- Email: [email protected]
The metabolism of fatty acids in hepatocytes isolated from triiodothyronine-treated rats
Rmhimicu
et BtophyWu
Am,
71 I (1982)
Elsevier Biomedical
90- 100
Press
BBA 5 1069
THE METABOLISM OF FATTY ACIDS IN HEPATOCYTES ~II~DOT~RONINE-BARD RATS JACOB
A. STAKKESTAD
Iwtitute
of Medid
(Received September (Revised manuscript
ISOLATED
FROM
and JON BREMER
Brochemisfy,
Unioersi?v
fst, 1981) received December
of Oslo, Oslo (Nonvq~)
2lst,
1981)
1. The effect of triiodothyronine on the metabolism of palmitate, oleate and erucate in isolated rat hepatocytes was studied. 2. In triiodothyronine-treated rats increased oxidation and decreased triacyiglycerol fo~ation from p~mitate and oleate was observed. For erucate ~ii~othyronine caused increased oxidation, but had no significant effect on esterification. 3. Glueagon had no effect on the fatty acid metabolism in hepatocytes from triiodothyronine-treated rats, whereas it stimulated the oxidation in hepatocytes from normal rats. Still, after treatment with triiodothyronine, the oxidation of fatty acids was significantly higher than in giucagon-stimulated norma hepatocytes. 4. In isolated rat liver mitochodria ~ii~othyronine raised the activity of the outer camitine palmitoyl~~sfer~e (EC 2.3.1.21). The activity of the total carnitine palmitoyltranferase was elevated only slightly in isolated mitochondria from triiodothyronine-treated rats. These effects were similar to those seen in fasted rats. S.Triiodothyronine had no significant influence on the concentration of long-chain acyl-CoA or cu-glycerophosphate in isolated rat hepatocytes.
Introduction
influence on the metabolism of fatty acids. With perfused rat livers, Keyes and co-workers [ 12,131 and Laker and Mayes 1141 found increased oxidation and decreased esterification of oleate in the hyperthyroid state. Paradoxically, a simultaneous stimulation of the lipogenesis [ 15- 181, chain elongation [17,19] and desaturation activities [15] has been found in thyrotoxic animals. The activity of the carnitine palmitoyltransferase is raised in hyperthyroidism [20]. The intention of the present study was to investigate the influence of t~iodothyronine on some of the known mechanisms regulating oxidation and esterification of long-chain and very-longchain fatty acids (palmitic, oleic and erucic acids) in isolated rat hepatocytes. Also the influence of glucagon on the fatty acid metabolism after treatment with triiodothyronine was investigated. Possible mechanisms behind the regulation of
In normal hepatocytes the oxidation of fatty acids seems to be regulated by the activity of the outer carnitine palmitoyltransferase (EC 2.3.1.21) and its inhibition by malonyl-CoA [l-5]. Addition of glucagon to perfused rat livers or to isolated rat hepatocytes has regulatory effects on fatty acid metabolism, causing increased oxidation and decreased esterification [6,7]. This may be caused by lowering the concentration of a-glycerophosphate [8] and of malonyl-CoA [9]. The metabolism of very-long-chain fatty acids is different from shorter-chain (erucate C,,:~,,) fatty acids (C,,_,,). Its metabolism seems to depend on a chain shortening by the peroxisomal ~-oxidation system [lO,l l] and its metabolism is not influenced by glucagon [lo]. Alterations in the thyroid status have extensive MOS-2760/82/oooO-0/$02.75
0 1982 Elsevier
Biomedical
Press
91
fatty acid metabolism gested.
by triiodothyronine
are sug-
Materials and Methods Materials [U-‘4C]Palmitate was obtained from the Radiochemical Centre, Amersham, U.K. [ 10-‘4C]Oleate and [14-‘4C]erucate were obtained from Commissariat a 1’Energie Atomique, Grif sur Yvette, France. [ 10-‘4C]Oleate and [ 14-‘4C]erucate were purified routinely by thin-layer chromatography using hexane/diethyl ether/glacial acetic acid (80 : 20: 1, v/v). The band corresponding to free fatty acid was scraped off and extracted with chloroform/methanol (2 : 1, v/v). Unlabeled palmitate, oleate and erucate were obtained from Fluca AG, Buchs SG, Switzerland. 3,3’$TriiodoL-thyronine (sodium salt), glucagon, essentially fatty acid-free bovine serum albumin, palmitoylCoA, malonyl-CoA, glycerol-3-phosphate dehydrogenase (EC 1.l.l.Q dithiotreitol and other biochemicals were purchased from Sigma Chemicals Co., St. Louis, MO, U.S.A. Purified 2-oxoglutarate dehydrogenase (EC 1.2.4.2), prepared according to the method of Billington et al. [21], was a kind gift from Dr. H. Osmundsen, Ph.D., Institute of Medical Biochemistry, University of Oslo. [methyl3H]( - )Carnitine was prepared according to the method of Stokke and Bremer 1221. Other reagents were commercially available products of analytical grade. Ready prepared silica gel plates (0.2 mm) were obtained from Machercy-Nagel, 5 160 Diiren, F.R.G. (DC-Fertigplatten Sil G-25). Packard Instage1 II from Packard-Becker B.V., Ulgersmaveg 47, 9731 BK Groningen, The Netherlands, was used as scintillation medium. Animals Albino male Wistar strain-derived rats (185240 g) had free access to a standard pellet diet and water until they were killed, unless stated otherwise. Thyreotoxic rats were prepared by injecting the animals intraperitoneally twice with 0.33 mg triiodothyronine per kg body weight 24 and 48 h before isolation of liver cells. Triiodothyronine was dissolved in 0.1% Na,CO, prior to injection. Treatment of the rats for more than 2 days seemed to have no significant additional effect in our
experiments, while only a single injection 24 h before preparation of the liver cells showed less effect. The food intake of the triiodothyronine-treated rats was compared carefully with rats given corresponding injections of 0.1% Na,CO,. The food intake was 21 k 2 g/24 h and 23 5 2 g/24 h respectively. Vehicle-injected animals showed no significant change of fatty acid oxidation and esterification when compared with uninjected animals. Thus, the observed changes in fatty acid metabolism were not due to fasting. Isolation of liver cells The parenchymal liver cells were prepared and purified according to the method of Seglen [23] except that Krebs-Henseleit bicarbonate buffer containing 0.5 mM CaCl, was used as the suspension and incubation medium. The cell suspensions were always examined for trypan blue exclusion. A high percentage of unstained cells (over 95%) was observed routinely. Preparation of liver mitochondria Liver mitochondria from triiodothyroninetreated rats and from fed and 24-h-fasted normal rats were prepared according to the method of Bremer [ 51. Incubation of liver cells Krebs-Henseleit bicarbonate buffer containing 0.5 mM CaCl, and 3.5% fatty acid-free bovine serum albumin was used as cell suspension medium. Pre-incubations and incubations were performed under oxygenation (95% 0,/5% CO,) at 37°C. The cells were pre-incubated for 20 min with 1.0 mM (-)-carnitine with or without 8 . 10 -8 M glucagon. Immediately prior to incubation with the substrates additional 4 - 10 -’ M glucagon was added. The incubation was started by the addition of 1.0 ml of the pre-incubated cell suspension to 1.O ml of the medium containing [U“C]palmitate, [ 10-‘4C]oleate or [ 14-‘4C]erucate. The incubation time was 30 min. The final incubation mixture contained 0.5 or 1.0 mM of fatty acid, 0.5 mM fatty acid-free bovine serum albumin, 6 . 10 -8 M glucagon and liver cells corresponding to at most 5 mg of cell protein per ml (in most experiments between 2 and 3.5 mg/ml) to
92
avoid anaerobiosis during incubation. At the end of the incubation period 1.0 ml of the incubation mixture was pipetted into 0.25 ml of 2N HClO, for determination of acid-soluble radioactivity, CXglycerophosphate and long-chain acyl-CoA. The acid-soluble radioactive reaction products included mainly ketone bodies and small amounts of citric acid cycle intermediates and acetylcarnitine [7]. Long - chain acyl - CoA and long - chain acylcarnitine were precipitated with the proteins [24]. For extraction and separation of lipids, 0.6 ml incubation mixture was pipetted into 10.0 ml of chloroform/methanol (2 : 1, v/v). Assay procedures Acid-soluble radioactivity (as a measure of the rate of /?-oxidation) was measured in the HClO, extracts after neutralisation with saturated K&O, and with methyl orange as indicator. The chloroform/methanol mixture was filtered and treated according to the method of Folch et al. [25]. The chloroform phase was evaporated to dryness and the lipid residue dissolved in 0.20 ml hexane. A sample of 40 ~1 was chromatographed with hexane/diethyl ether/glacial acetic acid (80 : 20 : 1, v/v), on silicic acid thin-layer plates. The lipid spots were made visible in an iodine vapour chamber. The spots corresponding to phospholipids (plus long-chain acylcarnitines), diacylglycerols, free fatty acids and triacylglycerols were scraped from the plates into counting vials and 10 ml of scintillation fluid was added. Separate experiments with radioactive palmitoylcarnitine showed that long-chain acylcarnitines remain in the chloroform phase and are found in the phospholipid spot in the thin-layer chromatogram. It is difficult to separate long-chain acylcarnitines from phospholipids, especially from lecithin, in thin-layer chromatographic procedures [26]. In hepatocytes incubated with 1.0 mM carnitine under the conditions used, long-chain acylcarnitines may go up to 3-4 nmol per mg of protein [7]. Long-chain acylcarnitines, therefore, may represent a significant fraction of the radioactivity recovered in the phospholipid spots in some experiments. Radioactivity was measured in a Packard TriCarb liquid scintillation spectrometer Model 3255. Fatty acid esterification products were determined
as ((A -B/P). C) nmol/mg protein where A = nmol of fatty acid in the incubation mixture, B = nmol of fatty acid metabolized to acid-soluble products, C = fraction of the total lipid radioactivity on the thin-layer plate found in the appropriate spot and P = mg cell protein in the incubation mixture. The formation of radioactive CO, was disregarded since only a minor fraction of the radioactivity (under 5%) of fatty acids labeled at high numbered positions is recovered as CO, under the conditions used [lo]. Long-chain acyl-CoA was determined in the protein precipitates after alkaline hydrolysis by an enzymatic method with 2-oxoglutarate dehydrogenase [27]. Palmitoyl-CoA was used as a standard with a reproducible recovery of approximately 84%. The hydrolysis was performed by washing the precipitate once with 0.4 M HClO,. Then 0.4 ml 10 mM dithiotreitol and 0.4 ml 2 N KOH were added. The mixture was incubated for exactly 30 min at 37°C then cooled in ice-water and acidified dropwise with 0.3 ml 4 N HClO, during vortex mixing. After centrifugation the mixture was neutralized with saturated KHCO, and with methyl orange as indicator. a-Glycerophosphate was determined in the neutralized HClO, extracts with glycerol-3-phosphate dehydrogenase [28]. The outer and total carnitine palmitoyltransferase of the isolated liver mitochondria were assayed by n-butanol extraction as described previously by Bremer [5]. The cell protein was determined according to the method of Lowry et al. [29]. The data represent the mean of results with 3-8 different hepatocyte preparations or 2-4 different preparations of liver mitochondria. In tables and figures the results are given as mean 2 S.D. Wilcoxons two-sample rank test was used for statistical treatment of the results. Results Effects of triiodothyronine on fatty acid metabolism Table I shows the gross metabolism of the longchain fatty acids palmitate, oleate and erucate in isolated liver cells from control rats and rats treated with triiodothyronine. Triiodothyronine increased the oxidation of all fatty acids to acid-soluble products significantly (palmitate, P -C 0.01; oleate
OF TRIIODOTHYRONINE
TREATMENT
ON THE METABOLISM
acid esterified
Fatty
53.8
“0.01
from control.
a.b Different
k8.5
kO.13 b 58.1
0.40
k12.1
10.18 109.0
0.43
15.1 ~8.4 b
6.6
*3.0b
76.7 )8.8b 16.9
*3.9b
*1.8b
42.2 “5.7b 10.2
b P
* 16.2
k9.0
1.82 *0.40 126.1
bolized
3.08
k 1.37 55.1
* 8.9
23.7
k3.8
k6.2
45.0 -5.8 27.0
k4.4
14.8 k4.5 17.2
oxidized Fatty acids meta-
to di- and triacylglycerols Ratio esterified
to phospholipids+ long chain acylcarnitines Fatty acid esterified
acid oxidized
a
b k5.0
kO.33 36.9
1.49
~2.4
14.8
* 1.9
15.3 k2.2 7.5
0.5 mM
0.5 mM
0.5 mM 1.0 mM
Control
Triiodothyroninetreated
Control
1.0 mM
Oleate
Palmitate
* 10.9
kO.41 81.1
I .36
k6.9
32.2
k4.2
35.3 25.6 14.4
1.0 mM
FATTY
k5.6
*0.18a 40.7
0.32
k4.7
6.0
to.9
a
32.2 i- 1.7 a 4.1
0.5 mM
=
k6.0
kO.27 a 87.8
0.41
t10.1
15.0
k-3.0
62.5 26.3 9.4
2.64 kO.52 17.6 k4.0
k2.4
7.8
i 1.9
4.9 * 1.1 5.2
0.5 mM
Control
Erucate
fatty
2.73 c 1.19 31.2 *3.1
13.1 k4.3
k3.1
8.8 L2.1 8.2
1.0 mM
acid/mg
IN ISOLATED
as nmol
ACIDS
1.0 mM
are expressed
Triiothyroninetreated
The results
OF LONG-CHAIN
conditions and assay procedures were as described in Materials and Methods. from eight (palmitate) or four (oleate and erucate) different experiments.
Fatty
Incubation mean*S.D.
THE EFFECT
TABLE I
and presented
1.16 &0.68 B 23.9 k6.4
6.2 * 3.9
11.9 -co.8 a 1.3 k4.2
0.5 mM
1.32 *0.50 42.6 kl2.1
8.3 *4.9
18.0 *1.7= 15.3 k8.4
1.0 mM
Triiodothyroninetreated
protein
RAT HEPATOCYTES as
s
and erucate, 0.01
0.05). The effect of triiodothyronine on the total metabolic rate of palmitate and oleate was very small, while the metabolism of erucic acid increased approximately 37% (P > 0.05) mainly because of its increased oxidation. The ratio of esterified to oxidized fatty acids was decreased after treatment with triiodothyronine for all fatty acids investigated. This effect was most pronounced for palmitate (PC O.Ol), but also for oleate and erucate the changes were significant (0.01 < P < 0.05). The inclusion of small amounts of long-chain acylcarnitines in the phospholipid fraction evidently does not invalidate this conclusion. Effects of triiodothyronine on the action of glucagon on fatty acid metabolism Table II shows the same experiments as shown in Table I, now with the addition of 6. 10 -s M glucagon to the incubation medium. When Tables I and II are compared, it is striking that glucagon had no effect on the fatty acid metabolism in hepatocytes from triiodothyronine-treated rats. Still, after treatment with triiodothyronine the oxidation of fatty acids was significantly higher than in glucagon-stimulated normal hepatocytes (palmitate, P < 0.01; oleate and erucate, 0.01 < P < 0.05). The difference was most pronounced for the lower fatty acid concentration (0.5 mM). The oxidation of erucate was stimulated relatively more than the oxidation of palmitate or oleate. Triiodothyronine treatment caused a decreased esterification of palmitate to diand triacylglycerols (P < 0.01) and most likely to phospholipids (which includes long-chain acylcarnitines). A decreased esterification rate was also seen
for oleate, but the results were statistically insignificant (P > 0.05). For erucate an unaltered rate of esterification to di- and triacylglycerols was observed. Triiodothyronine increased the total rate of metabolism of erucate by 55-42% (P < 0.05 at 0.5 mM concn.), but not that of palmitate or oleate. In control rats addition of glucagon in vitro caused an increased oxidation and a decreased esterification, as previously described [6,7]; hence, a diminished ratio of esterified to oxidized fatty acids in all fatty acids investigated. This effect was strongest for palmitate, less for oleate and least and almost absent for erucate. Only for palmitate was the change statistically significant (P < 0.01 or 0.01 < P < 0.05). The total metabolic rates were not altered. Effects of triiodothyronine on long-chain acyl-CoA and a-glycerophosphate Table III and IV show the concentrations of long-chain acyl-CoA and a-glycerophosphate in isolated liver cells from controls and triiodothyronine-treated rats with or without the addition of glucagon in vitro. No significant change was found in treated rats compared with controls concerning these metabolites. For controls, addition of glucagon to the cells caused a significant decrease in the cY-glycerophosphate content (P < 0.01) as previously reported [8]. The data from the a-glycerophosphate determinations must be interpreted with great caution, however, because of a high intra- and interassay variation. Effects of triiodothyronine on carnitine palmitoyltransferase. The activity of the outer carnitine palmitoyltransferase is inhibited by malonyl-CoA [l]. We therefore measured the activity of this enzyme and its inhibition by malonyl-CoA in triiodothyronine-treated rats compared with the activity in fasted and fed normal rats (Fig. 1 and Table V). In triiodothyronine-treated rats the activity of the outer carnitine palmitoyltransferase was higher. The activity of the total carnitine palmityltransferase was only slightly elevated in triiodothyronine-treated rats compared with fed control rats (Table V). These results were similar to those found in fasted normal rats, although the
II
a Different b Different ’ Different d Different
from from from from
56.9 18.9
Fatty acid metabolized
c
control, 0.01
121.5 k26.4
1.05 kO.30 d
1.13 to.59
Ratio esterified oxidized
d
39.3 i9.1
16.0 26.3’
Fatty acid esterified to di + triacylglycerols
59.6 * 16.5’
22.1 k5.8
d
13.0 e4.0
28.4 “6.1
Fatty acid esterified to phospholipids + long-chain acylcamitine
Fatty acid oxidized
h
of glucagon of glucagon
58.3 i8.3
0.33 kO.09 h
6.1 23.1
8.7 * 1.2 B
44.6 k6.6h
to the medium to the medium
40.7 *6.
84.8 i 12.6
0.86 kO.24
27.4 -t 7.6
11.9 -‘3.5
46.3 * 7.4
1.0 mM
with 0.01
I
0.84 to.35
0.42 to.12 b
112.0 2 12.8
12.1 i4.3
6.1 * 1.7
22.9 r 4.9
0.5 mM
Control
15.9 k6.2 b
17.0 -t 2.7 a
79.0 2 12.4 h
1.0 mM
0.5 mM
0.5 mM
I.0 mM
Triiodothyroninetreated
Control
OF LONG-CHAIN
FATTY
ACIDS
IN ISOLATED
43.0 i3.0
0.32 -‘O.ll
6.3 22.9
4.0 i 1.1
32.X iO.6 a
0.5 mM
^,>_
82.2 23.6
0.35 co.19
11.7 k6.7
8.7 22.1
61.1 = 5.7
1.0 mM
Triiodothyroninetreated
14.4 t2.9
1.70 10.73
4.9 c2.2
27.X k3.5
2.22 -c 1.0x
10.4 * 5.7
8.0 23.0
9.2 2 2.6
1.O mM
-_____
4.5 * 1.6
5.x kO.9
0.5 mM
Control
Erucate
39.4 t 10.2 22.3 24.5
s
- _. _ _ - _.
1.40 iO.66
1.05 r 0.44
a
9.3 i6.2
4.2 k2.7
16.8 22.3’ 13.9 i 8.8
=
1.0 mM
7.4 k4.3
11.2 to.7
0.5 mM
as nmol
HEPATOCYTES
arc expressed
RAT
Triiodothyroninetreated
and Methods, The experiments are identical with those of Table I. Results or four (oleate and erucate) different experiments.
Oleate
and assay procedures were as described in Materials and presented as mean) SD. from eight (palmitate)
Palmitate
Incubation conditions fatty acid mg protein
THE EFFECT OF TRIIODOTHYRONINE TREATMENT ON THE METABOLISM WITH ADDITION OF GLUCAGON TO THE INCUBATION MEDIUM
TABLE
._
+ Glucagon
- Glucagon
0.5 mM
0.5 mM 0.25 ‘0.06 0.27 kO.06
1.0 mM
0.3 I kO.07 0.34 kO.12
0.5 mM
0.24 kO.05 0.28 kO.07
0.34 kO.07 0.35 io.13
0.24 “0.05 0.24 e0.04
Control
Triiodothyroninetreated
Control
1.0 mM
Oleate
PaImitate
0.36 i-o.1 0.31 ‘0.13
I
I .O mM
are expressed
0.22 2 0.03 0.22 2 0.04
0.5 mM
0.29 ‘-0.06 0.36 * 0.05
I.0 mM
Triiodothyroninetreated
The results
0.32 2 0.06 0.34 * 0.03
0.5 mM
Control
Erucate
0.33 * 0.06 0.3 I kO.07
1.0 mM
protein
0.31 *o.og 0.28 * 0.09
0.5 mM
I.0 mM 0.39 CO.18 0.39 eo.17
IN-
and presented
HEPATOCYTES
Triiodothyroninetreated
acyl-CoA/mg
IN ISOLATED RAT TO THE MEDIUM
as nmol long-chain
and Methods.
Incubation conditions and assay procedures were as described as meanCS.D. from three different hepatocyte preparations.
in Materials
CONTENT OF LONG-CHAIN ACYL-CoA OR WITHOUT ADDITION OF GLUCAGON
III
THE EFFECT OF TRIIODOTHYRONINE TREATMENT ON THE CUBATED WITH DIFFERENT LONG-CHAIN FATTY ACIDS WITH
TABLE
97
TABLE
IV
THE EFFECT OF TRIIODOTHYRONINE RAT HEPATOCYTES INCUBATED WITH GON TO THE MEDIUM
TREATMENT LONG-CHAIN
ON THE a-GLYCEROPHOSPHATE CONTENT IN ISOLATED FATTY ACIDS WITH OR WITHOUT ADDITION OF GLUCA-
Incubation conditions and assay procedures were as described in Materials and Methods, The results are expressed as nmol a-glycerophosphate/mg protein and presented as mean--t S.D. from 17- I8 incubations from three different hepatccyte preparations, each with three different fatty acids (palmitate, oleate or erucate). Results obtained from control incubations with glucagon were different from experiments without addition of glucagon to the medium, PcO.01 Control
- Ghtcagon + Glucagon
Triiodothyronine-treated
0.5 mM
1.0 mM
0.5 mM
1.0 mM
2.69kO.79 1.63’0.72
3.18*1.03 1.96* 1.01
2.26* 2.07*
2.75* 1.51 2.56*1.31
relative inhibition by malonyl-CoA was less pronounced and the increase in the activity of the uninhibited outer carnitine palmitoyltransferase more pronounced than in mitochondria from normal fasted rats [5]. In this connection it must be stressed that because of the complicated kinetics of the carnitine palmitoyltransferase [30] the measured relative activities of the outer and the total
1.2 1.32
carnitine palmityltransferase are not comparable directly. However, the changes in the absolute activities are comparable. In preliminary experiments we also checked the effects of glucagon on the activity of camitine palmitoyltransferase and its inhibition by malonylCoA, both in intact rats and in isolated hepatocytes. However, no effects corresponding to the
“:::::-:rr:1: -L 10
Malonyl
30
-CoA
50
QM)
Fig. 1. The effect of malonyl-CoA on the formation 24-h-fasted (m), fed (A) and triiodothyronine-treated pM palmitoyl-CoA; B, 70 pM palmitoyl-CoA
Malonyl
-CoA
(PM)
of palmitoylcamitine from palmitoyl-CoA and camitine by mitochondria (0) rats. Each point represent mean* S.D. from 2-4 different animals.
from A, 21
9x
TABLE V THE ACTIVITY MITOCHONDRIA TROL RATS The assay conditions different experiments
OF THE TOTAL AND THE FROM FED RATS TREATED
OUTER CARNITINE PALMITOYLTRANSFERASE IN RAT LIVER WITH TRIIODOTHYRONINE AND FROM FED AND FASTED CON-
were as described [5]. The results are expressed and are presented as mean* S.D.
as nmol palmitoylcarnitine
Totai transferase
Triiodothyronine-treated Fed control rats ( n=4)
rats (n = 3)
Fasted control rats (n = 2) Ratio triiodothyronine-treated/fed Ratio fasted/fed
formed/mg
Outer
protein
per min from 2-4
transfcrasc
21 PM palmitoyl-CoA
70 PM palmitoyl-CoA
21 PM palmi toyl-CoA
70 PM palmi toyl-CoA
17.03*7.12 16.75% 1.45
23.935 10.35 1X.75* 3.27
16.972 1.02 1.01
22.45 i- 3.22 1.28 1.20
2.50’0.13 1.63iO.12 2.37~0.14 1.53 I .45
5.73 i 0.2x 2.95-~0.15 4.XX”O.l2 I .94
1.95
effects of fasting or of triiodothyronine were observed (not shown). Thus, the short term effects of glucagon on fatty acid metabolism in the liver seem to be different from those of fasting or of triiodothyronine. Discussion The present study show that treatment of normally fed rats with high doses of triiodothyronine results in extensive metabolic changes in the metabolism of long-chain fatty acids in isolated liver cells. For palmitate, oleate and erucate the oxidation was increased significantly. Simultaneously, the esterification to diand triacylglycerols was decreased markedly for palmitate and oleate, while for erucate a nearly unaffected di- and triacylglycerol formation was observed. For palmitate and oleate the stimulated oxidation was balanced by a lowered esterification, rendering the total metabolic rate of these two fatty acids unchanged. De novo synthesis of fatty acids (lipogenesis) has been reported to be stimulated in hyperthyroidism [ 15- 181. This suggests an increased concentration of malonyl-CoA, which makes the increased oxidation of fatty acids seem paradoxical since malonyl-CoA is an inhibitor of fatty acid oxidation [l]. However, our finding of an increased activity of the outer carnitine palmityltransferase seem to resolve this paradox. The
I .65
that treatment with triresults suggest iodothyronine, as does fasting [5], ‘exposes’ more carnitine palmitoyltransferase on the outer surface of the inner membrane of the mitochondria, and this transferase may also be less sensitive to malonyl-CoA. The increased oxidation without a decrease in esterification of erucate in hepatocytes from triiodothyronine-treated rats also seems paradoxical. These effects on erucate metabolism are similar to, but much weaker than, those previously found in rats fed diets containing clofibrate [lo] or partially hydrogenated fish oil [ 111, which stimulate partial peroxisomal P-oxidation. Since the rate of erucate metaolism most likely depends mainly on a chain-shortening function of the peroxisomal /Ioxidation enzymes [ 10,111, our results suggest that triiodothyronine has a (weak) stimulating effect on the peroxisomal P-oxidation system. Direct measurement of the peroxisomal j&oxidation system is necessary to evamate this possibility. In our experiments we found no significant change in the long- chain acyl- CoA or a glycerophosphate contents in Iiver cells from triiodothyronine-treated rats compared with control rats, and glucagon had no additional effect. These results support the significance of changed enzyme activities in the hyperthyroid state, especially of the outer carnitine palmitoyltransferase and its inhibition by malonyl-CoA. However, our results are in contrast to those of other investigators who
99
have found a decreased a-glycerophosphate concentration in perfused hyperthyroid rat liver 1311. No effect of triiodothyronine on the liver glycerophosphate acyltransferase has been found [34. As previously reported [7,8], we found a decreased cu-glycerophosphate, an increased oxidation and a decreased esterification of palmitate and oleate in rat hepatocytes from control animals upon addition of glucagon to the incubation medium. At the same time we found no effect of glucagon on the carnitine palmitoyltransferase. Thus, the decreased cu-glycerophosphate content in glucagon-treated hepatocytes from control rats may contribute to the decreased esterification capacity under these circumstances. However, in the present study we could not reproduce with certainty the reported increased level of long-chain acyl-CoA in isolated hepatocytes incubated with palmitic acid and glucagon [7,33]. In our experiments glucagon gave only a marginal, nonsignificant (P> 0.05) increase in cells incubated with palmitate and had no effect with oleate or erucate. Glucagon belongs to a group of hormones which, at least in part, acts by increasing intracellular cyclic AMP production. The effects of these hormones can be modulated by the thyroidal status of the organism [34]. Sperling et al. [34] found no difference in glucagon receptor characteristics or cyclic AMP response between the hyperthyroid and the euthyroid state in rat liver. It is striking, therefore, that glucagon has no effect on fatty acid metabolism or ff-glycerophosphate concentration in cells from triiodothyronin-treated rats. Glucagon also has no effect on fatty acid oxidation in cells from fasted rats [7]. In both these conditions we have found an increased activity of the outer carnitine palmitoyltransferase, suggesting a dominating role of this enzyme for the rate of fatty acid oxidation. Altogether, different mechanisms may influence the metabolism of fatty acids in the liver. Changed metabolite concentrations ((Y-glycerophosphate and malonyl-CoA) 18,351, changed sensitivity of enzymes for inhibitors (malonyl-CoA on carnitine palmitoyltransferase) [2-51 and changed enzyme activities (outer carnitine palmitoyItransferase and peroxisomal P-oxidation enzymes) [5,36] can influence oxidation and esterification rates.
Our results suggest that an increased activity of the outer carnitine palmitoyltransferase may represent an important mechanism explaining the increased oxidation and the decreased esterification of long-chain fatty acids in the liver of triiodothyronine-treated rats. Acknowledgements This work was supported by the Norwegian Research Council for Science and the Humanities. The expert technical assistance of Mrs. June Taje Haviken is appreciated gratefully. References McGarry, J.D., Leatherman, G.F. and Foster, D.W. (1978) J. Biol. Chem. 253,4128-4136 Cook, G.A., Otto. D.A. and Cornell, N.W. (1980) Biochem. J. 192,955-958 Ontko, J.A. and Johns, M.L. (1980) B&hem. J. 192, 959962 Saggerson, E.D. and Carpenter, C.A. (1981) FEBS Lett. 129 (2), 225-228 Bremer, J. (198 1) Biochim. Biophys. Acta 665, 628-63 1 Heimberg, M., Weinstein, J. and Kohout, M. (1969) J. BioI. Chem. 244, 5131-5139 Christiansen, R.Z. (I 977) Biochem. Biophys. Acta 488, 249 -262 Lund, H., Borrebaek, B. and Bremer, J. (1980) Biochim. Biophys. Acta 620, 364-371 9Cook. G.A., Nielsen, RX., Hawkins, R.A., Mehlman, M.A., Lakshmanan, M.R. and Veech, R.L. (1977) J. Biol. Chem. 252,442 I-4424 IO Christiansen, 324
R.Z. (1978) Biochim
Biophys.
Acta 530, 3 14-
I 1 Christiansen,
R.Z., Christiansen, E.N. and Bremer, J. (1979) Biochim Biophys. Acta 573,417-429 12 Keyes, W.G., Heimberg, M. (1979) J. Clin. Invest. 64, 182-190 13 Keyes, W.G., Wilcox, H.G. and Heimberg, M. (1981) Metabolism 30 (2). 135-146 14 Laker, M.E. and Mayes, P.A. (1981) Biochem. J. 196, 241-255
15 Gompertz, D. and Greenbaum, A.L. (1966) Biochim. Biophys. Acta I 16,441-459 16 Diamant, S., Gorin, E. and Shafrir, E. (1972)Eur. J. Biothem. 26. 553-559 17 Landriscina, C., Gnoni, G.V. and Quagliarieiio, E. (1976) Eur. J. Biochem. 71, 135-143 18 Gnoni, G.V., Landriscina, C. and Quagliariello, E. (1980) Biochem. Med. 24, 336-347 19 Gnoni, G.V., Landriscina, C. and Quagliariello, E. ( 1978) FEBS Lett. 94 (I), t79- 182 20 Van Tol, A. (1971) Hepatic Fatty Acid Oxidation, Thesis, Department of Biochemistry I, Rotterdam Medical School, Rotterdam, The Netherlands
21 Billington, D., Osmundsen, 0. B&hem. Pharm. 27, 2879-2890 22 Stokke, 0. and Bremer, 218, 552-554
and
Sherratt,
J. (1970) Biochim.
23 Seglen, P.O. (1973) Exptl. Cell Research 24 Pearson, D.J. and Tubbs, P.K. (1964) Acta 84, 772-773
S.A. (1978) Biophys.
Acta
82, 391-398 B&him. Biophys.
25 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 26 Soderberg, J., Therriault, D.G. and Wolf, G. (1965) in Recent Research on Carnitine. Its Relation to Lipid Metabolism (Wolf, G., ed.), pp. 165-171, MIT, Cambridge 27 Wiliamson, J.R. and Corkey, B.E. (1969) Methods Enzymol. 13,434-513 28 Michal, G. and Lang, G. (I 974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 3, pp. 1415-1418, Academic Press, New York
29 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 30 Bremer, J. and Norum, K.R. (1967) J. Biol. Chem. 242, 1749- 1755 31 Schimassek, H., Mitzkat, H.J. and Biochem. Pharmacol. 15, 129- 136 32 Roncari, D.A.K. and Veeraraghavan, 33
34 35 36
Feuerstein.
J. (1966)
K.M. (1975) J. Biol.
Chem. 250 (I I), 4134-4138 Christiansen, R.Z. (1979) Regulatory Mechanisms in the Oxidation of Long-Chain Fatty Acids, Thesis, University of Oslo, Norway Sperling, M.A., Ganguli, S., Voina, S., Kaptein, E. and Nicoloff, J.T. (1980) Endocrinology 107 (3). 684-690 McGarry, J.D. and Foster, D.W. (1979) J. Biol. Chem. 254 (17), 8163-8168 Neat, C.E., Thomassen, M.S. and Osmundsen, H. (1980) Biochem. J. 86, 369-371