1824-P: Bioenergetic Advantage of Pyruvate Kinase Overcomes Mitochondrial OXPHOS in Beta-Cells to Initiate Insulin Secretion

by sugaradmin



Introduction and Objective: The high relative free energy of phosphoenolpyruvate (PEP) positions pyruvate kinase (PK) as the driver of insulin secretion in beta-cells by increasing the ATP/ADP ratio and closing KATP channels. In this model, PK depletes ADP for mitochondrial OxPhos, leading to mitochondrial hyperpolarization and OxPhos inhibition. This study explores how PK-driven PEP hydrolysis impacts mitochondrial membrane potential (ΔΨm).Methods: OxPhos reduction by PK was assessed by measuring the oxygen consumption rate (OCR) in permeabilized human islets using a Seahorse XF Pro, and ΔΨm was tracked in intact mouse and human islets with TMRE. Both assays were conducted with mitochondrial or small molecule PK modulators (mPKs). Verapamil and diazoxide were used to prevent plasma membrane depolarization.Results: At physiological concentrations, PEP reduced OxPhos-dependent OCR in a dose-dependent manner while raising the ATP/ADP ratio. This effect required PK to hydrolyze PEP to limit ADP availability, without further pyruvate metabolism, and was independent of substrate or ETC complex. mPKs further suppressed OxPhos. Regeneration of intramitochondrial ADP with creatine restored PEP-inhibited OxPhos, while a phosphocreatine clamp fully suppressed OxPhos across fixed ATP/ADP ratios. Under the same conditions, PK was able to raise the ATP/ADP ratio higher than OxPhos (16.58 vs. 1.48 µM ratios; p<0.0001). PEP suppressed ATP synthesis by OxPhos, and this effect was enhanced by PK activation. mPK also hyperpolarized mitochondria (above glucose or oligomycin) in intact mouse and human islets. ADP depletion by PK was also observed in INS-1 and C2C12 cells, adipocytes, and hepatocytes.Conclusion: PK modulation induces ΔΨm hyperpolarization beyond that of glucose and closer to state IV levels. Thus, PK increases the ATP/ADP ratio to initiate KATP channel closure. This sensing mechanism allows beta-cells and other cell types to fine-tune their response to substrate fluctuations.

Disclosure

I. Ruz-Maldonado: None. R.L. Cardone: None. M.J. Merrins: None. R. Kibbey: Consultant; Structure Therapeutics, Inc.

Funding

R01 DK127637



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