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Acute hormonal control of acetyl-CoA


carboxylase. The roles of insulin, glucagon,
and epinephrine.
G M Mabrouk, I M Helmy, K G Thampy and
S J Wakil
J. Biol. Chem. 1990, 265:6330-6338.

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THE JOURNAL OF BIOLWXCAL CHEMISTRY


0 1990 by The American Society for Biochemistry

Acute Hormonal
THE

ROLES

Vol. 265, No. 11, Issue of April 15, pp. 633%6338,199O


Printed in Il. S. A.

and Molecular Biology, Inc.

Control of Acetyl-CoA

OF INSULIN,

GLUCAGON,

AND

Carboxylase

EPINEPHRINE*
(Received

Gamal
From

M. MabroukS,
the Verna

and Marrs

Ihab
McLean

M. Helmy$,
Department

K. George
of Biochemistry,

* This work was supported


in part by Grant
DK-35419
from the
National
Institutes
of Health
and by a grant
from The Clayton
Foundation.
The costs of publication
of this article were defrayed
in
part by the payment
of page charges.
This article
must therefore
be
hereby marked
aduertisement
in accordance
with 18 USC.
Section
1734 solely to indicate
this fact.
$ Present
address:
Dept. of Biochemistry,
Ain Shams University,
Cairo, Egypt.
Present
address:
Dept.
of Biochemistry,
Indiana
University
School of Medicine,
Fort Wayne
Center for Medical
Education,
2101
Coliseum
Blvd., East, Fort Wayne,
IN 46805.
11To whom correspondence
should be addressed:
Verna and Marrs
McLean
Dept.
of Biochemistry,
Baylor
College
of Medicine,
One
Baylor
Plaza, Houston,
TX 77030. Tel.: 713-798-4528.

Baylor

and Salih
College

October

13,1989)

J. Wakiln

of Medicine,

Houston,

Texas

77030

vated the enzyme (specific activity


-8 unitslmg
protein
in the absence of citrate)
and polymerized
the protein
(A!& - 10 x 106).
These observations
indicate
that insulin
and glucagon, by altering
the phosphorylation
state of the acetylCoA carboxylase,
play antagonistic
roles in the acute
control
of its activity
and therefore
in the regulation
of fatty acid synthesis.

Acetyl-CoA
carboxylase
plays an important
role in the
metabolism of fatty acids. It catalyzes the committed step in
fatty acid synthesis, producing malonyl-CoA,
the donor of the
CZ units for the synthesis of long-chain
fatty acids (l-3).
Malonyl-CoA
is also the allosteric regulator of the carnitine
palmitoyltransferase
shuttle system responsible
for transporting fatty acyl groups through the mitochondrial
membrane and making them available for oxidation
by the /3oxidation enzymes (4). Hence, investigations
of the structurefunction of the carboxylase and its regulation
become essential to our understanding
of the process of fatty acid metabolism, especially since the latter plays a pivotal role in the
energy metabolism
of vertebrates. The carboxylase is under
both long-term control, involving changes in its mRNA levels
and in the rate of protein synthesis and degradation
(5-7),
and short-term
control, involving
allosteric modification
by
citrate and palmitoyl-CoA
and covalent modification
by phosphorylation.
The physiological
significance
of these modifications in regulating
the activity of acetyl-CoA
carboxylase
has been unclear and controversial
(8-12). Citrate is a required activator of animal acetyl-CoA carboxylase with halfmaximal activation
(I&,) observed at concentrations
of 2-10
mM (13-17), which are significantly
higher than intracellular
concentrations
of 0.17-0.45
mM
(18) and may not be an
indicator of the rate of fatty acid synthesis (19).
Phosphorylation/dephosphorylation
of the carboxylase has
presented another problem. The isolated enzyme is a phosphoprotein
containing
6-15 mol of Pi/m01 of subunit (20,21);
little is known about the kinase(s) and phosphatase(s)
that
are specific for acetyl-CoA
carboxylase. The role of the carboxylase-specific
kinase(s) and phosphatase(s)
in regulating
enzyme activity remains ambiguous, with several reports indicating a direct relationship
between phosphorylation
and
catalytic activity (8, 9, 22-30), while others could not demonstrate such an effect (10-12).
Diet,
especially
a fat-free
diet, induces the synthesis of
acetyl-CoA carboxylase and increases its activity. Starvation
or diabetes represses the expression of the carboxylase gene
and decreases the activity of the enzyme. Although
not much
is known about the regulation
of the carboxylase
gene, our
recent studies involving
dietary manipulations
in rats have
suggested an interrelationship
between catalytic activity, citrate requirement,
phosphorylation
state, and polymerization

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

Acetyl-CoA
carboxylase,
purified
from
rapidly
freeze-clamped
livers of rats maintained
on a normal
laboratory
diet and given O-5 units of insulin
shortly
before death, gives a major protein
band (Mr 265,000)
on sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis.
The carboxylase
from untreated
rats has relatively
low activity
(0.8 unit/mg
protein
when assayed
in the absence of citrate)
and high phosphate
content
(8.5 mol of Pi/m01 of subunit),
while the enzyme from
livers of rats that received
5 units of insulin
has higher
activity
(2.0 units/mg
protein)
and lower
phosphate
content
(7.0 mol of Pi/m01 of subunit).
Addition
of
citrate
activates
both preparations
with half-maximal
activation
(&.s)
at 1.0 and 0.6 mM citrate,
respectively.
The enzyme
from rats that did not receive
insulin is mainly
in the octameric
state (M= - 2 x 106),
while that from rats that received
insulin
is mainly
in
the polymeric
state (iUr - 10 x 10). Thus, short-term
administration
of insulin
results in activation
of acetylCoA carboxylase,
lowering
of its citrate
requirement,
and dephosphorylation
and polymerization
of the protein. The insulin-induced
changes
in the carboxylase
are probably
due to dephosphorylation
of the protein
since similar
changes
are observed
when the enzyme
from rats that did not receive insulin
is dephosphorylated by the Mn2+-dependent
[acetyl-CoA
carboxylase]
-phosphatase
2.
The effect of glucagon
or epinephrine
administration
on acetyl-CoA
carboxylase
was also investigated.
The
carboxylase
from fasted/refed
rats has a relatively
high specific
activity
(3.4 units/mg
protein
in the absence of citrate),
lower phosphate
content
(4.9 mol of
Pi/m01 of subunit),
and is present
mainly
in the polymeric state (M= - 10 x 106). Addition
of citrate
activates the enzyme with &.s = 0.2 mM citrate.
Glucagon
or epinephrine
injection
of fasted/refed
rats yielded
carboxylase
with lower
specific
activity
(1.4 or 1.9
units/mg,
respectively,
in the absence
of citrate),
higher
phosphate
content
(6.4 or 6.7 mol of Pi/m01 of
subunit,
respectively),
and mainly
in the octameric
state (Mr - 2 x 106). Treatment
of these preparations
with [acetyl-CoA
carboxylasel-phosphatase
2 reacti-

Thampyg,

for publication,

Hormonal

Control of Acetyl-CoA Carboxylase

dependent.

Moreover,

dephosphorylation

of the carboxylase

by this phosphatase results in its polymerization to the polymeric state of molecular weight 10 X lo6 (31). The present
studies were designed after our earlier investigations of the
effect of fasting and refeeding on the activity, phosphate
content, and aggregation state of the acetyl-CoA carboxylase
(31, 46). In this report we present our results on the changes
in these properties of the enzyme taking place in uiuo after
short-term administration of insulin, glucagon, or epinephrine
to rats. Our results show that insulin causes activation of the
hepatic acetyl-CoA carboxylase with a lower citrate requirement and dephosphorylation and polymerization of the protein. Glucagon or epinephrine administration, on the other
hand, lowers carboxylase activity, increases the phosphate
content, and depolymerizes the protein.
EXPERIMENTAL

PROCEDURES

Materials-HumulinTM
R and glucagon
were gifts from
Lilly.
NovolinTM
R was purchased
from Squibb Novo, Inc.. and eDineDhrine
(AdrenalinTM
chldride
solution)
fro& Parke-D&is.
?he so;rces
of all
other materials
were described
previously
(46). Sprague-Dawley
rats
(retired
female breeders)
weighing
200-300
g each were purchased
from Harlan
Sprague-Dawley,
Inc.
Animals,
Hormone
Administration,
and Blood Level Analvsis-Rats
were divided
into two groups with 14 animals
per group. One group
was injected
with the indicated
hormone
and the other with an equal
volume of saline as a control.
The first two groups were fed a normal
laboratory
diet and injected
intraperitoneally
with either insulin
or
saline. To study the proposed
activation
of acetyl-CoA
carboxylase

by insulin,
the logical choice would have been fasted animals
where
carboxylase
is known
to be relatively
inactive.
However,
due to low
levels of blood glucose in the fasted animals,
insulin
administration
was not possible
because
of accompanying
insulin
shock.
For this
reason, animals
on a normal
laboratory
diet were chosen.
To study glucagon-induced
or epinephrine-induced
inactivation
of
carboxylase,
if any, fasted and refed rats were chosen mainly because
the carboxylase
in such rats is known
to be highly active (31). Thus,
another
two groups of rats were fasted for 2 days and refed a highcarbohydrate,
low-fat
diet for 2 days. After a 2-h deprivation
of food,
these animals
were injected
intraperitoneally
with either glucagon
or
saline. This short period
of deprivation
of food was introduced
to
guard against high levels of blood glucose that would cause the release
of endogenous
insulin
which
in turn would
have antagonized
the
glucagon
action. Similar
groups of rats were maintained
on the same
regimen and used for the epinephrine
experiment.
About 10 min after hormone
administration
blood was drawn from
the hearts
of two rats from each of the groups.
The blood glucose
level was determined
using a Sigma diagnostic
kit (Trinder).
The
insulin
and glucagon
levels in the blood were determined
by radioimmunoassay
(47). The remaining
12 rats from each group
were
anesthetized
with pentobarbital
(60 mg/kg;
NembutalTM
sodium
solution,
Abbott)
and the livers
were excised
and freeze-clamped
as
described
previously
(46). The frozen
livers were pulverized
in liquid
nitrogen
and stored at -70 C! until used.
Preparation
of Carboxylase
and Superose
6 Gel Permeation-Acetyl-CoA
carboxylase
was purified
from frozen
liver powder
as described
by Thampy
and Wakil
(46). The polymeric
and octameric
forms of the enzyme were separated
by using a Superose
6 HR column
(1.0 X 30 cm) from Pharmacia
LKB Biotechnology
Inc. operated
with
an LKB GTi high-pressure
liquid chromatography
system.
The column wasequilibrated at room temperature with 20 mM Tris-HCl, pH
7.2, containing 1 mM EDTA and 100 mM NaCl. About 300-400 pg of
acetyl-CoA
carboxylase
was applied onto the column
in a volume
of
200 11 and the proteins
were eluted with the same buffer at a rate of
0.5 ml/min
(46).
Dephosphorylation
and Polymerization
of Acetyl-CoA
Carboxyluse
by [Acety-CoA
Carboxylusel-phosphntase
P-Acetyl-CoA
carboxylase

was incubated with purified phosphatase for 20 min at 37 C! at a

ratio of 2:l (w/w)


in a reaction
mixture
similar
to that described
earlier
(31, 46). The reaction
was stopped
by cooling
on ice and an
aliquot of 200 ~1 was applied
immediately
onto a Superose
6 column
and the enzyme
was eiuied as described
above. Another
aliquot
was
assayed
simultaneously
for acetyl-CoA
carboxvlase
activity.
The remaining
mixture
cant-tining
lob pg of acetyl-CoA
carboxylase
was
dialyzed
against 2 liters of 100 mM Tris-HCl,
pH 7.0, containing
10%
glycerol
and 1 mM EDTA
to remove
the inorganic
phosphate
generated by the phosphatase
action. The dialyzed
enzyme was then treated
with 1.0 N NaOH
and the liberated
phosphate
was determined
as
described
below.
Protein
Determination-The
bicinchoninic
acid method
(modified
Lowry)
(56) was used according
to the manufacturers
(Pierce
Chemical Co.) recommendations
to determine
the protein
concentration
of
the purified
acetyl-CoA
carboxylase.
Bovine
serum albumin
was used
as a standard.
Acetyl-CoA
Carboxyluse
Assay-The
carboxylase
was assayed
by
measuring
the incorporation
of [Clbicarbonate
into malonyl-CoA
(13). Each assay tube contained
0.05-0.2
pg of affinity-purified
enzyme in a final volume of 0.15 ml. One unit of activity
is defined as 1
pmol of malonyl-CoA
formed
per min at 37 C. The specific
activity
is defined as units/mg
of protein.
Phosphate
Determination-Affinity-purified
carboxylase
(20-40
pg) was hydrolyzed
in 1.0 N NaOH
(final volume
0.1 ml) at 80 C for
4 h. The hydrolysate
was cooled to room temperature,
acidified
with
2 eq of sulfuric
acid, and the phosphate
was determined
colorimetritally by the addition
of 1.0 ml of reagent
as described
earlier
(48).
The intensity
of color was measured
at 660 nm using
inorganic
phosphate
as a standard.
[Acetyl-CoA
Carboxykwel-phosphatase
2 Assay-The
phosphatase
was assayed by measuring
the rate of activation
of affinity-purified
acetyl-CoA
carboxylase
as described
earlier
(13, 46), except that the
Mn2+ concentration
was increased
to 3.0 mM.
Purification
of [Acetyl-CoA
Carboxylasel-phosphatase
2-The
supernatant
fluid (750-800
ml) obtained
from the second (5.5%) polyethylene
glycol precipitation
step during the purification
of the a,$CoA carboxylase
(46) from insulin-treated
rats was diluted
I-fold
with distilled
water
and applied
dropwise
onto a DEAE-Bio-Gel
column
(300 ml in a column
(9.0-cm
diameter
X 5.0-cm
height))

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

of acetyl-CoA carboxylase (31). Many studies aimed at understanding the effect of hormones (insulin, glucagon, epinephrine, and the like) on acetyl-CoA carboxylase have
yielded confusing and contradicting results. Although insulin
is known to stimulate fatty acid synthesis while glucagon
antagonizes its effects, both hormones have been reported to
induce phosphorylation of acetyl-CoA carboxylase in disrupted tissues or cells (32-37). While there is a general consensus about glucagon-induced phosphorylation and inactivation of carboxylase (35,37,38), there is much lessagreement
on the effects of insulin, even among reports from the same
laboratory. For instance, Witters et al. (39) have suggested
that insulin induces dephosphorylation of acetyl-CoA carboxylase in hepatocytes. However, later studies showed that
insulin induces phosphorylation by increasing 32Pincorporation into carboxylase of rat epididymal fat pads (32,34); and,
of late, dephosphorylation of the enzyme by decreasing 32P
labeling after exposure of Fao-Reuber hepatoma cells to insulin (40). Also, several investigators have reported insulininduced phosphorylation of carboxylase with either no accompanying activation (32,41), with activation (33,34,42-44), or
with activation and polymerization (45). In some of these
studies the insulin-induced phosphorylation survived purification of the carboxylase; the activation did not (34, 43).
These studies led Ha&es group (43) to suggest that although
insulin stimulates phosphorylation, the accompanying activation of carboxylase is unrelated and may be due to the
presence of an insulin-induced, and as yet unidentified, allosteric activator.
Dephosphorylation of acetyl-CoA carboxylase by an [acetyl-CoA carboxylasel-phosphatase isolated from rat epididyma1 fat pads resulted in its activation (8). The phosphatase
has broad substrate specificity and does not require a metal
ion. More recently, however, a Mn*+-dependent phosphatase
([acetyl-CoA carboxylase] -phosphatase 2) has been purified
from rat liver and shown to activate the carboxylase about
lo-fold (13). This phosphatase has high affinity for the carboxylase and renders it citrate-independent, when compared
to the phosphorylated form of the carboxylase that is citrate-

6331

6332

Hormonal

Control of Acetyl-CoA Carboxylase

at -70

C.
RESULTS

Intraperitoneal administration of insulin to rats resulted in


rapid hypoglycemia and a marked increase in the serum
insulin/glucagon molar ratio (Table I). Injection of glucagon,
on the other hand, had an opposite effect, resulting in hyperglycemia and a sharp decrease in the insulin/glucagon ratio.
Administration of epinephrine resulted in an increase in the
level of blood glucose as shown in Table I. Thus, having

demonstrated that the injected hormones were active in modifying blood glucose levels within the first 10 min after their
introduction, we proceeded to explore the effect of these
hormones on liver acetyl-CoA carboxylase.
Purity of Acetyl-CoA Carboxylase-The enzyme purified by
avidin-Sepharose chromatography
from rapidly freezeclamped livers of rats appeared as a major protein band
(290%) on polyacrylamide gel electrophoresis in the presence
of sodium dodecyl sulfate with no evidence of significant
proteolysis (Fig. 1). The various preparations of acetyl-CoA
carboxylase appeared indistinguishable based on their electrophoretic mobilities on such gels. The minor protein present
is usually pyruvate carboxylase (&fr 125,000), which in our
experience does not affect the assays. Using the chicken liver
fatty acid synthase (M, 262,000), myosin (Mr 215,000), and
pyruvate carboxylase (Mr 130,000) as standards, the subunit
molecular weight of the carboxylase protein band was calculated to be 260,000, which is close to the calculated molecular
weight of 265,220 (49) based on cDNA sequencing and the
predicted amino acid sequence of 2,345 amino acid residues.
Effect of Insulin Administration
on Actiuity, Phosphate Content, and Polymerization
State of Carboxylase-Acetyl-CoA

carboxylase purified from freeze-clamped livers of rats fed a


normal laboratory diet possessed an activity of 0.8 units/mg
when assayed in the absence of citrate. Addition of citrate
resulted in stimulation of this activity, with half-maximal
activation (K& observed at a concentration of 1.0 mM citrate
(Fig. 2). Although this citrate concentration is slightly higher
than physiological levels, it is significantly lower than that
reported for acetyl-CoA carboxylase prepared from unfrozen
tissues (13, 16, 17).
Injection of insulin into animals fed a normal laboratory
diet resulted in increased activity of the purified acetyl-CoA
carboxylase (Fig. 2). The increases in enzyme activity were
noted both in the absence and presence of citrate. However,
the addition of citrate further stimulated the activity of the
carboxylase, with half-maximal activation observed at about
0.6 mM citrate, which is near its physiological concentrations.
This activation was insulin dose-dependent and a maximum
2.5-fold increase in specific activity of the carboxylase was
observed after injection of 5 units of insulin per animal (Fig.
3).
Analyses of the carboxylase for alkali-labile phosphate content showed that the enzyme prepared from animals that did
not receive insulin had a relatively high phosphate content

TABLE
I
Effect of hormones
on blood glucose level and acetyl-CoA
carboxylase
activity
The blood glucose
level and the immunoreactive
insulin
and glucagon
were determined
as described
under
Experimental
Procedures.
The mean + S. E. of 10 blood samples
representing
five preparations
are shown. The
percentage
of the polymeric
form of the carboxylase
was estimated
from the areas under the peaks plotted
from
the chromatogram
of the Superose
6 column.
Animal
Diet

Carboxylase

treatment
Hormone injection
(per rat)

Blood glucase level


mg/100

Laboratory

Fasted/refed

a Molecular

Insulin
None
5 units

(0.19

mg)

Glucagon
None
1 unit (1.0 mg)
Epinephrine
None
1 w
weight 10 x 106.

Insulin/glucagon
molar ratio

ml

Activity

PolymeP

unitsfmg

148 -c 5
50 f 3

9f2
804 f 100

0.8 f 0.1
2.1 f 0.1

8
56

118 + 0.2
200 f 6

12 zk 3
0.04 f 0.01

3.4 k 0.1
1.4 + 0.04

68
24

120
204

3.4
1.9

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

equilibrated
with 20 mM Tris-HCl,
pH 7.1 (buffer
A). All steps were
carried
out at 4 C. The column
was washed with buffer A until no
280 nm-absorbing
materials
were released.
The proteins
were then
eluted from the DEAE-Bio-Gel
with buffer A containing
0.5 M NaCl.
The eluate was mixed with 50 ml of avidin-Sepharose
and left to
stand
for 1.5 h with gentle
agitation
to remove
any acetyl-CoA
carhoxylase
remaining
in the supernatant
fluid. The avidin-Sepharose
was removed
by filtration
and washed
with
100 ml of buffer
A
containing
0.5 M NaCl. The proteins
from the wash and the filtrate
were precipitated
by ammonium
sulfate
at 50% saturation.
The
precipitated
proteins
were removed
by centrifugation
and dissolved
in buffer A, dialyzed
against the same buffer for 2-3 h, and applied
onto a DEAE-Bio-Gel
column
(3 x 10 cm) equilibrated
with buffer
A. The flow-through
solution
was collected
and reapplied
onto the
column
to ensure total absorption
of the phosphatase.
The DEAEBio-Gel
was then washed
with buffer
A until no 280 nm-absorbing
materials
were released.
The column
was washed
with 100 ml of
buffer A containing
100 mM NaCl, then followed with a NaCl gradient
made up from 300 ml each of 100 mM and 250 mM NaCl in buffer A.
Fractions
of 4.0 ml were collected
and assayed for acetyl-CoA
carboxylase phosphatase
activity.
The fractions
exhibiting
phosphatase
activity
were pooled and the proteins
were precipitated
with ammonium sulfate
at 50% saturation.
The precipitate
was removed
by
centrifugation,
dissolved
in buffer
A (50-100
ml), diluted
20 times
with buffer A, and applied onto a Q-Sepharose
Fast Flow column
(3
x 10 cm) pre-equilibrated
with buffer
A. The column
was washed
with buffer
A until no more proteins
were detected
in the wash,
followed
by another
wash with 100 ml of buffer A containing
250 mM
NaCl. The phosphatase
was then eluted with a linear gradient
of 100
ml each of buffer
A containing
250 and 500 mM NaCl. Fractions
(3
ml) were collected
and assayed for the phosphatase.
Those fractions
containing
the highest
activity
were pooled and the proteins
were
precipitated
with ammonium
sulfate at 50% saturation.
The precipitated proteins
were removed
by centrifugation
and dissolved
in buffer
A containing
1 mM EDTA
and 100 mM NaCl (buffer
B) and applied
onto a Superose
12 HR lo/30 gel-filtration
column,
pre-equilibrated
with buffer B and connected
to an LKB fast-protein
liquid chromatography
system.
The proteins
were eluted with buffer
B and the
fractions
with the highest
activity
were pooled
and the proteins
precipitated
with ammonium
sulfate at 50% saturation.
The precipitate was removed
by centrifugation,
dissolved
in buffer B, and stored

Hormonal

6333

Control of Acetyl-CoA Carboxylase

.i

FIG. 1. Sodium
dodecyl
sulfatepolyacrylamide
gel electrophoresis
of acetyl-CoA
carboxylase
prepared
from
animals
after
treatment
with
insulin
or glucagon.
Lanes
1 and

4#k

2
contain enzymes (2 rg) purified from
livers of rats fed a laboratory diet and
injected with saline or insulin, respectively; lanes 3 and 4 contain enzymes (3
rg) purified from livers of fasted-refed
rats injected with saline or glucagon, respectively; and lanes labeled with S contain proteins as molecular weight markers (myosin, 215,000; macroglobulin,
170,000; P-galactosidase, 116,000; transferrin, 76,000; glutamic dehydrogenase,
53,000).

Insulin
FIG.
insulin

3. Activation
injections.

of acetyl-CoA

Carboxylase
maintained on a normal laboratory
with the indicated doses of insulin.
ylase were performed as described
0

[units]
carboxylase

in response

to

was isolated from livers of rats


diet and injected intraperitoneally
Preparations and assay of carboxunder Experimental Procedures.

10

5
Citrate

[mM]

FIG. 2. Citrate
dependence
of preparations
of acetyl-CoA
carboxylase
isolated
from
livers
of rats injected
with
insulin
or saline.
Acetyl-CoA carboxylase was assayed at the indicated

citrate concentrations as described under Experimental Procedures.


The enzyme was prepared from rats maintained on a laboratory diet
and injected with saline (A) or 5 units (-0.19 mg) of insulin (A) prior
to extraction of the livers. The same carboxylase preparations from
rats injected with saline (0) or insulin (e), respectively, were treated
with the [acetyl-CoA carboxylasel-phosphatase 2 as described under
Experimental Procedures and assayed at the indicated citrate concentrations. Points, mean; vertical bars, + S. E.

comparable to that of the carboxylase isolated from unfrozen


livers (46). Insulin administration led to a net decrease in the
phosphate content of the carboxylase from 8.5 to 7.0 ml of
Pi/m01 of subunit (Table II).
The enzyme preparation from animals that did not receive
insulin eluted from the Superose 6 column in two fractions.

A minor fraction, obtained in 8 ml of eluate, represents


proteins with an estimated molecular weight of 10 X lo6
(polymeric state) and a major fraction, collected in about lo12 ml of eluate, represents protein with an estimated molecular weight of 2 X lo6 (octameric state; Fig. 4B). Insulin
administration, however, led to a pronounced increase in the
polymeric form of the enzyme as shown by the shift from the
octameric to the polymeric form in the elution profile (Fig.
4C).

The insulin-induced activation of acetyl-CoA carboxylase,


its polymerization, and its higher citrate sensitivity may be
attributed to its lower phosphate content since dephosphorylation of the enzyme isolated from rats that did not receive
insulin by [acetyl-CoA carboxylasel-phosphatase 2 resulted
in similar transformations in activity of the carboxylase, its
response to citrate, and its polymeric states (Figs. 2 and 4).
The enzyme isolated from insulin-treated animals can
undergo further polymerization when treated with [acetylCoA carboxylasel-phosphatase 2 as evidenced by the complete

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

I
0

Hormonal

6334

Control of Acetyl-CoA Carboxylase


TABLE

II

Effect of hormones
(insulin,
glucagon,
or epinephrine)
on activity
and phosphate
content
of acetyl-CoA
carboxylnse
All the values given below represent
the mean f S. E. from four preparations,
except for those obtained
after
treatment
of the carboxylase
with phosphatase.
For the enzyme
prepared
from insulin-,
glucagon-,
or epinephrineinjected
animals,
the values represent
the means of two, four, and two determinations,
respectively.
Source of carboxylase
Animal
Diet

preparations

Specific

activity

maintenance
Hormone iniection

&

Phosphatase
treatment*

No citrate

Phosphate

content

mhf

mol P&no1 subunit

1.0 + 0.04
0.6 k 0.02
0.17 f 0.05

8.5 f 0.3
7.0 f 0.3
3.65 + 0.3

units/mg

Laboratory

Saline
Insulin
Insulin

No
No
Yes

0.8 + 0.13
2.1 * 0.07
9.0 + 0.27

3.8 f 0.3
4.8 + 0.1
12.5 + 0.25

Fasted/refed

Saline
Glucagon
Glucagon
Epinephrine

No
No
Yes
No

3.4 + 0.11
1.4 f 0.04
8.1 + 0.01
1.9

7.7 + 0.5
5.0 + 0.2
9.7 + 0.1
5.4

Five units of insulin,


1 unit of glucagon,
or 1 mg of epinephrine
were
and the animals
were killed 10 min later.
* Where indicated,
carboxylase
was treated
with phosphatase
as described
Food was withdrawn
from these rats 2 h before injection
with hormones.

injected
under

0.2
1.0
0.17
1.0

+
+
-c
+

0.03
0.06
0.05
0.1

intraperitoneally
Experimental

4.9
6.4
5.2
6.7

+
+
f
k

0.5
0.5
0.4
0.2

as indicated
Procedures.

7C). The carboxylase was similarly activated and polymerized


when treated with [acetyl-CoA
carboxylasel-phosphatase
2
(data not shown).
DISCUSSION

The acetyl-CoA carboxylase of animal tissues is a complex


multifunctional
enzyme. The subunit protein is large (Mr 265,000), containing
a biotin-binding
site, the catalytic domains for the carboxylation
of biotin and transfer
of the
carboxyl group to acetyl-CoA, and the regulatory sites for the
binding of allosteric effecters, citrate and palmitoyl-CoA,
and
numerous phosphorylation
sites. The enzyme also tends to
aggregate, especially after activation of the carboxylase, forming polymers of up to 10 X lo6 in molecular weight (10, 13,
14, 19, 31). Moreover, the protein subunit is susceptible to
proteolysis which also results in modification
of activity (50).
In recent studies of the acetyl-CoA
carboxylase, we related
citrate sensitivity,
phosphorylation,
and polymerization
to
each other and presented evidence that the enzyme undergoes
modifications
during its purification
from animal sources (31).
For instance, in the crude stages of preparation
the enzyme
may undergo proteolysis, rapid phosphorylation
(51) (presumably using cellular ATP and endogenous
kinases), and dephosphorylation
catalyzed by endogenous phosphatases
(13,
52). While proteolysis
can have a minor effect on catalytic
activity and may be contained by the use of protease inhibitors, changes in the phosphorylation
state affected activity,
citrate dependence, and polymerization
of the protein (31).
Consequently,
we developed a method of rapid purification
of
the enzyme by avidin-affinity
chromatography
after including
a variety of protease inhibitors
designed to minimize hydrolysis of the protein subunit in the medium (46). These methods
led for the first time to the isolation of the active, citrateindependent,
and polymeric
form of the carboxylase
from
animal tissues (46). Moreover, the earlier isolation of a specific
phosphatase,
[acetyl-CoA
carboxylasel-phosphatase
2, that
activates the carboxylase and renders it citrate-independent
made it possible to relate the phenomenon
of citrate activation
to phosphorylation
and polymerization
of the enzyme (13,
31). Based on these observations
we then studied the interrelationships
of changes in specific catalytic activity, citrate
dependence, phosphate content, and polymerization
state of
the carboxylase as a function of fasting and refeeding (31). In
the present investigation,
we extended these previous studies

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

conversion of the protein to the polymeric


form as noted in
the elution profile from the Superose 6 column (Fig. 40).
These changes were also accompanied by significant increases
in the citrate-independent
and citrate-dependent
activities of
the carboxylase (Fig. 2).
Glucagon-induced
Phosphorylation
and Inhibition
of Carbox&se-In
agreement with previous reports (9), acetyl-CoA
carboxylase purified from freeze-clamped
livers of fasted and
refed rats exhibited
high, citrate-independent
activity (3.5
units/mg), with a low & value for citrate activation and low
phosphate content. Treatment
of similarly
fasted and refed
animals with glucagon prior to extraction of the livers yielded
enzyme preparations
with
relatively lower activity (60% decrease in specific activity), a &fold increase in Koa values for
citrate activation, and a higher phosphate content (Table II).
Moreover, the specific activity of the carboxylase was lower
at all citrate concentrations
tested as shown in Fig. 5. Treatment of this carboxylase with [acetyl-CoA carboxylasel-phosphatase 2 increased significantly
the citrate-independent
activity as shown in Fig. 5.
Chromatography
of these preparations
on a Superose 6
column gave elution patterns suggesting that the enzyme from
fasted/refed
animals is comprised of two polymer species, a
major one (Mr - 10 X 106) and a minor one (Mr = 2 X 106;
Fig. 6A). Glucagon treatment
of similarly
fasted and refed
rats yielded an enzyme preparation
with an elution pattern
from the Superose 6 column significantly
different from the
enzyme isolated from saline-injected
animals. Glucagon increases the relative content of the octameric form (Fig. 6B).
Dephosphorylation
of this carboxylase preparation
by [acetylCoA carboxylasel-phosphatase
2 resulted in significant
activation of the carboxylase with a concomitant
decrease in the
& value for citrate (Fig. 5) and polymerization
of the protein
(Fig. 6C), suggesting that these changes are due to phosphorylation
of the carboxylase subunit.
Effect of Epinephrine
on Acetyl-CoA Carboxylose-Like
glucagon, injection of epinephrine
into fasted/refed rats resulted
in a decrease in carboxylase activity and a 5-fold increase in
& values for citrate activation
(Table II). Chromatography
of the carboxylase preparations
on a Superose 6 column gave
elution profiles similar to those obtained with carboxylase
preparations
isolated from the group of rats injected with
glucagon (Fig. 7). Treatment
of the carboxylase with citrate
resulted in activation and polymerization
of the enzyme (Fig.

citrate

Plus citrate
(10 nlM)

Hormonal

Control of Acetyl-CoA

Carboxylase

6335

10

Citrate

[mM]

carboxylase
with saline

j-

preparations isolated from animals injected with saline or glucagon,


respectively. A, shows citrate activation of carboxylase prepared from
livers of animals injected with glucagon as that used in experiments
depicted in curve 0, but with added treatment of the purified enzyme
with [acetyl-CoA carboxylasel-phosphatase
2 prior to assaying as
described under Experimental Procedures. Points, mean; vertical

bars, f S. E.

j-

of

FIG. 4. Elution
of

acetyl-CoA
with
insulin.

profile
from
carboxylase

:-I

4
Elution

umn
jected

dependence
of preparations
of acetyl-CoA
isolated
from
livers
of fasted/refed
rats injected
or glucagon.
0 and 0, citrate activation of carboxylase

Volumd
8

fml]

I
11

Superose
6 gel permeation
prepared
from
animals

colin-

Carboxylase preparations used were purified


by affinity chromatography from freeze-clamped livers of rats fed a
laboratory diet and injected with either saline or insulin 10 min before
killing. 1, point of application of the samples onto the column. A,
elution profile of a mixture of known proteins used as a reference
standard (peak 1, pyruvate dehydrogenase, M, 10 x 106;peak IZ, yeast
fatty acid synthase, M, 2.4 X 106;peak III, rat liver fatty acid synthase,
M, 525,000; peak IV, ferritin, M, 440,000; peak
V, bovine serum
albumin, M, 63,000); inset, linear plot of molecular weight uersu.s
elution volume; B, elution profile of acetyl-CoA carboxylase (400 rg)
prepared from rats injected with saline; C, elution profile of acetylCoA carboxylase (350 pg) purified from livers of rats injected with 5
units of insulin; D, elution profile of acetyl-CoA carboxylase (90 rg)
employed in the experiment represented in C, but after treatment
with acetyl-CoA phosphatase as described under Experimental Procedures.

to include the acute effects of insulin, glucagon, and epinephrine on the properties
of the carboxylase. This report represents the first direct demonstration
of the effects of these
important
hormones on the various properties
of the highly
purified acetyl-CoA carboxylase when they are administered
to the whole animal.
The hormones insulin or glucagon were injected intraperitoneally into rats and their effects on acetyl-CoA carboxylase
and blood glucose levels were evaluated and related to both
hormone dosage and the time lapsed before the animals were
killed. The injection of 5 units of insulin causes a 70% decrease
in the level of blood glucose, while the injection of 1 unit of
glucagon results in a near doubling of glucose concentration
(Table I). These levels of hormones were optimal for eliciting
significant
changes in blood glucose levels and the insulin/
glucagon ratios, and at the same time are tolerated
by the
animals. Also, changes in carboxylase activity are near maximal at these levels (Fig. 3). Concomitant
with changes in
carboxylase activity, there is alteration
in the polymeric form
of the enzyme. Insulin injection resulted in polymerization
of
the carboxylase,
while glucagon administration
resulted in
depolymerization
of the protein (Table I; Figs. 4 and 6). It is
important
to note here that maintenance
of the animals on
the indicated diet (laboratory
chow for animals used in the
insulin experiments
and starvation/refeeding
with a highcarbohydrate
diet for animals used in the glucagon experiments) is important
for the success of these experiments.
Acetyl-CoA
carboxylase is an inducible enzyme whose amount
and activity is regulated by diet. Hence, acetyl-CoA
carboxylase is reasonably
well expressed in rats maintained
on a
laboratory
diet but has a relatively low activity, making this
a suitable condition
to test stimulation
by insulin. Similarly,
rats maintained
on a high-carbohydrate
diet after a period of
starvation
have a highly expressed and active carboxylase;
glucagon administration
causes a quantitatively
significant

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

FIG. 5. Citrate

Hormonal

6336

Control of Acetyl-CoA

Carboxyhe

0.0

0.025

0.02

0.0
E
r
8
fi
a

0.02

i
2

0.0

1
L
IL 12
3
0

FIG. 6. Elution
of

profile
from
acetyl-CoA
carboxylase
with
glucagon.
The

Volume

FIG. 7. Elution

colin-

carboxylase preparations used were


purified from freeze-clamped livers of fasted-refed rats after injection
with saline or glucagon as described under Experimental
Procedures. J, points of application of the enzyme onto the column. A and
B, elution profiles of acetyl-CoA carboxylase (400 rg) prepared from
livers of rats injected with saline or glucagon, respectively; C, elution
profile of acetyl-CoA carboxylase (90 pg) used in the experiment
depicted in B, but treated with (acetyl-CoA carboxylasel-phosphatase
2 prior to its chromatography on a Superose 6 column,
decrease in activity (Table I). Administration
of insulin activated the carboxylase,
concomitantly
dephosphorylated
it,
increased its citrate sensitivity, and polymerized
the protein.
Thus, the short-term
effects of insulin on acetyl-CoA carboxylase mimic those of refeeding (31).
While the effects of insulin on carboxylase were coordinated
in the direction of increased enzyme activity, that of glucagon
or epinephrine
were in the opposite direction. Thus, the net
glucagon-induced
phosphorylation,
depolymerization,
and increased citrate dependence of acetyl-CoA carboxylase lowered
its activity. These effects of glucagon on acetyl-CoA carboxylase are very similar to those observed after brief fasting
(31). Therefore,
the insulinor glucagon-induced
changes
described here further support the hypothesis that acetyl-CoA
carboxylase
exists in two forms, an active, citrate-independent
polymeric
form and an inactive octamer, that are interconvertible
by phosphorylation/dephosphorylation.
These results, together with earlier reports, clearly establish the concept that dephosphorylation
of acetyl-CoA carboxylase leads
to activation while phosphorylation
leads to inactivation
(13,
19, 31, 37, 51). This conclusion
is also compatible
with the
general dogma that enzymes in the anabolic pathways are
inhibited
by phosphorylation
and stimulated
by dephosphorylation.

profile
from
acetyl-CoA
carboxylase
with epinephrine.
The

0
Volume

12

1t
[ml]

Superose
6 gel permeation
prepared
from animals

col-

incarboxylase preparations used were


purified from freeze-clamped livers of fasted/refed rats after subcutaneous injection with saline or epinephrine as described under Experimental Procedures. J, points of application of the enzyme onto
the column. A and B, elution profiles of carboxylase (400 pg) prepared
from the livers of rats injected with saline or epinephrine, respectively;
C, elution profile of the carboxylase (400 pg) used in the experiment
depicted in B, except that the enzyme was treated with 10 mM citrate
prior to chromatography.

umn
jected

[ml]

Superose
6 gel permeation
prepared
from
animals

Elution

Elution

umn
jected

of

Earlier reports on the insulin-induced


phosphorylation
and
activation
of acetyl-CoA
carboxylase (33, 34, 42-45) were in
apparent contradiction
with the aforementioned
conclusion.
These studies reported increased incorporation
of 32P into the
carboxylase protein. This may have been an artifact of isolation of the enzyme because acetyl-CoA carboxylase is rapidly
phosphorylated
immediately
after excision of liver from the
rat; a quick freeze-clamping
of the tissue was necessary to
preserve the enzyme in its active form (46). Thus, precautionary measures must be taken to avoid phosphorylation
of the
enzyme, which in addition to increasing phosphate
content,
may lead to inhibition
of activity, increased citrate dependence, and depolymerization
of the protein subunits, depending
upon the sites phosphorylated.
These changes may explain,
at least in part, why past studies detected increased phosphorylation
of carboxylase
without
activation
(32, 41), or
phosphorylation
and activation (33,34,42-44),
or phosphorylation, activation,
and polymerization
(45) in the presence of
insulin.
Our results are in agreement with a recent report on the
insulin-dependent
activation
and dephosphorylation
of acetyl-CoA
carboxylase
(40) and a number of reports on the
glucagon-induced
inhibition
and phosphorylation
of the enzyme (35, 37, 38). The major difference between the study
presented here and those of other investigators
is the consistent demonstration
of activation, dephosphorylation,
and polymerization
as induced by insulin,
and the inactivation,

Downloaded from http://www.jbc.org/ by guest on October 29, 2014

0.0

Hormonal

Control of Acetyl-CoA

established.

The

Mn*+-requiring

[acetyl-CoA

carboxylase]

phosphatase 2 has a high affinity for the carboxylase and may


be one such phosphatase. This phosphatase mimics the action
of insulin, promoting
activation
and polymerization
of the
carboxylase and rendering it more sensitive to citrate.
Acknowledgments-We
bers of the Diabetes
measurement
and

thank
Dr. Aubrey
E. Boyd, III and memCore Facility
at Baylor
College of Medicine,
for
analysis
of insulin
and glucagon,
and Pamela

6337

Powell for editorial


assistance
during the preparation
of this manuscript. One of us (I. M. H.) would like to thank
Professor
Salah Eid,
Department
of Biochemistry,
Ain Shams
University,
Cairo, Egypt,
for his support,
encouragement,
and advice, and Ain Shams University and the Egyptian government for scholarship support.
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phosphorylation,
and depolymerization
of carboxylase in affinity-purified
preparations
as induced by glucagon or epinephrine when administered
to whole animals. Our results
imply that, for enzymes involved in the regulation
of carboxylase, the ratio of phosphatase
activity to kinase activity is
modified
by these
hormones.
These
modifications
may be
achieved by selective activation
of one or more of the phosphatases, or inhibition
of one or more kinases, or both. In the
case of insulin action, we have detected a significant
increase
in the specific activity of [acetyl-CoA
carboxylasel-phosphatase 2 in purified fractions from livers of insulin-treated
rats
(data not shown); the involved mechanism is not clear. [Acetyl-CoA
carboxylasel-phosphatase
2 may be regulated
by
phosphorylation,
but supporting
data are lacking.
Finally, the recent cloning and sequence analysis of the
cDNA coding for the rat acetyl-CoA
carboxylase
made it
possible to predict the amino acid sequence of the protein
subunit (49). Assignment
of the phosphorylation
sites has
also been made based on the amino acid sequence of the
various phosphopeptides
isolated from the carboxylase after
treatment with different protein kinases under various in uiuo
and in vitro conditions
(30,42,43).
The rat carboxylase cDNA
codes for an open frame of 2345 amino acids with seven
phosphorylation
sites, six of which are located near the NH2
terminus at serine positions 23, 25, 29, 76, 77, and 95. The
seventh site is located at serine residue 1200 (54). Although
these sites may be phosphorylated
by some known protein
kinases in vitro, the identity and hormonal
regulation
of the
protein kinases that carry out the phosphorylation
in vivo are
not known. Among the protein kinases that are thought to be
involved in the phosphorylation
and, hence, the regulation
of
the carboxylase
are CAMP-dependent
protein
kinase (19),
Ca2+-dependent
protein kinase (19), CAMP-independent
protein kinase (25), AMP-dependent
protein kinase (30), protein
kinase C (42, 53, 54), calmodulin-dependent
protein kinase
(42), and casein kinase 2 (34). The inactivation
of the carboxylase by glucagon and epinephrine
is probably brought about
by phosphorylation
of the enzyme by CAMP-dependent
protein kinase or Ca*+-dependent
protein
kinase, respectively.
Whether these two hormones exert their effects through the
CAMP-independent
protein kinase or AMP-dependent
kinase
remains to be determined.
However, investigations
of the
effect of insulin in the activation of the carboxylase have been
difficult,
especially since the mechanism
of action of this
hormone
is not yet known and its metabolic
and cellular
effects are proving to be more and more complex. In one way
or another, several kinases (protein kinase C, casein kinase 2,
and calmodulin-dependent
protein kinase) have been implicated in the activation
of carboxylase by insulin (36, 42, 53,
54). On the other hand, incubation
of Swiss mouse 3T3-Dl
cells with insulin rapidly and transiently
activated a protein
phosphatase (55), which led the authors to suggest that some
of the intracellular
effects caused by insulin
and growth
factors are mediated
through
the activation
of a protein
phosphatase.
Our results are consistent with this conclusion.
Insulin may activate the carboxylase by dephosphorylating
the enzyme. Whether this represents activation of a phosphatase by insulin via reversible phosphorylation
remains to be

Carboxylase

Hormonal

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