Vitamin D-vitamin D receptor system down-regulates expression of uncoupling proteins in brown adipocyte through interaction with Hairless protein

Abstract

Our previous study showed that feeding mice with vitamin D deficiency diet markedly alleviated high-fat-diet-induced overweight, hyperinsulinemia, and hepatic lipid accumulation. Moreover, vitamin D deficiency up-regulated the expression of uncoupling protein 3 (Ucp3) in white adipose tissue (WAT) and brown adipose tissue (BAT). The present study aimed to further investigate the effects of vitamin D and vitamin D receptor (Vdr) on Ucp1-3 (Ucps) expression in brown adipocyte and the mechanism involved in it. Rat primary brown adipocytes were separated and purified. The effects of the 1,25(OH)2D3 (1,25-dihydroxyvitamin D3; the hormonal form of vitamin D) and Vdr system on Ucps expression in brown adipocytes were investigated in basal condition and activated condition by isoproterenol (ISO) and triiodothyronine (T3). Ucps expression levels were significantly down-regulated by 1,25(OH)2D3 in the activated brown adipocyte. Vdr silencing reversed the down-regulation of Ucps by 1,25(OH)2D3, whereas Vdr overexpression strengthened the down-regulation effects. Hairless protein did express in brown adipocyte and was localized in cell nuclei. 1,25(OH)2D3 increased Hairless protein expression in the cell nuclei. Hairless (Hr) silencing notably elevated Ucps expression in activated condition induced by ISO and T3. Moreover, immunoprecipitation results revealed that Vdr could interact with Hairless, which might contribute to decreasing expression of Vdr target gene Ucps. These data suggest that vitamin D suppresses expression of Ucps in brown adipocyte in a Vdr-dependent manner and the corepressor Hairless protein probably plays a role in the down-regulation.

Keywords: 1,25(OH)2D3; hairless protein; uncoupling proteins; vitamin D; vitamin D receptor.

Figure 1. Effects of 1,25(OH)₂D₃ on the expression of Vdr and Ucps

(A) The mRNA levels of Vdr and Ucps were determined using real-time RT-PCR. (B) The protein levels of Vdr and Ucps were measured using Western blot. Blots reflect representative data. (C) The level of target protein/β-actin was quantified. Data were expressed as means ± SEM (n=3). *P<0.05.

Figure 2. Effects of Vdr interference by siRNA on the expression of Ucps regulated by 1,25(OH)₂D₃

(A) The mRNA levels of Ucps were determined using real-time RT-PCR. (B) The protein levels of Vdr and Ucps were measured using Western blot. Blots reflect representative data. (C) The level of target protein/β-actin was quantified. Data were expressed as means ± SEM (n=3). *P<0.05, **P<0.01.

Figure 3. Effects of Vdr overexpression on the expression of Ucps regulated by 1,25(OH)₂D₃

(A) The mRNA levels of Ucps were determined using real-time RT-PCR. (B) The protein levels of Vdr and Ucps were measured using Western blot. Blots reflect representative data. (C) The level of target protein/β-actin was quantified. Data were expressed as means ± SEM (n=3). *P<0.05, **P<0.01.

Figure 4. Hairless expression in the primary brown adipocyte of mice

(A) Hr mRNA level was determined using real-time RT-PCR. (B) Hairless protein level was measured using Western blot. (C) Immunohistochemical staining of Hairless protein using anti-Hairless antibody.

Figure 5. Effects of Hr interference by siRNA on the expression of Ucps regulated by 1,25(OH)₂D₃

(A) The mRNA levels of Ucps were determined using real-time RT-PCR. (B) The protein levels of Hairless and Ucps were measured using Western blot. Blots reflect representative data. (C). The level of target protein/β-actin was quantified. Data were expressed as means ± SEM (n=3). *P<0.05, **P<0.01.

Figure 6. Effects of Hr overexpression on the expression of Ucps regulated by 1,25(OH)₂D₃

(A) The mRNA levels of Ucps were determined using real-time RT-PCR. (B) The protein levels of Hairless and Ucps were measured using Western blot. Blots reflect representative data. (C). The level of target protein/β-actin was quantified. Data were expressed as means ± SEM (n=3). *P<0.05, **P<0.01.

Figure 7. Immunoprecipitation of Vdr and Hairless protein

To test for the interaction of Vdr and Hairless in brown adipocyte treated with 1,25(OH)₂D₃, immunoprecipitation with anti-Vdr antibody was carried out. Immunoprecipitation (IP) and immunoblot (IB) detection after pull-down by antibodies against Vdr and Hairless in brown adipocyte.