Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes

Abstract

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein–protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.

Sorting through the extensive and confusing roles of sortilin in metabolic disease

“…It is likely that the mechanism by which sortilin is involved in GSV biogenesis is through the binding of GLUT4 by its luminal domain and the APs AP1 and GGA by its cytoplasmic domain to traffic GLUT4 into vesicles ( 148 , 304 , 305 , 306 , 307 ). Recently, one group has discovered that the abundance of an alternatively spliced sortilin variant that includes the alternative exon 17b (Sort 17b ) increases with insulin resistance in mouse 3T3L1 adipocytes, the implications of which are still to be determined ( 308 ). In C2C12 myotubes, saturated fatty acids induce downregulation of sortilin in a PKC-dependent manner.…”

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“…Other studies reported that deprivation of glucose attenuates Sortilin levels in skeletal muscle [284], and that insulin resistance induces hepatic degradation of Sortilin in mice [285], suggesting Sortilin's role also in glucose homeostasis. Indeed, Sortilin enables the biogenesis of glucose transporter 4 (GLUT4) storage vesicles by binding and targeting GLUT4 into maturing vesicles, and by controlling GLUT4 recycling [286][287][288][289][290]. When insulin levels decline, GLUT4-positive vesicles are retrieved from the plasma membrane to the endosomal compartment.…”

Regulation of Human Sortilin Alternative Splicing by Glucagon-like Peptide-1 (GLP1) in Adipocytes

“…An alternatively spliced human sortilin variant was identified in neurons with inclusion of an exon, named exon 17b, inserted between exons 17 and 18 in sortilin pre-mRNA [ 22 , 23 ]. Importantly, the mouse and human sortilin pre-mRNA sequences are different, and it has been demonstrated [ 22 , 23 , 24 ] that a longer sortilin splice variant is present in mouse cells, which is not seen in human cells. In neurons, sortilin functions as a receptor for progranulin, while in adipocytes, sortilin is a component of the GSVs and functions as a sorting protein.…”