Development of lipoprotein lipase in cultured 3T3-L1 cells

Vol. 78, No. 1, 1977

BIOCHEMICAL

DEVELOPMENT OF LIPOPROTEIN Robert

H. Eckel,

Division University Received

July

Wilfred

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LIPASE

IN CULTURED 3T3-Ll

Y. Fujimoto,

CELLS

John D. Brunzell

of Metabolism, Department of Washington, Seattle,

of Medicine Washington

1977

27,

Summary The 3T3-Ll mouse fibroblast resembles an adipocyte after reaching a confluent stage of growth. Lipoprotein lipase activity was released with heparin and was present in acetone-ether extracts of these cells. During the early post-confluent period both activities increased rapidly. A wide variation in enzyme activities was noted in subclones suggesting that spontaneous heritable change continues to take place in these cells. Since lipoprotein lipase activity was measurable before triglyceride accumulation, it may be the earliest marker of adipocyte expression in this line. This system appears to offer a unique opportunity to study the processes of cellular differentiation and fat metabolism in vitro. The 3T3-Ll

cell

tinguished

by the

of growth,

eventually

does not

occur

cells

higher

in these

lation

of triglyceride (3).

poration

More

into

of several carboxylase,

fatty

days after

the

pose tissue terized ing from

is

synthesis

membrane

exponential

growth

Green

than

the original

of fatty

acid

have reached

and is

bound

hydrolysis

These confluence

of lipoprotein

have into

line

the enzyme

transferred

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

stage

This

the rates

to affect

[

14

accumu-

by increased

occur

A further

coA

about

eight of adi-

is

characFollow-

(5).

activated endothelium

glycerol

activity

property

pH optimum

incor-

acetyl

an enzyme that

is apparently

adi-

Clacetate

enzyme changes

into

significantly

known

lyase,

lipase,

of incor-

this

(ATP-citrate

(4).

conversion

and that

that

is paralleled

to the capillary

of triglyceride

a confluent

are

and drugs

have shown

is dis-

by bromodeoxyuridine

triglyceride

3T3 cell

3T3,

(1).

shown that

by serum and by an alkaline

by the adipocyte,

adipocyte

cells

biosynthesis

synthetase).

the production

for

--et al.

by 3T3-Ll

reaching

arrested

by hormones

Mackall

mouse line

after

and is

and glucose

in

recently,

activated

established

in appearance

and Kehinde

is modulated

acid

cells

by being

the

adipocytes

triglyceride

enzymes

the

of triglyceride

palmitate

cells

from

resembling

(2).

of acetate,

pocytes

derived

accumulation

during

in stationary poration

line,

and free

upon release where

it

is

fatty

288 ISSN

0006-291X

Vol.

78,

acids

No.

BIOCHEMICAL

1, 1977

Although

(6,7).

the stromal-vascular culture

model

the potential gated

for

Van --et al. fraction

for

the

usefulness

BIOPHYSICAL

have measured

of human adipose

study

the presence

Materials

AND

of lipoprotein

of lipoprotein

lipoprotein tissue

lipase

of such an -__ in vitro lipase

RESEARCH

is

model,

COMMUNICATIONS

lipase

(8),

activity

no sustaining

available.

cell

Because

the 3T3-Ll

line

in

of

was investi

activity.

and Methods

3T3-Ll cells were obtained from Dr. Howard Green (Massachusetts Institute of Technology) and grown in Dulbecco-Vogt modified Eagle's medium supplemented with 10% fetal calf serum (Gibco) and gentamicin (50 ug/ml, Microbiological Associates). The cells were grown at 37" in a humidified atmosphere of 5% CO2 and 95% air. Medium was changed every two to three days. Confluent cells in 16 oz glass culture bottles were rinsed with phosphate buffered saline and incubated with 0.25% trypsin in phosphate buffered saline The detached cells were resuspended in medium and approximately 5 x lo6 cells dispersed into 16 oz culture bottles. Four days later, cells for lipoprotein lipase assays were harvested. Following rinsing with phosphate buffered saline, cells were removed by scraping the cell monolayer with a rubber policeman into Krebs-Ringer phosphate buffer and collected after centrifugation for 5 min at 400 X g and 4". The pellet was then resuspended in 5.0 ml KrebsRinger phosphate buffer. Additional culture bottles were rinsed with phosphate buffered saline, and cells were collected after incubation with trypsin solution and counted in a hemocytometer. Lipoprotein lipase was measured both as the activity releasable into the medium from cells incubated with heparin and as the activity present in the ammonium chloride (NH Cl) extract of an acetone-ether powder of cells. For 4 activity releasable with heparin, 1.2 ml of Krebs-Ringer phosphate buffer suspended cells were incubated with heparin (2 units/ml, Upjohn intestinal mucosa) for 45 min. The mixture was then centrifuged at 400 X g for 5 min at room temperature and 1.0 ml was taken off and incubated in a Dubnoff shaker with 0.2 ml substrate at 37" for 30-60 min. Activity in acetone-ether powders was assayed using 1.2 ml of the Krebs-Ringer phosphate buffer cellular suspension according to Nilsson-Ehle et al. (9). After drying with nitrogen, the cellular powders were homogenized in 0.4 ml of 0.05 M NH4C1 (pH 8.2) and then incubated with 0.2 ml of substrate in a Dubnoff shaker at 37" for 30-60 min. The reaction was terminated by adding 10 ml of Dole's extraction mixture and free fatty acids were extracted as outlined by PykYlistti The sub--et al. (10). strate was prepared yzing 100 ~1 of unlabeled triolein (25 mg/ml in benzene, Sigma), 0.5 ml of l[ Cltriolein (2 PCi in benzene, Amersham) and 10 ~1 egg lecithin (12 mg/ml in chloroform-methanol, 1:l) purified by W.C. Vogel by column chromatography (11). Emulsification of the triolein and lecithin was carried out with a mixture of 10% fatty acid-poor bovine serum albumin, normal human serum, 2M Tris HCl buffer and distilled water (4:1.5:5:9.5) for 100 seconds of sonification (10 seconds on, followed by 8 seconds rest for 10 cycles) using a Branson 125 Sonifier at 4". Results Enzyme activity powders

of cells

of incubation

releasable was found

(Fig.

1).

with

in 3T3-Ll The removal

heparin cells

from

and increased

of serum from

289

cells

and in acetone-ether linearly

the assay

with

decreased

length the

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

Minutes

Figure

1:

of

RESEARCH COMMUNICATIONS

Incubation

Heparin releasable and acetone-ether cellular lipase activities as a function of incubation demonstrated up to 60 minutes.

Table

I.

Heparin

lipoprotein serum.

releasable

lipase Control

and acetone-ether

activities 3T3-C

cells

powder time.

in

cellular

the presence

were

assayed

lipoprotein Linearity

is

powder

and absence

of

in the presence

of serum only. SERUM

CELL

Lipoprotein

Lipase

Heparin 3T3-Ll

+

3T3-Ll

+

3T3-C

activity

of the

presence

of serum was very

period

of time

accumulate

enzyme by 90% (Table

documenting

lipase

that

Releasable

Acids/hour/lo6

Acetone

50.3 45.5

12.0 10.5

2.3 2.3

0.9

3.8 4.0

2.0 1.7

I).

Lipolytic

in 3T3 control the

Fatty

activity

cells

enzyme is present

only

Ether

measured

confluent

cells)

for

in

the

a similar

in those

cells

that

triglyceride.

To determine tein

low

(nEq Free

activity,

if

the

stage

initial

of cellular cell

densities

290

growth

had any effect

of 5 x 104,

1 x lo5

on lipoproand 5 x lo6

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

5c

O

Figure

2:

Figure

3:

2

4

Days

Confluent

6

6

Heparin releasable and acetone-ether cellular powder lipoprotein lipase activities as a function of duration of confluent growth. On the abscissa, the points before 0 represent subconfluent cultures. Means + 1 S.D. of duplicate assays are indicated. Lipoprotein lipase activities in eleven subclones. Heparin releasable and acetone-ether cellular powder activities for each subclone are connected by a straight line.

cells/plate

(100 mm culture

harvested

by scraping

confluent

growth.

from about

Cell

0.1 x lo6 confluent

fluent

(Figure

cells

and that

Single subclones protein

per

assay

2).

were

Plastics)

of 0.25%

Minimal

present

per

assay

in subcon-

releasable

of cells

of

varied

to 3.0 x lo6

the activity

were

stages

lipase

were

powders

and cells

at different

cultures

in both

used

lipoprotein

activities

in acetone-ether

occurred

with between

growth.

isolated

activities

were

trypsin

in subconfluent

An increase

established.

lipase

Falcon

used to measure

cultures.

of confluent

cells were

numbers

present

12 and 48 hours

dishes,

in the presence

in seven-day

heparin

IO

Growth

from

a single

In these were

culture

subclones,

found

(Figure

a wide

dish

of 3T3-Ll

variability

cells

and

in lipo-

3).

Discussion Adipose sensitive lipase,

tissue

lipase, although

contains

several

phospholipase, only

the

latter

different

mono-

lipase

and diglyceride

appears

291

to be important

activities lipases in the

- hormoneand lipoprotein transfer

of

Vol. 78, No. 1, 1977

triglyceride

fatty

sue enzymatic activities forming pH is

BIOCHEMICAL

into

activity

hormone

assay

at pH 8.4,

for

the adipocyte

on triglyceride

of both the 7.0

acids

hormone

lipase

greater

activity

in the absence

The discovery

of lipoprotein

of lipoprotein

differentiation fatty

acid

in these

that

lipid.

growth

are not

chick

fibroblasts, contact

glycolytic

unique

These

is neither

cells

with

that

cells

of these

observations

a uniform

To further

and Kehinde

nor

evaluate

suggest a constant the

in adipocytes,

to adipocyte are activity cell

cell

the activity line

it

in

of these

of the

of the

rapidly

became

between

cells

between

described

of lipoprotein

292

in

in

The lipoprotein

varied

lipoprotein

characteristics

alterations

to consume more glucose

the presence

similarity

as intra-

For example,

was variable

also

of

expression,

with

line.

of

present.

now stored

cultures.

property

deve-

of adipocyte

of confluency

and activities that

In fact,

The amount

had previously (12),

further

(4).

increasing

cells

cells,

provides

later

lipase

different

(5,15).

and induction

growth

this

of 90% or

(16)

related

with

lipo-

the loss

line.

have been shown

in these

in these

in subclones

is

of triglyceride

measurable Green

cell

lipases,

lipase

cells

(optimum

change

enzyme or to this

as between

thus

on the degree

In working

as well

the other

manifestation

to occur

preferentially

accumulation

alterations

range.

Morphological

cells

cells, (17).

the

Since

measured

to this

inhibited

activities

logical

of this

in lipoprotein

replicating

any one culture

cultures.

properties

in exponential

The changes

that

lipase

in 3T3-Ll

inhibition

utilized

pathway

apparent

lipase

By per-

optimized

lipoprotein

may be the earliest

contact

the calories

cellular

than

activator,

is

tis-

reflect

lipase.

activity

lipoprotein

seems to be dependent

suggests

perhaps

lipase

appear

might

and lipoprotein

of serum favors

enzymes

of adipose

potentially

Unlike

cells.

biosynthetic

enzyme activity This

lipase

Assays

lipase)(l4).

a serum

of the adipocyte-like

lopment

lipase

lipoprotein

sensitive

requires

(10,13).

substrate

sensitive

protein

evidence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lipase were

similar

morpho-

activity

was

found

of this

different

over

a wide

enzyme activity

cultures. lipase induction

found

in 3T3-Ll

and hormonal

BIOCHEMICAL

Vol. 78, No. 1, 1977

regulation tein

tic

within

3T3-Ll

accumulation enzymes

offers

cells

and the previous

and induction

with

a unique

and fat

The localization

of the enzyme need to be determined.

lipase

eride

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

adipocyte

expression

opportunity

metabolism

of activity

to study

demonstration

of several

suggest

that

the processes

the

of triglyc-

fatty 3T3-Ll

of cell

of lipopro-

acid

biosynthe-

fibroblast

differentiation

in vitro. -~

Acknowledgment We thank Ms. Sharon AM-02456,

Ms. Martha

Kemp for AM-15312,

Kimura

preparing

for this

her

excellent

manuscript.

technical Supported

assistance

and

by NIH Grants

and HL-18687.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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293