during malignant transformation , cells undergo stages of gene expression reprogramming and mutagenesis that alter their metabolic phenotype(s ) [ 15 ] . initial stimuli ( not all known ) dysregulate information signaling and activate oncogenes and/or cancer stem cells , resulting in a partial glycolytic  warburg phenotype [ 15 ] in which pyruvate is diverted , at least to a certain extent , from oxidative phosphorylation ( oxphos ) . high proliferation and impaired angiogenesis subsequently cause hypoxia in certain regions within a growing tumor , and then hypoxia - mediated metabolic reprogramming ( such as that promoted by hypoxia - induced factor , hif [ 68 ] ) further intensifies the glycolytic phenotype and may nearly completely divert pyruvate from pyruvate dehydrogenase ( pdh ) , that is , from oxphos . the sustained high rate of cell proliferation , however , results in aglycemia , initiating the revival of oxphos in conjunction with the promotion of glutaminolysis [ 1 , 2 , 9 , 10 ] . the overall glutaminolysis provides cytosolic pyruvate / lactate and also yields nadph via citrate export from mitochondria and subsequent atp - citrate lyase and malic enzyme reactions . this compensates for the reduced net energy production by the glycolytic pathway and pentose phosphate pathway ( ppp ) . pyruvate imported into mitochondria is the precursor of not only acetyl - coa but also citrate , which is required for fatty acid synthesis and hence for phospholipid synthesis , so it is essential for cell growth [ 15 ] . the final established phenotype is exemplified by human glioblastoma cells , which , despite their low respiration , maintain a constant pyruvate flux through pdh and hence partial oxphos . for the purpose of this paper , we shall use the term  glutaminolysis  in a more general way than just the transformation of glutamine to 2-oxoglutarate ( 2og ) . if 2og resulting from glutamine acts in the forward krebs cycle ( despite possible ongoing citrate extrusion and truncation of the cycle so that aconitase and  classic  nad - dependent isocitrate dehydrogenase ( idh3 ) reactions are not required ) , we define the system of metabolic reactions involved as  oxphos glutaminolysis . this term points out to its essential dependence on succinate dehydrogenase ( complex ii ) and hence on respiration and oxphos . in contrast , when the reductive carboxylation of 2og by isocitrate dehydrogenase 2 ( idh2 ) ( in the counter krebs cycle direction ) consuming nadph follows glutaminolysis of glutamine to 2og and when the reverse aconitase reaction and citrate efflux are added , we define that system as  reductive carboxylation glutaminolysis  ( rcg ) , also referred to as the latter term denotes the absolute independence of oxygen ( respiration ) .   in general although it acts frequently in broad cancer types , glutaminolysis is not universal for all cancers [ 35 ] . in cancer cells employing oxphos glutaminolysis , glutamine can fully compensate for the lack of glucose in terms of energy generation and syntheses of precursors for anabolic pathways [ 35 ] . thus , to survive under conditions of limited glucose , highly glycolytic cancer cells may adapt to glutaminolysis , which in its oxphos mode restores oxphos and may restore also at least partial pdh function [ 1 , 3 , 1214 ] . in normal cells , mitochondrial glutaminase catabolizes glutamine to produce ammonia and glutamate , which is further transaminated by glutamate dehydrogenase into 2og to feed the krebs cycle . in malignant tumors , negative allosteric effectors , such as gtp , inhibit glutamate dehydrogenase , resulting in a move toward glutaminolysis , where glutamate and pyruvate are reactants in a transamination reaction that produces , for example , alanine and 2og by alanine aminotransferase ( transaminase ) . in cancer cells , 2og usually feeds the forward - running krebs cycle truncated after citrate synthase during citrate extrusion , so that aconitase and  classic  nad - dependent idh3 reactions are not required [ 1 , 5 ] . this oxphos glutaminolytic mode is strictly dependent on oxaloacetate and acetyl - coa , that is , on the citrate synthase reaction ; hence , it proceeds only in cells in which oxaloacetate is provided by malate dehydrogenase fed by the krebs cycle as well as by malate import from the cytosol , where malate originates from atp - citrate lyase reaction . likewise incomplete inhibition of pdh restores acetyl - coa in mitochondria ( the pyruvate pool is split between the pdh and transaminase reactions ) . also , the mitochondrial malic enzyme may contribute to this pool by producing pyruvate from malate . citrate is extruded from mitochondria and converted to oxaloacetate and acetyl - coa by atp citrate lyase . acetyl - coa is then used to produce fatty acids by fatty acid synthase and cholesterol for general lipid synthesis , which is essential for cancer cell proliferation [ 18 , 19 ] . in glioblastoma , if there is excess nadh in the cytoplasm ( produced by aerobic glycolysis ) , the cytosolic oxaloacetate is converted first to malate by malate dehydrogenase and then to pyruvate by the cytosolic malic enzyme , thus may also contribute to lactate production [ 9 , 20 ] . moreover , alanine that is released by transaminase is used for cytosolic amino acid transformations and protein synthesis [ 1 , 4 , 5 ] . hypothetically , malignant tumors may survive on intermittent oxphos - independent rcg ( also termed  anoxic  glutaminolysis ) in parallel with intermittent glycolytic periods [ 1 , 2 ] . rcg utilizes reductive carboxylation of 2og by the reverse reaction of mitochondrial idh2 at the expense of nadph , followed by the reverse aconitase reaction and citrate efflux from the matrix [ 13 , 2123 ] . nadph is provided by the malic enzyme converting malate to pyruvate and might also be provided by the mitochondrial transhydrogenase . the oxphos independence of this mode means that it may proceed at any level of hypoxia and even at anoxia , thus increasing malignancy [ 1 , 3 ] . the reductive carboxylation involves idh2 , which converts 2og to isocitrate , from which the reverse aconitase reaction produces citrate , which is again exported from the mitochondrial matrix to the cytosol for fatty acid and lipid synthesis . note that acetyl - coa , and hence the pdh reaction , is not required in this mode . idh3 encodes a mitochondrial matrix nad - dependent octameric idh3 ( 422 subunits ) that acts in the krebs cycle . idh3 is allosterically positively regulated by ca , adp , and citrate and negatively regulated by atp , nadh , and nadph . the two other idh genes , idh1 and idh2 , encode cytosolic and mitochondrial matrix nadp - dependent ( or nadph - dependent ) idh1 and idh2 , respectively , which are structurally and genetically unrelated to idh3   ( table 1 ) . idh3 irreversibly decarboxylates isocitrate to yield 2og while reducing nad to nadh , whereas idh1 and idh2 catalyze reversible reactions , either decarboxylating isocitrate to 2og while reducing nadp to nadph or acting in the reductive carboxylation reaction to convert 2og to isocitrate while oxidizing nadph to nadp . heterozygous mutations in idh2 at arg172 and at the analogous residue arg132 in idh1 are frequently found in grade 2 and 3 gliomas , secondary glioblastomas , and acute myeloid leukemia ( aml ) , but they occur less frequently in primary glioblastomas and other cancers [ 2837 ] . no homozygous deletions of idh1 and idh2 have been found , as has been observed for classic tumor suppressors . nevertheless , mutated idh1 and idh2 exhibit a neomorphic enzyme activity , reducing 2og to d-2-hydroxyglutarate while converting nadph to nadp[23 , 29 , 36 , 3840 ] . interestingly , the d-2-hydroxyglutarate thus formed further promotes neoplasia by competitive inhibition of histone demethylation and 5-methyl - cytosine hydroxylation , leading to genome - wide alternations in the methylation of histones and dna . it has also been reported that glioblastoma sf188 cells produce d-2-hydroxyglutarate , in spite of lacking the above - described mutations . moreover , idh2 , like ~20% of other mitochondrial enzymes [ 42 , 43 ] , is acetylated at lysines , which inactivates the enzymatic activity . in turn , deacetylation of idh2 by the mitochondrial matrix deacetylase sirtuin 3 ( sirt3 ) activates the enzyme to produce more nadph . in nonmalignant cells , the cytosolic idh1 is involved in lipid metabolism and glucose sensing . idh2 was traditionally considered to be involved in the regulation of oxphos and redox homeostasis   ( see section 4 ) , and its involvement in reductive carboxylation has been recognized only recently ( sections 2.4 and 3 ) . nadp - dependent oxidative decarboxylation of isocitrate to 2og , as the major function of idh2 in nonmalignant cells , contributes substantially to the control of mitochondrial redox balance and the prevention of oxidative damage [ 45 , 46 ] . idh2 contains an n - terminal mitochondrial addressing sequence and hence is imported to the mitochondrial matrix , although localization to nuclei has also been reported . idh2 expression in heart , skeletal muscle , and lymphocytes is quite substantial ; lower levels are found in liver , kidney , and lung [ 45 , 47 ] . idh2 has also been found in cultured rat neurons , astrocytes , oligodendrocytes , and microglia . unlike idh1 , the 94-kda idh2 ( ec 1.1.1.42 ) is a homodimeric enzyme of two 413-amino acid subunits , each 47  kda [ 50 , 51 ] ( figure 1 ) . idh2 function requires a divalent metal ion , and bound mn yields the maximum activity . the structure of the mn - isocitrate binding site was mapped from the solved crystal structure of porcine idh2 . within the site , thr78 , ser95 , and asn97 ( of the porcine sequence ) donate a hydrogen bond to the c3 carboxyl , whereas asp252 and asp275 coordinate mn . the nadp binding site was originally predicted from the e. coli idh structures , positioning the 2-hydroxyl - bound phosphate to interact with his315 and lys374 of porcine idh2 . porcine arg83 enhances nadp affinity by hydrogen bonding with the 3-oh of the nicotinamide ribose , and asn328 provides a hydrogen bond to the n1 of adenine . for efficient coenzyme site function , a hydroxyl group must be present at position 373 ( thr373 of the porcine sequence ) , whereas asp375 and lys260 contribute to coenzyme affinity and catalysis [ 54 , 55 ] .   within the numbering of the human idh2 sequence , mutations in arg172 ( an analog of frequently mutated arg132 of cytosolic idh1 ) are detected in gliomas [ 23 , 36 , 37 ] , and mutations in arg172 and arg140 ( which is adjacent in the active site to arg172 ) are found in aml . mutations are apparent after the transition from a normal cell to a clinically evident tumor . arg172 ( as well as arg132 of idh1 ) provides hydrogen bonds to the  and  carboxyls of isocitrate and may be important in the transition from an open to closed state of the active site . the porcine arg residue mutated to asn in a position analogous to human arg172 displays a twofold reduction in specific activity and a km for isocitrate that is two orders of magnitude higher   ( table 1 ) . likewise , in lysates of cells overexpressing idh2 , activity is reduced when arg172 is substituted with gly , lys , or met . nevertheless , the existence of exclusively heterozygous idh2 ( and idh1 ) mutations in gliomas and aml and the small possibility of dominant - negative mutations ( minor mutant fractions existing could not exert this role ) have led to the search for other consequences of idh1 and idh2 mutations . arg132 mutants of cytosolic idh1   ( table 1 ) and arg172 mutants of mitochondrial idh2 [ 23 , 36 , 38 , 39 ] possess the ability to reduce 2og to d-2-hydroxyglutarate while converting nadph to nadp . this is because the active closed state of the enzyme exhibits a higher affinity for nadph . mutant idh2 and idh1   are not supposed to allow the reductive carboxylation reaction of 2og to isocitrate . initially , the production of d-2-hydroxyglutarate in gliomas , secondary glioblastomas , and aml by mutant idh2 results in a decrease of 2og and hence depletion of succinate , fumarate , and malate from the rest of the krebs cycle [ 23 , 36 , 39 ] . interestingly , idh2 mutations also lead to increases in amino acid levels   as would be expected for ongoing glutaminolysis . thus , the formed d-2-hydroxyglutarate strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methyl - cytosine hydroxylation , leading to genome - wide alternations in histone and dna methylation . moreover , prolyl hydroxylase domain enzymes , which employ 2og as a cofactor for marking hypoxia - induced factor-1 ( hif1 ) by proline hydroxylation , are inhibited by d-2-hydroxyglutarate as well as by the lack of 2og . as a result , hif1 is stabilized ( if its inhibitor , aspartyl hydroxylase factor inhibiting hif , is not active ) even at normoxia and thus can elicit the otherwise hypoxic reprogramming of gene expression . glutathionylation of proteins by the reversible glutaredoxin ( thioltransferase ) reaction serves to protect against irreversible oxidation of cysteines . such protection has been demonstrated for idh2 in that oxidized glutathione can inactivate idh2 by forming a mixed disulfide bond with cys269 . the inactivated idh2 is reactivated by glutaredoxin 2 in the presence of reduced glutathione . also , idh2 in mouse heart may exist in complex with calcineurin , containing as well aconitase , malate dehydrogenase , and mnsod . posttranslational modifications of numerous mitochondrial proteins frequently occur via acetylation / deacetylation of lysine residues [ 42 , 43 , 60 , 61 ] . among seven sirtuin family members , sirt3 , sirt4 , and sirt5 are enriched in mitochondria , such as exemplified and well described for sirt3 up to date [ 42 , 43 , 62 ] . caloric restriction prevents age - related hearing loss by reducing oxidative dna damage , but such is not the case for mice lacking sirt3 . sirt3 directly deacetylates the idh2 lysines and thus activates idh2 , which in its forward mode could result in increased nadph levels and thereby maintain reduced glutathione levels in mitochondria . indeed , overexpression of sirt3 and/or idh2 leads to increased nadph levels and is protective against oxidative stress - induced cell death . lys212 , lys374 , and lys260 ( porcine sequence ) may be the prime candidates for acetylation / inactivation . it remains to be shown that sirt3 can deacetylate these residues , however , and it should be investigated which reaction modes are active and possible before and after such activation , specifically , whether the reverse , nadph - dependent , reaction and rcg could be activated . as predicted in 1994 by sazanov and jackson   ( see also ) , the reductive carboxylation reaction by native idh2 converts 2og to isocitrate while oxidizing nadph to nadp . the arg172 and arg140 mutants of idh2 [ 23 , 36 , 38 , 39 ] and glioblastoma sf188 cells under hypoxia   convert 2og to d-2-hydroxy - glutarate in this  reverse - reaction  mode . this reductive carboxylation would proceed better in vivo when followed by the reverse aconitase reaction and subsequent citrate export from the mitochondrial matrix . reductive carboxylation was demonstrated for idh2 in 2002   and was indicated for cancer cells in transformed brown adipocytes , pediatric glioma sf188 cells [ 23 , 41 ] , and uok262 cells ( derived from a renal tumor in a patient with hereditary leiomyomatosis , these cells are defective in respiration and devoid of fumarate hydratase activity ) . reductive carboxylation accompanied by citrate efflux has also been found in quiescent fibroblasts and is enhanced in contact - inhibited fibroblasts . idh2 silencing in sf188 cells results in diminished conversion of glutamine to citrate [ 23 , 41 ] . recently , reductive carboxylation was detected in human osteosarcoma 143b cells in which the mitochondrial dna encoded a loss - of - function mutation in respiratory chain complex iii ( cytb 143b cells ) . because only low - level reductive carboxylation was detected in wild - type 143b cells , the authors suggested that the impairment of oxphos , such as given by mutant mitochondrial dna , induces rcg . silencing of either idh1 or idh2 reduces the growth of both wild - type and cytb 143b cells . moreover , unlike in wild - type 143b cells , de novo fatty acid synthesis from glutamine as a precursor is prevalent in cytb 143b cells . reductive carboxylation in fumarate hydratase  devoid uok262 cells , which are defective in respiration , has also been identified in parallel with oxphos glutaminolysis . interestingly , inhibition of respiration in mouse embryonic fibroblasts via administration of antimycin , rotenone , or metformin induces a switch towards rcg . thus , these data provide additional support for the authors ' hypothesis that rcg is a common cellular response to impaired mitochondrial metabolism . the reverse idh2 reaction was also considered such that idh2 acts together with the forward reaction of idh3 in a dissipative isocitrate/2og cycle   ( see section 4.2 ) . the reductive carboxylation reaction and the overall rcg may indeed proceed together with the forward decarboxylation reaction [ 2 , 22 ] . the best evidence was obtained by tracking the metabolites of c - labeled glutamine , such as the appearance of c - label in citrate [ 2 , 22 , 23 , 41 ] . the first demonstrations of rcg in cancer cells [ 22 , 23 , 41 ] are consistent with the recent findings that mutant idh2 in gliomas and aml also produce d-2-hydroxyglutarate from 2og by  alternate  reduction . because these mutants are heterozygous , both rcg and the production of d-2-hydroxyglutarate might occur . the former reaction involves the nonmutant nadph - dependent idh2  reverse  reaction followed by isocitrate conversion to citrate and by citrate export . the mutant idh2 ( but maybe also wild - type idh2 , see ) acting in a reverse  mode also produces d-2-hydroxyglutarate , which can not be transformed by aconitase ; however , it further enhances the malignant phenotype . the importance of this neomorphic idh2 activity for the cancer phenotype is valid even without consideration of d-2-hydroxyglutarate interference with epigenetics and the hif pathway , because idh2 depletes 2og from the krebs cycle . the consumption of nadph in the matrix is a consequence of the altered homeostasis of reactive oxygen species ( ros ) in cancer cells ( see section 4.2 ) and is also possible due to nadph production by the mitochondrial malic enzyme [ 1 , 5 ] and transhydrogenase [ 24 , 63 , 67 ] . it is not known whether sirt3-based activation also affects this reverse ( nadph - dependent ) idh2 reaction . although nadh , rather than nad , accumulates in the mitochondrial matrix of highly glycolytic cancer cells in which oxphos is dormant ( figure 2(a ) ) , nad might be produced by the inner membrane h transhydrogenase from nadh with the simultaneous formation of nadph from nadp in the matrix , thereby activating sirt3-mediated idh2 deacetylation . moreover , the usual acetylation of proteins may be retarded in highly glycolytic cancer cells ; hence , no sirt3-mediated deacetylation would be required . unlike glycolysis , rcg does not form atp . hence , either rcg coexistence with glycolysis or intermittent glycolysis is expected under hypoxic and deep hypoxic conditions ; that is , rcg may help cancer cells survive aglycemia and hypoxia in malignant cells . as clearly demonstrated by the examples of gliomas and aml with the oncogenic metabolite d-2-hydroxyglutarate , the establishment of rcg , even concomitantly with oxphos glutaminolysis ( note that this mode does not require idh2 and aconitase reactions ) , helps to accelerate the malignant phenotype . recently , it has been demonstrated that hypoxia elevates rcg in sf188 cells in a hif - dependent manner . sf188 cells were able to proliferate at 0.5% o2 even if such hypoxic conditions substantially diminished glucose - dependent production of citrate , that is , oxphos and forward krebs cycle participation . a major function of idh2 in nonmalignant cells , when acting within the forward krebs cycle , is likely maintaining an adequate pool of reduced glutathione and peroxiredoxin by providing nadph . this function improves the mitochondrial redox balance and prevents oxidative damage [ 45 , 46 , 69 ] , including heat - shock - induced oxidative damage   and numerous consequent events of oxidative stress , such as ros - induced apoptosis [ 71 , 72 ] , apoptosis induced by ionizing radiation   and cadmium , and staurosporine - induced cell death . the lack of idh2 or its activity elevates cytosolic ros , lipid peroxidation , and oxidative dna damage and shortens cell survival after oxidant exposure [ 69 , 7173 ] . also , susceptibility to curcumin - induced apoptosis has been demonstrated upon idh2 silencing in hct116 cells . cardiac hypertrophy development is attributed to a decrease in idh2 activity owing to the lipoperoxidation product 4-hydroxynonenal and oxidative stress . idh2 is also protective for paraquat - mediated oxidative inactivation of aconitase in heart mitochondria . inactivation of idh2 activity by various ros insults   is an important factor that has to be accounted for in any consideration of oxidative stress in cells . the forward krebs cycle activity of idh2 is inactivated by 4-hydroxynonenal , singlet oxygen , hypochlorous acid , aluminum , nitric oxide , and peroxynitrite . peroxynitrite forms s - nitrosothiol adducts on cys305 and cys387 of idh2 under nitrosative stress , such as that established in the liver of ethanol - fed rats . idh2 activity first increases and then decreases with age in fibroblasts and liver , kidney , and testes tissues of rats fed ad libitum but not of those fed a calorie - restricted diet . recently , caloric restriction has been proven to act via idh2 deacetylation through sirt3 and thus promote an antioxidant role for idh2-produced nadph . it is not known whether sirt3-mediated deacetylation also activates the nadph - dependent  reverse  reaction , that is , reductive carboxylation . the dissipative isocitrate/2og cycle has been suggested based on the reductive carboxylation reaction of idh2 ( counter krebs cycle reaction direction , nadph dependent ) in conjunction with the forward idh3 reaction in the canonical krebs cycle . the cycle may manifests itself in the absence of citrate export from mitochondria , as normally occurs in non - malignant cells , since cycling is impossible when reversed aconitase reaction depletes isocitrate . isocitrate formed by the reductive carboxylation reaction of idh2 is processed back to 2og by idh3 . although in non - malignant cells complex i regenerates nadh to nad and nadp could be regenerated to nadph by , for example , mitochondrial malic enzyme , with increasing malignancy ( more dormant state of mitochondria and hence decreasing respiration ) , the mitochondrial inner membrane h transhydrogenase [ 24 , 63 , 67 ] may alternatively transfer electrons from nadh and nadp to nadand nadph at the expense of the proton - motive force . however , it remains to be determined , whether this cycle is possible with d-2-hydroxyglutarate . if d-2-hydroxyglutarate was metabolized by idh3 in the canonical krebs cycle , then the cycle would be automatically induced by the appearance of d-2-hydroxyglutarate at simultaneously active h transhydrogenase . nevertheless oxphos can not be completely dormant , since proton - motive force would be required for this normal  forward  transhydrogenase reaction . consider the situation in highly malignant cells in which energy is derived primarily from glycolysis disconnected from oxphos ( warburg phenotype ) and high reductive carboxylation glutaminolysis takes place ( figure 2(a ) ) . presumably , oxphos impairment   or deep hypoxia   may set up this metabolic pattern . in this case , higher glucose-6-phosphate dehydrogenase activity ( the first ppp enzyme ) produces more nadph . it may be erroneously considered as antioxidant action ; however , because the constitutively expressed nadph oxidase isoform-4 , nox4 [ 89 , 90 ] , can consume a major portion of the excess nadph and produce more superoxide and consequently release more h2o2 into the cytosol , the overall reaction scheme may be prooxidant ( figure 2(a ) ) . recently , nox4 was also suggested to have mitochondrial localization . probably , nox4 has km in the same order of magnitude as idh enzymes . the nox4 consumption of nadph leaves fewer redox equivalents for the reduction of cellular glutathione and other redox systems [ 9294 ] . the cytosolic oxidative stress is further intensified by the slow electron transport in low respiring ( dormant ) mitochondria of highly malignant cancer cells [ 15 , 68 ] , resulting in more superoxide release to the cytosol as well as the mitochondrial matrix from the respiratory chain . the ongoing maximum reductive carboxylation reaction further contributes to the oxidative stress by consuming nadph , thus leaving less nadph for maintenance of the reduced glutathione pool . moreover , as mentioned above , the accumulated nadh at slow respiration may lead to nad formation in the reversed h transhydrogenase reaction by concomitant nadph formation from nadp to further feed the nadph pool and hence reductive carboxylation . simultaneously , nad may lead to sirt3-mediated activation of idh2 , at least of its  forward mode  , but it is not known whether reductive carboxylation is also activated by deacetylation of idh2 . we next consider an intermediate warburg phenotype , characterized by the mixed use of sole glycolysis , that is , aerobic glycolysis producing lactate , and oxphos ( figure 2(b ) ) . the latter may be represented either by oxphos pyruvate metabolism and/or by oxphos glutaminolysis . under these conditions , considerable cytosolic oxidative stress is expected because of the elevated nox4 activity , as described above . however , a lower mitochondrial contribution to the cytosolic oxidative stress exists owing to an intermediate level of respiration and hence lower superoxide release from mitochondria to both the cytosolic and matrix compartments . there is also lower oxidative stress expected in the matrix owing to the possible ongoing dissipative isocitrate/2og cycle , which by decreasing the proton - motive force   decreases mitochondrial superoxide formation . this can be considered , however , only when citrate efflux from mitochondria is not dominant or when d-2-hydroxyglutarate would be cycling instead of isocitrate/2og . if this is the case , nad rather than nadh would accumulate , further feeding the isocitrate/2og cycle by simultaneous action of the forward idh3 reaction and reverse ( reductive carboxylation ) reaction of idh2 . the accumulated nad would also promote sirt3-mediated idh2 deacetylation , consequently accelerating the idh2 branch of the reaction cycle ( figure 2(b ) ) . finally , we consider the situation in non - malignant cells , in which oxphos predominates and the sole glycolysis and ppp activities are low ( figure 2(c ) ) . in this case , low oxidative stress in the cytosol is a consequence of the negligible nox4 activity and the low contribution of mitochondria to the cytosolic ros pool . under these normal conditions , we assume that the forward idh2 reaction generates nadph , which further improves the reduced state of the mitochondrial matrix glutathione and peroxiredoxin systems . indeed , ample evidence suggests that in cells with unattenuated oxphos , idh2 plays an important antioxidant role that is further strengthened by nad accumulation in highly respiring cells . nad then induces sirt3-mediated idh2 deacetylation , thus increasing its protective function in nadph formation for the maintenance of the reduced glutathione and peroxiredoxin systems and for self - maintenance by the reactivation of cystine - inactivated idh2 by glutaredoxin-2 . ( a ) situation in cancer cells with a prevalent warburg phenotype and high reductive carboxylation . in the cytosol , higher glucose-6-phosphate dehydrogenase activity ( g6pdh ) produces higher nadph within the pentose phosphate pathway ( ppp ) . nadph - oxidase isoform-4 ( nox4 ) thus produces more superoxide and consequently contributes to high levels of reactive oxygen species ( ros ) in the cytosol . the cytosolic oxidative stress is further intensified by the slow electron transport in low - respiring ( dormant ) mitochondria , leading to higher superoxide release to the cytosol and matrix compartments . the ongoing maximum reductive carboxylation reaction further contributes to oxidative stress by consuming nadph , thus leaving less for maintenance of the reduced glutathione pool . the accumulated nadh at slow respiration may lead to nad formation in the reverse h transhydrogenase ( th ) reaction ( due to low proton / motive force ) with concomitant nadph formation from nadp to further feed the nadph pool and hence reductive carboxylation . hypothetically , nad may lead to sirtuin 3 ( sirt3)-mediated deacetylation / activation ( + ) of idh2 , but it is not known whether reductive carboxylation is also activated by deacetylation of idh2 . ( b ) situation in cancer cells with an intermediate warburg phenotype and possible oxphos glutaminolysis . the major contribution to however , a lower mitochondrial contribution to cytosolic ros leads to intermediate oxidative stress under these conditions . indeed , an intermediate or high respiration leads to much lower superoxide production and release from the mitochondria to both the cytosolic and matrix compartments ( dashed arrows ) . lower oxidative stress is also expected in the matrix owing to the ongoing dissipative isocitrate/2og cycle , which decreases further mitochondrial superoxide formation by decreasing the proton - motive force . oxphos glutaminolysis may predominate under these conditions ; hence , rcg might not be completed and the isocitrate/2og cycle with forward h transhydrogenase reaction may be initiated . aerobic glycolysis and ppp activity are low ; consequently , low oxidative stress in the cytosol also results from the negligible nox4 activity and the low contribution of mitochondria to the cytosolic ros . the forward idh2 reaction thus forms nadph , which further improves the reduced state in the mitochondrial matrix glutathione and peroxiredoxin systems . as in ( b ) , the nad accumulation in highly respiring cells then induces sirt3-mediated deacetylation / activation of idh2 . as briefly described above , mutant idh2 as well as idh1 [ 96100 ] produce d-2-hydroxy - glutarate , which can initiate an hif - mediated  hypoxic type  of gene reprogramming even at normoxia . nevertheless , detail investigations of d-2-hydroxyglutarate effects on hif signaling are required , since recently an opposite effect , diminishing hif levels by inhibition of egln prolyl 4-hydroxylases , has been reported . also , non - mutated idh2 acting in the 2og / isocitrate cycle together with h transhydrogenase   could contribute to the modulation of the ros pool by initiating an impulse originating from complex iii to dissipate the proton - motive force , which reduces superoxide formation in the mitochondrial respiratory chain . in contrast to mutant idh2 activity , the ongoing dissipative 2og / isocitrate cycle in the absence of citrate efflux from mitochondria would retard hif signaling . the discovery of mutant idh2 and idh1 in certain gliomas and aml and their production of the oncogenic metabolite d-2-hydroxyglutarate have unraveled a fascinating story of cancer self - acceleration via intermittent episodes of genome instability and metabolic remodeling and namely via epigenomic alterations [ 98 , 100 ] . however , there are additional aspects to be clarified . first , the exact role of d-2-hydroxy - glutarate must be further investigated to determine whether it promotes the reverse carboxylation mode of glutaminolysis and whether it acts in the dissipative 2og / isocitrate cycle , which would then become the 2og / d-2-hydroxyglutarate cycle , as we can now only speculate . also , conditions under which d-2-hydroxyglutarate might be formed in non - mutant idh2 should be defined . second , the role of sirt3 has to be established to determine whether it prevents or accelerates malignancy via idh2 . finally , the role of idh2 in other cancer types distinct from aml , gliomas , and renal tumors of hereditary leiomyomatosis should be investigated .