Loss of CCR5 results in glucose intolerance in diet-induced obese mice

Abstract

Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C chemokine receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of proinflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5-deficient (CCR5(-/-)) mice and C57BL/6 (WT) controls were fed 10% low-fat (LF) or 60% high-fat (HF) diets for 16 wk. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5(-/-) mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1-associated gene CD11c, whereas M2 associated genes were not different between genotypes. In addition, HF feeding caused a twofold increase in CD4(+) T cells in the AT of CCR5(-/-) compared with WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However, in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRβ in CCR5(-/-) compared with WT mice. These findings suggest that whereas CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

Keywords: C-C chemokine receptor 5; T cells; adipose tissue; diet-induced obesity; inflammation; insulin resistance; macrophages.

Fig. 1.

C-C chemokine receptor 5 (CCR5) deficiency leads to impaired glucose tolerance during high-fat (HF) diet feeding. Wild-type (WT) and CCR5^−/− mice were maintained on a low-fat (LF) or HF diet for 16 wk. A: body weight curves (n = 10 LF WT; n = 8 LF CCR5^−/−; n = 22 HF WT; n = 23 HF CCR5^−/−). At 16 wk mice were fasted for 5 h, and blood collected. B and C: serum insulin (B) and blood glucose (C) were measured (n = 8–11 mice/group). D: after 16 wk of HF feeding, mice were fasted for 5 h, and basal blood glucose levels were measured (0 min) before intraperitoneal administration of a bolus of glucose according to lean body mass (1.0 g/kg). Blood glucose was assessed at 15, 30, 45, 60, 90, and 150 min after injection (n = 12–13 mice/group). Area under the curve was calculated using GraphPad Prism. Data are presented as means ± SE of 22–26 mice/group. Data in B–D were analyzed by 2-way ANOVA. Area under the curve in D was analyzed by Student's t-test. *P < 0.05.

Fig. 2.

CCR5 deficiency reduces M1 gene expression in adipose tissue (AT) during HF diet feeding. Following 16 wk of LF or HF diet, stromal vascular cells were collected from AT and analyzed by flow cytometry. A: representative flow plots of isolated AT stromal vascular cells gated for macrophages and lymphocytes (P1). Cells were further separated by DAPI staining for live and dead cells. Representative flow plot of live cells gated for AT macrophages identified by expression of CD11b⁺F4/80⁺ (n = 3 LF WT; n = 3 LF CCR5^−/−; n = 16 HF WT; n = 14 HF CCR5^−/−) and quantification of AT macrophages. Data in A were analyzed by 2-way ANOVA. B: AT was collected from WT and CCR5^−/− mice. Tissues were stained with an antibody to F4/80 (green) for macrophages and DAPI (blue) for nuclei. Tissues were analyzed by confocal microscopy using a ×40 objective. Sections were chosen from images representing mice with F4/80 gene expression close to the mean of their respective groups. C: AT was collected from WT and CCR5^−/− mice maintained on a HF diet for 16 wk. RNA was isolated and used for real-time RT-PCR analysis, as described in materials and methods. Data are the mean ± SE of the relative gene expression for 12 mice/group. Data in C were analyzed by Student's t-test. *P < 0.05.

Fig. 3.

CCR5 deficiency increases CD4⁺ T cells in AT during HF diet feeding. WT and CCR5^−/− mice were maintained on a HF diet for 16 wk. Flow cytometry was used to quantify CD4⁺ and CD8⁺ T cells in AT. A: representative flow plots of isolated AT stromal vascular fraction (SVF) gated for live cells (left) and then the live cells gated for TCRβ⁺ cells (right). B: representative flow plots of AT CD4⁺ and CD8⁺ T cells identified by expressing TCRβ⁺CD4⁺ and TCRβ⁺CD8⁺. C: quantification of AT CD4⁺ and CD8⁺ T cells. Data are the mean ± SE of 12 mice/group. Data in B were analyzed by Student's t-test. D: RNA from isolated AT was used for real-time RT-PCR analysis, as described in materials and methods. Data are the mean ± SE of the relative gene expression for 12 mice/group. Data in C and D were analyzed by Student's t-test. *P < 0.05.

Fig. 4.

CCR5 deficiency mildly impairs phosphorylation of Akt in AT during obesity. At 16 wk of HF diet, WT and CCR5^−/− mice were injected with saline or insulin (0.5 U/kg) 15 min prior to euthanization. A: protein levels of p-Akt Ser⁴⁷³, β-actin, and total Akt (t-Akt) were determined by Western blot. Blots were analyzed densitometrically to quantify p-Akt relative to t-Akt. β-Actin was used as a loading control. Data represent the mean ± SE of 3–4 mice/roup. Equal amounts of AT protein lysates were immunoprecipitated (IP) with anti-IRβ (B) or IRS-1 antibody (C), followed by Western blot analyses with anti-p-Tyr, anti-IRβ, or anti-IRS-1, as indicated. Data represent the mean ± SE of 3 mice/group. Data in A–C were analyzed by 1-way ANOVA. Open bars represent WT mice, and black bars represent CCR5^−/− mice. IB, immunoblot.

Fig. 5.

CCR5 deficiency does not impact liver immune cell infiltration or inflammation under obese conditions. WT and CCR5^−/− mice were maintained on a HF diet for 16 wk. Flow cytometry was used to quantify macrophages and T cells in liver. A: representative flow plot of isolated hepatic nonparenchymal (NP) cells gated for macrophages and lymphocytes (P1). Cells were further separated by DAPI staining for live and dead cells. B: representative flow plot of hepatic macrophages identified by expression of CD11b⁺F4/80⁺ and quantification. Data are the mean ± SE of 3–5 mice/group. C: representative flow plot and quantification of liver CD4⁺ and CD8⁺ T cells identified by expressing TCRβ⁺CD4⁺ and TCRβ⁺CD8⁺. Data are the mean ± SE of 12 mice/group. D: RNA was isolated and used for real-time RT-PCR analysis, as described in materials and methods. Data are the mean ± SE of the relative gene expression for 12 mice/group. Data in B–D were analyzed by Student's t-test. SSC, side scatter; FSC, forward scatter.

Fig. 6.

CCR5 deficiency mildly impairs phosphorylation of Akt in muscle during obesity. Muscle was collected from WT and CCR5^−/− mice maintained on a HF diet for 16 wk. A: RNA was isolated and used for real-time RT-PCR analysis, as described in materials and methods. Data are the mean ± SE of the relative gene expression for 10–11 mice/group. B–D: after 16 wk of HF diet, WT and CCR5^−/− mice were injected with saline or insulin (0.5 U/kg) for 15 min prior to euthanization. B: protein levels of p-Akt Ser⁴⁷³, β-actin, and t-AKT were determined by Western blot. Blots were analyzed densitometrically to quantify p-Akt relative to t-Akt. β-Actin was used as a loading control. Data represent the mean ± SE of 3–4 mice/group. Equal amounts of muscle protein lysates were immunoprecipitated with anti-insulin receptor-β (IRβ; C) or IRS-1 (D) antibody, followed by Western blot analyses with anti-p-Tyr, anti-IRβ, or anti-IRS-1. Data represent the mean ± SE of 3 mice/group. Data in A were analyzed by Student's t-test. Data in B–D were analyzed by 1-way ANOVA.

Fig. 7.

CCR5 deficiency impairment of glucose tolerance is positively correlated with lean body mass. Regression analysis of lean body mass vs. area under the curve (AUC) calculated from glucose tolerance test (GTT) in WT (n = 22; A) and CCR5^−/− mice (n = 26; B). Linear regression was calculated using GraphPad Prism. GTTs of WT and CCR5^−/− mice after 16 wk of HF feeding were separated based on lean body mass <23 g (C) and lean body mass >23 g (D) (n = 11–18 mice/group).