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Rab GTPase-Activating Protein AS160 Is a Major Downstream Effector of Protein Kinase B/Akt Signaling in Pancreatic β-Cells

From the Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland

Corresponding author: Dr. Karim Bouzakri, University Medical Center, Department of Genetic Medicine and Development, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland. E-mail: karim.bouzakri{at}medecine.unige.ch

Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; ERK1/2, extracellular signal–regulated kinase 1/2; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; hGH, human growth hormone; IRS, insulin receptor substrate; JDRF, Juvenile Diabetes Research Foundation; PI 3-kinase, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKC, protein kinase C; Rab-GAP, Rab GTPase-activating protein; RNAi, RNA interference; shRNA, small hairpin RNA; TUNEL, transferase-mediated dUTP nick-end labeling

OBJECTIVE— Protein kinase B/Akt plays a central role in β-cells, but little is known regarding downstream Akt substrates in these cells. Recently, Rab GTPase-activating protein AS160, a substrate of Akt, was shown to be involved in insulin modulation of GLUT4 trafficking in skeletal muscle and adipose tissue. The aim of this study was to investigate the expression and potential role of AS160 in β-cells.

RESEARCH DESIGN AND METHODS— AS160 mRNA expression was measured in mouse and human islets and fluorescence-activated cell sorted β-cells and compared in islets from control subjects versus individuals with type 2 diabetes. For knockdown experiments, transformed mouse insulin-secreting MIN6B1 cells were transfected with pSUPER-GFP plasmid encoding a small hairpin RNA against insulin receptor substrate (IRS)-2, AS160, or a negative control. Primary mouse islet cells were transfected with AS160 small interfering RNA.

RESULTS— AS160 was expressed in human and mouse pancreatic β-cells and phosphorylated after glucose stimulation. AS160 mRNA expression was downregulated in pancreatic islets from individuals with type 2 diabetes. In MIN6B1 cells, glucose induced phosphorylation of Akt and AS160, and this was mediated by insulin receptor/IRS-2/phosphatidylinositol 3-kinase independently of changes in cytosolic Ca²⁺. Knockdown of AS160 resulted in increased basal insulin secretion, whereas glucose-stimulated insulin release was abolished. Furthermore, β-cells with decreased AS160 showed increased apoptosis and loss of glucose-induced proliferation.

CONCLUSIONS— This study shows for the first time that AS160, previously recognized as a key player in insulin signaling in skeletal muscle and adipose tissue, is also a major effector of protein kinase B/Akt signaling in the β-cell.

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