporation. Exposure of myofibroblasts to leptin (24 h) significantly increased MMP-2 activity, while mRNA and protein levels remained unchanged. Leptin also significantly enhanced mRNA and protein expression of MT1-MMP, a known activator of MMP-2. Biotinylation assays indicated increased cell surface expression of MT1-MMP in response to leptin and use of a MT1-MMP inhibitor attenuated the leptin-mediated elevation of MMP-2 activity. Total cellular collagen synthesis was unaffected by leptin treatment, however intracellular procollagen-I protein was significantly increased in treated cells. Furthermore, extracellular soluble procollagen-I was increased, while a decrease in soluble procollagen-III protein was observed in conditioned media. In summary, these findings in isolated cardiac myofibroblasts support the suggestion that leptin may directly influence myocardial matrix metabolism, and this may represent a mechanism contributing to cardiac fibrosis in obese patients with elevated plasma leptin levels.

Differential impact of adipokines derived from primary adipocytes of wild-type versus streptozotocin-induced diabetic rats on glucose and fatty acid metabolism in cardiomyocytes

The causal relationship between obesity and cardiovascular disease is extensively acknowledged; however, the exact mechanisms linking obesity and heart failure remain unclear. Here, we investigated the influence of adipokines derived from primary adipocytes on glucose and fatty acid uptake and metabolism in isolated primary cardiomyocytes. Either co-culture of these cell types or incubation with adipocyte-conditioned medium significantly increased glucose uptake in cardiomyocytes. When streptozotocin-induced diabetic rats were used as a source of adipocytes, there was a lower ability to elicit glucose uptake in cardiomyocytes which corresponded with lower Akt and AMPK phosphorylation. The profile of glucose metabolism also differed with oxidation being favored upon co-culture with wild-type adipocytes whereas lactate production was strongly induced by adipocytes from diabetic rats. Examination of fatty acid uptake revealed that stimulation only occurred in response to adipokines secreted by wild-type rat adipocytes. Importantly, oxidation of fatty acids by cardiomyocytes was decreased by adipokines derived from diabetic rat adipocytes. Analysis of adipokine profiles in diabetic rat adipocyte-conditioned medium demonstrated the most significant decreases in adiponectin and leptin with increased IL6 expression. Taken together, these data suggest that the profile of adipokines secreted by adipocytes from diabetic rats have a deleterious influence on cardiomyocyte metabolism which may be of relevance in the pathophysiology of heart failure.

Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes.

OBJECTIVE Adiponectin is known to confer its cardioprotective effects in obesity and type 2 diabetes, mainly by regulating glucose and fatty acid metabolism in cardiomyocytes. Dynamic actin cytoskeleton remodeling is involved in regulation of multiple biological functions, including glucose uptake. Here we investigated in neonatal cardiomyocytes whether adiponectin induced actin cytoskeleton remodeling and if this played a role in adiponectin-stimulated glucose uptake. MATERIALS/METHODS Primary cardiomyocytes were treated with full-length and globular adiponectin (fAd and gAd, respectively). RESULTS Both fAd and gAd increased RhoA activity, phosphorylation of the Rho/ROCK signaling target cofilin and actin polymerization to form filamentous actin as determined by rhodamine-phallodin immunofluorescence and quantitative analysis of filamentous to globular actin ratio. Scanning electron microscopy also demonstrated structural remodeling. Adiponectin stimulated glucose uptake, was significantly abrogated in the presence of inhibitors of actin cytoskeleton remodeling (cytochalasin D) and Rho/ROCK signaling (C3 transferase, Y27632). We showed that adiponectin increased colocalization of actin and APPL1 and that actin remodeling, phosphorylation of AMPK, p38MAPK and cofilin, glucose uptake and oxidation were all attenuated after siRNA-mediated knockdown of APPL1. CONCLUSION We show that adiponectin mediates Rho/ROCK-dependent actin cytoskeleton remodeling to increase glucose uptake and metabolism via APPL1 signaling.

Metabolomic profiling in liver of adiponectin-knockout mice uncovers lysophospholipid metabolism as an important target of adiponectin action

Adiponectin mediates anti-diabetic effects via increasing hepatic insulin sensitivity and direct metabolic effects. In the present study, we conducted a comprehensive and unbiased metabolomic profiling of liver tissue from AdKO (adiponectin-knockout) mice, with and without adiponectin supplementation, fed on an HFD (high-fat diet) to derive insight into the mechanisms and consequences of insulin resistance. Hepatic lipid accumulation and insulin resistance induced by the HFD were reduced by adiponectin. The HFD significantly altered levels of 147 metabolites, and bioinformatic analysis indicated that one of the most striking changes was the profile of increased lysophospholipids. These changes were largely corrected by adiponectin, at least in part via direct regulation of PLA2 (phospholipase A2) as palmitate-induced PLA2 activation was attenuated by adiponectin in primary hepatocytes. Notable decreases in several glycerolipids after the HFD were reversed by adiponectin, which also corrected elevations in several diacyglycerol and ceramide species. Our data also indicate that stimulation of ω-oxidation of fatty acids by the HFD is enhanced by adiponectin. In conclusion, this metabolomic profiling approach in AdKO mice identified important targets of adiponectin action, including PLA2, to regulate lysophospholipid metabolism and ω-oxidation of fatty acids.