Genetic Perturbation of Glycolysis Results in Inhibition of de Novo Inositol Biosynthesis

Abstract

In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the “inositol-less death” phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis. In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the “inositol-less death” phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis. Bipolar disorder, also called manic-depressive illness, is a severe psychiatric illness with a prevalence of about 1.5% (1Weissman M.M. Leaf P.J. Tischler G.L. Blazer D.G. Karno M. Bruce M.L. Florio L.P. Psychol. Med. 1988; 18: 141-153Crossref PubMed Scopus (578) Google Scholar). Lithium and valproate (VPA) 3The abbreviations used are: VPAvalproateDHAPdihydroxyacetone phosphateG3Pglyceraldehyde 3-phosphateMIPmyo-inositol 3-phosphateG6Pglucose 6-phosphateOAAoxaloacetateYPDyeast extract/peptone/dextrose. are two FDA-approved drugs for the treatment of bipolar disorder. Neither drug is completely effective, but the development of new therapies is hindered by the fact that the mechanisms underlying the therapeutic effects of lithium and VPA are not known. The inositol depletion hypothesis has been proposed to explain the therapeutic effects of lithium in the treatment of bipolar disorder (2Berridge M.J. Downes C.P. Hanley M.R. Cell. 1989; 59: 411-419Abstract Full Text PDF PubMed Scopus (890) Google Scholar). This hypothesis is based on the evidence that inositol monophosphatase is inhibited by therapeutic concentrations of lithium, which can, thus, disrupt the phosphoinositide cycle. More recently, VPA has also been linked to inositol depletion. VPA was found to decrease the concentration of myo-inositol in rat brain after chronic administration (3O'Donnell T. Rotzinger S. Nakashima T.T. Hanstock C.C. Ulrich M. Silverstone P.H. Brain Res. 2000; 880: 84-91Crossref PubMed Scopus (127) Google Scholar). Both lithium and VPA cause a decrease in intracellular inositol in yeast (4Vaden D.L. Ding D. Peterson B. Greenberg M.L. J. Biol. Chem. 2001; 276: 15466-15471Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Moreover, a recent study showed that lithium, VPA, and carbamazepine, another mood-stabilizing drug, decreased growth cone collapse and increased growth cone area in sensory neurons in culture. These effects were abolished by the addition of inositol (5Williams R.S. Cheng L. Mudge A.W. Harwood A.J. Nature. 2002; 417: 292-295Crossref PubMed Scopus (554) Google Scholar). These studies suggested that inositol metabolism may be associated with the mechanism of action of lithium and VPA. Surprisingly, very little is known about the molecular control of inositol de novo biosynthesis in human cells. valproate dihydroxyacetone phosphate glyceraldehyde 3-phosphate myo-inositol 3-phosphate glucose 6-phosphate oxaloacetate yeast extract/peptone/dextrose. The de novo biosynthesis of inositol has been extensively characterized in the yeast Saccharomyces cerevisiae. Isolation of spontaneous mutants unable to grow in the absence of inositol in S. cerevisiae was first carried out three decades ago (6Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar). Inositol auxotroph mutants undergo inositol-less death, the abrupt decrease in viable cells when deprived of inositol (7Henry S.A. Donahue T.F. Culbertson M.R. Mol. Gen. Genet. 1975; 143: 5-11Crossref PubMed Scopus (48) Google Scholar). In the rate-limiting step of inositol synthesis, glucose 6-phosphate (G6P) is converted to myo-inositol 3-phosphate (MIP), catalyzed by the INO1-encoded MIP synthase (8Majumder A.L. Johnson M.D. Henry S.A. Biochim. Biophys. Acta. 1997; 1348: 245-256Crossref PubMed Scopus (175) Google Scholar). MIP is then dephosphorylated to myo-inositol by inositol monophosphatase (8Majumder A.L. Johnson M.D. Henry S.A. Biochim. Biophys. Acta. 1997; 1348: 245-256Crossref PubMed Scopus (175) Google Scholar). MIP synthase has been purified from a number of species (9Sherman W.R. Loewus M.W. Pina M.Z. Wong Y.H. Biochim. Biophys. Acta. 1981; 660: 299-305Crossref PubMed Scopus (14) Google Scholar, 10Johnson M.D. Sussex I.M. Plant Physiol. 1995; 107: 613-619Crossref PubMed Scopus (58) Google Scholar, 11Donahue T.F. Henry S.A. J. Biol. Chem. 1981; 256: 7077-7085Abstract Full Text PDF PubMed Google Scholar, 12Bachhawat N. Mande S.C. J. Mol. Biol. 1999; 291: 531-536Crossref PubMed Scopus (61) Google Scholar, 13Guan G. Dai P. Shechter I. Arch. Biochem. Biophys. 2003; 417: 251-259Crossref PubMed Scopus (41) Google Scholar, 14Chun J.A. Jin U.H. Lee J.W. Yi Y.B. Hyung N.I. Kang M.H. Pyee J.H. Suh M.C. Kang C.W. Seo H.Y. Lee S.W. Chung C.H. Planta. 2003; 216: 874-880Crossref PubMed Scopus (38) Google Scholar). Myo-2-inosose 1-phosphate, an intermediate in the reaction, is a strong competitive inhibitor of yeast MIP synthase (15Migaud M.E. Frost J.W. J. Am. Chem. Soc. 1995; 117: 5154-5155Crossref Scopus (15) Google Scholar). Further investigation also demonstrated that myo-2-inosose 1-phosphate analogues, including 2-deoxy-myo inositol 1-phosphate, 1-deoxy-1-(phosphonomethyl)-myo-2-inosose, and dihydroxyacetone 1-phosphate (DHAP), are MIP synthase inhibitors (16Migaud M.E. Frost J.W. J. Am. Chem. Soc. 1996; 118: 495-501Crossref Scopus (17) Google Scholar). The extensive genetic and biochemical characterization of inositol metabolism in yeast make it an excellent model for investigating the mechanisms of action of inositol-depleting drugs. Furthermore, MIP synthase is highly conserved from yeast to humans, as expression of human MIP synthase complements the inositol auxotrophy of yeast ino1 null mutants (17Ju S. Shaltiel G. Shamir A. Agam G. Greenberg M.L. J. Biol. Chem. 2004; 279: 21759-21765Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Consistent with the hypothesis that VPA depletes inositol by indirect inhibition of MIP synthase, human MIP synthase activity is decreased in vivo in the presence of VPA (17Ju S. Shaltiel G. Shamir A. Agam G. Greenberg M.L. J. Biol. Chem. 2004; 279: 21759-21765Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To determine how lithium and VPA affect the inositol metabolic pathway, we used a genetic approach to identify mutants hypersensitive to lithium and VPA in the absence of inositol. We identified a yeast mutant defective in TPI1, which encodes triose phosphate isomerase. This enzyme catalyzes the interconversion of DHAP and glyceraldehyde 3-phosphate (G3P). In humans, TPI deficiency is a rare multisystem disorder characterized by autosomal recessive inheritance (18Schneider A.S. Valentine W.N. Hattori M. Heins Jr., H.L. N. Engl. J. Med. 1965; 272: 229-235Crossref PubMed Scopus (110) Google Scholar). It is associated with chronic hemolytic anemia, recurrent infections, cardiomyopathy, progressive neurologic dysfunction, and death in childhood (18Schneider A.S. Valentine W.N. Hattori M. Heins Jr., H.L. N. Engl. J. Med. 1965; 272: 229-235Crossref PubMed Scopus (110) Google Scholar). The crystal structures of Tpi1p from different species indicate that the enzyme is a dimer of two identical subunits (19Wierenga R.K. Kalk K.H. Hol W.G. J. Mol. Biol. 1987; 198: 109-121Crossref PubMed Scopus (61) Google Scholar, 20Lolis E. Alber T. Davenport R.C. Rose D. Hartman F.C. Petsko G.A. Biochemistry. 1990; 29: 6609-6618Crossref PubMed Scopus (254) Google Scholar, 21Mande S.C. Mainfroid V. Kalk K.H. Goraj K. Martial J.A. Hol W.G. Protein Sci. 1994; 3: 810-821Crossref PubMed Scopus (124) Google Scholar, 22Delboni L.F. Mande S.C. Rentier-Delrue F. Mainfroid V. Turley S. Vellieux F.M. Martial J.A. Hol W.G. Protein Sci. 1995; 4: 2594-2604Crossref PubMed Scopus (114) Google Scholar). The subunit interface is of particular interest because it is critical for dimer stability (23Sun A.Q. Yuksel K.U. Gracy R.W. J. Biol. Chem. 1992; 267: 20168-20174Abstract Full Text PDF PubMed Google Scholar, 24Casal J.I. Ahern T.J. Davenport R.C. Petsko G.A. Klibanov A.M. Biochemistry. 1987; 26: 1258-1264Crossref PubMed Scopus (60) Google Scholar). In humans, 80% of TPI-deficient patients carry an inherited substitution of aspartate for glutamate at residue 104 (E104D). This mutation is located at the subunit interface of the Tpi1p dimer, which leads to loss of enzyme activity due to instability of the mutant dimer (25Arya R. Lalloz M.R. Bellingham A.J. Layton D.M. Hum. Mutat. 1997; 10: 290-294Crossref PubMed Scopus (40) Google Scholar). In this report we show that a single mutation (N65K) in the dimer interface region of TPi1p completely abolished enzyme activity and led to a 30-fold increase in the intracellular DHAP level. Interestingly, tpi1 exhibited the inositol auxotrophy phenotype. Consistent with inositol auxotrophy, MIP synthase was inhibited by DHAP, and other metabolites of carbohydrate metabolism also inhibited the enzyme. This is the first report indicating that the perturbation of glycolysis inhibits the inositol biosynthetic pathway. Materials—All chemicals used were reagent grade or better. Glucose, yeast extract, and peptone were bought from Difco. Amino acids, NAD, NADH, myo-inositol, G6P, imidazole, lysozyme, VPA, ampicillin, ethylmethane sulfonate, sodium thiosulfate, triethanolamine, DHAP, and G3P were from Sigma. [14C]G6P, [14C]inositol, and [32P]UTP were from PerkinElmer Life Sciences. Protease inhibitor tablets and glycerol-3-phosphate dehydrogenase were from Roche Applied Science. The Zymoprep Yeast Plasmid Miniprep kit was bought from Zymo Research. The pGEM-T EASY vector, Wizard Miniprep DNA purification system and Wizard SV gel and PCR Clean-Up system were from Promega. Strains—The yeast strains used in this study are shown in TABLE ONE.TABLE ONEYeast strains used in this studyStrainsCharacteristics or genotypeSource/commentsSMY15D273-10B derivative, met2, ura3-52, MATaM. Greenberg laboratorySMY10Isogenic to SMY15 except lys2, met2, MATαM. Greenberg laboratorytpi1Isogenic to SMY15 except tpi N65KThis study; original mutant out-crossed to SMY10BY4741his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, MATaInvitrogenBY4741 ino1Δhis3Δ1, leu2Δ0, met15Δ0, ura3Δ0, ino1Δ::KanMX, MATaInvitrogenN501-1Bleu2, mal, mel, gal2, MATαATCCN548-8A, pgk1pgk1, leu2, mal, me, gal2, MATaATCC Open table in a new tab Growth Media—YPD (complex medium) contained glucose (2% w/v), Bacto-peptone (2% w/v), yeast extract (1% w/v), and agar (2% w/v) for solid medium. Complete synthetic defined medium contained glucose (2% w/v), ammonium sulfate (2 g/liter), and the amino acids and nucleotides arginine (20 mg/liter), adenine (20 mg/liter), histidine (10 mg/liter), leucine (60 mg/liter), lysine (20 mg/liter), methionine (20 mg/liter), threonine (0.3 g/liter), tryptophan (20 g/liter), and uracil (40 mg/liter). The salts are the of yeast and the synthetic medium contained yeast extract, and and was with and was carried out to the of G. C.C. J. Genet. 1965; PubMed Scopus Google Scholar). were to for and cells were on and to synthetic medium with or or VPA. mutants were to and to medium or lithium, VPA, or myo-inositol in which to lithium and VPA was in the presence of inositol were for of in a single gene were by with a in the of yeast DNA or a and were by uracil The were lithium or VPA to for increased drug To that from the DNA was from yeast a Zymoprep yeast in a Wizard Miniprep DNA purification and the DNA of the were with that the The from the and were used to identify the were that the of the in the The at and the at The for the the the at and at was with The were by a in the Saccharomyces to for to the The TPI1 were from DNA of SMY15 and were to the of the TPI1 gene and of the was carried and and were Growth were in the presence of inositol for cells in inositol were and in medium for were to an of and to the growth number was by a and the of cells were on the was by and on Plasmid the TPI1 gene was from the by the and and the The the TPI1 with and A TPI1 was from yeast DNA the TPI1 and TPI1 The PCR were the pGEM-T EASY The was with for of the TPI1 of TPI1 and were and was by F.M. R. J.A. K. in and on an and to a The were with TPI or by a for the gene to for for were the system from with as as and The of were by Protein concentration was by the of M.M. Biochem. PubMed Scopus Google as the activity of triose phosphate was in as by and J. Biol. Chem. Full Text PDF PubMed Google Scholar). of DHAP of intracellular DHAP concentration was in as by F. 1996; Scholar). cells were by a and in The was in and on three of the extract was carried out with at The DHAP was in NADH, and glycerol-3-phosphate To the intracellular DHAP a yeast of of yeast was K. Biochem. 1992; PubMed Scopus Google Scholar). of MIP A.J. J.H. Biol. 2000; PubMed Scopus Google and human (17Ju S. Shaltiel G. Shamir A. Agam G. Greenberg M.L. J. Biol. Chem. 2004; 279: 21759-21765Abstract Full Text Full Text PDF PubMed Scopus (58) Google MIP were and purified as MIP well were used to the MIP synthase the of and J. Biol. Chem. Full Text PDF PubMed Google and the of D.L. Biochem. J. PubMed Scopus (104) Google Scholar). The of concentrations of DHAP on yeast MIP synthase was by the of and J. Biol. Chem. Full Text PDF PubMed Google because phosphate in concentrations of DHAP with the was in the presence of G6P, and of purified yeast MIP synthase and inhibition for DHAP, and oxaloacetate on yeast and human MIP synthase were by the of D.L. Biochem. J. PubMed Scopus (104) Google with contained of purified to for yeast MIP synthase and for human MIP for and inhibition for DHAP, and were by of enzyme in the with concentrations of and and G3P and and and of identify of lithium and VPA, we a genetic screen to mutants hypersensitive to both drugs in the absence of in which was by inositol. was carried out to the of G. C.C. J. Genet. 1965; PubMed Scopus Google Scholar). cells were and mutants and valproate were mutants were and were including in which mutants a growth phenotype. of the was characterized that is for inositol and The mutant was unable to grow at on synthetic but inositol The mutant was hypersensitive to lithium or VPA in the presence of inositol. with inositol growth of the mutant in the presence of lithium and VPA but not growth of the mutant at Further that the mutant exhibited inositol-less death, the decrease in when cells are for inositol (7Henry S.A. Donahue T.F. Culbertson M.R. Mol. Gen. Genet. 1975; 143: 5-11Crossref PubMed Scopus (48) Google Scholar). in cells from to to in which MIP synthase is mutant inositol-less death when for inositol. of and strains and were in to the of were and at to or were at the after the on and at for To the gene in the mutant was with and and were for the to grow at in the absence of in the presence of lithium or VPA, and at the of on synthetic uracil out A a DNA the TPI1 and a the TPI1 gene the mutant including drug inositol and The TPI1 gene was the and the of the TPI1 gene growth in the absence of in the presence of lithium and VPA, and at a single mutation in the TPI1 of in a to a single amino to at residue The residue is at the dimer interface and is conserved in species for which the gene has been The mutant be to as Tpi1p amino The was carried out the The the mutation in of Tpi1p and of DHAP in The activity of triose phosphate and intracellular DHAP were in of tpi1 and cells. in TABLE triose phosphate activity is not in tpi1 mutant which about 30-fold DHAP cells. This is with found in a identified tpi1 mutant F. 1996; Scholar). The presence of a TPI1 gene in tpi1 of enzyme activity and intracellular The loss of triose phosphate activity in the tpi1 mutant is not due to decreased expression of TPI1, the of which were in tpi1 and cells phosphate activity and intracellular DHAP concentration in phosphate of Open table in a new tab of DHAP MIP determine inositol auxotrophy be by inhibition of MIP synthase by DHAP, we the activity of purified yeast MIP synthase in the presence of in DHAP inhibits MIP synthase DHAP, the concentration in the tpi1 mutant, yeast MIP synthase activity was completely This result suggested that inositol auxotrophy of tpi1 is due to inhibition of MIP synthase by increased DHAP and that other glycolysis mutants that DHAP are also inositol The glycolysis mutant pgk1 is defective in the conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, and of DHAP has been demonstrated in this mutant M. I. J. PubMed Google Scholar). Consistent with this pgk1 was also unable to grow in the absence of inositol and exhibited inositol-less death when deprived of inositol Inositol auxotrophy of tpi1 not be by the to in is in the tpi1 mutant when deprived of inositol. was by of from cells after the from to expression was in tpi1 and cells Furthermore, when in the absence of DHAP, MIP synthase was in tpi1 These indicate that is in tpi1 and an pgk1 mutant is unable to grow in the absence of inositol and inositol-less of the pgk1 mutant and were in to the of cells were on or and at cells were and at to and were at the after the on and for of and MIP synthase in tpi1 is were in to the of at to for and expression was by of from cells and with for and the gene as the of three MIP synthase activity was in by the of and J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of three We to determine other of carbohydrate metabolism to DHAP MIP synthase at This indicate control of MIP synthase perturbation of carbohydrate The and the inhibited MIP synthase activity as well The for G6P, the of yeast MIP synthase, was in TABLE DHAP, and were competitive inhibitors of yeast MIP synthase with of and result is with the that DHAP is a competitive inhibitor of yeast MIP human MIP synthase, we The for was The for DHAP, and were and In patients with triose phosphate DHAP as as A. A. Lalloz M.R. R. B. J. J. Bellingham A.J. Layton D.M. 1999; PubMed Google or of S. I. L. M. M. V. T. Sci. S. A. 1995; PubMed Scopus Google been These be to which is in the that human MIP synthase activity in of MIP synthase by DHAP, and of MIP synthase for G6P, MIP synthase for G6P, Open table in a new tab The of inositol auxotrophy in the tpi1 mutant perturbation of glycolysis to the inositol biosynthetic pathway. DHAP increased about 30-fold in with DHAP for a tpi1 mutant F. 1996; Scholar). DHAP is a competitive inhibitor of MIP synthase, due to to the and phosphate of myo-2-inosose 1-phosphate, an intermediate in the MIP synthase In the tpi1 mutant the gene is and MIP synthase is when inositol is as shown in inhibition of MIP synthase by DHAP is the for inositol auxotrophy of Consistent with the glycolysis mutant which also accumulates DHAP, was also for inositol. yeast and human MIP were inhibited by DHAP as well as by other metabolites of carbohydrate including G3P and These that genetic perturbation of glycolysis leads to inhibition of inositol de novo biosynthesis by inhibition of MIP This report a between glycolysis and inositol biosynthesis and and from studies of carbohydrate to of is by the glucose pathway, an mechanism that expression of for of other glucose when glucose is and on when glucose is Genet. 1995; Full Text PDF PubMed Scopus Google Scholar, M. 1999; PubMed Scopus Google Scholar). In the glucose is by the and the which a of for of M. 1999; PubMed Scopus Google Scholar). glucose is the glucose is is by Interestingly, the glucose inositol at the of The and mutants of expression and are inositol S.A. F. 1992; PubMed Scopus Google Scholar, Genetics. 1999; PubMed Google the mutant increased expression of and has an inositol Genetics. 1999; PubMed Google Scholar, M. Henry S.A. Genetics. 1999; PubMed Google Scholar). expression of is when the to glucose is The for the pathology associated with TPI of well enzyme (18Schneider A.S. Valentine W.N. Hattori M. Heins Jr., H.L. N. Engl. J. Med. 1965; 272: 229-235Crossref PubMed Scopus (110) Google Scholar, R. Lalloz M.R. Bellingham A.J. Layton D.M. Hum. Mutat. 1997; 10: 290-294Crossref PubMed Scopus (40) Google Scholar, A. B. J. R. R. R. E. Am. J. 1995; PubMed Scopus Google Scholar). In to other TPI-deficient patients a severe neurologic disorder J. F. J. S. J. Biochem. Soc. 2002; PubMed Google Scholar). The may in a result of inositol The brain and in brain inositol concentrations been in a number of including bipolar disorder, and J. 2002; PubMed Scopus Google Scholar). A recent study that MIP synthase activity is inhibited by VPA in of human G. Shamir A. J. Ding D. E. M. Harwood A.J. Greenberg M.L. Agam G. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google which may explain the decrease in intracellular inositol in the brain (3O'Donnell T. Rotzinger S. Nakashima T.T. Hanstock C.C. Ulrich M. Silverstone P.H. Brain Res. 2000; 880: 84-91Crossref PubMed Scopus (127) Google Scholar). In patients with TPI DHAP of are as as A. A. Lalloz M.R. R. B. J. J. Bellingham A.J. Layton D.M. 1999; PubMed Google Scholar, S. I. L. M. M. V. T. Sci. S. A. 1995; PubMed Scopus Google Scholar). MIP synthase activity is inhibited by in the presence of DHAP not It is that inhibition of MIP synthase by increased DHAP in TPI deficiency leads to inositol depletion in the which may be by inositol It is in this that inositol has been to be an treatment for system as disorder, and disorder L. Med. 3: Google Scholar). This study may for responsiveness to the inositol lithium and VPA. is associated with a increase in DHAP and G3P N. R. Am. J. Physiol. PubMed Google Scholar, A. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. M.J. PubMed Scopus Google Scholar). The of both DHAP and G3P affect inositol de novo biosynthesis by inhibition of MIP synthase activity and thus, a with inositol drugs. Further of the between glycolysis and inositol de novo biosynthesis may to and metabolic We M. for and of the Greenberg for and