METABOLISM CLINICAL AND EXPERIMENTAL 62 (2013) Available online at Metabolism.

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1 METBOLISM CLINICL ND EXPERIMENTL 62 (213) vilble online t Metbolism Gender determines CTH recovery from hypercortisolemi in helthy older humns nimesh Shrm, MBBS, Pul oun, DO, PhD, Jen Wighm, RN, Sue Weist, RN, Johnnes D. Veldhuis, MD Endocrine Reserch Unit, Myo School of Grdute Medicl Eduction, Center for Trnsltionl Science ctivities, Myo Clinic, Rochester, MN 9, US RTICLE INFO BSTRCT rticle history: Received 11 June 213 ccepted 22 ugust 213 Keywords: ging Feedbck Humn Objective. vilble clinicl dt rise the possibility tht stress-dptive mechnisms differ by gender. However, this notion hs not been rigorously tested in reltion to cortisolmedited negtive feedbck. Mterils/Methods. Degree of CTH inhibition during nd recovery from n experimentl cortisol clmp ws tested in 2 helthy older subjects (ge 6 ± 2.2 y). Volunteers received orl plcebo or ketoconzole (KTCZ) to inhibit drenl steroidogenesis long with i.v. infusions of sline or low vs high physiologicl dose of cortisol in prospectively rndomized double-blind, plcebo-controlled design. CTH nd cortisol concentrtions were mesured every 1 min during the feedbck-clmp phse nd therefter (recovery or escpe phse). Corticosteroid-binding globulin (CBG) ws mesured, nd free cortisol concentrtions were clculted. Results. Gender did not determine men CTH concentrtions during the sline or cortisol feedbck-clmp phses per se. However, women hd mrkedly impired CTH recovery fter stopping both low- nd high-dose cortisol infusions compred with men (P =., KTCZ/low-dose cortisol rm; nd P =.6, KTCZ/high-dose cortisol rm). Decresed CTH recovery in women ws ccompnied by lower totl nd free cortisol concentrtions, pointing to heightened feedbck inhibition of hypothlmo-pituitry drive of CTH secretion s the min mechnism. Conclusions. In summry, gender or fctor relted to gender, such s sex steroids or body composition, determines recovery of CTH secretion from cortisol-enforced negtive feedbck. ttenuted CTH recovery in post-menopusl women my hve relevnce to sex differences in stress-relted dpttions. 213 Elsevier Inc. ll rights reserved. 1. Introduction Time-delimited stress-dptive responses of the corticotropic xis re criticl to sustin life nd regulte key metbolic processes [1]. Hypothlmo-pituitry responses re ctivted nd restrined by feedforwrd nd feedbck signls, which together medite homeosttic CTH fluctutions in pthophysiologicl sttes [2 ]. lbeit crucil to multicomponent bbrevitions: CTH, drenocorticotropic hormone; E 2, estrdiol; T, testosterone; KTCZ, ketoconzole; CRH, corticotropin-relesing hormone; VP, rginine vsopressin. Corresponding uthor. Tel.: ; fx: E-mil ddress: veldhuis.johnnes@myo.edu (J.D. Veldhuis) /$ see front mtter 213 Elsevier Inc. ll rights reserved.

2 182 METBOLISM CLINICL ND EXPERIMENTL 62 (213) regultion in neuroendocrine systems, feedbck regultion is less well studied thn feedforwrd drive [6,7]. Moreover, how gender, sex steroids, puberty, hypogondism nd ging supervise feedbck effects is lrgely unknown [8 1]. In prticulr for the stress xis, wheres gender nd sex steroids regulte CTH secretion strongly in nimls, investigtions in humn subjects re both limited nd discrepnt [11]. In the rodent, femle sex (nd estrdiol) nd mle sex (nd testosterone) respectively potentite nd ttenute stress responses [1,12,13]. However, in the humn, inferences on the role of gender in modulting hypothlmic-pituitrydrenl (HP) responses re inconsistent. In prt, inconsistencies my reflect the dose nd type of feedbck signl pplied (e.g. long-lived synthetic glucocorticoids or physiologicl cortisol), s well s confounding interctions mong gender, ge nd the degree nd type of stress [,14 16]. Inn initil effort to ddress these uncertinties, the present clinicl investigtion exmines, in given ge group, how gender impcts () feedbck inhibition of CTH secretion by vrying systemic cortisol concentrtions; nd (b) feedforwrd recovery of hypothlmo-pituitry drive of CTH secretion during subsequent hypocortisolemi. These issues re importnt, since the short-term consequences of blunted cortisol/ CTH feedbck include elevted glucocorticoid secretion nd heightened CTH responsiveness to CRH nd VP, while the long-term consequences of excessive CTH/cortisol feedforwrd include elevted blood pressure, suppressed immune function, impired insulin ction, viscerl-ft ccumultion, decresed muscle protein nd bone mss, nd possibly reduced memory nd hippocmpl neurogenesis [17 2]. Thus, understnding how gender regultes CTH secretion in older dults should provide insights into stress-dptive mechnisms in individuls t incresed risk of frilty, injury nd disbility. 2. Mterils nd methods 2.1. Overview The bsic postulte tested ws tht older men nd postmenopusl women differ in the degree of feedbck inhibition of CTH secretion by, nd subsequent escpe of CTH secretion from, controlled intrvenous cortisol exposure nd withdrwl. This ws n institutionl review bord-pproved, prospectively rndomized, double-blind, nd plcebo-controlled crossover study. The investigtionl protocol ws lso reviewed by the U.S. Food nd Drug dministrtion nd ssigned n investigtor new drug number, insmuch s ketoconzole (KTCZ) ws used off-lbel to block drenl steroidogenesis reversibly [21,22] Subjects Prticipnts were recruited from the community within the llowble ge rnge of 8 yers. Ten men nd ten women were enrolled. Women were postmenopusl by 2 yr or more. No volunteers were receiving hormone replcement. Subjects mintined conventionl work nd sleeping ptterns nd reported no recent (within 1 dys) trnsmeridin trvel, significnt ( 2 kg) weight chnge, psychosocil stress, substnce buse, neuropsychitric medictions or illness, or systemic disese. There were no concurrent or historicl neuropsychitric dignoses or relted drug tretments, nd no disorders of eting, behvior, nxiety, depression or psychosis, when ssessed s in erlier cortisol feedbck studies [23]. complete medicl history, physicl exmintion, nd screening tests of hemtologicl, renl, heptic, metbolic nd endocrine function were norml. Volunteers were dmitted to the Myo Center for Trnsltionl Science for 4 seprte inptient overnight study sessions, ech seprted by t lest 1 dys. The study ws double blind, using orl plcebo vs KTCZ cpsules, nd i.v. sline vs cortisol infusions. (We did not try to infuse cortisol under orl plcebo, since the levels would then be hrd to interpret under high uninhibited cortisol pulses). Overnight infusions nd next-morning smpling were chosen to exmine gender-relted regultion of the strong diurnl rise in CTH in the cortisol-feedbck prdigm. Lights were extinguished t 2 h. Subjects received either orl plcebo tblets or KTCZ pills (6 mg in men nd 4 mg in women) t 18 h. KCTZ ws given with nondiry snck to obvite dyspepsi. The dose ws selected in pilot studies to chieve comprble suppression of cortisol in the two sexes. KTCZ (t one-hlf the dose) or plcebo ws given gin t 22 h nd 6 h to continue to block drenl steroidogenesis. Concurrently, either sline (1 ml) or cortisol (s hydrocortisone succinte in sline 1 ml) t totl dose of 2. mg/m 2 (low dose) or 1 mg/m 2 (high dose) ws infused continuously i.v. over 8 h until 2 h. This creted 4 seprte cortisol clmps (tretment rms): 1) plcebo + sline ddbck; 2) KTCZ + sline ddbck; 3) KTCZ + low-dose cortisol (LDC) infusion; nd 4) KTCZ + high-dose cortisol (HDC) infusion (Fig. 1). Blood smples were obtined for lter mesurement of plsm CTH (1. ml) nd serum cortisol (. ml) every 1 min for 14 h from 22 h until 12 h the next dy. Subjects slept through the smpling until 6 h. Fourteen h of smpling ws to monitor CTH secretion during the lst 4 h of the constnt 8-h infusion clmp, nd for 1 dditionl h fter stopping i.v. cortisol or sline bruptly t 2 h. t 12 h, 2 mg hydrocortisone succinte ws dministered s n i.v. bolus for sfety resons Hormone ssys Plsm CTH ws ssyed vi highly sensitive nd specific, solid-phse sequentil immunossy (Siemens Helthcre Dignostics, Deerfield, IL). The detection threshold ws ng/l (to convert to SI units, divide by 4.). The intr-ssy coefficient of vritions (CV) were 6.4%, 2.7%, nd 3.2% t.7 ng/l, 28 ng/l, nd 42 ng/l, respectively. ws ssyed vi competitive binding immunoenzymtic ssy (Beckmn Coulter, Inc., Fullerton C). The detection limit ws.4 μg/dl (to convert to SI units, multiply by 27.6) nd intrssy CVs were 13%, 9.4%, nd 6.6% t 1.6 μg/dl, 2.8 μg/dl, nd 3 μg/dl, respectively. ll smples from given subject were ssyed in the sme btch. Sex steroids were ssyed by mss spectrometry, nd LH, FSH, TSH nd prolctin by immunochemiluminescence [24,2].

3 METBOLISM CLINICL ND EXPERIMENTL 62 (213) CBG ssy Serum corticosteroid binding globulin (trnscortin, RD R, BioVendor, sheville, NC) ws quntified by ELIS. Microplte wells precoted with polyclonl CBG ntibody re used to cpture the nlyte, nd biotin-lbeled monoclonl humn CBG ntibody conjugted with streptvidin-horserdish peroxidse is used for the indictor rection in substrte solution (3,, -tetrmethylbenzidine), s described by the vendor [26,27]. The product is yellow color, wherein bsorbnce t 4 nm is proportionl to the concentrtion of CBG. ssy rnge is 3 2 ng/ml with interssy nd intrssy coefficients of vrition of 2% nd 7%, respectively. Dilution linerity nd recovery were 98.% nd 1.3%. ll smples were run in the sme ssy. Mesurements of lbumin, CBG nd totl cortisol llowed clcultion of free cortisol by Coolens eqution, s modified by Mzer [28,29]. 2.. Sttisticl nlysis Men plsm CTH (nd serum cortisol) concentrtions served s the primry endpoints. The effects of gender (2 fctors) nd sline/cortisol feedbck-intervention type (4 fctors) on the nturl logrithm of the outcome mesures were evluted by two-wy nlysis of covrince (NCOV). Log trnsformtion ws used to stisfy the Shpiro-Wilks test of normlity. To djust for intrsubject correltions, the men CTH (nd cortisol) concentrtion during the plcebo/sline rm served s the model covrite. When the covrite ws not significnt, 2-wy NOV with prtilly repeted mesures ws employed. Post hoc nlysis utilized Tukey s Honestly Significntly Different (HSD) test. Responses were segmented into the initil clmp phse (lst 4 h of the continuous 8-h sline/cortisol infusion) nd the subsequent (1-h) recovery phse. secondry outcome during the infusion clmp ws the feedbck slope defined by regressing medin 4-h CTH concentrtions on corresponding medin 4-h cortisol concentrtions in ech volunteer bsed upon his/her four tretments. Slopes in women nd men were compred using the Mnn Whitney U test. Similr outcomes were obtined by unpired t test of the bsolute vlue of the slopes. nother secondry outcome ws the slope of the rising nd flling portion of two-segment piecewise regression model relting CTH concentrtions to time over the 1-h recovery period [3]. This permitted objective determintion of brekpoint in time, nd thereby seprte slopes for the initil rise (recovery before the brekpoint) nd delyed fll (recovery fter brekpoint) in CTH concentrtions, when cortisol infusions were stopped. Three-wy NCOV (gender, brekpoint, infusion type) ws pplied to ssess men CTH levels in reltion to ll 3 fctors. Escpe from suppression (recovery) ws confirmed by nonliner curve-fitting of time-vrying (medin) CTH concentrtions to 4-prmeter sigmoidl function with 9% confidence intervls. Experiment-wise P <. ws construed s significnt. Liner regression ws used in the group of 2 subjects to test the reltionship between CTH responses nd ge, T or E 2 levels. For ese of visuliztion, lthough P vlues reflect nlysis fter log trnsformtion, hormonl dt re presented s the men ± SEM, medin or 9% confidence intervls. 3. Results ll 2 subjects completed ll 4 rndomized infusions nd 14-h smpling sessions. norexi nd dyspepsi were dverse events. Men nd women hd comprble ge, body mss index (BMI), nd screening 8 h lbumin, cortisol, prolctin nd TSH concentrtions. Testosterone nd estrdiol were lower nd SHBG, LH nd FSH higher in women thn men, s expected: Tble 1. Screening (8 h) ws 21 ± 1.4 Feedbck Clmp: 8 h CTH : 1 h Plcebo/ KTCZ Plcebo/ KTCZ Plcebo/ KTCZ Infusion: Fig. 1 Schem of experimentl design. Ten helthy older men nd ten postmenopusl women ech received successive orl doses of either KTCZ (steroidogenesis inhibitor) or plcebo t regulr intervls during 4 seprte rndomly ordered inptient study sessions. To crete feedbck clmp, sline or cortisol s low dose (LDC) or high dose (HDC) ws infused continuously i.v. over 8 h (18 2 h). This prdigm yielded 4 intervention types: plcebo/sline (control), KTCZ/sline, KTCZ/LDC, nd KTCZ/ HDC. The infusion ws stopped bruptly t 2 h to permit CTH recovery. In ech session, 1-min blood smpling ws conducted over totl of 14 h, divided into n initil (4-h) feedbck clmp phse (lst 4 h of the 8-h sline or cortisol infusion, viz h), nd lter (1-h) CTH recovery phse (22 12 h). Time is represented in clock hours.

4 1822 METBOLISM CLINICL ND EXPERIMENTL 62 (213) Tble 1 Subject chrcteristics t screening. Subject chrcteristics Older men Older women P vlue Number (N) 1 1 ge (yers) 6 ± ± BMI (kg/m 2 ) 28 ±.7 26 ± 1.8 lbumin (g/l) 44 ±. 43 ±.7.29 (μg/dl) 12 ± ± Estrdiol (pmol/l) 84 ± ±. <.1 FSH (IU/L).4 ±. 74 ± 7. <.1 LH (IU/L) 2.9 ±. 26 ± 4.4 <.1 Prolctin (pmol/l) 287 ± ± SHBG (nmol/l) 37 ±.4 9 ± Testosterone (nmol/l) 18 ± 2.3. ±.1 <.1 TSH (miu/l) 1.8 ± ± ± ±.8.23 Free cortisol (μg/l) 1. ± ± CBG (μmol/l).44 ± ±.2.71 Dt re the men ± SEM, single mesures t 8 hr. To convert to SI units (nmol/l) multiply by in men nd 1.6 ±.8 in women (P =.23), wheres cortisol (μg/dl) ws 12. ± 1.1 in men nd 12. ± 1.2 in women (P =.97). To visulize the hormone time ptterns, Fig. 2 presents medin 14-h CTH nd cortisol concentrtion profiles for ll 4 tretment rms. vlidting hypothesis ws tht KTCZ/sline dministrtion should induce significnt hypocortisolemi, thereby unleshing CTH secretion. This ws confirmed by compring (men ± SEM) cortisol nd CTH concentrtions over the entire 14-h period in the KTCZ/sline nd plcebo/ sline groups; viz. cortisol: 6.7 ±.2 vs 7.9 ±.3 μg/dl (P =.7); nd CTH: 26.7 ± 3.2 vs 16. ± 1.9 ng/l (P =.1) [N = 2]. Men nd women lso did not differ significntly with respect to pek plsm CTH concentrtions over the 14 h: viz., plcebo/sline: 43 ± 6.1 (men) nd 3 ± 8.2 ng/l (women); or during KTCZ/ sline: 77 ± 18 (men) nd 62 ± 21 ng/dl (women). Men nd pek cortisol concentrtions likewise did not differ by gender over 14 h under the 2 vlidting clmps (see profiles in Fig. 2, right) Sline/cortisol clmp phse This period ws defined s the 8-h intervl from the strt of sline/cortisol infusion to its discontinution (18 h 2 h), with blood smpling over the lst 4 h (22 h 2 h). Liner regression nlysis (N = 2) disclosed tht men 4-h CTH concentrtions in the control session (plcebo/sline rm) significntly determined men 4-h CTH concentrtions during ech of the other 3 min tretment rms, viz. KTCZ/ sline [R 2 =.81; P <.1], KTCZ/low-dose cortisol (LDC) [R 2 =.84; P <.1] nd KTCZ/high-dose cortisol (HDC) [R 2 =.4; P <.1]). This vlidted the use of plcebo/sline CTH mens s covrites in NCOV. By NCOV nd post hoc Tukey s HSD test, sex did not determine men CTH concentrtions during ny of the 3 KTCZ sline/cortisol clmps (gender P =.98, gender*tretment P =.29) (Fig. 3). 8 Plcebo/Sline CTH 8 KTCZ/Sline 3 Plcebo/Sline CORTISOL 3 KTCZ/Sline Medin Sline Medin Sline Medin ( g/dl) Sline Medin ( g/dl) Sline 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: 8 KTCZ + Low-Dose 8 KTCZ + High-Dose 3 KTCZ + Low Dose 3 KTCZ + High Dose Medin Low-Dose Medin Plcebo/Sline High-Dose Medin ( g/dl) Low-Dose Medin ( g/dl) HIgh-Dose 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: 18: 21: : 3: 6: 9: 12: Fig. 2 Fourteen-h profiles of medin gender-ctegorized concentrtions of CTH (left pnel) nd cortisol (right pnel). nlyses were divided into n erlier 8-h feedbck clmp phse, with smpling over the lst 4 h of this intervl (Fig. 1) nd lter 1-h recovery phse. Ech pnel shows n individul interventionl group (plcebo/sline, KTCZ/sline, KTCZ/LDC, KTCZ/HDC). Dt from men (closed circles) nd women (open circles) re displyed seprtely. To convert CTH ng/l to pmol/l multiply by.222, nd cortisol μg/dl to nmol/l multiply by 27.6.

5 METBOLISM CLINICL ND EXPERIMENTL 62 (213) Men Men ( g/dl) Plcebo/Sline p=.42 p=.99 p=.97 p=.78 p=.32 Plcebo/Sline B B C KTCZ/Sline KTCZ/Low Dose Gender independence of CTH concentrtions ttined during clmped sline/cortisol feedbck ws confirmed by regression of men 4-h CTH on men 4-h cortisol concentrtions in ech of the 2 subjects to estimte feedbck slopes. Sttisticl comprison of slopes reveled no significnt difference in CTH-cortisol regression slopes between men nd women (.7 ±.19 [N = 1] vs.26 ±.4 [N = 1], P =.4; Mnn Whitney U test). This ws confirmed by unpired t-test of log-trnsformed bsolute slopes Post-infusion CTH recovery KTCZ/High Dose B C D p=1. KTCZ/Sline CTH Tretment rm p<.1 Tretment rm p<.1 p=.77 KTCZ/Low Dose Tretment rm p=.13 KTCZ/High Dose Fig. 3 bsence of gender effect on men CTH (top) nd cortisol (bottom) concentrtions in older men nd women during the lst 4 h of the 8-h KTCZ-sline/cortisol feedbck clmp in the 4 interventions. Overll infusion effects were significnt by NCOV t P <.1 for both CTH nd cortisol. Pired br grphs surmounted by brce brckets with unique (unshred) boldfce cpitl letters re significntly different independently of gender. Individul P vlues by post hoc testing under ech brcket show the lck of gender effects within intervention. Dt re men ± SEM (N = 1 men, N = 1 women). Post hoc testing ws by Tukey s HSD multiple-comprison test. ws defined s the 1-h intervl between discontinuing the sline/cortisol infusion nd cesstion of blood smpling (2 h 12 h). During 1-h recovery, men CTH concentrtions on the control dy (plcebo/ sline rm) predicted those on ech of the other 3 tretment dys, viz. KTCZ/sline (R 2 =.63; P <.1), KTCZ/LDC (R 2 =.63; P <.1) nd KTCZ/HDC (R 2 =.7; P <.1), thus justifying the use of NCOV (Methods). Two-wy NCOV showed (1) min tretment/infusion effect (P <.1), in which the men CTH concentrtion (ng/l) during recovery in the two genders (N = 2) ws significntly lower in the KTCZ/HDC rm (16 ± 2.2) thn in both the KTCZ/sline (31 ± 3.7) nd the KTCZ/LDC (29 ± 3.2) rms, but no different from tht in the control (19 ± 2.1) rm; nd (2) strong sex effect (P <.1), in which women s group hd lower 1-h men CTH concentrtion responses cross the 3 KTCZ interventions thn men (19.2 ± 1.8 ng/l vs 32.3 ± 3. ng/l, P <.1). Post hoc infusion-specific sex comprisons by Tukey s HSD test disclosed significntly lower 1-h men CTH recovery in women thn men in the KTCZ/HDC rm only (9. ±.87 ng/l vs 23 ± 3. ng/l, P =.4 by Tukey s HSD test). There ws similr trend-level effect in KTCZ/LDC: men CTH in women 18 ± 2. nd in men 37 ± 6.4 ng/l (P =.62). Visul inspection of the 1-h CTH recovery plots suggested possibly biphsic sex-dependent pttern, chrcterized by rpid post-infusion rebound CTH secretion nd delyed CTH suppression (Fig. 2). To test this hypothesis objectively, two-segment piecewise liner-regression nlysis ws pplied to 1-h CTH (nd cortisol) concentrtions in ech of the 4 tretment rms (ppendix Figure 1). The brekpoint so defined identifies the time of significnt chnge in slope of CTH recovery on time from positive (initilly rising CTH) to negtive (delyed suppression of CTH). Men CTH concentrtions in the combined genders (N = 2 subjects) were significntly higher before thn fter the brekpoint only in the KTCZ/LDC rm (3 ± 4.2 ng/l vs 23 ± 3.2 ng/l; P <.1), nd KTCZ/HDC rm (21 ± 3.4 ng/l vs 1 ± 1.7 ng/l; P <.1, by 2-wy NCOV), thus verifying sequentil escpe nd suppression of CTH (Fig. 4). Postbrekpoint men cortisol concentrtions were higher thn pre-brekpoint vlues in the KTCZ/LDC rm (8.7 ±.3 μg/dl vs 7.1 ±.3 μg/dl; P =.3), nd conversely in the KTCZ/HDC rm (.6 ±.3 μg/dl vs 1.1 ±. μg/dl; P <.1): ppendix Tble 1 (left columns). Three-wy NCOV corroborted tht the brekpoint in time serves s boundry for rising nd flling CTH concentrtions during 1-h recovery (P <.1 min effect). Sex-specific comprisons showed tht gender determines pre- nd post-brekpoint CTH levels (P <.1); nd infusion type does the sme (P <.1). The precise sex effects re discussed in the next two prgrphs (pre- nd post-brekpoint) Pre-brekpoint CTH nd cortisol by gender By 2-wy NCOV nd Tukey s HSD test, the men CTH concentrtion during the pre-brekpoint (initil) recovery phse in post-menopusl women ws significntly lower thn tht in older men (overll P <.1) in both the KTCZ/ LDC rm ( ± 1.3 women vs 3 ±.8 men; P =.), nd the KTCZ/HDC rm (.9 ±. ng/l women vs 14 ± 2.9 ng/l men; P =.6). This is shown in Fig. 4. In contrst, gender did not determine time of the brekpoint (P =.7 KTCZ/LDC, P =.8 KTCZ/HDC). Further NCOV demonstrted lower pek

6 1824 METBOLISM CLINICL ND EXPERIMENTL 62 (213) Plcebo/Sline 6 KTCZ/Sline b b b 1 1 <Brekpoint >Brekpoint <Brekpoint >Brekpoint 6 KTCZ + Low-dose cortisol B 6 KTCZ + High-dose cortisol b c c B 1 1 b <Brekpoint >Brekpoint <Brekpoint >Brekpoint Fig. 4 Effects of gender on 1-h men CTH concentrtions during the post-infusion recovery phse segmented into before nd fter the objective brekpoint in ech of the 4 interventions. Different (unshred) upper-cse letters within KTCZ/LDC s well s within KTCZ/HDC signify significntly different men (± SEM) CTH concentrtions mesured pre- vs post-brekpoints. Different lower-cse letters in the KTCZ/HDC rm denote significnt gender effects on men CTH concentrtions both before nd fter the brekpoint. nlysis is bsed upon NCOV nd Tukey s post hoc HSD testing. (single mximl) CTH concentrtions before the brekpoint in women thn men in the KTCZ/HDC rm (22 ± 1.4 women vs 41 ± 6 ng/l men; P =.2). In contrst, there were no gender differences in men lbumin or CBG nd totl or free cortisol concentrtions in ny of the 4 tretment rms before the brekpoint: Tble 2. Tble 2 Mesured nd clculted cortisol levels. Condition Men Women Mesured Clculted Mesured Clculted Totl cortisol (μg/dl) Free cortisol (μg/dl) Totl cortisol (μg/dl) Free cortisol (μg/dl) 8 h bseline 12. ± ± ± ±.27 Plcebo/sline 4.3 ± ± ± ±.11 KTCZ/sline 3.3 ±.2. ± ± ±.1 KTCZ/LDC-clmp.2 ± ± ± ±.16 KTCZ/LDC-recovery 9.3 ± ± ±.39.4 ±.21 KTCZ/HDC-clmp 14 ± ± ± ±.33 KTCZ/HDC-recovery 6.4 ±.4.36 ± ± ±.13 Dt re men ± SEM. Boldfce denotes P <. lower in women thn men; ll other P vlues were >.1 for sex difference. CBG medin (rnge) men:.439 (.369.1). CBG medin (rnge) women:.496 (.371.6) μmol/l. ssumed K for cortisol-cbg ws L/mol, nd for lbumin L/mol [29].

7 METBOLISM CLINICL ND EXPERIMENTL 62 (213) Post-brekpoint CTH nd cortisol by gender Two-wy NCOV reveled tht post-brekpoint men CTH concentrtions were significntly lower in women thn men (overll P <.1) in both the KTCZ/LDC rm (2 ± 2.4 ng/l vs 4 ± 7 ng/l; P =.24), nd the KTCZ/HDC rm (12 ±.9 ng/l vs 31 ±.2; P <.1) (Fig. 4). The post-brekpoint men totl nd free cortisol concentrtions were both lower in women thn men in the KTCZ/HDC rm: totl cortisol 4.2 ±.32 (women) vs 6.4 ±.4 μg/dl (men (P =.23); nd free cortisol.4 ±.2 (women) nd.62 ±.3 (men) μg/dl (P <.). levels lso were lower in women thn men under KTCZ/LDC (P <.) (Tble 2). Thus, lower CTH escpe in women ws not due to reltive hypercortisolemi in women. 3.. Four-prmeter sigmoid model: gender effect The gender differences in initil CTH recovery were verified independently of the brekpoint concept by mens of simplified 4-prmeter sigmoidl model of time-vrying CTH nd cortisol concentrtions. The nonliner model ws superior to liner fit t P <.1 by F-rtio testing. This model identified objective bseline (minimum) nd plteu (mximum) vlues for hormone-concentrtion recovery in the low nd high-dose cortisol tretment groups (Fig. ) (color). Sttisticl 9% C.I. for the fits re shown by interrupted lines. No CTH recovery ws detected in the plcebo/sline or KTCZ/sline interventions, s expected. Bseline CTH concentrtions exhibited no gender differences. Plteu CTH recovery ws lower in post-menopusl women thn older men in both the KTCZ/LDC nd KTCZ/HDC rms (P <.): ppendix Tble 1 (right columns). Plteu cortisol concentrtions were lso lower in women thn men in the KTCZ/LDC rm (8.2 μg/dl, 9% CI = μg/dl in women vs 9.3 μg/dl, 9% CI = μg/dl in men) nd the KTCZ/HDC rm (4.2 μg/dl, 9% CI = μg/dl in women vs 6.4 μg/dl, 9% CI = μg/dl in men). 4. Discussion Investigtions of gender differences in CTH regultion in helthy humns hve been conflicting, possibly due to different experimentl prdigms, selection criteri, ges, smpling schedules, stress contexts, comorbidities, nd glucocorticoid feedbck signls used (e.g. cortisol vs dexmethsone) [2,3,31 33]. Moreover, no humn studies hve specificlly ddressed how gender impcts the recovery of CTH secretion fter experimentlly controlled stress. In contrst, in the rt, estrdiol exerts repertoire of net stimultory effects on the HP xis, such s muting glucocorticoid negtive feedbck [34], incresing CRH gene expression 6 KTCZ + Low-Dose 6 KTCZ + High-Dose Medin CTH Medin CTH KTCZ + Low-Dose 2 KTCZ + High-Dose CORTISOL CORTISOL % Conf Bnd-Lower 9% Conf Bnd-Upper 9% Conf Bnd-Lower 9% Conf Bnd-Upper % Conf Bnd-Lower 9% Conf Bnd-Upper 9% Conf Bnd-Lower 9% Conf Bnd-Upper Fig. Time course of medin CTH (top) nd cortisol (bottom) concentrtions during recovery phse in the KTCZ/LDC rm (left) nd KTCZ/HDC rm (right) in older men (blue) nd women (red) evluted vi 4-prmeter sigmoidl-curve model. The nlysis yields estimtes of the minimum (floor) nd mximum (plteu) concentrtion long with 9% confidence bnds (interrupted lines). Time is shown in min. Zero time (x-xis) denotes 2 h.

8 1826 METBOLISM CLINICL ND EXPERIMENTL 62 (213) in the prventriculr nucleus [4], nd potentiting drenl responsiveness to CTH [3]. Testosterone hs inhibitory effects on the rt CRH/VP-CTH-drenl xis. However, vilble dt in humns re discrepnt [11,,21,36], nd possibly confounded by stressor specificity nd ge dependence of sex differences [,37,38]. In this regrd, Otte et l. in metnlysis reported n lmost 3-fold greter ge-relted increse in certin CTH/cortisol responses in women thn men []. Bsed upon these issues, we evluted CTHfeedbck control in the context of withdrwl nd ddbck of the nturlly occurring glucocorticoid, cortisol, in helthy comprbly ged older individuls. This required severl step prdigm comprising (i) reversible blockde of drenl steroidogenesis t the level of CYP11 (cholesterol side-chin clevge enzyme) using KTCZ in the bsence of metyrpone vilbility [21,22]; (ii) i.v. infusion of sline or either of two doses of cortisol (clmp phse); nd (iii) brupt cesstion of the infusions to llow CTH escpe (recovery phse). The clmp phse yielded cortisol concentrtion-dependent inhibition of CTH secretion, thus vlidting the experimentl design. The degree of overnight CTH inhibition during the cortisol clmps ws comprble in men nd women. Specificlly, there were gender-comprble men nd pek CTH concentrtions during the cortisol clmps (P =.98), nd gender-similr clmped cortisol-concentrtion feedbck slopes onto CTH levels (P =.4). This min outcome during controlled cortisol clmps my help clrify pst mbiguity on the role of gender in glucocorticoid feedbck inhibition, wherein studies reported ugmented [39], equl [4,41], nd ttenuted [42,43] feedbck sensitivity in women compred with men. Published inconsistencies my in prt reflect heterogeneity of ge, sex hormone levels, confounding by body composition nd/or type of feedbck effector, viz., nturl cortisol or synthetic glucocorticoids. Indeed, glucocorticoids differ in plsm hlf-lives, potency, brin permetion, CNS export nd CBG binding [3,4,21]. brupt cesstion of i.v. cortisol infusions permitted estimtion of CTH recovery fter feedbck suppression. puttively reflects the degree of mplifiction or suppression of norml diurnl feedforwrd onto CTH s the experimentl feedbck effect is withdrwn. This spect of CTH dynmics hs been only poorly studied. To cpture quntittive CTH dynmics, the effect of gender on CTH recovery ws nlyzed in three wys. First, erly (pre-brekpoint) nd delyed (post-brekpoint) recoveries of men CTH concentrtions were compred by gender. Women mnifested mrkedly impired erly recovery (pre-brekpoint) compred with men fter both low nd high-dose cortisol infusion, despite sex-comprble totl nd free cortisol levels during erly recovery. This could be due to heightened feedbck inhibition in women, insmuch s CTH recovery ws not only lower but lso delyed fter high-dose cortisol infusion. Second, pek (mximl) CTH concentrtions chieved during recovery were lso lower in women thn men, despite comprble totl nd free cortisol levels by sex. Third, delyed (post-brekpoint) CTH recovery ws decresed in women compred with men in reltion to men CTH nd ccording to liner, regression nd nonliner sigmoidl (4-prmeter) model. Notbly, impired lte (postbrekpoint) CTH recovery in women ws ssocited with simultneously lower totl nd free cortisol in femles thn mles. Thus, by multiple criteri, CTH escpe from cortisol suppression is ttenuted in older women compred with older men. This sex difference might be due to impired hypothlmic CRH/VP drive in women nd/or heightened direct corticotrope inhibition by cortisol. Implictions of this sex difference could include ttenuted metbolic nd stress responses in women compred with men [32,44]. In nimls, sex differences in the mechnistic regultion of the HP xis re substntilly explicble by mbient sexsteroid concentrtions. In prticulr niml models show n ctivtionl role for estrogens nd n inhibitory role for ndrogens [1,17,33]. Whether this is true in humns is less cler. lthough no significnt effects of T or E 2 could be detected by explortory liner regression in the present cohorts, the study ws not powered for this possibility. Becuse CTH ws reltively suppressed in women compred with men, the present dt suggest the possibility tht higher E 2 /T rtio in women impirs CTH recovery from cortisol suppression. Cvets include the reltively limited size of the study smple (2 dults in totl) nd the nrrow ge rnge (6 ± 2 y). Thus, these outcomes require confirmtion in lrger cohorts nd over wider ge rnge. However, power nlysis for the negtive inference of no gender effect on stedy-stte clmped cortisol feedbck onto CTH ws 8% (bet =.) for two-sided lph (P.) t n effect size of one stndrd devition by unpired two-smple comprisons. dditionlly, the conclusions of this study pply to helthy individuls [4]. Possible confounding issues could include ) incomplete inhibition of drenl steroidogenesis by KTCZ; however, CTH secretion ws twofold higher in the KTCZ/sline compred with plcebo/sline context; b) possible dependence of outcomes on time of dy (here, overnight nd morning), therefore ultimtely requiring dytime nd evening comprisons; c) n unknown role for body composition (not ssessed here) in influencing the effects of free cortisol in negtive feedbck; however, there re no direct experimentl dt in humns showing concentrtion-dependent effects of cortisol-binding globulin (CBG) on negtive feedbck, or n interction between body composition nd free cortisol feedbck per se. lthough free cortisol seems to be higher in women thn men in young dults, CBG levels do not differ by gender in older subjects (present dt nd [46]); d) 3 weeks of KTCZ dministrtion cn increse CBG concentrtions [47], but whether this occurs fter single-dy KTCZ dministrtion or differs by gender is not known; nd e) future studies should ultimtely ttempt to mimic pulstility of cortisol feedbck. In conclusion, ccording to the present preclinicl model, wheres gender does not mesurbly modulte CTH suppression under constnt intrvenous cortisol feedbck in older individuls, sex strongly determines the mgnitude of overnight CTH recovery fter removl of inhibition by stopping constnt cortisol infusion. These mechnisms my hve relevnce to understnding gender differences in stress rectivity in this ge group. The roles of sex steroids nd body composition in this sex differences will require further study, possibly under conditions illustrted here by cortisol concentrtion-dependent feedbck clmps.

9 METBOLISM CLINICL ND EXPERIMENTL 62 (213) uthor contributions ppendix Dr. nimesh Shrm: performed experiments, nlyzed dt, nd interpreted results of experiments, drfted mnuscript, pproved finl version of mnuscript. Dr. Pul oun: Performed experiments nd pproved finl version of mnuscript. Sue West: Performed experiments nd pproved finl version of mnuscript. Jen Wighm: Performed experiments nd pproved finl version of mnuscript. Dr. Johnnes Veldhuis: conception nd design of reserch, nlyzed dt, interpreted results of experiments, prepred figures, drfted mnuscript, edited nd revised mnuscript nd pproved finl version of mnuscript. cknowledgments We thnk Jill Smith for support of mnuscript preprtion; Sndr Cbrl for dt nlysis nd grphics; the Myo Immunochemicl Lbortory for ssy ssistnce; nd the Myo reserch nursing stff for implementing the protocol. Supported in prt vi DK73148 nd DK46 (Metbolic Studies Core of the Minnesot Obesity Center) from the Ntionl Institutes of Helth (Bethesd, MD). The project described ws supported by Grnt Number UL1 TR13 from the Ntionl Center for dvncing Trnsltionl Sciences (NCTS). Contents re solely the responsibility of the uthors nd do not necessrily represent the officil views of the Ntionl Institutes of Helth. ppendix Tble 1 Time-dependent CTH recovery by gender. Liner phse NCOV Men (± SEM) Nonliner Curve-fit men (9% CI) Men Women Men CTH concentrtion (ng/l). Men CTH concentrtion (ng/l) Men Women Pre-brekpoint Sigmoid floor KTCZ/LDC 3 ±.8 ± 1.3 KTCZ/ LDC KTCZ/HDC 14 ± ±.9 KTCZ/ HDC Post-brekpoint KTCZ/LDC 4 ± 7. 2 ± 2.4 KTCZ/ LDC KTCZ/HDC 31 ±.2 12 ±.9 KTCZ/ HDC B. Mximum CTH concentrtion (ng/l) KTCZ/LDC 71 ± ± 7.6 KTCZ/HDC 41 ± 6. 2 ± (6.7-) 4.8 (2.7-.9) Sigmoid plteu 39 (37 41) 27 (26 28) 8.9 (7 11) 4.4 (4.-.) 24 (23 2) 16 ( 17) Comprison of CTH (1-h) recovery in men nd women using NCOV of men line-segment vlues (left) nd 9% confidence intervls of nonliner curve fitting (4-prmeter sigmoid curve, right). NCOV evluted gender effects on men nd mximl (pek) CTH concentrtions during recovery both pre- nd postbrekpoint. Nonliner modeling ssessed recovery floor (minimum) nd plteu (mximum). Significnt gender differences re denoted by * (P <.). p <. for the gender contrst. Conflict of interest The uthors hve no conflicts to declre.

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