Tomoatsu Mune

Responses of plasma adrenocortical steroids to low dose ACTH in normal subjects

OBJECTIVE The standard ACTH test in clinical use employs a pharmacological dose of ACTH which assesses the maximum secretory capacity of the adrenal cortex. We have investigated the responses of plasma adrenocortical steroids including cortisol, aldosterone and dehydroepiandrosterone (DHEA) to physiological doses of ACTH (ACTH 1–24, tetracosactide, Cortrosyn) and determined the minimal dose which induces a response equivalent to that induced by a pharmacological dose of ACTH.

Apparent Mineralocorticoid Excess Genotype Is Correlated With Biochemical Phenotype

The syndrome of apparent mineralocorticoid excess is a form of hypertension inherited in an autosomal recessive manner. This disorder results from mutations in the HSD11K (HSD11B2) gene, which encodes the kidney isozyme of 11beta-hydroxysteroid dehydrogenase. This enzyme converts active glucocorticoids such as cortisol and corticosterone to their inactive metabolites cortisone and 11-dehydrocorticosterone. An elevated ratio of cortisol to cortisone metabolites in the urine (tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone [(THF+aTHF)/THE]) is considered pathognomic for this disorder. To determine whether the biochemical phenotype of this disorder is correlated with genotype, we expressed enzymes carrying each of the six known missense mutations in cultured cells. Only one mutant, R337C, had detectable activity in cell lysates, but five of six mutants were partially active in whole cells. Apparent kinetic constants for cortisol and corticosterone were determined in whole cells, and the apparent first-order rate constant, Vmax/Km, was used as a measure of enzymatic activity. The urinary (THF+aTHF)/THE ratio in patients carrying each mutation was strongly correlated with in vitro enzymatic activity of the corresponding mutant (r=.839, P=.001 with cortisol as the substrate). We conclude that the biochemical phenotype of the syndrome of apparent mineralocorticoid excess is largely determined by genotype.

Mutants of 11β-Hydroxysteroid Dehydrogenase (11-HSD2) With Partial Activity: Improved Correlations Between Genotype and Biochemical Phenotype in Apparent Mineralocorticoid Excess

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.

DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase

We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 μM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-α, -β, and -γ. DHEA provoked the translocation of PKC-β and -ζ from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12- O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

Molecular analysis of 11β-hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess

The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11 beta-hydroxysteroid dehydrogenase (11-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. The deficiency allows mineralocorticoid receptors to be occupied by cortisol, because these receptors themselves have similar affinities for cortisol and aldosterone. There are two isozymes of 11-HSD, a liver (L) or type 1 isozyme with a relatively low affinity for steroids, and a kidney (K) or type 2 isozyme with high steroid affinity. Mutations in the gene for the kidney isozyme of 11-HSD have been detected in all kindreds with AME. We expressed enzymes carrying all known missense mutations in cultured cells and determined their activity. For each patient with AME, we compared the enzymatic activity predicted by the genotype with the ratio of cortisol to cortisone metabolites in the urine, (THF + aTHF)/THE. These were strongly correlated, suggesting that the biochemical phenotype of AME is largely determined by genotype. The K isozyme of 11-HSD is also expressed in high levels in the placenta, where its function is unclear. AME patients often have low birth weight. By analogy with AME, low placental 11-HSD K activity in humans might be a risk factor for low birth weight and subsequent hypertension. However, we found that there was no significant correlation between 11-HSD activity, mRNA levels, and either fetal or placental weight.

11β-Hydroxysteroid Dehydrogenase and Its Role in the Syndrome of Apparent Mineralocorticoid Excess

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal tubules and cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine. Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may result in hypokalemia, suppressed plasma renin activity, and hypertension. The syndrome of apparent mineralocorticoid excess(AME) is an inherited form of hypertension in which 11β-hydroxysteroid dehydrogenase (11β-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. Because mineralocorticoid receptors themselves have similar affinities for cortisol and aldosterone, it is hypothesized that the deficiency allows these receptors to be occupied by cortisol, which normally circulates at levels far higher than those of aldosterone. We cloned cDNA and genes encoding two isozymes of 11β-HSD. The liver (L) or type 1 isozyme has relatively low affinity for steroids, is expressed at high levels in the liver but poorly in the kidney, and is not defective in AME. The kidney (K) or type 2 isozyme has high steroid affinity and is expressed at high levels in the kidney and placenta. Mutations in the gene for the latter isozyme have been detected in all kindreds with AME. Moreover, the in vitro enzymatic activity conferred by each mutation is strongly correlated with the ratio of cortisol to cortisone metabolites in the urine [tetrahydrocortisone (THF) + allo-THF]/THE. This suggests that the biochemical phenotype of AME is largely determined by genotype.

Glycyrrhizic acid suppresses type 2 11β-hydroxysteroid dehydrogenase expression in vivo

Licorice-derivatives such as glycyrrhizic acid (GA) competitively inhibit 11 beta-hydroxysteroid dehydrogenase(11 beta-HSD) type 2 (11-HSD2) enzymatic activity, and chronic clinical use often results in pseudoaldosteronism. Since the effect of GA on 11-HSD2 expression remains unknown, we undertook in vivo and in vitro studies. Male Wistar rats were given 30, 60 or 120 mg/kg of GA twice a day for 2 weeks. Plasma corticosterone was decreased in those given the 120 mg dose, while urinary corticosterone excretion was increased in those given the 30 and 60 mg doses but decreased in those given 120 mg GA. NAD(+)-dependent dehydrogenase activity in kidney microsomal fraction was decreased in animals receiving doses of 60 and 120 mg GA. The 11-HSD2 protein and mRNA levels were decreased in those given 120 mg GA. In contrast, in vitro studies using mouse kidney M1 cells revealed that 24h treatment with glycyrrhetinic acid did not affect the 11-HSD2 mRNA expression levels. Thus, in addition to its role as a competitive inhibitor of 11-HSD2, the chronic high dose of GA suppresses mRNA and protein expression of 11-HSD2 possibly via indirect mechanisms. These effects may explain the prolonged symptoms after cessation of GA administration in some pseudoaldosteronism patients.

Correlation between left ventricular mass and urinary sodium excretion in specific genotypes of CYP11B2.

Background Aldosterone has essential roles in regulating intravascular volume and blood pressure, and is suggested to influence cardiac structure. However, the association of polymorphisms in the aldosterone synthase gene (CYP11B2) with hypertension or cardiac hypertrophy remains controversial. Objective To evaluate the distribution of polymorphisms in the CYP11B2 gene and the possible associations between genotypes and blood pressure, urinary excretion of aldosterone or electrolytes and echocardiographic measurements, in a Japanese population. Methods and results We examined the association of two common diallelic polymorphisms within CYP11B2, one in the promoter −344T/C and the other an intron 2 gene conversion, with blood pressure, 24-h urinary excretion of aldosterone and electrolytes, and echocardiographic measurements, in a Japanese population. We confirmed significant linkage disequilibrium between these polymorphic loci and ethnic differences in frequency of the alleles. The −344C and −344T haplotypes apparently diverged before the intron conversion polymorphism was generated on the latter haplotype. Allele frequencies did not differ between 535 normotensive and 360 hypertensive individuals or between hypertensive individuals with higher and lower concentrations of renin. The only significant correlation was a positive correlation of left ventricular mass with 24-h urinary excretion of sodium, which occurred only in individuals with the −344CC genotype or the intron 2 conversion (−/−) genotype. Conclusions The −344CC or intron 2 conversion (−/−) genotype in CYP11B2 may be a risk factor for developing sodium-sensitive cardiac hypertrophy. Ethnic differences in the distribution of CYP11B2 genotypes combined with differences in salt intake might account for inconsistencies between previous reports.

Effect of fasting on PPARγ and AMPK activity in adipocytes

We investigated the effects of fasting on gene expression and intracellular signals regulating energy metabolism in adipose tissue. Following fasting for 15h or 39h, epididymal fat pads were isolated from Wistar rats. PPARgamma mRNA levels decreased in the adipose tissues isolated from rats fasted for 39h, whereas adipocyte lipid-binding protein (aP2) and lipoprotein lipase (LPL) mRNA levels increased. Overnight fasting increased the AMP/ATP ratio and AMP-activated protein kinase (AMPK) in adipose tissue, but not in muscle or liver tissue. In addition, the effect of 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) on PPARgamma expression in primary cultured adipocytes was investigated. AICAR reduced PPARgamma mRNA levels but increased aP2 and LPL mRNA levels. Thus, fasting-induced AMPK activation may affect on the regulation of gene expression in adipocytes.

Analysis of the human gene encoding the kidney isozyme of 11β-hydroxysteroid dehydrogenase

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of cortisol to cortisone. This activity may be deficient in the syndrome of apparent mineralocorticoid excess (AME). 11 beta-HSD L (Type I), isolated from liver, is widely expressed and utilizes NADP+ as a cofactor. The gene for 11 beta-HSD L was found to be normal in patients of AME. A second isoform, 11 beta-HSD K (Type II), isolated from kidney, is more tissue specific in expression and utilizes NAD+ as a cofactor. The cDNA clone encoding 11 beta-HSD K was isolated from sheep kidney. The cDNA is 1.8 kb in length and encodes a protein of 404 amino acid residues with a predicted M(r) 43,953. The recombinant enzyme functions as an NAD(+)-dependent 11 beta-dehydrogenase with very high affinity for steroids, but it has no detectable reductase activity. It is 37% identical in amino acid sequence to an NAD(+)-dependent isozyme of 17 beta-hydroxysteroid dehydrogenase. It is expressed at high levels in the kidney, placenta, adrenal and at lower levels in colon, stomach, heart and skin. The human 11 beta-HSD K gene consists of five exons spread over 6 kb. The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon. The promoter for 11 beta-HSD K gene lacks a TATA box and has a high GC base content, suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescent in situ hybridization of metaphase chromosomes with a positive bacteriophage P1 genomic 11 beta-HSD K clone localized the gene to chromosome 16q22. In contrast, the 11 beta-HSD L gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. Different transcriptional start sites are utilized in kidney and placenta.