Critical role of chemokine (C-C motif) receptor 2 (CCR2) in the KKAy + Apoe -/- mouse model of the metabolic syndrome

Abstract

Aims/hypothesis: Chemokines and their receptors such as chemokine (C-C motif) receptor 2 (CCR2) may contribute to the pathogenesis of the metabolic syndrome via their effects on inflammatory monocytes. Increased accumulation of CCR2-driven inflammatory monocytes in epididymal fat pads is thought to favour the development of insulin resistance. Ultimately, the resulting hyperglycaemia and dyslipidaemia contribute to development of the metabolic syndrome complications such as cardiovascular disease and diabetic nephropathy. Our goal was to elucidate the role of CCR2 and inflammatory monocytes in a mouse model that resembles the human metabolic syndrome.

Methods: We generated a model of the metabolic syndrome by backcrossing KKAy ( + ) with Apoe ( -/- ) mice (KKAy ( + ) Apoe ( -/- )) and studied the role of CCR2 in this model system.

Results: KKAy ( + ) Apoe ( -/- ) mice were characterised by the presence of obesity, insulin resistance, dyslipidaemia and increased systemic inflammation. This model also manifested two complications of the metabolic syndrome: atherosclerosis and diabetic nephropathy. Inactivation of Ccr2 in KKAy (+) Apoe ( -/- ) mice protected against the metabolic syndrome, as well as atherosclerosis and diabetic nephropathy. This protective phenotype was associated with a reduced number of inflammatory monocytes in the liver and muscle, but not in the epididymal fat pads; circulating levels of adipokines such as leptin, resistin and adiponectin were also not reduced. Interestingly, the proportion of inflammatory monocytes in the liver, pancreas and muscle, but not in the epididymal fat pads, correlated significantly with peripheral glucose levels.

Conclusions/interpretation: CCR2-driven inflammatory monocyte accumulation in the liver and muscle may be a critical pathogenic factor in the development of the metabolic syndrome.

Figure 1. Weight and glucose analysis

(A) Percentage of weight increase was determined by comparing the mean weight in each group, following the mice from week 10 to 18. CCR2 inactivation in the pathogenic KKAy⁺Apoe^−/− mice leads to a significant reduction in the percentage of weight gain. (B) Food intake during periods of 24 hr was quantified for 4 consecutive days; no difference was present between KKAy⁺Apoe^−/− and KKAy⁺Apoe^−/−Ccr2^−/− mice. Representative data from experiments performed three times is shown for A and B (n=3–5). (C and D) Significant decrease in insulin tolerance test (ITT) and glucose tolerance test (GTT) in KKAy⁺Apoe^−/−Ccr2^−/− mice compared to KKAy⁺Apoe^−/− mice (n=3–5 per group).

Figure 2. Effects of CCR2 null state in the development of atherosclerosis in MetS

(A and B) Representative aortic plaque size stained with Sudan IV and quantification. (C and D) Representative pictures of macrophage infiltration stained with ER-HR3 and quantification. Results show decreased plaque burden and macrophage infiltration in KKAy⁺Apoe^−/−Ccr2^−/− mice compared to KKAy⁺Apoe^−/− mice (n=3–5 repeated three times).

Figure 3. CCR2 inactivation and kidney involvement in MetS

Periodic acid-Schiff (PAS) staining shows glycogen deposition in all four groups of mice (age 25 ± 5 weeks). (A) Histopathological images, (B) quantification, and (C) nodular sclerosis present only in KKAy⁺Apoe^−/− mice. (D) Collagen deposition was assessed by Masson’s trichrome staining. (E) Percentage of capillary lumen area per glomerulus and (F) corrected by glomerular volume; were calculated as described in materials and methods. In all the cases, 50 – 80 glomeruli were analyzed per mouse. (G) CCR2 null state is associated with less macrophage infiltration in the kidneys. Pictures and data are representative of three experiments (n=3–5 per group).

Figure 4. FACS plots representing the percentage of inflammatory monocytes (IMo) of KKAy⁺ Apoe^−/− and KKAy⁺ Apoe^−/− Ccr2^−/− in different organs

All plots were gated on CD11b⁺, and specific populations for Gr-1 and Ly6C⁺ were selected as shown. (A) Infiltration of IMo in the liver, (B) epididymal fat pads, (C) pancreas, (D) muscle, (E) and blood (n=9–15 per group).