Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport.
The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
Klip
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Ramlal
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D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism; D004987:Ethers / Q000494:pharmacology; D006601:Hexoses / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D015759:Ionomycin; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*
D004987:Ethers; D006601:Hexoses; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D015759:Ionomycin; D002118:Calcium