Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization
Hajime Kanda, … , Jun-ichi Miyazaki, Masato Kasuga
Hajime Kanda, … , Jun-ichi Miyazaki, Masato Kasuga
Published February 1, 2005
Citation Information: J Clin Invest. 2005;115(2):291-301. https://doi.org/10.1172/JCI22681.
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Article Metabolism

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization

Abstract

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c−/− mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c−/− cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c−/− cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.

Authors

Hajime Kanda, Yoshikazu Tamori, Hiroaki Shinoda, Mari Yoshikawa, Motoyoshi Sakaue, Jun Udagawa, Hiroki Otani, Fumi Tashiro, Jun-ichi Miyazaki, Masato Kasuga

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Figure 10

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Normalization both of syntaxin4 expression and of the insulin sensitivit...
Normalization both of syntaxin4 expression and of the insulin sensitivity of GLUT4 externalization in Munc18c–/– adipocytes by restoration of Munc18c expression. (A) Syntaxin4 expression. Munc18c+/+ or Munc18c–/– adipocytes were infected with an adenoviral vector for Munc18c (adex-Munc18c) or with the corresponding empty vector (control adex), as indicated. After 2 days, cell lysates were subjected to immunoblot analysis with antibodies to Munc18c (upper panel) or to syntaxin4 (lower panel). (B) Immunofluorescence microscopic analysis of Munc18c in adipocytes. Munc18c+/+ adipocytes infected with control adex, Munc18c–/– adipocytes infected with control adex, or Munc18c–/– adipocytes infected with adex-Munc18c at an MOI of 5 were fixed, permeabilized, subjected to immunofluorescence staining with antibodies to Munc18c, and analyzed by confocal microscopy. Arrows mark the localizations of differentiated adipocytes, and the arrowhead indicates undifferentiated cells. Scale bar: 10 μm. (C and D) GLUT4 translocation and externalization. Cells infected as in A were assayed for the translocation (C) and externalization (D) of GLUT4 in response to insulin as described in Figure 8. Data are mean ± SE of values from 3 separate experiments. *P < 0.05 versus the value for Munc18c–/– cells infected with control adex and stimulated with 1 nM insulin (Student’s t test).