Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA.
Brehm Diabetes Research Center, University of Michigan Medical School, Ann Arbor, MI, USA.
J Physiol. 2023 Dec;601(24):5655-5667. doi: 10.1113/JP284982. Epub 2023 Nov 20.
Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (K ) 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 locally produces the ATP that inhibits K 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 . To test the relative contributions of local vs. mitochondrial ATP production, we recorded K activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of K 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 . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on K channel closure by glucose, further suggesting that PK is not an important regulator of K . In contrast, addition of mitochondrial inhibitors robustly increased K 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 in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit K activity in excised patches. PEP solutions only inhibit K activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on K activity. Mitochondrial inhibition, in contrast, robustly potentiates K activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close K . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the K activity of pancreatic beta cells.
胰岛β细胞在血糖的刺激下分泌胰岛素。三磷酸腺苷敏感钾通道（KATP）通过响应细胞内 [ATP]/[ADP] 的增加，将葡萄糖代谢与胰岛电活动联系起来。最近有人提出，在β细胞附近的丙酮酸激酶（PK）可以局部产生抑制 KATP 活性的 ATP。该假说主要基于以下观察结果：向质膜外侧的分离外翻膜片的细胞质侧施加磷酸烯醇丙酮酸（PEP）和 ADP，可抑制 KATP 通道的活性。为了测试局部和线粒体 ATP 生成的相对贡献，我们使用小鼠β细胞和 INS-1 832/13 细胞记录 KATP 活性。与之前的报道相反，我们无法复制 PEP+ADP 对 KATP 活性的抑制作用。然而，当 PEP 溶液的 pH 值未因加入 PEP 而校正时，由于众所周知的质子抑制 KATP 通道的作用，观察到强烈的通道抑制作用。在细胞贴附记录中，灌注 PK 激活剂或抑制剂对葡萄糖引起的 KATP 通道关闭几乎没有影响，进一步表明 PK 不是 KATP 的重要调节因子。相反，添加线粒体抑制剂可显著增加 KATP 活性。最后，通过测量小鼠β细胞中钙振荡引起的 [ATP]/[ADP] 反应，我们发现氧化磷酸化甚至可以在钙爆发沉默期 ADP 处于低谷时升高 [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 KATP 的主要控制器。关键点：PEP 加腺苷二磷酸不会抑制分离膜片中的 KATP 活性。只有当 pH 值不平衡时，PEP 溶液才会抑制 KATP 活性。调节丙酮酸激酶对 KATP 活性的影响很小。相反，线粒体抑制作用可显著增强细胞贴附片中的 KATP 活性。尽管在钙振荡的沉默期 ADP 水平下降，但线粒体仍可通过氧化磷酸化产生足够的 ATP 来关闭 KATP。因此，线粒体氧化磷酸化是抑制胰腺β细胞 KATP 活性的 ATP 的主要来源。
