A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling

Abstract

A new mouse model has been developed to study the localisation and trafficking of the glucose transporter GLUT4 in muscle. The mouse line has specific expression of a GFP and HA-epitope-tagged version of GLUT4 under the control of a muscle-specific promoter. The exofacial HA-tag has enabled fluorescent labelling of only the GLUT4 exposed at the external surface. A distinction between sarcolemma labelling and transverse-tubule labelling has also been possible because the former compartment is much more accessible to intact anti-HA antibody. By contrast, the Fab fragment of the anti-HA antibody could readily detect GLUT4 at the surface of both the sarcolemma and transverse tubules. Here, we have used this mouse model to examine the route taken by cardiomyocyte GLUT4 as it moves to the limiting external membrane surface of sarcolemma and transverse-tubules in response to insulin, contraction or activators of energy-status signalling, including hypoxia. HA-GLUT4-GFP is largely excluded from the sarcolemma and transverse-tubule membrane of cardiomyocytes under basal conditions, but is similarly trafficked to these membrane surfaces after stimulation with insulin, contraction or hypoxia. Internalisation of sarcolemma GLUT4 has been investigated by pulse-labelling surface GLUT4 with intact anti-HA antibody. At early stages of internalisation, HA-tagged GLUT4 colocalises with clathrin at puncta at the sarcolemma, indicating that in cells returning to a basal state, GLUT4 is removed from external membranes by a clathrin-mediated route. We also observed colocalisation of GLUT4 with clathrin under basal conditions. At later stages of internalisation and at steady state, anti-HA antibody labeled-GLUT4 originating from the sarcolemma was predominantly detected in a peri-nuclear compartment, indistinguishable among the specific initial stimuli. These results taken together imply a common pathway for internalisation of GLUT4, independent of the initial stimulus.

Measuring GLUT4 translocation in mature muscle fibers

“…Myotubes have been fixed at specific time points and labeled with a surface membrane impermeant anti-myc antibody in order to measure GLUT4-myc (with or without GFP) inserted into the plasma membrane by detecting the exofacial loop containing the myc eptitope (44). The tags have been combined, including GLUT4-myc-GFP, which allows simultaneously the detection of the GLUT4-GFP and the fraction of GLUT4-myc actually inserted into the membranes (15,44).…”

“…3). Another approach has been to isolate mature muscle fibers or cardiomyocytes from transgenic mice overexpressing tagged (myc, HA, and/or GFP) versions of GLUT4 and subsequently perform analysis in vitro (15,54). Alternatively, the use of various generations of membrane impermeable affinity photolabels that bind to endogenous glucose transporters have also provided important information about GLUT4 traffic in mature muscle (26) (Fig.…”

“…This means that GLUT4-GFP actually inserted in the plasma membranes cannot be separated from GLUT4-GFP in close vicinity of the membrane (within 200 nm). Despite this lack in resolution, recent studies using GLUT4-GFP with different exofacial tags have demonstrated that the fold increase in GFP fluorescence staining of the sarcolemma and T-tubules correlates well with the fold increase in the GLUT4 actually inserted into the membrane (assayed by antibody staining for the exofacial tags myc or HA) (15,54). Furthermore, compared with in vitro preparations of muscle, the extracellular environment that the muscle fiber is exposed to cannot be rapidly changed due to dependence on internal clearance of injected drugs by organs such as the liver and kidneys.…”

“…Muscle-specific transgenic overexpression of GLUT4-GFP with an HA tag in the exofacial membrane loop has been achieved recently, although potential effects on whole body glucose homeostasis were not reported (15). Also, musclespecific expression of GLUT4-myc in transgenic mice resulted in a threefold increase in GLUT4 levels in muscle and was in turn associated with increased glucose tolerance (54).…”

The many ways to regulate glucose transporter 4This paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process.

“…The extent of label remaining on the surface, or alternatively that internalized, is then quantified. Suitable labels for these types of experiments are nonpermeating affinity photolabels (Fazakerley et al 2009;Karlsson et al 2009) or antibodies to exofacially exposed epitopes (Antonescu et al 2008a;Kao et al 2000). Methodological details for the latter can be found in Antonescu et al 2008b andIshikura et al 2009.…”