Hexose-6-Phosphate Dehydrogenase and Redox Control of 11?-Hydroxysteroid Dehydrogenase Type 1 Activity
http://www.100md.com
内分泌学杂志 2005年第6期
Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: Paul M. Stewart, Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail: p.m.stewart@bham.ac.uk.
Abstract
Hexose-6-phosphate dehydrogenase (H6PDH) is a microsomal enzyme that is able to catalyze the first two reactions of an endoluminal pentose phosphate pathway, thereby generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) within the endoplasmic reticulum. It is distinct from the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), using a separate pool of NAD(P)+ and capable of oxidizing several phosphorylated hexoses. It has been proposed to be a NADPH regenerating system for steroid hormone and drug metabolism, specifically in determining the set point of 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) activity, the enzyme responsible for the activation and inactivation of glucocorticoids. 11?-HSD1 is a bidirectional enzyme, but in intact cells displays predominately oxo-reductase activity, a reaction requiring NADPH and leading to activation of glucocorticoids. However, in cellular homogenates or in purified preparations, 11?-HSD1 is exclusively a dehydrogenase. Because H6PDH and 11?-HSD1 are coexpressed in the inner microsomal compartment of cells, we hypothesized that H6PDH may provide 11?-HSD1 with NADPH, thus promoting oxo-reductase activity in vivo. Recently, several studies have confirmed this functional cooperation, indicating the importance of intracellular redox mechanisms for the prereceptor control of glucocorticoid availability. With the increased interest in 11?-HSD1 oxo-reductase activity in the pathogenesis and treatment of several human diseases including insulin resistance and the metabolic syndrome, H6PDH represents an additional novel candidate for intervention.
Introduction
REDOX REACTIONS involve the transfer of reducing equivalents (either electrons or hydrogen) from a donor molecule to an acceptor molecule. Biological redox reactions are important for numerous cellular phenomena including the vital bioenergetic functions of mitochondria and chloroplasts. Common electron acceptor/donor molecules within cells, particularly with respect to steroidogenesis, are the oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH). A major source of NADPH in the cytosol is derived from the pentose phosphate pathway. The first and rate-limiting step in this pathway is carried out by the enzyme glucose-6-phosphate dehydrogenase (G6PDH) [enzyme commission (EC) 1.1.1.49]. This catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, and in doing so, generates NADPH from NADP+. However, major controlling steps in steroid metabolism requiring NADPH are carried out by enzymes located in subcellular compartments such as the endoplasmic reticulum (ER) and mitochondria. Indeed, the first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system, a mechanism dependent on NADPH. Because pyridine nucleotides are unable to cross cellular membranes, it is becoming increasingly important to investigate redox control mechanisms of steroid metabolism at a subcellular level.
For this reason, interest has been renewed in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, EC 1.1.1.47) because it can generate NADPH from NADP+ specifically within the ER. Here we review H6PDH, in terms of its tissue distribution, gene structure and its recently discovered role in the regulation of 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1, EC 1.1.1.146).
H6PDH Biological Activity and Kinetic Properties
H6PDH is an autosomally linked, microsomal glucose dehydrogenase, distinct from the cytosolic sex-linked G6PDH. Microsomal localization was confirmed in several studies where G6PDH activity was latent in washed microsomal preparations and only observed upon detergent treatment (1, 2, 3, 4). Initially, H6PDH was reported to have dehydrogenase activity on G6P and galactose-6-phosphate, but further studies show that it has a broader substrate specificity incorporating other hexose-6-phosphates such as 2-deoxyglucose 6-phosphate, and also simple sugars such as glucose (1, 5). It has dual nucleotide specificity for NADP+ and NAD+ but under physiological conditions, within the microsomal environment, the native substrates for H6PDH are believed to be G6P and NADP+ (6). Supply of G6P is ensured by the glucose-6-phosphate transporter of the ER, which is specific for G6P (7). Because, as noted above, the membrane of the ER is relatively impermeable to pyridine nucleotides, the supply of NAD(P)+ must be maintained through a functional cooperation between H6PDH (and perhaps additional oxidases) and intraluminal reductases (6, 8, 9). In fact, it has long been suggested that the role of H6PDH is to supply reduced NAD(P)+ to ER reductases involved specifically with steroid and drug metabolism (2).
Sequence comparisons revealed the possibility that H6PDH is a bifunctional enzyme, and in addition to its oxidative activity on G6P it is thought to catalyze the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate (10), performing in effect the first two steps of a pentose phosphate pathway within the ER (11). The 6-phosphogluconolactonase (6PGL) activity was subsequently confirmed in purified native H6PDH from mice (12) and found to have a similar pH optimum but be more thermolabile than cytoplasmic 6PGL. Because there is a high level of sequence similarity between the murine and human H6PDH enzymes, it is reasonable to expect that the human protein also contains 6PGL activity. The murine enzyme has been proven to have bifunctional activity (13), but this has yet to be confirmed for human H6PDH. The kinetics of H6PDH have been examined within microsomes and an apparent Michaelis-Menten constant values of 2.61 x 10–5 M for NADP+, 4.93 x 10–5 M for glucose 6-phosphate, and 2.14 x 10–4 M for 2-deoxyglucose 6-phosphate have been documented (14).
Currently, little is known about the transcriptional or the translational regulation of H6PDH. Preliminary studies carried out in the 1960s indicated that the levels of H6PDH were higher in female mice than male, suggesting hormonal regulation of H6PDH; however, levels were not altered across pregnancy or lactation (15). A further study in 1970 (16) reported a rapid increase in H6PDH activity before birth in rats, with a decline to adult levels 30 d postpartum. H6PDH activity has also been shown to be induced by phenobarbitol in rat liver (2).
Tissue Distribution
H6PDH expression was first reported in rat tissues and was found to be present in liver, adrenals, spleen, kidney, heart, lungs, muscle, testes, ovaries, prostate, uterus, and intestine (4, 16). Subsequently, activity has been reported in many human tissues including liver, placenta, lymphoid, fibroblasts, adipose, white blood cells, kidney, thymus, pancreas, ovary, testis, skeletal muscle, and lung (3, 12, 17, 18), with the highest activity observed in liver and placenta. Additionally, activity was also observed in porcine kidney cortex (3). This localization to steroidogenic cells, liver cells, and renal tissue further supports the hypothesis that H6PDH may be involved in drug and steroid metabolism (4).
Protein and Gene Structure
The H6PDH amino acid sequence was first determined from rabbit liver microsomes (19), and thus far has provided the only protein sequence data for this enzyme. The rabbit enzyme is a 90-kDa protein consisting of 763 amino acids, in comparison to the cytosolic G6PDH, which has a monomer molecular mass of 56 kDa. Carbohydrate is attached to Asn-138 and Asn-263, in keeping with its endoluminal orientation (19, 20), although the protein lacks a conventional ER signal sequence and trans-membrane domain. Consequently, the trafficking and retention of this protein within the lumen of ER remains to be determined.
The N-terminal region of the H6PDH protein has high homology to the cytosolic G6PDH, particularly the two functional motifs of the coenzyme binding site and G6P binding site. The C-terminal 250 amino acids show homology to bacterial devB proteins, the N-terminal regions of Plasmodium falciparum and Plasmodium berghei G6PDH proteins (21) and the human cytosolic 6PGL protein (10).
The sequence information provided by rabbit H6PDH protein allowed the cloning and identification of the human H6PDH cDNA and gene (12). The human H6PDH gene is located on 1p36, spans 37 kb, and consists of 5 exons and 4 introns that predicts a protein of 89 kDa. Exon 5 encodes more than half of the protein (12). Murine H6PDH gene is localized to chromosome 4 (a region equivalent to that of human chromosome 1p36), and encodes a protein of 789 amino acids with a molecular mass of 89 kDa (12).
Regulation of 11?-HSD Type 1 Set Point
Glucocorticoids exert a diverse range of physiological roles, including carbohydrate and amino acid regulation, control of blood pressure, and maintenance of stress and inflammatory responses. Therefore, the manner in which they are regulated in various tissues is highly important. Two isozymes of 11?-HSD allow the interconversion of glucocorticoids from their inactive (cortisone, 11-hydrocorticosterone) to their active forms (cortisol, corticosterone), enabling the prereceptor regulation of glucocorticoids. 11?-HSD2, the kidney isoform, has predominately dehydrogenase activity inactivating cortisol, enabling aldosterone to preferentially activate the mineralocorticoid receptor in vivo (22, 23). In intact cells, 11?-HSD1 is a bidirectional enzyme, predominately displaying oxo-reductase activity (24, 25), but in cellular homogenates or in purified preparations, 11?-HSD1 acts as a dehydrogenase (26). Using purified enzyme preparations, Walker et al. (27) calculated the equilibrium constant (pH 7.0) for the 11?-HSD1 cortisone to cortisol reaction as 0.03 (Fig. 1). This implies that this reaction lies heavily toward cortisone and NADPH at equilibrium, indicating a natural tendency for the 11?-HSD1-catalyzed reaction to proceed in the dehydrogenase direction.
FIG. 1. Diagrammatic representation of the equilibrium constant (Keq) for the 11?-HSD1-catalyzed cortisone to cortisol reaction. The Keq of the reaction (strictly Keq[H+]) is expressed here as [NADP][cortisol]/[NADPH][cortisone], measured at equilibrium. The diagram indicates the effect of different Keqs on the cortisol/cortisone ratio (assuming equal amounts of the two cofactors at each point). Using purified, recombinant protein the equilibrium constant at pH 7.0 for the reaction has been calculated as 0.03. This implies that this reaction lies heavily toward cortisone and NADPH at equilibrium, indicating a natural tendency for the 11?-HSD1-catalyzed reaction to proceed in the dehydrogenase direction.
11?-HSD1 is highly expressed in the liver and in adipose tissue; here its oxo-reductase activity has been implicated in the pathogenesis of the metabolic syndrome through increased local generation of cortisol facilitating hepatic glucose output (28) and adipocyte differentiation (29). Inhibition of 11?-HSD1, using selective inhibitors, has resulted in improved insulin sensitivity and weight loss (30, 31), but the factors determining 11?-HSD1 set point and oxo-reductase activity per se have remained elusive.
Initial kinetic studies using recombinant 11?-HSD1 expressed in Vaccinia virus indicated that availability of cofactor was an important regulator of the reaction direction of 11?-HSD1 (32). In an artificial situation, removal of NADP+ using recombinant cytosolic G6PDH, significantly increased oxo-reductase activity. Conversely, oxo-reductase activity can be restored in vitro by the addition of G6PDH and NADP to regenerate NADPH (27, 32). However, due to the subcellular location of 11?-HSD1 within the ER lumen, cytosolic G6PDH is unable to perform this function in vivo. This was endorsed by Bujalska et al. (33), where differences in oxo-reductase-dehydrogenase activities of 11?-HSD1 in human omental and sc adipose stromal cells could not be explained by differences in G6PDH activity.
In vivo studies have also highlighted the bidirectional capacity of 11?-HSD1. Rat Leydig cells, depending upon their stage of differentiation and testosterone secreting ability, vary significantly in underlying oxo-reductase compared with dehydrogenase activities (34). This could be manipulated by altering key components within culture media including the omission of glucose, NAD+, NADH, NADP+ (35). Set point also differs within preadipocytes and adipocytes. Thus, after 14 d of culture under conditions that promote adipocyte differentiation, a switch occurs from 11?-HSD1 dehydrogenase to oxo-reductase activity in omental preadipocytes (36), despite no change in absolute levels of enzyme expression. Additionally, neuronal cells have also been shown to have bidirectional activity (37).
The evidence that H6PDH and not G6PDH allows the regeneration of NADPH and promotes 11?-HSD1 oxo-reductase activity (Fig. 2) has come from several sources. First, patients with a putative 11?-HSD1-deficient state—cortisone reductase deficiency—are unable to convert cortisone to its active form cortisol. Sequencing of the gene encoding 11?-HSD1 (HSD11B1) revealed intronic mutations that may mediate reduced levels of enzyme expression but these are present in up to 4% of normal subjects so that other candidates are likely to be involved (38). Subsequently, sequencing of the H6PDH gene revealed mutations in exon 5 of the gene in all affected patients. Heterozygosity was shown in one case for a 29-bp insertion in exon 5 (620ins29bp621), which caused the inclusion of three new amino acids and a stop codon, resulting in a truncated protein, 171 amino acids shorter than the native protein. Expression of this mutation in hepatic WRL68 cells revealed a null enzyme with total lack of H6PDH activity. In two other cases, homozygosity for R453Q, a nonconservative missense amino acid change, was shown to encode for an enzyme with approximately 25% activity compared with wild type. Thus, cortisone reductase deficiency appears to be a digenic disease requiring interacting mutations in HSD11B1 and H6PDH. Importantly, these data link H6PDH to function of the 11?-HSD1 enzyme.
FIG. 2. Schematic representation of the proposed interaction between H6PDH and 11?-HSD1 within the ER. H6PDH generates NADPH by conversion of glucose-6-phosphate (G6P), transported via the G6P translocase (G6PT), to 6-phosphogluconate (6PG) within the ER. 11?-HSD1 (T1) uses the NADPH as cofactor, allowing the conversion of cortisone to cortisol.
Secondly, these genetic studies have been endorsed through in vitro functional studies. Atanasov et al. (39) showed colocalization of myc-tagged H6PDH and FLAG-tagged 11?-HSD1 transfected in human embryonic kidney (HEK) 293 to the luminal side of the ER. H6PDH was able to determine the direction of 11?-HSD1 in HEK 293 and Chinese hamster ovary cells. Both cell types had bidirectional activity of 11?-HSD1, with a slight preference of dehydrogenase activity. However, coexpression of 11?-HSD1 with H6PDH caused a 5-fold increase in oxo-reductase activity and a 6-fold decrease in dehydrogenase activity without affecting the kinetic parameters. The effect on the maximum velocity (Vmax) and not Michaelis constant for both directions indicated that availability of cofactor does not alter substrate affinity of 11?-HSD1. Similar data have come from our own group. Bujalska et al. (40) have demonstrated that H6PDH increased 11?-HSD1 oxo-reductase activity, while reducing dehydrogenase activity, whereas the cytosolic enzyme G6PDH was unable to affect oxo-reductase activity. Additionally, H6PDH was unable to affect the direction of the 11?-HSD2 enzyme. Stably transfected cells with 11?-HSD1 and a H6PDH short interfering RNA (siRNA) showed a reduction in oxo-reductase activity, simultaneously increasing dehydrogenase activity (Fig. 3).
FIG. 3. Effect of suppression of 11?-HSD1 and H6PDH in HEK 293T1 cells by siRNA. A, HEK 293T1 were transfected with 11?-HSD1, H6PDH, and scrambled-siRNAs (Control) for 48 h. Compared with control, mRNA levels for 11?-HSD1 and H6PDH were suppressed in cells transfected with the respective siRNAs (mean ± SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001). B, 11?-HSD1 oxo-reductase and dehydrogenase activities were both reduced by 11?-HSD1-siRNA, whereas oxo-reductase activity was decreased and dehydrogenase activity simultaneously increased upon transfection with H6PDH-siRNA (mean ± SE; *, P < 0.05, **, P < 0.01; ***, P < 0.001).
In terms of redox potential within the ER lumen, Banhegyi et al. (41) have shown that the intralumenal pool of NADPH and substrate availability of glucose-6-phosphate are critical in regulating 11?-HSD1 direction toward cortisol production under the stimulation of H6PDH. In microsomal preparations, addition of G6P increased production of NADPH and stimulated 11?-HSD1 oxo-reductase activity (41). Inhibition of G6P uptake, using an inhibitor of microsomal G6P transport, decreased this effect.
These cell-based studies all support the concept that H6PDH is important in conveying oxo-reductase activity upon 11?-HSD1, an enzyme that in its purified state is preferentially a dehydrogenase. Together, these recent studies cement a role for H6PDH in regulating the set point of 11?-HSD1 activity. They should also raise increased awareness of the pitfalls of measuring only 11?-dehydrogenase activity in vitro and inferring from this that this is representative of 11-oxo-reductase activity in vivo (42).
Future Directions
The recent excitement over the putative use of 11?-HSD1 inhibitors to treat patients with insulin resistance and obesity-metabolic syndrome is entirely dependant upon 11-oxo-reductase activity in vivo. It is interesting to speculate that inhibition of H6PDH with concomitant reduction in ER NADPH concentrations might result in a similar beneficial effect. Further studies are required to investigate the role of H6PDH and ER redox potential in human disease.
Acknowledgments
The authors would like to thank Dr. Jon Ride for helpful discussions and Drs. Iwona Bujalska, Jeremy Tomlinson, Nicole Draper, and Gareth Lavery for their contributions to this work.
References
Romanelli A, St Denis JF, Vidal H, Tchu S, van de WG 1994 Absence of glucose uptake by liver microsomes: an explanation for the complete latency of glucose dehydrogenase. Biochem Biophys Res Commun 200:1491–1497
Hori SH, Takahashi T 1974 Phenobarbital-induced increase of the hexose 6-phosphate dehydrogenase activity. Biochem Biophys Res Commun 61:1064–1070
Barash V, Erlich T, Bashan N 1990 Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex. Biochem Int 20:267–274
Tanahashi K, Hori SH 1980 Immunohistochemical localization of hexose 6-phosphate dehydrogenase in various organs of the rat. J Histochem Cytochem 28:1175–1182
Beutler E, Morrison M 1967 Localization and characteristics of hexose 6-phosphate dehydrogenase (glucose dehydrogenase). J Biol Chem 242:5289–5293
Kulkarni AP, Hodgson E 1982 Mouse liver microsomal hexose-6-phosphate dehydrogenase. NADPH generation and utilization in monooxygenation reactions. Biochem Pharmacol 31:1131–1137
Chou JY, Matern D, Mansfield BC, Chen YT 2002 Type I glycogen storage diseases: disorders of the glucose-6-phosphatase complex. Curr Mol Med 2:121–143
Sawada H, Hayashibara M, Hara A, Nakayama T 1980 A possible functional relationship between microsomal aromatic aldehyde-ketone reductase and hexose-6-phosphate dehydrogenase. J Biochem (Tokyo) 87:985–988
Sawada H, Hara A, Hayashibara M, Nakayama T, Usui S, Saeki T 1981 Microsomal reductase for aromatic aldehydes and ketones in guinea pig liver. Purification, characterization, and functional relationship to hexose-6-phosphate dehydrogenase. J Biochem (Tokyo) 90:1077–1085
Collard F, Collet JF, Gerin I, Veiga-da-Cunha M, Van Schaftingen E 1999 Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway(1). FEBS Lett 459:223–226
Bublitz C, Steavenson S 1988 The pentose phosphate pathway in the endoplasmic reticulum. J Biol Chem 263:12849–12853
Mason PJ, Stevens D, Diez A, Knight SW, Scopes DA, Vulliamy TJ 1999 Human hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) encoded at 1p36: coding sequence and expression. Blood Cells Mol Dis 25:30–37
Clarke JL, Mason PJ 2003 Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity. Arch Biochem Biophys 415:229–234
Kimura K, Endou H, Sudo J, Sakai F 1979 Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship. J Biochem (Tokyo) 85:319–326
Shaw CR, Koen AL 1968 Glucose 6-phosphate dehydrogenase and hexose 6-phosphate dehydrogenase of mammalian tissues. Ann NY Acad Sci 151:149–156
Mandula B, Srivastava SK, Beutler E 1970 Hexose-6-phosphate dehydrogenase: distribution in rat tissues and effect of diet, age and steroids. Arch Biochem Biophys 141:155–161
Blume KG, Schmidt GM, Heissmeier HH, Lohr GW 1975 Hexose-6-phosphate dehydrogenase from human tissues: an electrophoretic study in health and disease. Experientia 31:496–498
King J, Cook PJ 1981 Glucose dehydrogenase polymorphism in man. Ann Hum Genet 45:129–134
Ozols J 1993 Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase. Proc Natl Acad Sci USA 90:5302–5306
Brands R, Snider MD, Hino Y, Park SS, Gelboin HV, Rothman JE 1985 Retention of membrane proteins by the endoplasmic reticulum. J Cell Biol 101:1724–1732
Clarke JL, Scopes DA, Sodeinde O, Mason PJ 2001 Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase. A novel bifunctional enzyme in malaria parasites. Eur J Biochem 268:2013–2019
Stewart PM, Murry BA, Mason JI 1994 Human kidney 11?-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform. J Clin Endocrinol Metab 79:480–484
Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS 1994 Cloning and tissue distribution of the human 11?-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 105:R11–R17
Ricketts ML, Shoesmith KJ, Hewison M, Strain A, Eggo MC, Stewart PM 1998 Regulation of 11?-hydroxysteroid dehydrogenase type 1 in primary cultures of rat and human hepatocytes. J Endocrinol 156:159–168
Jamieson PM, Chapman KE, Edwards CR, Seckl JR 1995 11?-Hydroxysteroid dehydrogenase is an exclusive 11?-reductase in primary cultures of rat hepatocytes: effect of physicochemical and hormonal manipulations. Endocrinology 136:4754–4761
Lakshmi V, Monder C 1988 Purification and characterization of the corticosteroid 11?-dehydrogenase component of the rat liver 11?-hydroxysteroid dehydrogenase complex. Endocrinology 123:2390–2398
Walker EA, Clark AM, Hewison M, Ride JP, Stewart PM 2001 Functional expression, characterization, and purification of the catalytic domain of human 11-?-hydroxysteroid dehydrogenase type 1. J Biol Chem 276:21343–21350
Jones CG, Hothi SK, Titheradge MA 1993 Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes. Biochem J 289(Pt 3):821–828
Hauner H, Schmid P, Pfeiffer EF 1987 Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 64:832–835
Alberts P, Nilsson C, Selen G, Engblom LO, Edling NH, Norling S, Klingstrom G, Larsson C, Forsgren M, Ashkzari M, Nilsson CE, Fiedler M, Bergqvist E, Ohman B, Bjorkstrand E, Abrahmsen LB 2003 Selective inhibition of 11?-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains. Endocrinology 144:4755–4762
Livingstone DE, Walker BR 2003 Is 11?-hydroxysteroid dehydrogenase type 1 a therapeutic target? Effects of carbenoxolone in lean and obese Zucker rats. J Pharmacol Exp Ther 305:167–172
Agarwal AK, Tusie-Luna MT, Monder C, White PC 1990 Expression of 11?-hydroxysteroid dehydrogenase using recombinant vaccinia virus. Mol Endocrinol 4:1827–1832
Bujalska IJ, Kumar S, Hewison M, Stewart PM 1999 Differentiation of adipose stromal cells: the roles of glucocorticoids and 11?-hydroxysteroid dehydrogenase. Endocrinology 140:3188–3196
Ge RS, Hardy DO, Catterall JF, Hardy MP 1997 Developmental changes in glucocorticoid receptor and 11?-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells. Endocrinology 138:5089–5095
Ferguson SE, Pallikaros Z, Cooke BA, Michael AE 1999 The effects of different culture media, glucose, pyridine nucleotides and adenosine on the activity of 11?-hydroxysteroid dehydrogenase in rat Leydig cells. Mol Cell Endocrinol 158:37–44
Bujalska IJ, Walker EA, Hewison M, Stewart PM 2002 A switch in dehydrogenase to reductase activity of 11?-hydroxysteroid dehydrogenase type 1 upon differentiation of human omental adipose stromal cells. J Clin Endocrinol Metab 87:1205–1210
Seckl JR, Morton NM, Chapman KE, Walker BR 2004 Glucocorticoids and 11?-hydroxysteroid dehydrogenase in adipose tissue. Recent Prog Horm Res 59:359–393
Draper N, Walker EA, Bujalska IJ, Tomlinson JW, Chalder SM, Arlt W, Lavery GG, Bedendo O, Ray DW, Laing I, Malunowicz E, White PC, Hewison M, Mason PJ, Connell JM, Shackleton CH, Stewart PM 2003 Mutations in the genes encoding 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency. Nat Genet 34:434–439
Atanasov AG, Nashev LG, Schweizer RA, Frick C, Odermatt A 2004 Hexose-6-phosphate dehydrogenase determines the reaction direction of 11?-hydroxysteroid dehydrogenase type 1 as an oxoreductase. FEBS Lett 571:129–133
Bujalska IJ, Draper N, Minakami S, Tomlinson JW, White PC, Chapman KE, Walker EA, Stewart PM, Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11?-hydroxysteroid dehydrogenase type 1. J Mol Endocrinol, published online 9 February 2005
Banhegyi G, Benedetti A, Fulceri R, Senesi S 2004 Cooperativity between 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum. J Biol Chem 279:27017–27021
Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DE, Johnson O, Walker BR 2001 Tissue-specific dysregulation of cortisol metabolism in human obesity. J Clin Endocrinol Metab 86:1418–1421(Kylie N. Hewitt, Elizabet)
Address all correspondence and requests for reprints to: Paul M. Stewart, Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail: p.m.stewart@bham.ac.uk.
Abstract
Hexose-6-phosphate dehydrogenase (H6PDH) is a microsomal enzyme that is able to catalyze the first two reactions of an endoluminal pentose phosphate pathway, thereby generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) within the endoplasmic reticulum. It is distinct from the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), using a separate pool of NAD(P)+ and capable of oxidizing several phosphorylated hexoses. It has been proposed to be a NADPH regenerating system for steroid hormone and drug metabolism, specifically in determining the set point of 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) activity, the enzyme responsible for the activation and inactivation of glucocorticoids. 11?-HSD1 is a bidirectional enzyme, but in intact cells displays predominately oxo-reductase activity, a reaction requiring NADPH and leading to activation of glucocorticoids. However, in cellular homogenates or in purified preparations, 11?-HSD1 is exclusively a dehydrogenase. Because H6PDH and 11?-HSD1 are coexpressed in the inner microsomal compartment of cells, we hypothesized that H6PDH may provide 11?-HSD1 with NADPH, thus promoting oxo-reductase activity in vivo. Recently, several studies have confirmed this functional cooperation, indicating the importance of intracellular redox mechanisms for the prereceptor control of glucocorticoid availability. With the increased interest in 11?-HSD1 oxo-reductase activity in the pathogenesis and treatment of several human diseases including insulin resistance and the metabolic syndrome, H6PDH represents an additional novel candidate for intervention.
Introduction
REDOX REACTIONS involve the transfer of reducing equivalents (either electrons or hydrogen) from a donor molecule to an acceptor molecule. Biological redox reactions are important for numerous cellular phenomena including the vital bioenergetic functions of mitochondria and chloroplasts. Common electron acceptor/donor molecules within cells, particularly with respect to steroidogenesis, are the oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH). A major source of NADPH in the cytosol is derived from the pentose phosphate pathway. The first and rate-limiting step in this pathway is carried out by the enzyme glucose-6-phosphate dehydrogenase (G6PDH) [enzyme commission (EC) 1.1.1.49]. This catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, and in doing so, generates NADPH from NADP+. However, major controlling steps in steroid metabolism requiring NADPH are carried out by enzymes located in subcellular compartments such as the endoplasmic reticulum (ER) and mitochondria. Indeed, the first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system, a mechanism dependent on NADPH. Because pyridine nucleotides are unable to cross cellular membranes, it is becoming increasingly important to investigate redox control mechanisms of steroid metabolism at a subcellular level.
For this reason, interest has been renewed in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, EC 1.1.1.47) because it can generate NADPH from NADP+ specifically within the ER. Here we review H6PDH, in terms of its tissue distribution, gene structure and its recently discovered role in the regulation of 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1, EC 1.1.1.146).
H6PDH Biological Activity and Kinetic Properties
H6PDH is an autosomally linked, microsomal glucose dehydrogenase, distinct from the cytosolic sex-linked G6PDH. Microsomal localization was confirmed in several studies where G6PDH activity was latent in washed microsomal preparations and only observed upon detergent treatment (1, 2, 3, 4). Initially, H6PDH was reported to have dehydrogenase activity on G6P and galactose-6-phosphate, but further studies show that it has a broader substrate specificity incorporating other hexose-6-phosphates such as 2-deoxyglucose 6-phosphate, and also simple sugars such as glucose (1, 5). It has dual nucleotide specificity for NADP+ and NAD+ but under physiological conditions, within the microsomal environment, the native substrates for H6PDH are believed to be G6P and NADP+ (6). Supply of G6P is ensured by the glucose-6-phosphate transporter of the ER, which is specific for G6P (7). Because, as noted above, the membrane of the ER is relatively impermeable to pyridine nucleotides, the supply of NAD(P)+ must be maintained through a functional cooperation between H6PDH (and perhaps additional oxidases) and intraluminal reductases (6, 8, 9). In fact, it has long been suggested that the role of H6PDH is to supply reduced NAD(P)+ to ER reductases involved specifically with steroid and drug metabolism (2).
Sequence comparisons revealed the possibility that H6PDH is a bifunctional enzyme, and in addition to its oxidative activity on G6P it is thought to catalyze the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate (10), performing in effect the first two steps of a pentose phosphate pathway within the ER (11). The 6-phosphogluconolactonase (6PGL) activity was subsequently confirmed in purified native H6PDH from mice (12) and found to have a similar pH optimum but be more thermolabile than cytoplasmic 6PGL. Because there is a high level of sequence similarity between the murine and human H6PDH enzymes, it is reasonable to expect that the human protein also contains 6PGL activity. The murine enzyme has been proven to have bifunctional activity (13), but this has yet to be confirmed for human H6PDH. The kinetics of H6PDH have been examined within microsomes and an apparent Michaelis-Menten constant values of 2.61 x 10–5 M for NADP+, 4.93 x 10–5 M for glucose 6-phosphate, and 2.14 x 10–4 M for 2-deoxyglucose 6-phosphate have been documented (14).
Currently, little is known about the transcriptional or the translational regulation of H6PDH. Preliminary studies carried out in the 1960s indicated that the levels of H6PDH were higher in female mice than male, suggesting hormonal regulation of H6PDH; however, levels were not altered across pregnancy or lactation (15). A further study in 1970 (16) reported a rapid increase in H6PDH activity before birth in rats, with a decline to adult levels 30 d postpartum. H6PDH activity has also been shown to be induced by phenobarbitol in rat liver (2).
Tissue Distribution
H6PDH expression was first reported in rat tissues and was found to be present in liver, adrenals, spleen, kidney, heart, lungs, muscle, testes, ovaries, prostate, uterus, and intestine (4, 16). Subsequently, activity has been reported in many human tissues including liver, placenta, lymphoid, fibroblasts, adipose, white blood cells, kidney, thymus, pancreas, ovary, testis, skeletal muscle, and lung (3, 12, 17, 18), with the highest activity observed in liver and placenta. Additionally, activity was also observed in porcine kidney cortex (3). This localization to steroidogenic cells, liver cells, and renal tissue further supports the hypothesis that H6PDH may be involved in drug and steroid metabolism (4).
Protein and Gene Structure
The H6PDH amino acid sequence was first determined from rabbit liver microsomes (19), and thus far has provided the only protein sequence data for this enzyme. The rabbit enzyme is a 90-kDa protein consisting of 763 amino acids, in comparison to the cytosolic G6PDH, which has a monomer molecular mass of 56 kDa. Carbohydrate is attached to Asn-138 and Asn-263, in keeping with its endoluminal orientation (19, 20), although the protein lacks a conventional ER signal sequence and trans-membrane domain. Consequently, the trafficking and retention of this protein within the lumen of ER remains to be determined.
The N-terminal region of the H6PDH protein has high homology to the cytosolic G6PDH, particularly the two functional motifs of the coenzyme binding site and G6P binding site. The C-terminal 250 amino acids show homology to bacterial devB proteins, the N-terminal regions of Plasmodium falciparum and Plasmodium berghei G6PDH proteins (21) and the human cytosolic 6PGL protein (10).
The sequence information provided by rabbit H6PDH protein allowed the cloning and identification of the human H6PDH cDNA and gene (12). The human H6PDH gene is located on 1p36, spans 37 kb, and consists of 5 exons and 4 introns that predicts a protein of 89 kDa. Exon 5 encodes more than half of the protein (12). Murine H6PDH gene is localized to chromosome 4 (a region equivalent to that of human chromosome 1p36), and encodes a protein of 789 amino acids with a molecular mass of 89 kDa (12).
Regulation of 11?-HSD Type 1 Set Point
Glucocorticoids exert a diverse range of physiological roles, including carbohydrate and amino acid regulation, control of blood pressure, and maintenance of stress and inflammatory responses. Therefore, the manner in which they are regulated in various tissues is highly important. Two isozymes of 11?-HSD allow the interconversion of glucocorticoids from their inactive (cortisone, 11-hydrocorticosterone) to their active forms (cortisol, corticosterone), enabling the prereceptor regulation of glucocorticoids. 11?-HSD2, the kidney isoform, has predominately dehydrogenase activity inactivating cortisol, enabling aldosterone to preferentially activate the mineralocorticoid receptor in vivo (22, 23). In intact cells, 11?-HSD1 is a bidirectional enzyme, predominately displaying oxo-reductase activity (24, 25), but in cellular homogenates or in purified preparations, 11?-HSD1 acts as a dehydrogenase (26). Using purified enzyme preparations, Walker et al. (27) calculated the equilibrium constant (pH 7.0) for the 11?-HSD1 cortisone to cortisol reaction as 0.03 (Fig. 1). This implies that this reaction lies heavily toward cortisone and NADPH at equilibrium, indicating a natural tendency for the 11?-HSD1-catalyzed reaction to proceed in the dehydrogenase direction.
FIG. 1. Diagrammatic representation of the equilibrium constant (Keq) for the 11?-HSD1-catalyzed cortisone to cortisol reaction. The Keq of the reaction (strictly Keq[H+]) is expressed here as [NADP][cortisol]/[NADPH][cortisone], measured at equilibrium. The diagram indicates the effect of different Keqs on the cortisol/cortisone ratio (assuming equal amounts of the two cofactors at each point). Using purified, recombinant protein the equilibrium constant at pH 7.0 for the reaction has been calculated as 0.03. This implies that this reaction lies heavily toward cortisone and NADPH at equilibrium, indicating a natural tendency for the 11?-HSD1-catalyzed reaction to proceed in the dehydrogenase direction.
11?-HSD1 is highly expressed in the liver and in adipose tissue; here its oxo-reductase activity has been implicated in the pathogenesis of the metabolic syndrome through increased local generation of cortisol facilitating hepatic glucose output (28) and adipocyte differentiation (29). Inhibition of 11?-HSD1, using selective inhibitors, has resulted in improved insulin sensitivity and weight loss (30, 31), but the factors determining 11?-HSD1 set point and oxo-reductase activity per se have remained elusive.
Initial kinetic studies using recombinant 11?-HSD1 expressed in Vaccinia virus indicated that availability of cofactor was an important regulator of the reaction direction of 11?-HSD1 (32). In an artificial situation, removal of NADP+ using recombinant cytosolic G6PDH, significantly increased oxo-reductase activity. Conversely, oxo-reductase activity can be restored in vitro by the addition of G6PDH and NADP to regenerate NADPH (27, 32). However, due to the subcellular location of 11?-HSD1 within the ER lumen, cytosolic G6PDH is unable to perform this function in vivo. This was endorsed by Bujalska et al. (33), where differences in oxo-reductase-dehydrogenase activities of 11?-HSD1 in human omental and sc adipose stromal cells could not be explained by differences in G6PDH activity.
In vivo studies have also highlighted the bidirectional capacity of 11?-HSD1. Rat Leydig cells, depending upon their stage of differentiation and testosterone secreting ability, vary significantly in underlying oxo-reductase compared with dehydrogenase activities (34). This could be manipulated by altering key components within culture media including the omission of glucose, NAD+, NADH, NADP+ (35). Set point also differs within preadipocytes and adipocytes. Thus, after 14 d of culture under conditions that promote adipocyte differentiation, a switch occurs from 11?-HSD1 dehydrogenase to oxo-reductase activity in omental preadipocytes (36), despite no change in absolute levels of enzyme expression. Additionally, neuronal cells have also been shown to have bidirectional activity (37).
The evidence that H6PDH and not G6PDH allows the regeneration of NADPH and promotes 11?-HSD1 oxo-reductase activity (Fig. 2) has come from several sources. First, patients with a putative 11?-HSD1-deficient state—cortisone reductase deficiency—are unable to convert cortisone to its active form cortisol. Sequencing of the gene encoding 11?-HSD1 (HSD11B1) revealed intronic mutations that may mediate reduced levels of enzyme expression but these are present in up to 4% of normal subjects so that other candidates are likely to be involved (38). Subsequently, sequencing of the H6PDH gene revealed mutations in exon 5 of the gene in all affected patients. Heterozygosity was shown in one case for a 29-bp insertion in exon 5 (620ins29bp621), which caused the inclusion of three new amino acids and a stop codon, resulting in a truncated protein, 171 amino acids shorter than the native protein. Expression of this mutation in hepatic WRL68 cells revealed a null enzyme with total lack of H6PDH activity. In two other cases, homozygosity for R453Q, a nonconservative missense amino acid change, was shown to encode for an enzyme with approximately 25% activity compared with wild type. Thus, cortisone reductase deficiency appears to be a digenic disease requiring interacting mutations in HSD11B1 and H6PDH. Importantly, these data link H6PDH to function of the 11?-HSD1 enzyme.
FIG. 2. Schematic representation of the proposed interaction between H6PDH and 11?-HSD1 within the ER. H6PDH generates NADPH by conversion of glucose-6-phosphate (G6P), transported via the G6P translocase (G6PT), to 6-phosphogluconate (6PG) within the ER. 11?-HSD1 (T1) uses the NADPH as cofactor, allowing the conversion of cortisone to cortisol.
Secondly, these genetic studies have been endorsed through in vitro functional studies. Atanasov et al. (39) showed colocalization of myc-tagged H6PDH and FLAG-tagged 11?-HSD1 transfected in human embryonic kidney (HEK) 293 to the luminal side of the ER. H6PDH was able to determine the direction of 11?-HSD1 in HEK 293 and Chinese hamster ovary cells. Both cell types had bidirectional activity of 11?-HSD1, with a slight preference of dehydrogenase activity. However, coexpression of 11?-HSD1 with H6PDH caused a 5-fold increase in oxo-reductase activity and a 6-fold decrease in dehydrogenase activity without affecting the kinetic parameters. The effect on the maximum velocity (Vmax) and not Michaelis constant for both directions indicated that availability of cofactor does not alter substrate affinity of 11?-HSD1. Similar data have come from our own group. Bujalska et al. (40) have demonstrated that H6PDH increased 11?-HSD1 oxo-reductase activity, while reducing dehydrogenase activity, whereas the cytosolic enzyme G6PDH was unable to affect oxo-reductase activity. Additionally, H6PDH was unable to affect the direction of the 11?-HSD2 enzyme. Stably transfected cells with 11?-HSD1 and a H6PDH short interfering RNA (siRNA) showed a reduction in oxo-reductase activity, simultaneously increasing dehydrogenase activity (Fig. 3).
FIG. 3. Effect of suppression of 11?-HSD1 and H6PDH in HEK 293T1 cells by siRNA. A, HEK 293T1 were transfected with 11?-HSD1, H6PDH, and scrambled-siRNAs (Control) for 48 h. Compared with control, mRNA levels for 11?-HSD1 and H6PDH were suppressed in cells transfected with the respective siRNAs (mean ± SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001). B, 11?-HSD1 oxo-reductase and dehydrogenase activities were both reduced by 11?-HSD1-siRNA, whereas oxo-reductase activity was decreased and dehydrogenase activity simultaneously increased upon transfection with H6PDH-siRNA (mean ± SE; *, P < 0.05, **, P < 0.01; ***, P < 0.001).
In terms of redox potential within the ER lumen, Banhegyi et al. (41) have shown that the intralumenal pool of NADPH and substrate availability of glucose-6-phosphate are critical in regulating 11?-HSD1 direction toward cortisol production under the stimulation of H6PDH. In microsomal preparations, addition of G6P increased production of NADPH and stimulated 11?-HSD1 oxo-reductase activity (41). Inhibition of G6P uptake, using an inhibitor of microsomal G6P transport, decreased this effect.
These cell-based studies all support the concept that H6PDH is important in conveying oxo-reductase activity upon 11?-HSD1, an enzyme that in its purified state is preferentially a dehydrogenase. Together, these recent studies cement a role for H6PDH in regulating the set point of 11?-HSD1 activity. They should also raise increased awareness of the pitfalls of measuring only 11?-dehydrogenase activity in vitro and inferring from this that this is representative of 11-oxo-reductase activity in vivo (42).
Future Directions
The recent excitement over the putative use of 11?-HSD1 inhibitors to treat patients with insulin resistance and obesity-metabolic syndrome is entirely dependant upon 11-oxo-reductase activity in vivo. It is interesting to speculate that inhibition of H6PDH with concomitant reduction in ER NADPH concentrations might result in a similar beneficial effect. Further studies are required to investigate the role of H6PDH and ER redox potential in human disease.
Acknowledgments
The authors would like to thank Dr. Jon Ride for helpful discussions and Drs. Iwona Bujalska, Jeremy Tomlinson, Nicole Draper, and Gareth Lavery for their contributions to this work.
References
Romanelli A, St Denis JF, Vidal H, Tchu S, van de WG 1994 Absence of glucose uptake by liver microsomes: an explanation for the complete latency of glucose dehydrogenase. Biochem Biophys Res Commun 200:1491–1497
Hori SH, Takahashi T 1974 Phenobarbital-induced increase of the hexose 6-phosphate dehydrogenase activity. Biochem Biophys Res Commun 61:1064–1070
Barash V, Erlich T, Bashan N 1990 Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex. Biochem Int 20:267–274
Tanahashi K, Hori SH 1980 Immunohistochemical localization of hexose 6-phosphate dehydrogenase in various organs of the rat. J Histochem Cytochem 28:1175–1182
Beutler E, Morrison M 1967 Localization and characteristics of hexose 6-phosphate dehydrogenase (glucose dehydrogenase). J Biol Chem 242:5289–5293
Kulkarni AP, Hodgson E 1982 Mouse liver microsomal hexose-6-phosphate dehydrogenase. NADPH generation and utilization in monooxygenation reactions. Biochem Pharmacol 31:1131–1137
Chou JY, Matern D, Mansfield BC, Chen YT 2002 Type I glycogen storage diseases: disorders of the glucose-6-phosphatase complex. Curr Mol Med 2:121–143
Sawada H, Hayashibara M, Hara A, Nakayama T 1980 A possible functional relationship between microsomal aromatic aldehyde-ketone reductase and hexose-6-phosphate dehydrogenase. J Biochem (Tokyo) 87:985–988
Sawada H, Hara A, Hayashibara M, Nakayama T, Usui S, Saeki T 1981 Microsomal reductase for aromatic aldehydes and ketones in guinea pig liver. Purification, characterization, and functional relationship to hexose-6-phosphate dehydrogenase. J Biochem (Tokyo) 90:1077–1085
Collard F, Collet JF, Gerin I, Veiga-da-Cunha M, Van Schaftingen E 1999 Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway(1). FEBS Lett 459:223–226
Bublitz C, Steavenson S 1988 The pentose phosphate pathway in the endoplasmic reticulum. J Biol Chem 263:12849–12853
Mason PJ, Stevens D, Diez A, Knight SW, Scopes DA, Vulliamy TJ 1999 Human hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) encoded at 1p36: coding sequence and expression. Blood Cells Mol Dis 25:30–37
Clarke JL, Mason PJ 2003 Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity. Arch Biochem Biophys 415:229–234
Kimura K, Endou H, Sudo J, Sakai F 1979 Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship. J Biochem (Tokyo) 85:319–326
Shaw CR, Koen AL 1968 Glucose 6-phosphate dehydrogenase and hexose 6-phosphate dehydrogenase of mammalian tissues. Ann NY Acad Sci 151:149–156
Mandula B, Srivastava SK, Beutler E 1970 Hexose-6-phosphate dehydrogenase: distribution in rat tissues and effect of diet, age and steroids. Arch Biochem Biophys 141:155–161
Blume KG, Schmidt GM, Heissmeier HH, Lohr GW 1975 Hexose-6-phosphate dehydrogenase from human tissues: an electrophoretic study in health and disease. Experientia 31:496–498
King J, Cook PJ 1981 Glucose dehydrogenase polymorphism in man. Ann Hum Genet 45:129–134
Ozols J 1993 Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase. Proc Natl Acad Sci USA 90:5302–5306
Brands R, Snider MD, Hino Y, Park SS, Gelboin HV, Rothman JE 1985 Retention of membrane proteins by the endoplasmic reticulum. J Cell Biol 101:1724–1732
Clarke JL, Scopes DA, Sodeinde O, Mason PJ 2001 Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase. A novel bifunctional enzyme in malaria parasites. Eur J Biochem 268:2013–2019
Stewart PM, Murry BA, Mason JI 1994 Human kidney 11?-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform. J Clin Endocrinol Metab 79:480–484
Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS 1994 Cloning and tissue distribution of the human 11?-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 105:R11–R17
Ricketts ML, Shoesmith KJ, Hewison M, Strain A, Eggo MC, Stewart PM 1998 Regulation of 11?-hydroxysteroid dehydrogenase type 1 in primary cultures of rat and human hepatocytes. J Endocrinol 156:159–168
Jamieson PM, Chapman KE, Edwards CR, Seckl JR 1995 11?-Hydroxysteroid dehydrogenase is an exclusive 11?-reductase in primary cultures of rat hepatocytes: effect of physicochemical and hormonal manipulations. Endocrinology 136:4754–4761
Lakshmi V, Monder C 1988 Purification and characterization of the corticosteroid 11?-dehydrogenase component of the rat liver 11?-hydroxysteroid dehydrogenase complex. Endocrinology 123:2390–2398
Walker EA, Clark AM, Hewison M, Ride JP, Stewart PM 2001 Functional expression, characterization, and purification of the catalytic domain of human 11-?-hydroxysteroid dehydrogenase type 1. J Biol Chem 276:21343–21350
Jones CG, Hothi SK, Titheradge MA 1993 Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes. Biochem J 289(Pt 3):821–828
Hauner H, Schmid P, Pfeiffer EF 1987 Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 64:832–835
Alberts P, Nilsson C, Selen G, Engblom LO, Edling NH, Norling S, Klingstrom G, Larsson C, Forsgren M, Ashkzari M, Nilsson CE, Fiedler M, Bergqvist E, Ohman B, Bjorkstrand E, Abrahmsen LB 2003 Selective inhibition of 11?-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains. Endocrinology 144:4755–4762
Livingstone DE, Walker BR 2003 Is 11?-hydroxysteroid dehydrogenase type 1 a therapeutic target? Effects of carbenoxolone in lean and obese Zucker rats. J Pharmacol Exp Ther 305:167–172
Agarwal AK, Tusie-Luna MT, Monder C, White PC 1990 Expression of 11?-hydroxysteroid dehydrogenase using recombinant vaccinia virus. Mol Endocrinol 4:1827–1832
Bujalska IJ, Kumar S, Hewison M, Stewart PM 1999 Differentiation of adipose stromal cells: the roles of glucocorticoids and 11?-hydroxysteroid dehydrogenase. Endocrinology 140:3188–3196
Ge RS, Hardy DO, Catterall JF, Hardy MP 1997 Developmental changes in glucocorticoid receptor and 11?-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells. Endocrinology 138:5089–5095
Ferguson SE, Pallikaros Z, Cooke BA, Michael AE 1999 The effects of different culture media, glucose, pyridine nucleotides and adenosine on the activity of 11?-hydroxysteroid dehydrogenase in rat Leydig cells. Mol Cell Endocrinol 158:37–44
Bujalska IJ, Walker EA, Hewison M, Stewart PM 2002 A switch in dehydrogenase to reductase activity of 11?-hydroxysteroid dehydrogenase type 1 upon differentiation of human omental adipose stromal cells. J Clin Endocrinol Metab 87:1205–1210
Seckl JR, Morton NM, Chapman KE, Walker BR 2004 Glucocorticoids and 11?-hydroxysteroid dehydrogenase in adipose tissue. Recent Prog Horm Res 59:359–393
Draper N, Walker EA, Bujalska IJ, Tomlinson JW, Chalder SM, Arlt W, Lavery GG, Bedendo O, Ray DW, Laing I, Malunowicz E, White PC, Hewison M, Mason PJ, Connell JM, Shackleton CH, Stewart PM 2003 Mutations in the genes encoding 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency. Nat Genet 34:434–439
Atanasov AG, Nashev LG, Schweizer RA, Frick C, Odermatt A 2004 Hexose-6-phosphate dehydrogenase determines the reaction direction of 11?-hydroxysteroid dehydrogenase type 1 as an oxoreductase. FEBS Lett 571:129–133
Bujalska IJ, Draper N, Minakami S, Tomlinson JW, White PC, Chapman KE, Walker EA, Stewart PM, Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11?-hydroxysteroid dehydrogenase type 1. J Mol Endocrinol, published online 9 February 2005
Banhegyi G, Benedetti A, Fulceri R, Senesi S 2004 Cooperativity between 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum. J Biol Chem 279:27017–27021
Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DE, Johnson O, Walker BR 2001 Tissue-specific dysregulation of cortisol metabolism in human obesity. J Clin Endocrinol Metab 86:1418–1421(Kylie N. Hewitt, Elizabet)