| Objectives:To investigate the pancreatic morphology and functional changes and their possible epigenetic mechanisms in the rat model of catch-up growth after food restriction.Methods:Rats were provided a 60% food-restricted diet determined by quantification of normal intake in the ad libitum-fed rats for 6 weeks, and then refed for 0,8,12,16 weeks respectively. In all rats, general physiological parameters, including food intake, body weight, free fatty acid (FFA), triglyceride (TG), cholesterol (TC) were measured. All rats were received hyperglycemic clamp and serum insulin level on 0,5,10,15,30,60min were measured at 0,8,16 weeks after refeeding. The pancreatic slides were stained with insulin antibody. The quantification of pancretic insulin-positive area ratio and pancreatic insulin content were analyzed. The apoptotic p-cells in islets were detected and quantified by the TUNEL technology. The mRNA expression of Bcl-2, Bax and Pdx-1 were measured by the real time PCR. ChIP analysis of cross-linked chromatin from islets of CUGFR and control animals at 0,8,16 weeks after refeeding were done with antibody to acetylated H3, H3K4me3, and H3K9me2. Input DNA represents PCR products without prior IP. The relative amount of acetylated H3-, H3K4me3-, and H3K9me2-bound Pdx-1 promoter was measured by real time PCR and normalized to input DNA.Results:There was no statistical significant differences between the body weight of catch-up growth after food restriction group (CUGFR) and normal control (NC) group after 14 weeks' refeeding. The CUGFR group had higher serum triglyceride and free fatty acid than the NC group (P<0.05, P<0.01 respectively). The first-phase insulin secretion declined in the CUGFR group (P<0.05) Compared with NC group, food-restriction in rats was characterized by remarkably decrease in the insulin-positive area ratio and pancreatic insulin content(P<0.05). The apoptosis ratio ofβ-cell is significantly increased in the CUGFR group at 8 and 16 weeks after food restriction (P<0.05). The mRNA expression of Bcl-2 decreased in the CUGFR group at 0,8,16weeks (P<0.05). Bcl-2/Bax ratio decreased in CUGFR at 8 and 16 weeks after refeeeding (P<0.05, P<0.01, respectively)Both the mRNA expression and the protein quantificative level of Pdx-1 decreased in the CUGFR group at 0,8,16weeks (P<0.05). CUGFR significantly reduced the abundance of acetylated H3 (P<0.05) and deacetylation progressed with age. In islets from control animals at all ages, there was a high association of H3K4me3 at Pdx-1. In CUGFR animals at 0,8 and 16weeks after refeeding, the abundance of H3K4me3 was significantly reduced (P<0.05). ChIP assays demonstrated that by H3K9me2 at the Pdx-1 promoter was present in CUGFR islets and H3K9me2 binding increased with age (P<0.05)Conclusion:The CUGFR model shows a completed catch-up growth and dyslipidemia. CUGFR decreases insulin first-phase secretion, the expression of Pdx-1 and leads to pancreatic apoptosis, potentially providing a mechanism for the increased risk of metabolic disorder in those with CUGFR. The changed histone modifications in the cross-linked chromatin from the CUGFR islets may be one of the epigenetic mechanisms responsible for the down-regulation of Pdx-1 gene. Chapter IStudy on the setting of the animal model of catch-up growth after food restriction and the metabolic characters of itObjectives:To explore characters of the rat model with catch-up growth after food restriction and the serum GH, IGF-1, ghrelin, leptin, CCK of it.Methods:Rats were provided a 60% food-restricted diet determined by quantification of normal intake in the ad libitum-fed rats for 6 weeks, and then refed for 0,8,12,16 weeks respectively. In all rats, general physiological parameters, including food intake, body weight, free fatty acid (FFA), triglyceride (TG), cholesterol (TC) were measured. Serum growth hormone (GH), insulin-like growth factor-1 (IGF-1), ghrelin, leptin, cholecystokinin (CCK) were quantitatively estimated by sandwich enzyme-linked immunoabsorbent assay (ELISA).Results:There was no statistical significant difference between the body weight of catch-up growth after food restriction group (CUGFR) and normal control (NC) group after 14 weeks' refeeding. The CUGFR group had higher serum triglyceride and free fatty acid than the NC group (P<0.05, P<0.01). There were no statistical significant differences of IGF-1, ghrelin, CCK between the two groups (P>0.05). But the GH increased (P<0.05) and leptin decreased (P<0.05) after the 6 weeks'food restriction.Conclusion:The CUGFR model shows a completed catch-up growth. GH and leptin are significantly changed in the CUGFR model and they maybe work together with other energy regulatory factors such as CCK in increasing body fat, decreasing insulin sensitivity. Chapter IIStudy on pancreatic morphology and functional change in the rat model of catch-up growth after food restrictionObjectives:To investigate the pancreatic morphology and functional change in the rat model of catch-up growth after food restriction.Methods:Animal model was prepared as chapter I. All rats were received hyperglycemic clamp and serum insulin level on 0,5,10,15,30,60min were measured at 0,8,16 weeks after refeeding. The pancreatic slides were stained with insulin antibody. The quantification of pancretic insulin-positive area ratio and pancreatic insulin content were analyzed. The apoptoticβ-cells in islets were detected and quantified by the TUNEL technology. The mRNA expression of Bcl-2, Bax were measured by the real time PCR.Results:1. Results of hyperglycemic clamp There is no difference in the fasting glucose and insulin between the two groups (P>0.05). The first-phase insulin secretion declined in the CUGFR group (P<0.05)2. Pancreatic morphology Compared with NC group, food-restriction in rats was characterized by remarkably decrease in the insulin-positive area ratio and pancreatic insulin content (P<0.05)3. Apoptosis rate ofβ-cell The apoptosis ratio of (3-cell is significantly increased in the CUGFR group at 8 and 16 weeks after food restriction (P<0.05)4. The mRNA expression of Bcl-2, Bax in pancreas. The mRNA expression of Bcl-2 decreased in the CUGFR group at 0,8,16weeks (P<0.05)..Bax mRNA didn't changed during the observation time (P>0.05). Bcl-2/Bax ratio decreased in CUGFR at 8 and 16 weeks after refeeding (P<0.05, P<0.01, respectively).Conclusion:CUGFR decreases insulin first-phase secretion and leads to pancreatic apoptosis, potentially providing a mechanism for the increased risk of metabolic disorder in those with CUGFR. ChapterⅢObjectives:To explore the expression of Pdx-1 and the histone 3 acetylation and methylation on its promoter which were responsible for Pdx-1 down-regulation by using chromatin immunoprecipitation (ChIP) in the CUGFR animal models.Methods:The mRNA expression of Pdx-1 was measured by the real time PCR. The protein level of Pdx-1 was detected by western blotting. ChIP analysis of cross-linked chromatin from islets of CUGFR and control animals at 0,8,16 weeks after refeeding were done with antibody to acetylated H3, H3K4me3, and H3K9me2. Input DNA represents PCR products without prior IP. The relative amount of acetylated H3-, H3K4me3-, and H3K9me2-bound Pdx-1 promoter was measured by real time PCR and normalized to input DNA.Results:Both the mRNA expression and the protein quantificative level of Pdx-1 decreased in the CUGFR group at 0,8,16weeks (P<0.05). CUGFR significantly reduced the abundance of acetylated H3 (P<0.05) and deacetylation progressed with age. In islets from control animals at all ages, there was a high association of H3K4me3 at Pdx-1. In CUGFR animals at 0,8 and 16weeks after refeeding, the abundance of H3K4me3 was significantly reduced (P<0.05). ChIP assays demonstrated that by H3K9me2 at the Pdx-1 promoter was present in CUGFR islets and H3K9me2 binding increased with age (P<0.05).Conclusion:The changed histone modifications in the cross-linked chromatin from the CUGFR islets may be one of the epigenetic mechanisms responsible for the down-regulation of Pdx-1 gene. |
