Elevated Toll-Like Receptor 4 Expression and Signaling in Muscle From Insulin-Resistant Subjects

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

Effect of lipopolysaccharide on inflammation and insulin action in human muscle.

Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = -0.46, P = 0.005) and LBP (r = -0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals

Short-Term Exercise Training Improves Insulin Sensitivity but Does Not Inhibit Inflammatory Pathways in Immune Cells from Insulin-Resistant Subjects

Background. Exercise has an anti-inflammatory effect against, and immune cells play critical roles in the development, of insulin resistance and atherosclerotic vascular disease (AVD). Thus, the goal of this study was to determine whether exercise improves insulin sensitivity in insulin-resistant subjects by downregulating proinflammatory signaling in immune cells. Methods. Seventeen lean, 8 obese nondiabetic, and 11 obese type 2 diabetic individuals underwent an aerobic exercise program for 15 days and an insulin clamp before and after exercise. Peripheral mononuclear cells (PMNC) were obtained for determination of Toll-like receptor (TLR) 2 and 4 protein content and mitogen-activated protein kinase phosphorylation. Results. Compared with that in lean individuals, TLR4 protein content was increased by 4.2-fold in diabetic subjects. This increase in TLR4 content was accompanied by a 3.0-fold increase in extracellular signal-regulated kinase (ERK) phosphorylation. Exercise improved insulin sensitivity in the lean, obese, and type 2 diabetes groups. However, exercise did not affect TLR content or ERK phosphorylation. Conclusions. TLR4 content and ERK phosphorylation are increased in PMNC of type 2 diabetic individuals. While exercise improves insulin sensitivity, this effect is not related to changes in TLR2/TLR4 content or ERK phosphorylation in PMNC of type 2 diabetic individuals

Plasma LPS, LBP and sCD14 levels in insulin resistant subjects.

<p>Plasma LPS (A), LBP (B) and sCD14 (C) concentrations were determined as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063983#s2" target="_blank">materials and methods</a>’. All values are the mean ± SEM of data obtained from 12 lean, 9 obese and 10 T2DM subjects. *<i>P</i><0.05 compared to lean controls.</p

TLR4 silencing protects against LPS-induced inflammation and insulin resistance in human myotubes.

<p>Cells were transfected with TLR4 siRNA and negative control siRNA as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063983#s2" target="_blank">materials and methods</a>’. TLR4 mRNA (A) and protein content (B) were determined by quantitative real time PCR and Western blotting, respectively. Transfected myotubes were either left untreated or treated with 100 ng/ml LPS for 12 h. Glucose uptake (C), JNK phosphorylation (D), and MCP-1 (E) and IL-6 (F) mRNA expression was determined as described above. The absolute basal glucose uptake rates were 30.1±3.2, 35.4±3.9, 30.0±2.1, and 30.7±1.7 pmol/mg,min in the negative control siRNA transfected cells without LPS, negative control siRNA transfected cells with LPS, TLR4 siRNA transfected cells without LPS and TLR4 siRNA transfected cells with LPS, respectively. Results for glucose uptake are expressed as the mean ± SEM of data obtained from 4 subjects. Other values are the mean ± SD of triplicate determinations. Similar experiments were repeated 4 times using cells isolated from different subjects, and representative results are shown. *<i>P</i><0.05; #<i>P</i><0.05 compared to basal.</p

LPS induces inflammation and inhibits insulin signaling in human myotubes in a time-dependent manner.

<p>Human myotubes were either left untreated or treated with 100 ng/ml LPS for a varying length of time. The time course of IκBα protein degradation (A), JNK phosphorylation (B), and insulin-stimulated IRS-1-tyr612 (E), Akt (F), and AS160 (G) phosphorylation following LPS treatment was determined by Western blotting. The time course of MCP-1 (C) and IL-6 (D) mRNA expression following LPS treatment was determined by quantitative real time PCR. All values are the mean ± SD of triplicates. *<i>P</i><0.05; #<i>P</i><0.05 compared to basal.</p

TAK-242 prevents LPS-induced inflammation and insulin resistance in human myotubes.

<p>Human myotubes were either left untreated or treated with 100 ng/ml LPS for 12 h. Prior to LPS exposure, cells were pre-treated with 1 µM TAK-242 or vehicle control for 1h. JNK phosphorylation (A), insulin-stimulated IRS-1-tyr612 (E), Akt (F), and AS160 (G) phosphorylation were determined by Western blotting. MCP-1 (B) and IL-6 (C) mRNA expression was determined by real time PCR. Glucose transport (D) was determined by measuring ³H-2-DG uptake. The absolute basal glucose transport rates were 43.2±3.2, 46.4±4.1, 46.7±5.6 and 46.5±6.8 pmol/mg,min in cells without LPS, cells with LPS, TAK-242-pretreated cells without LPS, and TAK-242-pretreated cells with LPS, respectively. Results for glucose transport are expressed as the mean ± SEM of data obtained from 5 subjects. All the rest of the values are the mean ± SD of triplicate determinations. Similar experiments were repeated 4 times using cells isolated from different subjects, and representative results are shown. *<i>P</i><0.05; #<i>P</i><0.05 compared to basal.</p

Clinical and laboratory characteristics.

<p>All values are mean ± SEM.</p>*<p><i>P</i><0.05 vs. Lean. BMI: body mass index; FPG: fasting plasma glucose; FPI: fasting plasma insulin.</p

Effect of acute exercise on glycogen synthase in muscle from obese and diabetic subjects

Insulin stimulates glycogen synthase (GS) through dephosphorylation of serine residues, and this effect is impaired in skeletal muscle from insulin-resistant [obese and type 2 diabetic (T2DM)] subjects. Exercise also increases GS activity, yet it is not known whether the ability of exercise to affect GS is impaired in insulin-resistant subjects. The objective of this study was to examine the effect of acute exercise on GS phosphorylation and enzyme kinetic properties in muscle from insulin-resistant individuals. Lean normal glucose-tolerant (NGT), obese NGT, and obese T2DM subjects performed 40 min of moderate-intensity cycle exercise (70% of V̇o(2max)). GS kinetic properties and phosphorylation were measured in vastus lateralis muscle before exercise, immediately after exercise, and 3.5 h postexercise. In lean subjects, GS fractional activity increased twofold after 40 min of exercise, and it remained elevated after the 3.5-h rest period. Importantly, exercise also decreased GS K(m) for UDP-glucose from ≈0.5 to ≈0.2 mM. In lean subjects, exercise caused significant dephosphorylation of GS by 50–70% (Ser(641), Ser(645), and Ser(645,649,653,657)), and phosphorylation of these sites remained decreased after 3.5 h; Ser(7) phosphorylation was not regulated by exercise. In obese NGT and T2DM subjects, exercise increased GS fractional activity, decreased K(m) for UDP-glucose, and decreased GS phosphorylation as effectively as in lean NGT subjects. We conclude that the molecular regulatory process by which exercise promotes glycogen synthesis in muscle is preserved in insulin-resistant subjects

Whole body overexpression of PGC-1α has opposite effects on hepatic and muscle insulin sensitivity

Type 2 diabetes is characterized by fasting hyperglycemia, secondary to hepatic insulin resistance and increased glucose production. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a transcriptional coactivator that is thought to control adaptive responses to physiological stimuli. In liver, PGC-1α expression is induced by fasting, and this effect promotes gluconeogenesis. To examine whether PGC-1α is involved in the pathogenesis of hepatic insulin resistance, we generated transgenic (TG) mice with whole body overexpression of human PGC-1α and evaluated glucose homeostasis with a euglycemic-hyperinsulinemic clamp. PGC-1α was moderately (∼2-fold) overexpressed in liver, skeletal muscle, brain, and heart of TG mice. In liver, PGC-1α overexpression resulted in increased expression of hepatocyte nuclear factor-4α and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. PGC-1α overexpression caused hepatic insulin resistance, manifested by higher glucose production and diminished insulin suppression of gluconeogenesis. Paradoxically, PGC-1α overexpression improved muscle insulin sensitivity, as evidenced by elevated insulin-stimulated Akt phosphorylation and peripheral glucose disposal. Content of myoglobin and troponin I slow protein was increased in muscle of TG mice, indicating fiber-type switching. PGC-1α overexpression also led to lower reactive oxygen species production by mitochondria and reduced IKK/IκB signaling in muscle. Feeding a high-fat diet to TG mice eliminated the increased muscle insulin sensitivity. The dichotomous effect of PGC-1α overexpression in liver and muscle suggests that PGC-1α is a fuel gauge that couples energy demands (muscle) with the corresponding fuel supply (liver). Thus, under conditions of physiological stress (i.e., prolonged fast and exercise training), increased hepatic glucose production may help sustain glucose utilization in peripheral tissues