| Diabetes mellitus, characterized by chronic hyperglycemia, is a collection of metabolic imbalances covering glucose, lipids, protein, fluid and electrolyte. The cause for diabetes is generally due to abnormal secretion or function of insulin. The global prevalence of diabetes is dramatically increasing over years, with type2diabetes mellitus (T2DM) makes up the majority of cases. Intrauterine growth retardation is defined as the intrauterine events leading to a weaken growth potential of fetus. IUGR is often considered as an important cause for a series of perinatal diseases, including fetal distress, neonatal asphyxia and perinatal death. In addition, the long-term effects of IUGR can be expanded to adulthood and result in physical and mental stunting, as well as increased susceptibility to metabolic syndromes, which brings about severe negative impacts on life quality. It has been demonstrated that IUGR is one of independent risking factors for glucose intolerance and T2DM.Adverse intrauterine environment, such as maternal or external conditions, contributes most to IUGR besides genetic factors. Common phenomenon depicted by impaired pancreatic development as well as altered programming of β cell mass and function has been found on IUGR models induced by some certain insults. These irreversible changes in utero may largely increase the risk of suffering glucose intolerance and T2DM in the mid-to-late life. Previous studies showed that both prenatal insufficient formation of pancreatic β cell mass and postnatal catch-up growth pattern under over-nutrition may accelerate the process of β cell failure and finally lead to T2DM. What’s more, the altered programming of glucose metabolism could be transmitted to several generations and presents a trans-generational effect.Caffeine is a xanthine alkaloid widely consumed in the form of coffee, tea, soft beverages, food and some analgesic drugs. Epidemiological studies reveal a close association between prenatal caffeine consumption and IUGR. Previously, we have demonstrated and firstly proposed the "neuroendocrine metabolic programming mechanism" responsible for caffeine-induced IUGR and consequent predisposition to metabolic syndromes. To be specific, prenatal caffeine exposure induces fetal over-exposure to high levels of maternal glucocorticoids (GC), and then changes the neuroendocrine metabolism of fetus:an inhibition of hypothalamic- pituitary-adrenal (HPA) axis functional development and altered peripheral glucose/lipid metabolism mediated by suppressed local insulin-like growth factor1(IGF-1) signaling pathway. These neuroendocrine metabolic changes could be maintained after birth, presenting low basal activity but hypersensitivity of HPA axis. Meanwhile, the concomitant enhanced hepatic IGF-1signaling and glucose/lipid metabolic alterations in several tissues may further increase the susceptibility to adult metabolic syndromes and relevant metabolic diseases (i.e. non-alcoholic fatty liver diseases). Moreover, the above neuroendocrine metabolic programming alteration is also characterized by transgenerational effect.GC have direct influences on pancreatic cell differentiation during embryonic stage. For example, a high level of GC intervention imbalances pancreatic differentiation and leads to decreased β cell mass, attenuated insulin expression and secretion. Pancreatic IGF-1signaling pathway not only promotes the rate of β cells proliferation, but also inhibits apoptosis of differentiated β cells. In addition, IGF-1signaling has been verified to facilitate expansion of β cell progenitors, which exerts an indirect effect on β cells differentiation. Taken together, GC and IGF-1signaling may play vital roles in pancreatic development and β cell mass determination. Basing on the above, we propose a series of questions as follow:May caffeine induced fetal over-exposure to maternal GC and consequent suppression of peripheral IGF-1signaling pathway result in abnormal morphogenesis and inhibited functional differentiation of pancreas in fetus? Is there any functional changes of glucose metabolism in adulthood for these IUGR offspring with impaired pancreatic development when experiencing postnatal catch-up growth? What about underlying intrauterine programming mechanisms and transgenerational effect? Furthermore, could high-fat diet induce the final development of diabetes? These questions are of great interest and significance to be elucidated.Therefore, the present study firstly aimed to investigate the effects of prenatal caffeine exposure on pancreatic morphogenesis and functional differentiation as well as glucose-insulin metabolic phenotype in IUGR fetal rats, and also try to address roles of fetal over-exposure to maternal GC and suppressed pancreatic IGF-1signaling pathway in regulation of pancreatic development cascade genes from molecular aspect. Secondly, we expect to verify if an increased susceptibility to diabetes in prenatal caffeine-exposed IUGR rats via observing postnatal growth trajectory and function of glucose metabolism of adult offspring fed by normal or high-fat diet. Finally, we cross-mated prenatal caffeine-exposed IUGR offspring with control and give birth to a second generation, then transgenerational effects of altered function of glucose metabolism with specific parental and gender heredity were examined.PART ONEEffects and Mechanisms of Prenatal Caffeine Exposure on Pancreatic Development and glucose Metabolic Phenotype in Fetal RatsObjective:To investigate the effects of prenatal caffeine exposure on pancreatic morphogenesis and functional differentiation as well as glucose-insulin metabolic phenotype in IUGR fetal rats, and to discuss the potential mechanisms.Methods:Pregnant rats were randomly divided into control and caffeine groups. From GD11to GD20, rats in the caffeine group were each administered caffeine120mg/kg day by oral gavage. Rats in control group were given the same volume of distilled water. On GD20, pregnant rats were sacrificed and live fetuses were quickly removed to weigh and distinguish gender. IUGR rates were also calculated. Gas chromatography-mass spectrometry (GC-MS) was used to detect caffeine concentration in maternal and fetal blood. Blood glucose concentration were measured by biochemical assay. Proinsulin and insulin levels in both blood and pancreas were measured by ELISA kit as well as blood IGF-1content. Hematoxylin-eosin (HE) staining was applied to histological examination and islet calculation, and ultrastructure of (3cells were observed under transmission electron microscope (TEM). Real-time PCR was used to quantify pancreatic mRNA expressions of genes, including:pancreatic and duodenal homeobox1(Pdx-1), neurogenin3(Ngn-3), neurogenic differentiation1(NeuroD1), Nk6homeobox1(Nkx6.1), paired box6(Pax-6), insulin1(INS-1), INS-2, prohormone convertase1/3(PC1/3), glucose transporter2(Glut-2), glucokinase (Gck), glucagon-like peptide receptor1(Glp-1r), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), CCAAT enhancer binding protein α and β (C/EBPa, β), IGF-1, insulin-like growth factor1receptor (IGF-1R), insulin receptor substrate1and2(IRS1/2), protein kinase B1and2(Aktl/2), adenosine receptor Al (Adoral), Adora2a, Adora2b. Immunohistochemistry (IHC) was employed to examine both Pdx-1and insulin protein expression, then further to calculate β cell fraction.Results:①Body weights and IUGR rates:The mean bodyweights of both male and female fetuses in caffeine group were lower than control while corresponding IUGR rates were reached to76.77%and77.58%, respectively (P<0.01).②Blood caffeine concentrations: Blood caffeine concentration for dam and fetus were49±1μg/ml and30±3μg/ml, respectively.③Glucose metabolic phenotype:Prenatal caffeine treatment elevated maternal blood glucose levels but decreased those of both male and female fetuses (P<0.01). The levels of serum proinsulin and insulin were only obviously higher in male but not female fetuses of caffeine group when compared to control (P<0.05), and the ratio of serum proinsulin to insulin were higher in both male and female fetuses from caffeine group than that in control (P<0.05).④Histology and ultrastructure:when compared with control, the islets of caffeine group were much more smaller and with fewer counts, however, no obvious structural damages were observed. The number of insulin secretory granules in β cell of caffeine group were less than control. Significant membrane fusion and degranulation of insulin secretory granules could be observed in caffeine group.⑤Pancreatic morphometric analysis and insulin biosynthesis: when compared with control, the total area of islets and β cells in caffeine group were significantly decreased (P<0.05, P<0.01) while the corresponding fractions to pancreas were also tended to be lower. Meanwhile, the pancreatic proinsulin and insulin contents in caffeine group were also lower than those of control (P<0.01, P=0.06).⑥mRNA and protein expression of pancreatic genes:when compared with control, the mRNA expression levels of pancreatic development cascade genes (Pdx-1, Ngn3, NeuroD1, Nkx6.1and Pax-6), insulin biosynthesis and modification genes (INS1, INS2and PC1/3), and insulin secretion regulators (Glut-2, Gck and Glp-1r) in caffeine group were basically decreased (P<0.05, P<0.01) or had decline tendency. IHC showed fewer and lighter Pdx-1positive-stained nucleuses presented in islet of caffeine group, and insulin positive stained areas were also lighter in color and smaller in size for caffeine-treated fetal islets.⑦GC effect-related gene expressions:When compared to control, both GR and C/EBPα mRNA expression levels in caffeine group were increased while that of C/EBPβ was decreased (P<0.01).⑧Serum IGF-1concentration and pancreatic IGF-1signaling expression:The serum IGF-1concentrations of caffeine group were lower than those of control, especially for female (P<0.05). The pancreatic IGF-1signaling (IGF-1R, IRS1and IRS2) mRNA expression were only significantly decreased in female (P<0.05, P<0.01) but not male when compared to control.⑨Pancreatic adenosine receptor mRNA expression:when compared to control, the mRNA expression of both Adoral and Adora2b, but not Adora2a, were increased (P<0.05, P<0.01) in male of caffeine group, while all of three adenosine receptors mRNA expression levels were decreased in female of caffeine group.Conclusion:Prenatal caffeine exposure impaired pancreatic development and altered glucose metabolic phenotype in IUGR fetal rats, the underlying mechanisms, on the one hand, might be attributed to both direct inhibition of pancreatic development and function cascade, and C/EBPs-mediated indirect suppression of pancreatic IGF-1signaling by caffeine-induced fetal over-exposure to maternal GC; on the other, caffeine stimulated insulin secretion via antagonizing pancreatic adenosine receptors may further alter fetal glucose metabolic phenotype.PART TWOAltered Function of Glucose Metabolism and Diabetic-like Symptom in Prenatal Caffeine-exposed Adult Offspring RatsObjective:To observe and compare the postnatal growth trajectory, pancreatic histology and function of glucose metabolism in adult offspring fed by normal or high-fat diet, and to analysis the underlying causes by combing the results of hepatic insulin signaling pathway expression.Methods:First batch of animals:The prenatal caffeine treatment were basically same as those described in Part1. The pregnant rats were subjected to spontaneously delivered, and the offspring were fed by normal diet after weaning. Intra-peritoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed on postnatal week (P W)20and21, respectively. Second batch of animals:The animal manipulations were same as the first batch of animals excepting for high-fat diet feeding after weaning. On P W20, oral glucose tolerance test (OGTT) was performed. The postnatal growth trajectories of both two batches of animals were recorded and body weight gain rates were calculated as well. On PW24, the offspring were sacrificed to collect liver and pancreas tissues. Biochemical assay and radioimmunoassay were applied to measure serum glucose and insulin levels, respectively. Pancreatic histology and ultrastructure were examined by HE staining and TEM. β cell fraction to total pancreas was calculated according to insulin IHC. Real-time PCR was used to quantify hepatic insulin receptor (IR), IRS2and Glut2mRNA expression.Results:(1) Offspring fed by normal diet:①Absolute body weights and corresponding gain rates:The mean bodyweights of both male and female offspring in caffeine group were lower than control on PW1(P<0.01). The bodyweights of offspring from caffeine group were also lower than control (P<0.05, P<0.01) but with a higher bodyweight gain rates (P<0.05, P<0.01) during PW4-PW24.②IPGTT:When compared to respective control, the basal levels (0min) of blood glucose and insulin in caffeine groups were similar to those of control as well as quantitative insulin sensitivity check index (QUICKI). After glucose challenge, blood glucose at120min and area under the curve (AUC) of female offspring from caffeine group were decreased (P<0.01), while decline tendencies were observed in male. Blood insulin levels of female but not male from caffeine group were higher than control (P<0.05). Meanwhile, blood insulin levels at15min were significantly higher than0min in all groups (P<0.05, P<0.01) except for male offspring from caffeine group. Furthermore, after a normalization of blood glucose and insulin values according to basal levels (0min), we found the relative blood glucose and insulin value variations at each time point were consistent with those before normalization.③ITT:When compared with control, the relative blood glucose at60and120min as well as AUC were all decreased (P<0.05, P<0.01).④Pancreatic histology and ultrastructure:There were no pathological injuries occurred to islet in caffeine group. Mitochondrial swelling could be seen in β cell of caffeine group while nucleolus was edged in male and enlarged golgi apparatus in female.④Pancreatic β cell fraction:when compared to control, both male and female offspring from caffeine group exhibited a decline tendency in pancreatic β cell fraction to total pancreas area.⑤Hepatic insulin signaling expression:when compared to control, only female offspring of caffeine group presented increased mRNA expressions of IR, IRS2and Glut2(P<0.05, P<0.01).(2) Offspring fed by high-fat diet:①Absolute body weights and corresponding gain rates: The mean bodyweights of both male and female offspring in caffeine group were lower than control on PW1(P<0.01). The bodyweights of male offspring from caffeine group were also lower than control (P<0.05, P<0.01), but female offspring presented similar bodyweights to control, both of male and female in caffeine group were with a higher bodyweight gain rates (P<0.05, P<0.01) during PW4-PW24.②OGTT:When compared to respective control, the basal levels (0min) of blood glucose and QUICKI were increased while and insulin were decreased in caffeine group, especially for female (P<0.01). After glucose challenge, blood glucose at120min was elevated but AUC had no changes in male offspring of caffeine group, while both blood glucose peak and AUC were all increased in female (P<0.01). Blood insulin levels at each time points were decreased as well as AUC, especially for female (P<0.05, P<0.01). Furthermore, after a normalization of blood glucose and insulin values according to basal levels (0min), we found the relative blood glucose and AUC in female of caffeine group were lower than those of control, which is opposite to results presented in absolute values.③Pancreatic histology:when compared to control fed by high-fat diet, islet of caffeine group showed much more serious fatty vesicular degeneration.④Hepatic insulin signaling expression:when compared to control, only female offspring of caffeine group presented increased mRNA expressions of IR, IRS2and Glut2(P<0.01)Conclusion:Prenatal caffeine-exposed IUGR adult offspring exhibited normal blood glucose metabolic phenotype but improved glucose tolerance and insulin sensitivity under normal diet. Furthermore, high-fat diet aggravated pancreatic injury and thus induced a phenotype with high blood glucose and low blood insulin, which is accompanied by relative improved glucose metabolism and presented diabetic-like symptom, especially for female.PART THREETransgenerational effects of prenatal caffeine exposure on function of glucose metabolism in offspring ratsObjective:To investigate effects of prenatal caffeine exposure on glucose metabolic phenotype and function in the second generation (F2) fetal and adult rats, and to discuss the transgenerational effects and features of altered glucose metabolic function in offspring rats.Methods:The pregnant rats manipulation was the same as Part1. The dams were subjected to spontaneously delivery, and all F1offspring were fed by normal diet after weaning. On PW16, the offspring from caffeine and control groups were cross-mated and F2offspring were named as follow:CC:control♂-control♀; CaC:caffeine♂-control♀; CCa:control♂-caffeine♀; CaCa:caffeine♂-caffeine♀. Some F1pregnant rats were sacrificed on GD20to obtain F2fetuses, the fetal bodyweights were recorded and blood samples were also collected. The rest pregnant rats were subjected to delivery and the F2pups were fed by normal diet after weaning. Postnatal growth trajectory and corresponding bodyweight gain rates were recorded. IPGTT and ITT were performed on PW20and21, respectively. On PW24, the adult F2offspring were sacrificed to collect blood samples. ELISA kits were used to measure serum corticosterone (CORT) and insulin concentrations. Blood glucose concentration was detected by biochemical assay.Results:①Absolute body weights and corresponding gain rates:when compared to CC group, only female but not male fetal bodyweights in CaC group were tended to be increased; The fetal bodyweights of both CCa and CaCa groups had declined tendencies. Within PW4-PW16, the overall bodyweights of CCa and CaCa groups were lower than CC (P<0.01, P<0.05).②Glucose metabolic phenotype:when compared to CC, no statistical variations were found in fetal CORT levels of all caffeine groups. Both CaC and CaCa group had an increasing of fetal blood glucose levels, especially for CaC (P<0.01), while fetal blood glucose were conversely lower in CCa group (P<0.01, P<0.05), all changes were without gender difference. The blood insulin levels in male fetuses of all caffeine groups were decreased, especially for CaC and CCa (P<0.01); for female fetuses, the blood insulin were elevated in CaC and CaCa but declined in CCa (P<0.01, P<0.05). On PW24, when compared to CC group, the CORT levels were increased in adult offspring from caffeine groups, especially for CCa male, CaC and CaCa female (P<0.05). Blood glucose levels in CaC and CCa groups were increased (P<0.01) or tended to be increased, only male but not female of CaCa group had lower blood glucose level. The blood insulin levels were elevated in male of all caffeine groups (P<0.05), for female, the insulin levels of only CaC and CaCa group were tended to be decreased.③Glucose tolerance: for F2male offspring, when compared to CC, blood glucose at120min after glucose challenge were increased (P<0.05) while AUC tended to be increased as well; blood glucose at15,30min and AUC tended to decreased in CCa group but significantly decreased in CaCa group (P<0.05, P<0.01). For female F2offspring, CaC group had higher blood glucose at0min but lower those at15,30min as well as AUC (P<0.05, P<0.01); blood glucose levels at all-time points except for120min were decreased as well as AUC in both CCa and CaCa groups (P<0.05, P<0.01). Furthermore, after a normalization of blood glucose and insulin values according to basal levels (0min), we found the relative blood glucose value variations at each time point were consistent with those before normalization.④ITT:For male F2offspring, when compared to CC, no obvious changes were observed in CaC group; relative blood glucose at30,60min as well as AUC were all increased in CCa group (P<0.05); relative blood glucose at15min were decreased in CaCa group (P<0.01). For female F2offspring, relative blood glucose at a majority of time points as well as AUC were increased in CaC and CaCa groups; AUC had a declined tendency in CCa group.Conclusion:The inhibition of prenatal caffeine exposure on offspring’s growth and development is characterized by a transgenerational effect. Meanwhile, caffeine-induced altered function of glucose metabolism could be transmitted to F2offspring, presenting by similar improved glucose tolerance as F1but impaired insulin sensitivity, all of the changes were characterized by a certain parental and gender difference. |
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