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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3169-3176
Copyright © 2000 by The Endocrine Society

Original Studies

Abnormal Cytokine and Adrenocortical Hormone Regulation in Myotonic Dystrophy1

Åsa Johansson, Kjell Carlström, Bo Ahrén, Kristina Cederquist, Eva Krylborg, Håkan Forsberg and Tommy Olsson

Departments of Medicine (Å.J., T.O.), Clinical Chemistry (Å.J.), and Clinical Genetics (K.Ce.), Umeå University Hospital, 901 85 Umeå; Department of Obstetrics and Gynecology and Clinical Research Center (K.Ca.), Karolinska Institute, Huddinge University Hospital, 141 86 Huddinge; Department of Medicine (B.A.), Malmö University Hospital, 205 02 Malmö; and Department of Internal Medicine (E.K., H.F.), Boden Hospital, 961 85 Boden, Sweden

Address correspondence and requests for reprints to: Åsa Johansson, Department of Clinical Chemistry, Umeå University Hospital, 901 85 Umeå, Sweden. E-mail: asa.johansson{at}medicin.umu.se


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Metabolic-endocrine dysfunctions, including hyperinsulinemia, hypertriglyceridemia, increased fat mass, and dysregulation of the hypothalamic-pituitary-adrenal axis, are common in myotonic dystrophy (MD). We hypothesized that increased production of interleukin-6 (IL-6) and tumor necrosis factor-{alpha} (TNF-{alpha}) may be important underlying mechanisms.

We studied the diurnal rhythmicity of cytokines and cortisol, ACTH, and dehydroepiandrosterone in 18 men with adult onset MD and 18 controls. Morning levels of androstenedione, 17-hydroxyprogesterone, testosterone, and insulin were also determined. Genetic analyses were performed, including calculation of allele sizes.

Median circulating 24-h levels of IL-6 (P < 0.001), TNF-{alpha} (P = 0.05), ACTH (P < 0.05), and cortisol (P < 0.05) were all significantly increased in MD, whereas dehydroepiandrosterone levels were decreased (P < 0.001). The diurnal rhythms of these cytokines/hormones were disturbed in patients. Morning testosterone levels were decreased and insulin levels increased (P < 0.01 for both). Patients with high body fat mass had significantly increased insulin levels and decreased morning levels of cortisol, ACTH, and testosterone.

IL-6 and TNF-{alpha} levels are increased and adrenocortical hormone regulation is disturbed in MD. Adiposity may contribute to these disturbances, which may be of importance for decreased adrenal androgen hormone production and metabolic, muscular, and neuropsychiatric dysfunction in MD.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
MYOTONIC DYSTROPHY (MD) (designated DM1 by the International Myotonic Dystrophy Consortium) is the most common inherited form of muscle dystrophy among adults, associated with muscle atrophy and the characteristic myotonia (1). The genetic defect causing MD is an expansion of a CTG triplet repeat at chromosome 19, encoding a protein kinase named DMPK, the function of which remains to be established (2). Striking endocrine abnormalities in MD include hypogonadism and hyperinsulinemia associated with insulin resistance, and MD is often associated with increased fat mass (1, 3).

We and others have reported abnormalities in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis in MD. These include an exaggerated response of ACTH to various CRH-mediated stimuli (4, 5, 6) and decreased circulating levels of adrenal androgens, including dehydroepiandrosterone sulphate (7, 8, 9, 10). Recently, we also reported a disturbed diurnal rhythm of salivary cortisol among men with MD (10).

A link between these abnormalities may be an increased production of proinflammatory cytokines, especially interleukin-6 (IL-6) and tumor necrosis factor (TNF)-{alpha}, because these cytokines may influence HPA axis function at several sites (11, 12). IL-6 has thus been implicated in cortisol regulation in other diseases with disturbed diurnal rhythmicity of cortisol (13, 14, 15). Both cytokines are produced in adipose tissue and are of importance for metabolic functions (16, 17). Recently, levels of TNF receptor 2 (TNFR2) were found to be increased in MD patients, but no changes in TNF-{alpha} circulating levels were found (18). However, TNF-{alpha} and IL-6 both exhibit significant diurnal rhythmicity (19, 20).

Our hypothesis was that the 24-h profiles of IL-6 and TNF-{alpha} were abnormal in subjects with MD and that these abnormalities were associated with dysregulation of cortisol and adrenal androgen secretion.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Eighteen men with adult onset MD, age 41.3 ± 13.8 (mean ± SD; range, 20–71 yr) were included from the Dystrophia Myotonica (DM) Centre in Boden, northern Sweden, where the prevalence of the disease is exceptionally high (21). They had overt myotonia and muscular dystrophy. The diagnoses were based on genetic analyses. Two patients were smokers, two used snuff, and two patients both smoked and snuffed tobacco. Mean body mass index (BMI), defined as weight divided by the square of height, for the patients was 24.4 ± 4.6 (mean ± SD; range, 18.2–36.4 kg/m2).

Severity of physical handicap was assessed according to the Assessment of Motor and Process Skills protocol (22), and patients were divided into three groups based on the results of the test (group 0, below normal limit on both motor and process skills; group 1, below normal limit on either motor or process skills; group 2, normal scores for both motor and process skills).

Eighteen male controls of mean age 41.2 ± 14.5 (range, 20–76 yr) were recruited from healthy volunteers. BMI of the controls was 24.8 ± 4.6 (range, 19.3–37.9 kg/m2).

None of the patients or controls were on any relevant medication; had clinical or laboratory signs of endocrinological dysfunction (including diabetes mellitus and thyroid disease), cardiac failure, renal or hepatic insufficiency, infection, or inflammation; and none were hospitalized at the time of the study. Furthermore, none of the patients or controls had a diagnosis or any symptoms of sleep disturbances.

This study was approved by the regional ethical committee, and all participants had given their informed consent to participate in this study.

Sampling and measurements

Peripheral venous blood samples were collected at 0700, 1100, 1600, and 2200 h. A new site of venopuncture was chosen for every occasion, preferably more proximal than the last one or in the opposite arm.

Body composition was measured by bioelectrical impedance analysis (Akern-RJL Systems BIA 101, EL-DOT K/S, Fredriksværk, Denmark).

Analytical methods

Serum concentrations of cortisol, corticosteroid binding globulin (CBG), testosterone, 17{alpha}-hydroxyprogesterone (17 OHP), 4-androstene-3,17-dione (A4), and plasma ACTH were determined in untreated samples by RIA using commercial kits obtained from Orion Diagnostica, Esbo, Finland (cortisol), Medgenix Diagnostics SA, Fleurus, Belgium (CBG), Diagnostics Products Corp., Los Angeles, CA, (testosterone, 17 OHP), INCSTAR Corp., Stillwater, MN (A4), and Nichols Institute Diagnostics, San Juan Capistrano, CA (ACTH). Plasma insulin concentrations were analyzed with a double-antibody RIA technique. Guinea-pig antihuman insulin antibodies, 125I-Tyr-human insulin as tracer, and human insulin standard (Linco Research, Inc., St. Charles, MO) were used. Free and bound radioactivity was separated by use of an anti-IgG (goat anti-guinea pig) antibody (Linco Research, Inc.).

Serum dehydroepiandrosterone (DHEA) levels were determined after extraction with diethyl ether using an in-house method (23). Serum sex hormone-binding globulin (SHBG) and plasma IL-6 and TNF-{alpha} were determined in untreated samples, by enzyme immunoassay, using commercial kits obtained from Medgenix Diagnostics SA, (IL-6, TNF-{alpha}) and Diagnostics Products Corp., Los Angeles, CA (SHBG). Serum albumin was determined by the clinical routine method used at the Department of Clinical Chemistry, Umeå University Hospital.

Apparent concentrations of free testosterone were calculated from values of total testosterone, SHBG, and albumin, using a computer program based on an equation system derived from the law of mass action, according to Södergård et al. (24).

Detection limits were: for cortisol, 7 nmol/L; for ACTH, 1 ng/L; for DHEA, 1.5 nmol/L; for IL-6, 2 ng/L; for TNF-{alpha}, 3 ng/L; for 17 OHP, 0.3 nmol/L; for SHBG, 3 nmol/L; for testosterone, 0.2 nmol/L; for albumin, 10 g/L; for A4, 0.4 nmol/L; for CBG, 0.25 ng/L; and for insulin, 12 pmol/L.

Genetic analyses

Genomic DNA was prepared from blood collected in EDTA-tubes according to standard procedures and digested with EcoRI or PstI according to manufacturer’s instructions. Southern blotting and hybridizations were performed according to standard (25). The probe used was pM10M6 (2), a 1.4-kb fragment that flank the expanded region of the MD Protein Kinase gene.

The allele sizes were calculated with the computer program DNAfrag, version 3.03.

Cognitive performance

A Mini Mental State Exam (MMSE) (26) was performed on all participants.

Statistical analyses

All statistics were performed using commercial computer programs from SAS Institute, Inc. (Cary, NC) for analysis of repeated-measurements and SPSS, Inc. (Chicago, IL) for all other statistical calculations. We used Spearman’s rank correlation test for correlation analyses and Mann-Whitney U-test exact P-value for comparisons between groups. Multiple regression and partial correlation analyses were also used. Diurnal curves were analyzed with repeated-measurement analysis, with the morning sample not included in the equation. As a post hoc test for individual timepoints, we used the Mann-Whitney U-test with Bonferroni corrections.

For correlation analyses, median 24-h levels of hormones/cytokines for each participant were used. In cases where the hormone/cytokine level was below the detection limit, the value was set to half the detection limit for statistical calculations. A P-value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Cytokines

Levels of IL-6 were significantly increased among MD patients at all timepoints, and consequently, median 24-h levels were markedly increased in patients [5.5 (2.0–19.0) vs. 1.0 (1.0–4.4) ng/L; P < 0.001, medians and 10th and 90th percentiles, respectively] (Fig. 1aGo). Median 24-h TNF-{alpha} levels were also significantly increased in patients [22.0 (14.0–29.8) vs. 19.0 (13.0–28.0) ng/L, P = 0.05], and the diurnal rhythm of TNF-{alpha} was abnormal, with an inverse pattern in the forenoon and evening, compared with controls (Fig. 1bGo). Repeated-measurement analyses showed that there were significant time/group interactions for both cytokines (P < 0.05 for IL-6 and P < 0.01 for TNF-{alpha}).



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  		Figure 1. Diurnal rhythms of IL-6 (top) and TNF-{alpha} (bottom) in 18 men with MD and 18 healthy men. Repeated-measurement analyses revealed significant time/group interaction for both cytokines (P < 0.05 for IL-6 and P < 0.01 for TNF-{alpha}). a, P < 0.0125 (Bonferroni correction for repeated analyses).

 
HPA axis hormones

Diurnal rhythm of serum cortisol was flattened, with significantly increased levels in the afternoon and evening in patients (Fig. 2aGo). This was associated with a similar abnormality in ACTH levels (Fig. 2bGo). DHEA diurnal rhythm was highly abnormal in patients, with decreased levels mainly in the morning (Fig. 2cGo). There was a significant time/group interaction for DHEA (P < 0.05), but not for cortisol or ACTH, when repeated-measurement analyses were performed.



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  		Figure 2. Diurnal rhythms of cortisol (top), ACTH (middle), and DHEA (bottom) in 18 men with MD and 18 healthy men. Repeated-measurement analyses revealed significant time/group interaction for DHEA (P < 0.05). a, P < 0.0125 (Bonferroni correction for repeated analyses).

 
These abnormalities resulted in significantly increased median 24-h levels of cortisol and ACTH in MD patients [364 (120–562) vs. 271 (83–570) nmol/L, P < 0.05; and 36.2 (12.7–69.3) vs. 25.9 (12.4–50.7) ng/L, P < 0.05]. Median 24-h levels of DHEA were significantly decreased in patients with MD [6.6 (2.5–18.0) vs. 10.5 (4.9–25.4) nmol/L, P < 0.001].

In contrast, there were no statistically significant differences regarding morning serum levels of A4, 17 OHP, CBG, or SHBG (Table 1Go).


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  		Table 1. Morning serum levels in 18 men with MD and 18 healthy men

 
Insulin, testosterone, and body fat mass

Morning serum levels of insulin were significantly increased in patients, whereas the levels of testosterone were significantly decreased. Free testosterone levels were significantly decreased in patients (Table 1Go). HOMA index [fasting glucose (mmol/L) x fasting insulin (mU/L)/22.5] was calculated for all participants as an indicator of insulin sensitivity. Patients had a significantly higher HOMA index than controls [2.3 (1.4–5.5) vs. 1.4 (0.90–3.5), P < 0.01]. Percent body fat was significantly increased in patients [34.4 (16.2–50.7) vs. 21.4 (15.0–31.0), P < 0.001].

Correlations between insulin, testosterone, and SHBG

In healthy controls, testosterone levels correlated positively to SHBG levels (rs = 0.60; P < 0.01). In MD patients, testosterone levels correlated negatively to insulin levels (rs = -0.57, P < 0.05).

Correlations between BMI, HOMA index, and disease parameters

Morning insulin levels correlated positively to BMI (rs = 0.68, P < 0.01) in patients and to body fat mass in both groups (rs = 0.72 and rs = 0.65, P < 0.01 for both, patients and controls, respectively). Testosterone morning levels correlated negatively to BMI (rs = -0.67, P < 0.01; and rs = -0.51, P < 0.05) and body fat mass (rs = -0.64 and rs = -0.57, P < 0.05) in both groups. HOMA index correlated positively to BMI (rs = 0.65, P < 0.01; and rs = 0.49, P < 0.05; patients and controls, respectively) and body fat mass (rs = 0.67 and rs = 0.66, P < 0.01 for both) and negatively to testosterone (rs = -0.67, P < 0.01; and rs = -0.48, P < 0.05) in both groups.

Increasing number of CTG triplet repeats correlated to increased severity of handicap (rs = 0.60, P < 0.05).

Correlations between hormone/cytokine levels

Correlations between morning basal levels of ACTH and adrenocortical hormones are shown in Table 2Go, and correlations between median 24-h levels of hormones and cytokines are shown in Table 3Go. In summary, median 24-h cortisol levels correlated to median DHEA levels in patients; whereas in controls, cortisol levels correlated to ACTH levels. There was a negative correlation between median 24-h cortisol and TNF-{alpha} levels in MD patients.


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  		Table 2. Correlations between morning levels of plasma ACTH, serum cortisol, DHEA, 17 OHP, and A4 in 18 men with MD and 18 healthy men

 

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  		Table 3. Correlations between median 24-h levels of plasma ACTH, serum cortisol and DHEA, and plasma IL-6 and TNF-{alpha} in 18 men with MD and 18 healthy men

 
In MD patients, median 24-h levels of DHEA correlated positively, and morning levels of 17 OHP negatively, to morning insulin levels (rs = 0.52, P < 0.05; and rs = -0.64, P < 0.01, respectively). In controls, morning TNF-{alpha} levels correlated positively to morning insulin levels and HOMA index (rs = 0.55 and rs = 0.48, P < 0.05). When controlling for BMI in a partial correlation analysis, these associations no longer were significant.

Cognitive performance

Patients scored significantly lower at the MMSE [27 (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) points vs. 30 (29, 30), median (min-max); P < 0.001]. Low MMSE scores correlated to increasing severity of handicap (rs = 0.54, P < 0.05) and to increased 2200 h levels of TNF-{alpha} (rs = -0.49, P < 0.05) in MD patients.

Subgroups

When dividing patients into two groups, using the median number of CTG triplet repeats (614 repeats) as the cut-off, there were no significant differences between the two patient groups. However, compared with controls, patients with more than 614 CTG repeats had significantly lower morning 17 OHP [3.8 (2.2–4.6) and 5.4 (3.0–10.2), P < 0.05; median (10th-90th percentile), patients and controls, respectively] and testosterone [11.5 (2.1–12.4) and 19.7 (11.0–25.5), P < 0.001] levels, and higher levels of insulin [84.0 (58.0–210.0) and 51.0 (34.0–106.7), P < 0.01)].

We also divided patients into two groups according to body fat mass (Table 4Go). In four MD patients, bioelectric impedance analyses were not possible to perform because of resistance above the range of the instrument. We found no technical explanation for this, and it was a consistent finding at repeated trials. For practical reasons, no other methods of body composition estimation were possible. These four patients were not included in the following subgroup analyses based on fat mass.


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  		Table 4. Comparison between MD patients with low and high percent body fat and controls

 
The diurnal curve of serum cortisol was strikingly different in the two patient groups with an abolished diurnal rhythm among patients with high body fat (Fig. 3Go). Repeated-measurement analysis showed no significant time/group interaction between the groups. Compared with patients with less body fat, patients with high body fat mass had significantly decreased levels of testosterone and increased morning levels of insulin (Table 4Go). Patients with high body fat also had a significantly higher HOMA index than patients with less body fat. In both patient groups, median 24-h levels of ACTH, cortisol, and IL-6 were significantly increased, compared with controls.



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  		Figure 3. Diurnal rhythms of cortisol in MD patients with lower (n = 7) and higher (n = 7) body fat mass and in controls (n = 18). Repeated-measurement analyses revealed no time/group interaction. a, P < 0.0125 between high fat MD patients and controls (Bonferroni correction for repeated analyses).

 
Multiple regression analyses

To isolate the effects, on cytokine levels of disease per se, age, body fat mass, and hormones, stepwise linear multiple regression analyses were performed. Median 24-h IL-6 levels were independently predicted by disease per se (P < 0.001) and body fat mass (P < 0.05). Median 24-h levels of TNF-{alpha} predicted independently decreased median 24-h levels of DHEA (P < 0.05). Median 24-h levels of cortisol were predicted by disease (P < 0.01) and median 24-h levels of ACTH (P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
A major finding of this study is the clearly abnormal regulation of proinflammatory cytokines in MD, with increased levels and disturbed diurnal rhythms of plasma IL-6 and TNF-{alpha}. Furthermore, diurnal rhythms of the HPA axis hormones ACTH, cortisol, and DHEA were also abnormal, with increased levels of ACTH and cortisol and decreased levels of DHEA in MD. Figure 4Go shows a proposed model of interaction between these cytokines and the HPA axis. An increased production of cytokines from several putative sources may thus influence the HPA axis in different ways. These immunoendocrine abnormalities may contribute to metabolic dysfunctions, including insulin resistance, and may also be of importance for muscular and neuropsychiatric symptoms, such as sleep disturbances, cognitive dysfunction, and fatigue in MD.



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  		Figure 4. A simplified model of cytokine interactions with the HPA axis. +, Stimulatory; -, inhibitory; +/-, both stimulatory and inhibitory action/s.

 
IL-6 and TNF-{alpha} are produced in a variety of tissues, including adipose tissue (17). The markedly increased body fat mass in our MD patients might contribute to the increased levels of cytokines. Accordingly, body fat mass and disease both independently predicted median 24-h IL-6 levels in the present study, also after adjustment for possible confounding factors. There was a trend toward increased TNF-{alpha} levels in patients with high body fat mass, compared with patients with less body fat. IL-6 levels are increased in states of sleep disturbances (20, 27); however, none of our patients reported any symptoms of disturbed sleep.

TNF-{alpha} levels in our patients were relatively less increased, compared with IL-6 levels, a finding that is in line with an earlier study where the gradient between venous effluent and arterial IL-6 concentrations in fat tissue far exceeded that for TNF-{alpha} (28). This may be attributable to the inhibition by IL-6 of the production of TNF-{alpha} and the stimulation of TNF-{alpha} on the release of IL-6. In addition, TNF-{alpha} may be more sensitive to glucocorticoid negative feed-back because IL-6-producing cells may actually be sensitized to catecholamine effects by glucocorticoids (29, 30).

Increased levels of cytokines in the periphery may induce cytokine production in the brain via the vagus nerve (31). IL-6, as well as TNF-{alpha}, might also enter the brain via active transport; physiological levels of IL-6, but not of TNF-{alpha}, have been suggested as relevant for this transport to occur (32).

MD patients exhibit exaggerated ACTH responses to CRH-mediated stimuli (5, 6). Proinflammatory cytokines are possible mediators for this increased reactivity, because IL-6 and TNF-{alpha} both stimulate the HPA axis at the hypothalamic and/or pituitary level (12). Thus, both cytokines stimulate the expression of the precursor to ACTH, the POMC gene in cell cultures, both directly and by potentiating the effect of CRH on POMC gene expression (33). These cytokines also interact with the HPA axis peripherally (12). IL-6 has been suggested to exert its chronic effects mainly through stimulation of steroidogenesis from the adrenal gland (34), whereas TNF-{alpha} inhibits ACTH-induced steroid hormone synthesis in the adrenal (35). This fits with our findings of a negative association between circulating levels of cortisol and TNF-{alpha} in our MD patients.

We have previously shown a disturbed diurnal rhythm of cortisol in saliva in MD patients (10), and the present study confirms and extends those findings. Our present study shows that the diurnal rhythms of ACTH, as well as cortisol, are disturbed, resulting in increased median 24-h levels of these hormones. Diurnal rhythmicity of the HPA axis is influenced by the hippocampus, which is known to be sensitive to glucocorticoids and cytokines (36, 37, 38). Interestingly, morning levels of cortisol (and ACTH) are distinctly decreased in patients with high body fat mass, compared with patients with less body fat, in the present study. This has also been reported earlier in obesity (39) and implies that adipose tissue-derived factors may be of importance for diurnal rhythmicity of the HPA axis. However, body fat mass did not predict median 24-h cortisol levels in the present study; cortisol levels were instead predicted by disease per se and median 24-h ACTH levels.

Increased levels of IL-6 have been suggested to stimulate cortisol production in other diseases with disturbed cortisol diurnal rhythm (13, 14). However, we found no such association between IL-6 and cortisol levels. This could be an effect of insufficient negative feedback by cortisol on IL-6, because glucocorticoids may sensitize cells to catecholamines, which then stimulate IL-6 secretion (30). IL-6 in turn will together with cortisol inhibit TNF-{alpha} production (12). Catecholamine levels are reported to be normal in MD (40). Alternatively, there may be a temporal relationship between IL-6 and cortisol, as has been implied in rheumatoid arthritis (14). To establish this relationship, more frequent blood sampling would be needed.

ACTH is believed to be the major releasing factor of cortisol and DHEA from the adrenal gland. However, ACTH is probably not the only releasing factor in MD patients, because serum cortisol and DHEA did not display similar diurnal patterns. Thus, cortisol levels were decreased in morning and forenoon and increased in the afternoon and evening, compared with controls, whereas DHEA levels were decreased at all times throughout the day. A similar diurnal pattern of cortisol, together with an impaired dexamethasone suppression test, has been reported in depressed patients (41). None of our patients were depressed, however, as judged from a depression rating scale and clinical evaluation. Increased levels of cortisol and concomitant decreased levels of DHEA are also present in trauma and chronic diseases (42). Our patients were all mildly to moderately affected by the disease and had not experienced any recent physical or psychological major trauma. Furthermore, the negative feedback system in MD patients seems intact (10). Therefore, other factors seem to be involved.

The product of the DM gene, DMPK, could be a possible link between steroid hormone abnormalities, as suggested by Buyalos et al. (9). In spite of the association between increasing severity of the disease and increasing number of CTG repeats, we found no consistent associations between the number of CTG triplet repeats and hormones/cytokines. However, there was a tendency towards lower adrenal androgens in patients with a greater number of CTG triplet repeats. Cytokines are also putative mediators of the steroid hormone disturbances. Thus, TNF-{alpha} inhibits the ACTH-induced stimulation of the 17,20 hydroxylase (cyp17), converting pregnenolone to 17-hydroxypregnenolone (35), a precursor of DHEA, and IL-6 may suppress production of DHEA because, in normal aging, decreasing levels of DHEA are accompanied by an increase in IL-6 levels (43). Median 24-h levels of DHEA were accordingly associated with median 24-h levels of TNF-{alpha} in our study group. The release of proinflammatory cytokines is inhibited by DHEA (44, 45). Thus, a vicious circle may be present in our patients, where increased levels of IL-6 and TNF-{alpha} inhibit the production of DHEA, and the resulting loss of DHEA inhibition on cytokine release may then further increase cytokine levels.

MD patients exhibit many of the features seen in the metabolic syndrome; a disturbed diurnal rhythmicity of cortisol, increased fat mass, hypertriglyceridemia, hyperinsulinemia, and insulin resistance (39). TNF-{alpha} levels are increased in obese persons and may worsen insulin resistance by inhibiting insulin receptor function (16, 46). IL-6 stimulates insulin release in vitro (47) and has been implied in the insulin resistance after surgery (48). These cytokines may also increase triglyceride levels (47). There are at least two TNF receptors, and TNFR2 is believed to signal metabolic actions (49). TNF-{alpha} is known to be a potent inducer of TNFR2; and recently, TNFR2 levels were shown to be increased in MD patients (18). A novel finding in our study is the abnormal diurnal pattern of TNF-{alpha}, emphasizing the need for careful selection of timepoints for sampling of TNF-{alpha}.

Testosterone levels in MD are markedly decreased (1), possibly as a result of the disturbed testicular function. In the present study, also free testosterone levels (probably because of decreased total testosterone levels), as well as SHBG levels, were decreased. Testosterone and SHBG levels are decreased in male individuals with the metabolic syndrome, obesity, and/or type II diabetes (50, 51). The production of testosterone is inhibited by TNF-{alpha} and IL-6 (52, 53), whereas insulin is a strong negative regulator of SHBG (54).

Cytokines may also influence cognitive dysfunction (27, 55). Evening levels of TNF-{alpha} correlated negatively to MMSE scores in our patients. Furthermore, IL-6 overproduction or administration produces fatigue, a common complaint from patients with MD; and, interestingly, suppression of IL-6 has been suggested to help alleviate fatigue (56). This implies new treatment strategies of these patients.

In conclusion, patients with MD show markedly increased levels of IL-6 and TNF-{alpha}, and disturbed diurnal rhythms of HPA axis hormones ACTH, cortisol, and DHEA, resulting in increased 24-h levels of ACTH and cortisol and decreased levels of DHEA. This may be of importance, not only for the metabolic dysfunctions frequently encountered in this disease but also for muscular and neuropsychiatric dysfunction.


   Acknowledgments
 
We thank statistician Hans Stenlund for invaluable support in statistical matters.


   Footnotes
 
1 Supported in part by grants from the Joint Committee of the Northern Medical Region. Back

Received November 9, 1999.

Revised February 25, 2000.

Revised April 5, 2000.

Accepted May 25, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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