Effect Of Maternal Vitamin D Deficiency On The Development Of Obesity And Immune Response In Male Mice Offspring

Objectives:To explore the effects and mechanism of insufficient intake of Vitamin D(VD)during maternal pregnancy and lactation on obesity in the male offspring.Methods:30 female C57BL/6J mice of SPF at 4-week old were randomly divided into 2groups.VD deficient diet(VD-D)and normal chow diet(VD control,VD-C)was given to each group of mice during pregnancy and lactation,respectively.The females were fed for 6 weeks,and then mated with 12-week-old C57BL/6J male mice at 2:1.After weaning,their male offspring were divided into two groups(n=10),and given fed with high-fat diet(HFD)and normal chow diet separately,which was VD normal high fat diet group(VD-C-HFD),VD lack high-fat diet group(VD-D-HFD),VD normal and normal chow diet(VD-C)group,VD lack and normal chow diet(VD-D)group.The following assay was applied.1.the body weight,food intake and energy intake of mice were monitored weekly.2.Oral Glucose Tolerance Tests(OGTT)Mice were fed for 13 weeks and received glucose(2.0g/kg)via gavage after 10 hours of fasting.Blood glucose samples from the tail vein were collected at 15,30,60,90and 120 minutes to test blood glucose.3.Insulin Tolerance Tests(ITT)After feeding for 14 weeks,mice were fasted for 2 hours and injected intraperitoneally with insulin(2IU/kg).Blood glucose samples from the tail vein were collected at 15,30,60,90 and 120 minutes to test blood glucose.4.Serum adipose metabolism assayAfter feeding for 16 weeks,the serum of each group of mice was collected,and the concentrations of calcium ion,25(OH)D3,triglyceride(TG)and total cholesterol(TC)were detected using biochemical kits.5.Epididymal white adipose tissue(e WAT)and inguinal white adipose tissue(i WAT)assayAfter feeding mice for 16 weeks,e WAT and i WAT were obtained and weighed.Histopathological tests were performed to analyze e WAT and i WAT.Hematoxylin and eosin(HE)staining were used to analyze the size of adipocytes,and the quantity of the macrophage was detected by F4/80 immunohistochemistry method.The stromal vascular cells(SVCs)in adipose tissue were isolated and then stained with fluorescence tagged antibodies to detect cell lineages by flow cytometry analysis.The cell lineages included M1 adipose tissue macrophage(M1ATM,CD45~+CD64~+CD11C~+),M2 adipose tissue macrophage(M2ATM,CD45~+CD64~+CD11C~-),adipose tissue dendritic cell(ATDC,CD45~+CD64~-CD11C~+),CD4~+T cells(CD45~+CD3~+CD4+),CD8~+T cells(CD45~+CD3~+CD8~+).The expressions of proinflammatory cytokines in the e WAT and i WAT(i NOS,TNF-ɑ,IFN-γ,IL-6,IL-1β,IL-4,and IL-10)was detected by Quantitative real-time polymerase chain reaction(q-PCR).Inflammatory cytokines including TNF-ɑ,IFN-γ,IL-6,IL-4 and IL-10 in the serum were tested by ELISA methods.Results:For recipient mice in normal chow diet groups(VD-D vs VD-C),the body weight,e WAT weight,i WAT weights and the volume of adipocytes were all significantly higher in VD-D group than that of VD-C group(P<0.05).The same results were presented in the high fat feed VD-D group(VD-D-HFD vs VD-C-HFD)(P<0.05).OGTT test results show that VD deficiency during pregnancy and lactation couldsignificantly increase the obesity rate of male offspring(VD-D vs.VD-C,VD-D-HFD vs.VD-C-HFD).The blood glucose levels and the area under the curve(AUC)in VD-deficient group was also significant increased(P<0.05).The results of ITT assay demonstrated that there was no statistical difference in blood glucose level after insulin injection.Insulin immunohistochemistry results showed that the number of insulin-positive cells of offspring pancreas in VD deficiency groups were significantly decreased(VD-D-HFD vs.VD-C-HFD,VD-D vs.VD-C)(P<0.05),and the amount of insulin secretion was also significantly reduced(P<0.05).Furthermore,the results of serum adipocyte metabolism assay displayed that in normal chow diet mice(VD-D vs.VD-C),the plasma TG level in VD-D group was significantly higher than it in the VD-C group(P<0.05)and the concentrations of TC,Calcium,25(OH)VD,and random blood glucose showed no statistical difference.In the obesity group(VD-D-HFD vs.VD-C-HFD),the contents of plasma TG and TC in VD-D-HFD group were significantly higher than that of the VD--C-HFD group(P<0.05),whereas the concentrations of Calcium,25(OH)VD,and random blood glucose showed no statistical differences in these two groups.In e WAT,the number of F4/80 positive macrophages in male offspring(VD-D-HFD vs.VD-C-HFD,VD-D vs.VD-C)was significantly increased in VD-deficient group(P<0.05).Flow cytometry results showed that the number of M1ATMs,ATDCs,CD4~+T cells,CD8~+T cells and M1/M2 ratio in e WAT of obese mice was significantly increased,while the number of M2ATM and M2/M1 ratio was decreased significantly(P<0.05),and there was no statistical difference in CD4~+T cells/CD8~+T cells ratio.In i WAT,the number of M1ATMs,M2ATMs,ATDCs,CD4~+T cells and the ratios of M1/M2 and CD4~+/CD8~+T cells were significantly increased(P<0.05).In the obese mice feeding with high-fat diet(VD-D-HFD vs.VD-C-HFD),the number of M1ATMs and ATDCs in e WAT and the M1/M2 ratio in VD-D-HFD group were increased,and the M2/M1 ratio was decreased(P<0.05).Moreover,in the mice fed normal chow diet(VD-D vs.VD-C),the number of M1ATMs,ATDCs and M1/M2 ratio in e WAT of mice in VD-D group were increased,and the M2/M1 ratio was decreased significantly(P<0.05).In i WAT,the number of M1ATMs,M2ATMs,ATDCs,CD4~+T cells,CD8~+T cells and M1/M2 ratio were significantly increased(P<0.05).Q-PCR results revealed that,compared with the VD-C group,the expression of pro-inflammatory factors(i NOS,IL-1β,TNF-α,INF-γ,IL-6)and the anti-inflammatory factors(IL-4 and IL-10)in e WAT and i WAT tissues of VD-D group were increased,whereas the expression of Arg-1 was obviously decreased(P<0.05).Moreover,in the obese group(VD-D-HFD vs.VD-C-HFD),the expression of pro-inflammatory factors(i NOS,IL-1β,TNF-α,INF-γ,IL-6)in e WAT was up-regulated,and the expression of anti-inflammatory factors(Arg-1,IL-4 and IL-10)was down-regulated(P<0.05).Consistently,the expression of INF-γwas up-regulated and the expression of Arg-1 was decreased in i WAT(P<0.05).In addition,the serum inflammatory factors in each group were measured by ELISA method.Compared with normal diet feeding mice(VD-D vs.VD-C),the expression of pro-inflammatory factors(TNF-α,INF-γ)in the serum of obese mice(VD-D-HFD vs.VD-C-HFD)was significantly increased(P<0.05),while the expression of anti-inflammatory factors(IL-4 and IL-10)was decreased(P<0.05).Consistent with these results,VD deficiency during pregnancy and lactation can increase the expression of TNF-αor INF-γin the serum of male obesity/normal offspring mice(VD-D-HFD vs.VD-C-HFD,VD-D vs.VD-C),and decrease the expression of IL-4 and IL-10(P<0.05).Conclusions:These results suggest that insufficient maternal VD intake promotes the development of obesity in male offspring by regulating the recruitment and polarization of immune cells.