| Objective:Although the development of type 2 diabetes is more complex and incompletely clear in etiology, it is usually associated with a combination of pancreaticβ-cell dysfunction and insulin resistance. Moreover,β-cell dysfunction has played a decisive role in the progress that diabetes come into being. Chronically elevated levels of glucose and lipids are known as the initiated factors and also the hallmark of diabetes. Glucotoxicity and lipotoxicity can cause the decrease of insulin through impairment of the expression of the pancreatic duodenal homeobox factor-1 (PDX-1), which is a pancreatic transcription factor that differentially expresses in pancreas when the development of body is mature. PDX-1 plays a central role in the regulation of pancreatic development and differentiation of insulin-secreting cells from progenitor cells in pancreas. It is required for maintaining the pancreatic islet functions by activating transcriptions of genes including insulin, glucose transporter 2, and glucokinase. The decreased capacity ofβ-cell production and secret of insulin as a result of impairing PDX-1 appears to worsen glucose and lipids metabolism. Understanding the regulation and expression of PDX-1 in diabetes can contribute to elucidate the pathogenesis and the development of diabetes and find novel drugs for the treatment of diabetes.Thiazolidinedine (TZD) is an insulin-sensitizing agent which can activate the transcriptional regulator peroxisom proliferator-activated receptor(PPAR)-γto exert its function. The role of TZD in insulin resistance had been extensively studied and applied in clinical treatment of diabetes. Recent studies have indicated that it might have protected isletβcell from glucose and lipids damaging, but the underlining mechanism remains unclear. In the present study, we investigated pancreatic expression of PDX-1 in rat Type 2 diabetes model induced by long-term high-fat feeding accompanied with intraperitoneal injection of streptozotocin (STZ). We also observed the effect of rosiglitazine on pancreatic expression of PDX-1, and the protection function of TZD on the development of type 2 diabetes.Methods: Forty-five healthy male Whistar rats, body weight 199±14g, age 8 weeks, were provided by Hebei Provincial Experimental Animal Center. The animals were randomly divided into 3 groups: Group A received standard laboratory chow diet. Group B and group C were fed with high fat diet, which includes 20% ripe porcine fat, 2% cholesterol and 0.5% cholalic salt. When the high fat diet animals in groups B and C appeared the insulin resistance evaluated by Li Guangwei HOMA index formula, they were followed with a single injection of STZ (30mg/kg). A glucose-lower drug rosiglitazine was given to animals of group C as positive control. Both group B and C were continuously fed with high fat diet for 2 month as the original prescription during experimental period. Five rats in each cage, temperature (20±3)℃, dampness 55%, lightness 12/24 hours.All the rats were weighted and blood was drawn at the beginning and the end of the experiments. The rats were fasted for 12 hours before each experiment, blood was drawn from the medial canthus of eyes and sera were separated and stored at -20℃. At the end of experiment, one third of pancreas was taken and stored in liquid nitrogen for the determination levels of mRNA expression of insulin and PDX-1. One third of pancreas was stored at -80℃for the measurement of insulin and PDX-1 protein expression by western blot analysis, and the remains was fixed for histological analysis.1 Measurements of blood levels of glucose and insulinGlucose oxidation method was used for the determination of FBG and radioimmunoassay for the determination of FINS. Homeostasis Model Assessment (HOMA-IR) and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin,ISI=1/(FINSXFBG) and they were expressed by logarithm.2 Blood fatTotal cholesterol (TC), triglyceride (TC) and high density lipoprotein-cholesterol (HDL) were measured by chronometry. FFAs were measured by the ratio of Cu2+ chrom.3 OGTTThe animals in each group were subjected to an oral glucose tolerance test (OGTT) at the end of the experiment. After 12h fast, the rats were orally given 2.0 gram of glucose per kg body weight. The blood samples were taken from the medial canthus of eyes before (0 minute) and after oral glucose tolerance test at 30,60,120 and 180 minute intervals. Blood glucose levels were determined immediately, and the remain sera were stored at–20℃for the measurement of blood insulin levels.4 Expression of insulin and PDX-1 mRNAs in pancreatic tissuesThe rat pancreatic tissue mRNA was extracted by the Trizol solution. The insulin, PDX-1 andβ-actin mRNAs were amplified respectively by RT-PCR. The PCR products were observed by agarose gel electrophoresis. The DNA fragments of insulin, PDX-1andβ-actin was scanned and the band density was determined. The relative expression quantity of specific band was represented as the ratio of band densitometry units of insulin, PDX-1 to that ofβ-actin.5 Expression of PDX-1 protein level in pancreatic tissueWhole cell lysis of the rat pancreatic tissues was extracted and the total protein concentrations were determined. PDX-1 protein expression was measured by western blot analysis. The relative expression quantity of specific band was represented as the ratio of band densitometry units of PDX-1 to that ofβ-actin.6 The morphological examination of pancreatic tissueThe tissue section was made by routine method for light microscopy examination. Immunohistochemistry was used to confirm the expression of PDX-1 protein.7 Statistical methodsAll data were treated with SPSS 11.0, and all continuous variables were presented as mean±SD. The statistic significance of between means was determined by T-test. One-way ANOVA was used to compare continuous variables among groups. The significance was indicated by p value. The difference which had the significance was indicated by P<0.05, and the difference which had the largest significance was indicated by P<0.01.Results1 The effect of the high-fat diet on the rat weightThere was no significant difference in body weight among each group at the beginning of the experiment. At the end of the experiment, the weight of untreated diabetic rats (398.67±26.83g) were significantly higher than those of the controls (370.93±16.60g) (P<0.01) Rosiglitazine-treated rats (388.87±17.86g) were lower than those of untreated rats, however, there was no significant difference in statistics(P>0.05).2 Blood lipidWhen the model was established, all levels of TG, TC, and FFA in diabetic rats were obviously higher than those of control rats. However, the blood HDL in diabetic rats was significantly lower than those of control rats. TG, TC, and FFA in rosiglitazine-treated rats was significantly lower than those of diabetic rats(P<0.01), however, still higher than that in control rats(P<0.05-0.01). HDL in rosiglitazine-treated rats was significantly higher than those of diabetic rats(P<0.01).3 Results of OGTT at the end of the experimentThe fast blood glucose (FBG) and the blood glucose concentrations at 30min,60min,120min and 180min time points after oral administration of glucose in untreated diabetic rats were significantly higher than those in control group(P<0.01). The blood glucose concentrations at 0min,30min,60min,120min and 180min time points in rosiglitazine treated rats were significantly lower than those in untreated diabetic rats (P<0.01). HOMA-IR in untreated diabetic rats(2.96±0.34) were significantly higher than those in control group(1.13±0.23)(P<0.01), however, ISI were significantly lower than those in control group (P<0.01). In rosiglitazine-treated rats HOMA-IR were obviously higher and ISI were lower than those in untreated diabetic rats (P<0.01).4 Results of GSIS at the end of the experimentsThe fasting blood insulin (FBIN) concentration in untreated diabetic rats(29.14±5.11 mIU/L)was significantly higher than that in control rat(15.08±2.16mIU/L )(P<0.01). The peak of insulin secretion in control rat(84.83±1.73 mIU/L) was at 60min after oral administration of glucose ,but in untreated diabetic rats (102.72±2.88 mIU/L)it was at 120min. FBIN in rosiglitazine treated rat(s18.04±2.30 mIU/L) was significantly lower than that in untreated diabetic rats, and the peak of insulin secretion ( 80.5±1.70 mIU/L ) was at 60min after oral administration of glucose .5 The pathological morphological change of pancreasUsing light microscope, we observed that there were no significantly difference among the three groups.6 ImmunohistochemistryThe percentage of insulin-positive area (β/T area) in untreated diabetic rats (12.51±3.06)was significantly lower than that in control rats(47.55±8.07) (P?0.01). In rosiglitazine treated group (26.18±2.72)it was obviously higher than that of untreated diabetic rats pancreatic tissue(P?0.01). There were no significantly differences in the optical density of insulin-positive area among three groups. The average optical density of PDX-1-positive area in untreated diabetic rats(0.240±0.051) was significantly lower than that in control rats(0.648±0.087) ( P?0.01 ) ,and in rosiglitazine treated (0.460±0.045)it was obviously higher than that of untreated diabetic rats.7 Expression levels of insulin and PDX-1 mRNAsExpression of insulin ,PDX-1 andβ-actin mRNAs in 4 groups were observed with RT-PCR. In untreated diabetic rats, both the expression of insulin mRNA(0.21±0.023) and the expression of PDX-1 mRNA(0.153±0.071) were remarkably lower than that in normal controls (0.527±0.025) (0.49±0.032) P<0.01. Rosiglitazine-treated(0.351±0.035) (0.370±0.029) were obviously improved, but still lower than that in control rats.8 Measuring PDX-1 protein level by western blot analysisThe expression of PDX-1 protein level in control rats(0.720±0.036) was obviously higher than those of untreated diabetic rat pancreatic tissues (0.253±0.028). PDX-1 protein levels in rosiglitazine treated(0.59±0.050) rats were improved, yet lower than those of control rats.Conclusion1.Long-term high fat feeding accompanied with intraperitoneal injection of lower doses of streptozotocin (STZ) can induce diabetic disease which presented increases of blood levels of glucose and lipid. The fasting blood insulin concentration in glucose stimulated insulin secretion was decreased and the peak of insulin secretion was delayed.2.Many pathological changes in pancreas of our diabetic rat model were associated with significant decreasing expressions of insulin and PDX-1mRNAs and proteins. These findings confirmed the major role of PDX-1 in the pathogenesis of diabetes.3.Treatment with rosiglitazine significantly increased the expressions of PDX-1 mRNA and protein levels in rat pancreas. Moreover, the pathological changes of pancreas were found to be ameliorated. These findings implicated the protective roles of PPAR-γagonist on islet cell function are subjected to up-regulation of expression of PDX-1.
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