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Home , Dehydroepiandrosterone, Testosterone, Testosterone (patch)

ORIGINAL ARTICLE

Effect of and Supplementation on Systemic Lipolysis

Ana E. Espinosa De Ycaza, Robert A. Rizza, K. Sreekumaran Nair, and Michael D. Jensen

Division of , Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905 Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021

Context: Dehydroepiandrosterone (DHEA) and T are advertised as antiaging, antiobe- sity products. However, the evidence that these hormones have beneficial effects on adipose is limited.

Objective: The objective of the study was to determine the effect of DHEA and T supplementation on systemic lipolysis during a mixed-meal tolerance test (MMTT) and an iv tolerance test (IVGTT).

Design: This was a 2-year randomized, double-blind, placebo-controlled trial.

Setting: The study was conducted at a general clinical research center.

Participants: Sixty elderly women with low DHEA concentrations and 92 elderly men with low DHEA and bioavailable T concentrations participated in the study.

Interventions: Elderly women received 50 mg DHEA (n ϭ 30) or placebo (n ϭ 30). Elderly men received 75 mg DHEA (n ϭ 30),5mgT(nϭ 30), or placebo (n ϭ 32).

Main Outcome Measures: In vivo measures of systemic lipolysis (palmitate rate of appearance) during a MMTT or IVGTT.

Results: At baseline there was no difference in suppression of lipolysis measured during MMTT and IVGTT between the treatment groups and placebo. For both , a univariate analysis showed no difference in changes in systemic lipolysis during the MMTT or IVGTT in the DHEA group and T group when compared with placebo. There was no change in the results after adjusting for the resting energy expenditure, except for a small, but significant (P ϭ .03) lowering of MMTT nadir palmitate rate of appearance in women who received DHEA.

Conclusion: In elderly individuals with concentrations of DHEA (men and women) or T (men) below the normal range for young adults, supplementation of these hormones has no effect on insulin suppression of systemic lipolysis. (J Clin Endocrinol Metab 101: 1719–1728, 2016)

he production of the dehydroepiandros- ever, association does not mean causality. Hence, replace- T terone (DHEA) and T declines with aging (1–3). ment of androgens in elderly men and women who have Whether this decline has detrimental consequences to plasma concentrations of DHEA and T below the normal overall health is unknown, but DHEA and T are being range for young adults may not improve fat distribution or used to improve muscle mass/strength and reduce body adipose function. fat. There are well-documented, -specific associations The studies that tested the effects of androgens on body between levels and body fat distribution; how- composition, adipose metabolism, adipocyte differentia-

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: AT, ; AUC, area under the curve; CV, coefficient of variation; Printed in USA DHEA, dehydroepiandrosterone; DHEAS, sulfated dehydroepiandrosterone; FFA, free fatty Copyright © 2016 by the Endocrine Society acid; IVGTT, iv glucose tolerance test; MMTT, mixed-meal tolerance test; Ra, rate of ap- Received November 24, 2015. Accepted February 12, 2016. pearance; REE, resting energy expenditure. First Published Online February 17, 2016

doi: 10.1210/jc.2015-4062 J Clin Endocrinol Metab, April 2016, 101(4):1719–1728 press.endocrine.org/journal/jcem 1719 1720 Espinosa De Ycaza et al Effect of DHEA and Testosterone on Lipolysis J Clin Endocrinol Metab, April 2016, 101(4):1719–1728

tion, and lipolysis have given inconsistent results (4–12). and an iv insulin profile. They first underwent an insulin-mod- Several human studies report that DHEA and T stimulate ified IVGTT followed by a second inpatient visit for MMTT. We 3 ␮ lipolysis (8, 12, 13). used a continuous infusion of [9,10- H]palmitate (0.3 Ci/min) during both IVGTT and MMTT to assess palmitate kinetics. The We conducted a randomized, double-blind, placebo- details of the protocol used for IVGTT and MMTT have been controlled trial to evaluate the effects on body composi- published (17). The (T patch, DHEA, and placebo) tion, quality of life, glucose, and fat metabolism of DHEA were continued during the inpatient visits and were given each replacement in elderly men and women and T replacement morning. Of the initially randomized volunteers, eight elderly in elderly men, with low concentrations of these hor- women and nine elderly men did not complete the studies of mones. We found no significant, clinically relevant effects palmitate kinetics. During these studies palmitate concentration and specific ac- on body composition, quality of life, and insulin sensitivity tivity were measured at baseline (before the meal or iv insulin) after 24 months of supplementation with DHEA or T (14, and every 30 minutes for 3 hours during the MMTT and at 20, Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 15). The group that received T had an increase in meal 31, 45, 90, and 120 minutes during the IVGTT. Suppression of fatty acid storage in the sc abdominal adipose tissue (AT) lipolysis during either test is reported as the nadir palmitate rate compared with femoral sc AT (16). Although we could not of appearance (Ra) or the area under the curve (AUC) for the detect an effect of DHEA or T on postabsorptive lipolysis palmitate Ra. The AUC for palmitate concentration and AUC for Ra was the AUC for these parameters estimated throughout the (16), we cannot infer the same for postprandial lipolysis. entire duration of the MMTT or IVGTT. Furthermore, the differences in meal fatty acid storage The techniques used for each of the concentration, previously reported (16) could be offset by androgen-in- substrate, and palmitate assays have been previously reported duced changes in insulin suppression of lipolysis in the (16, 17). Nonsteady-state formulas were used to calculate palmi- postprandial state. As part of this 2-year study of DHEA tate Ra for the postprandial and IVGTT studies (18). and T replacement in elderly men and women, we col- The methods used to measure hormone levels are described below. lected data to test the hypotheses that T or DHEA sup- Total T was measured using a competitive chemiluminescent plementation had an effect on systemic lipolysis during a immunoassay on the DxI 800 automated immunoassay system mixed-meal tolerance test (MMTT) or an insulin-modi- (Beckman Instruments). Intraassay coefficients of variation fied iv glucose tolerance test (IVGTT). (CVs) are 6.5% at 69 ng/dL and 3.3% at 862 ng/dL. Interassay CVs are 7.4% at 116 ng/dL, 8.6% at 407 ng/dL, and 4.0% at 761 ng/dL. Bioavailable T was measured by the differential precipitation Materials and Methods of SHBG by ammonium sulfate after the equilibration of the serum sample with tracer amounts of tritium-labeled T. The re- Herein we report pre- and postintervention postprandial and sults are expressed as the percentage of T free or albumin bound insulin-suppressed free fatty acid (FFA) data from 135 elderly (not precipitated with SHBG) compared with an albumin stan- adults (Ͼ60 y) who participated in a published trial (14) assess- dard. The product of this percentage and the total T measure- ing the effect of 2 years of placebo vs supplementation with ment is the total bioavailable T. Intraassay CV was 5.4 at 14.1% DHEA or T on body composition, glucose metabolism, and of bioavailable T. Interassay CVs are 7.5 at 28.4%, 4.9 at 41.9%, density. This study was approved by the Mayo Institutional Re- and 4.9 at 50.8% of bioavailable T. view Board. Participants providing informed consent included SHBG was measured by a solid phase, two-site chemilumi- 60 elderly women with sulfated DHEA levels less than 0.95 nescent assay on the Siemens Immulite 2000 automated immu- ␮g/mL and 92 elderly men with bioavailable T levels less than noassay system (Siemens Healthcare Diagnostics). Intraassay 103 ng/dL and sulfated DHEA levels less than 1.57 ␮g/mL. Bio- CVs are 2.7% and 3.1% at 5.5 and 95.9 nmol/L, respectively. available T levels of less than 103 ng/dL correspond to levels less Interassay CVs are 4.0% at 5.4 and 5.9% at 74 nmol/L. than 2 SD of the mean, normal concentration for young men, and DHEA sulfate was measured by a solid-phase, competitive DHEA levels less than 1.57 ␮g/mL, corresponding to levels less chemiluminescent immunoassay (EIA) on the Siemens than 1.5 SD of the mean, normal concentration for young men Immulite 2000 automated immunoassay system (Siemens (20–30 y of age). Women were randomly assigned to receive DHEA (n ϭ 30) or Healthcare Diagnostics). The intraassay CVs are 5.7%, 5.7%, ␮ placebo (n ϭ 30), and men were randomly assigned to receive 5.7%, and 5.1% at 26.4, 77, 203, and 493 g/dL. The inter- DHEA (n ϭ 30), T (n ϭ 30), or placebo (n ϭ 32) (14). T was given assay CVs are 5.2%, 3.6%, and 5.4% at 80.1, 211, and 515 ␮ as a 5-mg patch applied daily and thus resulted in continuous g/dL, respectively. delivery of T to the men. DHEA was taken as tablets each morn- ing, 50 mg for women and 75 mg for men. The FFA flux study Statistical analysis procedures have been previously described in detail (16, 17). Baseline characteristics and descriptive data are presented as Briefly, participants underwent outpatient and inpatient visits at median (interquartile range) for continuous variables and pro- baseline and 24 months after replacement with placebo, DHEA, portion for categorical variables. Differences in baseline char- or T. The outpatient visits included dual-energy x-ray absorpti- acteristics between the treatment groups was determined by an ometry and single-slice abdominal computed tomography for ANOVA. The Pearson coefficient was used for the correlation of body composition. The participants underwent two inpatient continuous variables when normally distributed, and the Spear- visits to measure the suppression of lipolysis in response to a meal coefficient was used when the data were not normally dis- doi: 10.1210/jc.2015-4062 press.endocrine.org/journal/jcem 1721

Table 1. Baseline Characteristics of the 135 Elderly Men and Women Who Participated in the Lipolysis Studies

Elderly Women Elderly Men

Placebo DHEA Placebo DHEA T (26 ؍ n) (28 ؍ n) (29 ؍ n) (25 ؍ n) (27 ؍ Characteristic (n Age, y 69 (65–75) 68 (66–69) 67 (64–73) 69 (67–72) 66 (62–72) Weight, kg 74 (65–80) 70 (59–79) 86 (79–98) 84 (74–92) 86 (78–92) BMI, kg/m2 28.3 (26–30.4) 26.6 (24.7–29.5) 27.4 (26–29.8) 27 (24.4–29.1) 28.2 (25.7–30.2) Body fat, % 46 (41–48) 44 (40–49) 30 (25–34) 27 (23–31) 28 (25–33) Fat-free mass, kg 37.9 (35.5–40.1) 36 (34–39.4) 58.2 (53.6–61.8) 56.2 (52.1–60.4) 56.1 (53.8–60.6) REE, kcal/d 1318 (1241–1442) 1301 (1206–1401) 1662 (1589–1811) 1635 (1524–1719) 1645 (1527–1802)

Serum triglycerides, 123 (99–145) 121 (68–144) 112 (78–150) 103 (74–138) 127 (87–154) Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 mg/dL Abbreviation: BMI, . Data are expressed as median (interquartile range). tributed. The Kruskal-Wallis test was used to analyze the differ- sample size. We used data from previous studies to develop ences in the palmitate concentration or palmitate Ra (expressed post hoc power calculations. Under conditions comparable as either nadir levels or AUC) between the treatments groups by with these experiments, we reported that the intrasubject vari- sex. A multiple linear regression analysis was used to adjust for ability of overnight postabsorptive palmitate Ra was 14% Ϯ potential confounders for the relationship between the suppres- 8% (19); when insulin is given, it is 8% Ϯ 4% (20). In previous sion of lipolysis and treatment received. Two-sided statistical animal and studies, the change of lipolysis after re- tests and an alpha Ͻ.05 was used to determine statistical signif- placement with androgens varies from 35% to 80% (8, 13, icance for all analyses. All analyses were performed using JMP 21). With a sample size of 26 per group, a power of 80%, and software (version 10.0.0; SAS Institute, Cary, NC, USA). a significance level of 0.05, we could have detected a difference in nadir palmitate Ra of 10 ␮mol/min. Based upon the liter- Sample size ature, differences less than 10 ␮mol/min are unlikely to be Because the effect on T and DHEA on the suppression of clinically relevant, considerably larger differences exist be- lipolysis was not the primary outcome related to fatty acid tween insulin-sensitive and insulin-resistant states (22, 23). metabolism of this study, we did not perform power calcula- Palmitate Ra was expressed in micromoles per minute and tions for this analysis a priori. To estimate the statistical adjusted for resting energy expenditure (REE) for statistical anal- power, we assessed the minimal statistically significant dif- ysis. Some investigators express free fatty acid flux per kilogram ference we could detect between the groups based on our of fat-free mass; however, we have previously demonstrated that

Table 2. DHEA, SHBG, and T Values at Baseline and After 2 Years of Supplementation With DHEA or T

Younga Elderly Elderly Males

Hormone Values F M Placebo DHEA Placebo DHEA T

SHBG, nmol/L Baseline 72.2 (45.6, 144.5)b 23.4 (19, 29)c 50 (36.6, 73.8) 52.4 (34.9, 71.4) 41.7 (35.1, 50.8) 33.1 (28.5, 36.8) 36.8 (30.2, 40.8) Year 2 54.2 (37, 67.7) 37.8 (24.9, 45.9) 41.2 (36.6, 49.4) 31.2 (26.4, 36.6) 36.6 (31.3, 44.4) Changed Ϫ1(Ϫ8.5, 5.4) Ϫ13.6 (Ϫ27.2, Ϫ4.5)e 0.4 (Ϫ5, 6) Ϫ2.4 (Ϫ5, 2.6)f Ϫ0.2 (Ϫ5.1, 3.4)f DHEAS, ␮g/mL Baseline 1.4 (1, 2)b 2.75 (1.9, 3.7)c 0.3 (0.3, 0.4) 0.3 (0.3, 0.5) 0.62 (0.42, 1) 0.65 (0.4, 0.9) 0.6 (0.3, 0.9) Year 2 0.3 (0.3, 0.4) 3.66 (2.7, 6) 0.55 (0.3, 0.8) 3.96 (3.1, 6.3) 0.5 (0.3, 0.7) Changed 0(Ϫ0.04, 0) 3.9 (2.9, 5)g Ϫ0.1 (Ϫ0.2, 0.02) 3.9 (2.8, 5.3)e Ϫ0.07 (Ϫ0.17, 0) Total T, ng/dL Baseline 543 (470, 675)c 387 (294, 460) 373.5 (254, 440) 362 (295, 459) Year 2 383 (331, 527) 348 (276, 451) 494 (421, 637) Changed 24 (Ϫ14.5, 67.5) 1.5 (-23, 32.8) 111 (39, 252)e Bioavailable T, ng/dL Baseline 174.4 (125, 238)c 52.7 (43.6, 63.6) 63.5 (51.6, 76.5) 57.1 (45.7, 65.7) Year 2 52.9 (38.6, 65.1) 65.3 (53.4, 88.1) 82.8 (65, 115.1) Changed Ϫ0.78 (Ϫ7.7, 11.6) 3.5 (Ϫ5.7, 19) 30.5 (Ϫ0.9, 62.5)h

Abbreviations: F, ; M, male. Data are expressed as median (interquartile range). a Thirty-eight healthy young women and 37 healthy young men baseline hormonal values obtained compared with values in the elderly groups. b P Ͻ .001 young females vs elderly females at baseline. c P Ͻ .001 young males vs elderly males at baseline. d Change in hormone levels (year 2 Ϫ baseline). e P Ͻ .001 vs placebo. f No significant change compared with placebo. g P Ͻ .0001 vs placebo. h P Ͻ .01 vs placebo. 1722 Espinosa De Ycaza et al Effect of DHEA and Testosterone on Lipolysis J Clin Endocrinol Metab, April 2016, 101(4):1719–1728 fat-free mass does not correlate with palmitate Ra in adult men increased to an average of approximately 4 ␮g/mL in both and women (19), whereas REE is the best predictor and posi- groups assigned to DHEA (Table 2). The baseline median tively correlates with free fatty acid flux (16, 19, 24). total T levels in men assigned to T was 362 ng/dL and increased to 494 ng/dL 24 months after therapy (14) (Ta- ble 2). There was no statistically significant difference in T Results or DHEA concentrations measured throughout the study Baseline characteristics of the participants who completed in the groups assigned to placebo. After 2 years of hor- both pre- and posttreatment FFA flux studies are pre- mone supplementation, there was a small increase in fat- sented in Table 1. There were no significant differences in free mass for elderly men on T (14) but no other significant weight, age, body composition, baseline levels of sulfated changes in body weight or body composition between the DHEA (DHEAS), total T, or bioavailable T between el- groups. Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 derly men in the three treatment groups or elderly women The pre- and posttreatment palmitate Ra responses to in the two groups. The baseline median DHEA levels were the MMTT and the IVGTT for the placebo groups are 0.6 and 0.3 ␮g/mL for men and women, respectively, and shown in the top panels of Figure 1. The palmitate Ra

Figure 1. Palmitate Ra at baseline and after 2 years of hormone replacement (follow-up) according to treatment group during the MMTT (left panel) and during the IVGTT (right panel). doi: 10.1210/jc.2015-4062 press.endocrine.org/journal/jcem 1723

Table 3. Change in Systemic Lipolysis at 24 Months in Men and Women According to Treatment Group

Men Women

Change in Palmitate Ra P P P P (Baseline to 24 mo) Placebo DHEA T Valuea Valueb Placebo DHEA Valuea Valueb

MMTT Nadir Ra, ␮mol/min 4 (Ϫ2, 8) 3 (Ϫ7, 11) 3 (Ϫ2, 12) .82 .51 D 1(Ϫ4, 4) 3 (Ϫ2, 10) .16 .03 .65 T AUC Ra, ␮mol/min 1924 (93, Ϫ3824) 1341 (419, 4165) 1722 (Ϫ400, 3704) .92 .92 D 509 (Ϫ329, 1713) 780 (Ϫ518, 3040) .46 .09 .79 T IVGTT Nadir Ra, ␮mol/min 4 (Ϫ7, 16) 8 (Ϫ8, 18) 4 (Ϫ11, 17) .74 .59 D 8(Ϫ3, 14) 5 (Ϫ3, 14) 1.00 .71 .57 T

AUC Ra, ␮mol/min 794 (Ϫ1048, 2622) 1470 (Ϫ901, 2239) 1029 (Ϫ1395, 2357) .88 .3 D 1443 (Ϫ139, 3618) 1536 (Ϫ301, 3186) .63 .65 Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 .62 T

Abbreviations: D, P value of the DHEA group compared with placebo; T, P value of the T group compared with placebo; Nadir Ra, nadir palmitate rate of appearance; AUC Ra, area under the curve of palmitate rate of appearance. Data are expressed as median (interquartile range). a Unadjusted P value for any difference between the three groups in men and two groups in women. b Adjusted P value for change in REE. responses to DHEA (men and women) and T (men) before DHEA were no longer statistically significant (Figure 3, and after treatment are depicted in the middle and bottom right panel). panels of Figure 1, respectively. We found an overall trend Univariate analysis of data from the MMTT and for decrease in the nadir palmitate Ra at 24 months during IVGTT did not detect statistically significant differences the MMTT and IVGTT in men and women in all groups between changes in systemic lipolysis in women that re- (Table 3). There was a median decrease in MMTT nadir ceived DHEA or in men that received DHEA or T com- palmitate Ra of 4 ␮mol/min (Ϫ2, 10 ␮mol/min) and 1 pared with placebo (Figure 4). The results were similar ␮mol/min (Ϫ3, 6 ␮mol/min) for men and women, respec- whether lipolysis was expressed as the nadir palmitate Ra tively. The IVGTT nadir palmitate rate of appearance de- or AUC of the palmitate Ra. The lack of statistical signif- creased by a median of 5 ␮mol/min (Ϫ9, 17) for men and icance persisted after adjusting for the changes in either the 7 ␮mol/min (Ϫ3, Ϫ14) for women. Although most of these fat-free mass or REE, except for a small but statistically changes were statistically significant (except for women significant difference in nadir palmitate Ra in women re- after a mixed meal), the absolute changes were quite small ceiving DHEA compared with placebo during the MMTT of unknown physiological relevance. when the REE was included in the multivariate model (P ϭ During the pretreatment MMTT, there were no differ- .03) (Table 3). ences in the nadir palmitate concentrations for women or men. For women, the median (interquartile range) nadir palmitate concentrations were 9 ␮mol/L (8–12) and 9 Discussion ␮mol/L (8–11) for the DHEA and placebo groups, respec- tively (P ϭ .98). For men, the median nadir palmitate con- In this randomized, placebo-controlled trial of supple- centrations were 13 (10–19), 12 (9–17), and 13 (10–18) mentation of DHEA in elderly men and women and T in ␮mol/L in the T, DHEA, and placebo groups, respectively elderly men, we evaluated the effects of these hormones on (P ϭ .57). There were similar patterns during the IVGTT the suppression of lipolysis during an IVGTT and a mixed- (data not shown). There was no statistically significant meal tolerance test. We have previously reported that nei- difference in the basal and peak insulin concentrations ther DHEA nor T alters meal fatty acid metabolism or achieved in the pretreatment and posttreatment studies postabsorptive lipolysis (15, 16), but we were unable to between the groups (Figure 2). include the lipolysis suppression data in that already We analyzed the data from the pretreatment studies lengthy report. Subsequent analysis revealed that post- and found a significant, positive correlation between the prandial suppression of lipolysis is a better predictor of REE and the MMTT nadir of systemic lipolysis as mea- some aspects of metabolic dysregulation than postabsorp- sured by palmitate Ra (Figure 3). This association was tive lipolysis (17). We now report that neither DHEA nor present in the 24-month data in all groups (Figure 3, left T replacement for 24 months has a clinically significant panel). We also found a positive correlation between REE effect on postprandial or iv insulin suppression of and nadir IVGTT palmitate Ra at baseline in all groups. lipolysis. However, at 24 months the correlations between the nadir Previous studies indicate that DHEA replacement de- palmitate Ra and REE for men on placebo and women on creases body fat by 6%–30% (4, 6). It has been postulated 1724 Espinosa De Ycaza et al Effect of DHEA and Testosterone on Lipolysis J Clin Endocrinol Metab, April 2016, 101(4):1719–1728 Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021

Figure 2. Insulin concentrations in the participants in these studies. P, placebo. that this effect is mediated by the stimulation of lipolysis imal (25) or in vitro studies (21), which may not reflect the (8) via activation of hormone sensitive lipase (25) and effects of DHEA in AT in vivo. adipose triglyceride lipase (21). In contrast, we did not find In the case of T, there are conflicting results reported of an effect of DHEA on body composition or on systemic the effects on AT lipolysis. In cultured human adipocytes lipolysis. We found a small, statistically significant differ- and preadipocytes from young healthy men and women, ence in change in the nadir palmitate Ra for women on exposure to T had either no effect on lipolysis (11) or DHEA compared with placebo during MMTT. However, suppression of catecholamine-stimulated lipolysis (26). we did not find a difference when lipolysis was measured However, in another study, lipolysis was stimulated in as an AUC palmitate Ra or during IVGTT, which suggests preadipocytes cultured with DHT during differentiation that the former difference is probably not clinically rele- vant; furthermore, we cannot exclude a type 1 statistical (12). Increased in vivo lipolysis was also seen after 6 weeks error due to multiple comparison testing. There are other of treatment with T in middle-aged men (13). The meth- differences between the aforementioned studies and ours; odological differences between these studies and ours the studies that found differences in body composition make it difficult to directly compare the results. We believe were short-term studies of 6 months’ duration or less and our data provide strong evidence that T administration to the effect on lipolysis was predominantly evaluated in an- elderly men with mildly reduced levels at baseline does not doi: 10.1210/jc.2015-4062 press.endocrine.org/journal/jcem 1725 Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021

Figure 3. Correlations between REE and palmitate release rate during a MMTT and IVGTT. r, Pearson’s correlation coefficient; rs, Spearman’s correlation coefficient. alter systemic lipolysis in the absence of substantial losses groups. However, we should note that insulin suppression of body fat. of lipolysis was not the primary outcome of this random- The study design we used controls for potential selec- ized trial, and, however unlikely, it is possible that our tion bias or confounders for the association between the negative findings are the result of a type 2 statistical error. interventions and systemic lipolysis. The longer duration That said, the absolute differences in the changes of palmi- of therapy with T and DHEA avoids flawed results caused tate Ra we identified between the treatment groups were by regression toward the mean. However, this phenome- below the levels that might be physiologically relevant. non may explain why some of the short-term studies with Different body fat depots may contribute disproportion- DHEA on body composition (4, 6, 7) or T on lipolysis (13) ately to systemic lipolysis under basal (27, 28) and insulin- showed significant differences between the treatment suppressed (22, 29) conditions. We have previously 1726 Espinosa De Ycaza et al Effect of DHEA and Testosterone on Lipolysis J Clin Endocrinol Metab, April 2016, 101(4):1719–1728

the above-outlined metabolic vari- ables. We cannot say that these hor- mones have no effect on AT metab- olism because other measures in adipose tissue were not performed, such as AT , adipokine production, etc. However, we think our study adds more relevant infor- mation to help us understand the role of these hormones in AT

metabolism. Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 The definitions of low plasma DHEAS concentrations used for in- clusion in our study (Ͻ1.57 ␮g/mL for men and Ͻ0.95 ␮g/mL for women) were based on concentra- tions found in young, healthy men and women (1.5 SD below the mean). However, the age-specific reference range for DHEAS in adult women above the age of 60 is less Figure 4. Unadjusted change in nadir palmitate Ra at 24 months (baseline follow-up) according than 0.15–1.57 ␮g/mL and for el- to treatment group. The dashed lines indicate the change in palmitate Ra of Ϯ10 ␮mol/min derly men 0.25–1.31 ␮g/mL. None (from post hoc power calculation) in the treatment groups compared with placebo, necessary to detect a statistically significant difference. of our participants had DHEAS con- centrations below the lower end of their age-specific reference range. shown that upper body sc fat is the main source of excess Our results indicate that DHEA supplements given to el- FFA, and the splanchnic bed (visceral fat) accounts for derly adults with plasma DHEAS concentrations in the 15%–30% of systemic FFA (27). It is possible that DHEA age-specific normal range have negligible effects on adi- or T altered the relative contributions of FFA from the pose tissue metabolism. various fat depots. For example, these hormones could The assays for measuring T have changed during recent alter FFA release from visceral depot and this would not be years. In this study we used a chemiluminescence immu- reflected in our measures of systemic lipolysis. However, noassay, whereas currently mass spectrometry is the pre- changes in FFA delivery to the would be expected to ferred method. However, there is a good correlation between alter hepatic insulin sensitivity with regard to glucose T levels by chemiluminescence immunoassay compared with and/or triglyceride metabolism; we found no effects of mass spectrometry (31). The assay used in our study when these hormones on insulin-regulated glucose production compared with mass spectroscopy by liquid chromatogra- (15, 30) or plasma triglyceride concentrations (14). Re- phy mass spectrometry were well correlated (r ϭ 0.9 for gardless of hypothetical different effects these hormones total T levels and 0.95 for bioavailable T) (32). might have on specific fat depots, the lack of change in We defined low T levels using bioavailable T criteria systemic lipolysis is consistent with the finding that sup- because it is considered to be the fraction readily available plementing these hormones in the elderly also does not to cells. Our elderly male participants had moderately re- change peripheral insulin sensitivity (15, 30). duced bioavailable T concentrations, and some men had Abnormalities of AT lipolysis causes excess FFA, which total and bioavailable T concentrations in the lower part can result in hepatic and muscle insulin resistance, in- of the normal range. The lower end of the reference range creased very low-density lipoprotein-triglyceride-level for total T in adults is 300 ng/dL and 40 ng/dL for bio- production, abnormal vascular constriction, and excess available T for the assays used in our study; 24% and 11% insulin secretion. Our study was a mechanistic study with of the male participants had concentrations below these the goal of identifying whether DHEA or T have effects on levels for total T and bioavailable T, respectively. There- adipose tissue metabolism by measuring changes in sys- fore, the results of our study should be interpreted in the temic lipolysis. The lack of effect of these hormones on appropriated context and may not be generalizable to men systemic lipolysis suggests that these hormones may not with frank . Moreover, although the dose have significant effects on AT metabolism as it relates to of T we used caused a significant increase in bioavailable doi: 10.1210/jc.2015-4062 press.endocrine.org/journal/jcem 1727

T and fat-free mass as well as reductions in FSH and LH, 7. Villareal DT, Holloszy JO. Effect of DHEA on abdominal fat and the median increase did not reach the middle of the normal insulin action in elderly women and men: a randomized controlled trial. JAMA. 2004;292:2243–2248. range. This is the goal recommended by Endocrine Society 8. Hernández-Morante JJ, Pérez-de-Heredia F, Luján JA, Zamora S, guidelines for patients with symptomatic hypogonadism. Garaulet M. Role of DHEA-S on body fat distribution: Gender- and Nevertheless, the significant increment in T levels in our depot-specific stimulation of adipose tissue lipolysis. . 2008; 73:209–215. study was not associated with significant changes in sys- 9. Allan CA, Strauss BJG, Burger HG, Forbes EA, McLachlan RI. Tes- temic lipolysis. tosterone therapy prevents gain in visceral adipose tissue and loss of In summary, DHEA or T supplementation has no sig- in nonobese aging men. J Clin Endocrinol Metab. 2008;93:139–146. nificant effect on the suppression of systemic lipolysis 10. Kenny AM, Prestwood KM, Gruman CA, Marcello KM, Raisz LG. measured by after either a mixed meal or an IVGTT in Effects of testosterone on bone and muscle in older men Downloaded from https://academic.oup.com/jcem/article/101/4/1719/2804555 by guest on 24 September 2021 elderly women with low concentrations of DHEA or el- with low bioavailable testosterone levels. J Gerontol. 2001;56: derly men, with concentrations of DHEA and T below M266–M272. 11. Blouin K, Nadeau M, Perreault M, et al. Effects of androgens on those observed in young men. adipocyte differentiation and adipose tissue explant metabolism in men and women. Clin Endocrinol (Oxf). 2010;72:176–188. 12. Gupta V, Bhasin S, Guo W, et al. Effects of on differentiation and proliferation of human mesenchymal stem cells Acknowledgments and preadipocytes. Mol Endocrinol. 2008;296:32–40. 13. Rebuffe-Scrive M, Marin P, Bjorntorp P. Effect of testosterone on We thank the volunteers who participated in this study. We also abdominal adipose tissue in men. Int J Obes. 1991;15:791–795. thank Jean Feehan, Barbara Norby, and the members of the 14. Nair KS, Rizza RA, O’Brien P, et al. DHEA in elderly women and Mayo Clinic Clinical Research Unit nursing, dietary, and sup- DHEA or testosterone in elderly men. N Engl J Med. 2006;355: port laboratory staff for technical assistance in performing the 1647–1659. 15. Basu R, Dalla Man C, Campioni M, et al. Two years of treatment study. with dehydroepiandrosterone does not improve insulin secretion, insulin action, or postprandial glucose turnover in elderly men or Address all correspondence and requests for reprints to: women. Diabetes. [Erratum (2007) 56(5):1486] 2007;56:753–766. Michael D. Jensen, MD, Mayo Clinic, Endocrine Research Unit, 16. Koutsari C, Ali AH, Nair KS, et al. Fatty acid metabolism in the 200 First Street SW, Room 5-194 Joseph, Rochester, MN 55905. elderly: effects of dehydroepiandrosterone and testosterone replace- E-mail: [email protected]. ment in hormonally deficient men and women. J Clin Endocrinol This study had a registration number of Metab. 2009;94:3414–3423. NCT00254371. 17. Bush NC, Basu R, Rizza RA, Nair KS, Khosla S, Jensen MD. Insulin- mediated FFA suppression is associated with triglyceridemia and This work was supported by Grants DK07352, PO1 insulin sensitivity independent of adiposity. J Clin Endocrinol AG14283, DK40484, and RR00585 from the US Public Health Metab. 2012;97:4130–4138. Service and the Mayo Foundation. 18. Jensen MD, Heiling V, Miles JM. Measurement of non-steady-state Disclosure Summary: The authors have nothing to disclose. free fatty acid turnover. Am J Physiol. 1990;258:E103–E108. 19. Nielsen S, Guo Z, Albu JB, Klein S, O’Brien PC, Jensen MD. 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