Eur. J. Biochem. 156,603-607 (1986) 0 FEBS 1986

Evidence that the development of hepatic fatty acid oxidation at birth in the rat is concomitant with an increased intramitochondrial CoA concentration Fernando ESCRIVA, Pascal FERRE, Danielle ROBIN, Pierre ROBIN, Jean-FranGois DECAUX and Jean GIRARD Centre de Recherches sur la Nutrition, Centre National de la Recherche Scientifique, Meudon-Bellevue

(Rcceived November 20, 1985/January 31, 1986) - EJB 85 1259

The development of hepatic fatty acid oxidation during the perinatal period in the rat was studied using isolated mitochondria, Ketone body synthesis from substrates entering at different levels of ,%oxidation was 2-3 times lower in mitochondria isolated from term-fetal liver than in 16-h-old newborn or adult liver mitochondria. The low rate of palmitoyl-L-carnitine oxidation in term-fetal mitochondria was linked neither to the low capacity of the respiratory chain nor to the removal of acetyl-CoA in the hydroxymethylglutaryl-CoA synthase pathway. The 2.5-times lower concentration of CoA found in term-fetal liver mitochondria when compared to 16-h-old or adult liver mitochondria might be the factor responsible for the low rate of fatty acid oxidation in term-fetal liver mitochondria.

In newborn rats the capacity of the liver for fatty acid oxidation and ketone production is increased during the first day of life [l]. Previous studies [2, 31 had suggested that an increase in the capacity of liver mitochondria for fatty acid oxidation could be at the origin of the enhanced capacity of the liver to perform ketogenesis after birth in the rat.

The enzymes of fatty acid oxidation and of ketogenesis increase at birth in the liver of the rat [l, 4, 51. However, no clear-cut evidence indicates that these enhanced enzymatic activities are related to the increase in fatty acid oxidation. Yeh and Zee [6] have studied fatty acid oxidation in isolated liver mitochondria of term-fetal and newborn rats by a polarographic technique. They concluded that the increased ketosis of suckling animals was linked to an increased rate of fatty acid oxidation. However, the possible steps involved in the process were not investigated.

Fatty acid oxidation in the liver of fetal animals could be limited (a) by the low activities of specific enzymes of fatty acid oxidation and specially carnitine acyltransferase, which increases at birth [7]; (b) by a reduced availability of necessary cofactors (carnitine, CoA, NAD', FAD); (c) by the removal of end-products of fatty acid oxidation, i.e. acetyl-CoA via the citric acid cycle and the hydroxymethylglutaryl-CoA synthase pathway, NADH and FADHz via the respiratory chain. The aim of the present study was to investigate which of these factors was responsible for the development of the capacity for fatty acid oxidation at birth in the rat.

MATERIALS AND METHODS

Animals

Wistar, rats bred in our laboratory, were used. Mothers were housed at 24 "C with light from 0.7: 00 h to 19 : 00 h. They

Correspondence to P. Ferre, Centre de Recherches sur la Nutrition du CNRS, 9 Rue Jules Hetzcl, F-92190 Meudon-Bellevue, France

Enzymes. Acyl-CoA synthetase (EC 6.2.1.3), carnitine acyltrans- ferase I (EC 2.3.1.21), citrate synthase (EC 4.1.3.7), hydroxymcthyl- glutaryl-CoA synthase (EC 4.1.3.5), malate dehydrogenase (EC 1.1.1.37), panthotcnate kinase (EC 2.7.1.33), succinate dehydro- genase (EC 1.3.99.1).

had free access to water and chow pellets (65% carbohydrate, 11 YO fat and 24% protein of total energy). Pups were delivered by cesarean section at 21.5 days of gestation (term-fetal rats). They were either immediately killed or placed in an incubator (37"C, 70% relative humidity) and starved for 16 h (newborn rats). Adult rats in the postabsorptive state were also used.

Isolation of liver mitochondria

Mitochondria were isolated according to Aprille and Asimakis [S]. For term-fetal and newborn rats, 15 - 20 livers were pooled. Protein was estimated by the method of Lowry et al. [9] using crystalline bovine serum albumin, fraction V as standard.

Polurographic measurements

They were carried out with an oxygraph Cilson (model 5/6H) equipped with a 2-ml water-jacketed chamber maintained at 30°C and using a Clark oxygen electrode. For the measurement of coupled and uncoupled respiratory ca- pacity, mitochondria (1 mg protein) were added to a respirato- ry medium which consisted of 225 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM K2HP04/KH2P04, 5 mM MgCI2 and 10 mM Tris/HCl, pH 7.4 [8]. After the addition of substrate, succinate (10 mM), glutamate + malate (5 mM + 5 mM), the rates of oxygen consumption were measured in the absence (state 4) and in the presence of titrated ADP (90 pM) (state 3) [lo]. Uncoupled rates of oxygen consumption were deter- mined by adding 2,4-dinitrophenol (40 pM). The respiratory control ratio was taken as state 3/state 4 oxygen consumption [111.

Pulmitoyl-L-curnitine oxidation

The respiratory medium was according to Osmundsen and Sherratt [12]. After addition of mitochondria (2-3 mg protein), the oxidation of palmitoyl-L-carnitine was con- ducted in the presence of either malonate (10 mM) or malate (2.5 mM), both in the presence of 2,4-dinitrophenol(O.1 mM) (uncoupling conditions). Addition of palmitoyl-L-carnitine

604

(10 pM) initiated the reaction. The rate of oxygen consump- tion due to the oxidation of palmitoyl-L-carnitine was taken as the linear rate of oxygen consumption in the presence of this substrate and 2,4-dinitrophenol minus the oxygen con- sumption in the presence of 2,4-dinitrophenol alone. Ketone body production during palmitoyl-L-carnitine oxidation was also determined by sampling 1 ml chamber mixture 1 min after the addition of an excess (80 nmol) of palmitoyl-L- carnitine, during the linear phase of oxidation. This rate was corrected for the ketone body production in the absence of palmitoyl-L-carnitine. Ketone bodies were assayed according to Williamson et al. [13].

The ratio of oxygen consumed to palmitoyl group utilized (dO/d palmitoyl ratio) was calculated according to Garland et al. [14,15]. The theoretical value of this ratio in the presence of malonate is 14 since acetyl-CoA is channeled exclusively towards acetoacetate formation. In the presence of malate and dinitrophenol, citrate is the main end-product and the theoretical ratio is 22 [14, 151. Rates of fatty acid utilization were calculated by dividing the rate of oxygen consumption measured in the presence of palmitoyl-L-carnitine by the dOjd palmitoyl ratio.

Determination of CoA derivatives on isolated mitochondria or fresh liver

Mitochondria (6- 8 mg protein) were added either direct- ly into 250 pl ice-cold HC104 (1 M) and 50 p1 dithiothreitol (0.1 M) (state 1) or into the polarograph chamber containing the respiratory medium used for the measurement of palmitoyl-L-carnitine oxidation in the presence of malonate and 2,4-dinitrophenol (see above). 1 ml of the chamber content was sucked off and added to the HClO,/dithiothreitol mixture either 2 min after the addition of 2,4-dinitrophenol (state 2) or during the linear phase of oxygen consumption after the addition of palmitoyl-L-carnitine (40 pM). Long- chain fatty acyl-CoA were determined in the washed pellets of HC104-insoluble material by hydrolyzing the thioesters as described by Garland et al. [16]. The CoA, in the form of free CoA plus acetyl-CoA, was determined in the HC104-soluble material and free CoA, acetyl-CoA plus short-chain acyl- CoA were determined in the HClO,-soluble material after hydrolysis as described by Ingebretsen et al. [17]. Total mitochondrial CoA was obtained by adding CoA contained in hydrolysed HC104-soluble and insoluble fractions. Frequent controls were done by hydrolysing directly the whole acid sample and the results always agreed within

Liver total CoA was determined after killing the animals and rapid freeze-clamping of the tissue. The frozen material was homogenized in ice-cold HClO, (1 M) and dithiothreitol (0.01 M). Extraction of CoA and its thioesters was performed as described by Garland et al. [16] and Ingebretsen et al. [17].

CoA was assayed on the various extracts by the cycling assay of Michal and Bergmeyer [18].

5%.

Ketone body production by liver mitochondria during incubations

Incubations of liver mitochondria were done in 25-ml conical flasks in a final volume of 2.5 ml modified Krebs- Henseleit buffer, pH 7.4 containing 4 mM ATP, 1 mM ADP, 50pM CoA, 250pM reduced glutathione and 200pM L-carnitine [19]. The following substrates were studied: oletate (100 pM), oleyl-CoA (100 pM), octanoylcarnitine (200 pM) and octanoate (200 pM). All these substrates were previously

bound to fatty-acid-free dialyzed albumin, to reach a molar ratio fatty acid/albumin of 3.5. The flasks were gassed with 95% 02, 5% COz, capped and maintained at 30°C for 10 min. Then mitochondria (1 mg protein) were added, the flasks were gassed again with 0 2 / C 0 2 and shaken at 30°C during 15 min in a water bath. Incubation was ended by the addition of 0.4 ml 5% (w/v) HC104. The rate of ketone body production was the amount of ketone body formed in the presence of the substrate minus the amount formed in absence of the substrate during the same period. It was checked in preliminary experi- ments that the rates of ketone body production were linear with time.

Chemicals

Enzymes, substrates and cofactors of the best available grades were obtained from Boehringer (Meylan, France) and bovine serum albumin fraction V (fatty-acid-free) was purchased from Sigma (St Louis, MO, USA) and dialysed before use.

Statistics

cance of differences was assessed by the Student's t-test. Results are presented as means & SEM. Statistical signifi-

RESULTS AND DISCUSSION

Respiratory rates from succinate and glutamate f malate

In order to assess the intactness of mitochondria we have used the respiratory control ratio, i.e. the ratio of oxygen utilization in presence of substrate and ADP (state 3) to the utilization in presence of substrate alone (state 4). As seen from Table 1, mitochondria isolated from the liver of 16-h- old newborn or adult rats exhibit respiratory control ratios with succinate and glutamate + malate, greater than 5 , in- dicating that mitochondrial membranes are intact. In mito- chondria from term-fetal animals, the respiratory control ratio is much lower (Table 1). This does not result from an artefactually high oxygen utilization in state 4, since it is similar to the rate of oxygen utilization in state 4 observed in mitochondria from adult rats, but from a low rate of oxygen utilization in state 3, as previously described [8,20,21]. More- over, the oxygen utilization in the presence of succinate or glutamate + malate in mitochondria from term-fetal rats can be dramatically increased by 2,4dinitrophenol, when com- pared to state 3. This confirmed that the oxygen utilization in state 3 was limited at one of the steps of oxidative phos- phorylation [8].

Ketone body synthesis in incubated mitochondria

Ketone body synthesis from substrates (oleate, oleyl-CoA, octanoylcarnitine and octanoate) that enter the fatty acid oxidation pathway at different levels, was studied. Whatever the substrate used, ketone body synthesis was lower in term- fetal liver mitochondria when compared to 16-h-old and adult liver mitochondria (Fig. 1). This indicates that in term-fetal mitochondria the limiting step for fatty acid oxidation and ketone body synthesis involves and enzymatic step or a cofactor common to all fatty acid substrates entering the mitochondria and that carnitine acyltransferase I does not play a major role in the development of fatty acid oxidation in the newborn rat. In the guinea-pig, experiments performed

605

Table 1. Respiratory function in liver mitochondria isolated from term-fetal, 16-h-old newborn or adult rats Values are means f SEM for 12-20 determinations (succinate) and 4 determinations (glutamate + malate). For further details, see the text

Substrate ~

Group State 4 State 3 Uncoupled Respiratory control ratio

nmoI O min- mg protein-'

Succinate term-fetal 42 2 88k 4 177 f 11 2.1 f 0.1 16-h-old 46 f 2 250 f 12" 271 f 19" 5.3 f 0.05" adult 38 2 231 f 6" 280 f 18" 6.1 50.1"

adult 17 f 0.5 149 10" 220f l l a 8.3 f 0.5" Glutamate + malate term-fetal 20 * 2 5 1 k 2 119f 9 2.7 f 0.3

* Difference significant for P < 0.001 when compared to mitochondria from term-fetal animals.

Term-fetal

0 16-h

Adult

..

Oleate Oleyl-CoA

..

It Octonoylcarnitine Octonoote

Fig. 1. Rate of ketone body production from various fa t ty acid substrates by incubated liver mitochondria from term-jetal, 16-h-old newborn and adult rats. Results are presented as means k SEM of 6- 13 determinations. *, ** difference significant for respectively P < 0.05 and P < 0.001 when compared to term-fetal mitochondria. Incubations were carried out in Krebs Henseleit buffer, pH 7.4, containing 4 mM ATP, 1 mM ADP, 50 pM CoASH, 250 pM reduced glutathione 200 pM L-carnitine. Oleate and oleyl-CoA concentrations were 100 pM, and octanoyl carnitine and octanoate concentrations were 200 pM

on livcr homogenates [22] or isolated mitochondria [23] show also that, in addition to carnitine acyltransferase, a second limiting step exists, common to a variety of fatty acid sub- strates.

Oxidution of pulmitoyl-L-curnitine

In order to investigate the limiting steps of b-oxidation and ketone body synthesis in term-fetal liver mitochondria, experiments were done using a polarographic technique. Palmitoyl-L-carnitine was chosen as respiratory substrate to test ,boxidation since it bypasses the step catalysed by carnitine acyltransferase I and is, thus, a better index of fatty acid oxidation per se.

Acetyl-CoA, formed from jl-oxidation, might either enter the hydroxymethylglutaryl-CoA synthase pathway, and form ketone bodies, or the citric acid cycle. Since the removal of acetyl-CoA in one or the other pathway might influence the rate of fatty acid oxidation, oxidation of palmitoyl-L-carnitine was measured in conditions where the end-products were well- defined. The first series of experiments was performed in the presence of malonate, an inhibitor of succinate dehydro- genase, which also exchanges for intramitochondrial malate. Thus oxaloacetate is not available for citrate formation and the end-product of B-oxidation is acetoacetate [14].

We have first examined whether the low capacity of the respiratory chain in term-fetal liver mitochondria (Table 1)

impairs the reoxidation of NADH and FADH2 originating from /&oxidation and leads to an inhibition of fatty acid oxidation. Palmitoyl-L-carnitine oxidation was then studied in presence of 2,4-dinitrophenol, which greatly enhances the capacity of the respiratory chain of term-fetal liver mitochondria (Table 1). In the presence of 2,4-dinitrophenol and malonate (Table 2), the rate of oxidation of palmitoyl-L- carnitine as well as the rate of acetoacetate production was lower in term-fetal liver mitochondria than in adult liver mitochondria. No production of P-hydroxybutyrate could be detected under these conditions. Since during oxidation of palmitoyl-L-carnitine in term-fetal liver mitochondria in the presence of 2,4-dinitrophenol, oxygen consumption is far from reaching the levels observed with other substrates (compare Tables 1 and 2), this clearly eliminates the possibility that the low capacity of the respiratory chain in the fetal state is responsible for the low rate of fatty acid oxidation.

Next we have examined whether a limited capacity for the utilization of acetyl-CoA in the hydroxymethylglutaryl-CoA synthase pathway might exist in term-fetal animals compared to 16-h-old or adult rats. This might lead to an accumulation of acetyl-CoA and to the subsequent inhibition of fatty acid oxidation. Thus, acetyl-CoA was directed towards citrate syn- thesis by incubating mitochondria in the presence of malate (a donor of oxaloacetate) and 2,4-dinitrophenol.

As seen from Table 2, acetoacetate and P-hydroxybutyrate production were undetectable under these experimental con-

606

Table 2. Palmitoyl-I.-carnitine oxidation in the presence of 2,4-dinitrophenol and mahte or malonate in term7fetal, 16-h-old newborn and adult liver mitochondria Results are presented as mean + SEM for 5-9 determinations. Incubation medium contained 10 mM malonate or 2.5 mM malate, 0.1 mM 2,4-dinitrophenol (DNP), 10 pM palmitoyl-L-carnitine

Group Condition O2 consumption A o / A palmitoyl Palmitoyl-~-carniline Aceloacetatc utilization production

nmol 0 min ' nmol/nmol nmol min ~ ' mg protein - ' mg protein -

Term-fetal malonate, DNP 30k 2 12.6 k 0.2 2.3 f 0.1 7.54 & 0.15 16-h-old malonate, DNP 94+ 8" 13.8 k 0.3" Adult malonate, DNP 58+ 3" 14.0 0.2" 4.2 f 0 . 3 " ~ ~ 16.07 + 0.23" Term-fetal malate, DNP 70+ 5 21.3 + 0.5 3.3 f 0.1 undetectable 16-h-old malate, DNP 185+ Y a 20 k0.3 9.3 * 0.5"~' undctectable Adult malate, DNP 164 & loa 21.4 f 0.2 7.7 * 0.5"~' undetectable

6.8 0.6" -

~ ~~ ~~

Difference significant for P < 0.01 when compared to term-fetal mitochondria. Difrerencc significant for P < 0.01 when compared to 16 h-old mitochondria.

' Difference significant for P < 0.01 when compared to the rcspective incubations in presence of malonatc.

ditions and the AO/A palmitoyl obtained in each of our assay was close to the theoretical value of 22. This indicates that citrate is the end-product of palmitoyl-L-carnitine oxidation [24- 261 and that in term-fetal liver mitochondria, neither malate dehydrogenase nor citrate synthetase was rate-limit- ing; indeed, this would have led to an overflow of acetyl-CoA in the hydroxymethylglutaryl-CoA synthase pathway [27], to a decrease in the AOjA palmitoyl ratio and to a production of ketone bodies.

Under conditions where the major end-product of fatty acid oxidation is citrate, the oxidation of palmitoyl-L-carni- tine is enhanced by respectively 2.5 nmol min-' mg-' and 3.5 nmol min-' mg-' (Table 2) in adults and 16-h-old mito- chondria, suggesting that formation of ketone bodies from acetyl-CoA might be limiting when compared to fatty acid oxidation, as previously suggested [28]. In term-fetal liver mitochondria the oxidation of palmitoyl-L-carnitine remains lower than that in 16-h-old or adult mitochondria whatever the end-product of fatty acid oxidation: citrate or acetoacetate (Table 2). This suggests that removal of acetyl-CoA in the hydroxylmethylglutaryl-CoA synthase pathway is not the rate-limiting step in fatty acid oxidation in term-fetal liver mitochondria.

In the rat, in view of the present results, two hypotheses can then be proposed to explain the increase of fatty acid oxidation at birth.

a) An increase in the activity of enzyme of fatty acid oxidation. However, they show a substantial activity at birth and an increase which occurs mainly between 1 and 5 days of age, i.e. at a stage when fatty acid oxidation is already well- developed.

b) A low CoASH content in term-fetal liver mitochondria. Intramitochondrial CoASH is a necessary cofactor for an acyl group to enter p-oxidation and its availability might be of importance for fatty acid oxidation [29].

We have then measured free CoA and CoA ester concen- tration during palmitoyl-L-carnitine oxidation in the presence of dinitrophenol and malonate. In the absence or presence of 2,4-dinitrophenol, the distribution pattern between the HClO,-soluble fraction (free CoA + acetyl-CoA) or the hy- drolyzed HC104-soluble fraction (free CoA, acetyl- CoA + short-chain acyl-CoA) and the hydrolyzed HC104- insoluble fraction (long-chain fatty acyl-CoA) is similar in the mitochondria of the three groups of rats (Fig. 2). However,

concentrations of free CoA and CoA esters are 2.5 lower in mitochondria isolated from term-fetal liver than in adult or 16-h-old liver mitochondria. During the oxidation of pal- mitoyl-L-carnitine there is a large increase in long-chain acyl- CoA concentrations and a decrease in the HClO,-soluble and hydrolyzed HC104-soluble fractions. However, concentra- tions of long-chain acyl-CoA in term-fetal mitochondria re- main 2.5 times lower than in adult or 16-h-old liver mito- chondria.

During palmitoyl-~-carnitine oxidation in liver mito- chondria, the long-chain fatty acyl-CoA pool is composed of palmitoyl CoA and of Cl0- CI4 saturated thioesters [30], the substrates and products of the first turns of /l-oxidation. Thus, during palmitoyl-L-carnitine oxidation, if one of the steps of p- oxidation p e r se were rate-limiting in term-fetal mitochondria, one could expect an accumulation of fatty acyl-CoA when compared to adult or 16-h-old liver mitochondria. This happens, for instance, in adult rat liver mitochondria when palmitoyl-L-carnitine is added in the absence of ADP [16]. The lower concentration of acyl-CoA (Fig. 2) thus suggests that fatty acid activation rather than oxidation is the rate- limiting step of fatty acid metabolism in term-fetal mitochondria. Although we cannot totally rule out the possibility of a low amount of acyl-CoA synthetase, it is likely that the decreased fatty acid oxidation is linked to a limited mitochondria1 availability of CoA.

In order to test whether the lower total CoA concentration in term-fetal liver mitochondria might have been an artefact linked to CoA leakage during the procedure of mitochondria isolation, the total CoA content was measured on freeze- clamped term-fetal and fed adult rat liver. Again the CoA content was 2 - 3 times lower in term-fetal than in adult liver [respectively 119 5 (n = 4) and 274 + 18 (n = 4) (P < 0.001) nmol CoA/g wet weight]. Intramitochondrial CoA is not imported from the cytosol as such but in the form of precursor, 4'-phosphopantetheine, which is then adenylated and phosphorylated inside the mitochondria [31, 321. The last two steps require the presence of ATP. It has been shown in the rat [S] and in the guinea-pig [23] that the ATP content is low in term-fetal mitochondria. This might impair intramito- chondrd CoA synthesis and thus fatty acid activation, until a normal ATP content is achieved, which in the newborn rat takes several hours [8]. Moreover, it has been suggested that a low insulin/glucagon ratio might activate pantothenate

607

7 B

* * *

* *

* *

: \ : i - o J , 1

1 2 3

C

f /I- / , 1 J u i 1 2 3 1 2 3

Fig. 2. Content of CoA and derivatives in liver mitochondria. (1) state 1, (2) uncoupled state without palmitoyl-L-carnitine, (3) uncoupled state in presence of palmitoyl-L-carnitinc during the linear phase of oxidation. Polarographic measurements were done in presence of malonate 20 pM, palmitoyl-L-carnitine 40 pM; 6-8 mg mitochondria1 proteins were added. Results are presented as means SEM for 4-5 determinations. (A) HC104-soluble fraction = free CoA (reduced and oxidised form) + acetyl-CoA. (B) Hydrolysed HCIOd-solublc frac- tion = free CoA, acetyl-CoA and short-chain acyl-CoA. (C) Hydrolysed HC104-insoluble fraction = long-chain acyl-CoA. (D) Total CoA. *,** difference significant for respectively P < 0.05 and P < 0.001 when compared to term-fetal mitochondria. (m) Adult; ( 0 ) term-fetal; (A) 16-h-old

kinase, the flux-generating reaction in CoA synthesis [33 - 351. Since at birth in the rat the insulin/glucagon ratio decreases abruptly [36], it might explain the increased concentration of CoA in liver mitochondria of 16-h-old rats. Finally in the guinea-pig, as in the rat, there is a large rise in liver CoA during the perinatal period (371, which might explain in part the increased liver capacity for fatty acid oxidation.

We thank Dr C. Godinot for her help in the setting of the isolation procedure for mitochondria and I. Coquelet for the preparation of the manuscript. This work was supported in part by grants from the Ministhre de l'lndustrie et de la Recherche (ATP 53-82; 83-C-0448).

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