Klip A, Ramlal T
J Biol Chem. 1987 Jul 5;262(19):9141-6.
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.
在3T3-L1细胞从成纤维细胞分化为脂肪细胞的过程中，利用荧光钙指示剂喹啉2或indo-1对细胞进行悬浮和负载，从而测定细胞内游离钙离子（[Ca2+]i）的浓度。在未分化的成纤维细胞以及直至第9天的分化脂肪细胞中，[Ca2+]i稳定在170 nM左右，仅在老化的脂肪细胞（第12天）中显著升高。在分化过程中，胰岛素对葡萄糖摄取的刺激作用从百分之几增加到数倍。添加激素10分钟后，摄取刺激就已明显，10 nM胰岛素在30分钟内产生最大刺激作用。向负载喹啉2或indo-1的悬浮脂肪细胞中添加胰岛素（10(-6) M），至少10分钟内对[Ca2+]i没有可检测到的影响。相反，添加全身麻醉剂氟烷在3分钟内使[Ca2+]i从172 nM增加到251 nM。在乙二醇双（2-氨基乙基醚）四乙酸（EGTA）溶液中，钙离子载体离子霉素引发细胞内储存钙的释放，导致[Ca2+]i短暂升高。添加20微克/毫升磷脂酸会导致细胞内储存钙释放较少但可测量的量（使[Ca2+]i增加20 nM）。相反，胰岛素不会引起细胞内储存钙的任何可检测到的释放。在EGTA存在的情况下（EGTA过量于培养基中的钙离子浓度），将3T3-L1脂肪细胞与胰岛素一起孵育，不会阻止激素对己糖摄取的刺激，这表明细胞外钙离子在胰岛素反应中不起作用。此外，在暴露于胰岛素期间，在EGTA培养基中用喹啉2/AM孵育细胞不会阻止对己糖摄取的刺激。在这些条件下证明，即使细胞内储存钙释放到细胞质中，细胞内喹啉2也足以螯合细胞质中的钙离子。因此，喹啉2有效降低[Ca2+]i而不损害胰岛素作用。得出的结论是，胰岛素不会引起[Ca2+]i的变化，螯合细胞内钙离子不会阻止胰岛素对己糖摄取的刺激。这些结果表明，[Ca2+]i的变化不太可能在3T3-L1脂肪细胞中胰岛素刺激葡萄糖摄取的信息转导中起作用。
