Abstract The failure of metabolic tissues to appropriately respond to insulin (“insulin resistance”) is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employed phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults triggering insulin resistance, we observed marked rewiring of the insulin signaling network. This included both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying signaling changes common to multiple insults revealed subnetworks likely containing causal drivers of insulin resistance. Focusing on defective GSK3 signaling initially observed in a relatively small subset of well-characterized substrates, we employed a pipeline for identifying context-specific kinase substrates. This facilitated robust identification of widespread dysregulated GSK3 signaling. Pharmacological inhibition of GSK3 partially reversed insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that encompasses dysregulated GSK3 activity.

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance