TLR4 links innate immunity and fatty acid-induced insulin resistance

Abstract

TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. Here we show that nutritional fatty acids, whose circulating levels are often increased in obesity, activate TLR4 signaling in adipocytes and macrophages and that the capacity of fatty acids to induce inflammatory signaling in adipose cells or tissue and macrophages is blunted in the absence of TLR4. Moreover, mice lacking TLR4 are substantially protected from the ability of systemic lipid infusion to (a) suppress insulin signaling in muscle and (b) reduce insulin-mediated changes in systemic glucose metabolism. Finally, female C57BL/6 mice lacking TLR4 have increased obesity but are partially protected against high fat diet-induced insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat. Taken together, these data suggest that TLR4 is a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.

Figure 1. FFAs activate TLR4 signaling.

(A) FFAs activate TLR4 signaling in transfected 293T cells (n = 6; *P < 0.01). 293T cells were transiently transfected with TLR4/MD-2 expression vectors, with or without dominant negative MyD88 (MyD88-DN), and an NF-κB luciferase reporter and were then treated with a 200 μM oleate/palmitate mixture or 100 ng/ml LPS as a positive control. (B) FFAs cause IκBα degradation and JNK phosphorylation in WT but not TLR4-deficient macrophages. Peritoneal macrophages were isolated and precultured for 4 days before treatment. Cells were treated with 500 μM palmitate over the time course indicated. NS, nonspecific. (C and D) FFAs induce TNF-α and IL-6 mRNA in peritoneal macrophages in WT but not TLR4^–/– mice (n = 4; *P < 0.01). Peritoneal macrophages were treated with 200 μM FFA mixture for 8 hours. Real-time RT-PCR was used to measure mRNA levels. Data are expressed as mean ± SEM.

Figure 2. FFAs stimulate cytokine expression in macrophages.

(A) Saturated FFAs induce IL-6 mRNA in the RAW264.7 macrophage cell line. Cells were treated with the indicated FFAs (200 μM) for 8 hours. Saturated fatty acids include C12:0, C14:0, C16:0, and C18:0. AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid. (B) Palmitate stimulates TNF-α expression in a dose-dependent manner. RAW264.7 cells were treated with the indicated doses of palmitate or LPS for 8 hours. (C) The polyunsaturated fatty acid DHA blocks saturated FFA-induced TNF-α expression. RAW264.7 cells were pretreated with 200 μM DHA for 2 hours and were then treated with 200 μM of various saturated FFAs for 8 hours. Real-time RT-PCR was used to measure mRNA levels. Data are expressed as mean ± SEM.

Figure 3. Adipocytes express functional TLR4, and TLR4 expression is increased in adipose tissue of obese models.

(A) Adipocytes express TLR4. Northern blotting was used to detect TLR4 mRNA expression. Lane 1: 3T3-L1 preadipocytes; lane 2: 3T3-L1 adipocytes; lane 3: stromal-vascular cells; lane 4: mouse adipocytes; lane 5: mouse adipose tissue; lane 6: RAW264.7 macrophages. (B and C) TLR4 mRNA expression is increased in fat pads from DIO and ob/ob and db/db mice (n = 4; *P < 0.05). TLR4 mRNA was measured using real-time RT-PCR. (D) The TLR4 agonist LPS induces TLR2 expression in adipocytes. (E) LPS but not zymosan stimulates IL-6 mRNA expression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 100 ng/ml LPS or 40 μg/ml zymosan for 8 hours. Real-time RT-PCR was conducted to measure TLR2 and IL-6 mRNA levels. Data are expressed as mean ± SEM; n = 6, *P < 0.05.

Figure 4. FFAs cause inflammatory response via TLR4 in adipocytes.

(A–C) Generation of an adipocyte model with specific TLR4 knockdown. 3T3-L1 preadipocytes were infected with retroviral short hairpin RNA interference (shRNAi) to knock down TLR4 (TLR4-KD), and cells were selected and then differentiated into adipocytes. TLR4 mRNA (A) and protein (B) and TLR2 mRNA (C) levels were evaluated by real-time RT-PCR and immunoblotting. Data are expressed as mean ± SEM; n = 6; *P < 0.05. (D and E) FFAs stimulate IL-6 and TNF-α mRNA expression in 3T3-L1 adipocytes via TLR4. TLR4-knockdown and scramble control adipocytes were treated with 400 μM FFA (palmitate and oleate mixture) or 100 ng/ml LPS for 12 hours. Real-time RT-PCR was conducted to measure the mRNA levels. n = 4; *P < 0.05. (F and G) FFAs stimulate TNF-α and IL-6 mRNA in WT but not in TLR4^–/– adipocytes (n = 4; *P < 0.05). (H and I) FFAs stimulate TNF-α and IL-6 protein secretion in WT but not in TLR4^–/– adipocytes (n = 4; *P < 0.05). Mouse adipocytes were isolated and precultured for 6 hours and then were treated with 400 μM FFA mixture for 16 hours. Real-time RT-PCR was used to measure mRNA levels. Data are expressed as mean ± SEM.

Figure 5. Lipid infusion activates TLR4 signaling in adipose tissue in vivo.

(A) Schematic diagram of lipid infusion and inflammatory signaling studies. (B) Lipid infusion increases NF-κB DNA binding in adipose tissue of WT but not TLR4^–/– mice. Mice were fasted overnight and infused with lipid (coupled with heparin) for 8 hours. EMSA was conducted to examine the NF-κB DNA binding activity. (C) Lipid infusion increases NF-κB DNA binding to the IL-6 promoter. ChIP assays were conducted to quantify NF-κB DNA binding to the IL-6 promoter. SYBR Green quantitative PCR was used to measure the immunoprecipitated DNA (n = 3; *P < 0.01). (D–F) Lipid infusion increases TNF-α, IL-6, and MCP-1 mRNA expression in adipose tissue of WT but not TLR4^–/– mice (n = 4; *P < 0.05). Data are expressed as mean ± SEM.

Figure 6. TLR4 deficiency prevents impaired insulin signaling in skeletal muscle caused by lipid infusion.

(A) Schematic diagram of lipid infusion and in vivo insulin signaling studies. (B) Mice preinfused with lipid for 8 hours were i.v. injected with 10 U/kg BW of human insulin (Ins) or saline. Muscle lysates were immunoprecipitated and then immunoblotted with antibodies as indicated. (C) Mice were infused with lipid for 8 hours. Muscle lysates were immunoblotted with antibodies as indicated. Ser, serine; Tyr, tyrosine.

Figure 7. TLR4 deficiency prevents lipid-induced insulin resistance.

Mice were fasted overnight and preinfused with lipid for 5 hours. (A) Schematic diagram of lipid infusion and hyperinsulinemic-euglycemic clamp studies. Hyperinsulinemic-euglycemic clamps were conducted to examine the effect of lipid infusion on insulin sensitivity in TLR4^–/– and WT mice. (B) Glucose infusion rate. (C) Insulin-stimulated glucose turnover rate. (D) Insulin-stimulated glucose uptake in skeletal muscle (gastrocnemius). (E) Insulin-stimulated glucose uptake in white adipose tissue (epididymal fat). (F) Insulin-stimulated whole-body glycolysis. All data are expressed as mean ± SEM; n = 4–10; *P < 0.05.

Figure 8. Female mice lacking TLR4 show increased obesity but are partially protected against high-fat diet–induced (HFD-induced) insulin resistance, and HFD does not induce inflammatory gene expression in fat and liver in these mice.

(A) Body weights of WT and TLR4^–/– mice on HFD or chow diet. (B) Lean and fat tissue weight as assessed by dual-energy x-ray absorptiometry at 26 weeks on HFD. (C) Cumulative food intake. Daily food intake was measured for 1 week after 22 weeks on diet. (D) Insulin tolerance test. Insulin (1 mU/g BW) was administered to mice after 36 weeks on diet. HFD induces inflammatory gene expression in fat (E) and liver (F) in WT but not TLR4-knockout mice. Data are expressed as mean ± SEM (n = 7–9). *P < 0.05, WT versus TLR4^–/–; ^#P < 0.05 between groups as indicated.