IDH2 Deficiency Research Articles

MIR204 POTENTIALLY PROMOTES NON-ALCOHOLIC FATTY LIVER DISEASE BY INHIBITION OF CPT1A IN MOUSE HEPATOCYTES

Objective: Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigated the functional significance of miR204 in the evolution of NAFLD. Design and method: NAFLD was induced in isocitrate dehydrogenase (IDH2) knockout (KO) mice by feeding them with a high-fat diet (HFD). Blood parameters and hepatic fat accumulation were evaluated in IDH2 KO mice and wild-type (WT) mice. A miR204 inhibitor was subcutaneously injected into mice using an osmotic pump. The expression of miR204 was analyzed by quantitative PCR (qPCR). Results: IDH2 KO mice fed a normal diet (ND) or HFD showed significantly increased body weight, epididymal fat-pad weight, cholesterol, triglyceride, and macrophage marker (CD68), and inflammatory cytokine [tumor necrosis factor-a (TNF-a), interleukin (IL)-6 and IL-1b] expression compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 was significantly increased in mice with IDH2 deficiency. miR204 regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid b-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes—peroxisomal acyl-coenzyme A oxidase 1 (ACOX-1) and peroxisome proliferator-activated receptor alpha (PPARa)—by activating CPT1a expression, which decreases inflammatory cytokines, epididymal fat pad weight, cholesterol, low-density lipoprotein (LDL), and triglycerides. Conclusions: miR204 promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic metabolism and inflammation.

MicroRNA 101 Attenuated NSCLC Proliferation through IDH2/HIFα Axis Suppression in the Warburg Effect.

Lung cancer is the most diagnosed and deadly cancer in China. MicroRNAs are small noncoding RNA gene products that exhibit multifunctional regulation in cancer cell progressions. MiR-101 loss was illustrated in about 29% of lung cancer patients, and sophisticated mechanisms of miR-101 regulation in NSCLC are eager to be disclosed. Here, using specimens from NSCLC patients and Dural-luciferase reporter assay, we got a clue that miR-101 correlated with IDH2. MiR-101 overexpression and IDH2 deficiency both suppressed NSCLC tumor growth in mice. Moreover, in NSCLC, miR-101 suppressed IDH2 expression levels, further increased α-KG concentration, and finally inhibited the Warburg effect under hypoxic conditions through downregulating HIF1α expression by promoting HIF1α hydroxylation and degradation. In conclusion, miR-101 attenuated the Warburg effect and NSCLC proliferation through IDH2/HIF1α pathway.

MiR204 potentially promotes non-alcoholic fatty liver disease by inhibition of cpt1a in mouse hepatocytes

Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigate the functional significance of miR204 in the evolution of NAFLD. IDH2 KO mice feed a normal diet (ND) or HFD increased body weight, epididymal fat-pad weight, lipid droplet in liver, blood parameter and inflammation compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 is increased in mice with IDH2 deficiency. Increased miR204 by IDH2 deficiency regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid β-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes by activating CPT1a expression, which decreases lipid droplet in liver, inflammatory cytokines, epididymal fat pad weight, blood parameters. Increased miR204 by IDH2 deficiency promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic fatty acid metabolism and inflammation.

IDH2 Deficiency Is Critical in Myogenesis and Fatty Acid Metabolism in Mice Skeletal Muscle.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate into α-ketoglutarate with concurrent reduction of NADP+ to NADPH. However, it is not fully understood how IDH2 is intertwined with muscle development and fatty acid metabolism. Here, we examined the effects of IDH2 knockout (KO) on skeletal muscle energy homeostasis. Calf skeletal muscle samples from 10-week-old male IDH2 KO and wild-type (WT; C57BL/6N) mice were harvested, and the ratio of skeletal muscle weight to body and the ratio of mitochondrial to nucleic DNA were measured. In addition, genes involved in myogenesis, mitochondria biogenesis, adipogenesis, and thermogenesis were compared. Results showed that the ratio of skeletal muscle weight to body weight was lower in IDH2 KO mice than those in WT mice. Of note, a noticeable shift in fiber size distribution was found in IDH2 KO mice. Additionally, there was a trend of a decrease in mitochondrial content in IDH2 KO mice than in WT mice (p = 0.09). Further, mRNA expressions for myogenesis and mitochondrial biogenesis were either decreased or showed a trend of decrease in IDH2 KO mice. Moreover, genes for adipogenesis pathway (Pparg, Znf423, and Fat1) were downregulated in IDH2 KO mice. Interestingly, mRNA and protein expression of uncoupling protein 1 (UCP1), a hallmark of thermogenesis, were remarkably increased in IDH2 KO mice. In line with the UCP1 expression, IDH2 KO mice showed higher rectal temperature than WT mice under cold stress. Taken together, IDH2 deficiency may affect myogenesis, possibly due to impairments of muscle generation and abnormal fatty acid oxidation as well as thermogenesis in muscle via upregulation of UCP1.

Reactive oxygen species-mediated senescence is accelerated by inhibiting Cdk2 in Idh2-deficient conditions.

Among the many factors that promote cellular senescence, reactive oxygen species (ROS) are a focus of intense research because of their critical role in accelerating cellular senescence and initiating senescence-related diseases that can be fatal. Therefore, maintaining the proper balance of ROS in cells is a key method to alleviate senescence. Recent studies have found that isocitrate dehydrogenase 2 (IDH2), a critical enzyme of the tricarboxylic acid cycle, participates in ROS generation and in cellular dysfunction that is induced by excessive levels of ROS. Loss of IDH2 induces mitochondrial dysfunction that promotes excessive ROS generation and the development of several diseases. The results of this study suggest that Idh2 plays an important role in cellular senescence. Idh2 deficiency resulted in senescence-associated phenotypes and increased levels of senescence marker proteins in mouse embryonic fibroblasts and tissues. Furthermore, excessive ROS were generated in Idh2-deficient conditions, promoting cellular senescence by inducing cell cycle arrest through cyclin-dependent kinase 2. These results indicate that loss of Idh2 is a critical factor in regulating cellular senescence. Taken together, our findings contribute to the field of senescence research and suggest that IDH2 is a potential target of future anti-senescence studies.

IDH2 deficiency increases bone mass with reduced osteoclastogenesis by limiting RANKL expression in osteoblasts.

Mitochondria are not only responsible for cellular energy production but are also involved in signaling, cellular differentiation, cell death, and aging. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the decarboxylation of isocitrate to α-ketoglutarate, accompanied by NADPH production. IDH2 plays a central role in mitochondrial function in multiple cell types and various organs, including the heart, kidneys, and brain. However, the function of IDH2 in bone tissue is yet to be elucidated. Here, we report that disruption of IDH2 in mice results in high bone mass due to decreased osteoclast number and resorption activity. Although IDH2 played no cell-intrinsic role in osteoclasts, IDH2-deficient animals showed decreased serum markers of osteoclast activity and bone resorption. Bone marrow stromal cells/osteoblasts from Idh2 knockout mice were defective in promoting osteoclastogenesis due to a reduced expression of a key osteoclastogenic factor, receptor activator of nuclear factor-κB ligand (RANKL), in osteoblasts in vivo and in vitro through the attenuation of ATF4-NFATc1 signaling. Our findings suggest that IDH2 is a novel regulator of osteoblast-to-osteoclast communication and bone metabolism, acting via the ATF4-NFATc1-RANKL signaling axis in osteoblasts, and they provide a rationale for further study of IDH2 as a potential therapeutic target for the prevention of bone loss.

IDH2 deficiency impairs cutaneous wound healing via ROS-dependent apoptosis

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue that play a pivotal role in cutaneous wound healing by synthesizing fibronectin (a component of the extracellular matrix), secreting angiogenesis factors, and generating strong contractile forces. In wound healing, low concentrations of reactive oxygen species (ROS) are essential in combating invading microorganisms and in cell-survival signaling. However, excessive ROS production impairs fibroblasts. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates the mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. In the present study, the downregulation of IDH2 expression resulted in an increase in cell apoptosis in mouse skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also delayed cutaneous wound healing in mice and impaired dermal fibroblast function. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO alleviated the apoptosis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in cutaneous wound healing in association with mitochondrial ROS.

IDH2 deficiency exacerbates acetaminophen hepatotoxicity in mice via mitochondrial dysfunction-induced apoptosis

Acetaminophen (APAP)-induced hepatotoxicity is a major factor in liver failure and its toxicity is associated with the generation of reactive oxygen species (ROS), decreased levels of reduced glutathione (GSH) and overall oxidative stress. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) was demonstrated as an essential enzyme for mitochondria to maintain their antioxidant system by generating NADPH, which is an essential reducing equivalent for GSH turnover in mitochondria. Here, we investigated the role of IDH2 in APAP-induced liver injury with IDH2 deficient (idh2−/−) mice. Hepatotoxicity was promoted through apoptotic cell death following APAP administration in IDH2 deficient hepatocytes compared to that in wild-type hepatocytes. Apoptosis was found to result from the induction of ER stress and mitochondrial dysfunction as shown by the blocking the effect of phenylbutyrate and Mdivi1, respectively. In addition, mito-TEMPO, a scavenger of mitochondrial ROS, was seen to ameliorate APAP-induced hepatotoxicity in idh2−/− mice. In conclusion, IDH2 deficiency leads to a fundamental shortage of GSH that increases susceptibility to ROS generation and oxidative stress. This leads to excessive mitochondrial dysfunction and ER stress induction in response to APAP administration. Our study provides further evidence that IDH2 has a protective role against APAP-induced liver injury and emphasizes the importance of the elaborate linkages and functions of the antioxidant system in liver health.

Therapeutic potential of the mitochondria-targeted antioxidant MitoQ in mitochondrial-ROS induced sensorineural hearing loss caused by Idh2 deficiency

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major NADPH-producing enzyme which is essential for maintaining the mitochondrial redox balance in cells. We sought to determine whether IDH2 deficiency induces mitochondrial dysfunction and modulates auditory function, and investigated the protective potential of an antioxidant agent against reactive oxygen species (ROS)-induced cochlear damage in Idh2 knockout (Idh2−/−) mice. Idh2 deficiency leads to damages to hair cells and spiral ganglion neurons (SGNs) in the cochlea and ultimately to apoptotic cell death and progressive sensorineural hearing loss in Idh2−/− mice. Loss of IDH2 activity led to decreased levels of NADPH and glutathione causing abnormal ROS accumulation and oxidative damage, which might trigger apoptosis signal in hair cells and SGNs in Idh2−/− mice. We performed ex vivo experiments to determine whether administration of mitochondria-targeted antioxidants might protect or induce recovery of cells from ROS-induced apoptosis in Idh2-deficient mouse cochlea. MitoQ almost completely neutralized the H2O2-induced ototoxicity, as the survival rate of Idh2−/− hair cells were restored to normal levels. In addition, the lack of IDH2 led to the accumulation of mitochondrial ROS and the depolarization of ΔΨm, resulting in hair cell loss. In the present study, we identified that IDH2 is indispensable for the functional maintenance and survival of hair cells and SGNs. Moreover, the hair cell degeneration caused by IDH2 deficiency can be prevented by MitoQ, which suggests that Idh2−/− mice could be a valuable animal model for evaluating the therapeutic effects of various antioxidant candidates to overcome ROS-induced hearing loss.

IDH2 Deficiency Aggravates Fructose-Induced NAFLD by Modulating Hepatic Fatty Acid Metabolism and Activating Inflammatory Signaling in Female Mice.

Fructose is a strong risk factor for non-alcoholic fatty liver disease (NAFLD), resulting from the disruption of redox systems by excessive reactive oxygen species production in the liver cells. Of note, recent epidemiological studies indicated that women are more prone to developing metabolic syndrome in response to fructose-sweetened beverages. Hence, we examined whether disruption of the redox system through a deletion of NADPH supplying mitochondrial enzyme, NADP+-dependent isocitrate dehydrogenase (IDH2), exacerbates fructose-induced NAFLD conditions in C57BL/6 female mice. Wild-type (WT) and IDH2 knockout (KO) mice were treated with either water or 34% fructose water over six weeks. NAFLD phenotypes and key proteins and mRNAs involved in the inflammatory pathway (e.g., NF-κB p65 and IL-1β) were assessed. Hepatic lipid accumulation was significantly increased in IDH2 KO mice fed fructose compared to the WT counterpart. Neutrophil infiltration was observed only in IDH2 KO mice fed fructose. Furthermore, phosphorylation of NF-κB p65 and expression of IL-1β was remarkably upregulated in IDH2 KO mice fed fructose, and expression of IκBα was decreased by fructose treatment in both WT and IDH2 KO groups. For the first time, we report our novel findings that IDH2 KO female mice may be more susceptible to fructose-induced NAFLD and the associated inflammatory response, suggesting a mechanistic role of IDH2 in metabolic diseases.

P-28 - Idh2 deficiency exacerbates acrolein-induced lung injury through mitochondrial redox environment deterioration

Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of IDH2 in acrolein-induced lung injury using idh2 short hairpin RNA (shRNA)-transfected Lewis lung carcinoma (LLC) cells and idh2-deficient (idh2–/–) mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2–/– mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

P-101 - IDH2 deficiency exacerbates cisplatin nephrotoxicity

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is a major producer of NADPH in the mitochondria and plays a critical role in the redox balance. Cisplatin is an effective anticancer drug, however, its nephrotoxicity, by the breaking of redox balance, limits its use. Here, we investigated whether the cisplatin nephrotoxicity is associated with IDH2. IDH2 gene-deleted (IDH2-/-) and wild type (IDH2+/+) mice were administrated cisplatin with or without Mito-TEMPO, a mitochondria-specific antioxidant. Cisplatin reduced IDH2 activity and expression and NADPH levels in the kidney together with mitochondrial oxidative injury. Mitochondrial damage after cisplatin injection was greater in the IDH2-/-than IDH2+/+ kidneys. These changes were more pronounced in the IDH2-/- than IDH2+/+ kidneys, showing greater renal functional and morphological impairments in the IDH2-/-mouse. Mito-TEMPO reduced those cisplatin-induced kidney injuries both in IDH2-/- and IDH2+/+ mouse. This reduction in the IDH2-/- mouse was more prominent than that in the IDH2+/+ mouse. These results indicate that IDH2 deficiency worsens cisplatin nephrotoxicity by increasing oxidative stress, suggesting that cisplatin-induced nephrotoxicity is associated with mitochondrial IDH2.

Mitochondrial NADP+-dependent isocitrate dehydrogenase deficiency increases cisplatin-induced oxidative damage in the kidney tubule cells

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) plays an important role in the formation of NADPH, which is critical for the maintenance of mitochondrial redox balance. Cis-diamminedichloroplatinum II (cisplatin), an effective anticancer drug, induces oxidative stress-related nephrotoxicity, limiting its use. Therefore, we investigated whether IDH2, which is a critical enzyme in the NADPH-associated mitochondrial antioxidant system, is involved in cisplatin nephrotoxicity. Idh2 gene-deleted (Idh2−/−) mice and wild-type (Idh2+/+) littermates were treated with cisplatin, with or without 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-T), a mitochondria-specific antioxidant. Cisplatin-induced renal functional and morphological impairments were greater in Idh2−/− mice than in Idh2+/+ mice. Mito-T mitigated those impairments in both Idh2−/− and Idh2+/+ mice and this mitigation was greater in Idh2−/− than in Idh2+/+ mice. Cisplatin impaired IDH2 function in the mitochondria, decreasing mitochondrial NADPH and GSH levels and increasing H2O2 generation; protein, lipid, and DNA oxidation; mitochondrial damage; and apoptosis. These cisplatin-induced changes were much more severe in Idh2−/− mice than in Idh2+/+ mice. Mito-T treatment attenuated cisplatin-induced alterations in both Idh2−/− and Idh2+/+ mice and this mitigation was greater in Idh2−/− than in Idh2+/+ mice. Altogether, these data demonstrate that cisplatin induces the impairment of the mitochondrial IDH2-NADPH-GSH antioxidant system and IDH2 deficiency aggravates cisplatin-induced mitochondrial oxidative damage, inducing more severe nephrotoxicity. This suggests that the mitochondrial IDH2-NADPH-GSH antioxidant system is a target for the prevention of cisplatin-induced kidney cell death.

IDH2 deficiency accelerates skin pigmentation in mice via enhancing melanogenesis

Melanogenesis is a complex biosynthetic pathway regulated by multiple agents, which are involved in the production, transport, and release of melanin. Melanin has diverse roles, including determination of visible skin color and photoprotection. Studies indicate that melanin synthesis is tightly linked to the interaction between melanocytes and keratinocytes. α-melanocyte-stimulating hormone (α-MSH) is known as a trigger that enhances melanin biosynthesis in melanocytes through paracrine effects. Accumulated reactive oxygen species (ROS) in skin affects both keratinocytes and melanocytes by causing DNA damage, which eventually leads to the stimulation of α-MSH production. Mitochondria are one of the main sources of ROS in the skin and play a central role in modulating redox-dependent cellular processes such as metabolism and apoptosis. Therefore, mitochondrial dysfunction may serve as a key for the pathogenesis of skin melanogenesis. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. Downregulation of IDH2 expression resulted in an increase in oxidative DNA damage in mice skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also promoted pigmentation on the dorsal skin of mice, as evident from the elevated levels of melanin synthesis markers. Furthermore, pretreatment with mitochondria-targeted antioxidant mito-TEMPO alleviated oxidative DNA damage and melanogenesis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in skin melanogenesis in association with mitochondrial ROS and suggest unique therapeutic strategies for the prevention of skin pigmentation.

IDH2 Deficiency Aggravates Fructose‐Induced NAFLD by Activating Inflammatory Signaling in Female Mice

Fructose is a strong risk factor of non‐alcoholic fatty liver disease (NAFLD), resulting from disruption of redox systems by excessive reactive oxygen species production in the liver cells. Importantly, recent epidemiological studies indicated that women are more prone to develop metabolic syndrome in response to fructose‐sweetened beverages. Here, we examined whether disruption of redox system exacerbates fructose‐induced NAFLD conditions in female mice. The redox system was disrupted by blocking NADPH supply through mitochondrial NADP+‐dependent isocitrate dehydrogenase knock‐out (IDH2 KO) in C57BL/6 female mice. After, wild type (WT) and IDH2 KO mice were treated with either water or 34% fructose water over six weeks. NAFLD phenotypes and key proteins and mRNAs involved in inflammatory pathway (e.g., NF‐κB p65 and IL‐1β) were assessed. As results, hepatic lipid accumulation was significantly increased in IDH2 KO mice fed fructose compared to its WT counterpart. Neutrophil infiltration was observed only in IDH2 KO mice fed fructose. Further, phosphorylation of NF‐κB p65 and expression of IL‐1β was remarkably upregulated in IDH2 KO mice fed fructose, and expression of IκBα was decreased by fructose treatment in both WT and IDH2 KO groups. For the first time, we report that IDH2 KO significantly aggravates fructose‐induced NAFLD phenotypes (fatty liver and neutrophil infiltration) and related inflammatory responses (i.e., NF‐κB p65 pathway). Collectively, this study provides evidence that IDH2 deficiency may have implications on fructose‐induced NAFLD through suppression of fatty acid β‐oxidation and/or activation of the NF‐κB pathway. Given our significant phenotypes in response to fructose intervention, the present study will provide a good justification for further characterization thus warrants additional investigations to clarify mechanistic roles of mice IDH2 in metabolic diseases.Support or Funding InformationThis work was supported by the University of Arkansas, VPRED Start‐up fund (KJK, and PJH). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Idh2 deficiency accelerates renal dysfunction in aged mice

The free radical or oxidative stress theory of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species (ROS) that are produced as by-products of normal metabolic processes in mitochondria. The oxidative stress may arise as a result of either increased ROS production or decreased ability to detoxify ROS. The availability of the mitochondrial NADPH pool is critical for the maintenance of the mitochondrial antioxidant system. The major enzyme responsible for generating mitochondrial NADPH is mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2). Depletion of IDH2 in mice (idh2-/-) shortens life span and accelerates the degeneration of multiple age-sensitive traits, such as hair grayness, skin pathology, and eye pathology. Among the various internal organs tested in this study, IDH2 depletion-induced acceleration of senescence was uniquely observed in the kidney. Renal function and structure were greatly deteriorated in 24-month-old idh2-/- mice compared with wild-type. In addition, disruption of redox status, which promotes oxidative damage and apoptosis, was more pronounced in idh2-/- mice. These data support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice, and thus support the oxidative stress theory of aging.

P 197 - Role of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) on cisplatin-induced nephrotoxicity

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) is a major producer of NADPH, which is a critical factor for the maintenance of intracellular redox balance, in the mitochondria. Nephrotoxicity of cisplatin, an anticancer drug, limits the use of cisplatin by impairing glutathione (GSH)-mediated antioxidant system in the cell. Here, we investigated the involvement of IDH2 in cisplatin-induced nephrotoxicity using IDH2 gene-deleted (IDH2-/-) mice. IDH2 deficiency aggravated renal functional and morphological impairments after cisplatin administration. Cisplatin decreased the level of NADPH which is required for the reduction of oxidized glutathione (GSSG) by glutathione reductase (GR) in the kidney. This reduction of NADPH levels were greater in the IDH2-/- mouse kidneys than wild type (IDH2+/+) mouse kidneys. Cisplatin-induced reactive oxygen species (ROS) production and oxidative stress was greater in the IDH2-/- mouse kidneys than IDH2+/+ mouse kidneys as evaluated by hydrogen peroxide level, lipid peroxidation, and GSSG/total GSH ratio. In addition, mitochondrial fragmentation and renal cell death after cisplatin injection were greater in the IDH2-/- than IDH2+/+ mouse kidneys. These results indicate IDH2 deficiency worsens cisplatin-induced nephrotoxicity by increasing mitochondrial and cellular oxidative stresses.

Increased susceptibility of IDH2-deficient mice to dextran sodium sulfate-induced colitis

Inflammatory bowel disease (IBD) is a group of chronic, relapsing, immunological, inflammatory disorders of the gastrointestinal tract including ulcerative colitis (UC) and Crohn's disease (CD). It has been reported that UC, which is studied using a dextran sodium sulfate (DSS)-induced colitis model, is associated with the production of reactive oxygen species (ROS) and the apoptosis of intestine epithelial cells (IEC). Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme in the mitochondrial antioxidant system via generation of NADPH. Therefore, we evaluated the role of IDH2 in DSS-induced colitis using IDH2-deficient (IDH2-/-) mice. We observed that DSS-induced colitis in IDH2-/- mice was more severe than that in wild-type IDH2+/+ mice. Our results also suggest that IDH2 deficiency exacerbates PUMA-mediated apoptosis, resulting from NF-κB activation regulated by histone deacetylase (HDAC) activity. In addition, DSS-induced colitis is ameliorated by an antioxidant N-acetylcysteine (NAC) through attenuation of oxidative stress, resulting from deficiency of the IDH2 gene. In conclusion, deficiency of IDH2 leads to increased mitochondrial ROS levels, which inhibits HDAC activity, and the activation of NF-κB via acetylation is enhanced by attenuated HDAC activity, which causes PUMA-mediated apoptosis of IEC in DSS-induced colitis. The present study supported the rationale for targeting IDH2 as an important cancer chemoprevention strategy, particularly in the prevention of colorectal cancer.

Idh2 Deficiency Exacerbates Acrolein‐Induced Lung Injury through Mitochondrial Redox Environment Deterioration

Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (idh2) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of idh2 in acrolein-induced lung injury using idh2 short hairpin RNA- (shRNA-) transfected Lewis lung carcinoma (LLC) cells and idh2-deficient (idh2−/−) mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In addition, acrolein-induced toxicity in idh2 shRNA-transfected LLC cells and in idh2 knockout mice was ameliorated by the antioxidant, N-acetylcysteine, through attenuation of oxidative stress resulting from idh2 deficiency. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2−/− mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

Abstract 433: Mitochondrial Nadp-dependent Isocitrate Dehydrogenase is a Key Regulator in Vasomotor Function

Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) plays an essential role protecting cells against oxidative stress-induced damage. A deficiency in IDH2 leads to mitochondrial dysfunction and the production of reactive oxygen species (ROS) in cardiomyocytes and cancer cells. However, the function of IDH2 in vascular endothelial cells is mostly unknown. In this study the effects of IDH2 deficiency on mitochondrial and vascular function were investigated in endothelial cells. IDH2 knockdown decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III, which lead to increased mitochondrial ROS (mtROS). In addition, the levels of fission and fusion proteins (Mfn-1, OPA-1, and Drp-1) were significantly altered and MnSOD expression also was decreased by IDH2 knockdown. Furthermore, knockdown of IDH2 decreased eNOS phosphorylation and nitric oxide (NO) concentration in endothelial cells. Interestingly, treatment with Mito-TEMPO, an mtROS-specific scavenger, blunted mitochondrial fission, fusion and mtROS production, and reduced the IDH2 knockdown-induced decrease in MnSOD expression, eNOS phosphorylation and NO production in endothelial cells. Endothelium-dependent vasorelaxation was impaired, and the concentration of bioavailable NO decreased in the aortic ring in IDH2 knockout mice. These findings suggest that IDH2 deficiency induces endothelial dysfunction through the induction of dynamic mitochondrial changes and impairment in vascular function.