OBJECTIVE— Tall-like receptor (TLR)4 has been implicated in the pathogenesis of free fatty acid (FFA)-induced insulin resistance by activating inflammatory pathways, including inhibitor of κB (IκB)/nuclear factor κB (NFκB). However, it is not known whether insulin-resistant subjects have abnormal TLR4 signaling. We examined whether insulin-resistant subjects have abnormal TLR4 expression and TLR4-driven (IκB/NFκB) signaling in skeletal muscle. RESEARCH DESIGN AND METHODS— TLR4 gene expression and protein content were measured in muscle biopsies in 7 lean, 8 obese, and 14 type 2 diabetic subjects. A primary human myotube culture system was used to examine whether FFAs stimulate IκB/NFκB via TLR4 and whether FFAs increase TLR4 expression/content in muscle. RESULTS— Obese and type 2 diabetic subjects had significantly elevated TLR4 gene expression and protein content in muscle. TLR4 muscle protein content correlated with the severity of insulin resistance. Obese and type 2 diabetic subjects also had lower IκBα content, an indication of elevated IκB/NFκB signaling. The increase in TLR4 and NFκB signaling was accompanied by elevated expression of the NFκB-regulated genes interleukin (IL)-6 and superoxide dismutase (SOD)2. In primary human myotubes, acute palmitate treatment stimulated IκB/NFκB, and blockade of TLR4 prevented the ability of palmitate to stimulate the IκB/NFκB pathway. Increased TLR4 content and gene expression observed in muscle from insulin-resistant subjects were reproduced by treating myotubes from lean, normal-glucose-tolerant subjects with palmitate. Palmitate also increased IL-6 and SOD2 gene expression, and this effect was prevented by inhibiting NFκB. CONCLUSIONS— Abnormal TLR4 expression and signaling, possibly caused by elevated plasma FFA levels, may contribute to the pathogenesis of insulin resistance in humans.

Activation of AMP-activated protein kinase (AMPK) by exercise induces several cellular processes in muscle. Exercise activation of AMPK is unaffected in lean (BMI ∼25 kg/m²) subjects with type 2 diabetes. However, most type 2 diabetic subjects are obese (BMI >30 kg/m²), and exercise stimulation of AMPK is blunted in obese rodents. We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator–activated receptor coactivator (PGC)-1α, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% Vo_2max) and moderate (70% Vo_2max) intensities, with a 4–6 week interval. Exercise had no effect on LKB1 activity. Exercise had a time- and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise-induced increases in PGC-1 expression. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects.

The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle.

Aims/hypothesis TNF-α levels are increased in obesity and type 2 diabetes. The regulation of TNF-α converting enzyme (TACE) and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), in human type 2 diabetes is unknown. Methods We examined TACE/TIMP3 regulation: (1) in lean and obese normal glucose tolerant (NGT) individuals and in type 2 diabetes patients; (2) following 6 h of lipid/saline infusion in NGT individuals; and (3) in cultured human myotubes from lean NGT individuals incubated with palmitate. Insulin sensitivity was assessed by a euglycaemic clamp and TACE/TIMP3 was evaluated by confocal microscopy, RT-PCR, western blotting and an in vitro activity assay. Circulating TNF-α, TNF-α-receptor 1 (TNFR1), TNF-α-receptor 2 (TNFR2), IL-6 receptor (IL-6R), vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) levels were evaluated. Results TIMP3 levels were reduced and TACE enzymatic activity was increased in type 2 diabetes skeletal muscle. TACE expression, and TACE, TNF-α, TNFR1 and IL-6R levels were increased in type 2 diabetes, and positively correlated with insulin resistance. A 6 h lipid infusion into NGT individuals decreased insulin-stimulated glucose metabolism by 25% with increased TACE, decreased expression of the gene encoding TIMP3 and increased IL-6R release. Palmitate induced a dramatic reduction of TIMP3 and increased the TACE/TIMP3 ratio in cultured myotubes. Conclusions/interpretation TACE activity was increased in skeletal muscle of obese type 2 diabetes patients and in lipid-induced insulin resistance. We propose that dysregulation of membrane proteolysis by TACE/TIMP3 of TNF-α and IL-6R is an important factor for the development of skeletal muscle insulin resistance in obese type 2 diabetes patients by a novel autocrine/paracrine mechanism.

This study was undertaken to test the hypothesis that short-term exposure (4 h) to physiological hyperinsulinemia in normal, healthy subjects without a family history of diabetes would induce a low grade inflammatory response independently of glycemic status. Twelve normal glucose tolerant subjects received a 4-h euglycemic hyperinsulinemic clamp with biopsies of the vastus lateralis muscle. Microarray analysis identified 121 probe sets that were significantly altered in response to physiological hyperinsulinemia while maintaining euglycemia. In normal, healthy human subjects insulin increased the mRNAs of a number of inflammatory genes (CCL2, CXCL2 and THBD) and transcription factors (ATF3, BHLHB2, HES1, KLF10, JUNB, FOS, and FOSB). A number of other genes were upregulated in response to insulin, including RRAD, MT, and SGK. CITED2, a known coactivator of PPARα, was significantly downregulated. SGK and CITED2 are located at chromosome 6q23, where we previously detected strong linkage to fasting plasma insulin concentrations. We independently validated the mRNA expression changes in an additional five subjects and closely paralleled the results observed in the original 12 subjects. A saline infusion in healthy, normal glucose-tolerant subjects without family history of diabetes demonstrated that the genes altered during the euglycemic hyperinsulinemic clamp were due to hyperinsulinemia and were unrelated to the biopsy procedure per se. The results of the present study demonstrate that insulin acutely regulates the levels of mRNAs involved in inflammation and transcription and identifies several candidate genes, including HES1 and BHLHB2, for further investigation.

Insulin resistance in skeletal muscle is a prominent feature of obesity and type 2 diabetes. The association between mitochondrial changes and insulin resistance is well known. More recently, there is growing evidence of a relationship between inflammation, extracellular remodeling, and insulin resistance. The intent of this review is to propose a potentially novel mechanism for the development of insulin resistance, focusing on the underappreciated connections among inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle. Several sources of inflammation, including expansion of adipose tissue resulting in increased lipolysis and alterations in pro- and anti-inflammatory cytokines, contribute to the insulin resistance observed in obesity and type 2 diabetes. In the experimental model of lipid oversupply, an inflammatory response in skeletal muscle leads to altered expression extracellular matrix-related genes as well as nuclear encoded mitochondrial genes. A similar pattern also is observed in “naturally” occurring insulin resistance in muscle of obese nondiabetic individuals and patients with type 2 diabetes mellitus. More recently, alterations in proteins (including α-actinin-2, desmin, proteasomes, and chaperones) involved in muscle structure and function have been observed in insulin-resistant muscle. Some of these cytoskeletal proteins are mechanosignal transducers that allow muscle fibers to sense contractile activity and respond appropriately. The ensuing alterations in expression of genes coding for mitochondrial proteins and cytoskeletal proteins may contribute to the mitochondrial changes observed in insulin-resistant muscle. These changes in turn may lead to a reduction in fat oxidation and an increase in intramyocellular lipid, which contributes to the defects in insulin signaling in insulin resistance.