Hepatocyte Toll-like receptor 4 regulates obesity-induced inflammation and insulin resistance

Abstract

Chronic low-grade inflammation is a hallmark of obesity and thought to contribute to the development of obesity-related insulin resistance. Toll-like receptor 4 (Tlr4) is a key mediator of pro-inflammatory responses. Mice lacking Tlr4s are protected from diet-induced insulin resistance and inflammation; however, which Tlr4-expressing cells mediate this effect is unknown. Here we show that mice deficient in hepatocyte Tlr4 (Tlr4LKO) exhibit improved glucose tolerance, enhanced insulin sensitivity and ameliorated hepatic steatosis despite the development of obesity after a high-fat diet (HFD) challenge. Furthermore, Tlr4LKO mice have reduced macrophage content in white adipose tissue, as well as decreased tissue and circulating inflammatory markers. In contrast, the loss of Tlr4 activity in myeloid cells has little effect on insulin sensitivity. Collectively, these data indicate that the activation of Tlr4 on hepatocytes contributes to obesity-associated inflammation and insulin resistance, and suggest that targeting hepatocyte Tlr4 might be a useful therapeutic strategy for the treatment of type 2 diabetes.

Figure 1. Generation of mice deficient in hepatocyte Tlr4

(a) PCR analysis of genomic DNA isolated from primary hepatocytes of Tlr4^fl/fl and Tlr4^LKO mice. (b) qPCR analysis of Tlr4 mRNA expression in primary hepatocytes isolated from chow-fed mice. (c) Lipopolysaccharide (LPS, 1mg kg⁻¹ body weight) was injected intraperitoneally to chow-fed mice. Blood was collected 1.5 h after LPS administration and plasma Tnfα levels were measured by the MILLIPLEX Mouse Cytokine/Chemokine assay (n = 5–8). (d) Levels are presented as a percentage relative to Tlr4^fl/fl levels. *p < 0.05, compared between Tlr4^LKO and Tlr4^fl/fl mice (Student’s t-test). All data are presented as means ± s.e.m.

Figure 2. Hepatocyte Tlr4 promotes HFD-induced insulin resistance in mice

(a) Glucose tolerance test (GTT, 1.2 mg g⁻¹ BW, n = 9–11) in 5-h fasted HFD-fed mice. (b) Insulin tolerance test (ITT, 1.5mU g⁻¹ BW, n = 8–10) in 5-h fasted HFD-fed mice. (c) Glucose stimulated insulin secretion in overnight fasted mice after 8 weeks of HFD feeding. Glucose (2 mg g⁻¹ BW) were injected intraperitoneally and plasma insulin concentrations were measured at the indicated time points (n = 6–7). (d–f) After 16 weeks HFD feeding, hyperinsulinemic-euglycemic (10 mU kg⁻¹ min⁻¹, 150 mg dl⁻¹, respectively) clamps of 120 minutes were performed in conscious, chronically catheterized, 4- to 5-hour-fasted Tlr4^fl/fl and Tlr4^LKO mice (n = 6–7). (d) Glucose infiltration rate (GIR) during the 120-minute clamp experiment. (e–f) Basal and insulin-stimulated (clamp steady-state [t = 80–120 minutes]) hepatic glucose production (e, HGP) and glucose disposal rate (f). (g) Western blotting of insulin stimulated Akt phosphorylation in epididymal fat pad from HFD-fed Tlr4^fl/fl and Tlr4^LKO mice. Representative western blot images are shown. *p < 0.05, **p<0.01, ***p<0.001, compared between Tlr4^fl/fl and Tlr4^LKO mice (Student’s t-test). All data are presented as means ± s.e.m.

Figure 3. Hepatocyte Tlr4 stimulates obesity-related tissue and systemic inflammation in mice

(a) qPCR analysis of genes in epididymal white adipose tissue (WAT) of HFD-fed mice (n = 4–6). (b) Mac2 staining of epididymal adipose tissues sections prepared from mice on HFD and quantitation of crown-like structures. Scale bars, 100 µm. (c) qPCR analysis of genes in the liver of HFD-fed mice (n = 4–6). (d) Plasma Tnfa, IL-6, IL-1β and Mcp1 levels in mice fed HFD (n = 12–16 for Tnfα and Mcp1; n = 9–10 for IL-1β and IL-6). *p < 0.05, compared between Tlr4^fl/fl and Tlr4^LKO mice (Student’s t-test). All data are presented as means ± s.e.m.

Figure 4. Hepatocyte Tlr4 promotes the development of hepatic steatosis and liver injury in mice

(a) Liver weight in mice fed either chow (n = 8) or HFD (n = 10) for 12 weeks. (b) Liver triglyceride contents (n = 8–9). (c) Liver cholesterol contents (n = 8–9). (d) Relative hepatic mRNA levels of genes in HFD-fed mice (n = 4–6). (e) Plasma ALT and AST levels (n = 10) from HFD-fed Tlr4^fl/fl and Tlr4^LKO mice. *p < 0.05, compared between Tlr4^fl/fl and Tlr4^LKO mice on the same diet; ^#p < 0.05, compared between different diets for mice of the same genotype (Student’s t-test). All data are presented as means ± s.e.m.

Figure 5. Generation of mice lacking Tlr4 in myeloid cells

(a) PCR analysis of genomic DNA isolated from the indicated tissues of 6-week-old Tlr4^ΔmΦ mice. (b) qPCR analysis of Tlr4 mRNA expression in peritoneal macrophage (PM) isolated from chow-fed mice. (c) Lipopolysaccharide (LPS, 1mg kg⁻¹ body weight) was injected intraperitoneally to chow-fed mice. Blood was collected 1.5 h after LPS administration and plasma Tnfα levels were measured (n = 5–6). (d) Levels are presented as a percentage relative of Tlr4^fl/fl levels. *p < 0.05, ***p<0.001, compared between Tlr4^ΔmΦ mice and Tlr4^fl/fl mice (Student’s t-test). All data are presented as means ± s.e.m.

Figure 6. Macrophage Tlr4 deficiency does not protect mice from diet-induced obesity or insulin resistance

(a) Body weight curves in Tlr4^fl/fl and Tlr4^ΔmΦ mice on a chow (n = 9–11) or high fat diet (HFD, n = 15) for 12 weeks. (b–c) Blood glucose (b) and plasma insulin (c) concentrations (n = 6–8) in Tlr4^fl/fl and Tlr4^ΔmΦ mice after 8 weeks of diet feeding under either fed or overnight fasting states. (d) Glucose tolerance test (GTT, 1.5 mg g⁻¹ BW, n = 5–9 for chow fed mice; n = 8 for mice on HFD) in overnight fasted mice. (e) Glucose stimulated insulin secretion in overnight fasted HFD-fed mice. Glucose (1.5 mg g⁻¹ BW) were injected intraperitoneally and plasma insulin concentrations were measured at the indicated time points (n = 8–9). (f) Insulin tolerance test (ITT, 1.5 mU g⁻¹ BW, n = 5–8 for chow fed mice; n = 11–13 for mice on HFD) in mice fasted for 5 h. *p < 0.05, compared between Tlr4^fl/fl and Tlr4^ΔmΦ mice on the same diet; ^#p < 0.05, ^##p < 0.01, compared between different diets for mice of the same genotype (Student’s t-test). All data are presented as means ± s.e.m.

Figure 7. Macrophage Tlr4 deficiency results in elevated circulating inflammatory cytokines in obese mice

(a) qPCR analysis of genes in epididymal white adipose tissue (WAT, n = 5–7). (b) Plasma Tnfa, IL-6, IL-1β and Mcp1 levels in mice fed HFD for 12 weeks (n = 10–13 for Tnfα, IL-1β and Mcp1; n = 6–7 for IL-6). (c) qPCR analysis of genes involved in M2 macrophage activation in epididymal white adipose tissue (WAT, n = 5–7). (d) Plasma IL-10 level in mice on HFD. *p < 0.05, compared between Tlr4^fl/fl and Tlr4^ΔmΦ mice (Student’s t-test). All data are presented as means ± s.e.m.

Figure 8. Elevated Tlr4 mRNA expression in stromal vascular fractions of adipose tissues from Tlr4^ΔmΦ mice

(a–b) qPCR analysis of Tlr4 mRNA expression in the adipocytes (a) and stromal vascular fractions (b, SVF) isolated from epididymal white adipose tissue of Tlr4^fl/fl and Tlr4^ΔmΦ mice fed HFD (n = 3–4). (c) qPCR analysis of Tlr4 mRNA expression in the peritoneal macrophage isolated from Tlr4^fl/fl and Tlr4^ΔmΦ mice fed HFD for 12 weeks (n = 4). ***p < 0.05, compared between Tlr4^fl/fl and Tlr4^ΔmΦ mice (Student’s t-test). All data are presented as means ± s.e.m.