Effect of lipopolysaccharide on inflammation and insulin action in human muscle

Abstract

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.

Figure 1. Plasma LPS, LBP and sCD14 levels in insulin resistant subjects.

Plasma LPS (A), LBP (B) and sCD14 (C) concentrations were determined as described under ‘materials and methods’. All values are the mean ± SEM of data obtained from 12 lean, 9 obese and 10 T2DM subjects. *P<0.05 compared to lean controls.

Figure 2. LPS induces inflammation and inhibits insulin signaling in human myotubes in a time-dependent manner.

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. *P<0.05; #P<0.05 compared to basal.

Figure 3. TAK-242 prevents LPS-induced inflammation and insulin resistance in human myotubes.

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. *P<0.05; #P<0.05 compared to basal.

Figure 4. TLR4 silencing protects against LPS-induced inflammation and insulin resistance in human myotubes.

Cells were transfected with TLR4 siRNA and negative control siRNA as described under ‘materials and methods’. 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. *P<0.05; #P<0.05 compared to basal.