Skeletal muscle 11ßHSD1 activity of nondiabetic subjects is unaltered in central obesity-associated insulin resistance.

Local activation of glucocorticoids in insulin target tissues by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) has been implicated in the etiology of the metabolic syndrome. In obesity, adipose tissue 11ßHSD1 is upregulated, leading to the generation of higher tissue levels of cortisol, which may increase insulin resistance. However, skeletal muscle is the predominant site of insulin-mediated glucose disposal, which is known to be reduced in obesity. We aimed to determine if there is any relationship between skeletal muscle 11ßHSD1 and markers of central adiposity and insulin resistance in nondiabetic subjects. 20 nondiabetic volunteers (8 males and 12 females, mean age 55 ± 13 years, body mass index 21.5-47.6, mean 30.4 ± 1.6 kg/m (2)) underwent a single fasting blood sample followed by a muscle biopsy of vastus lateralis under local anesthetic. Fasting glucose, insulin and adiponectin were measured in serum. Skeletal muscle 11ßHSD1 oxoreductase activity was determined by measuring the conversion of radiolabelled (3)H-cortisone to cortisol by thin layer chromatography. When subjects were categorised according to abdominal obesity (waist circumference ≥ 102 cm in men, ≥ 88 cm in women), there was no difference between the groups in skeletal muscle 11ßHSD1 activity. There was no correlation between body mass index or waist circumference and 11ßHSD1 activity or between HOMA and 11ßHSD1 activity. Skeletal muscle 11ßHSD1 oxoreductase activity is not altered in nondiabetic subjects with central obesity-associated insulin resistance. It is therefore unlikely that the in vivo insulin resistance observed in skeletal muscle of centrally obese subjects is mediated by alterations in 11ßHSD1.

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11beta-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11beta-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11beta-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11beta-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11beta-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11beta-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11Beta-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11beta-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20-30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of > 60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11beta-HSD domains A-D to a cleft in the protein structure (cofactor binding domain), two parallel beta-sheets, and an alpha-helix (active site), respectively.

Serines at the active site of 11 beta-hydroxysteroid dehydrogenase type I determine the rate of catalysis.

Short chain alcohol dehydrogenases have an invariant YXXXK motif at the active site. Database analysis of 116 superfamily members showed that 92% also contain a Serine or Threonine residue at the Y + 1 or Y + 3 positions, a pattern we previously described as the ST rule. In the present study we have mutated Serines in the active site, YSASK, motif of 11 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studies were facilitated by the generation of a new specific polyclonal antibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34 kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active site Serines resulted in modest increases of Km values for corticosterone from 325 nM for 11 beta HSD1 to 512 nM-588 nM for the S1 (YAASK), S2 (YSAGK) and S3 (YAAGK) mutants in homogenates of transfected CHOP cells. However, far greater effects were observed on the first order rate constants with mutants displaying 10%, 1% and 1% of the wild type activity, respectively. When the oxidoreductase reaction was studied in whole cells mutagenesis again had a minimal effect on the Km value but dramatically lowered first order rate constants to 34%, 5% and 6%, respectively, of the wild type. These data show that Serines at the active site of 11 beta HSD1 play an important role in determining the rate of catalysis. Coexpression of wild type and mutant enzymes did not lower wild type activity suggesting that the active site of the multimeric enzyme is not a composite of active site Serines from different subunits.

A genetic defect resulting in mild low-renin hypertension.

Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (HSD11B2) encoding the 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD2) cause AME. Typical patients with AME have defective 11beta-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-3H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the HSD11B2 gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately elevated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0-6% in typical patients with AME whereas the normal conversion is 90-95%. Molecular analysis of the HSD11B2 gene of this patient showed a homozygous C-->T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation. In vitro expression studies showed an increase in the Km (300 nM) over normal (54 nM). Because approximately 40% of patients with essential hypertension demonstrate low renin, we suggest that such patients should undergo genetic analysis of the HSD11B2 gene.

Truncation of the N- and C-terminal regions of the human 11beta-hydroxysteroid dehydrogenase type 2 enzyme and effects on solubility and bidirectional enzyme activity.

The 11beta-hydroxysteroid dehydrogenase type II enzyme (11betaHSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala73 on the cytoplasmic side of the membrane, suggesting that 11betaHSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11betaHSD2 deltaN) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11betaHSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11betaHSD2 deltaC) resulted in an enzyme with a Km of 215 nM for cortisol in whole cell assays, while 11betaHSD2 and 11betaHSD2 deltaN displayed a Km of 62 and 74 nM, respectively. In homogenates 11betaHSD2 and 11betaHSD2 deltaC displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11BetaHSD2 was more stable than 11betaHSD2 deltaC in the presence of NaSCN, suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11betaHSD2 deltaN, while 11betaHSD2 deltaC and 11betaHSD2 displayed a Km of 135 and 46 nM, respectively. Although 11betaHSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids.

Oxoreductase and dehydrogenase activities of the human and rat 11beta-hydroxysteroid dehydrogenase type 2 enzyme.

The 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11betaHSD2) metabolizes glucocorticoids into their inactive 11-keto metabolites. Although the type 1 enzyme (11betaHSD1) displays both oxidative and reductive activity, to date 11betaHSD2 has been shown to have dehydrogenase activity only. In this study we compared both dehydrogenase and reductase characteristics of the cloned rat 11betaHSD1 and rat and human 11betaHSD2 for three different 11-hydroxysteroid substrates, cortisol (F), corticosterone (B), and dexamethasone (Dex), and the corresponding 11-keto metabolites, cortisone (E), 11-dehydrocorticosterone (A), and 11-dehydrodexamethasone (DH-Dex), respectively. In cell homogenates expressing either the rat or the human 11betaHSD2, the relative potency for the dehydrogenase reaction was B > F > Dex. Although there was no reduction of A or E, DH-Dex was readily converted to Dex with an equilibrium far on the side of the 11-hydroxy metabolite. DH-Dex reduction in homogenates was inhibited by both glycyrrhetinic acid and carbenoxolone, with a 50% inhibition at 80 and 100 nM, respectively. In intact cells transfected with rat 11betaHSD1, the equilibrium was on the reductase side for all substrates. Dehydrogenation of B or F was more potent with rat 11betaHSD2 than with rat 11betaHSD1. There was no detectable 11betaHSD1 oxidation of Dex. These data indicate that both the cloned human and rat 11betaHSD2 reduce DH-Dex and do this more readily than they oxidize Dex. Thus, 11betaHSD2 seems also to be a bidirectional enzyme, although no reduction of the physiological compounds A and E was observed.

Mutations in the 11 beta-hydroxysteroid dehydrogenase type II enzyme associated with hypertension and possibly stillbirth.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol into cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Placental 11 beta HSD2 is also thought to protect the fetus from the high maternal circulating levels of glucocorticoids. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME)--a low renin form of hypertension. Recently, a mutation has been identified in a family with AME and in which there is a high incidence of stillbirths. In this study we have expressed the R374X mutation and show that the mutant is devoid of enzyme activity in intact mammalian cells expressing a significant level of the truncated protein. While this observation elucidates the cause of AME in this family the degree to which R374X also contributes to the higher incidence of failed pregnancies remains to be determined.

Point mutations abolish 11 beta-hydroxysteroid dehydrogenase type II activity in three families with the congenital syndrome of apparent mineralocorticoid excess.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol into mineralocorticoid receptor inactive cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME), although proof of mutant protein synthesis was not provided. In the present study we have examined the metabolism of cortisol in mammalian cells transfected with plasmids expressing the wild type and mutant enzymes from three additional families of patients with mutations in the HSD11B2 gene. These studies revealed that the mutants were enzymatically inactive in intact mammalian cells expressing significant levels of both full length and truncated proteins. This is the first study to definitively show that point mutations in the HSD11B2 gene abolish 11 beta HSD2 enzymatic activity in the syndrome of AME.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme: biochemical consequences of the congenital R337C mutation.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol to cortisone, allowing the non-selective mineralocorticoid receptor to bind aldosterone. When the activity of this enzyme is compromised, as occurs in licorice intoxication or in the congenital syndrome of apparent mineralocorticoid excess (AME), there is marked sodium retention, hypokalemia, and hypertension. The first proof that this enzyme was defective in AME came from the identification of the R337C mutation in a number of siblings with the syndrome. Subsequent expression studies showed that the mutant had a Km one order of magnitude higher than the wild-type enzyme while in the cell-free system it was without detectable activity. In the present work we have extended our studies on this mutant and provide evidence that the mutant protein may also partially inhibit the wild-type enzyme in heterozygotes. Furthermore, experiments incorporating the protein synthesis inhibitor cycloheximide show that the mutant enzyme is less stable than the wild-type activity in intact cells. These results suggest that mutations in the 11 beta HSD2 enzyme may have multiple consequences for the mineralocorticoid target cell.

The human 11 beta-hydroxysteroid dehydrogenase type II enzyme: comparisons with other species and localization to the distal nephron.

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

The R337C mutation generates a high Km 11 beta-hydroxysteroid dehydrogenase type II enzyme in a family with apparent mineralocorticoid excess.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) inactivates glucocorticoids in the kidney and thus permits aldosterone to occupy the non-selective mineralocorticoid receptor in epithelial tissues. We have recently described a C to T transition in the HSD11B2 gene which results in an arginine to cysteine mutation (R337C) in the 11 beta HSD2 enzyme in a consanguineous family with three siblings suffering from Apparent Mineralocorticoid Excess (AME). In the present study we have examined the metabolism of cortisol in mammalian cells transfected with plasmids expressing the wild type and mutant enzymes. In whole cells the Km of the normal enzyme was 110nM, while the enzyme containing the R337C mutation displayed a Km of 1010nM. Further experiments revealed that the mutant was totally inactive in cell free preparations, suggesting that it has additional properties which may compromise its activity in whole cells.

A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess.

A mutation in the HSD11B2 gene has been discovered in a consanguineous Iranian family with three sibs suffering from Apparent Mineralocorticoid Excess (AME). Sequence data demonstrate a C to T transition resulting in an R337C mutation.

Cloning of the 11 beta HSD type II enzyme from human kidney.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to receptor inactive metabolites. Two isoforms of the enzyme exist. 11 beta HSD1 is a low affinity NADP dependent enzyme, while 11 beta HSD2 is a high affinity NAD dependent species thought to be responsible for endowing specificity on the mineralocorticoid receptor and for protecting the fetus from high circulating levels of maternal glucocorticoids. We have recently cloned the human renal 11 beta HSD2 enzyme. In this report we show that 11 beta HSD2 potently inactivates the synthetic glucocorticoid dexamethasone, producing a single product thought to be the 11-dehydrodexamethasone metabolite. Sequence analysis shows that the new isoform is a member of the short-chain alcohol dehydrogenase superfamily (SCAD), most closely related to 17 beta HSD2 and distantly related to 11 beta HSD1.

Cloning and tissue distribution of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) is thought to protect the non-selective mineralocorticoid receptor from occupation by glucocorticoids, and to modulate access of glucocorticoids to glucocorticoid receptors resulting in protection of the fetus and gonads. A ubiquitous low affinity NADP+ dependent enzyme (11 beta HSD1) and a tissue specific, high affinity NAD+ dependent form (11 beta HSD2) of 11 beta HSD exist. We now report the isolation of a cDNA coding for human 11 beta HSD2. The new isoform is NAD+ dependent, exclusively dehydrogenase in directionality, inhibited by glycyrrhetinic acid and metabolizes the synthetic glucocorticoid dexamethasone; it displays Km values for corticosterone and cortisol of 5.1 nM and 47 nM, respectively. Sequence alignment shows that 11 beta HSD2 shares 35% identity with 17 beta HSD2, but is only 14% identical with 11 beta HSD1. The 11 beta HSD2 gene is highly expressed in kidney, colon, pancreas and placenta and the message is also present in the ovary, prostate and testis. These data suggest that 11 beta HSD2 plays an important role in modulating mineralocorticoid and glucocorticoid receptor occupancy by glucocorticoids.

Isozymes of 11 beta-hydroxysteroid dehydrogenase: which enzyme endows mineralocorticoid specificity?

Intracellular enzymes which interconvert circulating hormones between active and inactive forms aid in regulating the biological activity of the ligand in a cell-specific manner. This is particularly important in mineralocorticoid target tissues where glucocorticoids and mineralocorticoids have equivalent affinity for the mineralocorticoid receptor. Inactivation of glucocorticoids at the 11-hydroxyl position by the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) permits the occupation of the mineralocorticoid receptor by aldosterone in the presence of much higher levels of circulating cortisol. The suppression of dehydrogenase activity allows glucocorticoids to activate the mineralocorticoid receptor, leading to classical mineralocorticoid type effects such as sodium retention and potassium excretion. A number of 11 beta-OHSDs are currently candidate protectors of the mineralocorticoid receptor. This review examines the attributes of these 11 beta-hydroxysteroid dehydrogenase isozymes and suggests reasons why a high affinity, NAD-dependent enzyme appears to be the most likely candidate to endow specificity on the mineralocorticoid receptor.