Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

STIMULATION OF TRIGLYCERIDESYNTHESIS IN MAMMALIANLIVER AND ADIPOSE TISSUE BY TWOCYTOSOLICCOMPOUNDS* Daniel Institute

A.K.

Roncari+

of Medical Department

Science

September

Y.W. Mack

and Toronto

of Medicine, Toronto,

Received

and Esther

Western

University

Hospital,

of Toronto,

Canada M5S lA8

22,1975

SUMMARY Two cytosolic compounds which stimulate neutral glyceride synthesis catalyzed by microsomal enzymes have been isolated from both rat liver and adipose tissue. In their absence, diacyl-sn-glycero-3-phosphate was the major product. They were separated from each other by Sephadex G-100 chromatography; in the case of each tissue, one had a molecular weight greater than 150,000, while the other was in the range 8,000-16,000. Both were inactivated by trypsin and the smaller one lost only 25% of its activity after incubation at pH 8.3, 90°, one hour. While Mg2+ also increased neutral glyceride synthesis, the cytosolic factors were active under optimal concentrations of these ions. These compounds may play a critical role in the regulation of neutral glyceride synthesis. The regulation understood. supernatant

of glycerolipid

A stimulatory fraction)

effect

compound(s)

intestine

the

is poorly

cytosol

synthesis

for and is

the

effect

the

subject

(l-3).

tissue, The nature

of the of

(100,000 catalyzed

in mammalian adipose

has been reported

responsible

has been investigated

of

on triglyceride

membranous enzymes present and small

biosynthesis

cytosolic

this

X g by liver of the fraction

report.

*This work was supported by The Medical Research Council of Canada Grant MA-4428, The Ontario Heart Foundation, The Atkinson Charitable Foundation and The Banting Research Foundation. +Scholar of the Medical Research Council of Canada. To whom reprint requests should be addressed at the Medical Sciences Building, Room 7238, University of Toronto, Toronto, Canada M5S lA8. Copyright o I9 75 by Academic Press, Inc. All rights of reproductim in any form reserved.

790

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Materials. Palmitic acid, rat-glycerophosphate, ATP (Na salt), CoA (Li salt), horse muscle myoglobin (Type I) (Sigma), lipid standards (Sigma and Serdary Research Laboratories), [U-14C]-snglycero-3-phosphate (120 Ci/mol) (New England Nuclear), Blue Dextran 2,000 and Sephadex G-100 (fine) (Pharmacia) were purchased from the designated sources. Livers and epididymal fat pads were obtained from Methods. At the time fasted-refed (48-48 hours) male albino Wistar rats. Homogenization was carried of sacrifice, they weighed 190-230 g. out at 0-4O in 0.005 M ammonium acetate adjusted to pH 8.3 with the homogenizing solution consisted NH4OH. In some experiments, of 0.01 M potassium phosphate, pH 7.0, 0.15 M KCl, 0.25 M sucrose and 0.01 M 2-mercaptoethanol. Livers were homogenized with a Waring Blendor, while epididymal fat pads were homogenized with a motor-driven Potter-Elvehjem apparatus (Teflon pestle). In the case of each tissue, after initial centrifugation at 15,000 X g for 20 min, the microsome-rich and the cytosolic (supernate) fractions were obtained at 100,000 X g for 60 min (all centrifugations at 0-4O). Incubations were carried out using optimal concentrations of known substrates (sn-glycero-3-phosphate and potassium palmitate) and cosubstrates (ATP and CoA) as previously reported (1) except for the fact that the radioactive substrate was [U-14C]-sn-glycero-3-phosphate (1 uCi per assay), the concentration of potassium phosphate was 0.1 M, 2-mercaptoethanol (0.01 M) was used and, unless otherwise specified, the concentration of MgC12 was 0.2 mM, in a total volume of 0.5 ml. Microsome-rich fractions of epididymal adipose tissue or liver were used in the presence or absence of adipose tissue or liver cytosol or partially purified fractions of the latter. Incubations were conducted at 25" for 15 min. Lipid extraction was carried out by the method of Bligh and Dyer (4) using methanol which contained 0.01 M rac-glycerophosphate and 0.1 N HCl instead of water. The lipids were separated by thin-layer chromatography on Silica Gel H as reported Fifty ug of authentic lipid were used as "carrier" for each (1). glycerolipid formed. After visualization with 12, the Silica containing the various lipid fractions was scraped and the radioactivity counted in a Nuclear-Chicago Mark II liquid scintillation spectrometer model 6847 using Bray's mixture. Protein concentrations were determined by a microbiuret procedure (5). Magnesium concentrations were determined by atomic absorption spectrometry (6). The liver cytosolic factors were resolved by chromatography on 2.5 X 60 cm Sephadex G-100 (fine) columns (Glenco Scientific, Inc.) equilibrated and eluted with 0.005 M ammonium acetate, pH 8.3, at O-4". Appropriate eluate fractions were concentrated by rotary evaporation prior to assay. RESULTS The effect glycerolipids Fig.

1.

some-rich

of

the

formed

Various

cytosolic

by microsomal

combinations

and cytosolic

fraction

of

fractions

on the

enzymes liver

illustrated

or adipose

were used.

791

is

composition

tissue

of in

micro-

Under the experi-

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7:

AM AMtLS

Fig. 1. Effects of liver or adipose tissue cytosol on glycerolipid synthesis catalyzed by liver or adipose tissue microsome-rich fractions. In the appropriate incubation mixtures, the following quantities of protein were used: liver cytosol (IS) 5.0 mg, adipose tissue cytosol (AS) 3.0 mg, liver microsome-rich fraction (LM) 0.8 mg and adipose tissue microsome-rich fraction (AM) 1.0 m1,2-diacyl-sn-glycero-3phosphate, m-- diglyceride, - triglyceride.

mental

conditions

catalyzed

used in this

predominantly

phosphate.

the

Diglyceride

products,

while

and adipose 7- and 3-fold

of

liver

microsomal

the

total

radioactive

was negligible.

fraction

resulted

respectively,

enzymes

1,2-diacyl-sn-glycero-3-

4% of

synthesis

cytosolic

increase,

the

synthesis

constituted

triglyceride

tissue

study,

in the

The liver

in an approximately diglyceride

frac-

tion. In the case of the

adipose

diacyl-sn-glycero-3-phosphate formed,

but

products

in appreciable ride

fraction

1).

constituted Both

adipose

enhancement and the

microsome-rich

was also

triglyceride

(Fig.

tissue

about tissue

of both

latter

the major 20% of

and liver

glycerolipid the radioactive cytosol

the diglyceride

became the major

fraction,

resulted

and triglyce-

glycerolipid

form-

ed. The resolution capable

of

enhancing

adipose

tissue

of two factors neutral

microsomal

derived

glyceride

enzymes 792

is

from liver

synthesis illustrated

cytosol

catalyzed in Fig.

and

by 2.

The

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P 2

n x ,"

IOO-

g 2 0

80-

.i P E =

60-

40-

z if 8

zo-

0

I 80

40

I 120

I 160

I 200

Elude

I 280

240

I 320

360

(ml)

Fig. 2. Resolution of liver cytosolic factors which stimulate neutral glyceride synthesis by Sephadex G-100 chromaOne ml of concentrated cytosol was applied onto a tography. 2.5 X 60 cm column equilibrated and eluted with 0.005 M ammonium acetate, pH 8.3, at 4O. The fractions of the eluate between the horizontal T symbols were pooled and concentrated. In separate assays, the partially purified factors I and II (20 and 11 mg of total protein, respectively) were tested in the presence of 0.3 mg of adipose tissue microsomal protein. Vo-column exclusion volume, Ve myoglobin-elution volume of myoglobin added as a marker, Vi-internal column volume ("salt fraction"). m1,2-diacyl-snglycero-3-phosphate, m -- diglyceride, 0 - triglyceride.

column eluting the

quantity

obtained Fig.

"salt

volume I;

smaller

it

glyceride

contained enhanced

factor(s)

portion regions

which

proportion

be referred

increment proportion

was reproduced

in the

of

to

the eluate.

will

eluate

as factor

diglyceride (Fig. II)

fraction, The effect (Fig.

repro-

be referred

fraction

of diglyceride.

793

to those

The column exclu-

of both

triglyceride

in 30 experiments

presence,

of the

synthesis.

a factor(s) the

in its were similar

by the microsome-rich (will

on the

(Vi)

two distinct

neutral

formed

an appreciable no effect

(Vo)

composition

fraction"

that

enhanced

triglyceride

factor

the

2 illustrates

as factor

was used as control;

and glycerolipid

with

ducibly sion

solution

2).

to and

2).

A

resulted

in

but

had

of each

Vol. 67, No. 2, 1975

BIOCHEMICAL

No Added

AND BIOPHYSICAL RESEARCH COMMUNiCATlONS

Mg’+

02mM

MgCIz

3mM M&I, I I/

0

Fig. 3. Effects of liver cytosolic factors on glycerolipid synthesis catalyzed by adipose tissue microsome-rich fractions in the presence of different concentrations of added M$+. After resolution by Sephadex G-100 chromatography, the partially purified factors I and II (34 and 15 mg of total protein, respectively) were assayed in the presence of 0.3 mg of adipose tissue microsomal protein. m - 1,2-diacyl-sn-glycero3-phosphate, m - diglyceride, 0 - triglyceride.

The molecular 150,000,

weight

while

that

The effects

of

of

the

of the

larger smaller

compound was greater

than

one was in the

8,000-

range

16,000.

composition some-rich of

ride

cytosolic

of glycerolipids fraction

and triglyceride.

persisted required

in for

the

The total

endogenou;

Sephadex

G-100 eluate

range

0.3-0.4

3.

Three

fraction

were the

for

resulted

relative

formed

factor which

were

when 10 mM

same as those

shown for

either

0.2 mM MgC12, increment

794

diglyce-

synthesis;

associated

factor

When an equimolar

MnC12 was substituted

micro-

mM MgC12 resulted

of newly

magnesium concentration containing

tissue

of each cytosolic

glyceride

results

on the

concentrations

of MgCl2 concentrations

neutral

meq/liter.

in a similar

adipose

of different

The effect

maximal

I and II

by the

in Fig.

in the

the presence

MgC12 was used,

presence

illustrated

in an increase

factors

formed

in the

added MgC12 is

by itself

liver

I or II

concentration

each cytosolic in the neutral

with

3 mM. the

was in the of compound glyce-

BIOCHEMICAL

Vol. 67, No. 2, 1975

ride of

fraction. soybean

enzymes, of the

trypsin

prior

to

and cosubstrates

by trypsin;

incubation

addition

with

prevented

membranous

any further

effect

protease. cytosolic

factor

I was inactivated

M ammonium acetate,

was stable vity

was inactivated

inhibitor

substrates

Liver 0.005

Each factor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

under

these

when preincubated

pH 8.3,

at 60" for

conditions

and lost

at

1 hour.

90'

by preincubation

for

1 hour: only

in

factor

25% of

II

its

acti-

DISCUSSION The current liver

study

cytosolic

thesis

compounds which

catalyzed

which

similar

or identical

viously

presented

fraction 3.4) as its

reports

3, 8).

heat

an activator larger these

The properties

catalytic

remains

the

tissue

protein

syn-

enzymes.

in nature,

adipocyte

effect

the

of

that

smaller

it

is

not

The precise

to be elucidated.

triglyceride

glyceride

factors

the

are pre-

cytosolic

phosphohydrolase"

activity.

liver

two rat

(7).

of

indicate

of

compound,

such

an enzyme,

but

nature

of the

The relationship

cytosolic synthesis

(EC 3.1.

factor

reported

from diglyceride,

of to be is not

(9). It

for

laboratory

phosphatidate

compounds to the for

partly

two cytosolic

have ascribed

stability, of

factor

required clear

by this

to a "soluble (2,

the

neutral

and adipose

are at least

to

the presence

stimulate

by membranous liver

These compounds,

Earlier

has demonstrated

has been reported

the effect

phosphatidate in addition neutral centrations

of

the

that

in rat

cytosolic

fraction

phosphohydrolase to Mg*+,

glyceride

(10).

two larger

synthesis;

adipose

by activating This

cytosolic

they

of Mg'+.

795

tissue

are active

study

Mg2+ account microsomal

has revealed

that,

compounds stimulate at

saturating

con-

Vol. 67, No. 2, 1975

Hence, ride

two cytosolic

synthesis

tissue.

BIOCHEMICAL

compounds which

have been isolated

They may play

synthetic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

enhance

from both

a critical

role

neutral

liver

glyce-

and adipose

in regulating

this

bio-

process.

ACKNOWLEDGEMENTS The authors are grateful tance. They are also thankful the magnesium concentrations.

to Mr. Henry Wang for to Dr. H. Husdan for

his assisdetermining

REFERENCES 1.

Roncari, D.A.K., Acta Biophys.

2.

Hiibscher, (1967)

G., Brindley, D.N., Nature G,-449-453.

3.

Johnston, (1967)

J.M., Lipids

4.

Bligh, 37,

5.

Munkres, K.D., and Richards, Biophys. 109, 466-479.

6.

Pybus,

7.

Roncari,

8.

Smith, M.E., Sedgwick, B., Brindley, D.N., (1967) European J. Biochem. 3, 70-77.

9.

Manley, E.R., Skrdlant, H.B., Hansbury, E., and Scallen, (1974) Biochem. Biophys. Res. Commun. 58, 229-235.

10.

Jamdar, 524.

and Hollenberg, 137, 446-463.

Rao, G.A., 2,.14-20;

E.G., and Dyer, 911-917.

J.

(1968) D.A.K.

S.C.,

Clin. (1974)

and Fallon,

C.H. Smith,

Lowe, P.A.,

W.J.

(1967) M.E.,

and Sedqwick,

and Schwarz,

(1959 ) Can. J. F.M.

(1965)

Biochem.

Arch.

Chim. Acta

23,

309-317.

Fed.

33,

1525.

Proc.

H.J.

796

(1973)

Biochim.

J.

B.E. Physio 1.

Biochem.

and Hiibscher,

Lipid

B.

Res. 14,

G. T.J. 517-