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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 10 4077-4084
Copyright © 2006 by The Endocrine Society

Age or Factors Associated with Aging Attenuate Testosterone’s Concentration-Dependent Enhancement of the Regularity of Luteinizing Hormone Secretion in Healthy Men

Peter Y. Liu, Paul Y. Takahashi, Pamela D. Roebuck and Johannes D. Veldhuis

Endocrine Research Unit (P.Y.L., P.D.R., J.D.V.) and Department of Internal Medicine (P.Y.T.), Mayo Clinic School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Endocrine Research Unit, Mayo Clinic School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Healthy older men have reduced testosterone (Te) production and frequent, small irregular LH pulses. Which is cause and which is effect are not known.

Rationale: In model systems, frequent and irregular LH pulses reflect attenuated feedback inhibition by Te.

Hypothesis: Factors associated with aging impair negative feedback by Te.

Subjects and Setting: Healthy men at an academic medical center were studied.

Methods: The study used quantification of the regularity of LH release patterns during blockade of gonadal steroidogenesis and graded transdermal Te addback in 18 healthy men aged 18–65 yr.

Results: In the cohort as a whole, stepwise Te repletion repressed LH concentrations (P = 0.001) and enhanced the quantifiable orderliness of LH release patterns (P < 0.001). By regression analysis, age attenuated the capability of increasing Te concentrations to regularize LH secretion patterns (P = 0.019). However, after a fixed GnRH stimulus, the effect of Te on LH regularity was invariant of the age factor (P = 0.16), thus pointing to a hypothalamic locus of impaired Te feedback.

Summary: Aging disrupts the capability of systemic Te concentrations to maintain orderly LH secretion under endogenous, but not exogenous, GnRH drive.

Conclusions: Age or factors associated with increased age reduce negative feedback by any given total Te concentration on hypothalamopituitary GnRH-LH outflow, thus contributing to disorderly LH secretion.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CROSS-SECTIONAL AND LONGITUDINAL studies have documented progressive androgen depletion in the aging male (1, 2, 3, 4, 5, 6, 7, 8, 9). In particular, testosterone (Te) concentrations fall by 30–50% between young adulthood and later decades of life in healthy community-dwelling men. The precise cause of declining Te production is not known but may be multifactorial (10). The question is important because epidemiological analyses correlate hypoandrogenemia with insulin resistance, dyslipidemia, sarcopenia, osteopenia, reduced physical stamina, sexual dysfunction, depressive mood, and cognitive impairment (11, 12, 13, 14, 15, 16). Conversely, androgen supplementation trials suggest that Te administration may ameliorate certain features of physical frailty in older men (17, 18, 19, 20, 21, 22). Clinical investigations of the mechanistic bases of aging-associated Te deficiency have identified at least tetrapartite regulatory deficits; viz., accelerated LH pulse frequency, reduced LH pulse size, disorderly release of LH and Te, and impaired Leydig cell responsiveness to human chorionic gonadotropin and LH (23, 24, 25, 26, 27, 28, 29, 30). Earlier studies have documented essentially unchanged LH pulse frequency with age (Refs. 31, 32 and reviewed in Ref. 30). However, previous methodological limitations included less frequent blood sampling, less sensitive assays, and the lack of deconvolution techniques. In experimental paradigms and mathematical feedback models, reduced Te concentrations in healthy young men evoke frequent small and irregular LH pulses, thereby mimicking aging-related patterns of LH secretion (33, 34, 35, 36, 37, 38). Thus, lower Te concentrations in older men may be responsible for quantifiably more irregular LH pulses. Alternatively, factors associated with aging may impair Te-dependent negative feedback on GnRH-LH secretion at any given Te concentration. Which of these hypotheses applies is not known.

Studies using exogenous androgens have reported normal, accentuated, or impaired inhibition of mean LH concentrations in older compared with young men (31, 32, 39, 40, 41). On the other hand, analytical estimates of endogenous Te-dependent suppression of LH secretion have forecast impaired negative feedback in aging individuals (33, 42). An ensemble model of GnRH-LH-Te interactions supported the latter prediction (37, 38). Given that Te concentrations vary among individuals and generally decline with age, available data cannot distinguish whether presumptive androgenic feedback failure in the elderly male reflects relative Te deficiency or a diminished capability of any given Te concentration to enforce negative feedback.

To address the foregoing basic mechanistic questions, the present investigation implements a combined strategy of short-term inhibition of Leydig-cell steroidogenesis and graded transdermal repletion of Te to enforce four echelons of negative feedback in healthy men aged 18–65 yr. The a priori postulate was that age, or factors associated with it, would impair Te’s feedback actions, as monitored by a sensitive (>90%) and specific (>90%) measure of feedback-enforced pattern regularity in neuroendocrine systems (43).


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

Eighteen men aged 19–65 yr (mean 41, two to four men per decade) participated in the study after providing written voluntary informed consent approved by the Mayo Clinic Institutional Review Board. Participants were healthy community-dwelling men within 20% of ideal body weight. None had undertaken recent transmeridian travel (within 10 d) or consumed alcohol, caffeine, or systemic medications within 5 biological half-lives. Detailed medical inventory excluded a history of infertility, systemic disease, recent weight change (exceeding 2 kg in the preceding 6 wk), Te therapy, or psychoactive drug use. Outpatient screening was unremarkable in relation to medical history (particularly libido and erectile function); physical examination (including testis size); and fasting morning (0800 h) biochemical tests of renal, hepatic, hematological, endocrine, and metabolic function [plasma glucose and serum T4, prolactin, IGF-I, LH, FSH, Te, and estradiol (E2)].

Intervention

The overall protocol time line is given in Fig. 1Go.


Figure 1
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  		FIG. 1. Time line of interventions and sampling schedule. Subjects were exposed to placebo or active Te gel in randomized double-blind order at least 10 d apart. The time line applies to any one of the four study sessions conducted in each subject.

 
Volunteers were admitted to the Mayo General Clinical Research Center (GCRC) for four separate overnight inpatient sampling studies scheduled at least 10 d apart. On an outpatient basis, the cholesterol side-chain cleavage (CYP11A) inhibitor, ketoconazole (1000 mg initial loading dose and then 400 mg four times each day) and glucocorticoid replacement (dexamethasone 0.75 mg twice each day) were begun orally and continued for 48 h (28, 35, 44). Concomitant Te addback entailed nightly (2200 h) application of transdermal gel containing any one of 0 (placebo), 2.5, 5, or 7.5 g Te (Androgel, impregnated with 1% steroid of which 10% is absorbed). This gel is known to result in near steady-state testosterone absorption (45). Gel was applied on 3 consecutive nights in a prospectively randomized, double-blind, within-subject, crossover fashion. The ketoconazole regimen reduces total Te concentrations to less than 100 ng/dl without depleting estrogen (28, 35, 44). Twice the above dexamethasone replacement dose administered for over thrice the duration exerts no detectable effect on 24-h mean, pulsatile, or GnRH-stimulated LH release in normal young men (46).

Sampling protocol

Subjects were admitted to the GCRC on the third night of the outpatient regimen. Ketoconazole and dexamethasone were continued, and transdermal gel was applied at 2000 h. Blood samples (1.0 ml) were withdrawn every 10 min beginning at 2200 h for a total of 14 h through a forearm iv catheter. After 12 h, a single iv pulse of GnRH (100 ng/kg) was administered to test pituitary responsiveness. Blood was allowed to clot at room temperature, and sera were frozen at –20 C for later assay of serum LH and Te concentrations.

Assays

LH concentrations were measured in duplicate by automated immunochemiluminometry (ACS Corning; Bayer, Tarrytown, NY) using the Second International Reference Preparation as standard (28, 47). Concentration-dependent intraassay coefficients of variation (CVs) averaged 5.5, 4.7, 3.5, and 3.8% and interassay CVs 6.5, 5.2, 3.7, and 4.7% at LH concentrations of 1.3, 4.4, 18, and 38 IU/liter, respectively. Procedural sensitivity was 0.05 IU/liter. Te was quantitated in the same assay system, wherein median intra- and interassay CVs were 6.8 and 8.3%, respectively, and the sensitivity was 18 ng/dl. Te measurements obtained by immunochemiluminometry correlate strongly (r2 = 0.98) with those determined by gas chromatography-mass spectrometry (48). E2 was measured by double-antibody RIA using reagents purchased from Diagnostic Systems Laboratories (third-generation DSL-39100; Webster, TX), as reported (49). Sensitivity was 3 pg/ml and intra- and interassay CV’s were 4.7 and 6.8%, respectively.

Analytical methods

Cluster analysis was used to estimate traditional pulse characteristics (50). Conservative pulse-detection parameters (<5% false-positive errors) for LH included 2-by-1 test cluster sizes and a threshold of t = 2.0 to identify consecutive upstrokes and downstrokes in the time series (24, 51). The relevant end points were mean, absolute peak (maximum), incremental amplitude (peak minus nadir), peak area (integral of peak above mean of preceding and subsequent nadir values), and interpulse nadir concentrations of LH and Te.

The approximate entropy (ApEn) statistic was used to quantitate the degree of irregularity, or disorderliness, of each time series (43, 52, 53). Technically ApEn is defined as the summed logarithmic likelihood that templates (of length m) of patterns in the data that are similar (within r) remain similar (within the same tolerance r) on next (m + 1) incremental comparison, as validated elsewhere (54). ApEn of any given time series is a single nonnegative number. The statistic confers an ensemble estimate of relative process randomness, wherein larger ApEn values denote greater irregularity. ApEn calculations assumed m = 1 and r = 20, 35, 50, or 75% of the SD of each data set for series of 12, 6, 4, or 2 h duration, respectively. These parameters afford sensitive, specific, valid, and statistically well-replicated regularity measures (52, 55, 56). For the 2-h post-GnRH LH pulse, data were initially first differenced [the ith element of the time series is converted into its algebraic difference from the (i – 1)th element] (57).

Statistics

Repeated-measures one-way ANOVA was used to test the overall postulate that transdermal Te doses (four factors) determine ApEn in the cohort as a whole (n = 18 subjects). The primary hypothesis was that age attenuates the effects of graded Te feedback. This was assessed in two ways. First, linear regression analysis was applied to evaluate the relationship between LH ApEn and age at each of the four transdermal Te doses. Significance was assessed by Pearson’s correlation coefficient at multiple-comparison protected P < 0.0125 (58). And second, repeated-measures two-way ANOVA was used to assess the individual and combined impact of Te dose and age on LH ApEn, assuming a compound symmetrical variance-covariance matrix. Post hoc comparisons of means were by Bonferroni adjustment at experiment-wise P < 0.05 (59).

The sensitivity of LH regularity to Te (Te sensitivity) was calculated in each individual by regressing ApEn linearly on the mean Te concentration in the four separate overnight visits. The resultant 18 slopes were then regressed on age to examine the influence of age on Te sensitivity. To explore the sampling dependence of detecting feedback effects, the same analyses were applied arbitrarily to ApEn values calculated during the last 6, 4, or 2 h of the total 12-h time period (60). Analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC). Data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Subjects ranged in age from 19 to 65 yr (mean 41, two to four men per decade). Their mean ± SEM body mass index was 26 ± 0.7 kg/m2. Linear-regression analysis revealed that baseline (prestudy) E2 and bioavailable and free Te declined with age (Fig. 2Go).


Figure 2
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  		FIG. 2. Regressions of E2 and total, bioavailable, and free Te concentrations on age. Data were obtained before any intervention.

 
The transdermal Te addback regimen yielded four strata of total Te concentrations (P < 0.001 by repeated-measures ANOVA). All differed (P < 0.005) except for the two associated with the 5- and 7.5-mg Te doses (Table 1Go). Mean values for the cohort (n = 18) spanned the castrate (30 ng/liter), low- (280 ng/dl), and midphysiological (410 and 560 ng/dl) morning reference range in healthy young men (365–870 ng/dl). Age had no effect on total Te concentrations (P > 0.50 by linear regression at each dose level; P = 0.79 by repeated-measures ANOVA). Te dose but not age increased 12-h mean E2 concentrations (Table 1Go). Figure 3Go illustrates paired 14-h LH and Te concentration profiles in three subjects (young, middle, and older) for each of the four Te addback strata. Age per se did not influence 12-h mean LH concentrations (Table 1Go). Age determined LH peak increment and number, whereas Te dose affected LH peak increment and nadir concentrations (Table 1Go).


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  		TABLE 1. Te dose and age effects on 12-h mean Te and LH concentrations and LH regularity (ApEn)

 

Figure 3
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  		FIG. 3. Illustrative serum LH and total Te concentration profiles in young, middle-aged, and older men, who underwent blood sampling every 10 min for 14 h overnight (2200–1400 h). Each subject received ketoconazole and glucocorticoid orally and placebo or Te transdermally for 48 h before and then during the study session at the indicated doses of 0, 2.5, 5.0, and 7.5 mg/d. A single pulse of GnRH (100 ng/kg) was injected 2 h before the end of sampling.

 
Te repletion reduced (12-h mean) LH concentrations and LH ApEn dose dependently in the cohort as a whole (both P < 0.001 by repeated-measures ANOVA) (Table 1Go). Linear regression of LH ApEn on age in the 18 subjects at each dose of Te applied showed significant positive correlations for the 2.5- and 7.5-mg Te doses (P = 0.007 and P = 0.02, respectively). Age did not modify LH regularity when Te concentrations were less than 85 ng/dl, viz., during placebo (zero-dose Te) addback. Age also did not modulate Te’s suppression of mean LH concentrations (P = 0.20) but significantly attenuated Te’s reduction of LH ApEn (P = 0.018), denoting feedback failure (Table 1Go).

Linear regression of LH ApEn on Te concentrations yielded an inhibition slope, here defined as a Te feedback-sensitivity estimate, in each subject (Fig. 4Go). Negative slopes denote enhancement of LH regularity (lower ApEn) at higher Te concentrations. Older subjects exhibited regression slopes lower in absolute value, in some cases approaching zero (ApEn then being independent of Te concentration), as quantified by regressing Te feedback-sensitivity values (n = 18) linearly on age. Failure of LH ApEn to decline comparably in older men given Te signifies that age impairs feedback-induced orderliness (R = 0.55, P = 0.019 by repeated-measures ANOVA). The consistency of the age effect was evaluated by examining LH regularity (ApEn) over 12-, 6-, 4-, and 2-h time blocks. Both 12- and 6-h time windows were sufficient to detect age-related erosion of feedback (Fig. 5Go).


Figure 4
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  		FIG. 4. Regression of LH regularity (ApEn) on total Te concentrations in 18 individual men. The age in years of each man is shown in the box. LH ApEn and mean total Te concentrations were determined from 10-min measurements made over 12 h before GnRH injection (Fig. 1Go). Higher ApEn denotes greater irregularity (disorderliness) and lesser feedback. Te lowers ApEn, especially in young men.

 

Figure 5
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  		FIG. 5. Age reduces the capability of increasing Te doses to enhance the orderliness (reduce the irregularity, ApEn) of LH secretion. The two panels evaluate the minimal sampling duration required to detect age-related reductions in Te’s feedback-dependent enhancement of LH regularity. Each panel shows the slopes of LH ApEn regressed on total Te concentrations as a function of age (n = 18 volunteers). Pearson’s correlation coefficient and P value are given.

 
Analogous analyses were performed on the 2-h post-GnRH injection time period (Table 2Go). Te feedback-sensitivity estimates for GnRH-induced LH release were not affected by age (Fig. 6Go).


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  		TABLE 2. Lack of age effect on postexogenous GnRH 2-h mean Te, E2, and LH concentrations and LH regularity

 

Figure 6
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  		FIG. 6. Age does not alter the capability of increasing Te doses to enhance the orderliness (reduce the irregularity, ApEn) of exogenous GnRH-stimulated LH secretion. The slopes of LH ApEn regressed on total Te concentrations after GnRH administration (analogous to Fig. 2Go) are here regressed on age (n = 18 volunteers).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present investigation used an experimental protocol of androgen depletion with graded transdermal Te replacement to test whether age or factors associated with aging determine Te’s concentration-dependent negative feedback on LH regularity in healthy men. Statistical analyses established that the steady-state Te clamp suppresses 12-h mean LH concentrations in a dose-dependent but age-independent fashion. Mechanistic assessments demonstrated that graded strata of total Te concentrations from near-castrate to midphysiological young adult values reduce the quantifiable irregularity (ApEn) of LH release. The capability of a feedback signal to enforce more orderly patterns of hormone secretion is consistent with both empirical and theoretical models (35, 37, 52). Increasing age over a five-decade range significantly attenuated Te’s feedback-dependent regularization of LH secretion. From these data, we infer that age or factors associated with aging impair expected feedback by systemic Te concentrations on the hypothalamopituitary unit in healthy men studied in the morning when outflow of the gonadal axis is maximal (10). These data show the utility of a regularity statistic (ApEn) to probe dynamic feedback regulation and complement other work showing feedback effects of testosterone on LH pulse characteristics (introductory section).

Previous studies disagree on whether pharmacological and high physiological amounts of Te or 5{alpha}-dihydrotestosterone decrease LH concentrations to a greater (31, 32, 39) or lesser (40, 41) extent in older compared with young men. Earlier limitations include the lack of evaluation of negative feedback in the same subject over a span of Te concentrations; comparison of responses at two age extrema instead of over an age range; variability among cohort selection criteria; and analysis of mean LH concentrations rather than regulated properties of LH secretion. Addressing these issues in the present study required evaluating feedback responses over a greater than 18-fold range of total Te concentrations (30–560 ng/dl), including healthy unmedicated subjects ages 18–65 yr, and using ApEn as a sensitive and specific validated measure of negative feedback (52). Thus, most prior reports can be related to the current data only with respect to asking how age alters the capability of the highest Te dose to suppress mean LH concentrations.

The paradigm of steroidogenic inhibition applied here was initially validated in young men, in whom 24-h LH secretion patterns become markedly irregular (elevated ApEn) during experimental Te depletion (35, 44). By way of validation, iv infusion of a fixed amount of Te restored secretory orderliness as putatively mediated via estrogen receptor-{alpha}, androgen receptor, and/or nongenomic pathways that regulate hypothalamic neurotransmission, GnRH neurons, and gonadotropes (10). The fundamental basis for the inferred reduction in negative feedback by any given Te concentration in older men is not known because many factors are associated with aging. However, in the present analysis, E2 concentrations were comparable by age, but intrasubject values differed by 9 ± 1.5 pg/ml between the lowest and highest Te strata. The small difference may reflect the variation in serum Te, which is the substrate for aromatization rather than any effect on aromatase per se because the in vitro effect of ketoconazole on aromatase is inhibitory and the in vivo effect is unclear (61). Studies in the aged rodent point to decreased hypothalamopituitary expression of both androgen and estrogen receptors (62, 63, 64). Whether this accounts for dysregulation of the LH response to Te, as observed here, is not known. Furthermore, how these molecular changes impact gonadal axis signaling will remain unclear until ligand and receptor concentrations within the hypothalamopituitary unit are directly quantified. This is because local aromatization of testosterone to E2 may be important in feedback regulation and might be affected by age (65, 66).

As an indirect probe of direct pituitary feedback by different Te concentrations, LH regularity was also estimated after injection of a submaximal GnRH stimulus (100 ng/kg). The orderliness of GnRH-induced LH release rose (ApEn fell) with higher total Te concentrations, but the factor, age, in the statistical analysis had no effect on GnRH action. Thus, if GnRH is the primary stimulus to LH pulses (10, 67, 68, 69), our data point to an effect of age on the Te-modulated release (amount or regularity) of hypothalamic GnRH rather than GnRH action. Direct monitoring of GnRH secretion patterns would be required to test this postulate.

Qualifications include the need to: 1) ultimately extend the generality of these inferences to larger and older cohorts; 2) assess the impact of alternate modes of Te addback on GnRH outflow and LH stimulation on Te secretion; and 3) establish the exact age dependence of inferred feedback failure in populationally based longitudinal studies.

In summary, an experimental paradigm of steroidogenic blockade and glucocorticoid replacement with graded transdermal Te repletion in healthy men aged 18–65 yr indicates that total Te concentrations within the physiological range enforce orderly LH secretion patterns and age or factors associated with aging impair Te’s feedback-dependent regularization of LH secretion without altering its suppression of mean LH concentrations. These data introduce the hypothesis that age or factors associated with aging impair the feedback effectiveness of any given Te concentration acting on the hypothalamic outflow of GnRH.


   Acknowledgments
 
We thank Solvay Pharmaceuticals for donating Androgel without a priori stipulations.


   Footnotes
 
This work was supported in part by the National Center for Research Resources (Rockville, MD) Grant M01 RR00585 to the General Clinical Research Center of the Mayo Clinic and Foundation, the National Institutes of Health (Bethesda, MD) Grants RO1 AG23133 and DK60717. P.Y.L. was supported by fellowships from the National Health and Medical Research Council of Australia (Grant 262025) and the American Australian Association. P.Y.T. was supported by a Mayo Institutional Medicine Innovation Development and Advancement System Award.

Current address for P.Y.L.: Division of Endocrinology, Harbor-University of California, Los Angeles Medical Center, Torrance, California 90509-2910.

Disclosure: I accept responsibility for the conduct of this study, and I have seen and approve the final manuscript. No portion of this article will be published or submitted elsewhere before appearing in JCEM. All authors have nothing to declare.

First Published Online July 25, 2006

Abbreviations: ApEn, Approximate entropy; CV, coefficient of variation; E2, estradiol; GCRC, General Clinical Research Center; Te, testosterone.

Received December 23, 2005.

Accepted July 17, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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P. Y. Liu, A. Iranmanesh, D. M. Keenan, S. M. Pincus, and J. D. Veldhuis
A noninvasive measure of negative-feedback strength, approximate entropy, unmasks strong diurnal variations in the regularity of LH secretion
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1409 - E1415.
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