Biochimica et Biophysics Acta 835 (1985) 147-153

Elsevier

147

BBA 51925

Flux of palmitate through the peroxisomal and mitochondrial /3-oxidation

systems in isolated rat hepatocytes

Jens Kondrup * and Paul B. Lazarow **

The Rockefeller University, 1230 York Avenue, New York, NY 10021 (U.S.A.)

(Received October lOth, 1984)

(Revised manuscript received January 17th, 1985)

Key words: Fatty acid oxidation: Peroxisome; Mitochondrial/3-oxidation; Metabolic flux; Pahnitate; (Rat hepatocyte)

Peroxisomes catalyze the B-oxidation of fatty acids but their quantitative role in fatty acid catabolism in the intact hepatocyte is not yet clarified. In the present study peroxisomal p-oxidation of (1-14C]palmitate was quantitated in hepatocytes without the use of metabolic inhibitors. It was assumed that acetyl-CoA formed by peroxisomal /S-oxidation enters the cytosolic pool of acetyl-CoA, whereas that from mitochondrial fi-oxida- tion enters the mitochondrial pool. The labeling of the two acetyl-CoA pools was assessed by measuring the incorporation of radioactivity into cholesterol (from cytosolic acetyl-CoA) and CO, (from mitochondrial acetyl-CoA). The system was calibrated with [ l-‘4C]acetate and [ l-l4 Clbutyrate because butyrate undergoes &oxidation only in mitochondria, whereas acetate forms acetyl-CoA primarily in the cytosol. The labeling ratio, ([ “C]cholesterol X 100)/(]14C]cholesterol + [ “C]C0,), reflects the site of formation of acetyl-CoA. This ratio was 0.51 for butyrate, 1.39 for acetate and 0.79 for palmitate. The difference between palmitate and butyrate was statistically significant (P < 0.02). This indicates that not all of the palmitate was oxidized in mitochondria. By linear interpolation it was estimated that approximately 32% of the [l-‘4C]palmitate oxidation began in peroxisomes.

Introduction

Peroxisomes from rat liver oxidize fatty acids by a /3-oxidative process [1,2]. The substrates of peroxisomal P-oxidation include the CoA deriva- tives of saturated and unsaturated fatty acids [l-6], dicarboxylic fatty acids [7], and the side chain of cholesterol [8,9]. The role of peroxisomal P-oxida- tion of these various substrates in the intact cell is incompletely elucidated. It has been suggested that peroxisomes may play a predominant role in the

* Present address: Rigshospitalet, Department of Medicine

A, Division of Hepatology, Blegdamsvej 9, DK-2100

Copenhagen, Denmark. ** To whom correspondence should be addressed.

P-oxidation of long-chain unsaturated (20 : 1, 22 : 1) [lO,ll] and very-long-chain saturated (24 : 0 and 26 : 0) [5,6] fatty acids. The physiological con- tribution of peroxisomes to palmitate oxidation is still a matter of controversy. When the per- oxisomal oxidation of palmitate is assessed by measuring H,O, production, only a negligible contribution by the peroxisomes has been found [12,13]. In contrast, in the presence of inhibitors of mitochondrial respiration or mitochondrial /3- oxidation, ketogenesis has been found to proceed at about 25550% of control rates [12,14].

In the present study, the metabolic fate of the carbon atoms of palmitate was investigated. It was assumed that radioactive acetyl-CoA formed dur- ing peroxisomal P-oxidation enters the cytosolic

0005-2760/85/%03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

148

pool of acetyl-CoA, and that acetyl-CoA resulting from mitochondrial P-oxidation enters the mito- chondrial acetyl-CoA pool. Radioactive labeling of these two pools may be distinguished experimen- tally, since CO, is formed from mitochondrial acetyl-CoA and cholesterol is formed from cyto- solic acetyl-CoA [15,16]. Radioactivity appearing in these two products from an exogenous substrate reflects the specific radioactivity of the two acetyl- CoA pools and, hence, the site of formation of the acetyl-CoA. There is interchange between the mitochondrial and cytosolic acetyl-CoA pools. Therefore, the system was calibrated with acetate which is activated to acetyl-CoA primarily in the cytosol[17,18], and with butyrate which is oxidized to acetyl-CoA in the mitochondria and which is not a substrate for peroxisomal P-oxidation [2].

Freshly isolated hepatocytes were incubated for various lengths of time with a mixture of palmi- tate, acetate and butyrate; each of these substrates was l-‘4C-labeled in one of triplicate flasks at each time point. The ratio of rates of incorpora- tion of label into the products, (cholesterol x

lOO)/(cholesterol + CO,), allows an estimation by linear interpolation of the peroxisomal and mitochondrial contributions to palmitate /Loxida- tion. In addition, the number of cycles of per- oxisomal P-oxidation is discussed on the basis of parallel experiments with [U-i4C]- and [16- i4C]palmitate and with [l-‘4C]- and [2-i4C]acetate. Some of these results have been published in a preliminary form [19].

Materials and Methods

Materials. 200 g female Wistar rats fed ad libi- turn were used. They were anesthesized at about 11 a.m. by intraperitoneal injection of 20 mg of sodium pentothal.

All radioactive compounds were from New England Nuclear, Boston, MA, U.S.A. They were purified as described below. Collagenase, type II, was from Worthington Biochemical Corporation, Freehold, NJ, U.S.A. Albumin (Fraction V from bovine plasma) was from Reheis Chemical Com- pany, Phoenix, AZ, U.S.A. It was purified to remove fatty acids by the method of Chen [20].

Hepatocytes. Hepatocytes were prepared as de- scribed by Dich et al. [21]. All media contained 20 mM glucose [22] and were aerated with a mixture

of O,/CO, (95 : 5). The average yield of cells was about 200. lo6 cells/100 g rat. The average per- centage stained after incubation for 1 min in 0.25% Trypan blue at room temperature was 15 (range, 9-22). These results were similar to those reported earlier [23].

Preparations of substrates. The commercial [l- “C]palmitate contained a radiochemical impurity that comigrated with cholesterol in thin-layer chromatograms, thus interfering with the measure- ments of radioactivity in cholesterol (see below). The [1-i4C]palmitate was diluted with unlabeled palmitate to a specific radioactivity of 63000 dpm/nmol, and dissolved in 2.5 mM NaOH/ H,0/95 % ethanol, (1 : 1.5 : 2.5. v/v). This solu- tion was washed twice with hexane which removed the impurity. The aqueous/ethanolic phase was acidified with HCl, the palmitate was re-extracted into hexane, the hexane was evaporated, and the residue was re-dissolved in 40 mM NaOH to a concentration of 7.2 mM. For each experiment. this [1-i4C]palmitate was added dropwise to 5 vol. of Krebs-Henseleit bicarbonate buffer (pH 7.4) containing defatted albumin to give final con- centrations of 1.2 mM palmitate and 0.57 mM albumin. Unlabeled palmitate-albumin complex was prepared at the same concentrations, but without prior extraction of the palmitate.

[l-‘4C]Butyrate in ethanol was dried under a stream of N,, dissolved in H,O, and diluted with unlabeled butyrate to a final specific activity of 1600 dpm/nmol and a final concentration of 20 mM. It was then washed with hexane. [l- 14C]Acetate was similarly prepared and washed. Its final specific activity and concentration were 1300 dpm/nmol and 40 mM, respectively. Un- labeled butyrate (20 mM) and acetate (40 mM) were also prepared. Other substrates, in which the 14C was located elsewhere than in the first carbon, were prepared in an identical manner and with the same specific radioactivity.

Six chemically identical mixtures of palmi- tate/butyrate/acetate (2 : 1 : 1, v/v) were pre- pared. Three of these mixtures were made with [l-i4C]-, [16-‘4C]- or [U-i4C]palmitate and un- labelled butyrate and acetate, one mixture with [1-i4C]butyrate (unlabeled palmitate and acetate) and two mixtures with [1-i4C]- or [2-14C]acetate (others unlabeled).

149

Incubation conditions. Hepatocytes (3.7 . lo6 in 0.5 ml) were mixed in 50 ml Erlenmeyer flasks with 1.5 ml Krebs-Henseleit bicarbonate buffer containing 2 mM unlabeled pahnitate (complexed to 0.76 mM albumin) and 20 mM glucose. Center wells each contained a piece of filter paper. The flasks were gassed with O,/CO, (95 : 5), capped, and preincubated at 37°C for 10 min in a shaking-water bath at 90 gyrations/mm. This pre- incubation allowed the cells to enter a steady-state fatty acid metabolism. In preliminary experiments it was found that the pahnitate concentration at the end of the 10 min period had dropped to 1.2 mM (determined by using labeled pahnitate as substrate and measuring residual radioactive palmitate after extraction [24] and thin-layer chro- matography [25].

At the end of the pre-incubations with un- labeled palmitate (time-point zero in Fig. l), each flask received 200 ~1 of one of the labeled sub- strate mixtures described above, and was regassed and recapped. Hepatocytes were then incubated for 0, 10, 20 or 30 min. Six flasks were used for each time-point; one of the three substrates was labeled in each flask. All flasks had identical chemical compositions at the start of the incuba- tions: 1.2 mM palmitate, 0.45 mM butyrate, 0.90 mM acetate, 0.57 mM albumin, 20 mM glucose and 1.7 - lo6 hepatocytes/ml in Krebs-Henseleit buffer, pH 7.4 (total volume 2.2 ml).

The incubations were stopped by cooling the flasks in an ice/water slurry. Flasks for zero-time values were cooled immediately after the addition of radiactivity.

Analysis of radioactivity in products. After cool- ing, 250 ~1 of 1.25 M potassium hydroxide was injected through the rubber cap into the center well and then 200 ~1 of a 0.2 M glycine buffer (pH 3) and 125 ~1 of 1 M perchloric acid were added to the hepatocyte suspension. The flasks were left in the ice slurry for 60 min for the 14C0, to diffuse into the filter paper. The filter paper was trans- ferred to a counting vial and the well was rinsed with 1 ml water which was added to the vial. Radioactivity was measured with 10 ml Formula 963 (New England Nuclear, Boston, MA, U.S.A.). By using [‘4C]bicarbonate, this procedure was found to give a recovery of about 95%.

Cholesterol was extracted with 2 pmol of car-

rier into alkaline ethanol, precipitated with water, and dissolved in hexane as described by Weis and Dietschy [26]. The hexane was back-washed twice with a mixture consisting of 50% ethanol, 0.29 M potassium hydroxide, 2.5 mM oleate, 1 mM butyrate and 2 mM acetate. The hexane was evaporated, the residue was redissolved in chloro- form/methanol (2 : 1) and 2 ~1 of 70 mM oleate was added as a carrier for any remaining radioac- tive fatty acids. The sample was applied to Gel- man SG thin-layer chromatography plates and developed in hexane/ether/acetic acid (80 : 20 : 1) [25]. The bands were visualized with iodine vapor. The chromatogram was cut into small pieces which were counted in 10 ml Formula 963.

By this extraction method, 98 & 9% of the [r4C]cholesterol added to incubation flasks con- taining cells and substrates was recovered in the back-washed hexane phase. After thin-layer chormatography, the recovery was 82 + 8% (mean k S.D., n = 11). The radioactivity in the cholesterol band was corrected by multiplying by the total radioactivity applied to the chromato- gram and dividing by the radioactivity recovered in the total chromatogram. Radioactivity in water-soluble products was determined after hexane extraction essentially according to Man- naerts et al. [27].

Radioactivity was counted in a Packard Tri- Carb scintillation counter with an efficiency of 60%. The results are expressed in nmol of radioac- tive carbon atoms per 10’ hepatocytes, which are approximately equivalent to 1 g wet liver weight [28]. Student’s t-test for paired observations was used for analysis of statistical significance.

Results

Oxidation of [1 -‘4C]palmitate After a lag of approx. 5 min, the incorporation

of radioactivity from the 1-14C-labeled substrates into CO, and cholesterol was linear with time (Fig. l), indicating that the system was in a metabolic and isotopic steady state. Labeling of cholesterol, relative to CO,, was higher for acetate than for butyrate and for palmitate it was intermediate.

The rates of incorporation of 14C into cholesterol and CO, were calculated for each ex- periment by linear regression analysis by the

150

vinutes

Fig 1. Metabolism of [l-‘4C]butyrate, palmitate and acetate to

cholesterol and CO,. Hepatocytes from fed rats were incubated

with 1.2 mM palmitate, 0.45 mM butyrate and 0.90 mM

acetate. In each flask, one substrate was labeled as indicated in

the figure. At the time-points shown, the radioactivity in CO1

and cholesterol was determined as described m the text. The

symbols represent the mean of three experiments and the lines

were drawn according to linear regression analysis.

least-squares method. The ‘cytosolic index’, the ratio of labeling rates of (cholesterol x loo)/

(cholesterol + CO,), is an arbitrary quantitative measure of the cytosolic generation of acetyl-CoA.

The cytosolic index for palmitate was higher than

that for butyrate in a total of six experiments (the

three experiments in Fig. 1 plus three preliminary

experiments employing only [ 1 -14 C]palrnitate and [l-‘4C]butyrate). The cytosolic index for palmitate was calculated as the percentage of the index for

butyrate in each of these experiments: the mean was 173% (S.E. = 20%; P vs 100% < 0.020). This

result indicates that the specific radioactivity of

the cytosolic acetyl-CoA, relative to that of mitochondrial acetyl-CoA, was higher when the 14C was in palmitate than when it was in butyrate.

Since the system was in a metabolic and isotopic

steady state, this finding demonstrates that palmi- tate is not oxidized to acetyl-CoA exclusively in

the mitochondria. In all three experiments of Fig. 1, the cytosolic

index was higher for acetate than for butyrate, and

for palmitate it was intermediate: the mean values

are given in Table I. The fraction of [l-14C]palmi-

tate oxidized to acetyl-CoA in peroxisomes was estimated in each experiment by linear interpola- tion as follows:

cytosolic index for palmitate - cytosolic index for butyrate

cytosolic index for acetate - cytosolic index for butyrate x 100

The individual results in the three experiments were 48, 30 and 17% (mean, 32%; S.E., 9%; P vs. zero’% i 0.05). These values apply only to [l- “C]palmitate, i.e., the first cycle of /3-oxidation.

Oxidution of (lJ-‘4C]palmitate

The oxidation of the remainder of palmitate was investigated in parallel incubations with [U-

14C]- and [16-‘4C]palmitate (Table II). The total

formation of labeled products from the first and

last carbon atoms of palmitate was approximately equal and total products from [U-‘4C]palmitate was about 16-times higher. The distribution be-

tween CO, and water-soluble products, however. depended on the position of the label. As a per-

centage of total oxidation, 14C0, accounted for

20, 2, and 10% with [1-‘4C]-, [16-‘4C]- and [U-

“C]palmitate, respectively. Calculations of the cytosolic index analogous to

the one above were not feasible since butyrate labeled in different positions was unavailable.

Calibration with [1-14C]butyrate would lead to

TABLE I

METABOLISM OF l-r4C-LABELED SUBSTRATES TO CO1 AND CHOLESTEROL

The experiments are the same as those presented in Fig. 1. The rates in each individual experiment were calculated by linear regression analysis of the data. The results are expressed as means f S.D. (n = 3).

I-“‘C-labeled substrate

Palmitate

Butyrate

Acetate

Rate of labeling

(nmol labeled carbon atoms/mm

per 10’ cells)

co2 Cholesterol

24+ 2.8 0.19 + .08

1 32544 0.69 f 0.37

27kll 0.35 5 0.07

Cytosolic index

(cholesterol)x lOO/(cholesterol+ CO, )

0.79 + 0.45

0.51 kO.24

1.39 f 0.46

151

TABLE 11

RATE OF OXIDATION OF VARIOUSLY LABELED SUB-

STRATES

The experiments are the same as those presented in Table 1.

The results are means i S.D. (n = 3) of nmol labeled carbon

atoms/min per 10s cells.

Labeled substrate CO, Water-soluble Total

products oxidation

[l-‘4C]Palmitate 24k 2.8 97* 21 121k 23

[16-‘4C]Palmitate 2* 0.2 121* 19 123k 19

[U-‘4C]Palmitate 204 f 24 1742 f 565 1946 + 580

[I-‘4C]Acetate 27k11 _ _

[2-14C]Acetate 8+ 0.5 _ _

Calculated a 227 1713 1940

’ Expected results for [U-‘4C]palmitate if its oxidation equals

seven (Cl, 2) units of palmitate and one (C15. 16) unit of

palmitate. correcting for the different metabolism of odd and

even carbons atoms determined with acetate.

erroneous results because odd and even carbon

atoms are metabolized differently [29-311. We ob- served that the formation of 14C02 from [2-

i4C]acetate in control flasks was only 8/27 or 30%

of that from [l-i4C]acetate (Table II). In addition, carbon atoms 15 and 16 of palmitate are partially converted directly to ketone bodies [32,33], instead

of equilibrating with the acetyl-CoA pool. These differences presumably account mostly, if not en- tirely, for the fact that labeling of CO, from

[16-i4C]palmitate was only 2/24 or 8% of that from [l-‘4C]palmitate (Table II).

We calculated what would be expected from the

oxidation of [U-i4C]palmitate if the first seven C2 units were all metabolized identically to the first C2 unit (last line of Table II). The calculated and experimental values agree within 10 and 2% for

CO, and water-soluble products, respectively, which is within the experimental error. This is

compatible with the possibility that the 32% of palmitate beginning P-oxidation in peroxisomes is completely oxidized by the peroxisomes. It is inter- esting to note that if the peroxisomal P-oxidation

only proceeded for five cycles (the maximum that has been observed in vitro [2]), hexanoyl-CoA (which is poorly oxidized by peroxisomes [2]) or hexanoyl-carnitine or hexanoate might accumulate as water-soluble products. In this case, 27 fewer nmol of i4C02 would form, which is close to the measured deficit of 23. Our results do not exclude

the possibility that more limited chain-shortening

[34] might occur in peroxisomes with completion of the P-oxidation in mitochondria. Experiments with other labeled substrates are required to assess

the number of cycles of peroxisomal /3-oxidation

in intact hepatocytes.

Discussion

In previous studies, attempts have been made to estimate peroxisomal P-oxidation of added fatty

acids in isolated cells or perfused liver by measure- ment of the concomitant increase in H,O, produc- tion [12,13]. From these studies it was concluded

that peroxisomes play an insignificant role in /?-

oxidation of palmitate in the intact cell. However,

the lack of increased net production of H,O, could

be due in part to a suppression of P-oxidation of endogenous fatty acids by palmitate added. In

addition, the H,O, produced could be consumed,

directly or indirectly, by re-oxidation of NADH generated in the third peroxisomal P-oxidation

reaction (see Ref. 35). In other experiments, non-

mitochondrial P-oxidation in intact cells was estimated by measuring ketogenesis in the pres- ence of inhibitors of mitochondrial respiration or

mitochondrial P-oxidation [12,14,36]. In these studies 25550% of ketogenesis was found to re-

main in the presence of inhibitors. If mitochondrial inhibition were complete and if the remaining

ketogenesis stemmed from /_&oxidation, it may re-

flect peroxisomal /3-oxidation; the contribution agrees with the results of this study. However, in

inhibitor studies, the peroxisomal P-oxidation could be artefactually too high because of a diver-

sion of substrate to peroxisomes caused by bloc-

kage of mitochondrial P-oxidation, or too low due to other pertubations of cell metabolism. In the

present study, peroxisomal P-oxidation was mea-

sured concomitantly with the rnitochondrial con- tribution in uninhibited cells.

The distribution of [1-i4C]palmitate between mitochondria and peroxisomes was apparently not

affected by the presence of butyrate and acetate; in their absence, the metabolism of [1-‘4C]palmi- tate to CO, and cholesterol was doubled but the cytosolic index was the same (data not shown). In the presence of butyrate and acetate, the total oxidation of [l- “C]palmitate (120 nmol/min per

152

10’ cell, Table II) falls within the range of values

measured in isolated hepatocytes by others [12,36,37] and it is almost identical to the total

oxidation in hepatocytes incubated under similar

conditions without butyrate and acetate (107

nmol/min per 10’ cells) [38]. Christiansen [36] also found that the total oxidation of [l-r4C]- and

[16-r4C]palmitate was the same, and she reported a distribution between CO, and water-soluble

products for the two differently labeled species

that was similar to our results. The main finding of the present investigation is

that palmitate and butyrate are not converted to

acetyl-CoA exclusively in the same subcellular compartment. We have estimated that 17-48s of

[1-r4C]palmitate is oxidized in peroxisomes, which is in accordance with the inhibitor studies men-

tioned above. If it is assumed that peroxisomes perform five

cycles of /?-oxidation in the intact cells, and that

e.g., hexanoate accumulates, the peroxisomal rate of acetyl-CoA production from exogenous [U-

“C]palmitate would be 0.32 x 1946/2 x 5/8 = 195 nmol acetyl-CoA/min per 10s cells. The

mitochondrial production rate would be 0.68 X 1946/2 = 662 nmol per 10’ cells. These flux rates

may be compared with the capacity for P-oxida-

tion measured in the isolated organelles. Expressed per g wet weight, which is equivalent to 10’ cells, the peroxisomal capacity is about 1000 nmol/min

[l] and the mitochondrial capacity is about 1000-1400 nmol/min (calculated from data given

by Osmundsen and Bremer) [39]. The contribution of peroxisomal P-oxidation

would be an overestimation in the present study if

a significant fraction of acetate were activated in the mitochondria; according to the subcellular dis- tribution of acetyl-CoA synthetase and. the kinetic

constants of the cytosolic and mitochondrial en- zymes, only 5.10% of the total activation of acetate would be expected to take place in the mitochondria [17,18]. The contribution of per- oxisomes would be an underestimation if a signifi- cant amount of radioactivity in cholesterol originated from reutilization of ketone bodies. In liver perfused with oleate, about 10% of cholesterol carbon atoms stemmed from ketone bodies [40].

Acetyl-CoA formed by P-oxidation in the glyoxysome (a type of peroxisome) in germinating

castor bean endosperm is used anabolically for the

synthesis of glucose [41]. Little is known about the

utilization of the acetyl-CoA produced by liver peroxisomes. It may be used for cholesterol bio-

synthesis and for acetylation reactions or it may be

transferred to the mitochondria for ketone body formation and CO, production [35].

Acknowledgements

The excellent technical assistance of Peter Eise- mann and Beth Schorr, and stimulating discus-

sions with Dr. Christian de Duve and Dr. Niels Grunnet are gratefully acknowledged. The re-

search was supported by NSF grants 80-08713 and 82-08315, by a stipend from the University of

Copenhagen to JK, and by an Established Fellow- ship from the New York Heart Association to

PBL.

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