Biochimica et Biophysics
Aria, 1042 (1990) 182-187 Elsevier
Metabolism of dicarboxylic acids in rat hepatocytes Steinar Bergseth I, Jean-Pierre ’ Institute of Medical Biochemistry,
* and Jon Bremer
University of Oslo, Oslo (Norway)
(Received 12 May 1989) manuscript received 29 August
acid; Beta oxidation;
[ curboxyf-14C]Dodecanedioic acid (DC,,) is metabolized in hepatocytes at a rate about two thirds that of (1-‘4C]palmitate. Shorter dicarboxylates (sebacic (DC,,), suberic (DC,), and adipic (DC,) acid) are formed, mainly DC,, less DC, and only a little DC,,. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the /l-oxidation proceeds all the way to succinate and acetyl-CoA. [curboxyl“C]Suberic acid (DC,) is oxidized at a rate only one fifth that of dodecanedioic acid. ( +)-Decanoylcamitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of lcurboxyf-‘4C]dodecanedioic acid or of (l-‘4C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are /l-oxidized mainly in the peroxisomes. Introduction Dicarboxylic acids are o-oxidation products of medium chain monocarboxylic acids. They are shortened by P-oxidation and excreted in small amounts in the urine [1,2]. Studies on the oxidation of dicarboxylic acids in mitochondria and peroxisomes suggest that the peroxisomes are more active than mitochondria in the oxidation of these acids [3-51. However, the dominating role of the peroxisomes has not been confirmed on intact cells. It is also striking that dicarboxylic acids are found in the urine of patients with Zellweger’s disease . These patients lack peroxisomes in their cells. Recently, we  and others [4,8] have shown that dodecanedioic acid (DC,,) is relatively efficiently metabolized in the rat. None is excreted in the urine, but depending on the dose, lo-50% is excreted as shorter dicarboxylic acids, mainly DC, and DC,, and other metabolites. Suberic acid (DC,) is relatively poorly
* Visiting Scientist from: Laboratoire de Physiologic Animale et de la Nutrition, Fact&t des Sciences Mirande, University of Dijon, Dijon, France. Abbreviations: ASA, acetylated sulfanilamide; DC, dicarboxylic; DC,, adipic acid; DC,, suberic acid; DC,,, sebacic acid; DC,,, dodecanedioic acid; DTT, dithiothreitol; TEA, triethanolamine; TFA, triffuoroacetic acid. Correspondence: S. Bergseth, Institute of Medical Box 1112, Blindem, 0317 Oslo 3, Norway. ooO5-2760/90/$03.50
0 1990 Elsevier Science Publishers
metabolized, and it is actively excreted in the urine by the kidneys . In the present study we have compared the oxidation of dicarboxylic acids and fatty acids in isolated hepatocytes. Sulfanilamide acetylation by the cytosolic N-acetyltransferase (EC 220.127.116.11)  has been used as a measure for the labeling of the extramitochondrial acetyl-CoA pool. Our results support the view that dicarboxylic acids are P-oxidized mainly in the peroxisomes in the intact cell and that DC,, is more efficiently metabolized than DC, when presented externally to the organelles. Materials and Methods MateriaIs [ carboxyf-‘4C]Suberic acid and [ carboxyl-‘4C]dodecanedioic acid were synthesized as described . [li4C]Palmitic and [2-14C]adrenic acids were obtained from Amersham International, U.K.. Unlabeled palmitic, sebacic, suberic and adipic acids and clofibrate (ethyl 2-(4-chlorophenoxy)2-methylpropionate), were from Fluka. Unlabeled dodecanedioic acid, collagenase and fatty-acid-free bovine serum albumin (BSA) were from Sigma (St. Louis, MO, U.S.A.). Mebumal was from Aphothekerenes laboratorium (Norway). ( + )-Decanoylcarnitine was synthesized according to Bohmer and Bremer [lo]. All other chemicals and solvents were of analytical grade. The Nacetyltransferase (EC 18.104.22.168) was partially purified as described [ 111. Division)
Male, albino W&tar rats (250 g) from Mollegaards Breeding Laboratory (Ejby, Denmark) were used. They had free access to a standard pellet, high carbohydrate diet 1121 until they were killed. Clofibrate-treated rats were fed the same diet with 0.3% (w/w) clofibrate for 3 weeks. The clofibrate diet was prepared as described ]131. Liver cell preparation and incubation coltditions
The rats were anaesthetized with 5% Mebumal (100 &‘lOO g), and hepatocytes were prepared as described by Seglen  with minor modifications . Incubation conditions and sample treatment were mainly as described previously [ 121. The perchloric acid quenching of the incubations precipitated about 95% of the DC,,, leaving the metabolites in the acidic supematant. To avoid dilution of the sample, solid NaHCO, was used for neutralization. Metabolite analyses
Metabolites from the hepatocyte experiments were separated and analyzed by HPLC. A Spherisorb ODS column (5 pm, 4.6 x 250 mm, Supelco) eluted at 0.8 ml/mm with methanol/water gradients containing 30 mM TFA adjusted to pH 3 with TEA (except for Fig. 1). The column was connected to a Spectra-Physics SP8800 gradient pump and a variable wavelength ultraviolet detector (Spectra-Physics, model 770) followed by a Raytest RamonoS-LS radioactivity detector (Raytest, Straubenhart, F.R.G.). The detector was equipped with a 1 ml flow-cell, where sample and liquid scintillator (Pica Aqua, Packard Instruments) were mixed 1 + 5. This was linked to a data collection program (Nuclear Interface, Mtinster, F.R.G.) running on an IBM compatible personal computer. Incubations with sulfanilamide were eluted isocratically with 10% methanol/30 mM TFA (pH 3) to separate acetylated sulfanilamide (ASA) from labeled adipic acid, sulfanilamide and other ultraviolet-absorbing compounds. Their retention times were 22, 12 and 8 min, respectively. The acetylsulfanila~de was measured at 260 nm, and the area of the peak was read against a standard curve to estimate the amount formed. The N-acetyltransferase was assayed as described 1151. 14C02 measurements were done as described 17,161. The Student’s t-test (unpaired, two tailed) was used for statistical treatment of the data. Results Metabolism in hepatoeytes from normal rats
Fig. 1 shows that there is a linear formation of metabolites from DC,, in hepatocytes from normal
Fig. 1. Time curves for metabolite formation from dodecanedioic acid incubated with hepatocytes from normal rats. Hepatocytes (7 mg/mI in incubation medium), were incubated with 1 mM [carboxylt4C]dodecanedioic acid (135 Bq/nmol) and analyzed on a reversedphase HPLC column as described. The column was initially eluted with 100% water containing 30 mM TFA adjusted to pH 2.5 with TEA for 5 mm. At 5 mm the gradient dropped to 90% of the water mixture and 10% methanol, containing 30 mM TFA, 24 mM TEA in 0.1 min. Thereafter, a linear gradient reaching 100% of the methanol mixture at 20 min was run. The metabolites eluted as follows: acetate +, 8 min; ketone bodies Cl, 11.1 min; DC, 0, 16.2 mm; DC, A, 19.4 min; DC,, X, 21.5 min; DC,,, 23.5 min. The points are means og hepatocyte preparations from two rats each incubated in parallel.
rats. Acetate and ketone bodies make up to about half of the radioactivity recovered in the metabolites, and there is no significant change in the metabolite pattern with time. Labeled succinate which could be formed either by direct &oxidation of DC,, or from acetyl-CoA through the tricarboxylic acid cycle was not detected. Succinate elutes in the same peak as the ketone bodies, but rechromatography of this peak on an Arninex HPX-87 column at 60°C , revealed only ketone bodies (not shown). Fig. 2, trace A, shows that DC,, DC, and DC,, were formed when DC,, was incubated with isolated hepatocytes from a normal rat. Relatively, more DC,
Fig. 2. Radio-HPLC chromatograms from hepatocyte incubations with dodecanedioic acid. Hepatocytes (5 mg/ml in incubation medium) from a normal rat (trace A) and from a clofibrate-treated rat (trace B) were incubated with 1 mM Icurboxyi-t4C]dodecanedioic acid (110 Bq/nmol) for 40 min and metabolites analyxed by HPLC as described. A programmed gradient, changing from 14% methanol at injection to 100% methanol after 15 min was used. PT: Pass-through fraction (see text for definition).
incubations with different substrates
Hepatccytes from normal and clofibrate-fed rats (5 mg/ml in incubation medium) were incubated with [l-‘4C]palmitate carboxylic acids (1 mM) for 40 min. Competing substrates are also 1 mM. See Materials and Methods for further details, give animals in each group. The results are given as nmol substrate oxidized. Substrate
[l-‘4C]Palmitate [I-‘4C]Pahnitate+( [I-‘4C]Pabnitate+DCs
[ catboxyl-‘4CjDC,, [corboxyl-“C]DC,, [ curboxyZ-‘4C]DC,,
+ ( + )-dec.cam. + palmitate
[ carboxyl-‘4 C]DC, [ carboxyl-‘4C]DCs + (+)-dec.cam. [ cnrboxy/-‘4C]DCs + pahnitate [2-14C]Adrenate /2-‘4CjAdrenate+(
26.9+ 1.9 (5) 8.9 + 3.7 a (4) 28.1 f 2.8 (3) 16.4*2.1 = (4)
60.9 + 4.0 a (6) 51.5 + 6.0 (4) X.2-14.6 (4) 58.7 + 6.0 (4)
7.5 + 1.7 b (3)
19X& 2.6 17.5 + 1.7 14.9 + 3.5
(5) (5) (4)
28.3 _t 2.1 2L(6) 33.9k1.8 (4) 25.4k1.2 (3)
6.6 + 0.7 b (3)
(3) (3) (3)
4.0 f 0.5 a (7) 5.1/4.8 5.2 I 1.1 (4)
a P i 0.05; significant difference from normal cell incubation, b P i 0.1 between normal and clofibrate group.
palmitate (Table I). The rate of radioactive CO, formation was similar from both substrates (Table I). The slower oxidation of the short dicarboxylic acids was confirmed in experiments with DC,, which was much more slowly oxidized than DC,2 (Table I). However, the slow oxidation of DC, may also be explained by a slow rate of uptake in the cells or by its slow activation to the corresponding CoA-ester . To test the importance of the peroxisomes in the oxidation of dicarboxylic acids, their oxidation was compared with that of fatty acids in hepatocytes preincubated for 20 min with 1 mM (-}-carnitine to maximise the mitochondrial oxidation, or with 2 mM (+)-
than DC, and DC,,, was formed. The two peaks called the pass-through fraction (FT) are chromatographic artefacts probably due to the intermediate ionization of acetate at pH 3. More than 95% of the radioactivity in these peaks could be evaporated, pointing towards acetate as the major constituent. This as evidently formed mainly from the acetyl-CoA produced in the P-oxidation and from succinate, which may be the final P-oxidation product of DC, in peroxisomes. The relative accumulaton of DC, over that of DC, and DC,, shows that the oxidation of the dicarboxylic acids slows down as the chain is shortened. It is striking that DC,, was metabolized at a rate more than two-thirds that of
and [carboxyl-‘4C]diNumbers in parentheses
( %) after incubation with k&led
Hepatocytes (5 mg,/mi in incubation medium) were incubated with [l-‘4C]pal~tate and [carboxyl- “C]d~ecanedioate (1 mM) and metabolites analyzed by HPLC. See Materials and Methods for details. Metabolites are given as percent of total acid-soluble metabolites rt SE.. Numbers in parentheses give animals in each group. Pass-through
11.2*1.2 6.0 + 0.6 a
3.7+ 1.5 1.9*0.4
Dodecanedioic Normal Cfofibrate
acid 58.7 f 2.4 80.8 + 3.0 ’
25.6kO.8 7.7 + 2.3 a
Suberic acid Normal Clofibrate
66.5 f 3.1 81.4+2.8 a
26.4 + 4.5 11.4+2.4 a
a P < 0.05 compared to normal * See text for definition.
9.1+ 4.8 4.2kl.O a
185 decanoylcarnitine, which is a competitive inhibitor of the camitine-dependent mitochodmial oxidation of fatty acids [19-211. Table I shows that no significant inhibition of the oxidation of dicarboxylic acids was observed. The oxidation of palmitate was 70% inhibited in these cells, while the oxidation of adrenic acid was only 40% inhibited by the ( + )-decanoylcarnitine as previously observed [19,22]. Table I also shows the mutual influence of palmitate and dicarboxylic acids on their oxidation rates. DC,, significantly decreased the rate of palmitate oxidation, while palmitate had no significant effect on the oxidation of DC,,. The oxidation of suberic acid was not affected by any of the additions (Table I). The rate of radioactive CO, formation was also little affected by ( + )-decanoylcamitine (not shown). Metabolism in hepatocytes from clofibrate-treated rats We also studied the oxidation of dicarboxylic acids in hepatocytes isolated from clofibrate-fed rats (Table I). In agreement with previous studies, palmitate oxidation was more than doubled and the ( +)-decanoylcamitine effect was almost eliminated in these hepatocytes . The oxidation of dicarboxylic acids also increased, relatively less, but significantly. Fig. 2, trace B and Table II show that the distribution of the metabolites formed was significantly changed. Total radioactivity in the early, most polar products greatly exceeded that of the sum of the shortened dicarboxylic acids (DC, + DC, + DC,,), showing that a significant fraction of the DC,, presumably had been completely b-oxidized to acetyl groups and succinate. The formation of radioactive CO, also increased, and it is striking that the ratio of labelled COJacid-soluble oxidation products for DC,, increased more (2.7 times) than for palmitate (1.8-times), in going from normal to clofibrate (Table I). This might be indicative of the different compartmentalization in the oxidation of these two substrates.
Fig. 3. Sulfanilamide acetylation. Hepatocytes from normal and clofibrate-treated rats (5 mg/ml in incubation medium) were incubated with 0.5 mM sulfanilamide and [l-‘4C]palmitate or [carboxyl“C]DC,2 for 20 min as shown, and analyzed by HPLC as described. The points are means of two different experiments. The incorporation of radioactivity is calculated as [14C]carboxyl groups in ASA. 0, Total acetylated sulfanilamide with pahnitate as substrate; A, total acetylated sulfanilamide with dodecanedioate as substrate; 0, labeling of acetylated sulfanilamide with pahnitate as substrate; A, labeling of acetylated sulfanilamide with dodecanedioate as substrate.
tion of fatty acids at 0.1 mM concentrations is linear with time for more than 30 min . Fig. 3A shows that high concentrations of DC,, inhibited sulfanilamide acetylation in normal hepatocytes, while palmitate did not. However, at low, noninhibitory concentrations (0.1-0.2 mM) the [ carboxyli4C]dicarboxyl acid was much more efficient than [l14CIPalmitate in labeling the acetylsulfanilamide. From Table III we calculated that the specific labeling of the acetylsulfanilamide pool was more than 6-times higher when DC,, was used, compared to when palmitate was used as a substrate. (In the calculations for Fig. 3 and Tale III, we assumed both carboxyl groups to be labeled and the specific activity of the acetyl-CoA formed by
Cytosolic acetyl groups To obtain further support for the role of the peroxisomes in the oxidation of dicarboxylic acids, we compared [1-‘4C]palmitate and [ carboxyl-‘4C]DC,, as precursors for the acetyl-CoA groups used for the acetylation of sulfanilamide in isolated hepatocytes. Sulfanilamide is acetylated by a N-acetyltransferase (EC 22.214.171.124) in the cytosol of hepatocytes , and this is the major metabolic pathway for sulfanilamide . Acetyl-CoA formed in the peroxisomes should enter the cytosolic pool used for this acetylation more directly than acetyl-CoA formed in the mitochondria. Kondrup and Lazarow  used a comparable approach to study the importance of the peroxisomes in fatty acid oxidation. Sulfanilamide, at least up to 4 mM, does not influence fatty acid oxidation (not shown) and oxida-
Formation of aceiylaied sulfanilamide Hepatocytes (5 mg/ml in incubation medium) were incubated with 0.5 mM sulfanilamide and 0.15 mM [I-t4C]palmitate and [carboxyl14C]dodecanedioate for 20 min. ASA was analyzed by HPLC as described. Numbers in parentheses give animals in each group. ASA formation (pmol.mg-’
carboxyl groups in ASA
22* 5 113*X18
Normal Palmitate Dodecanedioate
1902&157 1533 f 101
Clofibrate Palmitate Dodecanedioate
1370+215 1765 f 278
’ P < 0.05 compared
186 P-oxidation therefore to be half of the specific activity of the dicarboxylic acid used.) In hepatocytes from clofibrate-fed rats, the inhibition of sulfanilamide acetylation by high concentrations of the DC,2 was much less pronounced (Fig. 3B) and the difference in specific activity of the acetylsulfanila~de formed at low (0.1-0.2 m&l) substrate concentrations was no longer present.
The strong in~bition of palmitate oxidation by (+)decanoylc~itine in normal hepatocytes show that this fatty acid is oxidized mainly in the mitochondria . In comparative experiments, Hagve and Christophersen  studied the effect of (+)-decanoylcamitine on the oxidation of different fatty acids in hepatocytes and have concluded that the initial chain-shortening of adrenic acid is mainly a peroxisomal reaction. In the present study we have confirmed the effects of ( + )-decanoylcamitine on the oxidation of palmitate and adrenic acid and found that dodecanedioic acid oxidation is unaffected by this inhibitor. This indicates that the chain-shortening of DC,, is almost exclusively peroxisomal. Another indication is the 15550-fold higher K, values DC,, has for the mitochondrial P-oxidation enzymes [2,5] and the poor oxidation of free DC-acids and their camitineand CoA-esters in isolated mitochondria (5,8,25]. The studies on the acetylation of sulfanilamide with low concentrations of [l-‘4C]palmitate or [ carboxyf14C 1dodecanedioate as precursors also support this conelusion. The higher specific activity of the acetylsulfanilamide when dodecanedioic acid was the precursor, can be explained by a more direct formation of extra~tochond~al, cytosolic acetyl-CoA. Hemmelgam et al.  also obtained results indicating that P-oxidation from the w-end leads to formation of acetyl-CoA that is more effective in cytosolic acetylations. These results do not support the idea that there is considerable mobility of acyl groups between different cell compartments. The fact that we are able to trap the cytosolic acetyl-CoA in acetylated sulf~la~de more efficiently from DC,, than from palm&ate, suggests that the equilibration is slow compared to the formation of acetyl groups in the peroxisomes. Apparently, the mitochondrial acetyltransferase do not equilibrate the inside and outside acetyl-CoA pools. In recent studies we have also found that DC,, is oxidized mainly to free acetate in hepatocytes from bez~ibrate-treated rats, while it is a very poor precursor for ketone bodies (271. As shown herein (Fig. l), the same is true in hepatocytes from normal rats. Presumably, the peroxisomal acetyl-CoA is hydrolyzed to free
acetate by a peroxisomal or cytosolic acetyl-CoA hydrolase . Since clofibrate easily increases peroxisomal oxidation of fatty acids S-lo-fold , cells from clofibratefed rats might be expected to show a similar increase in the oxidation of dodecanedioic acid. However, Draye and Vamecq [8,18] have found that the dicarboxylylCoA synthetase shows little increase in the liver of clofibrate-fed rats. Presumably, activation is rate-limiting in cells from these rats (Table I). The decreased accumulation of shorter DC acids (Fig. 2B, Table II) can then be explained most simply by an increased oxidation capacity in the cells from clofibrate-fed rats, where the uptake or activation step becomes rate-limiting. As we have shown previously in perfused kidney  and now in hepatocytes, the fraction of metaboiites more polar than DC, make up more than half of the metabolites formed. In cells from clofibrate-treated rats, this fraction amounts to as much as 80% of the recovered metabolites (Table II). This is in good agreement with the reduced urinary excretion of shortened DC acids seen in clofibratetreated rats 181. From these studies it is evident that most of the DC,, is completely metabolized. This would be in agreement with the observations that DC acids are gluconeogenic in vivo [29,30]. Dicarboxylic acids can also be esterified and incorporated into the tissue lipids [71. The in~bition of sulfanila~de acetylation (Fig. 3A) and of palmitate oxidation (Table I) at high concentrations of dodecanedioic acid, suggests that its CoA-ester accumulates in the cytosol of normal cells, thus lowering the free CoA pool. Free CoA has been reported to be an inhibitor of the N-acetyltransferase . However, in separate experiments on a partially purified enzyme preparation, we did not see any effect of free CoA, pal~toyl-CoA or free DC acids (results not shown). Presumably, the more rapid peroxisomal P-oxidation from clofibrate-fed rats preof DC,, in hepatocytes vents the accumulation of its CoA-esters and explains that the inhibition of sulfanilamide acetylation has disappeared in these clels. The acetylsulfanilamide also was less efficiently labeled in cells from clofibrate-fed rats. Since uptake and/or activation of the [curbuxyl“C]DC,, seems to be rate-limiting in these cells, the increased peroxisomal formation of acetyl groups from endogenous, unlabeled fatty acids presumably lowers the specific activity of the cytosolic acetyl-CoA pool. Altogether, our results indicate that dicarboxylic acids normally are P-oxidized in the peroxisomes. The relative oxidation rates decrease as the chain-length decreases. Part of the shorter CoA-esters therefore presumably are hydrolyzed and excreted in the urine. However, even normal hepatocytes and kidneys  have a significant capacity for total oxidation of dicarboxylic acids.
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