| Epidemiological investigations show that, between IUGR fetus and normal individual, the incidence of metabolic syndrome when grown up is2.53versus1, which suggests that there is a fetal origin of metabolic syndrome. Xenobiotics, such as ethanol, nicotine, and caffeine, have been confirmed to be the most specific factors of IUGR. Our previous studies have found that during pregnancy, ethanol, nicotine, and caffeine as well as dexamethasone exposure could cause fetal over-exposure to maternal GC and inhibit the development of HPA axis in fetuses. After birth, the IUGR offspring induced by nicotine and caffeine show low basic activity and enhanced sensitivity of HPA axis to chronic stress, and accompanying with alterations of glucose and lipid metabolism depending on GC. These results indicate an underlying mechanism of "HPA axis-associated neuroendocrine metabolic programmed alteration" by xenobiotics.The scholar suggests that intrauterine programming of the HPA axis is the most likely mechanism of adult metabolic syndrome. The study testified more maternal GC entered the female fetus than the male fetus passing through the placenta barrier, and high GC was one of key hormones which affected the HPA axis of fetus. It suggested that the effects of maternal stress on the HPA axis in female offspring were more serious than male offspring. Our previous studies testified the low basic activity and enhanced sensitivity of HPA axis presented in IUGR adult offspring induced by prenatal xenobiotics exposure fed with normal diet or high-fat diet, and associated glucose and lipid metabolic alterations. How about is the function of the HPA axis in IUGR old female offspring with a long-term high-fat diet and diet adjustment after birth? What are the changes of the function of the glycolipid metabolism? Whether IUGR old female offspring have susceptibility to the metabolic diseases? What’s the mechanism? So far, the related reports have yet to see.In the present study, the IUGR rat models induced by prenatal xenobiotics (alcohol, nicotine, caffeine) exposure were established. The female offspring were weaned at postnatal week4, and given a four-month high-fat diet imitating hypernutritional environment, and followed by normal diet until the end of the experiment. We observed the effects on the function of HPA axis, glucose and lipid metabolism, morphology of related tissues (like hippocampus, hypothalamus, pituitary, adrenal glands, pancreas, liver, kidney and fat) in old female offspring with chronic stress. We hope to know the fuction of HPA axis and the changes of glycolipid metabolism in IUGR old female rats induced by prenatal xenobiotis exposure, and further explore the mechanism of susceptibility to metabolic disease in IUGR old female rats. In addition, we hope to further confirm that the effect induced by prenatal xenobiotics exposure on the offspring is not limited to fetus, newborn, childhood and even adulthood, some effects can be continued to old age, and provide the theoretical ang experimental basis for a reasonable lifestyle after birth of the special groups.PART ONE:Changes of body weight, ethology, structure and function of the HPA axis in old female offspring induced by prenatal xenobiotics exposure with long-term high-fat diet and diet adjustmentObjective:The enchanced sensitivity of HPA axis in IUGR offspring will speed up the process of the development of the metabolic syndrome. IUGR, catch-up growth and following obesity are closely related to metabolic syndrome, cardiovascular disease, type2diabetes and non-alcoholic liver disease in later stage. On the basis of previous studies in our laboratory, we observed further the changes of body weight, ethology determination, the morphology of HPA axis related tissues and the function of HPA axis and hippocampus in IUGR old female offspring induced by prenatal xenobiotics (ethanol, nicotine and caffeine) exposure fed with long-term high-fat diet and following diet adjustment (prenatal xenobiotics intervention model), and hoped to confirm the function of HPA axis in IUGR old female rats and the developmental mechanism of metabolic diseases.Method:Pregnant rats were randomly divided into four groups:control group, ethanol group, nicotine group and caffeine group. Starting from GD11until term delivery, the xenobiotic groups were respectively given ethanol4g/kg.d (intragastric administration, once a day), nicotine2mg/kg.d (subcutaneous injection, twice a day), and caffeine120mg/kg.d (intragastric administration, once a day). The control group was given the same volume of distilled water. At parturition, the dams in each group and their offspring were fed ad libitum. On postnatal day1,8-10female offspring were in each group to assure adequate and standardized nutrition until weaning. After weaning (postnatal week4, PW4), the offspring were fed with high-fat diet, and then fed with normal diet from PM5.5to the end of the experiment. High-fat diet previously reported by our lab contained88.0%corn flour,11.5%lard, and0.5%cholesterol, which provided18.9%kcal from protein,61.7%kcal from carbohydrate and19.4%kcal from fat. And the standard rodent chow purchased from the Experimental Centre of Hubei Medical Scientific Academy contained21%kcal from protein,68.5%kcal from carbohydrate and10.5%kcal from fat.Body weights of the female offspring were measured monthly. At postnatal month (PM) 9, the animals were taken blood by drawn from vena caudalis to detect the levels of serum adrenocorticotropic hormone (ACTH), corticosterone (CORT), glucose, insulin, triglyceride (TG), total cholesterol (TCH), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C). Oral glucose tolerance test (OGTT) and ethology determination including sugar preference experiment, maze experiment and open field test were also carried. After one week, rats were exposed to an unpredictable chronic stress procedure. Briefly, the rats were isolated, raised in a single cage, and then randomly assigned to one type of stress every day for14days. The following day of the last stress (PM9.75), we carried OGTT and ethology determination again. At the end of experiment, rats were anesthetized with isoflurane and decapitated in a room separate from that the other animals were kept, and the sampling of blood was carried again. Serum from blood was prepared by centrifugation at12000rpm,4℃for15min, and stored at-80℃until used for detecting the indexes again. The hypothalamus, hippocampus, pituitary gland, adrenal gland, pancreas, liver, fat and kidney were dissected and randomly selected from each group, fixed in4%paraformaldehyde solution for histological examination. RT-PCR was used to determine the mRNA expression, including Hippocampal11β-hydroxy steroid dehydrogenase-1(11β-HSD-1), glucocorticoid receptor (GR) and mineralcorticoid receptor (MR). Serum ACTH was determined using a radioimmunoassay kits, and CORT were detected by using ELISA kits following the manufacturer’s protocol before and after stress. Hematoxylin-eosin stain (HE) was used for morphologic observation of Hippocampus, hypothalamus, pituitary and adrenal gland.Result:①body weight. The birth weights of female offspring in ethanol group, nicotine group and caffeine group at PW1were significantly lower than those of the control (P<0.01), and less than two standard deviations, so the IUGR models were successed to establish. At the end of experiment, the body weight of IUGR old female offspring in ethanol group, nicotine group and caffeine group couldn’t reach or exceed the body weight of the control; the body weight in ethanol group increased quickly from the fourth month, and the gain rates of body weight significantly increased cpmpared with the control at the sixth month (P<0.01); the body weight in nicotine group increased quickly from the fifth month, and the gain rates of body weight significantly increased cpmpared with the control at the seventh month (P<0.01); the body weight in caffeine group increased quickly from the fifth month. It suggested that "catch-up growth" of body weight presented in prenatal xenobiotics intervention model with overnutrition, and the growth of body weight had difference among the xenobiotics. With diet adjustment, the body weights in each group were gradually stable, and the curves of body weight presented plateau phase, it suggested that diet adjustment could reduce the gain of the body weight.②the morphology and function of hippocampus:Hippocampal neuron cells decreased, astrocytes had focal vacuolar degeneration, liquefaction, necrosis in ethanol group, nicotine group and caffeine group. The mRNA expression of hippocampal11β-HSD-1and GR in xenobiotic groups had a tendency of increasing comparing with the control group, and the mRNA expression of hippocampal MR in xenobiotic groups had a tendency of decreasing, and the ratio of MR/GR was decreased.③ethology examination:Ethology determinations including sugar preference experiment, electric maze experiment and open field test were testified that there had no significant difference in between the xenobiotic groups and the control group.④the morphology of HPA axis associated tissues:Compared with the control group, in ethanol group, nicotine group and caffeine group, hypothalamic cells were edema, and neurons were decreased. Glial cells presented focal vacuolar degeneration, liquefaction and necrosis. Sinusoids of pituitary gland narrowed or disappeared, and cells were swelling, ballooning or vacuolar degeneration in ethanol group, nicotine group and caffeine group. The ethanol group could also seen multifocal liquefaction and secondary fibrosis. In ethanol group, nicotine group and caffeine group, adrenocortical cells were swelling and ballooning, and cells of adrenal fasciculata were fat metaplasia, the injury in the ethanol group was the most serious, followed by caffeine group and nicotine group. It suggested that prenatal xenobiotics exposure aggravated the injuries of HPA assciated tissues induced by high-fat diet. The function of HPA axis:the level of serum ACTH and CORT:Compared with the control group, the levels of serum ACTH in ethanol group and caffeine group had a tendency of decreasing before stress, but not in nicotine group. The levels of serum CORT in ethanol group and nicotine group had a tendency of decreasing before stress, but not in caffeine group. The levels of serum ACTH in ethanol group and caffeine group were higher significantly than the control group after stress (P<0.01), and the levels of serum ACTH in nicotine group were similar to those in control group. The levels of serum CORT of xenobiotic groups were also higher than the control group. In addition, the levels serum ACTH and CORT in xenobiotic groups were increased significantly after stress than before (P<0.01), except for the level of serum CORT in caffeine group. It suggested that there was an enhanced sensitivity of HPA axis in IUGR old female offspring.Conclusion:IUGR female offspring induced by prenatal xenobiotics exposure with the overnutrition after birth presented "catch-up growth" of body weight. With diet adjustment, the gain of body weight slowed down. Prenatal xenobiotics exposure aggravated the injuries of HPA assciated tissues and the injury and dysfunction of the hippocampus induced by high-fat diet. The enhanced sensitivity of HPA axis in IUGR old female rats presented, and the ethology related to the hippocampus had no change.PART TWO:Morphology of metabolic associated tissues and the function of glycolipid metabolism in old female offspring induced by prenatal xenobiotics exposure with long-term high-fat diet and diet adjustmentObjective:Epidemiological investigation prompts, the odds of occurrence of impaired glucose tolerance and insulin resistance will increase in the IUGR offspring in adulthood. The study finds that lipid disorders have toxic effect on cells, which can cause or aggravate insulin resistance and damage the function of β-cell. At the same time, insulin resistance and inadequate insulin secretion further aggravates the lipid disorders, accordingly, these can trigger a series of metabolic disorders and related diseases, such as type2diabetes, fatty liver, cardiovascular and cerebrovascular diseases. In the present study, we observed the morphology of metabolic associated tissues (pancreas, liver, fat and kindney) and the changes of the function of glycolipid metabolism before and after stress in IUGR old female offspring with long-term high-fat diet and followed by diet adjustment, in order to clarify the role of HPA axis programming in disorders of glycolipid metabolism and the possible mechanism of susceptibility to metabolic diseases in IUGR old female rats.Method:The methods were shown in the first part. The levels of serum glucose were detected with glucose oxidase kit. The levels of serum insulin were measured by using Radioimmunoassay kit. The levels of serum TG were measured by using Glycerol-3phosphate oxidase test. The levels of serum TCH were detected with cholesterol oxidase test. The levels of serum HDL-C and LDL-C were measured by using selective precipitation. The metabolic associated tissues (pancreas, liver, fat and kindney) were respectively fixed in4%paraformaldehyde solution overnight, processed by the paraffin slice technique. The morphological examinations of the metabolic associated tissues were observed under the light microscope.Result:①the glucose metabolism:Before stress, the levels of serum glucose in ethanol group, nicotine group and caffeine group were similar to those in control group. However, the levels of serum insulin in the caffeine group were significantly higher than those in control group (P<0.01), and the IRI in caffeine group were significantly higher than those in control group, it suggested that the insulin releasing increased in caffeine group. The levels of insulin in ethanol group and nicotine group were similar to those in the control group, and the AUC of glucose is the same as the control group. After stress, the levels of serum glucose had a tendency of increasing in xenobiotic groups comparing with the control group, and the levels of insulin were significantly lower than the control group (P<0.01). It suggested that there was the failure of pancreas releasing insulin in xenobiotic groups, followed by the decreasing IRI in IUGR old female rats. OGTT:Before stress, the highest point of glucose curve moved to60min, but it returned to the initial level of blood glucose at120min. After stress, the curve of blood glucose was similar to that of before stress, except at120min, the level of blood glucose was higher than the control, and it was unable to return to the level of blood glucose at Omin. it suggested that IUGR old female rats in ethanol group had decreased ability to clear glucose load before stress. After stress, their function of glucose metabolism further weakened, and impaired glucose tolerance presented. The changges of glucose metabolism in nicotine group and caffeine group were not typical. Compared with the control group, the relative value of blood glucose AUC in xenobiotic group had no significant difference before and after stress.②lipid metabolism: Before stress, the levels of TG, TCH had no difference between the xenobiotic groups and the control group; the levels of serum LDL-C in ethanol group were significantly higher than those in control group (P<0.01), and the levels of serum LDL-C in nicotine group and caffeine group had a tendency of increasing. The levels of serum HDL-C in ethanol group and nicotine group were significantly lower than those in control group (P<0.01), and the levels of serum HDL-C in caffeine group had a tendency of decreasing. After stress, the levels of serum TG in the xenobiotic groups increased significantly compared with before stress, the levels of serum TG in ethanol group and nicotine group increased significantly compared with the control (P<0.05), there had a tendency of increasing in caffeine group. The levels of serum TCH in ethanol group and caffeine group increased significantly compared with the control (P<0.01, P<0.05), except the nicotine group. The levels of serum LDL-C in ethanol group were significantly higher than those in control group (P<0.05), and the levels of serum HDL-C in nicotine group significantly decreased than those in control group (P<0.05), and the levels of serum HDL-C in caffeine group significantly decreased compared with before stress (P<0.01). The results suggested that prenatal xenobiotics exposure aggravated disorder of lipid metabolism induced by high-fat diet, and there had difference among the xenobiotics. the ratio of blood lipid:Before and after stress, the ratios of LDL-C/HDL-C and TCH/HDL-C in ethanol group and nicotine group were higher than those in the control group (P<0.01, P<0.05). Before stress, the ratios of TG/HDL-C in xenobiotic groups were similar to the control, but the ratios of TG/HDL-C in ethanol group and nicotine group increased significantly compared with the control after stress (P<0.05). The ratios in caffeine group were similar to those in the control before and after stress.③the morphology of metabolic associated tissues: Pancreatic islets in xenobiotics groups presented reduction or disappearance, acinar was swelling, deposition of lipid droplets, extensive liquefaction and necrosis were also seen in ethanol group. Change of water sample, ballooning degeneration, varying degrees of steatosis and hepatic necrosis were diffused in liver cells in xenobiotics groups. In particular, in the ethanol group, infiltration of inflammatory cells and proliferation of kupffer cells were also visible in the portal area. Glomerular endothelial cells and renal tubular epithelial cells were swelling, renal tubular epithelial cells presented vacuolar degeneration, partial disintegration and shedding in xenobiotic groups. Cell proliferation presented in the adipose tissue. The sizes of cells were not uniform, and partial cell fusions turned to large cells, especially in ethanol group. Tips, Prenatal xenobiotics exposure aggravated the injuries of metabolic associated tissues induced by high-fat diet.Conclusion:IUGR old female rats induced by prenatal xenobiotics exposure presented obvious dysfunction of glycolipid metabolism, injuries and lipid deposition in metabolic associated tissues induced by high-fat diet. Combined with our previous study, we suggested that the diet adjustment could reduce the disorders of glucose and lipid metabolism and lipid deposition in metabolic related tissues induced by high-fat diet, but the abnormal of releasing of insulin and glucose metabolism were exacerbated. The enhanced sensitivity of HPA axis induced by prenatal xenobiotics exposure increases the susceptibility to the metabolic diseases in IUGR old female offspring. |
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