Liver acetyl CoA carboxylase and fatty acid synthetase: Relative activities in the normal state and in hereditary obesity

Vol. 28, No. 5, 1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

LIVER ACETYL CoA CARBOXYLASE AND FATTY ACID SYNTHETASE: RELATIVE ACTIVITIES IN THE NORMAL STATE AND IN HEREDITARY OBESITY*

Huei-Che

Chang,

Departments

Irving

Seidman, ** George Teebor,

of Biochemistry Medicine,

and M . Daniel Lane+

and Pathology, New York University New York, New York 10016

School of

Received July 17, 1967 The reactions are catalyzed complex

of fatty acid biosynthesis

by acetyl

(Wakil,

Lowenstein,

1962) indicates

level of acetyl

activated

1960;

1961).

and Wakil,

is activated

to be rate-limiting

obese hyperglycemic

occurs at the

by citrate

and

in fatty acid synthesis

no condition

level per se account

. Hepatic

(Numa

1962; Kallen and

of fatty acid synthesis

However,

multienzyme

from many laboratories

The carboxylase

is alleged

in carboxylase

rate of fatty acid synthesis the genetically

that regulation

Numa & a.,

in which changes

Evidence

1962; Waite

CoA carboxylase.

even when fully (Ganguly,

and Vagelos,

CoA in higher organisms

and a fatty acid synthetase

1964).

19 63; Vagelos,

1961; Martin

al -et -*,

CoA carboxylase

from acetyl

fully

fatty acid synthesis

has been reported for changes in the

is 6-8 times higher in

mouse than in normal mice (Jansen fi al.,

1967). The initial carboxylase

objective

and/or

of this investigation

synthetase

levels

vated rate of fatty acid biosynthesis carboxylase

and fatty acid synthetase

was to determine

are increased in obese mice. levels

whether

commensurate Both hepatic

hepatic

with the eleacetyl

were found to be elevated

CoA to about

*Supported by research grants AM-09116 from the National Institutes of Health, y:S. Public Health Service and GB-5699 from the National Science Foundation. Career Scientist of the Health Research Council of the City of New York (I-275) +Career Development Awardee of the U. S. Public Health Service (Research Career Program Award K3-AM-18487).

682

Vol. 28, No. 5, 1967

BIOCHEMICAL

It was found that in livers

the same extent. in the chicken

and rat, the capacity

approximately

equals the capacity

into long chain fatty acids. malonyl

Procedure.

of the carboxylase of the synthetase

were obtained

Wayne Lab Blox ad libitum.

Sephadex G-25 in the homogenization boxylase

assays involved

locally.

minutes

in a medium (final

3 mM GSH,

of the gel-filtered

pH 7 .O at 37”) containing

the presence or absence of 5 mM citrate dition

of the following

trations

components

(specific

activity,

Reaction was for 5 minutes CoA was determined reaction 0.025 purified;

mixtures mM acetyl

(K+). Carboxylation

serum albumin at 37”

as described

l

by Gregolin

ati..

(final pH 7 at 37”) containing CoA,

0.10 mM 2-14 C-malonyl

0.2 PC per pmole),

EDTA and high-speed

1.2 mM NADPH,

supernatant

at 37” for 30

(0.6 mg per ml) in was initiated

solution

683

by adconcen-

10mM KH14C03

CoA, 3 mM GSH, 0.1 mM

and 5 mM citrate

Incorporation

Car-

60 mM Tris (Cl-) buffer,

serum albumin

0.2 mM acetyl

on

assays.

cytosol

0.05

(cytosol)

to gel filtration

2mM ATP, 8mM MgC12,

0.2 PC per pmole),

EDTA, 0.6 mg per ml bovine

and fed

(final pH 7.0 at 37”) to produce final

of: 60mM Tris (Cl-) buffer,

weeks

0.15 M KCl,

medium prior to carboxylase

0.1 mM EDTA, bovine

8 mM MgC12,

role of

Livers were ho-

solution

assays or subjected

preincubation

CoA

lo-14

from the Jackson Laboratory

(PH 7.2 at 25”) containing

for synthetase

as

CoA

malonyl

the possible

M Tris (Cl-) and 0.1 mM EDTA. The 105, 000 x g supernatant was either used directly

malonyl

mice (C576J/ob)

Laying hens were obtained

of a medium

as well

is discussed.

Male obese-hyperglycemic

in 3 volumes

to generate

to incorporate

of fatty acid synthesis

of age and lean littermates

mogenized

of obese and normal mice,

In view of the latter finding,

CoA as an inhibitor

Experimental

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(K+) as indicated.

of 14C-activity (1966a).

into malonyl Synthetase

100 mM phosphate

assay

(Kf) buffer,

CoA (chromatographically 2 mM dithiothreitol,

were incubated

for 10 minutes

3 mM at 37”.

BIOCHEMICAL

Vol. 28, No. 5, 1967

of 14C-activity

Incorporation

scribed by Guchhait

--et al.

Results and Discussion.

activities tivities

higher in livers sol activities carboxylase

TABLE I.

with the hepatic

elevated

per mg of cytosol

higher.

was observed

Hepatic

in the obese mouse.

3.1 2.6 4.8 3.1 Average 3.4 Obese mouse 10.1 16.8 9.1 9.6 Average 11.4 Laying hen 7.3 6.7 10.0 6.9 8.7 12.8 Average 8.7

(0.2)+ (0.1) (0.2) (0.2) (0.2) (0.2) (0.4) (0.1) (0.2) (0.2) (0.2) (0.1) (0.2) (0.2) (0.1) (0.2) (0.2)

to lean siblings.

in all cases

474 316 648 424 465 3600 6100 2100 2940 3700

-

of the

and synthetase The specific

ac-

of both enzymes were about 3-fold

A nearly

Acetyl CoA Carboxylase

CoA carboxylase

characteristic

Both carboxylase

from the obese mice compared were 6- to 8-fold

of acetyl

hyperlipogenesis

protein)

Carboxylase Specific Total activity activity x x m units per m units per mg protein liver Lean mouse

of the levels

mouse was investigated.

activity

as de-

(1966).

(Table I) were markedly (milliunits

RESEARCH COMMUNICATIONS

into long chain fatty acids was determined

The correlation

and fatty acid synthetase, obese-hyperglycemic

AND BlOPHYSlCAL

absolute

citrate

(Table I, values

Total liver cytorequirement in parentheses).

and Fatty Acid Synthetase

Synthetase Specific Total activity activity m units** per m units** per mg protein liver 4.7 6.0 7.3 8.3 6.6 16.9 18.5 15.5 19.5 17.6 10.5 15.8 13.2 15.6 18.1 11.8 14.2

700 746 980 1030 864 6100 6800 3600 5900 5600

for

Activities Specific activity ratio synthetase/ carboxylase 1.5 2.3 1.5 2.7 1.9 1.7 1.1 1.7 2.0 1.5 1.4 2.4 1.3 2.2 2.1 0.9 1.6

Protein was determined by the biuret method (Layne, 1957). *One milliunit = 1 mpmole of ‘acetyl CoA carboxylated/minute. **One milliunit = 1 mpmole of 2-14 C-malonyl CoA incorporated into long chain fatty acids per minute. +Values in parentheses are activities in the absence of citrate.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 5, 1967

Kornacker

and Lowenstein

zyme (expressed fold compared

protein)

of all principal

An unexpected

finding

er than carboxylase mine whether

enzymatic

components

of these activities

and synthetase

that observed

activities

to carboxylase

in the mouse.

synthetase.

Hence,

carboxylase margin.

Preliminary

Earlier investigations

low carboxylase

was only 1.5 to 1.9-fold

and synthetase

hepatic the

similar

liver car-

pH 6.8 (37”), for the chicken

(Ganguly,

liver

pH of 7.4 (37”) liver acetyl

to exceed.fatty

acid synthetase

activity

1960 and Numa etd.,

can be attributed

to suboptimal

to

were assayed

pH 7.5 (37”), for the chicken

CoA by a small

1961) indicated

was 20-80 times greater than carboxylase

activities

great-

To deter-

in the rat have yielded

that carboxylase

at the more physiologic

activity

of the carboxylase

hens were also determined;

studies

above the optimum,

would be expected

that synthetase

acid synthesis.

was found (Table I) to be similar

at pH 7 (37”) which is below the optimum, and slightly

of fatty

was unique to the mouse,

of laying

activity

. It should be emphasized

boxylase

with a coordinated

in the activities

activity

3-

rate of fatty

in both obese and normal mice (Table I).

the similarity

en-

in obese mice is increased

was the similarity

activity

ratio of synthetase

cleavage

mouse is associated

At pH 7 (37”) synthetase

and synthetase.

of citrate

Thus, it appears that the elevated

in the obese-hyperglycemic

rise in the levels

findings

that the level

per mg of liver cytosol

to lean mice.

acid synthesis

carboxylase

(1964) reported

activity;

carboxylase

these

assay con-

ditions . The fact that liver carboxylase suggests

that under certain

and synthetase

circumstances

may be sufficient

to depress carboxylase

hibitor

liver acetyl

of chicken

ing a Ki of about 1 OB5M. with respect to both acetyl

are nearly

the malonyl CoA concentration activity.

CoA carboxylase

Furthermore,

activities

malonyl

CoA and isocitrate

685

Malonyl (Gregolin

in vivo --

CoA is a potent in-

&al.,

CoA inhibition (or citrate).

equal

1966b) exhibitis competitive

In the case of acetyl

Vol. 28, No. 5, 1967

BIOCHEMICAL

CoA (Km = 1-4 x 10” M) malonyl type. kinetic

Evidence

of an equilibrium citrate

(or citrate)

nyl CoA.

Inhibition

safeguard

against

thereby

providing

CoA inhibition

has been presented

relationship

between of an active

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

malonyl

(Gregolin

is of the classical &al.,

excessive fine control

CoA carboxylase acetyl

(or citrate)

form of the carboxylase

and a catalytically-inactive of acetyl

196633) that the competitive

CoA and isocitrate

polymeric

is the result

promoted by iso-

protomeric

form promoted

by malonyl

CoA could provide

CoA utilization

over the committed

competitive

for malonyl

by maloa

CoA production,

step of fatty acid biosynthesis.

Reference s

J., Biochim. Biophys. Acta, 40, 110 (1960). Gregolin, C., Ryder, E., Kleinschmidt, A.K., Warner, R.C., and Lane, M.D., Proc. Natl. Acad. Sci., U.S., 56. 148 (1966a). Gregolin, C., Ryder, E., Warner, R. C., Kleinschmidt, A.K., and Lane, M.D., Proc. Natl. Acad. Sci., U.S., 56. 1751 (1966b). Guchhait, R. B., Putz, G.R., and Porter, J.W., Arch. Biochem. Biophys., 117, 541 (1966). Jansen, G.R., Zanetti, M.E., and Hutchison, C.F., Biochem. J., 102, 870 (1967). Kallen, R., and Lowenstein, J. M . , Arch. Biochem. Biophys., 96, 188 (1962). Kornacker, M . S., and Lowenstein, J. M . , Science, 144, 1027 (1964). Layne, E., Methods in Enzymology, & 450 (1957). Martin, D.B., and Vagelos, P.R., J. Biol. Chem., 237, 1787 (1962). Numa, S., Matsuhashi, M., and Lynen, F., Biochem. Z., 334, 203 (1961). Waite, M ., and Wakil, S. J., J. Biol. Chem., 237, 2750 (1962). Wakil, S. J., Ann. Rev. Biochem., 31. 369 (1962). Vagelos, P.R., Ann. Rev. Biochem., 33. 139 (1964). Gawuly,

686