Hormoual regulation of acid cholesterol ester hydrolase activity: effects of triiudothyronine and 17a-ethynylestradiol

I ~ A V I[) 1,. S ~ V ~ K S O N , ' L. JOS~I 'H HAYDEK, AND k kit^ FL,ETCHF.,R Deptrrtmcjnl c/fPharwrac~cr/ogy uric1 Ther-(~pecatics, F(rc*ul!j1 ofL44rclicitzu, CJr~iversh~ of&'cilgc~r-\~, Cialgcri-y. .~liba., C~~lclcki 12N //%I4

Received May 2, 1983

SEVERSON, D. L. . L. 5 . HAYUEV, and T. FLETCHER. 1984. Horrnonal regulation of acid cholesterol ester hydrolasc activity: effects of triiodothyronine and I7tr-ethynylestradiol. Can. J . Physiol. Pharmacol. 62: 244-247.

Cholesterol ester hyairolase activity was rneasured in isolated rat hepatocytes and adipocytes. Ad~ninistration of tri- iodothyronine to rats resallted in a specific and selective increase in lysosomal acid (pH 4.5) cholesterol ester hydrolase activity in hepatocytes. Since thc majority of lipoprotein degradation occurs in liver parenchymal cells (hcpatocytes). the stimulration of liver (hepatocyte) :acid cholesterol ester hyc'irolase activity by ta-iiodothyronine could cc9ntribute to the hypocholesterolemic action of thyroid hor~alones. 'T'reatrncnt of rats with 17a-ethynylestraciiol to increase the hepatic degradation o t lipoproteins did not change acid cholesterol ester hydrolase activity in liver, iia~dicating that the thyroid hormone induced stimulation of acid cholesterol ester hydrolase activity in lmepatocytes is not a secondary effect owing to the illcreased hepatic catabolism of low density lipoproteins (LIIL). Bra coaltrast to the results with hcpatocytes, hyperthyroidism did not increase acid cholesterol ester hydrolase activity in rat adipocytes.

SEVERSON, D. L., L. J . HAYDEN et T. FI~EI'CIIEW. 1984. Hormonal regulation of acid cholesterol ester hydrolase activity: effects of triiodothyronine and I7a-ethyny lestradiol. Can. J . Physiol. Pharanacc~!. $2: 244-247.

On a dkterrnink I'activitC hydn)lasiyue des esters cholest6riqaies dans les ht5patocytes et les adipocytes isoles de rats. 12'adrninistration de triiodothyronine aux rats provoqua une augnmcntation spkcifique et sklective de I'activitk hydrc~lasique des esters cholestCriques acides (pH 4,s) %ysosorniaux des idpatocytes. La dkgrndntion des lipoprotkines se produisant n140ri- tairernent dans Ies cellu[es parenchynnateuses (h6patocytes), la stirnulation de I'activitC iaydrolasique des esters cholest6riyues acides (hkpatocyte) du foie par Ia triiodothyronine pourrait contribuer ii I'action hppochulcst&rolksr~ique des tlor~ancpnes thyroi'diennes. Lc traitenlent des rats avec du 17~-@thynyloestradiol pour augrnenter la d6gradaticsn htpatique des lipoprotkines me n~odifia pas I'activitd hydrolasique des csters clmolest&riques asides d~a h i e ; ceci indique que la stirrlulation de l'activite hydrolasique des esters cholest6riqucs aciaies, induite dans les hkpatocytes par tes horlnones thyroi'diennes, n'eai pas utn effct secondaire rksailtant d'une augmentatiota du catakolisrne hkpatique des lipoprstkines de basse densite (LDB,). Contraircrncnt aux resultats obtenus avec Ies hdpatc?cytcs, I'hyperthyro'idisane w'augmenta pas I'activite hycSrc~lasiqa~e des esters cholestCriyues acides des adipocytes de rats.

['B'raduit par le journal 1

Introduction Hypothyroidism results in a decrease in the rates of low

density lipoprotein (LDL) catabolism (Sykes et al. 198 1 ; Katz- Feigenbaurn et al. 1981); this decrease in lipoprotein deg- radation can be prevented by the administration of thyroid horrnones (Katz-Feigenbaum et aB. 8 98 1) . Furthermore. it has been shown that thyrold hormones specifically stirnulate the receptor-naediated catabolism of LDL (Kat7-Feigenbaum et al. 1981), which involves the binding of LDL t43 cell-surhce re- ceptors, internalization of LDL, and the lysosomal degradation of the cholesterol ester component of thc lipoprotein particle by an acid cholesterol ester hydrolase (Brown et al. 1988). In rats, the liver accounts for approximately two-thirds of the total degradation of LDL; kidney armd adipose tissue represent wme of the important extrahepatic catabolic sites (Pittman et aB. 1982). Recently, the administration of triiodothyronine to rats has been shown to increase lysosoarmal acid cholesterol ester hydrolase activity in liver (Katz-Feigenbaum et al. 198 1 ; Severson and Fletcher 198 1 ), kidney (Katz-Feigcnbaun-a et al. 198 l ) , and epididymal fat pads (Severson and Fletcher 198 B ). These results suggest that the stinaulation of lysosomal acid cholesterol ester hydrolase activity by thyroid hormones at several tissue sites may be linked to increases in the catabolism of LDL (Wolinsky 1980), and thus could contribute to the hypocholesterolsmic effect of thyroid hormones. The effects of thyroid hormones on acid cholesterol ester hydrolase activity were tissue specific as the induction of hypothyroidisln and the administration of triiodothyronine did not inflarence acid cho-

' ~ u t h o r to whom correspondence should be addressed.

Iesterol ester hydrolase actlvity in the heart ((Severson and Fletcher 198 I ) , a tissue that does not contribute significantly to thc catabolism of IdDIJ (Pittman et al. 19821.

The majority (estimates ranging from 65 to 92%)) of the degradation of lipoproteins by the liver has been repc~rted to occur in parenchymal cells (hepatocytes) (b'ittrnan et al. 1982; Groot ca al. 19811; Lippiello et al. 198 I ; Van I'ol and Van Berkel 1980). Kupffer cells, for example, c%t, not possess a high-affinity binding site fix LDL (Goltfstein et al. 1979), and Inore than 85% of the receptor-independent catabolism of rnethylated LDL has also keen attributed to hepatocytes (Carew et al. 1982). However, the specific activity of acid cholcstend ester hydro%ase has been reported to be more than 10-fold higher in nonpmenchyrnal cells than in parenchymal cells frona liver (Van Berkel et al. 1980) so that approximately one-half of the total acid cholcsterol ester hydrolase activity in liver is present in nonparenchy~nal cells. Clearly, if thc effect of thy- roid horrnones on liver acid cholesterol ester hydrolase activity (Matz-Feigenbaatm et al. 198 B : Se\rcrson and FBctcher 198 1 ) in to have the potential for physiological significance in relation to lipoprotein degradation (Wolinsky 1980), then it is irn- portant to show that thyroid hormones can regulate the acid cholesterol ester hydrolase in isolated hepatocytes . SinniHarly , the binding, intemalizatiom, and degradation of LDll has only been reporfed in isolated adipocytes (Angel et al. 19791, but the effects of thyroid hormone on acid cholesterol eqter hydrolasc activity were observed with preparations from cpididy~nal fdt pads (Severson and Fletcher 198 1 B. Therefore, the objective of this investigation was to establish if thyroid hormones could increase acid cholesterol ester hydrolase activity in isolated rat

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hcpatocytes and adipoc ytes. The specificit) of the hormonal regulation of Iiver acid cholesterol ester hydrolase by thyroid horr-r-nones was also examined by determining the effect of es- trogens on liver acid chole~terol cster hydrolase activity, since the administration of 17e~-et%1ynylestradiol to rats has also keen shown to stimulate the hepatic degradation of LDE owing to an increase in LDE receptors (Chao et al. 1979; Kcrvanen et al. 1979).

Experiinental procedures Male Sprague-IIawIey rats were treated with subcutaneous injec-

tions of triiodottayronia~e (0.5 mg/kg body weight; Calbiochern, A grade) for 3 days as described previously (Severson and Fletcher 198 1 ) . Hepatic parenchymal cells were isc~latcd using Seglc~m's col- lageraasc perfusion teckaniq~ie (Seglen 1'676). Following a 10- to 30-min digestion with 0.33 mgjn~l , collagenase (\$'c~rthington: type II) in Krebs- Heanseleit bicarbonate buffer, the liver was minced and incubated for 10 min with 60 rn%., of oxygenated Krcbs-Henselcit bicarbonate buffer ~ ih ich contained I f % bovine albumin. 'The dis- persed cells were filtered through a 100-mesh nylon screen and washed three times; viability, determined by 0.4% trypan blue exclu- sion, was greater than 9UYh. Hyperthyroidisan had no effect on the yield or viability of hepatocytes. Adipocytes were prepared with mi- nor modifications to the method described by Kodbell (1'664). Hepa- tocytes and adipc~cytes were homogenized in an isotonic sucrose buffer (0.25 M sucrose - I rra,a/r EIIT.4 - 10 mr21 HEPES. pH 7.5) at 4°C for 18-20 s with a Polytron FIT- I 0 honaogenizer (Severson and Fletcher 198 1 ). Adipocyte homc~genates werc centrifuged at 1000 X g and the resulting infranatant fraction was used Tor enzyme assays.

In some experiments. rats were treated for 5 days with 17cu-ethynyl- estradiol ( , I 0 n ~ g j k g body weight: Sigma Chemical Co.) as described by Chao et al. ( 1 979). Livers were removed, homogenized as de- scribed previously (Severson and Fletcher 198 I ) . and then centrifuged at 1000 X g for 20 nnin to obtain a Bow-speed supernatant fraction. Serum samples collected from these rats were analyzed for cholesterol by the spectrophotornetric procedure outlined in the Sigma 'Technical Bulletin No. 350.

Glycerol-dispersed cholesterol oleate substrates werc prepared as described by Severson and Fletcher ( 198 B j. Routinely, appropriate quantities of cholesterol [ I -'"C]oleate and lecithin were dried under Nz and then dispersed into anhydrous glycerol with a Polytron hornoge- nizer. Aliquots from this stock solutic~n were then diluted with the appropriate buffer and cholesterol ester hydrolase activity was mea- sured as described previously (Severson and Fletcher 198%); assays were linear with respect to time and prcstcin.

Protein (Wobrish et a%. 1998), DNA (Thornas and Farquhar 19981, and the Iysosotnal marker enzytne N-acetylglucosaminidase (Peters et al. 1 972) were measured by flut~ronietric techniques. Cholesterol ester hydrolase activity was calculated as both units per rnilligram protein and as units per milligram DNA; units of enzyme activity were arbitrarily defined as given in Severson and Fletcher ( 198 1 ) . 'I'he DNA content of adipocyte homogenates was not detectable; consequently, enzyme activity En adipocyte preparations is expressed only as units per milligram protein.

Results Cholesterol ester hydrolase activity, determined with a

glycerol-dispersed cholesterol oleate substrate preparation con- taining lecithin, in a low-speed supernatant (1008 x g ) fraction from hepatocytes had an acid pH optiillu~n of 4.5. A pH opti- rnunn of 4.0 has been reported for hepatocyte homogenates by Van Berkel et al. (1986)); this discrepancy is likely due to differences in the substrate preparations. Sonne cholesterol es- ter hydrolase activity could also be measured at neutral and alkaline pH values. but this was much less (one-third to one- quarter) than the acid activity. Acid cholesterol ester hydrolase activity (measured at pH 4.5) in subcellular fractions obtained

renates was by differential centrifugation of hepatocyte homo& localized to particulate fractions and the distribution was very similar to that for It1-acetylglucosami~~idase, a lysosomal marker enzyme (results not shown). Nilsson et 21. (1973) have concluded previously that the acid cholesterol ester hydrolase in hepatocytes was of lysosomal origin. In contrast to tlae acid cholesterol ester hydrolase, the neutral cholesterol ester hydro- lase (assayed at pH 7) was localized in the sol~~ble fraction after centrifugation at 100000 X g .

The pH dependency of cholesterol cster hydrolase in isolated adipocytes was similar to that observed in epididyrnal fat pad preparations (Severson and Fktcher 1981). in that although enzyme activity could be measured at acid pH values, a distinct acid pH optinaum was obscured by the comsidcrable activity in the neutral-alkaline pH range. Coi-asequently, acid cholesterol e$ter hydrolase activity in adipcxyte infr-anatant preparations was also routinely determined at pH 4.5.

Previous results (Severson and Fletcher 198 1 ) have reported that acid cholestero8 ester hydrolase activity was reduced in liver preparations froin thyroidectornized rats. indicating that physiological levels of thyroid hormones can regulate the acid cholesterol ester hydrolase activity. However, since laypo- thyroidis111 (thyroidectomized rats) rcsulted in a marked cie- crease in liver wet weight and liver weight as a percentage of body weight (Severson and Fletcher I98 1). it was decided to measure acid cholesterol ester hydrolasc activity only in hepa- tocytes isolated from control and hyperthyroid rats. The admin- istration of triiodothyronine to euthyroid control rats for 3 days produced a two- to three-fold imcreasc of acid cholesterol ester hydrolase activity in hepatocyte homogenates (Table 1): this ressilt extends the previous observation that the treatment of control or hypothyroid rats with triiodothyronine prodeaced an increase in acid cholesterol ester hydrolase activity in whole liver preparations (Severson and Fletcher B 98 1 ). Hyper- thyroidism did not change either 1%'-acety1glucosan1inidase (Table 1) or neutral cholesterol cster hydrolase (results not shown) activity in these hepatocyte preparations.

The specificity sf the thyroid horrnorne-induced increase in acid cholesterol ester hydrolase activity in liver (Katz- Feigenbaurn et al. 198 1 ; Severson and Fletcher 198 1 ) and hepa- tocyte (Table 1) preparations was further examined by deter- mining if the increased hepatic catabolism of I,b>l, owing to the administration of estrogens (Chao et al. 1979; Kovanen et al. 1979) was also associated with an increase in acid lysosomaH cholesterol ester hydrolase activity. Preparations from the whole liver were studied to determine if estrogens might influ- ence cholesterol ester hydrolase activity in either won- parenchymal cells and (or) hepatocytes, and to avoid any po- tential effects of the experimental treatment on the isolation of hepatocytes. As shown in Tablc 2, adrninistratio~-a of pharma- cologic doses of estrogens (170-ethynylestraciiol) for 5 days resulted in a reduction in total serum cholesterol to 16%) of control levels hut had no effcct on liver acid cholesterol ester hydrolase activity.

Previously it was reported that thyroidectcarny resulted in a decrease in acid cholesterol ester hydrolase activity in rat epi- didymal fat pad preparations, and that the ai%ministration sf triiodothyronine to both control and hypothyroid rats increased fat pad acid hydrolase activity when results were expressed as units per milligram DNA (Severson and Fletcher 1981). In contrast, hyperthyroidism had no significant effect on either acid cholesterol ester hydrolase or 1%'-acetylglucosaminldare activity in isolated adipocytes (Table 1). Furthermore, the ad-

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TARLF 1 . Effect of triaodothqroninc oaj acid ctmlesterol e\tcr hydrolase and A'- ,~cstylglucosasnrrlicIa~e activity in iwlateal rat Imepatocytes and adipocytes

Acid cholesterol ester N-accty I- tiydrolase gluccssanliniaiase

-- Preparation r1 LJ/rng protein U/mg I > N h tl/rng prc~tsin ~~~~~

Hepaaocyte~ control 4 l3.OLO.8 3 8 + 3 9 0.30?0.07 Traiodothqrunane-treatcti 4 37.3? 3.0' * 69B I-76" 0 -40?0.01 hei~pocl tes control 13 33 49t4.B -. 0.08 tbi.01 'Tr~~od<bthyroni~se-treated 13 38 9 24 .8 -- 0.08 20.01

nor^: tijitirnlasr activity was rne:~slsred at pH 4.5 Result. are exprcsbed ttit. mean ? SE h ~igtiii'ici~nt tfilkr-ence Irorn the appropriaie contrtil is indicated by .\ .: 0 0 I : .';:'. 0.00 1 .

r . I ABL.L; 2. Effect of' 17tx-ethynylestrac%ia~i on acid cholestemil e5ter hydrolase activaty in rat llvcr

Serum Acid ct~ole\tt.rol cstsr ctmolcstersl hydrolase

~xpcrimental groiap mg/tlL l J / ~ n g protein Ll/rng DNA

Control (4) 69+6 47.450.8 255 2 3 3 F3qtrogen-treated (1) $ l * 2.p 7.6L0.3 t '74 + 27

Nor I . : Hytirolarc ilctivily \va< ~rieasurccl , ~ t pH 5 . Result\ arr c r p r e c d as tile mean -+ SL Stir. the nurnher o f prep;rr;ltiorls inciicatect ir: parenthest5 ;I signrfic.:~~~t cliUkr.t;rlcc i ' r o ~ ~ ~ tile ;ipl9rclpriate contrch is i~ rd~c ;~ tcd by ? I . . / J .: O . O ( i l .

ministration o f triiodi~thyrcsalirng. had raa significant effect on neutral cholesterol ester hydrolase activity rneasured at pH 7 3 (coratrol, 60.h t 10.3 U/mg protein; hyperthyroid. 79.8 -+ 1'2.5 U/rng protein; f l = 12).

The hepatic degradation of lipsprotein~s is inhibited by chloroquine, ~~adicating that Iysosornal enzymes are involved in the catabolic process (Stein et al. 1977: Floren and Wilssi~n 1977). 'H'he hydrolysis of ck~oiesterol esters bound to lipo- proteins and lipoprotein remnants by the lysosc~~nal acid cholesterol ester hydrolase provides a source of extrgenoias cholesterol that inhibits the biosynthesis of cholesterol through a red~~ction in 3-hydrcsxy-3-methy1glutary1 CoA reductase activity (Lakshmaanan ct al. 1981). Thus, reglalation of acid cholecterol ester hydrolasc activity may play an imporhalt role in the hepatic metabolism of cholecterol.

Since the liver rs the major organ involved in the degradation of LDI, and as the ma-jcsrity of this hepatic catttbolism of lipo- protei~lh ixcurs in parenchymal ceHs (hepatocytes) (Iqittman et al. 19821, it was irnporta~~t to demonstrate that the increase in raciti cholesterol ester hydrolase activity observed in whole liver preparations after the atirn-ninistration of triiodothyronine to rats (Katz-Feigenbaurn et al. I98 1 ; Severson and Fletcher 198 1 ), could also be observed in l-nepatocyte preparations if this change in tissue (cclltalar) mmetabolic activity is tcr be linked to the dynamic clearance of lipoproteins (Wolinsky 19801. Thlc iguestion 1s particularly relevant given that acid cholesterol ester hydrolase activity is very high in the nonpcarcmchy~g~al cells (Van Berkel et al. 1980) that do not contribute substantially to the hepatic degradation of lipoproteins (Pittman ct al. 8982: Groot et al. 198 I ; Lippiello et al. 198 1; &'an To1 and Vsrn Berkel 1980; Goldstein et al. 1979; Care-J et al. 1982).

1%'-acetylglta~cssan1inidase~ a lyxasonlal marker enLyme. This ~uggests that the thyroid llonnaone-induced increase in N-acetylglucos:aminidase activity observed in whole liver (Severson and Fletcher 1983) nnay have been restricted to nonparenchyrnal cells. Hyperthyroidi.;rn also did not influence the activity of the neutral cholesterol ester hydrolase in hepatocytes.

'Thyroid harmones specifically enhalace the r-eceptor- dependent degradrttion of LDL (Katz-Feige~ab:iun~ et al . 19): 1 ), probably by incrcabing the ~lrnmber of high-affinity B,Ill, recep- tors (Brown et al. 1981). Therefore, the stimulation crf the lyhosorrsal acid cholesterol ester hydrolase in hepatocytes by triiodotlryronine coulct be part of a coordinated response of the liver which also includes effect\ oan the hepatic catabolism of cholesterol (Miettenen 1968). Thc regulatory sigrrif'icmnce of these thyroid hcsrmc4ne-iwdtncec;1nce-inde effects will depend on tlme rate- limiting step ira the sequence of bicscherraical events invc~lvcd in the hepatic criltabolisrn of EI)II and cholesterol. I t could he argued that the effcct o f triii~dotl~yrtsnine c~n lysosornal acid sholesteroi ester Inydrola5e activity in hepatocytes might be a secc~ndary cpr conspensatcsry response to the iracreased receptor- depcrldent degradation of I,B112 owing icr the increased ssun-nber of I D & receptors on the hepatocyte (Kat7-Feigerabr~um et al. 198 1 ; Brotvn et al. 198 1 ) . 'This possibility was teeted by ckter- mining if the ad~ninistration of estrogens to rats wouBtl also increase Iiver acid c8-lolesteroI e ~ t e r hydrolasc activity. a \ cs- trogcns also sti~nulate the hepatic degradation of LDll- as a consequence of an increase in high-affinity binding \itex on liver cell memkrancs (Chao et al. 1979; Kovanen et al. 1979). Since the treatment of rats for 5 day\ with 17u-ethynylestradiol did rmot change acid choiestercpl hydrolase act~viay in Iiver prep- arations despite a reduction in pla\rna cisolesterol levels, thi5 rnay be taken as additional evidence that the thyroid hormone- induced response is specific and selective. Akhougtn Chait et al. ( 1979) havc reported that the addition of triiodothyroni~ae to the culture a-nediurn increased 1,DL binding and ctegrradrttion in cultured fibroblasts, to date the presence of varying con- centrations of triiodothyronine in the incr~bation or culture ~ncdiurn has not prclduccd an it1 vttr-61 effect on acid cb~olesterol ester hydrolasc activity in either freshly imlatecf or cult~ired lnepatocytes brT. Fletcher and L. I. IIaydcn, unpubli\hed a~kservatic~ns).

A variety of extrahcpatic tissues also conatribute to the deg- radation of LDI, (lPittnaan et al. 1982). Adipocyte5 l-navc been B F ~ P ~ W ~ to bind rand degrade I,IlI, (Aamgcl et al. 1979j, but the administration of triiodothyrr~n~ne cfiei not increase the acid

Hyperthyrc~idisrra did produce a11 increase in lysc?sor-raal acid cholesterc~l ester hydrolace ;activity in iaolated rat adipocytcs. It cholecterol e5ter hydrolase activ~ty in hepatocytes, which was should be noted that ahc spccific activity of the acid cholesterol specific since triiodothyroninc did not increasc tlne activ~ty of ester hydrolase in adipocytes ('['able l j ie much Icss ts3an in

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NOTES 247

intact fat pads (Sevcrso11 and Fletcher. 198 11, which would be consistent with a substantial arar-rount of cnzyrl-ae activity being present in stro~nal-\!ascular cells. 'T'hercfc>re, thyroid hormone- induced increases in acid kycircsla\e activity ~~rev ious ly oh- served in epididymal Fdt pad preparations (Sevcr , \o~~ and Fletcher 198 1 ) probably occam-ed in cell typcs other than adi- pocytes. Consequently, this effect crn the f i t pad acid choles- terol ester hydrolase would prohably not contribute to any enhancement of LDL, degradation 01% ing to thyroid hornnones. T . I he asseswlent of the physiological slgnificirnce of increases in acld hydrolase in the aorta and kidney 111 response to thyroid horrnoraec (Katz-Feigent~aunn et al. 198 11 will require addi- tional experimentation.

ticknowledgements This work was suppc~rtcd by a grant from the Alberta Hcarb

Foundation. D.L.S. was a Scholar of thc medical Research d 10n. Council of Canada during thc pericacl of this investig, t'

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