Accumulation of depolarized mitochondria within b-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (Dw m ) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of nonfusing mitochondria that were found to have reduced Dw m and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1 K38A or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose Dw m and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.

OBJECTIVEPrevious studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we investigate β-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients.RESEARCH DESIGN AND METHODSUsing matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in β-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in β-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on β-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity.RESULTSβ-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated β-cell, a β-cell within an islet, and an INS1 cell. Under noxious conditions, we find that β-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis.CONCLUSIONSThese data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced β-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.

Assembly of mitochondria into networks supports fuel metabolism and calcium transport and is involved in the cellular response to apoptotic stimuli. A mitochondrial network is defined as a continuous matrix lumen whose boundaries limit molecular diffusion. Observation of individual networks has proven challenging in live cells that possess dense populations of mitochondria. Investigation into the electrical and morphological properties of mitochondrial networks has therefore not yielded consistent conclusions. In this study we used matrix-targeted, photoactivatable green fluorescent protein to tag single mitochondrial networks. This approach, coupled with real-time monitoring of mitochondrial membrane potential, permitted the examination of matrix lumen continuity and fusion and fission events over time. We found that adjacent and intertwined mitochondrial structures often represent a collection of distinct networks. We additionally found that all areas of a single network are invariably equipotential, suggesting that a heterogeneous pattern of membrane potential within a cell's mitochondria represents differences between discrete networks. Interestingly, fission events frequently occurred without any gross morphological changes and particularly without fragmentation. These events, which are invisible under standard confocal microscopy, redefine the mitochondrial network boundaries and result in electrically disconnected daughter units.

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 mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated 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-Weshow that mitochondria display a wide heterogeneity in ⌬⌿ and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing 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 uncoupling protein 2 are not significant contributors to ⌬⌿ heterogeneity. 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 ⌬⌿ heterogeneity in ␤-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target. Diabetes