Abstract
Pathways linking activation of the insulin receptor to downstream targets of insulin have traditionally been studied using a candidate gene approach. To elucidate additional pathways regulating insulin activity, we performed a forward chemical-genetics screen based on translocation of a glucose transporter 4 (Glut4) reporter expressed in murine 3T3-L1 adipocytes. To identify compounds with known targets, we screened drug-repurposing and natural product libraries. We identified, confirmed, and validated 64 activators and 65 inhibitors that acutely increase or rapidly decrease cell-surface Glut4 in adipocytes stimulated with submaximal insulin concentrations. These agents were grouped by target, chemical class, and mechanism of action. All groups contained multiple hits from a single drug class, and several comprised multiple structurally unrelated hits for a single target. Targets include the β-adrenergic and adenosine receptors. Agonists of these receptors increased and inverse agonists/antagonists decreased cell-surface Glut4 independently of insulin. Additional activators include insulin sensitizers (thiazolidinediones), insulin mimetics, dis-inhibitors (the mTORC1 inhibitor rapamycin), cardiotonic steroids (the Na+/K+-ATPase inhibitor ouabain), and corticosteroids (dexamethasone). Inhibitors include heterocyclic amines (tricyclic antidepressants) and 21 natural product supplements and herbal extracts. Mechanisms of action include effects on Glut4 trafficking, signal transduction, inhibition of protein synthesis, and dissipation of proton gradients. Two pathways that acutely regulate Glut4 translocation were discovered: de novo protein synthesis and endocytic acidification. The mechanism of action of additional classes of activators (tanshinones, dalbergiones, and coumarins) and inhibitors (flavonoids and resveratrol) remains to be determined. These tools are among the most sensitive, responsive, and reproducible insulin-activity assays described to date.
Highlights
Pathways linking activation of the insulin receptor to downstream targets of insulin have traditionally been studied using a candidate gene approach
The screening assay has two key features: the insulin sensitivity/responsiveness of the cells and the sensitivity, speed, and accuracy of the detection system. These screens identify compounds that have acute effects in adipocytes pretreated with insulin
The candidate genes tested were found either biochemically or by analogy to other trafficking processes. The role of these candidate genes in glucose transporter 4 (Glut4) trafficking and insulin signaling was tested using a combination of chemical reagents and DNA-based reagents
Summary
A high-throughput chemical– genetics screen in murine adipocytes identifies insulin-regulatory pathways. We identified, confirmed, and validated activators and inhibitors that acutely increase or rapidly decrease cell-surface Glut in adipocytes stimulated with submaximal insulin concentrations These agents were grouped by target, chemical class, and mechanism of action. This paper describes assays to identify and validate small molecules that enhance or inhibit insulin action in adipocytes using changes in cell-surface Glut as a marker for insulin activity. These high-throughput assays are among the most sensitive, responsive, and reproducible insulin-activity assays that have been reported. Confirming the power of the chemical genetics approach, both novel activators and inhibitors of insulin action were discovered These hits revealed previously unidentified regulatory pathways for insulin signaling as well as for Glut trafficking. The potential role of inhibition of Glut translocation in these adverse drug affects is discussed
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