Effects Of Dietary Energy Sources On Puberty Onset And The Mechanism Investigation In Rats

This study aimed to explore the impact of different energy sources diets on the rats’ puberty onset and metabolic hormones in vivo. Proved that energy regulate KISS1/GPR54expression which are the key signaling proteins at puberty onset in ARC nuclear through the metabolic hormone. And further investigated the molecular mechanisms of leptin regulated the expression of KISS1/GPR54in ARC neuron cells. This study consists of four parts as followings:Exp.1Effect of dietary energy source on rat puberty onset and KISS1/GPR54expression in hypothalamus-pituitary-gonad axis (HPG)60Rats were randomly assigned to starch, oil or AIN93treatments with20replicates in each treatment and feeding through weaning to puberty, to study the effects of dietary energy source on rat puberty onset and KISS1/GPR54expression in HPG. The results showed that:1. The puberty onset time in oil-rich group was earlier than control and starch-rich group (P<0.05). Rat abdominal fat weight and abdominal fat percentage in oil-rich group were significant higher than the starch-rich and control group (P<0.05).2. Serum free fatty acid, triglyceride content were increased in the oil-rich group than the starch-rich and control group (P<0.05).3. Serum leptin concentration was enhanced in the oil-rich group than the starch-rich and control group (P<0.05).4. Serum GnRH, LH and E2concentration were higher in the oil-rich group than the control and starch-rich group at the time of puberty (P<0.05), but FSH, P4had no significant difference in each group.5. OB-Rb gene expression in ARC increased in the oil-rich group at day28and puberty (P<0.05), but KISS1gene expression in ARC increased only at day28in the oil-rich group (P<0.05).6. KISS1, GnRH-R gene expression increased in the AVPV, pituitary and ovaries at the time of puberty in the oil-rich group (P<0.05). The oil-rich group enhanced the expression of GPR54in hypothalamus (ARC nuclear) and pituitary but not detected in the ovary at the time of puberty. LH-R、FSH-R gene expression in pituitary also increased at the time of puberty in the oil-rich group (P<0.05).These results indicated that dietary energy source has significant effects on the rat puberty onset. Rats had younger age at puberty, heavier and larger abdominal fat pad and higher expression of leptin in the oil treatment. Oil treatment increased the expression of KISS1in the ARC nuclear at day28, and KISS1expression of the HPG axis and LH-R, FSH-R expression at the time of pituitary.Exp.2Effects of Insulin on the expression of KISS1/GPR54mRNA in ARC neuron in vitroExprement one found oil treatment has significant effects on the rat puberty onset. Rats had younger age at puberty and higher expression of leptin with oil treatment, in the same time increased the expression of KISS1in the ARC nuclear at day28. This experiment study the dose and timing effects of insulin on the expression of KISS1, GPR54and GnRH in ARC neuron of rat hypothalamus. There were15treatments, and three replicates in each treatment in this study. Under the same reaction time, according to the insulin concentration of1～10mg/ml which is the concentration at physiological conditions in rats to design the adding gradient, and final concentration of insulin were0,5,10,20,40,80and100mg/ml. Also the effects of insulin on the expression of KISS1, GPR54and GnRH at time0,2,4,6,8,10and12h at the same insulin concentration were explored. We used about320embryonic rats in this test. The results showed that:1. Successfully cultured ARC neurons in vitro.2. KISS1mRNA expression were suppressed in the higher insulin concentration (80ng/ml to100ng/ml) and longer (10,12h) treating time (P<0.05). GPR54mRNA expression were increased in20ng/ml and40ng/ml insulin concentration (P<0.05).3. mRNA expression of GnRH、GnRH-R and hormone secretion of GnRH significantly improved in the neuronal cells by the insulin treatments (P<0.05).4. OB-Rb mRNA expression were reduced and IR mRNA expression were increased by insulin treatment (P<0.05).These results indicated that insulin does not promote KISS1expression, which are the key genes of puberty onset in animals, but could increase the secretion of GnRH in ARC neurons.Exp.3Effects of Leptin on the expression of KISS1/GPR54mRNA in ARC neuron in vitroExprement one found in oil treatment rats had younger age at puberty and higher expression of leptin. This experiment evaluated the dose and timing effects of leptin on the expression of KISS1, GPR54and GnRH in ARC neuron of hypothalamus at embryonic rat. There were16treatments, and three replicates in each treatment in this study. Under the same reaction time, according to the leptin concentration of0.7-2.1ng/ml which is the concentration at physiological conditions in rats to design the adding gradient, and final concentration of leptin were0,0.15,0.3,0.6,1.25,2.5,5,10and20ng/ml. Also the effects of leptin on the expression of KISS1, GPR54and GnRH at time0,2,4,6,8,10and12h at the same insulin concentration were explored. We used about360embryonic rats in this test. The results showed that:1. KISS1mRNA expression were increased by leptin in both time and does treatments (P<0.05). GPR54mRNA expression were increased in0.3ng/ml leptin concentration (P<0.05).2. mRNA expression of GnRH、GnRH-R and Ob-Rb mRNA significantly improved in the neuronal cells by0.3ng/ml leptin treatments (P<0.05).3. IR mRNA expressions were reduced by leptin treatment (P<0.05).4. Secretion of GnRH was significantly improved in the neuronal cells by0.3ng/ml leptin treatments (P<0.05).These results indicated that the leptin effects on ARC neuron is directly. Leptin significantly improved KISS1, GPR54and Ob-Rb expressions, and increase the secretion of GnRH in ARC neurons.Exp.4The intracellular signaling pathway in the leptin regulation of Kisspeptin/GPR54systemThis experiment aimed to investigate whether leptin regulation KISS1/GPR54system through the JAK2/STAT3pathway. AG490can block the activation of signal molecular JAK2. We used AG490blocking JAK2/STAT3signal pathway, to investigate the influence of KISS1gene and protein expression in ARC neurons by leptin. We used about200embryonic rats in this test. The results showed that:1. Challenge by5μM AG490, ARC neurons reduces the pJAK2protein expression significantly (P<0.05).2. pJAK2、pSTST3、kisspeptin protein expression were increased by0.3and0.6ng/ml leptin treatments (P<0.05),5uM pJAK2inhibitor AG490blocks leptin stimulation of pJAK2、pSTST3、kisspeptin protein expression in ARC neurons,down regulation them to untreatment level.3. KISS1mRNA expression were increased by0.3and0.6ng/ml leptin treatments (P<0.01),5uM AG490down regulation the expression of KISS] mRNA, droped it to untreatment level.4. GnRH hormone secretions were increased by0.3and0.6ng/ml leptin treatments (P<0.05),5uM AG490down regulation the secretion of GnRH hormone, drop it to untreatment level.These results demonstrated that:leptin regulation kisspeptin protein’s expression through the JAK2/STAT3pathway.Taken together, dietary energy sources had significant effects on the rats’ puberty onset. Rats had earlier age at puberty and heavier, larger abdominal fat pad, higher expression of leptin in the oil treatment. Oil treatment increased the expression of KISS1in the ARC nuclear at day28, and KISS1expression of the HPG axis and LH-R、FSH-R expression in the pituitary. ARC is a start trigger of puberty onset; the other regions of the hypothalamus are the maintaining sites for puberty onset.Leptin has directly effects on the ARC nucleus cells, then increase OB-Rb, KISS1and GPR54gene expression, thus enhancing mRNA expression of GnRH, GnRH-R and hormone secretion of GnRH. Leptin regulate kisspeptin protein’s expression through JAK2/STAT3signal pathway.