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First published online 10 February 2009
doi: 10.1242/jcs.041178

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

¹ Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
² Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (54K): [in this window] [in a new window]   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.

View larger version (54K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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.

View larger version (57K): [in this window] [in a new window]   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.

View larger version (76K): [in this window] [in a new window]   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.

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