| ObjectiveGlucose is the primary factor for insulin secreted by pancreatic β - cells. Short - term exposure of β - cells to supraphysiologic concentration of glucose triggers the synthesis and secretion of insulin. But, long - term effection of high concentration of glucose would cause β - cells dysfuntion. It is shown that glucose enhance synthesis and secretion of insulin stimulated by the insulin secreted by β - cells trigged by glucose itself . As similar of surrounding target tissues of insulin , there are insulin receptors and the series of proteins of insulin sinaling cascade in p - cells, of which the phosphatidylinostiol - 3 kinase (PI - 3K) pathway is mainly involved in insulin gene transcription and secretion. Insulin regulates itself' s gene transcription by PI - 3K pathway . Inhibition of PI -3Kactivation deregulate activation of transactive factor of insulin gene such as IDX - 1 which cause decreasing of insulin gene expression stimulated by insulin., We have a hypothsis that the mmechanism of glutoxic effection is related to decrese of PI - 3 k activation and to deregulation of IDX - 1 activation, which caused reducing of insulin synthesis. Our study will observe the expression modle of β - cells dysfuntion by evalue of β - cells function after long - term of ex-porsed in high concentration of glucose. Then we analyses dysfunctional β - cells using molecular biological technology. We observe effection of long - term high concentration of glucose to PI -3K and IDX - 1 activation and expression to delight molecular mechanism of β- cells dysfunction caused by glucose.Method1. Wistar rat islets isolation and primary culture and conduction of 3 - cells dysfunctional model . Using the Shewade's modified method to isolate male Wistar rat( 150g -250g) pancreas and primary culture islets. Set three groups which is A group cultured in 5.5mM glucose,B group in 11. lmM glucose ;C group in 22. 2mM glucose . At the end of the first .second .third fouth week, give them a stimulation with 16. 7mM glucose or lOOnM insulin . After lOmin and lh( for glucose) or 5min ( for insulin) assay the insulin level in the supernatant and assay insulin mRNA level with semi - quantity RT - PCR (n = 4).2. Protein levels of p85 subunit of phosphatidylinositol 3 - kinase ( PI -3K) .was determined in total lysates by western immunoblot and determination the protein level of interaction of P85 with IRS - 1/ -2 and the protein of phos-phorylation of PKB were performed by immnoprecipitation, following with western immunoblot.3. To determined mRNA levels of IDX - 1 by RT - PCR at the end of the third and the fouth week.4. Indirct immunofluorescence and immunocytochemistry staining was processed to observe the translocation of IDX - 1 in islets treated with lOOmM insulin during a period of four weeks5. Western blot analysis were employed the protein expression levels of IDX - 1 at the end of the fouth week.Result1. Islets were observed on binocular dissecting microscope ( x20) with bac-ground green illumination with white side lighting. Assay islets yield by counting islets in three aliquota of 10% of the islets pellet and multiplying the mean number of islets in these aliquot by 10 ( n = 10) .which result is 1299. 22 ±116. 37 islets/ pancrease . Assessment of islets viability by Trypan Blue Dye Exclusion Test which result is 94. 41 ± 12. 2% .and specificity of islets was determind bydithiozone (DTZ) staining. The islets responded well in vitro to release insulin with high glucose stimulation . The basal and glucose - stimulated insulin release was 46 7. 33uU/10 islets ;291 ±24uU/10 islets the later of which is about 5 times of the former . ( n = 6; p < 0. 05).2. The releasing insulin levels of the three groups responding to 16. 7mM glucose at the end of first , second , third , fouth week were changed with prelong of culture.At the end of the first week : The Insulin levels of three groups were no significant difference . At the end of the second week they were as similar . As to the end of the third week ,the C groups first phasic(10 minute ) secretion levels was lower than that of B or C group( p <0. 05). The second phasic (lh ) secretion levels were no significant change . At the end of the fouth week there was a decreasing of the first phasic secretion in the three groups with the graded increasing of glucose concentration , that is C < B < A. While the second phasic secretion levels of C group was lower than that of A and B group . During the A B C group ,the four week'levers of insulin were no significant in A group . The first phasic secretion of B group at the end of the fouth week was lower than those of the former three week in the B group . In the C group the first phasic secretion levels was decreasing in dependent of the prolong of exposure to glucose . As to the second phasic secretion levels was decreased only at the end of the fourth week.3. Just the levels of C group responding to 5 minutes stimulation of insulin at the end of the fourth week was decreased ( p <0. 05).4. Semi - quantitative RT - PCRrevealed the insulin mRNA levels of the three group were no significant difference during the first two weeks . At the end of third week insulin mRNA levels of C group was lower than that of A group (p<0.001) and decreased compared with the C group at the end of first weeks. To the fourth week the levels of mRNA of the three groups were decreasing dependent of increasing of glucose concentration . and downregulated with increasing of culture time( p <0.01).5. Western blot analysis showed at the end of fouth week ,the level of p85 protein expreesion of C group was slightly but significantly lower than those of Aor B group (p <0.01).6. Western blot analysis showed at the end of fouth week the levels of p85 bonding to IRS - lin the three groups was downregulated by increasing of glucose concentration cultured . , while the level of p85 bonding to IRS - 2 was slightly decreased just in C group ( p <0. 01).7. Western blot analysis showed the phosphated - PKB ( p - PKB) levels at the end of fouth week was slightly decreased only in C group ( p <0.01)8. Western blot analysis showed the expression level of IDX - 1 protein was slightly decreased in the C group following four week of culture in 22. 2mM glucose . ( n = 4, p < 0. 05, vs A group)9. High concentration of glucose downregulated the expression levels of IDX- 1 mRNA after three weeks . At the end of third week , the level of C group was lower than that of A or B group (p<0.01,n=4).To the fouth week the level of B group was also decreased (p <0.01 ,vs A group ) , while that of the C group was decreased even more ( p <0. 01 ,p <0. 01 ,vas A group and B group n =4).10. Immunocytochemistry staining and indirect immunofluorescence showed IDX -1 was mainly located in cytosolic slightly in the basical station . and tans-located to nucleoplasmic greatly followed by stimulated with lOOnM insulin . At the first two cultural week the translocating rates in the three groups were as that of in normal station . To third week , this translocating rate of B or C group were decreased. (n=6,p <0.01,vs A group). To the fouth week the rate of the three groups were decreasing gratedly which was Cgroup < B group < A group , ( p < 0.01or0.05,n=6).Conclusion1 Long - term exposure of (3 - cells to high concentration of glucose cause(3- cells dysfunction that induced lower secretion and synthesis ability of (3 -cells.2 The first and second phasic secretion of insulin of 3 - cells were both impaired by long - term exposure to high concentration of glucose. During which , the first phasic secretion was damaged at first and the second phasic secretion... |
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