Evidence for transcript-specific epigenetic regulation of glucocorticoid-stimulated skeletal muscle 11ß-hydroxysteroid dehydrogenase-1 activity in type 2 diabetes.

BACKGROUND: The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) converts inactive cortisone into active cortisol in insulin target tissues. In people with type 2 diabetes, skeletal muscle (SkM) 11ßHSD1 is upregulated by the potent glucocorticoid dexamethasone. The HSD11B1 gene has two promoters designated P1 and P2. CCAAT/enhancer-binding protein beta (C/EBPß) is known to regulate expression of 11ßHSD1 via the P2 promoter. In this study, we investigated the potential role of altered DNA methylation of the P1 and P2 promoters in the observed dexamethasone-induced upregulation of SkM 11ßHSD1 oxoreductase activity in human diabetic subjects. SkM biopsies from 15 people with type 2 diabetes were collected before and after treatment with oral dexamethasone 4 mg/day for 4 days and SkM 11ßHSD1, C/EBPß and P1 and P2 promoter region mRNA levels were measured by quantitative RT-PCR. 11ßHSD1 oxoreductase activity was quantified by measuring the conversion of radiolabeled 3H-cortisone to cortisol by thin layer chromatography. Analysis of HSD11B1 promoter methylation (P1 and P2) was performed using Sequenom MassARRAY EpiTYPER analysis. RESULTS: Dexamethasone treatment resulted in a significant increase in 11ßHSD1 mRNA levels (P = 0.003), oxoreductase activity (P = 0.017) and C/EBPß mRNA (P = 0.015), and increased expression of both the P1 (P = 0.008) and P2 (P = 0.016) promoter regions . The distal P1 promoter region showed a significant reduction in methylation following dexamethasone (P = 0.026). There was a significant negative correlation between the change in methylation at this site and the increment in 11ßHSD1 oxoreductase activity (r = -0.62, P = 0.014). CONCLUSIONS: Our findings of reduced methylation in the HSD11B1 P1 promoter in association with increased 11ßHSD1 oxoreductase activity implicate complex multi-promoter epigenetic mechanisms in the regulation of 11ßHSD1 levels in SkM.

Dexamethasone administration inhibits skeletal muscle expression of the androgen receptor and IGF-1--implications for steroid-induced myopathy.

CONTEXT: Glucocorticoids are a well-recognized cause of muscle weakness. The early effects of glucocorticoids on skeletal muscle (SkM) androgen and IGF-1 pathways have not been previously investigated in human subjects. OBJECTIVE: To determine if administration of the potent glucocorticoid dexamethasone down-regulates SkM androgen receptor and the IGF-1 signalling pathway. METHODS AND SUBJECTS: Twenty-four subjects (12 men and 12 women), including 12 with type 2 diabetes and 12 nondiabetics were enrolled. Venous blood sampling and biopsy of vastus lateralis were performed before and after administration of oral dexamethasone 4 mg/day for 4 days. MAIN OUTCOME MEASURES: Changes in plasma testosterone and IGF-1, SkM androgen receptor mRNA, SkM IGF-1mRNA and SkM IGF-1 receptor mRNA by quantitative RT-PCR after dexamethasone. RESULTS: Relative expression of SkM androgen receptor was similar in male (1.63 +/- 0.37) vs. female (1.57 +/- 0.30) subjects, despite the significant difference in plasma testosterone levels. Plasma IGF-1 and SkM expression of IGF-1 and IGF-1 receptor were also similar between males and females. Following dexamethasone, there was a significant down-regulation of SkM androgen receptor (1.60 +/- 0.23 vs. 1.11 +/- 0.16, P < 0.05) and IGF-1 (1.72 +/- 0.29 vs. 1.06 +/- 0.14, P < 0.05) mRNA, but no change in expression of the IGF-1 receptor. Plasma testosterone fell significantly in both sexes (male: 15.0 +/- 1.3 vs. 11.3 +/- 1.2 nmol/l, P < 0.01, female: 1.8 +/- 0.5 vs. 0.5 +/- 0.1 nmol/l, P < 0.05). CONCLUSIONS: Exogenous steroid excess results in relative androgen deficiency at two levels, reduced circulating testosterone and SkM androgen receptor mRNA, along with reduced SkM IGF-1 mRNA. These defects may contribute to the development of steroid-induced myopathy.

Skeletal muscle 11beta hydroxysteroid dehydrogenase type 1 activity is upregulated following elective abdominal surgery.

OBJECTIVE: Cortisol has been traditionally implicated in the causation of peri-operative skeletal muscle (SkM) insulin resistance, but cortisol levels return to normal within 72 h of surgery. Tissue cortisol bioactivity may be prolonged by local upregulation of the enzyme 11betaHSD1. We aimed to investigate the changes of SkM 11betaHSD1 enzyme activity and mRNA expression, relative to plasma cortisol, insulin and glucose levels following elective abdominal surgery. PATIENTS AND DESIGN: Eight non-diabetic subjects (two male, six female) underwent serial plasma hormone sampling and muscle biopsy of vastus lateralis at baseline and on day 5 following elective laparoscopic cholecystectomy. METHODS: SkM 11betaHSD1 and H6PDH mRNA levels were measured by quantitative RT-PCR and enzyme activity by % conversion of (3)H cortisone to cortisol. Plasma glucose, insulin, free fatty acids (FFA), tumour necrosis factor-alpha and cortisol by standardised assays. RESULTS: Compared with baseline, SkM 11betaHSD1 activity was significantly increased on day 5 after surgery (14.7+/-2.1 vs 20.4+/-3.2%, P=0.005). Neither 11betaHSD1 nor H6PDH mRNA levels were altered after surgery. Plasma cortisol (P=0.027), FFA (P=0.01) and glucose (P=0.004) rose rapidly following surgery and had returned to baseline values by 24 h post-surgery. There was no significant change in plasma insulin. CONCLUSIONS: This is the first study to demonstrate an upregulation of SkM 11betaHSD1 activity in response to a physiological stressor. Sustained activation of this enzyme may increase tissue cortisol bioactivity.

Adiponectin, skeletal muscle adiponectin receptor expression and insulin resistance following dexamethasone.

OBJECTIVE: Skeletal muscle is a major site of adiponectin action and of glucocorticoid-induced insulin resistance. Little human data exist however, regarding the impact of exogenous glucocorticoids on adiponectin receptors in skeletal muscle. DESIGN AND PATIENTS: Twelve subjects with type 2 diabetes and 12 controls underwent blood sampling and muscle biopsy of vastus lateralis before and after 4 days of 4 mg dexamethasone. MEASUREMENTS: (i) Total and high molecular weight (HMW) plasma adiponectin, glucose and insulin; (ii) Skeletal muscle adiponectin receptor AdipoR1 and AdipoR2 mRNA levels by quantitative real time RT-PCR. RESULTS: Baseline total adiponectin (8.0 +/- 0.89 vs. 12.5 +/- 1.46 microg/ml, P = 0.013), HMW adiponectin (2.8 +/- 0.44 vs. 5.9 +/- 1.04 microg/ml, P = 0.014) and AdipoR2 mRNA levels (mean DeltaC(T )14.71 +/- 0.35 vs. 13.37 +/- 0.28, P = 0.017) were significantly lower in diabetic subjects. After dexamethasone, AdipoR2 mRNA fell in the controls but there was no change in the diabetic group, while there was a significant increase in total (P = 0.002) and HMW adiponectin (P < 0.001) across both groups. Total and HMW plasma adiponectin correlated with clinical and biochemical measures of insulin sensitivity. However following dexamethasone which increased insulin resistance, the relationship between adiponectin and the biochemical measures was lost. CONCLUSIONS: Plasma adiponectin and skeletal muscle AdipoR2 mRNA expression are reduced in subjects with diabetes; both are likely to contribute to the observed insulin resistance. Dexamethasone inhibits AdipoR2 mRNA expression in nondiabetic subjects, while there is a small rise in plasma adiponectin levels. The close relationship between plasma adiponectin and biochemical measures of insulin sensitivity is lost in the setting of glucocorticoid-induced insulin resistance.

Altered activity of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in skeletal muscle confers metabolic protection in subjects with type 2 diabetes.

CONTEXT: There is little information regarding the regulation of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes in skeletal muscle in the setting of type 2 diabetes. OBJECTIVE: Our objective was to investigate whether there is differential mRNA expression and enzyme activity of 11beta-HSD1 and 11beta-HSD2 in the skeletal muscle of diabetic subjects compared with controls at baseline and in response to dexamethasone. DESIGN: Participants underwent muscle biopsy of vastus lateralis at baseline and after dexamethasone. SETTING: The study took place at a university teaching hospital. PARTICIPANTS: Twelve subjects with type 2 diabetes and 12 age- and sex-matched controls participated. INTERVENTION: Subjects were given oral dexamethasone, 4 mg/d for 4 d. MAIN OUTCOME MEASURES: We assessed 11beta-HSD1, 11beta-HSD2, and H6PDH mRNA levels by quantitative RT-PCR and enzyme activity by percent conversion of [(3)H]cortisone and [(3)H]cortisol, respectively. RESULTS: At baseline, mRNA levels were similar in diabetic and control subjects for 11beta-HSD1, 11beta-HSD2, and H6PDH. 11beta-HSD1 activity was reduced in diabetic subjects (percent conversion of [(3)H]cortisone to [(3)H]cortisol was 11.4 +/- 2.5% vs. 18.5 +/- 2.2%; P = 0.041), and 11beta-HSD2 enzyme activity was higher in diabetic subjects (percent conversion of [(3)H]cortisone to [(3)H]cortisol was 17.2 +/- 2.6% vs. 9.2 +/- 1.3%; P = 0.012). After dexamethasone, 11beta-HSD1 mRNA increased in both groups (P < 0.001), whereas 11beta-HSD2 mRNA decreased (P = 0.002). 11beta-HSD1 activity increased in diabetic subjects (P = 0.021) but not in controls, whereas 11beta-HSD2 activity did not change in either group. At baseline, there was a significant negative correlation between 11beta-HSD1 and 11beta-HSD2 enzyme activity (r = -0.463; P = 0.026). CONCLUSIONS: The activities of skeletal muscle 11beta-HSD1 and 11beta-HSD2 are altered in diabetes, which together may reduce intracellular cortisol generation, potentially conferring metabolic protection.

11Beta hydroxysteroid dehydrogenase type 1 is expressed and is biologically active in human skeletal muscle.

OBJECTIVE: No data exist regarding the distribution and oxoreductase enzyme activity of 11beta hydroxysteroid dehydrogenase type 1 (11beta HSD-1) in fresh human skeletal muscle. We aimed to investigate the mRNA and protein expression of 11beta HSD-1 in fresh skeletal muscle, confirm its biological activity and determine its relationship with hexose-6-phosphate dehydrogenase (H6PDH). We also examined the muscle fibre localization of 11beta HSD-1. DESIGN: Eleven non-diabetic community volunteers underwent muscle biopsy of vastus lateralis. MEASUREMENTS: (i) 11beta HSD-1 and H6PDH mRNA expression by quantitative reverse transcription polymerase chain reaction (RT-PCR); (ii) protein localization and fibre type specificity by immunohistochemistry; and (iii) enzyme oxoreductase activity by percentage conversion of 3H cortisone to cortisol. RESULTS: 11beta HSD-1 mRNA was expressed at low levels compared to human liver. Mean DeltaCT of skeletal muscle in 11 subjects was 19.57 (range 18.40-20.79) compared to DeltaCT of 12.75 in human liver, which equates to an approximate 100-fold higher level of expression. H6PDH mRNA was also detected with a mean DeltaCT of 14.46 (range 13.13-16.60), approximately 35-fold more abundant than 11beta HSD-1 in skeletal muscle. There was a significant correlation between 11beta HSD-1 and H6PDH (r = 0.67, P = 0.03). 11beta HSD-1 immunostaining was present in all muscle specimens, with similar distribution among fast and slow twitch fibres. 11beta HSD-1 oxoreductase activity was demonstrated, with mean conversion of cortisone to cortisol of 17.7% per 200 mg of muscle per 24 h (range 7.1-29.5%). CONCLUSIONS: 11beta HSD-1 mRNA and protein is expressed in fresh human skeletal muscle along with readily demonstrable oxoreductase activity. 11beta HSD-1 localization is not muscle fibre type specific. High levels of skeletal muscle H6PDH should ensure that oxoreductase activity predominates in vivo.

Aromatase-deficient (ArKO) mice have reduced blood pressure and baroreflex sensitivity.

Aromatase-deficient (ArKO) mice are deficient in estrogens due to deletion of the aromatase gene. We hypothesized that there may be changes in the cardiovascular system of ArKO mice because of evidence linking estrogens with improved cardiovascular outcomes and the induction of the glucocorticoid-metabolizing enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), gene in the kidney, which is important for the regulation of blood pressure (BP). BP and baroreflex sensitivity (BRS) in female conscious ArKO mice were compared with those in age- and weight-matched wild-type (WT) mice. Power spectral analysis was used to determine cardiovascular variability and BRS. Although systolic BP was similar in the two groups, diastolic and mean BPs were lower in the ArKO mice (-6.3 +/- 1.9 and -4.6 +/- 2.1 mm Hg, respectively). Heart rate (HR) was greater in the ArKO mice (+36 +/- 6 beats/min). The mean BP in WT mice was 105 mm Hg, and the HR was 481 beats/min. In the autonomic frequency range, BP variability was 74% greater, and HR variability was only 26% that in WT mice. The BRS of ArKO mice was 46% of the value observed in WT mice. 11betaHSD2 levels were unaltered in ArKO mice, except in the kidney, where they were only 10% of WT levels. Estradiol administration to ArKO mice restored renal 11betaHSD2 to WT levels. The results show that ArKO mice have lower diastolic BP, but increased BP variability, perhaps due to an impaired BRS. Thus, aromatase activity is critical for normal autonomic control of the heart and, hence, for reducing the deleterious effects of high BP variability.