Gene Expression Profiling on Insulin Resistance and Obes

为了鉴定可能导致胰岛素抵抗的新基因，我们使用1)包含6800个基因和35000个表达序列标签（est）的人类寡核苷酸微阵列和2)差异显示PCR检测了非糖尿病血统皮马印第安人骨骼肌基因表达谱。我们还与Ulf Smith博士（瑞典Sahlgrenska大学医院）和Samuel Cushman博士（NIDDK）合作，对选定的胰岛素抵抗和胰岛素敏感的瑞典受试者的骨骼肌和脂肪细胞样本进行了基因表达谱分析。差异表达基因/ESTs在胰岛素抵抗中的潜在作用将通过以下方法进行评估：1)它们在相关代谢途径中的功能，以及对皮马人样本的检测；2)它们在皮马人群全基因组扫描中相对于胰岛素作用和糖尿病连锁峰的染色体位置。采用实时荧光定量PCR进一步检测所选基因在骨骼肌中的表达水平。这个项目的手稿已经提交了。以往的研究表明，原代培养的骨骼肌细胞的生化特性与供体的临床参数有很强的相关性。为了确定胰岛素抵抗和胰岛素敏感受试者肌肉组织中哪些差异表达基因可能是原发性异常，而不是继发性异常，我们还建议比较同源培养成肌细胞和肌管的基因谱。我们已经开始进行类似的实验，利用新鲜分离的脂肪细胞和培养的脂肪前细胞，从瘦弱和肥胖的皮马印第安人身上识别肥胖易感基因。利用寡核苷酸微阵列，我们比较了10个瘦皮马印第安人和10个肥胖皮马印第安人的脂肪细胞的表达谱。我们正在为这个项目分析现有的数据并收集更多的样本。

To identify novel genes that may contribute to insulin resistance, we examined skeletal muscle gene expression profiles in non-diabetic full-blooded Pima Indians using 1) human oligonucleotide microarrays containing 6800 genes and 35000 expressed sequence tags (ESTs), and 2) differential display PCR. We have also performed gene expression profiling in skeletal muscle and adipocyte samples from selected insulin resistant and insulin sensitive Swedish subjects in collaboration with Dr. Ulf Smith (Sahlgrenska University Hospital, Sweden)and Dr. Samuel Cushman (NIDDK). The potential roles of the differentially expressed genes/ESTs in insulin resistance will be evaluated by examining 1) their functions in relevant metabolic pathways, and additionally for Pima samples: 2) their chromosomal locations relative to peaks of linkage to insulin action and diabetes in a genome-wide scan in the Pima population. Skeletal muscle expression levels of selected genes were further investigated using real-time PCR. The manuscript on this project has been submitted. Previous studies have indicated strong correlations between biochemical characteristics of primary cultured skeletal muscle cells and clinical parameters of the donors. To determine which of the differentially expressed genes in muscle tissue of insulin resistant vs. insulin sensitive subjects may be primary, rather than secondary abnormalities, we propose also to compare the gene profiles of the cognate cultured myoblasts and myotubes. We have started to perform similar experiments to identify obesity susceptibility genes using freshly isolated adipocytes and cultured pre-adipocytes from lean and obese Pima Indians. Using oligonucleotide microarrays, we compared the expression profiles of adipocytes from 10 lean vs. 10 obese Pima Indians. We are analyzing the current data and collecting more samples for this project.