Effects And Mechanism Of Long-Term High-Protein Diet On The Pancreatic Islet In Diet-Induced Obesity Rats

Background:With the development of type 2 diabetes,studies have attached great importance for the effect of high-protein diet on glucose metabolism and insulin secretion.But the results from different studies are not in common.Several studies have shown high-protein diet can enhance insulin secretion,and improve glycaemic control, but epidemiological studies indicate that high protein intake might even increase the incidence of type 2 diabetes.Moreover,the mechanism of high protein diet on pancreatic isletβcell function has not been elucidated.Consequently,we aimed to investigate the effects of high protein diet on insulin secretion of pancreatic isletβcell,also to explore the mechanism involved in it.Except for postprandial elevations of plasma amino acids stimulating insulin secretion,the incretin hormone ---glucagon-like peptide-1(GLP-1),which secreted from intestinal endocrine L cells,can also increase insulin gene transcription, biosynthesis and secretion.Many studies had shown GLP-1 release was lower in type 2 dibetes,and that was an important factor of impairedβcell function.Pancreas duodenum homeobox 1(Pdx-1),a transcription factor essential for pancreatic development andβ-cell function,also plays a central role in mediating the actions of GLP-1 on insulin gene transcription and secretion.Studies have shown GLP-1 can increase Pdx-1 gene transcription and the binding of Pdx-1 to the insulin gene promote.GLP-1 secretion from intestinal L-cells is stimulated by a variety of nutrient,neural,and endocrine factors.Up to now,numerous studies in vitro and in vivo have demonstrated that chronic exposure to glucose or fatty acid is able to suppress pancreatic/duodenal homeobox-1(PDX-1) expression,leading to decreased insulin secretion,and many short-term studies had exerted evidence that high protein diet can promote glucagon-like peptide-1 secretion,but there had no long-term high protein diet studies about that.So these promoted us to speculate if the change of insulin secretion in high protein diet is related with GLP-1 and PDX-1.Objective:The aim of this study is to investigate the effects of long-term isocaloric high-protein diet intake on body weight,blood glucose,serum levels of insulin, plasma GLP-1 in diet-induced obesity rats,combining the mRNA and protein expression of GLP-1 in ileum,insulin and PDX-1 in pancreas,so as to probe the effect of long-term isocaloric high-protein diet intake on insulin secretory function ofβcell,and also its mechanism involved in it.Meanwhile,we observed the impacts of long-term high-protein diet on kidney in obesity rats,in order to explore the safety of dietary protein.Methods:Diet-induced obesity rat models were established by feeding Wistar rats(21 days old) high calorie and high fat diet for 14 weeks.The obese male rats were randomly divided into three groups:the high-protein diet group(HP,n=12),the high-fat diet group(HF,n=11),and the normal chow group(NC,n=11).The total calorie ingestion of each rat per day was similar and was maintained for 24 weeks.Body weight and fasting blood glucose(FBG) were monitored during the feeding period. After 24 weeks,fasting plasma GLP-1,insulin(FINS),blood urea nitrogen (BUN),creatinine(Cr) and 24 hour urine albumin(ALB) were assayed.To evaluate the insulin secretory function ofβcells,serum levels of insulin were determined in an intravenous glucose tolerance test(IVGTT).Visceral fat mass was measured after executing the rats.The mRNA and protein expression of GLP-1 in ileum,insulin and PDX-1 in pancreas were studied by immunohistochemical method and RT-PCR respectively,and the renal morphology was observed by HE under microscope.Results:1.Body weight and visceral fat mass:Compared with NC group,there was no significant difference in body weight among the three groups after 8 weeks.From 16th week to 24th week,body weight in HP rats were significantly lower than those in NC rats[(490.92±39.47) g vs(545.55±31.08) g,P＜0.01].The visceral fat mass in HP rats was also significantly lower[(22.42±7.04) g vs(32.33±9.27) g,P 1 ＜0.05].But those indexs were both higher[(656.01±58.49 )g vs(545.55±31.08) g, (55.33±17.81) g vs(32.33±9.27) g,both P＜0.01]in the HF group.2.FBG,FINS and IVGTT:There were no differences in FBG levels among the three groups at 12th and 24th week(P＞0.05).As to IVGTT,there were no differences in blood glucose among the three groups.Compared with NC group,the serum insulin levels in 5min and 10 min were significantly lower in HP than in NC [5min(91.56±21.72) mIU/L vs(121.29±34.03) mIU/L;10min(58.62±15.80) mIU/L vs(81.12±24.36) mIU/L,both P＜0.05].The area under the curve(AUC) of insulin was lower in HP than in NC[0～10min(132.46±33.42)uIU/mL vs(175.59±58.33) uIU/mL,P＜0.05;0～120min(227.64±54.34) uIU/mL vs(287.91±76.58) uIU/mL, p=0.086].As to HF group,the insulin was significantly increased at 0min [(40.21±14.12) mIU/L vs(27.48±11.31) mIU/L,P＜0.05],then reaching peak at 5min,but the peak value was slightly lower than in NC group[(110.52±28.93) mIU/L vs(121.29±34.03) mIU/L,p=0.12],and 10min insulin levels were significantly higher than in NC[(98.15±27.58) mIU/L vs(81.12±24.36) mIU/L,P＜0.05].Also there was an increased tend of AUC-INS in the HF group[0～120min (315.92±82.54) uIU/mL vs(287.91±76.58) uIU/mL,p=0.16].3.Fasting plasma GLP-1:Plasma GLP-1 level was significantly lower in HP rats[(0.52±0.13)μg/L vs(0.71±0.19)μg/L,P＜0.05],and there was an increased tend in the HF group[(1.03±0.28)μg/L vs(0.71±0.19)μg/L,p=0.11]compared with the NC group.4.BUN,Cr and 24 hour urine ALB:The BUN level in HP rats were significantly higher than in NC rats[(7.02±0.86) mmol / L vs(5.61±0.46) mmol / L,P=0.001].There were no differences in Cr and 24 hour urine ALB levels among the three groups(P＞0.05).5.The image analysis:(1) The insulin expression Compared with NC rats,there were no differences in total pancreatic islet area and positive area of isletβcell in HP rats[(658944±103586) um~2 vs(697419±123143)um~2,(48.42±5.03)%vs(50.28±4.15)%,both P＞0.05],also was the grey level[(102±15)vs(120±18),P＞0.05].As to HF group,total pancreatic islet area became significantly larger[(1250560±347510) um~2 vs(697419±123143)um~2 P＜0.05],but the ratio of insulin positive area to the islet was lower[(40.35±5.16)%vs(50.28±4.15)%,P＜0.05],and grey level was higher[(157±23) vs(120±18),P＜0.01].(2) The PDX-1 expression Compared with NC rats,there were no differences in grey level in HP rats[(135±13) vs(130±15),P＞0.05],but it had an declined trend of the ratio of PDX-1 positive area of isletβcel[(24.42±3.42)%vs(32.87±6.35) %,P=0.072].In HF group,there was a significantly lower ratio of PDX-1 positive areato the islet[(18.58±4.38)%vs(32.87±6.35)%,P＜0.05)],and a higher grey level[(163±20) vs(130±15) P＜0.01].(3) The GLP-1 expression Compared with NC rats,there were no differences in optical density(OD) in HP rats[(0.389±0.023.) vs(0.415±0.034),P＞0.05],but it had an declined trend of integrated optical density(IOD)[(27431±5813) vs (36601±5083),P=0.081].OD and IOD were both significantly lower in HF group[(0.341±0.056) vs(0.415±0.034),(20928±4378) vs(36601±5083),both P＜0.05].(4) After HE dye,there were no differences in the kidney morphology between two groups under microscope.6.RT-PCR results:Compared with NC rats,INS mRNA expression in HP group was significantly suppressed[(0.45±0.16)vs(0.73±0.08),P＜0.05)],and it had a declined trend in PDX-1 mRNA expression[(0.70±0.12) vs(0.87±0.09), P=0.102].But as in HF group,PDX-1 mRNA expression was significantly suppressed[(0.58±0.17) vs(0.87±0.09),P＜0.05],and there were no difference in INS mRNA expression in HF group[(0.64±0.13) vs(0.73±0.08),P＞0.05)].Also there were no difference in GLP-1 mRNA expression among the three groups.Conclusion:1.Long-term isocaloric high-protein diet may reduce the secretion of insulin but did not change the morphology of pancreatic islet.2.In HP rats,Plasma GLP-1 level was significantly lower,and it had an declined trend of GLP-1 protein expression,indicating GLP-1 can be suppressed by high-protein diet.3.It had an declined trend of PDX-1 mRNA level and protein expression in HP rats,indicating PDX-1 expression is proberbly suppressed by high- protein diet.4.The body weight and visceral fat mass in HP rats were both significantly lower,indicating high- protein diet has an effect of weight loss.5.The decreased insulin secretion in HP may be related with the suppression of GLP-1 and PDX-1,also with the loss of weight.6.There is no evidence that showed the damage of high- protein diet on kidney...