Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

Aging is associated with myocardial insulin resistance and mitochondrial dysfunction

Aging is associated with insulin resistance, often attributable to obesity and inactivity. Recent evidence suggests that skeletal muscle insulin resistance in aging is associated with mitochondrial alterations. Whether this is true of the senescent myocardium is unknown. Twelve young (Y, 4 years old) and 12 old (O, 11 years old) dogs, matched for body mass, were instrumented with left-ventricular pressure gauges, aortic and coronary sinus catheters, and flow probes on left circumflex artery. Before surgery, all dogs participated in a 6-wk exercise program. Dogs underwent measurements of hemodynamics and plasma substrates before and during a 2-h hyperinsulinemic-euglycemic clamp to measure whole body and myocardial glucose and nonesterified fatty acid uptake. Following the protocol, myocardial and skeletal samples were obtained to measure components of the insulin-signaling cascade and mitochondrial structure. There was no difference in plasma glucose (Y, 90 +/- 4 mg/dl; O, 87 +/- 4 mg/dl), but old dogs had higher (P < 0.02) nonesterified fatty acids (Y, 384 +/- 48 micromol/l; O, 952 +/- 97 micromol/l) and plasma insulin (Y, 39 +/- 11 pmol/l; O, 108 +/- 18 pmol/l). Old dogs had impaired total body glucose disposition (Y, 11.5 +/- 1 mg x kg(-1) x min(-1); O, 8.0 +/- 0.5 mg x kg(-1) x min(-1); P < 0.05) and insulin-stimulated myocardial glucose uptake (Y, 3.5 +/- 0.3 mg x min(-1) x g(-1); O, 1.8 +/- 0.3 mg x min(-1) x g(-1); P < 0.05). The impaired insulin action was associated with altered insulin signaling and glucose transporter (GLUT4) translocation. There were myocardial mitochondrial structural changes observed in association with decreased expression of uncoupling protein-3. Aging is associated with both whole body and myocardial insulin resistance, independent of obesity and inactivity, but involving altered mitochondrial structure and impaired cellular insulin action.     

SIRT1 attenuates high glucose-induced insulin resistance via reducing mitochondrial dysfunction in skeletal muscle cells

Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes mellitus (T2DM). Sustained high glucose is an important extracellular environment that induces insulin resistance. Acquired insulin resistance is associated with reduced insulin-stimulated mitochondrial activity as a result of increased mitochondrial dysfunction. Silent information regulator 1 (SIRT1) is one member of the SIRT2 (Sir2)-like family of proteins involved in glucose homeostasis and insulin secretion in mammals. Although SIRT1 has a therapeutic effect on metabolic deterioration in insulin resistance, it is still not clear how SIRT1 is involved in the development of insulin resistance. Here, we demonstrate that pcDNA3.1 vector-mediated overexpression of SIRT1 attenuates insulin resistance in the high glucose-induced insulin-resistant skeleton muscle cells. These beneficial effects were associated with ameliorated mitochondrial dysfunction. Further studies have demonstrated that SIRT1 restores mitochondrial complex I activity leading to decreased oxidative stress and mitochondrial dysfunction. Furthermore, SIRT1 significantly elevated the level of another SIRT which is named SIRT3, and SIRT3 siRNA-suppressed SIRT1-induced mitochondria complex activity increments. Taken together, these results showed that SIRT1 improves insulin sensitivity via the amelioration of mitochondrial dysfunction, and this is achieved through the SIRT1–SIRT3–mitochondrial complex I pathway.     

Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.     

NOD2 activation induces oxidative stress contributing to mitochondrial dysfunction and insulin resistance in skeletal muscle cells

Nucleotide-binding oligomerization domain protein-2 (NOD2) activation in skeletal muscle cells has been associated with insulin resistance, but the underlying mechanisms are not yet clear. Here we demonstrate the implication of oxidative stress in the development of mitochondrial dysfunction and insulin resistance in response to NOD2 activation in skeletal muscle cells. Treatment with the selective NOD2 ligand muramyl dipeptide (MDP) increased mitochondrial reactive oxygen species (ROS) generation in L6 myotubes. MDP-induced ROS production was associated with increased levels of protein carbonyls and reduction in citrate synthase activity, cellular ATP level, and mitochondrial membrane potential, as well as altered expression of genes involved in mitochondrial function and metabolism. Antioxidant treatment attenuated MDP-induced ROS production and restored mitochondrial functions. In addition, the presence of antioxidant prevented NOD2-mediated activation of MAPK kinases and the inflammatory response. This was associated with reduced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and improved insulin-stimulated tyrosine phosphorylation of IRS-1 and downstream activation of Akt phosphorylation. These data indicate that oxidative stress plays a role in NOD2 activation-induced inflammatory response and that MDP-induced oxidative stress correlates with impairment of mitochondrial functions and induction of insulin resistance in skeletal muscle cells.     

Human skeletal muscle mitochondrial uncoupling is associated with cold induced adaptive thermogenesis

Background

Mild cold exposure and overfeeding are known to elevate energy expenditure in mammals, including humans. This process is called adaptive thermogenesis. In small animals, adaptive thermogenesis is mainly caused by mitochondrial uncoupling in brown adipose tissue and regulated via the sympathetic nervous system. In humans, skeletal muscle is a candidate tissue, known to account for a large part of the epinephrine-induced increase in energy expenditure. However, mitochondrial uncoupling in skeletal muscle has not extensively been studied in relation to adaptive thermogenesis in humans. Therefore we hypothesized that cold-induced adaptive thermogenesis in humans is accompanied by an increase in mitochondrial uncoupling in skeletal muscle.

Methodology/Principal Findings

The metabolic response to mild cold exposure in 11 lean, male subjects was measured in a respiration chamber at baseline and mild cold exposure. Skeletal muscle mitochondrial uncoupling (state 4) was measured in muscle biopsies taken at the end of the respiration chamber stays. Mild cold exposure caused a significant increase in 24h energy expenditure of 2.8% (0.32 MJ/day, range of −0.21 to 1.66 MJ/day, p<0.05). The individual increases in energy expenditure correlated to state 4 respiration (p<0.02, R² = 0.50).

Conclusions/Significance

This study for the first time shows that in humans, skeletal muscle has the intrinsic capacity for cold induced adaptive thermogenesis via mitochondrial uncoupling under physiological conditions. This opens possibilities for mitochondrial uncoupling as an alternative therapeutic target in the treatment of obesity.     

Free fatty acids and skeletal muscle insulin resistance

PURPOSE OF REVIEW: Acute exposure to fatty acids causes insulin resistance in muscle, and excess dietary lipid and obesity are also strongly associated with muscle insulin resistance. Relevant mechanisms, however, are still not fully elucidated. Here we examine the latest evidence as to why lipids might accumulate in muscle and the possible mechanisms for lipid-induced insulin resistance. RECENT FINDINGS: Muscle lipid metabolites such as long chain fatty acid coenzyme As, diacylglycerol and ceramides may impair insulin signalling directly. Crosstalk between inflammatory signalling pathways and insulin signalling pathways, mitochondrial dysfunction and oxidative stress have also been put forward as major contributors to the development or maintenance of lipid-induced insulin resistance in muscle. Several animal models with gene deletions in pathways of fatty acid synthesis and storage also show increased metabolic rate, reduced intramuscular lipid storage and improved insulin action when challenged with a high lipid load. SUMMARY: Studies in genetic and dietary obese animal models, genetically modified animals and humans with obesity or type 2 diabetes suggest plausible mechanisms for effects of fatty acids, lipid metabolites, inflammatory pathways and mitochondrial dysfunction on insulin action in muscle. Many of these mechanisms, however, have been demonstrated in situations in which lipid accumulation (obesity) already exists. Whether the initial events leading to muscle insulin resistance are direct effects of fatty acids in muscle or are secondary to lipid accumulation in adipose tissue or liver remains to be clarified.     

A polyphenol rescues lipid induced insulin resistance in skeletal muscle cells and adipocytes

Skeletal muscle and adipose tissues are known to be two important insulin target sites. Therefore, lipid induced insulin resistance in these tissues greatly contributes in the development of type 2 diabetes (T2D). Ferulic acid (FRL) purified from the leaves of Hibiscus mutabilis, showed impressive effects in preventing saturated fatty acid (SFA) induced defects in skeletal muscle cells. Impairment of insulin signaling molecules by SFA was significantly waived by FRL. SFA markedly reduced insulin receptor β (IRβ) in skeletal muscle cells, this was affected due to the defects in high mobility group A1 (HMGA1) protein obtruded by phospho-PKCε and that adversely affects IRβ mRNA expression. FRL blocked PKCε activation and thereby permitted HMGA1 to activate IRβ promoter which improved IR expression deficiency. In high fat diet (HFD) fed diabetic rats, FRL reduced blood glucose level and enhanced lipid uptake activity of adipocytes isolated from adipose tissue. Importantly, FRL suppressed fetuin-A (FetA) gene expression, that reduced circulatory FetA level and since FetA is involved in adipose tissue inflammation, a significant attenuation of proinflammatory cytokines occurred. Collectively, FRL exhibited certain unique features for preventing lipid induced insulin resistance and therefore promises a better therapeutic choice for T2D.     

AMP kinase activation ameliorates insulin resistance induced by free fatty acids in rat skeletal muscle

We examined whether acute activation of 5'-AMP-activated protein kinase (AMPK) by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) ameliorates insulin resistance in isolated rat skeletal muscle. Insulin resistance was induced in extensor digitorum longus (EDL) muscles by prolonged exposure to 1.6 mM palmitate, which inhibited insulin-stimulated glycogen synthesis to 51% of control after 5 h of incubation. Insulin-stimulated glucose transport was less affected (22% of control). The decrease in glycogen synthesis was accompanied by decreased glycogen synthase (GS) activity and increased GS phosphorylation. When including 2 mM AICAR in the last hour of the 5-h incubation with palmitate, the inhibitory effect of palmitate on insulin-stimulated glycogen synthesis and glucose transport was eliminated. This effect of AICAR was accompanied by activation of AMPK. Importantly, AMPK inhibition was able to prevent this effect. Neither treatment affected total glycogen content. However, glucose 6-phosphate was increased after inclusion of AICAR, indicating increased influx of glucose. No effect of AICAR on the inhibited insulin-stimulated GS activity or increased GS phosphorylation by palmitate could be detected. Thus the mechanism by which AMPK activation ameliorates the lipid-induced insulin resistance probably involves induction of compensatory mechanisms overriding the insulin resistance. Our results emphasize AMPK as a promising molecular target for treatment of insulin resistance.     

Histopathological changes in rat pancreas and skeletal muscle associated with high fat diet induced insulin resistance

The effects of a high fat diet on the development of diabetes mellitus, insulin resistance and secretion have been widely investigated. We investigated the effects of a high fat diet on the pancreas and skeletal muscle of normal rats to explore diet-induced insulin resistance mechanisms. Forty-four male Wistar rats were divided into six groups: a control group fed standard chow, a group fed a 45% fat diet and a group fed a 60% fat diet for 3 weeks to measure acute effects; an additional three groups were fed the same diet regimens for 8 weeks to measure chronic effects. The morphological effects of the two high fat diets were examined by light microscopy. Insulin in pancreatic islets was detected using immunohistochemistry. The homeostasis model assessment of insulin resistance index and insulin staining intensity in islets increased significantly with acute administration of high fat diets, whereas staining intensity decreased with chronic administration of the 45% fat diet. Islet areas increased significantly with chronic administration. High fat diet administration led to islet degeneration, interlobular adipocyte accumulation and vacuolization in the pancreatic tissue, as well as degeneration and lipid droplet accumulation in the skeletal muscle tissue. Vacuolization in the pancreas and lipid droplets in skeletal muscle tissue increased significantly with chronic high fat diet administration. We suggest that the glucolipotoxic effects of high fat diet administration depend on the ratio of saturated to unsaturated fatty acid content in the diet and to the total fat content of the diet.     

Respiratory chain dysfunction in skeletal muscle does not cause insulin resistance

Insulin resistance in skeletal muscle is a characteristic feature of diabetes mellitus type 2 (DM2). Several lines of circumstantial evidence suggest that reduced mitochondrial oxidative phosphorylation capacity in skeletal muscle is a primary defect causing insulin resistance and subsequent development of DM2. We have now experimentally tested this hypothesis by characterizing glucose homeostasis in tissue-specific knockout mice with progressive respiratory chain dysfunction selectively in skeletal muscle. Surprisingly, these knockout mice are not diabetic and have an increased peripheral glucose disposal when subjected to a glucose tolerance test. Studies of isolated skeletal muscle from knockout animals show an increased basal glucose uptake and a normal increase of glucose uptake in response to insulin. In summary, our findings indicate that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in DM2.     

Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance

Fatty acid-induced apoptosis occurs in pancreatic beta-cells and contributes to the metabolic syndrome. Skeletal muscle insulin resistance is mediated by fatty acid oversupply, which also contributes to the metabolic syndrome. Therefore, we examined whether fatty acids induce apoptosis in skeletal muscle myotubes, the proapoptotic signaling involved, and the effects on insulin sensitivity. Exposure of L6 myotubes to palmitate induced apoptosis, as demonstrated by increased caspase-3 activation, phosphatidylserine exposure on the plasma membrane, and terminal deoxynucleotide transferase dUTP nick end labeling and DNA laddering, both markers of DNA fragmentation. Ceramide content was concomitantly increased, indicating a potential role for ceramides in palmitate-induced apoptosis. Supporting this notion, reducing stearoyl-CoA desaturase-1 (SCD-1) protein content with short interfering RNA resulted in ceramide accumulation and was associated with increased apoptosis in the absence of palmitate. Furthermore, the membrane-permeable C(2)-ceramide enhanced apoptosis in myotubes, whereas the ceramide synthase inhibitor, fumonisin B(1), abrogated the proapoptotic effects of palmitate. Insulin-stimulated glucose uptake was inhibited by palmitate treatment, whereas the addition of effector caspase inhibitors [Ac-DEVD-aldehyde (DEVD-CHO), Z-DQMD-FMK] independently restored >80% of the insulin-stimulated glucose uptake. These effects were observed independently from changes in the protein content of insulin signaling proteins, suggesting that proteosomal degradation is not involved in this process. We conclude that lipoapoptosis occurs in skeletal muscle myotubes, at least partially via de novo ceramide accumulation, and that inhibiting downstream apoptotic signaling improves glucose uptake in vitro.     

Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production

Reactive oxygen species (ROS), especially mitochondrial ROS, are postulated to play a significant role in muscle atrophy. We report a dramatic increase in mitochondrial ROS generation in three conditions associated with muscle atrophy: in aging, in mice lacking CuZn-SOD (Sod1(-/-)), and in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ROS generation in muscle mitochondria is nearly threefold higher in 28- to 32-mo-old than in 10-mo-old mice and is associated with a 30% loss in gastrocnemius mass. In Sod1(-/-) mice, muscle mitochondrial ROS production is increased >100% in 20-mo compared with 5-mo-old mice along with a >50% loss in muscle mass. ALS G93A mutant mice show a 75% loss of muscle mass during disease progression and up to 12-fold higher muscle mitochondrial ROS generation. In a second ALS mutant model, H46RH48Q mice, ROS production is approximately fourfold higher than in control mice and is associated with a less dramatic loss (30%) in muscle mass. Thus ROS production is strongly correlated with the extent of muscle atrophy in these models. Because each of the models of muscle atrophy studied are associated to some degree with a loss of innervation, we were interested in determining whether denervation plays a role in ROS generation in muscle mitochondria isolated from hindlimb muscle following surgical sciatic nerve transection. Seven days post-denervation, muscle mitochondrial ROS production increased nearly 30-fold. We conclude that enhanced generation of mitochondrial ROS may be a common factor in the mechanism underlying denervation-induced atrophy.     

Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells

Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Hyperinsulinaemia and hyperlipidaemia are hallmarks of the insulin-resistant state. We sought to determine the contributions of high insulin and saturated fatty acid exposure to mitochondrial function and biogenesis in cultured myocytes. Differentiated C2C12 myotubes were left untreated or exposed to chronic high insulin or high palmitate. Mitochondrial function was determined assessing: oxygen consumption, mitochondrial membrane potential, ATP content and ROS (reactive oxygen species) production. We also determined the expression of several mitochondrial genes. Chronic insulin treatment of myotubes caused insulin resistance with reduced PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) signalling. Insulin treatment increased oxygen consumption but reduced mitochondrial membrane potential and ROS production. ATP cellular levels were maintained through an increased glycolytic rate. The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced. In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38. Fatty acids reduced oxygen consumption and mitochondrial membrane potential while up-regulating the expression of mitochondrial ETC (electron chain complex) protein subunits and UCP proteins. Mfn-2 expression was not modified by palmitate. Palmitate-treated cells also showed a reduced glycolytic rate. Taken together, our findings indicate that chronic insulin and fatty acid-induced insulin resistance differentially affect mitochondrial function. In both conditions, cells were able to maintain ATP levels despite the loss of membrane potential; however, different protein expression suggests different adaptation mechanisms.     

Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells

Here we report that in skeletal muscle cells the contribution to insulin resistance and inflammation of two common dietary long-chain fatty acids depends on the channeling of these lipids to distinct cellular metabolic fates. Exposure of cells to the saturated fatty acid palmitate led to enhanced diacylglycerol levels and the consequent activation of the protein kinase C/nuclear factor kappaB pathway, finally resulting in enhanced interleukin 6 secretion and down-regulation of the expression of genes involved in the control of the oxidative capacity of skeletal muscle (peroxisome proliferator-activated receptor (PPAR)gamma-coactivator 1alpha) and triglyceride synthesis (acyl-coenzyme A: diacylglycerol acyltransferase 2). In contrast, exposure to the monounsaturated fatty acid oleate did not lead to these changes. Interestingly, co-incubation of cells with palmitate and oleate reversed both inflammation and impairment of insulin signaling by channeling palmitate into triglycerides and by up-regulating the expression of genes involved in mitochondrial beta-oxidation, thus reducing its incorporation into diacylglycerol. Our findings support a model of cellular lipid metabolism in which oleate protects against palmitate-induced inflammation and insulin resistance in skeletal muscle cells by promoting triglyceride accumulation and mitochondrial beta-oxidation through PPARalpha- and protein kinase A-dependent mechanisms.