Suppression of Endogenous Testosterone in Young Men Increases ...

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0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 10 Printed in U.S.A.

Suppression of Endogenous Testosterone in Young Men Increases Serum Levels of High Density Lipoprotein Subclass Lipoprotein A-I and Lipoprotein(a)* ARNOLD VON ECKARDSTEIN, SABINE KLIESCH†, EBERHARD NIESCHLAG, ALI CHIRAZI, GERD ASSMANN, AND HERMANN M. BEHRE Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium (A.v.E., G.A.), and Institut fu¨r Reproduktionsmedizin (S.K., E.N., H.M.B.), Westfa¨lische Wilhelms Universita¨t Mu¨nster; and Institut fu¨r Arterioskleroseforschung an der Universita¨t Mu¨nster (A.C., G.A.), Munster, Germany ABSTRACT We investigated the effect of testosterone suppression on lipoprotein metabolism in men. After a baseline period of 14 days, 12 healthy young men received over a period of 3 weeks daily sc injections of Cetrorelix, an antagonist of GnRH. The volunteers were then followed-up for 10 additional weeks. Administration of Cetrorelix suppressed testosterone significantly up to day 35, after which values returned to baseline. Suppression of testosterone was associated with significant and consistent increases in mean serum levels of high density lipoprotein (HDL) cholesterol by 20% (P , 0.0001), apolipoprotein A-I (apoA-I) by 10% (P 5 0.0032), apoA-II by 7% (P 5

0.0112), HDL subclass lipoprotein A-I (LpA-I) by 23% (P 5 0.002), and plasma lecithin:cholesterol acyltransferase by 7% (P , 0.001). Serum levels of HDL subclass LpA-I/LpA-II changed insignificantly. Moreover, suppression of testosterone significantly increased the median of lipoprotein(a) [Lp(a)] levels from 5.5 to 8.5 mg/dL (P , 0.0001). The increase in Lp(a) levels was positively correlated with baseline levels of Lp(a) (r 5 0.91; P , 0.001) and amounted to 40 – 60% in individuals with baseline levels of Lp(a) higher than 3 mg/dL. We conclude that endogenous testosterone is involved in the regulation of HDL cholesterol and Lp(a) levels and may thereby influence cardiovascular risk. (J Clin Endocrinol Metab 82: 3367–3372, 1997)

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suppression on lipid metabolism in healthy young men who received the GnRH antagonist Cetrorelix (19, 20) over a period of 3 weeks. To elucidate mechanisms of regulatory effects of testosterone on HDL metabolism, we paid special attention to changes in serum concentrations of apolipoproteins and HDL subclasses as well as plasma activities of the cholesterol-esterifying enzyme lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) (21, 22). As experiments in mice transgenic for human apolipoprotein(a) [apo(a)] have indicated that testosterone down-regulates the expression of the apo(a) gene and thereby suppresses serum concentrations of lipoprotein(a) [Lp(a)] (23), we also analyzed the time course of Lp(a) levels in this study.

EN SUFFER much more frequently from atherosclerotic vessel diseases than similarly aged premenopausal women. These sex-specific differences are usually explained by the antiatherogenic effects of estrogens on cardiovascular risk factors, especially on lipoprotein metabolism (1). The effects of testosterone on lipid metabolism in men are controversial. Several clinical studies found a positive correlation between plasma levels of testosterone and high density lipoprotein (HDL) cholesterol (2). Moreover, substitution of testosterone in hypogonadal men was associated with increases in HDL cholesterol levels in some studies (3, 4), with decreases (5, 6) or no change (7, 8) in others. Application of androgen-like anabolic steroids or supraphysiological amounts of testosterone, however, were consistently found to decrease HDL cholesterol (9 –15). Clinical experience with the suppression of endogenous testosterone production also indicates that testosterone has a HDL cholesterol-lowering effect. Antagonists of the GnRH suppress testosterone levels and cause increases in HDL cholesterol dependent on dosage and time (16 –18). In this study we investigated the effect of testosterone

Received May 5, 1997. Revision received June 19, 1997. Accepted June 27, 1997. Address all correspondence and requests for reprints to: Dr. Arnold von Eckardstein, Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfa¨lische Wilhelms Universita¨t Mu¨nster, Albert-Schweitzer Strasse 33, D-48129 Munster, Germany. * This work was supported by grants from Interdisziplina¨res Klinisches Forschungszentrum (Munster, Germany; to A.v.E. and H.M.B.). † Present address: Klinik and Poliklinik fu¨r Urologie, Westfa¨lische Wilhelms Universita¨t Mu¨nster, Albert-Schweitzer Strasse 33, D-48129 Munster, Germany.

Subjects and Methods Subjects We analyzed the effects of testosterone suppression on lipoprotein metabolism during a study on the effects of Cetrorelix on gonadotropic hormones and testosterone that has been described previously (20). Briefly, 12 men whose age ranged between 21–27 yr participated in this study after giving informed consent. The trial was approved by the ethics committee of the State Medical Board and the University of Munster. All participants were healthy according to medical history, physical examination, and screening laboratory tests. None had dyslipidemia. All had normal body weight (21.3 6 1.5 kg/m2) and exercised regularly but not vigorously. None of them abused alcohol; 4 of 12 volunteers smoked (10 – 40 cigarettes/day).

Protocol of the study Volunteers were divided into 3 groups of 4 individuals who received Cetrorelix (Asta-Medica, Frankfurt am Main, Germany) at different maintenance dosages (1 mg/24 h, 1 mg/12 h, and 2 mg/24 h). The trial

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consisted of 4 periods. A baseline period of 14 days was followed by a loading period of 5 days, during which volunteers received daily sc injections of 10 mg Cetrorelix. During the subsequent treatment period of 16 days, the volunteers received Cetrorelix at the doses and time intervals indicated above. Finally, the participants were followed-up for 10 weeks, during which time they did not take the GnRH antagonist. As no differences in hormonal suppression were detected among the 3 study groups (20), data from the 12 volunteers were pooled for further analysis.

Blood samples Blood samples for the laboratory tests were collected after overnight fasting on days 14 and 7 before treatment and on days 7, 14, 21, 28, 35, 49, 63, and 91 after treatment and were cooled immediately on ice. Plasma and sera were obtained by centrifugation at 4 C (800 3 g, 15 min), divided into aliquots, and frozen at 270 C. Serum was used for the quantification of hormones, lipids, lipoproteins, apolipoproteins, and HDL subfractions. LCAT and CETP activities were determined in ethylenediamine tetraacetate-plasma. All parameters were analyzed in series after each volunteer had finished his protocol.

Laboratory tests Serum concentrations of testosterone and 17b-estradiol were measured by RIA as described previously (19, 20). Serum concentrations of triglycerides, cholesterol, HDL cholesterol, apoA-I, and apoB were quantified enzymatically (Boehringer Mannheim, Mannheim, Germany). Low density lipoprotein cholesterol was calculated using the Friedewald formula (24). Lipoprotein A-I (LpA-I) was quantified using a commercially available differential electroimmunoassay (Hydragel LpA-I, Sebia, Paris, France). The concentration of LpA-I/A-II was calculated as the difference between total apoA-I and LpA-I (25). Lp(a) was measured by electroimmunodiffusion using antisera from Behringwerke (Marburg, Germany) and standards from Immuno (Vienna, Austria) (26). Plasma LCAT and CETP activities were determined by radiometric assays as described previously (27).

Statistical analysis Statistical analyses were performed using the statistical package for the social sciences (SPSS-X) (28). Because of its non-Gaussian frequency distribution, Lp(a) levels are summarized as medians. To clarify possible effects of testosterone suppression on lipoprotein metabolism, we performed two kinds of analyses. ANOVA was performed by Friedman’s two-way ANOVA. Data at the indicated time points were compared with either the mean values of the data on days 7 and 14 pretreatment or on day 91 posttreatment by paired Wilcoxon U test. In the latter case we corrected for multiple comparisons by Bonferroni adjustment. As eight comparisons per parameter were made, we defined the level of significance as P , 0.0064. Correlations between baseline values and relative changes in Lp(a) levels were calculated by Pearson’s test.

Results Effect of Cetrorelix on levels of testosterone and estradiol

As described previously in detail (20), sc administration of 10 mg Cetrorelix over 5 days induced a significant decrease in testosterone levels from 7.56 6 1.26 ng/mL (21.6 6 3.6 nmol/L) to the castrate range with serum levels of 0.67 6 0.53 ng/mL (1.9 6 1.5 nmol/L). Subsequent maintenance dose injections of Cetrorelix resulted in continuously suppressed testosterone levels in all volunteers. After completion of treatment, testosterone values remained suppressed below 12 nmol/L until day 28. On day 35, testosterone levels were still significantly lower than baseline (4.87 6 3.92 ng/mL; 13.9 6 11.2 nmol/L). By day 49, testosterone levels had returned to baseline values (Fig. 1). In parallel with testosterone, estradiol levels decreased from 28.1 6 8.4 pg/mL (104 6 31 pmol/L) at baseline to 10.5 6 2.2 pg/mL (39 6 8

FIG. 1. Effects of the administration of the GnRH antagonist Cetrorelix for 3 weeks (days 0 –20) on serum levels of testosterone. A baseline period (days 214 and 27) was followed by a 5-day period during which 12 volunteers received daily sc injections of 10 mg Cetrorelix. Groups of 4 probands then received Cetrorelix for 16 days at doses of 1 mg/24 h, 1 mg/12 h, or 2 mg/24 h. After this treatment period of 21 days, volunteers were followed-up for 10 weeks until day 91. The curve and symbols indicate the time course of mean testosterone values in all volunteers. The broken line indicates the lower normal limit. The arrow indicates the period of 21 days during which volunteers were treated with Cetrorelix. For transformation of nanomoles per L into nanograms per mL, multiply by 0.35.

nmol/L) on day 7, remained suppressed until day 28, and reached normal values on day 42 (Table 1). Effect of testosterone suppression on lipid metabolism

Treatment with Cetrorelix did not lead to significant variations in levels of total cholesterol (x2 5 10.8), low density lipoprotein cholesterol (x2 5 7.5), triglycerides (x2 5 13.0), or apoB (x2 5 12.5; Table 1). By contrast, HDL cholesterol levels varied significantly (P 5 0.0001). Until day 28, the mean HDL cholesterol level increased significantly by 20%. In nine volunteers the increase in HDL cholesterol amounted to at least 10%. Four weeks after completion of treatment (i.e. day 49), HDL cholesterol levels had returned to baseline (Fig. 2 and Table 1). To differentiate the effects of suppressed testosterone levels on HDL metabolism, we measured the 2 predominant HDL-associated apolipoproteins, apoA-I and apoA-II, as well as HDL subclasses that either contain apoA-II (LpA-I/ A-II) or are devoid of apoA-II (LpA-I; Table 1 and Fig. 3). In seven individuals, apoA-I levels increased steadily during treatment with Cetrorelix by at least 10% until day 21. At this point, the mean apoA-I level was 10% higher than at baseline (Table 1). Thereafter, apoA-I levels fell to values below baseline (Fig. 3A; P 5 0.0032). ApoA-II levels rose less significantly (by 7%) than those of apoA-I (P 5 0.0112). Only 5 volunteers experienced a 10% or more increase in apoA-II levels (Fig. 2B). In analogy to the changes in apoA-I and apoA-II levels, suppression of testosterone significantly increased levels of LpA-I by 23% (Fig. 3C; P 5 0.0020), but did not significantly change levels of LpA-I/A-II (Fig. 3D). Treatment with Cetrorelix increased LpA-I levels by more than 10% in 10 volunteers, but elevated LpA-I/A-II levels in only 4 individuals. Again, posttreatment values of LpA-I and LpA-I/A-II (day 91) were lower than the respective pretreatment values.

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TABLE 1. Influence of suppressed testosterone on various parameters of lipid metabolism A: 27/214 daysa

Testosterone ng/mL nmol/L Estradiol pg/mL pmol/L Cholesterol (mg/dL) Triglycerides (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Lp(a) (mg/dL)g ApoA-I (mg/dL) ApoA-II (mg/dL) ApoB (mg/dL) LpA-I (mg/dL) LpA-I,A-II (mg/dL) LCAT (nmol/hzmL) CETP (nmol/hzmL)

B: 21 daysa

C: 91 daysa

7.56 6 1.26d 21.6 6 3.6d

0.6 6 0.21 1.7 6 0.6

8.09 6 1.75d 23.1 6 5.0d

28.1 6 8.4d 104 6 31d 170.2 6 41.0e 91.2 6 40.6 46.2 6 7.8f 105.7 6 34.8 5.25f 126.6 6 17.4e 37.7 6 7.1 61.6 6 19.2 43.6 6 9.5f 82.6 6 13.9 18.0 6 2.6f 82.3 6 18.5e

10.8 6 2.7 40 6 10 180.7 6 46.0 77.5 6 49.5 55.3 6 8.9 109.7 6 38.4 6.5 140.1 6 18.9 40.2 6 6.6 62.8 6 20.3 53.4 6 12.0 86.7 6 12.3 19.2 6 2.6 92.8 6 23.5

29.4 6 4.86d 109 6 18d 165.9 6 48.0f 71.5 6 29.1 42.3 6 8.1f 109.0 6 41.5 5.5f 111.6 6 21.2f 33.1 6 7.2f 56.8 6 20.0 40.4 6 7.1f 71.2 6 15.2f 18.6 6 2.0e 88.6 6 20.6

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Mean value of days 7 and 14 before treatment with cetrorelix. Twenty-one days after start of treatment. Ninety-one days after start of treatment. d P , 0.001 (as calculated by paired Wilcoxon U test). e P , 0.05 (as calculated by paired Wilcoxon U test). f P , 0.01 (as calculated by paired Wilcoxon U test). g Because of its non-Gaussian frequency distribution, Lp(a) is presented as the median. b c

correlated with baseline values of Lp(a) (r 5 0.9; P , 0.001; Fig. 4B); Lp(a) levels remained almost unchanged in five individuals with baseline Lp(a) levels below 5 mg/dL. In seven others, Lp(a) levels increased by 40 – 60%. In three participants with baseline levels of Lp(a) below 22 mg/dL, suppression of testosterone thus resulted in Lp(a) levels that exceeded the cardiovascular risk threshold value of 30 mg/dL. Discussion

FIG. 2. Effects of testosterone suppression by Cetrorelix on serum concentrations of lipids and lipoproteins. For details of the clinical study protocol, see Materials and Methods and Fig. 1. The curve and symbols indicate the time courses of mean values and SDs for HDL cholesterol (n 5 12). The arrow indicates the 21-day period during which volunteers were treated with Cetrorelix. The hatched area indicates the time interval in which testosterone levels were significantly suppressed (cf. Fig. 1). Significant differences between the mean data at the indicated time points and the mean data at baseline (mean of days 27 and 214; *) and posttreatment (day 91; #; by paired Wilcoxon U test and Bonferroni adjustment, P , 0.0064) are shown. The P value was calculated using Friedman’s two-way ANOVA.

Suppression of testosterone with Cetrorelix was associated with a significant 7% increase in LCAT activity on day 21 (P , 0.001), which returned to baseline on day 91 (Table 1). CETP activity also increased during treatment with Cetrorelix (P , 0.05), but did not fall significantly after treatment (Table 1). Treatment with Cetrorelix was associated with a significant increase in Lp(a) (Fig. 4; P , 0.0001). The maximum median of Lp(a) was reached 14 days after treatment, when testosterone levels had already returned halfway to normal values (Fig. 4A). The increase in Lp(a) levels was significantly

To investigate the effect of endogenous testosterone in men on lipoprotein metabolism, we took advantage of the reversible suppression of endogenous testosterone through treatment with GnRH antagonists. Similar to two previous studies, we observed that treatment with the GnRH antagonist Cetrorelix results in a 20% increase in HDL cholesterol (11, 12). Restoration of testosterone levels after withdrawal of treatment with Cetrorelix resulted in normalization of HDL cholesterol. We did not prove directly that the increase in HDL cholesterol was due to the suppression of testosterone and not exerted by the drug itself or by suppression of GnRH, gonadotropins, or estradiol (20). Bagatell and colleagues, however, have previously shown that injection of testosterone in parallel with treatment with the GnRH antagonist Nal-Glu restores HDL cholesterol (17). Moreover, although supraphysiological doses of testosterone suppress GnRH and gonadotropins, these treatments were found to decrease HDL cholesterol (9–15). Finally, estrogens are known to increase HDL cholesterol (1). Thus, it is very unlikely that the absence of GnRH, gonadotropins, or estradiol is responsible for the increases in HDL cholesterol associated with Cetrorelix. The absence of testosterone is hence the most likely reason for our observations. In extension of previous studies we found that the rise in HDL cholesterol is caused predominantly by an increase in the HDL subclass LpA-I. LpA-I is predominantly found in

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FIG. 3. Effects of testosterone suppression by Cetrorelix on serum concentrations of HDL-associated apolipoproteins and HDL subclasses. For details of the clinical study protocol, see Materials and Methods and Fig. 1. Curves and symbols indicate the time courses of mean values and SDs for apoA-I (A), apoA-II (B), LpA-I (C), and LpA-I/A-II (D; n 5 12). Arrows indicate the 21-day period during which volunteers were treated with Cetrorelix. The ellipses indicate the time interval in which testosterone levels were significantly suppressed (cf. Fig. 1). Significant differences between the mean data at the indicated time points and the mean data at baseline (mean of days 27 and 214; *) and posttreatment (day 91; #; by paired Wilcoxon U test and Bonferroni adjustment, P , 0.0064) are shown. P values were calculated using Friedman’s two-way ANOVA. n.s., Not significant.

the density or size range of HDL2 (29, 30). Our finding is hence in agreement with the previous observations that high dose injections of testosterone cause decreases in HDL2 cholesterol and LpA-I (8, 31). Suppression of testosterone also caused significant increases in plasma activities of LCAT and CETP. Whereas LCAT activity returned to baseline values after treatment, CETP activity remained elevated. Thus, the effect of testosterone on CETP appears questionable also because of the low level of significance for the differences in CETP activity between baseline and day 21. Our data do not allow any conclusion on the mechanism by which suppression of testosterone causes the increase in HDL cholesterol. It cannot be excluded that the small (7%), but statistically highly significant, increase in LCAT activity reflects the up-regulation of this gene by the removal of testosterone. To our knowledge, regulation of the LCAT gene by testosterone has not been investigated. However, the higher LCAT activity may also simply reflect the higher number of particles that can carry LCAT and thus increase LCAT activity in plasma. Other mechanisms by which suppression of testosterone can increase HDL cholesterol levels have not been investigated by us. An important candidate is hepatic lipase, whose activity was previously found to be increased by administration of testosterone to men (5, 32, 33). Suppression of GnRH, gonadotropins, endogenous testosterone, and estradiol by Cetrorelix was also associated with a pronounced increase in Lp(a) levels by 40 – 60%. Although not proven directly in our study, it is very likely that the lack of testosterone, rather than direct effects of Cetrorelix or suppression of GnRH, gonadotropins, and estradiol, caused the rise in Lp(a). Frazer and colleagues observed that Lp(a) levels decrease in male, but not in female, apo(a)-

transgenic mice after sexual maturation. Castration of male animals restored initial Lp(a) levels, which again decreased upon application of dihydrotestosterone (23). Likewise, orchidectomy of men with prostate cancer resulted in a highly significant increase in Lp(a) levels (34, 35). In these studies, however, it was not ruled out that the disease itself caused the rise in Lp(a) levels. This may also explain why suppression of endogenous testosterone in patients with prostate cancer by the GnRH analog buserelin led to a decrease in Lp(a) levels (36). Administration of testosterone to orchidectomized patients with prostate cancer (35) as well as administration of supraphysiological doses of testosterone enanthate to healthy men were associated with significant 25–59% decreases in Lp(a) (9, 10). Similar to the findings of our study, the decrease in Lp(a) was correlated to baseline levels of Lp(a), i.e. men with the highest initial Lp(a) levels experienced the greatest decrease in Lp(a) levels (9, 10). Thus, as supraphysiological dosages of testosterone suppress GnRH and gonadotropins but decrease Lp(a), we exclude that Cetrorelix elevates Lp(a) via suppression of gonadotropins. As Cetrorelix also suppresses estradiol and as treatment of women with estradiol was repeatedly found to decrease Lp(a) (1, 37), one may argue that lack of estradiol rather than lack of testosterone caused the increase in Lp(a). However, testosterone was also found to suppress Lp(a) levels when it was administered in combination with testolactone, which inhibits the aromatization of testosterone to estradiol (10). Therefore, it is also unlikely that Cetrorelix induces a rise in Lp(a) through suppression of estradiol. Thus, our study provides further evidence in healthy men that testosterone is significantly involved in the regulation of Lp(a) levels. Testosterone must hence be added to the list of

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Acknowledgments We thank the volunteers of this study for their participation and interest. We gratefully acknowledge the excellent technical assistance of Cornelia Elsenheimer, Claudia Humpert, Gaby Klapdor, Iris Lange, and Bertram Tambyrajah. Dr. Helmut Schulte and Michael Stennecken gave advice for the statistical analysis. Susan Nieschlag, M.A., provided editorial input.

References

FIG. 4. Effects of testosterone suppression by Cetrorelix on serum levels of Lp(a). For details of the clinical study protocol, see Materials and Methods and Fig. 1. In A, the curve and symbols indicate the time course of median values for Lp(a) (n 5 12). The arrow indicates the 21-day period during which probands were treated with Cetrorelix. The ellipse indicates the time interval in which testosterone levels were significantly suppressed (cf. Fig. 1). Significant differences between median Lp(a) levels on day 35 and the median data at baseline (mean of days 27 and 214; *) and posttreatment (day 91; #; by paired Wilcoxon U test and Bonferroni adjustment, P , 0.0064) are indicated. The P value presented as text was calculated using Friedman’s two-way ANOVA. B demonstrates the relationship between Lp(a) levels at baseline [mean values of Lp(a) on days 214 and 27] and the maximal increase in Lp(a) levels after suppression of testosterone [mean values of Lp(a) on days 21, 28, and 35]. The correlation between levels of Lp(a) at baseline and the increase in Lp(a) after suppression of testosterone was significant (slope 5 2.05; intercept 5 1.68; r 5 0.91; P , 0.001; by Pearson’s test).

hormones that reduce Lp(a) levels. Postmenopausal replacement therapy with either estrogens alone or in conjunction with gestagens was shown to reduce median levels of Lp(a) by 15–50% (1, 37). Treatment of men with prostate cancer by estrogens was associated with a 50% decrease in Lp(a) levels (36). Substitution of GH in deficient patients increased Lp(a) by 40 –100% (38 – 40). Correction of hyperthyroidism was associated with 20 – 60% increases and correction of hypothyroidism with 10 – 40% decreases in median Lp(a) levels (41– 43). More generally, as levels of sex hormones, GH, and T4 vary inter- and intraindividually, for example due to sexual maturation, growth, and disease, we assume that previous genetic studies underestimated the contribution of nongenetic factors to the variation in Lp(a) levels in the population (44). In view of the negative and positive associations of HDL cholesterol and Lp(a), respectively, with coronary risk (45– 47), Cetrorelix-induced increases in both HDL cholesterol and Lp(a) suggest that testosterone exerts both proatherogenic and antiatherogenic effects. These opposite effects of testosterone on atherogenic risk factors may explain the equivocal outcomes of historical, epidemiological, and experimental investigations on the relationships between testosterone and arteriosclerosis (48 –50).

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