チアゾリジン化合物のPPARγ非依存性経路を介した新規インスリン作用増強剤の開発

2013 Fiscal Year Annual Research Report

• Project/Area Number: 13F03089
• Research Institution: The University of Tokyo
• Principal Investigator: 高橋 仲一郎 東京大学, 大学院農学生命科学研究科, 准教授
• Co-Investigator(Kenkyū-buntansha): YU Bu-chin 東京大学, 大学院農学生命科学研究科, 外国人特別研究員
• Keywords: IRS-associated protein / DGKζ / PIP5k1α / GKAP42 / Insulin resistance / Adipocytes / Glucose uptake / GLUT4 translocation

Research Abstract

In the first year, our purpose is to investigate the regulatory mechanism of IRS-associated proteins, diacylglycerol kinase ζ(DGKζ) and 42-kDa cGMP-dependent protein kinase anchoring protein (GKAP42), in response to insulin stimulation.
DGKζ has been reported to phosphorylate diacylglycerol (DG) to produce phosphatidic acid (PA). DGKζ was also reported to interact with phosphatidylinositol 4 phosphate 5 kinase 1 α (PIP5K1α), which is activated by PA. Therefore, we evaluated the roles of DGKζ and PIP5K1α in insulin-induced glucose uptake and GLUT4 translocation in 3T3-L 1 adipocytes.
Our results indicated that when DGKζ was knocked down in 3T3-L1 adipocytes by siRNA, insulin-induced GLUT4 translocation and glucose uptake were inhibited, even though insulin-dependent IRS-1 tyrosine phosphorylation and activation of the downstream signaling were not affected. Next, we used PIP5K1α knockdown to investigated whether PIP5K1α involve in the DGKζ-mediated signal in 3T3-L1 adipocytes. Our data su ggest a novel regulatory mechanism of glucose uptake and GLUT4 translocation in adipocytes in which DGKζ regulates GLUT4 translocation and glucose uptake through PIP5K1α activation.
We also elucidated the roles of GKAP42 in LPS-induced insulin resistance in 3T3-L1 adipocytes. In 3T3-L1 adipocytes treated with LPS, we observed an increase in GKAP42 protein levels and inhibition of insulin-dependent glucose uptake. To test the effects of increases in GKAP42 on GLUT4 translocation to the plasma membrane, we performed myc-GLUT4-GFP translocation assay using 3T3-L1 adipocytes transfected with both GKAP42 and myc-GLUT4-GFP expression vectors.
Overexpression of GKAP42 inhibited GLUT4 translocation to the plasma membrane with insulin stimulation, whereas GKAP42 overexpression enhanced it under the basal condition. In our models, LPS increased GKAP42 protein levels leading to impairment of insulin-dependent glucose uptake through disruption of GLUT4 translocation to plasma membrane in adipocytes.
Taken together with previous results that GKAP42 knockdown impaired IRS-1-mediated insulin signal, leading to insulin resistance, our data suggest that GKAP possesses multiple functions such as maintenance of IRS1-mediated insulin signals at low expression levels and inhibition of GLUT4 translocation to the plasma membrane at high expression levels.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Base on the present results, we established the regulatory mechanism of DGKζ and GKAP on the GLUT4 translocation and glucose uptake. Therefore, we will continue to find the low molecular weight substances, which enhance the dissociation of DGKζ from IRS1 or the association of GKAP with IRS, for enhancing the insulin signaling or treating the insulin resistance.

Strategy for Future Research Activity

In the second year, using split ELISA, high-throughput screening of chemical library (RIKEN, NPDepo) or food factor library is performed for the low molecular weight substances that show activity to dissociate the interaction between IRS-1 and DGKζ or to enhance the interation between IRS-1. And then, we will evaluate the affinity of the obtained low molecular weight substances with IRS-1, GKAP42 or DGKζ by using Biacore TM system. That technology is based on surface plasmon resonance (SPR), an optical phenomenon that enables detection of protein-protein interaction in real time. The Biacore TM system can be used in determination of active concentration as well as characterization of molecular interactions in terms of both affinity and chemical kinetics. Therefore, the Biacore TM system is performed for the low molecular weight substances that show activity to dissociate DGKζ from IRS1 or associate GKAP42 with IRS1 to enhance insulin signaling in glucose uptake and GLUT4 translocation. We then evaluate the effects of obtained low molecular weight substances to restore TNFα-induced repression in insulin signaling followed by insulin-dependent glucose uptake and GLUT4 translocation.