Novel aspects of adipocyte-induced skeletal muscle insulin resistance

Insulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one of the major contributing factors. Conditioned medium (CM) from differentiated human adipocytes impairs insulin signalling in human skeletal muscle cells. Recent data on adipocyte-induced insulin resistance in skeletal muscle cells describes underlying mechanisms of this process. Skeletal muscle insulin resistance involves multiple pathways and irreversible changes in the expression level of critical proteins. Furthermore, the reversibility of insulin resistance could be demonstrated. Several strategies to combat insulin resistance have been developed. One recent approach to treat obesity and the metabolic syndrome is the use of endocannabinoid receptor antagonists such as rimonabant. These compounds might also reduce insulin resistance in type 2 diabetes with effects on adipose tissue and liver and possibly skeletal muscle.     

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.     

Glucosamine and glucose induce insulin resistance by different mechanisms in rat skeletal muscle

It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy. J Biol Chem 276: 20101-20107, 2001), we found that insulin resistance developed even when the increase in UDP-Hex-NAcs was prevented. Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation, as well as the rat hemidiaphragm, to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D-inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3 h resulted in an approximately fivefold increase in UDP-HexNAcs, an approximately 50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3 h or with 10 mM glucosamine for 5 h also caused large decreases in insulin responsiveness of glucose transport but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine-induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the binding of PI 3-kinase to IRS-1 were decreased approximately 60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.     

Lipid oversupply,selective insulin resistance,and lipotoxicity: Molecular mechanisms

The accumulation of fat in tissues not suited for lipid storage has deleterious consequences on organ function, leading to cellular damage that underlies diabetes, heart disease, and hypertension. To combat these lipotoxic events, several therapeutics improve insulin sensitivity and/or ameliorate features of metabolic disease by limiting the inappropriate deposition of fat in peripheral tissues (i.e. thiazolidinediones, metformin, and statins). Recent advances in genomics and lipidomics have accelerated progress towards understanding the pathogenic events associated with the excessive production, underutilization, or inefficient storage of fat. Herein we review studies applying pharmacological or genetic strategies to manipulate the expression or activity of enzymes controlling lipid deposition, in order to gain a clearer understanding of the molecular mechanisms by which fatty acids contribute to metabolic disease.     

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.     

Rapid hyperpolarization of rat skeletal muscle induced by insulin

It has been proposed that the increase produced by insulin in electrical potential differences across membranes of target cells may be a mechanism by which the cell surface insulin-receptor complex causes at least some of the metabolic effects of insulin. If insulin-induced hyperpolarization is a transducer of common effector responses it must precede those responses. The problem has not been addressed previously, so that rapid responses to insulin have not been sought. Two methods were used. In one method, the bathing solution was changed rapidly so as to include insulin in supramaximal concentrations, and a series of measurements of membrane potentials, E_r, were made. Insulin hyperpolarized by 9.4 mV within 1 min. In the other method, nanoliter amounts of highly concentrated insulin solution were ejected from a micropipette onto the surface of an impaled muscle fiber. In 21 out of 32 insulin injections, hyperpolarization occurred within 1 s; in 11 control injections there was no change. This is the most rapid response to insulin yet reported, and is consistent with the hypothesis that insulin-induced hyperpolarization may transduce effector responses.     

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.     

Effect of pioglitazone on skeletal muscle lipid deposition in the insulin resistance rat model induced by high fructose diet under AMPK signaling pathway

To study the changes of lipid deposition in skeletal muscle of insulin resistance rat and the effect of pioglitazone intervention on the expression of AMPK pathway related genes in rat, a rat model of insulin resistance was induced and constructed by high fructose diet as an test group, and normal rats were used as a control group. First, the effect of pioglitazone intervention on serum lipids-related indicators and mRNA expression levels of fat-related genes in skeletal muscle in rats was investigated. Then skeletal muscle sections were made and stained with oil red O to investigate the effect of pioglitazone intervention on lipid deposition in skeletal muscle of rats. Finally, the effects of pioglitazone intervention therapy on the mRNA and protein expression of related genes in the AMPK signaling pathway in skeletal muscle tissue of rat were explored by real-time quantitative PCR (qRT-PCR) and Western-blotting technology. The results showed that the blood glucose (BG), insulin (INS), adiponectin (ADPN), free fatty acid (FFA), triglyceride (TG), and cholesterol (TC) levels in serum of the test group were higher than the control group (P < 0.05); the visceral fat weight and abdominal fat index of the test group were significantly higher than the control group (P < 0.01); after the pioglitazone intervention, all blood lipid-related indexes in the rat model were significantly lower than before the intervention (P < 0.05); skeletal muscle section staining results showed that the number of lipid droplets in skeletal muscle of rat model was significantly reduced after pioglitazone intervention; and pioglitazone intervention can significantly increase the mRNA and protein expression levels of p-ACC, GLUT7, PGC-1α, and CPT1 genes in the skeletal muscles of experimental rats (P < 0.05). Accordingly, it can be concluded that pioglitazone can play a role in treating insulin resistance by regulating the expression of related genes of AMPK, ACC, etc. in the AMPK signaling pathway.     

CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK

Ciliary neurotrophic factor (CNTF) induces weight loss and improves glucose tolerance in humans and rodents. CNTF is thought to act centrally by inducing hypothalamic neurogenesis to modulate food intake and peripherally by altering hepatic gene expression, in a manner similar to that of leptin. Here, we show that CNTF signals through the CNTFRalpha-IL-6R-gp130beta receptor complex to increase fatty-acid oxidation and reduce insulin resistance in skeletal muscle by activating AMP-activated protein kinase (AMPK), independent of signaling through the brain. Thus, our findings further show that the antiobesogenic effects of CNTF in the periphery result from direct effects on skeletal muscle, and that these peripheral effects are not suppressed by diet-induced or genetic models of obesity, an essential requirement for the therapeutic treatment of obesity-related diseases.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

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.     

Maternal obesity-impaired insulin signaling in sheep and induced lipid accumulation and fibrosis in skeletal muscle of offspring

The prevalence of maternal obesity is increasing rapidly in recent decades. We previously showed that maternal obesity affected skeletal muscle development during the fetal stage. The objective of this study was to evaluate the effects of maternal obesity on the skeletal muscle properties of offspring. Ewes were fed a control diet (100% energy requirement, Con) or an obesogenic diet (150% energy requirement, OB) from 2 mo before pregnancy to weaning. After weaning, the offspring lambs were fed a maintenance diet until 19 mo of age and then ad libitum for 12 wk to measure feed intake. At 22 mo old, the longissimus dorsi (LD) muscle was biopsied. The downstream insulin signaling was lower in OB than Con lambs as shown by reduction in the phosphorylation of protein kinase B, mammalian target of rapamycin, and 4-E binding protein 1. On the other hand, the phosphorylation of protein kinase C and insulin receptor substrate 1 was higher in OB compared to Con lambs. More intramuscular adipocytes were observed in OB compared to Con offspring muscle, and the expression of peroxisome proliferator-activated receptor gamma, an adipocyte marker, was also higher, which was consistent with the higher intramuscular triglyceride content. Both fatty acid transport protein 1 and cluster of differentiation 36 (also known as fatty acid translocase) were increased in the OB group. In addition, higher collagen content was also detected in OB compared to Con offspring. In conclusion, our data show that offspring from obese mothers had impaired insulin signaling in muscle compared with control lambs, which correlates with increased intramuscular triglycerides and higher expression of fatty acid transporters. These data clearly show that maternal obesity impairs the function of the skeletal muscle of offspring, supporting the fetal programming of adult metabolic diseases.     

Mitofusion-2-mediated alleviation of insulin resistance in rats through reduction in lipid intermediate accumulation in skeletal muscle

Background

Increased lipid accumulation and mitochondrial dysfunction within skeletal muscle have been shown to be strongly associated with insulin resistance. However, the role of mitofusion-2 (MFN2), a key factor in mitochondrial function and energy metabolism, in skeletal muscle lipid intermediate accumulation remains to be elucidated.

Results

A high-fat diet resulted in insulin resistance as well as accumulation of cytosolic lipid intermediates and down-regulation of MFN2 and CPT1 in skeletal muscle in rats, while MFN2 overexpression improved insulin sensitivity and reduced lipid intermediates in muscle, possibly by upregulation of CPT1 expression.

Conclusions

MFN2 overexpression can rescue insulin resistance, possibly by upregulating CPT1 expression leading to reduction in the accumulation of lipid intermediates in skeletal muscle. These observations contribute to the investigations of new diabetes therapies.     

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.     

Prevention of skeletal muscle insulin resistance by dietary cod protein in high fat-fed rats

In the present study, we tested the hypothesis that fish protein may represent a key constituent of fish with glucoregulatory activity. Three groups of rats were fed a high-fat diet in which the protein source was casein, fish (cod) protein, or soy protein; these groups were compared with a group of chow-fed controls. High-fat feeding led to severe whole body and skeletal muscle insulin resistance in casein- or soy protein-fed rats, as assessed by the euglycemic clamp technique coupled with measurements of 2-deoxy-D-[(3)H]glucose uptake rates by individual tissues. However, feeding cod protein fully prevented the development of insulin resistance in high fat-fed rats. These animals exhibited higher rates of insulin-mediated muscle glucose disposal that were comparable to those of chow-fed rats. The beneficial effects of cod protein occurred without any reductions in body weight gain, adipose tissue accretion, or expression of tumor necrosis factor-alpha in fat and muscle. Moreover, L6 myocytes exposed to cod protein-derived amino acids showed greater rates of insulin-stimulated glucose uptake compared with cells incubated with casein- or soy protein-derived amino acids. These data demonstrate that feeding cod protein prevents obesity-induced muscle insulin resistance in high fat-fed obese rats at least in part through a direct action of amino acids on insulin-stimulated glucose uptake in skeletal muscle cells.     

Re-evaluating lipotoxic triggers in skeletal muscle: relating intramyocellular lipid metabolism to insulin sensitivity

Ectopic fat accumulation has been linked to lipotoxic events, including the development of insulin resistance in skeletal muscle. Indeed, intramyocellular lipid storage is strongly associated with the development of type 2 diabetes. Research during the last two decades has provided evidence for a role of lipid intermediates like diacylglycerol and ceramide in the induction of lipid-induced insulin resistance. However, recently novel data has been gathered that suggest that the relation between lipid intermediates and insulin resistance is less straightforward than has been previously suggested, and that there are several routes towards lipid-induced insulin resistance. For example, research in this field has shifted towards imbalances in lipid metabolism and lipid droplet dynamics. Next to imbalances in key lipogenic and lipolytic proteins, lipid droplet coat proteins appear to be essential for proper intramyocellular lipid storage, turnover and protection against lipid-induced insulin resistance.Here, we discuss the current knowledge on lipid-induced insulin resistance in skeletal muscle with a focus on the evidence from human studies. Furthermore, we discuss the available data that provides supporting mechanistic information.     

Differing endoplasmic reticulum stress response to excess lipogenesis versus lipid oversupply in relation to hepatic steatosis and insulin resistance

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by ～60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (～40-60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (～60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance.     

Signalling and the control of skeletal muscle size

Skeletal muscle is highly adaptive to environmental stimuli and can alter its mass accordingly. This tissue is almost unique in that it can increase its size through two distinct mechanisms. It can grow through a cellular process mediated by cell fusion, or it can increase its size simply by increasing its protein content. Understanding how these processes are regulated is crucial for the development of potential therapies against debilitating skeletal muscle wasting diseases. Two key signalling molecules, Insulin like Growth Factor (IGF) and GDF-8/myostatin, have emerged in recent years to be potent regulators of skeletal muscle size. In this review we bring together recent data highlighting the important and novel aspects of both molecules and their signalling pathways, culminating in a discussion of the cellular and tissue phenotypic outcomes of their stimulation or antagonism. We emphasise the complex regulatory mechanisms and discuss the temporal and spatial differences that control their action, understanding of which is crucial to further their use as potential therapeutic targets.