Download effects of insulin and anchorage on hepatocytic protein metabolism

J. Cell Sri. 48, 1-18 (1981)
Printed in Great Britain © Company of Biologists Limited 1981
EFFECTS OF INSULIN AND ANCHORAGE ON
HEPATOCYTIC PROTEIN METABOLISM AND
AMINO ACID TRANSPORT
ALESSANDRO POLI*, PAUL B. GORDON, PER E. SCHWARZE,
BJ0RN GRINDE AND PER O. SEGLENf
Department of Tissue Culture, Norsk Hydro's Institute for Cancer Research,
The Norwegian Radium Hospital, Montebello, Oslo 3, Norway
SUMMARY
Insulin partially inhibits endogenous protein degradation in isolated hepatocytes. The
inhibition seems to specifically affect the lysosomal pathway of degradation, since it is not
additive to the effects of lysosome inhibitors such as propylamine and leupeptin. The insulin
effect is potentiated by intermediate concentrations of amino acids, but is largely abolished
at high amino acid concentrations which suppress degradation maximally, suggesting that
the hormone may exert its effect indirectly by acting upon the more basal amino acid control
mechanism. Glucagon, which stimulates protein degradation, similarly displays its effect only
in the presence of intermediate amino acid concentrations.
The insulin inhibition is not affected by the aminotransferase inhibitor, aminooxyacetate,
indicating that it is not due to interference with amino acid metabolism. Protein synthesis
furthermore does not seem to be required, since a significant insulin effect can be seen in the
presence of the protein synthesis inhibitor, cycloheximide. The issue is, however, complicated
by the fact that cycloheximide itself inhibits protein degradation to approximately the same
extent as does insulin.
Insulin stimulates uptake of the amino acid a-aminoisobutyrate (AIB), but not the uptake
of valine, indicating a specific stimulation of 'A'-type transport. Cycloheximide similarly
stimulates AIB uptake, without completely obfuscating the transport effect of insulin.
Neither protein synthesis, protein degradation, amino acid transport, nor the effects of
insulin were affected by cell-to-substratum anchorage (attachment and spreading) in any
detectable way.
INTRODUCTION
Hepatocytic protein degradation has been shown to be subject to regulation by the
pancreatic hormones - insulin and glucagon - both in the perfused rat liver (Seglen,
1968; Mortimore & Mondon, 1970; Woodside, Ward & Mortimore, 1974; Schworer
& Mortimore, 1979) and in isolated rat hepatocytes (Gunn et al. 1977; Hopgood,
Clark & Ballard, 1980; Grammeltvedt & Berg, 1976; Seglen, Gordon & Poli, 1980).
The hormones seem to act at the level of cellular autophagy, glucagon stimulating
and insulin suppressing the formation of autophagic vacuoles (Ashford & Porter,
1962; Deter, 1971; Pfeifer, 1978; Schworer & Mortimore, 1979).
Autophagy in hepatocytes is also controlled by the inhibitory effect of amino acids
• Present address: Institute of Comparative Anatomy, University of Bologna, 40100
Bologna, Italy.
f To whom correspondence should be addressed.
2
A. Poli and others
(Woodside & Mortimore, 1972; Mortimore & Schworer, 1977; Hopgood, Clark &
Ballard, 1979; Seglen et al. 1980a), and there are strong indications for the stimulation
by glucagon being mediated by a decrease in intracellular amino acid levels (Schworer
& Mortimore, 1979). In the present report, we have investigated the possible role
of amino acids in the inhibition of protein degradation by insulin in isolated
hepatocytes, examining several of the processes which influence intracellular amino
acid levels (transport, metabolism, protein synthesis). Since some of these processes
have been reported to be anchorage-dependent in cultured cells (Otsuka &
Moskowitz, 1975, 1978; Profit & Strauss, 1977; Benecke, Ben-Ze'ev & Penman, 1978)
we have, furthermore, looked at the role of anchorage for basal as well as for insulinaffected protein metabolism and amino acid transport.
MATERIALS AND METHODS
Isolated hepatocytes were prepared from the liver of i6-h-starved, male Wistar rats
(250-300 g) by the method of collagenase perfusion (Seglen, 1976a). r o - i ^ x i o 1 cells
(8-10 mg wet wt) were suspended in 2 ml attachment buffer, i.e. suspension buffer (Seglen,
1976a) fortified with 20 raM pyruvate, 18/iM (iomg/ml) garamycin and Mg 1+ to a final
concentration of 2 mM. The cells were seeded in 6-cm polystyrene tissue-culture dishes, and
incubated at 37 CC for up to 8 h. In some experiments a balanced amino acid mixture (Seglen,
19766) was added to the medium at various multiples of the 'normal' concentration.
Incubations were terminated by the addition of 0-5 ml ice-cold perchloric acid (10%, w/v)
or by replacement of the medium with 4 ml ice-cold buffer or 09 % NaCl (in the case of
transport studies).
Unless otherwise indicated, culture dishes pretreated with collagen (Gjessing & Seglen, 1980)
were used. Collagen (Sigma type I) adsorbs readily to polystyrene, giving a substratum which
will support the attachment and spreading of hepatocytes in a protein-free medium. Similarly
prepared substrata of albumin, asialofetuin, gelatin, polylysine or serum, which have different
attachment-supporting properties (Gjessing & Seglen, 1980), were used in experiments on
anchorage dependence.
Protein degradation was measured as the release of [14C]valine from protein pre-labelled
in vivo 24 h before cell isolation (Seglen, Grinde & Solheim, 1979), and protein synthesis as
the incorporation of [14C]valine of constant specific radioactivity (Seglen & Solheim, 1978 a).
The uptake of amino acids ([a- 14 C]aminoisobutyrate, 1 mM and 63-125 nCi/ml; [ u C]valine,
I mM and 250 nCi/ml) was measured as the accumulation of intracellular acid-soluble
radioactivity (Seglen & Solheim, 19786).
[14C]valine (CFB 75) and [a- 14 C]aminoisobutyrate (CFA 203) were purchased from the
Radiochemical Centre, Amersham, Bucks, England. Garamycin was from Schering, Kenilworth, N.J., U.S.A.; and all biochemicals from Sigma Chem. Co., St Louis, MO, U.S.A.
RESULTS
Amino acid-dependent inhibition of protein degradation by insulin
Insulin had a moderate inhibitory effect on protein degradation in hepatocyte
monolayers (Fig. 1), as previously reported (Gunn et al. 1977; Hopgood et al. 1980).
Inhibition was maximal at io~7 M (100 nM); at higher concentrations the hormone
seemed to be less effective. The inclusion of albumin (1-5 %) in the medium shifted
the dose-response curve about an order of magnitude to the left, without changing
the maximum effect (not shown), indicating that considerable destruction of the
hormone takes place in these serum-free, freshly seeded cultures.
Protein metabolism in hepatocytes
3
The insulin effect displayed in Fig. 1 was obtained in a medium containing amino
acids (7-5 x normal plasma concentrations), which by themselves suppress protein
degradation (Woodside & Mortimore, 1972; Seglen et al. 1980 a). Hopgood et al.
(1977) found the effects of insulin and amino acids to be approximately additive,
and in Fig. 2 it is shown that insulin inhibits degradation over a wide range of amino
acid concentrations. However, the insulin effect is significantly smaller at very high
and very low amino acid concentrations, suggesting that it is somehow dependent
upon amino acids.
10"
10"*
5
1CT
10"
Insulin concentration, M
Fig. 1. Concentration-dependent inhibition of hepatocytic protein degradation by
insulin. Isolated hepatocytes were incubated in collagen-treated tissue culture dishes
for 3 h at 37 °C in the presence of amino acids (7-5 x normal concentrations, cf. Seglen,
19766) and insulin at the concentration indicated. The mean rate of protein degradation was measured as the net release of ["Cjvaline from radioactive protein during
the entire incubation period, and expressed as % relative to the hormone-free control
(at 2 o %/h). Each value is the mean ± s.E. of 6 cell samples from 3 different experiments.
In addition to the complete amino acid mixture used in Figs. 1 and 2, a variety
of more simple amino acid combinations have been tried together with insulin.
Seven amino acids (leucine, tyrosine, phenylalanine, histidine, tryptophan, asparagine
and glutamine) are particularly active as inhibitors of protein degradation (Seglen
et al. 1980 a), and insulin has been found to potentiate the effect of all inhibitory
combinations of these, except when the maximally obtainable inhibition is approached.
Some examples are given in Table 1. The potentiation by insulin of the effect of
leucine alone is particularly striking, cf. also Table 3.
4
A. Poli and others
Stimulation of protein degradation by glucagon is also amino acid-dependent
Schworer & Mortimore (1979) found that the stimulation of protein degradation
in the perfused liver by glucagon was strongly amino acid-dependent, and Table 2
shows that this is also the case in isolated hepatocytes. Glucagon alone ( I O ~ 7 M )
did not stimulate degradation at all, but reduced the inhibitory effect of an amino
acid mixture ('old' mixture 10x normal plasma levels). Insulin (io~7 M) had the
5
10
15
Amino acid concentration, x N
Fig. 2. Influence of amino acids on the inhibition of protein degradation by insulin.
Isolated hepatocytes were incubated for 3 h with various amino acid concentrations
(multiples of the normal concentration given by Seglen, 19766) in the presence ( • ) or
absence (O) of insulin, io~7 M. The rate of protein degradation during the incubation
was measured as the release of [14C]valine from radioactive protein, and expressed
as %/h. Each value is the mean of 6 cell samples from 2 different experiments.
opposite effect, which was not significantly reduced by the simultaneous presence
of glucagon at an equimolar concentration. Hopgood et al. (1980) found that a 10-fold
molar excess of glucagon was required to prevent the effect of io" 8 M insulin.
As was the case with insulin, the effect of glucagon tended to disappear at very
high amino acid concentrations. The 'new' amino acid mixture in Table 2 contains
particularly large amounts of the 7 most degradation-inhibitory amino acids and
with this mixture none of the hormones produced any significant effect. Glucocorticoid hormone (dexamethasone) was ineffective under all conditions tested.
Methylamine, a lysosomotropic amine, and leupeptin, a protease inhibitor, are
Protein metabolism in hepatocytes
5
effective and relatively specific inhibitors of lysosomal protein degradation (Seglen
et al. 1979). As shown in Table 2, protein degradation in the hepatocytes was strongly
inhibited by these two agents, and the remaining degradation, thought to be nonlysosomal (Seglen et al. 1979), was not affected by insulin or glucagon. The pancreatic
hormones therefore seem to act selectively upon the lysosomal pathway of protein
degradation, in accordance with their known effect on cellular autophagy (Deter,
1971; Pfeifer, 1978).
Table 1. Effect of insulin on inhibition of protein degradation by various ammo acid
combinations
Amino acids present
Aminn flcifl
concentration
Inhibition of protein
degradation, %
(
Leu
Tyr
Phe
o
—
IO X
10 X
+
+
+
+
—
+
—
+
+
+
+
—
+
+
+
+
—
—
+
+
+
His
Trp
Asn
Gin
IS
—
-
—
—
—
—
—
— Insulin + Insulin
Expt. i
IOX
IOX
Expt. 2
o
5 mM
i mM
IOX
S mM
—
—
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
—
—
+
+
+
—
—
+
+
+
—
—
+
+
+
—
—
+
+
+
—
—
+
+
+
—
—
+
—
0
48
57
60
7i
9
65
72
74
82
0
12
16
39
67
69
76
46
SO
68
Hepatocytes were incubated in collagen-treated tissue culture dishes for 3 h at 37 °C with
or without insulin (io~7) and with different combinations of amino acids, either at 10 x the
concentrations of the physiological mixture (Seglen, 19766) or at the molarity indicated.
Protein degradation was measured as the net release of [l*C]valine from radioactive protein.
The effect of the amino acids, or amino acids plus insulin, was expressed as % inhibition of
the control degradation in unsupplemented medium (4-2 %/h in expt. 1; 4-6 %/h in expt. 2).
Each value is the mean of 2 cell samples.
Inhibition of protein degradation by insulin may be independent of amino acid metabolism
It has been suggested that the stimulation of hepatocytic protein degradation by
glucagon reflects an enhanced metabolic utilization of amino acids (Schworer &
Mortimore, 1979). To assess the role of amino acid metabolism in the mechanism
of action of insulin, the aminotransferase inhibitor aminooxyacetate (Rognstad &
Clark, 1974; Seglen & Solheim, 1978 a) was used. This drug will block the initial
step in the metabolism of the majority of amino acids, and has been found to inhibit,
e.g., hepatocytic gluconeogenesis (Rognstad & Clark, 1974). However, as shown in
Table 3, aminooxyacetate had no effect on either the basal protein degradation or the
inhibition by amino acids and/or insulin. It is therefore unlikely that amino acid
metabolism is involved in the response to insulin; indeed the general lack of effect
6
A. Poli and others
Table 2. Effects of glucagon, insulin and ghicorticoid hormone on protein degradation in
the presence of amino acids and lysosome inhibitors
Inhibition of protein degradation, %
Amino acids
Expt. 1
Control
Dexamethasone, IO'M
Glucagon, io~7 M
Insulin, io~7 M
Insulin + glucagon
Expt. 2
Control
Glucagon
Insulin
Insulin + glucagon
Methylamine, Leupeptin,
Old mixture
New
None
(10 x)
mixture
10 mM
0-25 mM
0
76
10
65
63
44
82
81
82
74
73
75
73
83
2
7
82
14
77
0
56
39
75
0
8
4
.
64
65
68
80
72
70
7i
Isolated hepatocytes were incubated in collagen-treated tissue culture dishes for 3 h at 37 °C
with hormones and inhibitors as indicated. Two types of amino acid mixture were used: the
'old' mixture, 10 x concentrated (Seglen, 19766) or a new mixture, containing additionally
elevated levels of leucine, 2-5 mM; asparagine, 5 mM; glutamine, 5 mM; phenylalanine, 2 mM;
tyrosine, 2 HIM; and histidine, 1 mM. The control rate of protein degradation in unsupplemented medium, measured as the release of [14C]valine from radioactive protein, was 3-8 %/h
in expt. 1 and 3'7%/h in expt. 2. The total effect of the various additions was expressed as
% inhibition of the control degradation. Each value is the mean of 2 cell samples.
Table 3. Effect of the aminotransferase inhibitor, aminooxyacetate, on the inhibition
of protein degradation by insulin
Inhibition of protein degradatior 1,
— Aminooxyacetate
%
+ Aminooxyacetate
— Insulin
+ Insulin
— Insulin
+ Insulin
None
0
7
0
Gly
Pro
Gin
Leu
0
9
3
7
7
0
13
13
0
10
0
28
32
81
7
15
26
29
Amino acids
0
3
Pro + Met + Phe + Trp
14
67
67
78
Complete amino acid mixture (10 x )
Isolated hepatocytes were incubated in collagen-treated tissue culture dishes for 3 h at 37 °C
with various combinations of insulin, I O " ' M ; aminooxyacetate, 5 mM; and amino acids,
individually or in combination, at 10 x the concentrations of the mixture previously given
(Seglen, 19766). Protein degradation was measured as the release of [14C]valine from radioactive protein, and the effect of amino acids and insulin expressed as percentage inhibition
of the control degradation in the absence (4-6 %/h) or presence (49 %/h) of aminooxyacetate.
The effect of the latter alone was regarded as non-significant. Each value is the mean of 2 cell
samples.
Protein metabolism in hepatocytes
1
2
Incubation time, h
3
4
Fig. 3. Time-course of inhibition of protein degradation by insulin and cycloheximide.
Isolated hepatocytes were incubated at 37 °C in collagen-treated tissue culture dishes
for the length of time indicated, and protein degradation measured as the release of
[14C]valine from radioactive protein. D, No additions; O, amino acids (5 x normal);
Ai amino acids + insulin, io~7 M; 0 , amino acids + cycloheximide, io~4 M; A, amino
acids + insulin + cycloheximide; • , propylamine, 10 mM. Each value is the mean
of 3 cell samples.
Table 4. Effect of the protein synthesis inhibitor, cycloheximide, on protein degradation
in the presence and absence of insulin
% Inhibition of protein degradation by
Expt.
no.
Amino
acids
Insulin
Cycloheximide
Insulin +
cycloheximide
Significance
1 + Cw.C
P < o-ooi
27 ± 2 (5)
46 ±2 (5)
P < o-ooi
46 ±2 (5)
54 ±2 (5)
29 ±3 (S)
39 ±3 (5)
o-01 < P < O'O2
SX
3
N.S.
44 ±3 (3)
43 ± 3 (3)
4
5*
Isolated hepatocytes were incubated in collagen-treated tissue culture dishes for 3 h at 37 CC
witlvinsulin (io~7 M), cycloheximide (io~s M) and intermediate concentrations of amino acids
(5 x normal, cf. Seglen, 19766) as indicated. Protein degradation was measured as the release
of ["CJvaline from radioactive protein. The control degradation rates without hormone and
inhibitor were 46 and 3-8 %/h in the absence of amino acids (expts. 1 and 2, respectively), and
2-8 and 33 %/h in the presence of amino acids (expts. 3 and 4, respectively). The effects of
insulin and cyclohexamide were expressed as % inhibition of the respective controls. Each value
is the mean ± S.E. of the no. of cell samples given in parentheses. The significance of the insulin
(I) effect in the presence of cycloheximide (C) has been calculated by the use of Student's t-test.
N.S. means not significant.
1
0
2
0
13 ±2 (5)
13 ±4 (5)
30 ±2 (5)
28 ± s (3)
8
A. Poli and others
of aminooxyacetate may suggest that amino acid metabolism is usually not a ratelimiting factor for hepatocytic protein degradation.
The role of protein synthesis: effect of cycloheximide
Cycloheximide, a strong inhibitor of hepatocytic protein synthesis (Seglen, 1977),
was previously found not to affect protein degradation in i-h experiments with
isolated hepatocytes (Seglen et al. 1979); however, in long-term experiments with
perfused livers (Woodside, 1976; Khairallah & Mortimore, 1976) or hepatocyte
monolayers (Hopgood et al. 1980) an inhibitory effect was observed. As shown in
Fig- 3, cycloheximide inhibited protein degradation significantly, but only after
a 60-min lag. At the concentration used here (io~3 M), cycloheximide inhibited protein
synthesis without a lag, and essentially completely (more than 95 %, as tested in
separate experiments). Insulin had an effect on protein degradation very similar to
that of cycloheximide; furthermore, an additional inhibition by insulin was evident
even in the presence of cycloheximide. The 2 compounds together inhibited protein
degradation almost as strongly as did the lysosomotropic inhibitor propylamine,
suggesting a virtually complete suppression of the lysosomal pathway (Seglen et al.
1979)The magnitude of the cycloheximide effect varied considerably from experiment
to experiment; unlike the insulin effect it was prominent both in the presence of
moderate amounts of amino acids and in an amino acid-free medium (Table 4). In
most experiments an additional effect of insulin was observed; however, the latter
tended to disappear when the total inhibition became large. The inhibition of protein
degradation by insulin thus does not appear to require protein synthesis, but the
hormone and the synthesis inhibitor seem to affect degradation by related mechanisms.
The cycloheximide effect, like the insulin effect, is not seen in the presence of lysosomotropic amines or at a very high amino acid concentration (A. Kovacs & P. O.
Seglen, unpublished experiments), suggesting an amino acid-mediated inhibition of
the lysosomal degradation pathway. The common denominator for insulin and
cycloheximide could therefore very well be suppression of cellular autophagy by
amino acids, as suggested by Hopgood et al. (1980).
Effect of insulin and cycloheximide on amino acid transport
It has been shown that the addition of insulin as well as glucagon to hepatocyte
suspensions or cultures selectively stimulates amino acid uptake by the 'A' system,
as exemplified by the use of the non-metabolizable model substrate a-aminoisobutyrate (AIB) (Kletzien et al. 1976; Pariza et al. 1976; Fehlmann, Le Cam &
Freychet, 1979). The stimulation of AIB uptake by insulin is demonstrated in Fig. 4A,
while Fig. 4B shows that the hormone had no effect on the uptake of valine, which
is transported by the ' L' system.
AIB uptake is inhibited by a mixture of amino acids whether these are present
outside (cis inhibition) or inside (trans inhibition) the cells (Kelley & Potter, 1978),
as shown in Table 5. It is therefore conceivable that a reduced formation of amino
Protein metabolism in hepatocytes
120
180
240
Incubation time, min
10
15
Fig. 4. Effect of insulin on amino acid transport. Isolated hepatocytes were preincubated in collagen-treated tissue culture dishes for 3 h at 37 °C in the presence
( • ) or absence ( O) of insulin (io~7 M), then re-incubated under the same conditions
for the length of time indicated. The latter period was used for the measurement of:
A, transport of a-aminoisobutyrate (AIB), measured as the continuous uptake of
[14C]AIB (1 mM; i2SnCi/ml) into the cells; andB, transport of valine, measured as the
uptake of ["C]valine (1 mM; 250 nCi/ml). Each value is the mean of 2 cell samples.
Table 5. cis and trans inhibition of a-atninoisobutyrate (AIB) uptake by amino acids
AIB uptake (relative)
Preincubation ][
i
h
— Amino acids
+ Amino acids (iox)
— Amino acids + Amino acids( 1 0 x )
IOO± 2
52 ± I
50 ±3
After 3 h in culture (collagen-treated dishes at 37 °C) hepatocytes were preincubated for
1 h in the absence or presence of amino acids (10 x normal); then washed 3 x at o°C and
re-incubated 1 h with or without amino acids. [UC]AIB (1 mM; nCi/ml) was added during the
last hour, and its uptake measured as the accumulation of acid-soluble radioactivity in washed
(3 x at o °C) cells. Uptake rates are expressed as % of the control (preincubated and incubated
without amino acids), which was 928 cpm/sample (106 mg cells). Each value is the mean ± S.E.
of s cell samples.
70 ±2
acids (e.g. by suppression of proteolysis) might play a part in the stimulation of AIB
transport by insulin, but this possibility has not been further examined.
Cycloheximide has been reported to inhibit the effect of insulin on hepatocytic
AIB uptake (Kletzien et al. 1976; Fehlmann et al. 1979). As shown in Table 6, we
found the effect of cycloheximide to be rather complex. The inhibitor alone stimulated
AIB uptake in all the experiments. The stimulation by insulin was unaffected or
slightly reduced; however, because of the stimulation by cycloheximide itself, the
relative effect of insulin was strongly, but not completely, suppressed. The insulin
io
A. Poli and others
effect would therefore seem not to require protein synthesis per se, but interference
with protein synthesis apparently affects AIB transport so strongly as to partially
obscure the hormone effect. The possibility should be considered that cycloheximide
and insulin may have related mechanisms of action, as was suggested for their effect
on protein degradation.
Table 6. Stimulation of a-aminoisobutyrate (AIB) uptake by insulin and cycloheximide
AIB uptake (relative)
Incubation
time,
h
Expt. i
5
Expt. 2
2
7
Expt. 3
2
7
— Cycloheximide
+ Cycloheximide
— Insulin
+ Insulin
— Insulin
+ Insulin
Significance
I + Ctt.C
ioo±4
334 ± H
235 ±9
382 ±11
P < o-ooi
ioo±4
IOI ± 2
162 ± 3
234 ±13
I38±3
120 ±3
I39±5
165 ± 4
N.S.
p < 0001
ioo±s
8S±3
165 ± 1
207 ± 1
142 ±6
i38±5
176 ±2
I7i±3
p < 0005
p < 0-005
Isolated hepatocytes were incubated in collagen-treated (expts. 1 and 2) or albumin-treated
(expt. 3) tissue culture dishes at 37 °C for the length of time indicated (up to 7 h), with or
without insulin (io~7 M) and cycloheximide (io~* M). [ M C ] A I B (I ITIM; 63 nCi/ml) was added
during the last hour, and its uptake measured as the accumulation of acid-soluble radioactivity
in the cells. The uptake rates are expressed as % of the values in insulin- and cycloheximide-free
controls at the earliest time points measured (this control uptake averaged 1688 cpm per
10-mg sample, and did not change significantly with incubation time). Each value is the
mean ± s.E. of 4-5 cell samples. The significance of the insulin effect in the presence of
cycloheximide has been calculated using Student's t-test.
The effects of insulin and cycloheximide both appeared to be independent of
cell-to-substratum anchorage, since qualitatively similar results were obtained with
cells attached and spread on collagen (expts. 1 and 2 of Table 6) and with nonattached cells on an albumin substratum (expt. 3 of Table 6). To investigate further
to what extent the action of insulin might be anchorage-independent, the influence
of several different substrata on protein degradation and amino acid transport was
studied.
Anchorage-independence of insulin effects on protein degradation and amino acid transport
Hepatocytes in short-term suspension have been found to respond poorly to
insulin (Seglen, 1977; Hopgood et al. 1979), whereas monolayers or long-term
suspensions of cellular aggregates respond well (Jeejeebhoy et al. 1975; Crane &
Miller, 1977; Tanaka, Kishi & Ishihara, 1979; Hopgood et al. 1980). This could
have a trivial cause, such as proteolytic destruction of insulin and/or its receptors
by proteases released from damaged cells in the vigorously shaken short-term
suspensions (cf. the demonstration of such a phenomenon in fat cell suspensions;
Protein metabolism in hepatocytes
II
Fig. 5. Morphology of hepatocytes on different substrata. Isolated hepatocytes were
incubated for 4 h at 37 °C in tissue culture dishes pretreated with: A, bovine serum
albumin (cells not attached); B, polylysine (cells attached, but not spread); c, foetal
calf serum; or D, calf skin collagen, x 360.
Gliemann & Sonne, 1978). However, it would also seem possible that insulin effects
might be dependent on anchorage of the cells, either to each other (in aggregates) or
to a substratum. Indications for the anchorage-dependence of amino acid transport
as well as of protein degradation have been found in other cell types (Otsuka &
Moskowitz, 1975, 1978; Pofit & Strauss, 1977) and the reported time-dependent
increase in the amino acid uptake capacity of freshly seeded hepatocyte cultures
(Kletzien et al. 1976) might be accordant with anchorage control. It is known that
internalization and degradation of insulin-receptor complexes (down-regulation of
receptors) takes place in isolated hepatocytes (Le Cam, Maxfield, Willingham &
Pastan, 1979; Carpentier et al. 1979), and the anchorage of receptors to a substratum
could be a mechanism by which to prevent such a decrease in hormone sensitivity.
By using protein substrata with different abilities to support hepatocyte attachment
and spreading (Gjessing & Seglen, 1980), the short-term effect of anchorage on
various hepatocytic properties can be investigated. We have used adsorbed monomolecular layers of bovine serum albumin (Fig. 5 A), to which hepatocytes do not
attach; gelatin, asialofetuin and polylysine (Fig. 5 B), which support attachment, but
A. Poli and others
12
•a
o
4 -
2 -
Incubation time, h
Fig. 6. Anchorage-independent inhibition of protein degradation by insulin. Isolated
hepatocytes were incubated at 37 °C with amino acids (5 x normal), in the absence
(open symbols) or presence (filled symbols) of insulin, io~7 M. The tissue culture
dishes were pretreated with albumin (O, • ) , polylysine (A, A) or collagen (D, • )
to provide non-attached, attached but non-spread, and well-spread cells, respectively.
Protein degradation was measured as the release of [14C]valine from radioactive
protein. Each value is the mean of 2 cell samples.
not cell spreading; fibronectin in the form of foetal calf serum (Fig. 5c), and collagen
(Fig. 5 D), both of which support attachment as well a9 spreading.
As shown in Fig. 6, protein degradation proceeded at the same rate in hepatocytes
cultured on either albumin, polylysine or collagen, and insulin stimulated degradation
to the same extent on all 3 substrata. Hepatocytes therefore do not appear to have
anchorage-dependent control of protein degradation, at least not on a short-term
basis.
Similarly, the uptake of AIB was stimulated 2- to 3-fold by insulin on all substrata
tested (Table 7), i.e. independently of anchorage. To see if quantitative differences
could be detected, non-attached (on albumin) and well-spread cells (on collagen)
were compared in a series of experiments. The basal AIB uptake (i.e. in the absence
of insulin) was found to be similar on the 2 substrata, and insulin, continuously
present for 5 h, stimulated uptake to the same extent in both cases (Table 7).
Hormonal stimulation of AIB uptake is a reversible process, and upon removal
of the hormone the uptake rate gradually returns to the basal value (ParLza et al. 1976).
To see if anchorage to the substratum might retard intemalization and degradation
of the insulin-receptor complex, and thus maintain the hormone effect for a longer
period, a 2-h pulse of insulin was given, followed by a 3-h chase in hormone-free
Protein metabolism in hepatocytes
13
medium. As shown in Table 7, some effect of insulin persisted after this regimen,
but no significant difference between non-attached (on albumin) and well-spread
cells (on collagen) could be detected. Our studies on AIB uptake therefore fail to
provide any evidence for anchorage-dependent short-term control of amino acid
transport in hepatocytes.
Table 7. Influence of cell-substratum anchorage on the stimulation of a-aminoisobutyrate
{AIB) uptake by insulin
AIB uptake (relative)
Type of anchorage
Substratum
Albumin
Polylysine
Serum
Collagen
Attached
+
Spread
;
No
hormone
Insulin
continuously
Insulin
pulse
IOO
IOO
IOO
IOO
242 ± 29 (6)
298
260
257 ± 24 (6)
135 ±19 (5)
154 ±12 (5)
Isolated hepatocytes were incubated for 5 h on various protein substrata, i.e. in tissue
culture dishes prerreated with the protein indicated. Insulin (io~7 M) was either absent,
present throughout the incubation, or given as a pulse during the first 2 h and removed for
the remainder of the incubation. The uptake of [14C]AIB (1 HIM; 63 nCi/ml) was measured
during the last 2 h of incubation. The basal rate of uptake on each substratum was defined
as 100% (no significant difference between the various substrata could be found), and the
uptake in the presence of insulin expressed as % relative to the hormone-free controls. The
values for polylysine and serum are taken from a single experiment (each value being the
mean of 3 cell samples), whereas the values for albumin and collagen are the means of 5-6
different experiments with 3-5 parallel samples in each experiment (s.E. determined on the
basis of the experimental means only).
Anchorage-independence of protein synthesis
Protein synthesis has been reported to be an anchorage-dependent process in
certain cultured cells (Otsuka & Moskowitz, 1978; Benecke et al. 1978). We therefore
measured the rates of protein synthesis after incubation of hepatocytes on different
substrata for various lengths of time. The synthesis rates were constant during the
first 8 h in culture, but fell during the next 8 h in the relatively simple, sub-optimal
medium used. No significant rate differences could be observed between substrata
which gave no attachment (albumin), attachment without spreading (asialofetuin and
gelatin) or both attachment and spreading (fibronectin and collagen) (Table 8). Thus,
as in the case of protein degradation and amino acid transport, our experiments provide
no evidence for any short-term anchorage-dependence of hepatocytic protein synthesis.
DISCUSSION
Evidence from several sources suggests that the primary control of hepatocytic
autophagy may be exerted by amino acids (Woodside & Mortimore, 1972; Mortimore
& Schworer, 1977; Schworer & Mortimore, 1979; Hopgood et al. 1979, 1980; Seglen
et al. 1980a). Only a limited number of amino acids are involved in such control, and in
14
A. Poli and others
our previous work with isolated hepatocytes we found the 7 amino acids leucine,
phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine to be particularly effective (Seglen et al. 1980a). Their effects were to a large extent additive, suggesting a complex mechanism of regulation, although certain simple combinations
such as asparagine plus leucine could apparently elicit a nearly complete response.
Intracellular amino acid levels can be altered by interference with protein synthesis,
protein degradation, amino acid metabolism or amino acid transport, and it is very
likely that several of these processes are involved in the secondary control of
autophagy, e.g. by hormones. Glucagon, which stimulates hepatic autophagy, appears
to do so by depressing the intracellular level of glutamine (Schworer & Mortimore,
1979) one of the most active degradation-inhibitory amino acids (Seglen et al. 1980a).
Table 8. Protein synthesis in hepatocytes cultured on different substrata
Rate of protein synthesis, % / h
Non-attached
Attached, non-spread
Culture time,
h
Albumin
Asialofetuin
Gelatin
2
070
4
8
O7S
073
043
0-69
063
078
066
073
047
061
16
o-8o
Attached, spread
Fibronectin
0-69
072
070
047
Collagen
063
076
070
058
Hepatocytes were cultured for up to 16 h, in medium supplemented with amino acids
(7 x normal concentration, cf. Seglen, 1976ft) on substrata of various proteins adsorbed to
polystyrene tissue-culture dishes. At the times indicated, the total protein content as well as
the incorporation of ["C]valine (10 mM; 50 mCi/mol) into protein during 1 h was measured,
and the rate of protein synthesis calculated and expressed as %/h. Each value is the mean
of 3 dishes.
Although the exact biochemical mechanism remains a conjecture, glucagon is known
both to stimulate gluconeogenesis, for which glutamine is an effective substrate,
and to activate hepatic glutaminase (Joseph & McGivan, 1978). The stimulatory
effect of glucagon on amino acid uptake (Pariza et al. 1976; Fehlmann et al. 1979) is
apparently insufficient to counter its effect on glutamine metabolism.
Insulin inhibits hepatic autophagy (Pfeifer, 1978) and the amino acid dependence
of this inhibition would seem to indicate that, like the glucagon effect, it might be
mediated by some of the processes which influence intracellular amino acid concentrations. However, neither insulin nor glucagon have any effects on over-all
protein synthesis during the time intervals used in these experiments (Seglen et al.
19806), and the lack of an effect of the aminotransferase inhibitor aminooxyacetate
suggests that at least the metabolic pathways starting with a transamination are not
involved in the action of insulin. The stimulation of amino acid transport by insulin
has been shown to be due to an increase in the amounts of 2 different transport
proteins with A-system characteristics (Fehlmann et al. 1979); however, this induction
is protein synthesis-dependent and therefore completely blocked by cycloheximide.
Protein metabolism in hepatocytes
15
Since at least part of the inhibition of protein degradation by insulin appears to be
cycloheximide-resistant, stimulation of amino acid uptake by known mechanisms
cannot fully account for the effect of the hormone.
Interpretation of insulin effects in the presence of cycloheximide is complicated
by the fact that cycloheximide itself both inhibits protein degradation and stimulates
amino acid uptake under the experimental conditions used. The effect on degradation
disappears at high amino acid concentrations, and could therefore conceivably be
mediated by amino acids. Inhibition of protein synthesis by cycloheximide might be
expected to result in some elevation of intracellular amino acid levels; however, under
the present conditions this effect would be very small because the synthesis rate is
only about one-tenth of the degradation rate (Seglen et al. 1979). In the perfused liver,
cycloheximide was found not to elevate intracellular amino acid concentrations under
conditions where it inhibited protein degradation (Woodside, 1976). The fact that
inhibition of degradation by cycloheximide is seen even in the absence of amino acids
further suggests that it is not secondary to the stimulation of amino acid uptake by
the drug. The inverse relationship would seem more likely: other investigators have
found that cycloheximide either inhibits or has no effect on AIB transport (Kletzien
et al. 1976; Pariza et al. 1976; Fehlmann et al. 1979); the paradoxical stimulation
seen in our experiments may therefore be related to the particularly high rate of
protein degradation. The amino acid efflux thus created may exert a significant
/rani-inhibitory effect on AIB uptake, which can be partially relieved when cycloheximide reduces the degradation rate. A similar mechanism may conceivably
contribute to the effect of insulin on AIB uptake.
While the mechanisms of insulin's effects on protein degradation and AIB transport
remain uncertain, our results indicate clearly that neither of these is dependent on
cellular anchorage. Some influence of anchorage on the effects of insulin might have
been expected on morphological grounds: the patchy alignment along active stress
fibres of both the coated pits involved in insulin receptor internalization (Anderson
et al. 1978; Goldstein, Anderson & Brown, 1979) and the fibronexuses involved in
cellular attachment (Hynes & Destree, 1978; Singer, 1979) suggest that the
2 organelles may be identical or related. On the other hand, since transmission of the
hormone signal does not require receptor internalization (Le Cam et al. 1979),
interference with the latter process would be expected to have only moderate long-term
effects (reduced 'down-regulation' of receptors). The absence of such effects of
anchorage in the present experiments may indicate either that anchorage does not
significantly retard receptor internalization, or that the effect is not of sufficient
magnitude to affect the hormone response.
As for the anchorage-independence of basal protein synthesis, protein degradation
and amino acid transport, our experiments can only exclude the operation- of a shortterm regulatory mechanism. The anchorage-dependence demonstrated in other cell
types (Otsuka & Moskowitz, 1975, 1978; Pofit & Strauss, 1977; Benecke et al. 1978)
may become evident only after long-term culture and may possibly be related to
cellular growth. Examination of the latter possibility would require the establishment of
proliferating hepatocyte cultures, a goal yet waiting to be achieved (Seglen et al. 19806).
16
A. Poli and others
This work was supported by grants from the Norwegian Cancer Society and the Norwegian
Council for Science and the Humanities, and by a fellowship to A. Poli from the North
Atlantic Treaty Organization (NATO).
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[Received 14 August 1980)