Effect Of High Fat Diet Induced Obesity On Hypothalamic-pituitary-testicular Gonadal Axis In Male SD Rats And Its Mechanism

Section one:The effects of high-fat diet-induced obesity on SD male rat metabolism by using bioinformatics analysisObjective:To investigate the effect of high-fat diet-induced obesity on metabolism in obese male ratsMethods:The gene expression profiles of GSE8700 were downloaded from GEO database,and it was analyzed separately using R software(LIMMA R package)and finally identifies the DEGs lists.In order to analyze the differentially expressed genes(DEGs)at the functional level,GO enrichment and KEGG pathway analysis were performed using DAVID online tool.Protein-protein interaction(PPI)network of the DEGs was constructed by Search Tool for the Retrieval of Interacting Genes(STRING)database.Results:A total of 1014 genes were identified after the analyses of GSE8700;of which,544 were up-regulated and 470 were down-regulated.GO analysis results showed that up-regulated DEGs were significantly enriched in biological processes(BP),including oxidation-reduction process,axonogenesis,response to peptide hormone,response to glucocorticoid.the down-regulated DEGs were significantly enriched in biological processes,including female pregnancy,response to organic substance,proteolysis,response to steroid hormone,triglyceride metabolic process,etc.Go cell component(CC)analysis only displayed in down-regulated DEGs,including extracellular space,cytoplasm,blood microparticle.For molecular function(MF),it can be observed only from the down-regulated DEGs which were enriched in serine-type endopeptidase activity,fatty acid binding,phospholipid binding,and scavenger receptor activity.Up-regulated DEGs did not enrich any pathway by KEGG analysis,and down-regulated DEGs were enriched in PPAR signaling pathway,fat digestion and absorption,pancreatic secretion.The top 10 hub genes included heat shock protein 90 alpha family class B member 1(Hsp90ab1),calcium-sensing receptor(Casr),chemokine(C-C motif)ligand 9(Ccl9),receptor-interacting serine-threonine kinase 4(Ripk4),dimethylglycine dehydrogenase(Dmgdh),fatty acid binding protein 1(Fabpl),sedoheptulokinase(Shpk),apolipoprotein H(Apoh),cholecystokinin A receptor(Cckar),glutamate metabotropic receptor 3(Gnn3).Conclusions:This study shows that high-fat diet induced obesity can influence the body's nutrient metabolism,especially lipid metabolism disorders,which have been regarded as one of the key characteristics of obesity.The GO term analysis and the pathway by KEGG analysis showed that obesity may affect the reproduction of male rat,and its mechanism might be related to the metabolic abnormality of steroids hormones.Section two:Obesity is harmful to male fertility:an experimental study of obese male rats and identification of possible mechanismObjective:To investigate the effect of high-fat diet-induced obesity on male fertility and possible mechanismMethods:Forty-five male rats were randomly divided into the control(N=15,normal diet)and test groups(N=30,high-fat diet)and fed for 16 weeks.The rats in the lower quartiles for weight gain(N=8)at the 16th week were defined as obesity resistant and excluded from the test group,and the remaining three-quarters of the rats were classified as the obesity group(N=22).Both of control and obesity group were sacrificed,and body weight and organ indexes were determined.Serum metabolic parameters and reproductive hormones were assayed.Indicators of reproductive function,including apoptosis of spermatogenic cells,oxidative stress level,and sperm count and motility were determined.Results:The general growth status and metabolic parameters of the two groups were compared at the end of the 16th week.The results showed that the weight,Lee index,fat coefficient,serum leptin,INS,and TG levels of the nutritional obese rats significantly increased(P<0.05),while the testis coefficient and kidney coefficient significantly decreased(P<0.05).No obvious differences were found in body length,serum GIU and TC concentrations between the two groups(P>0.05).Serum GnRH,the ratio of GnRH/GnIH,LH,T,SHBG,cFT concentrations were decreased(P<0.05),serum E2 levels and the ratio of E2/TT were increased in the obesity group(P<0.05).No statistically significant differences were found in serum GnIH and FSH between the two groups(P>0.05).Histological examination of the rat testes with HE staining demonstrated vacuolation in the seminiferous tubules and structural dysfunctions in the spermatogenic cells or detachment of germ cells from the basement membrane in the obesity group.No significant difference was found in Johnsen score between the two groups(P>0.05),and the apoptosis index of the spermatogenic cells was increased significantly in the obesity group(P<0.05).SOD concentration decreased significantly in the obesity group(P<0.01),while MDA concentration increased with statistical significance in the obesity group(P<0.05).The sperm concentrations extracted from the epididymis were found no significant difference between the two groups(P>0.05).The sperm motility decreased significantly in the obese group(P<0.01).Conclusions:The obese rats induced by high-fat diet developed dysfunctions in lipid metabolism,reproductive hormones,decreased sperm motility as well as increased oxidative stress levels in the testis tissues.Thus,on the basis of above fact and mechanism,it can be proposed that obesity affect physiological function of male reproductive system and lead to a decline in fertility.Section three:Expression of Kisspeptin-GnRH system is down-regulated in hypothalamic of male obese rats with high-fat-dietObjective:To investigate the potential effect of obesity on Kisspeptin/Kiss1 R-GnRH/GnIH system in hypothalamic of male ratsMethods:Forty-two male rats were randomly divided into control group(N=18,nonnal diet)and test group(N=24,high-fat diet).After 16 weeks,the rats in the lower quartiles for weight gain(N=6)at the 16th week were defined as obesity resistant and excluded from the test group,and the remaining three-quarters of the rats were classified as the obesity group(N=18).The rats in each group were sacrificed for lung hypothalamus tissue,and immunohistochemistry was used to determine the localizations of Lep R,Kisspeptin,Kissl R,GnRH,and GnIH(N=6).The protein levels of Lep R,Kisspeptin,Kiss1 R,GnRH and GnIH were measured by Western blot(N=6).QRT-PCR was used to determine Lep R,Kiss1-,Kiss1 R,GnRH and GnIH-mRNA levels(N=6).Results:Our results showed that the obesity model was successfully established.Lep R,Kisspeptin,Kiss1 R,GnRH,and GnIH-IR cell bodies could be detected in the hypothalamic of male rats in the both groups.The Kisspeptin,Kiss1 R,GnRH,and GnIH-IR cells in the hypothalamic of male rats were located in the cytoplast.The positive expression of Lep R is pale brown in cell nucleus.Both protein and mRNA levels of Lep R,Kisspeptin and GnRH were decreased significantly in the hypothalamic of high-fat diets(HFD)induced obese rats than controls(P<0.05).There was no obvious difference in both protein and mRNA expression levels of Kiss1 R and GnIH between the two groups(P>0.05).Conclusions:A high-fat diet induced obesity lead to male rat hypothalamus Lep R decrease and leptin resistance,eventually leading to Kisspeptin-GnRH production decline.During the process,high-fat-diet induced obesity play a negative regulatory role in the hypothalamus reproductive central.However,this negative effect may be achieved by decreasing GnRH secretion rather than increasing GnIH secretion.