Exercise improves insulin sensitivity. As AMP-activated protein kinase (AMPK) plays an important role in muscle metabolism during exercise, we investigated the effects of the AMPK activator 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) on insulin action in insulinresistant high-fat-fed (HF) rats. Rats received a subcutaneous injection of 250 mg/kg AICAR (HF-AIC) or saline (HF-Con). The next day, euglycemic-hyperinsulinemic clamp studies were performed. Glucose infusion rate during the clamp was enhanced (50%) in HF-AIC compared with HF-Con rats. Insulin-stimulated glucose uptake was improved in white but not in red quadriceps, whereas glycogen synthesis was improved in both red and white quadriceps of HF-AIC rats. HF-AIC rats also showed increased insulin suppressibility of hepatic glucose output (HGO). AICAR-induced responses in both liver and muscle were accompanied by reduced malonyl-CoA content. Clamp HGO correlated closely with hepatic triglyceride content (r ‫؍‬ 0.67, P < 0.01). Thus, a single dose of AICAR leads to an apparent enhancement in whole-body, muscle, and liver insulin action in HF rats that extends beyond the expected time of AMPK activation. Whether altered tissue lipid metabolism mediates AICAR effects on insulin action remains to be determined. Follow-up studies suggest that at least some of the post-AICAR insulin-enhancing effects also occur in normal rats. Independent of this, the results suggest that pharmacological activation of AMPK may have potential in treating insulin-resistant states and type 2 diabetes.

OBJECTIVEMedium-chain fatty acids (MCFAs) have been reported to be less obesogenic than long-chain fatty acids (LCFAs); however, relatively little is known regarding their effect on insulin action. Here, we examined the tissue-specific effects of MCFAs on lipid metabolism and insulin action.RESEARCH DESIGN AND METHODSC57BL6/J mice and Wistar rats were fed either a low-fat control diet or high-fat diets rich in MCFAs or LCFAs for 4–5 weeks, and markers of mitochondrial oxidative capacity, lipid levels, and insulin action were measured.RESULTSMice fed the MCFA diet displayed reduced adiposity and better glucose tolerance than LCFA-fed animals. In skeletal muscle, triglyceride levels were increased by the LCFA diet (77%, P < 0.01) but remained at low-fat diet control levels in the MCFA-fed animals. The LCFA diet increased (20–50%, P < 0.05) markers of mitochondrial metabolism in muscle compared with low-fat diet–fed controls; however; the increase in oxidative capacity was substantially greater in MCFA-fed animals (50–140% versus low-fat–fed controls, P < 0.01). The MCFA diet induced a greater accumulation of liver triglycerides than the LCFA diet, likely due to an upregulation of several lipogenic enzymes. In rats, isocaloric feeding of MCFA or LCFA high-fat diets induced hepatic insulin resistance to a similar degree; however, insulin action was preserved at the level of low-fat diet–fed controls in muscle and adipose from MCFA-fed animals.CONCLUSIONSMCFAs reduce adiposity and preserve insulin action in muscle and adipose, despite inducing steatosis and insulin resistance in the liver. Dietary supplementation with MCFAs may therefore be beneficial for preventing obesity and peripheral insulin resistance.

Aims/hypothesis We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. Methods Prkce −/− and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes.Results While fat-fed WT mice became glucose intolerant after 1 week, Prkce −/− mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce −/− animals. Liver triacylglycerols were increased in fat-fed Prkce −/− mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce −/− mice. Prkce −/− hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce −/− mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. Conclusions/interpretation Prkce deletion ameliorates dietinduced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.

Lipid-induced insulin resistance is associated with intracellular accumulation of inhibitory intermediates depending on the prevalent fatty acid (FA) species. In cultured myotubes, ceramide and phosphatidic acid (PA) mediate the effects of the saturated FA palmitate and the unsaturated FA linoleate, respectively. We hypothesized that myriocin (MYR), an inhibitor of de novo ceramide synthesis, would protect against glucose intolerance in saturated fat-fed mice, while lisofylline (LSF), a functional inhibitor of PA synthesis, would protect unsaturated fat-fed mice. Mice were fed diets enriched in saturated fat, n-6 polyunsaturated fat, or chow for 6 wk. Saline, LSF (25 mg/kg ⅐ d), or MYR (0.3 mg/kg ⅐ d) were administered by mini-pumps in the final 4 wk. Glucose homeostasis was examined by glucose tolerance test. Muscle ceramide and PA were analyzed by mass spectrometry. Expression of LASS isoforms (ceramide synthases) was evaluated by immunoblotting. Both saturated and polyunsaturated fat diets increased muscle ceramide and induced glucose intolerance. MYR and LSF reduced ceramide levels in saturated and unsaturated fat-fed mice. Both inhibitors also improved glucose tolerance in unsaturated fat-fed mice, but only LSF was effective in saturated fat-fed mice. The discrepancy between ceramide and glucose tolerance suggests these improvements may not be related directly to changes in muscle ceramide and may involve other insulin-responsive tissues. Changes in the expression of LASS1 were, however, inversely correlated with alterations in glucose tolerance. The demonstration that LSF can ameliorate glucose intolerance in vivo independent of the dietary FA type indicates it may be a novel intervention for the treatment of insulin resistance. (Endocrinology 151: 4187-4196, 2010)

Aims/hypothesis This study aimed to determine whether protein kinase C (PKC) δ plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCε in the generation of insulin resistance in skeletal muscle. Methods Prkcd−/−

Our aim was to determine the importance of changes in phosphorylation of key insulin signaling intermediates in the insulin resistance observed in skeletal muscle of rats fed diets high in saturated or n-6 polyunsaturated fat. We used phospho-specific antibodies to measure the time course of phosphorylation of key components of the insulin signaling pathway by immunoblotting during the initial stages of a physiological elevation in the circulating insulin concentration. The phosphorylation of insulin receptor at Tyr1162/1163 (IR Tyr1162/1163) increased over 20 min of insulin infusion, whereas the downstream phosphorylation of insulin receptor substrate-1 Tyr612 (IRS-1 Tyr612) peaked at 5 min and declined thereafter. Interestingly, phosphorylation of IRS-1 at Tyr895 continued to increase over the 20-min period, and protein kinase B (PKB) phosphorylation at Ser473 reached a plateau by 5 min, demonstrating that different profiles of phosphorylation are involved in transmission of the insulin signal despite a constant level of insulin stimulation. In muscle from rats fed high n-6 polyunsaturated or saturated fat diets, however, there was no insulin-stimulated increase in IRS-1 Tyr612 phosphorylation and a temporal difference in PKB Ser473 phosphorylation despite no difference in IR Tyr1162/1163 phosphorylation, IRS-1 Tyr895 phosphorylation, and ERK phosphorylation. These results demonstrate that under conditions of increased insulin, similar to those used to assess insulin action in vivo, chronic high-fat feeding impairs insulin signal transduction related to glucose metabolism at the level of IRS-1 Tyr612 and PKB Ser473 and that these effects are independent of the type of fat used in the high-fat diet.

Thiazolidinediones lower lipids, but it is unclear whether this is essential for their insulin-sensitizing action. We investigated relationships between lipid-lowering and insulin-sensitizing actions of a thiazolidinedione. Normal rats were pretreated with or without Pioglitazone (Pio, 3 mg/kg.d) for 2 wk. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp with elevation of free fatty acids (FFA) by Intralipid/heparin infusion over 6 h. In untreated rats insulin sensitivity decreased by 46% over 3-6 h of elevated FFA, whereas it remained normal but with a 50% increase in FFA clearance in Pio-treated rats. After matching plasma FFA, insulin sensitivity was still partially (30%) protected in Pio-treated rats, substantially by maintaining insulin suppressibility of hepatic glucose output. This was associated with lower hepatic long-chain acyl-coenzyme A. Plasma adiponectin was increased 2-fold in Pio-treated rats and was negatively correlated with hepatic glucose output (r2 = 0.70, P < 0.001) and liver long-chain acyl-coenzyme A (r2 = 0.39, P < 0.005). Pio-induced muscle insulin sensitization was largely diminished after matching plasma FFA elevation, but insulin-stimulated protein kinase B phosphorylation was protected. We conclude that thiazolidinediones can protect against lipid-induced insulin resistance with a significant component (mainly liver) of the protective effect not requiring lipid lowering. This may be related to chronic elevation of adiponectin by thiazolidinediones.

Regular physical exercise is well known to improve glucose and lipid metabolism in skeletal muscle. However, the transcription factors regulating these adaptive changes are not well-characterised. Recently the nuclear orphan receptor nur77 was shown to be induced by exercise and linked to regulation of metabolic gene expression in skeletal muscle. In this study we investigated the regulation of nur77 in muscle by different exercise-activated pathways. Nur77 expression was found to be responsive to adrenergic stimulation and calcium influx, but not to activation of the AMP dependent kinase. These results identify the adrenergic-cyclic AMP-PKA pathway to be the most potent activator of nur77 expression in muscle and therefore the likely cause of increased expression after exercise. We also identified nur77 expression to be reduced in the muscle of obese/insulin resistant rats after high fat feeding. Furthermore exposure to fatty acids, insulin or inflammation was not the cause of decreased nur77 expression in insulin resistant muscle. This suggests a reduced responsiveness to adrenergic stimulation as the likely cause of diminished nur77 expression in muscle of high fat fed rats, which has been observed in obese/insulin resistant individuals. Our results suggest adrenergic stimulation as the most important stimulus for nur77 expression and point to a significant role for this transcription factor in adaptive changes in muscle after exercise and in insulin resistant states.