Cooperativity between 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum

The functional coupling of 11beta-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 11beta-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.     

Contribution of hexose-6-phosphate dehydrogenase to NADPH content and redox environment in the endoplasmic reticulum

Abstract

Background: Hexose-6-phosphate dehydrogenase (H6PD) has been considered to be a main source of NADPH in the endoplasmic reticulum. It provides reducing equivalents to 11-hydroxysteroid dehydrogenase type 1 for in situ re-activation of glucocorticoids. H6PD null mice indeed show signs of glucocorticoid deficiency, but also suffer from a skeletal myopathy mainly affecting fast twitch muscles, in which the unfolded protein response (UPR) is activated. Thus, H6PD may have additional functions in muscle.

Materials and methods: To determine the contribution of H6PD to total microsomal NADPH content, we measured NADPH in microsomes from liver and quadriceps, gastrocnemius and soleus muscles. To evaluate the effect of H6PD deficiency on microsomal thiol-disulfide redox environment, we measured reduced and oxidized glutathione and free protein thiols.

Results and conclusions: H6PD deficiency decreased but did not eliminate NADPH content in liver and soleus microsomes. Thus there must be other sources of NADPH within the endoplasmic/sarcoplasmic reticulum. Levels of reduced glutathione and free protein thiols were decreased in gastrocnemius muscle from null mice, indicating a more oxidative environment. Such alterations in redox environment may underlie the myopathy and UPR activation in H6PD null mice.

General significance: H6PD plays a role in maintaining normal NADPH levels and redox environment inside the endoplasmic reticulum. Intrinsic differences in ER metabolism may explain the differing effects of H6PD deficiency in different tissues.     

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.     

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.     

Appropriate function of 11beta-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase

By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11beta-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys(5) by Ser in 11beta-HSD1, but not of the analogous Lys(4) by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu(25)/Glu(26) in 11beta-HSD1 and Asp(25) in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys(35)/Lys(36) and Glu(25)/Glu(26) on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11beta-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11beta-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.     

Expression of hexose-6-phosphate dehydrogenase in rat tissues

Hexose-6-phosphate dehydrogenase (H6PD) is the main NADPH generating enzyme in the lumen of the endoplasmic reticulum. H6PD is regarded as an ancillary enzyme in prereceptorial glucocorticoid activation and probably acts as a nutrient sensor and as a prosurvival factor. H6PD expression was determined in a variety of rat and human tissues by detecting mRNA and protein levels, and by measuring its dehydrogenase and lactonase activities. It was found that H6PD was present in all investigated tissues; both expression and activity remained within an order of magnitude. Correlation was found between the dehydrogenase activity and protein or mRNA levels. The results confirmed the supposed housekeeping feature of the enzyme.     

Microsomal hexose-6-phosphate dehydrogenase in injured liver.

Hexose-6-phosphate dehydrogenase (H6PD) in rat liver microsomes was clearly differentiated kinetically, immunologically and electrophoretically from glucose-6-phosphate dehydrogenase (G6PD) localized in liver supernatants. Although the soluble G6PD activity increased upon liver injuries induced by CCl4 and thioacetamide, the H6PD activity decreased markedly 1-2 days following administrations of these hepatotoxins. The specific activity of H6PD remained fairly constant under other experimental conditions where the levels of the soluble G6PD activity increased.     

Piperidine amides as 11beta-hydroxysteroid dehydrogenase type 1 inhibitors

A series of piperidine amide inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were identified via modifications of the HTS hit compound 1. The synthesis, in vitro biological evaluation, and structure-activity relationship of these compounds are presented.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

PPARalpha agonists reduce 11beta-hydroxysteroid dehydrogenase type 1 in the liver

11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Oxazolones as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

2,5,5-Trisubstituted oxazolones were identified as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The synthesis, structure-activity relationship and metabolic stability of these compounds are presented.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

Glucocorticoids, 11 beta-hydroxysteroid dehydrogenase type 1, and visceral obesity

Glucocorticoids are implicated as a pathophysiological mediator of obesity and its accompanying metabolic and cardiovascular complications. Obese patients exhibit normal circulating cortisol levels, related to increased glucocorticoid production and degradation. However, it has been demonstrated that local production of active cortisol from inactive cortisone driven by 11 beta-hydroxysteroid dehydrogenase type 1 is exaggerated in adipose tissue of obese subjects. Such local hypercortisolism may be responsible for increased adipocyte differentiation and enhanced secretion of free fatty acids and other substances involved in the metabolic and cardiovascular complications observed in obesity.     

Bis-aryl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

3-Aryl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). They are active in both in vitro and an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.