Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.     

Manifold effects of palmitoylcarnitine on endoplasmic reticulum metabolism: 11β-hydroxysteroid dehydrogenase 1, flux through hexose-6-phosphate dehydrogenase and NADPH concentration

With the exception of the oxidation of G6P (glucose 6-phosphate) by H6PDH (hexose-6-phosphate dehydrogenase), scant information is available about other endogenous substrates affecting the redox state or the regulation of key enzymes which govern the ratio of the pyridine nucleotide NADPH/NADP. In isolated rat liver microsomes, NADPH production was increased, as anticipated, by G6P; however, this was strikingly amplified by palmitoylcarnitine. Subsequent experiments revealed that the latter compound, well within its physiological concentration range, inhibited 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), the bidirectional enzyme which interconnects inactive 11-oxo steroids and their active 11-hydroxy derivatives. Notably, palmitoylcarnitine also stimulated the antithetical direction of 11β-HSD1 reductase, namely dehydrogenase. This stimulation of H6PDH may have likewise contributed to the NADPH accretion. All told, the result of these enzyme modifications is, in a conjoint fashion, a sharp amplification of microsomal NADPH production. Neither the purified 11β-HSD1 nor that obtained following microsomal sonification were sensitive to palmitoylcarnitine inhibition. This suggests that the long-chain amphipathic acylcarnitines, given their favourable partitioning into the membrane lipid bilayer, disrupt the proficient kinetic and physical interplay between 11β-HSD1 and H6PDH. Finally, although IDH (isocitrate dehydrogenase) and malic enzyme are present in microsomes and increase NADPH concentration akin to that of G6P, neither had an effect on 11β-HSD1 reductase, evidence that the NADPH pool in the endoplasmic reticulum shared by the H6PDH/11β-HSD1 alliance is uncoupled from that governed by IDH and malic enzyme.     

Metabolic Coupling Determines the Activity: Comparison of 11β-Hydroxysteroid Dehydrogenase 1 and Its Coupling between Liver Parenchymal Cells and Testicular Leydig Cells

Background

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) interconverts active 11β-hydroxyl glucocorticoids and inactive 11keto forms. However, its directionality is determined by availability of NADP+/NADPH. In liver cells, 11β-HSD1 behaves as a primary reductase, while in Leydig cells it acts as a primary oxidase. However, the exact mechanism is not clear. The direction of 11β-HSD1 has been proposed to be regulated by hexose-6-phosphate dehydrogenase (H6PDH), which catalyzes glucose-6-phosphate (G6P) to generate NADPH that drives 11β-HSD1 towards reduction.

Methodology

To examine the coupling between 11β-HSD1 and H6PDH, we added G6P to rat and human liver and testis or Leydig cell microsomes, and 11β-HSD1 activity was measured by radiometry.

Results and Conclusions

G6P stimulated 11β-HSD1 reductase activity in rat (3 fold) or human liver (1.5 fold), but not at all in testis. S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity. We compared the extent to which 11β-HSD1 in rat Leydig and liver cells might be coupled to H6PDH. In order to clarify the location of H6PDH within the testis, we used the Leydig cell toxicant ethane dimethanesulfonate (EDS) to selectively deplete Leydig cells. The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter). H6pd mRNA level and H6PDH activity were barely detectable in purified rat Leydig cells. In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells.     

Elevation of glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant Saccharomyces cerevisiae

To increase the NAD(P)H-dependent xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis, the activity of glucose 6-phosphate dehydrogenase (G6PDH) encoded by the ZWF1 gene was amplified to increase the metabolic flux toward the pentose phosphate pathway and NADPH regeneration. Compared with the control strain, the specific G6PDH activity was enhanced approximately 6.0-fold by overexpression of the ZWF1 gene. Amplification in the G6PDH activity clearly improved the NAD(P)H-dependent xylitol production in the recombinant S. cerevisiae strain. With the aid of an elevated G6PDH level, maximum xylitol concentration of 86 g/l was achieved with productivity of 2.0 g/l h in the glucose-limited fed-batch cultivation, corresponding to 25% improvement in volumetric xylitol productivity compared with the recombinant S. cerevisiae strain containing the xylose reductase gene only.     

Modification of microsomal 11beta-HSD1 activity by cytosolic compounds: glutathione and hexose phosphoesters

11beta-Hydroxysteroid dehydrogenase1(11beta-HSD1) can serve either as an oxo-reductase or dehydrogenase determined by the redox state in the endoplasmic reticulum (ER). This bidirectional enzyme governs paracrine glucocorticoid production. Recent in vitro studies have underscored the key role of cytoplasmic glucose-6-phosphate (G6P) in controlling the flux direction of 11betaHSD-1 by altering the intraluminal ER NADPH/NADP ratio. The hypothesis that other hexose phosphoesters or the plentiful cellular oxidative protector glutathione could also regulate microsomal 11betaHSD-1 activity was tested. Fructose-6-phosphate increased the activity of 11beta-HSD1 reductase in isolated rat and porcine liver microsomes but not porcine fat microsomes. Moreover, oxidized glutathione (GSSG) attenuated 11beta-HSD1 reductase activity by 40% while reduced glutathione (GSH) activated the reductase in liver. Fat microsomes were unaffected because they lack glutathione reductase. Nonetheless, another oxidizing agent, hydrogen peroxide (0.5mM), inhibited both fat and liver 11beta-HSD1 reductase. Consistent with the major role of the redox state, 2.5mM GSSG and hydrogen peroxide augmented the 11beta-HSD1 dehydrogenase, antithetical to the reductase, by 20-30% in liver microsomes. Given the key role of reactive oxygen species and hexose phosphate accumulation in the pathoetiology of obesity and diabetes, these compounds might also modify 11beta-HSD1 in these conditions.     

Cellular and genetic models of H6PDH and 11β‐HSD1 function in skeletal muscle

Glucocorticoids are important for skeletal muscle energy metabolism, regulating glucose utilization, insulin sensitivity, and muscle mass. Nicotinamide adenine dinucleotide phosphate‐dependent 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1)‐mediated glucocorticoid activation in the sarcoplasmic reticulum (SR) is integral to mediating the detrimental effects of glucocorticoid excess in muscle. 11β‐Hydroxysteroid dehydrogenase type 1 activity requires glucose‐6‐phosphate transporter (G6PT)‐mediated G6P transport into the SR for its metabolism by hexose‐6‐phosphate dehydrogenase (H6PDH) for NADPH generation. Here, we examine the G6PT/H6PDH/11β‐HSD1 triad in differentiating myotubes and explore the consequences of muscle‐specific knockout of 11β‐HSD1 and H6PDH. 11β‐Hydroxysteroid dehydrogenase type 1 expression and activity increase with myotube differentiation and in response to glucocorticoids. Hexose‐6‐phosphate dehydrogenase shows some elevation in expression with differentiation and in response to glucocorticoid, while G6PT appears largely unresponsive to these particular conditions. When examining 11β‐HSD1 muscle‐knockout mice, we were unable to detect significant decrements in activity, despite using a well‐validated muscle‐specific Cre transgene and confirming high‐level recombination of the floxed HSD11B1 allele. We propose that the level of recombination at the HSD11B1 locus may be insufficient to negate basal 11β‐HSD1 activity for a protein with a long half‐life. Hexose‐6‐phosphate dehydrogenase was undetectable in H6PDH muscle‐knockout mice, which display the myopathic phenotype seen in global KO mice, validating the importance of SR NADPH generation. We envisage these data and models finding utility when investigating the muscle‐specific functions of the 11β‐HSD1/G6PT/H6PDH triad.     

The molecular basis of type 1 glycogen storage diseases

Glycogen storage disease type 1 (GSD-1), also known as von Gierke disease, is a group of autosomal recessive metabolic disorders caused by deficiencies in the activity of the glucose-6-phosphatase (G6Pase) system that consists of at least two membrane proteins, glucose-6-phosphate transporter (G6PT) and G6Pase. G6PT translocates glucose-6-phosphate (G6P) from cytoplasm to the lumen of the endoplasmic reticulum (ER) and G6Pase catalyzes the hydrolysis of G6P to produce glucose and phosphate. Therefore, G6PT and G6Pase work in concert to maintain glucose homeostasis. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Both manifest functional G6Pase deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-1b patients also suffer from chronic neutropenia and functional deficiencies of neutrophils and monocytes, resulting in recurrent bacterial infections as well as ulceration of the oral and intestinal mucosa. The G6Pase gene maps to chromosome 17q21 and encodes a 36-kDa glycoprotein that is anchored to the ER by 9 transmembrane helices with its active site facing the lumen. Animal models of GSD-1a have been developed and are being exploited to delineate the disease more precisely and to develop new therapies. The G6PT gene maps to chromosome 11q23 and encodes a 37-kDa protein that is anchored to the ER by 10 transmembrane helices. A functional assay for the recombinant G6PT protein has been established, which showed that G6PT functions as a G6P transporter in the absence of G6Pase. However, microsomal G6P uptake activity was markedly enhanced in the simultaneous presence of G6PT and G6Pase. The cloning of the G6PT gene now permits animal models of GSD-1b to be generated. These recent developments are increasing our understanding of the GSD-l disorders and the G6Pase system, knowledge that will facilitate the development of novel therapeutic approaches for these disorders.     

Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate

Methods for 2-deoxyglucose (2-DG) and 2-deoxyglucose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH), but at a rate 1000-fold slower than for glucose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates. Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG, glucose, DG6P, and glucose 6-P can all be separately measured. To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase.     

Mutations in the glucose-6-phosphate transporter (G6PT) gene in patients with glycogen storage diseases type 1b and 1c.

Glycogen storage diseases type 1 (GSD 1) are a group of autosomal recessive disorders characterized by impairment of terminal steps of glycogenolysis and gluconeogenesis. Mutations of the glucose-6-phosphatase gene are responsible for the most frequent form of GSD 1, the subtype 1a, while mutations of the glucose-6-phosphate transporter gene (G6PT) have recently been shown to cause the non 1a forms of GSD, namely the 1b and 1c subtypes. Here, we report on the analysis by single-stranded conformation polymorphism (SSCP) and/or DNA sequencing of the exons of the G6PT in 14 patients diagnosed either as affected by the GSD 1b or 1c subtypes. Mutations in the G6PT gene were found in all patients. Four of the detected mutations were novel mutations, while the others were previously described. Our results confirm that the GSD 1b and 1c forms are due to mutations in the same gene, i.e. the G6PT gene. We also show that the same kind of mutation can be associated or not with evident clinical complications such as neutrophil impairment. Since no correlation between the type and position of the mutation and the severity of the disease was found, other unknown factors may cause the expression of symptoms, such as neutropenia, which dramatically influence the severity of the disease.     

Glycogen storage disease type 1b: genetic disorder involving the transport system of intracellular membrane

Glycogen storage diseases (GSD) type 1b is the first example of a genetic disorder involving the transport system of an intracellular membrane. It was revealed that the primary defect in GSD type 1b was a deficiency in the microsomal glucose-6-phosphate (G6P) translocase, based on the findings that the glucose-6-phosphatase activity was highly latent in the fresh liver homogenates. Further evidence of this defect in GSD type 1b has been provided by a membrane filter method which measures the uptake of 14C-G6P by microsomes. The clinical symptoms and enzymatic studies in our patients suggest that there is genetic heterogeneity in GSD 1b and the clinical severity depends on the level of residual activities of G6P translocase.     

A glucose-6-phosphate hydrolase, widely expressed outside the liver, can explain age-dependent resolution of hypoglycemia in glycogen storage disease type Ia

A fine control of the blood glucose level is essential to avoid hyper- or hypo-glycemic shocks associated with many metabolic disorders, including diabetes mellitus and type I glycogen storage disease. Between meals, the primary source of blood glucose is gluconeogenesis and glycogenolysis. In the final step of both pathways, glucose-6-phosphate (G6P) is hydrolyzed to glucose by the glucose-6-phosphatase (G6Pase) complex. Because G6Pase (renamed G6Pase-alpha) is primarily expressed only in the liver, kidney, and intestine, it has implied that most other tissues cannot contribute to interprandial blood glucose homeostasis. We demonstrate that a novel, widely expressed G6Pase-related protein, PAP2.8/UGRP, renamed here G6Pase-beta, is an acid-labile, vanadate-sensitive, endoplasmic reticulum-associated phosphohydrolase, like G6Pase-alpha. Both enzymes have the same active site structure, exhibit a similar Km toward G6P, but the Vmax of G6Pase-alpha is approximately 6-fold greater than that of G6Pase-beta. Most importantly, G6Pase-beta couples with the G6P transporter to form an active G6Pase complex that can hydrolyze G6P to glucose. Our findings challenge the current dogma that only liver, kidney, and intestine can contribute to blood glucose homeostasis and explain why type Ia glycogen storage disease patients, lacking a functional liver/kidney/intestine G6Pase complex, are still capable of endogenous glucose production.     

Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11beta-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11beta-HSD1 oxoreductase activity and add a new dimension to the investigation of 11beta-HSD1 as a therapeutic target in patients with the metabolic syndrome.     

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

H6PDH interacts directly with 11β-HSD1: Implications for determining the directionality of glucocorticoid catalysis

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11β-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11β-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11β-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11β-HSD1, and with cell lysates expressing H6PDH and 11β-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11β-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11β-HSD1 physically as well as modulate 11β-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11β-HSD1 activity.     

Deletion of the gene encoding the ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein (UGRP)/glucose-6-phosphatase catalytic subunit-beta results in lowered plasma cholesterol and elevated glucagon

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-beta. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by approximately 50% in homogenates of UGRP(-/-) mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP(-/-) mice exhibit growth retardation as do G6Pase(-/-) mice and patients with GSD type 1a. However, in contrast to G6Pase(-/-) mice and patients with GSD type 1a, UGRP(-/-) mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP(-/-) mice are not hypoglycemic, female UGRP(-/-) mice have elevated ( approximately 60%) plasma glucagon and reduced ( approximately 20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP(-/-) mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo.     

Liver and colon pro- and anti-oxidant enzyme activities in rats after long-term ethylnitrosourea exposure

Liver and colon pro- and anti-oxidant enzyme activities were investigated in rats treated with ethylnitrosourea (ENU) (i.p.) (4 mg/kg body wt) for 6 months. The pro-oxidant enzymes (NADPH cytochrome c reductase, NADH cytochrome c reductase, NADH cytochrome b5 reductase and cytochrome P-4502E1and the antioxidant enzyme, superoxide dismutase (SOD) exhibited significantly increased activity in liver and colon. Glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase (GST) showed enhanced activity in liver, but decreased activity in colon. Glutathione peroxidase (GP) and glutathione reductase (GR) activities were significantly increased in colon, but decreased in liver. Catalase (CAT) activity while showed a significant increase in liver, exhibited only marginal increase in colon. Malondialdehyde (MDA) level was significantly elevated in both tissues.     

Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes.

The hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Inoculation of hepatocytes from starved rats into primary cultures resulted in a 4-5-fold increase in G6PDH activity in 48 h in the absence of hormones. Parallel cultures treated simultaneously with glucocorticoids and insulin exhibited a 12-15-fold increase during the same time. Glucocorticoids by themselves did not elevate G6PDH activity, whereas insulin alone significantly stimulated enzyme activity. Thus the glucocorticoids acted in a 'permissive' role to amplify the insulin stimulation of G6PDH. Elevated concentrations of glucose in the culture medium increased enzyme activity in both the control cultures and those treated with hormones. Ethanol was found to potentiate G6PDH activity in cultures treated with glucocorticoids and insulin. The effect of ethanol was time- and dose-dependent. These results establish that insulin, glucocorticoids, glucose and ethanol interact in some undefined manner to regulate hepatic G6PDH activity.