Investigations of intracellular Ca2+ concentrations in direct vicinity of GLUT4-storage-vesicles in adipocytes

Background and Aims: The presence of calcium in the submembrane space is a fundamental requirement for the regulated fusion of intracellular vesicles with the plasma membrane. This is true for insulin granules but also for GLUT4 vesicles in insulin-sensitive cells like adipocytes or striated muscle cells. Since adipocytes are not electrically excitable and do not express voltage-dependent calcium channels the role of calcium for the fusion of GLUT4 vesicle has gained very little attention. However, it is conceivable that calcium released from intracellular calcium storages contributes to the fusion of GLUT4 vesicles with the plasma membrane and that disturbances contribute to the deficient fusion of GLUT4 vesicles in states of insulin resistance. To test this hypothesis, a tool to measure perivesicular calcium in the submembrane space is needed.

Methods: GCaMP6m-XC, a genetically engineered calcium indicator (GECI) was fused to the cytosolic N-terminal domain of insulin-responsive amino peptidase (IRAP), located in the membrane of the GLUT4-storage-vesicles (GSV) in adipocytes. Adipocytes were differentiated from 3T3-L1 fibroblasts using an established protocol, then adenovirally transduced with GCaMP6m-XC-IRAP and measured before and after stimulation with 1 µM Ionomycin and Calcium. Images were taken with TIRF microscopy.

Results: The transduction efficiency was high, approximately 90% of the adipocytes expressed the GECI. When imaged by TIRF microscopy the GLUT4 vesicles appeared as small spherical structures, which were evenly distributed throughout the entire cytosol of the 3T3-L1 adipocytes. Live-cell imaging showed occasional vesicle movement under basal condition, however, the fluorescent label was prone to photobleaching upon repeated imaging.

Intensity increases of the label were brought about by increases of the cytosolic Ca2+ concentration as generated by use of the Ca2+ ionophore Ionomycin.

Conclusion: The newly generated fusion protein, consisting of the GECI and the vesicle membrane anchor IRAP, enables live-cell imaging of GLUT4 vesicles and shows potential as a tool for studying intracellular changes in calcium concentration and their impact on the movement of GLUT4-storage-vesicles in adipocytes.

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Artikel online veröffentlicht:
18. April 2024