G protein-coupled receptor 21 deletion improves insulin sensitivity in diet-induced obese mice

Abstract

Obesity-induced inflammation is a key component of systemic insulin resistance, which is a hallmark of type 2 diabetes. A major driver of this inflammation/insulin resistance syndrome is the accumulation of proinflammatory macrophages in adipose tissue and liver. We found that the orphan GPCR Gpr21 was highly expressed in the hypothalamus and macrophages of mice and that whole-body KO of this receptor led to a robust improvement in glucose tolerance and systemic insulin sensitivity and a modest lean phenotype. The improvement in insulin sensitivity in the high-fat diet-fed (HFD-fed) Gpr21 KO mouse was traced to a marked reduction in tissue inflammation caused by decreased chemotaxis of Gpr21 KO macrophages into adipose tissue and liver. Furthermore, mice lacking macrophage expression of Gpr21 were protected from HFD-induced inflammation and displayed improved insulin sensitivity. Results of in vitro chemotaxis studies in human monocytes suggested that the defect in chemotaxis observed ex vivo and in vivo in mice is also translatable to humans. Cumulatively, our data indicate that GPR21 has a critical function in coordinating macrophage proinflammatory activity in the context of obesity-induced insulin resistance.

Figure 1. Quantification of Gpr21 expression.

q-PCR analysis of WT Gpr21 mRNA expression in (A) various tissues and BM-derived macrophages (BMDM), (B) brain regions, (C) SVF and adipocytes of NC- and HFD-fed mice, and (D) M1-like (F4/80⁺CD11b⁺CD11c⁺) and M2-like (F4/80⁺CD11b⁺CD11c^–) macrophages and T cells. WAT, white adipose tissue. Data (mean ± SEM) are shown as fold induction of gene expression normalized to housekeeping gene (n = 3 per group).

Figure 2. Effect of Gpr21 KO on BW gain, food intake, glucose tolerance, and insulin sensitivity.

(A) BW gain and (B) weekly food intake of NC-fed Gpr21 KO and WT littermates (n = 22–25 per group). (C) i.p. GTT (1 g/kg) on Gpr21 KO and WT mice (n = 8 per group). (D) Insulin concentration at the indicated time points. (E) ITT on HFD-fed Gpr21 KO and WT mice (n = 8 per group), shown as percent change from basal blood glucose. (F–I) Hyperinsulinemic-euglycemic clamp study in Gpr21 KO and WT littermates (n = 5–6 per group). (F) GIR. (G) Basal HGP. (H) HGP suppression by insulin. (I) IS-GDR. (J) Insulin-induced FFA suppression. *P < 0.05 vs. WT, repeated-measures 2-way ANOVA (A and C–E) or Student’s t test (B and F–J).

Figure 3. Effect of Gpr21 deletion on BW gain, food intake, and glucose tolerance.

(A) BW gain and (B) weekly food intake of 12-week HFD-fed Gpr21 KO and WT littermates (n = 38–40 per group). (C) i.p. GTT (1 g/kg) on BW-matched Gpr21 KO and WT mice fed HFD for 11 weeks (n = 5–6 per group). (D) Insulin concentration at the indicated time points. (E–I) Hyperinsulinemic-euglycemic clamp study on Gpr21 KO and WT littermates (n = 6 WT, 12 Gpr21 KO, 6 Gpr21 KO BW-matched to WT). (E) GIR. (F) Basal HGP. (G) HGP suppression by insulin. (H) IS-GDR. (I) Insulin-induced FFA suppression. (J) EE in Gpr21 KO and WT littermates over a 24-hour period. (K) Average VO₂ (ml/kg/h) and (L) average VCO₂ in the light and dark cycles. (M) CBT of Gpr21 KO and WT mice in a 24-hour period, averaged across 3 days. (N) Average activity in the dark and light cycles. (O) q-PCR analysis of BAT expression of Prdm16, Pgc1α, and Ucp1. (P) BAT weight. (Q) q-PCR analysis of inflammatory gene expression in BAT. ^#P < 0.1 (NS), *P < 0.05 vs. WT, repeated-measures 2-way ANOVA (A–D), 2-way ANOVA (E–I), or Student’s t test (J–Q).

Figure 4. Inflammatory state of liver and eWAT in 12-week HFD-fed Gpr21 KO mice.

(A) Liver weight from BW-matched mice. (B) Liver TG determination. (C) H&E stain of liver sections from Gpr21 KO and WT mice. Original magnification, ×50. (D) Quantitation of glycogen in the liver. (E) Inflammatory gene expression in liver. (F) eWAT weight. (G) MRI measurement of subcutaneous and visceral fat mass. (H) Inflammatory gene expression in eWAT. ^#P < 0.1 (NS), *P < 0.05 vs. WT, Student’s t test.

Figure 5. Decreased macrophages in adipose tissue of Gpr21 KO mice.

(A) Confocal merged images from epididymal fat pads of HFD-fed Gpr21 KO and WT mice costained with anti-F4/80 (cyan) and anti–caveolin-1 (blue) antibodies. Original magnification, ×200. (B) Number of crown-like structures (CLSs) per field in adipose tissue sections. (C) MCP-1 protein level in plasma of BW-matched HFD-fed mice. (D) FACS analysis of M1-like and M2-like ATM subsets in stromal vascular cells (SVCs). (E) Migratory capacity of i.p. macrophages, as measured by in vitro transwell chemotaxis assay using CM from 3T3L1 adipocytes, MCP-1 (100 ng/ml), or LTB4 (10 nM). (F) Gpr21 KO macrophage exhibited dysfunctional cytoskeletal organization. i.p. macrophages were treated with LPS (10 ng/ml) or MCP-1 (100 ng/ml) for 10 minutes, and phalloidin–Alexa Fluor 488 was used to stain F-actin. Scale bar: 50 μm. (G) Migratory capacity of human U-937 monocytes after human GPR21 was knocked down by RNAi, measured using an in vitro transwell chemotaxis assay. (H and I) Migratory capacity of Gpr21 macrophages in vivo to eWAT (H) and liver (I), assessed by PKH26-labeled monocytes. Data are mean ± SEM of 3 independent experiments in triplicate. ^#P < 0.1 (NS), *P < 0.05 vs. WT. All images are representative of results from 3 independent experiments.

Figure 6. BMT studies.

(A) BW gain and (B) daily food intake of 12-week HFD-fed BMT-WT and BMT-KO mice. (C) GTT (n = 9–11 mice per group). (D) Insulin concentration. (E) ITT. (F–J) Hyperinsulinemic-euglycemic clamp studies in BMT-KO and BMT-WT littermates (n = 5–6 per group). (F) GIR. (G) Basal HGP. (H) HGP suppression by insulin. (I) IS-GDR. (J) Insulin-stimulated FFA suppression. (K) Plasma protein expression of inflammatory markers. q-PCR analysis of inflammatory gene expression in (L) eWAT and (M) liver. *P < 0.05 vs. WT, repeated-measures 2-way ANOVA (A–E) or Student’s t test (F–I).