-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels Jakob D. Wikstrom, 1 Shana M. Katzman, 1 Hibo Mohamed, 1 Gilad Twig, 1 Solomon A. Graf, 1 Emma Heart, 2 Anthony J.A. Molina, 1 Barbara E. Corkey, 2 Lina Moitoso de Vargas, 2 Nika N. Danial, 3 Sheila Collins, 4 and Orian S. Shirihai 1 OBJECTIVE—-Cell response to glucose is characterized by mitochondrial membrane potential () hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mito- chondrial hyperpolarization accompanies the concentration-de- pendent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the inte- grated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single -cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of -cell mitochondria. RESULTS—We show that mitochondria display a wide hetero- geneity in  and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increas- ing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased  heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in  of individual mitochondria, ATP-hydrolyzing mitochondria, and un- coupling protein 2 are not significant contributors to  hetero- geneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity. CONCLUSIONS—We suggest that mitochondrial  heteroge- neity in -cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target. Diabetes 56:2569–2578, 2007 M itochondria play essential roles in pancreatic -cell function and dysfunction (1,2). They generate secretagogues for insulin secretion and produce factors that function to induce and propagate apoptosis. Essential to these processes is mitochondrial energy state, in the form of an electrochem- ical gradient, commonly termed the mitochondrial mem- brane potential (). This gradient influences ATP-to- ADP ratio, redox state (3– 6), and reactive oxygen species (ROS), as well as calcium sequestration (2). A growing body of evidence suggests that mitochondrial dysfunction plays a role in the pathophysiology of type 2 diabetes, both in the insulin secretion failure of -cells and insulin resistance in peripheral tissues such as fat and skeletal muscle. Mitochondrial ATP production is cardinal in the pathway leading to insulin secretion, which is demonstrated in the diabetic phenotype of patients with mitochondrial DNA mutations (1,2). It is not known whether mitochondria in healthy or dysfunctional -cells are metabolically homogeneous, thus having uniform , or whether they exist as subpopulations with different levels of , therefore contributing unevenly to ATP synthesis and insulin secretion. In theory, a heterogeneous population may represent the existence of subpopulations of mitochondria that have relatively reduced capacity to generate secretagogues and thus constitute a therapeutic target for increased insulin secretion. To date, the phe- nomenon of mitochondrial heterogeneity has been studied in several cell types (7–12) but not in -cells.  reflects mitochondrial fuel availability, Kreb’s cycle and respiratory chain activity, and processes that consume the proton gradient, including uncoupling and ATP synthe- sis. Glycolytic pathways supply the mitochondria with anapleurotic intermediates that participate in fuel-stimu- lated insulin secretion (1). BAD, a proapoptotic BCL-2 family member, has recently been implicated in cellular respiration (13). BAD is thought to interact with the mitochondrial glucokinase (hexokinase IV) complex, and BAD knock-out rodents exhibit reduced mitochondrial glucokinase activity (13). The uncoupling protein 2 (UCP2) has been shown to be a regulator of  in -cells (14). Alterations in UCP2 expression levels have been shown to modify -cell  (15,16), ATP levels, and the secretory response to glucose (17,18,19). To test whether -cell mitochondria constitute a meta- bolically diverse population, we developed a methodology that enabled the quantification of  heterogeneity. By using this approach, we also determined its physiological relevance and systematically examined possible sources From the 1 Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts; the 2 Obesity Research Center, Boston University School of Medicine, Boston, Massachu- setts; the 3 Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; and the 4 Division of Translational Biology, Endocrine Biology Program, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina. Address correspondence and reprint requests to Orian S. Shirihai, Tufts University, Department of Pharmacology and Experimental Therapeutics, 136 Harrison Ave., Boston, MA 02111. E-mail: orian.shirihai@tufts.edu. Received for publication 3 June 2006 and accepted in revised form 3 July 2007. Published ahead of print at http://diabetes.diabetesjournals.org on 8 August 2007. DOI: 10.2337/db06-0757. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-0757. S.M.K., H.M., and G.T. contributed equally to this study. , mitochondrial membrane potential; FFA, free fatty acid; FI, fluores- cence intensity; GLT, glucolipotoxicity; JC-1, tetrachloro-1,1',3,3'-tetraethyl- benzimidazol-carbocyanine-iodide; MeS; mono-methyl-succinate; MTG, MitoTracker Green; OM, oligomycin; PA-GFP mt , matrix-targeted photo-acti- vatable green fluorescent protein; ROS, reactive oxygen species; TMRE, tetramethylrhodamine-ethyl-ester-perchlorate; UCP2, uncoupling protein 2. © 2007 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. 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