Role of Intrinsic Disorder in Alternatively Spliced Sortilin Variants in 3T3L1 Adipocytes

Abstract

Type 2 diabetes mellitus (T2DM) is a metabolic disorder defined by systemic insulin resistance affecting more than 400 million people worldwide. Adipose tissue is an important regulator of glucose metabolism as well as an endocrine organ secreting hormones and cytokines. Defective signaling in insulin resistant adipocytes leads to impaired trafficking of main glucose transporter 4 (Glut4) thus leading to reduced glucose uptake and hyperglycemia. Notably, trafficking protein sortilin has an important role in regulating Glut4 movement in adipocytes thereby affecting glucose levels. We demonstrate that in mouse 3T3L1 adipocytes, alternative splicing generates a sortilin splice variant with an included exon 17b (Sort17b) expressed along with SortWT. Insulin resistance induced by high serum increases expression levels of Sort17b. Further, our results demonstrate that increased Sort17b decreases Glut4 translocation and glucose uptake. Using bioinformatic analysis we show inclusion of exon 17b introduces a novel intrinsic disorder region in a crucial ligand binding regulation site. Since intrinsic disorder is a measure of lack of protein folding potentially affecting ligand binding and protein interactions, we performed molecular modeling of both sortilin variants (Sort17b and SortWT) and analyzed flexibility of protein backbone (RMSD) and individual residues (RMSF). Our results show increased flexibility in Sort17b within the amino acids of exon 17b. Using co-immunoprecipitation assays we demonstrate that Sort17b binds robustly to Glut4 and the binding efficiency is increased in insulin resistant adipocytes. Overall, the results demonstrate that the novel splice variant of sortilin in mouse 3T3L1 adipocytes contains an intrinsically disordered region that affects glucose metabolism and may be a potential therapeutic target in T2DM.