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...

Dietary antioxidant compounds such as bioflavonoids may offer some protection against the early stage of diabetes mellitus and the development of complications. We investigated the effect of citrus bioflavonoids on blood glucose level, hepatic glucose-regulating enzymes activities, hepatic glycogen concentration, and plasma insulin levels, and assessed the relations between plasma leptin and body weight, blood glucose, and plasma insulin. Male C57BL/KsJ-db/db mice (db/db mice, 5 wk old), an animal model for type 2 diabetes, were fed a nonpurified diet for 2 wk and then were fed an AIN-76 control diet or the control diet supplemented with hesperidin (0.2 g/kg diet) or naringin (0.2 g/kg diet). Hesperidin and naringin supplementation significantly reduced blood glucose compared with the control group. Hepatic glucokinase activity and glycogen concentration were both significantly elevated in the hesperidin- and the naringin-supplemented groups compared with the control group. Naringin also markedly lowered the activity of hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase compared with the control group. Plasma insulin, C-peptide, and leptin levels in the db/db mice from the 2 bioflavonoid-supplemented groups were significantly higher than those of the control group. Furthermore, plasma leptin was positively correlated with plasma insulin level (r = 0.578, P < 0.01) and body weight (r = 0.541, P < 0.05), and was inversely correlated with the blood glucose level (r = -0.46, P < 0.05). The current results suggest that hesperidin and naringin both play important roles in preventing the progression of hyperglycemia, partly by increasing hepatic glycolysis and glycogen concentration and/or by lowering hepatic gluconeogenesis.

The by-product of lipid peroxidation, 4-hydroxynonenal (HNE), was shown to cause apoptosis in PC12 cells. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in these cells. Specifically, we determined the effect of HNE on the activities of mitogen-activated protein (MAP) kinases involved in early signal transduction. Within 15 to 30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before it returned to control level at 1 h post-treatment. In contrast, activities of extracellular signal-regulated kinase and p38 MAP kinase remained unchanged from their baseline levels. Stress-activated protein kinase kinase (SEK1), an upstream kinase of JNK, was also activated within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 and apoptosis signal-regulating kinase 1 (ASK1), an upstream kinase of SEK1, was demonstrated by the transient transfection of cDNA for wild-type SEK1 or ASK1 together with JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when either of the dominant negative mutant of SEK1 or ASK1 was cotransfected with JNK. Pretreatment of PC12 cells with a survival-promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, neither caused apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the selective JNK activation by HNE is critical for the apoptosis of PC12 cells and that the HNE-mediated apoptosis is likely to be mediated through the activation of the ASK1-SEK1-JNK pathway without activation of extracellular signal-regulated kinase or p38 MAP kinase.

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-kappaB signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-kappaB activation through stabilization of the NF-kappaB/IkappaB complex and IkappaB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-kappaB/IkappaB signal transduction pathways.

Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-beta1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.

A facile method of preparing triple‐modality, optical–nuclear–magnetic imaging probes using radiolabeled superparamagnetic nanoparticles is developed. Cerenkov luminescence imaging based on radionuclides showed great potential as a new optical imaging modality. The complementary nature of the optical/PET/MR hybrid nanoprobes facilitated non‐invasive differentiation between tumor‐metastasized sentinel lymph nodes (SLNs) and tumor‐free SLNs.

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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.