二甲双胍对大鼠胰岛素瘤细胞内质网应激影响的研究
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摘要
目的探讨二甲双胍对大鼠胰岛素瘤(INS-1)细胞内质网应激(ERS)的保护作用及其作用机制。方法体外培养INS-1细胞,分为二甲亚砜对照组(DMSO)、二甲双胍组(MET)、毒胡萝卜素组(TG)和毒胡萝卜素+二甲双胍(TG+MET)组。qRT-PCR检测C/EBP同源蛋白(CHOP)mRNA水平。Western blot检测CHOP的蛋白水平,c-Jun氨基端激酶(JNK)活性测定试剂盒检测JNK的磷酸化水平。放射免疫法测定INS-1细胞的胰岛素分泌水平。结果与DMSO组比较,TG组CHOP mRNA(1. 00±0. 17 vs 2. 67±0. 55,P<0. 01)、蛋白和JNK磷酸化水平升高(1. 00±0. 21 vs2. 29±0. 26,P<0. 01),INS-1细胞的胰岛素分泌降低[(8. 33±1. 67)vs(3. 82±1. 09)μmol/L,P<0. 05]。与TG组比较,TG+MET组CHOP mRNA、蛋白和JNK磷酸化降低[(2. 67±0. 55)vs(1. 63±0. 31),(2. 29±0. 26)vs(1. 53±0. 19),P<0. 05],INS-1细胞的胰岛素分泌升高[(3. 82±1. 09)vs(5. 96±0. 87)μmol/L,P<0. 05]。结论 INS-1细胞中二甲双胍可抑制TG诱导的ERS,改善胰岛素分泌水平,可能通过抑制JNK激活实现。
Objective To detect the protective effect of metformin(Met)on endoplasmic reticulum stress in INS-1 cells and to explore the underlying mechanism.MethodsINS-1 cells were cultured in vitro and divided into DMSO control(DMSO)group,metformin(MET)group,thapsigargin(TG)group and thapsigargin + metformin(TG + MET)group. The mRNA or protein levels of C/EBP homologous protein(CHOP)were detected by qRT-PCR or Western blot. The phosphorylation levels of c-Jun N-terminal kinase(JNK)was detected by kits. The insulin secreted by INS-1 was detected by radioimmunoassay.ResultsTG significantly increased the mRNA(2. 67±0. 55 vs 1. 00±0. 17,P<0. 01)and protein levels of CHOP,promoted the phosphorylation of JNK(2. 29±0. 26 vs 1. 00±0. 21,P<0. 01)and reduced the insulin secretion in INS-1 cells[(3. 82 ± 1. 09)vs(8. 33 ± 1. 67)μmol/L,P<0. 05]. Compared with TG group,metformin reduced the mRNA[(1. 63±0. 31)vs(2. 67±0. 55),P<0. 05]and protein levels of CHOP,inhibited JNK phosphorylation[(1. 53±0. 19)vs(2. 29±0. 26),P<0. 05]and increased the insulin secretion in INS-1 cells[(5. 96±0. 87)vs(3. 82±1. 09)μmol/L,P<0. 05].ConclusionMetformin can inhibit the endoplasmic reticulum stress induced by TG and improve the secretion of insulin in INS-1 cells possibly by inhibiting the activation of JNK.
Objective To detect the protective effect of metformin(Met)on endoplasmic reticulum stress in INS-1 cells and to explore the underlying mechanism.MethodsINS-1 cells were cultured in vitro and divided into DMSO control(DMSO)group,metformin(MET)group,thapsigargin(TG)group and thapsigargin + metformin(TG + MET)group. The mRNA or protein levels of C/EBP homologous protein(CHOP)were detected by qRT-PCR or Western blot. The phosphorylation levels of c-Jun N-terminal kinase(JNK)was detected by kits. The insulin secreted by INS-1 was detected by radioimmunoassay.ResultsTG significantly increased the mRNA(2. 67±0. 55 vs 1. 00±0. 17,P<0. 01)and protein levels of CHOP,promoted the phosphorylation of JNK(2. 29±0. 26 vs 1. 00±0. 21,P<0. 01)and reduced the insulin secretion in INS-1 cells[(3. 82 ± 1. 09)vs(8. 33 ± 1. 67)μmol/L,P<0. 05]. Compared with TG group,metformin reduced the mRNA[(1. 63±0. 31)vs(2. 67±0. 55),P<0. 05]and protein levels of CHOP,inhibited JNK phosphorylation[(1. 53±0. 19)vs(2. 29±0. 26),P<0. 05]and increased the insulin secretion in INS-1 cells[(5. 96±0. 87)vs(3. 82±1. 09)μmol/L,P<0. 05].ConclusionMetformin can inhibit the endoplasmic reticulum stress induced by TG and improve the secretion of insulin in INS-1 cells possibly by inhibiting the activation of JNK.
引文
[1]李延兵.2型糖尿病病程进展与胰岛素强化治疗.中国糖尿病杂志,2016,24:286-288.
[2]Biden TJ,Boslem E,Chu KY,et al.Lipotoxic endoplasmic reticulum stress,βcell failure,and type 2 diabetes mellitus.Trends Endocrinol Metab,2015,25:389-398.
[3]Gardner BM,Pincus D,Gotthardt K,et al.Endoplasmic reticulum stress sensing in the unfolded protein response.Cold Spring Harb Perspect Biol,2013,5:a013169.
[4]Marciniak SJ,Ron D.Endoplasmic reticulum stress signaling in disease.Physiol Rev,2006,86:1133-1149.
[5]Guan Z,Wang F,Cui X,et al.Mechanisms of sphingosine-1-phosphate-mediated vasoconstriction of rat afferent arterioles.Acta Physiologica,2017,222:e12913.
[6]SalvadóL,Palomer X,Barroso E,et al.Targeting endoplasmic reticulum stress in insulin resistance.Trends Endocrinol Metab,2015,26:438-448.
[7]Aguayo RLB,Brito GM.Metformin:an old but still the best treatment for type 2 diabetes.Diabetol Metab Syndr,2013,5:6.
[8]Haberzettl P,Hill BG.Oxidized lipids activate autophagy in a JNK-dependent manner by stimulating the endoplasmic reticulum stress response.Redox Biol,2013,1:56-64.
[9]Oyadomari S,Mori M.Roles of CHOP/GADD153 in endoplasmic reticulum stress.Cell Death Differ,2004,11:381-389.
[10]Sano R,Reed JC.ER stress-induced cell death mechanisms.Biochim Biophys Acta,2013,1833:3460-3470.
[11]Hetz C,Chevet E,Harding HP.Targeting the unfolded protein responseindisease.NatRevDrugDiscov,2013,12:703-719.
[12]Senft D,Ronai ZA.UPR,autophagy,and mitochondria crosstalk underlies the ER stress response.Trends Biochem Sci,2015,40:141-148.
[13]Rozpedek W,Pytel D,Mucha B,et al.The role of the PERK/eIF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress.Curr Mol Med,2016,16:533-544.
[14]Mihailidou C,Papavassiliou AG,Kiaris H.A crosstalk between p21 and UPR-induced transcription factor C/EBP homologous protein(CHOP)linked to type 2 diabetes.Biochimie,2014,99:19-27.
[15]Sánchez AM,Martínez-Botas J,Malagarie-Cazenave S,et al.Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells:a microarray study.Biochem Biophys Res Commun,2008,372:785-791.
[16]Zhou J,Xu G,Bai Z,et al.Selenite exacerbates hepatic insulin resistance in mouse model of type 2 diabetes through oxidative stress-mediated JNK pathway.Toxicol Appl Pharmacol,2015,289:409-418.
[17]Hirosumi J,Tuncman G,Chang L,et al.A central role for JNKin obesity and insulin resistance.Nature,2002,420:333-336.
[18]Bachar E,Ariav Y,Cerasi E,et al.Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis.Diabetologia,2010,53:2177-2187.
[2]Biden TJ,Boslem E,Chu KY,et al.Lipotoxic endoplasmic reticulum stress,βcell failure,and type 2 diabetes mellitus.Trends Endocrinol Metab,2015,25:389-398.
[3]Gardner BM,Pincus D,Gotthardt K,et al.Endoplasmic reticulum stress sensing in the unfolded protein response.Cold Spring Harb Perspect Biol,2013,5:a013169.
[4]Marciniak SJ,Ron D.Endoplasmic reticulum stress signaling in disease.Physiol Rev,2006,86:1133-1149.
[5]Guan Z,Wang F,Cui X,et al.Mechanisms of sphingosine-1-phosphate-mediated vasoconstriction of rat afferent arterioles.Acta Physiologica,2017,222:e12913.
[6]SalvadóL,Palomer X,Barroso E,et al.Targeting endoplasmic reticulum stress in insulin resistance.Trends Endocrinol Metab,2015,26:438-448.
[7]Aguayo RLB,Brito GM.Metformin:an old but still the best treatment for type 2 diabetes.Diabetol Metab Syndr,2013,5:6.
[8]Haberzettl P,Hill BG.Oxidized lipids activate autophagy in a JNK-dependent manner by stimulating the endoplasmic reticulum stress response.Redox Biol,2013,1:56-64.
[9]Oyadomari S,Mori M.Roles of CHOP/GADD153 in endoplasmic reticulum stress.Cell Death Differ,2004,11:381-389.
[10]Sano R,Reed JC.ER stress-induced cell death mechanisms.Biochim Biophys Acta,2013,1833:3460-3470.
[11]Hetz C,Chevet E,Harding HP.Targeting the unfolded protein responseindisease.NatRevDrugDiscov,2013,12:703-719.
[12]Senft D,Ronai ZA.UPR,autophagy,and mitochondria crosstalk underlies the ER stress response.Trends Biochem Sci,2015,40:141-148.
[13]Rozpedek W,Pytel D,Mucha B,et al.The role of the PERK/eIF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress.Curr Mol Med,2016,16:533-544.
[14]Mihailidou C,Papavassiliou AG,Kiaris H.A crosstalk between p21 and UPR-induced transcription factor C/EBP homologous protein(CHOP)linked to type 2 diabetes.Biochimie,2014,99:19-27.
[15]Sánchez AM,Martínez-Botas J,Malagarie-Cazenave S,et al.Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells:a microarray study.Biochem Biophys Res Commun,2008,372:785-791.
[16]Zhou J,Xu G,Bai Z,et al.Selenite exacerbates hepatic insulin resistance in mouse model of type 2 diabetes through oxidative stress-mediated JNK pathway.Toxicol Appl Pharmacol,2015,289:409-418.
[17]Hirosumi J,Tuncman G,Chang L,et al.A central role for JNKin obesity and insulin resistance.Nature,2002,420:333-336.
[18]Bachar E,Ariav Y,Cerasi E,et al.Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis.Diabetologia,2010,53:2177-2187.