Metabolism of Adrenal Cholesterol in Man

Transcription

1 Metbolism of Adrenl Cholesterol in Mn II. IN VITRO STUDIES INCLUDING A COMPARISON OF ADRENAL CHOLESTEROL SYNTHESIS WITH THE SYNTHESIS OF THE GLUCOCORTICOSTEROID HORMONES ABRAHAM BoRuowsmI, CLAUDE DELOROIX, nd SAM LEViN From the Service of Medicine nd Clinicl Investigtion, Institut Jules Bordet, Centre Anti-Cnce'reux de l'universite' Libre de Bruxelles, nd Centrl Lbortory of Nucler Medicine, Brussels, Belgium A B S T R A C T The synthesis of drenl cholesterol, its esterifiction nd the synthesis of the glucocorticosteroid hormones were studied in vitro on humn drenl tissue. It ws found tht the synthesis of drenl cholesterol my normlly be smll in the zon "fscicult," prticulrly when compred with the synthesis of the glucocorticosteroid hormones, tht it is severl times higher in the zon "reticulris" where esterified cholesterol is less bundnt, nd tht under ACTH stimultion it increses strikingly nd proportionlly to the degree of esterified drenl cholesterol depletion. On the other hnd, the reltive rte of esterifiction s well s the concentrtion of free drenl cholesterol re remrkbly stble: they do not differ ccording to the drenl zontion nd re unffected by ACTH. Furthermore, from qulittive point of view, the reltive proportions of A1 nd A2 cholesteryl esters formed in situ re similr to those nticipted from their reltive concentrtions, suggesting tht the chrcteristic ftty cid distribution of the drenl cholesteryl esters results from n in situ esterifiction rther thn from selective uptke of the plsm cholesteryl esters. Besides, the in vitro esterifiction revels propensity to the formtion of the most unsturted cholesteryl esters. Regrding hydrocortisone nd corticosterone, their synthesis tends to be more elevted in the zon "fscicult." Despite its higher cholesterol concentrtion the zon "fscicult" should not therefore be viewed s Received for publiction 9 November 1971 nd in revised form 15 Februry quiescent functionl complement to the zon "reticulris" nd the corticl distribution of glucocorticosteroid hormone synthesis is quite distinct from tht of drenl cholesterol synthesis. INTRODUCTION The concentrtion of drenl cholesterol is quite vrible from species to species (1); the synthesis of drenl cholesterol might vry ccordingly. In vitro studies in the squirrel monkey (2) indicte tht in the primte the rte of cholesterol synthesis might normlly be much lower in the drenl cortex thn in the liver but n immedite conversion of newly synthesized drenl cholesterol into steroid hormones ws not excluded. As fr s mn is concerned, the in vivo kinetic study of plsm nd drenl cholesterol equilibrtion under control conditions lso suggests tht drenl cholesterol comes minly from plsm nd tht the synthesis of cholesterol by the drenl cortex is reltively smll; it rises furthermore the problem of possible differences between the zon fscicult nd the zon reticulris (3). On the other hnd, ccording to the sme kinetic study in mn, esterified drenl cholesterol is produced within the drenl cortex by the esterifiction of free drenl cholesterol nd does not result from direct nd selective ccumultion of the plsm cholesteryl esters. Apprently, it is this locl esterifiction, together with the hydrolysis of the lrge pool of esterified drenl cholesterol which precisely regultes the mounts of free di enl cholesterol vilble for the synthesis of the steroid hormones nd for the stbility of the cellulr membrnes. The Journl of Clinicl Investigtion Volume 51 July

2 Briefly stted, esterified drenl cholesterol normlly serves s buffer to free drenl cholesterol nd constitutes rpid source of supply ccording to the immedite functionl demnds wheres the inflow of plsm cholesterol serves s purveyor to both free nd esterified drenl cholesterol but functions in more continuous nd less flexible fshion; s for the locl synthesis it seems, under control conditions, to ply secondry role. The purpose of the work reported here is to test the vlidity of this scheme nd to bring some further light on the functionl significnce, mode of regultion nd zonl distribution of drenl cholesterol synthesis. It constitutes n in vitro study, in humn drenl glnds removed for the tretment of generlized mmmry crcinom, of cholesterol synthesis, esterifiction, intrcellulr equilibrtion, nd conversion into steroid hormones. METHODS A. In vitro synthesis of drenl cholesterol from cette-2- '4C. The drenl glnds obtined in the operting room were immeditely plced in solution of Krebs-Ringer-bicrbonte nd processed in the cold room s previously described (3, 4). The slices of drenl tissue were then incubted, during 3 hr, t 370C, in metbolic incubtor, under constnt gittion nd in n tmosphere of 95% oxygen nd 5%o crbon dioxide. Ech incubtion flsk contined mg of drenl cortex in 3 ml of Krebs-Ringer-bicrbonte solution t ph 7.4, 60 uci of cette-2-"c (SA 55 mci/mmole; Amershm, Englnd), nd gmole of sodium cette. The concentrtion of glucose in the medium ws 2 g/liter. The incubtion ws stopped on ice nd fter homogenistion in the medium, the tissues were extrcted with 25 vol of cetone-ethnol 1: 1 (5). The precipitted proteins were seprted by centrifugtion nd their nitrogen content ws mesured by stndrd Kjeldhl procedures (6) wheres the corresponding concentrtions of free nd totl cholesterol were mesured in precise frction of the cetone-ethnol extrct; in the rest of the extrct, free nd esterified drenl cholesterol were seprted from ech other by thin-lyer chromtogrphy (7), the vrious drenl cholesteryl esters were frctionted further on silic gel impregnted with 5% Ag NO3, nd the specific ctivities (SA) were determined on the digitonides fter purifiction by the method of Schwenk nd Werthessen (8). The dequcy of this purifiction ws confirmed by the demonstrtion of the constncy of drenl cholesterol digitonides specific ctivities fter thin-lyer chromtogrphy nd two successive dibromintions: these specific ctivities were not significntly modified by two supplementry crystllistions of cholesterol in methnol. The synthesis of cholesterol ws clculted from the number of dpm of lbeled cholesterol obtined per milligrm of protein nitrogen, i.e. from the product of cholesterol concentrtions with cholesterol specific ctivities. This synthesis ws expressed in pmoles of cette-2-14c incorportion per milligrm of protein nitrogen during the durtion of the incubtion. The bsolute vlue of cholesterol synthesis could in turn be clculted from the bsolute mount of methyl crbon incorported nd from the contribution of this crbon to cholesterol moleculr weight (9) A. Borkowski, C. Delcroix, nd S. Levin The drenl glnds were obtined from eight control ptients, from six ptients who hd been treted by ACTH (Cortrophine Z; Orgnon Inc., Hollnd) t dose of U i.m. per dy for the 2-3 dys preceeding surgery, nd from six ptients who hd been given 4 mg of dexmethsone per dy during the 2 dys preceeding surgery. B. In vitro synthesis of hydrocortisone, corticosterone, nd ldosterone. The drenl tissue ( mg per flsk) ws incubted s described in A, but the incubtion lsted 2 hr nd followed 1 hr preincubtion, nd no lbeled cette ws dded to the milieu. The secretion nd the tissue concentrtion of hydrocortisone, corticosterone, nd ldosterone were mesured by cetyltion with tritited cetic nhydride (Amershm-SA 100 /uci/,umole) ccording to the method of Klimn nd Peterson (10) s modified by Dvis, Burwell, Csper, nd Brtter (11) ; besides, fter the chromic cid oxidtion, sixth chromtogrphy ws crried out using the E1 or the E4 system of Eberlein nd Bongiovnni (12). The correction for losses ws obtined by the ddition to the medium, or to the tissue t the time of homogenistion, of trcer mounts of the corresponding 14C-lbeled steroids previously purified by pper chromtogrphy in Bush 5 system (13) (Amershm, Englnd; hydrocortisone-4-14c: SA 56.8 mci/mmole, corticosterone-4-14c: SA 56.7 mci/mmole, nd ldosterone- 4-14C: SA 56.7 mci/mmole). C. Incorportion of cette-2-'4c in newly synthesized hormones. The drenl slices were cut into smll pieces nd divided into two equivlent homogeneous frctions: () The first frction ws preincubted nd incubted s described in B for the mesurement of hydrocortisone nd corticosterone secretion per milligrm of protein nitrogen; the concentrtion of cette dded to the Krebs-Ringerbicrbonte solution ws of jimole/ml. (b) The second frction ws preincubted nd incubted identiclly except for the ddition of 60 gci of cette-2-14c to the incubtion medium. The synthesis of cholesterol-14c nd the incorportion of cette-2-'4c into hydrocortisone were mesured seprtely. Hydrocortisone secreted in the medium ws isolted by chromtogrphy in Bush 5 (13) system, ws cetylted therefter with cold cetic nhydride nd chromtogrphed gin in system of cyclohexnebenzene-methnol-wter ( ) ; trcer mounts of tritited hydrocortisone were dded to the medium for correction of losses resulting from extrction nd purifiction nd the trcer itself ws purified before use by pper chromtogrphy in Bush 5 system. The incorportion of cette-2-14c into hydrocortisone ws obtined from the secretion of hydrocortisone per milligrm of protein nitrogen in the first frction of drenl tissue nd from the number of '4C dpm secreted s hydrocortisone in the second frction of the sme drenl tissue. D. Ittrcellulr equilibrtion of newly synthesized cholesterol. After incubtion with cette-2-'4c the drenl tissue ws frctionted s previously described (4) in order to follow the intrcellulr equilibrtion of newly synthesized cholesterol. The isolted subcellulr frctions were further chrcterized by their content of DNA, RNA, nd cytochrome oxidse per milligrm of protein nitrogen. RNA, DNA, nd proteins were extrcted by the method of Schmidt nd Thnnhuser (14). DNA ws mesured s described by Ceriotti (15). The bsorption spectrum of the extrcted RNA ws verified in Cry 15 spectrophotometer nd the concentrtion determined therefter from the bsorption t 260 nm. In known frction of the subcellulr orgnelles, the cytochrome oxidse ws mesured by the

3 method of Smith (16), cytochrome C being first reduced on column of Duolite Sio s described by Chntrenne (17). RESULTS A. In vitro synthesis of drenl cholesterol from cette-2-"c Incorportion of cette-2-"c into cholesterol ccording to cette concentrtion. As shown by Fig. 1, for sme number of microcuries of lbeled cette per milligrm of incubtion medium (20.sCi/ml) nd in sme drenl tissue, the incorportion of cette-2-14c into free nd esterified drenl cholesterol mesured fter 3 hr incubtion remins firly constnt when the concentrtion of cette in the medium is mde to vry from /moles/ml to umole/ml; in other words, since the in vitro synthesis of cholesterol does not fll t lower cette concentrtions, it ppers to be rection of zero order, in the concentrtion rnge studied. Comprison of cholesterol synthesis in the zons "reticulris" nd "fscicult" control conditions. Our results, obtined from eight different drenl glnds (Tble I), indicte tht everything else being equl the synthesis of cholesterol is severl times more importnt in the zon reticulris thn in the zon fscicult (P < 0.001). This synthesis is lso quite vrible from glnd to glnd, prticulrly in the so-clled zon "reticulris," perhps becuse of vrible degrees of contmintion of one zone by the other. The zonl distribution of drenl cholesterol synthesis is the opposite of tht of drenl cholesterol concentrtion nd more precisely of tht of esterified drenl cholesterol concentrtion which is significntly higher (P < 0.01) in the zon fscicult thn in the zon reticulris. A 600' Acette.2.14C incorportion into cholesterol (Ipmoles/mg N/3hr ) { A free drenl cholesterol Cl esterified drenl cholesterol I, 23 A, 4 UtS Acette concentrtion (pnmoles/ml) A FIGURE 1 Acette concentrtion nd cholesterol synthesis. The incorportion of cette-2-14c into free (A) nd esterified (El) drenl cholesterol remins firly constnt when the concentrtion of cette in the incubtion medium is mde to vry from to umoles/ml (Krebs-Ringerbicrbonte; 20,Ci cette-2->c/ml. TABLE I Norml Adrenl Glnds Acette-2-'4C incorportion into cholesterol* Cholesterol concentrtions Rtio of Esteri- esteri- Esteri- Study Zone: Free fied Totl fied/free Free fied Pmoles/mg nitrogen Per 3 hr mg/mg nitrogen 1 R F R F R F R F R F R , F R 3, , F R 7, , F , Mens R 1, , F * 3 hr incubtion in Krebs-Ringer-bicrbonte medium; sodium cette, jtmoles/ml; cette-2-14c, 20 A.Ci/ml. t R, reticulris; F, fscicult. Cholesterol synthesis in Tble I is expressed in terms of pmoles of cette-2-14c incorported into cholesterol per milligrm of protein nitrogen during 3 hr incubtion; when converted in terms of weight it ctully ppers to be very low with men vlue of 0.05 l'g of cholesterol per milligrm of protein nitrogen in the zon reticulris nd of sag in the zon fscicult. Interestingly enough, s lso shown in Tble I, the concentrtion of free drenl cholesterol nd its rte of esterifiction re quite similr in the two drenl zones. Influence of ACTH stimultion nd of dexmethsone suppression. Under the influence of ACTH stimultion the drenl cortex becomes delipidted nd the morphologicl differences between the drenl zones tend to dispper (18). Correspondingly (Tble II) we find tht the concentrtion of esterified drenl cholesterol flls significntly in our six ptients (P < 0.01) nd becomes similr in the inner nd outer cortex. The modifictions of totl cholesterol synthesis (Tble II) re symmetricl to those of esterified cholesterol concentrtion: the totl drenl cholesterol synthesis increses strikingly (P < in the zon "reticulris" nd P < in the zon "fscicult") nd in proportion to the degree of esterified drenl cholesterol depletion (Fig. 2); it lso becomes similr in the inner nd outer cortex. Study of Adrenl Cholesterol In Vitro 1681

4 TABLE II Adrenl Glnds under A CTH or Dexmethsone Acette-2-'4C incorportion into cholesterol* Cholesterol concentrtions Zone: Free Esterified Totl Rtio of esterified/free Free Esterified pmoles/mg nitrogen/3 hr mg/mg nitrogen Adrenl glnds under ACTH (n = 6) (n = 6) R Mens 10,714 2,204 12, Rnges (2,480-22,814) (344-3,770) (2,824-26,230) ( ) ( ) ( ) F Mens 8,153 1,312 9, Rnges (1,256-19,763) (188-2,238) (1,444-21,714) ( ) ( ) ( ) Adrenl glnds under dexmethsone (n = 6) (n = 8) R Mens undetectble Rnges ( ) ( ) F Mens undetectble Rnges ( ) ( ) * 3 hr incubtion in Krebs-Ringer-bicrbonte medium; sodium cette; 6.365,gmoles/ml; cette-2-14c, 20,Ci/ml. R, reticulris; F, fscicult. Despite this striking stimultion which suggests tht cette is good precursor of drenl cholesterol in our experimentl system, when expressed in bsolute terms, the in vitro cholesterol synthesis remins moderte with 0 Acette.2.14C incorportion into Cholesterol ( p moles/mg N /3 hr) o zon fscicult * zon reticulris mg N mg esterlf.cholesterol FIGURE 2 Cholesterol synthesis nd cholesterol concentrtion under ACTH. In delipidted drenl glnds the incorportion of cette-2-14c into cholesterol is proportionl to the degree of esterified cholesterol depletion. The correltion coefficient is men figure of Ag in the zon reticulris," of /Ag in the zon "fscicult," per milligrm of protein nitrogen nd during 3 hr incubtion; however, in the most delipidted drenl glnds, this in vitro cholesterol synthesis cn rech itg/mg N/3 hr, figure which is still low but prcticlly reches the rnge of the glucocorticosteroid hormone secretion obtined in vitro in the zon reticulris of control drenl glnds (see below-section B of Results). Finlly, it is remrkble tht under ACTH stimultion the concentrtion of free drenl cholesterol nd the rte of free drenl cholesterol esterifiction remin very constnt nd re prcticlly identicl to those found under control conditions (Tbles I nd II). Under dexmethsone (Tble II) the synthesis of drenl cholesterol becomes undetectble. With regrd to esterified drenl cholesterol concentrtion in the eight ptients investigted, it is slightly elevted in the zon "reticulris" (P < 0.05) but not significntly elevted in the zon "fscicult" when compred with the control drenl glnds. Qulittive spects of the esterifiction of cholesterol synthesized in vitro. In four control drenl glnds nd in three which hd been stimulted by ACTH the vrious cholesteryl esters were seprted from ech other fter incubtion with cette-2-14c in order to compre their specific ctivities. For the purpose of presenttion, these specific ctivities were normlized to tht of the sturted or Ao form: it cn be seen in Tble III tht they re firly similr to ech other except, s in vivo, for tendency to increse progressively with the unsturtion of the ftty 1682 A. Borkowski, C. Delcroix, nd S. Levin

5 cid moiety nd except for considerble increse in the most unsturted cholesteryl esters. It is remrkble in this regrd tht the specific ctivities of cholesterol in the Al nd A2 esters re prcticlly identicl to ech other, i.e., tht the reltive mounts of free drenl cholesterol esterified in situ with A1 nd A2 ftty cids re nerly equivlent to the reltive concentrtions of the corresponding drenl cholesteryl esters which ccumulte in vivo (19). It is lso remrkble (Fig. 3) tht the reltive proportions of the vrious drenl cholesteryl esters to be formed in situ do not vry with time during the incubtion: thus there is no evidence of ny interconversion. Finlly, it is noteworthy (Tble III) tht the reltive concentrtions of the vrious drenl cholesterol esters re not modified by ACTH despite vrible nd sometimes considerble degrees of drenl cholesterol depletion. Intrcellulr equilibrtion of cholesterol synthesized in vitro. As shown in Tble IV, except for the frction which is trpped in the lipid droplets nd which is less redily vilble for exchnge, free cholesterol equilibrtes rpidly within the drenl cell; the esterifiction must tke plce on the subcellulr orgnelles lthough most of the esterified drenl cholesterol is found in the superntnt nd in the lipid droplets. With regrd to the lter two subcellulr frctions it should be noted tht their distinction is prticulrly rbitrry since the superntnt nd the lipid droplets were thoroughly mixed with ech other during the homogenistion of the drenl tissue. Tble IV lso gives some indictions s to the purity of the other subcellulr frctions investigted: in terms of DNA, RNA, nd cytochrome oxidse TABLE III Frctiontion of the Adrenl Cholesteryl Esters fter Incubtion* Control drenl glnds Adrenl glnds under (n =4) ACTH (n = 3) Specific Concen- Specific Concenctivitiest trtions ctivities* trtions A4 Mens Rnges ( ) ( ) ( ) ( ) As Mens Rnges ( ) (8.6-17) ( ) ( ) A2 Mens Rnges ( ) (9-15) ( ) ( ) Al Mens Rnges ( ) ( ) ( ) ( ) Ao Mens Rnges ( ) ( ) Concentrtions of the esters, mg cholesterol/mg N Mens Rnges ( ) ( ) * Conditions of incubtion s in Tble I. t Normlized to Ao nd expressed in dpm/mg cholesterol. Number of double bonds of the ftty cid moiety. 500 cholesterol minutes FIGURE 3 Influence of time on cholesteryl esters specific ctivities. During 3 hr incubtion the reltive specific ctivities of the vrious drenl cholesteryl esters remin constnt (The whole corticl tissue of two drenl glnds ws combined for this nlysis). per milligrm of protein nitrogen these frctions re not pure but my be viewed predominntly s nuclei, microsomes, or mitochondri. B. In vitro synthesis of glucocorticosteroid nd minerlocorticosteroid hormones under control conditions As shown in Tble V the bsolute vlues of hydrocortisone nd corticosterone secretion re much higher thn those of the corresponding cholesterol synthesis lthough this comprison might not be perfectly vlid since the steroid hormones re produced from cholesterol which is lredy bundntly present within the drenl cell wheres cholesterol is synthesized de novo from cette: such comprison therefore does not tke into ccount the reltive durtion of the two syntheses nd the problem, of the efficiency of cette s precursor to cholesterol. In ny cse, the corticl distribution of hydrocortisone nd corticosterone secretion is certinly different from tht of cholesterol synthesis, since, everything else being equl, this secretion tends to be more importnt in the zon fscicult thn in the zon reticulris (P < 0.01). On the other hnd the in vitro secretion of the drenl steroid hormones undoubtedly reflects new synthesis since the concentrtions of these hormones in the drenl tissue re reltively low nd do not vry significntly during the incubtion. Indeed the men hydrocortisone, corticosterone, nd ldosterone concentrtions mesured before incubtion in the zon "reticulris" of the first four pirs of drenl glnds shown in Tble V mount respectively to: Study of Adrenl Cholesterol In Vitro 1683

6 TABLE IV Intrcellulr Equilibrtion of Choksterol Synthesized in Situ* Specific ctivities of drenl cholesterol Chrcteristics of the subcellulr frctionst Loo Dehi Lul Esteri- Cyto- Free fied Esteri- Esteri- Esteri- chrome choles- choles- Frction Free fled Free fied Free fied DNA RNA oxidse terol terol dpm/mg dpm/mg dpm/mg mg/ mg/ k/ mg/ mg! mgn mgn mgn mgn mgn nd nd % of % of totl totl Nuclei 51, ,259 2, ,464 29, Mitochondri 45, ,250 2, ,724 89, Microsomes 66,676 1,459 70,023 3, ,808 89, Superntnt 29,336-38,409 2, , Lipid droplets 3, , * 3 hr incubtion-sme conditions s Tble I. DNA, RNA, cytochrome oxidse: mens from three different drenl glnds where the concentrtions of unfrctionted DNA nd RNA were, respectively, nd 0.161/mg N; cholesterol: mesured in one drenl glnd where the concentrtions of unfrctionted free nd esterified cholesterol were, respectively, 0.19 mg/mg N nd 2.95 mg/mg N. Loo nd Deh: control studies; Lul: under ACTH. lk = I - log (Cyt++) nd 0.01 Ag/mg of protein nitrogen; in the zon "fscicult" we find correspondently: nd 0.04 olg/mg of protein nitrogen. After the incubtion in the sme tissues the bove vlues become: nd 0.00 ug in the zon "reticulris," nd 0.00 /Lg in the zon "fscicult." C. Direct comprison of glucocorticosteroid secretion with cholesterol synthesis under control conditions Four pirs of drenl glnds in which n pproprite seprtion of the drenl zones could not be obtined were subdivided into two identicl prts: the first of these two prts ws incubted with cold cette for the mesurement of the hormone secretion, the second prt ws incubted with lbeled cette for the mesurement of cholesterol synthesis nd of cette-2-14c incorportion into the steroid hormones. As confirmed in Tble VI, the bsolute synthesis of cholesterol is negligible when compred with the corresponding hormone secretion. Besides Tble VI indictes tht there is no importnt elective incorportion of cette-2-"c into the steroid hormones: in other words nd consequently, the low mgnitude of the in vitro cholesterol synthesis nd its chrcteristic zonl distribution re not ttributble to n elective incorportion of lbeled cette into the steroid hormones. DISCUSSION The extrpoltion of in vitro biosynthetic dt to the whole living orgnism is dngerous, prticulrly when cette is utilized s metbolic precursor since its ctivtion to cetyl CoA might vry from tissue to tissue. However, our in vitro observtions suggest very strongly tht drenl cholesterol synthesis must be low under norml conditions, much lower thn the needs for steroid hormone production, prticulrly in the zon fscicult, wheres under pproprite ACTH stimultion it cn become s efficient s the norml heptic cholesterol synthesis. Indeed, if one milligrm of protein nitrogen corresponds to mg of drenl tissue (reference 18 nd personl observtions), our dt indicte tht the men in vitro conversion of cette-2-14c to cholesterol within the zon fscicult is normlly of the order of 5,000-3,500 pmoles/g per 3 hr. These figures re prcticlly identicl to those found by Bhttthiry nd Siperstein (20) in norml heptic tissue obtined by liver biopsy in ptients submitted to highcholesterol diet, i.e., when the humn heptic cholesterol synthesis is lmost completely suppressed. On the other 1684 A. Borkowski, C. Deicroix, nd S. Levin

7 hnd, under ACTH stimultion we find tht the conversion of cette-2-uc to drenl cholesterol increses considerbly nd cn even exceed 300,000 pmoles/g per 3 hr, figure which is now prcticlly identicl to those obtined by Bhttthiry nd Siperstein (20) in the liver from ptients submitted to norml cholesterol diet: indeed, under these circumstnces the heptic incorportion of cette-l-c into cholesterol ws found to verge 282,000 pmoles/g per 2 hr. Furthermore, the norml rtes of cette incorportion into drenl nd heptic cholesterol of mn re both of the sme order of mgnitude s those obtined by Dietschy nd Wilson (2) in the drenl glnds nd in the liver of the squirrel monkey, respectively. Our experimentl observtions indicte tht however smll the usul rte of drenl cholesterol synthesis, it is severl times higher in the zon reticulris thn in the zon fscicult. They indicte lso tht this low rte of TABLE V In Vitro Secretion of the Adrenl Steroid Hormones* Study Zonet Aldosterone Corticosterone Hydrocortisone pg/mg protein N/2 hr Volv R F DeN R F DeV R F Nic R F Jon R F Sprt R F Rez R F Behn R F Sur R F ACTH ACTH Fu R F Moe R F Duch R p F * Mens R F * 2 hr incubtion fter 1 hr preincubtion in Krebs-Ringerbicrbonte medium. I R: "reticulris"; F: "fscicult". 3 U of ACTH per ml of incubtion medium (Cortrosyn: 24 first minocids of ACTH). TABLE VI Comprison of Cholesterol Synthesis with the Synthesis of the Glucocorticosteroid Hormones* Acette-2-14C Hormone incorportion Acette-2- Cho- synthesis into cholesterol 14C incor- lesportion terol Hydro- Corti- Esteri- into hydro- syn- corti- coster- Study Free fled Totl cortisone$ thesis sone one p moles/mg N/2 hr p moles/ ng/mg pg/mg N/2 hr mg NI N/2 hr 2 hr Leu Dm Jn Mens * 2 hr incubtion in Krebs-Ringer-bicrbonte medium; cette, pmole/ml; cette-2-4c, 20,gCi/ml; the incubtion followed 1 hr preincubtion without lbeled cette; no lbeled cette either for the mesurement of hormone synthesis. t Purified by pper chromtogrphy on Bush 5 (fter cetyltion plus n dditionl chromtogrphy the remining counts re insufficient for n ccurte mesurement). drenl cholesterol synthesis is not n rtifct due to preferentil nd immedite conversion of newly synthesized cholesterol into steroid hormones; neither does the zonl difference in drenl cholesterol synthesis result from n opposite zonl difference in this preferentil conversion: the ltter is too smll to interfere with the clcultion of the former. Wht then regultes drenl cholesterol synthesis, wht might be its functionl significnce nd why such corticl grdient? With regrd to the regultion nd to the corticl grdient our results indicte tht the rte of drenl cholesterol synthesis is not direct function of the needs for cholesterol s precursor to the glucocorticosteroid hormones. In fct, cholesterol synthesis is much more importnt in the zon reticulris lthough the ltter, everything else being equl, tends to produce less hydrocortisone nd less corticosterone thn the zon fscicult. On the other hnd, one is impressed by the reltionship which is repetedly found between drenl cholesterol synthesis nd drenl cholesterol concentrtion: under norml conditions the synthesis is higher in the zon reticulris which contins less cholesterol, nd under ACTH stimultion the synthesis increses in direct proportion to the degree of drenl cholesterol depletion. These observtions might indicte insted tht the synthesis of cholesterol in the drenl cortex is regulted s in the liver by its intrcellulr concentrtion through mechnism of feedbck inhibition (21, 22). Study of Adrenl Cholesterol In Vitro 1685

8 With regrd to functionl significnce the synthesis of free drenl cholesterol seems complementry to the hydrolysis of esterified drenl cholesterol when, under the influence of ACTH stimultion, the stores of the cholesteryl esters become progressively depleted. However, if the in vitro equivlence of cholesterol synthesis in the most delipidted drenl glnds with tht of norml heptic tissue is extrpolted to the living mn on whole orgn bsis (23) it cn be clculted tht the over-ll rte of cholesterol synthesis by the drenl cortex, under optiml conditions, should not exceed 20 or t most 30 mg/dy, which is still moderte when compred with the steroid hormone production under intense ACTH stimultion (24); our in vitro finding tht the highest syntheses of drenl cholesterol under intense ACTH stimultion hrdly rech the lowest control vlues for hydrocortisone secretion support the sme inference. While, s lredy mentioned, such extrpoltions hve definite limittions, they suggest tht even in delipidted glnds the pssive inflow of plsm cholesterol remins the most importnt source of drenl cholesterol; the dvntge of moderte locl cholesterol synthesis might then reside in greter flexibility nd sensibility of djustment to the instntneous demnds t time when the depleted stores of esterified drenl cholesterol cn no longer ply this role of buffer. Our dt lso provide some informtion with regrd to intrcellulr equilibrtion nd esterifiction of drenl cholesterol. Like free drenl cholesterol coming from plsm (4), free drenl cholesterol synthesized in situ is found to equilibrte rpidly within the drenl cell, except for the pool which is trpped in the lipid droplets nd seems less redily vilble for intrcellulr exchnge; in ddition, it is rpidly esterified nd, interestingly enough, the percentge of esterifiction is the sme in the two drenl zones nd remins constnt despite ACTH stimultion; the importnce of the esterifiction therefore ppers to be constnt function of free drenl cholesterol concentrtion nd seems independent of the levels of free cholesterol synthesis, esterified cholesterol concentrtion, nd steroid hormone production. This lck of regultion of cholesterol esterifiction by esterified cholesterol concentrtion suggests tht esterified cholesterol could ccumulte within the drenl cells when for some metbolic or mechnicl reson incresed mounts of free plsm cholesterol re mde vilble to the intrcellulr esterifying enzymes without correspondently incresed hydrolysis; such might be the cse under ACTH plus minogluthetimide nd explin the observtions of Dexter, Fishmn, Ney, nd Liddle (25); such might lso be the cse in drenl lipoid hyperplsi where congenitl defect in cholesterol side-chin clevge induces presumbly ACTH hypersecretion A. Borkowski, C. Deicroix, nd S. Levin From qulittive point of view nd with the exception of the most unsturted cholesteryl esters (A4) the pttern of the in vitro esterifiction is comprble to tht previously found in vivo (4). Indeed, the specific ctivities of cholesterol in the vrious esters re firly similr to ech other, indicting tht the distribution of the newly formed cholesteryl esters is similr to tht lredy present before incubtion; this is prticulrly striking for the A1 nd A2 cholesteryl esters (essentilly olete nd linolete) which re the most bundnt nd re found in reversed proportions in plsm (26) nd in the drenl cortex (19). Furthermore, despite their similrity, the specific ctivities of cholesterol in the vrious esters tend to increse progressively with the unsturtion of the ftty cid moiety: this ws lso found in vivo. Finlly, it is remrkble tht the reltive vlues of the cholesterol specific ctivities in the different esters do not vry with time. The correspondnce between the in vivo nd in vitro observtions suggests, s previously discussed tht the chrcteristic distribution of the drenl cholesteryl esters, results from n in situ esterifiction rther thn from selective nd direct uptke of the plsm cholesteryl esters, the differences of specific ctivity ccording to the unsturtion of the ftty cid moiety perhps being due to differences of ffinity ccording to the ftty cid involved in this esterifiction. On the other hnd, the constncy in time of the reltive specific ctivities rgues ginst the hypothesis of non selective uptke of the plsm cholesteryl esters followed by intr-drenl interconversions (27). The in vitro system revels striking propensity for the ccumultion of the A4-unsturted cholesteryl esters of which rchidonte is certinly the most representtive (19). We hve no explntion for the difference between the in vitro nd the in vivo observtions with regrd to the ltter point. Some experimentl evidence suggests tht the ftty cid moiety of the drenl cholesteryl esters formed in situ is provided by n intrcellulr pool of free ftty cids (28): it is then possible tht in vitro, becuse of the bsence of free ftty cid uptke from plsm, the composition of this intrcellulr pool is modified. In ny cse, such propensity to form the most unsturted cholesteryl esters must explin their reltive bundnce in the drenl cortex. Furthermore, the ccumultion of these esters might be mens of storing rchidonic cid which is precursor of the prostglndins (29); the ltter in turn might regulte the drenl vsculture nd blood perfusion; by their ACTH-like effects the prostglndins might lso exert more direct control on steroid hormone production (30). Finlly, with regrd to the functionl significnce of the chrcteristic ftty cid distribution of the drenl cholesteryl esters it is remrkble tht this distribution is

9 not modified by ACTH. In other words, under ACTH stimultion there is no preferentil utiliztion of ny of the mny drenl cholesteryl esters for steroid hormone synthesis. ACKNOWLEDGMENTS We re grteful to Prof. W. Smets nd Dr. W. Mttheiem for their collbortion in this work. The id of Mr. G. Vmecq in the sttisticl nlysis of our dt is lso grtefully cknowledged. We re indebted to Mrs. Vivine Keymolen nd Ver Verhs for expert technicl ssistnce. This work ws supported by Contrct Eurtom-ULB- Pis-BIAC nd the Fondtion Cncerologique de l Cisse Generle d'eprgne et de Retrite. REFERENCES 1. Cook, R. P Cholesterol-Chemistry, Biochemistry nd Pthology. Acdemic Press, Inc., New York Dietschy, J. M., nd J. D. Wilson Cholesterol synthesis in the squirrel monkey: reltive rtes of synthesis in vrious tissues nd mechnisms of control. J. Clin. Invest. 47: Borkowski, A., C. Delcroix, nd S. Levin Metbolism of drenl cholesterol in mn. I. In vivo studies. J. Clin. Invest. 51: Borkowski, A., S. Levin, C. Delcroix, nd J. Klstersky The equilibrtion of plsm nd drenl cholesterol in mn. J. Appl. Physiol. 28: Sperry, W. M., nd M. Webb A revision of the Schoenheimer-Sperry method for cholesterol determintion. J. Biol. Chem. 187: Archibld, R Nitrogen by the Kjeldhl method. In Stndrd Methods of Clinicl Chemistry. D. Seligson, editor. Acdemic Press, Inc., New York. 2: Christophe, A., nd F. Mtthijs New method for the determintion of the ftty cid pttern of serum lipid clsses. Clin. Chim. Act. 16: Schwenk, E., nd N. Werthessen Studies on the biosynthesis of cholesterol. III. Purifiction of '4C-cholesterol from perfusions of livers nd other orgns. Arch. Biochem. Biophys. 40: Cornforth, J. W., G. D. Hunter, nd G. Popjk Studies of cholesterol biosynthesis. II. Distribution of cette crbon in the ring structure. Biochem. J. 54: Klimn, B., nd R. Peterson Double isotope derivtive ssy of ldosterone in biologicl extrcts. J. Biol. Chem. 235: Dvis, W. W., L. R. Burwell, A. T. Csper, nd F. C. Brtter Site of ction of sodium depletion on ldosterone biosynthesis in the dog. J. Clin. Invest. 47: Eberlein, W. R., nd A. M. Bongiovnni New solvent systems for the resolution of corticosteroids by pper chromtogrphy. Arch. Biochem. 59: Bush, I. E Methods of pper chromtogrphy of steroids pplicble to the study of steroids in mmmlin blood nd tissue. Biochemn. J. 50: Schmidt, G., nd S. J. Thnnhuser A method for the determintion of desoxyribonucleic cid, ribonucleic cid, nd phosphoproteins in niml tissues. J. Biol. Chem. 161: Ceriotti, C A microchemicl determintion of desoxyribonucleic cid. J. Biol. Chem. 198: Smith, L Cytochrome,,, 2 nd s. Methods Enzymol. Vol. II. 17. Chntrenne, H Peroxydses induites pr l'oxygene chez l levure. Biochim. Biophys. Act. 18: Griffiths, K., J. K. Grnt, nd T. Symington A biochemicl investigtion of the functionl zontion of the drenl cortex in mn. J. Clin. Endocrinol. Metb. 23: Diley, R. C., L. Swell, H. Field, Jr., nd C. R. Tredwell Adrenl cholesterol ester ftty cid composition of different species. Proc. Soc. Exp. Biol. Med. 105: Bhttthiry, E. M., nd M. D. Siperstein Feedbck control of cholesterol synthesis in mn. J. Clin. Invest. 42: Tomkins, G. M., H. Shepprd, nd I. L. Chikoff Cholesterol synthesis by liver. III. Its regultion by ingested cholesterol. J. Biol. Chem. 201: Siperstein, M. D., nd V. M. Fgn Feedbck control of mevlonte synthesis by dietry cholesterol. J. Biol. Chem. 241: Chobnin, A. V., B. A. Burrows, nd W. Hollnder Body cholesterol metbolism in mn. II. Mesurement of the body cholesterol miscible pool nd turnover rte. J. Clin. Invest. 41: Liddle, G. W., D. Islnd, nd C. K. Medor Norml nd bnorml regultion of corticotropin secretion in mn. Recent Progr. Hormone Res. 18: Dexter, R. N., L. M. Fishmn, R. L. Ney, nd G. W. Liddle An effect of drenocorticotropic hormone on drenl cholesterol ccumultion. Endocrinology. 81: Goodmn, DeW. S., nd T. Shirtori Ftty cid composition of humn plsm lipoprotein frctions. J. Lipid Res. 5: Moore, J. H., nd D. H. Willims Studies on the cholesterol esters of the drenl glnds nd other tissues of the rbbit. Biochem. Biophys. Act. 125: Shyml, G., W. J. Lossow, nd I. L. Chikoff Esterifiction of cholesterol by rt drenl glnd homogentes nd subcellulr components. Biochem. Biophys. Act. 116: Bergstrom, S., nd B. Smuelsson Prostglndins. Annu. Rev. Biochem. 34: Flck, J. D., R. Jessup, nd P. W. Rmwell Prostglndin stimultion of rt corticosteroidogenesis. Science (Wshington). 163: 691. Study of Adrenl Cholesterol In Vitro 1687