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

Traffic. 2020 Oct;21(10):636-646. doi: 10.1111/tra.12760.

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.

Keywords: SNARE; exocytosis; glucose transporter type 4; insulin signaling; membrane fusion; membrane protein; membrane trafficking; tomosyn; vesicle fusion.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

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