Thomas T P, Pek S B
Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0678.
Endocrinology. 1992 Oct;131(4):1985-92. doi: 10.1210/endo.131.4.1396342.
We reported previously that in pancreatic islet cells, certain diacylglycerols (DGs) evoke increases in cytosolic calcium ([Ca2+]i), mainly by intracellular mobilization. We now examined the effects of DGs on the increase in [Ca2+]i due to Ca2+ influx. In the insulin-secreting HIT T-15 islet cell line, cell membrane depolarization using 40 mM KCl evoked a 2- to 3-fold increase in [Ca2+]i, which lasted several minutes. A cell-permeable DG, 1,2-dioctanoylglycerol (DiC8; 10 microM) induced a 12 +/- 4% rise in [Ca2+]i, which did not occur in the absence of extracellular Ca2+ or in the presence of verapamil; this effect was not protein kinase-C (PKC) dependent, because it was not altered by the addition of the PKC inhibitor staurosporine or by using PKC-depleted cells. When DiC8 was added first, the KCl-induced increase in [Ca2+]i was inhibited in a dose-dependent manner (100% at 10-15 microM DiC8); this effect was PKC independent. At a concentration of 10 microM, other synthetic DGs, 1,2-dihexanoylglycerol (DiC6), 1,2-didecanoylglycerol (DiC10), or 1-oleoyl-2-acetylglycerol, inhibited the KCl-induced rise in [Ca2+]i to 15 +/- 4%, 47 +/- 7%, and 51 +/- 5% of the control value, respectively. R59022 (10 microM), which inhibits DG kinase and causes accumulation of endogenous DGs, inhibited the KCl-induced rise in [Ca2+]i to 2 +/- 0.2% of the control value; this inhibition was not affected by staurosporine. In anchored cells, KCl stimulated insulin release (959 +/- 88 microU/mg protein above the control value); 20 microM DiC6 or DiC8 attenuated KCl-induced insulin release by 68% and 31% of the control value, respectively; DiC10 or 1-oleoyl-2-acetylglycerol had no effect. R59022 inhibited KCl-induced insulin release by 90% of the control value. We conclude that in HIT T-15 cells, DGs may serve as positive and negative modulators of [Ca2+]i, apparently by complex and PKC-independent mechanisms. These divergent actions of DGs on islet cell Ca2+ balance together with the accompanying activation of PKC affect insulin release in a complex manner.
我们之前报道过，在胰岛细胞中，某些二酰基甘油（DGs）主要通过细胞内动员引起胞质钙（[Ca2+]i）升高。我们现在研究了DGs对因Ca2+内流导致的[Ca2+]i升高的影响。在分泌胰岛素的HIT T-15胰岛细胞系中，用40 mM KCl使细胞膜去极化会引起[Ca2+]i升高2至3倍，持续几分钟。一种可透过细胞的DG，1,2 - 二辛酰甘油（DiC8；10 microM）使[Ca2+]i升高12±4%，在无细胞外Ca2+或存在维拉帕米时不会出现这种情况；这种作用不依赖蛋白激酶 - C（PKC），因为添加PKC抑制剂星形孢菌素或使用PKC缺失的细胞不会改变这种作用。当首先添加DiC8时，KCl诱导的[Ca2+]i升高以剂量依赖方式受到抑制（在10 - 15 microM DiC8时为100%）；这种作用不依赖PKC。在浓度为10 microM时，其他合成DGs，1,2 - 二己酰甘油（DiC6）、1,2 - 二十二酰甘油（DiC10）或1 - 油酰 - 2 - 乙酰甘油，分别将KCl诱导的[Ca2+]i升高抑制至对照值的15±4%、47±7%和51±5%。R59022（10 microM）抑制DG激酶并导致内源性DGs积累，将KCl诱导的[Ca2+]i升高抑制至对照值的2±0.2%；这种抑制不受星形孢菌素影响。在贴壁细胞中，KCl刺激胰岛素释放（比对照值高959±88微单位/毫克蛋白）；20 microM DiC6或DiC8分别将KCl诱导的胰岛素释放减弱至对照值的68%和31%；DiC10或1 - 油酰 - 2 - 乙酰甘油无作用。R59022将KCl诱导的胰岛素释放抑制至对照值的90%。我们得出结论，在HIT T - 15细胞中，DGs可能作为[Ca2+]i的正负调节剂，显然是通过复杂且不依赖PKC的机制。DGs对胰岛细胞Ca2+平衡的这些不同作用以及伴随的PKC激活以复杂方式影响胰岛素释放。
