Toll-like receptor 4 modulates skeletal muscle substrate metabolism

Abstract

Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.

Fig. 1.

Activation of toll-like receptor 4 (TLR4) in C₂C₁₂ skeletal muscle cells modulates glucose and fatty acid (FA) metabolism. Radiolabeled substrates (glucose and palmitate) were used to measure glucose and FA metabolism in C₂C₁₂ cells following 2 h of lipopolysaccharide (LPS) treatment (500 ng/ml). In response to LPS, there was an increase in glucose uptake (A), glucose oxidation (B), and lactate presence (C) in the media. LPS treatment also resulted in a decrease in total FA oxidation {complete (CO₂) and incomplete [acid soluble metabolites (ASMs)]} (D), increased FA partitioning to neutral lipid depots (E), and reductions in citrate synthase (CS; F) and β-hydroxyacyl-CoA dehydrogenase (β-HAD; G) activities. Data are presented as means ± SE. *P < 0.05, control vs. LPS.

Fig. 2.

Activation of TLR4 in human primary skeletal muscle cells modulates glucose and FA metabolism. Radiolabeled substrates (glucose and palmitate) were used to measure glucose and FA metabolism in human primary skeletal muscle cells following 2 h of LPS treatment (500 ng/ml). In response to LPS, there was an increase in glucose oxidation (A), a decrease in FA oxidation (B), and an increase partitioning of FAs toward neutral lipid depots (C). Data are presented as means ± SE. *P < 0.05, control vs. LPS.

Fig. 3.

LPS dose and time course experiments. Radiolabeled substrates (glucose and palmitate) were used to measure glucose and FA metabolism in C₂C₁₂ cells treated with either 50 or 500 ng/ml of LPS for 2, 6, 12, and 24 h. In response to either dose of LPS, glucose oxidation was increased (A) and FA oxidation was reduced (B) with no dose effects. Data are presented as means ± SE. *P < 0.05, control vs. LPS.

Fig. 4.

Activation of TLR4 via ultra pure LPS and lipid A also modulates glucose and FA metabolism. C₂C₁₂ cells were treated with ultra pure LPS (500 ng/ml) or lipid A (1 μg/ml), the active constituent of LPS, for 2 h. Both ultra pure LPS and lipid A resulted in an increase in glucose oxidation (A and D), a decrease in FA oxidation (B and E), and increased FA partitioning to neutral lipid depots (C and F). Data are presented as means ± SE. *P < 0.05, control vs. LPS.

Fig. 5.

Overexpression of TLR4 in C₂C₁₂ skeletal muscle cells results in an enhanced LPS-mediated shift in substrate metabolism. C₂C₁₂ cells were generated with a stable overexpression of the TLR4/MD2 and treated (500 ng/ml) with LPS for 2 h, and oxidation of [U-¹⁴C]glucose and [1-¹⁴C]palmitic acid was assessed. Relative to empty vector controls, the stable cell line possessed higher TLR4 mRNA (A) and protein (B) levels, and the LPS-induced percent changes in glucose and FA metabolism were more robust (C). Data are expressed as means ± SE. *P < 0.05, control vs. LPS.

Fig. 6.

The effect of TLR4 activation on substrate metabolism is partially due to activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). C₂C₁₂ cells were treated with either 50 pg/ml (A and B) or 500 ng/ml (C and D) of LPS for 2 h in the presence and absence 20 μM of the IκB kinase (IKK) inhibitor parthenolide. The presence of parthenolide either partially or completely blocked the effects of LPS on glucose oxidation and FA oxidation. Data are expressed as means ± SE. *P < 0.05, control vs. LPS.

Fig. 7.

High dose of LPS, but not low dose, results in increased expression of proinflammatory cytokines in human primary myotubes. Human primary myotubes were treated with either 50 pg/ml or 500 ng/ml of LPS for 2 h and then harvested for gene expression analysis of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP1). mRNA levels of IL-6 and MCP1 were increased ∼4- and ∼5-fold, respectively, in response to 500 ng/ml of LPS (A and B); however, TNF-α mRNA remained unchanged. There was no effect 50 pg/ml on IL-6, MCP1, or TNF-α mRNA. Human primary myotubes were then treated with either 50 pg/ml or 500 ng/ml of LPS for 2 h. At the 2-h time point, medium was collected to measure TNF-α, IL-6, and MCP1 concentrations. Cells were washed, and medium was then sampled at 3, 6, 12, and 24 h after the initial treatment with LPS. There was no effect of either low- or high-dose LPS on TNF-α concentration at any time point following treatment (C). Media concentrations of IL-6 (D) and MCP1 (E) were significantly increased at all time points following the high-dose LPS treatment. There was no effect on IL-6 or MCP1 concentrations at any time point following low-dose LPS treatment. Data are expressed as fold change relative to control treatments ± SE. P < 0.05, control vs. LPS (*) and low- vs. high-dose LPS (#).

Fig. 8.

Potential mechanism(s) for TLR4 regulation of substrate metabolism. C₂C₁₂ cells were treated with LPS as described above, cell lysates were harvested, and Western blotting was performed. Treatment with LPS resulted in no changes in peroxisome proliferator-activated receptor (PPAR)α (57 kDa) and PPARβ (50 kDa), liver X receptor (LXR)α (50 kDa) and LXRβ (56 kDa), CCAAT-enhancer-binding protein (C/EPB)β (37 kDa) and C/EBPδ (29 kDa), and β-actin (45 kDa).

Fig. 9.

Loss of TLR4 function is associated with an increased capacity to oxidize FAs in skeletal muscle. Gastrocnemius skeletal muscles were extracted from control (C3HeB/FeJ, n = 9) and TLR4-mutant (C3H/HeJ, n = 8) mice, and whole muscle homogenates were prepared for measures of [1-¹⁴C]palmitic acid oxidation and enzyme activities of CS and β-HAD. FA oxidation (A), CS activity (B), and β-HAD activity (C) were all higher in the TLR4-mutant mice relative to control mice. Serum free fatty acids (D) and triacylglycerols (E) were lower in the TLR4-mutant mice. Data are presented as means ± SE. *P < 0.05.

Fig. 10.

LPS-induced shift in muscle substrate metabolism occurs in vivo and is TLR4 dependent. At 8 wk of age, control (C3HeB/FeJ, n = 14) and TLR4-mutant (C3H/HeJ, n = 14) mice were injected with either saline (n = 7/group) or 1 mg/kg (∼25 μg/mouse) of LPS (n = 7/group) and killed 4 h postinjection. Gastrocnemius skeletal muscle was harvested and dissected into red and white portions. LPS treatment resulted in robust increases and decreases in LPS-induced percent changes in glucose (A) and FA (B) oxidation, respectively, which was completely blocked in the TLR4-mutant animals. These effects coincided with an increase in phosphofructokinase (PFK) activity (C) and decreased in CS (D) and β-HAD (E) activities in control mice, which were also blocked in the TLR4-mutant animals. Serum triacylglycerols (G) were also significantly increased following LPS injection in the control animals, which was blocked in the TLR4-mutant animals. Finally, although not significant, there was a trend for a decrease in serum free fatty acid (F) and an increase in plasma glucose (H) in the control animal. Data are presented as means ± SE. *P < 0.05.