Insulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

胰岛素是人类胚胎干细胞 (hESC) 存活和自我更新的重要生长因子。虽然它最广为人知的是促进体细胞糖酵解的主要激素，但胰岛素在 hESC 能量代谢中的作用仍不清楚。在这份报告中，我们证明了胰岛素对于维持 hESC 线粒体呼吸至关重要，这种呼吸在胰岛素去除后迅速下降。胰岛素依赖的线粒体呼吸是干细胞特异性的，主要依赖丙酮酸和谷氨酰胺，而葡萄糖抑制过度氧化磷酸化。药理学和基因操作表明，连续的胰岛素信号通过 PI3K/AKT 激活和下游 GSK3 抑制来维持线粒体呼吸。我们进一步表明，胰岛素通过抑制 GSK3 来抑制半胱天冬酶激活和拯救细胞存活。这项研究揭示了 AKT/GSK3 通路在调节线粒体呼吸和细胞存活中的关键作用，强调胰岛素是准确评估 hESC 线粒体呼吸的重要因素。