| With the change of diet and lifestyle, diabetes has become the major threats to human health disease following cancer and cardiovascular diseases, which can lead to the damage of heart, brain, kidney and other organ, and has become the main reason for death and disability in patients. In 2011, International Diabetes Federation (IDF) showsthe latest statistics that the global diabetic patientswere about 366 million, and by 2030, the totality of diabetes patients will reach 552 million. In China, an epidemiology of diabetes in 2007-2008 survey shows that the number of diabetes patients reached 97.2 million, while the number of patients pre-diabetes was to reach 145 million. The latest survey shows that the prevalence of diabetes has reached 11.7%, Thepatients with diabetes have more than 100 million people. The patients with pre-diabetes is type 2 diabetes reserves, the study of the pathogenesis of pre-diabetes, seeking early and effective strategies to prevent and treat pre-diabetes, and for improving the health and quality of life, reducing the economic burden of patients has important significance.In past years, people believed in the pathogenesis of pre-diabetes were basedon insulin resistance. However, in recent years, with the increase researchof pancreatic P-cell, more and more researchers began to realize that the number of pancreatic P-cell declined in the early development of type 2 diabetes andplayed an important role in diabetes. Animal experiments show thatthe number of islet P cell in db/db mice declinewith the increasing of blood sugar. An epidemiological survey, islet function has decreased before 10-12 yearsin the diagnosis of type 2 diabetes, and with elevated blood, the diabeteswas diagnosis.Islet function has been reduced to about 50% of normal. The autopsyto the patients of pre-diabetesshows that the number of pancreatic P cell is about 60% of normal, and is reduced to 40% of normalin patients of type 2 diabetes.Diet factors have been thought to be an important environmental factors leading to the development of type 2 diabetes. More research is focused on the effect high-fat and high-glucose diet on insulin resistance and islet function caused by diabetes. Fructose has especially attracted the attention of researchers as a factor contributing to the development of type 2 diabetes. With the introduction of high fructose corn syrup (HFCS) into the food industry, the intake of fructose in the populations has increased year by year. Animals’studies indicated the over-intake of fructose is related to overweight, dyslipidemia, insulin resistance, higher blood pressureandhyperuricemia. Therefore, the harmfulness of high-fructose-diet has been increasingly recognized. It is widely used to study the pathogenesis of diabetes. Existing literature mostly concentrated in the impact high-fructose to insulin resistance, but its effect on islet function remains unclear.Many studies have shown that stress caused glucolipotoxicity, such as oxidative stress and endoplasmic reticulum stress led to a decline in islet function. The endoplasmic reticulumin β cell synthesizes large amounts of insulin. Under the state of insulin resistance, insulin secretion is increased significantly. Persistent insulin synthesis and secretion will cause endoplasmic reticulum stress in β cells, which will cause β cell failure and even death, resulting in reduced glucose tolerance from normal todevelop into diabetes finally. Many studies have confirmed that endoplasmic reticulum stress plays an important role in the cause of insulin resistance in fructose. It remains unclear if high fructose induces islet dysfunction and endoplasmic reticulum stress participates the mechanism of islet dysfunction.In the present study, we first observed the change of islet function in patients with impaired glucose regulation and high triglycerides, to understand the role of glucolipotoxicityin islet functionwith pre-diabetes.Next, we observed the change of islet function and insulin sensitivity in Wistar rats after 8 weeks high-fructose-feeding and pancreatic endoplasmic reticulum stress-related protein expression and apoptosis pathway genes. In addition, we observed the relationship between islet function and endoplasmic reticulum stress pathway and used endoplasmic reticulum stress inhibitor 4-phenyl butyric acid feedthese rats for 8 weeks and observedthe change of islet function.Part One The studyof islet functionand insulin sensitivity in patients with impaired glucose tolerance and hypertriglyceridemiaObjective:To observe the changes of insulin sensitivity andislet function in patients with impaired glucose tolerance and hypertriglyceridemia and to explore the impact of pre-diabetes and lipid disorders in insulin resistance and islet function.Methods:All of subjects are outpatientsfrom January 2010 to January 2011 in Cangzhou Central Hospital, Hebei Province People’s Hospital and aged between 20-65 years old. According to 1999 World Health Organization (WHO) diagnostic criteria for impaired glucose tolerance(IGT): 6.1mmol/L<fasting blood glucose (FBG)<7.0mmol/L and oral glucose tolerance (OGTT) after 7.8mmol/L≤glucose load after 2h (2h BG) <11.1mmol/L, according to the USA Third Cholesterol Education Program diagnostic criteria:, diagnostic criteria for hypertriglyceridemia:triglyceride (TG)>2.3mmol/L.40 patients were randomly selected for IGT (IGT group), 37 patients were IGT combined with hypertriglyceridemia (IGT-HTG group) and normal control group (NGT group) 40 cases. All subjects’ height, body weight and body mass index (BMI). FBG, triglyceride (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glycated hemoglobin (HbA1c), insulin (FPI) and free fatty acid (FFA) were detected. HOMA-IR is for evaluatinginsulin resistance; HOMA-β is for evaluating basic islet function. Intravenous glucose tolerance test (IVGTT) is for evaluating first phase insulin secretion (FPIR). To compare all indexes in three groups, a complete random way ANOVA is used.Using linear correlation analysis impact factors of islet function and insulin resistance. Result:1 Comparisonof clinical data in three groupsNo differences were observed in sex, age, BMI, TC, LDL-C, HDL-C, and blood pressure in the three groups (P> 0.05); FBG,2 h-BG, HbA1c and FPI were all significantly higher in the IGT and IGT-HTG groups compared with the NGT group (P< 0.01 or P< 0.05), but there were no significant differences between the IGT and IGT-HTG groups(P>0.05); TG and FFA levels were highest in patients with IGT-HTG, and lowest in those with NGT (P<0.01 or P< 0.05).2 Comparison of insulin resistance and islet function evaluation in threegroup’sindexHOMA-IR was significantly higher than NGT group in patients with IGT and patients with IGT-HTG (P<0.01), but no significant difference was observed in IGT group and IGT-HTG group(P> 0.05); HOMA-β was lower than NGT group and IGT group in patients with IGT-HTG (P<0.01), but no significant difference was observed in IGT group and NGT group(P> 0.05); FPIR was lowest in patients with IGT-HTG, and highest in those with NGT (P <0.01 or P< 0.05).3 Correlations between islet function and various parametersSpearman correlation analysis between HOMA-IR, HOMA-β, FPIR and the various parameters examined. HOMA-P showed a negative correlation withFBG (r=-0.431, P= 0.008), HbA1c (r=-0.382, P=0.024), FPI (r=-0. 486, P= 0.005) and TG (r=-0.305, P= 0.034); FPIR showed a negative correlation with2h-BG (r=-0.435, P= 0.007), HbA1c (r=-0.408, P=0.009), FPI (r=-0.462, P= 0.005) and TG (r=-0.438, P= 0.006). HOMA-IR showed a positive correlation with FBG (r= 0.422, P= 0.008),2h-BG (r 0.427, P=0.007), HbA1c (r=0.453, P=0.006), FPI(r= 0.487, P=0.005) and TG (r=0.394, P=0.015). It indicated that glucose and lipid metabolism disorders involved in islet dysfunction and insulin resistance directly.Conclusion:1 exist insulin resistance and islet dysfunction and which was more apparent in patients with IGT and hypertriglyceridemia.2 The islet dysfunction was correlated with hyperglycemia and hyperlipidemia.Part Two The effects of high-fructose diet on islet function and glucose-regulated protein (GRP78) in Wistar ratObjective:To explore the early change and mechanism of islet function n high fructose diet-induced insulin resistance rat.Method:Ninety Wistar rat weighed from 180 to 200g were used in the study (Provided by Experimental Animal Center of Hebei Medical University). The animals were kept in a temperature-controlled room (18-26℃), and humidity(40%-60%)in animal feeding center on a 12-h light/dark cycle. After a week of acclimation feeding, the rats were divided into 2 groups:Normal control group (NC) (n=36) and high-fructose group (HF) (n=54). NC group were fed with standard lab chow dietwith the energy contents as follows:71% calories from carbohydrate,8% calories from fat, and 21% calories from protein with total energy of 260kcal/100g; HF group were fed with a high-fructose-diet with the energy contents as follows:70% calories from carbonhydrates with 35% of fructose,9% calories from fat and 21% calories from protein with total energy of 260kcal/100g;Sixrats were randomly chosen from each group after 8 week of feeding. Oral glucosetolerance test (OGTT) was performed in the 6 rats and plasma insulin and glucose were measured to calculate HOMA-IR, HOMA-β and ΔI30/AG30 evaluating islet function in rats. Twelverats were randomly chosen from each group.Blood samples were taken from 12 rats in every group and collected after centrifuged in refrigerated centrifuge for detection of plasma biochemical index. The islets of rats were Isolated and purified for Western-blot of glucose-regulated protein (GRP78) protein levels.Result:1 The changes of body weight and biochemical index in the two groups ratsAfter 8 week feeding by different diets, there are no significant difference in body weight and fasting blood glucose between 2 groups(P>0.05); Butthe plasma fasting insulin, proinsulin and TG were significantly higher than NC group (P<0.01).2 Evaluation of insulin resistance and pancreatic function in two group’s ratsCompared with the NC group, blood glucose at 30min,60min was significantly higher in HF group, the difference was statistically significant (P <0.05 or P<0.01); fasting insulin was significantly higher in HF group (P <0.05), but insulin secretion peak was lower than NC group. The difference was statistically meaningful (P<0.01).Compared with the NC group, HOMA-IR and fasting proinsulin/insulin ratio were significantly higher in HF group (P<0.01), ΔI30/ΔG30 for reflecting the early phase insulin secretion function was significantly lower in HF group (P<0.01), but no significant difference was observed in two group (P>0.01); These result showed that rats appeared insulin resistance early phase insulin secretion dysfunction after 8 weeks of feeding high fructose.3 The expression of four groups of rat islet protein GRP78Compared with the NC group, islet GRP78 protein expression was significantly higher in HF group, the difference was statistically significant (P<0.01).The result showed that the islet of rats appeared endoplasmic reticulum stress after 8 weeks of high fructose feeding.Conclusion:1 The rats showed insulin resistance and early islet dysfunction after 8 weeks of high fructose feeding2 High fructose-fed Wistar rat islet dysfunction may be related to endoplasmic reticulum stress. Part Three The mechanism of endoplasmic reticulum stress cause damage to islet function in high-fructose diet ratsObjective:To evaluate the effect of long-term high-fructose diet on β cell apoptosis in rats and further clarify the action of endoplasmic reticulum stress in β cell apoptosis.Method:We began the part of the experiment from the ninth weeks. NC group (n=18) continued to feed normal diet, HF group were randomly divided into a control group fed a high-fructose (HF-C group) (n=18) and high fructose feeding plus 4-phenyl butyric acid (4-PBA) treatment group (HF-PBA group) (n=18). HF-PBA group was given 4-PBA 0.5g/kg/d in distilled water orally from the ninth weeks. NC group and HF-C group were given the same amount of distilled water. After 8 weeks, six rats were randomly chosen from each group. Oral glucosetolerance test (OGTT) was performed in the 6rats and plasma insulin and glucose were measured to calculate HOMA-IR, HOMA-β and ΔI30/AG30 evaluating islet function in rats. Rats were anesthetized by 2.5% pentobarbital (50mg/kg) byintraperitoneal injection and were sacrificed. Blood samples were collectedand used formeasurement oftriglyceride and proinsulin, the pancreas of rats were isolatedfor TUNEL apoptosis, transmission microscope specimen and TG levels in the pancreas. The remaining pancreatic islet in ratswere isolated by collagenase digestion and used for RNA or protein extraction respectively, which were collected and stored in-70℃.The mRNA level of spliced XBP-1 was measured by real-time quantitative PCR (Real-time PCR) method. Protein expression of markers of islet ERS including the phosphorylated proteins of pancreatic ER kinase (PERK), inositol-requiring kinase la (IRE-1a), eukaryotic translation initiation factor 2a (eIF2a), c-Jun N-terminal kinase (JNK), CCAAT/enhancer-binding protein homologous protein (CHOP), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (bax), and caspase-12 were measured by Western Blot.Result:1 The changes in body weightAfter 16 weeks of feeding, the weight of each group of rats fed has increased. Although the weight in HF-C group was slightly higher than the NC group and HF-PBA group, no significant differences among the three groups (P>0.05).2 The changes in fasting glucose, insulin and proinsulinAfter 16 weeks of feeding, fasting blood glucose in HF-C group and HF-PBA group is slightly higher than NC group, but no significant difference among three groups; fasting insulin and proinsulin in HF-C group was significantly higher than the NC group (P<0.01). After 4-PBA treatment, fasting insulin and proinsulin were decreased significantly (P<0.01).3 The change of blood glucose levels and blood isulin after OGTT in three groups of rats fed for 16 weeksHOMA-IR in HF-C group was significantly higher than the NC group (P<0.01), proinsulin/insulin, Δ30/ΔG30 and HOMA-β in HF-C group was significantly lower than the NC group (P<0.01). After administration of 4-PBA of treatment, HF-PBA group 30min and 60min blood glucose lower than HF-C group, the difference was statistically significant (P<0.05 or P<0.01); Compared with HF-C group, HOMA-IR in HF-PBA group was lower, proinsulin/insulin, ΔI30/ΔG30 and HOMA-β were significantly higher than HF-C group, the difference was statistically significant (P<0.01).4 The change in plasma and pancreas TG contentsPlasma and pancreas TG contents in HF-C group were significantly higher than NC group (P<0.01). After treatment of 4-PBA, plasma and pancreas tissue TG in HF-PBA group lower than HF-C group, and.5 The Change in TUNEL assayCompared with the NC group, the percentage of TUNEL-positive cells in islets was significantly increased (38±4.2)% vs (10±1.1)%(38 vs.10%, P<0.01) in the HF-C group. The percentage of TUNEL-positive cells In the HF-PBA groupdecreased in islets to 15%. The difference was statistically significant (P<0.01).6 The changes of islet cell electron microscopeUnder the electron microscopein NC group, it can be seen oval nuclei, nucleoli clear, uniform nuclear chromatin oval clear nucleolus, nuclear chromatin, normal rough endoplasmic reticulum; Secretory granules were visible electron density dense core, the outer wrap bounded, there is a gap between the core and the limiting membrane round. Cytoplasm also was seen scattered in the mitochondria in the NC group. Well granulated cytoplasm, well developed endoplasmic reticulum, increase in the number of immature granules in the cytoplasm with no clear signs of cell injury were found in the islet beta cells of diabetic treated rats. Cytoplasm loose, swelling in mitochondria and endoplasmic reticulum, degranulation state, reducing the mature secretory granules, increased immature secretory granulesand surrounding nuclear chromatin condensationwere found in the islet beta cells of HF-C group rats. After the 4-PBA treatment, mature secretory granulesincreased, improving mitochondrial swelling and endoplasmic reticulum were seen in HF-PBA group.7 The effect of 4-PBA on endoplasmic reticulum stress signaling pathwaysCompared with the NC group, GRP78 protein expression further increased in HF-C group, the difference was significant (P<0.01); the phosphorylated proteins of upstream regulatory protein PERK increased in HF-C group (P<0.01). The phosphorylation of downstream factor eIF2a also increased in HF-C group (P<0,01); another upstream regulating protein phosphorylation of IRE-1a (p-IRE-1a) increased significantly (P<0.01); Compared with the HF-C group, The phosphorylation of PERK, eIF2a and IREla protein expression in HF-PBA group were significantly decreased, the difference was statistically significant (P<0.01).Compared with the NC group, the mRNA level spliced XBP1 (XBP-1s) increased significantly in HF-C group (P<0.01). Compared with the HF-C group, XBP-1s levels in HF-PBA group rats were decreased significantly after 4-PBA intervention, the difference was significant (P<0.01). It indicated IREla activated downstream of XBP-1 and cause endoplasmic reticulum stress.4-PBA reduced endoplasmic reticulum stress by inhibiting IRE1a-XBP-1 signaling pathway.8 The effect of 4-PBA on apoptosis by mediated endoplasmic reticulum stress signaling pathwaysFurther testing of ER stress-mediated apoptosis markers CHOP, caspase-12, phosphorylation of JNK(p-JNK) and Bcl-2, bax protein, we found HF-C group CHOP, caspase-12, p-JNK and bax protein expression was significantly increased compared with the NC group, the difference was significant (P<0.01), decreased Bcl-2 protein expression was significantly (P<0.01). Compared with HF-C group, after 4-PBA intervention CHOP, caspase-12, p-JNK and bax protein expression in HF-PBA group was significantly down-regulated, the differences were statistically significant (P<0.01), Bcl-2 protein expression was significantly increased (P<0.01).Conclusion:1 A long-term high-fructose diet induced endoplasmic reticulum stress signaling pathways mediated apoptosis in Wistar rat islets and islet dysfunction.24-PBA improved islet function by relieving islet β cell apoptosis by mediated endoplasmic reticulum stress signaling pathways. |
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