Vitamin D/VDR regulates peripheral energy homeostasis via central renin-angiotensin system

Abstract

Introduction: Some epidemiological studies have revealed that vitamin D (VD) deficiency is closely linked with the prevalence of obesity, however, the role of VD in energy homeostasis is yet to be investigated, especially in central nervous system. Given that VD negatively regulates renin in adipose tissue, we hypothesized that central VD might play a potential role in energy homeostasis.

Objectives: The present study aims to investigate the potential role of VD in energy homeostasis in the CNS and elaborate its underlying mechanisms.

Methods: This study was conducted in Cyp27b1^-/- mice, VD-treated and wild-type mice. After the intraventricular injection of renin or its inhibitors, the changes of renin-angiotensin system (RAS) and its down-stream pathway as well as their effects on metabolic rate were examined.

Results: The RAS activity was enhanced in Cyp27b1^-/- mice, exhibiting a increased metabolic rate. Additionally, corticotropin-releasing hormone (CRH), a RAS-mediated protein regulating energy metabolism in the hypothalamus, increased significantly in Cyp27b1^-/- mice. While in VD-treated group, the RAS and sympathetic nerve activities were slightly inhibited, hence the reduced metabolic rate.

Conclusion: Collectively, the present study demonstrates that the VD/vitamin D receptor (VDR) has a significant impact on energy homeostasis through the modulation of RAS activity in the hypothalamus, subsequently altering CRH expression and sympathetic nervous activity.

Keywords: Central nervous system; Corticotropin-releasing hormone; Energy homeostasis; Renin-angiotensin system; Vitamin D.

Graphical abstract

Fig. 1

Cyp27b1^−/− mice exhibit a lean phenotype with increased energy expenditure. (A) Gross morphology; (B) Body weight; (C) Body fat mass; (D) Lean mass; (E) Average basal rectal temperature; (F) Food intake; (G) Water intake; (H) Oxygen consumption; (I) Respiratory exchange ratio (RER). WT: wild-type mice group; KO: knockout mice group. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05.

Fig. 2

The phenotype of Cyp27b1^−/− mice in peripheral adipose tissue. (A) Representative images of H&E staining of abdominal WAT sections (×100); (B) FASN mRNA in WAT; (C) HSL mRNA in WAT; (D) Representative images of H&E staining of BAT sections (×100); (F) UCP1 protein in BAT; (G) Ucp1 mRNA in BAT. All studies were carried out in male control mice and Cyp27b1^−/− mice fed a standard diet. WT: wild-type mice group; KO: knockout mice group; WAT: white adipose tissue; BAT: brown adipose tissue; UCP1: uncoupling protein 1; HSL: hormone sensitive lipase; FASN: fatty acid synthase. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05.

Fig. 3

VD-treated mice exhibit an obese phenotype with decreased energy expenditure. (A) Gross morphology; (B) Body weight; (C) Body fat mass; (D)Lean mass; (E) Average basal rectal temperature; (F) Food intake; (G) Water intake; (H) Oxygen consumption; (I) Respiratory exchange ratio (RER); WT: wild-type mice group. WTD group was treated by supplying cholecalciferol cholesterol emulsion (CCE) in the drinking water (CCE: water = 10 μl: 100 ml) for 2 weeks. KO group was the Cyp27b1^−/− mice. VD: vitamin D. VO₂: oxygen consumption. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05.

Fig. 4

The phenotype of VD-treated mice in peripheral adipose tissue. (A) Representative images of H&E staining of abdominal WAT sections (×100); (B) FASN mRNA in WAT; (C) HSL mRNA in WAT; (D) Representative images of H&E staining of BAT sections (×100); (E) UCP1 mRNA in BAT; All studies were carried out in male control mice and VD-treated mice fed a standard diet. WTD group was treated by supplying cholecalciferol cholesterol emulsion (CCE) in the drinking water (CCE: water = 10 μl: 100 ml) for 2 weeks. WT: wild-type mice group; KO: knockout mice group; VD: vitamin D; WAT: white adipose tissue; BAT: brown adipose tissue; UCP1: uncoupling protein 1; HSL: hormone sensitive lipase; FASN: fatty acid synthase. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05.

Fig. 5

Intracerebroventricular administration of renin significantly enhanced the ablation of VD signaling system-stimulated energy expenditure. (A) Representative images of immunofluorescence staining of renin of frozen hypothalamus sections (×200). (B) Serum ANG II levels were determined by enzyme linked immunosorbent assay (ELISA); (C) VDR protein in hypothalamus; (D) Body weight; (E) Oxygen consumption; (F) Respiratory exchange ratio (RER); (G) Average basal rectal temperature; (H) Blood pressure; (I) Heart rate. WTD group was treated by supplying cholecalciferol cholesterol emulsion (CCE) in the drinking water (CCE: water = 10 μl: 100 ml) for 2 weeks. WT: wild-type mice group; KO: knockout mice group; VD: vitamin D; VDR: vitamin D receptor; VO₂: oxygen consumption; ANG: angiotensin. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05; ^**P < 0.01.

Fig. 6

VD system in the central nervous system regulates energy homeostasis via RAS pathways. (A) Summary data of relative protein levels; (B) showed CRH protein expressions in hypothalamus using western blot analysis. (C) The relative protein levels are shown as relative to GAPDH protein levels determined using Graphpad Prism 5. (D) Serum ANG Ⅱ levels were determined by enzyme linked immunosorbent assay (ELISA); (E) Functional interaction between VDR and renin promoter was evaluated by Chromatin Immunoprecipitation (CHIP) assay; (F) Representative images of immunofluorescence staining of CRH of frozen hypothalamus sections (×200). WTD group was treated by supplying cholecalciferol cholesterol emulsion (CCE) in the drinking water (CCE: water = 10 μl: 100 ml) for 2 weeks. WT: wild-type mice group; KO: knockout mice group; VD: vitamin D; VDR: vitamin D receptor; RAS: renin-angiotensin system (RAS); ANG II: angiotensin II; CRH: corticotropin-releasing hormone. (+): intracerebroventricular (ICV) injection of 1 μl renin (0.2 × 10^-3 μg/μl), (−): ICV injection of 1 μl PBS. Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05; ^**P < 0.01.

Fig. 7

The VD signaling system regulates energy homeostasis via altered sympathetic nerve activity. (A) Representative electrograms from sympathetic nerves; (B) Adrb3 mRNA in WAT. (C) Adrb3 mRNA in BAT; (D) showed UCP1 and PGC1α proteins in BAT; (E) demonstrated PPARγ proteins in WAT; (F) The relative protein levels are shown as relative to GAPDH protein levels determined using Graphpad Prism 5. (G) Oxygen consumption; (H) Respiratory exchange ratio (RER); (I) Summary data of relative protein levels were determined by western blot. WT group was the control group of wild-type mice. WTD group was treated by supplying cholecalciferol cholesterol emulsion (CCE) in the drinking water (CCE: water = 10 μl: 100 ml) for 2 weeks. KO group is the Cyp27b1^−/− mice. WTA group, WTDA group, KOA group were treated by Aliskiren respectively (0.2 mg/ml, 1 μl/d) (Renin inhibitor). VD: vitamin D; WAT: white adipose tissue; BAT: brown adipose tissue; UCP1: uncoupling protein 1; PPARγ: peroxisome proliferators-activated receptor γ; VO₂: oxygen consumption; ANG II: angiotensin II; CRH: corticotropin-releasing hormone; ADRB3:Beta 3 adrenergic receptor; PGC1α: peroxlsome proliferator-activated receptor-γ coactlvator-1α; AT1: angiotensin II type I receptor; AT2: angiotensin II type II receptor. (+): intracerebroventricular (ICV) injection of alisiken (0.2 mg/ml, 1 μl/d) for seven days, (−): ICV injection of 1 μl PBS; Values are the mean ± s.e.m (n = 5–7 rats in each group), *P < 0.05; ^**P < 0.01; ^***P < 0.001.

Supplementary figure 1

Replicates of all western blots have been shown in this figure. Replicates of (A) fig.2F, (B) fig.5C, (C) fig.6A, (D) fig.6B, (E) fig.7D, (F) fig.7E, and (G) fig.7I.