Increased production of mitochondrial reactive oxygen species (ROS) by hyperglycemia is recognized as a major cause of the clinical complications associated with diabetes and obesity [Brownlee, M. (2001) Nature 414, 813–820]. We observed that dynamic changes in mitochondrial morphology are associated with high glucose-induced overproduction of ROS. Mitochondria undergo rapid fragmentation with a concomitant increase in ROS formation after exposure to high glucose concentrations. Neither ROS increase nor mitochondrial fragmentation was observed after incubation of cells with the nonmetabolizable stereoisomer l -glucose. However, inhibition of mitochondrial pyruvate uptake that blocked ROS increase did not prevent mitochondrial fragmentation in high glucose conditions. Importantly, we found that mitochondrial fragmentation mediated by the fission process is a necessary component for high glucose-induced respiration increase and ROS overproduction. Extended exposure to high glucose conditions, which may mimic untreated diabetic conditions, provoked a periodic and prolonged increase in ROS production concomitant with mitochondrial morphology change. Inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure. These results indicate that the dynamic change of mitochondrial morphology in high glucose conditions contributes to ROS overproduction and that mitochondrial fission/fusion machinery can be a previously unrecognized target to control acute and chronic production of ROS in hyperglycemia-associated disorders.

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.

One of the main causes of cardiovascular complications in diabetes is the hyperglycaemia-induced cell injury, and mitochondrial fission has been implicated in the apoptotic process. We investigated the role of mitochondrial fission in high glucose-induced cardiovascular cell injury. We used several types of cultured mouse, rat, and bovine cells from the cardiovascular system, and evaluated mitochondrial morphology, reactive oxygen species (ROS) levels, and apoptotic parameters in sustained high glucose incubation. Adenoviral infection was used for the inhibition of the fission protein DLP1. We found that mitochondria were short and fragmented in cells incubated in sustained high glucose conditions. Under the same conditions, cellular ROS levels were high and cell death was increased. We demonstrated that the increased level of ROS causes mitochondrial permeability transition (MPT), phosphatidylserine exposure, cytochrome c release, and caspase activation in prolonged high glucose conditions. Importantly, maintaining tubular mitochondria by inhibiting mitochondrial fission in sustained high glucose conditions normalized cellular ROS levels and prevented the MPT and subsequent cell death. These results demonstrate that mitochondrial fragmentation is an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions. These findings indicate that the fission-mediated fragmentation of mitochondrial tubules is causally associated with enhanced production of mitochondrial ROS and cardiovascular cell injury in hyperglycaemic conditions.

The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pβ and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pβ caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.