Biochimica et Biophysica Acta, 1004 (1989) 187-195 Elsevier

187

BBALIP 53163

fl-Oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

Erik Christensen, Tor-Arne Hagve, Morten Gmnn and Bjorn O. Christophersen Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo (Norway)

(Received 30 March 1989)

Key words: Lauric acid; Myristic acid; Octanoic acid; Fatty acid oxidation; Mitochondrion; (Rat hepatocyte); (Peroxisome)

The p-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fruetose-refed rats. The t-oxidation of laurie acid (12: 0) was less inhibited by fructose ~efeeding and by (+)-deeanoyl- earnitine than the oxidation of oleie acid was, suggesting a peroxisomal t-oxidation of lauric acid. Li~le lauric acid was esterified in iriacylglyeerol fraction, except at high substrate concentrations or in the fmetose-refed state. With [l-14C]myristie acid (14: 0), [l-t4C]laurie acid (12:0), [l-14C]octano|e acid (8: 0) and i2- a4Cladrenic acid (22:4(n - 6)) as substrate for hepatoeytes from carbohydrate-refed rats, a large fraction of the 14C-labelled est.erified fatty acids consisted of newly synthesized palmitie acid ( i6:0) , stearic acid (i8: 0) and oieie acid (i8: i) while intact [|-:4C]oieic acid substrate was esterified directly. With [9,10-3H]myristie acid as the ~ubstrate, small amounts of shortened 3H.iabelled/t-oxidation intermediates were found. With [U-14C]palmitie acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the c~bohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal t-oxidation followed by synthesis of C 16 anti C 16 fatty acids which can then stored as triaeyig|ycerol.

introduction

Medium-chain fatty acids are abundant in some di- etary fast. In cows' milk fat, myristic (14:0), lauric (12 : 0), decanoic (10: 0), octanoic (8 : 0) and hexanoic (6:0) acids together constitute approx. 20 weight% of total fatty acids. In hydrogenated coconut oil, laurie acid (12:0) accounts for nearly 50% of the ~Jtal fatty acid content. In previous studies, we have used diets with 15 wt.% hydrogenated coconut oil to induce essen- tial fatty acid deficiency in rats [1]. Laurie acid was not detectable in liver triacylglycerol or phospholipids after feeding diets with up to 7 wt.% laurie acid for prolonged periods.

The oxidation of medium-chain fatty acids is rapid in vivo and is little influenced by the hormonal and nutri- tional state of the animal [2,31. Tfiese fatty acids, witfi some differences depending on chain length, can be fl-oxidized both by isolated mitochondria [4] and per- oxisomal preparations [5].

It has been proposed that intramitochondrial activa- tion of medium and short-chain fatty acids to acyl-CoA,

Correspondence: E. Christensen, Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, N-0027 Oslo 1, Norway.

which bypasses the carnitine-d,.pendent transfer mecha- nism, is important in explaining why the metabolism of medium and short (C~0-C4) fatty acid is subjected to little regulation by the hormonal and nutritional state of the animal [6]. The shorter medium-chain fatty acids from butyrate (4:0) to decanoate and possibly laurate can thus pass the mitochondrial membrane as free fatty acids and be activated to their acyl-CoA esters in the mitochondrial matrix prior to mitochondria2 fl-oxida- tion [71.

Since peroxisomal fatty acid fl-oxidation is not sub- ject to a strict metabolic control, oxidation in this particle offers an alternative explanation as to why the oxidation of medium-chain fatty acids is not regulated by hormonal and nutritional factors. This would require an efficient extramitochondrial synthesis of medium- chain acyl-CoA. Decanoic (10 : 0), laurie (12 : 0) and myristic acid (14:0) can be activated by long-chain acyl-CoA synthetase as efficiently as the longer C~6 and C18 fatty acid substrates [8-11]. This enzyme is local- ized both in the endoplasmatic reticulum [8], outer mitochondrial membrane [12,13] and peroxisomes [14,15]. Hexanoate (6 : 0) and octanoate (8:0), as well as very-long-chain monoenic fatty acids such as eicosenoic acid (20:1) and erucic acid ( 2 2 : 1 ( n - 9)) can also be activated by long-chain acyl-CoA synthetase, although at a slower rate [11].

0005-2760/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

188

The aim of the present work was to study the relative roles of mitochondrial and peroxisomal ~-oxidation in the oxidation of medium-chain fatty acids in hepato- cytes from fasted, fed and fructose-refed rats. In the present study, the metabolism of laurie acid was studied in detail while myristic acid and octanoic acid were also used as substlrates in some of the experiments.

Matedah and Methods

Chemicals [l.14C]Lauric acid (12: 0), [1-14C]myristic acid (14: 0)

and [2-14C]adrenic acid (22: ~ n - 6 ) ) were from the Am~sham International, Amersham, U.K. [1-~4C]Oc - tanoic acid (8:0), [9,10-~H]myristic acid (14:0), [U- t4C]palmitic acid (16:0) and [l-14C]oleic acid (18: l(n -9)) were from New England Nuclear, Boston, MA, U.S.A.

Unlabelled laurie acid, myristic acid and octanoic acid were from Fluka A.G., Buchs, Switzerland. Clofibrate was obtained from ICI Industrial Company, Macclesfield, U.K. (+)-Decanoylc~rn~tine was synthe- sized according to the method of Bt~hmer and Bremer [16]. Essentially fatty acid-free bovine serum albumin, Hepes, collagenase type I and unlabelled oleic acid and glucagon were from Sigma Chemical Co., St. Louis, MO, U.S.A. Docosatetraenoic acid was fron~ Nu Chek Prep, Elysian, MN, U.S.A. 2,6-Di-tert-butyl.p-cresol was from Koch-Light Ltd., Colnbrook, Bucks, U.K. ( - ) - Carnitine was from Lonza AG, Basle, Switzerland. N- Nitrosomethylurea was from ICN Biomedicals. Inc., Plainview, NY, U.S.A.

Animals Preparation of liver cells. Male weanling rats of the

Wistar strain were from MolleBaard Laboratory (Den- mark). The animals were fed standard pellets and had free access to food when not stated otherwise. In some experiments, the animals were fasted for 48 h before preparation of liver cells. Animals which had ~ n fasted for 48 h and then refed with a 15% (w/v) fructose solution and white bread for 24 h were a;so used. The clofibrate was dissolved in aceterle and mixed with standard pellets (0.3 g clofibrate/100 g of pelle, s). The solvent was blown off by air during shaking. Ttle rats were fed the clofibrate containing diets for 9 days when stated.

Parenchymal liver cells were prepared and purified according to the method of Seglen [17], except that only 0.03 M Hepes buffer was used in the suspension medium. About (100-300) 106 cells were obtained from each liver, and 90-95~ were viable, as measured by resis- tance to uptake of Trypan blue.

Incubation The concentration of cells in the preparation was

approx. 6-10 6 cells/ml and 1 ml of this suspension was

used (with a total volume of 2 ml of incubation medium in 25 ml Erlenmeyer flasks). Cells were incubated at 37°C in an oxygenated suspension medium [18] with 1.5~; (w/v) bovine serum albumin with 200 nmol of ~4C-labelled fatty acid when not stated otherwise.

The specific activity of all ~4C-labelled fatty acids substrates was 7 mCi/mmol. The specific activity of 3H-labelled myristic acid was 70 mCi/mmol. When indicated, the hepatocytes were preincubated with 1 mM (-)-carnitine, 1 mM (+)-decanoylcarnitine or 0.4 pM glucagon for 20 min.

Analytical methods The extraction of lipids and the measurement of

radioactive acid-soluble products (as a measure of the rate of ~-oxidation) and of radioactive CO2 were per- formed as described by Christiansen [19].

The lipids were extracted by the method of Folch et al. [20] and separated on either silicic acid thin-layer plates (Stahl H ÷) (hexane/diethyl ether/glacial acetic acid, 70: 30: 1, v/v) or when stated on Bond-Elut amino-propyl column (Analytichem International, Harbor City, CA, U.S.A.) into three fractions (neutral iipids, free fatty acids and phospholipids) [21]. Aliquots of the total lipid extracts and the free fatty acid, triacyl- glycerol and phospholipid fractions were transmethyl- ated [221.

Short- and medium-chain fatty acids were extracted with diethyl ether twice after initial hydrolysis and acidification. The ether extract was dried over anhydrous Na2SO 4 and methylation was performed in a solution of methanol/diethyi ether, 10:90 (v/v) with an excess of diazomethane liberated from N-nitrosomethylurea. These fatty acids were separated by high-performance liquid chromatography on a Spectra-Physics apparatus using a Supelcosil LC-18 (250 mm× 4.6 mm/O) re- versed-phase column (Supelco Inc., Bellefonte, PA, U.S.A.). The mobile phase was acetonitdle/H20 (90:10, v/v). The flow rate was 1.5 ml/min, the pres- sure 560 lb/in and the temperature 20°C. Fractions were collected every 30 s, and measured in a P~:~ckard Tri-Carb 300 C liquid scintillation counter.

In experiments with 14C-labelled fatty acids, the lipid fractions were analyzed by radio-gas chromatography using a Varian 2100 gas chromatograph connected to an ESI nuclear radioactivity detector with a 1:10 outlet splitter. Fatty acid methyl esters were separated at 165 and 200 °C using 10% SP-2340 on Supelcoport 100/120 (Supelco Inc. Bellefonte, PA, U.S.A.). The peaks were identified on the basis of the retention time compared with the standards.

The solutions used for lipid extraction and thin-layer chromatography contained 2,6-di-tert-butyl-p-cresol (50 mg/l) as an antioxidant and the lipid extracts were stored under nitrogen gas in the dark at - 2 0 ° C to prevent peroxidation of unsaturated fatty acids.

Cellular protein was determined according to the method of Lowry et al. [23].

R e s u l t s

Hepatocytes from fasted rats Fig. la shows that laurie acid (12 : 0) is oxidized very

rapidly in hepatocytes from fasted rats. 80-90% of the 12:0 substrate added was metabolized to acid-soluble products after less than 10 rain of incubation under the conditions used. Oleic acid was oxidized more slowly (Fig. 1). After a longer incubation period (120 min), approx. 80% of this substrate was also metabolized to acid-soluble products. 14C02 was also formed more rapidly from labelled laurie acid than with oleic acid, while after longer incubation periods, approx, the same fraction (10% of each substrate) was recovered as t4C02 (Fig. lb).

Fig. 2 shows a very small and transient incorporation of [14C]lauric acid in the triacylglycerol fraction after a short incubation period with h~patocytes from fasted rats. After longer incubation periods [t4C]lauric acid virtually disappeared from the triacylglycerol fraction. Distinctly more [14C]oleic acid was recovered in the triacylglycerol fraction. However, after longer incuba- tion periods (120 rain), also [t4Cloleic acid was removed from the tnacylglycerol fraction. The continued oxida- tion of [14C]oleic acid during long incubation periods was thus partly due to the oxidation of oleic acid liberated by hydrolysis of triacylglycerol initially formed. Only small amounts of 12 : 0 and 18 : 1 were esterified in the phospholipid fraction of hepatocytes from fasted rats (Fig. 2b). The amounts of labelled fatty acids

!89

"~ 2 0 ~ ..o_ ~ C

~ . ~ 0 "-- b

• r_ 10

" - ' ~ 0 0 40 80 0

c i 200 :~ ~,

~ 100 =, ¢,

"4' ~1

0 " - ' = 40 80

T i m e ( m i n ) Fig. 2. Esterification of 14C-labelled fatty acids and disappearance of the substrate with hepatocytes from fasted rats incubated with [1- 14C]iauric acid (0) or [lJ4C]oleic acid (O). The incubation conditions were as described in the legend to Fig. 1. (a) Esterification in triacylglycerol and (b) in phospholipids. (c) Disappearance of labelled fatty acid substrate. The results are expressed as nmol of labelled fatty

acid esterified or remaining as free fatty acids.

initially incorporated in the phospholipids diminished during long incubation periods.

The oxidation of lauric acid to acid-soluble products increased nearly linearly with increasing substrate con- centrations in hepatocytes from fasted rats (Fig. 3a).

Little lauric acid was recovered in the triacylglycerol fraction at low substrate concentrations (Fig. 3b). At higher substrate concentrations, a marked increase in the esterification of lauric acid in the triacylglycerol fraction was found. Distinctly less lauric acid than oleic acid was however esterified in triacylglycerol also at high substrate concentrations (Fig. 3b).

Fig. 4 shows that (+)-decanoylcarnitine, which is an inhibitor of the transport of long-chain acylcarnitine into the mitochondria, inhibits the oxidation of lauric

a

"6 E 150

• o 100 .'g_ o x 50 .'g_ 0 m 0

~ 2o

• .T I

" - " 40 80 120

Time (rain)

Fig. 1. Oxidation of 14C-labelled fatty acids in hepatocytes from fasted rats incubated with [1J4C]lauric acid (0) or [1J4C]oleic acid fin). The incubation conditions were as described in Materials and Methods. 0.1 mM labelled fatty acid, 1 mM (-)-carnitine and hepatocytes (15.2 mg of proteins) were used. (a) Oxidation to acid- soluble products and (b) to CO2. The results are expressed as nmol of

lal':, ~ed fatty acid oxidized.

P S 1000 a

500

300~- ,b , , i i

~ lO

" - " 0 0,2 0,6

~"C] fatty acid substrate (raM)

Fig. 3. Oxidation and esterification of 14C-labelled fatty acids in hepatocytes from fasted rats incubated with increasing concentrations of [l-~4Cllauric acid (o) or [1J4Cloleic acid (~). The incubation conditions were as descnbed in Materials and Methods with 1 mM (-)-carnitine present. Hepatocytes (17.3 mg of proteins) were in- cubated for 60 min. (a) Oxidation to acid.soluble products and (b) esterification in triacylglycerol. The results are expressed as nmol of

labelled fatty acid oxidized or esterified.

190 . m . m . m , . , , . . - - - , - "

o a "i5 150 E

.~ 100

.= 50 a l

.o 0 "~ b e 100 .>,

"G' so =z

!

0 0 40 80 120

Time train)

Fig. 4, Effec! of (+),decanoylcamitine on the oxidation and estedfi. cation of 14C, labeiled fatty acids in hepatocytes from fasted rats incubaled with [l-t4C]lauric acid (o,®) or [l-t4C]olei¢ acid (O, B), ( - ),Camitine (open symbols) or ( + ).decanoylcamitine (closed sym- bols) was added, The incubation conditions were as described in materials and Methods. 0.1 mM labelled fatty acid and hepatocytes (25,2 ms of proteins) were used, (a) Oxidatio,1 to acid-soluble prod- ucts and (b) estedfication in tdacylglycerol. The results are expressed

as nmol of labelled fatty acid oxidized or ester±fled.

acid distinctly less than observed with oleic acid as the substrate for hepatocytes from fasted rats. After an initial delay in the oxidation of laurie acid by (+)-de- canoylcamitine during the first 10-20 min of incuba- tion, nearly equal amounts of acid-soluble oxidation products were formed in the presence and the absence of (+)-decanoyicarnitine. With oleic acid as the sub- strafe, (+)-decanoylcarnitine causes a strong and pro- longed inhibition of the formation of acid.soluble oxidation products. On the other hand, (+)-decanoyl- cam±fine stimulates the esterification of laurie acid and even more of oleic acid in the triacylglycerol fraction (Fig. 5 and Table I).

100

0

m 40

0 20 40

Time (rain)

Fig. 5. Oxidation and esterification of 14C-labelled fatty acids in hepatocytes from fed rats incubated with [1-14C]iauric acid (o) or [l-14C]oleic acid (O). The incubation conditions were as described in Materials and Methods. 0.1 mM labelled fatty acid, 1 mM ( - )-earn±- fine and hepatocytes (23,2 mg of proteins) were used. (a) Oxidation to acid.soluble products and (b) esterification in triacylglycerol. The results are expressed as nmol of labelled fatty acid oxidized or

ester±fled.

Hepatocytes from fed rats When hepatocytes from fed rats were used, less of

the laurie acid substrate was oxidized to acid-soluble products and more was recovered in the triacylglycerol fraction than in experiments with hepatocytes from fasted rats (Table I and Fig. 5). Also in hepatocytes from fed rats, laurie acid was oxidized to acid-soluble products to a greater extent than with oleic acid as the substrate (Fig. 5). After prolonged incubation periods, approx. 60f~ of ~4C from laurie acid was recovered in acid-soluble products compared to 30% as acid-soluble products with oleic acid as the substrate.

In experiments with laurie acid as the substrate for hepatocytes from fed rats, significant amounts of the labelled fatty acid was ester±lied in the triacylglycerol fraction when all the free fatty acid substrate had been

TABLE I

Tke ef/ect~ of (-).camitine and of t + ),decanoylcamitine on the oxidation and esterification of laurate (12:0) and oleate ¢18:1) in hepatocytes from fasted rats and of latwate in ~patocytes from fed and clofibrate-fed rats

The incubation conditions were as des~bed in Materials and Methods. Labelled fatty acids (0.1 raM) and hepatocytes (13.5-17.2 mg of protein) were in ,ba ted for 60 rain. The results are expressed as nmol of labelled fatty acids oxidized or ester±fled. Mean + S.D. of two parallel incubations from three different livers are given.

Feeding condition of ani~tls, fatty acid substrate and additions

fasted. 12:0 . . . . fasted,' 18 :'i . . . . . . . . . . . . fed, 12":0 "clofibrate, 12:0

(-)-carni- (+)-decanoyi. (-).cam±- (+)-decanoyl- (-)-earn±- (+)-decanoyl- (-)-earn±- (+)-decanoyl fine cam±fine fine cam±fine fine cam±fine tine carnitine

Acid-soluble products 160.5+2.3 145.9:t:3.8 112.7+12.8 58.6+1.0 113.0+7.3 99.3+6.7 113.2+13.3 124.4+6.9

CO-z 16.6+0.5 20.0+3J 12.7+ 4.2 18.5+2.1 29.1+2,5 21.3+2.6 25.2+ 2.2 24.0+4.1 Ttiacylglyceroi Z3+0.7 10.64-4,5 11.6+ 2.9 60.64-6.9 12.7:t:3.2 62.1:!:2.7 16.54- 4 .21 21.1+_4.3 Phosphofipids 13.5:!:1.5 4.24-0,7 11.14- 1.9 17.84-3.6 10.1+3.4 11.9+1.0 49.0:!: 4.9 46.9+2.4

m

o 60 E _= -o /,0 N

2o

N |20 - u lU

~ 80

0 ,~0 80

Time (rain) Fi E, 6. Oxidation and esterification of 14C-labelled fatty acids in hepatocytes from fructose-refed rats incubated with [1 ol'~ C]lauric acid (0), [2-14C]adrenic acid (zx) or [l-14C]oleic acid (O). The incubation conditions were as described in Materials and Methods. 0.1 mM labelled fatty acid, 1 mM (-)-carnitine and hepatocytes (15.2 mg of proteins) were used. (a) Oxidation to acid-soluble products and (b) esterification in triacylglycerol. The results are expressed as nmol of

labelled fatty acid oxidized or esterified.

metabolized. Still distinctly more oleic acid was esteri- fled in the triacylglycerol fraction than with lauric acid. The quantity of both lauric acid and oleic acid initially esterified in the triacylglycerol fraction remained nearly

191

constant throughout long incubation periods with the hepatocytes from fed rats (Fig. 5).

When hepatocytes from clofibrate-fed animals were used, lauric acid substrate was oxidized to approxi- mately the same extent as with cells from fed rats. (+)-Decanoylcarnitine did not inhibit the oxidation of lauric acid when hepatocytes from clofibrate-fed animals were used (Table l).

Hepatocytes from fructose-refed rats In experiments with hepatocytes from fructose-refed

rats, lauric acid is also rapidly oxidized to acid-soluble products (Fig. 6 and 'fable If). The fraction of the substrate oxidized to acid-soluble products is, however, lower than with hepatocytes from fasted or fed rats. The oxidation of oleic acid to acid-soluble products is strongly suppressed in hepatocytes from fructose-refed rats (Fig. 6 and Ref. 24). We have previously shown that [2-~ac]adrenic acid (22 : 4(n - 6)) is to a significant extent oxidized to 14C-labelled acid-soluble products in hepatocytes from fructose-refed rats [24]. Fig. 6 shows that laurie acid is oxidized even more rapidly to acid- soluble products than with adrenic acid as the substrate. Approximately the same total amounts of ~4C-!abelled acid-soluble products were formed with both lauric acid and adrenic acid as substrates in experiments without added carnitine (Fig. 6).

Table II shows that (+)-decanoylcarnitine sup- presses the oxidation of lauric acid and of adrenic acid

TABLE II

Formatt~n of /4C-labelled C;4-CI8 fatty acids from [l-t4C]lauric acid (12:0), [2-J4C]adrenic acid (22 :9(n - 6)) and [l-14C]oleic acid (18: l(n - 9)) in iioer cells from fructose.refed rats

The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 raM) and hepatocytes (15.2-18.3 mg of protein) were incubated for 90 rain. The cells were premeubated with 4.10 -7 M glucacon when stated. The synthesized fatty acids were measured by radio-gas chromatography of the total lipid extract. The distribution of i,IC.labelled fatty acids between the triacylglycerol-, phospholipid- and free fatty acid fraction was determined by thin-layer chromatography. The results are expressed as nmol ~4C-labelled fatty acid synthesized, oxidized or esterified. Means + S.D. of two parallel incubations from two different livers are given. Results < 1 nmoi are not given.

Fatty acid ~ubstrate and additions

12:0 22:4(n -6 ) 18: l (n - 9 )

(-)-Carni- (+)-decanoyl- glucagon tine carnitine

(-)-carni- (+)-decanoyl- glucagon tine carnitine

(-)-carni- (+)-decanoyl- glucagon tine carnitine

14:0 9.3±2.7 12.6±1.6 1.4± 0.6 2.5±0.5 8.4± 0.2 1.8±0.3 16:0 30.1±4.8 30.3±0.9 4.7± 2.3 13.2±1.2 35.5± 2.3 6.4±0.8 16:1 4.4±1.0 4.9±1.5 - 4.5±0.7 8.2± 0.5 1.8±0.3 18:0 5.2±1.4 2.9±1.1 - 1.6±0.3 3.4± 0.1 - 18:1 8.7±1.3 3.8±1.8 - 7.1±0.6 5.3± 1.1 2.1±0.2

Sum synthe- sized fatty acids 57.7 54.5 6.1 29.0 60.8 12.1

Acid-soluble products 87.4±6.9 63.1±1.8 146.2±10.2 92.0±6.7 30.1± 3.5 132.5±4.7

CO 2 ~ .4±3.0 21.7±7.9 30.0± 2.9 4.2±0.2 6.8± 1.0 6.8±1.1 Triacyl~y~rol 60.6±9.8 99.0±9.7 13.2± 5.4 91.2±4.6 135.0±11.6 51.5±4.5 P h o s p h o ~ s 22.0±5.7 13.3±2.2 3.5± 1.2 11.0±3.0 19.6± 3.7 5.8±0.5 F~e fa t~ac ids 5.6±4.1 2.9±2.8 7.1± 5.4 1.6±1.6 8.5± 4.2 3.4±2.0

2.2±0.1 6.8±0.3 2.7±0.7

2.2 6.8 2.7

22.8±1.8 11.4±1.8 7.2±0.8 2.3±1.2

135.1±8.0 159.9±7.5 18.3±3.5 11.7±2.8 16.628.3 14.7~6.2

78.9±3.7 9.0±0.8

85.9±8.4 16.5±£.4 9.7±4.7

192

200'~ =" ]-\

'°°I '- I . 80

"d'

~ o 0 40 80 120

Time (rain) Fig, 7. Esterification or t4C-labelled palm±tic acid ((3) and lauric acid (0) in hepatocytes from fmctose--~fed rats incubated with [l- t4Cllauric acid, laC-labelled free ratty acids (w). The incubation conditkms were as described in Materials and Methods, 0.1 mM [l.t4Cllauric acid, ~mitine and hepatocytes (15,2 m8 of protein) were used, The lipids were separated by Bond-Elut amino-propyl column in ester fractions and a free fatty acid fraction. The t4C-labelled fatty acids in the total ester fraction were analyzed by radio-sas chromatog- raphy. The results are expressed as nmol of labelled fatty acids

esterified or remaining as free fatty acid.

to acid-soluble products approximately to the same extent compared to experiments with added cam±fine. Still significant amounts of acid-soluble products are formed from both substrates in the presence of (+)-de- canoylcarnitine.

Table I1 also shows that glucagon strongly stimulates the oxidation of all the three substrates used, lauric acid, adrenic acid and oleic acid in hepatocytes from fructose-refed rats. Except for the experiments with glucagon, significant amounts of t4C.labeled triacyl- gly~rol were formed from all the three fatty acid sub-

strates in hepatocytes from fructose-refed rats. With lauric acid and adrenic acid as substrates, a large frac- tion of the esterified fatty acids consisted of [14C]paimitic acid (16:0) and smaller amounts of 14C- labelled myristic acid (14:0), palmitoleic acid (16: l (n -7 ) ) , stearic acid (18:0) and oleic acid (18:1) (Table II). In contrast to this, nearly all the ~4C-labelled fatty acid ester±fled consisted of intact olei¢ acid when this fatty acid was used as the substrate.

Fig. 7 shows that the ester±fled [t4C]lauric acid frac- tion rapidly increases to reach a maMmum after 15-20 rain of incubation while the substrate, measured as free fatty acid falls to a very low level. The ester±fled [t4C]lauric acid thereafter gradually decreases while [t4C]palmitic acid and the other synthesized fatty acids in the ester fraction gradually increase during long incubation periods of up to 120 miv. This remodelling of the pattern of ester±fled fatty acids must be the result of continuous acylation and deacylafion reactions.

With hepatocytes from rats which had free access to food (standard pellets), t4C-labelled C!4-Cls fatty acids were formed with some preparations of hepatocytes, but not in all. This may be due to the fact that the activity of acetyl-CoA carboxylase, which regulates fatty acid synthesis, is stimulated by feeding and is rapidly re- duced after a few hours of fasting. With hepatocytes from fructose-refed animals, a large fraction of the t4c label from both laurie acid and adrenic acid was repro- ducibly recovered in C14-C~s fatty acids.

Also with [1-14C]myristic acid (14:0) and [1-t4C]oc - tanoic acid as substrates, significant amounts of ~4C- labelled palm±tic acid (16:0) and smaller amounts of palmitoleic acid (16: l ( n - 7), stearic acid (18:0) and oleic acid (18: 1) were formed in hepatocytes from fructose-refed rats (Table Ill). With [l -14 C]octanoate as

TABLE ill

Fommtlon of t~C.labclled Cl6-Cis fatty acids from [l.l~C]myristic acid (14:0), [9,10-JHImyristic acid and [l./VC]octanoic acid (8:0) in liver cells f , ~ fm.ose-ref,~ rats

The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 raM) and hepatocytes (15.2 ± 18.3 mg of protein) were incubated for 90 rain. The results are expressed as nmol talC-labelled fatty acid synthesized, oxidized or ester±fled. Means±S.D. of two parallel incubations from two different livers are given.

Fatty acid substrate and additions [lt'lC]i4:0 [1-34 C]8: 0 [9,10- 3 H]14: 0

( - )~arrdtine ( + )-decanoylcarnitine ( - )-carnitine ( - )-decanoylcarnitine 8:0 0 0 12.1+3.1 0 10:0-12:0 0 0 0 2.6+1.1 14 • 0 22,6 ± 3,3 82,8 ± 11,4 2.6 ± 1.3 65.9 + 6.7 16: 0 + 16: I 17.0 ± 0.8 23.8 :t: 3,0 44.8 + 6.6 19.1 ± 4.5 18:0+ 18:1 4,3 ±1.1 6.9+ 1.2 17.4± 1.4 4.3± 1.2 Sum synthesized fatty acids 21.3 30.7 64.8 23.4 Acid-soluble products 125.1 + 2.0 56.2 ± 1.5 108.5 ± 9.2 89.9 :t: 6.2 CO2 27.5 ± 2.7 16.1 + 3.5 41.7 ± 4.9 Triacyiglyceroi 32.8±2.8 107.9± 9.1 39A+';.6 101.2± 1.9 Phospholipids 13.1 + 1.6 13.6 ± 1.5 14.6 + 2.8 2.3 + 0.3

substrate, the newly synthesized fatty acids accounted for nearly all the 14C-labelled fatty acids estefified.

[9,10-3H]Myristic acid (14:0) was used as the sub- strate to detect possible shortened (12:0-6:0) fl-oxida- tion intermediates. Only small amounts of 3H-labelled C~2-C6 fatty acids were detected in hepatocytes from fed rats (not shown) and from fructose-refed rats when short incubation periods (3-15 min) or longer incuba- tion periods (Table III) were used. With [U-~4C]palmitie acids as the substrate for hepatocytes from fed or fructose-refed rats (not shown), no shortened fatty acids were detected.

Discussion

Medium-chain fatty acids can probably be fl-oxidized in liver cells by three different mechanisms: (1) carni- tine-dependent mitochondrial fl-oxidation; (2) carni- tine-independent mitochondrial oxidation after activa- tion to acyl-CoA esters in the nfitochondrial matrix and (3) by peroxisomal B-oxidation.

The role played by the carnitine-dependent mito- chondrial oxidation is regulated according to the feed- ing state of the animal, mainly by the level of malonyl- CoA, which inhibits the outer carnitine palmi- tyltransferase [251.

In the present e,,periments with hepatocytes from fasted rats, (+)-decanoylearnitine inhibits the oxidation of laurate distinctly less than observed with oleate. This may suggest that only a minor fraction of laurate is oxidized by the earnitine-dependent mitochondrial pathway in the cells. It can, however, not be excluded that carnitine-dependent oxidation of laurate plays a major role in cells in the absence of the inhibitor. A reduction in the rate of carnitine-dependent oxidation caused by (+)-decanoylearnitine may be compensated for by an increased oxidation by other pathways. The delay in the oxidation of laurate caused by (+)-de- canoylcarnitine (Fig. 4) may be explained by such a mechanism. The finding that (-)-carnitine increased the oxidation of laurate to acid-soluble products in hepatocytes from fasted animals also supports the view that mitochondrial earnitine-dependent oxidation plays a significant role. This stimulatory effect of carnitine on laurate oxidation can, however, also be caused by a stimulation of peroxisomal oxidation via a regeneration of free CoA due to the transfer of aeetyl groups from CoA to carnitine.

In hepatocytes from fructose-refed animals, the earnitine-dependent oxidation is strongly suppressed as shown b~ ~ the very low oxidation of oleate. The high rate of oxidation of laurate in these ceils must be due to carnitine-independent fl-oxidation either in the per- oxisomes or in the mitoehondria after activation of the free fatty acid to acyl-CoA ester in the mitochondrial matrix.

193

Experiments with different acyl-CoA esters as sub- strates for isolated peroxisomal fractions support the view that medium-chain fatty acids can be oxidized efficiently in the peroxisomes [5,26,27]. With liver per- oxisomes, high rates of peroxisomal fl-oxidation were obtained with the CoA esters of myristic acid (14:0), palmitic acid (16:0), laurie acid (12:0) and decanoie acid (10:0) in decreasing order [5]. In experiments with subcellular fractions from brown adipose tissue, per- oxisomes showed the lowest K m for medium chain (9:0-10:0) acyl-CoA and the highest oxidation rates with lauroyl-CoA [26].

Peroxisomal fatty acid oxidation requires extramito- ehondrial activation of the substrate by long-chain acyl- CoA synthetase, which is located both in the endo- plasmatic reticulum [8], the outer mitochondrial mem- brane [12,13] and in the peroxisomes [14,15]. Studies on the substrate specificity of long-chain acyl-CoA syn- thetase in isolated subcellular fractions support the view that laurie acid, myristic acid and decanoic acid are activated to acyl-CoA as efficiently as with palmitate and oleate as substrates [8-11].

The present finding that laurate is esterified in tri- aeylglyeerol under certain experimental conditions sup- ports the view that it can be efficiently activated to lauroyl-CoA by long-chain acyl-CoA synthetase. Laurate was thus esterified in triacylglycerol in cells from fasted animals at high substrate concentrations (Fig. 3) and in the presence of (+)-decanoylcarnitine (Fig. 4). In hepatoeytes from fed rats, laurate was also esterified in triacylglycerol (Fig. 5). In hepatocytes from fructose- refed animals, laurate was esterified, although the esterified fatty acids in this case also consisted of palmitate and other ~4C-labelled fatty acids.

The labelling of [x~C]palmitic acid and smaller amounts of 14C-labelled myristic acid, palmitoleic acid, stearic acid and oleic acid from laurie acid, myristie acid and octanoie acid as substrates in hepatocytes from fruetose-refed rats, could either the caused by chain elongation of the ~4C-labelled substrates or by de novo fatty acid synthesis from 14C-labelled acetate after ini- tial fl-oxidation of the fatty acids. With [~4Cladrenic acid as the substrate, only de novo synthesis can explain the formation of [14C]palmitie acids and the other ~4C- fatty acids formed (Tables II and III). With both laurie acid and adrenic acid substrates, [14C]palmitic acid is the main fatty acid formed. The pattern of the other 14C-labelled fatty acids was also remarkably similar, whether the initial substrate was laurie acid or adrenic acid. Although chain elongation of the substrates may play some role, these findings suggest that a major fraction of 14C-labelled fatty acids from [~4C]lauric acid, myristic acid and octanoic acid are formed by de novo synthesis. The sum of the 14C label recovered in newly synthesized fatty acids and in acid-soluble prod-

194

ucts can be used as a measure of the total fl-oxidation in hepatocytes from fructose-refed rats.

Since it ~ n be excluded that adrenic acid is activated to acyl-CoA inside the mitochondria, it is probable that most of the/t-oxidation of this fatty acid in hepatocytes from fructose-refed rats is caused by peroxisomal oxida- tion. Previous experiments with [2-14C]adrenic acid (22: 4(n - 6)) and [1-14C]arachidonic acid (20: 4(n - 6)) also showed that the first cycle of/i-oxidation of adrenic acid to arachidonic acid occurs faster and is less in- hibited by fructose refeeding and by (+)-decanoyl- carnitine t~an is the subsequent /l-oxidation of arAchidonic acid [24,28]. it was therefore concluded that the rapid oxidation of adrenic acid by hepatocytes from fructose.refed rats represents peroxisomAl chain-shor- tening and that a subsequent mitochondrial oxidation of arachidonic acid is regulated by the nutritional and hormonal state of the animal. It is interesting that it has been shown that fibroblasts from patients who lack peroxisomes (Zellweger syndrome) are unable to shor- ten erucic acid (22: l (n -9 ) ) , while normal human fibroblasts [29] as well as hepatocytes shorten this sub- strafe mainly by two cycles of/I-oxidation to oleic acid (18: l(n - 9) ) .

Only small amounts of shortened [3H]Cs-Cn fatty acid intermediates were found with [9,10-3H]myristic acid (14:0) as the substrAte (Table III). This could be explained either by a complete peroxisomal/i-oxidation of medium-chain fatty acids in the intact cell or by an incomplete peroxisomal oxidation with a subsequent rapid mitochondrial oxidation of any shortened inter- mediates.

The de nero synthesis of 14C.labelled fatty acids from [2.Ct4]adrenic acid is probably formed by fatty acid synthesis from the extramitochondriai pool of acetyI-CoA formed by peroxisomal /i-oxidAtion. It is assumed that any camitine-dependent mitochondrial oxidation of Adrenic acid is as suppressed as the oxida- tion of oleic acid in hepAtoCytes from fructose.refed rat. The formation of t4C-IAbelled Acid-soluble products from adrenic acid can probably be explained by trans- port of labelled acetyl groups from the extramitochon- drial acetyI-CoA pool to the intramitochondrial pool followed by ketogenesis.

If laurate was mainly oxidized in the mitochondria after activation of the free fatty acids in the mito- chondria matrix in hepatocytes from fructose-refed animal~, a higher ratio of 14C.labelled acid-soluble products to t4C-labelled synthesized fatty acid should ~ expected with laurate as the substrate than with Adrenic acid as the substrate. The finding that this ratio is rather similar both ~ t h lauric acid and with adrenic acid as substrates (Table ll) supports the view that laurate is to a large extent oxidized by peroxisomal /l-oxidation in the cells. It is interesting that Foerster et al. [30] found that perfusion of liver with octanoate or

hexanoate stimulated the formation of hydrogen per- oxide, which they used as measure of peroxisomal /~- oxidation while perfusion with palmitate produced no detectable H202. Leighton et al. [31] found that lauric acid produced distinctly more hydrogen peroxide in isolated liver cells than observed with palmitic acid or oleic acid as substrates.

The fatty acid specificity of glycerophosphate- acylating enzymes also determines the metabolic fate of individual fatty acids in isolated liver cells. An im- portant reason for the small extent of regulation of short- and medium-chain fatty acid oxidation is that glycerophosphate-acylating enzymes show a preference for long-chain fatty acids [32]. Octanoate is thus not used directly for triacylglycerol synthesis [8,33]. The glycerophosphate-acylating activity of rat liver micro. somes was found to be 3-times higher with palmityl- carnitine than with laurylcarnitine as the precursor of the acyl-CoA substrates, while the activity was very low with octanoylcarnitine [32]. The incorporation of oleic acid into triacylglycerol has been found to be 3-times faster than the incorporation of stearate in hepatocytes in culture [34]. The substrate specificity of the glycero- phosphate--acylating enzymes may channel palmitate and oleate into triacylglycerol when the carnitine-de- pendent mitochondrial p-oxidation is inhibited by malonyl-CoA in livers from carbohydrate-refed animals. Since the glycerophosphate-acylating enzymes dis- criminate against medium.chain fatty acids as well as against very-long-chain fatty acids [35] such as erucic acid (22: l ( n - 9)), peroxisomal /i-oxidation may he important in the metabolism both of medium-chain and of very-long-chain fatty acids. In the case of medium- chain fatty acids, peroxisomt'/]-oxidation may play a role when the camitine-dependent mitochond~.ial oxida- tion is inhibited. After an initial peroxisomal/i-oxida- tion to acetyl-CoA followed by de nero synthesis, the medium-chain fatty acids can then be retailored into long-chain fatty acids and stored as triacylglycerol.

Aeknowledgements

E.C. is Fellow of the Norwegian Cancer Society. The technical assistance of Mette Gregersen, Yngvar Johan- sen and Siri Tverdal and the secretarial assistance of Maxit S.K. Thoresen is greatly appreciated.

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