Genetic evidence for an inhibitory role of tomosyn in insulin-stimulated GLUT4 exocytosis.

Abstract

Exocytosis is a vesicle fusion process driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulin-stimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR-Cas9 genome editing to delete the two tomosyn-encoding genes in adipocytes. We observed that both basal and insulin-stimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α-SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin-stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn-arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.

• Authors: Shifeng Wang, Yinghui Liu, Lauren Crisman, Chun Wan, Jessica Miller, Haijia Yu, Jingshi Shen
• Journal: Traffic (Copenhagen, Denmark) (Traffic) Vol. 21 Issue 10 Pg. 636-646 (10 2020) ISSN: 1600-0854 [Electronic] England
• PMID: 32851733 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chemical References

• Glucose Transporter Type 4
• Insulin
• Nerve Tissue Proteins
• R-SNARE Proteins
• STXBP5 protein, human

Topics

• Cell Membrane (metabolism)
• Diabetes Mellitus, Type 2 (metabolism)
• Exocytosis
• Glucose Transporter Type 4 (genetics, metabolism)
• Humans
• Insulin (metabolism)
• Nerve Tissue Proteins (genetics, metabolism, physiology)
• Protein Transport
• R-SNARE Proteins (genetics, physiology)