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Departments of Public Health and Clinical Medicine (Å.J., T.O.) and Medical Biosciences (Å.J., K.C.), Umeå University Hospital, SE-901 85 Umeå, Sweden; Department of Internal Medicine (H.F.), Boden Hospital, SE-961 85 Boden, Sweden; and Department of Medical Sciences (R.A., B.R.W.), University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
Address all correspondence and requests for reprints to: Tommy Olsson, M.D., Department of Public Health and Clinical Medicine, Medicine, Umeå University Hospital, SE-901 85 Umeå, Sweden. Tommy. Olsson{at}medicin.umu.se
Abstract
Dysfunction of the hypothalamic-pituitary-adrenal axis might contribute to metabolic disturbances frequently encountered in myotonic dystrophy. We hypothesized that abnormal adrenocortical sensitivity to ACTH and/or glucocorticoid metabolism could be important in myotonic dystrophy.
We assessed diurnal rhythmicity of saliva cortisol, adrenocortical reactivity by a low-dose (1 µg) Synacthen test, and glucocorticoid metabolism in blood and urine in 42 myotonic dystrophy patients (22 males) and 50 controls (27 males). CTG triplet repeat expansions were quantified by Southern blot.
Diurnal rhythmicity of saliva cortisol was flattened in both men and
women with myotonic dystrophy, with significantly increased
afternoon/evening levels (P < 0.013). The cortisol
response to ACTH was associated with increased (CTG)n
expansions in myotonic dystrophy men and women (P
< 0.01). Male myotonic dystrophy patients also had increased
activation of cortisol from cortisone by 11ß-hydroxysteroid
dehydrogenase type 1. Both men and women with myotonic dystrophy had an
increased 5
/5ß-reductase ratio (P < 0.05 and
P < 0.01, respectively). Cortisol metabolites were
related to the genetic defect in myotonic dystrophy men
(P < 0.05), whereas ratios reflecting
11ß-hydroxysteroid dehydrogenase type 1 activity in myotonic
dystrophy women were positively associated with obesity
(P < 0.05).
Increased 11ß-hydroxysteroid dehydrogenase type 1 activity and adrenocortical reactivity to ACTH are related to the genetic defect in myotonic dystrophy men, whereas abnormal glucocorticoid metabolism is associated with alterations in body composition in female myotonic dystrophy patients. These disturbances may explain altered circulating cortisol levels and contribute to features of the metabolic syndrome in myotonic dystrophy.
MYOTONIC DYSTROPHY (DM1) is the most common inherited form of muscle dystrophy among adults, associated with muscle atrophy and the characteristic myotonia (1). The genetic defect causing DM1 is an expansion of a CTG triplet repeat at chromosome 19, encoding a protein kinase named myotonic dystrophy protein kinase (2). The number of CTG repeat expansions has been associated with some clinical features, notably cognitive dysfunction and male hypogonadism (3, 4, 5).
Features of the metabolic syndrome, including insulin resistance and hypertriglyceridemia, in conjunction with hyperinsulinemia and increased fat mass, are present in DM1 (1, 6, 7, 8, 9). Abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis has been suggested to be associated with these metabolic abnormalities (10). Earlier studies in DM1 patients show multiple abnormalities of the HPA axis, including an increased ACTH response to CRH-mediated stimuli (11, 12, 13, 14). Recently, we have reported increased median 24-h cortisol levels with a concomitant increase in proinflammatory cytokines in DM1 (15).
An enhanced adrenal responsiveness to ACTH stimulation could contribute to increased HPA axis activity, but earlier studies of adrenal responsiveness in DM1 patients have yielded conflicting results (11, 16, 17, 18, 19, 20, 21). Most studies suffer from methodological weaknesses such as a small number of patients, lack of controls, supraphysiological doses, and/or im administration of ACTH (Synacthen), and almost exclusively measurements of urinary 17-hydroxycorticosteroids rather than plasma cortisol. Whether an increased sensitivity of the adrenal cortex to "physiological" ACTH stimulation is present in DM1 has not, to our knowledge, been studied.
Alternatively, altered circulating cortisol levels in DM1 may reflect
abnormal metabolism of glucocorticoids. Glucocorticoids are metabolized
by several enzymes, including irreversible inactivation by A-ring
reductases (5- and 5ß-reductases) and reversible interconversion
between active cortisol and inactive cortisone by 11ß-hydroxysteroid
dehydrogenases (11ßHSDs) (Fig. 1
). Alterations in peripheral
metabolism of glucocorticoids have been proposed to influence cortisol
secretion and circulating levels in other syndromes including obesity
(22, 23).
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Materials and Methods
Subjects
Clinical data of the subjects are summarized in Table 1. Twenty-two men and 20 women
with adult onset DM1 were recruited from the Dystrophia Myotonica
Center in Boden, northern Sweden, where the prevalence of the disease
is exceptionally high (24). All patients included had
clinically overt myotonia and muscular dystrophy. The diagnoses were
based on genetic analyses. Three male and 10 female patients were
smokers, and 1 patient used snuff. Twenty-seven male and 23 female
controls were recruited from healthy volunteers. Two female controls
were smokers, two female and three male controls used snuff, and one
male and one female control both smoked and used snuff.
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This study was approved by the regional ethical committee, and all participants had given their informed consent to participate.
Sampling and measurements
All participants collected urine during 24 h before the blood sampling. Saliva samples were collected at 1100, 1600, 2200, and 0700 h. In nine male patients and eight male controls, saliva was collected also at 0200 h.
After an overnight fast, baseline blood samples were collected at 0700 h. An indwelling cannula was inserted at least 15 min before the baseline sample. A low-dose Synacthen test was performed with 1 µg Synacthen that was injected iv at 0700 h. Blood samples for analysis of cortisol was collected at 30 and 40 min, as described by Rasmuson et al. (25).
At 1000 h the subjects took 25 mg cortisone actetate (Cortone) orally, and blood samples for analysis of cortisol were collected every 15 min for 2 h.
Body composition was measured by bioelectrical impedance analysis (BIA; Akern-RJL Systems BIA 101, EL-DOT K/S, Fredriksværk, Denmark). The BIA failed to measure seven patients due to too high resistance (>999 ohm). One patient denied to undergo BIA measurements.
Analytical methods
Saliva concentrations of cortisol were determined in untreated
samples by RIA using commercial kits obtained from Orion Diagnostica
(Esbo, Finland). Serum cortisol, dehydroepiandrosterone sulfate
(DHEAS), T, 17-hydroxyprogesterone (17 OHP), and
4-androstene-3,17-dione (A4) concentrations were analyzed by
immunoassays from Diagnostics Products (Los Angeles, CA)
and INCSTAR Corp. (Stillwater, MN). Serum insulin
concentrations were analyzed by an immunoassay from Abbott Diagnostics
(Abbott Park, IL).
Urine cortisol, cortisone, and their metabolites were measured by gas chromatography and electron impact mass spectometry following Sep-Pak C18 extraction, hydrolysis with ß-glucuronidase, and formation of methoxime-trimethylsilyl derivative, as described previously (26). Epi-cortisol and epi-tetrahydrocortisol were used as internal standards that were added to samples before extraction. Peaks of interest were quantified by the ratio of (area under the peak)/(area under internal standard peak). Ratios were compared against standard curves for each steroid included in every assay batch.
Detection limits were: saliva cortisol, 0.19 nmol/liter; serum cortisol, 7 nmol/liter; A4, 0.4 nmol/liter; 17 OHP, 0.2 nmol/liter; DHEAS, 0.05 µmol/liter; T, 0.2 nmol/liter; insulin, 1.0 mU/liter; urine steroids, 1 µg/liter.
Genetic analyses
Genomic DNA was prepared from blood collected in EDTA tubes according to standard procedures and digested with EcoRI or PstI according to the manufacturer’s instructions. Southern blotting and hybridizations were performed with standard methodology (27). The probe used was pM10M6 (2), a 1.4-kb fragment that flank the expanded region of the myotonic dystrophy protein kinase gene.
The allele sizes were calculated with the computer program DNAfrag, version 3.03.
Data interpretation
The HPA axis was evaluated by several indices: 1) diurnal
variation of salivary cortisol; 2) stimulation of cortisol release by
exogenous ACTH; and 3) cortisol production rate via estimation of total
cortisol metabolite excretion [i.e. the sum of the daily
excretion of the principal urinary metabolites of cortisol and
cortisone: 5ß-tetrahydrocortisol (ß-THF), 5-tetrahydrocortisol
(-THF), tetrahydrocortisone (THE), - and ß-cortols, and -
and ß-cortolones] (28).
Metabolism of glucocorticoids was estimated by: 1) ratios of urinary
metabolites of cortisol, from which 11ßHSD activities are reflected
in the (-THF + ß-THF)/THE ratio, and the balance of 5 and 5ß
reductase activities are reflected in the -THF/ß-THF ratio; 2) the
accumulation of cortisol in peripheral serum following oral
administration of cortisone, which predominantly reflects hepatic
11ßHSD1 activity (29, 30).
Statistical analyses
All statistics were performed using a commercial computer program, SPSS (SPSS, Inc., Chicago, IL). We used Spearman’s rank correlation test for correlation analyses and Mann-Whitney U test exact P value for comparisons between groups. As post hoc test for individual time points in tests with repeated measurements we used the Mann-Whitney U test with Bonferroni correction for relevant time points.
To adjust for different baselines when comparing responses in the Synacthen and the conversion tests, we used the centered cumulative response [CCR = (area under the curve) - (baseline level x total minutes of sampling)].
For subgroup analyses we divided DM1 patients into two groups according to the number of CTG repeats, using the median as cut-off.
In cases where the hormone level was below the detection limit, the value was set to half the detection limit for statistical calculations. A P value of less than 0.05 was considered significant.
Results
CTG triplet repeat expansions
The median number of CTG triplet repeat expansions in the entire group was 679 and was used as cut-off for subgroup analyses. Median CTG repeat length was for men 666 (409–1168; 10th and 90th percentiles, respectively) and for women 691 (71–1100).
Gender differences
Serum cortisol levels at all sampling times after cortisone
intake, and accordingly the area under the curve response, were higher
in female controls compared with male controls (Fig. 2A). Female controls also had
significantly lower total urinary glucocorticoid metabolite excretion
(Table 2) and lower urinary levels of
-THF, ß-THF, and THE than male controls (P <
0.001, P < 0.001, and P < 0.05,
respectively). There were no other gender differences in controls or
patients. Given these differences between men and women, all data were
analyzed separately for each gender group.
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Total urinary glucocorticoid metabolite excretion did not differ
significantly between male or female patients and controls (Table 2
).
In DM1 men, diurnal rhythmicity of saliva cortisol was abnormal with
increased levels from noon onward (Fig. 3A) and increased median 24-h levels
compared with male controls
(P < 0.01).
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).
In DM1 women, the diurnal rhythmicity of cortisol was abnormal with
increased late evening cortisol levels (Fig. 3B). Median 24-h levels
were, however, not increased, and there were no relationships to the
number of CTG triplet repeat expansions.
Adrenocortical sensitivity to ACTH
There were no significant differences between men with DM1 and
healthy men regarding adrenal reactivity to ACTH. However, the response
to iv Synacthen was significantly increased in males with long CTG
repeat expansions compared with male controls and to DM1 males with
short CTG repeat expansions (Fig. 4A). In
DM1 men, increasing number of CTG repeats correlated significantly to
increased serum cortisol levels at 30 and 40 min after Synacthen
administration (rs = 0.58 and
rs = 0.60; P < 0.01).
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). However, serum cortisol levels
at 30 min after Synacthen administration correlated significantly and
positively to increasing number of CTG triplet repeat expansions
(rs = 0.62; P < 0.01), whereas
the area under curve response to Synacthen correlated to body mass
index (BMI) (rs = 0.47; P <
0.05) in DM1 women. Glucocorticoid metabolism
The generation of cortisol from cortisone was markedly increased
in DM1 men (Fig. 2B). When divided into two groups based on CTG repeat
numbers, there was a significant increase in cortisone
cortisol
conversion in men with long CTG repeats vs. controls (Fig. 2C). Conversion of cortisone to cortisol did not differ between female
DM1 patients and female controls (Fig. 2D).
Urinary glucocorticoid metabolite excretion and metabolite ratios are
summarized in Table 2 and Fig. 5 whereas
correlations between BMI/insulin and urinary glucocorticoid metabolite
ratios are shown in Table 3.
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-reduced metabolites
(-THF) in both men and women with DM1 (Table 2). There were trends
toward positive associations between 5/5ß-THF ratios and obesity
or hyperinsulinemia, but these did not reach statistical significance.
Urinary ratios reflecting 11ß-HSD activities (cortisol/cortisone and
THFs/THE ratios) were not different between DM1 patients, and controls
but were positively associated with BMI and fasting insulin levels in
DM1 women (Table 3).
The number of CTG repeats correlated negatively to -THF
(rs = -0.55; P = 0.01) and to
ß-THF (rs = -0.52; P < 0.05)
among male patients.
Androgens
Serum levels of T (P < 0.001; Table 1) and DHEAS
[2.0 (0.4–5.4) vs. 5.0 (2.2–7.9); P <
0.001, patients and controls, respectively] were decreased in DM1 men
compared with healthy men. In DM1 women, serum levels of T
(P < 0.01; Table 1), androstenedione [4.2 (2.5–9.0)
vs. 8.1 (4.2–14.9); P < 0.001], and DHEAS
[1.0 (0.4–2.8) vs. 3.3 (1.6–6.2); P <
0.001] were decreased. There were no differences in 17 OHP levels
(data not shown).
There were no significant correlations between cortisol data and adrenal/gonadal androgens (androstenedione, DHEAS, 17 OHP, or T) in DM1 patients.
Body composition
Fat mass was significantly increased in DM1 patients. There were no significant differences in BMI or body fat mass between patients with long and short CTG repeat expansions.
No association between urinary glucocorticoid metabolites or adrenocortical reactivity, on one hand, and body fat mass, on the other, was seen in DM1 males, except for the E/THE ratio (rs = -0.54; P < 0.05). In females, the pattern of correlations paralleled those for BMI in large.
In multiple regression analyses, body fat mass was an independent predictor of urinary F/E levels; otherwise no significant associations were found between glucocorticoid measures and body fat mass when gender, age, and disease per se were included in the multivariate models.
Discussion
This study of a large cohort of male and female DM1 patients contributes to the understanding of previous reports on disturbed diurnal rhythmicity and increased circulating 24-h levels of cortisol in male DM1 patients (14, 15). The main findings of this study are an association between the reactivity of the adrenal cortex to ACTH and the number of CTG repeat expansions, together with alterations in glucocorticoid metabolism in DM1, including an increased reactivation of cortisone to cortisol. These abnormalities are present mainly in males with DM1.
Previous studies have documented alterations in hypothalamic control of ACTH secretion in DM1 (12, 13, 31). In the current study, we show, using a physiological dose of ACTH, that adrenal responsiveness is increased in males with long CTG repeat expansions. In line with results from a recent study showing an unaltered cortisol response to naloxone (stimulating hypothalamic CRH secretion) in DM1 women (31), we found no difference between our DM1 women and healthy females.
We found no increase in 24-h total cortisol metabolite excretion in urine, suggesting that cortisol production may not be increased and the elevated circulating cortisol concentrations in DM1 may reflect impaired metabolism of cortisol.
Glucocorticoid metabolism and clearance has earlier been reported
mainly normal in DM1 (17, 18, 20, 21, 32), based on
analyses of either urinary 17-hydroxycorticosteroids or 17-ketogenic
steroids, methods that also measure noncortisol metabolites
(33). Using specific gas chromatography and electron
impact mass spectometry assays, we found evidence for altered
A-ring reduction of cortisol in both men and women with DM1. This would
be consistent with impaired 5ß-reductase activity in DM1. The
observation is complicated, however, by the previous findings in men
and women that idiopathic obesity is associated with enhanced
5-reductase activity (23, 34). DM1 patients have a
greater proportion of body fat, which may have contributed to the
changes in urine metabolite excretion. However, in multiple regression
analyses, only the urinary F/E ratio was independently associated with
increased fat mass. Furthermore, we can not exclude differences in
other metabolic pathways, such as 6ß-hydroxylation or side-chain
cleavage.
An alternative explanation for impaired peripheral clearance of cortisol in DM1 men is the increased reactivation of cortisone to cortisol that we show here for the first time. Enhanced regeneration of the inactive glucocorticoid cortisone to the active compound cortisol suggests an increased 11ßHSD1 activity in male DM1 patients. Increased 11ßHSD1 activity has been postulated to be important for the development of insulin resistance by increasing cortisol concentrations and gluconeogenesis in the liver (35), and it has been shown that knockout of this enzyme in mice impairs gluconeogenesis, thereby lowering circulating glucose levels (36). However, 11ßHSD1 activity was recently reported to decrease in the liver (i.e. decreased conversion of cortisone to cortisol) with increasing BMI (37, 38). In contrast, fat cells have been suggested as an important target for reactivation of cortisone (38, 39).
Our study emphasizes gender differences in 11ßHSD1 activity and
suggests hormonal regulation of this enzyme. Possible hormonal
mediators of 11ßHSD1 activity include gonadal/adrenal androgens,
insulin, and GH (40, 41, 42, 43, 44, 45). No associations to circulating
levels of androgens or insulin were found in the present study, but
these hormones could explain the gender-specific differences in
11ßHSD1 activity, which was lower in control men than in control
women and DM1 patients of either gender. The genetic defect of DM1 may
indirectly, through one of these hormonal regulators, account for
"feminization" of 11ßHSD1 activity in DM1 men. In line with this,
gender-specific differences have recently been shown in the expression
of genes from the DM1 locus (46). Other factors seem to
affect 11ßHSD1 activity in women, including a more potent effect of
obesity, reflected in the relationship between obesity/hyperinsulinemia
and urinary cortisol/cortisone metabolite ratios shown in Table 3.
The activity of 11ßHSD1 is also influenced by cytokines, including
TNF- and IL-1ß (47). Circulating 24-h TNF- levels
are increased in DM1 (15) and may increase 11ßHSD1
activity in the liver and fat cells (48). Whether fat cell
production of TNF- is increased in DM1 remains to be studied.
TNF- production might, thus, constitute a link between the profound
insulin resistance, increased fat mass, and increased tissue-specific
glucocorticoid concentrations in DM1 patients.
In conclusion, adrenal cortex reactivity to ACTH and enhanced 11ßHSD1 activity in DM1 are related to the number of CTG repeat expansions in men. In DM1 women, these abnormalities are less striking and altered glucocorticoid metabolism is associated with body composition. Increased concentrations of cortisol in the circulation and in key target tissues including liver and fat may contribute to the features of the metabolic syndrome in DM1, notably insulin resistance and hypertriglyceridaemia.
Acknowledgments
We thank research nurse Eva Krylborg for excellent practical assistance and statistician Hans Stenlund for valuable help in statistical matters.
Footnotes
This study was supported with grants from the Northern County Councils Cooperation Committee (Visare Norr) and the Swedish Association of Neurologically Disabled (NHR). T.O. is a Senior Research Fellow for the Swedish Medical Research Council. B.R.W. is a British Heart Foundation Senior Research Fellow.
Abbreviations: A4, 4-Androstene-3,17-dione; BIA, bioelectrical
impedance analysis; BMI, body mass index; CCR, centered cumulative
response; DHEAS, dehydroepiandrosterone sulfate; DM1, myotonic
dystrophy; HPA, hypothalamic-pituitary-adrenal; 11ßHSD,
11ß-hydroxysteroid dehydrogenase; 17 OHP,
17-hydroxyprogesterone; THE, tetrahydrocortisone; THF,
tetrahydrocortisol.
Received October 10, 2000.
Accepted May 29, 2001.
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, n = 27) and healthy
women (
, n = 23) (CCR, P < 0.001); B, male
DM1 patients (
, n = 22) and male controls (
60 min, P < 0.05, 
, n = 11) and short (
, n = 11) CTG repeat expansions
(more or less than 679 CTG repeat expansions) and in male controls
(
, P < 0.05; 
, P < 0.05; 
, P
< 0.05 (short vs. long repeats). Please note the
different scales on the y-axes in A and C compared with B and D.