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.

Fig. 1.

Translocation of HA-GLUT4-GFP to the sarcolemma of insulin-, contraction- and hypoxia-stimulated cardiomyocytes. Isolated cardiomyocytes expressing HA-GLUT4-GFP were maintained in a basal unstimulated state or incubated with 30 nM insulin for 30 minutes, electrically induced to contract for 5 minutes or incubated under hypoxic conditions for 15 minutes. All cells were viewed in approximately the same focal plane. Bars, 20 μm. GFP (top panels) was detected between 505 and 530 nm, HA antibody (middle panels) was detected using Alexa-633 secondary antibody between 657 and 753 nm. Merged images are in the bottom panels. Results shown are from representative images from ∼25 examined cells in five experiments.

Fig. 2.

Translocation of HA-GLUT4-GFP to the transverse tubules of insulin-, contraction- and hypoxia-stimulated cardiomyocytes. Isolated cardiomyocytes expressing HA-GLUT4-GFP were either maintained in the basal unstimulated state (A) or incubated with 30 nM insulin for 30 minutes (B), electrically induced to contract for 5 minutes at 100 V, 10 Hz, pulse width 1 msecond (C) or incubated under hypoxic conditions for 15 minutes (D). Data from the transverse-tubule GFP (top panels) and HA labelling (middle panels) were analysed in regions of interest (defined as described in Materials and Methods) as transverse intensity profiles (lower panels). Results shown are from representative images from ∼25 examined cells in three experiments.

Fig. 3.

Quantitative analysis and comparison of GLUT4 translocation to sarcolemma and transverse-tubule membranes of cardiomyocytes. Average GFP and anti-HA fluorescence intensity was determined from regions representing the sarcolemma and transverse-tubule membrane (as defined in the Materials and Methods). (A) Sarcolemma GFP (black bars) and anti-HA (white bars) signals measured under insulin, contraction and hypoxia stimulation. All fluorescence signals were normalised to the maximum values obtained under insulin stimulation. (B) Transverse-tubules GFP (black bars) and anti-HA (white bars) signals measured under insulin, contraction and hypoxia stimulation. (C) Ratio of anti-HA to GFP signals at sarcolemma and transverse tubules. Error bars are s.e.m. of 24-27 sarcolemma values and 40-90 transverse-tubules values from seven to ten cells per condition, from a single representative experiment. ^*P<0.05 versus basal; ^**P<0.05 versus hypoxia.

Fig. 4.

Colocalisation of internalising GLUT4 with clathrin. (A) Isolated cardiomyocytes expressing HA-GLUT4-GFP were maintained in the unstimulated, basal state (top panels), stimulated with 10 nM insulin (middle panels) or in hypoxic buffer for 15 minutes (bottom panels). During treatment, cells were incubated with anti-HA antibody (5 μg/ml) at 37°C for 15 minutes. Following removal of the stimulus and washing to remove excess antibody, cells were incubated for 0-40 minutes. Images shown are from 10-minute time points. Results shown are representative images. All cells were viewed in approximately the same focal plane. Bars, 10 μm in large images; 5 μm in smaller images. (B) Similar data were obtained with anti-clathrin antibody, for which Alexa-568 conjugated goat anti-rabbit IgG antibody was used. Arrowheads indicate colocalisation of the GFP tag and clathrin. Results shown are representative of ∼20 cells per condition examined in three experiments.

Fig. 5.

Reversal of stimulation and internalisation of sarcolemma-tagged GLUT4 in cardiomyocytes. Isolated cardiomyocytes were either maintained in the unstimulated basal state (top panels), incubated with 10 nM insulin for 30 minutes (second row panels), electrically induced to contract (100 V, 10 Hz, pulse width 1 ms) for 5 minutes (third row panels) or incubated under hypoxic conditions for 15 minutes (bottom panels). During these treatments, cells were incubated with anti-HA antibody (5 μg/ml) at 37°C for 15 minutes. Following removal of the stimulus and washing to remove excess antibody, cardiomyocytes were incubated for a further 40 minutes to return to the basal state. All cells were viewed in approximately the same focal plane. Bars, 20 μm. Results shown are representative of ∼25 cells examined in three experiments.