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Depletion and regeneration of fatty acid-absorbing capacity of adipose tissue and liver particles
BIOCHIMICA
456 BBA
ET BIOPHYSICA
ACTA
55276
DEPLETION CAPACITY
L. RESHEF
AND
REGENERATION
OF ADIPOSE
AND
AND
LIVER
ACID-ABSORBING PARTICLES
B. SHAPIRO
Department qf Biochemistry, (Received
TISSUE
OF FATTY
Hebrew University-Hadassah
Medical Schcol, Jerusalem (Israel)
May 6th, 1966)
SUMMARY
The absorption
of fatty
acids from a serum-albumin
solution by adipose-tissue
particles and liver mitochondria at o” and pH 7.4 is markedly activated by pretreatment of the particles with buffer solutions of pH 4.0. Following one contact of adipose-tissue particles with a solution of serum albumin, the particles showed very low activity on subsequent tests with a fatty acidserum albumin complex. With liver mitochondria such a depletion of activity by albumin occurred only with particles pretreated at pH 4.0. This inactivation by pretreatment with serum albumin
removed from the par-
ticles free fatty acids as well as another lipid compound, which behaved on thin-layer chromatography like lysolecithin. The fatty acid-absorbing capacity of depleted adipose-tissue and liver particles could be regenerated process. Reactivation
by incubation
at 37’. Pretreatment
at pH 4.0 accelerated
could also be achieved by adding the lipid extract
this
from the albu-
min supernatant that had been in contact with active or regenerated particles, but not with those in contact with depleted particles. The free fatty acids present in these extracts could not account for the reactivation. Addition of lysolecithin was able to produce partial regeneration of activity. Lysolecithin at higher concentrations could be shown to displace fatty acids from the serum-albumin complex even in the absence of particles.
INTRODUCTION
In a previous communication1 it was demonstrated that liver and adipose-tissue particles remove [%]palmitic acid from a serum-albumin solution. The uptake of palmitic acid took place at o0 and was independent of its esterification by the particles. The acid was firmly bound by an insoluble protein in the particles. The fatty acid partition between serum albumin and the particle protein was dependent on the relative amounts of these proteins and not on the amount of the acid. Concurrently with this uptake of radioactive palmitic acid from the medium, Biochim. Biophys. Acta, 125 (1966) 456-464
FATTY ACIDABSORPTION
457
a release of fatty acids from the particles took place. The net result of fatty acid movement was dependent on the relative amounts of fatty acid in the medium and in the particles. In subsequent experiments it was observed that when the level of fatty acids in the particles was decreased by prewashing with serum albumin, the subsequent release could be reduced or abolished. However, at the same time, with adiposetissue particles such a pretreatment also diminished the uptake of fatty acids from a serum albumin-fatty acid solution. The exhaustion of the fatty acid-binding capacity of the particles by albumin treatment and its regeneration is the subject of the present investigation. The effect of pretreatment of the particles with various buffer solutions is also reported. MATERIALSAND METHODS Preparation and treatment of particles Rat-adipose tissue particles and rat-liver mitochondria were prepared as described previously l. “Acidified particles”. These were prepared by suspending the mitochondria in a solution of 0.05 M sodium citrate buffer (pH 3.9) in 0.15 M KC1 (I mg protein per ml solution). After 15 min at o” the suspension was centrifuged at IIOOO x g for 15 min. The particles were washed with twice the volume of 0.15 M KCI-0.05 M Tris (pH 7.4) and once more centrifuged under the same conditions. These particles were used on the same day for the experiment. Albumin pretreatment of particles. Bovine serum albumin was added to a suspension of mitochondria in KU-Tris solution (pH 7.4) at a ratio of 20 mg albumin to I mg mitochondrial protein. The mixture was kept in ice for 20 min and then centrifuged at IIOOO x g for 15 min. The particles were then washed with an equal volume of KU-Tris (pH 7.4). When acidified particles were pretreated with albumin this procedure took place after the acidification and washing. These particles were hereafter referred to as “albumin-pretreated particles” or “acidified albumin-treated particles” respectively and used for the experiment on the same day. Egg lecithin was prepared by the method of PANGBORN~.This had an ester: P ratio of 2.0 and did not reveal any impurities on thin-layer chromatography. Lysolecithin was obtained by treating egg lecithin with Crotalus adamenteus phospholipase A (EC 3.1.1.4, Sigma). Special care was taken to purify it of traces of fatty acids by dissolving the lysolecithin in a small amount of alcohol and precipitating with a large volume of ether. A perfectly purified lysolecithin was obtained as shown by thin-layer chromatography. Potassium lysophosphatidate was prepared from lysolecithin by means of treatment with phospholipase D (EC 3.1.1.4, Biihringer) according to LONGS. Ester:P:N ratio was 1:1:o.16 indicating the presence of some lysolecithin. Phosphatidic acid was prepared from egg lecithin with phospholipase C (EC 3.1.1.3, Bohringer) according to their specifications. The ester:P ratio was 1.77: I, indicating the presence of some lysophosphatidic acid. Albumin-palmitic acid complex was prepared as described previously’. Bovine serum albumin (Pentex; fatty acid poor) was used. It contained 0.03 pmole titratable fatty acids per pmole albumin. Biochim.
Biophys.
Acta,
125
(1966) 456-464
L. RESHEF,
458
B. SHAPIRO
Protein was determined by the method of LOWRY~. Fatty acids were titrated by the method of DOLES. Radioactivity was measured as described previously’. RESULTS
Pretreatment with buffer at various pH values
Rat-liver mitochondria and adipose-tissue particles were treated with various buffer mixtures as detailed in Fig. I. After the treatment, the buffer was washed ‘~4
Uptake
A lB
PH
Fig. I. Effect on fatty acid-absorbing capacity of particles after pretreatment with various buffer mixtures. Particles were suspended in a 0.15 M KC1 solution of 0.05 M acetate or citrate buffer, kept for so min at o” and centrifuged at I I ooo x g. They were washed with twice the volume of KCl-Tris solution (pH 7.4), centrifuged as before and resuspended in KCl-Tris (pH 7.4). Fatty acid absorption was then tested as in Table I. Concentration of particulate protein from adipose tissue was 0.1 mg/ml; final volume, 5 ml; from liver, I mg/ml; final volume, so ml. A. Liver particles. x __ x , citrate buffer; O-O, acetate buffer; n, Tris buffer. B. Adipose-tissue particles. x __ x , citrate buffer; A, Tris buffer.
away with 0.15 M KCl-0.05 M Tris-HCl buffer (pH 7.4) and the fatty acid-absorbing capacity was tested by dispersing the particles in a [14C]palmitate-serum albumin solution. Such a pretreatment had a marked influence on the fatty acid-absorbing capacity of the particles. Optimal activity was found with citrate buffer at a pH around 4. The same optimum pH was also found with acetate buffer, but the activity obtained with this buffer was considerably lower than with citrate (Fig. I). Pretreatment with serum albumin
With adipose-tissue particles, uptake of radioactive palmitate from the medium decreased markedly following pretreatment with serum albumin (Table I). With liver particles, on the other hand, pretreatment with albumin did not deplete the fatty acid-absorbing capacity of the tissues but even caused some increase (Table II). However, when the particles were first “acidified” by suspension in citrate buffer at pH 3.9, and the citrate washed away (as in Fig. I), subsequent albumin treatment caused depletion of fatty acid-absorbing capacity in both adipose tissue and liver particles (Tables I and II). Regeneration of absorbing capacity
Incubation of particles so depleted at 37” caused partial recovery of the absorbing capacity, as compared with similar particles stored at 0’. A time curve of regeneration is presented in Fig. 2. Both acidified and non-acidified particles gradually regained a considerable part of their original activity. The regeneration was more rapid with acid-treated particles. Biochim.
Biophys.
Acta,
125 (1966)
456-464
459
FATTY ACID ABSORPTION TABLE
I
EFFECT
OF
RAT
FR~TREAT~ENT
ADIPOSE-TISSUE
WITH
SERUM
ALBUMIN
ON
THE
FATTY
ACID-ABSORBING
CAPACITY
OF
PARTICLES
Adipose-tissue mitochondria were suspended in KCl-Tris (pH 7.4) and treated as specified. I mg protein of the particulate fraction was mixed with 0.1 ml of a solution containing 0.14 pmole albumin and 0.21 pmole [Wlpalmitic acid (27000 countslmin) ; final volume adjusted to r4 ml with KCl-Tris. After IO min at o” the mixture was centrifuged at 105000 x g for 60 min. The precipitate was washed with KCl-Tris, centrifuged once more and resuspended in KCl-Tris, and aliquots were taken for radioactivity estimations. Acidification or albumin treatment of the particles was carried out as described under METHODS. The acid-treated particles were again subjected to albumin treatment under the same conditions as described for the non-acidified particles. Per cent uptake b a _-.. 17.6 “% 4.0 44 74 14.7. 3.6
PLWtiCleS Expt.
-... Untreated Albumin-treated Acidified Acidified, albumin-treated
TABLE EFFECT RAT-LIVER
87.0 26.7
d
e
51.5
82.2
8.0
10.2
-
II OF PRETREATMENT
WITH
SERUM
ALBUMIN
ON
THE
FATTY
ACID-ABSORBING
CAPACITY
OF
MITOCHONDRIA
Experimental Particles
-
--. c
details as in Table I. Protein content of particles 20 mg. b a Exet.
Untreated Albumin-treated Acidified Acidified, albumin-treated
d
c
18.3
12.1
21.0
IS.2 20.2
6.8 -_-
22.0
7.0
26.0
7.0
‘/a
Uptake I
0'
60
30 Time
90
120
(mid
Fig. 2. Time curve of regeneration of depleted adipose-tissue particles. I mg protein per 5 ml KCl-Tris (pH 7.4) was incubated at 37O for various times as indicated in the figure and cooled to 0’. Absorbing capacity test was carried out as described in Table I. O, acidified and albumintreated ; x , albumin-treated.
While heating the particles to boiling did not completely abolish fatty acid absorption, albumin treatment of boiled particles almost completely annulled their activity and very little uptake could be detected even following incubation at 37” (Table III). The substances removed from the particles by treatment with serum albumin were analyzed by subjecting the chloroform-methanol (a.:I, v/v) extract of the supernatant albumin solution to thin-layer chromatography. It was found that, in addition Biocla%~a. Biophys. Acta, 125 (1966) 456-464
TABI.E UPTAKE
III AND
REGENERATION
OF ACTIVITY
IN BOILED
ADIPOSE-TISSUE
PARTICLES
Bailed particles were obtained by heating a ICCX-Tris particuIate suspension (I mg protein per ml solution) at IOO’ for 20 min followed by acidification as described under MATERIALS AND METKODS. In all cases the assay was carried out at oO. Other experimental conditions as in Table I,
Acidified Acidified, albumin-treated Acidified, albumin-treated and incubated for 30 min at 37” ICroiled,acidified Hailed, acidified, albumin-treated and incubated for 30 min at 37”
66.4
82.2
x0.2 36.0
19.0
33.0 5.0
6.2
to free fatty acids, the albumin extract contained a Iipid which migrated on the plates like lysoleecithin. No compound of this kind could be detected in the extract of a KC&-Tris supernatant in the absence of albumin. This finding raised two questions: (I) Can the absorbtive capacity of depleted particles be renewed by the addition of the lipids present in the supernatant albumin solution? (2) Are the lipid compounds migrating out of the particles into the albumin solution responsible for the transfer of the radioactive fatty acids from the albumin solution to the particles ? In fact, it was found that the fatty acid-Fhos~holipid (Iysoiecithin ?) mixture leaving the particles, when added to the fatty arid-albumin solutions, had a pronounced influence on the partitioning of the free fatty acids between the albumin and the particles. This could be demonstrated by adding the lipid extract from the albumin in which particles had been washed, and also with defined lipids or fatty acids. In Table IV experiments are shown in which lipid extracts, obtained from albumin supernatants, of both “depleted” and “regenerated” particles, were added
ACTIVITY
OF THE LIPIDS
EXTRACTED
FROM
ACfDIFIED
LIVER
MITOCHONDRIA
WITH
ALBUMIN
Acidified rat-liver mitochoadria were first depleted of most of their fatty acid-absorbing capacity by washing with albumin (as described under METHODS). One portion of these particles was allowed to “regenerate” by incubating at 37” for 60 min and the other part W&S kept at 0’. Both types (20 mg protein) were then mixed with a solutian of 0.14 pmole albumin and 0.21 ,umole non-labeled palmitic acid under conditions identical with those used for fatty acid-uptake examinations. The supernatants were collected and extracted with chloroform-methanol (2 : I, v/v). The lipid extract was evaporated and suspended in 5 ml KCI-Tris solution by sonication in a glass tube for I min. 2 ml of each of these suspensions, i.e. E (the extract obtained from the depleted particies which were kept at o”) and ER (that obtained from the “regenerated” particles) were added to a suspension of depleted particles (zo mg protein) and an absorption-capacity test was carried out as in Table 1. ~---““_l_. I-lll._...Frse fatty acid Per,csnt uptake Particles Additions -pp content of lipid bc d a &fact tpmoles) d c -1--IO rg 2f 22 Acidified, albumin-treated None 0.08 0.X 18 24 W-5 Acidified, albumin-treated 2 mf E ‘3-4 0,os $7 58 26.3 21.3 0.1 zmlER Acidified, aibumin-treated Preincubaied at 37’ for 60 min None 5r 54 50 Biochinz.Biophys.
Acta, rzfi (7966) 456-464
461
FATTY ACID ABSORPTION
to “depleted” liver-particle suspensions. Only the extracts obtained from regenerated particles had an activating effect on the absorption of 14C-labeled fatty acid. In Expts. c and d, the free fatty acid content of the lipid extracts was titrated and the extracts were diluted to the same fatty acid content. Even then the extracts of the regenerated particles caused marked activation, while those from the depleted particles had no or negligible effect. The addition of palmitic acid or of a mixture of fatty acids (at concentrations obtained in experiments presented in Table IV) to the depleted particles caused no significant increase of ~~C~palmitate uptake by the particles (Table V). When lysophospholipids (lysolecithin, lysophosphatidate) were added to a depleted particulate suspension, increased uptake of [‘*C]palmitic acid was demonstrated (Table VI). In the presence of lysolecithin, the addition of carrier palmitic acid caused some synergistic effect on the absorbing capacity. TABLE
V
ADDITIONo?? FATTY
ACIDS
TO ACIDIFIED,
ALBUMIN-PRETREATED
LIVER
PARTICLES
The mixtures of the fatty acids, as potassium salts, were suspended in KG-T& (pH 7.4) solution by sonication and added to the experimental tubes containing liver particles, acidified and albumin depleted. procedure of the experiment as indicated in Table I. Particles
Additions
Acidified and albumin-treated Acidified and albumin-treated
None 0.08 ;cmole of a mixture of stearic acid, oleic acid and linoleic acid in equal amounts 0.09 /&mole of palmitic acid
Acidified and albumin-treated The same preincubated at 37” for 60 min
TABLE ADDITION
Per cent uptake of [W]palmitic acid
None
7.4
a:: 51.5
VI OF KNOWN
LIPlDS
TO ACIDIFIED,
ALBUMIN-PRETREATED
PARTICLES
Various lipids as specified in the table were suspended by sonication in KCl-Tris and added to the test mixtures containing 1.6 mg protein of adipose-tissue particles or zo mg protein of liver particles and tested for fatty acid uptake as in Tables I and II. Additions
Per cent uptake Adipose-tissue R A
None Lysolecithin, 0.25 @mole Lysolecithin 0.25 pmole + potassium palmitate 0.1 pmole Lysolecithin 0.5 pmole Lysolecithin 0.5 pmoie + potassium palmitate 0.2 ~umole Lysophosphatidic acid 0.25 ,umole + potassium palmitate 0.1 pmole Lysophosphatidic acid 0.5 pmole Lysophosphatidic acid 0.5 ymole + potassium palmitate 0.2 pmole Potassium palmitate 0.1 pmole Potassium palmitate 0.2 @mole -. --
19.5
Liver pa&G-
particles -._--
A
B
4.5
21.0
IQ.0
34.4
40.9
23.0
9.1
45.5 40.5
43.2 45.2
31.0
21.6
51.5
57.0
27.0
8.2
32.0 34.2
26.2 30.0
46.0 23.8 35.6
43.0 28.3 35.0
29.0 X7.4 19.4
11.2
5.0 5.6
Biochim. 3~o~h~s. Acta,
125
(1966) 456-464
L. RESHEF,
462
B. SHAPIRO
The ability of lysolecithin to remove [l*C]palmitic acid from its complex with albumin at o” was tested in the absence of mitochond~a. Table VII shows that when a solution of [r4C]palmitate-serum albumin complex was mixed with lysolecithin, [lPC]palmitate was precipitated by high-speed centrifugation. No such effect was observed with lecithin. With palmitic acid some precipitation occurred, but only at high TABLE
VII
PRECIPITATIONOF [W]PALMITATE FROM ALBUMIN-FATTY
ACID
COMPLEX BY LYSOLECITHIN
Sonicated suspensions of the lipid substrates in amounts indicated were mixed with 0.1 ml of a solution containing 0.14 pmole albumin and 0.14 ymole [“C]palmitic acid (27000 counts~m~n). The mixture was brought to a final volume of 14.5 ml with 0.15 M KCl-0.05 M Tris (pH 7.4), and centrifuged at Ioooooo x g for 90 min. The precipitates were extracted with alcohol-ether (3: I, v/v) and aliquots from supernatants and precititates were counted.
Substrate
added
p&es
Egg lecithin
Lysolecithin
II.0
1.0
*I.0
I,5
0.5 I.0 2.0
0.5
4.0 8.0 Potassium
palmitate
Per cent of radioactivity
0.5 I.0 2.0
4.0
precipitated
1.4 9.2 22.2 38.0 0.7 I.7 3.0
12.5
concentration. The amounts of lysolecithin required for the above-mentioned effect were 8-16 times higher than those inducing a ir*C]palmitate / uptake by depleted particles. DISCUSSION
Results in this and in our previous communication1 show that the uptake of fatty acids from a serum-~bumin solution by cell particles is a non-enzymatic process, proceeding at o0 and being comparatively heat resistant. Uptake seems to be due to binding to a protein acceptor in the particle membrane. Lipid constituents of the particles also play a role in this process. It has now been shown that some lipids diffuse out into the albumin media and as a result the fatty acid-absorbing capacity of the particles becomes exhausted. There was a direct relationship between the initial activity and the extent of exhaustion. Adipose-tissue particles, which are highly active, were also easily depleted. Liver particles, which are much less active, did not lose activity. Only when liver particles were activated by pretreatment with buffer at pH 4.0 did considerable depletion occur on contact with serum-albumin solutions. Such “acidified” particles were also more rapidly and extensively regenerated by incubation at 37”. The nature of the change brought about by the acid treatment is not clear. As it took place at 0’ it seems most likely that it is based on changes in the ionization of some membrane constituents, although an enzymatic change cannot be excluded. The results point to the conclusion that during contact with serum albumin, Biochim.
Bio@~.
Acta,
125 (1966) 456-464
E ATTY ACID ABSORPTION
463
the particles lost certain constituents which are participating in the process of fatty acid uptake. It could be shown that, in addition to free fatty acids, albumin removed a phospholipid which behaved chromatographically like lysolecithin. In fact the mixture of lipid substances extracted from the albumin supernatant could reactivate the particles. This occurred only with albumin solutions which had been in contact with active particles, i.e. fresh or regenerated ones, but not with depleted particles. The free fatty acids alone could not explain the activating potency of these lipid extracts. Even when the free fatty acid concentrations in extracts obtained from regenerated and non-regenerated particles were identical, only the former exhibited activity. No activity was evolved by the addition of various fatty acids at concentrations present in the active extracts. Lysophosphatides, on the other hand, could induce fatty acid translocation from the albumin solution to the particles. The combined action of both lysophosphatides and free fatty acids may be responsible for the extraction of the fatty acids from the albumin complex. When used at a ro-fold concentration, lysolecithin can remove fatty acids from their albumin complex, even in the absence of tissue particles. Contrary to the non-enzymatic nature of the uptake itself, the regeneration of this capacity does seem to involve enzymatic processes. Regeneration does not take place at o’, nor is it brought about in heat-treated particles. The nature of the enzymatic processes bringing about regeneration, and of the chemical compounds formed, is not yet clear. Some indication may be found in the activation of the process by acid buffer treatment and in the fact that the free fatty acid concentration in the particles increases during regeneration. This makes it likely that a phospholipase activity is responsible for this process. The mechanism of fatty acid uptake by particles seems to involve interaction between the fatty acid-albumin complex and lipids (fatty acid and lyso compounds) present on the particle membrane. In our experiments in vitro these lipids actually diffuse out into the medium. The possibility may be considered that, under physiological conditions, lyso compounds serve as factors facilitating fatty acid transport into the particles, by loosening the fatty acid-albumin complex.Following the uptake of the fatty acid by the particles, esterification of the lyso compound takes place forming the phospholipid. This may then release the fatty acid into the particle by Outside
I
I
Fatty ocidalbumin complex
I-
Membrane
Inside
Lysophospholipid
I
+
Fatty acid
Phospholipld
Scheme I. Hypothetic
scheme of fatty acid uptake under physiological Biochim. Biophys.
conditions. Acta,
125
(1966) 456-464
464
L. RESHEF,
B. SHAPIRO
phospholipase action (Scheme I). The rapid turnover of the fatty acids in the p position of lecithin has been repeatedly demonstrated and may be part of such a transport system. The release of free fatty acids seen in vitro may be an artifact, caused by disruption of the organisation which directs the acids into the particle and by the conditions of the experiment, i.e. o’, which do not permit the re-esterification of the lysophospholipid. REFERENCES L. RESHE~ AND B. SHAPIRO, Biochim. Biophys. Acta, 98 (1965) 73. M. PANGBORN, J. Biol. Chem., 188 (1951) 471. C. LONG, R. ODAVIC AND E. J. SARGENT, B&hem. J., 85 (1962) 33. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 265. 5 V. P. DOLE, J. C&z. Invest., 35 (1~56) 150. I 2 3 4
Biochim.
Biophys.
Ada,
125 (1966)
456-464
193 (1951)
456 BBA
ET BIOPHYSICA
ACTA
55276
DEPLETION CAPACITY
L. RESHEF
AND
REGENERATION
OF ADIPOSE
AND
AND
LIVER
ACID-ABSORBING PARTICLES
B. SHAPIRO
Department qf Biochemistry, (Received
TISSUE
OF FATTY
Hebrew University-Hadassah
Medical Schcol, Jerusalem (Israel)
May 6th, 1966)
SUMMARY
The absorption
of fatty
acids from a serum-albumin
solution by adipose-tissue
particles and liver mitochondria at o” and pH 7.4 is markedly activated by pretreatment of the particles with buffer solutions of pH 4.0. Following one contact of adipose-tissue particles with a solution of serum albumin, the particles showed very low activity on subsequent tests with a fatty acidserum albumin complex. With liver mitochondria such a depletion of activity by albumin occurred only with particles pretreated at pH 4.0. This inactivation by pretreatment with serum albumin
removed from the par-
ticles free fatty acids as well as another lipid compound, which behaved on thin-layer chromatography like lysolecithin. The fatty acid-absorbing capacity of depleted adipose-tissue and liver particles could be regenerated process. Reactivation
by incubation
at 37’. Pretreatment
at pH 4.0 accelerated
could also be achieved by adding the lipid extract
this
from the albu-
min supernatant that had been in contact with active or regenerated particles, but not with those in contact with depleted particles. The free fatty acids present in these extracts could not account for the reactivation. Addition of lysolecithin was able to produce partial regeneration of activity. Lysolecithin at higher concentrations could be shown to displace fatty acids from the serum-albumin complex even in the absence of particles.
INTRODUCTION
In a previous communication1 it was demonstrated that liver and adipose-tissue particles remove [%]palmitic acid from a serum-albumin solution. The uptake of palmitic acid took place at o0 and was independent of its esterification by the particles. The acid was firmly bound by an insoluble protein in the particles. The fatty acid partition between serum albumin and the particle protein was dependent on the relative amounts of these proteins and not on the amount of the acid. Concurrently with this uptake of radioactive palmitic acid from the medium, Biochim. Biophys. Acta, 125 (1966) 456-464
FATTY ACIDABSORPTION
457
a release of fatty acids from the particles took place. The net result of fatty acid movement was dependent on the relative amounts of fatty acid in the medium and in the particles. In subsequent experiments it was observed that when the level of fatty acids in the particles was decreased by prewashing with serum albumin, the subsequent release could be reduced or abolished. However, at the same time, with adiposetissue particles such a pretreatment also diminished the uptake of fatty acids from a serum albumin-fatty acid solution. The exhaustion of the fatty acid-binding capacity of the particles by albumin treatment and its regeneration is the subject of the present investigation. The effect of pretreatment of the particles with various buffer solutions is also reported. MATERIALSAND METHODS Preparation and treatment of particles Rat-adipose tissue particles and rat-liver mitochondria were prepared as described previously l. “Acidified particles”. These were prepared by suspending the mitochondria in a solution of 0.05 M sodium citrate buffer (pH 3.9) in 0.15 M KC1 (I mg protein per ml solution). After 15 min at o” the suspension was centrifuged at IIOOO x g for 15 min. The particles were washed with twice the volume of 0.15 M KCI-0.05 M Tris (pH 7.4) and once more centrifuged under the same conditions. These particles were used on the same day for the experiment. Albumin pretreatment of particles. Bovine serum albumin was added to a suspension of mitochondria in KU-Tris solution (pH 7.4) at a ratio of 20 mg albumin to I mg mitochondrial protein. The mixture was kept in ice for 20 min and then centrifuged at IIOOO x g for 15 min. The particles were then washed with an equal volume of KU-Tris (pH 7.4). When acidified particles were pretreated with albumin this procedure took place after the acidification and washing. These particles were hereafter referred to as “albumin-pretreated particles” or “acidified albumin-treated particles” respectively and used for the experiment on the same day. Egg lecithin was prepared by the method of PANGBORN~.This had an ester: P ratio of 2.0 and did not reveal any impurities on thin-layer chromatography. Lysolecithin was obtained by treating egg lecithin with Crotalus adamenteus phospholipase A (EC 3.1.1.4, Sigma). Special care was taken to purify it of traces of fatty acids by dissolving the lysolecithin in a small amount of alcohol and precipitating with a large volume of ether. A perfectly purified lysolecithin was obtained as shown by thin-layer chromatography. Potassium lysophosphatidate was prepared from lysolecithin by means of treatment with phospholipase D (EC 3.1.1.4, Biihringer) according to LONGS. Ester:P:N ratio was 1:1:o.16 indicating the presence of some lysolecithin. Phosphatidic acid was prepared from egg lecithin with phospholipase C (EC 3.1.1.3, Bohringer) according to their specifications. The ester:P ratio was 1.77: I, indicating the presence of some lysophosphatidic acid. Albumin-palmitic acid complex was prepared as described previously’. Bovine serum albumin (Pentex; fatty acid poor) was used. It contained 0.03 pmole titratable fatty acids per pmole albumin. Biochim.
Biophys.
Acta,
125
(1966) 456-464
L. RESHEF,
458
B. SHAPIRO
Protein was determined by the method of LOWRY~. Fatty acids were titrated by the method of DOLES. Radioactivity was measured as described previously’. RESULTS
Pretreatment with buffer at various pH values
Rat-liver mitochondria and adipose-tissue particles were treated with various buffer mixtures as detailed in Fig. I. After the treatment, the buffer was washed ‘~4
Uptake
A lB
PH
Fig. I. Effect on fatty acid-absorbing capacity of particles after pretreatment with various buffer mixtures. Particles were suspended in a 0.15 M KC1 solution of 0.05 M acetate or citrate buffer, kept for so min at o” and centrifuged at I I ooo x g. They were washed with twice the volume of KCl-Tris solution (pH 7.4), centrifuged as before and resuspended in KCl-Tris (pH 7.4). Fatty acid absorption was then tested as in Table I. Concentration of particulate protein from adipose tissue was 0.1 mg/ml; final volume, 5 ml; from liver, I mg/ml; final volume, so ml. A. Liver particles. x __ x , citrate buffer; O-O, acetate buffer; n, Tris buffer. B. Adipose-tissue particles. x __ x , citrate buffer; A, Tris buffer.
away with 0.15 M KCl-0.05 M Tris-HCl buffer (pH 7.4) and the fatty acid-absorbing capacity was tested by dispersing the particles in a [14C]palmitate-serum albumin solution. Such a pretreatment had a marked influence on the fatty acid-absorbing capacity of the particles. Optimal activity was found with citrate buffer at a pH around 4. The same optimum pH was also found with acetate buffer, but the activity obtained with this buffer was considerably lower than with citrate (Fig. I). Pretreatment with serum albumin
With adipose-tissue particles, uptake of radioactive palmitate from the medium decreased markedly following pretreatment with serum albumin (Table I). With liver particles, on the other hand, pretreatment with albumin did not deplete the fatty acid-absorbing capacity of the tissues but even caused some increase (Table II). However, when the particles were first “acidified” by suspension in citrate buffer at pH 3.9, and the citrate washed away (as in Fig. I), subsequent albumin treatment caused depletion of fatty acid-absorbing capacity in both adipose tissue and liver particles (Tables I and II). Regeneration of absorbing capacity
Incubation of particles so depleted at 37” caused partial recovery of the absorbing capacity, as compared with similar particles stored at 0’. A time curve of regeneration is presented in Fig. 2. Both acidified and non-acidified particles gradually regained a considerable part of their original activity. The regeneration was more rapid with acid-treated particles. Biochim.
Biophys.
Acta,
125 (1966)
456-464
459
FATTY ACID ABSORPTION TABLE
I
EFFECT
OF
RAT
FR~TREAT~ENT
ADIPOSE-TISSUE
WITH
SERUM
ALBUMIN
ON
THE
FATTY
ACID-ABSORBING
CAPACITY
OF
PARTICLES
Adipose-tissue mitochondria were suspended in KCl-Tris (pH 7.4) and treated as specified. I mg protein of the particulate fraction was mixed with 0.1 ml of a solution containing 0.14 pmole albumin and 0.21 pmole [Wlpalmitic acid (27000 countslmin) ; final volume adjusted to r4 ml with KCl-Tris. After IO min at o” the mixture was centrifuged at 105000 x g for 60 min. The precipitate was washed with KCl-Tris, centrifuged once more and resuspended in KCl-Tris, and aliquots were taken for radioactivity estimations. Acidification or albumin treatment of the particles was carried out as described under METHODS. The acid-treated particles were again subjected to albumin treatment under the same conditions as described for the non-acidified particles. Per cent uptake b a _-.. 17.6 “% 4.0 44 74 14.7. 3.6
PLWtiCleS Expt.
-... Untreated Albumin-treated Acidified Acidified, albumin-treated
TABLE EFFECT RAT-LIVER
87.0 26.7
d
e
51.5
82.2
8.0
10.2
-
II OF PRETREATMENT
WITH
SERUM
ALBUMIN
ON
THE
FATTY
ACID-ABSORBING
CAPACITY
OF
MITOCHONDRIA
Experimental Particles
-
--. c
details as in Table I. Protein content of particles 20 mg. b a Exet.
Untreated Albumin-treated Acidified Acidified, albumin-treated
d
c
18.3
12.1
21.0
IS.2 20.2
6.8 -_-
22.0
7.0
26.0
7.0
‘/a
Uptake I
0'
60
30 Time
90
120
(mid
Fig. 2. Time curve of regeneration of depleted adipose-tissue particles. I mg protein per 5 ml KCl-Tris (pH 7.4) was incubated at 37O for various times as indicated in the figure and cooled to 0’. Absorbing capacity test was carried out as described in Table I. O, acidified and albumintreated ; x , albumin-treated.
While heating the particles to boiling did not completely abolish fatty acid absorption, albumin treatment of boiled particles almost completely annulled their activity and very little uptake could be detected even following incubation at 37” (Table III). The substances removed from the particles by treatment with serum albumin were analyzed by subjecting the chloroform-methanol (a.:I, v/v) extract of the supernatant albumin solution to thin-layer chromatography. It was found that, in addition Biocla%~a. Biophys. Acta, 125 (1966) 456-464
TABI.E UPTAKE
III AND
REGENERATION
OF ACTIVITY
IN BOILED
ADIPOSE-TISSUE
PARTICLES
Bailed particles were obtained by heating a ICCX-Tris particuIate suspension (I mg protein per ml solution) at IOO’ for 20 min followed by acidification as described under MATERIALS AND METKODS. In all cases the assay was carried out at oO. Other experimental conditions as in Table I,
Acidified Acidified, albumin-treated Acidified, albumin-treated and incubated for 30 min at 37” ICroiled,acidified Hailed, acidified, albumin-treated and incubated for 30 min at 37”
66.4
82.2
x0.2 36.0
19.0
33.0 5.0
6.2
to free fatty acids, the albumin extract contained a Iipid which migrated on the plates like lysoleecithin. No compound of this kind could be detected in the extract of a KC&-Tris supernatant in the absence of albumin. This finding raised two questions: (I) Can the absorbtive capacity of depleted particles be renewed by the addition of the lipids present in the supernatant albumin solution? (2) Are the lipid compounds migrating out of the particles into the albumin solution responsible for the transfer of the radioactive fatty acids from the albumin solution to the particles ? In fact, it was found that the fatty acid-Fhos~holipid (Iysoiecithin ?) mixture leaving the particles, when added to the fatty arid-albumin solutions, had a pronounced influence on the partitioning of the free fatty acids between the albumin and the particles. This could be demonstrated by adding the lipid extract from the albumin in which particles had been washed, and also with defined lipids or fatty acids. In Table IV experiments are shown in which lipid extracts, obtained from albumin supernatants, of both “depleted” and “regenerated” particles, were added
ACTIVITY
OF THE LIPIDS
EXTRACTED
FROM
ACfDIFIED
LIVER
MITOCHONDRIA
WITH
ALBUMIN
Acidified rat-liver mitochoadria were first depleted of most of their fatty acid-absorbing capacity by washing with albumin (as described under METHODS). One portion of these particles was allowed to “regenerate” by incubating at 37” for 60 min and the other part W&S kept at 0’. Both types (20 mg protein) were then mixed with a solutian of 0.14 pmole albumin and 0.21 ,umole non-labeled palmitic acid under conditions identical with those used for fatty acid-uptake examinations. The supernatants were collected and extracted with chloroform-methanol (2 : I, v/v). The lipid extract was evaporated and suspended in 5 ml KCI-Tris solution by sonication in a glass tube for I min. 2 ml of each of these suspensions, i.e. E (the extract obtained from the depleted particies which were kept at o”) and ER (that obtained from the “regenerated” particles) were added to a suspension of depleted particles (zo mg protein) and an absorption-capacity test was carried out as in Table 1. ~---““_l_. I-lll._...Frse fatty acid Per,csnt uptake Particles Additions -pp content of lipid bc d a &fact tpmoles) d c -1--IO rg 2f 22 Acidified, albumin-treated None 0.08 0.X 18 24 W-5 Acidified, albumin-treated 2 mf E ‘3-4 0,os $7 58 26.3 21.3 0.1 zmlER Acidified, aibumin-treated Preincubaied at 37’ for 60 min None 5r 54 50 Biochinz.Biophys.
Acta, rzfi (7966) 456-464
461
FATTY ACID ABSORPTION
to “depleted” liver-particle suspensions. Only the extracts obtained from regenerated particles had an activating effect on the absorption of 14C-labeled fatty acid. In Expts. c and d, the free fatty acid content of the lipid extracts was titrated and the extracts were diluted to the same fatty acid content. Even then the extracts of the regenerated particles caused marked activation, while those from the depleted particles had no or negligible effect. The addition of palmitic acid or of a mixture of fatty acids (at concentrations obtained in experiments presented in Table IV) to the depleted particles caused no significant increase of ~~C~palmitate uptake by the particles (Table V). When lysophospholipids (lysolecithin, lysophosphatidate) were added to a depleted particulate suspension, increased uptake of [‘*C]palmitic acid was demonstrated (Table VI). In the presence of lysolecithin, the addition of carrier palmitic acid caused some synergistic effect on the absorbing capacity. TABLE
V
ADDITIONo?? FATTY
ACIDS
TO ACIDIFIED,
ALBUMIN-PRETREATED
LIVER
PARTICLES
The mixtures of the fatty acids, as potassium salts, were suspended in KG-T& (pH 7.4) solution by sonication and added to the experimental tubes containing liver particles, acidified and albumin depleted. procedure of the experiment as indicated in Table I. Particles
Additions
Acidified and albumin-treated Acidified and albumin-treated
None 0.08 ;cmole of a mixture of stearic acid, oleic acid and linoleic acid in equal amounts 0.09 /&mole of palmitic acid
Acidified and albumin-treated The same preincubated at 37” for 60 min
TABLE ADDITION
Per cent uptake of [W]palmitic acid
None
7.4
a:: 51.5
VI OF KNOWN
LIPlDS
TO ACIDIFIED,
ALBUMIN-PRETREATED
PARTICLES
Various lipids as specified in the table were suspended by sonication in KCl-Tris and added to the test mixtures containing 1.6 mg protein of adipose-tissue particles or zo mg protein of liver particles and tested for fatty acid uptake as in Tables I and II. Additions
Per cent uptake Adipose-tissue R A
None Lysolecithin, 0.25 @mole Lysolecithin 0.25 pmole + potassium palmitate 0.1 pmole Lysolecithin 0.5 pmole Lysolecithin 0.5 pmoie + potassium palmitate 0.2 ~umole Lysophosphatidic acid 0.25 ,umole + potassium palmitate 0.1 pmole Lysophosphatidic acid 0.5 pmole Lysophosphatidic acid 0.5 ymole + potassium palmitate 0.2 pmole Potassium palmitate 0.1 pmole Potassium palmitate 0.2 @mole -. --
19.5
Liver pa&G-
particles -._--
A
B
4.5
21.0
IQ.0
34.4
40.9
23.0
9.1
45.5 40.5
43.2 45.2
31.0
21.6
51.5
57.0
27.0
8.2
32.0 34.2
26.2 30.0
46.0 23.8 35.6
43.0 28.3 35.0
29.0 X7.4 19.4
11.2
5.0 5.6
Biochim. 3~o~h~s. Acta,
125
(1966) 456-464
L. RESHEF,
462
B. SHAPIRO
The ability of lysolecithin to remove [l*C]palmitic acid from its complex with albumin at o” was tested in the absence of mitochond~a. Table VII shows that when a solution of [r4C]palmitate-serum albumin complex was mixed with lysolecithin, [lPC]palmitate was precipitated by high-speed centrifugation. No such effect was observed with lecithin. With palmitic acid some precipitation occurred, but only at high TABLE
VII
PRECIPITATIONOF [W]PALMITATE FROM ALBUMIN-FATTY
ACID
COMPLEX BY LYSOLECITHIN
Sonicated suspensions of the lipid substrates in amounts indicated were mixed with 0.1 ml of a solution containing 0.14 pmole albumin and 0.14 ymole [“C]palmitic acid (27000 counts~m~n). The mixture was brought to a final volume of 14.5 ml with 0.15 M KCl-0.05 M Tris (pH 7.4), and centrifuged at Ioooooo x g for 90 min. The precipitates were extracted with alcohol-ether (3: I, v/v) and aliquots from supernatants and precititates were counted.
Substrate
added
p&es
Egg lecithin
Lysolecithin
II.0
1.0
*I.0
I,5
0.5 I.0 2.0
0.5
4.0 8.0 Potassium
palmitate
Per cent of radioactivity
0.5 I.0 2.0
4.0
precipitated
1.4 9.2 22.2 38.0 0.7 I.7 3.0
12.5
concentration. The amounts of lysolecithin required for the above-mentioned effect were 8-16 times higher than those inducing a ir*C]palmitate / uptake by depleted particles. DISCUSSION
Results in this and in our previous communication1 show that the uptake of fatty acids from a serum-~bumin solution by cell particles is a non-enzymatic process, proceeding at o0 and being comparatively heat resistant. Uptake seems to be due to binding to a protein acceptor in the particle membrane. Lipid constituents of the particles also play a role in this process. It has now been shown that some lipids diffuse out into the albumin media and as a result the fatty acid-absorbing capacity of the particles becomes exhausted. There was a direct relationship between the initial activity and the extent of exhaustion. Adipose-tissue particles, which are highly active, were also easily depleted. Liver particles, which are much less active, did not lose activity. Only when liver particles were activated by pretreatment with buffer at pH 4.0 did considerable depletion occur on contact with serum-albumin solutions. Such “acidified” particles were also more rapidly and extensively regenerated by incubation at 37”. The nature of the change brought about by the acid treatment is not clear. As it took place at 0’ it seems most likely that it is based on changes in the ionization of some membrane constituents, although an enzymatic change cannot be excluded. The results point to the conclusion that during contact with serum albumin, Biochim.
Bio@~.
Acta,
125 (1966) 456-464
E ATTY ACID ABSORPTION
463
the particles lost certain constituents which are participating in the process of fatty acid uptake. It could be shown that, in addition to free fatty acids, albumin removed a phospholipid which behaved chromatographically like lysolecithin. In fact the mixture of lipid substances extracted from the albumin supernatant could reactivate the particles. This occurred only with albumin solutions which had been in contact with active particles, i.e. fresh or regenerated ones, but not with depleted particles. The free fatty acids alone could not explain the activating potency of these lipid extracts. Even when the free fatty acid concentrations in extracts obtained from regenerated and non-regenerated particles were identical, only the former exhibited activity. No activity was evolved by the addition of various fatty acids at concentrations present in the active extracts. Lysophosphatides, on the other hand, could induce fatty acid translocation from the albumin solution to the particles. The combined action of both lysophosphatides and free fatty acids may be responsible for the extraction of the fatty acids from the albumin complex. When used at a ro-fold concentration, lysolecithin can remove fatty acids from their albumin complex, even in the absence of tissue particles. Contrary to the non-enzymatic nature of the uptake itself, the regeneration of this capacity does seem to involve enzymatic processes. Regeneration does not take place at o’, nor is it brought about in heat-treated particles. The nature of the enzymatic processes bringing about regeneration, and of the chemical compounds formed, is not yet clear. Some indication may be found in the activation of the process by acid buffer treatment and in the fact that the free fatty acid concentration in the particles increases during regeneration. This makes it likely that a phospholipase activity is responsible for this process. The mechanism of fatty acid uptake by particles seems to involve interaction between the fatty acid-albumin complex and lipids (fatty acid and lyso compounds) present on the particle membrane. In our experiments in vitro these lipids actually diffuse out into the medium. The possibility may be considered that, under physiological conditions, lyso compounds serve as factors facilitating fatty acid transport into the particles, by loosening the fatty acid-albumin complex.Following the uptake of the fatty acid by the particles, esterification of the lyso compound takes place forming the phospholipid. This may then release the fatty acid into the particle by Outside
I
I
Fatty ocidalbumin complex
I-
Membrane
Inside
Lysophospholipid
I
+
Fatty acid
Phospholipld
Scheme I. Hypothetic
scheme of fatty acid uptake under physiological Biochim. Biophys.
conditions. Acta,
125
(1966) 456-464
464
L. RESHEF,
B. SHAPIRO
phospholipase action (Scheme I). The rapid turnover of the fatty acids in the p position of lecithin has been repeatedly demonstrated and may be part of such a transport system. The release of free fatty acids seen in vitro may be an artifact, caused by disruption of the organisation which directs the acids into the particle and by the conditions of the experiment, i.e. o’, which do not permit the re-esterification of the lysophospholipid. REFERENCES L. RESHE~ AND B. SHAPIRO, Biochim. Biophys. Acta, 98 (1965) 73. M. PANGBORN, J. Biol. Chem., 188 (1951) 471. C. LONG, R. ODAVIC AND E. J. SARGENT, B&hem. J., 85 (1962) 33. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 265. 5 V. P. DOLE, J. C&z. Invest., 35 (1~56) 150. I 2 3 4
Biochim.
Biophys.
Ada,
125 (1966)
456-464
193 (1951)