Inverse relationship between plasma epinephrine and testosterone levels during acute glucoprivation in healthy men

Inverse relationship between plasma epinephrine and testosterone levels during acute glucoprivation in healthy men

Life Sciences 68 (2001) 1889–1898 Inverse relationship between plasma epinephrine and testosterone levels during acute glucoprivation in healthy men ...

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Life Sciences 68 (2001) 1889–1898

Inverse relationship between plasma epinephrine and testosterone levels during acute glucoprivation in healthy men Igor Elmana,*, David S. Goldsteinb, Caleb M. Adlerc, Susan E. Shoafd, Alan Breiere,f a

Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA Clinical Cardiology Section, Building 10, Room 6N252, National Institute of Neurological Disorders and Stroke, 9000 Rockville Pike, Bethesda, MD 20892-2655, USA c Experimental Therapeutics Branch, National Institutes of Mental Health, 9000 Rockville Pike, Bethesda, MD 20892-2655, USA d Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville Pike, Bethesda, MD 20892-2655, USA e Lilly Research Laboratories, Lilly Corporate Center Drop code 0510, Indianapolis, IN 46285, USA f Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN 46202, USA b

Received 15 May 2000; accepted 23 August 2000

Abstract In healthy men, a decrease in plasma testosterone levels was observed in the context of metabolic stress. While physiological mechanisms underlying this response are unclear, there are several lines of evidence suggesting circulating epinephrine’s influence on plasma testosterone levels. The purpose of this study was to directly relate stress-induced changes in plasma testosterone and epinephrine. The stressor used was blockade of glucose metabolism with pharmacological doses (40mg/kg) of 2 deoxyglucose (2DG). Arterial plasma samples from 10 healthy males were assayed at 20 minutes intervals for 60 minutes for the concentrations of testosterone, epinephrine and related biochemicals. Bolus administration of 2DG resulted in progressive decline in testosterone and increases in epinephrine and norepinephrine plasma levels (mean change from baseline: 29, 2530 and 186%, respectively). Inverse correlation was detected between both absolute (rs520.72; df58; p50.017) and baseline-corrected testosterone concentrations at the 60 minute time point and epinephrine area under the curve values. Our results suggest that adrenomedullary activation may be involved in stress-induced testosterone effects. The implications of these data for the understanding of the role of catecholamines in glucoprivic stress response are discussed. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Deoxyglucose; Stress; Catecholamines; Norepinephrine; HVA; 5-HIAA

* Corresponding author. Addiction Services, Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, 15 Parkman St WACC-812, Boston, MA 02114. Tel.: (617) 724-9385; fax: (617) 248-0070. E-mail address: [email protected] (I. Elman) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 0 9 8 2 -1

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Introduction In healthy men, a decrease in plasma testosterone levels was observed in association with acute glucose deprivation whether elicited by administration of insulin [1] or by blockade of glucose metabolism with pharmacological doses of 2 deoxyglucose (2DG) [2]. The physiological mechanism underlying this response remains unclear. The opposite effects on blood glucose levels produced by insulin and 2DG, with the former resulting in hyperglycemia [3] and the later in hypoglycemia renders the direct action of insulin or blood glucose levels per se unlikely options. Other factors known to control plasma testosterone levels such as gonadotopins and sex hormone binding globulin [4, 5] were excluded in our previous study as their levels were not significantly affected by the 2DG administration [2]. Also, although 2DG administration was associated with an activation of the hypothalamic-pituitary-adrenocortical (HPA) axis there was no correlation between changes in plasma ACTH, cortisol and testosterone concentrations [2]. HPA axis activation, evoked by metabolic challenge posed by 2DG, is only part of a constellation of homeostatic adjustments, with epinephrine secretion predominating those of ACTH, cortisol and other counterregulatory hormones [6–8]. While there are several lines of preclinical and clinical data suggesting that circulating epinephrine may influence plasma testosterone levels, the direction of this response is inconsistent. Intravenous infusion of epinephrine resulted in a decrease in plasma testosterone in men [9] and rats [10, 11]. Similarly, immobilization stress in rats produced substantial increases in plasma catecholamines and the associated testosterone decreases were abolished by a sympathetic neurotoxic agent, b2 blockade or adrenalectomy [12]. In contrast to those reports, physical activity in man [13] or immobilization stress in dominant primates [14] elicited elevations in both catecholamines and testosterone levels and the later were prevented by sympathetic blockade [14, 15]. Lastly, intra-arterial infusion of epinephrine into dog testis also produced increases in testosterone concentration in venous blood [16]. The reasons for these discrepant findings are unclear and may be related to differences in species, social rank [14], functional status of the testis [17], type of stressor, exogenous or endogenous source and amount of epinephrine. There are currently no studies directly relating in vivo 2DG-induced changes in plasma testosterone and epinephrine. To address this issue, arterial plasma samples of the subjects previously studied concerning the 2DG effects on plasma testosterone and progesterone levels [2] were assayed for the levels of epinephrine. In exploratory fashion we measured additional peripheral indices of monoaminergic function, e.g., norepinephrine, dihydroxyphenylacetic acid (DOPAC), dihydroxyphenylalanine (DOPA), dihydroxyphenylglycol (DHPG), dopamine, homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), that may potentially affect plasma testosterone levels during exposure to stress [18]. To characterize the responses to 2DG, we also report physiologic (temperature, blood pressure, heart rate) and behavioral (self rating of hunger and thirst) measures. Methods Subjects Ten men (mean age6SEM: 32.362.0; weight: 84.764.8 kg) were recruited from the volunteers pool of the National Institute of Health (NIH), Bethesda, MD. The subjects were in

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good physical health, as evidenced by physical exam, SMAC, thyroid function tests, CBC, urinalysis, HIV antibody test, toxicology screen and ECG. The subjects and their first-degree relatives had no past or current psychiatric history, including substance abuse and alcohol dependence as determined by the Structured Clinical Interview for DSM-III-R (SCID). The subjects denied concurrent use of anabolic steroids. The subjects gave written consent to this NIH Institutional Review Board approved protocol. Clinical protocol On the morning of the procedure the subjects were admitted to the 4E Unit of the Clinical Center, NIH after having fasted and refrained from alcohol, tobacco, caffeine, or physical activity for at least 10 hours. With the patient in supine position an arterial catheter was inserted percutaneously after local anesthesia of the overlying skin. An intravenous catheter was placed into the antecubital fossa of the contralateral arm and was kept patent with a slow isotonic (0.9) saline drip. After a 90 minute rest period, 2DG (40mg/kg) in 50 ml of isotonic saline solution was administered as an intravenous bolus. Continuous cardiac monitoring was performed throughout the course of the study. Oral temperature, blood pressure and heart rate were collected immediately prior to (0 minutes) and at 20, 40 and 60 time points. Self-ratings of hunger and thirst were determined at baseline (before 2DG administration) and at the end of the study with a self-report visual analog rating scale, scored in millimeters (from the left side of 100-mm line to a perpendicular mark made by the subjects at the point corresponding to their subjective impression). The scale items ranged from 0 mm (not at all) to 100 mm (extremely). Biochemical variables Blood samples were collected in heparinized tubes at 30 minutes before (230), immediately prior to bolus (0) and at 120, 140 and 160 minutes following the bolus and were placed on wet ice. After separation by refrigerated centrifugation, the plasma was stored at 2808 C. Testosterone was measured with radioimmunoassay (the intra- and interassay coefficient of variation 7.5 and 7.8% respectively) [19]. Liquid chromatography with electrochemical detection after batch alumina extraction [20] was used to measure plasma epinephrine (the intra-and interassay coefficient of variation was 3.0 and 11.4% respectively), norepinephrine (the intra-and interassay coefficient of variation was 1.9 and 6.5% respectively), DOPAC (the intra-and interassay coefficient of variation was 3.9 and 11.6% respectively), DOPA (the intra-and interassay coefficient of variation was 3.8 and 5.9% respectively), DHPG (the intra-and interassay coefficient of variation was 7.6 and 8.4% respectively) and dopamine (the intra-and interassay coefficient of variation was 8.1 and 11.6% respectively). Plasma concentrations of HVA (the intra-and interassay coefficient of variation was 5.0 and 7.0% respectively) and 5-HIAA (the intra-and interassay coefficient of variation was 3.0 and 5.0% respectively) were determined by liquid chromatographic method with electrochemical detection using an internal standard, 3-ethoxy-4-hydroxyphenylglycol [21]. Statistical analyses Data were analyzed using statistical package Statistica (StatSoft, Inc., Tulsa OK). Hormonal levels at 230 min and 0 time points were averaged to constitute a single baseline

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value. To determine effects of glucoprivation on neuroendocrine (arterial plasma levels of epinephrine, norepinephrine, DHPG, DOPAC, DOPA, HVA and 5HIAA) and physiological (heart rate, blood pressure and oral temperature) variables, a one-way analysis of variance (ANOVA) was conducted with time (baseline, 20, 40 and 60 minutes) as the within subjects factor. When the time effect was significant, post hoc Newman-Keuls t-tests were performed. Area under the concentration-time course curve (AUC) was computed using trapezoidal integration. The non-parametric Spearman coefficient was used for correlation analyses. All analyses were two-tailed. A p value less than 0.05 defined statistical significance. Group data were summarized as mean6standard error of the mean (SEM). Results 2DG-induced neuroendocrine changes Bolus administration of 2DG resulted in significant decreases in testosterone (F55,65; df53,9; p,0.01; mean change from baseline: 29%) and increases in epinephrine (F525.5; df53,9; p,0.001; mean change from baseline: 2530%) and norepinephrine (F511.53; df53,9; p,0.001; mean change from baseline: 186%) plasma levels (Figure 1). Post hoc NewmanKeuls t-tests revealed that testosterone plasma levels were significantly lower (p,0.01) than the baseline values at 60 minutes. Epinephrine elevations were significant at 20 and 40 minutes (p,0.001) and peaked at 60 minutes (p,0.001). Norepinephrine levels were significantly increased at 20 minutes (p,0.001), peaked at 40 minutes (p,0.001) and persisted till 60 minutes (p,0.001) (Figure 1). For DOPAC and DOPA, 2DG produced marginal effects, while no significant effects were observed for DHPG, dopamine, HVA and 5-HIAA plasma levels (Table 1). Physiological and behavioral variables Physiological (Figure 2) and behavioral (Table 2) variables were examined to characterize the responses to 2DG. 2DG produced significant decreases in oral temperature (F515.15; df53,9; p,0.001) and diastolic blood pressure (F59.04; df53,9; p,0.001) but no significant changes in systolic blood pressure (F51.30; df53,9; p50.30) and the heart rate (F51.13; df53,9; p50.36). Oral temperature changed continuously over time, while diastolic blood pressure decreased below resting levels at 20 minutes (p,0.01) and did not change significantly thereafter. Subjects reported significant increases in hunger and thirst. Correlation analyses Across individuals, inverse correlation were detected between epinephrine AUC values and absolute (rs520.72; df58; p50.017) (Figure 3), percent change scores (160 minute minus baseline/baseline 3 100) for the 60 minute time point testosterone concentrations (rs520.78; df58; p50.008), and testosterone AUC values (rs520.84; df58; p50.002). No significant relationship was observed between norepinephrine AUC values and absolute (rs520.06; df58; p50.99) (Figure 3), baseline-corrected (rs520.1; df58; p50.78) the 60 minutes time point testosterone concentrations, and testosterone AUC values (rs520.09; df58; p50.80).

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Fig. 1. Effect of 2DG administration on plasma testosterone, epinephrine, and norepinephrine in healthy males (N510). Values represent mean 6SEM. Statistical differences were determined using a one-way analysis of variance (ANOVA) with time (baseline, 20, 40 and 60 minutes) as within subjects factor and post hoc Newman-Keuls t-tests. Significant effects for testosterone (F55,65; df53,9; p,0.01), epinephrine (F525.5; df53,9; p,0.001) and norepinephrine (F511.53; df53,9; p,0.001). * p,0.01

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Table 1 The effects of pharmacological doses of 2DG (40mg/kg) on arterial plasma concentrations (mean 6 SEM) of selected catechol- and indolamine metabolites in healthy males (N510) Biochemical (pmol/ml) DOPAC DOPA DHPG Dopamine HVA 5HIAA a

Baseline

20 minutes

40 minutes

60 minutes

F (df53,9)

pa

6.3761.20 5.6560.84 3.9060.88 0.2660.02 50.32651.24 20.6567.61

6.9560.96 6.1460.62 3.9260.83 0.3260.03 37.50612.39 21.8166.21

7.0861.02 6.1160.62 4.1160.88 0.3060.03 41.63619.44 21.7866.16

7.4761.21 6.3360.80 4.0560.77 0.2660.02 49.34631.17 21.9665.90

2.76 2.63 1.08 2.34 0.9 1.89

0.06 0.07 0.38 0.10 0.45 0.16

Time effect.

Discussion This is to our knowledge the first report on the influence of acute metabolic stress on arterial plasma levels of monoamines and their metabolites. Bolus administration of pharmacological doses of 2DG produced three different response patterns: decreases in testosterone,

Fig. 2. The effects of pharmacological doses of 2DG on oral temperature, blood pressure and heart rate in healthy males (N510). Values represent mean6SD. Statistical differences were determined using a one-way analysis of variance (ANOVA) with time (baseline, 20, 40 and 60 minutes) as within subjects factor and post hoc NewmanKeuls t-tests. Significant effects for oral temperature (F515.15; df53,9; p,0.001) and diastolic blood pressure (F59.04; df53,9; p,0.001). No significant changes in systolic blood pressure (F51.30; df53,9; p50.30) and the heart rate (F51.13; df53,9; p50.36). * p,0.01 compared to baseline values.

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Table 2 2DG-induced changes (mean 6 SEM) in behavioral self ratings (mm) Score Hunger Thirst

Baseline

End point

t (df51,9)

p

5.3760.73 4.2260.85

7.6860.67 7.6760.72

3.66 4.88

0.006 0.001

elevations in epinephrine and norepinephrine and no significant changes in DOPA, HVA and 5HIAA plasma levels. The results for DOPAC and DOPA must be regarded as tentative because there was only a trend for significant effects. The 2DG-induced changes in vital signs and behavioral ratings replicate our previous findings [6, 7]. This negative correlation found under in vivo condition between both absolute and baseline-corrected plasma testosterone and epinephrine levels suggests that adrenomedullary hormonal system activation may be involved in mediating testosterone decreases. The correlational nature of our results does not yet prove direct epinephrine-testosterone interaction. Thus, it can not be determined from our results whether testosterone levels decreased as a direct result of the increase in epinephrine or both were a function of glucoprivation. Further studies exploring potential sympathetic blockade-induced disruption of the relationship be-

Fig. 3. Scatterplots relating individual 60 minutes time point testosterone concentrations and epinephrine and norepinephrine AUC values using Spearman coefficient (N510). Significant correlation between testosterone concentrations and epinephrine AUC values (rs520.72; df58; p50.017), but not with norepinephrine AUC values (rs520.06; df58; p50.99).

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tween testosterone and epinephrine [12, 14, 15] may help to clarify this issue. Nevertheless, our data are in agreement with some preclinical and clinical studies demonstrating suppression of testosterone production by exogenous or endogenous epinephrine [9–12]. Other studies, however, implied an opposite, stimulatory action of epinephrine on plasma testosterone [13, 14, 16, 17]. Possible reasons for these inconsistencies may include differences of experimental designs, e.g., in vivo versus in vitro, stress paradigms, species or combination. The almost 2.5-fold increase in norepinephrine levels was unexpected given that previous studies with 2DG found only slight increases (30%) in the face of profound activation of epinephrine secretion similar to that observed in our study [6, 7]. Direct comparisons between these studies and the present one is complicated by methodological nuances, bolus versus infusion 2DG administration, and sampling of arterial rather than venous blood. NE concentrations in mixed venous blood may be unreliable indicators of total body sympathetic nervous activity [8, 22]. The advantage of arterial sampling is that it is less affected by local peripheral metabolism, as suggested by studies demonstrating that tissues of the arm remove a substantial proportion of the radioactively-labeled NE in the arterial plasma [23]. The mechanism of 2DG-induced NE elevation is unclear. Plasma levels of any endogenous biochemical represent the ratio of the rate of release of the substance into the bloodstream (spillover) and clearance of the substance from the bloodstream. 2DG-induced elevations in plasma NE levels therefore could result from increased spillover, decreased clearance or combination of both. Assuming that plasma clearance of NE was not decreased during 2DG-induced glucoprivation, the increments of NE were due to increase in spillover which is determined by the rate of release from sympathetic nerves, the adrenal medulla, or both. Since adrenal medulla can account for only about 30–45% of plasma NE during stress [7, 24], the remaining 55%–70% is probably contributed by sympathetic neurons. The finding of a trend for significant elevations of plasma DOPA would support this sort of mechanism, because DOPA reflects enhanced sympathoneural NE synthesis with only a negligible proportion of it derived from adrenals [8]. Also, perfuse sweating and pallor consistently observed during our 2DG experiments may be another expression of sympathetic hyperactivity. Consistent with this view, clinical studies with insulin-induced hypoglycemia also indicated stimulation of the sympathoneural as well as adrenomedullary component of the sympathoadrenal system [25–28]. The lack of significant increases in the plasma levels of the intraneuronal NE metabolite DHPG may however argue against a simple sympathoneural involvement in the NE elevations. Concurrent inhibition of neuronal NE uptake due to decreased ATP/ADP ratio [29–31] could explain this phenomenon. Nonetheless, a study formally assessing NE kinetics during 2DG-induced glucoprivic stress may be warranted. Plasma HVA and 5HIAA levels may reflect changes in the brain activity of dopamine [32] and serotonin [33, 34] respectively. Both neurotransmitters have been implicated in the pathophysiology mechanisms of schizophrenia. In addition, 2DG-induced metabolic stress was associated with greater increases in venous plasma HVA levels in schizophrenic patients than healthy controls [35]. It would be of interest to examine 2DG effects on arterial plasma levels of HVA and 5HIAA in schizophrenic patients to further illuminate neurobiological correlates to impaired stress response in patients with schizophrenia. A few caveats should be considered in interpreting our results. First, 2DG produces a distinct type of metabolic stress and it is unknown if other stressors would result in similar data.

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