Role And Mechanism Of Chronic Restraint Stress In Regulating Energy Metabolism And Reproductive Function Through Hypothalamic Kisspeptin Neurons

Objectives:Stress activates the hypothalamic-pituitary-adrenal(HPA)axis,affecting energy homeostasis and reproductive function.Previous studies have confirmed the role of Kisspeptin in reproductive function.Recent researches found that Kisspeptin mainly expressed on the arcuate nucleus of the hypothalamus and it was also a factor regulating energy balance.Our previous research found that dexamethasonecan inhibit the expression of Kiss1 m RNA and Kisspeptin protein in GT1-7 cells,which both expressed Gn RH and Kisspeptin,which could be reversed byglucocorticoid receptor antagonist RU486,suggesting that the hypothalamic Kisspeptin neurons might be a new important central target in stress affecting energy metabolism and reproductive function.Therefore,the purpose of this study was to investigate whether stress affected energy metabolism and reproductive function through the glucocorticoid receptor(GR)on Kisspeptinneurons in the hypothalamus.Methods:1.The first part of this study focused on the effects of chronic restraint stress on energy metabolism and reproductive function in mice.We constructed a chronic restraint stress mouse model,monitored the body weight and food intake of mice daily,and performed vaginal smears on female mice to observe the estrous cycle.After 28 days of restraint stress,intraperitoneal glucose tolerance test was performed and tissues were obtained.The ratio of white adipose tissue,brown adipose tissue and body weight was calculated.Serum sex hormone levels were measured,including LH,FSH and estrogen levels in female mice and testosterone level in male mice.Paraffin-embedded ovaries were used for HE staining to observe ovarian morphology.2.The second part of this study focused on the effects of chronic restraint stress on the expression of serum cortisol and prolactin and the expression of GR and Kiss1 genes in the hypothalamus.3.Based on the above research,we constructed Kisspeptin specific glucocorticoid receptor knock out(KGRKO)mice,collected blood samples from the orbital canthus vein blood of mice in the morning and afternoon,and measuredserum cortisol levels to assess the rhythm of cortisolby ELISA.The mice were subjected to restraint stress intervention,the body weight of the mice was monitored every day,and the vaginal smear was performed on the female mice to observe the estrous cycle.After 28 days of restraint stress,serum was collected to detect serum sex hormone levels,including LH,FSH and estrogen levels in female mice and testosterone level in male mice by ELISA.Results:1.The effects of restraint stress on energy metabolism in mice were investigated.The body weight and energy intake of the restraint stress group were lower thanthose of the control group,high-fat diet group,and high-fat diet+restraint stress group.The high-fat diet+restraint stress group showed lower body weight and energy intake than the high-fat diet group.Glucose tolerance test suggested thatthere was no statistical difference in the glucose tolerance between the restraint stress group and the control group both in male and female mice with normal diet.While the case of a high-fat diet,the high-fat diet+restraint stress group showed impaired glucose tolerance compared with the high-fat diet group.2.The effects of restraint stress on thereproductive function of mice were investigated.After restraint stress,the female mice showed irregular estrous cycle.LHand FSH levels in the stress group were significantly decreased compared with the control group.No differences were observedin estrogen level and ovarian morphology in female mice.No differences were observed in testosterone levels and sperm morphology in male mice.3.After restraint stress,the levels of serum cortisol and prolactin in male and female mice were significantly higher than the control group,and the hypothalamus Kiss1 gene m RNA expression and Kisspeptin protein expression were significantly decreased.4.We constructed Kisspeptin neuron-specific GR knockout mice(KGRKO).Compared with GR^flox/flox/Kiss1^Cre-control mice,there were no differences observed in the nadir and peak levels of serum cortisol in male GR^flox/flox/Kiss1^Cre+KGRKO mice.In female mice,compared with GR^flox/flox/Kiss1^Cre-control mice,the nadir serum cortisol increased significantly in the morning in GR^flox/flox/Kiss1^Cre+KGRKO mice,however,there was no significant difference in the serum peak cortisol in the afternoon.Compared with the control group,the restraint stress group and the KGRKO+restraint stress group had a significant weight loss compared with the KGRKO group.Compared with the restraint stress group,the KGRKO+restraint stress group had a reduced weight loss,suggesting that restraint stress might partially affect the energy metabolism through GR on Kisspeptin neurons.In terms of reproductive function,the restraint stress group and the KGRKO+restraint stress group showed missingpre-estrus period or prolonged estrous cycles.Serum LH and FSH in KGRKO+restraint stress group decreased significantly compared with KGRKO group.There was no significant difference in the level of serum testosterone among the restraint stress group,KGRKO group and KGRKO+restraint stress group.Conclusions:Chronic restraint stress induced weight loss in mice and reversedbody weight gain induced by high-fat diet.Chronic restraint stress played a negative role in regulating reproductive function.The effects of chronic restraint stress on energy metabolism and reproduction were partially mediated by GR on Kisspeptin neurons inthe hypothalamus.This study provided a central mechanism for chronic restraint stress in affecting energy metabolism and reproductive function,and was of greatsignificance in revealing the relationship between the HPA axis with energy metabolism and reproduction.