No 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 employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

No abstract

Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, hampering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.