Download Relationship between Protein Synthesis and Secretion in Liver Cells

Aust. J. Bioi. Sci., 1979,32,299-307
Relationship between Protein Synthesis
and Secretion in Liver Cells and the State
of the Adenine Nucleotide System
Kaylene Edwards, Jorg Urban and Gerhard Schreiber
Russell Grimwade School of Biochemistry,
University of Melbourne, Parkville, Vic. 3052.
Abstract
Adenine nucleotide levels could be precisely and reproducibly adjusted in liver cell suspensions by
partially depleting the ATP pool with D-fructose or glycerol. Thus, it was possible to quantitatively
correlate rates of protein synthesis and secretion with intracellular levels of ATP and with derived
parameters, such as the adenylate energy charge. Half the maximum rate of incorporation of leucine
into protein was observed at an energy charge of 0·80, a ratio of ATP to ADP of 2·6, and an ATP
level of 1·05 pmol per g of wet cells. Proteins were secreted with half the maximum rate at an
energy charge of 0·85, a ratio of ATP to ADP of 3·1 and an ATP concentration of 1 ·1 pmol per g
of wet cells. Protein secretion dill not depend on continued synthesis. Inhibitors of oxidative
phosphorylation inhibited protein secretion in addition to protein synthesis, in contrast to observations by other authors on liver slices.
Introduction
Little is known about the relationship in liver between the actual rate of protein
synthesis and the intracellular concentrations of ATP, the ratio of ATP to ADP, and
the adenylate energy charge. Studies on the relationship between energy supply
and protein secretion are scarce and contradictory. Secretion of proteins by pancreatic
tissue, for example, was shown to require an intact energy-supplying system (Coore
and Randle 1964; Jamieson and Palade 1968), whereas 2,4-dinitrophenol did not
seem to inhibit albumin secretion in liver slices (Judah and Nicholls 1971).
One method for studying ATP involvement in biochemical processes is to deplete
the ATP pool with physiological metabolites such as fructose and glycerol (for
review see Farber 1971, 1973). This is less drastic than the interruption of oxidative
phosphorylation with inhibitors. Injection of fructose (Coore and Randle 1964;
Rose and Warms 1967; Maenpaa et al. 1968; Raivio et al. 1969; Burch et al. 1969,
1970; Woods et al. 1970; Sestoft and Fleron 1973; Hultman et al. 1975) or glycerol
(Burch et al. 1970) into living rats (Maenpaa et al. 1968; Burch et al. 1969, 1970),
humans (Hultman et al. 1975), perfused rat liver (Raivio et al. 1969; Woods et al.
1970; Sestoft and Fleron 1973) or rat hepatocyte suspensions (Sestoft and Fleron
1973) has been reported to lead to a decrease of ATP concentration (Maenpaa et al.
1968; Raivio et al. 1969; Burch et al. 1969, 1970; Woods et al. 1970; Sestoft and
Fleron 1973; Hultman et al.1975) and to an inhibition of protein synthesis (Maenpaa
et al. 1968).
In recent years, liver cell suspensions prepared by continuous perfusion of the
liver in situ with collagenase and hyaluronidase (Berry and Friend 1969) have been
optimized for protein synthesis (Schreiber and Schreiber 1973). The suspended
300
K. Edwards, J. Urban and G. Schreiber
liver cells secrete protein (Schreiber and Schreiber 1973), in particular albumin
(Weigand and Otto 1974), into the medium. They are also able to convert proalbumin
into albumin (Edwards et al. 1976; Schreiber et al. 1976).
In this paper, we show first that it is possible to adjust adenine nucleotide levels
in liver cell suspensions in a precise and reproducible way utilizing methods described
above for influencing intracellular adenine nucleotide levels in mammalian liver
in vivo. Protein synthesis and secretion are then correlated with adenylate energy
charge and ATP/ADP ratio. Finally, the energy dependence of protein synthesis
and secretion in liver cell suspensions is demonstrated in experiments with inhibitors
of oxidative phosphorylation. Two relevant abstracts have been published previously
(Schreiber et al. 1977; Edwards et al. 1978).
Materials and Methods
Chemicals and Isotopes
L-[1- 14CJleucine (59--62 Ci/mol) was from the Radiochemical Centre (Amersham, England).
Sodium pyruvate, phosphoenolpyruvate (potassium salt), IX-oxoglutaric acid, ADP (disodium salt),
AMP (disodium salt), P-NADH (disodium salt), P-NADP (disodium salt), hexokinase from yeast
(EC 2.7.1.1), myokinase from rabbit muscle (EC 2.7.4.3), pyruvate kinase from rabbit muscle
(EC 2.7.1.40), glucose-6-phosphate dehydrogenase from yeast (EC 1.1.1.49), and lactate dehydrogenase from rabbit muscle (BC 1.1.1.27) Were purchased from Boehringer-Mannheim (Germany).
Glycerol and 2,4-dinitrophenol were obtained from British Drug Ho.uses Chemicals Ltd (Melbourne,
Australia). Rotenone (Grade II), P-o( - )fructose and the disodium salt of ATP (Grade I) were
purchased from Sigma Chemical Co. (St Louis, U.S.A.). The sources of all other cheJ;Ilicals have
been given previously (Schreiber and Schreiber 1972). All reagents were of analytical grade.
Stock solutions of rotenone and 2,4-dinitrophenol in methanol were added to the suspensions
such that the final concentration of methanol was 1 ·25 % (v/w) or less. This concentration of methanol
does not interfere significantly with protein synthesis in liver cell suspensions (Schreiber and Schreiber
1973). All other compounds added to the suspensions had been dissolved in incubation medium
before addition to the suspensions. The pH of all solutions was adjusted to 7·3 prior to addition
to the suspensions.
Preparation of Cell Suspensions
Cell suspensions were prepared with a modification (Weigand et al. 1971; Schreiber and Schreiber
1973) of the method of Berry and Friend (1969) from the livers of male non-starved Buffalo rats
(160--270 g body weight) fed ad libitum with a diet containing about 20% protein (Barastoc Mouse
Breeder Ration, Barastoc Products, Melbourne, Australia). The dissociation medium consisted
of calcium-free Hanks solution (Hanks and Wallace 1949), pH 7·8, containing 0·05 % collagenase.
Incubation of Cell Suspensions
Volumes of 1·0--2·0 m! were incubated in 18 by 100 mm glass centrifuge tubes, and glass
Erlenmeyer flasks of 25 and looml were used for incubation of 2·0--5·0 and to·Om! volumes
respectively. Cells, suspended in incubation medium (Edwards et al. 1976), were shaken at 3JOC
at an amplitude of 9 em and at the lowest frequency sufficient to keep the cells in suspension.
Incubation was terminated by rapid cooling to 2°C.
Determination of Radioactivity in Total Protein
Radioactivity in total protein was determined on chromatography filter paper discs according
to Mans and Novelli (1961). The procedure involves precipitation of protein with 10% trichloroacetic acid and washing with hot and cold trichloroacetic acid and ether-ethanol (1 : 1, v/v).
Determination of ATP,ADP and AMP
Cell suspension samples, either 1 or 3 ml (about 5 x 106 cells/m!), were rapidly mixed with an
equal volume of ice-cold 10% perchloric acid. The precipitate was removed by centrifugation. The
Energy Supply and Protein Synthesis and.Secretion
301
supernatant fraction was neutralized with 5 M K 2CO a and the resulting precipitate again removed
by centrifugation. In a portion of the supernatant fraction, ATP was determined with hexokinase
and glucose-6-phosphate dehydrogenase (Lamprecht and Trautschold 1974). No ATP was detected
in the incubation medium obtained after removal of cells by centrifugation, indicating that all ATP
measured in the suspension was located intracellularly.
AMP and ADP were determined in other portions of the supernatant according to Jaworek et al.
(1974).
Effect of Rotenone, 2,4-Dinitrophenol, Fructose and Glycerol on the Structural Integrity of Suspended
Cells
The effect of rotenone, 2,4-dinitrophenol, fructose and glycerol on the release into the medium
of lactate dehydrogenase and glutamate dehydrogenase, enzymes of the cytoplasm and mitochondria,
respectively, was assayed. Enzyme activities were monitored in both total cell suspension and
incubation medium. Prior to measuring the enzymes in total cell suspensions, the suspensions were
homogenized in 4-15 volumes of 50 mM potassium phosphate buffer (pH 7·5) in a Sorvall Omnimix
homogenizer for 1 min at full speed. Insoluble material was removed by spinning for 15 min at
1000 g. For the separation of incubation medium from the cells, the cell suspensions were centrifuged
for 15 min at 1000 g using the separation tubes described by Hems et al. (1975).
Lactate dehydrogenase was measured as described by Bergmeyer and Bernt (1974). Glutamate
dehydrogenase was measured as described by Schmidt (1974). From 0·9 to 1 ·9% of total glutamate
dehydrogenase and from 27 to 55 % of total lactate dehydrogenase activity were found in the medium
of different preparations of cells. In each of the preparations the ratio of extracellular to total activity
of either enzyme did not change throughout the incubation or with increasing inhibitor concentrations.
Furthermore, the decrease in the amount of extracellular radioactive protein observed for increasing
concentrations of the four inhibitors indicated that cells were not damaged in such a way as to allow
leakage of newly synthesized proteins. A 'healthy' state of the cells was particularly well documented
in the experiments with fructose by the high values measured for the level of ATP and for the adenylate
energy charge (see Results section).
Results
Adjustment of Adenine Nucleotide Levels in Liver Cell Suspensions
The adenine nucleotide content of liver cells, incubated in a medium optimized
for protein synthesis (Schreiber and Schreiber 1973), was found to range from 1·6
to 3·3 ,umoIATP, from 0·34 to 0·53 ,umoIADP, and from 0·058 to 0·069 ,umolAMP
per g of cells, wet weight. This agrees with values reported in the literature (Phillips
et al. 1974; Hems et al. 1975; Jeejeebhoy et al. 1975; Hofmann et al. 1976).
When liver cell suspensions were incubated with fructose, the ATP content of the
cells decreased. Concentrations of 3-8 mM fructose lowered the ATP content of
cells to half the value measured in incubations without fructose.
Fructose did not have a marked effect on the level of ADP, which was about
0·4 ,umol per g cells, wet weight. The content of AMP in the cells rose from about
0'06,umol per g cells, wet weight, at 0-4 mM fructose to a maximum of 0·16 ,umol
AMP per g cells, wet weight, at 10 mM fructose. An example of the relationship
between the level of ATP, ADP and AMP and the concentration of fructose in the
hepatocyte suspensions is depicted in Fig. 1.
. When liver cell suspensions were incubated with glycerol, rotenone or 2,4-dinitrophenol, the ATP content of the cells was lowered by 50 % at concentrations of
9 mM, 6 and 50,uM respectively.
Adenylate Energy Charge and the ATP/ADP Ratio
Adenylate energy charge and the ATP/ADP ratio in liver cells incubated for
25 min under conditions optimized for protein synthesis (Schreiber and Schreiber
. K. Edwards, J. Urban and G. Schreiber
302
1973) were found to be 0·90 and 6·0 respectively. Addition of fructose decreased
the level of ATP. The ratio of ATP to ADP remained between 5·5 and 6·5 and the
energy charge stayed at 0·90 for fructose concentrations between 0 and 4 mM,
indicating a satisfactory supply of ATP. An energy charge of 0·85 was reached at
about 5-6 mM fructose. At 10 mM fructose the energy charge dropped to O· 70 and the
ATP/ADP ratio decreased to 1·6 .
.-... 2·5
f
~
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Fig. I. Relationship between the
adenine nucleotide levels of liver cells in
suspension and the concentration of
fructose in the medium. Hepatocytes
(5·8 x 106 cells/ml) were incubated at
37°C in 5·0 ml portions of incubation
medium containing various concentrations of fructose. ATP (0), ADP
( .) and AMP (.) were determined
after 25 min as described under
Materials and Methods .
2·0
1·5
~
8
ial
E,
.
<J
0'5
~
«~
-c
0
•
...
,.
4
6
8
10
Fruclose concentration (mM)
Relationship Between Protein Synthesis and Energy Supply
Protein synthesis was studied by measuring the incorporation of L-[1-14C]leucine
into protein in liver cell suspensions in which the adenine nucleotide system was
manipulated by adding either fructose, glycerol, rotenone or 2,4-dinitrophenol.
The incorporation of L-[1-14C]leucine into protein was lowered by 50% by 4-8 mM
fructose, 2-3 mM glycerol, 2-4 fLM rotenone, and 10-30 fLM 2,4-dinitrophenol.
When cells were incubated in the presence of fructose, the incorporation of
L-[1-14C]leucine into protein was lowered within 5 min. The reduced rate of·incorporation of L-[1-14C]leucine into protein in the presence of fructose was observed
throughout the 45-min incubation period.
The correlation between radioactivity in protein after an incubation period of
45 min with theATP content of the cells determined after 25 min is given in Fig. 2a.
The ATP content at 25 min was taken to reflect the average ATP content of the cells
over the entire incubation period. The concentration of ATP in the cells had been
decreased by adding fructose up to a concentration of 10 mM. A half-maximal
incorporation of L-[1-14C]leucine into protein was observed at about 1·05 fLmol
ATP per g cells, wet weight. The relationships between protein synthesis and the
ratio of ATP to ADP, and between protein synthesis and the energy charge of the
adenine nucleotide system are described in Figs 2b and 2c respectively. Half-maximal
incorporation was measured at an ATP to ADP ratio of about 2·6 and an energy
charge of O· 80. At an energy charge of 0·85 the rate of protein synthesis was 70 %
of the value observed at an energy charge of O· 90, the maximum value measured.
Energy Supply and Protein Synthesis and Secretion
303
Relationship Between Protein Secretion and Energy Supply
Protein synthesis in liver cell suspensions can be inhibited by cycloheximide
(Schreiber and Schreiber 1973) or puromycin (Schreiber et al. 1974) without interfering with protein secretion. These inhibitors can therefore be used to study protein
secretion independently of synthesis.
After incubation of cell suspensions from rat liver with L-[IJ4C]leucine, further
synthesis of protein was interrupted by adding 22·9 J1M cycloheximide. The increase
of radioactivity in protein in the medium during further incubation was measured to
determine protein secretion.
l'
240
f(a)
~ ~
o..c
a.
180
l-
;.,.0
120
I-
.~
A
~
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I
/L
24Or(b)
180
l-
120
l-
60
l-
I
240
•
f(c)
180
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.-:::
;:::s
"
C
.-"
~ta
>
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--
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0..
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/
601-4
01
0·6
I
1·2
1·8
ATP content of liver cells
(J.Lmoifg cells wet weight)
0
I
e
I
120
60
0
[ATP)/[ADP]
0·6
0·7
0·8
o·g
Energy charge
Fig.2. Correlation between protein synthesis and (a) ATP content, (b)
ratio of ATP to ADP, and (c) adenylate energy charge in liver cells in
suspension. Cells were incubated at 3TC in 5 ·O-ml volumes (5·8 x 106
cells/ml) with 0·27 IlCi/ml L-[p4C]leucine in the presence of various
concentrations of fructose from 0 to 10 mM. After 25 min incubation,
3·0 ml of cells was withdrawn for determination of ATP, ADP and
AMP. The remainder was incubated for a further 20 min, cooled to
2°C and protein radioactivity was determined in portions of the
suspension (containing cells plus medium.)
Addition of either fructose, glycerol, rotenone or 2,4-dinitrophenol to the cell
suspensions at the time of cycloheximide addition led to a decrease in the secretion of
radioactive protein, concentrations ranging from 10-40 J1M rotenone, 20-70 J1M
2,4-dinitrophenol, 4-20 mM fructose and about 7 mM glycerol, lowering protein
secretion by 50 %. Figs 3 and 4 show the decrease in the amount of radioactive
protein secreted from the cells during incubation with 2,4-dinitrophenol and fructose
respectively. For comparison the effect of 2,4-dinitrophenol on protein synthesis is
also depicted (Fig. 3).
The relationship between protein secretion and the level of ATP, the ratio of
ATP to ADP and the energy charge was similar to that between protein synthesis
and the same parameters. Half-maximal protein secretion was observed at about
1 . 1 J1mol ATP per g cells, wet weight, a ratio of ATP to ADP of about 3· 1 and an
adenylate energy charge of 0·85.
Discussion
The energy charge of the adenylate system (Atkinson and Walton 1967),
([ATP]+0'5[ADPD/([ATP]+[ADP]+[AMPD, has been proposed as an important
304
K. Edwards, J. Urban and G. Schreiber
regulatory parameter for energy-consuming and energy-producing metabolic systems
(Atkinson· 1968, 1977). In living cells it is stabilized at about 0·85, when reactions
either producing or consuming ATP operate at half their maximum rates (Atkinson
1968, 1972). In growing E. coli cells the adenylate energy charge is between 0·8
18
120
Fig. 3~ Synthesis and secretion of protein
by suspensions of liver cells in the presence
o
of 2,4-dinitrophenol. For studies on
~
protein synthesis (0), cell suspensions
80
(1·2x 106 cells/ml) were incubated at 37°C
for 50 min with 1 JlCi L-[J-14C]leucine per
3! 60
ml of incubation medium in the presence
1
of varying concentrations of 2,4-dinitro.ill 40
phenol.
Radioactivity in protein was
Jlc
determined in each sample and expressed as
~ 20
a percentage of the value measured in the
control sample, which did not contain
o.
"""
!I~
2,4-dinitrophenol. 100% corresponds to
10
100
224 000 dpm/ml of cell suspension.
2,4-Dinitrophenol concentration (I'M)
For studies on protein secretion (.),
cell suspensions (5 ·6x 106 cells/ml) were incubated at 37°C for 25 min with 1 JlCi of L-[1-14C]leucine
per mI of incubation medium. Cells were then cooled to 2°C and 22·9 JlM cycloheximide was added.
One portion was kept at 2°C as zero sample and the remainder dispensed into tubes containing
varying concentrations of 2,4-dinitrophenol to give a final concentration of 2·8 X 106 celis/mI.
Incubation was then continued at 37°C for 30 min and terminated by rapid cooling. Cells were
separated from the medium by centrifuging for 15 min at 200 g and radioactivity in protein in the
supernatant was determined. The radioactivity in protein which appeared in the medium during
the 30 min incubation is plotted as a percentage of the value measured in the control sample which
contained no 2,4-dinitrophenol. 100% corresponds to 42000 dpm/mI of medium.
100
.j
1
240
Fig. 4. Secretion of protein by liver
cells in suspension in the presence of
fructose. Cell suspensions (5·9 x 106
c.~
] Ii ISO
cells/ml) were incubated at 37°C for
~ s:
35 min with 3 JlCi of L-[J-14C]leucine
S! •.8
c II 120
per mI of incubation medium.
.- B
Cells were then cooled to 2°C and
.~.5
22·5 JlM cycloheximide was added.
60
One portion was kept at 2°C as zero
'g-%
sample and the remainder dispensed
cx;'-""
into 25-ml Erlenmeyer flasks
10
6
8
o
4
containing varying concentrations of
Fructose concentration (mM)
fructose to give a final concentration
of 5·4 X 106 cells/ml. Incubation was then continued at 37°C for 85 min and terminated by rapid
cooling. Cells were separated from the medium by centrifuging for 45 s at 30 g. The supernatant
was then centrifuged for 15 min at 1000 gand radioactivity in protein was determined in the 1000 g
supernatant. The radioactivity in protein which appeared in the medium during the 85 min incubation
is plotted as a function of concentration of fructose in the incubation medium.
.S;
e"' .......E
·i i
and 0·9. Maintenance, but not growth, is possible between 0·5 and O· 8, while
below O· 5 metabolism becomes disorganized resulting in cell death (Atkinson 1972).
The values of 0·90 found for the adenylate energy charge in the cells incubated under
conditions optimized for protein synthesis indicated that the energy-generating
systems in the hepatocytes provided more ATP than needed for maintenance of
Energy Supply and Protein Synthesis and Secretion
305
cellular metabolism. A decrease of ATPconcentration by fructose or glycerol could
be produced in liver cell suspensions in a very precise and quantitatively reproducible
way. The ratio of ATP to ADP and the energy charge of the adenine nucleotide
system were found to decrease accordingly. A decrease in the incorporation of
L-[1-14C]leucine into protein in the liver cell suspensions was observed as the ATP
concentration, the ATP to ADP ratio and the adenylate energy charge were decreased.
It is unlikely that fructose, glycerol, rotenone or 2,4-dinitrophenol influence
the incorporation of L-[1-14C]leucine into protein by affecting the size of the leucine
pool in the cell, since a rapid equilibrium exists between intracellular and extracellular amino acid pools in liver cells in suspension (Schreiber and Schreiber 1972,
1973). All preparations of cells used in this work were washed twice with leucine-free
incubation medium before use, this effectively depleting the cell of intracellular
leucine (Schreiber and Schreiber 1972). Hence the only leucine available for protein
synthesis is the 'added L-[1-14C]leucine (4·5-16·7 JlM) and any leucine arising from
protein degradation. However, since rotenone and 2,4-dinitrophenol have been
shown to inhibit protein degradation in liver cells in suspension (Hopgood et al. 1977),
the use of these inhibitors may result in an increase in the specific radioactivity of
the leucine pool in the incubated cells. Thus, for a given rate of protein synthesis,
more radioactive leucine would be incorporated into protein in cells incubated in
the presence of these inhibitors than in cells incubated without inhibitors. Despite
this, a reduction in the incorporation of L-[1-14C]leucine into protein was observed
when cells were incubated with fructose, glycerol, rotenone or 2,4-dinitrophenol.
. The data available on the rehitionship between energy supply and protein secretion
are contradictory. Anoxia and 250 JlM 2,4-dinitrophenol have been reported to
inhibit the secretion of insulin by pieces of pancreatic tissue (Coore and Randle 1964).
The transport of secretory proteins in pancreatic slices was blocked by N 2 , cyanide,
antimycin A, dinitrophenol and oligomycin (Jamieson and Palade 1968). However,
no effect of 100 JlM 2,4-dinitrophenol was seen on the appearance of label in iritraor extracellular albumin in liver slices incubated with radioactive amino acids (Judah
and Nicholls 1971). The data presented in this paper indicate that an intact energysupplying system is necessary for secretion of protein from liver cells.
AckOowledgment
The authors thank the National Health and Medical Research Council for financial
support ..
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Manuscript received 12 October 1978