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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5507-5515
Copyright © 2002 by The Endocrine Society

Other Original Article

Poorly Controlled Type I Diabetes Mellitus in Young Men Selectively Suppresses Luteinizing Hormone Secretory Burst Mass

Juan Carlos López-Alvarenga, Teresa Zariñán, Aleida Olivares, Jorge González-Barranco, Johannes D. Veldhuis and Alfredo Ulloa-Aguirre

Department of Endocrinology and Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición SZ (J.C.L.-A., J.G.-B.); Research Units in Reproductive Medicine (T.Z., A.U.-A.) and Developmental Biology (A.O.), Instituto Mexicano del Seguro Social, Mexico D.F., Mexico; and Department of Internal Medicine, University of Virginia Health Sciences Center (J.D.V.), Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Alfredo Ulloa-Aguirre, M.D., D.Sc., Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, Apartado Postal 99-065, Unidad Independencia, C.P. 10101, Mexico City D.F., Mexico. E-mail: aulloaa{at}servidor.unam.mx.

Abstract

Alterations in the reproductive axis function are present to a variable extent in patients with type 1 diabetes mellitus (IDDM). Results from studies in IDDM men have yielded discrepant findings, which may reflect nonuniform patient selection criteria, age, diabetic status, duration of the disease and differences in sampling protocols. To more clearly define the impact of early diabetic alterations in the male reproductive axis, we applied a combined strategy of patient selection restricted to young men with relatively short duration of IDDM, dual control groups, multiparameter deconvolution analysis to assess LH secretory activity, and assessment of time-dependent changes in human chorionic gonadotropin (hCG)-stimulated serum testosterone concentrations. Three groups of subjects were studied: 11 young men with poorly controlled IDDM, 9 well controlled diabetics, and 9 healthy men. All volunteers underwent blood sampling at 10-min intervals before and after 2 consecutive iv pulses of 10 µg GnRH. On a separate day, 40 IU/kg hCG were given im, and blood samples were collected before hCG administration, every 60 min thereafter for 6 h, and then 24, 48, and 72 h after the injection. Mean serum LH concentrations across the basal 6-h sampling period were significantly (P < 0.05) decreased in men with poorly controlled IDDM (11 ± 1.6 IU/liter) compared with those in well controlled diabetics (19 ± 1.8 IU/liter) and healthy controls (19 ± 1.5 IU/liter). Multiple parameter deconvolution analysis revealed a 50% reduction in the mass of LH secreted per burst and the pulsatile LH secretion rate in poorly controlled IDDM (mass of LH secreted/burst, 7 ± 1.1 vs. 12 ± 2.1 and 13 ± 1.5 IU/liter; LH secretion rate, 47 ± 6.3 vs. 78 ± 10 and 87 ± 11 IU/liter·6 h; poorly controlled vs. well controlled IDDM and healthy controls, respectively; P < 0.05 for both parameters). Uncontrolled IDDM patients had significantly (P < 0.05) lower integrated serum LH concentrations after the first and second GnRH pulses (first GnRH pulse, 4460 ± 770 vs. 7250 ± 1200 and 5120 ± 910 IU/liter; second pulse, 4700 ± 615 vs. 7640 ± 881 and 7100 ± 1230 IU/liter; poorly controlled vs. well controlled IDDM and healthy men, respectively) and markedly attenuated LH secretory burst mass after the second GnRH stimulus (49 ± 8.8 vs. 90 ± 13 and 83 ± 19 IU/liter; poorly controlled vs. well controlled IDDM and healthy controls, respectively). The biological to immunological ratio of LH released in baseline conditions was higher in uncontrolled IDDM patients (0.81 ± 0.10) than in controlled IDDM (0.37 ± 0.08) and healthy controls (0.48 ± 0.06; P < 0.01), whereas LH released in response to exogenous GnRH exhibited comparable ratios among the three study cohorts. Baseline serum testosterone levels as well as absolute and incremental responses to exogenous hCG did not differ by degree of metabolic control. Collectively, these results indicate that the function of the hypothalamic-gonadotrope axis is compromised in young men with poorly controlled IDDM, such that the amplitude of spontaneous pulsatile and exogenous GnRH-stimulated LH secretion is attenuated. This central hypogonadotropism is paradoxically associated with the presence in the circulation of gonadotropin molecules with enriched biological activity, which is evidently sufficient to temporarily maintain normal total testosterone concentrations in the earlier stages of IDDM.

INSULINOPENIC DIABETES MELLITUS disrupts reproductive hormone outflow consistently in the experimental animal (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) and affects the gonadal axis to a variable extent in the human (15, 16, 17, 18, 19). For example, in the rodent, acute insulin withdrawal damps LH pulsatility and impairs acute gonadotrope secretory responsiveness to GnRH (10, 13, 14). Insulin-driven reductions in brain stem glucose availability likewise repress pulsatile GnRH secretion (13, 14). In the human, poorly controlled type I diabetes mellitus (IDDM) may delay the onset of sexual maturation in children (20) and impair menstrual cyclicity in young women (21, 22, 23, 24). Amenorrhea in the latter circumstance appears to reflect hypothalamic hypogonadotropism, as infusion of GnRH stimulates normal or increased LH release (21, 22, 23, 24).

IDDM men maintain normal or low serum testosterone (T) and LH concentrations and exhibit variable gonadotrope responses to exogenous GnRH (16, 17, 18, 19, 25, 26). Discrepant findings in men with IDDM may reflect nonuniform patient selection criteria, age, degree of metabolic control, duration of diabetes, and extent of comorbid complications.

To examine the nature of early diabetic alterations in the male gonadal axis, the present clinical investigation applies a combined strategy of 1) patient selection restricted to young men with a shorter duration of poorly controlled IDDM without macrovascular, renal, or neurological complications or erectile dysfunction; 2) dual control groups comprising patients with well controlled IDDM and healthy volunteers of comparable age and body mass index; 3) multiparameter deconvolution analysis to quantitate pulsatile LH secretion and LH half-life before (baseline) and after two consecutive iv pulses of a submaximally effective dose of GnRH; and 4) time-dependent increases in the serum T concentration after a single maximal human chorionic gonadotropin (hCG) stimulus.

Subjects and Methods

A total of 20 patients with IDDM selected from our out-patient clinic agreed to participate. The study was approved by the local human ethics committee. Written informed consent was provided before participation. The initial criteria for eligibility included age between 20–30 yr, IDDM for less than 7.0 yr, poor (but nonketotic) or optimal/suboptimal metabolic control (defined as glycated hemoglobin levels >10% or <8%, respectively) over at least the preceding 2 months, and body mass index of 19–27 kg/m2. The primary cohort comprised 11 men with IDDM, with a median duration of disease of 3.7 yr (range, 2–6.7 yr) and age of 22 yr (range, 20–29 yr); these subjects exhibited poor metabolic control and presented mean ± SEM glycated hemoglobin concentrations of 12.2 ± 1.6% (normal range, 4.0–6.0%; IDDM-U group). Daily insulin doses [administered in a 2-injection/d mixed (intermediate- plus fast-acting insulin) regimen] ranged from 0.38–0.72 U/kg (0.56 ± 0.14 U/kg). A disease-specific control group included 9 well controlled type I diabetic patients (IDDM-C), with a median disease duration of 5.0 yr (range, 2–6.5 yr) and a median age of 23 yr (20–27 yr; P = NS vs. IDDM-U). Glycated hemoglobin values were 5.4–7.9% (6.9 ± 0.8%), and daily insulin (administered in a multiple, 3- or 4-injection/d regimen, including an intermediate-acting insulin dose at bedtime) requirements were 0.44–0.76 U/kg (0.63 ± 0.15 U/kg). Patients in this group exhibited fasting plasma glucose concentrations below 7.1 mmol/liter and 2-h postprandial levels less than 10 mmol/liter on 3 different occasions over a 2-month observation period. None of the patients in the IDDM-C group had ever presented glycated hemoglobin concentrations above 10%. A healthy control cohort (NDC) included 9 men with a median age of 25 yr (21–28 yr) and normal glycated hemoglobin concentrations (5.6 ± 0.6%). The mean body mass index was comparable in diabetic patients (IDDM-U, 22.7 ± 2.9; IDDM-C, 22.7 ± 1.4 kg/m2) and healthy subjects (NDC, 24.7 ± 1.2 kg/m2). Physical activity history was similar in the 3 study groups.

Exclusion criteria included the use of drugs and/or alcohol, hypertension, renal failure or microalbuminuria, proliferative retinopathy, macrovascular disease, fixed neurological deficits, autonomic dysfunction, or impotence. One IDDM-U patient had mild acral sensory loss. Physical examination (including penis and testis size) and screening laboratory tests of hepatic, renal, and hematological function were normal. All subjects were biochemically euthyroid. None had ever received (noninsulin) hormonal treatment or neuropsychotropic medications.

Volunteers were admitted to the metabolic research ward at 0730 h, and an indwelling heparinized catheter was placed in an antecubital vein. Subjects remained recumbent and were provided light meals at 0900 and 1400 h. Beginning at 0800 h, blood samples were obtained every 10 min for 10 h. The first 6-h segment was used to monitor endogenous GnRH-driven (spontaneous) LH secretion. After 6 and 8 h of blood sampling, a pulse of 10 µg GnRH (Serono de Mexico, Mexico D.F., Mexico) was injected by iv bolus. Subjects were readmitted on a separate day at 0730 h, and at 0830 h they were given hCG 40 IU/kg, im (Organón Mexicana S.A. de C.V., Mexico City, Mexico). This hCG dose provokes a maximal early (i.e. within 1–6 h) and late (i.e. within 24–72 h) rise in serum T concentrations in normal individuals (27). Blood samples were collected before hCG administration, every 60 min thereafter for 6 h, and then 24, 48, and 72 h after the injection. Sera were frozen at -20 C until later assay.

Immunoassays

The LH RIA employed 125I-radiolabeled NIH LH-I3 (specific activity, 70–90 µCi/µg protein), antiserum to human LH-2 (final dilution, 1:800,000), and LER-907 as standard (1 mg LER-907 = 277 IU Second International Reference Preparation of human menopausal gonadotropin) (28). All samples from a given subject were analyzed together in duplicate. The detection threshold was 0.7 IU/liter. Intra- and interassay coefficients of variation were 4.1–6.2% and 6.1–11.3%, respectively, over displacement ranges of 15–23%, 45–59%, and 75–84%. FSH was assayed by RIA of pooled sera (6-h baseline) using the Second International Reference Preparation of human menopausal gonadotropin standard, as previously described (29). PRL was determined by ELISA (Immunometrics, London, UK) using WHO International Reference Preparation 84/500. Assay sensitivity was 20 mIU/liter, with an absolute range of 40–310 mIU/liter. Estradiol (E2) was measured by solid phase RIA (Diagnostic Products, Los Angeles, CA), SHBG by immunofluorometry (Delfia, Wallac, Inc., Turku, Finland), and cortisol by RIA (WHO Matched Reagent Program, Geneva, Switzerland). T concentrations were quantitated by immunochemiluminometry (Immulite Total Testosterone, Diagnostic Products). T bioavailability was estimated as the molar ratio of T to SHBG. Intraassay coefficients of variation averaged less than 8% (FSH and cortisol), less than 5% (PRL, E2, and T), and less than 3.5% (SHBG).

In vitro bioassay of LH

In vitro LH bioassay and RIA were applied separately to the single baseline (6-h) and the two GnRH-stimulated (2-h) serum pools (denoted segments 1, 2, and 3 in Fig. 1Go). Both assays employed highly purified human recombinant LH produced by Chinese hamster ovary cells (provided by Organon International BV, Oss, The Netherlands) as standard for the corresponding reference curves. The LH bioassay monitors cAMP production by a human embryonic kidney cell line stably transfected with the full-length human LH receptor cDNA (provided by Dr. Aaron J. W. Hsueh, Stanford University, Palo Alto, CA). Total (intra- plus extracellular) cAMP concentrations were determined by RIA after acetylation, as described previously (30). To equalize volume, samples were diluted with sera collected from women treated with oral contraceptives, wherein LH immunoreactivity and bioactivity were undetectable. Each sample was assayed at three dilutions (12.5, 25, and 50 µl) in triplicate. The final concentration (vol/vol) of serum was less than 10%. Assay sensitivity was 0.075 mIU (LER-907)/tube. Inter- and intraassay coefficients of variation were less than 18% and less than 10%, respectively, at the 50% effective dose.



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  		Figure 1. Immunoreactive LH concentrations determined in serum samples collected every 10 min for 6 h before and for 4 h after two consecutive iv pulses of GnRH administered 2 h apart (arrowheads) in men with poorly controlled (n = 11) and well controlled (n = 9) IDDM and in healthy controls (n = 9). Sera were pooled over successive 6-, 2-, and 2-h intervals (delineated by the vertical broken lines) for later analysis by in vitro bioassay. Data represent the mean ± SEM.

 
Deconvolution analysis of spontaneous and GnRH-induced LH release

Deconvolution analysis was applied to quantitate the frequency, amplitude, and mass of LH secretory bursts and the apparent LH half-life from the 6-h (basal) and 2-h (post-GnRH) serum LH concentration-time series (31, 32). The mass of an LH secretory burst is the analytical time integral of the deconvolution-resolved secretory impulse.

Assessment of monohormonal (LH) pattern regularity

Univariate approximate entropy (ApEn) was employed to quantitate the pattern regularity of serial LH measurements (33). This statistical approach allows measurement of relative orderliness in time series data, with minimal dependence on mean pulse amplitude, interpulse baseline, or subthreshold sample uncertainty, and thus complements conventional pulse detection (34, 35). In the present analysis, m (i.e. the pattern comparison window size) was assigned a value of 1, which serves to evaluate the statistical consistency of contiguous (sample by sample) data patterns; the parameter r (i.e. the de facto statistical tolerance for testing pattern recurrence) was set at 35% as appropriate for a shorter time series (36). The foregoing ApEn parameters, designated by ApEn (1,35%), provide a replicable ApEn statistic with an approximate SD of 0.06–0.08 (36, 37, 38). A normalized ratio of observed to random ApEn was calculated for each time series as the mean ratio of observed to random ApEn values calculated by shuffling the original data series times 1000 times (35). ApEn ratios below unit denote more orderly patterns of hormone release and vice versa.

Statistical analysis

ANOVA was applied to compare serum hemoglobin A1c, LH, FSH, E2, and T concentrations, incremental bioactive LH response to exogenous GnRH ({Delta}LH; difference between exogenous GnRH-stimulated bioactive LH concentrations and baseline concentrations), maximal early (1–6 h post-hCG) and late (d 2–4 post-hCG) T peaks, and the incremental T response to hCG [{Delta}T was defined as the difference between the T peak and preceding T nadir (early) or basal T (late)] (27). Post hoc contrasts used the Newman-Keuls critical ranks test and the Tukey highest significant difference criterion. Dynamic LH measures (frequency, mass, amplitude, half-duration and half-life) were transformed logarithmically to equalize residual variance before ANOVA. Values are reported as the mean ± SEM or range. P < 0.05 was considered statistically significant.

Results

Baseline hormone measurements

Table 1Go summarizes mean serum concentrations of LH, FSH, PRL, cortisol, and E2 and the molar T/SHBG ratio in the three cohorts studied. LH concentrations were comparable in IDDM-C and NDC, but were significantly lower in men with poorly controlled diabetes mellitus (IDDM-U).


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  		Table 1. Serum concentrations of FSH, PRL, E2, and cortisol and the molar T/SHBG ratio in men with IDDM and normal controls

 
Spontaneous LH secretion

Figure 1Go depicts group mean (±SEM) serum LH concentrations profiles sampled every 10 min over 6 h (spontaneous LH release). Deconvolution analysis was applied to evaluate the mechanisms underlying reduced integrated serum LH concentrations in IDDM-U (Table 2Go). The number of LH secretory bursts was comparable in all three cohorts. However, men with poorly controlled diabetes mellitus showed a 50% reduction in the mass of LH secreted per burst and, hence, in the pulsatile LH secretion rate. This was due to selective attenuation of LH secretory burst amplitude (i.e. a reduced maximal rate of LH secretion attained per burst). The foregoing contrasts were specific, as LH secretory burst half-duration (duration of the secretory event at half-maximal amplitude), basal/nonpulsatile LH release, and LH half-life were similar in the three groups. The synchrony outflow of LH in IDDM-U was statistically indistinguishable from those in IDDM-C and NDC (Table 2Go). Figures 2Go and 3Go illustrate individual LH concentration and secretory profiles from each cohort.


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  		Table 2. Deconvolution-based comparisons and ApEn (1,35%) ratios of pulsatile LH secretion and half-life in patients with IDDM and healthy men

 


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  		Figure 2. Illustrative serum immunoreactive LH concentration profiles in two healthy men and two patients with poorly vs. well controlled IDDM. Each panel shows the observed data and corresponding deconvolution-predicted fits (continuous lines). Vertical bars associated with each value denote the LH concentration-dependent within-assay SD.

 


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  		Figure 3. Illustrative deconvolution-resolved LH secretory rates in the two normal men and two patients with well vs. poorly controlled IDDM, whose concentration data are shown in Fig. 2Go.

 
GnRH-stimulated LH release

LH concentrations peaked 25 min (median) after each GnRH injection. NDC and IDDM-C subjects responded to GnRH stimulation equivalently (Table 3Go). IDDM-U patients had significantly lower integrated serum LH concentrations after the first and second GnRH pulses and markedly attenuated LH secretory burst mass after the second GnRH stimulus.


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  		Table 3. Secretory response to two consecutive GnRH stimuli in men with IDDM and normal controls

 
In vitro LH bioactivity

Serum bioactive LH concentrations (international units per liter; pooled over the 6-h baseline interval) averaged 18 ± 2 (IDDM-U), 12 ± 2 (IDDM-C), and 14 ± 1 (NDC; P < 0.05, IDDM-U vs. IDDM-C and NDC). Given the reduction in immunoreactive LH concentrations (above), baseline ratios of LH bioactivity to immunoreactivity (B/I) were significantly higher in IDDM-U (0.81 ± 0.10) than in IDDM-C (0.37 ± 0.08) and NDC (0.48 ± 0.06; P < 0.01). Bioactive LH concentrations were higher after GnRH than at baseline in all three study cohorts (Table 3Go). Although the mean bioactive LH responses to the first and second exogenous GnRH pulses were reduced in IDDM-U (mean bioactive LH responses to the first and second GnRH pulses, 15 ± 4 and 19 ± 5, respectively) compared with IDDM-C (22 ± 7 and 21 ± 4) and NDC (23 ± 7 and 24 ± 6), the differences did not reach statistical significance. The B/I remained similar to baseline (not shown).

Serum T response to hCG

IDDM-C and IDDM-U volunteers had normal serum T concentrations at baseline (Table 4Go). hCG stimulated a biphasic rise in T concentrations (Fig. 4Go). Absolute and incremental responses did not differ by disease state or degree of metabolic control (Table 4Go).


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  		Table 4. Impact of hCG administration on serum T concentrations in men with IDDM

 


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  		Figure 4. Early (1–6 h) and late (24, 48, and 72 h) serum total T concentrations after a single im injection of hCG (40 IU/kg; arrow). Data are from well and poorly controlled patients with IDDM and normal subjects. Each point represents the mean ± SEM.

 
Discussion

Several studies in experimentally induced and spontaneously occurring diabetes indicate that uncontrolled diabetes may potentially alter the function of the reproductive axis (10, 11, 13, 14, 39). However, studies applying extended sampling protocols to assess more precisely the nature of the alterations in LH and/or T secretion in men with DM-1 are rather scarce. The present study appraises spontaneous (endogenous) and exogenous GnRH-stimulated LH secretion in healthy men and patients with well and poorly controlled IDDM. Deconvolution analysis of baseline time series revealed that LH secretory burst frequency was normal in both diabetic cohorts and comparable to independent estimates in healthy young men (40, 41). On the other hand, patients with poorly controlled IDDM exhibited a 50% reduction in the mass and amplitude of LH secretory bursts, and consequently lower mean serum LH concentrations. Reduced LH pulse amplitude is consistent with observation in the streptozotocin-treated diabetic male rat (10). However, experimentally induced diabetes also suppresses LH pulse frequency (10, 12). The precise basis for the latter contrast with the human is not known. Plausible explanations include 1) interspecies differences in mechanisms governing gonadotropin synthesis and secretion; 2) underlying pathophysiology, including severity and duration of hypoinsulinism and/or hyperglycemia as well as presence of secondary metabolic disturbances (such as ketone body formation) that may accompany and potentially aggravate the effects of insulin deprivation on the hypothalamus and/or the pituitary (19, 39, 42); and 3) use of different sampling protocols and pulse analysis methodological approaches (43, 44). In addition, isolated suppression of LH pulse amplitude in men with uncomplicated IDDM may denote less severe disruption of GnRH/LH output, akin to the low amplitude pattern evident in aging, uremia, and short-term fasting (45, 46, 47, 48, 49). Thus, alterations in the amplitude of LH secretory events may represent the first detectable alteration of LH secretory dynamics within the context of the natural history of reproductive failure in diabetic men. In this setting, abnormalities in both LH pulse amplitude and frequency may result from long-standing uncontrolled disease, as suggested by studies documenting time-dependent morphological alterations in the hypothalamus and gonadotropes of streptozotocin-treated male rats (42). Longitudinal studies would be required to establish the nature of progressive hypothalamo-pituitary pathophysiology in IDDM (50).

To evaluate the mechanism(s) underlying the reduction in LH secretory burst mass in poorly controlled IDDM, we infused two consecutive submaximally stimulatory doses of GnRH (51, 52). Deconvolution analysis documented a marked (50%) reduction in LH secretory burst mass after the second GnRH pulse. This inference is compatible with some, but not all, findings in insulinopenic animals, in which the heights of single GnRH-stimulated LH peaks are reportedly normal (2, 5, 7, 16, 26) or decreased (3, 8, 15, 53). Comparison of LH secretory burst mass after GnRH stimulation revealed marked attenuation of LH release after the second stimulus in men with poorly controlled IDDM. This distinction could denote that poorly controlled IDDM initially impairs the de novo bioynthesis of LH, which is required to replete gonadotrope cell stores after the first GnRH stimulus. Impaired gonadotrope priming by endogenous GnRH and/or enhanced gonadotrope susceptibility to down-regulation might account for the present findings. Hypothalamic hypogonadotropism is inferable in young women with poorly controlled IDDM. In one study such patients evinced paradoxically heightened LH secretion after a single submaximally effective GnRH stimulus and reduced LH pulse frequency (23). If findings in the female are corroborated in further studies, one could postulate that gender, the sex steroid milieu, and/or the severity and duration of diabetes mellitus contribute to gonadotrope sensitivity.

The design of the present study does not allow us to ascertain whether the changes observed in LH secretion may be reversible with metabolic control. Although studies in experimentally induced diabetes have shown that the effects of short-term insulin withdrawal on LH secretion may be reversed by acute insulin resupplementation (13, 39), longitudinal studies are required to establish the potential reversibility of central hypogonadotropism in temporarily uncontrolled IDDM.

Based on the results obtained in the present study, a single definitive mechanism through which LH secretion is impaired in poorly controlled diabetic men cannot be postulated. The presence of an insulin-deprived milieu may play a pivotal role in the development of gonadotrope dysfunction in uncontrolled IDDM; in fact, insulin can enhance gonadotrope cell sensitivity to GnRH in vitro (54). Further, studies in both streptozotocin-treated male rats and male diabetic db mutant mice have demonstrated a reduced pituitary LH content as well as the presence of morphological changes at the gonadotrope level (2, 42, 55), lending additional support to the possibility that relative hypoinsulinism may lead to changes in pituitary function in diabetic individuals. On the other hand, insulin has systemic access to and binding sites within certain regions of the basal hypothalamus (including the median eminence and the arcuate nucleus), facilitates GnRH secretion in the sheep, and promotes the proliferation of murine GTI-7 neurons directly (13, 24, 56, 57). Thus, relative insulin deficiency, like that present in uncontrolled IDDM, may potentially reduce the strength of the GnRH impulse and consequently attenuate GnRH-dependent pituitary feedforward signaling. Finally, metabolic stress in experimental animals also drives hypothalamic CRH and dopamine outflow, both of which can restrain pulsatile GnRH release (24, 58, 59). As indirect markers of such putative stress responses, we measured serum cortisol and PRL concentrations in baseline (6-h) pools, which were normal in men with poorly controlled IDDM.

In vitro bioassay of LH revealed elevated values in men with poorly controlled diabetes mellitus. Infusion of GnRH failed to completely stimulate bioactive LH secretion equivalently in such patients, in agreement with the RIA result. However, the baseline B/I was significantly elevated in poorly regulated IDDM. This unexpected finding could reflect altered posttranslational processing of LH. Indeed, glycosylation, sialylation, and sulfation of terminal oligosaccharide residues control the in vivo metabolic clearance and in vitro bioactivity of human LH (28, 60, 61). A glycation-dependent mechanisms(s) also influences the cellular recognition, uptake, and degradation of glycoproteins by hepatic reticuloendothelial receptors for asialo-glycoprotein molecules (62). Accordingly, the present findings of normal bioactive, but reduced immunoreactive, LH concentrations in men with poorly controlled IDDM could signify either relatively increased secretion or decreased removal of biologically active compared with immunoreactive LH. Alternatively, as may occur with other proteins (62, 63), a sustained exposure to a glucose-enriched endogenous milieu may promote nonenzymatic glycation of LH molecules, rendering glycosylation variants with altered functional and/or biochemical properties. Further studies are needed to identify the mechanism(s) by which metabolically unstable IDDM may potentially disturb the biosynthetic pathway involved in gonadotropin glycosylation.

A single dose of hCG stimulates T secretion initially after 2–4 h (early) and further after 48–120 h (late) (27, 64, 65). The biphasic response is attenuated in GnRH deficiency states, e.g. hypogonadotropic hypogonadism of either physiological (prepubertal) or pathological (Kallmann’s syndrome) origin (27, 66, 67). In the diabetic subjects studied here, baseline serum T concentrations and T/SHBG ratios were normal. Moreover, both the early and delayed actions of hCG were preserved. In other studies, ketosis-prone, systemically complicated, and older diabetic patients exhibit variable degrees of hypoandrogenemia before and after hCG administration (16, 17, 18, 19). In the experimental animal, severe insulinopenia and hyperglycemia impair hCG binding in the testis and reduce Leydig cell responsiveness to a lutropic stimulus (1, 5, 14, 23, 68, 69). The basis for this difference from the present clinical data is not known. We speculate that retention of LH bioactivity and/or intragonadal (paracrine) regulation of Leydig cell function may be relevant to maintaining T secretion in the earlier stages of IDDM (70).

In summary, poorly controlled, but systemically uncomplicated, IDDM in young men suppresses the amplitude of spontaneous (endogenous) pulsatile and exogenous GnRH-stimulated LH secretion, leading to central hypogonadotropism. This hyposecretory state more greatly affects LH immunoactivity than LH bioactivity. The presence in the circulation of LH molecules with enriched bioactivity is evidently sufficient to maintain normal total T concentrations, the molar T/SHBG ratio, and Leydig cell responsiveness to hCG. Together the present data suggest that the attenuated GnRH-dependent drive of LH secretion in poorly controlled, but otherwise uncomplicated, IDDM may be subserved by a dual mechanism involving both pituitary responsiveness to GnRH and hypothalamic system control of the strength of the GnRH impulse. It is suggested that both alterations result from relative insulin deficiency and that other metabolic disturbances and/or complications that accompany long-standing diabetes aggravate the reproductive axis dysfunction eventually found in the majority of men with IDDM.

Acknowledgments

Reagents were generously provided by the NIDDK (Bethesda, MD) through Dr. A. F. Parlow from the National Hormone and Pituitary Program (Torrance, CA).

Footnotes

This work was supported by Grants 117643 from the Consejo Nacional de Tecnología, Mexico (to J.C.L.-A.), FP0038-1261 from the FOFOI-Instituto Mexicano del Seguro Social, Mexico (to A.U.-A.), and support of the General Clinical Research Center of the University of Virginia and the Mayo Clinic from the National Center for Research Resources, Bethesda, MD (to J.D.V.).

J.C.L.-A. and T.Z. contributed equally.

J.C.L.-A. is a postgraduate student from the Facultad de Medicina, Universidad Nacional Autónoma de México (Mexico City, Mexico).

Abbreviations: ApEn, Approximate entropy; B/I, ratio of LH bioactivity to immunoreactivity; E2, estradiol; hCG, human chorionic gonadotropin; IDDM, insulin-dependent diabetes mellitus; T, testosterone.

Received May 24, 2002.

Accepted August 16, 2002.

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