Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects

Abstract

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 kappaB (IkappaB)/nuclear factor kappaB (NFkappaB). 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 (IkappaB/NFkappaB) 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 IkappaB/NFkappaB 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 IkappaBalpha content, an indication of elevated IkappaB/NFkappaB signaling. The increase in TLR4 and NFkappaB signaling was accompanied by elevated expression of the NFkappaB-regulated genes interleukin (IL)-6 and superoxide dismutase (SOD)2. In primary human myotubes, acute palmitate treatment stimulated IkappaB/NFkappaB, and blockade of TLR4 prevented the ability of palmitate to stimulate the IkappaB/NFkappaB 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 NFkappaB. CONCLUSIONS- Abnormal TLR4 expression and signaling, possibly caused by elevated plasma FFA levels, may contribute to the pathogenesis of insulin resistance in humans.

FIG. 1.

TLR4 and TLR2 gene expression and protein content. TLR4 mRNA expression (A) and protein content (B) were measured in lean, obese, and type 2 diabetic (T2DM) subjects. Data are means ± SE in 7 lean, 8 obese, and 14 type 2 diabetic subjects in A and 6 lean, 8 obese, and 10 type 2 diabetic subjects in B (protein extracts were not available for all subjects). C: Correlation between TLR4 content and HOMA-IR. D: TLR2 mRNA expression in 7 lean, 8 obese, and 14 type 2 diabetic subjects. *P < 0.05 vs. lean. E: Direct Western blotting in muscle from three lean and three type 2 diabetic subjects using an antibody against human TLR2 (eBiosciences). A band corresponding to TLR2 was detected in mononuclear cells (MNCs) isolated from two subjects.

FIG. 2.

TLR4 signaling. IκBα protein abundance (A) and mRNA expression of IL-6 (B) and SOD2 (C) were measured in lean, obese, and type 2 diabetic (T2DM) subjects. Data are means ± SE. *P < 0.05; †P = 0.05 vs. lean.

FIG. 3.

Effect of FFAs on IκB and IKK activity. Human myotubes were treated with palmitate (Palm), and IκBα content (A) and phosphorylation (B) were measured by Western blotting. IKK activity was measured after myotubes were treated with 400 μmol/l palmitate, 100 ng/ml LPS, or 20 ng/ml human TNF-α for 1 h (C). IκBα phosphorylation was measured after myotubes were treated with 400 μmol/l stearate (Stear) or 400 μmol/l linoleate (Lino) for 1 h (D). Data are means ± SE, n = 6–9 per group. *P < 0.05 vs. BSA.

FIG. 4.

Effect of TLR neutralizing antibodies. Human myotubes were incubated with TLR4 (A) or TLR2 (B) neutralizing antibodies for 1 h and then stimulated with 400 μmol/l palmitate (Palm) for 1 h. Data are means ± SE, n = 6–9 per group. *P < 0.05 vs. BSA of isotype control group, †P < 0.05 vs. palmitate of isotype control group, and ‡P < 0.05 vs. BSA of anti-TLR2 group.

FIG. 5.

Palmitate (Palm) increases TLR4 content and gene expression. Human myotubes were incubated with 200 μmol/l palmitate for 3 days (A) or 6 h (B), and TLR4 protein content (A) and mRNA expression (B) were measured by Western blotting and real-time PCR, respectively. TLR4 gene expression was measured after treating myotubes with synthetic monophosphoryl lipid A (10 μg/ml) for 6 h (C). Data are means ± SE, n = 6–9 per group. *P < 0.05 vs. BSA/control.

FIG. 6.

Palmitate induces an inflammatory response via NFκB. Human myotubes were transduced with adenovirus-IκB supperrepressor (SR)-HA or adenovirus-GFP for 4 h, and 48 h later, cells were stimulated with 200 μmol/l palmitate for 6 h. IκBα was detected by Western blotting (A), NFκB p65 activity was measured by ELISA (B), and IL-6 (C), SOD2 (D), and TLR4 (E) gene expression were quantitated by real-time PCR. Data are means ± SE, n = 9 per group. *P < 0.05 vs. BSA of GFP group and †P < 0.05 vs. palmitate of GFP group.