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1128(1992) 181- !85 © 1992 Elsevier Science Publishers B.V. All rights reserved {1005-276|)/t)2/$05.1}0

Biochimica et Biophy,sica Acta.

BBALIP 54014

Opposite regulation of hepatic lipase and lecithin" cholesterol acyltransferase by glucocorticoids in rats H a n s J a n s e n a,b, A r i e v a n T o l b, J o h a n A u w e r x

:'~, G r e t h e

S k r e t t i n g d, B a r t S t a e l s ~

Departments of "btteraal Medicine ill and "Biochemist~'. Erasmus Unirt'rsitv Rotterdam (NetherlandsL " Laborato~" o[ E.rpt'rimetttal Medicine. Kathotieke Unil'ersitt'it. Leer'ca (Belgium) and ,t 13iott,chnok)k~". Ct',iter of Oslo, Unit'e,-sity of Oslo, Oslo (Norway)

(Received 10 April t992)

Key words: Hepatic lipase" Lecithin : cholesterol acyhransferase: Lipase: Regulation: CAucocortieoid:High-densitylipoprotein Rats were treated with hydrocortisone, dexamcthasone or triamcinolone for 4 days. The effect of treatment on hepatic iipase and lecithin:cholesterol acyltransferase (LCAT) mRNA levels and catalytic activities was determined. Hepatic lipase mRNA was not affected by hydroeortisone, but was decreased after dexamethasone ( - 28%) and triamcinolone ( - 54%). Hepatic iipase activity followed the same pattern, it was not affected by hydrocortisone and lowered by dexamethamne ( - 3 8 % ) antl u-iamcinolone ( - 7 P % ) The LCAT mRNA level in the liver wa,~ also not affected by hydrocortisone, but increased upon treatment with dexamethasone (+ 22%) and triamcinolone (+ 72¢'~). Plasma LCAT, determined with an cxce~ exogenous substrate (designated I.CAT-II), tended to decrease after hydrocortisone treatment (-119~) and was higher after dexamethasone (+2I%) and triamcinotoae (+ 22%). The plasm:~ cholesterol esterifieation rate (designated LCAT-I), determined by incubation of the plasma at 37°C, followed the same pattern. The activity ratio of hepatic lipase/LCAT-II decreased from I in the contro:s to 0.51 after deaamethasone and 0.25 in the triamcinolone-treate~l animals, The plasma HDL cholesterol concentration in the different groups changed oppositely to the hepatic lipase/LCAT activity ratio. It is concluded that HDL cholesterol is raised by" synthetic glucocorticoids due, among other factors, to a lowered hepatic lipase and an increased plasma LCAT activity. The influence of glucocorticolds on these enzymes is, at least partly, explained by the effects on the hepatic mRNA contents.

Introduction Glucocorticoids are widely used as anti-inflammatory agents. An accelerated development of atherosclerosis has been neted during long-term treatment with cortis';eroids and has been suggested to be a complication of the treatment [1], The maj:~r reason for this adverse effect is probably the influence of ghtcoeorticoids on lipid metabolism. Plasma triacyiglyccroi and cholesterol in very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins ( H D L ) was found to increase in humans treated with prednisone [2-5]. In laboratory animals also, administration of glucocorticoids affects

Correspondence to: H. Jansen, Department of Biochemistry, Erasmus University Rotterdam, POB 1738, 3000 DR Rotterdam, Netherlands. t Present address: Laboratoire de Biologic des Regulateurs des Eucaryotes. Centre de Biochimie, Pare Valrose. 06108 Nice, France. Abbreviations: VLDL, very-low-density iivoproteins; HDL, highdensity lipoproteins; LDL, tow-density lipoproteias; L C A T , iecithin:choles,terol aeyltransferase: AU, arbitrary units: RAU, relative absorbance units.

plasma cholesterol a n d / o r triacylglyccrois [6-11]. Using three different glucocorticoid preparations, we observed i ~ g ~ variations in the concentration of total plasma cholesterol in ~ t s [1 l l. These effects may be exerted via several mechanisms on different levels. On one hand, corticosteroids may affect lipopro,ein synthesis. Treatment of rats with different glucocorticoid preparations leads to changes in hepatic .,po A-l, A-II and A-IV m R N A levels [11,12]. V L D L secretion is increased by corticosteroids in rats as well as in rat hepatocyte cultures [7,8]. On the other hand, lipoprotein catabolism is also affected. Plasma triacylglycerol clearing in corticosterone-treated rats may be impaired due to a lowering of the adipose tissue Iipoprotein lipase activity [7-10,13]. Since dexamethasone was shown to decrease the binding and catabolism of lowdensity lipoprotein (LDL) to hepatocytes in vitro [14], plasma cholesterol removal is probably also affected by glucocorticoids. The mechanisms leading to changes in H D L cholesterol are not cle, r. To study the mechanism by which glucocorticoids may effect plasma H D L levels, we determined the iniluence of different synthetic glucocorticoid preparations on the m R N A and catalytic activity of two important determinants of H D L

182 metabolism, lecithin •cholestcroI acyltransferase {LCAT, EC 2.3.I.43) and hepatic lipase (HL, EC 3.1.1.3). Methods and Materials

,4nhntds Male rats from the Wistar strain were used. They were kept under controllcd conditions (lights on 7.00 a.m.-7.(10 p.m.) with free access to food and tap water, unless noted otherwise.

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plasma level of active LCAT was determined in frozen plasma using excess exogenous substrate (LCAT-II) [21]. Incubations wcre for 6 h at 37°C in a total volume of 0.145 ml. The reaction was stopped by addition of 0.30 ml of cold methanol. The lipids were extracted twice with 0.4 ml hexane. Esterificd and unesterified cholesterol were separated using disposable silica columns. ['~H]cholesterol esters were eiuted with 3.0 ml hexanc/dicthyl ether (6" 1, v/v) as described [22]. The measured LCAT activities were linear with the amount of plasma used. The within-day coefficient of variation was 4.5%. The activities were related to the activity in a human plasma pool and expressed as the percen[age

The rats were injected d:dly between 5.00 p.m.-5.15 p.m. for 4 days by subcutaneous injection in the suprascapular rcgion with one of three different commercially available glucocorticoid preparations in equipotent doses: hydrocortisonc (l(}O /.tg/g bodyweight), dcxamethasone phosphate (3.7 # g / g bodyweight) or triamcinolone acetate (211 /zg/g bodyweight). Control animals we're ini,_',:t,~,_t. wi!h l(lO p.l 0.15 M NaCI. On the 4th dtty ~.itc food was withdrawn. The rats were killed by decapitation on the next day between 2.00 p.m.-3.00 p.m.

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RNA analysis

Materials

Briefly, RNA was prepared by the quanidine isothiocyanatc/ccsium chloride procedure from livers of individual aninmls [15]. Dot blot hybridizations of total cellular RNA was performed as described previously [16]. LCAT and hcpatic lipase mRNA levels were dctcrmincd using human LCAT [I 7] and hepatic lipase [18] cDNA probes. The probes were labelled by random primed labeling (Boehringer-Mannheim). Filters were hybridized to 1.5.1t) ~' c p m / m l of each probe, as described [16]. They were washed in 500 ml of 1).5 >" SSC and 0.I% SDS for 10 ,,~in at room ten~per'-3t0rc and twice tk~r 311 mill at 65°C and subsequciitly exposed to X-ray film (X-OMAT-AR. Kodak). Autoradiograms werc analyzed by quantitative scanning dt:n.~iiomeiry (I.KB 2202 U!tr~;scan Laser Densitometer).

Hydrocortisonc sodium succinate was from Upjohn (Solu-Cortef, 100 mg/ml), dexamethasone sodiumphosphate from Chibrct (Decadron, 20 m g / m l ) and triamcinolone diacctate from Cyanamid Benelux (Ledercort, Parent. Forte, 40 mg/ml).

Enzyme ass ay,v The livers wcrc Imzcn in liquid N: immediately after the decapitation of the animals and stored at -80°C until use. Hepatic lipase activity was determined in liver homogcnatcs, using a gum accacia stabilizcd [3H]triolcoylglycerol substrate in the presence of 1 M NaCI, ::s dcscribcd bcforc [19]. Lecithin' cholcstcrol acyltransferase (LCAT) activity was determi:~cd in two ways. The estcrification rate of endoge"

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Plasma lipid and iipoprotein concentrations Plasma triacyiglycerol and cholesterol contents were determined with enzymatic methods, using commercially available kits (Bochringer, Mannhcim, Oermany). Phospholipids were assaye0 enzymatically, using kits from Wako Chemicals (Neuss, Germany). HDL components were determined after precipitation of apo-B containing lipoproteins with a phosphotungstic a c i d / magnesium chloride mixture [23].

Analysis" of the data The statistical significance of the effect of treatment was calculated with one-way analysis of variance (ANOVA) followed by the method of Bonferroni for comparison of groups. Results

Effects o, hepatk" lipase and LCA T Glucoeorticoid treatment of rats led to significant effects on hepatic lipase activity and hepatic lipase mRNA levels. The different glucocorticoids affected hepatic lipase activity per gram liver and hepatic lipase mRNA levels in the liver differently (Table I). Hydrocortisone had no significant effect on either variable ( - 4 % each). Dexamethas:me and triamcinolone lowered hepatic lipase mRNA by 28% and 54% respectively. The catalytic activity of hepatic lipase was lowered by 38% and 70% Since the liver weights were only a,~;,,t.> ,,,,,.~-,,,., by ,,,,. drug t r e a t m e n t s (6.7 + ,

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7.0-t-0.1 g, 7.0 + 0. l g and 6.5 + 0.6 g in the controls, hydrocortisone-, dexamethasone- and triamcinolonetreated animals, respectively), the hepatic lipase activity/liver showed the same pattern as the activity/g

183 TABLE !

TABLE I I I

The t'ffect o[ hydrocortisone, dcxamclhasrmc and triamcinolonc trcatttlent on hepath" lipaxe acticity and hepatic lipase mR:~:4 in rat lirer

7-he e]fi,t t Of hydr~wortixone, dexamethasont, aml triamt'bl~hmt, tr¢'tttmcnt wl phtsma dr,his and tlDL choh'ster~d in the rat

Means +_S.D.. Values statistically significant from controls arc indicated with asterisks. * denotes a P < I).05. ** P < i).l)t)l. Lipasc aclivily was determined in seven animals in each group, hepatic lipase mRNA in three animals per group. RAU = relative absorbancc unit.,, (percentage of the control).

Me:ms± S.D. Values statistically si.~lnificant from '2onlrols ;.fie ip.dicared with asterisks, ' dent, los P < o.05. * ~ P < 0.t)t. Lipids were determined in four animals in each group.

Treatment None ]tydrocorlisone Dexamcthasonc T. r.;.:.l m . .r.i.n.n. l.,.l n. .~... . . . . . .

Hop,,tic lipasc activity (mU/g)

mRNA (RAU)

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l).01 ±0.22 11.31 ÷ ll.ll3 I).83 _+.1).06

rriamcinolone

3.1 ±11.3 **

0.52_+11.11

li.96 +_It. 15 (I.74 ± t). 16 i.-15 +_11.13 * ~ 1.62 _+11.117 * *

1.3s; + I).(}9 m M ( n = 4, P < 0 . 0 1 ) . t r i a m c i n o l o n e 1.38 4_: 0.06 m M ( n = 4, P < 0.01 ). liver. G l u c o c o r t i c o i d t r e a t m e n t also a f f e c t e d h e p a t i c LCAT mRNA concentrations (P

Opposite regulation of hepatic lipase and lecithin: cholesterol acyltransferase by glucocorticoids in rats.

Rats were treated with hydrocortisone, dexamethasone or triamcinolone for 4 days. The effect of treatment on hepatic lipase and lecithin:cholesterol a...
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