1093-OR: Metabolite-Driven Reversal of Adipocyte Insulin Resistance Identified by Reliance, a Novel Live Lipolysis Screening Platform

Introduction and Objective: Lipolysis is a central regulator of metabolic homeostasis, and impaired insulin-mediated suppression of lipolysis contributes to insulin resistance and type 2 diabetes. Gut microbiome-associated metabolites are emerging modulators of host energy metabolism and insulin sensitivity, but their direct effects on lipolysis remain poorly defined. No high-throughput platform currently exists to assess insulin action on lipolysis. We developed RELIANCE, a live-cell, real-time platform for high-throughput assessment of insulin action on lipolysis, and applied it to identify metabolites that suppress lipolysis and enhance insulin sensitivity in adipocytes. Methods: RELIANCE was established in mouse 3T3-L1 adipocytes and validated in primary human adipocytes. Insulin resistance was induced in 3T3-L1 adipocytes using dexamethasone, and lipolysis was monitored in real time under basal, stimulated, and insulin-suppressed conditions, with rosiglitazone included as a positive control. A library of 182 microbiome-associated metabolites was screened for insulin-sensitizing activity. Lead candidates were further characterized by analysis of hormone-sensitive lipase (HSL) phosphorylation and insulin signaling pathways. Results: RELIANCE robustly detected impaired antilipolytic insulin action and its restoration by rosiglitazone and selected metabolites. Twenty-nine compounds improved insulin-mediated suppression of lipolysis at least as effectively as rosiglitazone, including kaempferol, curcumin, naringenin, 4-methylcatechol, dopamine, and the omega-3 fatty acids EPA and DHA. Dopamine, EPA, and DHA consistently enhanced insulin sensitivity in independent experiments, whereas other metabolites primarily reduced lipolysis via modulation of HSL phosphorylation. Conclusion: Using RELIANCE, we identified multiple metabolites capable of reversing adipocyte insulin resistance and highlight omega-3 fatty acid combinations as potent and safe modulators of insulin action. Disclosure B. Diel: None. E. Lorza-Gil: None. A.L. Birkenfeld: None. R.J. von Schwartzenberg: None. G. Sancar: None.