Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP ؉ -dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.Cell damage induced by oxidative stress and reactive oxygen species (ROS) 1 has been implicated in several human diseases including aging, alcohol-mediated organ damage, neurodegenerative diseases, many types of cancers, cardiovascular diseases, and UV-mediated skin disorders (1). As one of the major sources of ROS (2), mitochondria are highly susceptible to oxidative damage. ROS can damage mitochondrial enzymes directly (3), and they can cause mutation in mitochondrial DNAs (4). At the same time, ROS can change the mitochondrial transmembrane potential (⌬m), which is indicative of mitochondrial membrane integrity (5) and precedes cell death induced by various toxic compounds and cytokines (6). Recent reports indicate that mitochondrial ROS cause apoptosis (7, 8) by activating various apoptotic effectors such as cytochrome c release, procaspase-2, procaspase-9, procaspase-3, and latent apoptosis-inducing factor, which is released from the mitochondria during apoptosis (9 -11). Another report also suggested that mitochondrial ROS directly caused apoptosis of T cells (12). It was also reported that tumor necrosis factor ␣ causes a rapid production of mitochondrial ROS (13) and that ceramide, an apoptotic stimulus, also plays a crucial role in tumor necrosis factor ␣-induced mitochondrial ROS generation (14). Furthermore, several other investigators demonstrated that ROS are involved in the signaling pathway of certain growth factors (15) and cytokines (16). In addition, mitochondrial ROS, under hypoxic conditions, activate the transcription of the genes for glycolytic enzymes as well as erythropoietin and vascular endothelial growth factor by upregulating a transcriptional factor, hypoxia-inducible factor 1 (17), suggesting that mitochondrial ROS mediate cross-talk b...
NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP ؉ -dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP ؉ -dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor ␥ were markedly elevated. The hepatic and epididymal fat pad contents of acetylCoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.Abnormal lipid metabolism is frequently associated with obesity and hyperlipidemia. In fat and cholesterol biosynthesis, NADPH is an essential cofactor for numerous enzymes. For instance, 3-L-hydroxylacyl-coenzyme A dehydrogenase and enoyl-coenzyme A reductase in fatty acid synthesis and hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, require NADPH for their enzyme activities. It has been demonstrated that glucose-6-phosphate dehydrogenase (G6PDH), 1 6-phosphogluconate dehydrogenase, and malic enzyme are considered as the major enzymes producing cytosolic NADPH (1). Nevertheless, the activities of these enzymes were markedly lower than that of cytosolic NADP ϩ -dependent isocitrate dehydrogenase (IDPc) in the rat liver (1, 2). Consistent with this observation, McLean and co-workers (3) reported that certain adaptive changes in the pentose phosphate pathway dehydrogenases did not take place in parallel with fat synthesis in adipose tissue and suggested that a major source of NADPH for fat synthesis could be IDPc. It is worthy of note that IDPc is expressed mainly in lipogenic tissues such as liver and adipocytes, whereas G6PDH and 6-phosphogluconate dehydrogenase are expressed ubiquitously (4, 5). These data indicate that NADPH-producing IDPc may ...
To understand the interactions and functional role of each of the three mitochondrial NAD؉ -dependent isocitrate dehydrogenase (IDH) subunits (␣, , and ␥), we have characterized human cDNAs encoding two  isoforms ( 1 and  2 ) and the ␥ subunit. Analysis of deduced amino acid sequences revealed that  1 and  2 encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The ␥ cDNA encodes 354 amino acids and is almost identical to monkey IDH␥. Northern analyses revealed that the smaller  2 transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger  1 mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDH gene indicates that the difference in the C-terminal 28 amino acids between  1 and  2 proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, ␣␥, ␣, and ␣␥ combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and ␥ showed no detectable activity. These data suggest that the ␣ is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of  1 with  2 in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain ( 1 prevalent; optimal pH 8.0) versus heart ( 2 prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle-and heartspecific  2 isoform. Taken together, these data indicate a regulatory role of IDH isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.
The protective role of IDPc and IDPm against gamma-ray-induced cellular damage can be attributed to elevated NADPH, reducing equivalents needed for recycling reduced glutathione in the cytosol and mitochondria. Thus, a primary biological function of the ICDHs may be production of NADPH, which is a prerequisite for some cellular defence systems against oxidative damage.
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