1、线粒体与2型糖尿病Chen-Yu Zhang MD, PhD State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China,糖尿病(Diabetes mellitus)是一类以长期高血糖为主要特征的代谢病。 2型糖尿病是环境与遗传因素共同作用所引起的慢性复杂性状疾病，以胰岛素抵抗、胰岛b细胞功能障碍和全身代谢紊乱所导致的血糖持续升高为特征。,线粒体损伤与胰岛素抵抗：在正常的生理状态下，细胞的能量代谢（糖代谢，脂代谢）是通过线粒体的作用而实现的。糖和脂
2、肪在线粒体内氧化，产生ATP，作为细胞功能所需的能量。线粒体损伤必然导致细胞功能缺陷。 近来针对人类的磁共振光谱研究表明，细微的线粒体功能损伤可导致胰岛素抵抗和2型糖尿病发病。Petersen等人研究发现，健康无肥胖的老年人与年轻人的对照组相比，线粒体氧化活性的下降以及线粒体内三磷酸腺苷生成的减少与肌肉胰岛素抵抗正相关20, 21。 另有研究发现，在父母均为2型糖尿病患者，自身患有胰岛素抵抗的年轻病人中（这类胰岛素抵抗人群更易发展为糖尿病），也发现有相似的线粒体活性的降低以及肌细胞内脂肪含量的增加22。 与对胰岛素敏感的对照者相比，胰岛素抵抗的研究对象体内1型肌纤维与2型肌纤维的比例更低。1型
3、肌纤维大多是氧化性的，与2型相比含有更多的线粒体22。可以想象，这些个体肌细胞线粒体的含量更少。 2型糖尿病患者线粒体氧化酶的活性较低23这些患者NADH:O(2)的活性也较低24。这表明线粒体脂肪酸氧化磷酸化障碍导致细胞内脂肪酸代谢产物（酯酰辅酶A和二酯酰甘油）的增多，影响胰岛素信号转导通路，从而引起胰岛素抵抗。,前沿进展,1) 线粒体上表达的蛋白的功能对线粒体功能的影响及对肝细胞功能的影响。2) 原本不在线粒体上表达的蛋白，但在线粒体上形成蛋白功能复合物，进而影响线粒体功能和肝细胞功能。3) 原本不在线粒体上表达，但通过调节相关基因如NRF1等从而调控线粒体数量和功能的蛋白。 4) 线粒体
4、蛋白组学的研究。,From: Langin D. Diabetes, Insulin Secretion and the Pancreatic Beta-Cell. NEJM 345: 1772-4, 2001,1) 线粒体上表达的蛋白的功能对线粒体功能的影 响及对细胞功能的影响。,From: Nature 424, 896 - 897 (21 August 2003); Cell biology: Metabolism meets death JULIAN DOWNWARD,2) 原本不在线粒体上表达的蛋白，但在线粒体上形成蛋白功能复合物，进而影响线粒体功能和肝细胞功能。,3) 原本不在线粒
5、体上表达，但通过调节相关基因如NRF1等从而调控线粒体数量和功能的蛋白。,From:Cell. 2003 Nov 26;115(5):629-40. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Mootha VK, et, al,4) 线粒体蛋白组学的研究。,A UCP2 specific inhibitor stimulates insulin secretion in isolated islets and in vivo.C
6、hen-Yu Zhang MD, PhD State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China,Mitochondria: Fuel ATP,UCP1: a Mitochondrial Uncoupling Protein,Heat,Cloning of UCP Homologues,UCP2 - Fleury, et al., Nat. Genet. 15, 269, 1997. Gimeno, et al., Diabetes 46, 9
7、00, 1997. UCP3 - Boss, et al., FEBS Lett. 408, 39, 1997. Vidal-Puig, Solanes, Grujic, et al., BBRC 235, 79, 1997.Gong, et al., JBC 272, 24129, 1997.Larkin et al., BBRC 240, 222, 1997.Matsuda et al., FEBS Lett. 418, 200, 1997.Liu et al., Gene 207, 1, 1998.,Amino Acid Sequence Identity to UCP1,Uncoupl
8、ing Protein-1 100%Uncoupling Protein-2 56% Uncoupling Protein-3 56%BMCP1 34% Dicarboxylate Carrier 32% Oxoglutarate/malate Carrier 31% Uncoupling Protein-4 29%Carnitine Carrier 26% Citrate Carrier 22% Ornithine Carrier 22% ADP/ATP Carriers 20% Phosphate Carrier 17%,UCPs: Sites of Abundant Expression
9、,UCP1: Brown adipose tissue.UCP2: Spleen, thymus, lung, gastrointestinal tract,white adipose tissue, and pancreatic islets.UCP3: Skeletal muscle and brown adipose tissue.,UCP2 mRNA,UCP2 Protein,UCP2 Gene Knockout Mice,Mitochondrial Membrane Potential and ATP Levels in Resting Thymocytes,WT,KO,*,*,*,
10、= p0.05,*,= p0.05,UCP2 and Regulation of Insulin Secretion,From: Langin D. Diabetes, Insulin Secretion and the Pancreatic Beta-Cell. NEJM 345: 1772-4, 2001,Glucose (mM),0,2,4,6,5.5,25,Glucose (mM),KO,WT,*,*,0,1,2,3,4,5,.,5,11,*,WT,KO,*,ATP (pmol/islet),Insulin (ng/islet/hr),Incubated Pancreatic Isle
11、ts (ATP content and Insulin Release),Serum Insulin and Blood Glucose in UCP2 KO Mice,Hyperglycemic Clamp,Basal Glycemia, Glucose 50%,glucose infusion (140-250 M/min/kg),-60 min. 0 min. + 90 min.,Measure: Serum Insulin,Collaboration with Pascale Perret and Gerald Shulman Yale University,UCP2 is Upreg
12、ulated in Pancreatic Islets of ob/ob Mice,UCP2 mRNA in Islets,+/+,ob/ob,Blood Glucose and Insulin,UCP2 protein in Islets of,+/+,ob/ob,-/-,Glucose Tolerance Test (1g/kg/i.p.),Glucose (mg/dl),Insulin (ng/ml),Obesity,Insulin Resistance in Target Tissues (muscle, fat, liver),-Cell Hyperplasia,Increased
13、-Cell Mass,Type 2 Diabetes Insulin resistance Hyperinsulinemia with impaired glucose-stimulated insulin secretion Hyperglycemia, UCP2 in -Cells,-Cell Dysfunction,Zhang CY et al. CELL 105 (6): 745-755 2001,Nature 415: 96-99, 2002,Addition of exogenous superoxide activates UCP1-, UCP2- and UCP3-mediat
14、ed proton leak.,1) Does Endogenously Produced Superoxide Stimulate UCP2-Mediated Proton Leak? 2) Does this Pathway Regulate Glucose-Stimulated Insulin Secretion?,Modified from: Langin D. Diabetes, Insulin Secretion and the Pancreatic Beta-Cell. NEJM 345: 1772-4, 2001,O2-,Superoxide is a Normal Bypro
15、duct of Mitochondrial Electron Transport Chain Activity Superoxide is created when O2 reacts with radicals in the electron transport chain,Conclusion: Endogenous levels of superoxide activateUCP2-mediated proton leak.,Effect of reducing superoxideWT KO1)Proton leak No effect 2)Membrane potential No
16、effect3) ATP level No effect(J Clin Invest. 2003;112(12):1831-42),Obesity Hyperglycemia,-Cell Dysfunction,Obesity Hyperglycemia, UCP2 Protein, Superoxide, UCP2 Activity,Obesity Hyperglycemia, UCP2 Protein, Superoxide, UCP2 Activity,-Cell Dysfunction,Insulin Secretion (% of Insulin Content),Chronic H
17、yperglycemia (25 mM) and -Cell Dysfunction,0,2,4,6,Chronic 5.5 mM,Chronic 25 mM,Chronic 25 mM + MnSOD,Chronic 25 mM + GPX1,*,*,*,*,#,#,#,Insulin Secretion (% of Insulin Content),Chronic Hyperglycemia (25 mM) and -Cell Dysfunction,0,2,4,6,Chronic 5.5 mM,Chronic 25 mM,Chronic 25 mM + MnSOD,Chronic 25
18、mM + GPX1,*,*,*,*,#,#,#,Effect of removing O2 (+SOD),Insulin Secretion (% of Insulin Content),Chronic Hyperglycemia (25 mM) and -Cell Dysfunction,0,2,4,6,Chronic 5.5 mM,Chronic 25 mM,Chronic 25 mM + MnSOD,*,*,*,*,#,#,Effect of UCP2 Knockout,#,*,Chronic 25 mM,*,ob/ob,ob/ob + SOD,ob/ob + UCP2 KO,0,2,4
19、,6,Insulin Secretion (% of Insulin Content),Obesity and -Cell Dysfunction (ob/ob Islets),Obesity and -Cell Dysfunction (ob/ob Islets),Obesity and -Cell Dysfunction (ob/ob Islets),Zhang CY et al, J. CLIN. INVEST. 112 (12): 1831-1842 2003,UCP2 is a negative regulator of insulin secretion.Endogenous su
20、peroxide actives UCPs.Interrupting the pathway of superoxide-mediated activation of UCP2 in islets prevents the loss of GSIS that normally results from chronic hyperglycemia, or obesity.,Inhibition of UCP2 could be a viable strategy to restore b-cell dysfcuntion in type 2 diabetes,A UCP2 specific in
21、hibitor stimulates insulin secretion in isolated islets and in vivo.,proton leak (nmol H+/min per mg protein),Effect of 50uM genipin on superoxide-induced proton leak in isolated kidney mitochondria (UCP2),wildtype,UCP2 KO,Mitochondrial membrane potential (mV),control,X/XO,X/XO + genipin,0,10,20,30,
22、40,50,60,70,80,160,180,200,220,control,X/XO,X/XO + genipin,0,10,20,30,40,50,60,70,80,160,180,200,220,Mitochondrial membrane potential (mV),Proton leak rate (nmol O/min per mg),Effect of 50uM genipin on superoxide-induced proton leak in isolated skeletal muscle mitochondria (UCP3),wildtype,UCP3 KO,Ef
23、fect of genipin on mitochondrial integrity substrate transport and oxidative phosphorylation,Fundamental question: how does superoxide activate UCP2?,Proton leak rate (nmol H+ / min / mg protein),Mitochondrial membrane potential (mV),wildtype,UCP2 KO,Effect of 50uM genipin on HNE-induced proton leak
24、 in isolated kidney mitochondria (UCP2),Effect of genipin on TMRM fluorescence in dispersed islet cells,Effect of genipin on insulin secretion in WT and UCP2 KO islets (glu:5.5mM),Chronic Hyperglycemia (25 mM) and -Cell Dysfunction,0,4,8,12,16,Insulin (,ng,/islet/hr),*,*,*,*,Chronic,5.5 mM,Chronic,2
25、5 mM,Chronic,25,mM,+ G,Chronic Hyperglycemia (25 mM) and -Cell Dysfunction,0,4,8,12,16,Insulin (,ng,/islet/hr),*,*,*,*,Chronic,5.5 mM,Chronic,25 mM,Chronic,25,mM,+ G,Effect of UCP2 KO,Chronic Hyperglycemia (25 mM) and -Cell Dysfunction,0,4,8,12,16,Insulin (,ng,/islet/hr),*,*,*,*,Chronic,5.5 mM,Chron
26、ic,25,mM,+ G,Obesity and -Cell Dysfunction (ob/ob islets),Obesity and -Cell Dysfunction (ob/ob islets),Obesity and -Cell Dysfunction (ob/ob islets),Zhang CY et al, Cell Metabolism in press,Acknowledgements,Bradford B. Lowell Stefan KraussGyrgy Baffy Olivier Boss Jia Yu Ying XuLouise Dalgaard Jennife
27、r Wade,O. Peroni, Y.-B. Kim, B. Kahn, BIDMC Xin Xiao Zheng, Yan Tian, Div. of Immunology, BIDMC Gerald Shulmans Lab, YaleGary Cline - NMRPascale Perret - Clamp studies Michael Wheeler, Domenica De Leo, Jamie Joseph, Univ. of Toronto Cathy Chan, Univ. of Prince Edward Island Shan T. Grey - BIDMC Luca Scorrano - DFCI Julie St. Pierre - DFCI John Porco, Jr., Ruichao Shen and Tony Lin, Boston University,What, you dont know genipin?,
