Mechanisms Of The Energy Homeostasis Mediated By Vitamin D/VDR In Hypothalamus In Mice

Objective:In addition to maintaining Ca homeostasis and skeletal health,Vitamin D is closely associated with cardiovascular diseases,inflammation,tumor,etc.It is shown that vitamin D deficiency may contribute to the pathogenesis of a number of metabolic disorders,including obesity,type 2 diabetes and metabolic syndrome.Animal studies in vivo have revealed that VDR knocked out mice display lean phenotype with reduced body weight,decreased white adipose tissue mass,increased β-oxidation rate,increased energy expenditure.Interestingly,uncoupling protein 1（UCP1）expression in white adipose tissue of VDR^-/-mice is increased considerably.Additionally,in transgenic VDR mice,VDR over-expresses in white adipose tissue,leading to reduced lipolysis andβ-oxidation rate with the outcome of obesity.Conversely,in mouse 3T3-L1 preadipocytes,1,25-（OH）₂D₃ inhibits adipogenesis by acting on multiple targets suppressing C/EBPα and PPARγ expression.The discrepancy of the results in vivo and in vitro still remains unclear.The central nervous system（CNS）,especially the hypothalamus,plays a key role in the regulation of energy homeostasis.It is validated that VDR expresses in certain neurons.Renin-angiotensin system（RAS）is well recognized for its varied roles in cardiovascular physiology.It is thought that RAS may regulate energy metabolism.Grobe has demonstrated that stimulation of the brain RAS by transgenic activation,intracerebro-ventricular infusion（ICV）of ANG increases resting metabolic rate through AT1-dependent mechanisms.Moreover,it is confirmed that vitamin D is a negative endocrine regulator of the RAS.Therefore,we speculate that Vitamin D/VDR may not only play a role in homeostasis through impacting on adipose tissues,but also partially affect energy metabolism via negative control of RAS in the hypothalamus.Methods:8-10 week old male CYP27B1^-/-mice on ICR background and its littermates were enrolled in our study.They were randomized into WT group,vitamin D intervention（WTD）group and KO group（n=10）,and the mice in WTD group was treated by supplying cholecalciterol cholesterol emulsion（CCE）in the drinking water（CCE:water=10 μl:100 ml）for 2 weeks.Half of the three groups as control groups were ICV injected PBS 1 μl for consecutive 4 days,the other half mice were ICV injected renin 1μl(0.2×10^-3 μg/μl)for consecutive 4 days as well.Body weight,rectal temperature,food intake and water intake were recorded at 3 p.m.（basal metabolic state）.Volume of O2 consumption and CO₂ production and metabolic rate were examined for 24 hours in a comprehensive laboratory animal monitoring system.Before sacrificing the mice,blood pressure,heart rate and sympathetic nerve activity were measured.In addition,8-10 week old male WT mice were randomly divided into WT group,aliskiren group,perindopril and Telmisartan group（n=5）.They were treated by ICV injections of PBS,aliskiren,perindopril and Telmisartan 1 μl for continuous 14 days respectively.Body weight,rectal temperature,food intake and water intake were recorded at 3 p.m.each day.After the scarification,the hypothalamus,brown adipose tissue and white adipose tissue were harvested.Next,newborn WT and KO mice were sacrificed and the hypothalamus tissue was harvested for tissue culture.The hypothalamus tissue were cut into mince and cultured in high glucose DMEM,and the WTD group was add in paricalcitol(2×10^-8 M/L)for 2 hours.Then renin was added to reach the concentration of 10^-9 M/L.The expressions of VDR,renin receptor,renin,AT1,AT2,Ang,CRH in the hypothalamus,the expressions of UCP1,PGC-1α in brown adipose tissue and PPAR-γ in white adipose tissue were examined by Western blot.The POMC,NPY and AgRP mRNA in the hypothalamus,the UCP1,UCP2,UCP3 and ADRB3 mRNA in brown adipose tissue,the FASN,LPL,HSL,CPT1 and ADRB3 mRNA in white adipose tissue were determined by RT-PCR.Serum Ang Ⅱ,glucose,Ca²⁺,cholesterol,NEFA were assayed by ELISA.The expressions of CRH and renin were examined by immunofluorescence staining.Mice body composition was measured using NMR imaging system.The binding abilities of renin promoter with VDR were evaluated by CHIP.Results:1.Compared with the WT mice,CYP27B1^-/-mice displayed lean phenotype with reduced body weight,decreased white adipose tissue mass,high volume of water intake and food intake,lower levels of blood glucose,serum Ca²⁺,cholesterol,NEFA,triglycerides and high levels of metabolic rate.In contrast,WTD mice showed obese phenotype with increased body weight,white adipose tissue mass,low volume of water intake and food intake,higher levels of blood glucose,serum Ca²⁺,cholesterol,NEFA,triglycerides and lower levels of metabolic rate.2.CYP27B1^-/-mice showed high serum AngⅡ levels,whereas WTD mice were with lower level of AngⅡ.Moreover,more renin was found in the PVN of hypothalamus in CYP27B1^-/-mice,while there was less in WTD mice.3.After the ICV injection of renin,mice in all three groups showed lower levels of food and water intake,reduced body weight,increased rectal temperature,blood pressure,blood glucose,and decreased levels of cholesterol,triglycerides and NEFA,however,the extent changed in different groups varied.4.The levels of AT1,AngⅡ and renin were expressed higher in the hypothalamus in CYP27B1^-/-mice,while lower in WTD mice.After the ICV injection of renin,they were expressed even higher,however the increased levels were lower in WTD group than in CYP27B1^-/-mice.5.The expressions levels of CRH were similar with AngⅡ,so did the sympathetic nerve activity.6.CHIP assay demonstrated that the binding abilities of renin promoter with VDR in WTD group were significantly lower than that in the WT group.7.After the ICV injection of RAS blockers,we found that compared with WT group,there was a decrease in rectal temperature and food intake,an increase in body weight in the aliskiren and perindopril group.ANGⅡ,AT1 and CRP expression levels were reduced in the aliskiren and perindopril group.8.In the brown adipose tissue,UCP1,UCP2 and UCP3 mRNA were higher in CYP27B1^-/-mice and lower in WTD mice.UCP1 and its upstream regulator PGC-1α were up-regulated after the ICV injection of renin.9.In the white adipose tissue,FASN and LPL mRNA were lower and HSL and CPT1 mRNA were higher in CYP27B1^-/-mice.The expression levels of PPAR-γ were higher in CYP27B1-/mice.Conclusions:1.Under different vitamin D states,the energy metabolism is different.2.Under different vitamin D conditions,RAS activities vary in the hypothalamus.3.Increased RAS activities may give rise to up-regulated CRH and promote sympathetic nervous activity.4.Vitamin D/VDR negatively regulates the renin expressions in the hypoythalamus.5.Lack of vitamin D/VDR,the thermogenesis was enhanced in the brown adipose tissue,while the lipolysis was strengthened and the adipogenesis was weakened in the white adipose tissue.6.Vitamin D/VDR may regulate energy homeostasis at least in partial via mediating RAS activity in the hypothalamus in mice.