| Background:Long-term and massive high-fat diet(HFD)-induced obesity has become one of the main causes of testosterone deficiency(TD).However,the concrete mechanism has not been clear.Testosterone biosynthesis is a complex and multi-step process in which many intermediate substances conversion are involved.The key regulatory molecules are steroidogenic acute regulatory protein(StAR),cytochrome P450 side-chain cleavage enzyme(P450scc)and 3?-hydroxysteroid dehydrogenase(3?-HSD).As the first and the most important rate-limiting molecule in the process of testosterone synthesis,StAR assists cholesterol to cross the outer mitochondrial membrane(OMM)and the inner mitochondrial membrane(IMM)into the mitochondrial matrix.Mitochondrial homeostasis plays an important role in maintaining the function of StAR.Cyclophilin D(CypD)plays a crucial role in regulating mitochondrial function.Objective:In this experiment,a HFD feeding mouse model was first established to explore the effects of HFD on testosterone levels in serum and testes,StAR and CypD expression levels,as well as mitochondrial structure and function.Subsequently,combining with the CypD overexpression model and global CypD gene knockout model,we further clarified the role of CypD in HFD-induced StAR expression downregulation.Methods:1.Construction of a HFD mouse model:C57BL/6 mice were fed with normal diet(ND)to 8 weeks of age and randomly divided into two groups.One group was continued to be fed with ND and the other group was given HFD and then was fed for 16 weeks.During this period,body weight was recorded every two weeks.At the end of the 16 weeks,the distribution of body fat in whole body and the perineum in mice were examined via X-ray.Body somatotype,testis size and fat mass around epididymis of mouse were observed.Serum lipid profiles were determined by biochemical analyzer.The levels of male sex hormones(total testosterone,TT and luteinizing hormone,LH)in serum and testes were detected by corresponding enzyme-linked immunosorption assay(ELISA)kit of TT and LH respecctively.Lipid accumulations in the testis were observed via oil red staining.2.At the 16th week fed with HFD,the difference in the mRNA expression levels of StAR,P450scc and 3?-HSD between the HFD and ND group was detected via RT-PCR.3.The effects of HFD on the protein expression of StAR was further detected by Western Blot technique and immunofluorescence staining.4.The effects of HFD on the mitochondria in Leydig cells were examined via transmission electron microscopy.5.In the mouse model of HFD,the effects of HFD on CypD mRNA and protein expression levels in testes were investigated by RT-PCR and Western Blot technique.6.Construction of a CypD overexpression mouse model:8-week-old C57BL/6 mice were injected with adenovirus(Ad-PPIF)containing CypD DNA(ppif)or an empty adenovirus(Ad-EGFP)via tail vein once a week for 2 weeks.RT-PCR and Western Blot techniques were respectively used to check the changes of CypD and StAR mRNA and protein expression in mouse testes.7.Construction of a CypD gene full-knockout(Ppif-/-)mouse model:Ppif-/-mice and C57BL/6 wild type(Ppif+/+)mice were hybridized for 10 generations to produce heterozygote(Ppif+/+)male and female Ppif+/-mice,as parents,then they were mated to produce two groups of mice in a littermate,which are respectively Ppif-/-and Ppif+/+.At 8 weeks of age,all these mice were given HFD for 16 weeks.The expression changes of CypD and StAR protein in mouse testis were detected by Western Blot.Results:1.Construction of a HFD mouse model:The trend of weight gain in HFD group was more pronounced than that in ND group;and from the 4th week,there was a statistically significant difference in body weight between the two groups within the same week(p<0.05).The body fat distribution of the HFD group was more than that of the ND group both in the whole body and the perineum,and the difference was statistically significant(p<0.05).The mice in HFD group were much more obese than those in ND group;the testicular size of HFD group was slightly smaller than that of ND group,whereas the fat mass around the epididymis were significantly more than those of ND group.The analysis via biochemical analyzer showed that only triglyceride(TG)concentrations were not statistically significant between the two groups;the levels of total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)in serum were obviously elevated in the HFD group,and all these differences were statistically significant(p<0.05).The results of ELISA kits showed that although both T and LH concentrations in the HFD group were reduced compared with ND group,there was no statistically significant in the two sex hormones between these two groups;the intratesticular testosterone levels in the HFD group were dramatically reduced compared with ND group(p<0.05).Representative oil red staining showed that the lipidosis in interstitial tissue in testes of mouse in the HFD group was significantly increased compared with ND group.2.The results of RT-PCR showed that at the 16th week fed with HFD StAR mRNA expression levels were significantly down-regulated in the HFD group compared with the ND group(p<0.05).P450scc mRNA levels were only numerically increased but not statistically significant;while the mRNA expression levels of 3P-HSD were significantly increased in the HFD group compared with the ND group(p<0.05).3.The gray analysis of Western Blot showed that the expression levels of StAR pretein in the HFD group were markedly lower than those in the ND group(p<0.05).Representative immunofluorescence staining showed that green fluorescence representing StAR protein in the HFD group was more feeble than that in the ND group.4.Representative transmission electron microscopic images showed that compared with the ND group,intermembrane space of mitochondria of Leydig cells in the testicular interstitium was enlarged in HFD group.5.The expression levels of CypD mRNA and protein were much higher than those in the ND group(p<0.05).6.Construction of a CypD overexpression mouse model:Both CypD mRNA and protein expression levels were significantly elevated in Ad-PPIF group relative to Ad-EGFP group(p<0.05).The expressions of StAR mRNA and protein were significantly down-regulated in Ad-PPIF group relative to Ad-EGFP group(p<0.05).7.Construction of a CypD gene full-knockout(Ppif/-)mouse model:Western Blot and gray analysis showed that testicular CypD protein expression was completely inhibited in Ppif-/-HFD group compared with Ppif+/+ HFD group.There was a slight increase in the expression levels of StAR protein in Ppif-/-HFD group but not statistically significant(p>0.05).Summary and conclusions:HFD may induce mitochondrial dysfunction by activation of CypD overexpression,which may be involved in the down-regulation of StAR expression induced by HFD.This study provides a possible and new idea for the treatment of testosterone deficiency induced by HFD.In addition,the study described the link between long-term HFD and testosterone deficiency,leading people to pay attention to unhealthy eating habits and reminding people to maintain a moderate diet. |
PDF Full Text Request