Highlights:• Loss of mitochondrial phosphoenolpyruvate (PEP) impairs insulin release in vivo.• Pyruvate kinase (PK) activators stimulate beta-cells in preclinical diabetes models.• PEP cycling in vivo depends on PK and mitochondrial PEPCK (PCK2) for insulin release. • Acute and 3-week oral PK activator amplifies insulin release during hyperglycemia. eTOC Blurb:Abudukadier et al. show that small molecule pyruvate kinase activation in vivo and in vitro increases insulin secretion in rodent and human models of diabetes. The phosphoenolpyruvate (PEP) cycling mechanism and its amplification are dependent on mitochondrial PEPCK (PCK2). SummaryThe mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle is an anaplerotic-cataplerotic mitochondrial shuttle utilizing mitochondrial PEPCK (PCK2) and pyruvate kinase (PK). PEP cycling stimulates insulin secretion via OxPhos-independent lowering of ADP by PK. We assess in vivo whether islet PCK2 is necessary for glucose sensing and if speeding the PEP cycle via pharmacological PK activators amplifies insulin secretion. Pck2 -/mice had severely impaired insulin secretion during islet perifusion, oral glucose tolerance tests and hyperglycemic clamps. Acute and chronic pharmacologic PK activator therapy improved islet insulin secretion from normal, high-fat diet (HFD) fed, or Zucker diabetic fatty (ZDF) rats, and glucolipotoxic or diabetic humans. A similar improvement in insulin secretion was observed in regular chow and HFD rats in vivo.Insulin secretion and cytosolic Ca 2+ during PK activation were dependent on PCK2.These data provide a preclinical rationale for strategies, such as PK activation, that target the PEP cycle to improve glucose homeostasis.

Determination of metabolic fluxes by measurement of time-dependent sampling of isotopic enrichments during the administration of labeled substrates provides rich information.Because such experiments are resource-intensive and frequently push the limits of sensitivity of the measurement techniques, optimization of experiment design can improve feasibility with respect to financial and labor costs, time to completion, and increase precision and accuracy of the results. Here we used a previously published set of data acquired in cultured insulinoma cells to evaluate contributions to the sensitivity and variability of the rate of citrate synthase (CS). Specifically, we calculated changes in uncertainty in CS if sample times were dropped or new ones were added, and we observed that some sampling times can be dropped with little effect, while improvements can be made with a strategic choice of when to add samples. We measured the contributions of data sampled at different times on the sensitivity of CS, finding that CS had greater sensitivity at early time points. We tested the concept that if two estimated parameters are correlated significantly, then refining one might constrain the other. In this case, the rate of Beta-oxs was found to be correlated with CS, and narrower variability in Beta-ox did indeed improve the sensitivity of CS. The tests described here might be applied at the initial design stage and then after a pilot phase to improve sensitivities of targeted fluxes and the reduction of materials, time, labor, and other experimental resources. The correlation analyses can be used to consider what orthogonal measurements might be beneficial for further improvement of measurements. While this study used a specific example of a set of time-dependent kinetic isotopic measurements, the results illustrate some generalizable behaviors that can be tested in other experimental systems.