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Original Studies |
and ß Isoforms and Type I 11ß-Hydroxysteroid Dehydrogenase Expression in Human Skeletal Muscle Cells: A Key Role in the Pathogenesis of Insulin Resistance?
Endocrinology and Metabolism Unit, Southampton General Hospital School of Medicine (C.B.W., S.J.D.), and Department of Chemical Pathology (P.J.W.) and Medical Research Council Environmental Epidemiology Unit (D.I.W.P.), Southampton General Hospital, Southampton, United Kingdom SO16 6YD; and Division of Biomedical Sciences, University of Portsmouth (S.J.D.), Portsmouth, United Kingdom P01 2UP
Address all correspondence and requests for reprints to: Dr Christopher B. Whorwood, Endocrinology and Metabolism Unit, Level D, South Block, Southampton General Hospital School of Medicine, Tremona Road, Southampton, United Kingdom SO16 6YD. E-mail: c.whorwood{at}soton.ac.uk
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Abstract |
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and GRß) and 11ß-hydroxysteroid
dehydrogenase type I (11ßHSD1)-mediated intracellular synthesis of
active cortisol from inactive cortisone. We have analyzed the
expression of GR, GRß, and 11ßHSD1 and their hormonal regulation
in skeletal myoblasts from men (n = 14) with contrasting levels of
adiposity and insulin resistance. Immunohistochemical, Northern blot,
and Western blot analysis indicated abundant expression of GR and
11ßHSD1 under basal conditions. The apparent Km and
maximum velocity for the conversion of cortisone to cortisol were
440 ± 14 nmol/L and 75 ± 7 pmol/mg protein·h and 437
± 16 nmol/L and 33 ± 6 pmol/mg protein·h (mean ±
SEM; n = 4) in the presence and absence of 20% serum.
Incubation of myoblasts with increasing concentrations of
glucocorticoid (50–1000 nmol/L) resulted in a dose-dependent decline
in GR expression and a dose-dependent increase in GRß expression.
11ßHSD1 activity was sensitively up-regulated by increasing
concentrations of glucocorticoid (50–1000 nmol/L:
P < 0.05). Abolition of these effects by the GR
antagonist, RU38486, indicates that regulation of GR, GRß, and
11ßHSD1 expression is mediated exclusively by the GR
ligand-binding variant. In contrast, 11ßHSD1 was down-regulated by
insulin (20–100 mU/mL: P < 0.01) in the presence
of 20% serum, whereas incubation with insulin under serum-free
conditions resulted in a dose-dependent increase in 11ßHSD1 activity
(P < 0.05). Incubation with insulin-like growth
factor I resulted in a similar pattern of 11ßHSD1 activity. Although
neither testosterone nor androstenedione (5–200 nmol/L) affected
11ßHSD1 activity, incubation of myoblasts with dehydroepiandrosterone
(500 nmol/L) resulted in a decline in 11ßHSD1 activity
(P < 0.05). These data suggest that glucocorticoid
hormone action in skeletal muscle is determined principally by
autoregulation of GR, GRß, and 11ßHSD1 expression by the
ligand-binding GR isoform. Additionally, insulin and insulin-like
growth factor I regulation of 11ßHSD1 may represent a novel mechanism
that maintains insulin sensitivity in skeletal muscle tissue by
diminishing glucocorticoid antagonism of insulin action. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-sectional studies have revealed strong positive associations
among circulating levels of cortisol, blood pressure, glucose
intolerance, and hypertriglyceridemia and have led to suggestions that
chronic activation of the hypothalamic-pituitary-adrenal (HPA) axis may
underlie this relationship (15, 16). However, in most
obese, insulin-resistant subjects, circulating levels of cortisol are
normal or may even be slightly decreased (17).
Furthermore, there is evidence to suggest that metabolic clearance of
cortisol may be enhanced in obese subjects as a consequence of an
increase in 5-reductase activity in hepatic and adipose tissues
(18) accompanied by an increase in HPA drive to compensate
for lower plasma cortisol concentrations (19, 20). These
contradictory data have led to the suggestion that relatively modest
changes in the regulation of tissue sensitivity to glucocorticoid may
result in increased levels of glucocorticoid hormone action, which, in
turn, promote impaired insulin sensitivity, glucose intolerance, raised
blood pressure, and other features of the metabolic syndrome.
Levels of circulating glucocorticoid are determined principally by the
rate of cortisol secretion, which is regulated by ACTH under the
control of the HPA axis and its rate of metabolic clearance. In
contrast, tissue sensitivity to glucocorticoid is determined not only
by the levels of circulating cortisol, but also by the abundance of
glucocorticoid receptor (GR) and the availability of physiologically
active glucocorticoid in the intracellular compartment. Two isoforms of
the GR have been described that comprise splice variants of the
same gene (21). GR is able to bind ligand, whereas the
truncated ß isoform (GRß), which is unable to bind ligand, is
thought to act as a dominant negative inhibitor of glucocorticoid
hormone action through heterodimerization with GR (21, 22). Moreover, the expression of GR is regulated by its own
ligand, such that cortisol induces down-regulation of GR messenger
ribonucleic acid (mRNA) expression and stability and increases the
posttranslational turnover of GR protein (22, 23).
Similarly, two isoforms of 11ß-hydroxysteroid dehydrogenase
(11ßHSD) have been characterized and cloned. 11ßHSD1, largely
expressed in classical glucocorticoid target tissues, encodes
predominantly low affinity NADP(H)-dependent 11-oxoreductase (11-OR)
activity, generating physiologically active cortisol from its inactive,
11-oxo derivative, cortisone, and is colocalized with the GR
(24). In contrast, 11ßHSD2 encodes a high affinity
unidirectional NAD(H)-dependent 11-dehydrogenase that catalyzes the
conversion of cortisol to cortisone and is localized to
mineralocorticoid target tissue such as the renal distal convoluted
tubule and collecting duct, where it is believed to convey aldosterone
specificity upon the mineralocorticoid receptor (MR) (25).
Moreover, renal 11ßHSD2 activity is considered to contribute to the
metabolic clearance of cortisol and to be the principal source of
circulating cortisone in man (26, 27).
Compelling evidence suggests that the intracellular conversion of
cortisone to cortisol by 11ßHSD1 represents an important mechanism
for the prereceptor regulation of tissue sensitivity to glucocorticoid
in insulin-sensitive tissues by modulating the availability of active
ligand for binding with the GR (28). Moreover, recent
human studies point to key roles for both the GR and 11ßHSD1 in the
etiology of insulin resistance, hyperglycemia (11, 29),
and central obesity (30), and this has been supported by
animal studies in which pharmacological blockade of the GR abolishes
high fat diet-induced adiposity, glucose intolerance, and insulin
resistance (31), and deletion of the 11ßHSD1 gene
attenuates gluconeogenesis despite high circulating glucocorticoid
levels (28). Thus, the tissue-specific expression and
predominant 11-OR activity of 11ßHSD1 in glucocorticoid target
tissues may be considered to play a key role in the regulation of
tissue sensitivity to glucocorticoid through two interdependent
mechanisms: the direct regulation of intracellular levels of cortisol,
and the corollary of increased intracellular levels of cortisol upon
the regulation of GR.
The tissue-specific regulation of isoforms of 11ßHSD is poorly understood. However, several studies have produced evidence in support of the hypothesis that the regulation of 11ßHSD may be mediated through the actions of a number of hormones and growth factors. However, much of this research is contradictory and either predates the discovery of 11ßHSD2 or fails to characterize the species- and tissue-specific mechanisms that underlie the regulation of 11ßHSD in an isoform-specific manner. Nevertheless, these data have served to highlight key elements underlying the hormonal regulation of 11ßHSD, including glucocorticoids (30, 32, 33), insulin (34, 35), GH (35), thyroid hormones (36), and the sex steroids (37, 38) and support the hypothesis that dysregulation of enzyme activity may underlie the etiology of a spectrum of diseases, including essential hypertension (29), insulin resistance and glucose intolerance (11, 39), and central obesity (30).
Skeletal muscle represents a key target tissue for insulin-stimulated glucose uptake, metabolism, and utilization (40). Moreover, previous studies have shown that in vivo levels of insulin sensitivity and glycogen synthase activity are maintained in skeletal muscle cells grown in vitro (41). Although the molecular mechanisms of insulin-dependent glucose uptake in skeletal muscle have been extensively investigated, there are no reports of the molecular mechanisms underlying glucocorticoid hormone action in this tissue. Thus, the aims of this investigation were to characterize the regulation of glucocorticoid hormone action in skeletal muscle and to investigate the role of glucocorticoids in the development of the metabolic syndrome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Study subjects
Fourteen adult men comprising both lean-moderately overweight subjects (body mass index, 25–30 kg/m2; n = 7) and obese subjects (body mass index, >30 kg/m2; n = 7) as defined by WHO criteria were recruited for the study. Two subjects had type 2 diabetes, but none had evidence of other disease and were not receiving therapy. The experimental protocol was approved by the combined ethical committee of Southampton and Southwest Hampshire National Health Service Trust, and written informed consent was obtained from each subject. All of the nondiabetic subjects had normal glucose tolerance defined by a fasting glucose below 126 mg/dL and a 2-h glucose level following a standard 75-g oral glucose tolerance test of less than 140 mg/dL. At least 4 weeks before each component of the study, any hypoglycemic or related agents were withdrawn. Each subject was admitted to the Clinical Research Facility at Southampton General Hospital, where they consumed a standard weight maintenance diet comprising 55% of calories as carbohydrate, 30% as fat, and 15% as protein for at least 24 h before the studies.
Hyperinsulinemic-euglycemic clamp
Insulin resistance indexes were measured using the hyperinsulinemic euglycemic glucose clamp technique. All subjects were fasted for 12 h overnight before the procedure. Insulin and glucose infusions were administered into an antecubital vein. Blood sampling was performed from a dorsal vein on the opposite hand. This hand was warmed to enable sampling of arterialized blood. After a priming infusion of insulin, a continuous infusion of insulin was started at a rate of 60 mU/m2·min. The infusion was continued for 2 h. Plasma glucose was maintained at 5 mmol/L by variable glucose infusion. The amount of glucose to maintain euglycemia was taken as the amount of glucose metabolized (M). The mean plasma insulin (I) during steady state euglycemia (60–120 min) was calculated. The M/I ratio (milligrams per m2·min/microunits per mL) was used as the measure of tissue sensitivity to insulin.
Skeletal muscle biopsy and cell culture
Tissue was obtained by Bergstrom needle biopsy (26–143 mg) of
the vastus lateralis muscle in the thigh of 14 Caucasian men, the
characteristics of whom are shown in Table 1
. Two to 3 biopsies of muscle tissue
were obtained from each subject during the procedure and immediately
microdissected free of any fat or connective tissue at 4 C. Aliquots
were immediately snap-frozen in liquid nitrogen and stored at -80 C
for subsequent morphological and molecular studies. The majority of
material was, however, prepared for isolation of viable proliferating
skeletal muscle satellite cells. We have optimized techniques for the
isolation of viable satellite cells and the dispersal and proliferation
of skeletal muscle cells from them using significantly smaller biopsies
than have previously been reported.
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Immunohistochemical analyses
Cells were washed with PBS, air-dried, and fixed with either
10% formol saline or 4% paraformaldehyde. Five or six circles (20-mm
diameter) per dish of cells were marked out using a hydrophobic resin
to allow multiple parallel immunostaining with different antisera.
Nonspecific immunostaining was diminished by incubation with blocking
solution (5% serum from the species in which the secondary antisera
was raised; obtained from DAKO Corp., Copenhagen, Denmark)
in PBS for 1 h at room temperature, followed by washing with PBS
(three times) and incubation with primary antiserum for human GR (hGR)
at 1:100 to 1:1000 dilution (affinity-purified rabbit polyclonal
antibody raised against a 16-amino acid peptide corresponding to the
amino-terminus of GR common to both the 95-kDa GR and GRß isoforms
(obtained from Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), skeletal muscle desmin antisera at 1:10 to 1:100 dilution, mouse
monoclonal sarcomeric -actinin antisera at 1:100 to 1:1000 dilution,
mouse monoclonal antihuman fibroblast surface protein antisera at 1:500
to 1:2000 dilution, and mouse monoclonal anti -smooth muscle actin
at 1:500 to 1:2000 dilution for either 2 h at room temperature or
overnight at 4 C in a humidified box, as previously described
(42). Working dilutions of antisera were prepared using
PBS/0.05% Tween-20. Omission of primary antisera, absorption of
primary antisera with appropriate purified proteins, and the use of
nonimmune sera (DAKO Corp.) served as negative controls of
specific immunostaining (data not shown). After further washing with
PBS (three times), cells were incubated with horseradish
peroxidase-conjugated antirabbit IgG or antigoat IgM secondary antisera
at 1:1000 to 1:2500 dilution for an additional 1 h at room
temperature, and immunostaining was detected using brief incubation
with diaminobenzadine and visualized under light microscopy.
Estimation of 11ßHSD activity
Triplicate 25-cm2 flasks of subconfluent cells were incubated with 5 mL DMEM containing insulin (20–100 µU/mL), cortisol (50–1000 nmol/L), and cortisone (50–1000 nmol/L), separately and in combination for 48–96 h before assay of 11ßHSD activity. Cells were washed (three times) in hormone-free DMEM and incubated with 200 nmol/L cortisol for 24–48 h. Kinetic analyses of 11-OR and 11-dehydrogenase activities were conducted with concentrations of cortisone or cortisol in the range 31.25–1000 nmol/L for a period of 24–48 h. Aliquots of culture medium from each flask, before and after incubation with substrates for 11ßHSD activity, were removed for assay of cortisol and cortisone by high pressure liquid chromatography (HPLC). Briefly, steroids were quantitatively extracted from 4 mL culture medium (containing 80 µg dexamethasone as internal standard for the HPLC) through preconditioned Sep-Pak+ C18 cartridges. The steroids were eluted using 5 mL ethyl acetate/diethyl ether (4:1) and washed with 2 mL 1 mol/L NaOH saturated with Na2SO4, followed by 2 mL 1% acetic acid saturated with Na2SO4. The phases were separated, and the aqueous layer was discarded. The remaining organic layer was evaporated to dryness under a stream of dry nitrogen. The residue was reconstituted with 240 µL 20% acetonitrile/H2O, and 160 µL of this were injected onto a Waters Nova-Pak 60-angstrom 30-cm C18 reverse phase HPLC column. The steroids were detected by UV absorbance at 247 nm after programmed gradient elution with mobile phases comprising phase A (50 mmol/L KH2PO4 and 10 mmol/L acetic acid) and phase B (65% acetonitile in phase A) at a flow rate of 0.8 mL/min. Cortisol and cortisone levels were quantified against known internal standards.
Although the maximum absorbance for steroids with an
,ß-unsaturated ketone in the A ring can typically be found at 240
nm, the absorbance maximum for both cortisol and cortisone using this
technique (using several scanning UV spectrophotometers) was 247 nm.
This technique has been fully validated against fluorescence detection
and GC-MS (Donovan, S. J., et al., unpublished
observations). The limits of detection for this technique were
estimated to be 1.6 ± 0.8 nmol/L (i.e. approximately
2.4 ng steroid injected onto the column) for all steroids examined.
Mean analytical recoveries for cortisol and cortisone were 98.9 ±
2.5% and 96.5 ± 3.0%, respectively. Analytical imprecision
(CV%) was estimated to be 6.1 ± 1.2% (within-batch) and
8.3 ± 2.4% (between-batch) for a range of cortisol and cortisone
concentrations (60–500 nmol/L).
In addition to cortisol and cortisone, this technique was capable of
detecting all steroids with an unsaturated ring A and was validated for
the quantitative estimation of 6ß-hydroxycortisol,
20-dihydrocortisol, 20ß-dihydrocortisol, 20-dihydrocortisone,
20ß-dihydrocortisone, cortisol, cortisone, dexamethasone,
corticosterone, 11-deoxycortisol, 11- deoxycorticosterone,
17-hydroxyprogesterone, androstenedione, and progesterone. Although
more polar, tetrahydro metabolites of cortisol and cortisone would not
be detected by this technique, it is highly unlikely that this degree
of metabolism (more akin to hepatic than peripheral metabolism of
glucocorticoids) would occur in human skeletal myoblasts. Importantly,
the sum of cortisol and cortisone that was quantitatively extracted
from the cell culture medium subsequent to incubation was not
significantly different from that which had been added before
incubation (within the aforementioned limits of experimental error and
methodological imprecision). This is consistent with there being
undetectable levels of cortisol and cortisone in cell culture medium
containing FCS. As 11ßHSD1 behaved exclusively as an 11-OR in these
cells, any cortisol present in the cell culture medium remained
unmetabolized. This was confirmed by assay of cortisol concentrations
after incubation of cells with cortisol during assays of
11-dehydrogenase activity. To maintain first order kinetics for the
analysis of 11ßHSD1 activity in these cells, substrate
(i.e. cortisone) concentrations and sampling times were
established so that reaction rates were well within the linear part of
the reaction velocity vs. substrate concentration plot.
Western blot analysis
Cells were washed with ice-cold PBS (three times), gently scraped from flasks, briefly centrifuged (750 x g, 2 min), and suspended in either PBS/1 mmol/L phenylmethylsulfonylfluoride (for total cell protein) or in Nonidet P-40 lysis buffer (0.05% Nonidet P-40 in phosphate buffer, pH7.2) and subjected to differential centrifugation to enable isolation of nuclear and cytosolic protein. Varying concentrations of protein, assayed by the Bradford method using a commercially available kit (Bio-Rad Laboratories, Inc., Herts, Hemel Hempstead, UK), were mixed with 1 vol SDS-glycerol/ß-mercaptoethanol/bromophenol blue loading buffer, heated to 95 C for 5 min, and electrophoresed alongside molecular weight markers through 4% stacking and 10–12% resolving denaturing SDS-PAGE gels. After electroblotting (35 V/0.8 mA/cm2) onto Hybond C membranes (Amersham Pharmacia Biotech, Aylesbury, UK), samples were incubated with blocking solution (10% milk powder/PBS/0.05% Tween-20/1% goat serum) for 1 h at room temperature, followed by incubation with either rabbit antihuman hGR polyclonal antisera (Santa Cruz Biotechnology, Inc.) or sheep antihuman 11ßHSD1 polyclonal antisera (The Binding Site, Birmingham, UK) in 1% milk powder/PBS/0.05% Tween-20 for 3 h at room temperature. After washing with PBS (three times), membranes were incubated with either horseradish peroxidase-conjugated goat antirabbit IgG at 1:100 dilution for 1 h at room temperature (for human GR) or horseradish peroxidase-conjugated donkey antisheep IgG at 1:50,000 dilution (for human 11ßHSD1) for 1 h at room temperature. hGR and human 11ßHSD1 was visualized using the ECL Plus system (Amersham Pharmacia Biotech) and exposed to autoradiographic film within its linear range, and the signal was quantified by scanning laser densitometry.
RT-PCR
RNA was isolated from tissue and cultured myocytes using a
single step acidified phenol/chloroform extraction method (RNAzol B,
Biogenesis, Poole, UK). First strand DNA was synthesized
from 10 µg total RNA using reverse transcriptase-driven primer
extension from either random hexamers or 3'-antisense oligonucleotide
primers corresponding to human GR, GRß, 11ßHSD1, 11ßHSD2, MR,
and Na,K-adenosine triphosphatase 1-subunit, the sequences for which
are listed in Table 2, as we have
previously described (43). Briefly, RNA was heated to 65 C
for 5 min, snap-cooled to 4 C, mixed with reaction buffer (50 mmol/L
Tris-HCl, 50 mmol/L KCl, 10 mmol/L MgCl2, 10
mmol/L dithiothreitol, and 0.5 mmol/L spermidine, pH 8.3), ribonuclease
inhibitor (RNasin, Promega Corp., Southampton, UK),
deoxy (d)-NTPs (10 mmol/L each of dATP, dCTP, dGTP, and dTTP), 30 pmol
primers, and 200 U Superscript II (Pharmacia-Amersham Pharmacia Biotech) in diethylpyrocarbonate-treated water at a volume of 50
µL and incubated at 42 C for 1 h. Five to 10% of this reaction
served as a template for the PCR amplification of fragments of these
mRNAs using specific sense and antisense primers to generate DNA
products of the predicted sizes shown in Table 2. Complementary DNAs
(cDNAs) including pT7/T3 hGR cDNA fragment, pT7/T3 hGRß cDNA
fragment, pT7/T3-hGR full-length cDNA, 11ßHSD1/pcDNAI, and RT
products from previous assays served as positive controls for the PCR
step. A total RNA pool served as a positive control for the RT step.
Negative controls included PCR of nonreverse transcribed RNA samples
and omission of primers from both RT and PCR reactions.
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Total RNA (30–50 µg/lane) from tissue and cultured myocytes
was electrophoresed alongside RNA molecular weight markers
(Amersham Pharmacia Biotech) in a 1.5% agarose/15%
formaldehyde/1 x morpholinopropane-sulfonic acid (MOPS)
gel at 100 mA for 4–6 h, followed by transfer to Hybond
N+ membranes. Parallel dot-blot analyses of each
RNA preparation were also performed using a Hybridot apparatus
(Life Technologies, Inc.) to assist quantification of mRNA
recorded as arbitrary units in relation to those for 18S ribosomal RNA
by scanning laser densitometry. Membranes were hybridized with either
cDNA or complementary RNA (cRNA) probes for GR, GRß, 11ßHSD1,
and 18S in either 0.77 mol/L sodium phosphate/5 mmol/L ethylenediamine
tetraacetate/7% SDS/200 µg/mL denatured salmon sperm DNA (ssDNA)
buffer (pH 7.2) at 65 C for cDNA probes or 50% deionized
fomamide/2 x SSPE/5 x Denhardt’s solution/10%
dextran sulfate/0.1% SDS, 200 µg/mL ssDNA buffer at 42 C for cDNA
probes or 63 C for cRNA probes. Membranes were washed in 2 x SSC
(1 x SSC = 150 mmol/L NaCl and 15 mmol/L trisodium
citrate)-0.1% SDS (10 min at room temperature) and up to a maximum
stringency of 0.1 x SSC-0.1% SDS (30 min at 68 C). Hybridization
signals were analyzed using a PhosphorImager (model 850,
Molecular Dynamics, Inc., Amersham Pharmacia Biotech, Buckingham, UK) and were exposed to autoradiographic
film (DuPont-Cronex, Wilmington, DE) between intensifying screens at
-70 C for 1–10 days, such that the signal fell within the linear
range of the film. Before rehybridization with other probes, cDNA
probes were removed from membranes by washing with 1% SDS (3 h at 70
C), and cRNA and 18S ribosomal DNA probes were removed by washing with
0.1% SDS at room temperature.
Nucleic acid probes
cDNA probes for GR (44), GRß, h11ßHSD1
(45), and ribosomal 18S (46) were
radiolabeled with [32P]dCTP (3000 Ci/mmol) by
random priming of the excised cDNA fragment using commercially
available kits (Amersham Pharmacia Biotech). GR
isoform-specific probes were synthesized, as previously described
(23), from the 537-bp PstI/KpnI
fragment pT7/T3- cDNA by T7 RNA polymerase after linearization with
PstI (to detect GR mRNA alone) and from the 960-bp
PstI/SstI fragment pT7/T3-ß cDNA using T7 RNA
polymerase. After linearization with NsiI, a 581-bp probe
was generated (to detect GRß mRNA alone). Antisense h11ßHSD1 cRNA
probes (1.2 kb) were generated from the full-length h11ßHSD1 cDNA
(45), subcloned into pBluescript KS+
using T3 RNA polymerase after linearization with HindIII.
All cRNA probes were radiolabeled by incorporation of
[32P]UTP (3000 Ci/mmol), and synthesis of more
than 90% full-length cRNA probes was confirmed by autoradiography of
probes subjected to denaturing (7 mol/L urea) polyacrylamide (6%) gel
electrophoresis.
The hGR cDNAs were provided by Drs. Robert Oakley and John Cidlowski (NIH, Raleigh, NC), the h11ßHSD1 cDNA was provided by Prof. Perrin White (University of Texas Southwestern Medical Center, Dallas, TX), and the r18S ribosomal DNA was provided by Prof. Ira Wool (University of Chicago, Chicago, IL).
Statistical analyses
All data presented are expressed as the mean + SEM. Data were compared using unpaired Student’s t test. Where appropriate, this analysis was performed on log-transformed data. Values were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell isolation and growth protocols were optimized to yield
myoblasts of pure muscle origin (Fig. 1a). Serum deprivation induced the
formation of multinuclear myotubes. Induction of myotubes either
spontaneously or after serum deprivation coupled with positive
immunohistochemical staining for skeletal muscle desmin and sarcomeric
-actinin and negative staining for human fibroblast surface protein
and -smooth muscle actin confirmed yield and proliferation of
greater than 99% human skeletal myoblasts. All subsequent analyses
were performed on 95% confluent cells that were exclusively myoblasts
to ensure uniformity of experimental conditions across a large number
of tissue culture flasks. Immunohistochemical analyses of hGR and
11ßHSD1 staining revealed expression of hGR (Fig. 1b) and 11ßHSD1
(Fig. 1c) in all cells. hGR expression appeared to be distributed in
both nuclear and cytosolic compartments despite the absence of
glucocorticoid in culture medium containing 20% FCS (Fig. 1b). In
contrast, 11ßHSD1 expression was confined to the cytosol (Fig. 1c).
This was confirmed by Western blot analyses of hGR and 11ßHSD1
expression in nuclear and cytosolic protein fractions isolated by
differential centrifugation (Fig. 2).
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, GRß, and
11ßHSD1 expression
Analyses of gene expression by RT-PCR using primers specific for
GR, GRß, MR, and constitutively expressed Na/K-adenosine
triphosphatase 1-subunit (across a range of PCR cycle numbers)
revealed expression of GR and GRß, but no MR or 11ßHSD2, mRNA in
skeletal muscle biopsies (data not shown). Similar patterns of
expression were evident in cultured myoblasts under basal
glucocorticoid-free conditions, except that GRß mRNA was not
expressed in myoblasts from any of the subjects. The detection of GRß
mRNA in cells cultured in the presence of more than 100 nmol/L cortisol
suggests that GRß expression in skeletal myoblasts may be
up-regulated by glucocorticoid (47). This necessitated the
use of GR- and GRß-specific cRNA probes for quantitative analyses
of the expression of the ligand-binding GR and nonligand binding
GRß variant.
Northern blot analyses of total RNA isolated from human skeletal
myoblasts using a cRNA probe specific to GR indicated abundant
expression of GR mRNA comprising predominantly a 7.0-kb species
(Fig. 2a
). Detection of 95-kDa hGR protein by Western blot using
polyclonal hGR antisera in both nuclear and cytosolic protein fractions
isolated by differential centrifugation confirmed translation of GR
mRNA in these cells (Fig. 2b). Similarly, Northern blot analysis
indicated abundant expression of 1.4-kb 11ßHSD1 mRNA (Fig. 2c),
whereas Western blot analysis of total cell protein using a sheep
antihuman 11ßHSD1 primary antiserum revealed a single 34-kDa
immunoreactive protein that corresponded to an identical 11ßHSD1
protein species in human liver (Fig. 2d). No 11ßHSD1 protein
expression was detected in nuclear fractions (data not shown). In
intact cells, apparent Km and maximum velocity
(Vmax) for the conversion of cortisone to
cortisol were 440 ± 14 nmol/L and 75 ± 7 pmol/mg
protein·h and 437 ± 16 nmol/L and 33 ± 6 pmol/mg
protein·h (mean ± SEM; n = 4) in the presence
and absence of 20% FCS, respectively (Fig. 2e). Conversion of cortisol
to cortisone, indicative of 11-dehydrogenase activity, was
undetectable. Under basal, glucocorticoid-free conditions there were
marked between-subject differences in levels of GR and 11ßHSD1
mRNA expression, which were unaffected by cell passage number (passages
3–12) or cell density (up to 98% confluence; Fig. 3, a and c). Furthermore, the
between-subject differences in levels of GR and 11ß HSD1 mRNA
expression observed under basal conditions were maintained after
incubation with 500 nmol/L cortisol. GRß mRNA was not detected in
cultured myoblasts under basal, glucocorticoid-free conditions.
However, Northern blot analysis of total RNA from skeletal myoblasts
cultured in the presence of more than 100 nmol/L cortisol revealed
marked intersubject variability in levels of 6.5 kb GRß mRNA
expression (Fig. 3b). The variability attributable to sources of
methodological error was minor compared with the marked differences
evident between subjects. Thus, the percent coefficient of variation
analysis for both GR and 11ßHSD1 expression revealed that variability
possibly due to 1) intraflask variation was less than 5%; 2) temporal
variation (in nine subjects) was 8.7–19.7%, 3) intra-Northern blot
variation was 10.1–14.0%, 4) interpassage variation was 12.7–22.5%,
and 5) to cell density was 9.4–16.1%. In common with previous reports
of glycogen synthase activity and insulin sensitivity in skeletal
myoblasts (40, 41), in vitro variability in GR
and 11ßHSD1 expression is likely to be a true reflection of in
vivo variability between subjects.
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, GRß, and 11ßHSD1 by glucocorticoid
Removal of serum from the growth medium resulted in a marked
decline in the levels of GR and 11ßHSD1 mRNA expression and
11ßHSD1 11-OR activity. Incubation of cultured myoblasts isolated
from a representative lean insulin-sensitive subject with increasing
concentrations of cortisol in the presence and absence of 20% serum
resulted in a marked dose-dependent decline in both GR mRNA
expression and hGR protein (Fig. 4, a and
b). However, in the presence and absence of 20% serum, incubation of
skeletal myoblasts with a range of concentrations of cortisol (50–1000
nmol/L) resulted in a dose-dependent increase in 11ßHSD1 mRNA, 34-kDa
immunoreactive protein, and 11ßHSD1 11-OR activity (P
< 0.05; Fig. 4, c, d, and f). Importantly, incubation of cultured
myoblasts with cortisol under serum-free conditions enhanced 11ßHSD1
mRNA expression and 11-OR activity by a 2- to 3-fold greater extent
than when in the presence of serum (Fig. 4f). Furthermore, in keeping
with the changes in 11ßHSD1 mRNA and 34-kDa immunoreactive protein,
multiple kinetic analyses of enzyme activity revealed that incubation
of cultured myoblasts with 500 nmol/L cortisol had no significant
effect on the affinity of 11ßHSD1 for cortisone, but increased the
Vmax of the reaction by 40% (P
< 0.001). In marked contrast with the glucocorticoid-dependent
down-regulation of GR, incubation of cultured myoblasts with
increasing concentrations of cortisol resulted in a marked apparent
dose-dependent increase in GRß mRNA expression (Fig. 4e). Incubation
of cultured myoblasts with a range of concentrations of cortisone
(50–1000 nmol/L) also resulted in a dose-dependent increase in
11ßHSD1 mRNA, 34-kDa immunoreactive protein, and 11ßHSD1 11-OR
activity (Fig. 5, a and b). Importantly,
coincubation of myoblasts with cortisone and carbenoxolone abolished
the induction of 11ßHSD1 mRNA expression and 11ßHSD1 11-OR activity
observed after pretreatment of the cells with cortisone alone (Fig. 5, a and b), whereas incubation of cultured myoblasts with carbenoxolone
alone had no effect on 11ßHSD1 11-OR activity. GR mRNA expression
in cultured myoblasts was unaffected after incubation with cortisone or
carbenoxolone (Fig. 5c). Moreover, both down-regulation of GR and
up-regulation of 11ßHSD1 by cortisol were abolished by coincubation
with a 10-fold molar excess of the GR antagonist, RU38486 (Fig. 5, a
and c). These data suggest that the regulation of the basal expression
of GR, its regulation by cortisol, and the glucocorticoid-dependent
regulation of 11ßHSD1 expression in human skeletal myoblasts are
mediated exclusively by binding of cortisol to its receptor and that
the conversion of cortisone to cortisol by 11ßHSD1 11-OR activity may
represent the autoregulation of 11ßHSD1 11-OR activity by the product
of its own catalytic activity.
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and 11ßHSD1 by insulin, glucose, and
insulin-like growth factor I (IGF-I)
Northern blot analysis indicated that incubation of cultured
myoblasts with either insulin or increasing concentrations of glucose
had no effect on GR mRNA expression in these cells. Nevertheless,
although glucose had no effect on 11ßHSD1 mRNA expression or 11-OR
activity, incubation of cultured myoblasts in the presence of 20%
serum with increasing concentrations of insulin (20–100 µU/mL, which
approximate physiological levels of insulin in subjects with insulin
resistance) induced a marked reduction in 11ßHSD1 mRNA expression,
34-kDa immunoreactive protein, and 11ßHSD1 11-OR activity (Fig. 6, a, c, and d). In marked contrast,
however, incubation with increasing concentrations of insulin under
serum-free conditions induced a marked increase in 11ßHSD1 mRNA,
34-kDa immunoreactive protein, and 11ßHSD1 11-OR activity (Fig. 6, b–d). In common with previous experiments, multiple kinetic analyses
of 11ßHSD1 11-OR activity revealed that incubation of cultured
myoblasts with 100 µU/mL insulin had no effect on the affinity of
11ßHSD1 for cortisone (Km = 445 ± 6.1;
mean ± SEM), but caused a 19% decline in
Vmax in the presence of 20% FCS
(P < 0.01), whereas serum removal induced a 6%
increase in Vmax (P = <0.05).
Although incubation with GH (0.05–5 nmol/L) had no effect on the
expression of 11ßHSD1 mRNA or 11-OR activity, incubation of cultured
myoblasts with IGF-I (50–200 µg/L) in the presence and absence of
20% serum resulted in a pattern of 11ßHSD1 mRNA and 34-kDa
immunoreactive protein expression and 11ßHSD1 11-OR activity similar
to that observed in cultured myoblasts after incubation with insulin
(data not shown).
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In the presence of 20% serum the marked increase in 11ßHSD1
11-OR activity effected by incubation of cultured myoblasts with 200
nmol/L cortisol alone (1.3-fold over basal; P < 0.05)
was markedly attenuated by coincubation with insulin (20–100 µU/mL)
and cortisol (200 nmol/L) in combination (Fig. 7a). In contrast, under serum-free
conditions, the induction of 11ßHSD1 mRNA and 11ßHSD1 11-OR
activity in cultured myoblasts effected by 200 nmol/L cortisol alone
was markedly increased as a consequence of coincubation with 20 µU/mL
insulin and 200 nmol/L cortisol (2.1-fold over basal; P
< 0.0001; Fig 7, a and b). The markedly greater induction of 11ßHSD1
11-OR activity in skeletal myoblasts after incubation with 500 nmol/L
cortisol under serum-free conditions (2.4-fold over basal;
P < 0.0001) was also further enhanced (4-fold over
basal; P < 0.0001) as a consequence of
coincubation with 100 µU/mL insulin and 500 nmol/L cortisol (Fig. 7a). Northern blot analyses revealed parallel marked changes in 11ß
HSD1 mRNA (Fig. 7b).
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Incubation of cultured myoblasts with ACTH, GH,
T3, and a spectrum of the steroidogenic
precursors and metabolites of cortisol, cortisone, and aldosterone
(Table 3) had no effect on the expression
of 11ßHSD1 mRNA, 34-kDa immunoreactive protein, or 11ßHSD1 11-OR
activity in these cells. Incubation with 17ß-estradiol, progesterone,
testosterone, and androstenedione was similarly without effect.
However, incubation of cultured myoblasts with dehydroepiandrosterone
(DHEA; 500 nmol/L) in both the presence and absence of
20% serum induced a modest, but significant, decrease in 11ßHSD1
11-OR activity (P < 0.02; Fig. 8). However, changes in 11ßHSD1 11-OR
activity as a consequence of pre-treatment with DHEA at
concentrations below 500 nmol/L failed to achieve statistical
significance. Importantly, incubation of cultured myoblasts with the
more potent androgens, testosterone and androstenedione (5–200
nmol/L), had no effect on 11ßHSD1 11-OR activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study Northern blot analysis of total RNA from human skeletal
myoblasts cultured under basal, glucocorticoid-free conditions revealed
abundant gene expression of both GR and 11ßHSD1, whereas
immunohistochemical analysis of intact cells indicated positive
staining for hGR distributed throughout the cytoplasm, which was
colocalized with positive staining for 11ßHSD1. Importantly, kinetic
analysis indicates that 11ßHSD1 in intact human skeletal myoblasts
acts exclusively as an 11-OR, with kinetic constants consistent with
the cloned, native 11ßHSD1 isoform and rates of activity similar to
those reported in human hepatocytes (32), human adipose
stromal cells (30), and human colonic lamina propria cells
(48). These observations lend strength to the hypothesis
that 11ßHSD1 may play an important role in determining glucocorticoid
hormone action in skeletal muscle by regulating the level of active
glucocorticoid available for binding with functional GR. Importantly,
these observations also suggest that tissue sensitivity to
glucocorticoid hormone action in skeletal muscle, a key insulin target
tissue, is much greater than is generally recognized. Thus, as
glucocorticoids are potent antagonists of insulin action, it might be
predicted that the sensitive regulation of glucocorticoid hormone
action in skeletal muscle is an important prerequisite for the
preservation of insulin sensitivity in this tissue. Importantly, these
data also suggest that relatively small changes in glucocorticoid
hormone action may be accompanied by marked changes in glucocorticoid
sensitivity and consequently induce significant changes in levels of
insulin sensitivity in skeletal muscle.
Previous investigations have demonstrated that the regulation of
glucocorticoid hormone action and of tissue sensitivity to
glucocorticoid is achieved at least in part by the sensitive
down-regulation of GR expression in response to exposure to
glucocorticoid and that this is regulated at the level of mRNA
transcription and stability and to a lesser extent as a consequence of
increased protein turnover (22, 23). The close agreement
between levels of GR mRNA and protein and abolition of
glucocorticoid-mediated down-regulation by blockade with RU 38486
evident in our study is consistent with the predominantly
transcriptional regulation of GR expression in human skeletal
myoblasts and confirms that this is mediated exclusively by the
ligand-binding GR isoform. Importantly, this study represents the
first report of glucocorticoid-mediated up-regulation of the GRß
isoform in human skeletal myoblasts, and although the magnitude of
glucocorticoid-mediated GRß up-regulation is generally smaller than
the down-regulation of GR effected by the same concentration of
cortisol, it might be predicted that the combined effect of both
regulatory events would result in a marked decline in glucocorticoid
hormone action, in accordance with the law of mass action, as a
consequence of increased levels of GR/GRß heterodimerization. This
is supported by recent studies describing associations between
increased ratios of GRß/GR expression in peripheral blood
mononuclear cells and airway cells and both generalized glucocorticoid
resistance (49) and steroid-resistant asthma
(50).
It is widely accepted that 11ßHSD1 plays a significant role in the
maintenance of tissue sensitivity to glucocorticoid by modulating the
accessibility of active glucocorticoid to functional GR. In common with
previous in vitro studies using cultured human adipose
stromal cells (30, 33) and hepatocytes (51),
our data indicate that in human skeletal myoblasts, 11ßHSD1 mRNA,
immunoreactive protein, and 11-OR activity are sensitively up-regulated
by physiological concentrations of cortisol in a dose-dependent manner.
Furthermore, abolition of the glucocorticoid-dependent induction of
11ßHSD1 by RU38486 confirms that this effect is mediated exclusively
by GR. As a consequence, we propose that the magnitude of the
glucocorticoid-mediated induction of 11ßHSD1 expression in skeletal
myoblasts is likely to be tempered by the combined effects of
glucocorticoid-dependent down-regulation of GR and up-regulation of
GRß, which are themselves dependent upon intracellular levels
of active glucocorticoid. Indeed, as the level of cortisol within the
cell is dependent upon 11ßHSD1 11-OR activity, it is likely that
glucocorticoid-dependent up-regulation of 11ßHSD1 represents a
mechanism by which sensitivity to glucocorticoid, and hence
glucocorticoid hormone action, is maintained in skeletal muscle even
when levels of functional GR decline as a consequence of the
glucocorticoid-dependent fall in the GR/GRß ratio.
Thus, the regulation of glucocorticoid hormone action in skeletal
muscle may be considered to be the product of two GR-mediated
mechanisms characterized by 1) a decline in functional GR as a
consequence of the sensitive glucocorticoid-dependent down-regulation
of GR expression and up-regulation of GRß, accompanied by 2) the
sensitive glucocorticoid-dependent GR-mediated up-regulation of
11ßHSD1 11-OR activity. Indeed, it is likely that these two
mechanisms are mutually interdependent, because the failure to decrease
functional GR might be predicted to result in a greater increase in
GR-mediated 11ßHSD1 expression. Importantly, this might also be
predicted to result in higher levels of intracellular cortisol and a
persistent amplification of glucocorticoid hormone action. These
events, by analogy with the insulin resistance that accompanies
Cushing’s syndrome, might also be predicted to result in
tissue-specific glucocorticoid-mediated insulin resistance and may
represent a mechanism underlying the insulin resistance of the
metabolic syndrome.
The association between glucocorticoid excess, central obesity (14), and insulin resistance (52) and evidence for the regulation of 11ßHSD1 by insulin (34, 53) has prompted speculation that dysregulation of 11ßHSD1 activity may underlie the pathogenesis of glucocorticoid-dependent insulin resistance (11). Moreover, this hypothesis is strengthened by the observations detailed in the present study, which highlight a key role for 11ßHSD1 in the intracellular amplification of glucocorticoid hormone action. In conditions such as noninsulin-dependent diabetes mellitus and the metabolic syndrome, skeletal muscle is considered to represent the principal site of insulin resistance (40, 41), and data from the present study suggest that this tissue may also be considered to represent an important glucocorticoid target tissue. Importantly, our data indicate that although neither glucose nor insulin alters the expression of functional GR in cultured myoblasts, insulin does effect a marked in vitro decline in 11ßHSD1 11-OR activity in skeletal myoblasts in the presence of serum. This is in marked contrast with contemporary studies of human adipose stromal cells in which insulin appears to have no effect on 11ßHSD1 activity (33). However, these contrasting observations may be interpreted to represent the tissue- or cell type-specific differential regulation of 11ßHSD1 by insulin. Nevertheless, our data suggest a novel role for insulin in the regulation of glucocorticoid hormone action in skeletal muscle and, by analogy with the improved insulin sensitivity induced by pharmacological inhibition of 11ßHSD1 (39) or 11ßHSD1 gene knockout (28), suggest that down-regulation of 11ßHSD1 11-OR activity by insulin represents a mechanism of glucocorticoid antagonism that serves to maintain insulin sensitivity in skeletal muscle.
In contrast with previous reports (34), our data indicate
that insulin induces a marked increase in 11ßHSD1 mRNA and 11-OR
activity in cultured myoblasts after removal of serum from the growth
medium. Moreover, under serum-free conditions, coincubation of cultured
myoblasts with glucocorticoid and insulin in combination results in an
increase in 11ßHSD1 mRNA and 11-OR activity that is greater than the
sum of the increase induced by either glucocorticoid or insulin alone.
Importantly, these data suggest a role for unidentified serum factors
in defining the nature of the interaction between insulin and
glucocorticoid in skeletal myoblasts in vitro and are likely
to reflect similarly undefined interactions in vivo. In
support of this supposition, previous studies have described the potent
up-regulation of 11ßHSD1 11-OR activity by interleukin-1ß and tumor
necrosis factor- (TNF) (47, 54). Moreover, a marked
increase in circulating levels of TNF has been reported in obese
patients and in patients with noninsulin-dependent diabetes mellitus.
Importantly, serum levels of TNF correlate with decreased insulin
sensitivity in these subjects (55), and TNF is
overexpressed not only by adipose tissue, but also by skeletal muscle
in obese subjects (56).
Evidence from human and rodent in vivo studies supports the hypothesis that GH inhibits hepatic 11ßHSD1 activity (37, 57, 58). However, to date there have been few reports of the regulation of 11ßHSD1 by GH in tissues other than those of hepatic origin. Moreover, the role of IGF-I in the regulation of 11ßHSD1 has not previously been addressed, and data from the present study suggest that the regulation of this enzyme by GH may occur indirectly and be mediated through the action of IGF-I. The metabolic effects of GH are varied, but include the mobilization of fatty acids from triacylglycerols in adipose tissue and the stimulation of hepatic glycogenolysis. However, many of these effects are mediated by IGF-I, which is synthesized and secreted in the main by the liver in response to GH and to a lesser extent by skeletal muscle during periods of exercise (59). Our data indicate that GH has no effect on 11ßHSD1 activity in skeletal myoblasts per se. However, incubation of skeletal myoblasts with IGF-I in the presence and absence of serum results in changes in 11ßHSD1 11-OR activity and 11ßHSD1 immunoreactive protein that mirror those observed when the cells are exposed to insulin under similar conditions. Importantly, the metabolic actions of IGF-I are similar to those of insulin, and the expression of IGF receptor in skeletal muscle explains the in vivo effects of IGF-I in this tissue where it stimulates glucose uptake, glycolysis, and glycogen synthesis (60). Thus, by analogy with the proposed role for insulin in the antagonism of glucocorticoid hormone action in skeletal muscle, the paracrine secretion of IGF-I might be predicted to improve insulin sensitivity in this tissue not only through direct effects on carbohydrate metabolism, but also through the down-regulation of 11ßHSD1 11-OR activity.
Indirect evidence for the regulation of 11ßHSD1 by androgens has come from studies of the sexually dimorphic expression of isoforms of 11ßHSD1 in both rodent and human models (58, 61). Most studies imply that in vivo regulation of 11ßHSD1 occurs as a consequence of the potent androgenic effects of testosterone. Nevertheless, the present study indicates that the potent androgens, androstenedione and testosterone, have no effect on 11ßHSD1 11-OR activity in skeletal myoblasts, but exposure to DHEA, a relatively week androgen, attenuates 11ßHSD1 11-OR activity in these cells. These observations confirm previous reports of a DHEA-dependent decline in hepatic 11ßHSD1 in spontaneously hypertensive rats after treatment with DHEA sulfate (62). Furthermore, these observations suggest that the activity of steroid sulfatases may play a significant role in mediating the effects of administered DHEA sulfate on 11ßHSD1 activity. This leads to the intriguing possibility that despite the relatively low levels of DHEA in the general circulation, enzymatic cleavage of DHEA sulfate, which in man circulates at micromolar concentrations, by steroid sulfatase activity may play a role in the tissue-specific regulation of 11ßHSD1 activity in a variety of tissues. Moreover, recent reports have suggested that the age-related decline in DHEA sulfate levels is associated with an increased risk of cardiovascular disease and insulin resistance (63). Importantly, the failure of androstenedione and testosterone to effect the regulation of 11ßHSD1 11-OR activity in skeletal myoblasts suggests that regulation of 11ßHSD1 by DHEA is not mediated by the androgen receptor, and the mechanism underlying the regulation of 11ßHSD1 by DHEA awaits explanation.
In summary, abundant levels of functional GR and 11ßHSD1 expression
in human skeletal myoblasts indicate not only that skeletal muscle
represents a key site for insulin-stimulated glucose uptake, but also
that this tissue is an important glucocorticoid target tissue.
Importantly, glucocorticoid-dependent down-regulation of GR in
skeletal myoblasts is accompanied by glucocorticoid-dependent
up-regulation of GRß and a decline in the GR/GRß ratio. The
consequent decline in GR function, and hence glucocorticoid hormone
action in this tissue, may be tempered by a concomitant
glucocorticoid-dependent increase in 11ßHSD1 11-OR activity to
maintain intracellular levels of active glucocorticoid. Furthermore,
down-regulation of 11ßHSD1 11-OR activity by insulin and the
paracrine secretion of IGF-I may represent a mechanism that maintains
insulin sensitivity in skeletal muscle by diminishing the impact of
glucocorticoid antagonism of insulin action. Although the physiological
significance underlying the regulation of 11ßHSD1 by
DHEA remains unclear, the data presented in this study
suggest that regulation of 11ßHSD1 by DHEA may also
contribute to the regulation of glucocorticoid hormone action in human
skeletal muscle.
Received October 30, 2000.
Revised February 5, 2001.
Accepted February 7, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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