ATP and ADP levels are critical regulators of glucosestimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P i ) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P i was estimated as a function of time. ATP/ADP/P i increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P i . Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K ؉ -dependent and -independent mechanisms. Diabetes 53: [401][402][403][404][405][406][407][408][409] 2004 F ree ATP and ADP levels are understood to couple glucose metabolism with the closing of ATPsensitive K ϩ (K ATP ) channels and the ionic events leading to the exocytosis of insulin (1,2). Furthermore, the phosphorylation potential (ATP/ADP/P i ) may augment insulin secretion beyond that mediated by K ATP channels (3). Although it is well established that the critical ATP and ADP levels depend on islet substrate metabolism and mitochondrial electron transport (4 -6), it is less broadly appreciated that ADP stimulates electron transport and ATP production (7,8) as a feedback regulator of ATP/ADP/P i (9,10). Thus, mitochondrial ATP production, the ATP/ADP/P i , and ultimately the insulin secretory rate depend dually on both substrate supply as well as energy demand.Although the mechanisms by which mitochondria sense ADP levels and control ATP/ADP/P i are not fully established, a likely candidate is cytochrome c oxidase, the rate-limiting step in the electron transport chain (11). Wilson et al. (11,12) validated a mathematical model correlating substrate supply and energy demand (i.e., as phosphorylation potential) to the redox state and oxygen cons...