Altered adrenal gland cholesterol metabolism in the apoe-deficient mouse

Transcription

1 Altered drenl glnd cholesterol metolism in the poe-deficient mouse Fynne E. Thorngte,* Penelope A. Strockine,* Sndr K. Erickson, nd Dvid L. Willims 1, * Deprtment of Phrmcologicl Sciences,* University Medicl Center, Stte University of New York t Stony Brook, Stony Brook, NY 11794; nd Deprtment of Medicine, University of Cliforni t Sn Frncisco nd Veterns Affirs Medicl Center, Sn Frncisco, CA Astrct Previous studies suggest the hypothesis tht poe produced y drenocorticl cells modultes cellulr cholesterol metolism to enhnce the storge of esterified cholesterol (EC) t the expense of cholesterol delivery to the steroidogenic pthwy. In the present study, prmeters of drenl cholesterol metolism nd corticosteroid production were exmined in wild type nd poe-deficient (poe ) mice. Adrenl glnd EC content nd the EC/free cholesterol (FC) rtio in mice stressed y drenocorticotropin (ACTH) tretment or sline injection were reduced in poe / compred to poe / mice. Reltive to poe / mice, poe deficiency lso resulted in incresed levels of plsm corticosterone in the sl stte, in response to cute or long-term ACTH tretment, nd fter swiminduced neuroendocrine-directed stress test. Mesurements of drenl glnd scvenger receptor clss B, type I (SR-BI), LDL receptor, nd LDL receptor relted protein (LRP) levels nd the ctivities of ACAT or HMG-CoA reductse showed no difference etween genotypes. nd poe / mice showed similr quntittive increses in LDL receptors, SR-BI, drenl weight gin, nd ACAT ctivities in response to ACTH, nd oth genotypes hd similr sl plsm ACTH concentrtions. These results suggest tht the effects of poe deficiency reflect events t the level of the drenl glnd nd re specific to chnges in cholesterol ccumultion nd corticosterone production. Further, these findings support the hypothesis tht poe cts to enhnce drenocorticl EC ccumultion nd diminish corticosterone production. Thorngte, F. E., P. A. Strockine, S. K. Erickson, nd D. L. Willims. Altered drenl glnd cholesterol metolism in the poe-deficient mouse. J. Lipid Res : Supplementry key words lipoprotein steroidogenesis corticosterone scvenger receptor clss B type I SR-BI LDL receptor Mnuscript received 23 My 2002 nd in revised form 5 August Pulished, JLR Ppers in Press, August 16, DOI /jlr.M JLR200 Apolipoprotein E (poe) is prominent component of plsm lipoproteins nd serves to medite endocytic uptke of remnnt lipoproteins y memers of the LDL receptor fmily (1). In contrst to other polipoproteins, poe is expressed in mny peripherl tissues including rin, dipose, skin, kidney, lung, nd steroidogenic orgns such s drenl glnd, ovry, nd testis (2 8). Studies with humns, nonhumn primtes, nd rts show tht the level of poe gene expression in the drenl glnd is similr to tht in liver nd my ccount for s much s 0.5 1% of totl tissue protein synthesis (2, 8 11). ApoE mrna nd protein re expressed in drenocorticl zon fscicult nd zon reticulris cells, the sites of glucocorticoid production nd cholesteryl ester storge in the drenl cortex (12, 13). Electron microscopic immunolocliztion studies show poe intrcellulrly within vesiculr structures of the secretory nd endocytic pthwys nd prominently on the plsm memrne oth in cell surfce microvillr chnnels nd in non-microvillr regions of the memrne (13). The high expression of poe in drenocorticl cells nd its pttern of regultion suggest tht loclly-derived poe my fcilitte the cquisition of lipoprotein cholesterol, lter cellulr esterified cholesterol (EC) storge, or modulte the vilility of cholesterol for steroidogenesis. For exmple, in rt drenl glnd, poe expression is regulted in direct proportion to EC stores nd inversely to the level of steroid production (11, 12). In studies with cultured murine Y1 drenocorticl cells, constitutive expression of humn poe leds to enhnced EC ccumultion nd suppression of steroid production (14, 15). In ddition, inducile expression of poe in Y1 cells enhnces oth the endocytic nd selective uptke of LDL EC (16). The studies noted ove support the hypothesis tht locl poe production modultes cellulr cholesterol metolism to enhnce the storge of lipoprotein-derived EC t the expense of cholesterol delivery to the steroidogenic Arevitions: ACTH, drenocorticotropin; EC, esterified cholesterol; FC, free cholesterol; LRP, LDL receptor relted protein; SR-BI, scvenger receptor clss B, type I. 1 To whom correspondence should e ddressed. e-mil: dve@phrm.sunys.edu Copyright 2002 y Lipid Reserch, Inc Journl of Lipid Reserch Volume 43, 2002 This rticle is ville online t

2 pthwy. In the present study, prmeters of drenl cholesterol metolism nd corticosteroid production hve een exmined in wild type nd poe-deficient mice. The dt suggest tht poe deficiency leds to n ltered disposition of drenl cholesterol with more directed towrd steroid production nd less towrd EC storge. These dt otined with n in vivo mouse model support the hypothesis tht locl poe synthesis modultes cellulr cholesterol metolism. EXPERIMENTAL PROCEDURES Animls ApoE-deficient (poe / ) mice on mixed C57BL/6J 129 ckground were kindly provided y N. Med (Deprtment of Pthology, University of North Crolin, Chpel Hill, NC) (17). The mice were red s heterozgotes to C57BL/6J mice for four genertions, nd then red to homozygosity. Control (poe / ) mice were otined in the sme fshion. In severl experiments, control C57BL/6J mice were otined from the Jckson Lortory (Br Hror, ME), in which cse the mice were cclimted in the niml colony for t lest 1 week prior to experiments. Animls were mintined on chow diet with 12 h drk/12 h light cycle, nd ll experiments were initited t 2 h into the light cycle. The experiments reported here were crried out over period of 3.5 yers using mles tht rnged from 2 5 months of ge. Adrenl function is suject to vrition due to stress in the colony (niml cre procedures) tht is difficult to control perfectly over long period of time. Thus, it is not lwys possile to mke quntittive comprisons etween experiments performed t different times. However, considerle effort ws mde to control for potentil vriles within ny experiment y using 1) ge mtched nimls, 2) y inititing experiments t 2 h into the light cycle, nd 3) y lternting the hndling (leeding, scrifice, injections) of poe / nd poe / mice within n experiment. All reported comprisons re etween poe / nd poe / mice in the sme experiment. True control vlues (i.e., unstressed) were otined y killing nimls within minute upon removl from the cge. Controls in experiments involving injections or non-terminl leeding were, y necessity, suject to the stress of the experimentl protocol. Experimentl tretments For swim stress tests, mice were led from the retrooritl plexus into EDTA coted tues, swum for 3 min t 5 C, rested for 17 min t room temperture, nesthetized with n intrmusculr injection of 50 l ketmine-xylzine (3:2, v/v), nd led from the hert (18). For cute drenocorticotropin (ACTH) responsiveness, mice received n intrperitonel injection of either 0.1 ml sline or 0.1 ml sline contining 200 mu ACTH (Acthr, Rorer Phrmceuticls, Fort Wshington, PA). Mice were nesthetized s ove nd killed fter 40 min. For long term ACTH tretment, mice received injections of 4 U ACTH or sline s ove t 0 nd 12 h. At 24 h, mice were injected with sline or 200 mu ACTH nd were killed fter 40 min. Plsm ws prepred for corticosterone ssy nd drenl glnds were removed, trimmed of dipose tissue, nd frozen for susequent cholesterol mesurement. Adrenl glnd cholesterol Adrenl glnds from individul mice were homogenized in 0.2 ml PBS in ground glss tissue grinder tht ws rinsed with nother 0.2 ml PBS. The pooled homogente nd rinse were extrcted with 1.5 ml CHCl 3 -MeOH (1:2, v/v) for 15 min t room temperture, centrifuged for 15 min t 1,000 g, nd the superntnt ws removed to new tue. The pellet ws dissolved in 0.4 N NOH nd ssyed for protein y the method of Lowry (19). To the superntnt were dded 0.5 ml H 2 O nd 0.5 ml CHCl 3, the smple ws mixed, centrifuged to seprte phses, nd the CHCl 3 phse ws removed nd dried down under N 2. Free cholesterol nd totl cholesterol were mesured with enzymtic colorimetric ssys (Wko Chemicl) dpted to 96 well plte formt. Cholesteryl ester ws determined y difference. Plsm corticosterone nd ACTH Blood ws collected into EDTA rinsed syringes or cpillry tues, centrifuged twice to remove cells, nd stored t 80 C until ssy. For corticosterone mesurement, plsm ws mixed with 10 vol of ethnol for 10 min t room temperture, centrifuged t 10,000 g for 10 min to remove protein, nd liquots were dried nd ssyed for corticosterone y rdioimmunossy (Endocrine Sciences, ntiserum B3-163). The distriuton of corticosterone etween the lipoprotein nd non-lipoprotein frctions of plsm ws determined fter ultrcentrifugtion of pooled plsm smples djusted to 1.21 gm/ml with KBr (20). The 1.21 gm/ml nd the 1.21 gm/ml frctions were djusted to equl KBr concentrtions, liquots were diluted with 10 vol of ethnol, centrifuged to remove KBr, dried, nd ssyed for corticosterone. Plsm ACTH ws determined y ELISA using commercil kit ccording to the mnufcturer s protocol (Sngui Biotech, Snt An, CA). Enzyme ssys nd Western lots Adrenl glnds from mice were pooled nd homogenized in ice cold 0.6 ml 0.1 M sucrose, 0.05 M KCl, 0.04 M KH 2 PO 4, 0.03 M EDTA, 10 g/ml protinin, ph 7.4. Aliquots of the homogente were frozen in liquid N 2, nd stored t 80 C for Western lot nlysis. The reminder of the homogente ws centrifuged t 4 C t 12,000 g for 15 min, nd the superntnt ws centrifuged t 105,000 g for 45 min. The microsome pellet ws rehomogenized in 0.6 ml of the ove uffer, centrifuged t 105,000 g for 45 min, nd the wshed microsoml pellet ws resuspended in 0.3 ml uffer, frozen in liquid N 2, nd stored t 80 C. Liver homogente nd microsomes were prepred in the sme mnner except with 1 gm of liver homogenized in 5 ml. Microsomes were ssyed for HMG-CoA reductse ctivity nd ACAT s previously descried (21). ACAT ws mesured using oth endogenous nd exogenous sustrte. For Western lot nlysis tissue homogentes (50 g protein) were nlyzed y SDS 8% polycrylmide gel electrophoresis followed y trnsfer of the proteins to nitrocellulose memrne. Blots were proed with ntiodies ginst the LDL receptor nd the LDL receptor relted protein (LRP) (21, 22) nd with ntiody to scvenger receptor clss B, type I (SR-BI) (23). After incution with horserdish peroxidse-conjugted secondry ntiody, immunorective nds were visulized y enhnced chemiluminescence (Pierce) nd quntified y densitometry. Adrenl morphology nd ultrstructure For light microscopy, mice were perfused through the left ventricl with PBS followed y 4% formldehyde in PBS. Frozen sections were stined with oil red O nd lightly counterstined with hemtoxylin s descried (18). For electron microscopy, mice were perfused t constnt pressure (110 mm Hg) through the left ventricl first with 0.5 ml 0.1 M sodium ccodylte, ph 7.4, followed y 25 ml freshly prepred 2% glutrldehyde, 1% prformldehyde in 0.1 M sodium ccodylte, ph 7.4. Tissue preprtion, stining, electron microscopy, nd mesurement of microvillr chnnels were crried out with sections from two mice Thorngte et l. Cholesterol metolism in the poe-deficient drenl glnd 1921

3 of ech genotype (18). Tissue morphology ws lso evluted y light microscopy using toluidine lue-stined epon sections (0.5 m) of drenl glnd nd liver tissue. RESULTS Plsm nd drenl cholesterol metolism Previous studies indicted tht drenl poe expression is positively correlted with drenl EC ccumultion, suggesting potentil role for poe in the mintennce or cquisition of drenl EC stores (11). Accordingly, plsm nd drenl glnd cholesterol concentrtions were determined in poe / nd poe / mice under sl, unstressed, conditions nd in response to either sline or ACTH tretments. The dt in Tle 1 show the expected ccumultion of plsm cholesterol in the poe / mice in the sl stte nd in ll experimentl groups. Acute ACTH tretment for 40 min hd no effect on plsm cholesterol in the poe / or poe / mice. Long term ACTH tretment over 24 h period, however, elevted oth plsm free cholesterol (FC) nd EC in poe / mice nd reduced the rtio of EC/FC compred to poe / mice in the sl stte or fter cute ACTH or sline tretment. A similr trend towrd elevted plsm FC (15 20% incresed) ws seen with long term ACTH tretment of poe / mice, ut this did not rech sttisticl significnce. mice sujected to long term sline or ACTH tretments lso showed significnt reduction in the rtio of EC/FC compred to poe / mice in the sl stte or fter cute ACTH or sline tretments. In ddition to incresing corticosteroid production, long term ACTH tretment stimultes n increse in drenl glnd wet weight. As shown in Tle 2, long term ACTH tretment incresed drenl weight similrly in oth poe / nd poe / mice. No differences in drenl weight etween poe / nd poe / mice were seen in ny experimentl group. In sl unstressed mice, we oserved no significnt differences in totl drenl cholesterol or in EC etween poe / nd poe / mice, ut poe / mice hd reduced drenl FC (74 2 vs g/mg protein, P 0.01). The dt in Fig. 1 nd Tle 3 compre the drenl cholesterol responses of poe / nd poe / mice to cute nd long term ACTH tretments. With cute ACTH tretment, poe / mice showed n increse in drenl totl cholesterol tht ws due to EC ccumultion (Fig. 1A, B). In comprison to poe / mice, the poe / mice showed two differences. First, the drenl contents of totl cholesterol nd EC were less in the poe / mice with oth cute sline nd cute ACTH tretments (Fig. 1A, B). In ddition, the FC content ws lower in the cute sline treted poe / mice (Tle 3). Second, wheres the poe / mice showed n increse in drenl totl cholesterol nd EC in response to cute ACTH, the poe / mice did not. With long term sline or ACTH tretment, the poe / mice hd significntly reduced levels of drenl totl cholesterol nd EC in comprison to poe / mice (Fig. 1C, D). In ddition, drenl FC content ws lower in the poe / mice fter long term ACTH tretment (Tle 3). In contrst to the effects of cute ACTH tretment, poe / mice treted with ACTH for 24 h showed no increse in totl cholesterol or EC, ut did show n increse in drenl FC content (Tle 3). mice showed significnt reduction in EC in response to long term ACTH tretment (Fig. 1C, D), s well s the increse in drenl FC content (Tle 3). As compred to the poe / mice, the EC/FC rtio (Tle 3) ws considerly lower in the poe / mice in ll tretment groups nd ws reduced with long term ACTH tretment in oth poe / nd poe / mice. This result suggests greter moiliztion of drenl EC stores with long term, s compred to cute, ACTH tretment. The moiliztion of EC ws greter in the poe / mice. Adrenl receptor levels nd enzyme ctivities A vriety of receptor levels were determined y Western lotting of homogentes of drenl glnds from poe / nd poe / mice. As shown in Tle 4, little or no difference ws seen in the levels of SR-BI, LDL receptor, or the LRP etween poe / nd poe / mice in the sl stte. Both genotypes showed 3.5-fold increse in SR-BI nd 1.5-fold increse in LDL receptor levels in response to ACTH tretment for 24 h. Mesurements mde with liver homogentes lso showed no difference in receptor levels etween poe / nd poe / mice. Mesurements of drenl microsoml HMG-CoA reductse ctivity showed little or no difference etween poe / nd poe / mice efore or fter ACTH tretment (Tle 4). Similrly, no differences were noted etween genotypes in reductse ctivity in liver microsomes. Adrenl ACAT ctivity with either endogenous or exogenous su- TABLE 1. Effect of ACTH tretment on plsm cholesterol concentrtion Tretment Totl Cholesterol Free Cholesterol Ester Cholesterol EC/FC Totl Cholesterol Free Cholesterol Ester Cholesterol EC/FC mg/dl mg/dl Bsl Acute sline Acute ACTH Long term sline Long term ACTH n 20 mice/group except for sl poe / with n 19 nd sl poe / with n 30. Differs from the sline control group t P Journl of Lipid Reserch Volume 43, 2002

4 TABLE 2. Adrenl weight response to ACTH Tretment mg/drenl pir Acute sline Acute ACTH Long term sline Long term ACTH n 20 mice/group. Differs from the sline control group t P strte ws similr etween poe / nd poe / mice nd showed n pproximtely 2-fold increse in oth genotypes in response to ACTH. No difference ws noted etween genotypes in ACAT ctivity in liver microsomes. Plsm corticosterone ccumultion Plsm corticosterone levels were elevted in poe / mice in comprison to poe / mice in the sl stte (Fig. 2A, white r) s well s in response to oth cute (Fig. 2A, solid rs) nd 24 h ACTH tretments (Fig. 2B, solid rs). A trivil explntion for this difference is tht greter frction of the plsm steroid prtitions into the much lrger pool of plsm lipoproteins in the poe / mice ut tht the levels of free corticosterone nd tht ound to inding gloulins is the sme. To test this point, plsm ws pooled from stressed mice of ech genotype, nd seprted into 1.21 gm/ml nd 1.21 gm/ml frctions to mesure lipoprotein-ssocited nd lipoprotein-unssocited corticosterone. The results showed tht 75% of the corticosterone ws in the 1.21 gm/ml frction nd 25% in the 1.21 gm/ml frction in oth genotypes (dt not shown). Additionlly, no significnt correltion ws oserved etween totl plsm cholesterol nd plsm corticosterone in either poe / or poe / mice. Thus, the elevted plsm corticosterone in poe / mice ppers to reflect physiologicl difference tht is not explined y the much lrger plsm lipid pool in these mice. To determine whether elevted sl plsm corticosterone in poe / mice is n effect t the drenl glnd or reflects incresed ACTH stimultion through the hypothmic-hypophysel xis, plsm ACTH concentrtions were mesured. No significnt difference etween genotypes ws found (poe /, pg/ml, n 14; poe /, pg/ml, n 24). Additionlly, plsm corticosterone ccumultion in response to n cute swim stress test ws determined s mesure of centrl nervous systemdirected drenl response. As shown in Fig. 3, s compred to the poe / mice, corticosterone vlues were elevted in the poe / mice efore nd fter the swim stress test. The fold increse in corticosterone upon stress ws pproximtely the sme in oth genotypes (poe / 3.3 vs. poe / 2.6). Adrenl morphology nd HDL locliztion in microvillr chnnels Adrenl morphology ws evluted t the light microscopic level y y oil red O/hemtoxylin stining of cryosections nd y toluidine lue stining of thick sections of epon-emedded tissue prepred for electron microscopy. No significnt differences were noted etween poe / nd poe / tissue (dt not shown). Unlike the sitution in the skin of poe / mice where mcrophge fom cells Fig. 1. Adrenl glnd cholesterol content in response to drenocorticotropin (ACTH). Mice (n 20) were injected with sline or ACTH for either n cute (A nd B) or long term (C nd D) tretment s descried in Experimentl Procedures. Open rs, totl cholesterol; solid rs, esterified cholesterol. P 0.02 versus respective poe / group. P 0.05 versus respective sline-treted group. Thorngte et l. Cholesterol metolism in the poe-deficient drenl glnd 1923

5 TABLE 3. Adrenl glnd free cholesterol content in response to ACTH Tretment FC FC/EC FC FC/EC drenl cholesterol g/mg protein drenl cholesterol g/mg protein Acute sline Acute ACTH Long term sline P 0.01 P 0.01 Long term ACTH c 1.7 n 20 mice/group. P 0.02 versus respective poe / vlue. c P vlues in the tle refer to comprison etween ACTH nd sline groups. nd inflmmtory cells ccumulte (24), the drenl glnd showed no signs of inflmmtion or mcrophge fom cells (dt not shown). Oil red O stining ppered somewht less dense in some drenl glnds of poe / mice, ut this difference ws not consistently oserved. Liver sections from poe / mice, ut not poe / mice, showed mny lipid lden Kupffer cells, grnulom-like ccumultions of histiocytes in suendothelil loctions, nd heptocytes contining incresed numers of lipid droplets; the ltter point ws noted previously (25). Adrenl ultrstructure t the electron microscopic level lso ppered to e norml in poe / mice. The width of drenl microvillr chnnels (poe /, nm, n 51; poe /, nm, n 68) nd ppernce of HDL prticles in the chnnels were the sme in oth genotypes. DISCUSSION This study shows significnt differences in drenl glnd cholesterol metolism in poe / mice. The most prominent effects were oserved in drenl cholesterol content in ACTH-treted mice or mice stressed y sline injection. In oth groups, drenl EC content nd the EC/FC rtio were lower in poe / mice, suggesting reduced cpcity to mintin drenl cholesterol stores during stress. In some groups drenl FC content ws lso reduced in poe / drenl glnds. These results complement dt from previous study tht showed incresed FC nd EC ccumultion in murine Y1 drenl cells tht overexpressed poe (14). Additionlly, the present results complement previous studies in the rt tht demonstrted direct correltion etween the levels of poe mrna nd drenl EC content under conditions tht produced 15- fold rnge of these prmeters (11). Tken together with these previous studies, the present findings in the poe / mouse support physiologicl role for poe in mintining the EC content of drenocorticl cells. Becuse these chnges were seen only in drenl glnds from stressed mice, poe my ct to mintin drenl cholesterol stores in the fce of incresed cholesterol utiliztion for steroid production. The sence of poe lso resulted in incresed levels of plsm corticosterone in the poe / mouse in the sl stte, in response to cute or long-term ACTH tretment, nd fter swim-induced neuroendocrine-directed stress. Incresed plsm corticosterone in the sl stte or in response to long-term ACTH tretment could result from incresed drenl production or reduced plsm cler- TABLE 4. Receptor nd enzyme ctivities in poe / nd poe / mice Adrenl Glnd Bsl ACTH Liver Prmeter n n n SR-BI LDL receptor LRP ND c ND HMG CoA reductse pmoles/min/mg protein ACAT pmoles/min/mg protein Endogenous sustrte ND ND Exogenous sustrte n numer of homogente or microsome preprtions exmined. Ech preprtion ws derived from drenl glnds or liver smples pooled from mice. Receptor levels reflect densitometric units expressed s percent of the sl poe / smple set t 100. c Not determined Journl of Lipid Reserch Volume 43, 2002

6 Fig. 2. Plsm corticosterone in response to ACTH. Mice were untreted (A, sl, left r; poe / n 18; poe / n 31), or injected with sline (middle r) or ACTH (solid rs) for either n cute (A, n 20) or long term (B, n 10) tretment s descried in Experimentl Procedures. P 0.01 versus respective poe / group. P 0.07 versus respective poe / group. nce. The rpid increse in plsm corticosterone following 3-min swim or n cute ACTH tretment, however, suggests tht elevted corticosterone levels in the poe / mouse result from greter drenl hormone production. The present results do not distinguish etween n indirect systemic effect nd direct drenl effect of the sence of poe on steroid production. However, direct effect of poe on drenocorticl cell steroid production is suggested y previous studies tht showed suppression of steroid production in murine Y1 drenl cells tht constitutively over-express poe (15). Additionlly, the finding tht poe mrna concentrtions nd reltive poe synthesis rtes re very high in drenocorticl cells of humns, non-humn primtes, nd rodents supports locl effect of poe (2, 4, 5, 8, 10). This interprettion of the present findings lso is supported y the inverse reltionship etween drenl poe mrna content nd serum corticosteroids oserved in the rt (11). The mechnism through which steroid production is incresed in the poe / drenl glnd my reflect the filure of poe to dmpen responsiveness through the protein kinse A pthwy nd suppress expression of the mrna for the cholesterol side chin clevge enzyme. ApoE exhiits these effects when over expressed in cultured murine drenl cells (26, 27). The present results showing elevted corticosterone levels in poe / mice re in greement with recent study (28), lthough few differences were noted. Rer et l. (28) sw elevted corticosterone levels in 6-month-old poe / mice ut not in 3-month-old mice, wheres in the present study elevted corticosterone levels were seen in 2 5-month-old poe / mice. We oserved no correltion etween plsm corticosterone levels nd niml ge in control or ACTH treted mice. Rer et l. (28) lso reported incresed lipid droplets in the drenl cortex nd medull of poe / mice s judged y Nile red stining suggesting incresed EC ccumultion. In the present study we oserved decresed EC ccumultion in the drenl glnds of poe / mice y cholesterol mesurement, occsionl ut inconsistent decreses in oil red O stining in the drenl cortex, nd no oil red O stining in the drenl medull. These discrepncies with Rer et l. (28) my reflect methodologicl differences. Among the prmeters studied, the effects of poe deficiency re specific to drenl cholesterol ccumultion nd corticosterone production. No significnt differences were oserved in drenl LDL or HDL receptor levels, ACAT or HMG-CoA reductse ctivities, or in the response of these prmeters to ACTH tretment. Similrly, drenl weight gin in response to ACTH tretment ws similr in oth genotypes. Additionlly, the sence of morphologicl differences t the light nd electron microscopic levels nd the sence of inflmmtory cells in the drenl glnds of poe / mice suggest tht the effects of poe deficiency re reltively specific to chnges in cholesterol ccumultion nd corticosterone production. The finding of similr sl plsm ACTH levels in the poe / nd poe / mice suggests tht the oserved differences in drenl cholesterol metolism nd steroid production re due to poe effects on drenocorticl cells nd not to effects on neuroendocrine pthwy. This point is lso supported y the similr quntittive responses of oth genotypes in the ACTH stimultion of LDL receptors, SR- BI, drenl weight gin, nd ACAT ctivities wheres t the sme time poe / mice showed elevted plsm corti- Fig. 3. Plsm corticosterone in response to swim stress test. Plsm corticosterone ws mesured (n 18) efore (open rs) nd fter (solid rs) swim stress test s descried in Experimentl Procedures. P 0.01 versus respective poe / group. Thorngte et l. Cholesterol metolism in the poe-deficient drenl glnd 1925

7 costerone levels nd decresed cpcity to mintin drenl cholesterol stores when stressed. The mechnism y which poe expression lters drenl cholesterol metolism is uncler ut could involve chnges in lipoprotein cholesterol uptke or in cholesterol trfficking or utiliztion in drenocorticl cells. Previous studies in Y1 drenocorticl cells showed tht poe expression increses the selective uptke of LDL EC vi pthwy tht does not involve SR-BI (16, 29). A smller increse in the endocytic uptke of LDL EC ws lso seen upon poe expression. Similr effects of poe could contriute to the mintennce of drenl glnd EC during stress, lthough HDL, nd not LDL, ppers to provide most of the drenl glnd EC in vivo (18, 30). Further studies on the effect of poe on HDL nd LDL EC uptke into drenocorticl cells my e informtive on this issue. This reserch ws supported y Grnts HL32868 nd HL52069 from the Ntionl Institutes of Helth nd Merit Awrd from the Deprtment of Veterns Affirs. 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Hvekes Altered lipid metolism in polipoprotein E-deficient mice does not ffect cholesterol lnce cross the liver. Heptology. 24: Reylnd, M. E., M. M. Prck, nd D. L. Willims Incresed expression of protein kinse C in Y1 cells which express polipoprotein E decreses sl steroidogenesis y inhiiting expression of P450-cholesterol side chin clevge enzyme mrna. J. Biol. Chem. 267: Reylnd, M. E., nd D. L. Willims Suppression of campmedited signl trnsduction in mouse drenocorticl cells which express polipoprotein E. J. Biol. Chem. 266: Rer, J., S. F. Akn, S. Bhtngr, M. F. Dllmn, D. Wong, nd L. Mucke Hypothlmic-pituitry-drenl dysfunction in Apoe( / ) mice: possile role in ehviorl nd metolic ltertions. J. Neurosci. 20: Swrnkr, S., J. Beers, D. K. Stricklnd, S. Azhr, nd D. L. Willims The polipoprotein E-dependent low density lipoprotein cholesteryl ester selective uptke pthwy in murine drenocorticl cells involves chondroitin sulfte proteoglycns nd n 2-mcrogloulin receptor. J. Biol. Chem. 276: Glss, C., R. C. Pittmn, M. Civen, nd D. Steinerg Uptke of high-density lipoprotein-ssocited poprotein A-I nd cholesterol esters y 16 tissues of the rt in Vivo nd y drenl cells nd heptocytes in Vitro. J. Biol. Chem. 260: Journl of Lipid Reserch Volume 43, 2002