Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide

Kevin P. Foley,Amira Klip

doi:10.1242/bio.20147898

Abstract

ABSTRACTGLUT4 constitutively recycles between the plasma membrane and intracellular depots. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting GLUT4 to the plasma membrane from insulin-responsive vesicles. Muscle is the primary site for dietary glucose deposition; however, how GLUT4 sorts into insulin-responsive vesicles, and if and how insulin resistance affects this process, is unknown. In L6 myoblasts stably expressing myc-tagged GLUT4, we analyzed the intracellular itinerary of GLUT4 as it internalizes from the cell surface and examined if such sorting is perturbed by C2-ceramide, a lipid metabolite causing insulin resistance. Surface-labeled GLUT4myc that internalized for 30 min accumulated in a Syntaxin-6 (Stx6)- and Stx16-positive perinuclear sub-compartment devoid of furin or internalized transferrin, and displayed insulin-responsive re-exocytosis. C2-ceramide dispersed the Stx6-positive sub-compartment and prevented insulin-responsive re-exocytosis of internalized GLUT4myc, even under conditions not affecting insulin-stimulated signaling towards Akt. Microtubule disruption with nocodazole prevented pre-internalized GLUT4myc from reaching the Stx6-positive perinuclear sub-compartment and from undergoing insulin-responsive exocytosis. Removing nocodazole allowed both parameters to recover, suggesting that the Stx6-positive perinuclear sub-compartment was required for GLUT4 insulin-responsiveness. Accordingly, Stx6 knockdown inhibited by ∼50% the ability of internalized GLUT4myc to undergo insulin-responsive re-exocytosis without altering its overall perinuclear accumulation. We propose that Stx6 defines the insulin-responsive compartment in muscle cells. Our data are consistent with a model where ceramide could cause insulin resistance by altering intracellular GLUT4 sorting.

Highlights

The regulation of glucose transport into muscle and fat cells is essential to glucose homeostasis, and is mediated by the Glucose Transporter 4 (GLUT4) protein
By tracking the intracellular sorting of GLUT4 after pulse-labeling at the cell surface, we show that GLUT4 sorts into a perinuclear subcompartment that is positive for Stx6 and devoid of furin or transferrin receptor (TfR); we show that C2-ceramide prevents this sorting in parallel with inhibiting GLUT4 reexocytosis, even when signaling to Akt is allowed to resume
Insulin-responding GLUT4-containing vesicles have been imaged within 200 nm of the plasma membrane of adipocytes (Bai et al, 2007; Chen et al, 2012; Huang et al, 2007; Lizunov et al, 2005; Xiong et al, 2010) and muscle cells (Boguslavsky et al, 2012; Sun et al, 2014), it is unknown how or where GSV/IRV are constituted to segregate away from constitutive recycling, as at steady-state GLUT4 is visualized both in the perinuclear region and in cytosolic vesicles (Boguslavsky et al, 2012; Chen et al, 2012; Foley et al, 2011; Randhawa et al, 2008; Xiong et al, 2010)

Summary

Introduction

The regulation of glucose transport into muscle and fat cells is essential to glucose homeostasis, and is mediated by the Glucose Transporter 4 (GLUT4) protein. To increase glucose uptake into muscle and adipose tissue, insulin signals promote translocation of GLUT4 to the plasma membrane, but, surprisingly, the intracellular localization of the GLUT4-retaining, insulinresponding intracellular compartment (commonly termed GLUT4storage vesicles or insulin-responding vesicles, respectively GSV or IRV) remains unknown (Foley et al, 2011; Rowland et al, 2011). Instead, this compartment is defined functionally (Govers et al, 2004; Karylowski et al, 2004) and can be crudely recovered by subcellular fractionation (Kupriyanova et al, 2002), i.e. GSV/ IRV are terms given to subcellular fractions containing insulinresponsive GLUT4. Difficulty in defining the intracellular localization of the GSV/IRV has left untested the possibility that defective GLUT4 sorting may contribute to C2-ceramide induced insulin resistance

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Discussion

Conclusion