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- Title
Effect of Silencing Genes Involved in Type 2 Diabetes in Human T-Lymphocytes.
- Authors
Stentz, Frankie B.; Kitabchi, Abbas E.
- Abstract
Our studies have shown that certain genes associated with insulin signaling and carbohydrate metabolism are expressed at different levels in activated T-lymphocytes (T-cells) and muscle of type 2 diabetic(T2DM) subjects compared to normal controls. Using Affymetrix human microarrays we found the insulin receptor OR) is decreased over 2 fold in T2DM compared to normal subjects as is the GLUT4 receptor. Additionally, the TNFα gene expression and translation are greater than 2 fold in the T2DM compared to normal. To determine the effect of decreased gene expression on insulin signaling parameters such as GLUT 4 we used gene silencing. To silence the expression of the IR we used various concentrations of IR siRNA designed from Applied Biosystems(AB) incubated during the activation of T-cells and using quantitative reverse transcriptase real time-PCR (QRT-PCR) for determination of GLUT 4, IR and TNFα mRNA. The threshold values (Ct values) for IR and GLUT4 by QRT-PCR showed a 50, 87, and 97% reduction for IR mRNA using 0.1, 1 and 10 nM IR siRNA compared to baseline (0 nM IR siRNA or a negative IR siRNA); and 50, 75, and 84%, respectively, decrease in GLUT 4 mRNA compared to baseline. This demonstrates that the decrease in the expression of the IR caused a decrease in the expression of the GLUT4 gene. Thus, the decrease in the expression of the IR in the T-cells of diabetic subjects could be responsible for the decrease seen in the GLUT4 in these cells. We also studied the effect of silencing the TNFα gene in the T2DM T-cells using TNFα siRNA obtained from Applied Biosystems with the same conditions as above. The increasing amount of siRNA of 0.1, 1 and 10 nM TNFα siRNA resulted in decreases of 50, 75, and 84% respectively, compared to baseline (0 nM TNFα siRNA or a negative siRNA) for the TNFα mRNA and 50, 75 and 75%, increases for the GLUT 4 mRNA. Thus, demonstrating an increase in GLUT 4 expression with the decrease in TNFα expression in the T2DM T-cells. TNFα translated determined by ELISA also demonstrated a decrease in levels corresponding to the changes noted in the mRNA levels of TNFα. Conclusion, in T2DM silencing the IR gene decreases expression and translation of GLUT4; whereas, silencing the TNFα gene increases expression and translation of GLUT4 which demonstrates the effect of alteration of one gene involved in T2DM and the effect on other gene expressions and translations.
- Subjects
GENES; TYPE 2 diabetes; T cells; INSULIN; CARBOHYDRATE metabolism; MUSCLES
- Publication
Diabetes, 2007, Vol 56, pA665
- ISSN
0012-1797
- Publication type
Article