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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2704
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 9 3626-3632
Copyright © 2007 by The Endocrine Society

Age Attenuates Testosterone Secretion Driven by Amplitude-Varying Pulses of Recombinant Human Luteinizing Hormone during Acute Gonadotrope Inhibition in Healthy Men

Paul Y. Takahashi, Patrick Votruba, Mohammed Abu-Rub, Kristi Mielke and Johannes D. Veldhuis

Endocrine Research Unit, Department of Internal Medicine, General Clinical Research Center, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905

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


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Whether testosterone (Te) depletion in aging men reflects deficits in the testis, hypothalamus, and/or pituitary gland is unknown.

Objective: Our objective was to quantify the impact of age on gonadal Te secretion driven by amplitude-varying pulses of recombinant human LH (rhLH) in the absence of confounding by endogenous hypothalamo-pituitary signals.

Design: This was a double-blind, placebo-controlled study.

Setting: The setting was an academic medical center.

Subjects: Fifteen healthy community-dwelling men ages 22–78 yr were included in the study.

Intervention: Saline or four separate rhLH doses were each infused twice iv in randomized order as one pulse every 2 h over 20 h to stimulate Te secretion, after LH secretion was suppressed by a GnRH-receptor antagonist, ganirelix.

Main Outcome: LH and Te concentrations were determined in blood samples collected every 5 min. Maximal and minimal (as well as mean) Te responses were regressed linearly on age to reflect LH peak and nadir (and average) effects, respectively.

Results: The ganirelix/rhLH paradigm yielded serum LH concentrations of 4.6 ± 0.22 IU/liter (normal range 1–9). By regression analysis, age was associated with declines in rhLH pulse-stimulated peak and nadir (and mean) concentrations of total Te (P = 0.0068), bioavailable Te (P = 0.0096), and free Te (P = 0.013), as well as lower Te/LH concentration ratios (P < 0.005). Deconvolution analysis suggested that the half-life of infused LH increases by 12%/decade (P = 0.044; R2 = 0.28).

Conclusions: Infusion of amplitude-varying pulses of rhLH during gonadal-axis suppression in healthy men unmasks prominent age-related deficits in stimulated total (39%), bioavailable (66%), and free (63%) Te concentrations, and a smaller age-associated increase in LH half-life. These data suggest that age-associated factors reduce the efficacy of LH pulses.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
TESTOSTERONE (TE) IS the primary androgen that maintains anabolism and sexual function in the adult (1, 2). However, Te availability decreases significantly in older men studied cross-sectionally or longitudinally (3, 4, 5, 6, 7). Decreased Te availability in aging may contribute to sarcopenia, osteopenia, visceral adiposity, insulin resistance, reduced exercise capacity, cognitive changes, and increased cardiovascular risk (2, 8, 9, 10, 11, 12). Although the precise causes have not been clarified, lower Te concentrations in elderly men could in principle result from impairment in the hypothalamus, pituitary gland, and/or testis (13). Indirect evidence points to decreased GnRH drive of gonadotropes (14, 15, 16, 17). On the other hand, primary LH deficiency has been excluded by GnRH stimulation studies (18, 19). At the level of the testis, Leydig cell insufficiency may contribute significantly because injections of human chorionic gonadotropin (hCG) elevate Te concentrations less in older than young men (20, 21, 22, 23). Nonetheless, major limitations of hCG stimulation tests include the pharmacological doses used, rapid desensitization of Leydig cells (24, 25, 26, 27, 28), and the prolonged half-life of hCG, which is nonphysiological compared with that of LH (29, 30, 31).

Recent strategies to evaluate putative Leydig cell failure have used LH rather than hCG as the lutropin stimulus. One method was to deliver iv pulses of GnRH to stimulate LH pulses, and thereby secondarily induce Te secretion (18). A caveat was that this model assumed that pituitary LH has normal bioactivity in aging individuals, which is not established (32, 33). A second approach was to suppress gonadotropin secretion over several weeks with a potent GnRH agonist, and then inject repeated fixed pulses of recombinant human LH (rhLH) to stimulate Te secretion (34). This model was flawed because short-term suppression of the gonadal axis severely blunted subsequent testis responses to rhLH. A third strategy was to inhibit LH secretion acutely with a GnRH-receptor antagonist and then stimulate Leydig cells with iv pulses of a single fixed arbitrary dose of rhLH (35). This paradigm revealed that Te secretory responses to a particular train of identical LH stimuli are lower in older than young men (36, 37). However, criticisms of the last protocol are that it fails to explore the physiological LH concentration range and might down-regulate gonadal responses due to repeated fixed stimuli (38, 39, 40, 41, 42).

From this background, two fundamental questions occur. Does age determine testicular steroidogenic responses to amplitude-varying LH pulses? If so, does age reduce maximal or minimal (peak or nadir) Te responses to variable LH pulses?


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

There were 15 men ages 22–78 yr enrolled in the study after providing voluntary informed written consent. The study and the informed consent were reviewed and approved by the U.S. Food and Drug Administration, General Clinical Research Center (GCRC) Advisory Board and Mayo Clinic Institutional Review Board. All participants lived in the community and were in good health. Participants had not consumed alcohol, caffeine, or prescription drugs within 48 h, and had not traveled beyond three time zones within 10 d before the study visit day. Detailed medical inventory excluded a history of systemic disease, infertility, erectile dysfunction, psychoactive or recreational drug use, weight loss (>2 kg in 6 wk), or hormonal therapy. Physical examination was unremarkable for age. Biochemical tests, including renal, hepatic, and metabolic function (fasting glucose, thyroid function, electrolytes), were within normal limits. Volunteers had normal age-related fasting concentrations of prolactin, LH, FSH, total Te, free Te, and bioavailable (bio) Te before entry into the study. All subjects were compensated for time spent in the study according to a payment schedule determined by the institutional review board.

Sampling and infusion protocol

At 0730 h, blood was obtained for baseline hormone assays. Then, at 0800 h, ganirelix (Organon, Oss, The Netherlands), a potent selective antagonist of GnRH action (43), was administered at a dose of 2 mg sc. Volunteers remained in the GCRC. At 2000 h on the same day, subjects received another injection (1 mg dose) of ganirelix sc, followed by a train of iv pulses of saline or rhLH (Serono, Geneva, Switzerland). Each pulse comprised a square-wave bolus of saline or rhLH delivered over 6 min at a dose of 12.5, 25, 50, or 100 IU every 2 h in randomized order. Each dose (or saline) was infused twice, yielding a total of 10 separate pulses. The two injections of ganirelix are consistent with its plasma half-life of 15 ± 2 h and 20- to 28-h duration of inhibition of LH and Te concentrations (35, 43). LH doses were estimated from pilot dose-finding studies (34). Blood (1 ml) was withdrawn through a forearm iv catheter every 5 min for a total of 20 h beginning at 2000 h. Meals were served in the GCRC. Samples were allowed to clot at room temperature, and sera were frozen at –20 C for later assay of LH and Te concentrations.

Assays

Serum LH concentrations were measured in each sample in duplicate by automated chemiluminescence-based assay (Beckman Dxi 800 immunoassay; Beckman Coulter, Fullerton, CA) using the World Health Organization Second International Reference Preparation 80/552 as standard. Concentration-dependent intraassay coefficients of variation (CVs) averaged 10.1, 4.3, and 4.0% at LH concentrations of 0.2, 1.2, and 38 IU/liter, respectively. Interassay CVs averaged 8.1, 5.9, and 5.5% at LH concentrations of 1.2, 13, and 34 IU/liter, respectively. Sensitivity was 0.2 IU/liter. Te was quantified in the same assay system. Intraassay CVs were 14.5, 6.5, and 3.3% at Te concentrations of 28, 369, and 862 ng/dl, respectively. Interassay CVs were 4.2, 5.1, and 4.7% at Te concentrations of 123, 341, and 700 ng/dl, respectively. Sensitivity was 10 ng/dl. The correlation coefficient and slope for the regression of Te measured in this assay on Te quantified by mass spectrometry were reported as 0.95 and 0.96, respectively, with a y-intercept value of –10 ng/dl (44). Screening FSH and prolactin concentrations were measured by Mayo Medical Laboratories (www.mayoclinic.org, in 2006). SHBG was quantified using Immulite 2000 (Diagnostic Products Corp., Los Angeles, CA) (45). Intraassay CVs were 2.7 and 3.1% at SHBG concentrations of 5.5 and 96 nmol/liter, respectively. Interassay CVs were 4.0 and 5.9% at SHBG concentrations of 5.4 and 87 nmol/liter, respectively. Serum albumin was measured using the Roche/Hitachi 912 (Roche Diagnostics, Basal, Switzerland (46). Intraassay CVs were 1.7 and 1.0% at albumin concentrations of 2.5 and 4.6 g/dl respectively. Interassay CVs were 1.0 and 1.0% at albumin concentrations of 2.8 and 4.2 g/dl, respectively. bio and free Te concentrations were measured in baseline serum samples after 50% ammonium-sulfate precipitation and by equilibrium dialysis at 37 C, respectively, as described (18).

Calculation of free and bio-Te concentrations

Free and bio-Te concentrations were calculated in each 5-min sample using measured total Te concentrations, albumin, and SHBG. The equation system was adapted from Sodergard et al. (47). Association constants were first estimated empirically, as given in the supplemental Appendix, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.

Analytical measures

The primary analytical endpoints were peak and nadir LH and Te concentrations. The peak Te concentration was taken as a measure of the effect of the height of the LH pulse, and the nadir Te concentration as a measure of the effect of interpeak nadir LH concentration (48). Peak and nadir total, bio and free Te concentrations were determined in each of the 10 successive 2-h time blocks, one block for each pulse of saline or rhLH. To identify peaks and nadirs in a model-free manner, concentration profiles were first smoothed using a three-point moving average. As an index of mean responses, the foregoing peak and nadir values were averaged [(peak + nadir)/2] in each 2-h window, and then again over 20 h. The result was termed the "mean peak/nadir concentration" in each subject.

LH half-lives were estimated by deconvolution analysis of each 20-h serum LH concentration time series (49). Similar results were obtained by assuming that the published initial (rapid) half-life component of 18 min contributes 37% the decay amplitude (50), and then estimating the delayed (slow-phase) half-life (17, 51). Estimates were regressed linearly on age.

Statistical analysis

Linear regression was used to relate peak, nadir, and means of peak and nadir (mean peak/nadir) Te concentrations (dependent variable) to age (independent variable). Normality was tested by the Kolmogorov-Smirnov statistic (52). The interpretation of P was protected at 0.0167 due to analyses of three different Te fractions (53).

Secondary analyses asked whether age controlled an individual’s single absolute maximum or minimum LH or Te concentration achieved over the 20 h.

Data are presented as the mean ± SEM. Analysis was performed using SAS version 9.1 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The range of subject ages was 27–78 yr (mean 49 ± 4.4) and of body mass index (BMI) 21–32 kg/m2 (average 26 ± 0.82). BMI did not vary with age. Screening baseline concentrations of LH, total Te, bio-Te, free Te, SHBG, albumin, FSH, and prolactin were normal (Table 1Go). No screening baseline hormone concentration varied with age except bio and free Te, which decreased significantly and linearly (R2 = 0.44, P = 0.007; and R2 = 0.31, P = 0.029, respectively).


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  		TABLE 1. Baseline hormone concentrations in 15 men

 
Figure 1Go illustrates profiles of LH and total Te concentrations sampled every 5 min for 20 h in three representative volunteers ages 27, 48, and 69 yr. The linkage between LH and Te pulses is evident in individual subjects. Measurements were obtained beginning 12 h after the first dose of ganirelix concomitantly with iv infusion of saline and rhLH. For the cohort as a whole (n = 15 subjects), LH concentrations averaged 4.6 ± 0.22 IU/liter and total Te concentrations 226 ± 7 ng/dl (multiply Te values by 0.0347 for nmol/liter).


Figure 1
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  		FIG. 1. Top, Illustrative profiles of LH concentrations (Con) measured in samples collected every 5 min over 20 h in three healthy men ages 27, 48, and 69 yr old (yo). Volunteers received sc injections of ganirelix (a GnRH-receptor antagonist) and 10 consecutive iv pulses of randomly ordered doses of rhLH or saline. Bottom, Matching 20-h profiles of total Te concentrations.

 
Figure 2Go depicts the effect of age on peak and nadir LH concentrations identified in each 2-h block during the 20-h infusion and then averaged. Two-hour mean LH concentrations decreased within the normal young-adult concentration range (1–9 IU/liter). Linear regression analysis disclosed that increasing age predicted higher peak and higher nadir LH concentrations (P = 0.032, R2 = 0.31; and P = 0.014, R2 = 0.39, respectively). By extrapolation, mean LH concentrations increased by 11 ± 2%/decade over the age range 25–75 yr. Absolute (single extreme) minima and maxima of the 20-h LH concentration profiles in the cohort averaged 1.2 ± 0.37 and 13.7 ± 0.65 IU/liter, respectively. Single maximal LH, but not Te, values exceeded the normal range occasionally (18% of peaks were 10–15 IU/liter). These and rare extreme LH values (4% >15 IU/liter) were invariant of age (Table 2Go).


Figure 2
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  		FIG. 2. Mean peak and nadir LH concentrations regressed on age in 15 men administered ganirelix and pulses of rhLH and saline (see legend for Fig. 1Go). To obtain means, peak and nadir values were estimated in 2-h windows after each pulse, and the results were averaged over 20 h in each subject. The grand mean of the mean peak and nadir regressions is given by the interrupted line. Unadjusted R2 and P apply to the linear regressions.

 

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  		TABLE 2. Absolute maxima and minima of 20-h LH and Te concentration profiles

 
Supplemental Appendix Fig. 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org) presents estimates of equilibrium-association rate constants for Te-albumin and Te-SHBG, which were used to calculate bio and free Te concentrations in the 20-h time series. Linear regressions of calculated free (top) and bio (bottom) Te concentrations on directly measured free and bio-Te concentrations are shown for a cohort of 40 healthy men. Numerical values are the slope, y-intercept, R2, and iteratively optimized kas for the association of Te with SHBG and albumin.

To test the primary hypothesis that age reduces pulsatile Te responses to pulsatile LH stimulation, grand (20-h) mean peak/nadir Te concentrations (see Subjects and Methods) were regressed on age (Fig. 3Go). Statistical analysis indicated that total, bio, and free Te mean peak/nadir concentrations decrease linearly with age (P ≤ 0.013; R2 ≥ 0.39). Expressed as percentage decrements between projected ages 25 and 75 yr, mean peak/nadir concentrations declined by 39, 66, and 63% for total, bio, and free Te, respectively.


Figure 3
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  		FIG. 3. Mean peak/nadir concentrations of (top-to-bottom) total, bio, and free Te regressed on age. Each point is the grand mean of peak and nadir values (see definition in Fig. 2Go). The slope ± SEM, correlation coefficient (R2), and corresponding significance level are given in each panel.

 
A secondary question was whether age specifically diminishes one or both of mean 2-h peak and mean 2-h nadir Te concentrations. To this end, peak and nadir values were regressed on age (Fig. 4Go). The analysis revealed that age negatively predicts both peak and nadir concentrations of: 1) total Te (P = 0.008, R2 = 0.43; and P = 0.007, R2 = 0.44, respectively); 2) bio-Te (P = 0.0099, R2 = 0.41; and P = 0.0095, R2 = 0.42, respectively); and 3) free Te (P = 0.013, R2 = 0.39 for both regressions). Mean peak/nadir regression lines are plotted for comparison.


Figure 4
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  		FIG. 4. Separate peak and nadir concentrations of total Te (A), bio-Te (B), and free Te (C) regressed on age in men administered ganirelix and rhLH pulses. The interrupted line is the grand mean of the two regressions. See legend for Fig. 2Go.

 
Because LH concentrations increased with age even under the ganirelix/rhLH clamp (Fig. 2Go), the question emerged how much age affects maximally stimulated Te concentrations normalized for maximal LH concentrations, as a surrogate of LH efficacy. This point was examined by regressing the ratio of absolute maximal Te/absolute maximal LH concentrations on age (Fig. 5Go). Statistical analysis disclosed a strongly negative association between age and the ratios of maximal total, bio, and free Te to maximal LH concentrations (R2 ≥ 0.49; P ≤ 0.004). BMI had no effect on the relationships (R2 < 0.10; P > 0.10).


Figure 5
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  		FIG. 5. Age reduces normalized Te responses to rhLH pulses. Data are ratios of absolute maximal (mean of three consecutive highest values over 20 h) Te to matching absolute maximal LH concentrations. See format of Figs. 2Go and 4Go.

 
Deconvolution analysis yielded rapid and slow half-lives of rhLH of [median (range)] 54 (25–66) and 157 min (117–197), with a rapid/total fractional amplitude of 0.45 (0.23–0.65). Regressions of the rapid and slow phases of the biexponential half-lives of infused rhLH on age are shown in Fig. 6Go. Age accounted for 28% the variance in both values (P = 0.044).


Figure 6
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  		FIG. 6. Linear regression of rapid and slow-phase half-lives of infused rhLH on age. The fractional contribution of the rapid (to the total) decay amplitude was 0.45 (0.23–0.65) [median (range)], which was independent of age.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present study implemented a new investigative strategy of blocking pituitary GnRH receptors to lower LH secretion acutely overnight, and then infusing saline or amplitude-varying pulses of rhLH in randomized order to test the hypothesis that age reduces corresponding peak and nadir Te concentrations in healthy men aged 22–78 yr. Mean peak and nadir LH concentrations achieved by exogenous LH pulses were within the middle and upper normal range for young adults. Linear regression analysis revealed that age 75 vs. 25 yr predicts markedly (39–66%) lower stimulated peak as well as nadir as well as mean peak/nadir concentrations of total, bio, and free Te. Age accounted for at least 40% the statistical variance in Te responses in the cohort of 15 men studied here. Therefore, we infer that aging or factors associated with age impair testicular steroidogenic responses to amplitude-varying stimulation by biosynthetic LH. Reduced responses of each of the three major Te fractions to the highest doses of LH used strongly suggest that aging particularly decreases the efficacy of (maximal testis Te response to) LH pulses.

Regression analysis indicated that LH concentrations achieved in the experimental ganirelix/rhLH paradigm increased by about 12%/decade of age. The increase was not due to decreased pituitary suppression by ganirelix, given that absolute LH minima did not vary with age. Because identical doses of rhLH were infused in each subject, one may surmise that age reduces the metabolic clearance rate of LH, which is determined by both the distribution volume and plasma half-life of a hormone (50). Because the estimated half-lives of infused rhLH also increase linearly with age to the same fractional degree as LH concentrations, there is no need to postulate a decline in LH distribution volume with age. Median estimated half-lives of biosynthetic LH of 54 and 162 min exceeded those of pituitary purified and endogenous LH (18 and 90 min) in earlier investigations (35, 36, 42, 50). These distinctions could reflect posttranslational differences in ternary carbohydrate structure of secreted and recombinant glycoprotein (54). In contrast to this inference, age may decrease the distribution volume of rhGH (55).

The accompanying estimates of Te concentration fractions were based upon a technical innovation suggested by Giton et al. (56). The concept was to calculate a laboratory specific association constant (ka) for Te-SHBG by iterative regression of calculated on measured bio-Te concentrations. The motivation was that published dissociation constants for Te-SHBG vary over a 12-fold range (0.27–3.2 x 109 M–1) (56, 57, 58, 59, 60, 61). The present analyses extend this idea by estimating the association constant (reciprocal of dissociation constant) of Te for both SHBG and albumin based upon measured bio and free Te concentrations in 40 men. For the steroid and protein assays used here and the cohort of men sampled in Olmsted County, MN, the estimated association constant for Te-SHBG was 1.78 x 109 M–1. This value is comparable with that measured directly by Nisula and Dunn [viz. 1.6 x 109 M–1 (58)]. The corresponding association constant for Te-albumin was 1.80 x 104 M–1, similar to that described by the same authors and by Nisula and Dunn (58) and Moll and Rosenfield (62). The proposed merit of laboratory-specific estimates is to obviate the necessity to use association constants otherwise chosen arbitrarily from a wide literature range.

The pathophysiology of putative age-associated failure of Te synthesis is unclear. In the human, one mechanism may involve attrition of Leydig cells (63), which could explain the reduction in LH efficacy (maximal stimulation of Te secretion) inferred here. Low Leydig cell mass might be reversible given that pluripotent stem cells can acquire steroidogenic competence (64, 65, 66). Other possible mechanisms include decreased steroidogenic function of individual Leydig cells. In this regard, Leydig cells in the old brown Norway rat express lower concentrations of key steroidogenic enzymes, cholesterol-transport proteins, and antioxidant cofactors (67, 68), and higher concentrations of cyclooxygenase-2 and other cytokine-responsive inhibitory molecules (69, 70). Bypassing the LH receptor by stimulating Leydig cells from the aged rat with a cAMP analog can induce normal in vitro Te secretion, and prolonged suppression of LH secretion in young animals can prevent steroidogenic failure in the older animal (71, 72).

Caveats in interpreting the present outcomes include, first, the relatively small group of men studied (n = 15), thus requiring confirmation in larger cohorts. Second, the relatively short duration (20 h) of the amplitude-varying study does not address whether weeks or months of stimulation with LH pulses could normalize steroidogenesis in older men. Third, at the doses studied, absolute maximal LH concentrations (viz. 10–15 IU/liter) occasionally exceeded normal values. Whether such large pulses alter testicular steroidogenic responses is not known. Fourth, whereas an 8-fold range of LH doses was used, the majority of pulses appeared to stimulate nearly maximal Te responses. Thus, the maximal response to LH was accurately estimated, but sensitivity or potency could not be calculated. Finally, strong linearity of the regression of Te responses on age does not establish that the aging-related decline is linear or that aging per se causes Leydig cell failure. Longitudinal studies in a stable community setting would be necessary to document a prima facie role of aging and to discern the tempo of age-related decrements in testis responses.

In summary, a paradigm of amplitude-varying pulses of rhLH infused during acute suppression of gonadotropin secretion unveils a prominent (~65%) linear decline in exogenously stimulated concentrations of free and bio-Te in healthy men ages 22–78 yr, and a smaller (12%/decade) increase in rhLH half-life with age. Longitudinal studies based upon comparable paradigms could be used to evaluate the causal role of aging, tempo of the apparent age effect, and reversibility of inferred Leydig cell insufficiency in older men.


   Footnotes
 
Supported by a Mayo Institutional Award and Career Development Award (to P.Y.T.), the National Center for Research Resources (Rockville, MD) Grant M01 RR00585 to the General Clinical Research Center of the Mayo Clinic and Foundation, and the National Institutes of Health (Bethesda, MD) Grants R01 AG23133, R21 AG23777, and R21 AG29215 (to J.D.V.).

Disclosure Statement: The authors have nothing to declare.

First Published Online June 19, 2007

Abbreviations: bio, Bioavailable; BMI, body mass index; CV, coefficient of variation; GCRC, General Clinical Research Center; hCG, human chorionic gonadotropin; mean peak/nadir, means of peak and nadir; rhLH, recombinant human LH; Te, testosterone.

Received December 7, 2006.

Accepted June 13, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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