Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Niketa Patel, Ph.D.

Co-Major Professor

Robert Deschenes, Ph.D.

Committee Member

Denise Cooper, Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Subhra Mohapatra, Ph.D.

Keywords

Glut4, Glut4 Storage Vesicles, Insulin Resistance, SFRS10, Tra2β1

Abstract

Type 2 diabetes mellitus (T2DM) is a chronic and progressive metabolic disease defined by systemic insulin resistance. The inability of cells to respond to insulin and take up glucose results in elevated blood sugar that is detrimental to the overall health of patients. There is no cure for T2DM. Identification of new targets to alleviate hyperglycemia are crucial for treatment and prevention of metabolic diseases. Adipocytes play a central role in glycemic regulation by insulin stimulated glucose uptake, storage of excess nutrients, and secretion of adipokines. Major glucose transporter-4 (Glut4) is the main transporter responsible for postprandial clearance of glucose and lies in specialized vesicles called Glut4 Storage Vesicles (GSVs) in the cytoplasm of adipocytes. In healthy individuals, insulin signaling drives the translocation of Glut4 to the plasma membrane for glucose uptake. Sortilin is an important constituent of GSVs and sortilin’s C-terminal domain interacts with guiding proteins to determine GSV location in the trans-Golgi network.

We are the first the identify alternatively spliced sortilin variants in adipocytes and adipose tissue and show differential expression of the two splice variants in response to insulin resistance. The function and ligands of sortilin are tissue specific and effects of sortilin alternatively spliced variants are yet to be determined. We hypothesized that the alternatively spliced sortilin contributes to impaired Glut4 trafficking in diabetic adipocytes and aimed to characterize their behavior in murine 3T3L1 adipocytes and human adipose derived stem cells differentiated into mature adipocytes.

Murine 3T3L1 cells are the most used adipocyte cell model and has been critical in elucidating GSV development, GSV trafficking, and effects of insulin resistance. In this model, protein sortilin has been recognized as the major component responsible for GSV development and trafficking. Inclusion of exon 17b between exons 17 and 18 in murine sortilin pre-mRNA encodes 33 novel amino acids inserted between sortilin’s ectodomain and transmembrane domain. We demonstrated the presence of this alternatively spliced sortilin variant (Sort17b) and its enhanced expression with the induction of insulin resistance. 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 determined by 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.

Sortilin alternative splicing is species dependent. In murine 3T3L1’s inclusion of 99bp from exon 17b encoded 33 novel residues lengthening protein sortilin. However, in human adipocytes, insertion of exon 17b results in an in-frame stop codon and truncates the sortilin protein above the transmembrane domain (Sort_T). Our second aim was to identify and characterize the role of alternatively spliced sortilin in human adipose derived stem cells (hASCs) differentiated into adipocytes. Sort_T lacks the motif-rich cytoplasmic domain present in full length sortilin (Sort_FL) thus cannot participate in dynamic adapter binding and post translational modifications necessary for subcellular trafficking. We demonstrated elevated Sort_T expression in diabetic adipocytes and adipose tissue. To determine half-life, treatment with translation inhibitor cycloheximide demonstrated prolonged stability of Sort_T compared to Sort_FL. Overexpression of Sort_T in nondiabetic adipocytes resulted in reduced Glut4 levels, blunted insulin stimulated glucose uptake and enhanced soluble sortilin secretion into the cultured media. Molecular modeling and protein docking showed that Sort_T retained its ability to bind Glut4 and was confirmed using co-immunoprecipitation assays. We developed and verified a Sort_T specific antibody and using immunocytochemistry we confirmed increased colocalization of Sort_T with Glut4 in diabetic adipocytes. Further studies using immunocytochemistry and Sort_T overexpression in nondiabetic adipocytes demonstrated reduced colocalization of Glut4 to the plasma membrane and increased lysosomal targeting of Glut4. These studies identify a novel sortilin splice variant unknown in human adipocytes and characterized its involvement in glucose metabolism via Glut4 interaction and trafficking.

Incretin hormone glucagon-like peptide-1 (GLP1) is released in response to a meal and regulates glycemic control in a tissue specific manner. GLP1 is reduced in the serum of type 2 diabetic patients and is now widely used as a therapeutic to manage insulin resistance. Using human omental adipocytes, we demonstrated increased expression of Sort_T in diabetic adipocytes which was reversed with GLP1 treatment at physiological doses (8nM). A splicing minigene was generated by cloning human sortilin exon 17b between splice donor and splice acceptor sites and confirmed increased inclusion of exon 17b in diabetic adipocytes which was reduced with GLP1. We showed splice factor Tra2β1 as a novel regulator of sortilin alternative splicing in response to GLP1 signaling in adipocytes. Results were verified by sortilin exon 17b splicing minigene with knockdown and overexpression of Tra2β1. RNA immunoprecipitation assay was performed to confirm binding of Tra2β1 to sortilin pre-mRNA and reduced binding upon GLP1 treatment. Mutation of the Tra2β1’s consensus binding site in the sortilin minigene reduced exon 17b inclusion and custom Morpholino antisense oligonucleotides, masking Tra2β1 binding site, reduced Sort_T levels in diabetic adipocytes. Inhibition of PKCδ and PKA signaling pathways ablated GLP1’s ability to reduce Sort_T expression, implicating both pathways in regulation of sortilin alternative splicing in diabetic adipocytes. These results demonstrate the role of GLP1 and Tra2β1 in sortilin alternative splicing and thus glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of T2DM and metabolic syndrome.

The goal of this project is twofold: firstly, to determine the role of sortilin alternatively spliced variants in insulin resistance murine and human adipocytes and secondly, elucidate the role of GLP1, an incretin hormone and T2DM therapeutic, in the regulation of sortilin alternative splicing in adipocytes. This project pioneers research of hormone GLP1 regulating alternative splicing of proteins and will be the first to report truncated sortilin effects in human diabetic adipocytes. GLP1’s control over sortilin alternative splicing and ultimately major glucose transporter-4 location will bridge a gap in knowledge in reduced glucose uptake in insulin resistant adipocytes. These studies illustrate an undescribed mechanism in adipocytes that may aid in deciphering novel targets needed to develop more impactful therapeutics for T2DM.

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