Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP₇, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP₇ regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP₇. Although the inhibitors have diverse targets, they all perturbed IP₇ levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP₇ levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K_m for ATP makes IP₇ synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP₇ concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP₇ indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP₇’s role in cellular signal transduction is likely to have been underestimated.

肌醇焦磷酸盐已成为许多关键细胞过程的重要调节剂，从囊泡运输和细胞骨架重排到端粒长度调节和细胞凋亡。我们之前已经证明 5-二磷酸肌醇五磷酸 (IP ₇₎在胰腺 β 细胞中处于高水平，对胰岛素胞吐作用很重要。为了更好地了解β 细胞中的IP ₇调节，我们使用胰岛素分泌细胞系 HIT-T15 来筛选许多不同的肌醇代谢药理学抑制剂，以了解它们对细胞 IP ₇的影响。尽管抑制剂具有不同的靶点，但它们都干扰了 IP ₇水平。这让我们怀疑可能涉及抑制剂的间接、脱靶效应。众所周知，代谢毒物会降低IP ₇水平。肌醇六磷酸激酶 (IP6Ks)对 ATP具有高 K _m的事实使得 IP ₇合成可能容易受到 ATP 消耗的影响。此外，许多激酶抑制剂靶向激酶的 ATP 结合位点，但鉴于这些位点的相似性，难以实现高特异性。在这里，我们展示了 IP ₇HIT-T15 细胞中的浓度被 PI3K（渥曼青霉素，LY294002）、PI4K（Phenylarsine Oxide，PAO）、PLC（U73122）和胰岛素受体（HNMPA）的抑制剂降低。这些抑制剂中的每一种也降低了 ATP/ADP 比率。因此，损害能量代谢的试剂间接降低了 IP ₇。此外，PAO、U73122 和 LY294002 也直接抑制纯化 IP6K 的活性。这些数据对研究胰腺 β 细胞信号转导的人特别关注，但也强调了这样一个事实，即使用这些抑制剂可能错误地表明关键信号转导途径参与了各种细胞过程。相反，IP ₇在细胞信号转导中的作用可能被低估了。