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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 6 2203-2214
Copyright © 2000 by The Endocrine Society

From The Clinical Research Centers

Impact of Age on Cortisol Secretory Dynamics Basally and as Driven by Nutrient-Withdrawal Stress*

M. Bergendahl, A. Iranmanesh, T. Mulligan and J. D. Veldhuis

Departments of Pediatrics and Physiology (M.B.), University of Turku, Turku, Finland; Endocrine Section, Medical Service (A.I.), Veterans Affairs Medical Center, Salem, Virginia 24153; Gerontology Section (T.M.), Virginia Commonwealth University, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia 23249; and Division of Endocrinology, Department of Internal Medicine, General Clinical Research Center, Center for Biomathematical Technology (J.D.V.), University of Virginia School of Medicine, Charlottesville, Virginia 22908

Address correspondence and requests for reprints to: Dr. J. D. Veldhuis, Division of Endocrinology, Department of Internal Medicine, Box 202, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: JDV{at}Virginia.Edu


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
The present study tests the clinical hypothesis that aging impairs homeostatic adaptations of cortisol secretion to stress. To this end, we implemented a short-term 3.5-day fast as an ethically acceptable metabolic stressor in eight young (ages 18–35 yr) and eight older (ages 60–72 yr) healthy men. Volunteers were studied in randomly ordered fed vs. fasting sessions. To capture the more complex dynamics of cortisol’s feedback control, blood was sampled every 10 min for 24 h for later RIA of serum cortisol concentrations and quantitation of the pulsatile, entropic, and 24-h rhythmic modes of cortisol release using deconvolution analysis, the approximate entropy statistic, and cosine regression, respectively. The stress of fasting elevated the mean (24-h) serum cortisol concentration equivalently in the two age cohorts [i.e. from 7.2 ± 0.35 to 11.6 ± 0.71 µg/dL in young men and from 7.7 ± 0.39 to 12.6 ± 0.59 µg/dL in older individuals (P < 10-7)]. The rise in integrated cortisol output was driven mechanistically by selective augmentation of cortisol secretory burst mass (P = 0.002). The resultant daily (pulsatile) cortisol secretion rate increased significantly but equally in young (from 94 ± 6.3 to 151 ± 15 µg/dL·day) and older (from 85 ± 5.4 to 145 ± 7.3 µg/dL·day) volunteers (P < 10-4). Nutrient restriction also prompted a marked reduction in the quantifiable regularity of (univariate) cortisol release patterns in both cohorts (P < 10-4). However, older men showed loss of joint synchrony of cortisol and LH secretion even in the fed state, which failed to change with metabolic stress (P < 10-6). In addition, older individuals maintained a premature (early-day) cortisol elevation in the fed state and unexpectedly evolved an anomalous further cortisol phase advance of 99 ± 16 min during fasting (P < 10-5). Caloric deprivation in aging men also disproportionately elevated the mesor of 24-h rhythmic cortisol release (P = 10-7) and elicited a greater increment in the mean day-night variation in cortisol secretory-burst mass (P < 0.01 vs. young controls). Lastly, short-term caloric depletion in older subjects paradoxically normalized their age-associated suppression of the 24-h rhythm in cortisol interburst intervals.

In summary, acute metabolic stress in healthy aging men (compared with young individuals) unmasks distinct, albeit complex, disruption of cortisol homeostasis. These dynamic anomalies impact the feedback-dependent and time-sensitive coupling of pulsatile and 24-h rhythmic cortisol secretion. Nutrient-withdrawal stress in the older male heightens the cortisol phase disparity already evident in fed elderly individuals. Conversely, the stress of fasting in young men paradoxically reproduces selected features of the aging unstressed (fed) cortisol axis; viz., abrogation of joint cortisol-LH synchrony and suppression of the normal diurnal variation in cortisol burst frequency. Whether fasting would unveil analogous disruption of feedback-dependent control of the corticotropic axis in healthy aging women is not yet known.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE STRESS-ADAPTIVE corticotropic-adrenal axis mediates life-sustaining homeostatic and allostatic adjustments to internal and external stressors (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Circadian rhythmicity and pulsatile neurohormone secretion are physiological hallmarks of this dynamic feedback network (5, 6, 13). However, how the nyctohemeral and pulsatile time domains interface in health, stress, aging and/or disease is incompletely understood (14, 15, 16). Indeed, stress adaptations by this axis are typically orchestrated by combined adjustments in the nyctohemeral and pulsatile modes of ACTH-cortisol control (17, 18, 19, 20, 21). Although typical provocative or suppressive tests of a neuroendocrine axis usually impact only a single gland, the intact CRH/arginine vasopressin (AVP)-corticotropic-cortisol axis normally operates as an ensemble feedback-controlled network. Thus, the behavior of any one gland studied in isolation may not faithfully reflect the time-coordinated adaptations expected of an interactive control system (20, 22, 23). Accordingly, in an effort to capture dynamic adaptations of the integrated corticotropic axis, we here use several complementary and time-sensitive measures of within-axis feedback reactivity.

Few clinical studies have examined the specific impact of age on dynamic adjustments of the ACTH-adrenal axis to stress in men or women (24, 25, 26, 27, 28, 29). Aberrant homeostatic adaptations of the cortisol axis to stress in the elderly could have potentially significant clinical implications, because even short-term increases in target-tissue cortisol exposure can exacerbate the catabolism associated with stress (9, 12, 30, 31, 32, 33). For example, modest or relative glucocorticoid elevations within the population normative range, if extended over prolonged intervals in animals and humans, may impair neuronal dendritic plasticity, repress synaptic function, and reduce hippocampal cell survival (10, 18, 32, 34, 35). In this regard, higher serum cortisol concentrations in the elderly are associated epidemiologically with reductions in muscle and skeletal mass, greater visceral obesity, impaired cognitive function, and restricted quality of life (9, 12, 31, 32, 33, 34, 35, 36). Biological variability among older individuals in their stress responsiveness at the target-tissue level may further reflect molecular polymorphisms of the glucocorticoid receptor, thereby conferring unequal responses to ambient cortisol levels (10). Given these clinical implications, we have examined the pathophysiological control of (24-h) cortisol secretion in the elderly human subjected to mild or moderate stress.

To test the clinical hypothesis that aging disrupts dynamic stress-adaptive (feedback-dependent) control of the ACTH-cortisol axis, we have imposed the ethically feasible stressor of short-term fasting in healthy older and young men. To obviate species or gender confounds (37, 38) and to limit interindividual variability (7, 8, 39), we studied men using a within-subject, prospective and randomly ordered crossover design. Analytically, we quantitated each of the major feedback-dependent facets of daily cortisol secretion [i.e. nyctohemeral rhythmicity (2, 40, 41, 42, 43, 44, 45), pulsatile secretion (2, 4, 44, 45), pulsatile/nyctohemeral coupling (6, 39, 46), and the orderliness of the cortisol release process (47, 48, 49, 50)]. The last-mentioned feature of secretory regularity is assessed by the approximate statistic, which provides a sensitive and validated barometer of within-axis feedback coordination (51, 52, 53, 54). These collective strategies disclose distinctive age- and nutrient-dependent contrasts in the adaptive control of the ACTH-cortisol axis in healthy older and young men.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Clinical protocol

Sixteen healthy men within ±30% of normal body weight [overall mean body mass index, 22 ± 2.3 kg/m2 (range, 20–26)] and ages 18–35 yr (n = 8) and 60–72 yr (n = 8) were studied after provision of written informed consent approved by the Human Investigation Committee of the University of Virginia. No volunteer was receiving hormones or systemic medications, had undertaken recent transmeridian travel of more than three time zones within 10 days, or had an abnormal clinical history or physical examination. Each volunteer had normal screening biochemical tests of renal, hepatic, metabolic, and hematologic function and unremarkable (fasting) serum concentrations of T4, TSH, GH, insulin-like growth factor I, PRL, dehydroepiandrosterone sulfate, testosterone, estradiol, and immunoreactive LH and FSH, as assessed by RIA or immunoradiometric assay (14, 44, 55).

Volunteers were admitted to the General Clinical Research Center (GCRC) of the University of Virginia the night before blood sampling to allow overnight adaptation. Admissions were prospectively and randomly ordered in a crossover fashion at least 1 month apart to appraise cortisol dynamics by way of a paired within-subject fed vs. fasting design. Blood sampling was carried our during the last 24 h of the 3.5-day fast (see below). The LH data in this study were presented recently (55) but do not overlap with the current analysis of joint cortisol-LH synchrony.

In both the fed and fasting states, blood was sampled at 10-min intervals for 24 h beginning at 0800 h the morning after venipuncture. Samples (1.5 mL/sample) were withdrawn through an iv catheter placed earlier in a forearm vein. Samples were allowed to clot at room temperature. After centrifugation, the serum was frozen at -20 C for later cortisol assay. Subjects remained in the GCRC with bathroom privileges during sampling. In the fed state, three isocaloric meals were given per day at 0800, 1200, and 1800 h without intervening snacks. During the fast, volunteers received only water or other caffeine- and calorie-free liquids and slept in the GCRC. Lights were extinguished at 2300 h. Daytime naps were not permitted. Ambulation (but not vigorous exercise) was allowed in the GCRC. Urinary ketones were monitored each morning and evening to assess compliance with the fast. All patients exhibited consistent ketonuria on and after day 1 of the fast. Potassium chloride (40 milliequivalents) and water-soluble vitamins were administered orally daily.

Assays

Serum cortisol concentrations were measured in duplicate by a solid-phase RIA (Diagnostic Products, Inc., San Diego, CA), as described previously (44). The median inter- and intra-assay coefficients of variation were less than 5.6%. Serum concentrations of insulin, leptin, GH, PRL, and corticosteroid-binding globulin (CBG) were assayed in pooled (24-h) sera, as reported earlier (14, 39, 44, 55).

Deconvolution analysis

Multiparameter deconvolution analysis was used to quantitate subject-specific features of pulsatile cortisol secretion and its half-life (39, 46, 56). This technique resolves the serum cortisol concentration profile into constituent secretory pulses and simultaneously estimates the endogenous cortisol (monoexponential) half-life. The daily pulsatile cortisol secretion rate is the product of secretory burst frequency and the mean mass of cortisol released per secretory event. The latter is the analytical integral of the underlying secretory burst (46). Deconvolution analysis was carried out at 95% joint statistical confidence intervals (CIs) for all secretory burst amplitudes (56, 57). The technician was blinded to the randomized order of the fed vs. fasting admissions. A common cortisol secretory burst half-duration was calculated for each 24-h time series. No basal cortisol secretion was required to fit the present data (58).

Approximate entropy (ApEn)

ApEn was used as a scale- and model-independent statistic to quantitate the orderliness or regularity of consecutive serum cortisol concentrations over 24 h. Normalized ApEn parameters of m = 1 (test range) and r = 20% (threshold) of the intraseries SD were used, as described previously (54). This member of the ApEn family is, hence, designated ApEn (1, 20%) (48). The ApEn metric evaluates the consistency of recurrent subordinate (nonpulsatile) patterns in the data and, thus, yields information distinct from and complementary to both cosinor and deconvolution (pulse) analyses (47). Higher absolute ApEn values denote greater relative randomness of hormone secretion patterns (e.g. as observed for cortisol release in Cushing’s disease) (59). Cross-ApEn was calculated analogously after series standardization (z-score transformation) to assess the joint pattern synchrony of cortisol (present data) and LH (55) release, as described earlier for LH and testosterone (54).

Nyctohemeral (24-h) rhythmicity

Diurnal variations in serum cortisol concentrations were appraised by cosinor analysis, as reported earlier (6, 44). Ninety-five percent statistical CIs were determined for the 24-h cosine amplitude (50% of the nadir-zenith difference), mesor (mean), and acrophase (time of maximal value) (44). In addition, the 24-h rhythms of deconvolution-calculated cortisol secretory burst mass and interpulse intervals were evaluated for all subjects collectively within each of the four separate study sessions (6, 39, 46).

Statistical analysis

Because of nonnormal distributions of many nonlinearly derived parameters, deconvolution-calculated measures, ApEn values, and cosine coefficients were logarithmically transformed and then compared by one-way ANOVA. Statistical significance was construed for P less than 0.05. Post-hoc testing of group means was carried out via Duncan’s new multiple-range test. Mean and integrated (24-h) serum cortisol concentrations were evaluated without logarithmic transformation.

Data are presented as the mean ± SEM in the text and tables and in the figures as box-and-whisker plots (median, interquartile, and absolute range).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Table 1Go gives selected endocrine and metabolic measures made in 24-h serum pools prepared from the 145 blood samples collected from each volunteer fed and fasting. Leptin was higher at baseline (fed) in older than young men, but fell to a similar mean nadir value during fasting. There was no correlation between the fall in leptin and the rise in cortisol in either age cohort.


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  		Table 1. Selected endocrine and metabolic measures in fed and fasting young and older men

 
As depicted in Fig. 1Go, in the fed state, group mean serum cortisol concentrations in both age cohorts (n = 8 subjects/group) showed evident 24-h rhythmic variations. Visual inspection further suggested a fasting-associated increase in serum cortisol concentrations during the expected late-day nadir [i.e. at approximately 1800–0200 h clocktime (see quantitative analysis below)]. Fasting stress elevated the mean and integrated (24-h) serum cortisol concentrations significantly in both age groups (P < 10-7 and P < 10-8). Fig. 2AGo depicts the median, interquartile values, and absolute range of 24-h mean and integrated serum cortisol concentrations in the four study sessions (young and older men fed vs. young and older individuals fasting),which changed similarly in the two age cohorts.



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  		Figure 1. Group average (±SEM) 24-h serum cortisol concentration profiles in eight young and eight older men, each studied in randomly ordered fed and fasting sessions. Blood was sampled at 10-min intervals for 24 h in the fed state (three meals daily at 0800, 1200, and 1700 h clocktime) vs. 3.5-day fasting state. Serum cortisol concentrations were determined by RIA (see Patients and Methods). To convert µg/dL to nmol/L, multiply by 27.6.

 


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  		Figure 2. Integrated (top) and mean (bottom) 24-h serum cortisol concentrations in eight young and eight older men sampled in the randomly ordered fed vs. fasting states (see legend to Fig. 1Go). Data are box-whisker plots depicting the medians, interquartile values, and absolute range. P values denote the overall interventional effect. To convert cortisol concentration in µg/dL to nmol/L, multiply by 27.6. Unshared alphabetic superscripts denote significantly different group means, as assessed by ANOVA.

 
Deconvolution analysis was applied to quantitate the specific pulsatile mechanisms by which overall serum cortisol concentrations rose in response to fasting. As summarized in Table 2Go, certain dynamic features of cortisol secretion were unaffected by fasting [i.e. cortisol half-life, burst frequency (number of pulses per 24 h), interburst interval (min), and half-duration of cortisol release episodes]. Rather, metabolic stress specifically amplified cortisol secretory burst mass [i.e. the amount (µg) of cortisol secreted per unit (dL) cortisol distribution volume in both age groups (P = 0.002); Fig. 3AGo]. This reflected a higher cortisol secretory burst amplitude (maximal rate of cortisol secretion attained within each pulse) with no prolongation of secretory-event duration. The amplification of secretory burst mass during fasting was quantitatively similar in young and older individuals. As shown in Fig. 3BGo, the total 24-h pulsatile cortisol secretion rate rose significantly (and equivalently) in the two age groups during fasting (P = 0.000017).


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  		Table 2. Selected age- and fasting-independent measures of cortisol secretion and half-life in men

 


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  		Figure 3. Deconvolution-calculated cortisol secretory burst mass (µg/dL) (A) and daily pulsatile cortisol secretion (production) rates (µg/dL·24 h) (B) in young (n = 8) and older (n = 8) men during the fed state vs. a 3.5-day fast, assigned prospectively in randomized order. Multiparameter deconvolution analysis was used to estimate the cortisol secretory burst frequency, amplitude, mass (integral of the secretory burst), interpulse interval, and half-duration, as well as cortisol half-life (Table 1Go). The daily (pulsatile) cortisol secretion rate was calculated as the product of the mean cortisol secretory burst mass and frequency. To convert µg/dL to nmol/L, multiply by 27.6. Unshared alphabetic superscripts denote significantly different group means, as evaluated by ANOVA.

 
Fig. 4Go illustrates four individual 24-h (10-min sampled) serum cortisol concentration profiles (i.e. in young and older fed and fasting men). The corresponding deconvolutioncalculated cortisol secretory profiles are shown in Fig. 4BGo. The fasting-driven increase in cortisol secretory burst mass (and amplitude) is evident.



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  		Figure 4. Individual 24-h pulsatile profiles of serum cortisol concentrations (measured) and cortisol secretion rates (calculated) in two illustrative young and older men. Volunteers were studied in randomly ordered fed vs. 3.5-day fasting sessions, as described in the legend to Fig. 1Go. A, Measured serum cortisol concentrations (±intrasample SD) assayed in blood collected every 10 min for 24 h. The continuous curves through the observed data are predicted by deconvolution analysis (see Patients and Methods). B, Computed cortisol secretory profiles [µg of cortisol secreted per unit distribution volume (dL) per unit time (min)]. To convert µg/dL to nmol/L, multiply by 27.6.

 
ApEn was used to explore the impact of age and/or fasting on the pattern orderliness of serial serum cortisol measurements (Fig. 5Go). Cortisol ApEn values were similar in fed older and young subjects and increased during fasting in both age groups (P = 0.000003). Higher ApEn values signify greater irregularity (or reduced orderliness) of cortisol release patterns in fasting.



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  		Figure 5. ApEn (1, 20%) of 24-h serum cortisol concentration profiles in young (n = 8) and older (n = 8) men studied in randomly ordered fed vs. fasting sessions. Data are presented as depicted in the legend to Fig. 2Go. ApEn values rose significantly in fasting in both young and older individuals. Higher ApEn values denote greater irregularity or disorderliness of hormone release patterns. Unshared alphabetic superscripts reflect significantly different mean values by ANOVA.

 
Cosinor analysis was applied to appraise the diurnal rhythmicity of serum cortisol concentrations. As shown in Fig. 6AGo, in the fed state the mesor (24-h cosine average) was significantly higher in older subjects (P < 0.01 by unpaired Student’s t test). During fasting, the cortisol mesor rose in both young and older individuals (P < 10-7 by ANOVA), but to a greater absolute value in older subjects (P = 0.0045 by unpaired Student’s t test). Fig. 6BGo summarizes the amplitudes (50% of the difference between the zenith and nadir) of the 24-h rhythms in the serum cortisol concentration. Amplitudes were comparable in the fed state in young and older individuals and were unaltered by fasting. The acrophase (clocktime of the maximum of the 24-h serum cortisol concentration rhythm) was significantly age dependent (P < 10-5) and phase advanced by fasting in older men only (Fig. 6CGo). In particular, in the calorie-replete state, older men attained a maximal serum cortisol concentration approximately 168 ± 39 min before (earlier than) young men. More strikingly, during fasting, older (but not young) men sustained a significant additional phase advance, which resulted in an acrophase that was 267 ± 33 min earlier than that of fasting young adults. The incremental cortisol phase advance due to fasting in older volunteers averaged 99 ± 16 min.



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  		Figure 6. Twenty-four-hour (cosine) rhythmicity of serum cortisol concentrations in eight young vs. eight older men each studied in the fed and fasting states. Top, The cosine mesor, defined as the computed mean about which the 24-h sinusoidal rhythm oscillates. Middle, The cosine amplitude (one half the difference between the minimum and maximum of the daily rhythm in serum cortisol concentrations). Bottom, Cortisol acrophase (clocktime at which the maximum serum cortisol concentration occurs). Different alphabetic superscripts identify significantly different means.

 
Cosinor analysis was separately applied to the deconvolution-calculated cortisol secretory parameters [i.e. cortisol intersecretory burst interval (Fig. 7AGo) and cortisol secretory burst mass (Fig. 7BGo)]. The mean (mesor) of the interburst interval variation over 24 h was lower at 63 min (95% CI, 59–68) in fed older men compared with 76 min (95% CI, 68–83) in fed young men (Table 3Go). The amplitude of the rhythm in cortisol interburst intervals (related to the reciprocal of cortisol pulse frequency) was significantly nonzero in young (but not older) individuals in the fed state. Paradoxically, fasting reinstated a normal 24-h variation of the cortisol interburst interval in older individuals. Young and older fed volunteers both exhibited significant 24-h rhythms in cortisol secretory burst mass. The acrophase reached its maximum 161 min earlier, and the mean (mesor) was significantly lower in older men. The mesor rose more in older men during fasting, thereby equalizing this measure during fasting in the two age groups.



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  		Figure 7. Twenty-four-hour rhythms of deconvolution-calculated cortisol intersecretory burst intervals (A) and cortisol secretory burst mass (B) in young (n = 8) and older (n = 8) men. Volunteers were each studied twice in randomly ordered fed and fasting sessions. Pulsatile cortisol secretion was quantified by deconvolution analysis (see Patients and Methods). The interburst interval denotes the time (min) between consecutive secretory bursts, which data are regressed for each group of young or older men against clocktime in panel A. Cortisol secretory burst mass (defined as the integral of the calculated cortisol secretory pulse) is regressed against clocktime in panel B. NS, Nonsignificant (P > 0.05) by cosinor analysis of the ensemble data. To convert µg/dL to cortisol nmol/L, multiply by 27.6. Statistical analyses are summarized in Table 2Go.

 

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  		Table 3. Twenty-four-hour rhythms in deconvolution-estimated cortisol intersecretory burst interval and cortisol burst mass

 
The joint synchrony of cortisol and LH release patterns was assessed by cross-ApEn (see Patients and Methods). As shown in Fig. 8Go, the mean cross-ApEn value for cortisol/LH was significantly elevated in fed older men vs. fed young men (P = 0.029). This denotes reduced synchrony of bihormonal (cortisol-LH) release patterns in fed older individuals. Cross-ApEn rose in fasting young men, but failed to do so in fasting older volunteers.



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  		Figure 8. Cross-ApEn values to quantify the degree of joint synchrony between patterns of cortisol and LH release over 24 h in eight young and eight older men each studied fed and fasting. Elevated cross-ApEn denotes relative loss of bihormonal (cortisol/LH) pattern synchrony. Different alphabetic superscripts identify significantly different means.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The present clinical experiments support the hypothesis that healthy aging in men disrupts adaptive cortisol responses to a 2.5-day fast. This inference derives from a combination of intensive (10-min) and extended (24-h) blood sampling; a prospective, randomly ordered, within-subject crossover experimental design; and a 3-fold analytical strategy to quantitate endogenous cortisol dynamics. Such mapping of the feedback-sensitive control of cortisol secretion unmasked prominent age-dependent anomalies in neuroendocrine control. These age contrasts may be clinically relevant in older stressed individuals, because even relative glucocorticoid excess can exacerbate muscle protein catabolism during tissue-specific stress and may contribute to the relative visceral obesity, reduced quality of life, sarcopenia, osteopenia, cognitive impairment, and hippocampal neuronal attrition that tend to accompany advanced aging (18, 30, 31, 32, 33, 34, 35, 36).

From a mechanistic perspective, we could identify several strongly age-specific contrasts in cortisol dynamics: 1) an elevated mean (mesor) of the 24-h rhythm in serum cortisol concentrations in older men compared with young men, in both the fed and fasting milieus; 2) a marked (99 ± 16 min) additional advance in the cortisol acrophase (clocktime of the zenith in the 24-h rhythm of serum cortisol concentrations) during caloric depletion in older men, beyond the 168 ± 39-min phase advance evident in the unstressed nutrient-replete state; 3) the loss of the expected young-adult 24-h variation in cortisol intersecretory burst interval (the reciprocal of burst frequency) in fed older men; 4) stress-associated paradoxical restoration of the daily cortisol pulsefrequency rhythm in older volunteers; 5) a greater fasting-induced increment in the day-night variation in cortisol secretory burst mass in aging men; and 6) deterioration of the joint pattern synchrony of cortisol and LH release in fed older men, which (unlike the adaptive response in young controls) was unaltered by caloric restriction.

From a single-hormone perspective, the foregoing age-associated aberrations in the regulation of cortisol dynamics during fasting primarily involve the time-dependent control of pulsatile (ultradian) and nyctohemeral (circadian) cortisol secretion, and their expected (young adult) physiological coupling. From a two-hormone perspective, fed older men also showed erosion of joint cortisol and LH pattern synchrony. We, thus, infer that healthy aging likely disrupts neuroendocrine mechanisms that coordinate within-axis pulsatile and 24-h rhythmic cortisol release and also alters the interaxis mechanisms that link LH and cortisol release. Such regulatory aberrations emerge in healthy older individuals without any damping of the overall capacity of the cortisol axis to elevate total daily cortisol secretion. Akin to the present findings in fasting stress, other studies using pharmacological stimulation of the hypothalamo-pituitary-adrenal axis (e.g. insulin-induced hypoglycemia or metyrapone feedback blockade) also show normal maximal ACTH and cortisol release in aging men and women (11, 60, 61, 62), In contrast, one earlier analysis of glucocorticoid responses to thoracic or abdominal surgery in 47 patients aged 17–86 yr disclosed accentuated cortisol release in older subjects (62). These discrepancies might indicate that age-related contrasts in maximal ACTH/cortisol axis responsivity are also stressor specific.

Alterations in the time-sensitive regulation of cortisol secretion in aging men likely denote disruption of feedforward- and/or feedback-dependent regulation within the interactive CRH/AVP-ACTH-adrenal axis (22, 47, 51, 63, 64). Based on simplified principles of feedback network-control (23, 47, 51, 53), we postulate that such age-associated disparities may reflect more specifically loss of central nervous system (CNS) integrative control among neural sleep-wake/circadian centers (15, 16), the putative hypothalamic pulse generators for CRH and/or AVP (65, 66), anterior pituitary corticotropes, and the ACTH-responsive adrenal cortex. Although not well formalized, the strength and timing of the foregoing within-axis multisite interactions are assumed to supervise pulsatile cortisol secretion, dictate its 24-h rhythmicity, and couple ultradian-circadian dynamics (23, 28, 44, 54, 64). This notion of integrative within-axis control in health would predict, for example, that autonomous tumoral secretion of ACTH in Cushing’s disease would disrupt the pulsatile, entropic, and diurnal rhythmic properties of cortisol secretion, due to loss of physiological within-axis feedback control as, indeed, is observed (59, 67).

Although less well understood than the foregoing single-axis regulatory complexity, other neuroendocrine mechanisms act to sustain two-axis synchrony (e.g. coordinate patterns of cortisol and LH release). The present analysis identifies vivid cortisol-LH asynchrony in fed aging men and reveals that similar cortisol-LH asynchrony can be induced in young men by the stress of fasting. Reduced pattern synchrony of cortisol and LH secretion in fed older individuals thematically complements the age-dependent disruption of two-hormone synchrony reported for LH and testosterone (54), ACTH and cortisol (67), LH and FSH (68), LH and PRL (69), and LH and nocturnal penile tumescence oscillations (69). Insulin and GH secretion show disordered monohormonal patterns in aging men and women (54, 67, 68, 69, 70, 71, 72, 73, 74). The generality of these findings across several neuroendocrine axes allows the conjecture that aging [or one of its primary correlates (12)] impairs neuroregulatory feedback in the human. Because the expected 3-fold joint synchrony among CNS-governed pulsatile LH secretion, nocturnal penile tumescence oscillations, and sleep-stage transitions is also prominently disrupted in healthy older men (69), we can suggest that aging alters CNS-dependent neuroregulatory control of several axes (54).

A recent meta-analysis highlighted a consistent phase advance in the timing of the 24-h serum cortisol concentration rhythm in fed older men and women, as assessed principally by 20-min blood sampling (75). An analysis of fed septua- and nonagenarians revealed a progressive cortisol phase advance with increasing age even within this elderly cohort (76). Unlike the phase, the periodicity of the free-running circadian cortisol rhythm seems to be remarkably stable within the adult human lifetime (13). The present study, although not assessing the endogenous circadian period length, demonstrates that fasting stress further extends the (fed) age-associated phase advance by 1 and 1.5 h. Whereas the disparity in cortisol acrophase in the fed state may reflect body compositional, sleep and/or other behavioral differences in older and young individuals (75, 77, 78), the additional phase advance uncovered here by fasting would point to a loss of adaptive circadian cortisol control in aging men.

The foregoing unexpected and prominent cortisol phase-shift associated with fasting in older men holds possible analogy to the inordinate locomotor-activity shifts that can be induced in the aging hamster by certain (circadian phase-sensitive) photic stimuli (79). Altered circadian coupling in the latter species seems to reflect age-related changes in suprachiasmatic nucleus monoaminergic neurotransmission. Moreover, disruption of phase control can be overcome partially by transplantation of fetal hypothalamic suprachiasmatic nucleus grafts into older animals (80).

Age-related contrasts in the reactivity of cortisol dynamics to short-term fasting stress were highly specific with respect to secretory mechanisms. Thus, older and young men exhibited indistinguishable fed and fasting cortisol half-lives, secretory burst frequencies, interburst intervals, and pulse durations (Table 2Go). Age-related differences in cortisol dynamics were not explained by disparate metabolic changes during fasting, because the two age cohorts maintained similar pooled 24-h serum concentrations of insulin, PRL, and GH both fed and fasting, albeit divergent leptin levels fed (Table 1Go). Relatively elevated (fed) leptin values in older men could likely reflect their greater percentage (total and visceral) body fat (81).

The metabolic stress of fasting disrupted the orderly pattern of 24-h cortisol release in both age groups, as quantitated by way of the ApEn statistic (22, 47, 50, 52, 82). This scale-independent metric identified less regular cortisol secretion in fasting men, which complements the separate analyses of cortisol dynamics by peak detection and cosine regression (47, 48, 83, 84). Scale invariance of the entropy measure is pertinent, because it here ensures a valid inference of greater cortisol secretory disorderliness in fasting irrespective of higher serum cortisol concentrations. This statistical property is illustrated by the fact that the orderliness of daily ACTH profiles actually decreases significantly during the metyrapone-induced 20-fold elevation in plasma ACTH concentrations (85). Other "open-loop" clinical experiments also affirm that the ApEn of pituitary-hormone secretion is dictated by relevant negative feedback signaling [e.g. elevated ApEn of LH secretion is reversed by iv testosterone feedback inhibition during ketoconazole-induced blockade of steroidogenesis (86)] and the disorderly release of TSH in primary thyroidal failure is reversed by T4 replacement (85). GH secretion also becomes highly irregular during fastinginduced insulin-like growth factor I withdrawal and in aging (50, 70, 87). These clinical paradigms indicate that the ApEn metric monitors axis-specific feedback control (47, 48, 54, 83), an inference corroborated biomathematically by recent computer-assisted simulations of the GnRH-LH-testosterone feedback axis (53). Accordingly, the elevated ApEn of 24-h cortisol secretory profiles in fasting men argues strongly for stress-induced alterations in feedback control of the CRH/AVP-ACTH-cortisol axis.

Fasting stress primarily elevated cortisol release in the late afternoon and early evening hours in both young and older men. The mechanisms that subserve the normal late-day decline in 24-h serum cortisol concentrations and mediate the abrogation of this rhythmicity by stress are not well known. In the healthy unstressed adult, day-night variations in serum ACTH and cortisol concentrations are driven primarily by nyctohemeral control of the amplitude (or mass) of ACTH and cortisol secretory bursts (44, 45, 88). In the rodent, day-night differences are recorded in the hypothalamic expression of the CRH and AVP genes, the responsiveness of corticotroph cells to secretagogue input, the negative feedback efficacy of corticosterone and the sensitivity of the adrenal cortex to ACTH stimulation (40, 41, 43, 89, 90, 91, 92). Fewer analogous data are available in the human. However, in one autopsy study of 39 individuals aged 6–91 yr, significant 24-h variations in the suprachiasmatic nuclear expression of AVP immunoreactivity was evident in young but not older subjects (66). Diurnal rhythmicity can regulate adrenal responsivity to ACTH in healthy adults, wherein the acute serum cortisol increment observed 5 and 15 min after an iv bolus infusion of ACTH is higher in the afternoon or evening than morning (92), as are the rate of rise and maximal cortisol values achieved by sustained (8-h) iv infusion of ACTH (93). The maximal serum cortisol rise following CRH injection likewise is greater in the late day (at 2300 h) than in the morning, despite comparable increases in the plasma concentration of immunoreactive ACTH (41). Lastly, there may be a small 24-h rhythm in the impact of mineralocorticoid negative feedback on the corticotropic axis in young men (94). However, how and whether these nyctohemeral variations in normal ACATH-cortisol axis responsiveness are influenced by fasting and/or age is not known.

In limited earlier analyses of adrenal responsivity to ACTH in aging, one investigation of hospitalized octogenarians described a greater serum cortisol increment in 20 ill elderly patients compared with 50 healthy young subjects given an im injection of ACTH gel (11). However, two other controlled clinical studies conducted in healthy cohorts of young and older volunteers reported that synthetic ACTH’s stimulation of cortisol secretion is age invariant (26, 61). Exogenous CRH’s drive of ACTH and/or cortisol release was also comparable in young and older men in two separate assessments (27, 95). In contrast, another clinical study reported heightened pituitary and adrenal secretory responsiveness to CRH and/or lysine vasopressin infusions in healthy individuals aged 65–90 yr (78). These apparent inconsistencies illustrate the clinical challenge inherent in detecting age-related changes in ACTH-adrenal axis activity based on single-gland acute stimulation or inhibition tests, which isolate the studied locus from its expected within-axis feedback adjustments.

One dose-responsive analysis of dexamethasone-imposed negative feedback on ACTH release revealed comparable suppression in young and older men (27). However, two other studies, which together included a total of 68 individuals whose ages spanned 17–78 yr, reported a progressive age-related rise in postdexamethasone morning serum cortisol concentrations, consistent with accelerated clearance of synthetic glucocorticoid and/or impaired negative feedback in aging (96, 97). Further clinical analyses using iv hydrocortisone infusions to explore rapid and delayed glucocorticoid negative feedback in aging humans have disclosed selectively impaired feedback effects of cortisol on ACTH release in elderly individuals (24, 25). Thus, the results of the majority of human investigations suggest that glucocorticoid negative feedback is attenuated in aging, akin to data in the older rodent (98).

Serum CBG concentrations, as measured here and in two earlier clinical studies, were not affected by age (95, 99), which is reported also in the aging male rat (9). By deconvolution analysis, we predicted similar CBG levels in older and young men, inasmuch as mean estimated endogenous cortisol half-lives were no different in the two groups. This kinetic inference is based on the theoretical expectation that changes in plasma binding-protein concentrations strongly influence free and total ligand half-lives in vivo (100, 101, 102).

The foregoing investigations were carried out in healthy young and older men exposed to a particular metabolic stressor. Thus, our inferences may not apply equally to other types of stress, recurrent or chronic stress, or healthy children or women. Indeed, there are evident stressor-dependent and gender-based differences in the stress-adaptive reactivity of the ACTHcortisol axis (37, 38, 78, 83, 91). The present short-term fasting paradigm may be useful to further appraise the stress and/or gender specificity of dynamic cortisol adaptations.


   Acknowledgments
 
We thank Patsy Craig for assistance in manuscript preparation, Paula Azimi for statistical and deconvolution analysis and artwork, Brenda Grisso for performance of the immunoassays, and Sandra Jackson and the nursing staff at the University of Virginia Clinical Research Center for conduct of the clinical research protocols.

Received August 27, 1999.

Revised February 14, 2000.

Accepted February 27, 2000.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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