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The Study on the Regulation Pathway in Which CR Effects the Insulin Secretion of NIT-1 Cell

Author: YangJieHua
Tutor: ChenShenRen
School: Shantou University
Course: Endocrinology and Metabolism
Keywords: Calorie Restriction (CR) SIRT1 INS/IGF-1 signal pathway T2DM
CLC: R587.1
Type: Master's thesis
Year: 2008
Downloads: 99
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Abstract

As early as in 1930s, some researchers had studied the effect on rodents which were restricted food and found that it could extend their life span. Since main component of food which was restricted was calorie, researchers called it as calorie restriction(CR). In 2000, Lin SJ et al found that it could extend saccharomycetic life span for 30%-40%, when the glycose in medium was reduced from 2% to 0.5% or more. Weiss EP et al found that CR could also increase insulin sensitivity of tissue, decrease insulin resistance and then decrease insulin secretion of beta Cell of islet. It played an important role in prevention and therapy of T2DM.Clinic epidemiologic survey showed that when ages were increased, sugar tolerance was decreased with morbidity rate of T2DM and impaired glucose tolerance increasing. Animal experiment also showed that in mouse model which high fat diet induced,βcell senescence participated in morbidity of T2DM which diet induced. Therefore,βcell senescence participated in occurrence and development. But, the mechanism still remained unclear.Silent information regulator 2(SIR2) was an important gene which regulated aging proceeding of biosystem. It was found first in yeast cell, and participated in mating type gene silencing, telomere gene silencing, and rDNA recombination, to maintain genic stability. It had important relationship with substance metabolism and longevity and was deacetylase depending on NAD+. It could deacetylated many substrates to mediate kinds of biological effects.In addition, homologous genes of SIR2 in mammalian, SIRT1, could up-regulated insulin secretion ofβcell. Studies found that SIR2 had high correlation with CR. In many organisms (such as yeast, nematode, Drosophila melanogaster, mammalian), CR activated SIR2 and homologous genes, to increase their silencing activity, mRNA level, to increase the expression in cell and then to extend aging.Insulin/insulin-like growth factor pathway (INS/IGF-1pathway, INS/IGF-1-PI3K- AKT-FOXO) was an important regulation pathway which participated in ageing and metabolism. FOXO family was downstream of INS/IGF-1 pathway. Inβcell, FOXO1 was the effector of INS/IGF-1 pathway. The inactivation of FOXO1 could lead to up-regulation of Pdx1 expression and proliferation ofβcell and then impact its function. SIRT1could regulate downstream effect which FOXO mediated by deacetylating it,such as enhancing reaction of oxidative stress and heat stress ,leading to cell cycle arrest and enhancing DNA repair.Previous experiments in our laboratory topic have show thatβcell of rat cell senescence increased insulin secretion reduced with aging. The expression of SIRT1 inβcell of CR was more than which in control group. Inβ-gal stain which reflected ageing, the expression of CR group obviously less than which of control group. It demonstrated that CR induced expression of SIRT1 inβcell successfully. CR enhanced INS/IGF-1 pathway and deacetylase activity of SIRT1, inhibited activity of FOXO1 to degrade the expression of downstream aging- associated protein and apoptosis-associated protein of FOXO1 with elevating insulin sensitivity, reducing apoptosis ofβcell because of excessing secreting insulin, delaying aging ofβcell at last. Therefore , we supposed that inβcell of mammalian, CR could regulate PI3K, AKT, FOXO of INS/IGF-1 pathway by SIRT1, impacte the multiplication ofβcell and insulin secretion; CR could participate in insulin resistance and generation and development of T2DM. Our experiment showed that CR could induce SIRT1 hyperexpression of NIT-1 cells, and delay doubling generation time of NIT-1 cells. We detected the dependability between SIRT1 and the major factor of INS/IGF-1 pathway by image handling software. We explored the regulation pathway in which CR effected insulin secretion of NIT-1 cells to provid new experiment evidence and rationale for pathogenesy of T2DM.Content and MethodsNIT-1 cells were cultured to exponential growth phase by three different ways, one of which with high glucose medium, another of which with low glucose medium, the third of which with low glucose medium and nicotinamide (NIC), the suppressor of SIRT1. The expressions of PI3K, AKT, FOXO1 were detected by RT-PCR and Immunocytochemical assay. The secretion and expression of insulin were detected by radioimmunity and Immunocytochemical assay. The cell multiplication cycle was measured by cytometry.Result1. CR could extend the cell multiplication cycle and decrease insulin secretion in theβneoplastic cell NIT-1;2. CR could increase the expression and transcription of SIRT1 with affecting INS/IGF-1 signal pathway ;3. Nicotinamide could reverse the effects of CR above.Conclusion1. CR would effect the INS/IGF-1 signal pathway by inducing the hyperexpression of SIRT12. CR could extend the cell multiplication cycle to improve the function of insulin secretion

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