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Department of Endocrinology and Diabetes (C.J., V.R.O., W.J.I., F.P.A.), St. Vincent’s Hospital, Melbourne 3065, Australia; Department of Medicine (C.J., R.J.D., W.J.I., F.P.A.), University of Melbourne, Melbourne 3010, Australia; and Laboratory of Molecular Hypertension (Z.K.), Baker Heart Research Institute, Melbourne 3004, Australia
Address all correspondence and requests for reprints to: Christina Jang, M.B., B.S., FRACP, Department of Endocrinology and Diabetes, St. Vincent’s Hospital, Melbourne, 41 Victoria Parade, Fitzroy 3065, Australia. E-mail: christina.jang{at}svhm.org.au.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Objective: Our objective was to investigate whether there is differential mRNA expression and enzyme activity of 11ß-HSD1 and 11ß-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 11ß-HSD1, 11ß-HSD2, and H6PDH mRNA levels by quantitative RT-PCR and enzyme activity by percent conversion of [3H]cortisone and [3H]cortisol, respectively.
Results: At baseline, mRNA levels were similar in diabetic and control subjects for 11ß-HSD1, 11ß-HSD2, and H6PDH. 11ß-HSD1 activity was reduced in diabetic subjects (percent conversion of [3H]cortisone to [3H]cortisol was 11.4 ± 2.5% vs. 18.5 ± 2.2%; P = 0.041), and 11ß-HSD2 enzyme activity was higher in diabetic subjects (percent conversion of [3H]cortisone to [3H]cortisol was 17.2 ± 2.6% vs. 9.2 ± 1.3%; P = 0.012). After dexamethasone, 11ß-HSD1 mRNA increased in both groups (P < 0.001), whereas 11ß-HSD2 mRNA decreased (P = 0.002). 11ß-HSD1 activity increased in diabetic subjects (P = 0.021) but not in controls, whereas 11ß-HSD2 activity did not change in either group. At baseline, there was a significant negative correlation between 11ß-HSD1 and 11ß-HSD2 enzyme activity (r = –0.463; P = 0.026).
Conclusions: The activities of skeletal muscle 11ß-HSD1 and 11ß-HSD2 are altered in diabetes, which together may reduce intracellular cortisol generation, potentially conferring metabolic protection.
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Introduction |
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11ß-HSD1 is a bidirectional enzyme, most abundantly expressed in glucocorticoid target tissues such as liver and adipose. It displays predominant oxoreductase activity in intact cells, generating active cortisol from inactive cortisone (3). In adipose tissue, studies show increased oxoreductase activity in omental compared with sc adipocytes, suggesting that differential amplification of glucocorticoid in this tissue is a potential mechanism in the pathogenesis of central obesity (4). In contrast, 11ß-HSD2 has dehydrogenase activity only, converting cortisol to cortisone (3). It is expressed in mineralocorticoid target tissues (5), where its main role is to protect the mineralocorticoid receptor from cortisol, thereby conferring specificity of aldosterone to its receptor (6, 7), whereas expression in the placenta protects the fetus from elevated circulating maternal glucocorticoids (8, 9).
Skeletal muscle is a major site of insulin resistance and accounts for the majority of peripheral glucose disposal (10). There are few data, however, about the role of the 11ß-HSD enzymes and their impact on cortisol metabolism in skeletal muscle. We have recently demonstrated that 11ß-HSD1 is present and biologically active in fresh muscle of nondiabetic subjects (11). However, whether there is any alteration of 11ß-HSD1 activity in skeletal muscle of diabetic subjects is unknown. Previous studies examining muscle 11ß-HSD1 have been performed on myoblasts (12) or have examined 11ß-HSD1 mRNA expression without quantification of enzyme activity (13, 14). Such in vitro data may not be applicable in vivo, because enzyme expression and activity may change during cellular differentiation as demonstrated in adipose tissue (15). Furthermore, in the basal state, skeletal muscle 11ß-HSD1 mRNA levels do not correlate with oxoreductase activity (11).
The direction of 11ß-HSD1 activity depends on the provision of cofactor by hexose-6-phosphate dehydrogenase (H6PDH) (16, 17). Its location within the lumen of the endoplasmic reticulum (18) serves the crucial role of generating the cofactor reduced nicotinamide adenine dinucleotide (NAD) phosphate (NADPH), which is required for oxoreductase activity. Cotransfection of Chinese hamster ovary cells with 11ß-HSD1 and H6PDH increases oxoreductase activity and virtually eliminates dehydrogenase activity compared with 11ß-HSD1 alone. This highlights the importance of H6PDH in regulating the in vivo direction of 11ß-HSD1 conversion from cortisone to cortisol.
Previous in vitro studies in myoblasts have demonstrated that addition of glucocorticoid up-regulates 11ß-HSD1 mRNA and activity (19). To date, no study has examined whether exogenous glucocorticoid up-regulates 11ß-HSD1 activity in adult human skeletal muscle. The effect of glucocorticoids on H6PDH is also unknown. Increased local generation of active glucocorticoid within skeletal muscle may contribute to the pathogenesis of the metabolic syndrome and be a possible mechanism for glucocorticoid-induced insulin resistance.
The potential role of 11ß-HSD2 in glucocorticoid inactivation in muscle has similarly not been explored. Early studies using Northern blotting or conventional RT-PCR did not detect 11ß-HSD2 mRNA in skeletal muscle (14, 19, 20). However, to our knowledge, there have been no studies using the more sensitive technique of TaqMan-based quantitative real-time RT-PCR.
The aims of this study are therefore to determine whether 1) 11ß-HSD2 is present and biologically active in fresh skeletal muscle; 2) 11ß-HSD1 and 11ß-HSD2 mRNA expression and enzyme activities are altered in the skeletal muscle of diabetic compared with control subjects; 3) there is a relationship between skeletal muscle H6PDH and 11ß-HSD1 expression in these two groups; and 4) short-term administration of exogenous dexamethasone differentially affects muscle 11ß-HSD1 and 11ß-HSD2 mRNA expression and activities in human subjects with and without diabetes.
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Subjects and Methods |
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The protocol was approved by the institutional Human Research Ethics Committee. Twelve subjects with type 2 diabetes and 12 healthy age- and sex-matched control subjects gave written informed consent for participation. Control subjects had fasting blood glucose levels less than 6.0 mmol/liter, normal glycosylated hemoglobin (HbA1c), and no family history of diabetes. Subjects with type 2 diabetes were treated by diet alone (n = 4) or oral hypoglycemic agents (metformin, a sulfonylurea, or both; n = 8). No patient was using insulin or thiazolidinediones. Exclusion criteria included poorly controlled hypertension (systolic blood pressure 
160 mm Hg and/or diastolic 90 mm Hg), a history of liver or renal disease, and evidence of unstable angina or peripheral vascular disease.
Experimental design
After an overnight 10-h fast, subjects had venous blood samples drawn between 0800 and 0830 h for glucose and HbA1c. Body weight, body mass index (BMI) and waist-to-hip ratio (WHR) were determined for each subject (Table 1
). After sedation with iv midazolam and administration of local anesthetic, biopsy of vastus lateralis was performed using a 5-mm Bergstrom needle as previously described (21). Fresh muscle was rapidly dissected of visible adipose and connective tissue and placed in DMEM for activity studies. Additional samples were immediately snap frozen in liquid nitrogen for mRNA analysis and homogenate activity studies and in isopentane cooled in liquid nitrogen for immunohistochemistry and then stored at –70 C for later analysis. Within 28 d, subjects were administered oral dexamethasone 4 mg daily as a single evening dose for 4 d before repeat blood sampling and muscle biopsy. Subjects with type 2 diabetes monitored their capillary blood glucose levels at least twice daily during dexamethasone therapy and were contacted by an investigator on a daily basis. Blood glucose levels before and after dexamethasone were 8.6 ± 0.9 and 9.8 ± 1.5 mmol/liter, respectively.
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Plasma glucose was analyzed by a glucose oxidase method employing a YSI 1500 Sidekick analyzer (Yellow Springs Instrument Co., Yellow Springs, OH), with a coefficient of variation of 2.4%. HbA1c was measured by an HPLC assay.
Quantitative real-time RT-PCR
Relative skeletal muscle mRNA levels for 11ß-HSD1, 11-HSD2, and H6PDH were determined using quantitative real-time RT-PCR (TaqMan chemistry). One hundred milligrams of snap-frozen tissue were homogenized in buffer RLT (QIAGEN, Hilden, Germany) using Ika-ultraturrex T25 homogenizer (Janke and Kunkle, Stauten, Germany). Total RNA from skeletal muscle was then extracted using the RNeasy mini kit (QIAGEN). One microgram of total RNA was reverse transcribed to cDNA using TaqMan RT reagents according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). 11ß-HSD1-specific primers and a probe were designed using Primer Express 1.5 software (ABI, Foster City, CA): forward primer, 5'-GCAAAGGGATCGGAAGAGAGA-3'; reverse primer, 5'-GCT GAGGCTGCTCCAAGCT-3'; MGB FAM probe, 5'-CCA CAT GGG CTC CCA-3'. For 11ß-HSD2 and H6PDH, a specific assay-on-demand gene expression assay was obtained from Applied Biosystems. Singleplex reactions were performed and normalized to the endogenous control human 18S rRNA (Applied Biosystems). Relative 11ß-HSD1, 11ß-HSD2, and H6PDH mRNA levels were calculated using the 
CT methodology (2–CT, where CT is cycle threshold) (ABI User Bulletin no. 2). Total RNA from commercially obtained human skeletal muscle (BD Biosciences, Palo Alto, CA) was reverse transcribed to cDNA and was used as the calibrator in all experiments. The CT value for 11ß-HSD1, 11ß-HSD2, and H6PDH mRNA for each subject was expressed as a ratio with the CT measured from the commercially obtained skeletal muscle cDNA.
11ß-HSD enzyme activity
Oxoreductase enzyme activity of 11ß-HSD1 was determined by measuring the conversion of radiolabeled [3H]cortisone to [3H]cortisol by thin-layer chromatography performed within 4 h of the biopsy, as previously described (11). Sample transport difficulties resulted in missing data in one diabetic subject; therefore, results were obtained on 11 subjects for this group. Fresh muscle was weighed and incubated in serum-free, low-glucose DMEM containing [3H]cortisone (1 nM), 100 nM cold cortisone, and 5 µg insulin at 37 C for 24 h. The mean weight of muscle specimens collected from 48 muscle biopsies was 198 mg. We previously demonstrated that the relationship between muscle mass and oxoreductase enzyme activity is linear (11). To correct for the different mass of each biopsy specimen, oxoreductase activity results are expressed as a percentage conversion standardized to 200 mg muscle.
Additionally, total 11ß-HSD1 and 11ß-HSD2 dehydrogenase activities were determined by measuring the conversion of radiolabeled [3H]cortisol to [3H]cortisone in a cell-free system. Specificity in homogenates was conferred by the addition of the enzyme-specific cofactor in excess (NADP for 11ß-HSD1 and NAD for 11ß-HSD2) (22). Homogenates were also incubated with no cofactor to measure any activity due to endogenous cofactor and subtracted. Snap-frozen muscle samples were homogenized using Ika-ultraturrex T25 homogenizer (Janke and Kunkle) in 4 vol homogenizing/assay buffer (0.25 M sucrose, 10 mM sodium phosphate, 140 mM NaCl, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, pH 7.4). Protein concentrations were determined colorimetrically by the Bradford method (23). Activity was measured by incubating 3 mg/ml of homogenate for 3.5 h at 37 C in assay buffer in the presence of 10 nM [3H]cortisol and 1 mM cofactor. Human placenta, which expresses both 11ß-HSD1 (24) and 11ß-HSD2 (8) was used as a positive control. All reactions were terminated by the addition of 3 vol ethyl acetate. The steroids were extracted from either the media or assay buffer and were analyzed by thin-layer chromatography. The labeled steroid and its converted product were visualized by phosphoimager (Fujix BAS 1000; Fuji Film Co., Tokyo, Japan) scanning, and the result is expressed as a percentage conversion, after correcting each sample to 3 mg protein.
Immunohistochemistry
Assessment of skeletal muscle 11ß-HSD1 and 11ß-HSD2 distribution was performed using immunohistochemistry. We have previously demonstrated 11ß-HSD1 immunostaining in the skeletal muscle of nondiabetic subjects (11), and the present study was performed on two diabetic subjects before and after dexamethasone. 11ß-HSD2 immunohistochemistry was performed in two diabetic and five nondiabetic subjects, with human placenta as a positive control. Frozen sections of skeletal muscle were fixed in acetone at –20 C for 20 min. Sections were air dried and then blocked at room temperature for 30 min in Dako protein block serum-free ready-to-use solution (Dako Corp., Carpinteria, CA). For 11ß-HSD1, sections were incubated with a 1:100 dilution of sheep antihuman 11ß-HSD1 antiserum (The Binding Site, Birmingham, UK) in 5% donkey serum/PBS. For 11ß-HSD2, sections were incubated with a 1:100 dilution of rabbit antihuman antiserum (8) in 5% goat serum/PBS. For von Willebrand factor (vWF), sections were incubated with a 1:50 dilution of rabbit antihuman vWF (Dako). Primary antibody was incubated for 60 min followed by PBS washes. Secondary antibodies were donkey antisheep antibody for 11ß-HSD1 and goat antirabbit for 11ß-HSD2; both antibodies were conjugated to AlexaFluor 488 and vWF (Molecular Probes, Eugene, OR). The sections were then rinsed in PBS and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). A Bio-Rad MRC1024 confocal system was used to analyze the sections (Bio-Rad, Richmond, CA).
Statistical analysis
SPSS version 13.0 statistical software was used for statistical analysis (SPSS Inc., Chicago, IL). Data are expressed as mean ± SEM. A repeated-measures ANOVA was performed to compare the response to dexamethasone between the diabetic and nondiabetic groups. The outcome variables analyzed were 11ß-HSD1 and 11ß-HSD2 mRNA and enzyme activity and H6PDH mRNA levels.
The effect of dexamethasone was analyzed as a within-subjects effect; however, it should be noted that the difference in the response to dexamethasone in the two groups is a between-person effect. To test the relationships between different variables, Pearson correlation analysis was employed. The unpaired t test was used to compare baseline data in the control vs. diabetic group.
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Results |
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Baseline characteristics of the subjects are shown in Table 1
. Diabetic and control subjects were well matched for age and sex. As expected, there was a significant difference in HbA1c and fasting glucose.
mRNA expression
At baseline, there was no difference in mRNA levels as estimated by CT between diabetic and control subjects for 11ß-HSD1 (19.59 ± 0.18 vs. 19.52 ± 0.23, P = 0.838), 11ß-HSD2 (21.18 ± 0.41 vs. 20.78 ± 0.39, P = 0.497), or H6PDH (14.40 ± 0.37 vs. 14.43 ± 0.31, P = 0.948). There was a significant difference in the relative expression of 11ß-HSD1 vs. 11ß-HSD2 for the combined groups (CT, 19.55 ± 0.14 vs. 20.98 ± 0.28, P = 0.006). This equates to a 2.7-fold higher level of expression for 11ß-HSD1 in skeletal muscle compared with 11ß-HSD2. There were no gender differences in 11ß-HSD1 or 11ß-HSD2 mRNA levels. H6PDH was significantly more abundant than 11ß-HSD1 (CT, 14.41 ± 0.24 vs. 19.55 ± 0.14, P < 0.001), equating to a 35-fold higher expression in muscle for H6PDH.
To examine the effect of dexamethasone on mRNA levels, CT values were expressed as a ratio to the commercially available skeletal muscle cDNA library as described. After dexamethasone, relative 11ß-HSD1 mRNA levels increased significantly in both groups (Fig. 1A) to a similar degree (P < 0.001 by ANOVA). In contrast, 11ß-HSD2 mRNA levels were significantly reduced by dexamethasone (Fig. 2A) when the groups were analyzed together (P = 0.002 by ANOVA, Fig. 2A). When 11ß-HSD2 mRNA levels was analyzed in the diabetic and control groups separately, the response to dexamethasone was significant in the diabetic group only (P = 0.010) and approached significance in the control group (P = 0.059) due mainly to the wide scatter of the dexamethasone response in the control group. H6PDH expression was unchanged in response to dexamethasone.
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Basal 11ß-HSD1 oxoreductase activity in intact skeletal muscle was significantly lower in diabetic subjects (percent conversion of [3H]cortisone to [3H]cortisol was 11.4 ± 2.5% per 200 mg muscle/24 h in diabetics compared with 18.5 ± 2.2% per 200 mg muscle/24 h in controls, P = 0.041, Fig. 1B). When total 11ß-HSD1 dehydrogenase activity (cofactor NADP) was estimated in cell homogenates, very low levels of conversion from [3H]cortisol to [3H]cortisone were observed (mean, 4.3 ± 0.37%; range, 1.8–9.2%). 11ß-HSD1 dehydrogenase activity was significantly higher in the diabetic group (percent conversion of [3H]cortisol to [3H]cortisone was 5.3 ± 0.6% per 3 mg protein in diabetics compared with 3.3 ± 0.3% per 3 mg protein/3.5 h in controls, P = 0.007). Baseline 11ß-HSD2 enzyme activity (cofactor NAD) was significantly higher in the diabetic group compared with the control group (percent conversion [3H]cortisol to [3H]cortisone per 3 mg protein per 3.5 h was 17.2 ± 2.6% in diabetics vs. 9.2 ± 1.3% in controls, P = 0.012, Fig. 2B). 11ß-HSD2 activity in placenta was essentially 100%.
After dexamethasone, there was a significant increase in 11ß-HSD1 oxoreductase activity in the intact muscle system (P = 0.021, Fig. 1B). Oxoreductase activity was now similar in the diabetic and control groups after dexamethasone. There was no significant group effect (P = 0.489), but there was a significant group x treatment interaction (P = 0.041), indicating that the response to dexamethasone differed between the diabetic group and controls. As can be seen in Fig. 1B, the increase in 11ß-HSD1 activity was confined to the diabetic group. There was no significant change in 11ß-HSD1 dehydrogenase activity in the cell-free system or in 11ß-HSD2 activity in response to dexamethasone (Fig. 2B).
Correlations
There was no correlation between basal 11ß-HSD1 mRNA expression and oxoreductase (intact muscle) or dehydrogenase (homogenates) enzyme activity. 11ß-HSD2 mRNA expression also did not correlate with its enzyme activity. There was a highly significant correlation between basal H6PDH and 11ß-HSD1 mRNA expression (r = 0.65; P = 0.001) but not with 11ß-HSD1 oxoreductase activity (r = 0.08; P = 0.79). Across all subjects, a significant negative correlation between 11ß-HSD1 and 11ß-HSD2 enzyme activity was observed (r = 0.46; P = 0.03). There was no correlation between the incremental response to dexamethasone of 11ß-HSD1 mRNA and enzyme activity (r = 0.33; P = 0.12) or H6PDH mRNA levels for the combined groups (r = 0.24; P = 0.26). However, when the diabetic group was analyzed separately, there was a highly significant correlation between the 11ß-HSD1 mRNA increment and change in oxoreductase enzyme activity (r = 0.77; P = 0.006).
There were no significant correlations between 11ß-HSD1 or 11ß-HSD2 mRNA or enzyme activities with age, BMI, WHR, or fasting glucose.
Immunohistochemistry
11ß-HSD1 and 11ß-HSD2 immunoreactivity was observed within the muscle fibers of all specimens. Higher-intensity staining of 11ß-HSD1 was observed after dexamethasone treatment in the diabetic subject whose increase in oxoreductase activity was the largest (Fig. 3). 11ß-HSD2 immunostaining was observed, predominantly within the interstitium of the myocytes (Fig. 3) and vascular structures but also within the muscle fiber.
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Discussion |
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11ß-HSD1 oxoreductase activity was found to be significantly lower in diabetic subjects at baseline but increased significantly after dexamethasone. Although previous work has demonstrated that in myoblasts, 11ß-HSD1 oxoreductase activity is up-regulated in vitro by glucocorticoids (19), baseline differences between diabetic and control subjects were not examined. In a subsequent study employing myotubes established from diabetic and control subjects, increased expression of 11ß-HSD1 mRNA was described in the diabetic myotubes (14), but enzyme activity was not examined. Our study is the first to be done in fresh intact muscle, and the findings support the hypothesis that reduced oxoreductase enzyme activity in the basal state may represent a potential compensatory metabolic mechanism (2, 25, 26). Similarly, conversion of cortisone to cortisol, a marker of hepatic 11ß-HSD1 activity, was impaired in a group of 25 diabetic and/or glucose-intolerant lean subjects (27). Other studies have examined whole-body (hepatic) and adipose tissue 11ß-HSD1 metabolism in diabetic subjects, with variable results (28, 29).
The biological relevance of 11ß-HSD1 in skeletal muscle is incompletely understood. Our previous studies have demonstrated the presence of skeletal muscle 11ß-HSD1 in nondiabetic subjects, although the relative contribution of muscle cortisol production to whole-body levels appears to be small. Studies using in vivo measurements taken from forearm (30) and leg (31) circulations demonstrate minimal cortisol production at these sites, suggesting that cortisone-cortisol turnover in skeletal muscle is relatively low. Furthermore, carbenoxelone has also not been found to alter insulin-stimulated glucose uptake (32). Nevertheless, the fact that 11ß-HSD1 and 11ß-HSD2 enzyme activities were demonstrated in both our study and other in vivo studies (31) indicates a potentially important local effect of glucocorticoid within skeletal muscle. Glucocorticoids are well known to have a paracrine effect in adipose tissue; cortisol production within adipose stromal cells promotes their differentiation into mature skeletal muscle cells (33). In skeletal muscle, an autocrine/paracrine effect of cortisol may exist and contribute to insulin resistance.
Catheterization of hepatic and leg vasculature followed by infusion of isotopically labeled cortisol with 4 deuteriums in the steroid skeleton (d4-cortisol) has found that the splanchnic bed contributes significantly to plasma cortisol levels, whereas in the leg, there is net uptake of cortisol that tends to increase during hyperinsulinemic conditions (31). Increased d4-cortisol extraction in the leg implies 11ß-HSD dehydrogenase activity; in vivo, this is more likely to be from 11ß-HSD2. Furthermore, d3-cortisol generation (from 11ß-HSD1 oxoreductase activity) also increased with insulin infusion. These findings are consistent with those of our study; biologically active 11ß-HSD1 explains the generation of d3-cortisol, and 11ß-HSD2 activity explains net d4-cortisol uptake (by 11ß-HSD2 dehydrogenation). The net release of d3-cortisol may be underestimated because it can be metabolized to d3-cortisone by 11ß-HSD2 or irreversibly degraded via the 5
-reductase pathway to other metabolites (31). Our study also demonstrates a positive correlation between 11ß-HSD2 and fasting insulin levels, which may be consistent with the acute increase in leg d4-cortisol uptake observed after insulin infusion (31).
11ß-HSD1 oxoreductase activity depends on the generation of cofactor NADPH via the enzyme H6PDH in intact cells. Cell damage during tissue dispersal of the muscle specimen might underestimate the extent of 11ß-HSD1 activity. To investigate this possibility, we measured 11ß-HSD1 dehydrogenase activity in cell homogenates as a marker of total enzyme activity. However, muscle 11ß-HSD1 dehydrogenase activity using this method was low, whereas there was significant conversion in placenta, which was used as a positive control. Using this latter assay, it is difficult to quantitate biological activity of 11ß-HSD1 or interpret the small absolute difference between the diabetic and control group. There was also no change in dehydrogenase activity in response to dexamethasone. Therefore, fresh skeletal muscle oxoreductase activity appears to be a more reliable and direct measure of biological 11ß-HSD1 activity.
The novel finding of expression and functional enzyme activity of 11ß-HSD2 in skeletal muscle, albeit at low levels, is of considerable interest. Earlier studies that did not detect 11ß-HSD2 mRNA used less sensitive techniques such as Northern blotting (20) or RT-PCR with ethidium bromide-stained agarose gels (14, 19). Skeletal muscle is not a known mineralocorticoid target tissue where 11ß-HSD2 is abundantly expressed (5). We demonstrate that baseline 11ß-HSD2 activity is significantly higher in subjects with type 2 diabetes than control subjects. Within skeletal muscle, inactivation of glucocorticoid may impact on whole-body glucose metabolism. Furthermore, a significant negative correlation was observed between 11ß-HSD1 and 11ß-HSD2 enzyme activity. The findings of reduced 11ß-HSD1 oxoreductase activity with increased 11ß-HSD2 dehydrogenase activity suggest that the 11ß-HSD enzymes’ set-point appears to be directed toward reducing a diabetic individual’s exposure to active cortisol, serving as a protective mechanism in the metabolically adverse state.
Consistent with our previous findings, we found a lack of correlation between 11ß-HSD1 mRNA levels and enzyme activity. A similar lack of correlation was seen for 11ß-HSD2 mRNA and enzyme activity. Furthermore, for 11ß-HSD2, a decrease in mRNA levels with dexamethasone did not translate into a significant down-regulation of enzyme activity. These data support the notion that mRNA expression of the 11ß-HSD enzymes in skeletal muscle cannot be used as a surrogate marker for biological enzyme behavior.
Dexamethasone was used in this study as a stimulus for 11ß-HSD1 activity, inducing a metabolic state comparable to a physiological stress response. One striking observation was the up-regulation of 11ß-HSD1 oxoreductase activity in the diabetic group not seen in control subjects. The potential protective mechanism of reduced activity in diabetic subjects in the basal state is lost in the presence of dexamethasone and may be involved in the pathogenesis of glucocorticoid-induced hyperglycemia. After exogenous glucocorticoid administration, 11ß-HSD1 mRNA levels increased, whereas 11ß-HSD2 mRNA levels decreased. In contrast, only 11ß-HSD1 enzyme activity in the diabetic subjects was up-regulated in response to dexamethasone. This highlights the complex relationship between gene expression and functional activity of these two enzymes at a cellular level.
Our earlier immunohistochemical results demonstrated 11ß-HSD1 protein distribution to be most prominent around the periphery of the muscle cell in control subjects (11). In contrast, in the subject with a large increment in 11ß-HSD1 oxoreductase activity, myocyte immunoreactivity was increased throughout the muscle fiber. Protein expression of 11ß-HSD2 was also established in muscle by immunohistochemistry. Immunostaining for 11ß-HSD2 was most intense around the periphery of the muscle cell, suggesting that it is more abundant in interstitial cells including fibroblasts and vascular structures, although weak immunoreactivity was observed within the fiber.
In summary, muscle 11ß-HSD1 oxoreductase enzyme activity is reduced in diabetic subjects but significantly up-regulated in response to the administration of a potent glucocorticoid. This up-regulation is not associated with changes in H6PDH, which is present in excess, acting to steer 11ß-HSD1 strongly in the oxoreductase direction. 11ß-HSD2 is expressed and biologically active in human skeletal muscle. Its function is increased in the muscle of diabetic subjects, promoting inactivation of cortisol. Our results are consistent with the skeletal muscle 11ß-HSD enzyme system acting as a protective mechanism, reducing the adverse metabolic consequences of high tissue levels of cortisol in diabetic subjects. This protective mechanism is lost after dexamethasone, which may in part be a factor in the etiology of glucocorticoid-induced hyperglycemia. Specific inhibitors of 11ß-HSD1 may prove to be useful agents in the treatment of glucocorticoid or metabolic stress-induced insulin resistance.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: C.J. has received lecture fees from Novo Nordisk. W.J.I. has received lecture fees from Novartis, Novo Nordisk, and Eli Lilly. V.R.O., R.J.D., Z.K., and F.P.A. have nothing to disclose.
First Published Online May 22, 2007
Abbreviations: BMI, Body mass index; HbA1c, glycosylated hemoglobin; H6PDH, hexose-6-phosphate dehydrogenase; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADPH, reduced NAD phosphate; vWF, von Willebrand factor; WHR, waist-to-hip ratio.
Received December 11, 2006.
Accepted May 10, 2007.
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and ß isoforms and type I 11ß-hydroxysteroid dehydrogenase expression in human skeletal muscle cells: a key role in the pathogenesis of insulin resistance? J Clin Endocrinol Metab 86:2296–2308This article has been cited by other articles:
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  S. A. Morgan, M. Sherlock, L. L. Gathercole, G. G. Lavery, C. Lenaghan, I. J. Bujalska, D. Laber, A. Yu, G. Convey, R. Mayers, et al. 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Regulates Glucocorticoid-Induced Insulin Resistance in Skeletal Muscle Diabetes, November 1, 2009; 58(11): 2506 - 2515. [Abstract] [Full Text] [PDF]
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, Before dexamethasone; 
, after dexamethasone. *, P < 0.01 compared with baseline; 
, P < 0.05 compared with baseline; 
, P < 0.05 compared with control group.
