Fatty acid oxidation in embryonic chick tissues

376

BBA

BIOCHIMICA

ET BIOPHYSICA

ACTA

55896

FATTY

ACID OXIDATION

ELIZABETH

PUGH*

IN EMBRYONIC

AND J. B. SIDBURY,

Department ofPediatrics and Biochemistry, a7706 (U.S.A.) (Received

CHICK

TISSUES

JR.,

Duke Uniuevsity

School of Medicine,

Durham,

K.C.

March 8th, 1971)

SUMMARY

A study of fatty acid oxidation in chick embryos and hatched chicks has shown that the oxidation of long chain and short chain fatty acids conforms with the mechanism shown in adult mammalian tissue. Carnitine was required for maximal oxidation of long-chain fatty acids even though carnitine could be shown to be present in a relatively constant concentration throughout incubation. The rate of oxidation of fatty acids increased with time of incubation in heart tissue except for a transient decrease before hatching, whereas in liver there was a progessive decrease until hatching. The specific activities of enoyl-CoA hydratase (EC 4.2.1.17) P-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and acetyl-CoA acetyltransferase (EC 2.3.1.9) in the embryo heart mitochondria increased with time of incubation. The increase in rate of fatty acid oxidation by the embryo heart mitochondria was not accompanied by a similar increase in the rate of oxidation of cr-ketoglutarate or succinate. These findings suggested a change in heart mitochondrial composition or function with development.

INTRODUCTION

Little information is available on the mechanism of fatty acid oxidation by embryonic chick tissues. Previous attempts’ to demonstrate the oxidation of octanoate, butyrate, crotonate or L+hydroxybutyrate by embryonic chick liver mitochondria were unsuccessful. Recently 2+3,hoewever, carnitine acetvltransferase, one of the enzymes involved in transport of fatty acid across mitochondrial membranes, has been shown to be present in embryonic chick heart and brain as well as liver. We have therefore reinvestigated the problem of fatty acid oxidation in the embryonic chick. This paper describes an enzyme system in embryonic chick tissues that is capable of oxidizing both long chain and short-chain fatty acids. The oxidation of fatty acid has been demonstrated in whole homogenates and in mitochondrial fractions of embryonic tissues. Cofactor requirements for oxidation of long-chain fatty acid were ATP, CoA and carnitine. Furthermore, in the embryonic heart, fatty acids were shown to be oxidized by a mitochondrial b-oxidation system. * Present address:

B&him.

Department

Biophys. Acta, 239

of Chemistry,

(1971)

376-383

University

of Ottawa,

Ottawa,

Canada

FATTY

ACID

377

OXIDATION IN EMBRPONIC TISSCI’:

MATERIALS AND METHODS W-labeled fatty acids were purchased from New England Nuclear Corp., Boston Mass. and I-carnitine was purchased from Calbiochem, Los Angeles, Calif. @-Hydroxyacyl-CoA dehydrogenase (I-3-hydroxyacyl-CoA : NAD oxidoreductase, EC, 1.1.1.35) was a product of Boehringer and Soehne, Mannheim, Germany. All other reagents were commercial preparations and were used without further purification. AcetoacetplCo4 and crotonyl-CoA were prepared by reacting reduced CoA at pH 8.0 with diketene and crotonic anhydride, respectively. Unincubated embryonated eggs were obtained from a commercial supplier and incubated in a James incubator at 37.5 It: 0.5” and 60% relative humidity. The embryonic age was estimated from the wet weight of the embryos according to the method of LEVY AND PALMER”. Embryos were removed from the eggs, freed of extra-embryonic tissues and decapitated. The heart and liver were removed and washed with CaZ+-free KrebsRinger phosphate solution, pH 7.4. Tissues were homogenized with a Potter-Elvehjem homogenizer in the Krebs-Ringer phosphate solution or, in experiments in which mitochondria were to be isolated, in 0.25 M sucrose containing x mM EDTA. The homogenation time was r-3 min and the whole procedure was carried out at 4O. The whole homogenates were centrifuged at 7oo x g to remove unbroken cefls and large debris, and the cell-free extracts were used immediately. The amount of protein in the extracts was estimated by the method of LOWRY et al.5, using bovine serum albumin as a standard. Mitochondria were obtained from whole homogenates prepared in 0.25 M sucrose containing I mM EDTA. The homogenates were centrifuged and separated into three fractions (see Table I]. The fraction that sedimented at 12000 x g is referred to as the mitochondrial fraction. The mitochondriaf fraction was washed twice with 0.25 M sucrose and finally suspended in sucrose to 20-30 mg protein per ml without further treatment. TABLE

FYCZ&?%

I

Oxidation -.spCGi$C

of jr-WZ]palwitic ‘ZCtiVity

(nmolcs/30 min

per wigfwot&n)

74

acid

qf total

activity

Fatty acid oxidation was assayed by determining the amount of l*CO, formed from I-f4C-IabeIed fatty acid by the method of BRESSLER AND FRIEDBEW@. Each crude homogenate and each subcellular fraction were assayed at two different levels of protein (r-2 mg), a range over which the reaction was linear. The reaction mixture contained 1.0 ml freshly oxygenated Krebs-Ringer phosphate, pH 7.4; [r-%]-palmitic acid (Ioo nmoles, 12oooo counts/min); CoA, I pmole; ATP, I pmole; carnitine, I pmole; protein, 1-2 mg; and water to a final volume of 2 ml,

E. PUGH, J. B. SIDBURY, JR.

378

The activity of each enzyme was assayed at two different levels of protein. The isolated mitochondria were sonicated for 30 set before enzyme assay. The rate of reaction

of each enzyme was proportional

to the amount of proicin added. Enoyl-CoA

hydratase (L3-hydroxyacyl-CoA hydro-lyase, EC 4.2.1.1.17) : the assay used was essentially that described by WAKIL AND MAHLER’. The assay mixture consisted of diethanolamine buffer (pH g.5), 175 pmoles; EDTA, 2. pmoles; NAD, 0.3 pmole; /?-hydroxyacyl-CoA

dehydrogenase,

20 pg; crotonyl

CoA, 0.1 ,umole; and IO-5opg

of

mitochondrial protein in a volume of I ml. P-Hydroxyacyl-CoA dehydrogenase: The assay used was essentially that of WAI
EC

2.3.1.9):

The

LYNEN AND OCHOA~. The assay mixture

method

used was that

(I ml) contained

,umoles; M&l,, 5 pmoles; CoA, 0.2 pmole; Sodium thioglycolate, CoA, 0.085 ,umole; and 10-30 ,ug of mitochondrial protein. The rate of respiration apparatus

by a fixed oxygen

(oxygen uptake) electrode

was measured

(Yellow

Springs

described

by

(pH 8.1),

200

Tris-buffer 5 pmoles;

acetoacetyl-

in the Gilson Oxygraph

Instrument

Company)

im-

mersed in a small beaker containing the incubation mixture. The mixture was stirred continually with a magnetic stirrer. The assay mixture contained the medium of CHANCE.~NDWILLIA~IS~~.A typical assay mixture consisted of 16 mM phosphate buffer (pH 7.4),

12 mM NaF,

a-ketogultarate

26 mM NaCl, 58 mM KCl, 6 mM MgCl,, 4 pmoles

or succinate,

IO mg mitochondrial

volume was 2 ml; temperature, 25”. Carnitine was analyzed in tissue extracts

prepared

AND TUBBS”. Tissues were removed from embryos containing solid CO,. Frozen tissue was extracted carnitine

of either

protein and 0.6 pmole ADP. by the method

Total

of PEARSON

and frozen immediately in acetone with cold HClO,, and acid-soluble

was assayed by the method of MARQUIS AND FRITZ?. A typical assay mixture

contained Tris-HCl buffer (pH 7.5), IOO ,umoles; freshly prepared 5,5’-dithiobis(2-nitrobenzoic acid), 0.1 ,umole; acetyl-CoA, 0.05 pmole; carnitine acetyltransferase, 0.05 mg; tissue extracts, initiated

by the addition

minute. Values assay mixture.

obtained

0.05-0.1

ml; and water to a volume of I ml. The reaction

of enzyme

and absorbance

were proportional

readings

to the amount

were taken

of extract

was

after one

added to the

RESULTS Oxidation of fatt_y acids The rate of long-chain fatty acid oxidation was estimated by measuring the formation of 14C0, from [I-14Clpalmitic acid. Fatty acid oxidation could first be detected in the heart of the 6-day embryo and was found to be low during the first week of development. After the first week, the rate of oxidation increased in a linear fashion until just before hatching at which time a small but reproducible decrease in activity was observed (Fig. I). After hatching on the 2Ist day of incubation, the rate of oxidation continued to increase until adult levels were reached. The oxidation of [I-Xlpalmitic acid could also be demonstrated in cell-free extracts of embryonic liver. In contrast with the results obtained with extracts of Biochim. Biophys. Acta. 239 (1971) 376-383

FATTY ACID OXIDATION IN EMBRYONIC TISSUE

379

8-

f

I ZVI I

4

8

I

I

12 16 x, Age

I

,I# I

I

24 28” 50

1 4

H (days)

I 8

1 12

IId

1 16 20;

24

28

f 50

Age (days)

Fig. r. Oxidation of [r-‘*C]palmitic acid by cell-free e%tracts of embryonic chick heart. Each point represents the average of 4 to 8 experiments of pooled tissues. The vertical bars represents the range of activities observed. H indicates the day of hatching. Fig. 2. Oxidation of [r-Wlpalmitic acid by cell-free extracts of embryonic represents the average of 4 to 6 experiments.

chick liver. Each point

heart tissues, the rate of oxidation by the Iiver appeared to be high in the early embryos and to decrease with time of incubation (Fig. 2). The oxidation of short-chain fatty acids could also be demonstrated in homogenates of embryonic chick tissues. In the liver, the rate of oxidation of octanoic acid was higher in the early embryo than in the late embryo (Table II). Similar results were reported above (Fig. I) for the oxidation of palmitic acid. In fact, the rates of oxidation of long-chain and short-chain fatty acids were almost identical at the two ages tested in the liver. In the heart, lower activities were observed with the early embryos than with the late ones. EIowever, the rate of oxidation of short-chain acids was always less than the rate of oxidation of long-chain acids (Table I). Whole homogenates were separated into three subcellular fractions by centrifugation as described in MATERIALS AND METHODS, and each of the fractions was assayed for oxidation of long-chain fatty acids. The fatty acid oxidizing system was found in the particulate fractions of the cell (Table I). Most of the activity was in the fraction that sedimented at 12000 x g. This fraction appears to contain discrete particles analogous to the mitochondrial particles found in adult tissues and it is TABLE OXIDATION

II OF SHORT-CHAIN

FATTY

ACID

BY

CHICK

EMBRYONIC

TISSUES

The oxidation of short chain fatty acid was measured as described in MATERIALS AND METHODS except that [I-‘*C]octanoic acid (IOO nmoles, IOOOOOcounts/min) was substituted for [r-W]palmitic acid. Carnitine was omitted from the reaction mixture, Values given are for two independent experiments on different batches of embryos. __I--. TiSSUf2

~~ Liver Heart

of[r-'qJctanoic

Age of embryo

Oxidation

Cdwl

acid (nvnobs~go rPaim per W.g pro&&)

.._ 13 21

3.29, 3.34 0.10, 0.20

13

0.61, 0.29

21

2.50,

2.80

Bioclaim.

Biophys.

AC&Z, 239 (197rj

376-383

380

E. PUGH,

TABLE

J. B. SIDBURY,

JR.

III

CO-FACTOR REQUIREMENTs HEART ~_

OF THE

Co-$&or

IZ-DAY CHICK ~____

omitted

FOR

OXIDATION

OF PALMITIC

ACID

BY

EMBRYO

CELL-FREE-

EXTRACTS

OF THE

-

O%~dut~o~o+f [I-*“C]palmitic acid (nmoles~go min per mg protein) .-I____

None iZT1’ C.oA

2.17

0.03 0.39

Carnitine ATP, CoA

0.06 0.03

and carnitine

hereafter referred to as the mitochondrial fraction. A small but variable activity was also found in the fraction that sedimented at zooo x g.

amount

of

Co-factor requirements for oxidation of fatty acids Cell-free extracts of chick embryonic heart oxidized palmitic acid to CO, in the presence of various co-factors. Little activity was observed in the absence of either ATP or carnitine (Table III) and CoA was needed for optimal activity. NAD and succinate did not stimulate the oxidation under the conditions of the assay. Similar results were obtained with the embryonic liver. A requirement for ATP, CoA and carnitine could be demonstrated in both heart and liver at all ages of embryos tested. The oxidation of short-chain fatty acids by embryonic chick tissues was not dependent on the addition of carnitine although a partial requirement for CoA could be shown. The oxidation of long-chain fatty acids could not be demonstrated in crude extracts of embryonic heart in the absence of added carnitine. We therefore analyzed TABLE

IV

LEVELS

OF FREE

Tissue ___~__ Heart

Liver

* Average TABLE

Age

IN EMBRYONIC

CHICK

Age ofembryo (days)

Free carnitine* jpmoies/g dry wt.)

9 ‘5 *Cl

4.5 1.’ 4.3

9 ‘5 20

5.8 4.5 3.9

values

determined

from

several

TISSUES

independent

experiments.

V

SPECIFIC CHICK _

CARNITINE

ACTIVITIES

OF THE

ENZYMES

OF

@-OXIDATION

FROM

MIT~CHOPITDRIA

OF THE

HEART

Enzyme

(days)

specific

Fi&yE-C*A hydrutusc 6

1.21

8 ‘4 19

N.D. 7.35 13.1

20

20.0

Biochim.

activity (~molesjmin per mg protein) Acetyl-CoA ~-~ydY~Xy#Gy~~~~~~~~ransj~ya~e CoA dehyd~og~~as~

Biophys.

Acta. 239

(1971)

0.016 0.012

12.5

0.23 0.12 o.175

65.0 35.9 49.5

376-383

4.1

EMBRYONIC

381

FATTY ACID OXIDATION IN EMBRYONIC TISSUE

embryonic tissues for the presence of this co-factor. The amount of free (non-esterified) carnitine was taken to be the carnitine found when tissue extracts were examined without prior exposure to alkali. The contents of free carnitine found in embryonic heart and liver are given in Table IV. The results show that the amount of carnitine in embryonic heart remains relatively constant from 9 to 20 days of incubation. In the liver, free carnitine shows a gradual decrease with age of the embryo. Similar results have recently been reported by CASILLAS AND NEWBURGH~. Activities

of the enzymes of b-oxidation To determine whether the P-oxidation system is operating in the embryonic chick, extracts were assayed for several enzymes of the &oxidative pathway. Assays for enoyl-CoA hydratase, fi-hydroxyacyl-CoA dehydrogenase and acetyl-CoA acetyltransferase were carried out on mitochondrial fractions obtained from embryonic hearts. As shown in Table V, the presence of each of the enzymes could be detected. Furthermore, the specific activities of the three enzymes tested were found to increase with age of the embryo in a manner similar to that observed for over-all oxidation of long-chain fatty acids. Variation

of rate of respiration

with age of embryo

The rate of respiration of the embryonic mitochondria with a-ketoglutarate or succinate as substrate was measured by means of an oxygen electrode. The embryonic mitochondria incubated with substrate and orthophosphate showed a low level of respiration in the absence of ADP. The addition of ADP led to a considerable increase

d-Ketoglutarate &

I

, 4

I

I

8

12

Age of

Embryo

I

I

16

20

(days)

Fig. 3. Rate of respiration of mitochondria with a-ketoglutarate as substrate. Mitochondria were isolated from the heart of the 14.day chick embryo. At the points indicated by arrows IO mg mitochondrial protein, 4 pmoles cr-ketoglutarate and 0.6 /Amole ADP were added. Fig. 4. Rate of oxidation of long-chain fatty acid and rate of respiration in the presence of a-ketoglutarate as a function of age. Mitochondria were isolated from the hearts of embryonic chicks of various ages. Each mitochondrial preparation was assayed for both oxidation of palmitic acid (0 - 0) and respiration with a-ketoglutarate as substrate (q - q) Each point represents the mean of 2 to 3 experiments.

in the rate of respiration (Fig. 3). From 3- to s-fold increases were routinely observed for mitochondria isolated from 9-, 14- and zo-day chick hearts. Preparations giving acceptor control ratios of less than 3 were discarded. Biochim.

Biophys.

Acta,

239 (1971)

376-383

E.

382

PUGH,

J.

B. SIDBURY,

JR.

The rate of respiration with or-ketoglutarate as substrate was measured in mitochondria isolated from embryos of various ages. The rate of respiration was high in the early embryos and appeared to fall during the last z weeks of incubation (Fig. 4). During this period, the rate of oxidation of fatty acid by the embryonic ~llitocllondria, as shown in Fig. 4, increases. The data suggest that the variation in fatty acid oxidation results not from an increase in the quantity of mitochondria but rather from a qualitative change in the mitochondrial component of the embryonic tissue. DISCUSSION

The oxidation of fatty acid has been demonstrated in cell-free extracts of embryonic chick tissues in the presence of the appropriate cofactors, ATP, CoA and carnitine were required for the optimal oxidation of long-chain fatty acids, Little or no oxidation was observed in the absence of added carnitine although this cofactor could be detected in the embryonic extracts. Carnitine was not required for the oxidation of short-chain fatty acids. In contrast in mammalian fetal heart evidence was deduced for a marked limitation in fetal mitochondrial oxidation of palmityl-CoA with added carnitine when compared with calf heart mitochondria , suggesting that the formation of paimityl carnitine was limiting3. The oxidation appeared to proceed by the /3-oxidative pathway described in adult mammalian tissuesi3*14 and in bacteria 16+i6.Fatty acids were first activated with CoA to form acyl-CoA as shown by the requirement for ATP and CoA. It was also shown in separate experiments that acyl-Cob could substitute for fatty acid, ATP and CoA. The acyl-CoA was then presumably oxidized to z-enoylCoA by the action of acyl-CoA dehydrogenase. The remaining steps in the reaction sequence were demonstrated in embryonic mitochondria. The z-enoyl-CoA is hydrated to /ShydroxpacylCoA by the enzyme enoyl-CoA hydratase. The product of this reaction is then oxidized by p-keto acyl-CoA by the enzyme ,&hydroxyacyl-CoA dehydrogenase. Finally, the jS-ketoacyl-CoA is cleaved by the enzyme acetyl-C.oA acetyltransferase to acetyl-CoA and an acyl-CoA with z carbon atoms less than the original acyl-CoA. This acyl-CoA is then recycled through the same reactions until it is completely degraded to acetylCoA. The oxidation of long-chain fatty acids was studied as a function of the age of the embryo. In the heart, the rate of oxidation of palmitic acid increased from 6 to 18 days of age at which time a slight decrease in activity was observed. Thereafter, the rate of oxidation rose until adult levels were reached. The rise in activity is not attributed solely to changes in the mitochondrial content of the heart since a similar pattern was observed in isolated mitochondria. The activities of enoyl-CoA hydratase, @-hydroxyacyl-CoA dehydrogenase and acetpl-CoA acetyl transferase per mg of mitochondrial protein were also shown to increase with age of the embryo. Moreover, the rise in activity of the fatty acid oxidizing system was not accompanied by an increase in the rate of respiration with cx-ketoglutarate as substrate. In fact, the rate of respiration in the presence of cr-ketoglutarate appeared to be higher in the early embryos than in the late ones. The results suggest that the variation in fatty acid oxidation observed is due not to an increase in the quantity of mitochondria but to a change in mitochondrial composition or function. This point is currently under investigation. Rio&m. Biophys. A&z,

239 (1971)

376-383

FATTY ACID OXIDATION

IN EMBRYONIC

TISSUE

383

ACKNOWLEDGEMENT

This work was supported HE11307 and AMo6815.

by grants

from the National

Institutes

of Health,

REFERENCES I 2 3 4 5 6 7 8 g IO II 12 13 14 15

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29 (1967) 28.

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Biochim. Biophys. Acta, 239 (1971) 376-383