| Metabolic syndrome (metabolic syndrome, MS) is a collection of clinical syndrome that is determined by genetic and environmental factors while characterized as a complication of multiple metabolic diseases (such as central obesity, diabetes and impaired glucose tolerance, hypertension, dyslipidemia, and hyperuricemiapsychosis). In recent years, the incidence of MS increases significantly and makes a serious threat to human health. Intrauterine growth retardation (IUGR) refers to fetus whose gestational age is greater than37weeks but birth weight less than2500g or birth weight is two standard deviations less than the average weight. Studies have shown that malnutrition can result in IUGR with increased susceptibility to MS, but the next generation offspring’s susceptibility to MS has not well reported. In the present study, we established an IUGR rat model by prenatal ethanol exposure during middle and late gestation, then observed the susceptibility to MS in IUGR offspring (Fl and F2generation). and tried to investigate the mechanism from the perspective of neuroendocrine metabolic programming of hypothalamic-pituitary-adrenal (HPA) axis.PARTⅠEffects and mechanism of prenatal ethanol exposure on susceptibility to metabolic syndrome in IUGR ratsObjective:To demonstrate the phenomenon of metabolic syndrome (MS) susceptibility in adult IUGR offspring induced by prenatal ethanol exposure and to investigate the potential mechanism. Method:For mating, two female rats were placed together with one male rat overnight. The day at which the evidence of mating (i.e. vaginal plug or vaginal smear with sperm cells) was observed was designated as GD0. From GD9to GD20, the pregnant rats were orally administered4g/kg of ethanol to establish an IUGR rat model. The control group received the same volume of the vehicle. The dams were normally delivered. The weight of pups were weighed per week since postnatal week1(PW1). The pups were sub-grouped according to gender and the number of animal in each group, namely the female control group, female ethanol group, male control group, male ethanol group. The offspring were fed by high-fat diet containing89.5%of corn flour,10%of lard and0.5%of cholesterol from the day from PW4. Rectal temperature were measured in PW20; oral glucose tolerance test (OGTT) were performed in PW21; the animals were anesthetized and sacrificed at9-12AM in PW24, the serum and tissues were collected. The serum insulin were detected by radioimmunoassay; the biochemical and EL1SA assay were used to determine the following indices:serum glucose, triglyceride(TG), total cholesterol (TCh), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), corticosterone (CORT), and adrenocorticotrophic hormone (ACTH), insulin resistance index (IRI). Real-time PCR were employed to measure the mRNA expression ofsteroidogenic acute regulatory protein (StAR), P450cholesterol side chain cleavage (P450scc),11β-hydroxyl steroid dehydrogenase-1(11β-HSD1), glucocorticoids receptor (GR), corticotropin releasing hormone(CRH) and arginine vasopressin(AVP). Results:(1) postnatal body weight growth rate and basal body temperature:When being compared with control, the body weight of male ethanol group had no significant change but presented a significant increase in growth rate (P<0.01). the rectal temperature at11AM and11PM in female ethanol group were significantly raised (P<0.01), but male ethanol were not changed.(2) glucose metabolism phenotype and glucose tolerance:When being compared with control, basal glucose level in female ethanol group was increased (P<0.05), and the serum insulin and IRI exhibited a declining tendency, the glucose tolerance was enhanced (up-shifting of OGTT curve), however, the serum insulin level and IRI in male ethanol group were also tended to be decreased while the blood glucose and tolerance had no significantly change.(3) Lipid metabolism:When being compared with control, the level of TG and TCh were not changed in female ethanol group, but the level of LDL-c was significantly increased (P<0.01), the HDL-c content presented no significant alteration, and the ratio of TCh/HDL-c and of LDL-c/HDL-c were not altered; for male, the TCh and LDL-c were increased (P<0.05. P<0.01), TG and HDL-c remained unchanged, the ratio of TCh/HDL-c significantly increased (P<0.05) while that of LDL-c/HDL-c remained unchanged.(4) HPA axis activity and ACTH secretion rhythm:Compared with the control group, female ethanol group showed no significant change in content of ACTH and decreased trend of CORT content as well as decreased hypothalamic AVP mRNA expression (P<0.05), CRH mRNA expression as well as adrenal StAR and P450scc mRNA expression showed decreased tendency. No significant changes in ACTH circadian rhythm were observed the secretion at multiple time-point were varied, the secretion at9:00was decreased (P<0.01) while secretion at13:00secretion was increased (P<0.01); male ethanol group blood ACTH content was increased (P<0.01), but CORT was decreased (P<0.01), hypothalamic AVP and CRH mRNA expression were not changed significantly, the male adrenal StAR and P450scc mRNA expression was reduced (P<0.05), the ACTH level was similar with female.(5) Change in hippocampal function:Compared with the control group, the female ethanol group rat hippocampal11beta-HSD-1and GR mRNA expression showed no significant change, and male11beta-HSD-1and GR mRNA expression were increased (P<0.05, P<0.01). Conclusion: the adult IUGR offspring induced by prenatal ethanol exposure might be susceptible to MS with a sex-dependent manner. The mechanism might be attributed to the neuroendocrine metabolic programmed alteration in prenatal ethanol-exposed IUGR offspring, and the disorder of glucose and lipid metabolism induced by postnatal high-fat diet.PART IIThe susceptibility to metabolic syndrome in the offspring of prenatal ethanol-exposed rats and the potential mechanismObjective:to observe the phenomenon of MS susceptibility in prenatal ethanol exposure induced IUGR fetus (F0generation) and the offspring (F2generation) t, and to explore its mechanism from the perspective of HPA axis dysfunction. Methods:The animal protocol was same as previous part. After weaning, the pups were fed by high-fat diet until maturity. The adult offspring from different group were mated to give a birth to second generation. The day of F2generation pup’s birth was designated as postnatal day 0. The bodyweight were recorded since postnatal week1. The pups were weaned and separated according to gender since PW4, and subjected to four groups with10animals in each group:female control group, female ethanol group, male control group, male ethanol group. The animals were fed by normal diet. Rectal temperature were measured in PW20; OGTT were performed in PW21; ACTH secretion rhythm was determined in PW22; the chronic, unexpected and moderate stress (chronic stress) were performed on PW23:6different stimulus were given to animals for21days, including fasting for24h, water deprivation for24h,5min tail clamping,4℃water swimming,45℃warming for5min and reverse of day and night. One stimulus was randomly selected in each day in order to avoid the adaption of animal, animals were anesthetized and sacrificed at in PW27, the serum and tissues were collected. The serum insulin were detected by radioimmunoassay; the biochemical and ELISA assay were used to determine the following indices:serum glucose, TG, TCh, LDL-c, HDL-c, CORT, ACTH and IRI. Real-time PCR were employed to measure the mRNA expression of StAR, P450scc,11β-HSD1, GR, CRH and AVP. Results:(1) postnatal bodyweight growth rate and basal body temperature:When being compared with control, the body weight of ethanol group had no significant change but presented a decline in growth rate (P<0.01). The rectal temperature at11AM remained unchanged but was significantly raised at11PM in ethanol group (P<0.01).(2) glucose metabolism phenotype:When being compared with control, basal insulin level in ethanol group tended to be decreased, especially for female (P<0.05); the blood glucose level all presented decline trend, especially for female (P<0.05) the glucose tolerance had no significantly change.(3) lipid metabolism:When being compared with control, the level of TG, TCh, HDL-c and LDL-c were not changed in female ethanol group, and for the male the level of HDL-c and LDL-c was significantly decreased (P<0.05, P<0.01), the level of TG and TCh were not changed. The ratio of LDL-c/HDL-c were increased in male ethanol group (P<0.01), and the ratio of TCh/HDL-c was increased, however, the female ethanol group remained unchanged.(4) HPA axis activity and ACTH secretion rhythm:When being compared with the control group, both male and female ethanol group showed no significant change in content of ACTH, but female presented an increased trend of CORT content; both hypothalamic AVP mRNA expression of male and female ethanol group were increased (P<0.05, P<0.01). male CRH mRNA expression was also increased (P<0.05), adrenal P450scc mRNA expression in male showed decreased tendency., but no changes in StAR expression. Both male and female ethanol group showed disordered ACTH circadian rhythm, presenting by delayed summit and increased secretion at9:00.(5) hppocampal HPA axis regulation center:Compared with the control group, the female ethanol group rat hippocampal11beta-HSD-1and GR mRNA expression showed no significant change, and male11beta-HSD-1mRNA expression were increased (P<0.05) while GR remained unchanged. Conclusion:the offspring (F2generation) of IUGR rats induced by prenatal ethanol exposure might be susceptible to MS, presenting by decreased blood glucose, LDL-c and HDL-c, but increased ratio of LDL-c/HDL-c, the change was sex-dependent. The potential mechanism might be attributed to HPA axis high activity programmed alteration. |
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