| IntroductionObesity is pandemic in the modern world and continues to increase at an a-larming rate, especially, the rate is rapidly increasing among children and adolescents. Increased rates of hypertension, diabetes, and dyslipidemia, among other medical conditions, threaten to shorten the longevity of the populace by as much as 5 years. The incidence of depression is increasing and experts suggest this is linked with the increased prevalence of obesity. Novel multidisciplinary, preventive, and therapeutic approaches, and social changes are needed that address the complex interplay of biologic, genetic, and social factors that have created the current obesity epidemic.Hypothalamus plays an important role in the regulation of food intake and energy balance as well as body weight gain , considerable evidence has indicated that most hypothalamic peptides, such as neuropeptide Y ( NPY) , proopi-omelanocorein(POMC) and orexin, influence food intake as well as energy expenditure, so that the two effects synergise in modulating energy balance and body weight. POMC, one important target for leptin and insulin in the hypothalamus , is an anorexigenic peptide synthesized in the arcuate nucleus of the hypothalamus and plays an important role in promoting negative energy balance and preventing excessive fat deposition . the POMC - derived peptide, α - MSH, inhibits feeding via interaction with hypothalamic melanocortin receptors. There is considerable evidence that POMC is regulated by food intake and by changes in leptin and insulin levels, and that POMC mediate some of the central effects of leptin and insulin on energy balance. NPY, a 36 - amino acid peptide with strong orexigenic effects, is expressed by neurons in the hypothalamic arcuatenucleus projecting into the paraventricular, the dorsomedial, and the ventrome-dial hypothalamic nuclei, areas implicated in the control of food intake, injection of NPY into the cerebral ventricles induces a robust feeding response and body weight gain. Six different NPY receptor subtypes ( Yx) have been identified, among them five were cloned and characterized, the Yx and Y5receptor subtypes have been linked to the orexigenic action of NPY. Orexin - A and B are peptides derived by porteolytic cleavage from a 130 -amino acid precursor, prepro - orexin, which wejre exclusively distributed in the lateral hypothalamic area, the posterior hypothalamic area and the perifomical nucleus in rats. Orexin - A is fully conserved across mammalian species, whilst rat and human orexin - B differ by two amino acid, these peptides bind to two Gq - coupled receptors, termed orexin -1 and orexin -2. The receptors are 64% homologous and highly conserved across species. Orexin - A is equipotent at orexin - 1 and orexin - 2 receptors, whilst orexin - B displays moderate selectivity for orexin - 2 receptors. The distribution and pharmacology of the orexin peptides and their receptors indicate that they play a role in various regulatory systems including energy homeostasis and the regulation of feeding.Leptin is an anorectic ob gene product secreted by adipocytes, its receptors are present on some hypothalamic neurons, the hormone is transported through the blood stream and acts on the hypothalamus to regulate energy intake and expenditure, mounting evidence suggests that leptin activates the the anorexigenic POMC and CART neurons in the ARC and suppresses orexigenic NPY and GRP neurons, there may be interactions between the hypothalamic peptides and leptin which play an important role in the regulation of body weight. But the mechanism by which these hypothalamic peptides and leptin interact to control food intake and energy expenditure is still far from clear.In order to study how peripheral leptin and hypothalamic peptides change in different growing stage, the current research examined serum leptin concentration and hypothalamic peptides gene expression for NPY, orexin and POMC in Wistar rats of different weeks. The present study also examined the effects of high fat diet on serum leptin, insulin and glucose concentration as well as central gene expression for NPY, orexin and POMC as compared to standard diet.Furthermore, the current research also study the effects of fasting on obese rats induced by high fat diet through measurement of the above parameters.Materials and methodsAnimals;I. Male and female postnatal Wister rats ( n = 40, respectively) were housed individually in stainless steel hanging cages with the temperature maintained at 201 under a 12 - hour light/12 - hour dark cycle (lights on at 7: 00 am). Subjects were given ad libitum access to water and standard laboratory diet (10% calories from fat, 1% calories from saturated fat). All rats were weighed once every week. Subjects were sacrificed at different stage: 0 day, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks and 20 weeks (female n =5, male n = 5, every time respectively ).At the time of sacrifice, trunk blood was collected for leptin assay. Brain was rapidly excised, chilled in ice cold saline, whole hypothalamus was taken, samples were frozen in liquid nitrogen and stored at -70^! until analyzed.II. Male healthy weaning Wistar rats were treated with high fat chow (40% calories from fat, 20% calories from saturated fat) or standard laboratory chow as control (n = 35/group). Rats were housed individually in stainless steel hanging cages with the temperature maintained at 20^ under a 12 - hour light/ 12 -hour dark cycle (lights on at 6: 00 am). Subjects were given ad libitum access to water and standard laboratory diet or high fat diet . All rats were weighed once every week. Subjects were sacrificed at different stage: 0 week, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 16 weeks ( n =5/group, every time respectively ).At the time of sacrifice, trunk blood was collected for leptin , insulin and glucose assays. Brain was rapidly excised, chilled in ice cold saline, whole hypothalamus was taken, samples were frozen in liquid nitrogen and stored at -70^ until analyzed.III. 120 Male healthy weaning Wistar rats were used in the third experiment, 15 rats were treated with standard laboratory chow throughout the experi-? 7 ?ment as lean control group . 105 Male healthy weaning Wistar rats were placed on the high fat diet for 8 weeks to get obese rats , at the end of the eighth week, rats of the high fat diet group weighed twenty percent higher than that of lean control group were defined as obese rats ( n =61). Obese rats were feed with different diets : (1) obese control group: obese rats were treated with full quantity of high fat diet for 12 weeks (n = 17);(2) obese 80% high fat diet group: obese rats were treated with 80% quantity of high fat diet for 12 weeks (n = 10);(3) obese 60% high fat diet group: obese rats were treated with 60% quantity of high fat diet for 12 weeks (n = 10);(4) obese 100% standard diet group: obese rats were treated with 100% quantity of standard diet for 12 weeks (n = 10);(5) obese 80% standard diet group: obese rats were treated with 80% quantity of standard diet for 12 weeks (n = 10);(6) obese 60% standard diet group: obese rats were treated with 60% quantity of standard diet for 12 weeks (n = 10).At the beginning of the fasting experiment , rats from lean control group and obese control group ( n = 5, n = 7, respectively ) were sacrificed and the parameters above were measured. All rats were weighed once every week. Subjects were sacrificed at the end of 2 weeks or 12 weeks (n =5/group, every time respectively ).At the time of sacrifice, trunk blood was collected for leptin , insulin and glucose assays. Brain was rapidly excised, chilled in ice cold saline, whole hy-pothalamus was taken, samples were frozen in liquid nitrogen and stored at -10X. until analyzed.Hormone and metabolite determinationsSerum from trunk blood was assayed for insulin using assay kit from Chinese Peoples Army Hospital,for leptin using assay kit from Shanghai Shenxiong technoligical company, for glucose using assay kit from Shanghai Kehua bioligi-cal company. According to the manufacturers instructions.RT-PCRTotal cellular RNA was prepared from excised hapothalamus using an RNeasy Mini kit according to the manufacturers instructions. Procedures for reverse transcription (RT) -polymerase chain reaction (PCR) : mixing lOmL 2x? 8 ?single strand buffer;4mL MgSO4(25mM);lmL dNTP (lOmM);lmL AMV (22U/mL);lmL Oligo dT 15;0.5mL Rnase Inhibitor;0.5mL ddH2O, heating the mixture at 65 X for 1 minutes, cooling to 30 X and adding lmL reverse transcription enzyme into the mixture ,5 minutes later, incubating the mixture at 65 X for 30 minutes, then at 98X. for 5 minutes, 5 "^C for 5 minutes, store the cDNA at - 20T!. PCR amplification was performed with Taq polymerase (Qia-gen) for 35 cycles at 94 X for 3 minute, 54X for 1 minute, and 72 X for 1 minute.Data analysisAll values are expressed as mean sem, statistical analyses comparing the different measures for the subgroups were performed using an paired Student t -test. The criterion for the use of the term significant' in the text was that the probability value (p) for a given test be < 0.05.ResultPostnatal development of body weight, leptin, orexin, NPY, and POMC in ratsDuring the neonatal period, the body weight of the rats gradually increased throughout the postnatal period, the body weight increased rapidly from 0 week to 8 weeks, after 16 weeks the body weight increased slowly and became relatively consistent. The body weight of male rats is significantly higher than that of female rats at 8 weeks (p < 0.05 ).The concentration of serum leptin was extremely low in postnatal rats at 1 week and 2 week, the concentration of serum leptin significantly increased at 4 weeks and reached the highest level between 8weeks and 12weeks, the concentration of serum leptin was relatively consistent after 12 weeks, the concentration of serum leptin in female postnatal rats was significantly higher than that in male postnatal rats at 8 weeks ( p <0. 05 ).The pre - orexin mRNA expression in hypothalamus was weakly detected from days 0 to 7. The pre - orexin mRNA expression in hypothalamus markedly increased after 1 week and reached the highest level at 4 weeks, after 4 weeks,the pre - orexin mRNA expression decreased slightly and keeped relatively con-sistant.The level of NPY mRNA expression in hypothalamus of postnatal rats was high on day 0 and decreased slightly at 4 weeks, the expression of NPY mRNA was relatively unchanged after 4 weeks.The POMC mRNA expression in hypothalamus was not detected from weeks 0 to 2, but it was observed after 2 weeks and increased gradually, the POMC mRNA expression reached the highest level at 16 weeks and was consistent after 16 weeks.The effects of high fat diet on body weight, leptin, insulin, glucose, orexin , NPY, and POMC in Male healthy weaning Wistar ratsThe body weight of rats treated with high fat diet increased more rapidly than that of rats feed with standard laboratory diet, the difference was significant at 2 weeks( p <0. 05) , and it was more significant at 4 weeks ( p <0.001).The concentration of serum leptin showed no significant changes between groups from weeks 0 to 2, however, the leptin level of high fat diet rats was significantly higher than that of standard diet rats (8.7 ±2.42ng/ml, 12.4 ±2. 53 ng/ml, respectively, p < 0. 05 ) , the difference was more" significant after 8 weeks. The concentration of serum insulin and glucose of high fat diet rats was significantly higher than that of standard diet rats ( p <0.05).The expression of pre - orexin mRNA in the hypothalamus increased immediately after high fat diet and reached peak at 1 week and 2 week, the expression decreased slightly after 4 weeks and decreased more after 8 weeks.The expression of NPY mRNA in the hypothalamus of high fat diet rats increased after 2 weeks , reached peak from weeks 8 to 12 and decreased slightly afterl6 weeks.No difference between groups was seen in POMC mRNA level after 2 weeks of feeding, after 4 weeks, the high fat diet rats, compared to standard diet rats, showed down - regulation in POMC mRNA expression ( p < 0. 05 ) , the difference was more significant after 8 weeks (p <0.01).The effects of fasting on body weight, leptin, insulin, glucose, orexin, NPY, and POMC in Male healthy weaning Wistar rats?10-The fasting groups rats, especially obese standard diet group rats, displayed significantly reduced body weight, serum leptin, serum insulin and serum glucose.No differences between obese control group and obese 80% high fat diet group were seen in NPY, orexin and POMC mRNA levels at 2 weeks (p >0. 05). at 12 weeks, NPY, orexin and POMC mRNA expression of obese 80% high fat diet group showed significantly increase than that of obese control group.At 12 weeks, no difference between obese control group and obese 60% high fat diet group was seen in POMC mRNA levels ( p > 0.05 ). expression of NPY and orexin mRNA in obese 60% high fat diet group demonstrated significantly increase than that in obese control group.At 12 weeks, the orexin of obese 100% standard diet group and obese 80% standard diet group showed no significant difference with that of lean control group, the NPY mRNA expression was significantly lower than that of obese control group but higher than that of lean control group, the POMC mRNA expression was significantly higher than that of obese control group but lower than that of lean control group.At 2 weeks, no differences between obese 80% high fat diet group and obese 100% standard diet group were seen in NPY, orexin and POMC mRNA levels;at 12 weeks, compared to obese 80% high fat diet group, obese 100% standard diet group showed significant decrease of NPY expression and increase of orexin, POMC expre.ConclusionThe expression of NPY and orexin were prior to the expression of POMC and leptin.High fat diet induced leptin resistance, insulin resistance and obesity , as well as affected the expression of orexin, NPY and POMC. The disturbance of hypothalamic peptides which may be caused by over - activated orexin neurons contributed to obesity.. 11 .Adjust of diet construction reduced body weight of obese rats more effectively and long - lastingly than decrease of diet mount.
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