Chemerin deficiency regulates adipogenesis is depot different through TIMP1

Abstract

Adipocytes and immune cells are vital for the development of adipose tissue. Adipokines secreted by adipocytes regulate adipogenesis and body metabolism. Chemerin is one of the adipokines. However, the function and mechanism of chemerin in adipose tissue are not fully illuminated. Compared with wild type (WT) mice, Rarres2 ^-/- mice gained weight and significantly increased fat distribution in subcutaneous adipose tissue (SAT), rather than visceral adipose tissue (VAT) on high fat diet (HFD). PPARγ and C/EBPα, the master regulators of adipogenesis, were up-regulated in SAT and down-regulated in VAT in Rarres2 ^-/- mice comparing with WT mice. Inspite of chemerin deficiency or not, the ratio of adipocyte-progenitors to total cells and the differentiation capacity of adipocyte-progenitors were similar in SAT and VAT, but macrophage infiltration in VAT was more severe than in SAT in Rarres2 ^-/- mice. Furthermore, CD45⁺ immune cells supernatant from Rarres2 ^-/- SAT promoted the differentiation of adipocyte-progenitors and 3T3-L1 cells. Adipokine array assay of CD45⁺ immune cells supernatant revealed that metalloproteinase inhibitor 1 (TIMP1), an inhibitor of adipogenesis, was reduced in Rarres2 ^-/- SAT, but increased in Rarres2 ^-/- VAT. As we specifically knocked down chemerin in SAT, TIMP1 was down-regulated and adipogenesis was promoted with reducing infiltration of macrophages. The present study demonstrates that the effects of chemerin on adipose tissue is depot different, and specific knock down chemerin in SAT promote adipogenesis and improve glucose tolerance test (GTT) and insulin tolerance test (ITT). This suggests a potential therapeutic target for chemerin in the treatment of obesity related metabolic disorder.

Keywords: Adipogenesis; Adipose tissue depot; Chemerin; Inflammation; TIMP1.

Figure 1

Chemerin deficiency regulates adipogenesis differently in SAT and VAT. (A) Body weight of WT, Rarres2 heterozygous (Rarres2^+/−) and Rarres2 knockout (Rarres2^−/−) mice, fed with ND or HFD. (B) Comparison of SAT and VAT. (C) Fat index (ratio of fat weight to whole body weight) of indicated mice. (D) H&E and quantification of cell size. (E-F) Whole-body oxygen consumption rate (E) and the average values of RER (F). (G) GTT and ITT analysis. (H-I) mRNA expression levels of lipolysis and lipogenic genes. (J-K) Western blot analysis of PPARγ and C/EBPα in SAT (J) and VAT (K). All the mice were male and fed with HFD for 12 weeks if not indicated otherwise, n = 8–12/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

Chemerin had no effect on adipocyte-progenitors of SAT and VAT. (A-B) Representative flow cytometric analysis of adipocyte-progenitors in SAT (A) and VAT (B). (C) Percentage of adipocyte-progenitors measured by flow cytometry. (D-E) mRNA expression levels and Western blot analysis of Pref-1 in SAT and VAT. Oil red O staining (F), Western blot analysis of PPARγ and C/EBPα (G). All the mice were male and fed with HFD for 12 weeks if not indicated otherwise, n = 6–8/group.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 3

Supernatant of CD45⁺ cells from different depots have diverse effects on adipogenesis in vitro. 3T3-L1 (A-C) and adipocyte-progenitors sorted from 6-week-old mice (D-F) were induced by conditioned media (details in methods and materials). (A and C) Representative oil red O staining was photographed. (B and E) Oil red O staining in (A) and (C) were quantified. (C and F) Western blot analysis of PPARγ and C/EBPα. All the supernatants of CD45⁺ cells in adipose tissue of WT or Rarres2^−/− male mice fed with HFD for 12 weeks. n = 6–8/group. ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

TIMP1 secreted by macrophage is involved in the regulation of adipogenesis. (A) Concentration of TIMP1 in supernatant of CD45⁺ cells in indicated depots of mice. (B) Concentration of TIMP1 were quantified. (C) Concentration of TIMP1 in supernatant of CD45⁺ cells in indicated depots of mice. (D) Oil red O stain of 3T3-L1 in different concentration of TIMP1. (E) mRNA expression of TIMP1 in adipocyte and macrophage. (F) Representative IHC staining of F4/80 in SAT and VAT from WT and Rarres2^−/− mice. All the mice were male and fed with HFD for 12 weeks if not indicated otherwise, n = 6–8/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

Knock down of chemerin specific in SAT promote adipogenesis and improve GTT and ITT. Male mice were fed with HFD and infected with shLacZ or shRarres2 twice weekly via subcutaneous injection adjacent to inguinal SAT for 8 weeks. (A) Gene expression levels of chemerin in SAT. (B) Body weight of mice. (C) Comparison of SAT. (D) Ratio of fat weight to body weight. (E) Total DNA of SAT. (F and H) Adipocyte cell size of SAT. (F and G) IHC of TIMP1 in SAT. (I) mRNA expression levels of TIMP1 in SAT. (J) Western blot analysis of PPARγ and C/EBPα. (K) GTT and ITT analysis. (L-M) Representative flow cytometric analysis of adipocyte-progenitors (L) and macrophage (M) in SAT of indicated mice. n = 6/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.