| Metabolic syndrome (MS) is a cluster of metabolic abnormalities, including hypertension, hyperglycamia, dyslipidemia and obesity, which increase the risk of diabetes mellitus, hepatic steatosis and cardiovascular diseases. Intrauterine growth retardation (IUGR) is a condition in which the fetus does not reach its growth potential for a given gestational age. The worldwide prevalence of IUGR is approximately24%in developing countries and2%in the developed world. IUGR has often associated to the development of several features of MS in adulthood, increasing the risk of diabetes mellitus, hepatic steatosis and cardiovascular diseases. Epidemiological investigations have determined that the incidence of adult MS in IUGR foetuses (2.3%) is5.75-fold higher than normal foetuses (0.4%), suggesting that the MS may partly be original from foetal. Available data indicate that non-alcoholic fatty liver disease (NAFLD) also may be the hepatic manifestation of MS. NAFLD is the most common liver disease since its prevalence is between20-30%in general population in Western. Community prevalence ranges is estimated to be20%in China and15-45%in South Asia. Recently, IUGR has been reported to be an important risk factor for NAFLD. The prevalence of NAFLD in low-birth weight children was approximately fourfold higher compared with those in control subjects. It’s suggested that the NAFLD also partly be original from foetal.The fetal programming is the permanent alterations of tissue function induced by the alterations of intrauterine environment. The potencial mechanism of IUGR and the MS is still vague. The latest reports is suggested that the programmed alteration of hypothalamic-pituitary-adrenal (HPA) axis intrauterine may contribute to the original adult MS. Poor nutrition in utero is a common method used to induce IUGR, and intrauterine malnutrition may have long-term consequences on endocrine and metabolic function.A high-fat diet has been linked to hypertension, glucose intolerance, insulin resistance, type2diabetes, dyslipidemia, obesity and reproductive disorders in the adults. In the present study, we established a nutrient restriction-induced IUGR rat model to observed the function of fetal HPA axis and glucose/lipid metabolism; simulated overnutrition conditions after birth with a high-fat diet to induce adult MS and associated diseases, and then observed the basal activity and the reponse to stress (unpredictable chronic stress), and the related glucose/lipid metabolism; moreover, to observe the susceptibility of metabolic disease in liver (NAFLD).PART ONE Prenatal Food Restriction Induced the Alterations of Hypothalamic-pituitary-adrenocortical axis Function in Offspring Fetal RatsObjective:To observed the the alteration of HPA axis function of IUGR fetus prenatally food restriction, to comprehend the effects of intrauterine malnutrition.Methods:Pregnant rats were allowed feed ad libitum or put on a restricted diet (50%of the daily food intake of control rats, approximately60g/kg body weight) from (gestational day) GD11until term delivery. Pups from food-restricted dams were designated as food-restricted (FR) rats, whereas those from ad libitum-fed mothers were control (CN) rats. On GD20, after12h fasting, randomly selected pregnant rats from each group were anesthetized with isoflurane and euthanized.The fetuses of each group were quickly removed, weighed and randomly selected five male and five female fetuses from five different dams. Fetal blood and tissue of hippocampus, hypothalamus and adrenal gland were immediately frozen in liquid nitrogen followed by storage at-80℃for subsequent analyses.Results:①)Body weights, IUGR rate and blood CORT levels:The fetal body weights were dramatic lower (P<0.01, P<0.05) and the IUGR rate were significant higher (P<0.01) in FR fetus than those in CN fetus. The serum corticosterone (CORT) levels of FR fetus were higher (P<0.05) than those of CN fetus.②The activity of HPA axis:When compared with CN fetus, the corticotrophin releasing hormone (CRH) of FR fetus had no change, but arginine vasopressin (AVP) were significantly lower (P<0.01). When compared with CN groups, the mRNA expression of vesicular glutamate transporter2(VGLUT2) of FR male fetus were significantly increased (P<0.01), while in FR female fetus had no change; the mRNA expression of glutamate decarboxylase65(GAD65) were higer in FR male fetus (P<0.01) but deceased in FR female fetus (P<0.05), the VGluT2/GAD65ratio of both FR male and female rats were significantly increased (P<0.01); the NR1and NR2B of FR fetus were decersed (P<0.01, P<0.05); the y-Aminoburyric acid A receptor subunitβ1(GABAR-Aβ1) of FR male fetus were incersed (P<0.01), while FR female fetus had no change; and the GABAR-Aβ1of FR female fetus were decersed (P<0.05), while FR male fetus had no change. The steroidogenic acute regulatory protein (StAR) and cytochrome P450cholesterol side chain cleavage (P450scc) of FR fetus had no change, the mRNA expression of insulin-like growth factor receptor1(IGFIR) and protien kinase B (Aktl/PKB1) of FR adrenal gland were lower (P<0.01) than those of CN rats.③the fetal hippocampus function related gene expression:When compared with CN fetus, the expression of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) had no change in FR fetal hippocampus. The expression of hippocampus brain-derived neurotrophic factor (BDNF) of FR rats were decreased (P<0.05), the expression of cyclic adenosine monophosphate responsive element binding protein (CREB) and the tropomyosin receptor kinase B (TrkB) had no change. The hippocampus N-methyl-Daspartic acid receptor (NR)2B, synapsin1(Synl) and synaptosomal-associated protein25(SNAP25) were decerease (P<0.05), while NR1, NR2A and glutamate receptor (GluR2) had no change. The expression of cyclin dependent kinase2(CDK2) were increased (P<0.05), but cell division control protein42(Cdc42), ras-related C3botulinum toxin substrate1(Racl), ras homolog gene family member A (RhoA) and cell cycle protein A (CyclinA) had no change. The expression of b-cell lymphoma2(Bcl-2) had no change, while cysteine aspartyl acid protease (Caspase)8were significantly increased (P<0.05).Conclusion:Prenatal food restriction induces the fetus overexposure of maternal GC, inhibites the fuction of fetal HPA axis; the damage of hippocampus neuron and the increased excitatory potential of hypothalamus were observed in prenatal food restricted fetus, the reglulated function of hippocampus and hypothalamus were also decerased.PART TWO Prenatal Food Restriction Induced the Glucose/Lipid Metabolic Alteration in Offspring Fetal Rats Objective:To observed the the alteration of glucose/lipid metabolism of IUGR fetus prenatally food restriction.Methods:The treatment of pregnant rats and its offspring were according to Part1. Fetal blood and tissue of livers were immediately frozen in liquid nitrogen followed by storage at-80℃for subsequent analyses. The same part of the liver was fixed in a4%paraformaldehyde solution for histological examination or sliced into1mm3tissue blocks for transmission electron microscopy analysis. The rest livers were immediately frozen in liquid nitrogen followed by storage at-80℃for subsequent analyses.Results:①Liver morphology:The vacuoles increased but cytoplasm reduced in the liver parenchyma cell of FR rats; the ridge structure of liver mitochondria is clear, however, the swelling deformation were observed in liver mitochondria of FR rats, and plenty of glycogen particles were also found in liver of FR rats.②Serum glucose/lipid metabolic phenotypes: When compared with CN fetus, the blood glucose, insulin, triglyceride (TG), total cholesterol (TCH) and high density lipoprotein-cholesterol (HDL-C) of FR fetus had no change, the low density lipoprotein-cholesterol (LDL-C) of FR female fetus were decreased (P<0.05), while those of FR male fetus had no change. The serum IGF1levels were lower (P<0.05) in FR two groups compared to the CN fetuses.③Genes expression of hepatic glucose and lipid metabolic pathways:When compared with CN fetus, the expression of IGF1of male and female FR fetus were significantly decerased (P<0.01, P<0.05), the insulin receptor substrate1(IRS1) had decrease in FR male fetus, and decreased in FR female fetus (P<0.05), the insulin-like growth factor binding protein3(IGFBP3), IGF1R, insulin receptor (INSR) and IRS2expression of FR fetus were had no change. The glycogen synthase kinase3β (GSK3β) had no change; the Glucose-6-phosphatase (G6Pase) were up-regulated (P<0.05) in FR female fetus and didn’t change in FR male rats; the fork-head transcriptional factor01(FOXO1) of had decrease tenendcy in FR male rats and decreased in FR male rats (P<0.05). The adiponectin receptor2(AdipoR2), leptin receptor (LEPR), Janus kinase (JAK)2, adenosine monophosphate activated protein kinase a (AMPKa) and mammalian target of rapamycin complex2(mTORC2) expression of FR fetus were had no change. The sterol regulatory element binding protein-1c (SREBP1c) and fatty acid synthase (FASN) of FR male fetus were all increased (P<0.05), while its in FR female fetus had no change; the acetyl-CoA carboxylase a (ACCa) expression in FR fetus had increased tendency, the3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) had no change. The expression of carnitine palmitoyltransferase1a (CPT1α) of FR male fetus were decreased (P<0.05), while in FR female fetus had no change. The microsomal triglyceride transfer protein (MTTP) and the peroxisome proliferator activated receptor a (PPARa) of FR fetus were down-regulated (P<0.05); the apolipoprotein B (APOB) had deceased (P<0.05) in FR male fetus and had decrease tendency in FR female fetus. The hepatocyte nuclear factor4(HNF4) expression were up-regulated (P<0.01). Compared with the CN group, the fetal hepatic parenchyma cells of FR group exhibited the increased vacuolar and the reduced cellularity. The mitochondrial structures characterized by the swelling deformation were observed in FR rats, while it was normal in the CN rats. In addition, a mass of glycogen granules had accumulated in the cytoplasm of hepatic parenchyma cells in FR groups.Conclusion:Prenatal food restriction induces the changes of fetal glucolipid metabolism, that function of gluconeogenesis were up-regulated, while the function of lipid output and oxidation were down-regulated.PART THREE Prenatal Food Restriction Induces a Hypothalamic-pituitary-adrenocortical axis-associated Neuroendocrine Metabolic Programmed Alteration in Offspring Adult RatsObjective:To observed the the programmed alteration of HPA axis function and glucose/lipid metabolism of IUGR adult rats which prenatally food restriction and postnatally high-fat diet.Methods:Pregnant rats were allowed feed ad libitum or put on a restricted diet (50%of the daily food intake of control rats, approximately60g/kg body weight) from GD11until term delivery. Pups from food-restricted dams were designated as food-restricted (FR) rats, whereas those from ad libitum-fed mothers were control (CN) rats. On postnatal day (PD)1, the numbers of pups in each litter was selected to8randomly and balancing the gender ratio to ensure adequate and equal nutrition until weaning. At postnatal week (PW)4, eight male or female pups were selected randomly from eight different mothers were placed in each group. All of the pups were weaned to an ad libitum high-fat diet before being sacrificed. The offspring were weighed weekly. At PW16, the animals were fasted for12h and blood was drawn from the caudal vena cava within3min to detect the levels of adrenocorticotrophic hormone (ACTH), CORT, glucose, insulin, TG and TCH. At PW17, rats were exposed to the unpredictable chronic stress (UCS) procedure for3weeks. On the last day of UCS, rats were anesthetised with isoflurane and decapitated1h after swimming. Fetal blood and tissue of hippocampus, hypothalamus, adrenal gland and livers were immediately frozen in liquid nitrogen followed by storage at-80℃for subsequent analyses. The hypothalamus, pituitary gland and livers were dissected and fixed in a4%paraformaldehyde solution for histological examination.Results:①Body weight:Prenatal food restriction resulted in the decreased body weights of the FR male and female pups compared to the CN pups at PW1(P<0.01, P<0.05, respectively). The body weights of the FR rats continued to be lower than the CN rats, up to PW9for the males and PW4for the females (P<0.01, P<0.05, respectively), after which the difference between two groups disappeared. After the3-week UCS during PW17-PW20, the body weights of the male FR rats were normal, but the weights of the female FR rats were increased (P<0.05) compared to the CN rats.②the regulated functions of hippocampus: When compared with the CN group, after UCS, the hippocampus11β-hydroxysteroid dehydrogenase type-1(11β-HSD-1), GR and MR expression were all increased (P<0.05, P<0.01), the MR/GR ratio had decerased tendency.③The alterations of activity of HPA axis:When compared with the CN group, before UCS, the expression of hypothalamic CRH of FR rats were significant lower (P<0.05), after UCS, the CRH expression of FR male rats were increased (P<0.05) while femle rats had no change, however, the expression of AVP of FR rats were increased (P<0.01); before UCS, the serum ACTH levels of FR male and female rats were decerased, serum CORT levels were no change in FR male rats but increased in femle rats (P<0.01), after UCS, the serum ACTH and CORT levels of FR rats were all increased (P<0.01). After UCS, the hypothalamic MR expression of FR rats had no change, but the GR expression of FR male rats were increased (P<0.05). after UCS, the adrenal StAR and P450scc expression of FR rats were all increased (P<0.01).④the metabolic phenotype of circulating glucose before and after UCS:Prior to UCS, the fasting serum glucose concentrations were the same in the FR and CN groups. After UCS, serum glucose concentrations of the FR rats were significantly increased from baseline (P<0.01), and were significantly higher than the CN rats (P<0.05, P<0.01). Furthermore, the serum glucose level rate-of-gain in the FR groups was significantly higher than the CN group. Compared to the CN groups, the serum insulin concentrations before UCS were decreased significantly in the male FR group (P<0.05) but were slightly increased in the female FR group. After UCS, the serum insulin concentrations were significantly lower (P<0.05) in the male and female FR rats than the CN rats, and the insulin concentration rate-of-gain was significantly decreased or had a negative trend in the FR group (P<0.05) compared to the CN group.⑤the metabolic phenotype of circulating lipids before and after UCS:Serum TG and TCH levels in the FR male rats showed an increasing tendency or increased significantly (P<0.05) compared to the CN rats before UCS. After UCS, the TG and TCH levels in the FR male rats decreased to the levels in the CN rats. In the FR female rats, serum TG and TCH levels showed no change compared to the CN rats prior to UCS. After UCS, there was an increasing trend in TG and TCH levels as compared to the CN groups.⑥Pathological observations after UCS:Normal architecture of the hypothalamus, pituitary gland and liver were observed in the male and female CN rats. However, many lipid droplets were observed in the hypothalamus, pituitary gland and liver tissues of the FR rats, particularly in the female rats.Conclusion:In summary, we demonstrated that prenatal FR induces lower birth weight and future "caTCH-up" growth, lower basal activity but enhanced sensitivity of the HPA axis to chronic stress, altered glucose and lipid metabolism and steatosis of multiple organs in IUGR offspring fed a postnatal high-fat diet. The underlying mechanism may involve the programmed alteration of HPA axis-associated neuroendocrine metabolism. These data and underlying mechanisms may provide an explanation for the susceptibility to MS and associated diseases in the adult offspring with IUGR induced by prenatal FR.PART FOUR Prenatal Food Restriction Induces Increased Susceptibility to High-fat Diet-induced Non-alcoholic Fatty Liver Disease in Offspring Adult Rats Objective:To observed the susceptibility of prenatal food restricted adult rats to NAFLD after postnatal treated with high-fat diet.Methods:The treatment of pregnant rats and its offspring were according to Part3. All of the pups were weaned to an ad libitum high-fat diet before being sacrificed. The offspring were weighed weekly. Rectal temperature was measured by clinical thermometer at23:00and food intake of24h was detected at PW23. On PW24, after12h fasting, rats were anesthetised with isoflurane and decapitated. Blood were collected and separated to serum. The livers were dissected and the same part of liver which randomly selected from5rats of each group, were fixed in4%paraformaldehyde solution for histological examination. The rest livers were immediately frozen in liquid nitrogen, and then storage at-80℃for subsequent analyses.Results:①The body weight, food intake and rectal temperature:Prenatal food restriction resulted in the decreased body weights of the FR male and female pups compared to the CN pups at PW1(P<0.01); Whereas comparing with the CN rats, the significant differences of FR male and female rats were disappeared after PW12or PW8, respectively; the rectal temperature of FR rats were significantly higher than those of CN rats (P<0.01, P<0.05), the food intake was lower in FR male rats (P<0.01) and no significant difference in FR female rats compared with respective CN groups.②Serum metabolic phenotypes:Comparing with the CN rats, serum CORT levels were lower (P<0.01) while serum IGF1levels were higher (P<0.01) in the FR rats. The levels of serum glucose of FR rats had no change, while the serum TG concentrations in female was higher than that of CN rats (P<0.05).④Liver pathology:From the liver HE staining sections, sparse hepatocyte microvesicular steatosis was observed in the CN rats. Furthermore, there was prevalent macro vesicular steatosis in the FR rats, with the average Kleiner score surpass3, which indicated a diet-induced NAFLD in adult offspring rats.④The mRNA expression of hepatic IGF1pathway:The mRNA expression of IGF1, IGF1R, IRS-2and glucose transporter2(GLUT-2) of FR rats were higher (P<0.05, P<0.01) or had an increased tendency than those of CN rats, especially for the IGF1and IGF1R gene expression.⑤The expression of hepatic metabolic nuclear factors and key enzymes:The mRNA expression of G6Pase was significant higher in FR female rats than that of CN group (P<0.01). Lipid synthesis related gene SREBPlc was also increased in FR female rats (P<0.05). MTTP was decreased in FR female rats comparing with that of CN rats (P<0.01). CPT1a was lowerin FR male group (P<0.05).Conclusion:We demonstrated that the intrauterine origin of increased susceptibility to NAFLD in prenatal food restricted IUGR offspring rats. The "two-programming" may explain this phenomenon. The first programming is adverse intrauterine environments, which induced the programming alteration of hepatic glucose and lipid metabolism, lead to an increased susceptibility to NAFLD after birth. The second programming is postnatal caTCH-up growth, which triggered by the adaptive changes of GC-IGF1axis, could accelerate the occurrence of NAFLD. These data and underlying mechanisms may provide an elucidation for the high susceptibility to NAFLD in the adult offspring with IUGR induced by prenatal FR. |
PDF Full Text Request