RGC32 deficiency protects against high-fat diet-induced obesity and insulin resistance in mice

Abstract

Obesity is an important independent risk factor for type 2 diabetes, cardiovascular diseases and many other chronic diseases. Adipose tissue inflammation is a critical link between obesity and insulin resistance and type 2 diabetes and a contributor to disease susceptibility and progression. The objective of this study was to determine the role of response gene to complement 32 (RGC32) in the development of obesity and insulin resistance. WT and RGC32 knockout (Rgc32(-/-) (Rgcc)) mice were fed normal chow or high-fat diet (HFD) for 12 weeks. Metabolic, biochemical, and histologic analyses were performed. 3T3-L1 preadipocytes were used to study the role of RGC32 in adipocytes in vitro. Rgc32(-/-) mice fed with HFD exhibited a lean phenotype with reduced epididymal fat weight compared with WT controls. Blood biochemical analysis and insulin tolerance test showed that RGC32 deficiency improved HFD-induced dyslipidemia and insulin resistance. Although it had no effect on adipocyte differentiation, RGC32 deficiency ameliorated adipose tissue and systemic inflammation. Moreover, Rgc32(-/-) induced browning of adipose tissues and increased energy expenditure. Our data indicated that RGC32 plays an important role in diet-induced obesity and insulin resistance, and thus it may serve as a potential novel drug target for developing therapeutics to treat obesity and metabolic disorders.

Keywords: adipose tissue; insulin resistance; obesity; response gene to complement 32.

Figure 1

RGC-32 deficiency prevented HFD-induced obesity. (A) RGC-32 expression in adipose tissue of wild-type (WT) mice fed with normal chow or a 12-week high-fat diet (HFD) were detected by western blot and normalized to α-tubulin (n = 3). (B) Body weight of WT and RGC32^–/– mice fed with normal chow (n = 6) or HFD (n = 10). (C) Weights of epididymal fat from WT and RGC32^–/– mice fed on normal chow and HFD. (D) Representative H&E-stained images of epididymal fat from WT and RGC32^–/– mice fed on normal chow and HFD. (E) Quantitative analysis of the mean adipocyte area. The areas were normalized to the mean adipocyte area of WT mice fed on normal chow. (F) Energy intake of WT and RGC32^–/– mice fed on normal chow and HFD. (G) Body weight change of WT and RGC32^–/– mice after an 8-hour fast. **P<0.01 compared with WT chow group, ##P<0.01 compared with WT HFD group.

Figure 2

RGC-32 deficiency improved metabolic homeostasis in HFD-fed mice. (A) Serum triglyceride (TG), (B) high-density lipoprotein (HDL) cholesterol and low-density lipoprotein/very-low-density lipoprotein (LDL/VLDL) cholesterol concentrations in wild-type (WT) and RGC32^–/– mice fed on normal chow and HFD (n = 6). (C) Fasting blood glucose, (D) insulin concentration, and (E) homeostasis model assessment-insulin resistance (HOMA-IR= fasting glucose × fasting insulin/22.5) in WT and RGC32^–/– mice fed on normal chow and HFD (n = 6). **P<0.01 compared with WT chow group, ##P<0.01 compared with WT HFD group.

Figure 3

RGC-32 deficiency prevents HFD-induced insulin resistance in mice. (A and B) Insulin tolerance test (ITT) in wild-type (WT) and RGC32^–/– mice fed on normal chow (A) and HFD (B) (n = 6). (C) Inverse area under the curve (AUC) of ITT. (D and E) Glucose tolerance test (GTT) in WT and RGC32^–/– mice fed on normal chow (D) and HFD (E) (n = 6). (F) Quantification of the AUC of GTT. *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.

Figure 4

RGC-32 deficiency attenuated adipose tissue and systemic inflammation in HFD-fed mice.(A and B) mRNA expression of adiponectin, leptin, interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-12 in epididymal adipose tissue from wild-type (WT) and RGC32^–/– mice (n = 6) was measured by qPCR. (C and D) Protein concentration of adiponectin, leptin, IL-6, TNF-α and IL-12 in the serum from WT and RGC32^–/– mice (n = 6) was measured by cytometric bead array immunoassay. *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.

Figure 5

RGC-32 deficiency increased the expression of metabolic genes in adipose tissues. (A) mRNA expression of PPAR-α, HSL and PGC1α in epididymal adipose tissues from wild-type (WT) and RGC32^–/– mice (n = 6). (B and C) PPAR-α, HSL and PGC1α protein expression in epididymal adipose tissues from WT and RGC32^–/– mice were detected by western blot and normalized to α-tubulin (n = 6). (D and E) mRNA expression of PGC1α, UCP1, and Prdm16 in interscapular (D) and inguinal (E) fat tissues from WT and RGC32^–/– mice (n = 6). *P<0.05, **P<0.01 compared with WT chow group, #P<0.05, ##P<0.01 compared with WT HFD group.

Figure 6

RGC-32 had no effect on adipocyte differentiation. (A) mRNA expression of RGC-32, PPAR-γ and C/EBPα during 3T3-L1 preadipocyte differentiation at the indicated times. (B and C) RGC-32 protein expression during 3T3-L1 preadipocyte differentiation was detected by western blot and normalized to α-tubulin. *P<0.05, **P<0.01 compared with vehicle-treated group (0 day). (D-H) 3T3-L1 preadipocyte was transduced with Ad-GFP, Ad-shRGC32 or Ad-RGC32 for 24 h and then was induced for adipocyte differentiation. (D) mRNA expression of RGC-32, PPAR-γ and C/EBPα and (E and F) lipid droplet accumulation were determined at the indicated times. (G and H) PPAR-α, HSL, PGC1α, and RGC-32 expression was detected by western blot and normalized to α-tubulin. *P<0.05, **P<0.01 compared with Ad-GFP group. All results are representatives of at least three independent experiments.