Rat offspring prenatally exposed to alcohol display features of metabolic syndrome characterized by a low birth weight, catch-up growth, dyslipidemia, and insulin-resistant diabetes with increased gluconeogenesis, during adult life. Gluconeogenesis is partly regulated by cyclic AMP- and glucocorticoid-dependent mechanisms. Glucocorticoid action at the receptor level depends on its circulating concentrations and is amplified at the prereceptor level by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regenerates active glucocorticoids from inactive forms. To determine whether 11β-HSD1 is dysregulated in this rat model, we examined the expression and enzyme activity of 11β-HSD1 and its regulator enzyme hexose-6-phosphate dehydrogenase (H6PD) in the liver of postnatal day 7 (neonatal) and 3-mo-old (adult) rat offspring prenatally exposed to alcohol. Measurements of 11β-HSD1 and H6PD were also performed in the omental fat of adult rat offspring. In both neonatal and adult rats, prenatal alcohol exposure resulted in increased tissue corticosterone concentrations, increased expression, and oxoreductase activity of 11β-HSD1, and a parallel increase of H6PD expression. The data suggest that due to both transcriptional and posttranscriptional dysregulations, rats exposed to alcohol early in life have increased 11β-HSD1 activity, which may explain insulin-resistant diabetes in these animals later in life.
- prenatal exposure
several prenatal adverse factors have been implicated in the pathogenesis of chronic diseases in adulthood. The importance of these factors was first recognized by epidemiological studies describing associations between intrauterine growth restriction (IUGR) and insulin resistance, type 2 diabetes, and cardiovascular diseases later in life (6). Since the offspring exposed to such prenatal factors were small at birth, these abnormalities are considered to be a consequence of IUGR. In animal models of IUGR employing malnutrition (44), placental ischemia (53), glucocorticoid exposure (8), or diabetes (62) during pregnancy, the offspring develop insulin resistance, glucose intolerance, and obesity with aging.
Alcohol is a common component of human diet, about 7% of pregnant women are chronic alcoholics, and in some communities nearly 50% of women of reproductive age consume alcohol (40). Children of alcoholic mothers are born with multiple birth defects, mental retardation, and delayed growth collectively known as Fetal Alcohol Syndrome, and administration of alcohol to pregnant animals replicates these defects (7). Abnormalities of glucose homeostasis have also been reported in the offspring of humans (14) and animals in association with prenatal alcohol exposure (36, 63). More recently, we and others have demonstrated that alcohol consumption during pregnancy in amounts resulting in blood alcohol levels found in sober alcohol users (60) leads to IUGR and is associated with insulin resistance, hyperlipidemia, and glucose intolerance in the rat offspring (16–18, 22, 46). These rats show increased gluconeogenesis and may develop diabetes with fasting hyperglycemia during adulthood (68).
It has been suggested that adverse events during pregnancy increase the activity of fetal hypothalamic-pituitary-adrenal (HPA) axis. This is present in both humans and animals born with IUGR and is considered to be a mechanism programming the fetus to insulin resistance and diabetes in later life (47). Maternal alcohol consumption during pregnancy was also reported to program fetal HPA axis, leading to elevated glucocorticoid concentrations in the offspring throughout postnatal life (32). Glucocorticoids are potent antagonists of insulin action and, when in excess, can induce a cluster of metabolic disorders manifested by obesity, insulin resistance, glucose intolerance, type 2 diabetes, and dyslipidemia (9). Glucocorticoids promote adipocyte proliferation and differentiation (25), stimulate hepatic gluconeogenesis, and reduce the ability of insulin to inhibit hepatic glucose production (48).
Recent evidence suggests that glucocorticoid action on target tissues, such as liver and adipose tissue, depends not only on circulating glucocorticoid levels and the cellular expression of their receptors, but also on their prereceptor metabolism, which is regulated by the two isoforms of the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) (29, 59). These enzymes catalyze the interconversion of biologically active glucocorticoids (cortisol and corticosterone) and their inactive 11-oxometabolites (cortisone and 11-dehydrocorticosterone). The isoform 11β-HSD1 is a NADPH-dependent enzyme that resides within the lumen of the endoplasmic reticulum (ER) and expresses highly in liver, adipose tissue, and skeletal muscle (59). This isozyme acts predominantly as an oxoreductase in vivo (29). Activation of 11β-HSD1 results in the production of excess tissue glucocorticoids and induction of local glucocorticoid-mediated alterations of insulin action, adiposity, and glucose homeostasis, all of which are associated with visceral obesity and type 2 diabetes (34, 37). In contrast, reduction of 11β-HSD1 expression prevents regeneration of active glucocorticoids, attenuates hepatic and adipose tissue glucocorticoid action, and increases insulin sensitivity in humans and laboratory animals (2, 26, 30). These studies point to the importance of intrahepatic and intra-adipose 11β-HSD1-mediated regeneration of active glucocorticoids in the pathogenesis of type 2 diabetes.
Recent reports suggest that the oxoreductase activity of 11β-HSD1 is stimulated by hexose-6-phosphate dehydrogenase (H6PD) located in the lumen of the ER, where it regenerates the NADPH required for 11β-HSD1 reductase activity (1, 4, 5, 12). H6PD regenerates NADPH from NADP by catalyzing the conversion of glucose-6-phosphate to 6-phosphogluconate, corresponding to the first two steps of the pentose phosphate pathway (11). H6PD is distinct from the cytosolic glucose-6-phosphate dehydrogenase (G6PDH), as it is capable of acting on several phosphohexoses (20).
The purpose of the present study was to investigate whether prenatal exposure of rats to alcohol augments the expression and activity of 11β-HSD1 and H6PD in liver and adipose tissue, which could contribute to the pathogenesis of insulin resistance and diabetes observed in these animals.
MATERIALS AND METHODS
Ethyl alcohol was obtained from the pharmaceutical services of Health Sciences Centre (Winnipeg, MB, Canada). Electrophoresis and electroblotting consumables were from Bio-Rad (Hercules, CA). Corticosterone enzyme-linked immunoassay (EIA) strip plate kit and 11β-HSD1 polyclonal antibody were obtained from Cayman (Ann Arbor, MI). Anti-cytochrome enzyme cytochrome P-450–2E1 (CYP2E1) was from Chemicon International (Temecula, CA). Other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Penzberg, Germany). Enhanced chemiluminescence (ECL) kit was obtained from Amersham Pharmacia (Piscataway, NJ). Trizol, oligo(deoxythymidine) primers, SuperScript reverse transcriptase, and cDNA primers were obtained from Invitrogen (Carlsbad, CA). G6PDH and H6PD primers were designed using ABIprism software. Standard RNA (GeneAmplimer pAW109 RNA) and corresponding primers (primer 1 DM151 and primer 2 DM152) and SYBR Green PCR master mix were obtained from Applied Biosystems (Foster City, CA). All other chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada).
Rat offspring exposed to alcohol in utero were generated as described before (16, 17). Briefly, timed-pregnant Sprague-Dawley rats were randomly divided into two weight-matched groups. Throughout the gestation period, one group was given 2 g/kg alcohol (36%) by gavage twice daily from days 1 to 22 of gestation, while the other group was given equivalent volumes of tap water. With this method, we have obtained a peak alcoholemia of 115 mg/dl and 70 mg/dl at 2 and 4 h after ingestion, respectively (16), similar to levels found in sober alcohol users (60). The characteristics of the animal model have been reported elsewhere (16, 17). Feed intake of alcohol-treated dams was <10% that of controls, but weight gain during pregnancy, litter size, and perinatal mortality were similar to controls. At 7 days of age (neonatal), male offspring from both treatment groups were fasted for 2 h and killed. Livers were rapidly excised, frozen in liquid nitrogen, and stored at −80°C. The remaining pups (n = 5–6/litter) were kept with their mothers until weaning and later housed three per cage and fed a normal chow. At 12 wk of age (adult), male offspring from each group were fasted overnight for 15 h and, while being otherwise unstressed, blood was drawn from the saphenous vein before death. Livers and omental fat pads were immediately dissected out and stored at −80°C. Aliquots of serum were stored at −20°C until analysis. Only male offspring were used because in the vast majority of studies of IUGR rats, abnormalities of glucose metabolism were found only in males (22, 57). All of the animal studies were approved by the Committee for Animal Care and Use in Research and Teaching of the University of Manitoba.
Tissue protein extraction.
Frozen liver and adipose tissue samples were homogenized on ice for 1 min with five and three volumes, respectively, of homogenizing buffer (pH 7.6) containing (in mM) 20 Tris, 250 sucrose, 0.1 EDTA, 0.5 EGTA, 2 imidazole, 1 sodium fluoride, 1.15 sodium molybdate, 1 sodium orthovanadate, 4 sodium tartrate, and 0.1% Triton X-100 and 10 μl/ml protease inhibitors cocktail. Homogenates were centrifuged at 4°C at 12,000 g for 10 min, and the supernatants were collected. Protein concentrations were measured by the Bradford assay using bovine serum albumin (BSA) as standard.
Microsomes were prepared at 4°C as described by Raucy and Lasker (49). Briefly, livers were homogenized in 100 mM Tris·HCl buffer, pH 7.4, containing 100 mM KCl, 1 mM EDTA, 1 mM PMSF, and centrifuged at 10,000 g for 30 min. The supernatants were centrifuged at 100,000 g for 90 min, and the pellets (microsomes) were resuspended in 100 mM sodium pyrophosphate buffer, pH 7.4, and centrifuged at 100,000 g for 60 min. The microsomes were resuspended in 50 mM KPO4 buffer, pH 7.4, containing 0.1 mM EDTA, 0.1 mM dithiothreitol, and 20% glycerol, and frozen at −80°C until use.
Western blot analysis.
The samples were mixed with loading buffer, proteins were denatured by boiling for 5 min, and 25 μg of total protein were electrophoretically resolved on 12% SDS-polyacrylamide gels at 100 V for 1 h and then transferred for 1 h onto a nitrocellulose membrane by using a semidry blot apparatus (Trans-Blot SD Cell; Bio-Rad). After blotting, the membranes were blocked overnight at 4°C with 5% nonfat dry milk. The membranes were then washed three times for 10 min each with Tris-buffered saline-0.1% Tween (TBS-T) and incubated at room temperature for 1 h with rabbit anti-11β-HSD1 (2 μg/ml) or anti-CYP2E1 (1:1,000) antibody diluted with TBS-T in 5% BSA. Blots were then again washed three times for 10 min each with TBS-T and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000) for 1 h and washed with TBS-T. Immune complexes were detected using ECL detection kit after exposing the blots to a Kodak X-OMAT AR (XAR-5) film for 2 min. Quantitative image analysis was performed using NIH Image software (Image J) to determine the intensity of the protein signal, which was expressed relative to the amount of β-actin used as an internal control.
Enzyme activity assays.
The assay of oxoreductase activity of 11β-HSD1 was performed by immunoassay (5, 21, 38, 56) of the corticosterone produced from 11-dehydrocorticosterone using a sensitive EIA. Briefly, 20 μl of 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM EDTA, 1 M NaCl, 40% glycerol (wt/vol) and 0.4% Triton X-100 (wt/vol) was preincubated with 10 μl of NADPH (2.4 mM) and 10 μl of 11-dehydrocorticosterone (1 mM) for 3 min at 37°C. The reaction was started by adding 60 μl of the tissue extract (0.5 to 1.0 mg protein) or microsomes (2.0 mg protein), and the tubes were incubated for 1 h at 37°C. The reaction was terminated by dipping the tubes immediately into ice, and corticosterone was determined using EIA corticosterone kit following manufacturer's instructions. Blanks were tubes where neither NADPH nor 11-dehydrocorticosterone were added. Specific activities were expressed as picograms of corticosterone formed per minute per milligram of protein.
H6PD activity was measured as described by Clarke and Mason (20) after disrupting the microsomal membranes by preincubation at 4°C with 0.2% Triton X-100 for 30 min. The enzyme incubation mixture contained 0–3 mM glucosamine-6-phosphate, 100 mM glycine-NaOH buffer (pH 10.0), 1 mM NADP, 1% BSA, and 0.5 to 1.0 mg tissue protein or 50 μg protein from microsomes in a total volume of 1 ml. Incubations were started by the addition of 100 μl tissue homogenate or microsomes to 900 μl of a mixture that contained all other components and was already equilibrated at room temperature. The increase in absorbance at 340 nm was monitored during the 5-min incubation period using a spectrophotometer (Ultrospec 2000; Pharmacia Biotech). The amount of NADPH produced was calculated using 6.22 as the millimolar extinction coefficient for NADPH. Specific activities were expressed as micromoles of NADPH formed per minute per milligram of protein.
CYP2E1 activity was determined by spectrophotometric analysis of p-nitrophenol hydroxylation onto 4-nitrocatechol (15). Briefly, 50 μg of microsomes were incubated at 37°C in an assay buffer containing 50 mM potassium phosphate, pH 7.4, with a premixture of p-nitrophenol at variable concentrations, 26 mM NADP, 66 mM glucose-6-phosphate, 66 mM MgCl, and 40 U/ml of glucose-6-phosphate dehydrogenase in a total volume of 500 μl per tube. After 30 min, 100 μl of 20% TCA were added, and the mixture was vortexed and placed on ice for 30 min, followed by 5-min centrifugation at 10,000 g. The supernatant (500 μl) was mixed with 250 μl of 2 M NaOH, and the absorbance was determined at 535 nm.
Real-time quantitative RT-PCR.
Total RNA was extracted from 5-g frozen tissue by the Trizol method, treated with DNaseI, and first-strand cDNAs were synthesized from 5 μg total RNA and serial dilutions of standard RNA using superscript reverse transcriptase and oligo(deoxythymidine) primers. For each sample, the reverse transcription product (200 ng) was amplified by real-time PCR using specific primers for rat 11β-HSD1 (sense: 5′-GGAGCCCATGTGGTATTGACTGCTGCAAGGTCG-3′, antisense: 5′-GCTTCCTACTCTGCAAGCAAGTTTGCTCTG-3′), rat G6PDH (sense: 5′-CCTCTATGTGGAGAATGAACGG-3′, antisense: 5′-TCGGCTGCGATAGACATACG-3′) and the predicted sequence for rat H6PD (sense: 5′-GGGCTATGTTCGGATCTTGTTTA-3′, antisense: 5′-GTTCCGGCACCCAGTGTCT-3′), in a final volume of 30 μl containing 10 μl SYBR Green PCR master mix. For each standard dilution, 10 μl of cDNA reverse transcript was amplified using primer 1 and primer 2. The Applied Biosystem 7500 thermocycler was used to perform the PCR, with the following cycles: denaturation at 95°C for 5 min followed by 40 cycles of 1 min at 95°C, 1 min at 65°C, and 1 min at 72°C, and a final 7 min at 72°C. A standard curve generated with cDNA obtained from serial dilutions of standard RNA allowed us to calculate the concentrations of target sequences expressed as the number of copies in 200 ng cDNA.
Serum and tissue concentrations of corticosterone.
Serum and tissue corticosterone levels were measured with the EIA kit following the manufacturer's instructions. The assay detection limit was 25 pg/ml.
Statistical analyses were conducted with SPSS software for Windows (SPSS; Chicago, IL). Differences between two groups were evaluated by unpaired Student's t-test. mRNA levels were not normally distributed and were log-transformed prior to analysis. Differences in enzyme activity at several substrate concentrations were compared by using a two-way ANOVA with group and substrate concentration as fixed factors. Data are expressed as the means ± SE. P < 0.05 was considered significant.
Serum and tissue corticosterone.
Liver tissue concentrations of corticosterone were measured in both neonatal and adult rat offspring, whereas, due to sample size limitation, serum and adipose tissue concentrations of corticosterone were measured in adult rat offspring only. In adult offspring, serum corticosterone concentrations were about 10-fold smaller than liver and adipose tissue corticosterone concentrations and were not significantly increased by prenatal alcohol exposure (Fig. 1). A ∼35% (P < 0.01) increase in corticosterone was found in the liver of neonatal offspring exposed to alcohol compared with controls. A similar increase in corticosterone was also observed in the liver of alcohol-exposed adult offspring compared with controls (P < 0.01). On the other hand, a twofold increase in corticosterone was noticed in the adipose tissue of alcohol-exposed adult offspring compared with controls.
The expression of 11β-HSD1 protein was determined by Western blot analysis in neonatal rat liver and in adult rat liver and adipose tissue (Fig. 2). The levels of 11β-HSD1 protein increased by ∼20–30% (P < 0.05) in the liver of both neonatal and adult rats exposed to alcohol in utero compared with the control groups. In adipose tissue of adult rats, prenatal alcohol increased 11β-HSD1 protein by ∼30% (P < 0.05). Adult rat liver 11β-HSD1 mRNA determined by real-time RT-PCR was also increased by alcohol exposure (Fig. 2D).
The effect of gestational alcohol exposure on the NADPH-dependent oxoreductase activity of 11β-HSD1 was studied in liver of neonatal rats and in liver and adipose tissue of adult offspring. Alcohol-exposed neonatal offspring had an approximately twofold increase in 11β-HSD1 oxoreductase activity compared with control offspring (Fig. 3). A significant increase in 11β-HSD1 activity of ∼70% (P < 0.01) in liver and ∼35% (P < 0.025) in adipose tissue was observed in alcohol-exposed adult offspring compared with controls. In both alcohol-exposed and control offspring, the 11β-HSD1 activity was about fivefold greater in adipose tissue than in liver despite similar 11β-HSD1 protein content. Similar results were obtained when 11β-HSD1 activity was measured in liver microsomes (Fig. 3D).
We measured the enzymatic activity of H6PD by using 2 mM glucosamine-6-phosphate as substrate (Fig. 4, A–C). Prenatal alcohol exposure produced a ∼40% (P < 0.001) increase in H6PD enzyme activity in liver of neonatal rat offspring. A more significant increase (∼60%, P < 0.001) in H6PD activity was observed in liver of alcohol-exposed adult offspring compared with control offspring. Studies in adipose tissue of adult offspring showed a 35% (P < 0.05) increase in H6PD activity in alcohol-exposed offspring compared with controls. Similar to 11β-HSD1 activity, the H6PD activity was about threefold greater in adipose tissue than in liver in both alcohol-exposed and control offspring. The difference between alcohol-exposed and control offspring was also confirmed using several concentrations of glucosamine-6-phosphate in liver microsomes (Fig. 4D). When the expression of H6PD in the liver of adult rats was determined by real-time qPCR, we found it to be significantly increased in alcohol-exposed rats compared with controls (Fig. 5A).
Because of a recent report that NADPH required for 11β-HSD1 activity may be provided by G6PDH (38), we determined the expression of G6PDH by real-time RT-PCR. G6PDH expression was dramatically suppressed in alcohol-exposed rats (Fig. 5B) and would unlikely explain the increased 11β-HSD1 activity in these animals.
Because H6PD requires NADP for its activity, we determined by Western blot analysis the expression of CYP2E1, which is increased in chronic alcoholism and can generate NADP. Prenatal alcohol exposure increased CYP2E1 protein by ∼30% in liver of both neonatal (P < 0.01) and adult (P < 0.05) rats. A significant increase of CYP2E1 protein (∼50%, P < 0.01) was also found in adipose tissue of alcohol-exposed adult rat offspring compared with controls. When CYP2E1 enzymatic activity was determined in the liver at several concentrations of p-nitrophenol used as substrate, there was no difference in enzyme activity per milligram protein between the two rat treatment groups (not shown).
In the present study, we found that maternal consumption of alcohol during pregnancy increases tissue corticosterone concentration, 11β-HSD1, and H6PD expression in offspring liver and omental adipose tissues. To our knowledge, this is the first report of 11β-HSD1 alterations in alcohol-exposed offspring and the first study of H6PD expression in an IUGR model.
Recent evidence suggests that glucocorticoids play a fundamental role in the development of type 2 diabetes, acting through glucocorticoid receptor, which confers tissue-specific responsiveness to these hormones. Glucocorticoids increase hepatic glucose production and promote adipocyte proliferation and differentiation (25, 48), contributing to the pathogenesis of the metabolic syndrome. Patients with Cushing's syndrome have visceral obesity, insulin resistance, diabetes, and dyslipidemia, and these manifestations are also found in genetically obese rodents, which have increased glucocorticoid production (9, 23). Adverse events during pregnancy are thought to increase the activity of the fetal HPA axis and the resulting hypercortisolism is considered to be a mechanism programming the fetus to diabetes in later life (47). Maternal alcohol consumption during pregnancy may cause programmed activation of the fetal HPA axis, leading to elevated postnatal glucocorticoid concentrations (32). Administration of dexamethasone during pregnancy induces glucose intolerance and overexpression of gluconeogenic genes in rat offspring (41). As found in other IUGR studies (41, 53), alcohol-exposed rat offspring have increased expression of the hepatic rate-limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase (19, 68). Thus, an increase in corticosterone levels in alcohol-exposed offspring could provide an explanation for anomalies of glucose homeostasis that we (16–18) and others (22, 46) have previously reported. Collectively, these studies have shown that rat offspring prenatally exposed to alcohol may be born small, but undergo a period of catch-up growth in association with insulin resistance, dyslipidemia, and hyperglycemia. In the present study, however, the contribution of the HPA axis was negligible, because serum corticosterone concentrations were not increased by prenatal alcohol exposure. Furthermore, the fact that serum corticosterone concentrations were about 10-fold smaller than tissue corticosterone concentrations suggests that tissue corticosterone was increased by local factors.
Importantly, the tissue-specific action of glucocorticoids can be enhanced via activation of these hormones by 11β-HSD1 at the prereceptor level (1, 59). This enzyme acts predominantly as an oxoreductase in vivo, leading to glucocorticoid activation, whereas it may act both as an oxoreductase and a dehydrogenase in cellular homogenates and in purified preparations (29). Kinetic studies using recombinant 11β-HSD1 indicated that the NADPH-to-NADP ratio is an important regulator of the 11β-HSD1 reaction direction (1). In this study, we found a measurable amount of 11β-HSD1 oxoreductase activity in tissue homogenates of liver and adipose tissue, indicating that NADPH is available in the ER of rat offspring. We found a good correlation between 11β-HSD1 oxoreductase activity and protein levels in both liver and adipose tissues, indicating that the enzyme predominantly displayed oxoreductase activity in these tissues. In addition, 11β-HSD1 mRNA determined in the liver was increased in rats exposed to alcohol in utero. This increased 11β-HSD1 expression could be responsible for the increased tissue levels of corticosterone, increased gluconeogenesis, hyperlipidemia and glucose intolerance, or diabetes found in the alcohol-exposed offspring (19, 68).
Increased 11β-HSD1 expression is thought to be important for the pathogenesis of the metabolic syndrome in humans. Obese individuals show elevated 11β-HSD1 activity and mRNA levels in adipose tissue. Leptin-resistant db/db mice, which are hypercortisolemic, also have elevated hepatic 11β-HSD1 in association with increased phosphoenolpyruvate carboxykinase mRNA expression and high blood glucose (34), whereas transgenic mice overexpressing 11β-HSD1 selectively in adipocytes have increased adipose levels of corticosterone and develop the metabolic syndrome with visceral obesity and insulin-resistant diabetes (37). Increased expression of 11β-HSD1, specifically in mice liver, caused fatty liver, dyslipidemia, and insulin resistance, although no alterations of gluconeogenic enzymes (45). Conversely, reduction of 11β-HSD1 expression by pharmacological inhibition or targeted gene disruption attenuated hepatic and adipose tissue glucocorticoid action and increased insulin sensitivity in humans and laboratory animals (2, 26, 30). Thus, besides their elevated circulating levels, increased prereceptor activation of glucocorticoids by 11β-HSD1 may also explain the abnormalities in glucose and lipid metabolism reported in alcohol-exposed rat offspring.
Persistent postnatal alterations of 11β-HSD1 expression have not been reported before in offspring born with IUGR. However, maternal endometrial carunclectomy in sheep stimulated 11β-HSD1 expression in fetal liver (39), whereas hypoxemia stimulated 11β-HSD1 expression in fetal lungs (24). Similarly, neonatal lambs born to nutrient-restricted ewes had elevated 11β-HSD1 mRNA expression in perirenal adipose tissue (66). In the rat fetus, maternal dexamethasone treatment increased 11β-HSD1 expression in the lungs (27) and the hippocampus (64). In addition, overfeeding of postnatal rats upregulated 11β-HSD1 mRNAs in mesenteric adipose tissue (10). These studies show that the expression of 11β-HSD1 may be altered by perinatal insults, in line with the present study.
The effects of alcohol on 11β-HSD1 are not well known. However, alcohol has been reported to inhibit the isozyme 11β-HSD2, which acts exclusively as a dehydrogenase and is found in mineralocorticoid-sensitive organs, such as the kidney, colon, and placenta (50, 61). Zhang et al. (69) reported that alcohol increases corticosterone production in rat mesenteric arteries. Although not so stated by these authors, it could be inferred that alcohol activated 11β-HSD1. In the present study, we demonstrate for the first time that alcohol exposure in utero causes an elevation of 11β-HSD1 expression and oxoreductase activity in neonatal and adult rat offspring.
The evidence that H6PD (4, 5, 12) and G6PDH (38) regulate 11β-HSD1 oxoreductase activity prompted us to measure the expression of these two enzymes. H6PD is the microsomal counterpart of the cytosolic G6PDH and has broader substrate specificity. Both enzymes utilize glucose-6-phosphate and NADP to produce NADPH. However, H6PD also accepts other hexose-6-phosphates, such as galactose-6-phosphate, deoxyglucose-6-phosphate, and glucosamine-6-phosphate (20). Studies have shown that H6PD, by increasing the ER luminal NADPH-to-NADP ratio, directs the oxoreductase activity of 11β-HSD1, and these enzymes work together in functional cooperation (4, 5, 12). Mice with a targeted inactivation of the H6PD gene are unable to convert 11-dehydrocorticosterone to corticosterone but demonstrate increased conversion of corticosterone to 11-dehydrocorticosterone, consistent with a lack of 11β-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity (31). A recent report shows that, in addition to H6PD locally forming NADPH, G6PDH also regulates 11β-HSD1 activity by providing cytosolic-derived NADPH to ER (38). In the present work, we studied H6PD activity in total cellular fractions of liver and adipose tissue by using glucosamine-6-phosphate as a substrate, an assay that detects only H6PD activity (20). Therefore, the H6PD activity observed in our experiments was likely due solely to H6PD and not to G6PDH. Interestingly, G6PDH mRNA expression was suppressed, while H6PD expression was increased, in alcohol-exposed rats, indicating that H6PD, rather than G6PDH, was likely to promote 11β-HSD1 activity in these animals. The increase in H6PD activity in alcohol-exposed offspring by providing NADPH would increase the oxoreductase activity of 11β-HSD1.
We do not have a plausible explanation for the dissociation in the mRNA expression between G6PDH and H6PD. The activities of these enzymes tend to respond to similar physiological stimuli, being enhanced by oxidative stress, oxoglucocorticoids (20, 55), and estrogens (28, 52) but attenuated by DHEA (3, 38). However, H6PD is less affected by steroids and NADPH (43) and more affected by some hepatotoxins (65) than G6PDH, while G6PDH may be inhibited by oxidants and alcohol (42, 67).
The source of NADP required for H6PD activity in alcohol-exposed rats is currently controversial. Some investigators believe that NADP cannot cross the ER-membrane and must be generated inside the ER lumen (5, 58), while others believe that NADP may enter the ER from the cytosol (38, 51). A cytosolic source of NADP associated with chronic alcohol exposure is CYP2E1 (33). We found increased expression of CYP2E1 in liver and adipose tissues of alcohol-exposed rat offspring, which is reminiscent of increased CYP2E1 expression found in chronic alcoholics. However, CYP2E1 enzymatic activity per unit protein was not increased, consistent with the notion that the activity of cytochromes is determined by the protein level (54).
In this study, the enzymatic activity of 11β-HSD1 and H6PD appeared to be greater in adipose tissue than in liver. Although this may be at odds with reports showing greater expression of 11β-HSD1 in liver compared with adipose tissue (35), these studies used the 11β-HSD1 dehydrogenase assay, whereas we assayed the physiologically predominant reductase enzyme. Reductase assays seem to discriminate better between physiological events, such as tissue differentiation or sexual dimorphism in 11β-HSD1 activity in rodents. Bujalska et al. (13) used both the 11β-HSD1 dehydrogenase and reductase assays in adipocytes and found an increase of 11β-HSD1 reductase, but not dehydrogenase, after stimulation with glucocorticoids and insulin and subsequent differentiation. Similarly, Jamieson et al. (29) used both the 11β-HSD1 dehydrogenase and reductase assays in perfused rat liver and found decreased 11β-HSD1 reductase activity but increased dehydrogenase activity in female rats compared with males.
In conclusion, in utero alcohol exposure augments 11β-HSD1 expression in liver and omental fat during the neonatal period and adulthood, thereby catalyzing local amplification of active glucocorticoids. The elevated H6PD expression may further contribute to the enhanced oxoreductase activity of 11β-HSD1 by supplying its cofactor NADPH. These changes may lead to enhanced gluconeogenesis in rat offspring exposed to alcohol during pregnancy.
This work was supported by a grant from the Canadian Diabetes Association (to B. L. G. Nyomba).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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