Effect Of Different Energy Balance States On Reproductive Function Of Male Mice And Its Relationship With Nesfatin-1

ObjectiveEnergy balance is closely related to reproductive function,long-term obesity exerts negative impact on male reproductive function,but the mechanism is still unclear.Recent studies show that nesfatin-1 is not only a metabolic molecule that regulates appetite and energy balance,but also can promote male reproductive function and reduce inflammation.Chronic low-grade inflammation is one of the main reasons for the decline of male reproductive function in obesity.The relationship among obesity,nesfatin-1,inflammation and male reproductive function is still unclear.Long-term exercise and diet intervention can effectively down-regulate the energy balance in the body to lose weight,and can also improve the increase of inflammatory response caused by obesity and the decline of male reproductive function,but whether it is related to nesfatin-1 has not been reported.This study changes the energy balance through diet and exercise,to explore the relationship among male reproductive function,nesfatin-1 and inflammation during the energy variation,and to determine the effect of nesfatin-1 on the HPT axis in central and peripheral tissues,which may provide the experimental data for revealing the molecular mechanism underlying metabolism regulates reproductive function,which may also provides theoretical basis for exploring effective methods to improve the reproductive dysfunction caused by obesity.Methods145 newly weaned male C57BL/6J mice were randomly divided into normal control group?NC group,28 mice,fed with standard diet?and high-fat diet group?HFD group,117 mice,fed with high-fat diet?.After 10 weeks of high-fat diet,the mice were randomly divided into four groups:the obesity control group?OC,n=28?,the obesity and calorie restriction group?OR,n=28?,the obesity and moderate exercise group?OE,n=28?,and the obesity and calorie restriction combined with moderate exercise group?ORE,n=28?.The food intake of mice in the OR and ORE group was 70%of the OC group,and mice in OE and ORE groups were conducted an electric treadmill running for 8 weeks.Before sampling,mice in each group were divided into two groups,and saline and nesfatin-1?15ug/kgBW?were injected in each group,once a day,and the injection was conducted for continuous 3 days,then samples were collected.Cell count,H-E staining,ELISA,real-time PCR and western-blot were used to detect the relevant index in blood,hypothalamus and testis.ResultsPart 1:?1?Serum LH,FSH and T levels were significantly reduced and the serum E₂ level was significantly increased after male mice were fed a high-fat diet for 18 weeks.Obesity reduced the relative mass of gonads and caused the structural disorder,significantly decreased thickness and area of spermatogenic cell layer of testis,eventually leading to a significant decrease in sperm concentration and motility?P<0.05?.Serum nesfatin-1 and IL-10 levels were significantly decreased?P<0.05?,but hsCRP and IL-1?levels were significantly increased?P<0.05?in obese mice,which might be the mechanisms of the decreased reproductive function in male mice.After nesfatin-1 injection,serum nesfatin-1 and IL-10 levels were significantly increased in mice of the OC group?P<0.05?,but serum hsCRP and IL-1?levels were significantly decreased?P<0.05?,and LH,FSH and T concentrations were significantly increased in mice of the OC group?P<0.05?,which verified the abovementioned mechanisms.?2?The mRNA and protein levels of GnRH and nesfatin-1 in the hypothalamus were significantly decreased in obese mice?P<0.05?.Obesity also resulted in significantly decreased IL-10 mRNA expression in the hypothalamus?P<0.05?,increased TNF-and IL-1?mRNA and protein expression levels?P<0.05?,and significantly increased IKK?and NF-?B mRNA and protein expression levels?P<0.05?.After nesfatin-1injection,the mRNA and protein levels of nesfatin-1,IL-10 and GnRH in the hypothalamus of mice in the OC group were significantly increased?P<0.05?,the mRNA and protein levels of TNF-?and IL-1?were significantly decreased?P<0.05?,and the mRNA and protein levels of IKK and NF-?B were significantly decreased in the hypothalamus of mice in the OC group?P<0.05?.?3?The mRNA and protein levels of nesfatin-1 and SF-1,as well as the mRNA levels of P450scc in the testis of obese mice were significantly decreased?P<0.05?.Obesity also resulted in decreased IL-10 mRNA and protein levels in the testis?P<0.05?,increased TNF-?and IL-1?mRNA and protein levels?P<0.05?,and increased IKK?and NF-?B mRNA and protein levels in the testis of obese mice?P<0.01?.Nesfatin-1 injection significantly increased the mRNA and protein levels of nesfatin-1,IL-10,SF-1,StAR and P450scc in the testes of mice in the OC group?P<0.05?,decreased the mRNA and protein levels of TNF-?and IL-1?,and decreased the mRNA and protein levels of IKK?and NF-?B in the testes of mice in the OC group?P<0.05?.Part 2:?1?Calorie restriction and combined intervention significantly increased serum LH and T levels,but significantly reduced E2 levels in obese mice after 8 weeks intervention.Dietary control and combined intervention improved gonad morphology and spermatogenic cell layer structure of male mice,increased spermatogenic cell layer thickness and area,and increased sperm motility and concentration?P<0.05?.After diet control and exercise intervention,serum nesfatin-1 and IL-10concentrations were significantly increased,and the decrease of hsCRP,TNF-?and IL-10 concentrations might be the mechanism of the improvement of reproductive function in male mice.After nesfatin-1 injection,the concentrations of nesfatin-1 and IL-10 were significantly increased in each intervention group?P<0.05?,the levels of hsCRP and IL-1?were significantly decreased?P<0.05?,and the levels of LH,FSH,and T were increased,which verified the abovementioned mechanism.?2?Calorie restriction and exercise interventions significantly increased the mRNA and protein expression levels of GnRH and nesfatin-1 in the hypothalamus of OR group and ORE group,significantly increased the mRNA and protein expression levels of IL-10 in the hypothalamus of each interventional group,significantly decreased the mRNA and protein expression levels of TNF-?and IL-1?in the hypothalamus of each interventional group,and finally led to significantly decreased mRNA and protein expression levels of IKK?and NF-?B.After nesfatin-1 injection,the mRNA and protein expression levels of nesfatin-1,IL-10 and GnRH in the hypothalamus of mice in the diet and exercise group were significantly increased?P<0.05?,the mRNA and protein expression levels of IL-1?were significantly decreased?P<0.05?,and the mRNA and protein expression levels of IKK?and NF-?B were significantly decreased in each intervention group?P<0.05?.?3?Calorie restriction and exercise significantly increased the mRNA and protein expression of nesfatin-1,SF-1,StAR and P450scc in the testis,and also significantly increased the mRNA and protein expression of IL-10,decreased the mRNA and protein expression of TNF-?and IL-1?,and decreased the mRNA protein expression of IKK?and NF-?B?P<0.05?.Nesfatin-1 injection significantly increased the mRNA and protein levels of nesfatin-1,SF-1 and StAR in the testes of mice in the intervention group?P<0.05?,increased the mRNA level of P450scc in the intervention group?P<0.05?,and increased the protein expression levels of P450scc in the testes of mice in the ORE group?P<0.05?.The expressions of IL-10 mRNA and protein in each intervention group were significantly increased?P<0.05?,and the expressions of TNF-?mRNA in the testis of mice in each intervention group and TNF-?protein in the OE group were significantly decreased?P<0.05?,and the expressions of IL-1?mRNA and protein in the OR and ORE groups were significantly decreased?P<0.05?.The mRNA and pIKK protein levels of IKK?and NF-?B were decreased in each intervention group.Conclusions?1?Under different energy balance states,short-term injection of nesfatin-1 can stimulate the synthesis of GnRH in the hypothalamus and testosterone synthase in the testis,or up-regulate GnRH release and testosterone synthesis by inhibiting the inflammatory signaling pathway.?2?Long-term positive energy balance?obesity?induced hypogonadism in obese male mice,the expressions of nesfatin-1 in hypothalamus and testes were decreased,which directly and indirectly?by triggering inflammation?decreased GnRH expression in hypothalamus and testosterone synthesis in testis may be one of the mechanisms of hypogonadism in male mice.?3?Long-term diet and moderate load intervention effectively up-regulate the expressions of nesfatin-1,inhibit the inflammation in the hypothalamus and testes,which up-regulates the release of GnRH and the synthesis of testosterone,and eventually alleviate the hypogonadism caused by obesity in male mice.