89

Biochbnica et Biophysica Acta. 11186(1991) 89-94

~?, It)91 Elsevier Science Publishers B.V. All rights reserved 11005-2760/91/$03.51) A DONIS 0tX)52761)9i 11t)279J BBALIP 53753

Stearyl-CoA desaturase activity in cultured rat hepatocytes P h i l i p p e L e g r a n d l aild A n d r e B e n s a d o u n 2 t Laborut,;;re de Biochimi('. INRA Ecol(. Natumale Super{cur(" .~ ~rtmomique de Remws. Rc)mcs (France) and " Dicision of Nutritional Sciences and Bioh)gical Sciem'es. (~orll('ll Un~l't'r.$ily, Ithaca. N Y (USv4)

{Received 28 March itS91)

Key words: StearyI-CoA desaturase: Cultured hepatocyte: (Rat)

StearyI-CoA desaturase activity was measured as a function of time in culture in rat primary hepatocytes up to 90 h of culture, A decrease to half of the original activity occurred within 40 h of culture. Activity remained constant thereafter. StearyI-CoA decaturase activity increased with insulin molarity in the mediu~ and remained constv.nt as dexamethasone or triiod~thyronine coacentrations were in¢~ased in the medium. When fructose replaced glucose in the medium, the desaturase activity was enhanced. No stimulating effect of cholesterol on the enzyme activity was observed and no inhibF, ing effect of griseofulvin was shown. Finally, starvation (,~z h) of the donor rat followed by 60°7o sucrose refeeding (48 h) did not enhance/.l~-desaturase activity in the cultured hepatocytes compared with ad libitum nutritional status.

Introduction

The conversion of stearyI-CoA tn oleyI-CoA in animal tissues and in particular in rat liver parenchymal cells is catalyzed by the stearyI-CoA desaturase, (acylCoA desaturase; EC 1.14.99.5) which is located in the endoplasmic reticulum. This enzyme has been mostly studied with rat liver microsomes [1-6] and more recently in isolated rat hepatocytes [7-9], in 3T3-LI cells during adipose conversion [10], in HTC cells (hepatoma tissue culture cells) [11,12] and in avian liver explants [13]. Recent research performed in our laboratory on a'~-desaturase activity in two lines (fat and l e a n ) o f chicken [I4] led us to study the involvement of liver ag-desaturase in the regulation of VLDL output. The purpose of this investigation was to evaluate cultured primary rat hepatocytes as a model system to study d%desaturase regulation. This present paper describes such a primary heoatocyte culture system and its a'~-desaturase activity, as well as the response of the enzyme to hormones; insulin, dexamethasone, T 3 and the response of the enzyme to fructose which activates the desaturase in vivo when given in the diet after starvation [4,15]. Results are also presented on the influence of griseofulvin on the A'~-desaturase; this chemical has been

Correspondence: P. Legrand, Laboratoire de Biochimie, INRA ENSA, 65 rue de Saint-Brieuc, 35000 Rennes. France.

reported as an in vivo inhibitor of stearyl-CoA dcsaturase [16], The response of the stearyI-CoA desaturase to free cholesterol, which has been reported to increase the A'~-desaturase activity [17,18] is also investigated, Materials and Methods

Chemicals [t-)'*ClStearic acid (56 mCi/mmol) was obtained from Amersham. It was diluted with cold stearic acid in order to get a 6.1 mCi/mmol specific activity, which is used in all A'~-desaturase assays. Collagenase was purchased from Boehringer Mannheim. Porcine insulin was purchased form Eli Lilly and Co., dexamethasone, T 3, Percoll, amino acids, cholesterol, cofactors (NADH, coenzyme A, ATP) were provided by Sigma. Silica gel for thin-layer chromatography was obtained from Merck. Collagen was prepared from rat tail in the laboratory [10]. The Williams E medium (from GIBCO, formula #85-5015) was supplemented with: (final concentrations) methionine 10 -'~ M, glutamine 2.10 -3 M, leucine 5.7.10 -4 M, glucose 2 g/l, NaHCO 3 2.2 g/l, PSN antibiotic mixture (GIBCO) 1%, BSA (fatty acid free, from Sigma) 0.3%, Hepes 11.88 g/I, insulin 10-t' M, dexamethasone 10 -s M, final pH = 7.35, Only for plating medium, fetal bovine serum (FBS) from GIBCO (heat inactivated) was added at a final concentration of 15%. The Krebs buffer (calcium free) had the following composition: NaCI, 6.93 g/I; KCI, I).35 g/I;

90 KHzPO 4, 0.16 g/l; MgSO 4, 0.29 g/l; NaHCO 3, 2.1)9 g/! (this I,.tst compound was added the day of perfusion). The buffer was adjusted to a pH of 7.4.

lYeparation ~f cultured hepatocytes The method p;o.sently used is based on that of Berry [20] modified by Krebs [21] and adapted in this laboratory. Male Spraguc-Dawley rats (18(I-250 g) were fed rat ci~ow and wcrc given water, ad libitum. A rat was etherizcd briefly, injected with Nembutol (50 mg/kg) in the jugular vein. The fiver was first perfused in situ for 1{1rain (flow rate 5 ml,/min} via the portal vein with a calcium free Hepes Krebs buffer. This buffer was maintained at 37°C and continuously gassed with 95% oxygen/5% CO, in a glass lung, At the end of the pcrfusion the liver was removed and placed in a funnel. Thc digestion was then perfl}rmed with the same buffer containing collagcnase (I mg/ml buffer) for 8-12 min. At the end of the digestion, the liver was minced in the collagenase-containing buffer (10 ml). The cell suspension was then filtered twice through 2511 # nylon mesh filter and once through 11)5 # nylon mesh filter and collected in the hormone and serum free, 0.3% BSA culture medium. The ceils were then washed twice by slow centrifugation (100 x g min), and then spun for 3 min in a perco[I gradient in order to remove cell debris, damaged cells and non-parenchymal cells. The Percoll solution was prepared by mixing 9 volumes of Percoll with ! volume of a saline solution: NaCI, 69.3 g/I; KCI, 3.5 g/I; KHzPO ~, 1.6 g/l; MgSO4, 2.9 g/I. A mixture of pcrcoll solution/medium (1: 1, v/v) was added to the cell pellet). The final cell pellet was brought up in 15 ml of plating medium. Cells were counted using the Trypan bluc exclusion test. After counting, the cells were brought to a concentration of (I.83. I(Y' cells/ml, and plated on 60 mm diameter culture dishes with 3 ml of suspended cells. The dishes had been previously coated with coIlafcn {2 mi/dish of a collagen solution, 24.8 ~ g / m l ) and dried overnight at room temperature under a laminar flow hood with ultraviolet light on. After plating, the cells were maintained in a humidified incubator at 37°C under 5°/~ CO. in air. After 4 h the plating culture medium was changed to serum-free feeding medium {2 ml/dish). 12 h after this first rcfccding, the medium was changed again and every 24 h thereafter. The different treatments unless indicated began 4 h after plating (hormones, sugars, cholesterol, griseofulvin). The ._l%dcsaturase assays were performed at different times of treatment, as described in each case in the figure legends.

A'~-De.~aturase assay Culture dishes were rinsed twice with phosphatebuffered saline (PBS: 0.5 M NaCI, 5 mM sodium phosphate, pH = 7.4) and harvested with a rubber po-

liceman in PBS. Pooled cell suspensions (two dishes) were centrifuged at 800 × g for 4 min; the supernatant was aspirated and the cell pellet was resuspended in 2(}0 ttl of a (I.25 M sucrose, 0.05 M phosphate buffer solution (pH 7.4) and sonicated at 20 W for 15 s using a Braun-sonic 1510 probe sonicator equipped with a microprobe (4 mm diameter). Ap aliquot (80 ptl)was stored at 4°C for DNA analysis and the rest of the sonicated solution was centrifuged for 15 min at 100130 ×g. The supernatant was sucked up and used for A%dcsaturase assay. All incubations were performed in tubes placed in a shaking water bath at 37°C. Each incubation mixture contained 80 ~i of supernatant (about 0.6 mg protein), and in ~mol, phosphate buffer (pH 7.16): 11)2; MgCI2: 6; ATP: 7.2; coenzyme A: 0.54; NADH: 0.8; [1-I~C]stearie acid (spec. act. 6.1 C i / m o l ) : 0.02 in 2 y.I ethanol solution. The final incubation volume was ! ml. The reaction was initiated by the addition of stearic acid and was stopped 10 min later by adding 1 ml of 12% KOH in ethanol. Each assay mixture was heated for I h under reflux. The fatty acids were liberated by acidification, extracted with diethyl ether and taken to dryness. Fatty acid methyl esters were prepared by heating for I h with a methanol/H2SO 4 (10: 1, v/v) solution and then extracted with pentane. They were later separated by thin-layer chromatography on silver nitrate-impregnated silicagel G plates using a mixture diethyl e t h e r / hexane (I0:90, v/v) for development. Following their visualization by dichlorofluorescein, the spots corresponding to the saturated and monoene fractions were separately scraped off the plates, eluted with diethyl ether and subjected to liquid scintillation counting. From the amount of radioactivity found in the monoene fraction vs the radioactivity recovered from the saturated fraction, the enzyme activity could be determined and expressed as nmol stearic acid converted to oleic acid/min. The desaturase assay has been validated since the monoene spot has been identified as 18:1 ( n - 9) as followed: we collected the monoene spot and proceeded to an oxidative cleavage followed by a butylation of the monocarboxylic and diearboxylic acids, according to the method of Von Rudloff [22] and modified by Lemarchai [23]. The monobutylic esters and the dibutylic esters were separated and identified by reversed-phase liquid chromatography [24] and counted. No radioactivity was found in the monobutylic esters and 86% of the radioactivity was found in the C9 dibutylic esters showing that the monoene spot was mainly if not exclusively 18:1 ( n - 9). (The remaining 14% radioactivity was found in the C8 dibutylic esters, which is commonly observed w~Li~ tilts mci.hod because of the overoxidation of the C9 by permanganate). Moreover, the total recovery of added J'~C was between 80 and 90%, making any oxidatin.,., or" the labelled

91 s t e a r a t e f o l l o w e d by t h e e l o n g a t i o n o f p r e e x i s t i n g m o n o e n e s to r a d i o a c t i v e o l e a t e very unlikely.

DNA and protein D N A was m e a s u r e d by m i c r o f l u o r i m e t r i c analysis w i t h d i a m i n o b e n z o i c acid ( S i g m a ) [25]. P r o t e i n was d e t e r m i n e d by a m o d i f i e d Lowry p r o c e d u r e [26].

Results expression T h e activity o f t h e s t e a r y l - C o A d e s a t u r a s e is exp r e s s e d as n a n o m o l e s o f t r a n s f o r m e d s u b s t r a t e p e r m ; n

(9

25-

~ Z o

0-

fo-

5-

t/b

and per mg of DNA or per mg of protein. Each point in a figure is the mean of three or four assays. Results

.

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,

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i

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Fig. 1 p r e s e n t s t h e a m o u n t s o f D N A a n d t h e activity

of the stearyl-CoA desaturase in cultured hepatocytes as a function of culture time. After an initial drop in "v5

® 50 40.

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