糖脂代谢障碍对脑损伤的体内外研究
|
摘要
目的以糖尿病动物为模型研究持续高血糖,高血脂对认知功能,海马神经细胞损伤及其突触可塑性的影响。通过体外细胞模型,分别研究高糖及高脂对PC12细胞的损伤效应,这一损伤效应与细胞增殖以及自噬的关系。
方法
1、C57B/L小鼠按体重连续给予四天腹腔注STZ,持续高脂饮食。对照组给予等量的无菌柠檬酸/柠檬酸钠缓冲液腹腔注射,普通饲料喂养。一周后,从鼠尾采血分离血浆,测定葡萄糖浓度,血糖≥200mg/dL作为模型成功的标志。模型小鼠高脂饮食喂养一个月后做胰岛素敏感性实验。随后每个月小鼠尾部采血并采用葡萄糖氧化酶法连续监测各组小鼠血糖变化,采用酶比色法连续监测各组小鼠血液中甘油三酯及胆固醇变化,通过水迷宫实验检测动物学习记忆变化,通过Fluoro-Jade-C染色观察并计数海马区细胞密度及退变的神经细胞,并用免疫组织化学染色、ELISA法检测海马区β-淀粉样蛋白含量,通过Golgi-Cox染色结合体视学半定量技术分析海马CA1区锥体细胞树突棘密度变化,通过透射电子显微镜观察海马CA1区神经细胞自噬情况。
2、用葡萄糖体外培养PC12细胞,应用MTT法加入不同浓度葡萄糖以及不同作用时间检测细胞存活率,并确定合适的损伤模型；普通显微镜下观察细胞形态并进行细胞计数,Brdu掺入后采用免疫荧光显微技术观察细胞增殖的情况；采用流式细胞术测定细胞周期及增殖情况。用油酸诱导体外培养的PC12细胞,应用MTT法加入不同浓度油酸作用不同时间检测细胞存活率,并用Western blotting检测不同浓度油酸对细胞LC3-Ⅱ表达水平的影响,加入自噬抑制剂3-MA及自噬诱导剂雷帕霉素应用Western blotting检测处理后细胞内LC3-Ⅱ的表达水平,采用MTT法检测细胞存活率,应用MDC染色观察自噬囊泡。
结果
1、体内实验结果表明,2型糖尿病小鼠在一个月出现胰岛素抵抗,并伴随持续的高血糖及高胆固醇血症。2个月,4个月出现学习记忆能力下降(p<0.05)。通过Fluoro-Jade-C染色表明4个月糖尿病小鼠海马区退变的神经细胞数量增加。通过ELISA及免疫组化观察到β淀粉样蛋白表达增加。利用Golgi-Cox染色观察到海马CA1区锥体细胞树突棘密度显著下降(p<0.01)。
2、体外实验结果表明,随着葡萄糖浓度增加,细胞存活率逐渐下降,葡萄糖作用于PC12细胞48h,其有效致死量为150mM/L,选取此浓度建立损伤模型,显微镜下观察高糖作用组细胞数量与正常组比较明显减少(p<0.01)。Brdu掺入实验发现高糖抑制PC12细胞增殖。通过流式细胞仪检测细胞周期,表明高糖作用48h后PC12细胞被阻滞在G1期,S期细胞数量减少。给予不同浓度油酸可以诱导PC12细胞出现自噬,自噬相关基因LC3-Ⅱ表达水平明显升高,MDC染色荧光显微镜观察可见大量自噬泡出现。自噬抑制剂3-MA导致LC3-Ⅱ表达下调,同时细胞存活率下降。雷帕霉素促进LC3-Ⅱ的表达,同时也促进了细胞的存活。结论
1、体内研究结果表明2型糖尿病持续的高血糖、高血脂可能直接导致海马区神经细胞损伤,β-样淀粉蛋白沉积,突触可塑性下降,上述变化导致认知功能障碍。
2、高糖诱导PC12细胞后形态没有明显的变化,但增殖受到抑制。高脂能够诱导自噬的出现,给予自噬抑制剂后细胞损伤严重,而给予自噬诱导剂雷帕霉素后有保护效应。这说明高脂诱导的自噬活化是一种保护性代偿效应。
Objective
In vivo, our study focuses on the effects of hyperglycemia and hyperlipidemia on cognition, neural degeneration and synaptic plasticity in hippocampus. In vitro, our experiments study the changes of proliferation and autophagy on PC12 cell with high glucose or high oleic acid, respectively.
Methods
1. C57B/L mice were injected with STZ (30mg/kg, i.p) once a day for four days consecutively. The control mice were injected with the equal volume of sterile citric acid/sodium citrate buffer. After one week, blood plasma was harvest from tail vein, and the concentration of glucose was determined, and≥250mg/dL was used. Plasma glucose was measured by oxidase-methods and plasma cholesterol was test by Enzyme assay every month. Learning and memory was test using the moires water maze. And neural degeneration in hippocampus was valued through Fluoro-Jade C-staining.β- amyloid deposition in hippocampus was measured by ELISA and immunohistochemistry. Golgi-Cox staining together with semi-quantitative stereological method was used to detect the density of dendritic spine of pyramidal cells in CA 1 of hippocampus. Additionally, autophagosome of pyramidal cells in hippocampus were observed by transmission electron microscopy.
2. PC12 cells were cultured at different condition to mimicry high glucose or high lipid by adding the different concentration of glucose or oleic acid to medium. Cell viability was valuated by MTT assay. Cell morphology were observed by microscope and cell proliferation by immunofluorescence microscope with high glucose; Also cell cycle was determined by flow cytometry. Under high lipid condition, presenting of 3-MA, autophagy inhibitor, or rapamycin, autophagy inducer, respectively, the expression level of LC3-Ⅱwere detected by western blotting, cell viability were valuated by MTT.
Results
1. In vivo, our results indicates that serum insulin levels were lower and plasma insulin sensitivity were decreased in diabetic mouse compared with control group at 4 weeks after induced. The ability of learning and memory declined compared to the age-matched group after 2 mouth (p<0.05). Neural degeneration in hippocampus aggregated at 4 month compared with that of control. More deposition ofβamyloid in hippocampus of DM-mouse exits than control. The density of dendritic spine in hippocampus is lowed in DM-mouse compared to the control group (p< 0.01). Autophagysome were observed in cytoplasma of pyramidal cells in hippocampus in diabetic mouse by transmission electron microscopy.
2. In vitro, the viability of PC 12 cell was obviously inhibited with different concentrations of glucose in time- and dose-dependent manners, half of inhibition concentration(IC50) value was 150mM. The proliferation of PC 12 cells significantly were inhibited with high glucose by incorperation of Brdu. Most of cells were arrested at G1 phase, and S phase synthesis of were reduced after 48h with high glucose. Autophagy were induced in PC 12 cells with oleic acid, and the expression levels of autopha related protein LC3-Ⅱwere significantly increased and autophagy vesica was observed using fluorescent microscope. Giving autophagy inhibitors. MTT show the cell vibility and the expression of LC3-Ⅱlevels decreased significantly compared with single oleic acid, while the opposite effect of rapamycin.
Conclusion
1. Our study suggested persistant high glucose and lipid induce the neurodegeneration and synapse-loss,enhancement of beta amyloid deposition in hippocampus, which impaired the cognition in type 2 diabetes model.
2. PC 12 cells proliferation were inhibited by high glucose, autophagy was induced by oleic acid, also inhibit autophaby influence cell viability, but has protective effect of rapamycin.
方法
1、C57B/L小鼠按体重连续给予四天腹腔注STZ,持续高脂饮食。对照组给予等量的无菌柠檬酸/柠檬酸钠缓冲液腹腔注射,普通饲料喂养。一周后,从鼠尾采血分离血浆,测定葡萄糖浓度,血糖≥200mg/dL作为模型成功的标志。模型小鼠高脂饮食喂养一个月后做胰岛素敏感性实验。随后每个月小鼠尾部采血并采用葡萄糖氧化酶法连续监测各组小鼠血糖变化,采用酶比色法连续监测各组小鼠血液中甘油三酯及胆固醇变化,通过水迷宫实验检测动物学习记忆变化,通过Fluoro-Jade-C染色观察并计数海马区细胞密度及退变的神经细胞,并用免疫组织化学染色、ELISA法检测海马区β-淀粉样蛋白含量,通过Golgi-Cox染色结合体视学半定量技术分析海马CA1区锥体细胞树突棘密度变化,通过透射电子显微镜观察海马CA1区神经细胞自噬情况。
2、用葡萄糖体外培养PC12细胞,应用MTT法加入不同浓度葡萄糖以及不同作用时间检测细胞存活率,并确定合适的损伤模型；普通显微镜下观察细胞形态并进行细胞计数,Brdu掺入后采用免疫荧光显微技术观察细胞增殖的情况；采用流式细胞术测定细胞周期及增殖情况。用油酸诱导体外培养的PC12细胞,应用MTT法加入不同浓度油酸作用不同时间检测细胞存活率,并用Western blotting检测不同浓度油酸对细胞LC3-Ⅱ表达水平的影响,加入自噬抑制剂3-MA及自噬诱导剂雷帕霉素应用Western blotting检测处理后细胞内LC3-Ⅱ的表达水平,采用MTT法检测细胞存活率,应用MDC染色观察自噬囊泡。
结果
1、体内实验结果表明,2型糖尿病小鼠在一个月出现胰岛素抵抗,并伴随持续的高血糖及高胆固醇血症。2个月,4个月出现学习记忆能力下降(p<0.05)。通过Fluoro-Jade-C染色表明4个月糖尿病小鼠海马区退变的神经细胞数量增加。通过ELISA及免疫组化观察到β淀粉样蛋白表达增加。利用Golgi-Cox染色观察到海马CA1区锥体细胞树突棘密度显著下降(p<0.01)。
2、体外实验结果表明,随着葡萄糖浓度增加,细胞存活率逐渐下降,葡萄糖作用于PC12细胞48h,其有效致死量为150mM/L,选取此浓度建立损伤模型,显微镜下观察高糖作用组细胞数量与正常组比较明显减少(p<0.01)。Brdu掺入实验发现高糖抑制PC12细胞增殖。通过流式细胞仪检测细胞周期,表明高糖作用48h后PC12细胞被阻滞在G1期,S期细胞数量减少。给予不同浓度油酸可以诱导PC12细胞出现自噬,自噬相关基因LC3-Ⅱ表达水平明显升高,MDC染色荧光显微镜观察可见大量自噬泡出现。自噬抑制剂3-MA导致LC3-Ⅱ表达下调,同时细胞存活率下降。雷帕霉素促进LC3-Ⅱ的表达,同时也促进了细胞的存活。结论
1、体内研究结果表明2型糖尿病持续的高血糖、高血脂可能直接导致海马区神经细胞损伤,β-样淀粉蛋白沉积,突触可塑性下降,上述变化导致认知功能障碍。
2、高糖诱导PC12细胞后形态没有明显的变化,但增殖受到抑制。高脂能够诱导自噬的出现,给予自噬抑制剂后细胞损伤严重,而给予自噬诱导剂雷帕霉素后有保护效应。这说明高脂诱导的自噬活化是一种保护性代偿效应。
Objective
In vivo, our study focuses on the effects of hyperglycemia and hyperlipidemia on cognition, neural degeneration and synaptic plasticity in hippocampus. In vitro, our experiments study the changes of proliferation and autophagy on PC12 cell with high glucose or high oleic acid, respectively.
Methods
1. C57B/L mice were injected with STZ (30mg/kg, i.p) once a day for four days consecutively. The control mice were injected with the equal volume of sterile citric acid/sodium citrate buffer. After one week, blood plasma was harvest from tail vein, and the concentration of glucose was determined, and≥250mg/dL was used. Plasma glucose was measured by oxidase-methods and plasma cholesterol was test by Enzyme assay every month. Learning and memory was test using the moires water maze. And neural degeneration in hippocampus was valued through Fluoro-Jade C-staining.β- amyloid deposition in hippocampus was measured by ELISA and immunohistochemistry. Golgi-Cox staining together with semi-quantitative stereological method was used to detect the density of dendritic spine of pyramidal cells in CA 1 of hippocampus. Additionally, autophagosome of pyramidal cells in hippocampus were observed by transmission electron microscopy.
2. PC12 cells were cultured at different condition to mimicry high glucose or high lipid by adding the different concentration of glucose or oleic acid to medium. Cell viability was valuated by MTT assay. Cell morphology were observed by microscope and cell proliferation by immunofluorescence microscope with high glucose; Also cell cycle was determined by flow cytometry. Under high lipid condition, presenting of 3-MA, autophagy inhibitor, or rapamycin, autophagy inducer, respectively, the expression level of LC3-Ⅱwere detected by western blotting, cell viability were valuated by MTT.
Results
1. In vivo, our results indicates that serum insulin levels were lower and plasma insulin sensitivity were decreased in diabetic mouse compared with control group at 4 weeks after induced. The ability of learning and memory declined compared to the age-matched group after 2 mouth (p<0.05). Neural degeneration in hippocampus aggregated at 4 month compared with that of control. More deposition ofβamyloid in hippocampus of DM-mouse exits than control. The density of dendritic spine in hippocampus is lowed in DM-mouse compared to the control group (p< 0.01). Autophagysome were observed in cytoplasma of pyramidal cells in hippocampus in diabetic mouse by transmission electron microscopy.
2. In vitro, the viability of PC 12 cell was obviously inhibited with different concentrations of glucose in time- and dose-dependent manners, half of inhibition concentration(IC50) value was 150mM. The proliferation of PC 12 cells significantly were inhibited with high glucose by incorperation of Brdu. Most of cells were arrested at G1 phase, and S phase synthesis of were reduced after 48h with high glucose. Autophagy were induced in PC 12 cells with oleic acid, and the expression levels of autopha related protein LC3-Ⅱwere significantly increased and autophagy vesica was observed using fluorescent microscope. Giving autophagy inhibitors. MTT show the cell vibility and the expression of LC3-Ⅱlevels decreased significantly compared with single oleic acid, while the opposite effect of rapamycin.
Conclusion
1. Our study suggested persistant high glucose and lipid induce the neurodegeneration and synapse-loss,enhancement of beta amyloid deposition in hippocampus, which impaired the cognition in type 2 diabetes model.
2. PC 12 cells proliferation were inhibited by high glucose, autophagy was induced by oleic acid, also inhibit autophaby influence cell viability, but has protective effect of rapamycin.
引文
[1]Biessels, G. J., van der Heide, etal. Ageing and diabetes:implications for brain function[J]. Eur J Pharmacol.2002,441:1-14.
[2]Mijnhout, G. S., Scheltens, etal. Diabetic encephalopathy:A concept in need of a definition[J]. Diabetologia.2006,49:1447-1448.
[3]Tuzcu, M., and Baydas, G.. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats[J]. Eur J Pharmacol.2006,53:106-110.
[4]Reagan, L. P. Insulin signaling effects on memory and mood[J]. Curr Opin Pharmacol.2007,7:633-637.
[5]Ryan, Williams, Finegol.Cognitive dysfunction in adults with type 1 (insulin-dependent) diabetes mellitus of long duration:effects of recurrent hypoglycaemia and other chronic complications[J]. Diabetologia.1993,3:329-334.
[6]Spitzer, Barnow, Wallaschofski, Freyberger, etal. Association between depression and subclinical carotid atherosclerosis in patients with Type 1 diabetes [J].Diabet Med.2008,25:349-354.
[7]Mastrocola,Vercellinatto,Brignardello, etal. Oxidative and nitrosative stress in brain mitochondria of diabetic rats[J]. J Endocrinol.2005,187:37-44.
[8]Somfai, Ruzicska, Magyar, etal. Soluble semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation in streptozotocin-induced diabetic rats[J]. Neurochem Int.2006,48:746-752.
[9]Balasubramanyam,Hampe, Maldonado, M. Syndromes of ketosis-prone diabetes mellitus[J].Endocr Rev.2008,29:292-302.
[10]Planel, E., Tatebayashi, Y., Miyasaka, T,etal.Insulin dysfunction induces in vivo tau hyperphosphorylation through distinct mechanisms[J].J Neurosci.2007,27:13635-13648.
[11]Korf, E. S., White, L. R., Scheltens, P.,etal. Brain aging in very old men with type 2 diabetes: the Honolulu-Asia Aging Study[J].Diabetes Care.2006,29:2268-2274.
[12]Musen, G., Lyoo, I. K., Sparks, C. R., etal. Effects of type 1 diabetes on gray matter density as measured by voxel-based morphometry[J].Diabetes.2006,55:326-333.
[13]Park, C. R. Cognitive effects of insulin in the central nervous system[J]. Neurosci Biobehav Rev.2001,25:311-323.
[14]Perez, A., Morelli, L., Cresto, J. C.,etal. Degradation of soluble amyloid beta-peptides 1-40. 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains[J]. Neurochem Res.2000,25:247-255.
[15]White, M. F. The IRS-signaling system:a network of docking proteins that mediate insulin and cytokine action[J]. Recent Prog Horm Res.1998,53:119-138.
[16]Cole, G. M., Frautschy, S. A. The role of insulin and neurotrophic factor signaling in brain aging and Alzheimer's Disease[J]. Exp Gerontol.2007,42:10-21.
[17]Wertkin, A. M., Turner, R. S., Pleasure, S. J.,etal. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides[J].Proc Natl Acad Sci U S A. 1993,90:9513-9517.
[18]Tienari, P. J., Ida, N., Ikonen, E.,etal. Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons[J].Proc Natl AcadSci U S A.1997,94:4125-4130.
[19]Chyung, A. S., Greenberg, B. D., Cook, D. G.,etal. Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells[J]. J Cell Biol.1991,138:671-680.
[20]Xu, H., Sweeney, D., Wang, R.,etal. Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation[J].Proc Natl Acad Sci U S A.1997,94:3748-3752.
[21]Oh, D., Han, S., Seo, J.,etal. Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins[J]. JNeurosci.2010,30:14134-14144.
[22]Malone, J. I., Hanna, S., Saporta, S.,etal. Hyperglycemia not hypoglycemia alters neuronal dendrites and impairs spatial memory[J].Pediatr Diabetes.2008,9:531-539.
[23]Fernando, R. N., Albiston, A. L., Chai, S. Y. The insulin-regulated aminopeptidase IRAP is colocalised with GLUT4 in the mouse hippocampus--potential role in modulation of glucose uptake in neurones? [J].Eur J Neurosci.2008,28:588-598.
[24]Reiter, R. J. Oxidative processes and antioxidative defense mechanisms in the aging brain[J].FASEB J.1995,9:526-533.
[25]Dalal, P. M., and Parab, P. V. Cerebrovascular disease in type 2 diabetes mellitus[J].Neurol India.2002,50:380-385.
[26]Zochodne, D. W. Diabetic neuropathies:features and mechanisms[J].,Brain Pathol.1999,9:369-391.
[27]Shinozaki, K., Naritomi, H., Shimizu, T., etal. Role of insulin resistance associated with compensatory hyperinsulinemia in ischemic stroke[J].Stroke.1996,27:37-43.
[28]Pike, L. J. Lipid rafts:bringing order to chaos[J]. J Lipid Res.2003,44:655-667.
[29]Meer, G. Cell biology. The different hues of lipid rafts[J].Science.2002,296:855-857.
[30]Pfrieger, F. W. Role of cholesterol in synapse formation and function[J].Biochim Biophys Acta.2003,1610:271-280.
[31]Heverin, M., Meaney, S., Lutjohann, D.,etal. Crossing the barrier:net flux of 27-hydroxycholesterol into the human brain[J].J Lipid Res.2005,46:1047-1052.
[32]Puglielli, L., Tanzi, R. E., Kovacs, D. M. Alzheimer's disease:the cholesterol connection[J].Nat Neurosci.2003,6:345-351.
[33]Refolo, L. M., Malester, B., LaFrancois, J., etal. Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model[J].Neurobiol Dis. 2000,7:321-331.
[34]Sparks, D. L., Liu, H., Gross, D. R.,etal. Increased density of cortical apolipoprotein E immunoreactive neurons in rabbit brain after dietary administration of cholesterol[J].Neurosci Lett.1995,187:142-144.
[35]Wahrle, S. E., Jiang, H., Parsadanian, M.etal. Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease[J].J Clin Invest.2008,118:671-682.
[36]Wollmer, M. A., Streffer, J. R., Lutjohann, D., etal. ABCA1 modulates CSF cholesterol levels and influences the age at onset of Alzheimer's disease[J]. Neurobiol Aging.2003,24:421-426.
[37]Mizushima, N.,Klionsky, D. J. Protein turnover via autophagy:implications for metabolism[J].Annu Rev Nutr.2007,27:19-40.
[38]Lemasters, J. J., Qian, T., He, L.,etal. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy [J]. Antioxid Redox Signal.2002,4:769-781.
[39]Bellu, A. R., Kiel, J. A. Selective degradation of peroxisomes in yeasts[J].Microsc Res Tech.2003,61:161-170.
[40]Roberts, P., Moshitch-Moshkovitz, S., Kvam, E., etal. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae[J].Mol Biol Cell.2003,14:129-141.
[41]Iwata, A. Neurodegenerative disease and autophagy[J].Rinsho Shinkeigaku.2006,46:887-889.
[42]Cuervo, A. M., and Dice, J. F. (1996) A receptor for the selective uptake and degradation of proteins by lysosomes, Science 273,501-503.
[43]Ashford, T. P., Porter, K. R. Cytoplasmic components in hepatic cell lysosomes[J].J Cell Biol.1962,12:198-202.
[44]Biederbick, A., Kern, H. F., Elsasser, H. P. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles[J].,Eur J Cell Biol.1995,66:3-14.
[45]Mizushima, N., Yamamoto, A., Matsui, M., etal. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[J].Mol Biol Cell.2004,15:1101-1111.
[46]Kabeya, Y., Mizushima, N., Yamamoto, A.,etal. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-Ⅱ formation[J].J Cell Sci.2004,117:2805-2812.
[47]Seglen, P. O., Gordon, P. B.3-Methyladenine:specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes[J].Proc Natl Acad Sci U S A.1982,79:1889-1892.
[48]Blommaart, E. F., Krause, U., Schellens, J. P.,etal. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes[J].Eur J Biochem.1997,243:240-246.
[49]Tanida, I., Ueno, T., Kominami, E. LC3 conjugation system in mammalian autophagy[J]. Int J Biochem Cell Biol.2004,36:2503-2518.
50] Mizushima, N., Yamamoto, A., Hatano. M. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells[J].J Cell Biol.2001,152:657-668.
[51]Klionsky, D. J., Meijer, A. J., Codogno. P. Autophagy and p70S6 kinase[J]. Autophagy.2005,1:59-51.
[52]Kamada, Y., Funakoshi, T., Shintani, T.,etal. Tor-mediated induction of autophagy via an Apgl protein kinase complex[J].J Cell Biol.2000,150:1507-1513.
[53]Baehrecke, E. H. Autophagy:dual roles in life and death? [J].Nat Rev Mol Cell Biol.2005,6:505-510.
[54]Kim, J., Klionsky, D. J. Autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells[J].Annu Rev Biochem.2000,69:303-342.
[55]Furuya, D., Tsuji, N., Yagihashi, A.,etal. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity[J].Exp Cell Res.2005,307:26-40.
[56]Sooparb, S., Price, S. R., Shaoguang, J.,etal. Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus[J].Kidney Int.2004,65:2135-2144.
[57]Marsh, B. J., Soden, C., Alarcon, C., etal. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells[J].Mol Endocrinol.2007,21:2255-2269.
[58]Choi, S. E., Lee, S. M., Lee, Y. J.,etal. Protective role of autophagy in palmitate-induced INS-1 beta-cell death[J].Endocrinology.2009,150:126-134.
[59]Bandyopadhyay, U., Cuervo, A. M. Chaperone-mediated autophagy in aging and neurodegeneration:lessons from alpha-synuclein[J]. Exp Geronto.2007,42:120-128.
[60]Kaniuk, N. A., Kiraly, M., Bates, H., etal. Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy[J]. Diabetes.2007,56:930-939.
[61]Hoyer, S. The aging brain. Changes in the neuronal insulin/insulin receptor signal transduction cascade trigger late-onset sporadic Alzheimer disease (SAD) [J].J Neural Transm.2002,109:991-1002.
[62]Haan, M. N. Therapy Insight:type 2 diabetes mellitus and the risk of late-onset Alzheimer's disease[J].Nat Clin Pract Neurol.2006,2:159-166.
[63]Jager, J., Gremeaux, T., Cormont, M., etal. Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression[J].Endocrinology.2007,148:241-251.
[64]Hoyer, S. Risk factors for Alzheimer's disease during aging. Impacts of glucose/energy metabolism[J].J Neural Transm Suppl.1998,54:187-194.
[65]Abrous, D. N., Koehl, M., Le Moal, M. Adult neurogenesis:from precursors to network and physiology[J]. Physiol Rev.2005,85:523-569.
[66]Brazil, D. P., Hemmings, B. A. Ten years of protein kinase B signalling:a hard Akt to follow[J].Trends Biochem Sci.2001,26:657-664.
[67]Gagne, J., Milot, M., Gelinas, S., etal. Binding properties of glutamate receptors in streptozotocin-induced diabetes in rats[J].Diabetes.1997,46:841-846.
[68]Dwyer, D. S., Liu, Y., Miao, S., Bradley, R. J. Neuronal differentiation in PC 12 cells is accompanied by diminished inducibility of Hsp70 and Hsp60 in response to heat and ethanol[J].Neurochem Res.1996,21:659-666.
[69]Nakatogawa, H., lchimura, Y., and Ohsumi. Y.) Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion[J].Cell.2007,130:165-178.
[70]Kabeya, Y., Mizushima, N., Ueno. T.,etal. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[J].EMBO J.2000,19:5720-5728.
[71]Kovacs, A. L., Molnar, K., and Seglen, P. O. Inhibition of autophagic sequestration and endogenous protein degradation in isolated rat hepatocytes by methylated adenosine derivatives[J]. FEBS Lett.1981:134,194-196.
[72]Wullschleger, S., Loewith, R., Oppliger, W., etal. Molecular organization of target of rapamycin complex 2,J Biol Chem.2005,280:30697-30704.
[2]Mijnhout, G. S., Scheltens, etal. Diabetic encephalopathy:A concept in need of a definition[J]. Diabetologia.2006,49:1447-1448.
[3]Tuzcu, M., and Baydas, G.. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats[J]. Eur J Pharmacol.2006,53:106-110.
[4]Reagan, L. P. Insulin signaling effects on memory and mood[J]. Curr Opin Pharmacol.2007,7:633-637.
[5]Ryan, Williams, Finegol.Cognitive dysfunction in adults with type 1 (insulin-dependent) diabetes mellitus of long duration:effects of recurrent hypoglycaemia and other chronic complications[J]. Diabetologia.1993,3:329-334.
[6]Spitzer, Barnow, Wallaschofski, Freyberger, etal. Association between depression and subclinical carotid atherosclerosis in patients with Type 1 diabetes [J].Diabet Med.2008,25:349-354.
[7]Mastrocola,Vercellinatto,Brignardello, etal. Oxidative and nitrosative stress in brain mitochondria of diabetic rats[J]. J Endocrinol.2005,187:37-44.
[8]Somfai, Ruzicska, Magyar, etal. Soluble semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation in streptozotocin-induced diabetic rats[J]. Neurochem Int.2006,48:746-752.
[9]Balasubramanyam,Hampe, Maldonado, M. Syndromes of ketosis-prone diabetes mellitus[J].Endocr Rev.2008,29:292-302.
[10]Planel, E., Tatebayashi, Y., Miyasaka, T,etal.Insulin dysfunction induces in vivo tau hyperphosphorylation through distinct mechanisms[J].J Neurosci.2007,27:13635-13648.
[11]Korf, E. S., White, L. R., Scheltens, P.,etal. Brain aging in very old men with type 2 diabetes: the Honolulu-Asia Aging Study[J].Diabetes Care.2006,29:2268-2274.
[12]Musen, G., Lyoo, I. K., Sparks, C. R., etal. Effects of type 1 diabetes on gray matter density as measured by voxel-based morphometry[J].Diabetes.2006,55:326-333.
[13]Park, C. R. Cognitive effects of insulin in the central nervous system[J]. Neurosci Biobehav Rev.2001,25:311-323.
[14]Perez, A., Morelli, L., Cresto, J. C.,etal. Degradation of soluble amyloid beta-peptides 1-40. 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains[J]. Neurochem Res.2000,25:247-255.
[15]White, M. F. The IRS-signaling system:a network of docking proteins that mediate insulin and cytokine action[J]. Recent Prog Horm Res.1998,53:119-138.
[16]Cole, G. M., Frautschy, S. A. The role of insulin and neurotrophic factor signaling in brain aging and Alzheimer's Disease[J]. Exp Gerontol.2007,42:10-21.
[17]Wertkin, A. M., Turner, R. S., Pleasure, S. J.,etal. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides[J].Proc Natl Acad Sci U S A. 1993,90:9513-9517.
[18]Tienari, P. J., Ida, N., Ikonen, E.,etal. Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons[J].Proc Natl AcadSci U S A.1997,94:4125-4130.
[19]Chyung, A. S., Greenberg, B. D., Cook, D. G.,etal. Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells[J]. J Cell Biol.1991,138:671-680.
[20]Xu, H., Sweeney, D., Wang, R.,etal. Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation[J].Proc Natl Acad Sci U S A.1997,94:3748-3752.
[21]Oh, D., Han, S., Seo, J.,etal. Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins[J]. JNeurosci.2010,30:14134-14144.
[22]Malone, J. I., Hanna, S., Saporta, S.,etal. Hyperglycemia not hypoglycemia alters neuronal dendrites and impairs spatial memory[J].Pediatr Diabetes.2008,9:531-539.
[23]Fernando, R. N., Albiston, A. L., Chai, S. Y. The insulin-regulated aminopeptidase IRAP is colocalised with GLUT4 in the mouse hippocampus--potential role in modulation of glucose uptake in neurones? [J].Eur J Neurosci.2008,28:588-598.
[24]Reiter, R. J. Oxidative processes and antioxidative defense mechanisms in the aging brain[J].FASEB J.1995,9:526-533.
[25]Dalal, P. M., and Parab, P. V. Cerebrovascular disease in type 2 diabetes mellitus[J].Neurol India.2002,50:380-385.
[26]Zochodne, D. W. Diabetic neuropathies:features and mechanisms[J].,Brain Pathol.1999,9:369-391.
[27]Shinozaki, K., Naritomi, H., Shimizu, T., etal. Role of insulin resistance associated with compensatory hyperinsulinemia in ischemic stroke[J].Stroke.1996,27:37-43.
[28]Pike, L. J. Lipid rafts:bringing order to chaos[J]. J Lipid Res.2003,44:655-667.
[29]Meer, G. Cell biology. The different hues of lipid rafts[J].Science.2002,296:855-857.
[30]Pfrieger, F. W. Role of cholesterol in synapse formation and function[J].Biochim Biophys Acta.2003,1610:271-280.
[31]Heverin, M., Meaney, S., Lutjohann, D.,etal. Crossing the barrier:net flux of 27-hydroxycholesterol into the human brain[J].J Lipid Res.2005,46:1047-1052.
[32]Puglielli, L., Tanzi, R. E., Kovacs, D. M. Alzheimer's disease:the cholesterol connection[J].Nat Neurosci.2003,6:345-351.
[33]Refolo, L. M., Malester, B., LaFrancois, J., etal. Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model[J].Neurobiol Dis. 2000,7:321-331.
[34]Sparks, D. L., Liu, H., Gross, D. R.,etal. Increased density of cortical apolipoprotein E immunoreactive neurons in rabbit brain after dietary administration of cholesterol[J].Neurosci Lett.1995,187:142-144.
[35]Wahrle, S. E., Jiang, H., Parsadanian, M.etal. Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease[J].J Clin Invest.2008,118:671-682.
[36]Wollmer, M. A., Streffer, J. R., Lutjohann, D., etal. ABCA1 modulates CSF cholesterol levels and influences the age at onset of Alzheimer's disease[J]. Neurobiol Aging.2003,24:421-426.
[37]Mizushima, N.,Klionsky, D. J. Protein turnover via autophagy:implications for metabolism[J].Annu Rev Nutr.2007,27:19-40.
[38]Lemasters, J. J., Qian, T., He, L.,etal. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy [J]. Antioxid Redox Signal.2002,4:769-781.
[39]Bellu, A. R., Kiel, J. A. Selective degradation of peroxisomes in yeasts[J].Microsc Res Tech.2003,61:161-170.
[40]Roberts, P., Moshitch-Moshkovitz, S., Kvam, E., etal. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae[J].Mol Biol Cell.2003,14:129-141.
[41]Iwata, A. Neurodegenerative disease and autophagy[J].Rinsho Shinkeigaku.2006,46:887-889.
[42]Cuervo, A. M., and Dice, J. F. (1996) A receptor for the selective uptake and degradation of proteins by lysosomes, Science 273,501-503.
[43]Ashford, T. P., Porter, K. R. Cytoplasmic components in hepatic cell lysosomes[J].J Cell Biol.1962,12:198-202.
[44]Biederbick, A., Kern, H. F., Elsasser, H. P. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles[J].,Eur J Cell Biol.1995,66:3-14.
[45]Mizushima, N., Yamamoto, A., Matsui, M., etal. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[J].Mol Biol Cell.2004,15:1101-1111.
[46]Kabeya, Y., Mizushima, N., Yamamoto, A.,etal. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-Ⅱ formation[J].J Cell Sci.2004,117:2805-2812.
[47]Seglen, P. O., Gordon, P. B.3-Methyladenine:specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes[J].Proc Natl Acad Sci U S A.1982,79:1889-1892.
[48]Blommaart, E. F., Krause, U., Schellens, J. P.,etal. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes[J].Eur J Biochem.1997,243:240-246.
[49]Tanida, I., Ueno, T., Kominami, E. LC3 conjugation system in mammalian autophagy[J]. Int J Biochem Cell Biol.2004,36:2503-2518.
50] Mizushima, N., Yamamoto, A., Hatano. M. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells[J].J Cell Biol.2001,152:657-668.
[51]Klionsky, D. J., Meijer, A. J., Codogno. P. Autophagy and p70S6 kinase[J]. Autophagy.2005,1:59-51.
[52]Kamada, Y., Funakoshi, T., Shintani, T.,etal. Tor-mediated induction of autophagy via an Apgl protein kinase complex[J].J Cell Biol.2000,150:1507-1513.
[53]Baehrecke, E. H. Autophagy:dual roles in life and death? [J].Nat Rev Mol Cell Biol.2005,6:505-510.
[54]Kim, J., Klionsky, D. J. Autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells[J].Annu Rev Biochem.2000,69:303-342.
[55]Furuya, D., Tsuji, N., Yagihashi, A.,etal. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity[J].Exp Cell Res.2005,307:26-40.
[56]Sooparb, S., Price, S. R., Shaoguang, J.,etal. Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus[J].Kidney Int.2004,65:2135-2144.
[57]Marsh, B. J., Soden, C., Alarcon, C., etal. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells[J].Mol Endocrinol.2007,21:2255-2269.
[58]Choi, S. E., Lee, S. M., Lee, Y. J.,etal. Protective role of autophagy in palmitate-induced INS-1 beta-cell death[J].Endocrinology.2009,150:126-134.
[59]Bandyopadhyay, U., Cuervo, A. M. Chaperone-mediated autophagy in aging and neurodegeneration:lessons from alpha-synuclein[J]. Exp Geronto.2007,42:120-128.
[60]Kaniuk, N. A., Kiraly, M., Bates, H., etal. Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy[J]. Diabetes.2007,56:930-939.
[61]Hoyer, S. The aging brain. Changes in the neuronal insulin/insulin receptor signal transduction cascade trigger late-onset sporadic Alzheimer disease (SAD) [J].J Neural Transm.2002,109:991-1002.
[62]Haan, M. N. Therapy Insight:type 2 diabetes mellitus and the risk of late-onset Alzheimer's disease[J].Nat Clin Pract Neurol.2006,2:159-166.
[63]Jager, J., Gremeaux, T., Cormont, M., etal. Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression[J].Endocrinology.2007,148:241-251.
[64]Hoyer, S. Risk factors for Alzheimer's disease during aging. Impacts of glucose/energy metabolism[J].J Neural Transm Suppl.1998,54:187-194.
[65]Abrous, D. N., Koehl, M., Le Moal, M. Adult neurogenesis:from precursors to network and physiology[J]. Physiol Rev.2005,85:523-569.
[66]Brazil, D. P., Hemmings, B. A. Ten years of protein kinase B signalling:a hard Akt to follow[J].Trends Biochem Sci.2001,26:657-664.
[67]Gagne, J., Milot, M., Gelinas, S., etal. Binding properties of glutamate receptors in streptozotocin-induced diabetes in rats[J].Diabetes.1997,46:841-846.
[68]Dwyer, D. S., Liu, Y., Miao, S., Bradley, R. J. Neuronal differentiation in PC 12 cells is accompanied by diminished inducibility of Hsp70 and Hsp60 in response to heat and ethanol[J].Neurochem Res.1996,21:659-666.
[69]Nakatogawa, H., lchimura, Y., and Ohsumi. Y.) Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion[J].Cell.2007,130:165-178.
[70]Kabeya, Y., Mizushima, N., Ueno. T.,etal. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[J].EMBO J.2000,19:5720-5728.
[71]Kovacs, A. L., Molnar, K., and Seglen, P. O. Inhibition of autophagic sequestration and endogenous protein degradation in isolated rat hepatocytes by methylated adenosine derivatives[J]. FEBS Lett.1981:134,194-196.
[72]Wullschleger, S., Loewith, R., Oppliger, W., etal. Molecular organization of target of rapamycin complex 2,J Biol Chem.2005,280:30697-30704.