CCR2 modulates inflammatory and metabolic effects of high-fat feeding

Abstract

The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.

Figure 1

Expression of Ccr2 and its ligands in lean mice, obese mice, and obese mice treated with pioglitazone. Expression of Ccr2 and genes that encode 3 of its ligands, Ccl2, Ccl7 and Ccl8, were measured in lean mice (white bars), obese mice (black bars), and obese mice treated with the insulin sensitizer pioglitazone (gray bars). *P < 0.05, lean vs. obese; ^†P < 0.05 obese vs. obese pioglitazone-treated (n = 4). Values are expressed as mean ± SD.

Figure 2

Body mass of Ccr2^–/– mice. (A) C57BL/6J Ccr2^+/+ (black symbols) and Ccr2^–/– (gray symbols) mice were fed a low-fat (triangles) or a high-fat diet (squares) for 24 weeks. There was no significant difference in body mass between mice of each genotype on the low-fat diet (n = 5; P > 0.05). The mean body mass of Ccr2^–/– mice fed a high-fat diet was significantly lower than that of the Ccr2^+/+ mice (39.3 ± 6.9 g vs. 46.3 ± 4.1 g; n = 10; P < 0.05). (B) Average daily food intake was measured over a 6-week period for Ccr2^+/+ and Ccr2^–/– mice fed a high-fat diet. *P < 0.05 vs. Ccr2^+/+.

Figure 3

Insulin sensitivity in obese Ccr2^–/– and obese Ccr2^+/+ mice. Fasting plasma insulin (A) and blood glucose concentrations (B) were measured in lean Ccr2^+/+ (black bars) and Ccr2^–/– (gray bars) mice and mice of both genotypes made obese following 20 weeks of high-fat diet feeding. There were no significant genotype-dependent differences in fasting glucose or insulin concentrations in lean animals. However, fasting glucose and insulin concentrations were lower in obese Ccr2^–/– compared with obese Ccr2^+/+ mice despite similar degrees of adiposity (insulin: P < 0.005; glucose: P < 10^–4). (C) HOMA-IR values (expressed as IU-mg/dl) were significantly lower (P < 10^–4) among obese Ccr2^–/– than obese Ccr2^+/+ mice. (D) A plot of HOMA-IR values against body mass among all Ccr2^–/– (gray squares) and Ccr2^+/+ (black circles) mice reveals that the relationship between insulin sensitivity and body mass differs between mice dependent upon Ccr2 genotype. **P < 0.01 compared with wild type. Values are expressed as mean ± SD.

Figure 4

Glucose homeostasis in obese Ccr2^–/– and Ccr2^+/+ mice. (A) The response of fasted obese Ccr2^+/+ (black circles) and Ccr2^–/– (gray squares) mice following a single intraperitoneal injection of insulin (1.5 U/kg) was monitored by serially measuring blood glucose concentrations. The percent reduction in glucose concentration in obese Ccr2^–/– mice was significantly greater than that in obese Ccr2^+/+ mice at 75, 90, and 130 minutes (*P < 0.05). (B) Intraperitoneal injection of a bolus of glucose to fasted obese Ccr2^+/+ (black circles) and Ccr2^–/– (gray squares) mice lead to similar peak glucose concentrations but lower glucose concentrations at 45, 60, and 90 minutes. *P < 0.05 compared with wild type. Values are expressed as mean ± SD.

Figure 5

CCR2 deficiency lowers ATM content in obese mice. The fraction of F4/80-expressing macrophages was determined by immunohistochemical analysis of epididymal adipose tissue from obese Ccr2^+/+ (A) and Ccr2^–/– (B) mice with the macrophage-specific marker F4/80 (EMR1). (C) The fraction of ATMs (F4/80-stained cells/total cells) in periepididymal adipose tissue of obese Ccr2^+/+ mice (white bar) was significantly greater than the fraction of ATMs in lean Ccr2^+/+ mice (black bar) (P < 10^–4) and obese Ccr2^–/– mice (gray bar) (P < 0.005). (D) The average fraction of ATMs was also greater in subcutaneous adipose tissue of obese Ccr2^+/+ mice (black bar) compared with obese Ccr2^–/– mice (gray bar). Values are expressed as mean ± SD. **P < 0.01 compared with obese Ccr2^+/+ mice.

Figure 6

Expression of genes involved in inflammation. Quantitative RT-PCR was used to measure the expression in periepididymal adipose tissue of genes involved in macrophage function and inflammation (Tnfa, Cd68, Emr1, and Serpine1). Obese Ccr2^–/– (light gray bars) and obese Ccr2^+/+ (dark gray bars) mice with body mass greater than 40 g were studied, as were lean Ccr2^+/+ mice (black bars) and lean Ccr2^–/– mice (white bars). *P < 0.05, obese mice on high-fat chow compared with mice of the same genotype on low-fat chow; ^§P < 0.05, obese Ccr2^+/+ compared with obese Ccr2^–/– mice. Values are expressed as mean ± SEM.

Figure 7

Expression of genes involved in adipocyte function. Quantitative RT-PCR was used to measure the expression in periepididymal adipose tissue of genes involved in adipocyte function (Fabp4, Gpam, Lipe, Pparg, and Acdc). Obese Ccr2^–/– (light gray bars) and obese Ccr2^+/+ (dark gray bars) mice with body mass greater than 40 g were studied, as were lean Ccr2^+/+ mice (black bars) and lean Ccr2^–/– mice (white bars). *P < 0.05, mice with dietary obesity compared with lean mice of the same genotype; ^§P < 0.05, obese Ccr2^+/+ compared with obese Ccr2^–/– mice. ^#NS. Values are expressed as mean ± SEM.

Figure 8

Plasma adiponectin in lean and obese Ccr2^–/– and Ccr2^+/+ mice. Proteins in 1 μl of plasma drawn from lean (white bars) and adiposity-matched obese (black bars) Ccr2^–/– and Ccr2^+/+ mice were denatured, reduced, and separated using SDS-PAGE. We performed immunoblotting for adiponectin with an antibody specific for adiponectin. Values are expressed as mean ± SD. *P < 0.05.

Figure 9

Insulin sensitivity in CCR2 antagonist–treated mice. (A) Hyperglycemia after a daytime fast (6 hours) was reduced in high-fat diet–fed obese mice that received the selective CCR2 antagonist INCB3344 for 14 days (white bars) compared with high-fat diet–fed obese mice that received vehicle injections (black bars; n = 7 per group; *P < 0.05). (B) Following an overnight fast (14 hours), blood glucose concentrations of INCB3344 and vehicle-treated obese animals were not significantly different. However, fasting insulin concentrations and HOMA-IR values (expressed as IU-mg/dl) were significantly lower in the INCB3344-treated animals (n = 7 per group; *P < 0.05). (C) Following an intraperitoneal injection of glucose, obese mice treated with INCB3344 (9 days of daily injections; open circles) were significantly less hyperglycemic (n = 8 per group; **P < 0.01 at 10, 20, 30, and 60 minutes) than those treated with vehicle (filled squares). (D) Following an intraperitoneal injection of insulin (1.5 U/kg) the percentage reduction in blood glucose concentration was greater in obese mice treated with INCB3344 (14 days of daily injections) than in vehicle-treated controls (n = 7; *P < 0.05 at 30, 45, 75, 90, and 130 minutes). Values are expressed as mean ± SD.