The Relationship Between Gastric Emptying And Ghrelin And Obestatin In The Early Stages Of Streptozotocin-Induced Diabetic Rats

BACKGROUNDDiabetes mellitus is a metabolic disease characterized by chronic hyperglycemia because of defects in insulin secretion and/or defects in insulin action. The prevalence rate of diabetes mellitus increased year by year. As the high prevalence and high disability of diabetes mellitus, it not only affects the quality of life but also high medical costs. Diabetes mellitus results in serious social problems and economic problems and become global health issues that a threat to human health. Therefore, to enhance the study of diabetes mellitus has important significance.In recent years, the relationship between diabetes and gastrointestinal tract, especially gastric emptying was received more and more attention. On the one hand the speed of gastric emptying may affect the postprandial blood glucose level, on the other hand, gastrointestinal tract can secrete multiple glucoregulatory hormones involved in regulating blood glucose.It is well recognized that certain populations of diabetic patients have delayed gastric emptying called gastroparesis. Gastroparesis is a relatively rare complication that occurs late in diabetes. Diabetic gastroparesis is explained by the autonomic neuropathy associated with diabetes. However, rapid gastric emptying can be seen in subgroups of patients in the early stages of Type 2 diabetes, non-insulin-dependent diabetes mellitus (Type 2 diabetes), and neuropathy-free, insulin-dependent diabetes mellitus (Type 1 diabetes) It has also been reported that gastric emptying is accelerated in the early stages of streptozotocin-induced diabetes in rats and in spontaneously diabetic BB rats. Although a great deal of attention has been paid to diabetic gastroparesis, the mechanism of accelerated gastric emptying in the early stages of diabetes has not been fully studied.Ghrelin is a 28 amino acid peptide that originally was isolated from the rat stomach in 1999 by.Kojima et al. Ghrelin, an orexigenic peptide, is the endogenous ligand for the growth hormone secretagogue receptor(GHS-R). Ghrelin is produced by A-like cells in the in the oxyntic glands of the gastric mucosa and is released into circulation. The stomach, especially the fundus part, is the major source of circulating ghrelin. Besides the stomach, the peptide is also expressed at lower levels in many other tissues such as intestine, pancreas, pituitary, hypothalamus, liver and so on. Ghrelin has potent stimulatory effects on GH secretion and food intake. In addition, it was shown that ghrelin promotes gastric emptying and a positive energy balance, thus increasing adiposity and body weightIt is well established that ghrelin stimulates gastrointestinal motility. Ghrelin accelerates solid gastric emptying in humans, rats, and mice. Gastric emptying of nonnutrient liquid is also accelerated by ghrelin in rats and mice. Circulating levels of ghrelin rise before and decrease after a meal in normal-weight subjects. In contrast, the postprandial reduction of ghrelin is not obvious in obese subjects and patients with new onset childhood Type 1 diabetes In STZ-induced diabetic rats, postprandial ghrelin concentrations are higher than in control rats and return to fasted levels rapidly after feeding. Gastric ghrelin-immunoreactive cells are slightly decreased in STZ-induced diabetic rats, whereas preproghrelin mRNA levels are extremely higher in STZ rats than in control rats. The increased plasma ghrelin levels and the decreased gastric ghrelin cells in the diabetic rats may be due to an increase in ghrelin release from the stomach into the bloodstream.Based on these evidences, we hypothesize that ghrelin contributes to rapid gastric emptying in the early stages of STZ-induced diabetes.Obestatin is a newly discovered peptide of 23 amino acids isolated from rat stomach in 2005 by Zhang et al. It derived from the same precursor protein as ghrelin. Obestatin is derived from the carboxy-terminal part of proghrelin, whereas ghrelin is derived from the N-terminal part of the same precursor. The distribution of obestatin-and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats was characterized. Obestatin is proposed to be the endogenous ligand for GPR39. Notwithstanding the same precursor, the biological activity of obestatin is reportedly opposite to that of ghrelin. It has been reported that obestatin has inhibitory effects on feeding and digestive motility and thus antagonizes the stimulatory effect of ghrelin. Zhang et al first reported that obestatin can suppresse cumulative food intake, decrease body weight gain, and inhibited gastric emptying and jejunal muscle contraction in mice. Since then, however, the inhibitory effects of obestatin on food intake and gastrointestinal motility have remained controversial, and further studies are needed to determine the physiological function of obestatin. The role of obestatin in the gastric emptying in the early stages of streptozotocin-induced diabetic rats has not been studied.Objective:This study was conducted in streptozotocin-induced diabetic rats featuring with insulin deficiency and treated with insulin at the same time. The aim of our study was to investigate the gastric emptying, plasma levels of ghrelin and obestatin, the expression levels of ghrelin, obestatin and ghrelin mRNA in the fundus of stomach, levels of ghrelin and obestatin in the hypothalamus, the expression levels of GHSR mRNA and GPR-39 mRNA in the hypothalamus in the early stages of streptozotocin-induced diabetic rats, and explore the relation between them.Methods:Forty Wistar rats were randomly divided into four groups:normal control group(NC group), diabetic mellitus group(DM group), low-dose insulin-treated group(LD group) and high-dose insulin-treated group(HD group). Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ). Rats in LD group and HD group were received 4U/animal/day and 8U/animal/day recombinant human insulin (Humulin N) by subcutaneous injection beginning the 4th day after the STZ injection. Two weeks after the STZ injection, Gastric emptying was measured by intragastric administration of methylcelluose-phenol red solution. The levels of ghrelin and obestatin in the plasma and hypothalamus were measured by EISIA method. The expression levels of ghrelin and obestatin in the fundus of stomach were detected by immunohistochemistry The expression of ghrelin mRNA in the fundus of stomach and the expression levels of GHSR mRNA and GPR-39 mRNA in the hypothalamus were measured by semi-quantitative RT-PCR. Statistical analysis was performed by using SPSS11.0 statistical software. P values<0.05 were considered as significant.Results1.From there days to two weeks after the STZ injection, the body weight decreased significantly and the blood glucose levels increased significantly in DM group compared to those in NC group(P<0.01). Two weeks after the STZ injection,the blood insulin levels decreased significantly in DM group compared to those in NC group(P<0.01). Two weeks after the STZ injection, the liquid gastric emptying accelerated significantly in DM group compared to those in NC group(P<0.01).2.Two weeks after the STZ injection, the plasma ghrelin levels increased significantly in DM group compared to those in NC group(P<0.01). The ghrelin integral optical density in the gastric fundus decreased significantly in DM group compared to those in NC group(P<0.01). The ghrelin mRNA expression levels in the gastric fundus increased significantly in DM group compared to those in NC group(P<0.01). The ghrelin levels in the hypothalamus increased significantly in DM group compared to those in NC group(P<0.01). The GHSR mRNA expression levels in the hypothalamus increased significantly in DM group compared to those in NC group(P<0.01).3.Two weeks after the STZ injection, the plasma obestatin levels increased significantly in DM group compared to those in NC group(P<0.01).The obestatin integral optical density in the gastric fundus decreased significantly in DM group compared to those in NC group(P<0.01).The obestatin levels in the hypothalamus were no statistical differnce between DM and NC group(P> 0.05). There was no GPR-39 mRNA expression detected in the hypothalamus in DM and NC group.4. After insulin treatments, the body weight increased significantly in LD and HD group compared to those in DM group(P<0.01) and the body weight increased significantly in HD group compared to those in LD group(P<0.05). The blood glucose levels decreased significantly in LD and HD group compared to those in DM group(P<0.01) and increased significantly in LD and HD group compared to those in NC group(P<0.01). The blood glucose levels decreased significantly in HD group compared to those in LD group (P<0.01). The blood insulin levels increased significantly in LD and HD group compared to those in DM group(P<0.01) and increased significantly in HD group compared to those in LD group(P<0.01). The liquid gastric emptying slowed significantly in LD and HD group compared to those in DM group(P<0.01) and accelerated significantly in LD and HD group compared to those in NC group(P<0.01). The liquid gastric emptying slowed significantly in HD group compared to those in LD group(P<0.01).5.After insulin treatments, the plasma ghrelin levels decreased significantly in LD and HD group compared to those in DM group(P<0.01). The plasma ghrelin levels increased significantly in LD group compared to those in NC group(P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The ghrelin integral optical density in the gastric fundus increased significantly in LD and HD group compared to those in DM group(P<0.01) and decreased significantly in LD and HD group compared to those in NC group(P<0.01,P<0.05). The ghrelin integral optical density in the gastric fundus increased significantly in HD group compared to those in LD group(P＜0.01) The ghrelin mRNA expression levels in the gastric fundus decreased significantly in LD and HD group compared to those in DM group(P<0.05,P<0.01). The ghrelin mRNA expression levels in the gastric fundus increased significantly in LD group compared to those in NC group (P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The ghrelin levels in the hypothalamus decreased significantly in LD and HD group compared to those in DM group(P<0.01). The ghrelin levels in the hypothalamus increased significantly in LD group compared to those in NC group(P<0.01) and were no statistical differnce in HD group compared to those in NC group(P>0.05). The GHSR mRNA expression levels in the hypothalamus were no statistical differnce in LD and HD group compared to those in DM group(P>0.05) and increased significantly in LD and HD group compared to those in NC group (P<0.01).6.After insulin treatments, the plasma obestatin levels were no statistical difference in LD and HD group compared to those in DM group(P> 0.05) and increased significantly in LD and HD group compared to those in NC group(P＜0.01). The obestatin integral optical density in gastric fundus were no statistical difference in LD and HD group compared to those in DM group(P>0.05)and decreased significantly in LD and HD group compared to those in NC group(P<0.01). The obestatin levels in the hypothalamus were no statistical differnce among LD, HD, DM and NC group (P> 0.05). There was no GPR-39 mRNA expression detected in the hypothalamus in LD and HD group.Conclusionl.The liquid gastric emptying accelerated significantly in the early stages of STZ-induced diabetic rats.2.In the early stages of STZ-induced diabetic rats, the plasma ghrelin levels increased significantly, the ghrelin expression levels and the ghrelin mRNA expression levels in the gastric fundus increased significantly, the ghrelin levels and the GHSR mRNA expression levels in the hypothalamus increased significantly. 3.In the early stages of STZ-induced diabetic rats, the plasma obestatin levels increased significantly, the obestatin expression levels in the gastric fundus decreased significantly. The obestatin levels in the hypothalamus had no significant change and there was no GPR-39 mRNA expression detected in the hypothalamus.4.Treatments with insulin attenuated significantly increased blood glucose levels, decreased blood insulin levels and accelerated liquid gastric emptying in the early stages of STZ-induced diabetic rats.5.Treatments with insulin attenuated significantly increased plasma ghrelin levels, decreased ghrelin expression levels and increased ghrelin mRNA expression levels in the gastric fundus, increased ghrelin levels in the hypothalamus, but did not attenuate significantly increased GHSR mRNA expression levels in the hypothalamus in the early stages of STZ-induced diabetic rats.6.Treatments with insulin had no significant effect on increased plasma obestatin levels, decreased obestatin expression levels in the gastric fundus and the obestatin levels in the hypothalamus in the early stages of STZ-induced diabetic rats.7. Our study suggests that elevated endogenous ghrelin may play important role in the mechanism of accelerated liquid gastric emptying in the early stages of STZ-induced diabetic rats and the role of endogenous obestatin need further study.