Characterization of triglyceride lipase activities in rat skeletal muscle

Vol. 121, No. 1, 1984 May 31, 1984

BIOCHEMICAL

CHARACTERIZATION

OF TRIGLYCERIDE LIPASE RAT SKELETAL MUSCLE

IN Peter Dept.

Int.

Received

February

Strohfeldt

and

University

Med., 24,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 87-94

of

Christian

ACTIVITIES Heugel

Saarland,

HomburglSaar,

W.Germany

1984

SUMMARY: Triglyceride lipase activity was determined in particulate and soluble fractions from rat skeletal muscle homogenates. The fractions exhibited an acid (pH 5,0) optimum with an impressive enhancement in the combined P17/100 fraction. Methylamine inhibited this acid lipase activity.A further lipase was observed with maximal activity at pH 7,0 and only a small enhancement in the combined P17/100 fraction and inhibition by diethyl pnitrophenyl-phosphate but not by protamine sulfate. Lipoprotein lipase activity was identified by the following in vitro criteria: Stimulation of activity by serum, maximal activity at alkaline pH and inhibition of activity by NaCl and protamine (PH 8-5 - 9,o) sulfate. There was a definite enhancement of lipoprotein lipase activity in the combined P17/100 fraction after the lipase activity has been washed out from the capillary bed with heparin.

INTRODUCTION:

There

intracellular

TG stores

oxidizable

substrate

has

been

be

involved

Oscai for

given

et

in

that never

studies An

acid

found

in

the

intracellular

(7,8,9).

systematically determine

The look their

be

only

TGL

rat

of and

may

because heart

muscle,

of

soluble which

in

responsible

several

lysosomal

present

activities

subcellular

enzyme

presence

of the

of

which

muscle,

the

localization for

the

probably

of

lipases

In

microsomal

purpose

a source

TG stores(4,5,6).

skeletal

revealed lipase

the

contains attention

muscle

identified.

have

as

intracellular

may in

been

recent

and

to

of LPL

muscle

cells(l,2,3).Litt?e

skeletal

TG hydrolysis have

lipase

to

of

skeletal

be mobilized

musc?e

activities:

a LPL

can

study

a1.(6)suggest

a neutral

and

the

mobilization

discussion

that

which

the

lipases

however, lipase

evidence

wtthin

to

intracellular

other

is

fractions is

study rat

origin, still

under

therefore

skeletal

is

muscle

localization.

Abbreviations used: TG, triglyceride; bt-A f f tty acfd; LPL, lipoprotein DPN~P,r~~et~yl p-nitrophenylphosphate; minidase; R.S.A., relative specific

TGL, triglyceride lipase; lipase; MA, methylamine; NAGA, N-acetyl-flglucosaactivity. 0006-291X/84

87

$1.50

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 121, No. 1, 1984

MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS:

Vaterials:

Glycer 1 tri [1- 14C]oleate (45,7 mCi/mmol~ [1-14C(j o 1 eic acid (57,4 mCi/mmol), (8-1 C) adenosine 5' -monophosphate (58,o mCi/mmol) were purchased from Radiochemical Amersham. Unlabelled triolein, lecithin, heparin, protamine sulfate, methylamine, diethyl p-nitrophenyl-phosphate, p nitrophenyl N-acetyl-P-d-glu'cosaminide and adenosine 5'-monophosphate were obtained from Sigma Chemical Company. Bovine albumin (fraction V), which had been defatted as .described by Chen (lo), was obtained from Merck AG.All other chemicals were of reagent grade. The solvent for 1iquSd scintillation counting (Insta-Gel) was purchased from Packard. Serum was prepared from human blood and was heated Tt 55°C for 60 min to eliminate any endogenous lipase activity. Preparation of subcellular fractions: feaad libitum,were anaesthctized and thoroughly exsang-inated.

with

Male Sprague-Dawley pentobarbital,decapitated,

rats,

Muscle tissues (soleus consisting predominantly of slow-twitch red fibers, deep portions of vastus lateralis consisting predominantly of fast-twitch red fibers and superficial portions of vastus lateralis consisting predominantly of fast-twitch white fibers) were rapidly dissected out, trimmed of fat and frozen in liquid nitrogen, powdered in a ,qortar and allowed to thaw in buffer 1 (0,25 'JI silccrose, o,ol ?I Tris-HCl, 0,001 M EDTA, 20 % (v/v) glycerol, pH 7,5) for the assay of acid and neutral TGL activities or in buffer 2 (0,025 M NAOH/NH4Cl, 1,o U/ml heparin, pH 8,l) for the assay of LPC activity. All subsequent operations were carried out at 4OC. The crude muscle homogenate was further homogenized for 30 min in a "cell r'isruption bomb".The homogenate then was centrifuged at 5000 x g for 20 minutes. The pellet was resuspended in buffer 1 or q i? a volume corresponding to the original homogenate (fraction P5).The supernatant was centrifuged at 17000 x g for 20 min.The pellet was resusopnded in buffer 1 or 2 in a volume corresponding to one-fifth of tne original homogenate (fractic P17). The supernatant was centrifuged at looooo x g for Go min. The pellet was resuspended in buffer 1 or 2 to a volume corresponding to one-fifth of the original homogenate (fraction Ploo). The supernatant was called Sloo. o,l ml of the respective fractions was added to the test-system.Protein was determined by the method of Lowry et al. (11). Assay methods: NAGA was assayed as described by Brecher et a1.(12). !ir~fi?'r6?i~$&re activity was measured t)y the procedure described by Wang et al. (13). TGL assay was performed with dispersions of triolefn as substrate: 113 nmol unlabelled triolein with about 0,2 uCi 14C-labelled triolein and 40 ug lecithin were dispersed in chloroform-methanol (v/v 2:l) dried under N2 and resuspended in 0,7 ml of 50 mM sodium phosphate or sodium acetate bigffer. LPL activity was also assayed with dispersions of 14C-triolein and lecithin as substrate in 0,675 ml Tris/HCl-buffer, pH 8,5. In addition the substrate contained lo mY CaC12, 1,o U/ml heparin and 25 ul human serum. The further progress of the lipase assays was exactly as published by Severson 17). Lipase activity is expressed as nmol FFA released per h per mg protein. All enzyme assays were performed under conditions in which the assays were linear with respect to time and protein. Figures showing pH dependency of the enzyme activities are the results of one experiment. To ensure reproducibility of results, however, all experiments were repeated several times. 88

Vol.

121,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Perfusion experiments: Surgical preparation of the hindlimbs was -performed as &esby Strohfeldt (14).The hindlimbs were perfused (6 ml/min) with KHB buffer(pH 7,4, T=37,ooC) containing 6% bovine albumin, 5 mM glucose,5,o U/m 1 heparin and 20% (v/v)fresh and perfused in human blood.The medium was drawn from a .,,:servoir a nonrecirculatory manner. Samples of the medium were assayed for LPL activity at 1, lo and 60 minutes after the start of perfusion experiments. LPL in the P17/100 fraction of the soleus was determined after lo or 60 minutes of perfusion and correlated to LPL activity of soleus samples of nonperfused animals assayed the same day. The

Wilcoxon-Test

RESULTS: and

was

The

percentage

!i'-nucleotidase indicate

plasma

membranes and

that

Ploo

the

was

(P17/100)

fraction in

under

and

as

conditions

of

IN SUBCELLULAR

of

FRACTIONS

NAGA %distribution R.S.A.

5

5'-Nucleotidase

5

43.7 1;3

%distribution R.S.A.

various

1.2

given both

in of

TGL

t

Ploo

P17 of

skeletal

the

pH on TGL

muscles

of

SPECIFIC LIPASE

ACTIVITY ACTIVITIES

OF RAT SOLEUS MUSCLE

63::

37.6

soleus

I

Fraction P17

P5

rat

combined effect

AND RELATIVE AND VARIOUS

n

Enzyme

the

preparation.The

DISTRIBUTION 5'-NUCLEOTIDASE

NAGA

characterization with

fraction

of

sediment

I).Further

Plilloo

of

homogenization

structures

performed enzyme

enhancement fractions

TABLE PERCENTAGE OF NAGA,

analysis. and

lysosomal

therefore

the

statistical

subcellular

fraction(Table

activities activity

for

distribution in

muscle P17

used

11.7 696

Ploo

Sloe

lo,4

39.1

2,8 29.1

8,1

0.6 21,4 0.3

P17/100 TGL-pH5

3

Idistribution R.S.A.

TGL-pti

7.5

LPL-pH 8.5 Zdistribution R.S.A. LPL-pH 8,5a

17,8

6.1 092

3

%distribution R.S.A.

Xdistribution R.S.A.

57.9

38,9 a,5 69,3 190

:::

56,6

17,9

23.3 099

12

0.8

5.7

25,3 0.9

6 23.8

53.9 0,7

8.2

22.1 099

Data show the mean of the indicated number of experiments. = % of total enzymatic activity/% of total protein. R.S.A. a = LPL activity in soleus muscle after perfusion for 60 with heparin (5.0 U/ml). 89

min

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

. = Sdeus 0 = Red Vastus 0 = White 11

l

=

Soleus

0 = Red Vastus 0 = White 11

4,o c,s 5,o

5.5

6,0

6.5

7,0

7.5

8.0

8,s PH

PH

the

Fig.

1:

Effect of of various

pH on TGL activity in rat skeletal muscles.

the

P17/Ioo

fraction

Fig.

2:

Effect of of various

pH on LPL activity in rat skeletal muscles.

the

P17/100

fraction

an

ootimum

rat

is

shown

impressive Little an

activity optimum

P17/100

at

7,0.

fraction

total

activity

TGL

At acid fraction 5,5,

of

MA,

a lysosomal

neutral but

the lipases,

was

Alkaline

of

than

relative pH 5,

50

without lipase

pH values be

% of

and

total

inhibitor, assayed

activity

was

identified

8,5

with

for

LPL, alkaline

in

the

only

of

subcellular in

Table the

sedimented

I.

P17/100 At pH 7,5

with

the

for

the

the acid an inhibitor

TGL

fraction.

activity

acid

5.

et

recovered in this fraction.

markedly innibited at pH 7,5. DPNPP, the

to

shown

observed

P17/loo

lipase on

in was

the

inhibited effect

was

are

an at

distribution

activity 8,5

activity

enhancement in

active

Percentage

specific 7,5

6,5

optirnal

highly

- 9.

exhibited of

conditions

activity enhancement

activity

TGL

at

8,5

at

fractions

pH with

to

and

A clear lipase

stimulated

pH optima

The

acid

assay

observed

pH, 58 % of total with a definite more

2.

at

Under

soleus

P5 fraction. alkaline

1 and

determined

was

with

fractions

Fig.

activity was

pH values

and

in

lipase

assayed

lipase

activity as

LPL

at

-q

(Table by

the

but of

7,5

II).

following

BIOCHEMICAL

Vol. 121, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE EFFECTS LIPASE

OF MA, ACTIVITY

II

OPNPP. SERUM, NACL AND PROTAMINE AT PH 5.0, 7.5 AND 8.5 IN THE OF RAT SOLEUS MUSCLE

n

Lipase-activity

(% of PH

MA

(50

3 3

mM)

DPtiPP (0,58

PM)

MA (50 mM) DPNPP (0.58 PM) Protamine sulfate (0.5 mg/ml)

: 3

Without serum NaCl (500 mM) Protamine sulfate (0.5 mg/ml)

: 2

vitro

criteria:

protamine

sulfate

assayed

in

of

pH 8.5 15,2 6.9 0

from

perfusates specific soleus

were activity did

glyceride the

was

lipases

employed

in

single

the in

mobilization the

differential

from

study

for

fraction by

density

fractionation

are

not

to

characterize

The

study

the

(P17 has

identified

neutral

(pH

An acid

pH optimum

lysosomes.

of

7 Further

7,5)

be

gradient

an

triin

scheme fractions

expected

to or

in

alkaline

microsomes

that

this

evidence

that

the 91

TGL acid

results

a combined an 8,5 TGL

muscle

acid - 9)

activity was

tried particulate

skeletal

with (pH

suggests

a

We therefore

disrupted activity

by

yield

centrifugation,the (15).

3).

involved The

lysosomes

activities complete

(Fig.

for

be

wash the

time the perfused

look

may

the

to

muscle

subcellular

not

satisfactory

TGL and

U/ml) minutes same of

to

which

containing

lipase

+Ploo)

study

of

would

of

origin,

(5 60

At the fraction

this

and

activity

endo+,nelial

nonperfused

isolation

NaCl

LPL

triglycerides.

centrifugation

exclusively.Even

fraction

of

muscle

intracellular

subcellular

of of

skeletal

of

by

with heparin perfusion for

that

intent

of experiments activity (X + SX).

that

from LPL activity. LPL in the P17/100

differ

It

exclude was

perfused After beds.

free of

not

DISCUSSION:

fraction

were

capillary

number control

inhibition

To

II).

P17/100 rats

5,o

pR 7,5 147,9 64.8 116,2

activation,

(Table

the

hindlimbs LPL

Serum

control)

45,0 99.2

Data show the mean of the indicated and are expressed as percentage of

in

SULFATE UPON P17/100 FRACTION

(pH

5),

cells. a

pH optimum. originated of

lysosomal

in

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMAWNlCATlONS

“S

Rf.

1()

origin

3:

was

afforded (16).

lipase The

was

lofold pH 7-7,5

recent

studies

raised

as is

TGL

with

to

whether

II),

with

with

may

LPL was

lipase,its

be

any

In

the

the

Since

under

the

assay

or

92

one

neutral

shifted (Fig.

to

higher

l).This

They lipase

heavily

conditions

or

however,

et al.(g). neutral

has a low activity to the mobilization

small.

enzyme

system

LPL

been

inhibited

curve

assay

of Hiilsmann muscle "that

neutral lipase contribution

has

study,

LPL,neither

the

enhancement fraction.From

a separate

pH activity in

optimum

question

present

acid lipase.

an

definite

is

of

neutral with

the

lipase

(9).

the

the

subcellular

muscle,

heparin-non-releasable

enzyme". study

only

in

heart

a lysosomal of

lipase

uncertain.A

inhibitor nor

of

the

remained seen

MA,

activity

that of

to the findings with rat heart with

this

present acid

than

a phospholipid

in contrast from results identical

specific

neutral to

with

not rat

sulfate,an

(Table

values

was

related

protamine

maximum greater

activity

which

experiments

localization

at

this

by The

subcellular

activity of

LPL activity in the P17/100 fraction of rat soleus muscle (columns) and during perfusion with heparin (5.0 U/ml) for lo (n=6) and 60 (n=6) mln (curve). Results are expressed as the percentage of control activity measured without perfusion with heparin (n=6). The loo % value of LPL activity in the perfusion medium correspond to that measured 1 min after the start of perfuslon with heparin; n.s.: values are expressed as y + Sx. not significant;

Fig.

inhibitor

Time I min )

is

suppose is either

contaminated used

in

the

when compared to of stored lipid

Vol. 121, No. 1, 1984

BIOCHEMICAL

LPL

by

was

characterized

a considerable which

enhancement

was

even

activity

the

witit

et

releases

not

also of

being

found

heart

and

the

some

the

transported

from

surface. in

either

the

The

present

are

activity

of

be

al.

LPL

showed,

activity

be

rat

to

extracellularly

in

reproducable

be

heart.

paradox" with

which

is

the

inhibited et

rat

heart

60 (9)

muscle.

the LPL

by

% of

Triton

the

et heparin-

WR -

1339

the

so

used is

localized

This

perfusion

In

Borensztajn

Triton

LPL

of

considered of

al.

that

of

reported inhibitable

by

fraction

Activity

not about

It process

activity

(6)

only

hindlimb

P17/100

al. results

Hijlsmann

the

to

The

demonstrate

the

synthesis

Results et

that

can

the

origin.

Oscai

by

tissue.

represents

excluded.

however,

published

the

the

From

heparin in

of

the

I).

probably

interstitial

muscle

fraction

of

was

fraction, out

tissue that from

is

therefore

intracellularly.

isolated

"Calcium

can

skeletal

releasable the

site

conflicting.

located

(17)

its

P17/100 (Table

derived

enzyme

There

washed

muscle be

which

or/and

origin

literature

heparin

LPL

study

intracellular

endothelial

with

functional

of

the

having

can

criteria.

in

after skeletal

it

RESEARCH COMMUNICATIONS

mentioned

activity

bed

(17),

only

endothelial

of

emphasized

al.

releases

above

capillary

rat

Borensztajn

to

more

from

studies

the

AND 3lOPHYSlCAL

using called only

paradox

technique

was (date

not not

shown). The

maximum

correspond of

specific to

skelet.?:

Further

activities

are

skeletal

ACKNOWLEDGYENT:

required

to

of

these

enzymes

muscle

and

to

4.)

different

types

establish in

delineate

the

relative

intracellular the

TG

mechanism

This

work

was

supported

by

a grant

of

the

Forschungsgemeinschaft.

REFERENCES : 1.) Baldwin,K.M.,

3.)

lipases

the

activation.

Deutsche

2.)

various of

(1).

importance of

the

capacity

fibers

investigations

lipolysis

of

oxidative

muscle

physiological its

the

Reitman,J.S., Terjung,R.L., Winder,W.W., Holloszy,J.O. (1973) Am.J.Physio1.225, 1045-1050 Carlson,L.A., Ekelund,L.-G., Froberg,S.O. (1971) Eur.J.Clin.Invest.1, 248-254 Reitman,J.S., Baldwin,K.M., Holloszy,J.O. (1973) Proc.Soc.Exp.Biol.Med.142, 628-631 Merzel,M., Heller,M., Pinson,A. (1979) Biochim.Biophys.Acta 572, 218-224 93

of

Vol. 121, No. 1, 1984 5.)

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

!o.) 11.’

Gorin,E., Shafrir,E. (1968) Biochim.Biophys.Acta 152, 28-39 Oscai,L.B., Caruso,R.A., Uerqeles,A.C. (1982) J.Appl.Physioi.5i, 1059-1063 Severson,O.L. (1979) J.Mol.Cell.Card. 11, 569-583 Rosen,P., Budde,Th., Reinauer,H. (1981) J.Mof.Cell.Card.13, 539-550 Htilsmann,W.C., Stam,H., Breeman,W.A.P. (1982) Biochim.Biophys.Res.Commun.lo8, 371-378 Chen,R.F.. (1967) J.Biol.Chem. 242, 173-181 Cowry,O.H., Rosenbrough,N.J., Farr, A.L., Randal1,R.J.

12.)

Brecher,P.,

6.1 7.) 8.)

9.)

Biale,Y.,

(1951)

J.Biol.Chem.

193 265-275

14.) 15.)

Pyun,H.Y., Chobanian,A.V. (1977) J.Lip.Res. 18, 154-163 Wang,T.-W., Yenahan,L.A., Lech, J.J. (1977) J.Mol.Cell.Card. 9, 25-38 Strohfeldt,P., (1973) Res.exp.Med. 162, 7-16 Martin,F.C., Levi,A.J., Slavin,G., Peters,T.J.

16.)

Stam,H.,

13.)

(1983) 17.)

Eur.J.Clin,Invest.

13, 49-56

Htilsmann,W.C. (1982) Biochem.Biophys.Res.Commun.lo8, Borensztajn,J., Rone.Y.S., Kotlar,T.J.

(1376)

Bi0chem.J.

156,

94

539-543

333-340