The Effect Of DHT-induced Hypothalamus Insulin Resistance On The Pathogenesis Of Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS)is a common reproductive and metabolic disorder with heterogeneous phenotype,which is characterized by anovulation,hyperandrogenism and polycystic ovaries.The percentage of metabolic syndrome like abdominal adiposity,obesity,and insulin resistance(IR),is higher than the women with non-PCOS.Metabolic syndrome significantly impacts the long-term health risks and reproduction function of PCOS.The mechanism of metabolic disorder remains unclear,androgen excess may induce classic metabolic tissues(such as muscle,liver and fibroblasts)insulin resistance.There are emerging global data that PCOS patients have different baseline dietary energy intakes,the intake of energy and fat are higher in PCOS patients than common control.Despite the influence of body mass indexes,PCOS patients still have higher energy intake.In adolescent girls with PCOS,the eating-disorder was associate with androgen excess.The regulations of food intake and energy homeostasis are controlled by a complex regulatory network in the Arcuate nucleus(ARC)of the hypothalamus.NPY/Agrp neurons in the ARC synthesize the orexigenic peptides NPY and Agrp,which result in a significant increase in food-intake.By contrast,POMC neurons in the ARC,synthesize an anorexigenic peptide ?-MSH,which leads to a decrease in food intake.Any conditions which can impact on these neurons will lead to food-intake disorder.Some research had reported that the NPY level in both obesity and non-obesity PCOS patients is higher than non-PCOS women,which also indicate that the PCOS patients have eating disorder.But the mechanism underlines this phenomenon is still indistinct.Therefore,an improved understanding of the disorder may be critical to the pathogenesis of PCOS.In our study,a DHT-induced PCOS rat model was used to detect the effect of androgen excess on the hypothalamus insulin signal and the NPY/Agrp/POMC neurons in the ARC which can lead to food-intake disorder.In addition,we elucidated the possible role of hypothalamus insulin signal on GnRH expression.Part ?Objective To determine the effect of prepuberty androgen excess on the body weight,food intake,and metabolic parameter in female rat.Methods The 3weeks aged female rat was subcutaneously implanted a tube containing 7.5 mg DHT or an empty tube as a control.The body weight,food-intake,glucose and lipid metabolic parameters of the rat model were determined.The NPY/Agrp and POMC expression and also the insulin signal in the ARC of hypothalamus were detected.Results Prepuberty DHT treatment resulted in markedly elevated body weight and foodintake(P<0.01).The elevated foodintake was appeared earlier than the elevated body weight.The intraperitoneal glucose tolerance tests(IPGTTs)results showed that,although there was no difference between the two groups in glucose level on 8 weeks old(P>0.05),the insulin level was significantly higher in the DHT rat.The glucose levels at 15,30,60 and 120 min following the glucose administration were significantly higher in DHT rat on 13 weeks old(P<0.01).The intraperitoneal insulin tolerance tests(IPITTs)results showed that,the glucose level at 30 and 120 min following the insulin administration were significantly higher in DHT rat on 8 weeks old(P<0.05).Moreover,DHT induced significantly elevated glucose levels at 30,60 and 120 min after insulin administration on 13 weeks old(P<0.05).There was no difference of PCK1 expression between the two groups(P>0.05).The body fat was higher in the DHT rat,mainly in visceral fat(P<0.05).No differences were observed in the serum total cholesterol and free fatty acids levels in the DHT and control group,however the triglyceride level was increased in DHT rat(P<0.05).Comparing with control rat,the UCP1 mRNA expression was significantly lower in DHT rat.(P<0.05).The serum level of leptin was higher in the DHT rat(P<0.05),The mRNA levels of NPY and Agrp were higher in the DHT rat comparing to controls(P<0.05).Prepuberty DHT treatment remarkably decreased both the basal level and the insulin-stimulated phosphorylation of AKT and GSK in the hypothalamus(P<0.05)in both 6weeks old and 13weeks old rat.Part ?Objective To investigate the effect of hyperandrogen on the NPY/Agrp and POMC expression and on the insulin signal in hypothalamus,and to determine whether these effects were independent of obesity and the possible pathology.Methods To exclude the possible effect of obesity on the hypothalamus,we testified the NPY/Agrp and POMC expression and the insulin signal in the 6-week old rat model.We also constructed a pared-food intake DHT rat model,giving DHT rat the same amount of food in control rat,and detected the NPY/Agrp and POMC expression and the insulin signal in the pared-food intake DHT rat model.The effect of DHT on NPY/Agrp and POMC expression was detected in the primary culture hypothalamus neuron cell.We also use the GT1-7 cell to investigate the effect of DHT and androgen receptor on insulin signal.The TNFa expression and NF-KB signal were detected in the pared-food intake DHT rat model.The GT1-7 cell was treated with DHT,then the TNFa expression and NF-KB signal were investigated.Results The mRNA levels of NPY and Agrp were higher in the DHT rat comparing to controls(P<0.05).Prepuberty DHT treatment remarkably decreased both the basal level and the insulin-stimulated phosphorylation of AKT and GSK in the hypothalamus(P<0.05)in both 6weeks old rat.Prepuberty DHT treatment also reduced the phosphorylation of JAK2 and STAT3 in 6weeks old rat and pared-food intake DHT rat model(P<0.05).The serum level of insulin and leptin did not differ between the pared-food intake DHT rat model(P>0.05).Prepuberty DHT treatment increased the NPY/Agrp and POMC mRNA expression in the ARC of hypothalamus(P<0.05).In the primary culture hypothalamus neuron cell,DHT treatment significantly increased the NPY expression(P<0.05).In GT1-7cell,DHT suppressed the TNFa expression and the NF-KB signal.Using LY294002 to suppress insulin signal in GT1-7 cell can inhibit the NF-KB signal.Part ?Objective To investigate the effect of DHT on ovulation dysfunction and the mechanism of hypothalamus insulin signal involved in that effect.Methods The 3 weeks aged female rat was subcutaneously implanted a tube containing 7.5 mg DHT or an empty tube as a control.Then the estrus cycle and serum LH level were detected.6 weeks old SD female rat was intracerebroventricular injection of LY294002,the glucose metabolism and serum LH level were determined after that.Using GT1-7cell to testify whether DHT can affect the GnRH expression through insulin signal.Res?lts Comparing to control rats,DHT rats had irregular estrus cycle and suppressed LH secretion.DHT down-regulated the GnRH expression in GT1-7cell(P<0.05).Anti-androgen receptor treatment can suppress that effect(P<0.05).Intracerebroventricular injection of LY294002 can decrease the serum LH level and increase the glucose level at 15,30,60 and 120 min after glucose administration(P<0.05).Down-regulating the insulin signal in GT1-7 cell can inhibit GnRH expression(P<0.05).Conclusion1.The prepuberty DHT treated rat exhibited elevated body weight,elevated food-intake,reproductive and metabolic derangements,similar to the symptom of PCOS.2.We investigated the contribution of prepuberty androgen excess to the hypothalamus feeding center.The results showed that androgen excess induced hypothalamus insulin resistance and increased the expression of NPY and Agrp in hypothalamus,resulting a diet-dependent obesity.The effect of androgen excess on hypothalamus was independent of obesity.Androgen excess induced diet-dependent obesity through insulin signal in the hypothalamus.This could offer a new insight into the pathology of metabolic derangements in PCOS3.Androgen excess decreased TNFa expression and inhibited NF-KB signal both in vivo and in vitro,which was different from the over-nutrition induced hyperphagia.This offers a new insight to investigate the mechanism of DHT induced hyperphagia.4.DHT suppressed GnRH expression through androgen receptor and insulin signal in GT1-7 cell.5.Our results showed that prepuberty DHT treatment can induced hyperphagia and ovulation dysfunction.DHT-induced hypothalamus insulin resistance was involved in the possible mechanism of hyperphagia and ovulation dysfunction.This results could be valuable for investigating the pathology of PCOS.