GLUT1 Redistribution Assay

• Designed to assay compounds for their ability to modulate translocation of GLUT1 to the plasma membrane
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Glucose transporters (GLUT) are a family of membrane proteins which includes twelve members, numbered 1-12. A thirteenth member of the GLUT family is the myoinositol transporter, HMIT1. GLUT1 is highly expressed in erythrocytes and brain, while GLUT4 is responsible for insulin-regulated glucose transport in adipose tissues, heart muscles, and skeletal muscles. Insulin triggers translocation of GLUT1 from intracellular membrane compartments to the plasma membrane. This translocation involves the PI3K pathway, and GLUT1 translocation is inhibited by the specific PI3K inhibitor wortmannin.

Figure 1. Translocation of GLUT1-EGFP in response to insulin. Cells were treated with 100 nM insulin for 5 min (right panel) or untreated (DMSO control, left panel). Arrows indicate the GLUT1-EGFP translocation from the cytoplasm to the plasma membrane that is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the GLUT1 assay: A) Insulin concentration response in the GLUT1 agonist assay (n=16). The EC₅₀ is ~24 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with insulin for 5 min. Cells were then fixed and GLUT1 translocation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (1 μM insulin) and negative control (0.125% DMSO). B) Wortmannin concentration response in the GLUT1 assay run in antagonist format (n=16). Cells were preincubated for 20 min with a half log dilution series of test compound (wortmannin), followed by a 5 minute treatment with 100 nM insulin. Cells were then fixed and analyzed on the Cellomics ArrayScan V^TI Reader. % activity was calculated relative to the positive (150 nM wortmannin) and negative control (0.25% DMSO).The EC₅₀ of wortmannin is ~17 nM.