摘要
第一部分:PI3K/Akt通路在高糖毒性诱导的TC1-6细胞和胰岛α细胞胰岛素抵抗中发挥的作用
目的:探讨慢性葡萄糖毒性对α细胞胰高糖素分泌的影响及α细胞胰岛素抵抗对其影响。
方法:胰岛细胞和TC1-6细胞(α细胞株)分别培养于含低浓度(5.5mM)、中浓度(11.1mM)和高浓度(25mM)葡萄糖的培养基中1,3,5d,分别检测胰高糖素(Glc)分泌及其mRNA表达;加入不同浓度胰岛素6h后,观察对培养5d的原代胰岛和TC1-6细胞Glc分泌和基因表达的影响并用Western Blot方法检测高糖对α细胞Akt磷酸化的影响。
结果:(1)培养1d时,低、中和高浓度葡萄糖培养的原代胰岛细胞和TC1-6细胞Glc分泌没有显著差别,培养3d时,高糖培养的胰岛细胞胰高糖素分泌明显增加42%[(2252.64±41.71)pg/mg protein vs(3205.97±45.53)pg/mg protein,P<0.05)],而TC1-6细胞胰高糖素的分泌无明显变化,试验第5d,中浓度和高糖培养的原代胰岛细胞胰高糖素均较低糖培养明显升高,分别增加52%[(2452.22±67.89)pg/mg proteinvs(3735.44±54.89)pg/mg protein,P<0.05)和117%[(2452.22±67.89)pg/mg protein vs(5320.20±398.51)pg/mg protein,P<0.05)],而TC1-6细胞仅在高糖培养5d时胰高糖素分泌较低糖培养明显增加,为74%((78.62±4.72)ng/10~6 protein vs(136.8±10.94)ng/10~6 protein,P<0.05]。(2)与低糖培养1d相比,中、高浓度葡萄糖培养1d的原代胰岛细胞和TC1-6细胞Glc基因表达没有明显差别,但培养3d后,高糖培养的胰岛细胞胰高糖素基因表达较低糖培养升高125%(P<0.05),而TC1-6细胞胰高糖素基因表达此时没有明显变化;当培养5d时,25mM糖浓度培养的原代胰岛和TC1-6细胞Glc基因表达明显升高,比低糖培养分别增高272%和78%;(3)10~(-7)M胰岛素可以抑制低糖培养的原代胰岛和TC1-6细胞Glc的分泌,分别为60%[(2315.02±48.12)pg/mgprotein vs(915.99±32.54)pg/mg protein;P<0.05]和61%[(55.12±3.86)ng/10~6 proteinvs(21.59±1.30)ng/10~6 protein,P<0.05],但仅能轻微抑制高糖培养下原代胰岛和TC1-6细胞Glc的分泌;当胰岛素增至10~(-5)M时,高糖培养下原代胰岛和TC1-6细胞的Glc的分泌也明显受到抑制,分别为66%[(3872.34±29.66)pg/mg protein vs(1307.65±23.34)pg/mg protein;P<0.05]和61%[(118.61±10.68)ng/10~6 protein vs(46.55±3.72)ng/10~6 protein;(4)加入10(?)胰岛素2h后,高糖和低糖培养下原代胰岛和TC1-6细胞P-Akt水平分别升高137%和70%以及200%和180%,但高糖组明显低于低糖组,给予PI3K抑制剂-wortmannin,低糖培养的原代胰岛和TC1-6细胞P-Akt水平抑制率明显高于高糖组。±结论:高糖可以引发原代胰岛和TC1-6细胞胰高糖素的分泌升高,其可能的机制与原代胰岛和TC1-6细胞胰岛素介导的PI3K/Akt信号通路受阻而产生的原代胰岛和TC1-6细胞胰岛素抵抗有关。
第二部分:Syntaxin1A/SNAP-25蛋白在逆转α细胞高糖毒性中的作用
目的:探讨逆转高糖毒性对α细胞胰高糖素分泌和合成的影响以及可能的机制。
方法:TC1-6细胞(α细胞株)分别给予5.5mmol/L(低糖组)和25mmol/L(高糖组)葡萄糖的培养基培养10d,25mmol/L葡萄糖培养5d/5.5mmol/L葡萄糖培养5d(高糖.低糖组)以及5.5mmol/L葡萄糖培养5d/25mmol/L葡萄糖培养5d(低糖-高糖组),检测各组细胞胰高糖素分泌及其mRNA的表达;Western印迹检测持续高糖以及由高糖降至低糖后TC1-6细胞Snare蛋白(syntaxin1A和SNAP-25)的表达水平;结果:(1)与高糖组TC1-6细胞相比,高糖-低糖组TC1-6细胞Glc分泌下降29%[(2.68±0.21)ng/mg protein vs(3.74±2.99)ng/mg protein,P<0.05];(2)与高糖组TC1-6细胞相比,高糖-低糖组TC1-6细胞Glc mRNA表达下降52.6%±2.8%(P<0.05);(3)高糖组TC1-6细胞表达syntaxin1A和SNAP-25的水平较低糖组TC1-6细胞syntaxin1A和SNAP-25的表达分别升高36%和69%(P<0.05),而高糖-低糖组syntaxin1A和SNAP-25蛋白表达与高糖组相比明显下调,分别下降49%和56%(P<0.05)。
结论:解除高糖毒性α细胞胰高糖素分泌异常明显改善,促进胰高糖素分泌相关的syntaxin1A和SNAP-25蛋白表达相应降低。
PartⅠ
Involvement of the PI3K/Akt pathway in high glucose-induced insulin resistance in rat islets and TC1-6 cells
objective In order to clarify whether long-term exposure to high glucose affects a cell function and induces hyperglucagonemia via insulin resistance.
Method We intraperitoneal injected Sprague Dawley rats with dose of 100mg/kg streptozotocin and administrated the diabetic rats with NPH insulin to keep their glucose level from 5mmol/L-10mmol/L to establishβcell deleted rat model,then we isolated rat islet and cultured for 1d,3d and 5d at 37℃with RPMI 1640 medium containing 5.5mM,11.1mM and 25mM glucose,similarly,TC1-6 cells were exposed to DMEM media containing 5.5mM,11.1mM and 25mM glucose for 1d,3d and 5d.On the following experiment day,the secretion and gene expressions of glucagon were measured and we also detected the glucagon secretion when rat islets and TC1-6 cells cultured for 5 d stimulating with three different concentrations of insulin.Then we used Western Blot method to confirm the effect of high glucose on phosphorylated of Akt in rat islets and TC1-6 cells.
Result(1) There was no significant difference in glucagon secretion in response to the three glucose levels in the rat islets or in TC1-6 cells at the end of one day's high glucose exposure,At day 3,compared with the control group(5.5 mM),the glucagon secretion in rat islets exposed to 25 mM glucose was markedly increased by 42%(2252.64±41.71 vs 3205.97±45.53 pg/mg protein;P<0.05),but there was no difference in TC1-6 cells.After 5d exposure,compared with the control group(5.5mmol/l),the glucagon secretion in rat islets was increased by 52%in response to 11 mM glucose(2452.22±67.89 vs 3735.44±54.89 pg/mg protein;P<0.05) and by 117%in response to 25 mM glucose (2452.22±67.89 vs 5320.20±398.51 pg/mg protein;P<0.05).However,with respect to TC1-6 cells,compared with 5.5 mM glucose,glucagon secretion was increased by 74% at day 5 in response to 25 mM glucose(78.62±4.72 vs 136.8±10.94 ng/10~6 protein;P< 0.05).(2) Acute(1 day) exposure of TC1-6 cells and rat islets to high glucose had no effect on glucagon mRNA levels measured by RT-PCR relative to cells and islets incubated with 5.5 mM glucose(control group).However,at day 3,the glucagon mRNA levels of rat islets incubated with 25 mM glucose increased by 125%compared with the control group(P<0.05),whereas there was little change in glucagon mRNA levels in TC1-6 cells.Meanwhile,at day 5,the glucagon mRNA levels of rat islets and TC1-6 cells exposed to 25 mM glucose increased by 272%and 78%,respectively,relative to the control groups(both:P<0.05).(3) 10~(-7)mmol/L insulin can significantly inhibit glucagon secretion from rat islets and TC1-6 cells cultured with containing 5.5mM glucose by 60% (2315.02±48.12 vs 915.99±32.54 pg/mg protein;P<0.05) and 61%(55.12±3.86 vs 21.59±1.30ng/10~6 protein;P < 0.05),accordingly,but just can slightly inhibit glucagon secretion both in rat islets and TC1-6 cells containing 25mM glucose(P>0.05).when insulin concentration added to 10~(-5) mmol/L,glucagon secretion of rat islets and TC1-6 cells cultured with 25mM were both suppressed by 66%(3872.34±29.66 vs 1307.65±23.34 pg/mg protein;P<0.05)and 61%(118.61±10.68 vs 46.55 + 3.72 ng/10~6 protein;P<0.05);(4) when 10~(-5)mmol/L insulin were administrated for 2h,the phosphorylated ser473-Akt protein levels of rat islets and TC1-6 cells exposure to 5.5mM glucose increased by 200%and 180%accordingly(P<0.05),those of rat islets and TC1-6 cells exposure to 25mM glucose also increased by 137%and 70%in response to 5.5mM after 5d(P<0.05 ).While pretreatment with 10~(-5)mmol/L wortmannin, phosphorylated ser473-Akt protein expressions of rat islets and TC1-6 cells exposure to 5.5mM glucose and 25mM glucose were both down regulated(P<0.05),but the inhibition in low high group is more significant than high glucose group.
Conclusion Chronic glucose toxicity can cause hypersecretion of glucagon,which may be due toαcell insulin resistance induced by impaired activity of the a cell insulin signaling pathway mediated by glucose toxicity.
PartⅡThe role of Syntaxin1A/SNAP-25 proteins in reversing hyperglycemia-induced a cell toxicity
Objective To examine the effects of reversing hyperglycemia-induced a cell toxicity and explore the possible mechanisms.
Method TC1-6 cell were cultured 10 days in media containing 5.5mmol/L glucose(Low glucose groups,LG),10 days in media containing 25mmol/L glucose(High glucose groups,HG),5 days in 25mmol/L glucose plus 5 days in 5.5mmol/L glucose(HL groups), 5 days in 5.5mmol/L glucose plus 5 days in 25mmol/L glucose(LH groups),The secretion and gene expression of glucagon were measured,we also used Western Blot analysis to confirm the effects of high glucose and reversible glucose induced effects on the expression of Snare proteins(syntaxinl A and SNAP-25).
Result(1)compared with TC1-6 cells of HG group,HL group cells Glucagon secretion dropped by 29%(P<0.05);(2)compared with TC1-6 cells of HG group,HL group cells Glucagon mRNA was decreased by 52.6%±2.8%(P<0.05);(3)compared with TC1-6 cells of LG group,the expressions of syntaxin1A and SNAP-25 of HG group cells were increased by 36%and 69%,and HL group cells were decreased by 49%and 56% respectively(P<0.05);
Conclusionα-cells abnormal glucagon were ameliorated after removing glucose toxicity, also the expressions of syntaxinlA and SNAP-25 proteins which regulating secretion of glucagon were reduced accordingly.
引文
[1]Unger RH,Orci L.The essential role of glucagon in the pathogenesis of diabetes mellitus[J].Lancet.1975,1(7897):14-16.
[2]Unger RH.The Banting Memorial Lecture 1975.Diabetes and the alpha cell.Diabetes[J],1976 Feb;25(2):136-51.
[3]Buchanan KD,McCarroll AM.Abnormalities of glucagon metabolism in untreated diabetes mellitus[J].Lancet.1972 Dec 30;2(7792):1394-5.
[4]Marks V.Glucagon[J].Clin Endocrinol Metab.1972,1(3):829-45.
[5]Gerich JE,Langlois M,Noacco C,Karam JH,Forsham PH.Lack of glucagon response to hypoglycemia in diabetes:evidence for an intrinsic pancreatic alpha cell defect[J].Science.1973,182(108):171-3.
[6]Unger RH.Glucagon physiology and pathophysiology[J].N Engl J Med.1971,285(8):443-9.
[7]Gerich JE,Langlois M,Noacco C,Lorenzi M,Karam JH,Korsham PH.Comparison of the suppressive effects of elevated plasma glucose and free fatty acid levels on glucagon secretion in normal and insulin-dependent diabetic subjects.Evidence for selective alpha-cell insensitivity to glucose in diabetes mellitus[J].J Clin Invest.1976,58(2):320-5.
[8]Unger RH,Orci L.The role of glucagon in diabetes[J].Compr Ther.1982,8(1):53-59.
[9]Tsuchiyama N,Takamura T,Ando H,et al.Possible role of alpha-cell insulin resistance in exaggerated glucagon responses to arginine in type 2 diabetes[J].Diabetes Care.2007,30:2583-2587.
[10]杜瑞琴,李宏亮,杨文英,等.吡格列酮对胰岛α细胞胰岛素抵抗的影响[J].中华内分泌代谢杂志,2008,24:23-27.
[11]杜瑞琴,李宏亮,杨文英,等.胰岛α细胞胰岛素抵抗与炎症通路激活的关系及机制[J].中华内科杂志,2007,46:661-665.
[12]王昕,杨文英,萧建中,等.高脂饲养及罗格列酮干预对α细胞功能的影响[J].中华内科杂志,2005,44:601-605.
[13]Raskin P,Aydin I,Unger RH.The effects of acute and chronic insulin administration on plasma IRG levels in human diabetes[J].Metabolism.1976,25(11 Suppl 1):1531-3.
[14]Umpaichitra V,Bastian W,Taha D,Banerji MA,AvRuskin TW,Castells S.C-peptide and glucagon profiles in minority children with type 2 diabetes mellitus[J].J Clin Endocrinol Metab.2001,86(4):1605-9.
[15]Tsuchiyama N,Takamura T,Ando H,et al.Possible role of alpha-cell insulin resistance in exaggerated glucagon responses to arginine in type 2 diabetes[J].Diabetes Care.2007,30:2583-2587.
[16]Eizirik DL,Korbutt GS,Hellerstrom C.Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function[J].J Clin Invest,1992,90,1263.1268.
[17]Federici M,Hribal M,Perego L,Ranalli M,Caradonna Z,Perego C,Usellini L,Nano R, Bonini P,Bertuzzi F.High glucose causes apoptosis in cultured human pancreatic islets of langerhans:a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program[J].Diabetes,2001,50,1290-1301.
[18]Ling Z,Pipeleers DG.Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation[J].J Clin Invest.1996,98,2805-2812.
[19]Moran A,Zhang HJ,01son LK,Harmon JS,Poitout V,Robertson RP.Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line,HIT-T15[J].J Clin Invest,1997,99,534-539.
[20]Sharma A,Olson LK,Robertson R,Stein R.The reduction of insulin gene transcription in HIT-T15b cells chronically exposed to high glucose concentration is associated with the loss of RIPE3M and SFT-1 transcription factor expression[J].Mol.Endocrinol.1995,9,1127-1134.
[21]Poitout V,01son LK,Robertson RP.Chronic exposure of betaTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3M insulin gene transcription activator[J].J Clin Invest,1996,97,1041-1046.
[22]Dumonteil E,Ritz-Laser B,Magnan C,Grigorescu I,Ktorza A,Philippe J.Chronic exposure to high glucose concentrations increases proglucagon messenger ribonucleic acid levels and glucagon release from InR1G9 cells[J].Endocrinology.1999,140(10):4644-50.
[23]McGirr R,Ejbick CE,Carter DE,Andrews JD,Nie Y,Friedman TC,Dhanvantari S.Glucose dependence of the regulated secretory pathway in alphaTC1-6 cells[J].Endocrinology.2005,146(10):4514-23.
[24]Hribal ML,Perego L,Lovari S,Andreozzi F,Menghini R,Perego C,Finzi G,Usellini L,Placidi C,Capella C,Guzzi V,Lauro D,Bertuzzi F,Davalli A,Pozza G,Pontiroli A,Federici M,Lauro R,Brunetti A,Folli F,Sesti G.Chronic hyperglycemia impairs insulin secretion by affecting insulin receptor expression,splicing,and signaling in RIN beta cell line and human islets of Langerhans[J].FASEB J.2003Jul;17(10):1340-2.
[25]Li P,Koike T,Qin B,Kubota M,Kawata Y,Jia YJ,Oshida Y.A high-fructose diet impairs Akt and PKCzeta phosphorylation and GLUT4 translocation in rat skeletal muscle[J].Horm Metab Res.2008 Aug;40(8):528-32.
[26]Nakajima K,Yamauchi K,Shigematsu S,Ikeo S,Komatsu M,Aizawa T,Hashizume K.Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line,HepG2 cells[J].J Biol Chem,2000,Jul 7;275(27):20880-6.
[27]Aiaujo ER,Amaral ME,Souza CT,et al.Blockade of IRS-1 in isolated rat pancreatic islets improves glucose-induced insulin secretion[J].FEBS Lett,2002,531:437-432.
[28]Diao J,Asghar Z,Chan CB,et al.Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic alpha-cells[J].J Biol Chem.2005,280:33487-96.
[29]罗梅,李秀均,李军等.糖尿病大鼠胰岛α细胞胰岛素受体分布和含量的初步研究[J].中华糖尿病杂志,2005,3;196-198.
[30]Kaneko K,Shirotani T,Araki E,et al.Insulin inhibits glucagon secretion by the activation of PI3-Kinase in InR1G9 cells[J].Diabetes Res Clin Pract.1999,44:83-92.
[31]Hong J,Abudula R,Chen J,Jeppesen PB,Dyrskog SE,Xiao J,Colombo M,Hermansen K.The short-term effect of fatty acids on glucagon secretion is influenced by their chain length,spatial configuration,and degree of unsaturation:studies in vitro[J].Metabolism.2005 Oct;54(10):1329-36.
[32]Hong J,Chen L,Jeppesen PB,Nordentoft I,Hermansen K.Stevioside counteracts the alpha-cell hypersecretion caused by long-term palmitate exposure[J].Am J Physiol Endocrinol Metab.2006 Mar;290(3):E416-22.
[33]DU RQ,Li HL,Yang WY,Xiao JZ,Wang B,Lou DJ,Bai XP,Pan L.The relationship between islet alpha cell insulin resistance and inflammatory pathway activation and its mechanism[J].Zhonghua Nei Ke Za Zhi.2007 Aug;46(8):661-5.
[34]Hong J,Jeppesen PB,Hermansen K.Effects of elevated fatty acid and glucose concentrations on pancreatic islet function in vitro[J].Diabetes Obes Metab.2009Apr;11(4):397-404.
[35]陈晓波,秦杰,董维平,等.大鼠胰岛分离条件的优化[J].现代生物医学进展.2007,7(2):166-168.
[36]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods,2001Dec;25(4):402-8.
[37]邬云红,李秀钧,李宏亮,等.高脂饮食肥胖大鼠胰岛细胞胰岛素抵抗机理的探讨[J].中华医学杂志,2005,20;85(27):1907-10.
[38]Luo M,Li XJ,Li J,et al.Study on iusulin receptor content and distribution of islet a cell in diabetic rats;preliminary insight into mechanism of pancreatic a cell insulin resitance[J].Diabetes,2004,53(Suppl):A313.
[39]杜瑞琴,李宏亮,杨文英,等.胰岛素信号转导分子的表达与胰岛α细胞的胰岛素抵抗的实验研究[J].中华医学杂志,2006,36:2542-2546.
[40]Robertson RP,Harmon JS.Diabetes,glucose toxicity,and oxidative stress:A case of double jeopardy for the pancreatic islet β cell[J].Free Radical Biology &Medicine,2006,41:177-184.
[41]Salehi A,Vieira E,Gylfe E,Paradoxical stimulation of glucagon secretion by high glucose concentrations[J].Diabetes.2006,55:2318-23.
[42]Vieira E,Salehi A,Gylfe E,et al.Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells[J].Dibetologia,2007,50:370-379.
[43]Hamaguchi K,Leiter EH.Comparison of cytokine effects on mouse pancreatic α-cell and β-cell lines[J].Viability,secretory function,and MHC antigen expression.Diabetes,1990,39:415-425.
[44]Ravier MA,Rutter GA.Glucose or insulin,but not zinc ions,inhibit glucagon secretion from mouse pancreatic α-cells[J].Diabetes,2005,54:1789-1797.
[45]Ostenson CG.Regulation of glucagon release:effects of insulin on the pancreatic A2-cell of the guinea pig[J].Diabetologia.1979;17:325-330.
[46]Starke A,Imamura T,Unger RH.Relationship of glucagon suppression by insulin and somatostatin to the ambient glucose concentration[J].J Clin Invest 1987;79:20-24.
[47]Buchanan KD,Mawhinney WA.Insulin control of glucagon release from insulin-deficient rat islets[J].Diabetes.1973;22:801-803.
[48]Raskin P,Aydin I,Unger RH.The effects of acute and chronic insulin administration on plasma IRG levels in human diabetes[J].Metabolism.1976;25:1531-1533.
[49]Franklin I,Gromada J,Gjinovci A,Theander S,Wollheim CB.p-cell secretory products activate a-cell ATP-dependent potassium channels to inhibit glucagon release[J].Diabetes,2005,54:1808-1815.
[50]Hermansen K.Characterisation of the abnormal pancreatic D and A cell function in streptozotocin diabetic dogs:studies with D-glyceraldehyde,dihydroxyacetone, D-mannoheptulose,D-glucose,and L-arginine[J].Diabetologia 1981;21:489-494.
[51]Hermansen K,Schmitz O,Orskov H.Reversal of D-and A-cell insensitivity to glucose in alloxan-diabetic dogs by treatment with the artificial beta cell (Biostator) [J].Diabetes.1985;34:260-266.
[52]Reach G,Assan R.Glucagon and diabetes mellitus.Diabetes Metab 1979;5:63-70.
[53]Unger RH,Orci L.The role of glucagon in diabetes[J].Compr Ther 1982;8:53-59.
[54]Henkel E,Menschikowski M,Koehler C,Leonhardt W,Hanefeld M.Impact of glucagon response on postprandial hyperglycemia in men with impaired glucose tolerance and type 2 diabetes mellitus[J].Metabolism.2005 Sep;54(9):1168-73.
[55]Burant CF,Treutelaar MK,Buse MG.Diabetes-induced functional and structural changes in insulin receptors from rat skeletal muscle[J].J Clin Invest.1986,Jan;77(1):260-70.
[56]Ordonez P,Moreno M,Alonso A,Fernandez R,Diaz F,Gonzalez C,Insulin sensitivity in streptozotocin-induced diabetic rats treated with different doses of 17beta-oestradiol or progesterone[J].Exp Physiol.2007,Jan;92(1):241-9.
[57]Kisanuki K,Kishikawa H,Araki E,et al.Expression of insulin receptor on clonal pancreatic a cells and its possible role for insulin-stimulated negative regulation of glucagon secretion[J].Diabetologia.1995,38:422-429.
[1]Unger RH,Orci L.The essential role of glucagon in the pathogenesis of diabetes mellitus[J].Lancet.1975,1(7897):14-16.
[2]Korsgren O,Jansson L,Sandler S,et al.Hyperglycemia-induced B cell toxicity.The fate of pancreatic islets transplanted into diabetic mice is dependent on their genetic background[J].J Clin Invest,1990,86:2161-2168.
[3]Ogawa Y,Noma Y,Davalli AM,et al.Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted beta-cells[J].Diabetes,1995,44:75-79.
[4]Giaccari A,DeFronzo RA.Glucose toxicity[J].Diabetes Care.1990,Jun;13(6):610-30.
[5]Eizirik DL,Korbutt GS,Hellerstrom C.Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function[J].J Clin Invest,1992,90,1263.1268.
[6]Federici M,Hribal M,Perego L,Ranalli M,Caradonna Z,Perego C,Usellini L,Nano R, Bonini P,Bertuzzi F.High glucose causes apoptosis in cultured human pancreatic islets of langerhans:a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.Diabetes[J],2001,50,1290-1301.
[7]Ling Z,Pipeleers DG Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation[J].J Clin Invest.1996,98,2805-2812.
[8]Moran A,Zhang HJ,01son LK,Harmon JS,Poitout V,Robertson RP.Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line,HIT-T15[J].J Clin Invest,1997,99,534-539.
[9]Sharma A,Olson LK,Robertson R,Stein R.The reduction of insulin gene transcription in HIT-T15b cells chronically exposed to high glucose concentration is associated with the loss of RIPE3bl and SFT-1 transcription factor expression[J].Mol.Endocrinol.1995,9,1127-1134.
[10]Poitout V,Olson LK,Robertson RP.Chronic exposure of betaTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3b1 insulin gene transcription activator[J].J Clin Invest,1996,97,1041-1046.
[11]The Diabetes Control and Complications Trial Research Group.The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus[J].N Engl J Med,1993,Sep 30;329(14):977-86.
[12]The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group.Sustained effect of intensive treatment of type 1 diabetes mellitus on development and progression of diabetic nephropathy:the Epidemiology of Diabetes Interventions and Complications (EDIC)study[J].JAMA.2003,22;290(16):2159-67.
[13]Nathan DM,Cleary PA,Backlund JY,Genuth SM,Lachin JM,Orchard TJ,Raskin P,Zinman B.Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study Research Group.Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes[J].N Engl J Med.2005,22;353(25):2643-53.
[14]Ryan EA,Imes S,Wallace C.Short-term intensive insulin therapy in newly diagnosed type 2 diabetes[J].Diabetes Care,2004,27:1028-1032.
[15]Wheeler MB,Sheu L,Ghai M,Bouquillon A,Grondin G,Weller U,Beaudoin AR,Bennett MK,Trimble WS,Gaisano HY:Characterization of SNARE protein expression in P-cell lines and pancreatic islets[J].Endocrinology,1996,137:1340-1348.
[16]Gaisano HY,Ostenson CG,Sheu L,Wheeler WB,Efendic S:Abnormal expression of pancreatic islet exocytotic SNARE proteins in GK rats is partially restored by phlorizin and accentuated by high glucose treatment[J].Endocrinology,2002,143:4218-4226.
[17]Chan CB,MacPhail RM,Sheu L,Wheeler MB,Gaisano HY:p-Cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression[J].Diabetes,1999,48:997-1005.
[18]Brunzell JD,Robertson RP,Lerner RL,Hazzard WR,Ensinck JW,Bierman EL,Porte DJ.Relationships between fasting plasma glucose levels and insulin secretion during intravenous glucose tolerance tests[J].J Clin Endocrinol Metab.1976,42:222-229.
[19]Weyer C,Tataranni PA,Bogardus C,Pratley RE:Insulin resistance and insulin secretory dysfunction are independent predictors of worsening of glucose tolerance during each stage of type 2 diabetes development[J].Diabetes Care,2001,24:89-94.
[20]Leahy JL,Bonner-Weir S,Weir GC.P-Cell dysfunction induced by chronic hyperglycemia:current ideas on mechanism of impaired glucose-induced insulin secretion[J].Diabetes Care,1992,15:442-455.
[21]Gleason CE,Gonzalez M,Harmon JS,Robertson RP.Determinants of glucotoxicity and its reversibility in the pancreatic islet beta-cell line,HIT-T15[J].Am J Physiol Endocrinol Metab,2000,279:E997-E1002.
[22]Harmon JS,Gleason CE,Tanaka Y,Oseid EA,Hunter-Berger KK,Robertson RP.In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression[J].Diabetes,1999 Oct;48(10):1995-2000.
[23]Weng J,Li Y,Xu W,et al.Induction of long-term glycemic control in newly diagnosed type 2 diabetic patients is associated with improvement of beta-cell function[J].Diabetes Care,2004,27:2597-2602.
[24]张波,安雅莉,李光伟,等.短期胰岛素泵强化治疗诱导2型糖尿病患者长期缓解的预测因素[J].中华内分泌代谢杂志,2007,23:134-138.
[25]He QH,Zhou YS,Wang Z,et al.Improvement of the function of islet beta and alpha cells by intervention against glucotoxicity:experiment with rats[J].Zhonghua Yi Xue Za Zhi,2008,88:374-377.
[26]McGirr R,Ejbick CE,Carter DE.Glucose dependence of the regulated secretory pathway in alphaTC1-6 cells[J].Endocrinology,2005,146:4514-4523.
[27]Dumonteil E,Ritz-Laser B,Magnan C.Chronic exposure to high glucose concentrations increases proglucagon messenger ribonucleic acid levels and glucagon release from InR1G9 cells[J].Endocrinology,1999,140:4644-4650.
[28]Yang S-N,Larsson O,Bra~(¨)nstro~(¨)m R,et al.Syntaxin-1 interacts with the L(D)subtype of voltage-gated Ca(2+) channels in pancreatic beta cells[J].Proc Natl Acad Sci,1999,96:10164-10169.
[29]Kang YH,Huang XH,Pasyk EA,et al.Syntaxins-3 and -1A inhibit L-type calcium channel activity,insulin biosynthesis and exocytosis in beta-cell lines[J].Diabetologia,2002,45:231-241.
[30]Daniel S,Noda M,Straub SG,Sharp GWG.Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion[J].Diabetes,1999,48:1686-1690
[31]Zhang W,Khan A,Ostenson CG,Berggren PO,Efendic S,Meister B:Down-regulated expression of exocytotic proteins in pancreatic islets of diabetic GK rats[J].Biochem Biophys Res Commun.2002,291:1038-1044.
[32]Chan CB,MacPhail RM,Sheu L,Wheeler MB,Gaisano HY.β-Cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression[J].Diabetes,1999,48:997-1005.
[33]Ostenson CG,Gaisano H,Sheu L,Tibell A,Bartfai T.Impaired gene and protein expression of exocytotic soluble N-ethylmaleimide attachment protein receptor complex proteins in pancreatic islets of type 2 diabetic patients[J].Diabetes,2006,55:435-440.
[34]Nagamatsu S,Nakamichi Y,Yamamura C,Matsushima S,Watanabe T,Ozawa S,Furukawa H,Ishida H.Decreased expression of t-SNARE,syntaxin 1,and SNAP-25in pancreatic beta-cells is involved in impaired insulin secretion from diabetic GK rat islets:restoration of decreased t-SNARE proteins improves impaired insulin secretion[J].Diabetes.1999 Dec;48(12):2367-73.
[35]Dubois M,Vacher P,Roger B,et al.Glucotoxicity inhibits late steps of insulin exocytosis[J].Endocrinology,2007,148:1605-1614.
[36]Xia F,Leung YM,Gaisano G,et al.Targeting of voltage-gated K+ and Ca2+ channels and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins to cholesterol-rich lipid rafts in pancreatic alpha-cells:effects on glucagon stimulus-secretion coupling[J].Endocrinology,2007,148:2157-2167.
[1]Brunicardi FC,Kleinman R,Moldovan S,Nguyen TH,Watt PC,Walsh J,Gingerich R.Immunoneutralization of somatostatin,insulin,and glucagon causes alterations in islet cell secretion in the isolated perfused human pancreas[J].Pancreas,2001,23:302-308.
[2]Franklin I,Gromada J,Gjinovci A,Theander S,Wollheim CB.β-cell secretory products acitvate α-cell ATP-dependent potassium channels to inhibit glucagon release[J].Diabetes,2005,54:1808-1815.
[3]Gromada J,Ma X,Hoy M,Bokvist K,Salehi A,Berggren PO,Rorsman P.ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/-mouse alpha-cells[J].Diabetes,2004,53:S181-189.
[4]Mufioz A,Hu M,Hussain K,Bryan J,Aguilar-Bryan L,Rajan AS.Regulation of glucagon secretion at low glucose concentration:evidence for adenosine triphosphate-sensitive potassium channel involvement[J].Endocrinology.2005,Dec; 146(12):5514-21.
[5]Meier JJ,Kjems LL,Veldhuis JD,Lefebvre P,Butler PC.Postprandial suppression of glucagon secretion depends on intact pulsatile insulin secretion[J].Diabetes.2006, Apr;55(4):1051-6.
[6]Ravier MA,Rutter GA.Glucose or insulin,but not zinc ions,inhibit glucagon secretion from mouse pancreatic α-cells[J].Diabetes,2005,54:1789-1797.
[7]Evans ML,McCrimmon RJ,Flanagan DE,Keshavarz T,Fan X,McNay EC,Jacob RJ,Sherwin RS.Hypothalamic ATP-sensitive K+ channels play a key role in sensing hypoglycemia and triggering counterregulatory epinephrine and glucagon responses[J].Diabetes,2004,53:2542-2551.
[8]Kisanuki K,Kishikawa H,Araki E,Shirotani T,Uehara M,Isami S,Ura S,Jinnouchi H,Miyamura N,Shichiri M.Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion[J].Diabetologia,1995,38:422-429.
[9]Leung YM,Ahmed I,Sheu L,Gao,X,Hara M,Tsushima RG,Diamant NE,Gaisano HY.Insulin regulates islet α-cell function by reducing KATP channel sensitivity to adenosine 5'-triphosphate inhibition[J].Endocrinology,2006,147:2155-2162.
[10]Khan FA,Goforth PB,Zhang M,Satin LS.Insulin activates ATP sensitive K+ channels in pancreatic β-cells through a phosphatidylinositol 3-kinase-dependent pathway[J].Diabetes,2001,50:2192-2198.
[11]Mirshamsi S,Laidlaw HA,Ning K,Anderson E,Burgess LA,Gray A,Sutherland C, Ashford ML.Leptin and insulin stimulation of signalling pathways in arcuate nucleus neurones:PI3K dependent actin reorganization and K-ATP channel activation[J].BMC Neurosci,2004,5:54.
[12]Xu E,Kumar M,Zhang Y,Ju W,Obata T,Zhang N,Liu S,Wendt A,Deng S,Ebina Y,Wheeler MB,Braun M,Wang Q.Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system[J].Cell Metab,2006,3:47-58.
[13]Ishihara H,Maechler P,Gjinovci A,et al.Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells[J].Nat Cell Biol.2003,5:330-335.
[14]Franklin I,Gromada J,Gjinovci A,et al.p-cell secretory products activate α-cell ATP-dependent potassium channels to inhibit glucagon release[J].Diabetes,2005, 54:1808-1815.
[15]Bancila V,Nikonenko I,Dunant Y,Bloc A.Zinc inhibits glutamate release via activation of pre-synaptic K channels and reduces ischaemic damage in rat hippocampus[J].J Neurochem,2004,90:1243-1250.
[16]Bloc A,Cens T,Cruz H,Dunant Y.Zinc-induced changes in ionic currents of clonal rat pancreatic β-cells:activation of ATP-sensitive K+ channels[J].J Physiol,2000,529:723-734.
[17]Bancila V,Cens T,Monnier D,Chanson F,Faure C,Dunant Y,Bloc A.Two SUR1-specific histidine residues mandatory for zinc-induced activation of the rat KATP channel[J].J Biol Chem,2005,280:8793-8799.
[18]Wendt A,Birnir B,Buschard K,Gromada J,Salehi A,Sewing S,Rorsman P,Braun M.Glucose inhibition of glucagon secretion from rat α-cells is mediated by GABA released from neighboring β-cells[J].Diabetes,2004,53:1038-1045.
[19]01sen HL,Theander S,Bokvist K,Buschard K,Wollheim CB,Gromada J.Glucose stimulates glucagon release in single rat a-cells by mechanisms that mirror the stimulus-secretion coupling in β-cells[J].Endocrinology,2005,146:4861-4870.
[20]Winnock F,Ling Z,De Proft R,Dejonghe S,Schuit F,Gorus F,Pipeleers D.Correlation between GABA release from rat islet beta-cells and their metabolic state[J].Am J Physiol Endocrinol Metab,2002,282:E937-E942.
[21]Wang C,Kerckhofs K,Van de Casteele M,Smolders I,Pipeleers D,Ling Z.Glucose inhibits GABA release by pancreatic beta-cells through an increase in GABA shunt activity[J].Am J Physiol Endocrinol Metab,2006,290:E494-E499.
[22]Braun M,Wendt A,Birnir B,et al.Regulated exocytosis of GABA-containing synaptic-like micro vesicles in pancreatic beta-cells[J].J Gen Physiol,2004,123: 191-204.
[23]Xu E,Kurnar M,Zhang Y,et al.Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system[J].Cell Metab,2006,3:47-58.
[24]Ludvigsen E,Olsson R,Stridsberg M,Janson ET,Sandler S.Expression and distribution of somatostatin receptor subtypes in the pancreatic islets of mice and rats[J].J Histochem Cytochem,2004,52:391-400.
[25]Gromada J,Hoy M,Olsen HL,Gotfredsen CF,Buschard K,Rorsman P,Bokvist K.Gi2 proteins couple somatostatin receptors to low-conductance K+ channels in rat pancreatic a-cellsf J].Pflugers Arch,2001,442:19-26.
[26]Yoshimoto Y,Fukuyama Y,Horio Y,Inanobe A,Gotoh M,Kurachi Y Somatostatin induces hyperpolarization in pancreatic islet a cells by activating a G protein-gated K+ channel.FEBS Lett[J],1999,444:265-269.
[27]Gromada J,Hoy M,Buschard K,Salehi A,Rorsman P.Somatostatin inhibits exocytosis in rat pancreatic a-cells by Gi2-dependent activation of calcineurin and depriming of secretory granules[J].J Physiol,2001,535:519-532.
[28]Strowski M,Casher DE,Birzin ET,Yang L,Singh V,Jacks TM,Nowak K,Rohrer SP, Patchett AA,Smith RG,Schaeffer JM.Antidiabetic activityvof a highly potent and selective non-peptide somatostatin receptor subtype-2 agonist[J].Endocrinology,2006,146:4664-4673.
[29]Strowski MZ,Parmar RM,Blake AD,Schaeffer JM.Somatostatin inhibits insulin and glucagon secretion via two receptors subtypes:an in vitro study of pancreatic islets from somatostatin receptor 2 knockout mice[J].Endocrinology,2000,141:111-117.
[30]Ludvigsen E,Stridsberg M,Taylor JE,Culler MD,Oberg K,Janson ET,Sandler S.Regulation of insulin and glucagon secretion from rat pancreatic islets in vitro by somatostatin analogues[J].Regul Pept.2007,10;138(1):1-9.
[31]Ahren B.Sensory nerves contribute to insulin secretion by glucagon-like peptide-1 in mice[J].Am J Physiol Regul Integr Comp Physiol,2004,286:R269-R272.
[32]Dunning BE,Foley JE,Ahren B.Alpha cell function in health and disease:influence of glucagon-like peptide-1 [J].Diabetologia,2005,48:1700-1713.
[33]Creutzfeldt WO,Kleine N,Willms B,Orskov C,Hoist JJ,Nauck MA.Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide 1(7-36) amide in type I diabetic patients[J].Diabetes Care,2005,19:580-586.
[34]Behme MT,Dupre J,McDonald TJ.Glucagon-like peptide 1 improved glycemic control in type 1 diabetes[J].BMC Endocr Disord,2003,3:3.
[35]Li Y,Cao X,Li LX,Brubaker PL,Edlund H,Drucker DJ.β-cell Pdxl expression is essential for the glucoregulatory,proliferative,and cytoprotective actions of glucagon-like peptide-1 [J].Diabetes,2005,54:482-491.
[36]Preitner F,Ibberson M,Franklin I,Binnert C,Pende M,Gjinovci A,Hansotia T, Drucker DJ,Wollheim C,Burcelin R,Thorens B.Glucoincretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors[J].J Clin Invest,2004,113:635-645.
[37]Dunning BE,Foley JE,Ahren B.Alpha cell function in health and disease:influence of glucagon-like peptide-1 [J].Diabetologia,2005;48:1700-13.
[38]Salehi A,Vieira E,Gylfe E,Paradoxical stimulation of glucagon secretion by high glucose concentrations[J].Diabetes.2006,55:2318-23.
[39]vieira E,Salehi A,Gylfe E,et al.Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells[J].Dibetologia,2007,50:370-379.
[40]McGirr R,Ejbick CE,Carter DE,et al.Glucose dependence of the regulated secretory pathway in alphaTCl-6 cells[J].Endocrinology.2005,146:4514-23.
[41]Dumonteil E,Ritz-Laser B,Magnan C,Grigorescu I,Ktorza A,Philippe J.Chronic exposure to high glucose concentrations increases proglucagon messenger ribonucleic acid levels and glucagon release from InR1G9 cells[J].Endocrinology.1999,140(10):4644-50.
[42]Webb GC,Akbar MS,Zhao C,Steiner DF 2000 Expression profiling of pancreatic Pcells:glucose regulation of secretory and metabolic pathway genes[J].Proc Natl Acad Sci USA 97:5773-5778
[43]Xia F,Leung YM,Gaisano G,Gao X,Chen Y,Manning Fox JE,et al.Targeting of KV4,CaV1.2 and SNARE proteins to cholesterolrich lipid rafts in pancreatic alpha-cells:effects on glucagon stimulus-secretion coupling[J].Endocrinology,2007, 148:2157-67.
[44]Gremlich S,Bonny C,Waeber G,Thorens B.Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2,glucokinase,insulin,and somatostatin levels[J].J Biol Chem.1997 Nov 28;272(48):30261-9.
[45]Olofsson CS,Salehi A,G(o|¨)pel SO,Holm C,Rorsman P 2004 Palmitate stimulation of glucagon secretion in mouse pancreatic α-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium[J].Diabetes 53:2836-2843.
[46]杜瑞琴,李宏亮,杨文英,等.胰岛α细胞胰岛素抵抗与炎症通路激活的关系及机制[J].中华内科杂志.2007,46(8):661-665.
[47]Hong J,Abudula R,Chen J,et al.The short-term effect of fatty acids on glucagon secretion is influenced by their chain length,spatial configuration,and degree of unsaturation:studies in vitro[J].Metabolism.2005 Oct;54(10):1329-36.
[48]Hong J,Chen L,Jeppesen PB,Nordentoft I,Hermansen K.Stevioside counteracts the alpha-cell hypersecretion caused by long-term palmitate exposure[J].Am J Physiol Endocrinol Metab.2006,290(3):E416-22.
[49]Madsen,P,Knudsen LB,Wiberg FC,Carr RD.Discovery and structureactivity relationship of the first non-peptide competitive human glucagon receptor antagonists[J].J Med Chem,1998,17:5150-157.
[50]Qureshi SA,Rios Candelore M,Xie D,et al.A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects[J],Diabetes,2004 Dec;53(12):3267-73.
[51]Petersen KF,Sullivan JT.Effects of a novel glucagon receptor antagonist(Bay 27-9955) on glucagon-stimulated glucose production in humans[J].Diabetologia.2001,44(11):2018-24.
[52]S(?)rensen H,Brand CL,Neschen S,Holst JJ,Fosgerau K,Nishimura E,Shulman GI.Immunoneutralization of endogenous glucagon reduces hepatic glucose output and improves long-term glycemic control in diabetic ob/ob mice[J],Diabetes,2006,55:2843-2848.
[53]Liang Y,Osborne MC,Monia BP,Bhanot S,Gaarde WA,Reed C,She P,Jetton TL,Demarest KT.Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice[J].Diabetes,2004,53:410-417.
[54]Sloop KW,Cao JX-C,Siesky AM,et al.Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors[J].J Clin Invest,2004,113:1571-1581.