title: Development of a novel GLUT4 translocation assay for identifying potential novel therapeutic targets for insulin sensitization.
abstract: GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKC(theta) (protein kinase C theta). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.
authors.affiliation: Metabolic Disorders-Diabetes, Merck and Co., Inc., Rahway, NJ 07065, USA.
authors.forename: Franklin
authors.initials: F
authors.lastname: Liu
authors.forename: Qing
authors.initials: Q
authors.lastname: Dallas-Yang
authors.forename: Gino
authors.initials: G
authors.lastname: Castriota
authors.forename: Paul
authors.initials: P
authors.lastname: Fischer
authors.forename: Francesca
authors.initials: F
authors.lastname: Santini
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Taro E
authors.initials: TE
authors.lastname: Akiyama
authors.forename: Joel P
authors.initials: JP
authors.lastname: Berger
authors.forename: Bei B
authors.initials: BB
authors.lastname: Zhang
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Jiang
mesh_terms: D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007004:Hypoglycemic Agents / Q000302:isolation & purification / Q000627:therapeutic use*; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism; D007333:Insulin Resistance; D007527:Isoenzymes / Q000235:genetics; D048055:Mitogen-Activated Protein Kinase 8 / Q000235:genetics; D051059:PTEN Phosphohydrolase / Q000235:genetics; D019308:Palmitic Acid / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics; D000074121:Protein Kinase C-theta; D021381:Protein Transport; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics; D014162:Transfection
chemical_list: D005947:Glucose; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D048055:Mitogen-Activated Protein Kinase 8; D051059:PTEN Phosphohydrolase; D019308:Palmitic Acid; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D019893:Ribosomal Protein S6 Kinases; C495692:SLC2A4 protein, human; C076233:PRKCQ protein, human; C494929:PTEN protein, human