The Effects Of Leptin And Glucocorticoids On Metabolism And Fertility

Objective:Leptin,an adipocyte-derived hormone,plays a positive role in the daily life for normal reproductive function and energy homeostasis,while glucocorticoid(GC),a stress-induced hormone,plays an inhibitory role in the time of emergency.Previous studies demonstrate that leptin exerts its actions primarily on GABAergic neurons,then kissl neurons to up-regulate GnRH and gonadotropin secretion centrally in the female mice.Data is lacking on the leptin reproductive pathway of males.Furthermore,neuronal pathways that communicate between GC and leptin as metabolic signals to the HPG axis are poorly understood.Besides,high-dose glucocorticoids(HDG)are used in the treatment of autoimmune diseases.Glucocorticoids-induced hyperglycemia(GIH)is often described in elderly patients.In young patients with autoimmune diseases,however,the risk for GIH has not been well characterized.In this study,firstly we used transgenic mice model to unravel the leptin reproductive pathways in males and central interaction between GC and leptin,by cre-loxp techniques which selectively delete leptin receptor at GABAergic neurons in males(Vgat-Cre;Lepr lox/lox)and glucocorticoid receptor(GR)at leptin receptor expressing neurons in both females and males(Lepr-Cre;GR lox/lox),respectively.Secondly,we monitored the glucose levels of young inpatients with autoimmune diseases undergoing HDG therapy to assess the metabolic effects of GC in clinical settings.Methods:For basic research,we accessed their fecundity by the time of puberty onset(progression to vaginal opening in females and preputial separation in males)with continuing body weight after weaning(P21-P60),and fertility tests among Vgat-Cre;Lepr lox/lox males,Lepr-Cre;GR lox/lox females and males.Secondly,pair-feeding tests were done to evaluate the fertility of Vgat-Cre;Lepr lox/lox males.Thirdly,for Lepr-Cre;GR lox/lox female mice and controls,we performed 48-hour fasting tests;for Lepr-Cre;GR lox/lox male mice and controls,we performed 48-hour fasting tests and 6-wksHDGtherapy(100-200 pg/ml corticosterone)with 24-hour fasting tests at the 5th wk.We checked the body weight,glucose levels and LH levels before and after every test.Finally,Serum and hypothalamic tissue extraction,orchidectomy were performed to compare the LH and FSH levels,the kissl mRNA expression,and the pathology of testes and testes weight between Vgat-Cre;Lepr lox/lox males and controls.We also performed orchidectomy after HDG therapy to compare the testes weight and the weight of adipose around testes between Lepr-Cre;GR lox/lox males and controls and those without exposure to GC.For clinical research,we recruited 24 inpatients(median age,32 years;interquartile range,25-42)with exacerbations of autoimmune diseases,receiving 1-2mg/kg/day prednisone or equivalent methylprednisone.Fourteen subjects were naive to glucocorticoids(group 1)and 10 subjects were on glucocorticoid maintenance(≤15 mg/day prednisone at least 3 months)(group 2)prior to HDG.All subjects were monitored by continuous glucose monitoring system(CGMS)for 3 days.Results:For basic research,we found that the Vgat-Cre;Lepr lox/lox male mice had significant increased body weight and delayed puberty onset(38.4 ± 3.6 d vs 27.9 ± 2.0 d,P<0.0001).Pair-feeding tests couldn’t reverse their infertility(P<0.0001).Vgat-Cre;Lepr lox/lox males also had significant reduced testes weight after body weight correction(0.007 ± 0.004 vs 0.026 ± 0.014,p=0.014).Surprisingly,these males with deletion of leptin receptor at GABAergic neurons had similar amounts of kiss1 neurons in the ARC of the upper hypothalamus level,similar levels of LH and FSH in the pituitary level and similar amounts of spermatozoa and Leydig cells with controls in the lower gonad levels(p=not significant).The Lepr-Cre;GR lox/lox male mice showed a markedly lower body weight(P=0.0007).Both Lepr-Cre;GR lox/lox females and males showed regular puberty onset,however,the females had impaired estrous cycles(increased estrous phase,P=0.0001;decreased metestrus/diestrus phase,P=0.001)and reduced fertility(trend of increased time to interlitter intervals,25.0 ± 2.9 d vs 21.0 ± 1.4d,p=0.059;decreased numbers of pups in each litter,5.7±2.6 vs 9.0±1.3 pups/litter,p=0.015).Fasting tests and repeated fasting tests after HDGtherapy didn’t exhibit any significant difference in body weight,glucose levels and LH levels between Lepr-Cre;GR lox/lox female and male mice vs controls.Lepr-Cre;GR lox/lox males had remarkable increased body weight increment(p=0.0009)but not adipose weight around testes than controls after HDG therapy.For clinical research,GIH developed in 21(91%)subjects,11/13 in group 1 and 10/10 in group 2.The main peak of glucose excursion(128.7 ± 6.4 mg/dL,group 1;143.9 ± 10.0 mg/dL,group 2)occurred at 2 to 3 pm.Another peak occurred before sleep.Two-hour mean postprandial glucose levelswere normal in both groups:breakfast,105.0 ± 28.4 versus 125.6 ± 24.4 mg/dL,P=.065;lunch,115.7 ± 21.1 versus 135.9 ±29.0 mg/dL,P =.082;dinner,122.8 ± 18.5 versus 137.8 ±26.4 mg/dL,P =.144 in groups 1 and 2,respectively.There was a positive association between pretreatment hemoglobin A1C and peak glucose levels(P<.0001).Notably,35%of our subjects experienced early morning hypoglycemia(65.2 ± 2.8 mg/dL).Conclusions:Together,the current study provides substantial new information in the molecular pathways of neuroendocrine area and clinical guidance for glucose management.It implicates that leptin’s action through GABAergic neurons is necessary for metabolism and sufficient to modulate puberty onset and fertility in males.We also demonstrated that GC might inhibit leptin metabolic signaling in the central brain of males,and is critical for HPG axis functioning in females.Under stress,GC might not directly influence leptin to regulate HPG axis,or the effect is extremely mild in males.Finally,GC and leptin might act separately to control HPG axis in response to starvation in both females and males.In hospitalized young patients with autoimmune diseases,CGMS data revealed that short-term consistent HDG treatment(1-2mg/kg/day prednisone or equivalent)induced mild hyperglycemia,but early morning hypoglycemia was common.There is no difference about glucose levels among young patients with or without long-term low-dose GC maintenance.