Pancreatic Islet Injury Induced By High Lipid Implicated The Production Of IAPP:Studies Of Its Effects And Mechanisms

ObjectiveObesity is an important risk factor in type 2 diabetes mellitus (T2DM). Obesity is often accompanied with the hyperlipidemia, hyperinsulinemia and insulin resistance, which also are common features in T2DM. Islet amyloid protein (IAPP), as the chaperon, produced with the insulin secretion after the metablite stimulation. Oligomerized IAPP is easy to deposite in intracellular and extracellular areas in pancreatic islet. Deposite IAPP may damage the p-cell and aggravated in the T2DM. The blood from person with obesity and hyperlipidemia contain high IAPP. However, it is unclear whether or not the increased IAPP involved in the islet beta cell damage.Methods1. SD male rats weighing 250±20g were purchased from Experimental Animal Center, Lanzho University (lisence number:SCXK(gan) 2009-0004). Half of animals from were single injected with STZ (30mg/kg, i.p), and the plasma glucose were measured at 7 days after STZ injection. The rats whose plasma glucose increased by 50% were used in the following experiment. Hyperglycemic rats were randomly divided into two groups, one group was given regular diet (STZ); another group was given a high-fat diet (STZ+HD). Half of animals were injected with the equal volume of sterile citric acid buffer, and the plasma glucose was tested at 7days after injection, then the rats were randomly divided into two groups, one group was given regular diet (Normal); another group was given a high-fat diet (HD). Four-group animals were raised for 2 months, glucose tolerance and insulin sensitivity were evaluated. Animals were sacrificed, blood plasma was harvest from heart, and the plasam isolated by centrifugation. Plasma glucose and triglycerides were measured by enzymatic methods. Parease were desected and embedded with paraffin. The sections (5μm thick) were stained with HE to observe the islet morphology. The insulin receptor (IR), Amylin and P53 expression were analysised by immunohistochemistry combined with semi-quantitative method in the pacreatic islet, respectively.2. The rat insulin secreting beta cell derived line (INS-1) were cultured with RPMI 1640 containing 2 mmol glutamine and 10% FBS, and treated by different concentration of palmitic acid sodium (PA). Cell viability was valuated by MTT assay. The effect of PA-treatment on insulin release in INS-1 cells were detected by ELISA method. The insulin and amylin mRNA in INS-1 cell were measured by Q-RT-PCR after treatment with PA. The expression of IR and p53 in INS-1 cells were detected by Western blottig after treatment with PA.3. The synthesised hIAPP were dissolved with Hexafluoroisopropanol (HFIP) for final protein concentration of 250μmol/L, then evaporated under nitrogen condition at 22℃ for 5-24 hours to form the oligomer hIAPP. Oligmoer hIAPP was dissolved in dd-water to a final concentration of 500μmol/L. Lactate dehydrogenase (LDH) in INS-1 cells were detected by specific kit after treatment with oligomer hIAPP. Insulin secretion was evaluated by ELISA and INS mRNA were measured by Q-RT-PCR in INS-1 cells after oligomer hIAPP treatment. IR and p53 expression in INS-1 cell were tested by western blotting after treatment with oligmer hIAPP.Results1. Animal experimental results showed plasma glucose and triglyceride are higher in STZ-, HD-and STZ+HD groups than these in normal group. Among them, the plasma glucose is highest in STZ-group, and the plasma triglyceride is highest in HD-group, consistently, the insulin sensitivity and glucose tolerance significantly decreased in HD-group. The expression of IR in pacreatic islet decreased in STZ-, HD- and STZ+HD groups compared with normal group. The amylin expression in pareatic islet increased in HD- and STZ+HD groups compared with the normal group. The expression of p53 in pacreatic islet also increased in STZ-, HD- and STZ+HD groups compared with the normal group.2. In vivo, the results showed that INS-1 cell viability decreased after PA treatment with dose dependent manner. The insulin and Amylin mRNA increased after PA treatment. Additionally, the IR expression decreased and the p53 expression increased after PA treatment.3. In vivo, the results showed that the LDH levels increased after treatment with 20μmol/L, 30μmol/L and 50μmol/L oligomer hIAPP in INS-1 cell. Consistantly, the p53 expression also increased in INS-1 cell after treatment with oligomer hIAPP.Conclusion1. High fat diet can lead to the insulin resistance acompanied with the dysfunction of insulin signals, amylin secreation and βcell damage in SD rats. 2. Insulin and amylin higer expression were induced by PA dose-dependently. But the insulin signals were decreased by treatment with PA in INS-1 cells.3. The cell membrane were damaged after treatment with oligomer hIAPP in INS-1 cells.4. Amylin secreation and oligomerized partly contributed the pacreatic islet damaged under the hyperlipidemic conditions.