| ObjectiveGlucose is the primary factor for insulin secreted by pancreaticβ-cells.Short-term exposure ofβ-cells to high concentration of glucose triggers the synthesis and secretion of insulin. But,long-term effection of high concentration of glucose would causeβ-cells dysfunction.In typeⅡdiabetes mellitus,the concentration of free fatty acid (FFA) and triglycerine(TG) is generally high, especially in obesity.Long-term exposure ofβ-cells to high concentration of FFA will induceβ-cell ' s apoptosis.Many studies have shown that glucose and FFA effect synthesis and secretion of insulin stimulated by the insulin secreted byβ-cells trigged by themselves.AS similar of surrounding target tissues of insulin,there are insulin recrptors and the series of proteins of insulin sinaling cascade inβ-cells,of which the phosphatidylinositol-3kinase (PI-3K)pathway is mainly involved in insulin gene transcription and secretion.Insulin up-regulates itself's gene transcription by activation of transcription factor such as PKB/Akt,PDX-1 in PI3K pathway.Inhibition of PI3K activation deregulate activation of transactive factor of insulin gene which cause decreasing of insulin gene expression stimulated by insulin.We have a hypothsis that the mechanism of glutoxic and lipotocxic effection is related to decrease of PI3K activation and to deregulation of PDX-1 activation,which caused reducing of insulin synthesis,secretion and promoted apoptosis.Our study will observe the expression model ofβ-cells dysfunction by evalue ofβ-cells function after exposed to high concentration of FFA synergize with elevated glucose.Then we analyse dysfunction by morphology technology of apoptosis, insulin secretion detection and molecular biological technology to reveal molecular mechanism ofβ-cells dysfunction caused by high concentration of FFA synergize with elevated glucose.MethodWistar rat islets isolation and primary culture and establishment ofβ-cells dysfunctional model.Islets were isol;ated and purified according to modified Minnesota program.After intraductal infusion digestion,the islet were purifed by discontinous Ficoll density gradient (25%,23%,20.5%,11%).The degree of purity was defined as dithizone (DTZ) staining.Cells were cultured overnight and were divided into nine groups: A: 5.5mM Glucose; B: 5.5mM Glucose, 0.4mM PA; C: 5.5mM Glucose, 0.4mM OA; D: 11mM Glucose; E: 11mM Glucose, 0.4mM PA; F: 11mM Glucose,0.4mM OA; G: 25mM Glucose;H: 25mM Glucose,0.4mM PA;I: 25mM Glucose, 0.4mM OA.After 24h and 48h, to detect insulin level in the supernatant after sitimulated 1h by 16.7mM glucose with RIA method and to assay insulin mRNA level with semi-quantity RT-PCR(n=4)Protein levels of p85 subunit of PI3K,phosphorylation of PKB was determined in total lysates by western immunoblot. To determined mRNA levels of bax,myc and PDX-1 by RT-PCR at the end of 24h,48h cell culture. Western biot analysis was used to assay the protein expression levels of PDX-1 in different time.Indirect immunofluorescence and immunocytochemistry staining was processed to observe the translocation of PDX-1 in selets treated with 16.7mM glucose after cultured by high concentration of FFA synergize with elevated glucose.Result438±27 islets were procured from one rat.the degree of purity was 87.68±3.2%. Assessment of islets viability by Trypan Blue Dye Exclusion Test which results is 92.3±2.3%.Insulin secretin response to glucose challenge in vitro showed the mean value of insulin in the low-glucose medium was 39.74±2.73mM, while that of high-glucose medium was 128.94±8.72mM, the SI was 3.25±0.26, that means the islets functioned well.The releasing insulin levels of the nine groups responding to 16.7mM glucose at the end of 24h and 48h: At the end of the first 24h, the controls (A, D, G)secretion were similar in two phases, while the G group has increasing tendency;As for oleate groups, the base secretion have no difference,while the stimulated secretion was descending;As palmic acid as concerned,both base and sitimulated secretion was descending(p＜0.01) accompany with the elevated glucose. At the end of 48h, there was no difference in the controls (A, D, G)secretion;As for oleate groups, both base and sitimulated secretion was descending(p＜0.05) accompany with the elevated glucose;As palmic acid as concerned,the descending tendency was sharply (p＜0.01).In the same concentration of glucose, the oleate group has no statistical sense (p＞0.05) in the end of 24h; while the palmic acid group shew decreasing tendency (p＜0.05).At the end of 48h, both the oleate group and the palmic acid groups were decreased.Semi-quantitative RT-PCR revealed the insulin mRNA levels of the control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew no significant difference during the first 24h (p＞0.05), while there was statistical sense between C,F groups at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).In situ end labelling technique (TUUNEL) for apoptosis: There was no statistical sense in the control groups (p＞0.05),apoptosis cell population was increasing in oleate groups in 24h,and had distinction between C,H and I (p＜0.05)after 48h; while obviously apoptosis happened in the palmic acid groups (p＜0.01)in 24h,and even more after 48h(p＜0.01).The mRNA expression of Apoptosis related gene bax,myc in control groups and experiment groups: the control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew no significant difference during the first 24h (p＞0.05), while there was statistical sense between C,F groups at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).the expression of protein PI3K,phosphorylated-PKB,PDX-1: The control groups were significant difference neither in the first 24h nor in the end of 48h; The oleate groups shew decreasing tendency with the eleveated glucose and time extending, difference during the first 24h (p＞0.05), while there was significant statistical sense between C group and I group at the end of 48h (p＜0.05);the palmic acid groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).The mRNA expression of PDX-1 in different groups: There was no significant difference in control groups neither in the first 24h nor in the end of 48h; The oleate groups shew no statistics difference during the first 24h (p＞0.05), while there was statistical sense between C and I groups at the end of 48h (p＜0.05);While B,E,H groups were decreased early in 24h (p＜0.05),and decreased sharply after 48h (p＜0.01).Nuclear translocation of protein PDX-1 in different groups: Indirect immunofluorescence showed PDX-1 was mainly located in cytosolic slightly in the basical station, while translocated to nucleoplasmic greatly followed by stimulated with 16.7mM glucose. With the elevated glucose and time extending,the expression was lower and the translocating rates was decreased,which was Hgroup＜Egroup＜Bgroup,(p＜0.01 or 0.05).ConclusionShort-term exposure ofβ-cells to high concentration of glucose triggers the synthesis and secretion of insulin.But co-cultivated with FFA will damageβ-cells function, which shew that the synthesis and secretion ability of insulin descended both in base phase and stimulated phase. High concentration of FFA induced insulin mRNA level decreased, which lower accompany with the elevated glucose and time prolongation.High concentration of FFA induced insulin synthesis dysfunction before secretion impairment. In the same concentration level, impairment caused by saturated fatty acid exceeded those caused by unsaturated fatty acid, which increased accompany with the elevated glucose and time prolongation.High concentration of FFA inhibited the expression of phosphorylated-PKB protein and PDX-1protein by inhibiting the expression of PI3K signal transducer, which strengthened accompany with the elevated glucose and time prolongation. In the same concentration level, impairment caused by saturated fatty acid exceeded those caused by unsaturated fatty acid, which indicated that "lipotoxic"caused by saturated fatty acid was more harmful.High concentration of FFA up-regulated expression of apoptosis gene,which would promoteβ-cells apoptosis by inhibiting the expression of PI3K signal transducer, which strengthened accompany with the elevated glucose and time prolongation. |
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