| Type 2 diabetes is a major cause of blindness, kidney failure, ischemic heart disease, loss of limb, stroke, and overall mortality. This disease affects a total of 140 million people worldwide. Its prevalence in different populations ranges from >50% among Pima Indians to 2% among Indian tribes in Chile. Insulin resistance is commonly defined by reduced insulin sensitivity of peripheral tissues, such as skeletal muscle and fat, and represents the major abnormality of type 2 diabetes. Decreased insulin induced glucose disposal in skeletal muscle (insulin resistance) is a common pathophysiological trait implicated in the development of type 2 diabetes. Several lines of evidence clearly point to genetic factors as important determinants of insulin sensitivity. A number of candidate genes for insulin resistance have been evaluated, including insulin receptor substrate-1, glycogen synthetase, UCP-3, GLUT4, hexokinase II, phosphatidylinositol 3-kinase, serine-threonine kinase, Rad genes, and calpain-10. Although the expressions of several of these genes are altered in type 2 diabetes, none has emerged as the leading candidate for causing type 2 diabetes. Furthermore, it is not clear whether theses changes primarily induce insulin resistance by defects in insulin signaling or are simply secondary phenomenon of impaired insulin sensitivity. It is highly likely that type 2 diabetes results from interactions between many genetic factors and the environment. The identification of genes involved will hopefully allow modifications of lifestyle to prevent or delay the onset of diabetes. It is now possible to examine the transcript profile of several thousand genes simultaneously using microarray technology. The technique offers powerful tool to decipher the pathophysiology of type 2 diabetes at the molecular level, thereby leading to a better fundamental understanding of muscle insulin resistance and providing new therapeutic targets for treatment of this common disease.[ Objective ]To explore the molecular mechanism of insulin resistance, obtain differentially expressed genes in skeletal muscle between the IR and non-IR patients using cDNA microarray and identify 2 genes with Northern blot analysis.[Methods]K The clinical characteristics of the subjects are shown in Table 1. The control subjects (n=4) were matched for age, sex, and BMI with the diabetic subjects. The four diabetic subjects who were studied included one man and three women. All subjects were undergone lumbar disc herniation surgery and the skeletal muscle tissues were obtained during the operation. All of the samples were instantly frozen in liquid nitrogen and were divided into two parts, one for gene expression analysis; another for Northern blot.2^ Total RNAs were extracted from the skeletal muscle tissues after which were purified to mRNAs by oligotex. The PCR products of 4096 genes were spotted onto a chemical substance-coated glass plate in array using Cartesian Pixsys 7500. DNA was fixed onto the glass plate after series of treatments. Both mRNAs from the IR and non-IR normal skeletal muscle tissue were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray scanned for the fluorescent signals and showed differences between tissues. Then the differentially expressed genes were analyzed by ImaGeneS.O software.3-. Total RNAs were extracted from the skeletal muscle tissues. The primers of fatty acid binding protein 3(FABP3), cGMP-stimulated phosphodiesterase 2A(PDE2A) and GAPDH were designed and synthesized. After that, identify these 2 genes with Northern blot analysis.I Results}1 -. We obtain 37 differentially expressed genes to human insulin resistance through four times hybridizations and scanning. These 37 genes were all downregulated in type 2 diabetic skeletal muscle tissues. The genes are associated with metabolism, cell signal transduction, oncogene, anti-oncogene, ion tra...
|
PDF Full Text Request