Ian R. W. Ritchie

Adiponectin resistance precedes the accumulation of skeletal muscle lipids and insulin resistance in high-fat-fed rats

High-fat (HF) diets can induce insulin resistance (IR) by altering skeletal muscle lipid metabolism. An imbalance between fatty acid (FA) uptake and oxidation results in intramuscular lipid accumulation, which can impair the insulin-signaling cascade. Adiponectin (Ad) is an insulin-sensitizing adipokine known to stimulate skeletal muscle FA oxidation and reduce lipid accumulation. Evidence of Ad resistance has been shown in obesity and following chronic HF feeding and may contribute to lipid accumulation observed in these conditions. Whether Ad resistance precedes and is associated with the development of IR is unknown. We conducted a time course HF feeding trial for 3 days, 2 wk, or 4 wk to determine the onset of Ad resistance and identify the ensuing changes in lipid metabolism and insulin signaling leading to IR in skeletal muscle. Ad stimulated FA oxidation (+28%, P < or = 0.05) and acetyl-CoA carboxylase phosphorylation (+34%, P < or = 0.05) in control animals but failed to do so in any HF-fed group (i.e., as early as 3 days). By 2 wk, plasma membrane FA transporters and intramuscular diacylglycerol (DAG) and ceramide were increased, and insulin-stimulated phosphorylation of both protein kinase B and protein kinase B substrate 160 was blunted compared with control animals. After 4 wk of HF feeding, maximal insulin-stimulated glucose transport was impaired compared with control. Taken together, our results demonstrate that an early loss of Ads stimulatory effect on FA oxidation precedes an increase in plasmalemmal FA transporters and the accumulation of intramuscular DAG and ceramide, blunted insulin signaling, and ultimately impaired maximal insulin-stimulated glucose transport in skeletal muscle induced by HF diets.

IL-6 indirectly modulates the induction of glyceroneogenic enzymes in adipose tissue during exercise.

Background Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls. Methodology and Principle Findings Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6−/− mice. Conclusions and Significance Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.

Restoration of skeletal muscle leptin response does not precede the exercise-induced recovery of insulin-stimulated glucose uptake in high-fat-fed rats

Leptin administration increases fatty acid (FA) oxidation rates and decreases lipid storage in oxidative skeletal muscle, thereby improving insulin response. We have previously shown high-fat (HF) diets to rapidly induce skeletal muscle leptin resistance, prior to the disruption of normal muscle FA metabolism (increase in FA transport; accumulation of triacylglycerol, diacylglycerol, ceramide) that occurs in advance of impaired insulin signaling and glucose transport. All of this occurs within a 4-wk period. Conversely, exercise can rapidly improve insulin response, in as little as one exercise bout. Thus, if the early development of leptin resistance is a contributor to HF diet-induced insulin resistance (IR) in skeletal muscle, then it is logical to predict that the rapid restoration of insulin response by exercise training would be preceded by the recovery of leptin response. In the current study, we sought to determine 1) whether 1, 2, or 4 wk of exercise training was sufficient to restore leptin response in isolated soleus muscle of rats already consuming a HF diet (60% kcal), and 2) whether this preceded the training-induced corrections in FA metabolism and improved insulin-stimulated glucose transport. In the low-fat (LF)-fed control group, insulin increased glucose transport by 153% and leptin increased AMPK and ACC phosphorylation and the rate of palmitate oxidation (+73%). These responses to insulin and leptin were either severely blunted or absent following 4 wk of HF feeding. Exercise intervention decreased muscle ceramide content (-28%) and restored insulin-stimulated glucose transport to control levels within 1 wk; muscle leptin response (AMPK and ACC phosphorylation, FA oxidation) was also restored, but not until the 2-wk time point. In conclusion, endurance exercise training is able to restore leptin response, but this does not appear to be a necessary precursor for the restoration of insulin response.

Oral administration of a PPAR-δ agonist to rodents worsens, not improves, maximal insulin-stimulated glucose transport in skeletal muscle of different fibers.

Agonists targeting the nuclear receptor peroxisome proliferator-activated receptors (PPAR)-delta may be potential therapeutic agents for insulin-resistant related conditions, as they may be able to stimulate fatty acid (FA) oxidation and attenuate the accumulation of harmful lipid species in skeletal muscle. Several reports have demonstrated that PPAR-delta agonists improve whole body insulin sensitivity. However, whether these agonists exert their direct effects on glucose and FA metabolism in skeletal muscle, and specifically with different fiber types, is unknown. This study was undertaken to determine the effects of oral treatment with the PPAR-delta agonist, GW 501516, in conjunction with the administration of a high-saturated-fat diet on insulin-stimulated glucose transport in isolated oxidative (soleus) and glycolytic (epitrochlearis) rodent skeletal muscle in vitro. High-fat feeding significantly decreased maximal insulin-stimulated glucose transport in soleus, but not epitrochlearis muscle, and was associated with increased skeletal muscle diacylglycerol and ceramide content. Unexpectedly, treatment with the PPAR-delta agonist significantly reduced insulin-stimulated glucose transport in both soleus and epitrochlearis muscles, regardless of dietary fat content. The reduction in insulin-stimulated glucose transport induced by the agonist was associated with large increases in total muscle fatty acid translocase (FAT)/CD36protein content, but not diacylglycerol or ceramide contents. Agonist treatment did not alter the protein content of PPAR-delta, GLUT4, or insulin-signaling proteins (IRS-1, p85 PI3-K, Akt). Agonist treatment led to a small, but significant increase, in the oxidative capacity of glycolytic but not oxidative muscle. We propose that chronic treatment with the PPAR-delta agonist GW 501516 may induce or worsen insulin resistance in rodent skeletal muscle by increasing the capacity for FA transport across the sarcolemma without a sufficient compensatory increase in FA oxidation. However, an accumulation of diacylglycerol and ceramide, while associated with diet-induced insulin resistance, does not appear to be responsible for the agonist-induced reduction in insulin-stimulated glucose transport.

Submaximal ADP‐stimulated respiration is impaired in ZDF rats and recovered by resveratrol

•  Disparity exists within the literature surrounding mitochondrial dysfunction and insulin resistance and previous reports have primarily examined mitochondrial function as a capacity measurement. •  We show that submaximal ADP‐stimulated respiration rates are lower in ZDF rats, which coincides with decreased adenine nucleotide translocase 2 (ANT2) protein content. •  Supplementation of ZDF rats with resveratrol improves skeletal muscle insulin sensitivity, increases submaximal ADP‐stimulated respiration rates and increases ANT2 protein content. •  Improvements in the ability of ADP to attenuate mitochondrial reactive oxygen species (ROS) emission and cellular redox balance were also observed following resveratrol supplementation. •  These data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for ROS formation.

Rapid Loss of Adiponectin-Stimulated Fatty Acid Oxidation in Skeletal Muscle of Rats Fed a High Fat Diet Is Not Due to Altered Muscle Redox State

A high fat (HF) diet rapidly impairs the ability of adiponectin (Ad) to stimulate fatty acid (FA) oxidation in oxidative soleus muscle, but the underlying mechanism remains elusive. Mere days of HF feeding also increase the muscle’s production and accumulation of reactive oxygen species (ROS) and shift cellular redox to a more oxidized state. It seems plausible that this shift towards a more oxidized state might act as negative feedback to suppress the ability of Ad to stimulate FA oxidation and generate more ROS. Therefore, we sought to determine whether i) a shift towards a more oxidized redox state (reduction in GSH/2GSSG) coincided with impaired Ad-stimulated palmitate oxidation in oxidative and glycolytic rodent muscle after 5 days of HF feeding (60% kCal), and ii) if supplementation with the antioxidant, N-acetylcysteine (NAC) could prevent the HF-diet induced impairment in Ad-response. Globular Ad (gAd) increased palmitate oxidation in isolated soleus and EDL muscles by 42% and 34%, respectively (p<0.05) but this was attenuated with HF feeding in both muscles. HF feeding decreased total GSH (−26%, p<0.05) and GSH/2GSSG (−49%, p<0.05) in soleus, but not EDL. Supplementation with NAC prevented the HF diet-induced reductions in GSH and GSH/2GSSG in soleus, but did not prevent the loss of Ad response in either muscle. Furthermore, direct incubations with H2O2 did not impair Ad-stimulated FA oxidation in either muscle. In conclusion, our data indicates that skeletal muscle Ad resistance is rapidly induced in both oxidative and glycolytic muscle, independently of altered cellular redox state.

Recovered insulin response by 2 weeks of leptin administration in high-fat fed rats is associated with restored AS160 activation and decreased reactive lipid accumulation.

Leptin is an adipokine that increases fatty acid (FA) oxidation, decreases intramuscular lipid stores, and improves insulin response in skeletal muscle. In an attempt to elucidate the underlying mechanisms by which these metabolic changes occur, we administered leptin (Lep) or saline (Sal) by miniosmotic pumps to rats during the final 2 wk of a 6-wk low-fat (LF) or high-fat (HF) diet. Insulin-stimulated glucose transport was impaired by the HF diet (HF-Sal) but was restored with leptin administration (HF-Lep). This improvement was associated with restored phosphorylation of Akt and AS160 and decreased in reactive lipid species (ceramide, diacylglycerol), known inhibitors of the insulin-signaling cascade. Total muscle citrate synthase (CS) activity was increased by both leptin and HF diet, but was not additive. Leptin increased subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria CS activity. Total muscle, sarcolemmal, and mitochondrial (SS and IMF) FA transporter (FAT/CD36) protein content was significantly increased with the HF diet, but not altered by leptin. Therefore, the decrease in reactive lipid stores and subsequent improvement in insulin response, secondary to leptin administration in rats fed a HF diet was not due to a decrease in FA transport protein content or altered cellular distribution.

Adiponectin is sufficient, but not required, for exercise‐induced increases in the expression of skeletal muscle mitochondrial enzymes

Adiponectin is a regulator of skeletal muscle mitochondrial biogenesis. Previous research using the ob/ob mouse (leptin deficient, low adiponectin) has suggested that the presence of adipokines, including adiponectin, is necessary for exercise‐induced increases in mitochondrial content. In the current study, we examined the importance of adiponectin as a regulator of skeletal muscle mitochondrial content in response to exercise by comparing wildtype and adiponectin deficient mice. Adiponectin deficient mice showed no differences in resting VO2 , RER, or time to exhaustion during exercise when compared to wildtype mice. There were no differences in various protein markers of mitochondrial content. A single bout of treadmill running increased the mRNA expression of mitochondrial proteins similarly in wildtype and adiponectin deficient mice. Chronic exercise (8 weeks) also increased the protein content of mitochondrial markers similarly in wildtype and adiponectin deficient mice. We conclude that Ad is not required for exercise‐induced increases in muscle mitochondrial proteins.

Adiponectin is not required for exercise training‐induced improvements in glucose and insulin tolerance in mice

Adiponectin (Ad) is a potent insulin‐sensitizing adipokine that has been found to activate pathways involved in the adaptation to exercise. Therefore, we examined whether Ad is required for the increased insulin response observed following exercise training in Ad knockout mice (AdKO). Eight weeks of exercise training significantly increased glucose and insulin tolerance in both wild type (WT) and AdKO mice. There were no differences in glucose tolerance between genotypes but insulin tolerance was improved to a greater extent in AdKO compared to WT mice following exercise training (+26%, P < 0.05). There were no genotype differences in the insulin‐stimulated phosphorylation of AKT or AS160 in red or white gastrocnemius muscle (RG, WG). Exercise training increased total AKT and AS160 protein content in RG and total AS160 protein content in WG. There were no genotype differences in total AKT or AS160. However, exercise training induced a more robust increase in total AS160 in RG from AdKO (+44 ± 8%, P < 0.05) compared to WT mice (+28 ± 7%, P = 0.06). There were no differences in total GLUT4 or FAT/CD36 in RG or WG in WT or AdKO, with or without exercise training. Similarly, there were no differences in RER, VO2, or activity between any groups. Our results indicate the presence of Ad is not required for exercise‐induced increases in insulin response. Furthermore, it appears that exercise may improve insulin sensitivity to a greater extent in the absence of Ad, suggesting the presence of an unknown compensatory mechanism.