Regulation of ATP/ADP in Pancreatic Islets

Abstract

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...

A Novel Method for the Assessment of Cellular Composition and Beta-Cell Viability in Human Islet Preparations

“…Additional methods to predict islet function have been proposed, including analysis of oxygen consumption (28) and analysis of ATP levels (29,30). While the theoretical premises to utilize these methods are certainly strong, we feel that there could be difficulties in using them as single, straight-forward analytical tests of islet potency.…”

Mitochondrial regulation of insulin production in rat pancreatic islets

“…Glucose is converted via glycolysis to pyruvate, which enters the mitochondria and is metabolised in the tricarboxylic acid (TCA) cycle. Pyruvate carbon metabolism leads to generation of ATP and hence to an increased cytosolic ATP : ADP ratio, which is crucial for insulin secretion [4,5] and biosynthesis [3]. Thus, the energetic state of the beta cell is an important component of the stimulus-secretion coupling mechanism of glucose-induced insulin production and secretion.…”

Identification and characterization of a novel isoform of the vesicular γ-aminobutyric acid transporter with glucose-regulated expression in rat islets

“…One of us (I R S) has performed perifusion experiments using the INS-1 that we received and that were in the same passage number range as the cells used for the experiments described herein . These perifusion experiments demonstrated appropriate responses to glucose and epinephrine and as well as sulfonylurea (glibenclamide) binding INS-1 cells were cultured in medium with 11 mM glucose as described (Sweet et al 2004). Cell extracts were prepared in the same manner as islet extracts.…”