170
BBALIP
53790
Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells Morten Gr@nn, Erik Christensen,
Tor-Arne Hagve and Bj@-n 0. Christophersen
Itzstituie fi~r C‘linicul Biochcnrist~.
Urzir,rrsiry of Oslo. Riksho.sprtukt,
(Received
Key words:
Fatty acid oxidation;
Clofibrate;
Oslo (Rionr~uy)
6 May 199 I )
J’-Desaturase;
A”-Desaturase;
(Rat hepatocyte)
The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal &oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C,, and C,, fatty acids recovered, was observed when 18:2(n-6), 20:3(n-61, 18:3(n-3) and 20:5(n-31, but not when 20: 4(n-6) and 22: 4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial ace@-CoA produced from peroxisomal P-oxidation is more easily used for fatty acid new synthesis than ace@-CoA from mitochondrial &oxidation. The A 6 and A 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.
Introduction
Clofibrate (ethyl-p-chlorophenoxyisobutyrate) is an effective hypolipidernic agent which has been used in the treatment of human hyperlipidemias [1,2]. Other related fibrates have been introduced in recent years. Several studies have shown that clofibrate causes peroxisomal proliferation and increases peroxisomal fatty acid p-oxidation 5-lo-fold in rats [ 1,3,4]. The mitochondrial activity is also affected, but to a lesser extent [5,6]. The activity of palmitoyl carnitine CoA acyl transferase is increased 2-fold, thus clofibrate stimulates mitochondrial P-oxidation approximately 2-fold [7]. The total mitochondrial mass is also increased [8]. Other peroxisomal proliferating agents have, however, been shown to inhibit mitochondrial fatty acid oxidation [9-l 11. Clofibrate feeding in addition increases the activity of long chain acyl CoA synthetase, acyl-CoA dehydrogenase, d 4 enoyl CoA reductase and the concentration of fatty acid binding protein [12-151. Peroxisome proliferators are also thought to cause
Correspondence: M. Gwinn, Institute hospitalet, N-0027 Oslo I. Norway.
of Clinical
Biochemistry.
Riks-
proliferation of smooth endoplasmatic reticulum [16,17]. Recent studies have shown increased conversion of linoleic acid to arachidonic acid in liver microsomes isolated from rats fed clofibric acid and fenofibrate [ 18,191. Since the activity of the peroxisomal B-oxidation is stimulated distinctly more than the activity of other fatty acid metabolizing enzymes, clofibrate feeding has been used to study the role of peroxisomes in fatty acid oxidation [1,6,20]. In the present work clofibrate feeding is used to study the role of peroxisomal fatty acid oxidation in the metabolism of polyunsaturated fatty acids, and also the effect of clofibrate on esterification and on fatty acid desaturation and chain-elongation in isolated hepatocytes. Materials
and Methods
Chemicals [2-“C]Adrenic acid (22: 4(n-6)) was from Amersham International, Amersham, U.K. [U- “Cl- and [l“Cleicosapentaenoic acid (20 : 5(n-311, [I- ‘“Clarachidonic acid (20: 4(n-6)), [l-‘“C]dihomogammalinolenic acid (20: 3(n-6)), [l-‘4C]linolenic acid (18 : 3(n-3)) and [l-‘4C]linoleic acid (18 : 2(n-6)) were obtained from New England Nuclear, Boston, MA, U.S.A. Radiolabelled
171 substrates were analyzed by radio-gas chromatography or HPLC connected to a radioactive flow detector, and impurities were less than 3%. ( + 1- Decanoylcarnitine was synthesized according to the method of Bohmer and Bremer [21]. ( + 1 - Lactate, essentially fatty acidfree bovine serum albumin, 4-(2-hydroxyethyll-lpiperazine-ethane-sulphonic acid (HEPES), collagenase type I and unlabelled eicosapentaenoic, arachidonic, dihomogammalinolenic, linolenic and linoleic acid were from Sigma Chemical Co., St. Louis, MO, U.S.A. Unlabelled adrenic acid was from 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, Basel, Switzerland. Clofibrate was obtained from ICI Industrial Company, Macclesfield, U.K. Animals
Male weanling rats of the Wistar strain were from Mollegaard Laboratory (Denmark). The animals were fed standard pellets and had free access to food and water unless otherwise stated. Some animals were fed a clofibrate containing diet for 5 days when stated. The clofibrate diet was prepared by soaking the pellets in acetone containing clofibrate (0.3 g clofibrate/lOO g of pellets). The acetone was blown off by air under constant stirring. Preparation of liL\ercells
Parenchymal according to the 0.03 M HEPES medium. About from each liver, by resistance to
liver cells were prepared and purified method of Seglen [22] except that only buffer was used in the suspension (200-400) . 10h cells were obtained and 90-95% were viable, as measured uptake of Trypan blue.
incubation medium in 25 ml Erlenmeyer flasks). Cells were incubated at 37°C in an oxygenated suspension medium [23] with 1.5% (w/v) bovine serum albumin with 200 nmol of [14C]-labelled fatty acid. The specific activity of all [14C]-labelled fatty acid substrates was 7 mCi/mmol. When indicated the hepatocytes were preincubated with 10 mM (+)-lactate, 1 mM (-I-carnitine or 1 mM (+)-decanoylcarnitine for 30 min. Analytical methods
The measurement of radioactive acid-soluble products (as a measure of the rate of P-oxidation) and of radioactive CO, were performed as described by Christiansen [24]. The lipids were extracted by the method of Folch et al. [25] and separated on silicic acid thin-layer plates (Stahl Hf) [26]. Aliquots of the total lipid extracts and the free fatty acid, triacylglycerol and phospholipid fractions were transmethylated [27], and analyzed by radio-gas chromatography. Fatty acid methyl esters were separated at 170°C with 10 min initial hold followed by a programmed temperature increase of 4”C/min to 250°C using 10% SP-2340 on Supelcoport 100/120 (Supelco Inc. Bellefonte, PA, U.S.A.). 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 -20°C to prevent peroxidation of unsaturated fatty acids. Cellular protein was determined according to the method of Lowry et al. [28]. Data are reported as mean + S.D. Statistical comparisons were made using Students’ unpaired t-test. Statistical significance was taken as P < 0.05.
Incubations
Results
The concentration of hepatocytes in the preparation was approximately 10. lo6 cells/ml and 1 ml of this suspension was used (with a total volume of 2 ml of
Table I shows that clofibrate feeding strongly stimulates the oxidation of all the unsaturated fatty acids
TABLE
1
The effect of clofibrate on the oxidation and esterification in hepatocytes incubated with [I-‘.‘C]linoleic acid (18:2(n-6)). (18:3(n-3)). [l”‘C]dihomogammalinolenic acid (20:3(n-6)), [I-“Clarachidonic acid (20:4(n-6)), [I-“Cleicosapentaenoic [2- “Cladrenic
[l-‘JC]linolenic acid (20:5(n-3))
acid and
acid (22: 4(n-6))
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acid oxidized, esterified in triacylglycerol and phospholipids. Mean k S.D. of two parallel incubations of hepatocytes from three different livers are given Fatty acid substrate
Oxidation Control
products Clofibrate
Control
Clofibrate
Control
Clofibrate
18:2(n-6) 18 : 3(n-3) 20 : 3tn-6) 20 : 4tn-6) 20 : 5(n-3) 22 : 4tn-6)
32.3 * 5.0 36.6 + 2.0 30.4 + 5.3 32.1+ 6.9 55.9 + 8.3 91.5k6.5
66.1 f 6.8 121.5*7.3 72.0 k 9.1 87.3 k 9.7 128.4 f 7.5 148.3 + 6.1
87.9 f 2.9 122.0k9.2 70.2k5.2 49.4 f 5.9 90.4 + 7.3 75.Ok 8.9
29.7k5.7 33.6 f 2.8 29.7 + 5.5 9.0+2.0 20.5 + 4.4 20.6 + 4.8
79.4+8.3 35.7 * 6.8 90.2 + 4.3 111.7k3.4 48.6 + 9.0 24.6+ 1.1
99.9 f 37.6 k 93.3 k 109.7 + 48.1+ 26.6 +
Triacylglycerol
Phospholipid
7.3 9.4 6.5 6.0 7.9 3.8
172 used. With linolenic acid (18:3(n-3)) and eicosapentaenoic acid (20 : G-3)) as substrates approximately 65% of the substrate added was oxidized and with adrenic acid (22 : 4(n-6)) 75% of the substrate was oxidized to acid soluble products and CO, in hepatocytes from clofibrate treated rats compared to 20-30% and 45% in the controls respectively. With linoleic acid (18 : 2(n6)) dihomogammalinolenic acid (20 : 3(n-6)) and arachidonic acid (20: 4(n-6)) as substrates a smaller fraction, approximately 40%, of the substrates was oxidized in hepatocytes from clofibrate treated animals compared to 15% in the controls. Almost the total increase in the fatty acid oxidation in hepatocytes after clofibrate treatment compared to the controls could be accounted for by a corresponding decrease in the acylation in triacylglycerol (Table I). In hepatocytes from the standard pellets fed animals the two n-3 fatty acids linolenic acid (18 : 3(n-3)) and eicosapentaenoic acid (20 : 5(n-3)) were esterified in the triacylglycerol fraction to a particularly large extent (approximately 65% and 45% of the substrate used). In hepatocytes from clofibrate fed rats less than 15% of both the n-3 and n-6 fatty acid substrates were esterified in triacylglycerol. Although the individual unsaturated fatty acid substrates are esterified in the phospholipids to a very different extent (Table I), the incorporation into phosphohpids was remarkably constant in cells from clofibrate treated rats compared to the controls. Thus with linoleic acid (18 : 2(n-6)), dihomogammalinolenic acid (20: 3(n-6)) and arachidonic acid (20: 4(n-6)). a large fraction, 40-50% of the substrate, was esterified in the phospholipids both in hepatocytes from the clofibrate treated rat and from the controls. With adrenic acid (22: 4(n-6)) little of the substrate, less than 15%. was recovered in the phospholipids with both types of hepatocytes. Nearly all the fatty acid substrate was metabolized, either esterified or oxidized under the conditions used. ( +)-Decanoylcarnitine is known to be an efficient inhibitor of fatty acid oxidation in hepatocytes 126,291.
TABLE
Table II shows that the inhibitory effect on the fatty acid oxidation is much weaker with cells from clofibrate treated animals than in the control cells. Lactate has also been found to inhibit the fatty acid oxidation in hepatocytes [30]. The results presented in Table I are from cells incubated in the presence of lactate. The data on the oxidation of arachidonic acid (20:4(n-6)) and cicosapentaenoic acid (70 : 5(n-3)) presented in Table I compared to the data in Table II shows that lactate inhibits fatty acid oxidation in hepatocytes from clofibrate treated rats where ( + )-decanoylcarnitine has little inhibitory effect. In some experiments [U-‘“Cl eicosapentaenoic acid was used as the substrate for hepatocytes from clofibrate treated rats and ( + )-decanoylcarnitine was added to inhibit mitochondrial fatty acid oxidation (not shown). Under these conditions peroxisomal fatty acid oxidation should be expected to account for an important part of the high total oxidation of eicosapentaenoic acid. Shortened fatty acids which might have been formed by partial P-oxidation of the [U- “Cllabelled substrate were however not found. This observation was in contrast to the generally accepted view that peroxisomal fatty acid oxidation is incomplete and limited to a few cycles of P-oxidation. These results suggest that any shortened [‘“Cl fatty acid intermediate formed had been rapidly removed either by mitochondrial @-oxidation or possibly by a complete peroxisoma1 p-oxidation. Tables III and IV show that the [“Cl-1abelled polyunsaturated fatty acid substrates were subject to chain-elongation and desaturation in hepatocytes from clofibrate treated rats and from the controls. Some of the [‘“Cl from the [I-‘“CJ- or [2-‘3C]-labelled suhstrates was also recovered in newly synthesized fatty acids primarily as palmitic acid (16: 01, with smaller amounts of palmitoleic acid ( 16 : I ). stearic acid (18 : 0) and oleic acid (18 : 1) as previously observed with control hepatocytes [6,3 I]. Table III shows that little [2-“Cl adrenic acid
II
The effect of clofibrate feeding and addition of ( - )-carnitine and ( + )-decanoylcarnitine on the oxidation und esterification in heputocytes incubated with [I- “C]arachidonic acid and [I- “‘C]eicosapentaenoic mid The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min. The results are expressed as nmol of [‘4C]labelled fatty acid oxidized or esterified in triacylglycerol and phospholipids. Mean + SD. of two parallel incubations of hepatocytes from three different livers are given Fatty acid substrate
Additions
20:4(n-6)
Carnitine Decanoylcarnitine None Carnitine Decanoylcarnitine None
20:5(n-3)
Oxidation
products
Phospholipids
Triacylglycerol
Control
Clofibrate
Control
Clofibrate
Control
Clofihrate
66.3 f 2.6 43.5f3.6 55.5 * 5.‘) 88.7 k 5.6 48.4 k 3.6 63.2? 6.8
118.3 k 9.7 114.0 * 8.6 94.2+5.3 16X.7+ 8.7 155.9+ 1.2 160.1 f 9.9
48.3 k5.8 70.2 k 5.3 50.6 k 7.3 X3.6 + 6.X 130.5 rf-8.X 89.1 k 5.5
2.8i 10.4* 3.1 f 4.8+ 10.8+ 7.1+
80.5 *3.x 75.2 i 9.7 x9.7 * 5.3 33.2k5.6 18.2 f 3.5 29.0 f 3.5
76.4 + 4.6 74.2 2 5.0 YO.6 + 2.0 22.3t3.1 74.2+3.1 29.6 * 4.1
1.0 3.7 1.2 7.5 3.2 2.4
173 TABLE III The effect of clofbrate on the pattern of [14C]labelled fatty acids in isolated hepatocytes incubated with [I- 14C]linoleic acid, [1- “C]dihomogammalinolenic acid, [I - “C]arachidonic acid and [2- 14C]adrenic acid
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 mm in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acids. A 6 desaturase activity are expressed as sum of 18: 3,20: 3, 20:4,22:4 and 22:5 in % of these five + 18: 2. A 5 desaturase activity are expressed as sum of 20:4, 22: 4 and 22: 5 in % of these three + 20: 3. Chain-elongations were calculated as the sum of chain-elongated fatty acids formed in % of these and the substrate for elongation. Mean f S.D. of two parallel incubations of hepatocytes from three different livers are given Fatty acid in phospholipids and triacylglycerol
Diet and fatty acid substrate
18:2 22:5 2214 20~4 20:3 20:2 18:3 18:2 3 6 Desaturase activity A 5 Desaturase activity Newly synthesized fatty acids Chain-elongation of C,, to C,, and Caz Chain-elongation of Cza to c2z
Clofibrate
Control
4.6k 1.3 4.2If: 1.2 3.0 + 0.4 2.2 f 0.3 150.3 + 4.2 6.8 k 0.3 52.2 + 1.9 7.2k0.5
20:3 2.2kO.2 72.8 f 3.8 86.Ok4.0
46.5 + 2.5 2.4 f 0.9
20~4
2214
3.0+ 1.0 17.9 k 3.4 158.4 + 6.3
5.3* 1.0 76.9 f 2.8
3.6+ 1.2
44.7 + 5.6
7.2 1.4
18:2
9.7 + 2.7 5.6kO.9 2.5 + 0.4 1.9kO.3 110.7+5.6 13.45 1.0 b 63.3 k 4.2 a 11.6k2.0 a
20:3
20:4
1.5kO.3 62.1+ 7.0 50.4+ 1.0
1.3 * 0.9 112.5 k6.8
55.8k3.3 a 13.6+ 1.5 b
4.8 f 2.0
2214 1.3kO.2 23.5 + 1.0
28.7k4.6
13.7 11.7
1.3
1.1
a P < 0.05, b P < 0.001 compared to corresponding control.
(22 : 4(n-6)) remained as intact substrate after incubation of the substrate with hepatocytes from clofibrate treated rats. Newly synthesized [14Cl labelled C,, and
C,, fatty acids accounted for nearly 60% of the [14C] labelled fatty acids with [2- 14C] adrenic acid (22 : 4(n-6)) as the substrate for these cells. With hepatocytes from
TABLE IV The effect of clofibrate on the pattern of [‘JC]labelled pentaenoic acid
fatty acids in isolated hepatocytes incubated with [I-‘4C]l. zno1enic acid and [l-‘4C]eicosa-
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acids. A 6 desaturase activity are expressed as sum of 18:4, 20:4, 20: 5, 22:5 and 22:6 in % of these five + 18: 3. A 5 desaturase activity are expressed as sum of 20:5, 22 : 5 and 22: 6 in % of these three + 20 : 4. Chain-elongations were calculated as the sum of chain-elongated fatty acids formed in % of these and the substrate for elongation. Mean f SD. of two parallel incubations of hepatocytes from three different livers are given Fatty acid in phospholipids and triacylglycerol 22:6 22:5 20:5 2014 20:3 18:4 18:3 A 6 Desaturase activity A 5 Desaturase activity Newly synthesized fatty acids Chain-elongation of C,, to C,, and C,, Chain-elongation of C,, to caa
Diet and fatty acid substrate Control
Clofibrate
18:3
20:5
18:3
20:5
1.2kO.3 4.7kO.4 ll.lk1.9 4.8 + 0.4 2.7+0.6 2.9 f 0.2 151.0+6.1 14.0 + 0.9 78.0 f 5.3 5.0 * 2.2
3.2k 1.2 21.3+2.1 139.0 f 3.5
0.8kO.2 1.8kO.4 11.4+ 1.8 1.1 kO.2 1.5 + 0.3 3.7 + 0.3 43.6 k 6.2 30.1 k 2.4 b 92.7k3.5 a 15.454.2 =
2.250.7 8.0+ 1.0 46.6 f 9.8
6.3 f 2.4
26.0
13.7 24.1
a P < 0.05, b P < 0.001 compared to corresponding control.
18.4 k 4.1 a
15.0
15.7
18.0
174 the control rats larger amounts of the intact adrenic acid (22 : 4(n-6)) substrate were recovered and the newly synthesized fatty acids accounted for approximately 35% of the total [‘“Cl labelled fatty acids after the incubations. With [l-“Cl arachidonic acid as the substrate, more [“Cl-1abelled adrenic acid (22 : 4(n-6)) was recovered in control cells than in cells from clofibrate treated rats (Table III). With 20: 4(n-6) as substrate only small amounts of newly synthesized fatty acids, accounting for approximately 2% of the total fatty acids. were formed both in hepatocytes from the control and from clofibrate treated rats, respectively. Table III also shows that with 20 : 3tn-6) as substrate less [“Cllabelled 20 : 4(n-61 was recovered in cells from clofibrate fed animals compared to control fed. A more pronounced reduction in the amount of intact csterified 20 : 3(n-61 substrate was, however, observed in cells from clofibrate fed. This finding suggests that the -1 5 desaturase activity was stimulated by the clofibrate feeding. A markedly increased amount of newly synthesized fatty acids was recovered in the cells from clofibrate fed animals incubated with 20: Xn-6). S-6 times that found with control hepatocytes. With [l-‘JC] eicosapentaenoic acid as the substrate more newly synthesized [“Cl labelled C,, and C,, fatty acids were formed, 2.9 times that of controls, and less of the substrate was elongated to C,, fatty acids in the hepatocytes from clofibrate treated rats than from the controls (Table IV). With [1-“Cl linoleic acid as the substrate more arachidonic acid (20 : 4tn-61) and dihomogammalinolenic acid (20:3(n-61) were formed in cells from clofibrate treated rats than with cells from the controls, although more intact linoleic acid was recovered in the cells from the controls (Table III). This suggests that the J 6 desaturase activity was increased in the hepatocytes from the clofibrate treated rats. This view is supported by the finding with [ I-“Cllinolenic acid (18: 3(n-31) as the substrate (Table IV). Nearly the same amounts of [“Cllabelled eicosapentaenoic acid were recovered in both types of cells although more than three times more of the intact esterified linolenic acid remained in the control cell than in the hepatocytes from clofibrate treated rats. Less [“‘Cl-labelled C,, fatty acids were formed from [I-“Cl linolenic acid (18: 3(n-3)) in the clofibrate treated rats than in the controls (Table IV). With both C,, substrates, more newly synthesized [‘JCl-labelled C ,h and C ,x fatty acids were recovered in hepatocytes from clofibrate fed animals than control fed, 1.6 tlinoleatel and 3.1 tlinolenate) times that of controls. respectively. Discussion The present results show that clofibrate feeding has profound effects on the metabolism of polyunsaturated
fatty acids in liver cells. Fatty acid desaturation. chainelongation, oxidation and esterification are affected in a way which is specific for each individual n-3 and n-6 fatty acid. The increased /Soxidation induced by clofibrate feeding in the present study was considerable and in agreement with the view that clofibrate stimulates both mitochondrial and especially peroxisomal P-oxidation (Table Il. A greater increase in P-oxidation was observed when the two n-3 fatty acids. 1X:3 and 20:~~. were used as substrates compared to the n-6 fatty acids. 1X: 2, 30: 3 and 70: 1. In corresponding feeding experiments dietary n-3 fatty acids especially atimulated the peroxisomal /&oxidation when n-3 fatty acids were used as substrates [33]. These findings support the view that n-3 fatty acids arc oxidized in pcroxisomes to a greater extent than n-6 fatty acids arc. The main reason why t + l-decanoylcarnitine has little effect after clofibrate treatment (Table II) is probably that the peroxisomal fatty acid oxidation which is unaffected by ( + ) - dccanoylcarnitine. plays a more important role in fatty acid oxidation in these cells. The fact that clofibrate feeding stimulates long chain acyl carnitine CoA transferase and mitochondrial oxidation two fold and also increases the cndogenous carnitine concentration may also rcducc the inhibitory effect of ( + ) ~ decanoylcarnitine [5-71. Adrenic acid (72 : 4(n-6)) was efficiently oxidized in both types of hepatocytes. The percentage increase in P-oxidation caused by clofibrate was smallest with this substrate (Table Il. Clofibrate feeding reduced the intact adrenic acid remaining after incubation to approximately 25% of that in the control, while the newly synthesized [ ‘JC]-labelled fatty acids were less reduced. to 60% of the control. All this agrees with the view that adrenic acid (22: 4(n-6)) is predominantly oxidized in the peroxisomes, also in control cells 1-31,331. The newly synthesized fatty acids formed from [2-“Cl adrenic acid. C ,h and C,, fatty acids, may not bc rcmovcd by oxidation to the same extent after clofibrate feeding since peroxisomal oxidation is probably less important in the metabolism of these fatty acids. The acetate units produced by P-oxidation can either be oxidized to CO, and H,O. converted to ketone bodies or used for extra-mitochondrial synthetic purposes such as fatty acid synthesis. Acctyl-CoA produced by extra-mitochondrial /S-osidation should then be more easily used in fatty acid synthesis than acetylCoA from mitochondrial p-oxidation. In all cxperiments with 1X : Zn-61. 1X : 3(n-31, 20 : .i(n-6) and 20: S(n-3), a marked increase in the amounts of newly synthesized fatty acids in cells from animals fed clofibratc was observed (Tables III and IV). This increase in newly synthesized C,, and C,, fatty acids probably reflects an increased production of extra-mitochondrial acetyl-CoA caused by a stimulated peroxisomal fi-
175 oxidation [29]. The extent of increase in newly synthesized fatty acids induced by clofibrate observed in the present study, was consistent with previous reports. In a recent study with isolated rat liver microsomes where only 18: 2(n-6) was used as substrate, the increase in newly synthesized fatty acids was 3.8 times that of control rats [HI. The elongation of C,, to C,, fatty acids seemed to be stimulated by clofibrate, but less C,, products were formed from C,, fatty acids in hepatocytes from clofibrate fed animals (Tables III and IV). The increased conversion of C,, to C,, fatty acids may be the result of both an increased A 6 desaturase activity and an increased supply of extra-mitochondrial acetyl-CoA for chain-elongation. With both arachidonic acid and eicosapentaenoic acid as substrates any increased chain-elongation of C,, to C,, products caused by increased supply of acetyl-CoA was probably more than outweighed by the strongly increased retroconversion of C,, to C,, fatty acids which has been shown to be a peroxisomal function 134,351. Polyunsaturated fatty acids with different chain lengths and number and position of double bonds are incorporated in phospholipids with a very high fatty acid specificity. In the present work we found that the individual polyunsaturated fatty acids are esterified in the phospholipids to the same extent in the controls as in hepatocytes from clofibrate treated rats. This finding agrees with the view that the reactions incorporating polyunsaturated fatty acids in the phospholipid fractions have distinctly lower K, values than competing pathways such as fatty acid oxidation and triacylglycerol synthesis. The incorporation of polyunsaturated fatty acids in phospholipids is also little affected by fasting and feeding which strongly affects the distribution of fatty acids between oxidation and triacylglycerol synthesis [35,36]. With both C,, substrates the calculated A 6 desaturase activity was increased by 100% by clofibrate feeding, while the activity of the A 5 desaturase was increased by 20% compared to control cells. A greater reduction in the concentration of the intact substrate, caused by the increased P-oxidation, than in the content of the desaturated products could explain part of the apparent stimulating effect of clofibrate feeding on the A 6 and A 5 desaturase activities. With linoleic acid and linolenic acid as substrates the amounts of A 6 desaturated products were however increased with (18 : 2(n-6)) or remained unchanged with (18 : 3 (n-3)). Conclusively, clofibrate feeding caused a real induction of the A 6 desaturase activity with these two C,, fatty acid substrates. These findings with isolated hepatocytes were consistent with results from recent studies with isolated rat liver microsomes [18,19]. Peroxisomal proliferators like
clofibric acid and fenofibrate, were found to increase the conversion of linoleate to arachidonic acid by inducing the A 6 and A 5 desaturase activities. The extent of the increases in the A 6 and A 5 desaturase activities in the present experiments with clofibrate, were approximately the same as observed with isolated microsomes in the study with fenofibrate, but lower than that observed with clofibric acid [HI. The results indicate that induction of increased activity of the two desaturases is common to peroxisome proliferators [lS]. This effect is different from the effects of n-3 fatty acids. The feeding of eicosapentaenoic acid (20 : 5(n-3)) and docosahexaenoic acid (22 : 6(n-3)) stimulate peroxisomal fatty acid oxidation in isolated hepatocytes [32]. But at the same time and in contrast to clofibrate dietary n-3 fatty acids inhibit A 5 and A 6 desaturase activity in hepatocytes [32] and isolated microsomes [37-391. The effects of clofibrate and of n-3 fatty acid feeding are thus probably mediated by different mechanisms. Acknowledgements
The technical assistance of Mette Gregersen, Yngvar Johansen, Anne Marie Lund and Siri Tverdal and the secretarial assistance of Marit S.K. Thoresen are greatly appreciated. References 1 Lazarow, P.B. and de Duve, C. (1976) Proc. Natl. Acad. Sci. USA 73, 2043-2046. 2 Lazarow, P.B. (1977) Science 197, 580-581. 3 Svoboda, J., Grady, H. and Azarnoff, D.L. (1967) J. Cell. Biol. 3.5, 127-152. 4 Reddy, J.K., Azamoff, D.L., Svoboda, J. and Prasad, D. (1974) J. Cell. Biol. 61, 344-358. 5 Mannaerts, G.P., Thomas, J., Debeer, L.J.. McGarry, J.D. and Foster, D.W. (1978) Biochim. Biophys. Acta 529, 201-211. 6 Mannaerts, G.P., Debeer, L.J., Thomas, J. and DeSchepper, P.J. (19791 J. Biol. Chem. 254, 4585-4595. 7 Solberg, H.E., Aas, M. and Daae. L.N.W. (1972) Biochim. Biophys. Acta 280, 434-439. 8 Kurup, C.K.R., Aithal, H.N. and Ramasarma, T. (19701 Biochem. J. 116, 773-779. 9 Eacho, P.I. and Foxworthy, P.S. (19881 Biochem. Biophys. Res. Commun. 3, 1148-1153. 10 Foxworthy, P.S. and Eacho, P.I. (19881 J. Biochem. 252, 409-414. 11 Horie, S. and Suga, T. (19851 Biochem. Pharmacol. 34, 1357-1362. 12 Furuta. S., Miyazawa, S. and Hashimoto, T. (1982) J. Biochem. 90, 1751-1756. 13 Borrebaek, B., Osmundsen, H. and Bremer. J. (19801 Biochem. Biophys. Res. Commun. 93, 1173-1180. 14 Prasad, M.R., Nagi, M.N., Ghesquier, D.. Cook, L. and Cinti, D.L. (19861 J. Biol. Chem. 261, 8213-8217. 15 Kawashima, Y., Nakagawa, S., Tachibana, Y. and Kokuza, H. (1983) Biochem. Biophys, Res. Comm. 754, 21-27. 16 Svoboda, D.J. and Azarnoff, D.L. (1966) J. Cell. Biol. 30,442-450. 17 Reddy, J.K. and Krishnakantha, T.P. (19751 Science 190,787-789. 18 Kawashima, Y., Musoh, K. and Kokuza, H. (19901 J. Biol. Chem. 265. 9170-9175.
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Rwtvrit, 10X2. 57-Q
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BBALIP
53790
Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells Morten Gr@nn, Erik Christensen,
Tor-Arne Hagve and Bj@-n 0. Christophersen
Itzstituie fi~r C‘linicul Biochcnrist~.
Urzir,rrsiry of Oslo. Riksho.sprtukt,
(Received
Key words:
Fatty acid oxidation;
Clofibrate;
Oslo (Rionr~uy)
6 May 199 I )
J’-Desaturase;
A”-Desaturase;
(Rat hepatocyte)
The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal &oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C,, and C,, fatty acids recovered, was observed when 18:2(n-6), 20:3(n-61, 18:3(n-3) and 20:5(n-31, but not when 20: 4(n-6) and 22: 4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial ace@-CoA produced from peroxisomal P-oxidation is more easily used for fatty acid new synthesis than ace@-CoA from mitochondrial &oxidation. The A 6 and A 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.
Introduction
Clofibrate (ethyl-p-chlorophenoxyisobutyrate) is an effective hypolipidernic agent which has been used in the treatment of human hyperlipidemias [1,2]. Other related fibrates have been introduced in recent years. Several studies have shown that clofibrate causes peroxisomal proliferation and increases peroxisomal fatty acid p-oxidation 5-lo-fold in rats [ 1,3,4]. The mitochondrial activity is also affected, but to a lesser extent [5,6]. The activity of palmitoyl carnitine CoA acyl transferase is increased 2-fold, thus clofibrate stimulates mitochondrial P-oxidation approximately 2-fold [7]. The total mitochondrial mass is also increased [8]. Other peroxisomal proliferating agents have, however, been shown to inhibit mitochondrial fatty acid oxidation [9-l 11. Clofibrate feeding in addition increases the activity of long chain acyl CoA synthetase, acyl-CoA dehydrogenase, d 4 enoyl CoA reductase and the concentration of fatty acid binding protein [12-151. Peroxisome proliferators are also thought to cause
Correspondence: M. Gwinn, Institute hospitalet, N-0027 Oslo I. Norway.
of Clinical
Biochemistry.
Riks-
proliferation of smooth endoplasmatic reticulum [16,17]. Recent studies have shown increased conversion of linoleic acid to arachidonic acid in liver microsomes isolated from rats fed clofibric acid and fenofibrate [ 18,191. Since the activity of the peroxisomal B-oxidation is stimulated distinctly more than the activity of other fatty acid metabolizing enzymes, clofibrate feeding has been used to study the role of peroxisomes in fatty acid oxidation [1,6,20]. In the present work clofibrate feeding is used to study the role of peroxisomal fatty acid oxidation in the metabolism of polyunsaturated fatty acids, and also the effect of clofibrate on esterification and on fatty acid desaturation and chain-elongation in isolated hepatocytes. Materials
and Methods
Chemicals [2-“C]Adrenic acid (22: 4(n-6)) was from Amersham International, Amersham, U.K. [U- “Cl- and [l“Cleicosapentaenoic acid (20 : 5(n-311, [I- ‘“Clarachidonic acid (20: 4(n-6)), [l-‘“C]dihomogammalinolenic acid (20: 3(n-6)), [l-‘4C]linolenic acid (18 : 3(n-3)) and [l-‘4C]linoleic acid (18 : 2(n-6)) were obtained from New England Nuclear, Boston, MA, U.S.A. Radiolabelled
171 substrates were analyzed by radio-gas chromatography or HPLC connected to a radioactive flow detector, and impurities were less than 3%. ( + 1- Decanoylcarnitine was synthesized according to the method of Bohmer and Bremer [21]. ( + 1 - Lactate, essentially fatty acidfree bovine serum albumin, 4-(2-hydroxyethyll-lpiperazine-ethane-sulphonic acid (HEPES), collagenase type I and unlabelled eicosapentaenoic, arachidonic, dihomogammalinolenic, linolenic and linoleic acid were from Sigma Chemical Co., St. Louis, MO, U.S.A. Unlabelled adrenic acid was from 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, Basel, Switzerland. Clofibrate was obtained from ICI Industrial Company, Macclesfield, U.K. Animals
Male weanling rats of the Wistar strain were from Mollegaard Laboratory (Denmark). The animals were fed standard pellets and had free access to food and water unless otherwise stated. Some animals were fed a clofibrate containing diet for 5 days when stated. The clofibrate diet was prepared by soaking the pellets in acetone containing clofibrate (0.3 g clofibrate/lOO g of pellets). The acetone was blown off by air under constant stirring. Preparation of liL\ercells
Parenchymal according to the 0.03 M HEPES medium. About from each liver, by resistance to
liver cells were prepared and purified method of Seglen [22] except that only buffer was used in the suspension (200-400) . 10h cells were obtained and 90-95% were viable, as measured uptake of Trypan blue.
incubation medium in 25 ml Erlenmeyer flasks). Cells were incubated at 37°C in an oxygenated suspension medium [23] with 1.5% (w/v) bovine serum albumin with 200 nmol of [14C]-labelled fatty acid. The specific activity of all [14C]-labelled fatty acid substrates was 7 mCi/mmol. When indicated the hepatocytes were preincubated with 10 mM (+)-lactate, 1 mM (-I-carnitine or 1 mM (+)-decanoylcarnitine for 30 min. Analytical methods
The measurement of radioactive acid-soluble products (as a measure of the rate of P-oxidation) and of radioactive CO, were performed as described by Christiansen [24]. The lipids were extracted by the method of Folch et al. [25] and separated on silicic acid thin-layer plates (Stahl Hf) [26]. Aliquots of the total lipid extracts and the free fatty acid, triacylglycerol and phospholipid fractions were transmethylated [27], and analyzed by radio-gas chromatography. Fatty acid methyl esters were separated at 170°C with 10 min initial hold followed by a programmed temperature increase of 4”C/min to 250°C using 10% SP-2340 on Supelcoport 100/120 (Supelco Inc. Bellefonte, PA, U.S.A.). 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 -20°C to prevent peroxidation of unsaturated fatty acids. Cellular protein was determined according to the method of Lowry et al. [28]. Data are reported as mean + S.D. Statistical comparisons were made using Students’ unpaired t-test. Statistical significance was taken as P < 0.05.
Incubations
Results
The concentration of hepatocytes in the preparation was approximately 10. lo6 cells/ml and 1 ml of this suspension was used (with a total volume of 2 ml of
Table I shows that clofibrate feeding strongly stimulates the oxidation of all the unsaturated fatty acids
TABLE
1
The effect of clofibrate on the oxidation and esterification in hepatocytes incubated with [I-‘.‘C]linoleic acid (18:2(n-6)). (18:3(n-3)). [l”‘C]dihomogammalinolenic acid (20:3(n-6)), [I-“Clarachidonic acid (20:4(n-6)), [I-“Cleicosapentaenoic [2- “Cladrenic
[l-‘JC]linolenic acid (20:5(n-3))
acid and
acid (22: 4(n-6))
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acid oxidized, esterified in triacylglycerol and phospholipids. Mean k S.D. of two parallel incubations of hepatocytes from three different livers are given Fatty acid substrate
Oxidation Control
products Clofibrate
Control
Clofibrate
Control
Clofibrate
18:2(n-6) 18 : 3(n-3) 20 : 3tn-6) 20 : 4tn-6) 20 : 5(n-3) 22 : 4tn-6)
32.3 * 5.0 36.6 + 2.0 30.4 + 5.3 32.1+ 6.9 55.9 + 8.3 91.5k6.5
66.1 f 6.8 121.5*7.3 72.0 k 9.1 87.3 k 9.7 128.4 f 7.5 148.3 + 6.1
87.9 f 2.9 122.0k9.2 70.2k5.2 49.4 f 5.9 90.4 + 7.3 75.Ok 8.9
29.7k5.7 33.6 f 2.8 29.7 + 5.5 9.0+2.0 20.5 + 4.4 20.6 + 4.8
79.4+8.3 35.7 * 6.8 90.2 + 4.3 111.7k3.4 48.6 + 9.0 24.6+ 1.1
99.9 f 37.6 k 93.3 k 109.7 + 48.1+ 26.6 +
Triacylglycerol
Phospholipid
7.3 9.4 6.5 6.0 7.9 3.8
172 used. With linolenic acid (18:3(n-3)) and eicosapentaenoic acid (20 : G-3)) as substrates approximately 65% of the substrate added was oxidized and with adrenic acid (22 : 4(n-6)) 75% of the substrate was oxidized to acid soluble products and CO, in hepatocytes from clofibrate treated rats compared to 20-30% and 45% in the controls respectively. With linoleic acid (18 : 2(n6)) dihomogammalinolenic acid (20 : 3(n-6)) and arachidonic acid (20: 4(n-6)) as substrates a smaller fraction, approximately 40%, of the substrates was oxidized in hepatocytes from clofibrate treated animals compared to 15% in the controls. Almost the total increase in the fatty acid oxidation in hepatocytes after clofibrate treatment compared to the controls could be accounted for by a corresponding decrease in the acylation in triacylglycerol (Table I). In hepatocytes from the standard pellets fed animals the two n-3 fatty acids linolenic acid (18 : 3(n-3)) and eicosapentaenoic acid (20 : 5(n-3)) were esterified in the triacylglycerol fraction to a particularly large extent (approximately 65% and 45% of the substrate used). In hepatocytes from clofibrate fed rats less than 15% of both the n-3 and n-6 fatty acid substrates were esterified in triacylglycerol. Although the individual unsaturated fatty acid substrates are esterified in the phospholipids to a very different extent (Table I), the incorporation into phosphohpids was remarkably constant in cells from clofibrate treated rats compared to the controls. Thus with linoleic acid (18 : 2(n-6)), dihomogammalinolenic acid (20: 3(n-6)) and arachidonic acid (20: 4(n-6)). a large fraction, 40-50% of the substrate, was esterified in the phospholipids both in hepatocytes from the clofibrate treated rat and from the controls. With adrenic acid (22: 4(n-6)) little of the substrate, less than 15%. was recovered in the phospholipids with both types of hepatocytes. Nearly all the fatty acid substrate was metabolized, either esterified or oxidized under the conditions used. ( +)-Decanoylcarnitine is known to be an efficient inhibitor of fatty acid oxidation in hepatocytes 126,291.
TABLE
Table II shows that the inhibitory effect on the fatty acid oxidation is much weaker with cells from clofibrate treated animals than in the control cells. Lactate has also been found to inhibit the fatty acid oxidation in hepatocytes [30]. The results presented in Table I are from cells incubated in the presence of lactate. The data on the oxidation of arachidonic acid (20:4(n-6)) and cicosapentaenoic acid (70 : 5(n-3)) presented in Table I compared to the data in Table II shows that lactate inhibits fatty acid oxidation in hepatocytes from clofibrate treated rats where ( + )-decanoylcarnitine has little inhibitory effect. In some experiments [U-‘“Cl eicosapentaenoic acid was used as the substrate for hepatocytes from clofibrate treated rats and ( + )-decanoylcarnitine was added to inhibit mitochondrial fatty acid oxidation (not shown). Under these conditions peroxisomal fatty acid oxidation should be expected to account for an important part of the high total oxidation of eicosapentaenoic acid. Shortened fatty acids which might have been formed by partial P-oxidation of the [U- “Cllabelled substrate were however not found. This observation was in contrast to the generally accepted view that peroxisomal fatty acid oxidation is incomplete and limited to a few cycles of P-oxidation. These results suggest that any shortened [‘“Cl fatty acid intermediate formed had been rapidly removed either by mitochondrial @-oxidation or possibly by a complete peroxisoma1 p-oxidation. Tables III and IV show that the [“Cl-1abelled polyunsaturated fatty acid substrates were subject to chain-elongation and desaturation in hepatocytes from clofibrate treated rats and from the controls. Some of the [‘“Cl from the [I-‘“CJ- or [2-‘3C]-labelled suhstrates was also recovered in newly synthesized fatty acids primarily as palmitic acid (16: 01, with smaller amounts of palmitoleic acid ( 16 : I ). stearic acid (18 : 0) and oleic acid (18 : 1) as previously observed with control hepatocytes [6,3 I]. Table III shows that little [2-“Cl adrenic acid
II
The effect of clofibrate feeding and addition of ( - )-carnitine and ( + )-decanoylcarnitine on the oxidation und esterification in heputocytes incubated with [I- “C]arachidonic acid and [I- “‘C]eicosapentaenoic mid The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min. The results are expressed as nmol of [‘4C]labelled fatty acid oxidized or esterified in triacylglycerol and phospholipids. Mean + SD. of two parallel incubations of hepatocytes from three different livers are given Fatty acid substrate
Additions
20:4(n-6)
Carnitine Decanoylcarnitine None Carnitine Decanoylcarnitine None
20:5(n-3)
Oxidation
products
Phospholipids
Triacylglycerol
Control
Clofibrate
Control
Clofibrate
Control
Clofihrate
66.3 f 2.6 43.5f3.6 55.5 * 5.‘) 88.7 k 5.6 48.4 k 3.6 63.2? 6.8
118.3 k 9.7 114.0 * 8.6 94.2+5.3 16X.7+ 8.7 155.9+ 1.2 160.1 f 9.9
48.3 k5.8 70.2 k 5.3 50.6 k 7.3 X3.6 + 6.X 130.5 rf-8.X 89.1 k 5.5
2.8i 10.4* 3.1 f 4.8+ 10.8+ 7.1+
80.5 *3.x 75.2 i 9.7 x9.7 * 5.3 33.2k5.6 18.2 f 3.5 29.0 f 3.5
76.4 + 4.6 74.2 2 5.0 YO.6 + 2.0 22.3t3.1 74.2+3.1 29.6 * 4.1
1.0 3.7 1.2 7.5 3.2 2.4
173 TABLE III The effect of clofbrate on the pattern of [14C]labelled fatty acids in isolated hepatocytes incubated with [I- 14C]linoleic acid, [1- “C]dihomogammalinolenic acid, [I - “C]arachidonic acid and [2- 14C]adrenic acid
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 mm in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acids. A 6 desaturase activity are expressed as sum of 18: 3,20: 3, 20:4,22:4 and 22:5 in % of these five + 18: 2. A 5 desaturase activity are expressed as sum of 20:4, 22: 4 and 22: 5 in % of these three + 20: 3. Chain-elongations were calculated as the sum of chain-elongated fatty acids formed in % of these and the substrate for elongation. Mean f S.D. of two parallel incubations of hepatocytes from three different livers are given Fatty acid in phospholipids and triacylglycerol
Diet and fatty acid substrate
18:2 22:5 2214 20~4 20:3 20:2 18:3 18:2 3 6 Desaturase activity A 5 Desaturase activity Newly synthesized fatty acids Chain-elongation of C,, to C,, and Caz Chain-elongation of Cza to c2z
Clofibrate
Control
4.6k 1.3 4.2If: 1.2 3.0 + 0.4 2.2 f 0.3 150.3 + 4.2 6.8 k 0.3 52.2 + 1.9 7.2k0.5
20:3 2.2kO.2 72.8 f 3.8 86.Ok4.0
46.5 + 2.5 2.4 f 0.9
20~4
2214
3.0+ 1.0 17.9 k 3.4 158.4 + 6.3
5.3* 1.0 76.9 f 2.8
3.6+ 1.2
44.7 + 5.6
7.2 1.4
18:2
9.7 + 2.7 5.6kO.9 2.5 + 0.4 1.9kO.3 110.7+5.6 13.45 1.0 b 63.3 k 4.2 a 11.6k2.0 a
20:3
20:4
1.5kO.3 62.1+ 7.0 50.4+ 1.0
1.3 * 0.9 112.5 k6.8
55.8k3.3 a 13.6+ 1.5 b
4.8 f 2.0
2214 1.3kO.2 23.5 + 1.0
28.7k4.6
13.7 11.7
1.3
1.1
a P < 0.05, b P < 0.001 compared to corresponding control.
(22 : 4(n-6)) remained as intact substrate after incubation of the substrate with hepatocytes from clofibrate treated rats. Newly synthesized [14Cl labelled C,, and
C,, fatty acids accounted for nearly 60% of the [14C] labelled fatty acids with [2- 14C] adrenic acid (22 : 4(n-6)) as the substrate for these cells. With hepatocytes from
TABLE IV The effect of clofibrate on the pattern of [‘JC]labelled pentaenoic acid
fatty acids in isolated hepatocytes incubated with [I-‘4C]l. zno1enic acid and [l-‘4C]eicosa-
The incubation conditions were as described in Materials and Methods. Labelled fatty acids (0.1 mM) and hepatocytes (21.5-24.5 mg of protein) were incubated for 120 min in the presence of 10 mM lactate. The results are expressed as nmol of [‘4C]labelled fatty acids. A 6 desaturase activity are expressed as sum of 18:4, 20:4, 20: 5, 22:5 and 22:6 in % of these five + 18: 3. A 5 desaturase activity are expressed as sum of 20:5, 22 : 5 and 22: 6 in % of these three + 20 : 4. Chain-elongations were calculated as the sum of chain-elongated fatty acids formed in % of these and the substrate for elongation. Mean f SD. of two parallel incubations of hepatocytes from three different livers are given Fatty acid in phospholipids and triacylglycerol 22:6 22:5 20:5 2014 20:3 18:4 18:3 A 6 Desaturase activity A 5 Desaturase activity Newly synthesized fatty acids Chain-elongation of C,, to C,, and C,, Chain-elongation of C,, to caa
Diet and fatty acid substrate Control
Clofibrate
18:3
20:5
18:3
20:5
1.2kO.3 4.7kO.4 ll.lk1.9 4.8 + 0.4 2.7+0.6 2.9 f 0.2 151.0+6.1 14.0 + 0.9 78.0 f 5.3 5.0 * 2.2
3.2k 1.2 21.3+2.1 139.0 f 3.5
0.8kO.2 1.8kO.4 11.4+ 1.8 1.1 kO.2 1.5 + 0.3 3.7 + 0.3 43.6 k 6.2 30.1 k 2.4 b 92.7k3.5 a 15.454.2 =
2.250.7 8.0+ 1.0 46.6 f 9.8
6.3 f 2.4
26.0
13.7 24.1
a P < 0.05, b P < 0.001 compared to corresponding control.
18.4 k 4.1 a
15.0
15.7
18.0
174 the control rats larger amounts of the intact adrenic acid (22 : 4(n-6)) substrate were recovered and the newly synthesized fatty acids accounted for approximately 35% of the total [‘“Cl labelled fatty acids after the incubations. With [l-“Cl arachidonic acid as the substrate, more [“Cl-1abelled adrenic acid (22 : 4(n-6)) was recovered in control cells than in cells from clofibrate treated rats (Table III). With 20: 4(n-6) as substrate only small amounts of newly synthesized fatty acids, accounting for approximately 2% of the total fatty acids. were formed both in hepatocytes from the control and from clofibrate treated rats, respectively. Table III also shows that with 20 : 3tn-6) as substrate less [“Cllabelled 20 : 4(n-61 was recovered in cells from clofibrate fed animals compared to control fed. A more pronounced reduction in the amount of intact csterified 20 : 3(n-61 substrate was, however, observed in cells from clofibrate fed. This finding suggests that the -1 5 desaturase activity was stimulated by the clofibrate feeding. A markedly increased amount of newly synthesized fatty acids was recovered in the cells from clofibrate fed animals incubated with 20: Xn-6). S-6 times that found with control hepatocytes. With [l-‘JC] eicosapentaenoic acid as the substrate more newly synthesized [“Cl labelled C,, and C,, fatty acids were formed, 2.9 times that of controls, and less of the substrate was elongated to C,, fatty acids in the hepatocytes from clofibrate treated rats than from the controls (Table IV). With [1-“Cl linoleic acid as the substrate more arachidonic acid (20 : 4tn-61) and dihomogammalinolenic acid (20:3(n-61) were formed in cells from clofibrate treated rats than with cells from the controls, although more intact linoleic acid was recovered in the cells from the controls (Table III). This suggests that the J 6 desaturase activity was increased in the hepatocytes from the clofibrate treated rats. This view is supported by the finding with [ I-“Cllinolenic acid (18: 3(n-31) as the substrate (Table IV). Nearly the same amounts of [“Cllabelled eicosapentaenoic acid were recovered in both types of cells although more than three times more of the intact esterified linolenic acid remained in the control cell than in the hepatocytes from clofibrate treated rats. Less [“‘Cl-labelled C,, fatty acids were formed from [I-“Cl linolenic acid (18: 3(n-3)) in the clofibrate treated rats than in the controls (Table IV). With both C,, substrates, more newly synthesized [‘JCl-labelled C ,h and C ,x fatty acids were recovered in hepatocytes from clofibrate fed animals than control fed, 1.6 tlinoleatel and 3.1 tlinolenate) times that of controls. respectively. Discussion The present results show that clofibrate feeding has profound effects on the metabolism of polyunsaturated
fatty acids in liver cells. Fatty acid desaturation. chainelongation, oxidation and esterification are affected in a way which is specific for each individual n-3 and n-6 fatty acid. The increased /Soxidation induced by clofibrate feeding in the present study was considerable and in agreement with the view that clofibrate stimulates both mitochondrial and especially peroxisomal P-oxidation (Table Il. A greater increase in P-oxidation was observed when the two n-3 fatty acids. 1X:3 and 20:~~. were used as substrates compared to the n-6 fatty acids. 1X: 2, 30: 3 and 70: 1. In corresponding feeding experiments dietary n-3 fatty acids especially atimulated the peroxisomal /&oxidation when n-3 fatty acids were used as substrates [33]. These findings support the view that n-3 fatty acids arc oxidized in pcroxisomes to a greater extent than n-6 fatty acids arc. The main reason why t + l-decanoylcarnitine has little effect after clofibrate treatment (Table II) is probably that the peroxisomal fatty acid oxidation which is unaffected by ( + ) - dccanoylcarnitine. plays a more important role in fatty acid oxidation in these cells. The fact that clofibrate feeding stimulates long chain acyl carnitine CoA transferase and mitochondrial oxidation two fold and also increases the cndogenous carnitine concentration may also rcducc the inhibitory effect of ( + ) ~ decanoylcarnitine [5-71. Adrenic acid (72 : 4(n-6)) was efficiently oxidized in both types of hepatocytes. The percentage increase in P-oxidation caused by clofibrate was smallest with this substrate (Table Il. Clofibrate feeding reduced the intact adrenic acid remaining after incubation to approximately 25% of that in the control, while the newly synthesized [ ‘JC]-labelled fatty acids were less reduced. to 60% of the control. All this agrees with the view that adrenic acid (22: 4(n-6)) is predominantly oxidized in the peroxisomes, also in control cells 1-31,331. The newly synthesized fatty acids formed from [2-“Cl adrenic acid. C ,h and C,, fatty acids, may not bc rcmovcd by oxidation to the same extent after clofibrate feeding since peroxisomal oxidation is probably less important in the metabolism of these fatty acids. The acetate units produced by P-oxidation can either be oxidized to CO, and H,O. converted to ketone bodies or used for extra-mitochondrial synthetic purposes such as fatty acid synthesis. Acctyl-CoA produced by extra-mitochondrial /S-osidation should then be more easily used in fatty acid synthesis than acetylCoA from mitochondrial p-oxidation. In all cxperiments with 1X : Zn-61. 1X : 3(n-31, 20 : .i(n-6) and 20: S(n-3), a marked increase in the amounts of newly synthesized fatty acids in cells from animals fed clofibratc was observed (Tables III and IV). This increase in newly synthesized C,, and C,, fatty acids probably reflects an increased production of extra-mitochondrial acetyl-CoA caused by a stimulated peroxisomal fi-
175 oxidation [29]. The extent of increase in newly synthesized fatty acids induced by clofibrate observed in the present study, was consistent with previous reports. In a recent study with isolated rat liver microsomes where only 18: 2(n-6) was used as substrate, the increase in newly synthesized fatty acids was 3.8 times that of control rats [HI. The elongation of C,, to C,, fatty acids seemed to be stimulated by clofibrate, but less C,, products were formed from C,, fatty acids in hepatocytes from clofibrate fed animals (Tables III and IV). The increased conversion of C,, to C,, fatty acids may be the result of both an increased A 6 desaturase activity and an increased supply of extra-mitochondrial acetyl-CoA for chain-elongation. With both arachidonic acid and eicosapentaenoic acid as substrates any increased chain-elongation of C,, to C,, products caused by increased supply of acetyl-CoA was probably more than outweighed by the strongly increased retroconversion of C,, to C,, fatty acids which has been shown to be a peroxisomal function 134,351. Polyunsaturated fatty acids with different chain lengths and number and position of double bonds are incorporated in phospholipids with a very high fatty acid specificity. In the present work we found that the individual polyunsaturated fatty acids are esterified in the phospholipids to the same extent in the controls as in hepatocytes from clofibrate treated rats. This finding agrees with the view that the reactions incorporating polyunsaturated fatty acids in the phospholipid fractions have distinctly lower K, values than competing pathways such as fatty acid oxidation and triacylglycerol synthesis. The incorporation of polyunsaturated fatty acids in phospholipids is also little affected by fasting and feeding which strongly affects the distribution of fatty acids between oxidation and triacylglycerol synthesis [35,36]. With both C,, substrates the calculated A 6 desaturase activity was increased by 100% by clofibrate feeding, while the activity of the A 5 desaturase was increased by 20% compared to control cells. A greater reduction in the concentration of the intact substrate, caused by the increased P-oxidation, than in the content of the desaturated products could explain part of the apparent stimulating effect of clofibrate feeding on the A 6 and A 5 desaturase activities. With linoleic acid and linolenic acid as substrates the amounts of A 6 desaturated products were however increased with (18 : 2(n-6)) or remained unchanged with (18 : 3 (n-3)). Conclusively, clofibrate feeding caused a real induction of the A 6 desaturase activity with these two C,, fatty acid substrates. These findings with isolated hepatocytes were consistent with results from recent studies with isolated rat liver microsomes [18,19]. Peroxisomal proliferators like
clofibric acid and fenofibrate, were found to increase the conversion of linoleate to arachidonic acid by inducing the A 6 and A 5 desaturase activities. The extent of the increases in the A 6 and A 5 desaturase activities in the present experiments with clofibrate, were approximately the same as observed with isolated microsomes in the study with fenofibrate, but lower than that observed with clofibric acid [HI. The results indicate that induction of increased activity of the two desaturases is common to peroxisome proliferators [lS]. This effect is different from the effects of n-3 fatty acids. The feeding of eicosapentaenoic acid (20 : 5(n-3)) and docosahexaenoic acid (22 : 6(n-3)) stimulate peroxisomal fatty acid oxidation in isolated hepatocytes [32]. But at the same time and in contrast to clofibrate dietary n-3 fatty acids inhibit A 5 and A 6 desaturase activity in hepatocytes [32] and isolated microsomes [37-391. The effects of clofibrate and of n-3 fatty acid feeding are thus probably mediated by different mechanisms. Acknowledgements
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