Chronic muscle stimulation improves insulin sensitivity while increasing subcellular lipid droplets and reducing selected diacylglycerol and ceramide species in obese Zucker rats

Aims/hypothesis

Although insulin resistance has been associated with accumulations of specific intramuscular fatty acids and altered subcellular localisation of lipid droplets, these concepts remain controversial. Therefore, we aimed to identify specific intramuscular fatty acids and subcellular lipid localisations associated with improved insulin sensitivity following chronic muscle contraction.

Methods

In lean and insulin-resistant obese Zucker rats the tibialis anterior muscle was stimulated (6 h/day for 6 days). Thereafter, muscles were examined for insulin sensitivity, intramuscular lipid droplet localisation and triacylglycerol (TAG), diacylglycerol (DAG) and ceramide fatty acid composition.

Results

In lean and obese animals, regardless of muscle type, chronic muscle contraction improved muscle insulin sensitivity and increased intramuscular levels of total and most C14–C22 TAG fatty acids (p?<?0.05). Therefore, accumulation in subcellular lipid droplet compartments reflected the oversupply of lipids within muscle. In contrast, improvements in insulin sensitivity induced by muscle contraction were associated with reductions in specific DAG and ceramide species that were not uniform in red and white muscle of obese rats. However, these reductions were insufficient to fully normalise insulin sensitivity, indicating that other mechanisms are involved.

Conclusions/interpretation

Reductions in 18 C length DAG and ceramide species were the most consistent in red and white muscle and therefore may represent therapeutic targets for improving insulin sensitivity.     

Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.     

Gene expression profiling in skeletal muscle of Zucker diabetic fatty rats: implications for a role of stearoyl-CoA desaturase 1 in insulin resistance

Aims/hypothesis Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes. Therefore, we sought to identify and validate genes involved in the development of insulin resistance in skeletal muscle. Materials Differentially regulated genes in skeletal muscle of male obese insulin-resistant, and lean insulin-sensitive Zucker diabetic fatty (ZDF) rats were determined using Affymetrix microarrays. Based on these data, various aspects of glucose disposal, insulin signalling and fatty acid composition were analysed in a muscle cell line overexpressing stearoyl-CoA desaturase 1 (SCD1). Results Gene expression profiling in insulin-resistant skeletal muscle revealed the most pronounced changes in gene expression for genes involved in lipid metabolism. Among these, Scd1 showed increased expression in insulin-resistant animals, correlating with increased amounts of palmitoleoyl-CoA. This was further investigated in a muscle cell line that overexpressed SCD1 and accumulated lipids, revealing impairments of glucose uptake and of different steps of the insulin signalling cascade. We also observed differential effects of high-glucose and fatty acid treatment on glucose uptake and long-chain fatty acyl-CoA profiles, and in particular an accumulation of palmitoleoyl-CoA in cells overexpressing SCD1. Conclusions/interpretation Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Gender-related differences in the metabolic response to fasting

CONTEXT: Free fatty acids (FFA) may induce insulin resistance via synthesis of intramyocellular ceramide. During fasting, women have lower plasma glucose levels than men despite higher plasma FFA, suggesting protection from FFA-induced insulin resistance. OBJECTIVE: We studied whether the relative protection from FFA-induced insulin resistance during fasting in women is associated with lower muscle ceramide concentrations compared with men. MAIN OUTCOME MEASURES AND DESIGN: After a 38-h fast, measurements of glucose and lipid fluxes and muscle ceramide and fatty acid translocase/CD36 were performed before and after a hyperinsulinemic euglycemic clamp. RESULTS: Plasma glucose levels were significantly lower in women than men with a trend for a lower endogenous glucose production in women, whereas FFA and lipolysis were significantly higher. Insulin-mediated peripheral glucose uptake was not different between sexes. There was no gender difference in muscle ceramide in the basal state, and ceramide did not correlate with peripheral glucose uptake. Muscle fatty acid translocase/CD36 was not different between sexes in the basal state and during the clamp. CONCLUSION: After 38 h of fasting, plasma FFA were higher and plasma glucose was lower in women compared with men. The higher plasma FFA did not result in differences in peripheral insulin sensitivity, possibly because of similar muscle ceramide and fatty acid translocase/CD36 levels in men and women. We suggest that during fasting, women are relatively protected from FFA-induced insulin resistance by preventing myocellular accumulation of ceramide.     

Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment

Aims/hypothesis

An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

Methods

CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [¹⁴C]glucose into glycogen.

Results

Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

Conclusions/interpretation

Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.     

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.     

Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle

Aims/hypothesis A role for increased activity of the innate immune system in the pathogenesis of insulin resistance is supported by a number of studies. The current study assessed the potential role of the lipopolysaccharide receptor known as Toll-like receptor-4 (TLR-4), a component of the innate immune system, in mediating lipid-induced insulin resistance in skeletal muscle. Methods The effects of TLR-4 inhibition/deletion on lipid-induced insulin resistance was determined in skeletal muscle of TLR-4 null mice in vivo and in rat L6 myotubes in vitro. Results In mice, acute hyperlipidaemia induced skeletal muscle insulin resistance, but a deletion of TLR-4 conferred significant protection against these effects. In L6 myotubes, inhibition of TLR-4 activity substantially reduced the capacity of the saturated fatty acid palmitate to induce insulin resistance. Importantly, palmitate activated the nuclear factor κB (NFκB) pathway in L6 myotubes and mouse skeletal muscle, and these effects were blocked by inhibition of TLR-4 in L6 myotubes and absence of TLR-4 in skeletal muscle. Furthermore, inhibition of the NFκB pathway downstream of TLR-4 in L6 myotubes also protected against the induction of insulin resistance by palmitate. Conclusions/interpretation Inhibition or absence of TLR-4 confers protection against the detrimental effects of lipids on skeletal muscle insulin action, and these effects are associated with a prevention of the activation of the NFκB pathway by lipids. Importantly, inhibition of the NFκB pathway in myotubes downstream of TLR-4 also protects against lipid-induced insulin resistance, suggesting a mechanism by which reduced TLR-4 activity confers beneficial effects on insulin action.     

Angiopoietin 1 reduces rat follicular atresia mediated by apoptosis through the PI3K/Akt pathway

Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and α-ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells.     

Metabolic flexibility in the development of insulin resistance and type 2 diabetes: effects of lifestyle

Lipotoxicity in skeletal muscle plays a critical role in the aetiology of insulin resistance and type 2 diabetes mellitus by interference of lipid metabolites with insulin signalling and action. The dynamics of lipid oxidation and fine tuning with fatty acid uptake and intramyocellular triacylglycerol turnover may be very important to limit the accumulation of lipid intermediates. The use of metabolic inflexibility, defined as the impaired capacity to increase fat oxidation upon increased fatty acid availability and to switch between fat and glucose as the primary fuel source after a meal, does more justice to the complexity of changes in fuel oxidation during the day. Fatty acid availability, uptake and oxidation all play a role in metabolic flexibility and insulin resistance. During high fatty acid availability, fatty acid transporters may limit cellular and mitochondrial fatty acid uptake and thus limit fat oxidation. After a meal, when the demand for fatty acids as fuel is low, an increased fractional extraction of lipids from plasma may promote intramyocellular lipid accumulation and insulin resistance. Furthermore, defects in fuel switching cluster together with impaired mitochondrial content and/or function. Lifestyle changes in dietary fat intake, physical activity and weight loss may improve metabolic flexibility in skeletal muscle, and thereby contribute to the prevention of type 2 diabetes.     

Obesity-associated insulin resistance in skeletal muscle: Role of lipid accumulation and physical inactivity

An alarming increase in the prevalence of obesity, type 2 diabetes mellitus, and associated diseases can be observed world-wide during the past 20 years. In obesity, profound alterations in the secretion profile of adipokines and inflammatory markers as well as increased lipolysis occur, leading besides other events to elevated levels of free fatty acids, which in turn are distributed to nonadipose tissue such as skeletal muscle. While the amount of intramyocellular lipids can be used as a marker of insulin resistance in physical inactive individuals, these neutral triglycerides themselves are not thought to be harmful. However, they provide a source for the generation of harmful lipid metabolites such as diacylglycerol and ceramide, which are implicated in insulin resistance by perturbing insulin signaling pathways. In this review, we will discuss the role of lipid metabolites in insulin resistance and potential mechanism involved in accumulation of intramyocellular lipids. Furthermore, we will highlight the key role of PGC-1α, which is a master regulator of mitochondrial biogenesis and coordinates the activation of genes involved in oxidative energy production as well as genes involved in fiber type transformation. Finally, the role of exercise in stimulating PGC-1α activity and expression as well as the release of contraction-induced myokines is discussed.     

Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome

In this study, we defined the role of peroxisome proliferator-activated receptor beta/delta (PPARdelta) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPARdelta subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPARdelta controls fatty acid oxidation by regulating genes involved in fatty acid transport, beta-oxidation, and mitochondrial respiration. Similar PPARdelta-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid beta-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid beta-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob/ob mice. These data suggest that PPARdelta is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals.     

The role of stearoyl-CoA desaturase in body weight regulation

Obesity is a significant health problem due to its serious medical complications that include hypertension, insulin resistance, diabetes, coronary artery disease, and heart failure. This review addresses the hypothesis that stearoyl-CoA desaturase (SCD) is an important metabolic control point in body weight regulation. SCD is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. These products are the most abundant fatty acids found in triglycerides, cholesterol esters, wax esters, and phospholipids. Mice with a disruption in the scd1 gene (scd1(-/-)) have increased energy expenditure, reduced body adiposity, and increased insulin sensitivity, and are resistant to diet-induced obesity. The expression of several genes encoding enzymes of lipid oxidation is upregulated, whereas genes encoding enzymes of lipid synthesis are down regulated in the scd1-deficient mice. scd1 is also a component of the novel metabolic response to the hormone leptin. SCD therefore appears to be an important metabolic control point, and inhibition of its expression could be of benefit in the treatment of obesity, diabetes, cardiovascular disease, and other metabolic diseases.     

Direct demonstration of lipid sequestration as a mechanism by which rosiglitazone prevents fatty-acid-induced insulin resistance in the rat: comparison with metformin

Aims/hypothesis Thiazolidinediones can enhance clearance of whole-body non-esterified fatty acids and protect against the insulin resistance that develops during an acute lipid load. The present study used [³H]-R-bromopalmitate to compare the effects of the thiazolidinedione, rosiglitazone, and the biguanide, metformin, on insulin action and the tissue-specific fate of non-esterified fatty acids in rats during lipid infusion.Methods Normal rats were treated with rosiglitazone or metformin for 7 days. Triglyceride/heparin (to elevate non-esterified fatty acids) or glycerol (control) were then infused for 5 h, with a hyperinsulinaemic clamp being performed between the 3rd and 5th hours.Results Rosiglitazone and metformin prevented fatty-acid-induced insulin resistance (reduced clamp glucose infusion rate). Both drugs improved insulin-mediated suppression of hepatic glucose output but only rosiglitazone enhanced systemic non-esterified fatty acid clearance (plateau plasma non-esterified fatty acids reduced by 40%). Despite this decrease in plateau plasma non-esterified fatty acids, rosiglitazone increased fatty acid uptake (two-fold) into adipose tissue and reduced fatty acid uptake into liver (by 40%) and muscle (by 30%), as well as reducing liver long-chain fatty acyl CoA accumulation (by 30%). Both rosiglitazone and metformin increased liver AMP-activated protein kinase activity, a possible mediator of the protective effects on insulin action, but in contrast to rosiglitazone, metformin had no significant effect on non-esterified fatty acid kinetics or relative tissue fatty acid uptake.Conclusions/interpretation These results directly demonstrate the lipid steal mechanism, by which thiazolidinediones help prevent fatty-acid-induced insulin resistance. The contrasting mechanisms of action of rosiglitazone and metformin could be beneficial when both drugs are used in combination to treat insulin resistance.Abbreviations AMPK AMP-activated protein kinase - ¹⁴C-2DG [¹⁴C]-2-deoxyglucose - GIR glucose infusion rate - HGO hepatic glucose output - LCACoA long-chain fatty acyl CoA - PPAR peroxisome proliferator-activated receptor - TZDs thiazolidinediones     

GRP40介导游离脂肪酸对葡萄糖刺激的胰岛素分泌和β细胞内钙离子浓度的变化

目的 探讨G蛋白耦联受体40(GPR40)是否介导游离脂肪酸(FFAs)短期、长期作用对小鼠胰岛NIT-1 β细胞葡萄糖刺激的胰岛素分泌(GSIS)及胞内钙离子浓度的影响.方法 利用siRNA技术抑制GPR40在NIT-1细胞的表达,ELISA法检测棕榈酸、油酸短期(2 h)、长期(48 h)干预对NIT-1细胞GSIS的影响,激光共聚焦显微镜下观察GSIS过程中细胞内钙离子的变化.结果 FFAs短期干预促进空转染组、对照siRNA转染组GSIS水平(P＜0.01),而对GPR40 siRNA组GSIS无明显影响,该组细胞GSIS水平明显低于空转染组和对照siRNA转染组(P＜0.01).FFAs长期干预明显抑制空转染组、对照siRNA转染组细胞GSIS水平,对GPR40 siRNA组GSIS无明显影响.空转染组、对照siRNA组GSIS水平低于GPR40siRNA组(P＜0.05).激光共聚焦显微镜结果显示,在GSIS过程中,FFAs短期干预后,GPR40 siRNA组胞内钙离子浓度峰值明显低于对照siRNA组(棕榈酸:5.10 vs 7.02,油酸:4.27 vs 6.21,均P＜0.05);而FFAs长期干预后,GPR40 siRNA组胞内钙离子浓度峰值明显高于对照siRNA组(棕榈酸:3.24 vs 1.21,油酸:2.83 vs 1.18,均P＜0.05).结论 GPR40介导FFAs对GSIS和胞内钙离子浓度变化的短期刺激和长期抑制作用.     

Muscle adaptation to short-term fasting in healthy lean humans

CONTEXT: It has been demonstrated repeatedly that short-term fasting induces insulin resistance, although the exact mechanism in humans is unknown to date. Intramyocellular sphingolipids (i.e. ceramide) have been suggested to induce insulin resistance by interfering with the insulin signaling cascade in obesity. OBJECTIVE: Our objective was to study peripheral insulin sensitivity together with muscle ceramide concentrations and protein kinase B/AKT phosphorylation after short-term fasting. MAIN OUTCOME MEASURES AND DESIGN: After 14- and 62-h fasting, glucose fluxes were measured before and after a hyperinsulinemic euglycemic clamp. Muscle biopsies were performed in the basal state and during the clamp to assess muscle ceramide and protein kinase B/AKT. RESULTS: Insulin-mediated peripheral glucose uptake was significantly lower after 62-h fasting compared with 14-h fasting. Intramuscular ceramide concentrations tended to increase during fasting. During the clamp the phosphorylation of protein kinase B/AKT at serine(473) in proportion to the total amount of protein kinase B/AKT was significantly lower. Muscle ceramide did not correlate with plasma free fatty acids. CONCLUSIONS: Fasting for 62 h decreases insulin-mediated peripheral glucose uptake with lower phosphorylation of AKT at serine(473). AKT may play a regulatory role in fasting-induced insulin resistance. Whether the decrease in AKT can be attributed to the trend to higher muscle ceramide remains unanswered.     

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKCε (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKCε.     

Evidence for adipose-muscle cross talk: opposing regulation of muscle proteolysis by adiponectin and Fatty acids

Illnesses associated with insulin resistance exhibit increases in whole-body protein degradation and amino acid oxidation. However, the mechanisms stimulating muscle catabolism under these conditions are not clear. Because insulin resistance is associated with accumulation of lipids in muscle, we measured protein degradation in muscles of mice fed a high-fat diet. Muscle protein catabolism was accelerated on the high-fat diet, and this was associated with an increase in plasma free fatty acid and a decrease in plasma levels of the adipocyte-derived cytokine adiponectin. To evaluate how free fatty acids influence adiponectin-mediated changes in muscle protein breakdown we examined C2C12 skeletal muscle cells exposed to free fatty acids. Both saturated fatty acids (palmitate) and unsaturated fatty acids (oleate) increased protein degradation (25 and 18%, respectively) in part by activating the E3 ubiquitin ligases. Adenovirus-mediated overexpression of adiponectin blocked fatty acid-induced protein degradation in C2C12 cells. Palmitate activated the E3 ubiquitin ligases by suppressing insulin receptor substrate-1/Akt signaling in the C2C12 muscle cells, whereas adiponectin attenuated the E3 ubiquitin ligase activation by increasing both insulin receptor substrate-1 tyrosine phosphorylation and Akt Ser473 phosphorylation. In related experiments, adiponectin overexpression decreased TNFalpha and IL-6 expression in 3T3-L1 adipocytes, whereas exposure to free fatty acids had the opposite effect. We conclude that the balance between free fatty acids and adiponectin impacts muscle proteolysis in insulin-resistant conditions and suggest a role for adipose tissue-muscle cross talk in diabetes and obesity.     

Up-regulation of uncoupling protein 3 gene expression by fatty acids and agonists for PPARs in L6 myotubes

Uncoupling protein 3 (UCP3), which uncouples electron transport from ATP synthesis, is expressed at high levels in the skeletal muscle, an important organ in glucose and lipid metabolism. Because several reports proposed that fatty acids induced UCP3 gene expression in skeletal muscle in vivo, in the present study we examined the regulation of UCP3 gene expression by various fatty acids using L6 myotubes. UCP3 gene expression was increased in L6 myotubes by various fatty acids or by alpha-bromopalmitate, a nonmetabolized derivative of palmitic acid. Because fatty acids are also known as agonists for PPARs, we examined the involvement of PPARs in the regulation of the UCP3 gene expression. L-165041, a PPAR delta agonist, increased UCP3 gene expression in L6 myotubes, whereas neither Wy 14,643, a PPAR alpha agonist, nor Pioglitazone, a PPAR gamma agonist, increased it. Therefore, we conclude that UCP3 gene expression is increased by the activation of PPAR delta in L6 myotubes and postulate that PPAR delta mediates at least some part of the increased UCP3 gene expression by fatty acids in skeletal muscle in vivo.