Morand Jean-Paul F, Macri Joseph, Adeli Khosrow
Division of Clinical Biochemistry, and Structural Biology and Biochemistry, Department of Pediatric Laboratory Medicine, Research Institute, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada.
J Biol Chem. 2005 May 6;280(18):17626-33. doi: 10.1074/jbc.M413343200. Epub 2005 Mar 9.
Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold down-regulated, whereas alpha-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be down-regulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.
肝脏胰岛素抵抗和脂蛋白过量生成是代谢综合征和胰岛素抵抗状态的常见特征。最近开发了一种用果糖喂养的胰岛素抵抗仓鼠模型，以研究肝脏胰岛素抵抗的发展与致动脉粥样硬化脂蛋白过量生成之间的联系机制。在此，我们报告了对从用果糖喂养的仓鼠肝脏中分离出的内质网（ER）组分中的蛋白质表达谱进行的系统分析，目的是鉴定参与胰岛素抵抗和脂蛋白失调的肝脏并发症的新候选蛋白。我们使用二维凝胶电泳和质谱对正常饮食（对照）和果糖喂养（胰岛素抵抗）仓鼠的肝脏内质网相关蛋白进行了分析。总共使用了26块肝脏内质网的大规模二维凝胶，以鉴定34个差异表达的肝脏内质网蛋白点，观察到这些蛋白点在用果糖喂养和出现胰岛素抵抗时差异表达至少2倍。通过基质辅助激光解吸电离-四极杆飞行时间（MALDI-Q-TOF）、MALDI-TOF-源后衰变以及使用ProteinProspector MS-fit和MS-tag进行数据库挖掘，或使用聚焦的啮齿动物或哺乳动物搜索引擎PROWL ProFound进行搜索，来鉴定差异表达的蛋白质。结果显示，肝脏内质网蛋白ER60、ERp46、ERp29、谷氨酸脱氢酶和TAP1下调超过2倍，而α-葡萄糖苷酶、P-糖蛋白、纤维蛋白原、蛋白二硫键异构酶、GRP94和载脂蛋白E在果糖喂养的仓鼠肝脏内质网中均上调。在果糖喂养/胰岛素抵抗仓鼠的肝细胞中，肝脏内质网中的7种ER60同工型均显示下调至少2倍。本文讨论了已明确鉴定的蛋白因子差异表达在肝脏胰岛素抵抗和脂蛋白异常发展中的意义。
