Liver International ISSN 1478-3223 BASIC STUDIES Overexpression of 11b-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue and portal hypercortisolism in non-alcoholic fatty liver disease Roberto Candia1, Arnoldo Riquelme1, Rene Baudrand2, Cristian A. Carvajal2, Mauricio Morales2, Nancy Solı́s1, Margarita Pizarro1, Alex Escalona3, Gonzalo Carrasco5, Camilo Boza3, Gustavo Pérez3, Oslando Padilla4, Jaime Cerda4, Carlos E. Fardella2 and Marco Arrese1 1 2 3 4 5 Department of Gastroenterology, Pontificia Universidad Católica de Chile, Santiago, Chile Department of Endocrinology, Pontificia Universidad Católica de Chile, Santiago, Chile Department of Digestive Surgery, Pontificia Universidad Católica de Chile, Santiago, Chile Department of Public Health, Pontificia Universidad Católica de Chile, Santiago, Chile Pathology Department, Hospital de San Bernardo, San Bernardo, Chile Keywords 11beta-HSD1 – fatty liver – glucocorticoids – liver steatosis – non-alcoholic Abbreviations 11b-HSD1, 11b-hydroxysteroid dehydrogenase type 1; ALT, alanine aminotrasferases; AST, aspartate aminotrasferases; ATP III, adult treatment panel III; BMI, body mass index; CI, confidence interval; EAT, epididymal adipose tissue; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; hs-CRP, high-sensitivity C-reactive protein; LDL, lowdensity lipoprotein; MetS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; OR, odds ratio; ROC, receiver operating characteristics; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. Correspondence Marco Arrese MD, Department of Gastroenterology, Pontificia Universidad, Católica de Chile, Marcoleta #367833-0024, Santiago, Chile Tel: 56-2-6397780 Fax: 56-2-6397780 e-mail: marrese@med.puc.cl Abstract Background: The enzyme 11b-hydroxysteroid-dehydrogenase type 1 (11bHSD1) catalyses the reactivation of intracellular cortisol. We explored the potential role of 11b-HSD1 overexpression in visceral adipose tissue (VAT) in non-alcoholic fatty liver disease (NAFLD) assessing sequential changes of enzyme expression, in hepatic and adipose tissue, and the occurrence of portal hypercortisolism in obese mice. 11b-HSD1 expression was also assessed in tissues from obese patients undergoing bariatric surgery. Methods: Peripheral and portal corticosterone levels and liver histology were assessed in ob/ob mice at two time points (8–12 weeks of age). 11b-HSD1 tissue expression was assessed in by RT-pcr in ob/ob mice and in 49 morbidly obese patients. Results: Portal corticosterone serum levels were higher in obese mice with a 26% decrease between 8 and 12 weeks of age (controls: 78.3 ± 19.7 ng/ml, 8-week-old ob/ob: 167.5 ± 14.5 ng/ml and 12-week-old ob/ob: 124.3 ± 28 ng/ml, P < 0.05). No significant differences were found in peripheral corticosterone serum levels. Expression of 11b-HSD1 was lower in the liver [–45% at 8 weeks and –35% at 12-weeks (P = 0.0001)] and highly overexpressed in VAT in obese mice, compared to controls (128fold higher in 8-week-old ob/ob and 41-fold higher in 12-week-old ob/ob, P < 0.01). No significant differences were seen in the expression of 11b-HSD1 in subcutaneous adipose tissue. In multivariate analysis, human 11b-HSD1 expression in VAT (OR: 1.385 ± 1.010–1.910) was associated with NAFLD. Conclusion: Murine NAFLD is associated with portal hypercortisolism and11b-HSD1 overexpression in VAT. In humans, 11b-HSD1 VAT expression was associated with the presence of NAFLD. Thus, local corticosteroid production in VAT may contribute to NAFLD pathogenesis. Received 9 August 2011 Accepted 16 October 2011 DOI:10.1111/j.1478-3231.2011.02685.x Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disorders characterized by intrahepatic fat accumulation accompanied by varying degrees of hepatic necroinflammation and fibrosis that may evolve to cirrhosis in a proportion of affected individuals thus conveying the risk of developing end-stage liver disease and hepatocellular carcinoma (1, 2). NAFLD is Liver International (2011) © 2011 John Wiley & Sons A/S now considered the most common liver disease worldwide with estimated prevalence figures between 20 and 30% of the general population (3). Moreover, it has also been recognized that NAFLD is an independent risk factor for cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) stressing the fact that NAFLD is part of a systemic metabolic imbalance that 1 Candia et al. 11beta-HSD1 and NAFLD involves cardiovascular, metabolic and liver-related risks (4, 5). The pathophysiology of NAFLD is complex and not entirely elucidated (6). However, it is widely accepted that insulin resistance (IR) has a central role in disease development and progression (7, 8). Reduced wholebody insulin sensitivity and evidence of IR at the level of muscle, white adipose tissue and liver has been found in patients with NAFLD although it is not known which is the primary site of IR. Current views on NAFLD pathogenesis (6) considers that impaired peripheral insulin action leads to an uninhibited adipose tissue lipolysis resulting in an increased flux of fatty acids (FA) to the liver and to a compensatory hyperinsulinemia which in turn determines an enhanced de novo hepatic lipogenesis resulting in triglyceride accumulation in the liver. The latter itself determines metabolic disturbances resulting in hepatic insulin resistance and a pro-inflammatory and pro-thrombotic state (9, 10). Epidemiological data indicate that hepatic steatosis is associated with IR, dyslipidemia and obesity, especially central obesity (11). In clinical practice, the presence of these conditions define the so-called metabolic syndrome (MS), a medical condition with clustering of risk factors for cardiovascular disease and T2DM (12). Of note, NAFLD is considered by many authors as the hepatic manifestation of MS (13, 14). Interestingly, the severity of fatty liver is positively correlated with visceral adipose tissue (VAT) accumulation in both obese and non-obese subjects, suggesting that hepatic fat infiltration may be influenced by visceral fat adipokines or enzymes, regardless of body mass index (BMI) (15). Several authors have pointed to the phenotypic similarities between central obesity, MS and patients with endogenous or exogenous glucocorticoid excess. This has led them to propose that cortisol contributes, at least in part to pathogenesis of those abnormalities, despite the fact that patients with obesity and MS have consistently normal cortisol in plasma and urine (16–18). A plausible explanation for this phenomenon is to consider the MS a result of increased local glucocorticoid activity in certain organs suggesting that central obesity might be, as proposed by Bujalska decades ago, a ‘Cushing’s disease of the omentum’ (19). In connection with this concept, recent studies have showed that intracellular glucocorticoid action not only depends upon hypothalamo-pituitary-adrenal axis but also by local regulation at the pre-receptor level by the activity of two isoforms of the 11b-hydroxysteroid dehydrogenase enzyme type 1 and 2 (11b-HSD1 and 11b-HSD2) (20, 21). Enzyme type 1, 11b-HSD1, is a microsomal enzyme, expressed mainly in liver and adipose tissue, which acts mainly as a NADP (H)-dependent reductase converting inactive cortisone to active cortisol which locally activates glucocorticoid receptors (22). According to this view, progressive expansion of visceral fat would result in an increased production of cortisol by the action of 11b-HSD1, causing splanchnic and portal hypercortiso- 2 lism that could be a key in the pathogenesis of metabolic disorders, including NAFLD (23–25). Recently, our group demonstrated that 11b-HSD1 expression levels in liver and VAT, in morbidly obese patients, correlates with dyslipidemia and insulin resistance, suggesting that this enzyme might have a pathogenic role in obesity and related metabolic disorders (26). Since available data on 11b-HSD1 in NAFLD are scarce (23, 24), in the current study we explored the potential role of 11b-HSD1 overexpression in visceral adipose tissue (VAT) in non-alcoholic fatty liver disease (NAFLD) assessing sequential changes of enzyme expression, in hepatic and adipose tissue, and the occurrence of portal hypercortisolism in obese mice, an accepted model of hepatic steatosis (27). The 11b-HSD1 expression was also assessed in tissues from obese patients undergoing bariatric surgery exploring their correlations with clinical, anthropometric and biochemical values. Methods Animals Male ob/ob C57BL/6J mice (B6.V-Lepob/J) and agematched lean C57BL/6J mice were obtained at the age of 4 weeks from Jackson Laboratories (Bal Harbor, ME, USA). All animals were allowed a standard laboratory diet and water ad libitum and housed in transparent polycarbonate cages subjected to 12 h light/darkness cycles under a temperature of 21°C and a relative humidity of 50%. Obese and lean animals were intraperitoneally anesthetized with a dose of sodium pentobarbital (50 mg/kg body weight) at 8 or 12 weeks of age respectively. Blood samples from systemic circulation and portal system were obtained by puncture of the retro-orbital sinus with a glass capillary tube and portal vein at the moment of euthanasia respectively. Serum and tissue samples, including liver and adipose tissue were obtained at the same time in the morning and stored at –80°C until analysed. Animal experiments were approved by the local ethics review committee. Patients Morbidly obese patients undergoing bariatric surgery were prospectively recruited in our institution from January 2004 to February 2008. Only morbidly obese patients with no endocrine or genetic disease causing their obesity were enrolled. This study was approved by the Institutional Review Board Ethics Committee for Human Studies of the Pontificia Universidad Católica de Chile and informed consent was obtained from all participants. The screening protocol included a pre-coded questionnaire with socioeconomic data, medical history including previous known diagnosis of hypertension, type 2 diabetes, liver diseases, a detailed history of current alcohol consumption and estimation of daily alcohol intake in grams per day. The questionnaire included Liver International (2011) © 2011 John Wiley & Sons A/S Candia et al. a complete record of concomitant medications. Patients with known alcohol consumption over 20 g per day, based on the questionnaire previously described, and those with chronic hepatic disease of a known origin (chronic viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, hemochromatosis, Wilson’s disease, a-1 antitrypsindeficiency-associated liver disease) were excluded. Biochemical and clinical measurements Serum cholesterol and triglycerides were measured using kits from Human Gesselheit (Wiesbaden, Germany) and serum alanine aminotransferase (ALT) was quantified with Kovalent kit (Rı́o de Janeiro, Brazil). Serum corticosterone, the murine equivalent of cortisol, was measured using an Enzyme Immunoassay (EIA kit; Cayman, Ann Arbor, MI, USA) following the manufacturer’s instructions. Blood samples from each patient were obtained. Serum fasting glucose, serum aminotransferase levels (ALT, AST) and serum lipid profile measurements were performed in an automat zed Roche tm Hitachi Modular chemistry analyzer (Tokyo, Japan). Serum adiponectin was determined by ELISA technique (R&D Systems, Minneapolis, MN, USA). Hepatitis C virus (HCV) antibodies were detected by a third-generation immunoassay test, using the MEIA (Microparticle Enzyme Immunoassay) technique on the Abbott AxSYM tm (Abbott Park, IL, USA). Insulin serum level was measured with the Immulite 2000 equipment with DPC reactive (Diagnostics Product Corporation, Los Angeles, CA, USA). Insulin resistance was determined by the homeostasis model assessment-insulin resistance (HOMA-IR) method which has a good correlation with the clamp method to determine total glucose disposal and to assess insulin sensitivity (28). The HOMA-IR was calculated according to the formula: insulin (lU/ml) 9 fasting plasma glucose (mmol/L)/22.5. In Chilean population, a HOMA-IR value > 2.6 is indicative of insulin resistance in non-diabetic subjects according to Acosta et al. (29). Patients were classified as having MS if they had at least three of the following variables, according to modified NCEP-ATP III criteria: (a) waist circumference  40 inches (102 cm, men) or  35 inches (88 cm, women), (b) HDL cholesterol  1.03 mM (40 mg/dl, men) or  1.3 mM (50 mg/dl, women) or taking medication for reduced HDL cholesterol, (c) triglycerides  1.7 mM (150 mg/dl) or taking medication for elevated triglycerides, (d) systolic blood pressure (BP)  130 mmHg or diastolic BP  85 mmHg or taking antihypertensive medication and (e) fasting glucose  5.6 mM (100 mg/dl) or taking medication for hyperglycaemia (30). Abnormal aminotransferase levels were defined as ALT > 30 IU/L in men and 19 IU/L in women (31). All patients met the inclusion criteria for obesity surgery corresponding to a BMI  40 kg/m2 or a BMI  35 kg/m2 with significant co-morbid Liver International (2011) © 2011 John Wiley & Sons A/S 11beta-HSD1 and NAFLD conditions such as arterial hypertension, T2DM, sleep apnoea or dyslipidemia (32). Tissue samples Initially, and to establish the time course of fatty liver development in the obese mice, separate groups of mice (n = 2–3) were sacrificed a different time points (4-7-8 and 12 weeks of age). Then samples of liver, subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT) and visceral adipose tissue (VAT) were obtained from C57BL6 mice and ob/ob at 8 and 12 weeks of age (n = 3–5 per group). VAT was obtained from mesenteric adipose tissue (mesenteric root). Serum and tissue samples were frozen in liquid nitrogen and stored at –80°C. Human samples included the following: concomitant biopsies of liver, SAT, and VAT obtained from patients undergoing bariatric surgery. Liver samples were obtained by intra-operative biopsy, VAT samples were obtained from greater omental fat and SAT samples were obtained by biopsy from the abdominal port site insertion. Approximately, 400–500 mg of each fat tissue depot and 50 mg of liver tissue were retrieved and immediately stored with RNAlater® (Ambion, Austin, TX, USA), frozen in liquid nitrogen and stored at –80°C until further analysis. Liver biopsies were examined by a single pathologist (G.C.) and NAFLD was assessed following the recommendations developed by Kleiner et al. Total RNA isolation and quantification In the experimental model, RNA was isolated from liver, SAT, EAT and VAT samples obtained from ob/ob and C57BL6 male mice using SV Total RNA Isolation System (Promega, Madison, WI, USA). RNA integrity was assessed by electrophoresis on 1% (w/v) agarose gels, and quantity was determined spectrophotometrically in a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). cDNA synthesis was performed with one microgram of total RNA, then it was reverse transcribed in 25 ll total volume (Improm II system, Promega, Madison, WI, USA) and 150 pmol random hexamers according to the manufacturer’s guidelines. The reaction was terminated by heating the cDNA to 70°C for 5 min and stored at –80°C until required. A similar process was used for RNA isolation and quantification in liver, VAT and SAT samples from morbidly obese patients. 11b-HSD1 gene expression analysis The cDNA extracted from hepatic, SAT, EAT and VAT samples of ob/ob and C57BL6 mice were amplified by real time PCR, with gene-specific 11b-HSD1 primers obtained from IDT (Coralville, ID, USA) (sense primer: 5′-AGCCCATGTGGTATTGACTG-3′, antisense 3 11beta-HSD1 and NAFLD primer: 5′-ATGTCTTCCATAGTGCCAGC-3′), using Maxima SYBR Green/ROX qPCR Master Mix Kit (Fermentas, Hanover, MD, USA) in a 72-well disc Rotor Gene 6000 real-time Termocycler (Corbett, Australia). In human samples, a similar process for cDNA amplification was used with the 11b-HSD1 gene-specific primers and probe (11b-HSD1 sense primer 5′-AGGAAAG CTCATGGGAGGACTAG-3′, 11b-HSD1 antisense primer 5′-ATGGTGAATATCATCATGAAAAAGATTC-3′, and 11b-HSD1 probe 5′-6FAM-CATGCTCATTCTCA ACCACATCACCAACA-TAMRA-3′) and standardized against 18S RNA (18S sense primer 5′-AGGGAAT TCCCGAGTAAGTGC-3′, 18S antisense primer 5′-GCC TCACTAAACCATCCAATC-3′ and 18S probe 5′-JOECATAAGCTTGCGTTGATTAAGTCCCTGC-TAMRA-3′)35. Results were expressed as arbitrary units (AU) and normalized against 18S RNA expression. Statistical analyses All continuous variables are presented as mean ± standard deviation (SD) and were compared using the non-paired Student’s t-test for independent variables. Variables with non-parametric distribution were compared using the Kruskal–Wallis or Mann–Whitney non-parametric tests. Discrete variables are presented as percentages and were analysed using the chi-square test for categorical data. Analysis of liver biopsy findings was carried out in three different histological categories (i.e. Normal histology, NAFLD and NASH). Univariate and multivariate analysis were performed comparing morbidly obese patients with or without NAFLD and a similar approach was carried out comparing morbidly obese patients with or without liver fibrosis. To identify independent variables associated with NAFLD or liver fibrosis, a stepwise procedure for a multivariate logistic regression analysis was carried out which included variables that appeared significant in univariate analysis and 11b-HSD1 tissue expression. Highly correlated variables were excluded to avoid multicollinearity (i.e. insulin and HOMA-IR). All statistical analyses were performed with Microsoft excel (v.2007) or SPSS version 15.0 (standard version, SPSS Inc., Chicago, IL, USA) software. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Significant difference was set at P value < 0.05. Candia et al. ALT and total cholesterol serum levels compared with those in controls (Table 1). No differences in serum triglycerides were observed. Sequential assessment of 11bHSD1 expression in both liver and adipose tissue at both 8 and 12 weeks of age showed that both ob/ob mice and controls have a higher hepatic expression of 11b-HSD1 compared with adipose tissue expression (Table 1) and that obesity is associated to a significant decrease of 11b-HSD1 hepatic expression over the time [–45% at 8 weeks and –35% at 12-weeks (P = 0.0001)] and a marked increase in 11b-HSD1 VAT expression (128-fold higher in 8-week-old ob/ob and 41-fold higher in 12-week-old ob/ob, P < 0.01) (Fig. 2). There were no significant differences in 11b-HSD1 SAT and EAT expression in ob/ob mice compared with controls although a significant decrease in EAT expression was seen in 12-week-old ob/ob. Measurement of portal corticosterone serum levels in control C57Bl6 mice and in ob/ob mice at both 8 and 12 weeks of age showed that hormone levels were significantly higher in obese animals at two time points although 12-week-old ob/ob displayed significantly lower levels compared to 8-week-old ob/ob mice. This is in agreement with the observed reduction in 11b-HSD1 VAT expression in 12-week-old ob/ob compared with younger animals. Corticosterone concentrations in peripheral circulation showed no differences in both groups (Fig. 3). Clinical and histopathological features in obese patients Forty-nine patients were prospectively included, 35 of them were women (71.4%), with a mean age of 42.2 years (range 25–64 years), and a mean BMI 41.9 ± 6 kg/m2. Forty per cent of them had arterial hypertension, 73.5% insulin resistance based on Results Hepatic steatosis development, 11b-HSD1 tissue expression and portal and peripheral serum corticosterone levels in ob/ob mice Obese mice exhibited liver steatosis starting at 8 weeks of age with progressive worsening afterwards. At 12 weeks of age a significant macrovesicular steatosis was present in all obese mice (Fig. 1). This was associated with increased body and liver weight and higher 4 Fig. 1. Representative photomicrographs showing liver steatosis development in ob/ob mice. Hematoxylin-eosin stain, magnification 920. Lipidic macrovacuoles are visible since 8 weeks of age. Worsening of steatosis is appreciated at week 12 of life. Liver International (2011) © 2011 John Wiley & Sons A/S Candia et al. 11beta-HSD1 and NAFLD Table 1. Weight, serum parameters and 11b-HSD1 tissue expression in control and ob/ob mice Weight (g) Liver weight (g) ALT (IU/L) Triglycerides (mg/dl) Total cholesterol (mg/dl) 11b-HSD1 (liver expression) (AU) 11b-HSD1 (SAT expression) (AU) 11b-HSD1 (EAT expression) (AU) 11b-HSD1 (VAT expression) (AU) Control (C57BL6) 8 week-old ob/ob 12 week-old ob/ob 21.2 0.9 46.3 63.6 60.5 2232 122 169 2.55 37.7 1.9 157.1 52.4 123.6 1105 302 262 342 46.8 2.7 113 58.2 128.4 1222 332 128 103 ± ± ± ± ± ± ± ± ± 2.6 0.1 14.1 13.2 11.3 426‡ 1 51 0.22 ± ± ± ± ± ± ± ± ± 2.9* 0.1* 116* 8.5 9.24* 206‡ 77 30 84 ± ± ± ± ± ± ± ± ± 1.4† 0.2† 38.8 8.6 42.8 355‡ 87 14 41 Data are expressed as mean ± standard deviation. *P < 0.05 compared with controls. †P < 0.05 compared with 8 week-old ob/ob mice. ‡P < 0.05 compared with adipose tissue expression (SAT, EAT and VAT). ALT, alanine aminotrasferases; 11b-HSD1, 11 b-hydroxysteroid dehydrogenase type 1; EAT, epididymal adipose tissue; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. HOMA-IR> 2.6, 16.3% type 2 diabetes mellitus, 57.1% dyslipidemia and 63.3% met criteria for MS. Histological analysis showed that 25 (51%) of the patients had NAFLD and 19 (39%) had fibrosis, according to Kleiner classification. None of them had cirrhosis (Table 2). Human tissue expression of 11b-HSD1 Fig. 2. Relative 11b-hydroxysteroid dehydrogenase type 1 (11bHSD1) tissue expression in control C57BL6 mice and 8 and 12 week-old obese mice. Enzyme expression was determined by RT-pcr in liver tissue, subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT) and visceral adipose tissue (VAT). Data represent mean ± SD, *P < 0.05. In morbidly obese patients, 11b-HSD1 hepatic mRNA levels were significantly higher (31.03 ± 16.4 AU, range 2.6–90.4) than those seen in VAT (1.36 ± 1.09 AU, range 0.2–4.6, P < 0.001) and SAT (5.24 ± 4.89 AU, range 0.8–23.9, P < 0.001), with no gender-related differences. Variables associated with non-alcoholic fatty liver disease. In univariate analysis, eight variables were directly associated with NAFLD: Male gender, arterial hypertension (OR: 5.7, 95% CI 1.4–25.1), MS (OR: 12.2, 95% CI 2.4–70.4), BMI, AST, ALT, serum glucose and HOMAIR (Table 3). Serum adiponectin was inversely associated with NAFLD (Table 3). In multivariate analysis, Table 2. Liver histological features of patients undergoing bariatric surgery Fig. 3. Portal and peripheral serum corticosterone levels in control C57BL6 and 8 and 12 week-old obese mice. Serum levels of Corticosterone (ng/ml) were determined by a commercially available ELISA assay. Data represent mean ± SD, *P < 0.05. Liver International (2011) © 2011 John Wiley & Sons A/S Grading for NASH Normal Steatosis NASH NASH with fibrosis Fibrosis Stage 1 Stage 2 Stage 3 Stage 4 N (%) 24 25 20 19 49 51 41 39 15 2 2 0 78 11 11 0 NASH, non alcoholic steatohepatitis. 5 Candia et al. 11beta-HSD1 and NAFLD Table 3. Univariate analysis comparing morbidly obese patients with NAFLD vs. patients with normal liver histology Variables NAFLD (n = 25) Normal (n = 24) P value Gender (men/women)§ Age (years)† Arterial hypertension§ BMI (kg/m2)†,§ Diabetes mellitus Metabolic syndrome (MS)§ Glucose (mg/dl)†,§ Total cholesterol (mg/dl)† HDL cholesterol (mg/dl)† LDL cholesterol (mg/dl)† Triglycerides (mg/dl)† AST (IU/L)†,§ ALT (IU/L)†,§ HOMA-IR b, § Adiponectin (lg/ml)†, § 11b-HSD1 liver expression (AU)‡,§ 11b-HSD1 VAT expression (AU)‡,§ 11b-HSD1 SAT expression (AU)‡, § 13/12 43.76 ± 10.91 15 (60%) 43.9 ± 7.25 5 (20%) 22 (88%) 106.04 ± 22.28 193.96 ± 44.91 48 ± 13.73 124.13 ± 41.49 134.24 ± 56.17 37.74 ± 20.77 52.66 ± 31.77 5.4 (2.25–8.55) 3.4 ± 1.8 47 (5.4–107.5) 2.25 (0.3–8.35) 11.65 (1.4–26.4) 1/23 40.62 ± 9.19 5 (20.8%) 39.6 ± 4.05 3 (12.5%) 9 (37.5%) 89.5 ± 13.54 208.62 ± 37.13 50.86 ± 11.74 132.21 ± 38.22 133.29 ± 53.01 24.55 ± 10.1 35.02 ± 13.15 2.8 (1.9–4.4) 4.8 ± 2.1 33.4 (4.9–61.9) 1.2 (0.4–3.3) 7.16 (0.5–20.7) < 0.001* 0.284 0.009* 0.018* 0.702 <0.001* 0.003* 0.220 0.488 0.542 0.952 0.003* 0.016* 0.006* 0.012 0.224 0.109 0.285 †Mean ± SD. ‡Median (Q1–Q3). §Selected for Multivariate analysis. *P < 0.05 was considered statistically significant. NAFLD, nonalcoholic fatty liver disease; BMI, body mass index; ALT & AST, alanine and aspartate aminotrasferases; HOMA, homeostasis model assessment; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MS, Metabolic Syndrome; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; 11b-HSD1, 11 b-hydroxysteroid dehydrogenase type 1. serum glucose and 11b-HSD1 VAT expression were positively associated with NAFLD. On the other hand, liver and SAT 11b-HSD1 expression were not significantly associated with NAFLD. Variables associated with liver fibrosis In univariate analysis, five variables were directly associated with liver fibrosis: Male gender, MS, AST, ALT and serum glucose. In multivariate analysis, only serum glucose was independently associated with liver fibrosis (OR: 1.085, CI 1.008–1.168). The liver, SAT and VAT 11b-HSD1 expression were not associated with liver fibrosis. Discussion Dysregulation of 11bHSD1 expression in adipose tissue seems to be relevant in the molecular aetiology of obesity and obesity-related disorders. This is underscored by data generated in different genetically engineered mice that lack or overexpress the enzyme in the whole body or in selected tissues [reviewed in (33)]. For example, the transgenic mice overexpressing 11bHSD1 selectively in adipose tissue had increased adipose levels of corticosterone and develop visceral obesity, insulin resistance, diabetes and hyperlipidemia. In the present study, we aimed to explore the role of corticosteroids overproduction from VAT in experimental NAFLD by examining sequential changes in the expression of 11bHSD1 and 6 portal levels of corticosterone, the main glucocorticoid in rodents, in the ob/ob mice. Our findings confirm a strong induction (> 100 times) of gene expression in VAT in obesity, with no substantial differences in the other adipose tissues evaluated, which was associated to significantly elevated portal levels of corticosterone. Both phenomena occur early in life of obese mice (8 week of age) and, somewhat unexpectedly given that metabolic derangements are progressive in ob/ob mice, tended to attenuate in older animals. Thus, both VAT 11bHSD1 overexpression and portal hypercortisolism suggest that in early obesity the liver is exposed to higher-than-normal levels of corticosteroids originated in the intra-abdominal fat compartment. This exposure might have a pathogenic role in steatosis development since glucocorticoids have significant effects on both hepatic carbohydrate and lipid metabolism mainly promoting the de novo synthesis of glucose and increasing triglyceride synthesis (34, 35). Also, recent data from Lemke et al. (36) showing that the hepatic glucocorticoid receptor action is important in hepatic steatosis argues in favour of a relevant role of glucocorticoid action in liver steatosis development. These authors have shown that disruption of glucocorticoid receptor in mice improves the steatotic phenotype in fatty liver mouse models and normalizes hepatic triglyceride levels in these animals. Of note, our observation that the hepatic expression of 11bHSD1 decrease with age and is lower in obese mice compared with that in controls likely represent a hepatic response to excessive Liver International (2011) © 2011 John Wiley & Sons A/S Candia et al. corticosteroid exposure and suggest a possible mechanism of negative feedback of the enzyme secondary to increased glucocorticoids production in the splanchnic circulation or to increased insulin levels (26, 37). The occurrence of portal hypercortisolism secondary to VAT 11b-HSD1 overexpression in obese mice with NAFLD is in agreement with previous observations on enzyme expression levels in obese rodents and have relevance beyond the liver as it has been shown that 11bHSD1 overexpression in VAT determines a myriad of metabolic abnormalities. Human studies have shown a potential pathogenic role of 11b-HSD1 and local hypercortisolism in central or intra-abdominal fat distribution (38), type 2 diabetes (39), metabolic syndrome (16), hypertriglyceridemia, low HDL (40) and hypertension as published recently by our group (41). Also, it has been described that over nutrition, sedentary lifestyle, and sleep deprivation generates an hyper-responsive hypothalamo-pituitary-adrenal axis leading to slightly but inappropriately elevated cortisol secretion (42). Recent publications reporting result of studies conducted with stable isotopes suggest that the liver and not visceral adipose tissue accounts for a substantial portion of the conversion of cortisone to cortisol, leading to some controversy about the role of splanchnic cortisol production in humans (23, 43). However, these studies measured portal cortisol in a few patients including subjects with chronic liver disease which makes it difficult to interpret the data. In relation to cortisol and NAFLD, only a few studies have analysed altered cortisol metabolism as a pathogenic factor in humans. Recently, Konopelska et al. described that total cortisol metabolite excretion was increased in patients with fatty liver or NASH compared with healthy controls (24). This study also analysed hepatic 11b-HSD1 expression and prompted us to conduct the current study. Our assessment showed that hepatic 11b-HSD1 mRNA levels were higher than the expression levels seen in adipose tissue, and consistent with our previous reports, SAT exhibited higher 11bHSD1 mRNA levels than VAT (26, 44). We found no differences in hepatic or VAT 11bHSD1 expression, when we analysed this variable in relation to the presence or absence of NAFLD, but these may be owing to the fact that in the present study we included obese patients and did not count them with a normal weight control group. However, when analysing 11bHSD1 expression and fatty liver, in a multivariate analysis, we observed that VAT 11b-HSD1 expression, but not liver or SAT expression, was positively associated with the presence of NAFLD. This argues in favour of VAT 11bHSD1 expression as being a relevant player in steatosis development in obesity although no relation was found with inflammation or fibrosis. In conclusion, our results show that murine obesity is associated with an increase in portal glucocorticoid levels and 11b-HSD1 over-expression in VAT. Moreover, in morbidly obese humans, glucose and 11b-HSD1 VAT Liver International (2011) © 2011 John Wiley & Sons A/S 11beta-HSD1 and NAFLD expression were the variables associated with NAFLD. Both findings suggest an increase in the overall production rate of glucocorticoids within the visceral adipose tissue. This local cortisol production may contribute to the pathogenesis of obesity-related metabolic disorders as NAFLD and may account for the phenotypic similarities of central obesity and Cushing’s syndrome. Furthermore studies are needed to precisely define the role of 11b-HSD1 in NAFLD development. Our laboratory is currently exploring strategies of selective manipulation of 11b-HSD1 in both liver and adipose tissues at different stages of NAFLD in obese rodents. Also, the effects of treatment with specific 11b-HSD1 inhibitors (33) in NAFLD deserve exploration as these agents have the potential to improve insulin sensitivity (45) and may ultimately add to the treatment options available for this common liver condition. Acknowledgements This work was supported by grants from FONDECYT (Fondo Nacional de Ciencia y Tecnologı́a, Grant # 1110455 to MA) and FONDEF D08I1087 and Millenium Nucleus in immunology and Immunotherapy P07/ 088-F to CF). References 1. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002; 346: 1221–31. 2. Cheung O, Sanyal AJ. Recent advances in nonalcoholic fatty liver disease. Curr Opin Gastroenterol 2010; 26: 202–8. 3. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 2011; 34: 274–85. 4. Musso G, Gambino R, Cassader M, Pagano G. 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