Cooperativity between 11β-Hydroxysteroid Dehydrogenase Type 1 and Hexose-6-phosphate Dehydrogenase in the Lumen of the Endoplasmic Reticulum

Abstract

The functional coupling of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11β-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other. The functional coupling of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11β-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other. The endoplasmic reticulum is a separate subcellular compartment with properties different from those of the cytosol. Its lumen is characterized by an oxidative redox potential (1Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1571) Google Scholar), which is maintained by the relative impermeability of the endoplasmic reticulum membrane toward numerous redox-active compounds, including pyridine nucleotides.Because the oxidoreductases of the lumen have to use a separate NAD(P)+/NAD(P)H pool, a functional cooperation between intraluminal oxidases and reductases has been hypothesized (2Sawada H. Hara A. Hayashibara M. Nakayama T. Usui S. Saeki T. J. Biochem. (Tokyo). 1981; 90: 1077-1085Crossref PubMed Scopus (18) Google Scholar, 3Sawada H. Hayashibara M. Hara A. Nakayama T. J. Biochem. (Tokyo). 1980; 87: 985-988Crossref PubMed Scopus (12) Google Scholar, 4Kulkarni A.P. Hodgson E. Biochem. Pharmacol. 1982; 31: 1131-1137Crossref PubMed Scopus (16) Google Scholar, 5Draper N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google Scholar). Hexose-6-phosphate dehydrogenase (H6PDH) 1The abbreviations used are: H6PDH, hexose-6-phosphate dehydrogenase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; G6PT, glucose-6-phosphate transporter; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: H6PDH, hexose-6-phosphate dehydrogenase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; G6PT, glucose-6-phosphate transporter; MOPS, 4-morpholinepropanesulfonic acid. is supposed to have a key role in the generation of reducing equivalents, which can be used by several reductases present in the lumen that include, among others, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1).H6PDH is the microsomal counterpart of the cytosolic glucose-6-phosphate dehydrogenase; both enzymes utilize glucose-6-phosphate and NADP+. However, the microsomal enzyme accepts other hexose-6-phosphates (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google Scholar). The substrate supply of the enzyme is ensured by the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is specific for glucose-6-phosphate (7Chou J.Y. Matern D. Mansfield B.C. Chen Y.T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (221) Google Scholar). H6PDH is a bifunctional enzyme that catalyzes the first two reactions of the pentose phosphate pathway (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google Scholar, 8Ozols J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5302-5306Crossref PubMed Scopus (70) Google Scholar, 9Collard F. Collet J.F. Gerin I. Veiga-da-Cunha M. Van Schaftingen E. FEBS Lett. 1999; 459: 223-226Crossref PubMed Scopus (51) Google Scholar). It contains hexose-6-phosphate dehydrogenase and 6-phosphogluconolactonase activities on a single polypeptide chain. The dehydrogenase activity of the enzyme is latent in microsomal vesicles because of the limited membrane permeability to NADP+ (10Hori S.H. Takahashi T. Biochim. Biophys. Acta. 1977; 496: 1-11Crossref PubMed Scopus (24) Google Scholar, 11Takahashi T. Hori S.H. Biochim. Biophys. Acta. 1978; 524: 262-276Crossref PubMed Scopus (56) Google Scholar, 12Takahashi T. Hori S.H. Biochim. Biophys. Acta. 1981; 672: 165-175Crossref PubMed Scopus (6) Google Scholar).11β-HSD1 is mainly expressed in liver and adipose tissue (13Seckl J.R. Walker B.R. Endocrinology. 2001; 142: 1371-1376Crossref PubMed Scopus (559) Google Scholar). The enzyme can act either as a dehydrogenase or a reductase according to the local redox potential (possibly regulated by H6PDH). In vivo conditions favor reductase activity (which may suggest a high NADPH/NADP+ ratio in the endoplasmic reticulum lumen.) The physiological role of the enzyme is supposed to be the regulation of local glucocorticoid levels in the target tissues. In cortisone reductase deficiency, the activation of cortisone to is which results in a A. Walker B.R. White P.C. Connell J.M. J. 1999; PubMed Google Scholar). a role in can as and E. Biochem. Pharmacol. PubMed Scopus Google Scholar, M. J. Biochem. Mol. PubMed Scopus Google Scholar, I. E. 2003; Google Scholar). The of the enzyme is and the active can be in the lumen J. J. PubMed Scopus Google suggest that may be in the of The enzyme active from and a role in glucocorticoid which is in from a role for local glucocorticoid in and the accumulation and in on a high A. P.M. D. J.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). an increased enzyme activity to an to that in with and and H. H. J.M. H. S. J.R. J. 2003; PubMed Scopus Google Scholar). of can in the C. S. C. M. M. M. E. E. Endocrinology. 2003; PubMed Scopus Google Scholar). is a target for the of coupling of the activities of H6PDH and has been in on the of (2Sawada H. Hara A. Hayashibara M. Nakayama T. Usui S. Saeki T. J. Biochem. (Tokyo). 1981; 90: 1077-1085Crossref PubMed Scopus (18) Google Scholar, 3Sawada H. Hayashibara M. Hara A. Nakayama T. J. Biochem. (Tokyo). 1980; 87: 985-988Crossref PubMed Scopus (12) Google Scholar). was that in the of the two enzymes to cortisone reductase N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google Scholar). However, for the cooperation is The of the present was to demonstrate the and the of glucose-6-phosphate in rat liver microsomal of liver from as has been C. A. A. J. PubMed Scopus Google Scholar). in 1 and MOPS, and in The of the vesicles was by the of activity A. J. A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). was in microsomal The of was the with as a according to the of in H6PDH activity was by of in the of glucose-6-phosphate NADP+. by the addition of The was in the and by a The of was the of cortisone reductase activity of 11β-HSD1, in the by the addition of the was in with the and was by an in C. A. A. J. PubMed Scopus Google in the of glucose-6-phosphate the and with of and acid. was to the of glucose-6-phosphate the The radioactivity with by the was by In each was to the to the intravesicular and the The on and as The of radioactivity was and as and cortisone from was by was from was from from other of and and in activity glucose-6-phosphate as and cortisone reductase activity of in rat liver H6PDH on the of the reduction of was in intact vesicles In glucose-6-phosphate, a reduction of NADP+ was present The activity the of vesicles with cytosolic glucose-6-phosphate of the with the in an in the activity The of H6PDH was In glucose-6-phosphate, reduction of NADP+ was present glucose-6-phosphate is the microsomal membrane by G6PT, the is to the limited permeability to NADP+. to H6PDH, cortisone reductase activity stimulated by the addition of be increased by The of the enzyme was results that the active of the two enzymes in the lumen of the endoplasmic reticulum and that for the cytosolic pyridine be that a cortisone reductase which was on be in intact the addition of a was and which be by The is because the of a was in liver C. Biochem. J. PubMed Scopus Google Scholar). The that this is present in in the cortisone reduction in rat liver in cortisone in the or of glucose-6-phosphate by with for to was as of of H6PDH and by the of or Glucose-6-phosphate, H6PDH activity can supply with reducing that can NADP+ for H6PDH, enzyme activities in intact vesicles the addition of pyridine reductase activity was on the of the of H6PDH activity by the intravesicular NADP+ pool, radiolabeled glucose-6-phosphate was used as and the intravesicular accumulation of radioactivity was The other intravesicular glucose-6-phosphate was by the addition of C. A. A. J. PubMed Scopus Google Scholar). The radioactivity of the pentose phosphate because H6PDH the enzymes of the pathway present in the lumen of the vesicles S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). has been used in other I. Van Schaftingen E. FEBS Lett. 2002; PubMed Scopus Google Scholar, M. A. E. D. J. A. J. PubMed Scopus Google reduction of cortisone to was increased upon glucose-6-phosphate addition The addition of the inhibitor D. H. J. Google decreased the of glucose-6-phosphate, that the was to an intravesicular which is a substrate for H6PDH (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google is a for G6PT, the substrate for a microsomal dehydrogenase C. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), was in this cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled In the of the microsomal uptake of radioactivity to the of and intravesicular glucose-6-phosphate was by a of to an intravesicular the cortisone was expressed The was cortisone was and the of the stimulation was cortisone the the glucose-6-phosphate the the cortisone-dependent stimulation of the intravesicular accumulation of radioactivity was The addition of the inhibitor S3483, as in a accumulation of radioactivity both in the and of cortisone glucose-6-phosphate in rat liver in to activity. The uptake and intravesicular accumulation of radioactivity upon addition was by a of the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate in the or of the of cortisone on the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate of of the of cortisone on the uptake and accumulation of radioactivity upon the addition of and the cortisone-dependent of the uptake is of the inhibitor on the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate in the or of redox of to the of the two enzymes be in In the of the generation of by the microsomal H6PDH activity was decreased a and and of H6PDH and glucose-6-phosphate-dependent of cortisone reductase activity and the cortisone-dependent accumulation of radioactivity from glucose-6-phosphate from the of and However, the is in favor of in with the can be on the by the of the enzyme of the pentose phosphate dehydrogenase is to be present in the lumen of S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). the other H6PDH activity be because of the of glucose-6-phosphate and the of the pentose phosphate pathway S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar), which to the in the intravesicular accumulation of of cortisone-dependent glucose-6-phosphate and glucose-6-phosphate-dependent cortisone reduction from the of and the present the that and H6PDH in close cooperation with each other in the lumen of the endoplasmic It the N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google that H6PDH can a role in the of to In can be that is a potential may by reducing the substrate supply to by the of activity. the results that the of the oxidative redox potential in the endoplasmic reticulum lumen by the ratio be the redox potential of the intraluminal pyridine be in vivo to intraluminal reactions as The endoplasmic reticulum is a separate subcellular compartment with properties different from those of the cytosol. Its lumen is characterized by an oxidative redox potential (1Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1571) Google Scholar), which is maintained by the relative impermeability of the endoplasmic reticulum membrane toward numerous redox-active compounds, including pyridine the oxidoreductases of the lumen have to use a separate NAD(P)+/NAD(P)H pool, a functional cooperation between intraluminal oxidases and reductases has been hypothesized (2Sawada H. Hara A. Hayashibara M. Nakayama T. Usui S. Saeki T. J. Biochem. (Tokyo). 1981; 90: 1077-1085Crossref PubMed Scopus (18) Google Scholar, 3Sawada H. Hayashibara M. Hara A. Nakayama T. J. Biochem. (Tokyo). 1980; 87: 985-988Crossref PubMed Scopus (12) Google Scholar, 4Kulkarni A.P. Hodgson E. Biochem. Pharmacol. 1982; 31: 1131-1137Crossref PubMed Scopus (16) Google Scholar, 5Draper N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google Scholar). Hexose-6-phosphate dehydrogenase (H6PDH) 1The abbreviations used are: H6PDH, hexose-6-phosphate dehydrogenase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; G6PT, glucose-6-phosphate transporter; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: H6PDH, hexose-6-phosphate dehydrogenase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; G6PT, glucose-6-phosphate transporter; MOPS, 4-morpholinepropanesulfonic acid. is supposed to have a key role in the generation of reducing equivalents, which can be used by several reductases present in the lumen that include, among others, 11β-hydroxysteroid dehydrogenase type 1 H6PDH is the microsomal counterpart of the cytosolic glucose-6-phosphate dehydrogenase; both enzymes utilize glucose-6-phosphate and NADP+. However, the microsomal enzyme accepts other hexose-6-phosphates (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google Scholar). The substrate supply of the enzyme is ensured by the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is specific for glucose-6-phosphate (7Chou J.Y. Matern D. Mansfield B.C. Chen Y.T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (221) Google Scholar). H6PDH is a bifunctional enzyme that catalyzes the first two reactions of the pentose phosphate pathway (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google Scholar, 8Ozols J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5302-5306Crossref PubMed Scopus (70) Google Scholar, 9Collard F. Collet J.F. Gerin I. Veiga-da-Cunha M. Van Schaftingen E. FEBS Lett. 1999; 459: 223-226Crossref PubMed Scopus (51) Google Scholar). It contains hexose-6-phosphate dehydrogenase and 6-phosphogluconolactonase activities on a single polypeptide chain. The dehydrogenase activity of the enzyme is latent in microsomal vesicles because of the limited membrane permeability to NADP+ (10Hori S.H. Takahashi T. Biochim. Biophys. Acta. 1977; 496: 1-11Crossref PubMed Scopus (24) Google Scholar, 11Takahashi T. Hori S.H. Biochim. Biophys. Acta. 1978; 524: 262-276Crossref PubMed Scopus (56) Google Scholar, 12Takahashi T. Hori S.H. Biochim. Biophys. Acta. 1981; 672: 165-175Crossref PubMed Scopus (6) Google Scholar). is mainly expressed in liver and adipose tissue (13Seckl J.R. Walker B.R. Endocrinology. 2001; 142: 1371-1376Crossref PubMed Scopus (559) Google Scholar). The enzyme can act either as a dehydrogenase or a reductase according to the local redox potential (possibly regulated by H6PDH). In vivo conditions favor reductase activity (which may suggest a high NADPH/NADP+ ratio in the endoplasmic reticulum lumen.) The physiological role of the enzyme is supposed to be the regulation of local glucocorticoid levels in the target tissues. In cortisone reductase deficiency, the activation of cortisone to is which results in a A. Walker B.R. White P.C. Connell J.M. J. 1999; PubMed Google Scholar). a role in can as and E. Biochem. Pharmacol. PubMed Scopus Google Scholar, M. J. Biochem. Mol. PubMed Scopus Google Scholar, I. E. 2003; Google Scholar). The of the enzyme is and the active can be in the lumen J. J. PubMed Scopus Google Scholar). suggest that may be in the of The enzyme active from and a role in glucocorticoid which is in from a role for local glucocorticoid in and the accumulation and in on a high A. P.M. D. J.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). an increased enzyme activity to an to that in with and and H. H. J.M. H. S. J.R. J. 2003; PubMed Scopus Google Scholar). of can in the C. S. C. M. M. M. E. E. Endocrinology. 2003; PubMed Scopus Google Scholar). is a target for the of The coupling of the activities of H6PDH and has been in on the of (2Sawada H. Hara A. Hayashibara M. Nakayama T. Usui S. Saeki T. J. Biochem. (Tokyo). 1981; 90: 1077-1085Crossref PubMed Scopus (18) Google Scholar, 3Sawada H. Hayashibara M. Hara A. Nakayama T. J. Biochem. (Tokyo). 1980; 87: 985-988Crossref PubMed Scopus (12) Google Scholar). was that in the of the two enzymes to cortisone reductase N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google Scholar). However, for the cooperation is The of the present was to demonstrate the and the of glucose-6-phosphate in rat liver microsomal vesicles. of liver from as has been C. A. A. J. PubMed Scopus Google Scholar). in 1 and MOPS, and in The of the vesicles was by the of activity A. J. A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). was in microsomal The of was the with as a according to the of in H6PDH activity was by of in the of glucose-6-phosphate NADP+. by the addition of The was in the and by a The of was the of cortisone reductase activity of 11β-HSD1, in the by the addition of the was in with the and was by an in C. A. A. J. PubMed Scopus Google in the of glucose-6-phosphate the and with of and acid. was to the of glucose-6-phosphate the The radioactivity with by the was by In each was to the to the intravesicular and the The on and as The of radioactivity was and as and cortisone from was by was from was from from other of of liver from as has been C. A. A. J. PubMed Scopus Google Scholar). in 1 and MOPS, and in The of the vesicles was by the of activity A. J. A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). was in microsomal The of was the with as a according to the of in H6PDH activity was by of in the of glucose-6-phosphate NADP+. by the addition of The was in the and by a The of was the of cortisone reductase activity of 11β-HSD1, in the by the addition of the was in with the and was by an in C. A. A. J. PubMed Scopus Google in the of glucose-6-phosphate the and with of and acid. was to the of glucose-6-phosphate the The radioactivity with by the was by In each was to the to the intravesicular and the The on and as The of radioactivity was and as and cortisone from was by was from was from from other of and and in activity glucose-6-phosphate as and cortisone reductase activity of in rat liver H6PDH on the of the reduction of was in intact vesicles In glucose-6-phosphate, a reduction of NADP+ was present The activity the of vesicles with cytosolic glucose-6-phosphate of the with the in an in the activity The of H6PDH was In glucose-6-phosphate, reduction of NADP+ was present glucose-6-phosphate is the microsomal membrane by G6PT, the is to the limited permeability to NADP+. to H6PDH, cortisone reductase activity stimulated by the addition of be increased by The of the enzyme was results that the active of the two enzymes in the lumen of the endoplasmic reticulum and that for the cytosolic pyridine be that a cortisone reductase which was on be in intact the addition of a was and which be by The is because the of a was in liver C. Biochem. J. PubMed Scopus Google Scholar). The that this is present in in the of H6PDH and by the of or Glucose-6-phosphate, H6PDH activity can supply with reducing that can NADP+ for H6PDH, enzyme activities in intact vesicles the addition of pyridine reductase activity was on the of the of H6PDH activity by the intravesicular NADP+ pool, radiolabeled glucose-6-phosphate was used as and the intravesicular accumulation of radioactivity was The other intravesicular glucose-6-phosphate was by the addition of C. A. A. J. PubMed Scopus Google Scholar). The radioactivity of the pentose phosphate because H6PDH the enzymes of the pathway present in the lumen of the vesicles S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). has been used in other I. Van Schaftingen E. FEBS Lett. 2002; PubMed Scopus Google Scholar, M. A. E. D. J. A. J. PubMed Scopus Google reduction of cortisone to was increased upon glucose-6-phosphate addition The addition of the inhibitor D. H. J. Google decreased the of glucose-6-phosphate, that the was to an intravesicular which is a substrate for H6PDH (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google is a for G6PT, the substrate for a microsomal dehydrogenase C. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), was in this cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled In the of the microsomal uptake of radioactivity to the of and intravesicular glucose-6-phosphate was by a of to an intravesicular the cortisone was expressed The was cortisone was and the of the stimulation was cortisone the the glucose-6-phosphate the the cortisone-dependent stimulation of the intravesicular accumulation of radioactivity was The addition of the inhibitor S3483, as in a accumulation of radioactivity both in the and of cortisone glucose-6-phosphate in rat liver in to activity. The uptake and intravesicular accumulation of radioactivity upon addition was by a of the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate in the or of the of cortisone on the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate of of the of cortisone on the uptake and accumulation of radioactivity upon the addition of and the cortisone-dependent of the uptake is of the inhibitor on the uptake and accumulation of radioactivity upon the addition of glucose-6-phosphate in the or of redox of to the of the two enzymes be in In the of the generation of by the microsomal H6PDH activity was decreased a and and of H6PDH and glucose-6-phosphate-dependent of cortisone reductase activity and the cortisone-dependent accumulation of radioactivity from glucose-6-phosphate from the of and However, the is in favor of in with the can be on the by the of the enzyme of the pentose phosphate dehydrogenase is to be present in the lumen of S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). the other H6PDH activity be because of the of glucose-6-phosphate and the of the pentose phosphate pathway S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar), which to the in the intravesicular accumulation of of cortisone-dependent glucose-6-phosphate and glucose-6-phosphate-dependent cortisone reduction from the of and the present the that and H6PDH in close cooperation with each other in the lumen of the endoplasmic It the N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google that H6PDH can a role in the of to In can be that is a potential may by reducing the substrate supply to by the of activity. the results that the of the oxidative redox potential in the endoplasmic reticulum lumen by the ratio be the redox potential of the intraluminal pyridine be in vivo to intraluminal reactions as H6PDH and and in activity glucose-6-phosphate as and cortisone reductase activity of in rat liver H6PDH on the of the reduction of was in intact vesicles In glucose-6-phosphate, a reduction of NADP+ was present The activity the of vesicles with cytosolic glucose-6-phosphate of the with the in an in the activity The of H6PDH was In glucose-6-phosphate, reduction of NADP+ was present glucose-6-phosphate is the microsomal membrane by G6PT, the is to the limited permeability to NADP+. to H6PDH, cortisone reductase activity stimulated by the addition of be increased by The of the enzyme was results that the active of the two enzymes in the lumen of the endoplasmic reticulum and that for the cytosolic pyridine It be that a cortisone reductase which was on be in intact the addition of a was and which be by The is because the of a was in liver C. Biochem. J. PubMed Scopus Google Scholar). The that this is present in in the vesicles. of H6PDH and by the of or Glucose-6-phosphate, H6PDH activity can supply with reducing that can NADP+ for H6PDH, enzyme activities in intact vesicles the addition of pyridine reductase activity was on the of the of H6PDH activity by the intravesicular NADP+ pool, radiolabeled glucose-6-phosphate was used as and the intravesicular accumulation of radioactivity was The other intravesicular glucose-6-phosphate was by the addition of C. A. A. J. PubMed Scopus Google Scholar). The radioactivity of the pentose phosphate because H6PDH the enzymes of the pathway present in the lumen of the vesicles S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). has been used in other I. Van Schaftingen E. FEBS Lett. 2002; PubMed Scopus Google Scholar, M. A. E. D. J. A. J. PubMed Scopus Google Scholar). The reduction of cortisone to was increased upon glucose-6-phosphate addition The addition of the inhibitor D. H. J. Google decreased the of glucose-6-phosphate, that the was to an intravesicular which is a substrate for H6PDH (6Clarke J.L. Mason P.J. Arch. Biochem. Biophys. 2003; 415: 229-234Crossref PubMed Scopus (50) Google is a for G6PT, the substrate for a microsomal dehydrogenase C. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), was in this Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled In the of the microsomal uptake of radioactivity to the of and intravesicular glucose-6-phosphate was by a of to an intravesicular the cortisone was expressed The was cortisone was and the of the stimulation was cortisone the the glucose-6-phosphate the the cortisone-dependent stimulation of the intravesicular accumulation of radioactivity was The addition of the inhibitor S3483, as in a accumulation of radioactivity both in the and of cortisone The redox of to the of the two enzymes be in In the of the generation of by the microsomal H6PDH activity was decreased a and and of H6PDH and glucose-6-phosphate-dependent of cortisone reductase activity and the cortisone-dependent accumulation of radioactivity from glucose-6-phosphate from the of and However, the is in favor of in with the can be on the by the of the enzyme of the pentose phosphate dehydrogenase is to be present in the lumen of S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar, C. S. J. PubMed Google Scholar). the other H6PDH activity be because of the of glucose-6-phosphate and the of the pentose phosphate pathway S. J. Biochem. 1982; PubMed Scopus Google Scholar, S. J. PubMed Google Scholar), which to the in the intravesicular accumulation of In the present the that and H6PDH in close cooperation with each other in the lumen of the endoplasmic It the N. Walker E.A. Bujalska I.J. Tomlinson J.W. Chalder S.M. Arlt W. Lavery G.G. Bedendo O. Ray D.W. Laing I. Malunowicz E. White P.C. Hewison M. Mason P.J. Connell J.M. Shackleton C.H. Stewart P.M. Nat. Genet. 2003; 34: 434-439Crossref PubMed Scopus (252) Google that H6PDH can a role in the of to In can be that is a potential may by reducing the substrate supply to by the of activity. the results that the of the oxidative redox potential in the endoplasmic reticulum lumen by the ratio be the redox potential of the intraluminal pyridine be in vivo to intraluminal reactions as to for