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 fission͞ fusion machinery can be a previously unrecognized target to control acute and chronic production of ROS in hyperglycemiaassociated disorders.DLP1͞Drp1 ͉ mitochondrial fission ͉ dynamin ͉ diabetes ͉ obesity T he vascular and multiorgan complications in diabetes and obesity are causally associated with hyperglycemia-induced overproduction of reactive oxygen species (ROS) (1-4). In addition, surgical and intensive care patients often exhibit high blood glucose levels, which contributes to multiorgan injury (5, 6). Hyperglycemic conditions increase input of metabolic substrate into mitochondria and overwhelm the electron transport system, resulting in ROS overproduction (2).Mitochondria are dynamic organelles, frequently changing shape and distribution (7). Defined sets of proteins that mediate mitochondrial fission and fusion constitute the critical regulatory components controlling mitochondrial dynamics (8)(9)(10)(11)(12)(13)(14). The basic morphology of mitochondria in cells is tubular, and balanced frequency of fission and fusion determines mitochondrial morphology (15). Growing evidence indicates that maintaining correct mitochondrial morphology is critical for cell function (16). Recent findings that mutations in genes encoding fission and fusion proteins cause human diseases (17-20) signify the importance of mitochondrial dynamics.Mitochondrial function is reflected in their structure and morphology (13,14,(21)(22)(23)(24). A frequently observed characteristic in tissues of hyperglycemic patients and animals is mitochondrial deformation, most notably mitochondrial swelling or accumulation o...

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.

Increased production of reactive oxygen species (ROS) from mitochondria is the main cause of hyperglycemic complications. We previously showed that hyperglycemic conditions induce mitochondrial fragmentation that is causal for ROS overproduction. This study was to identify signaling components that induce mitochondrial fragmentation in high-glucose stimulation. We found that exposing cells to the high-glucose concentration evokes increases in cytosolic Ca(2+). Chelating Ca(2+) in the high-glucose medium prevented not only the Ca(2+) transient but also mitochondrial fragmentation and the ROS increase, indicating that the Ca(2+) influx across the plasma membrane is an upstream event governing mitochondrial fission and the ROS generation in high-glucose stimulation. We found that the high-glucose-induced Ca(2+) increase activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2). The Ca(2+) chelation prevented the ERK1/2 activation, and inhibition of the ERK1/2 phosphorylation decreased mitochondrial fragmentation as well as ROS levels in high-glucose stimulation. In addition, the level of the mitochondrial fission protein dynamin-like protein 1 in mitochondria increased in high-glucose incubation in a Ca(2+)-dependent manner. In vitro kinase assays showed that ERK1/2 is capable of phosphorylating dynamin-like protein 1. These results demonstrate that high-glucose stimulation induces the activation of mitochondrial fission via signals mediated by intracellular Ca(2+) and ERK1/2.

Mitochondrial fission is a highly regulated process mediated by a defined set of protein factors and is involved in the early stage of apoptosis. In mammals, at least two proteins, the dynamin-like protein DLP1/Drp1 and the mitochondrial outer membrane protein hFis1, participate in mitochondrial fission. The cytosolic domain of hFis1 contains six α-helices that form two tetratricopeptide repeat (TPR) motifs. Overexpression of hFis1 induces DLP1-mediated fragmentation of mitochondria, suggesting that hFis1 is a limiting factor in mitochondrial fission by recruiting cytosolic DLP1. In the present study, we identified two regions of hFis1 that are necessary for correct fission of mitochondria. We found that the TPR region of hFis1 participates in the interaction with DLP1 or DLP1-containing complex and that the first helix (α1) of hFis1 is required for mitochondrial fission presumably by regulating DLP1-hFis1 interaction. Misregulated interaction between DLP1 and hFis1 by α1 deletion induced mitochondrial swelling, in part by the mitochondrial permeability transition, but significantly delayed cell death. Our data suggest that hFis1 is a main regulator of mitochondrial fission, controlling the recruitment and assembly of DLP1 during both normal and apoptotic fission processes.

Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism-secretion coupling in pancreatic b-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications. Antioxid. Redox Signal. 14, 439-457.

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca2+) is one signal that has been shown to both regulate mitochondrial fission in various cells types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca2+ plays an important role in the adult cardiac muscle cells for excitation-metabolism coupling, little is known about whether Ca2+ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca2+ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca2+ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca2+ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca2+-mediated ROS generation in cardiac myocytes. These results suggest that Ca2+, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm(-2) with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm(-2) and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm(-2). Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H(2)DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab-on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.