Kowluru A, Li G, Rabaglia M E, Segu V B, Hofmann F, Aktories K, Metz S A
Medical and Research Services, William S. Middleton Memorial VA Medical Center, Madison, WI 53705, USA.
Biochem Pharmacol. 1997 Nov 15;54(10):1097-108. doi: 10.1016/s0006-2952(97)00314-6.
We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).
我们利用梭菌毒素（已知对GTP酶的抑制具有特异性）来确定候选GTP酶在β细胞生理性胰岛素分泌中的作用。将正常大鼠胰岛或分离的β（HIT-T15）细胞暴露于艰难梭菌毒素A和B，可催化β细胞内源性Rac、Cdc42和Rho的糖基化（从而使其失活）；同时，两种毒素均可降低大鼠胰岛和HIT细胞中葡萄糖或钾诱导的胰岛素分泌。用索氏梭菌致死毒素（LT；仅修饰Ras、Rap和Rac）处理β细胞也可降低葡萄糖或钾诱导的分泌。然而，梭菌毒素C3外切酶（仅对Rho进行ADP核糖基化并使其失活）对葡萄糖或钾诱导的胰岛素分泌均无任何影响。这些数据表明，葡萄糖或钾介导的分泌可能需要Cdc42、Rac、Ras和/或Rap（但不需要Rho）。这些毒素的作用似乎对刺激-分泌偶联具有特异性，因为在对照和毒素（A或LT）处理的β细胞之间，未发现代谢活力有差异（通过在营养物质代谢驱动的还原反应中定量四唑盐转化为甲臜的比色法评估）。毒素（A或LT）处理也未改变葡萄糖或钾介导的胞质游离钙浓度（[Ca2+]i）升高，这表明这些GTP酶参与了[Ca2+]i升高之后的步骤。最近的研究发现，仅葡萄糖可刺激Cdc42的羧基甲基化，而Rap（Kowluru等人，《临床研究杂志》98：540 - 555，1996）和Rac（本研究）的羧基甲基化则受葡萄糖或钾的调节。总之，这些发现首次提供了直接证据，表明GTP酶的Rho亚家族在胰岛素分泌中起关键调节作用，并且表明Cdc42可能是葡萄糖刺激胰岛素释放早期步骤所必需的，而Rap和/或Rac可能是刺激-分泌偶联级联反应后期步骤（即Ca2+诱导的胰岛素胞吐作用）所必需的。
