Edward W. Kraegen

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.

The Metabolic Syndrome, which includes obesity and type 2 diabetes, is reaching alarming proportions. A key factor is insulin resistance, defined as a reduced ability of insulin to stimulate glucose utilization and storage. Compelling evidence links insulin resistance with an excess fatty acid supply over energy need, resulting in lipid accumulation in non-adipose tissues. The AMPK pathway plays a key role in sensing and regulating tissue energy metabolism, influencing fuel metabolism in tissues including muscle and liver. A number of its actions could improve muscle insulin sensitivity at least partly by increasing fatty acid oxidation and diminishing synthesis of malonyl CoA, glycerolipids, ceramide and other molecules linked to insulin resistance, although the extent of these effects, particularly in the human context, is uncertain. Secondly, its activation could bypass the metabolic block associated with insulin resistance. Thirdly, it is possible that a dysregulation of the AMPK pathway may itself contribute to the metabolic derangement associated with insulin resistance. These issues are important in considering the AMPK pathway as a therapeutic target in insulin resistant states.

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. 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. 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.

Insulin resistance of skeletal muscle is a major defect in obesity and type 2 diabetes. Insulin resistance has been associated with a chronic subclinical inflammatory state in epidemiological studies and specifically with activation of the inhibitor κB kinase (IκBK)-nuclear factor-κB (NF-κB) pathway. However, it is unclear whether this pathway plays a role in mediating insulin resistance in muscle in vivo. We separately overexpressed the p65 subunit of NF-κB and IκBKβ in single muscles of rats using in vivo electrotransfer and compared the effects after 1 wk vs. paired contralateral control muscles. A 64% increase in p65 protein ( P < 0.001) was sufficient to cause muscle fiber atrophy but had no effect on glucose disposal or glycogen storage in muscle under hyperinsulinemic-euglycemic clamp conditions. Similarly, a 650% increase in IκBKβ expression ( P < 0.001) caused a significant reduction in IκB protein but also had no effect on clamp glucose disposal after lipid infusion. In fact, IκBKβ overexpression in particular caused increases in activating tyrosine phosphorylation of insulin receptor substrate-1 (24%; P = 0.02) and serine phosphorylation of Akt (23%; P < 0.001), implying a moderate increase in flux through the insulin signaling cascade. Interestingly, p65 overexpression resulted in a negative feedback reduction of 36% in Toll-like receptor (TLR)-2 ( P = 0.03) but not TLR-4 mRNA. In conclusion, activation of the IκBKβ-NF-κB pathway in muscle does not seem to be an important local mediator of insulin resistance.

The phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3β phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.

Glucose infusion in rats for 1–4 days results in insulin resistance and increased triglyceride, whole tissue long-chain fatty acyl-CoA (LCA-CoA), and malonyl-CoA content in red skeletal muscle. Despite this, the relation between these alterations and the onset of insulin resistance has not been defined. We aimed to 1) identify whether the changes in these lipids and of diacylglycerol (DAG) precede or accompany the onset of insulin resistance in glucose-infused rats, 2) determine whether the insulin resistance is associated with alterations in AMP-activated protein kinase (AMPK), and 3) assess whether similar changes occur in liver and in muscle. Hyperglycemia (17–18 mM) was maintained by intravenous glucose infusion in rats for 3 or 5 h; then euglycemia was restored and a 2-h hyperinsulinemic clamp was performed. Significant ( P < 0.01) muscle and liver insulin resistance first appeared in red quadriceps and liver of the glucose-infused group at 5 h and was associated with a twofold increase in DAG and malonyl-CoA content and a 50% decrease in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and AMPK activity. White quadriceps showed qualitatively similar changes but without decreases in AMPK or ACC phosphorylation. Triglyceride mass was increased at 5 h only in liver, and whole tissue LCA-CoA content was not increased in liver or either muscle type. We conclude that the onset of insulin resistance induced by glucose oversupply correlates temporally with increases in malonyl-CoA and DAG content in all three tissues and with reduced AMPK phosphorylation and activity in red muscle and liver. In contrast, it was not associated with increased whole tissue LCA-CoA content in any tissue or triglyceride in muscle, although both are observed at later times.

Agonists of peroxisome proliferator-activated receptors (PPARs) have emerged as important pharmacological agents for improving insulin action. A major mechanism of action of PPAR agonists is thought to involve the alteration of the tissue distribution of nonesterified fatty acid (NEFA) uptake and utilization. To test this hypothesis directly, we examined the effect of the novel PPARα/γ agonist tesaglitazar on whole-body insulin sensitivity and NEFA clearance into epididymal white adipose tissue (WAT), red gastrocnemius muscle, and liver in rats with dietary-induced insulin resistance. Wistar rats were fed a high-fat diet (59% of calories as fat) for 3 wk with or without treatment with tesaglitazar (1 μmol·kg−1·d−1, 7 d). NEFA clearance was measured using the partially metabolizable NEFA tracer, 3H-R-bromopalmitate, administered under conditions of basal or elevated NEFA availability. Tesaglitazar improved the insulin sensitivity of high-fat-fed rats, indicated by an increase in the glucose infusion rate during hyperinsulinemic-euglycemic clamp (P < 0.01). This improvement in insulin action was associated with decreased diglyceride (P < 0.05) and long chain acyl coenzyme A (P < 0.05) in skeletal muscle. NEFA clearance into WAT of high-fat-fed rats was increased 52% by tesaglitazar under basal conditions (P < 0.001). In addition the PPARα/γ agonist moderately increased hepatic and muscle NEFA utilization and reduced hepatic triglyceride accumulation (P < 0.05). This study shows that tesaglitazar is an effective insulin-sensitizing agent in a mild dietary model of insulin resistance. Furthermore, we provide the first direct in vivo evidence that an agonist of both PPARα and PPARγ increases the ability of WAT, liver, and skeletal muscle to use fatty acids in association with its beneficial effects on insulin action in this model.

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.