Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (K_ATP) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell K_ATP locally produces the ATP that inhibits K_ATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited K_ATP. To test the relative contributions of local vs. mitochondrial ATP production, we recorded K_ATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of K_ATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit K_ATP. In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on K_ATP channel closure by glucose, further suggesting that PK is not an important regulator of K_ATP. In contrast, addition of mitochondrial inhibitors robustly increased K_ATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of K_ATP in pancreatic beta cells.

胰腺 β 细胞响应血浆葡萄糖分泌胰岛素。ATP 敏感钾通道 （K_ATP） 通过响应胞质 [ATP]/[ADP] 增加，将葡萄糖代谢与这些细胞中的胰岛电活动联系起来。最近有人提出，靠近 β 细胞 K_ATP 的丙酮酸激酶 （PK） 局部产生抑制 K_ATP 活性的 ATP。该提案主要基于以下观察结果：将磷酸烯醇式丙酮酸 （PEP） 和 ADP 应用于切除的由内而外的贴剂的细胞质侧会抑制 K_ATP。为了测试局部与线粒体 ATP 产生的相对贡献，我们使用小鼠 β 细胞和 INS-1 832/13 细胞记录了 K_ATP 活性。与以前的报告相比，我们无法复制 PEP + ADP 对 K_ATP 活性的抑制。然而，当 PEP 溶液的 pH 值未因添加 PEP 而校正时，由于众所周知的质子抑制 K_ATP 的作用，观察到强烈的通道抑制。在细胞附着记录中，使用 PK 激活剂或抑制剂对葡萄糖的 K_ATP 通道闭合影响很小或没有影响，进一步表明 PK 不是 K_ATP 的重要调节因子。相比之下，添加线粒体抑制剂强烈增加了 K_ATP 活性。最后，通过测量小鼠 β 细胞中 [ATP]/[ADP] 对强加钙振荡的反应，我们发现即使 ADP 在突发沉默阶段处于最低点，氧化磷酸化也可以提高 [ATP]/[ADP]，这与我们的数学模型一致。这些结果表明，线粒体氧化磷酸化产生的 ATP 是胰腺 β 细胞中 K_ATP 的主要控制因子。