Alcohol dehydrogenase 1C*1 allele is a genetic marker for alcohol-associated cancer in heavy drinkers

Chronic alcohol consumption is associated with an increased risk for upper aerodigestive tract cancer and hepatocellular carcinoma. Increased acetaldehyde production via alcohol dehydrogenase (ADH) has been implicated in the pathogenesis. The allele ADH1C*1 of ADH1C encodes for an enzyme with a high capacity to generate acetaldehyde. So far, the association between the ADH1C*1 allele and alcohol-related cancers among heavy drinkers is controversial. ADH1C genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism in a total of 818 patients with alcohol-associated esophageal (n=123), head and neck (n=84) and hepatocellular cancer (n=86) as well as in patients with alcoholic pancreatitis (n=117), alcoholic liver cirrhosis (n=217), combined liver cirrhosis and pancreatitis (n=17) and in alcoholics without gastrointestinal organ damage (n=174). The ADH1C*1 allele and genotype ADH1C*1/1 were significantly more frequent in patients with alcohol-related cancers than that in individuals with nonmalignant alcohol-related organ damage. Using multivariate analysis, ADH1C*1 allele frequency and rate of homozygosity were significantly associated with an increased risk for alcohol-related cancers (p<0.001 in all instances). The odds ratio for genotype ADH1C*1/1 regarding the development of esophageal, hepatocellular and head and neck cancer were 2.93 (CI, 1.84-4.67), 3.56 (CI, 1.33-9.53) and 2.2 (CI, 1.11-4.36), respectively. The data identify genotype ADH1C*1/1 as an independent risk factor for the development of alcohol-associated tumors among heavy drinkers, indicating a genetic predisposition of individuals carrying this genotype.

Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.

Lactate, not glucose, up-regulates mitochondrial oxygen consumption both in sham and lateral fluid percussed rat brains

OBJECTIVE: Failure of energy metabolism after traumatic brain injury may be a major factor limiting outcome. Although glucose is the primary metabolic substrate in the healthy brain, the well documented surge in tissue lactate after traumatic brain injury suggests that lactate may provide an energy need that cannot be met by glucose. We hypothesized, therefore, that administration of lactate or the combination of lactate and supraphysiological oxygen may improve mitochondrial oxidative respiration in the brain after rat fluid percussion injury. We measured oxygen consumption (VO2) to determine what effects glucose, lactate, oxygen, and the combination of lactate and oxygen have on mitochondrial respiration in both injured and uninjured rat brain tissue. METHODS: Anesthetized Sprague-Dawley rats were intubated and ventilated with either 0.21 or 1.0 fraction of inspired oxygen (FIO2). Brain tissue from acute sham animals was subjected in vitro to 1.1 mM, 12 mM and 100 mM concentrations of glucose and L-lactate. In another group, injury (fluid percussion injury of 2.5 +/- 0.02 atmospheres) was induced over the left hemisphere. The VO2 of mug amounts of brain tissues were measured in a microrespirometry system (Cartesian diver). RESULTS: The VO2 was found to be independent of glucose concentrations, but dose-dependent for lactate. Moreover, the lactate dependent VO2s were all significantly higher than those generated by glucose. Injured rats on FIO2 0.21 had brain tissue VO2 rates that were significantly lower than those of shams or preinjury levels. In injured rats treated with FIO2 1.0, the reduction in VO2 levels was prevented. Injured rats that received an intravenous infusion of 100 mM lactate had VO2 rates that were significantly higher than those obtained with FIO2 1.0. Combined treatment further boosted the lactate generated VO2 rates by approximately 15%. CONCLUSION: Glucose sustains mitochondrial respiration at a low level "fixed" rate because, despite increasing its concentration nearly 100-fold, it cannot up-regulate VO2 after fluid percussion injury. Lactate produces a dose-dependent VO2 response, possibly enabling mitochondria to meet the increased energy needs of the injured brain.

Frequency of Malaria and Glucose-6-phosphate Dehydrogenase Deficiency in Tajikistan

Background During the Soviet era, malaria was close to eradication in Tajikistan. Since the early 1990s, the disease has been on the rise and has become endemic in large areas of southern and western Tajikistan. The standard national treatment for Plasmodium vivax is based on primaquine. This entails the risk of severe haemolysis for patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Seasonal and geographical distribution patterns as well as G6PD deficiency frequency were analysed with a view to improve understanding of the current malaria situation in Tajikistan. Methods Spatial and seasonal distribution was analysed, applying a risk model that included key environmental factors such as temperature and the availability of mosquito breeding sites. The frequency of G6PD deficiency was studied at the health service level, including a cross-sectional sample of 382 adult men. Results Analysis revealed high rates of malaria transmission in most districts of the southern province of Khatlon, as well as in some zones in the northern province of Sughd. Three categories of risk areas were identified: (i) zones at relatively high malaria risk with high current incidence rates, where malaria control and prevention measures should be taken at all stages of the transmission cycle; (ii) zones at relatively high malaria risk with low current incidence rates, where malaria prevention measures are recommended; and (iii) zones at intermediate or low malaria risk with low current incidence rates where no particular measures appear necessary. The average prevalence of G6PD deficiency was 2.1% with apparent differences between ethnic groups and geographical regions. Conclusion The study clearly indicates that malaria is a serious health issue in specific regions of Tajikistan. Transmission is mainly determined by temperature. Consequently, locations at lower altitude are more malaria-prone. G6PD deficiency frequency is too moderate to require fundamental changes in standard national treatment of cases of P. vivax.

Impaired 11 beta-hydroxysteroid dehydrogenase contributes to renal sodium avidity in cirrhosis: hypothesis or fact?

Exaggerated renal sodium retention with concomitant potassium loss is a hallmark of cirrhosis and contributes to the accumulation of fluid as ascites, pleural effusion, or edema. This apparent mineralocorticoid effect is only partially explained by increased aldosterone concentrations. I present evidence supporting the hypothesis that cortisol confers mineralocorticoid action in cirrhosis. The underlying molecular pathology for this mineralocorticoid receptor (MR) activation by cortisol is a reduced activity of the 11 beta-hydroxysteroid dehydrogenase type 2, an enzyme protecting the MR from promiscuous activation by cortisol in healthy mammalians.

Coffee inhibits the reactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1: a glucocorticoid connection in the anti-diabetic action of coffee?

Recent epidemiological studies demonstrated a beneficial effect of coffee consumption for the prevention of type 2 diabetes, however, the underlying mechanisms remained unknown. We demonstrate that coffee extract, corresponding to an Italian Espresso, inhibits recombinant and endogenous 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activity. The inhibitory component is heat-stable with considerable polarity. Coffee extract blocked 11beta-HSD1-dependent cortisol formation, prevented the subsequent nuclear translocation of the glucocorticoid receptor and abolished glucocorticoid-induced expression of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase. We suggest that at least part of the anti-diabetic effects of coffee consumption is due to inhibition of 11beta-HSD1-dependent glucocorticoid reactivation.

Why is 11beta-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11beta-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

The discovery of new 11beta-hydroxysteroid dehydrogenase type 1 inhibitors by common feature pharmacophore modeling and virtual screening

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the conversion of biologically inactive 11-ketosteroids into their active 11beta-hydroxy derivatives and vice versa. Inhibition of 11beta-HSD1 has considerable therapeutic potential for glucocorticoid-associated diseases including obesity, diabetes, wound healing, and muscle atrophy. Because inhibition of related enzymes such as 11beta-HSD2 and 17beta-HSDs causes sodium retention and hypertension or interferes with sex steroid hormone metabolism, respectively, highly selective 11beta-HSD1 inhibitors are required for successful therapy. Here, we employed the software package Catalyst to develop ligand-based multifeature pharmacophore models for 11beta-HSD1 inhibitors. Virtual screening experiments and subsequent in vitro evaluation of promising hits revealed several selective inhibitors. Efficient inhibition of recombinant human 11beta-HSD1 in intact transfected cells as well as endogenous enzyme in mouse 3T3-L1 adipocytes and C2C12 myotubes was demonstrated for compound 27, which was able to block subsequent cortisol-dependent activation of glucocorticoid receptors with only minor direct effects on the receptor itself. Our results suggest that inhibitor-based pharmacophore models for 11beta-HSD1 in combination with suitable cell-based activity assays, including such for related enzymes, can be used for the identification of selective and potent inhibitors.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity.

Readjusting the glucocorticoid balance: an opportunity for modulators of 11beta-hydroxysteroid dehydrogenase type 1 activity?

Glucocorticoids play a pivotal role in the regulation of most essential physiological processes, including energy metabolism, maintenance of electrolyte balance and blood pressure, immune-modulation and stress responses, cell proliferation and differentiation, as well as regulation of memory and cognitive functions. There are several levels at which glucocorticoid action can be modulated. On a tissue-specific level, glucocorticoid action is tightly controlled by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes. The conversion of inactive 11-ketoglucocorticoids (cortisone and 11-dehydrocorticosterone) into active 11beta-hydroxyglucocorticoids (cortisol and corticosterone) is catalyzed by 11beta-HSD1, which is expressed in many tissues and plays an important role in metabolically relevant tissues such as the liver, adipose tissue and skeletal muscles. Chronically elevated local glucocorticoid action as a result of increased 11beta-HSD1 activity rather than elevated systemic glucocorticoid levels has been associated with metabolic syndrome, which is characterized by obesity, insulin resistance, type 2 diabetes and cardiovascular complications. Recent studies indicate that compounds inhibiting 11beta-HSD1 activity ameliorate the adverse effects of excessive glucocorticoid concentrations on metabolic processes, providing promising opportunities for the development of therapeutic interventions. This review addresses recent findings relevant for the development and application of therapeutically useful compounds that modulate 11beta-HSD1 function.