318

Biochinzica cl Hiophysica 0 Els~vicr/North-Iiollatld

Acta.

489

(1977)

Biomedical

018-329

Press

BBA 57080

STUDIES OF THE FORMATION AND RELASE OF GLYCEROLIPIDS BY PRIMARY MONOLAYER CULTURES OF ADULT RAT HEPATOCYTES

ROBERT G. LAMB, CRAIG and HAROLD J. FALLON

K. WOOD, BARBARA

(Received March 30th, 1977) (Revised manuscript received August

lst,

M. LANDA,

PHILIP

S. GUZELIAN

1977)

Summary Primary monolayer culture of adult rat hepatocytes represents a relatively novel and potentially useful in vitro technique for studying various hepatic processes for extended periods of time. This study evaluates the potential of monolayers for studying hepatic glycerolipid biosynthesis. hepatocyte Hepatocyte monolayers synthesize ~lycerolipids from ~1,3-‘~C~glycerol and fatty acids and release them into the culture medium. Glycerolipid synthesis and release by monolayers declines 40 to 50% during the first 24 h of culture and these lower rates are maintained for at least ‘72 h. Triacylglycerol formation and release is dependent upon fatty acid concentration in the incubation medium although phospholipid is not. The release of triacylglycerol is greater in the presence of unsaturated acids (16 : 1, 18 : 1 and 18 : 2) than saturated acids (16 : 0 and 18 : 0) although saturated acids produce equal or greater amounts of intracellular triacylglycerol. Phospholipid and triacylglycerol release is reduced in monolayers exposed to colchicine for 24 h but only triacylglycerol release is lowered by the presence of cycloheximide. These results suggest release of triacylglycerol-containing lipoprotein by hepatocyte monolayers. However, we were unable to detect known apolipoprotein in the culture medium by immunologic techniques. Exposure of hepatocyte monolayers to ethanol and estradiol causes alterations in glycerolipid production and release. These changes in glycerolipid formation correlate well with corresponding alterations in glycerol kinase activity measured in monolayer homogenates. These experiments demonstrate the potential usefulness of hepatocytes in monolayer to study various aspects of glycerolipid metabolism. Their longer

Abbreviation:

MqSO,

dimrthyl

sulfoxide,

319

viability, days rather than hours, preparations (slices, homogenates, many types of experiments.

is an isolated

advantage over other in vitro hepatocytes and liver perfusion)

liver for

Introduction It is well recognized that glycerolipids are essential for various hepatic functions including; the formation of very low density lipoprotein; proliferation and repair of intracellular membranes; and the formation of biliary phospholipid. However, knowledge is still limited about the factors (hormones, enzymes, etc.) which regulate the liver’s ability to form glycerolipids. This is partially attributed to the absence of an in vitro technique which will maintain hepatic glycerolipid biosynthesis for extended periods of time and exhibit drug and hormone induced alterations in hepatic glycerolipid formation. Several techniques have been utilized previously to study hepatic glycerolipid biosynthesis including isolated hepatocytes [l-3], liver slices [ 441 and liver perfusion [ 7-83. The usefulness of these preparations in identifying regulatory factors is restricted because basic liver functions are maintained for short periods of time. The problem of short term viability can be overcome by maintaining liver cells in monolayer culture for prolonged periods of time. Nevertheless, rapidly proliferating cell lines may exhibit culture related modifications in cell metabolism [9,10]. Therefore, we have undertaken detailed studies of glycerolipid formation in non-proliferating monolayer cultures of adult rat hepatocytes [ 111. These preparations exhibit many functional [lo-141 and morphological [15] features of normal adult rat liver for periods of several days. Hence, hepatocyte monolayers should be useful in identifying factors which alter hepatic glycerolipid biosynthesis and elucidating their mechanisms of action.

Materials All lipid standards used in thin-layer chromatography and fatty acids (16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2) used in incubations were obtained from Applied Science Labs, Inc., State College, Pa. Colchicine, cycloheximide, estradiol, coenzyme A, ATP, Collagenase Type I and culture medium amino acids were purchased from Sigma Chemical Co., St. Louis, MO. Fatty acid-poor albumin Inc. and Eagle’s minimal essential (Fraction V) was obtained from Pentex, vitamins (100 times) from Grand Island Biological Co., Grand Island, N.Y. [ 1,3-‘4C]Glycerol, [1,3-‘4C]glycerol 3-phosphate and D-[ U-14C]glucose were purchased from New England Nuclear. All culture medium was prepared in our laboratory and sterilized by membrane filtration (Millipore 0.45 pm pore size). Each drug used in these studies except estradiol was dissolved in glass distilled water and sterilized by membrane filtration. Estradiol was dissolved in dimethyl sulfoxide (Me,SO) and this solution was not filtered. Aliquots of drug solutions (0.01-0.02 ml) and solvent were added to experimental and control plates respectively. There was no significant difference between the effect of water and Me,SO on monolayer glycerolipid formation and release.

320

Methods MonolaJjcr preparation Monolayers of adult rat hepatocytes were prepared as described by Bisscll et al. [lo] with slight modifications [ 12-131. Male Sprague-Dawley rats (200250 g) were subjected to a two-thirds hepatectomy [lo] and four or five days later the regenerated liver was pcrfuscd with 0.03%’ collagenase solution. The perfusion was carried out for 20-25 min at a flow rate of 17 ml/min without The softened liver was then removed and recirculation of the perfusate. incubated with 0.03% collagenase solution for 10 min at 37°C with shaking. Parenchymal cells were separated from other cellular components by centrifugation at 70 X R for 2 min. The isolated hepatocytes were washed three times in culture medium and aliquots of cells (l- 2 . 10h) were placed in 60 mm plastic tissue culture dishes containing 2.5 ml of medium [ 111. All culture dishes were precoated with rat tail collagen [ 141 24 h before cells were added. Cells were incubated 18-24 h under humidified air at 35°C. Unless stated otherwise the medium was changed at 24-h intervals after plating. The medium was similar to that described previously [ 131 except that each liter contained the following: 500 mg L-arginine, 250 mg L-histidinc, 14 mg Lisoleucine, 28 mg L-leucinc, 82 mg L-lysinr, 150 mg r1r.,-methionine, 250 mg DL-phenylalanine, 60 mg I)I,-threonine, 80 mg L-tryptophan, 26 mg L-valine and 24 mg L-proline. Glycerolipid formation by monolayers The formation of glyccrolipids and their release into the cultured medium was measured by replacing the standard medium with 2.5 mls of fresh medium containing 1.0 mM palmitoleate, 1% defatted bovine serum albumin 1161 and 0.5 mM ]1,3-‘4C]glycerol (0.4 PCi) or 5.0 mM [‘4CJglucose (0.4 PCi). The incubation with the labeled medium was carried out at 35°C for 90 min before thee reaction was terminated by placing the sample on ice and removing the media. Monolayers were washed twice with phosphate buffered saline (0.8% NaC1/0.02% KC1/0.135% sodium phosphate, pH 7.4). The cells were removed by scraping and placed in 4 ml of phosphate buffered saline. These cells were then sonicated for 20 s at a setting of 5 on a Branson Sonifier before aliquots were removed for protein determinations as described by Lowey et al. [17]. Media samples were centrifuged at 700 X g for 2 min to remove cells. Both media and cell glycerolipids were extracted by a modified Bligh and Dyer procedure [ 181. Chloroform lipid extracts were applied to small silicic acid columns and the neutral lipid and phospholipid fractions were separated by eluting first with 100% chloroform and then 1 : 1 CHCl,/MeOH containing lo%> 1 N HCl 1191. Individual species of neutral lipids [ 18,20,21] and phospholipids [20-221 were identified by thin-layer chromatography. Incorporation of [ 1,3-‘4C]glycerol and [ “C]glucose into cellular and media glycerolipid was taken as a measure of glycerolipid formation and release respectively. Enzyme assays Cells from washed plates were scraped into five ml of buffer 0.225 M sucrose, 0.05 M Tris . HCl pH 7.5 and 2.5 mM EDTA.

containing These cells

were sedimented by ~entrifugation at 700 X g for 2 min, buffer removed by aspiration and the cells resuspended in 2 ml of the sucrose/Tris/EDT_A buffer. The cells were homogenized for 15 s at a setting of 4 on a Technicon Homogenizer. This cell homogenate was used as the enzyme source for measuring glycerol kinase and sn-glycerol-3-phosphate acyltransferase activity. Glycerol kinase activity was measured by a modification of the procedure described by Newsholme et al. [23]. In the standard glycerol kinase assay, 3.6 mM ATP, 18.0 mM NaF, 1.4 mM dithiothreitol, 3.0 mM MgCl, 50 mM Tris . HCl pH 8.3, 1.0 mM EDTA and 0.6 mM ]1,3-‘4C]glycerol (0.2 FCi) were incubated with cell homogenate (0.05-0.10 mg protein) in a total volume of 0.35 ml. The reaction was initiated by addition of cell l~omogenate and terminated after 5 min incubation at 35°C with shaking, by addition of 0.1 ml ethanol and heating in boiling water for 2 min. Radioactivity in glycerol phosphate was collected on DEAE-cellulose disks and measured as described by Newsholme et al. [ 231. The control incubations were stopped at zero time, and radioactivity associated with the DEAE-cellulose disks at zero time subtracted from that of the five minute incubation. The formation of sn-glycerol 3-phosphate under these incubation conditions was linear with respect to incubation time and the amount of homogenate protein. sn-Glycerol-3-phosphate acyltransferase activity was measured by incubation of cell homogenates (0.05-0.1 mg protein) with 0.5 mM sn-[ 1,3-‘4C]glycerol 3-phosphate (0.1 PCi), 0.6 mM ammonium palmitate, 3.3 mM ATP, 0.7 mM dithiothreitol, 0.05 mM CoA, 3.3 mM MgCl and 1.25 mg albun~in in a total volume of 0.4 ml. Reactions were started by addition of homogenate, incubated for 15 min at 37°C with shaking and terminated by adding 3 ml of 1 : 2 chloroform/methanol. Lipids were extracted and identified as described previously for monolayers. These incubation conditions were optimum for total glycerolipid formation and identical to those described previously for rat liver homogenates [ 20,241, Results Fig. 1 shows the effect of incubation time on the incorporation of [ 1,3-‘4C]glycerol into gly~erolipids by 24 h old hepatocyte monolayers under the standard incubation conditions. Triacylgly~erol, phosphatidyl~holine and phosphatidyl ethanolamine formations were linear for 90 min. Under these incubation conditions, the rate of total glycerolipid formation ranged from 0.2 to 0.4 nmol/min/mg protein. This rate of glycerolipid formation is similar to that reported for normal rat liver homogenates [ 201. The only neutral lipids formed from [ 1,3-“Cl glycerol were diacylglycerol and triacylglycerol, with diacylglycerol representing less than 10% of the labeled neutral lipid formed at 90 min. Diacylglycerol radioactivity was constant after the first 10 minutes of incubation. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidate comprised 79, 18 and 3% respectively, of the phospholipid formed during 90 minute incubations. Freshly isolated hepatocytes incubated under these same conditions exhibited identical characteristics with respect to the type and percentage composition of neutral lipid and phospholipid formed and the time course of glycerolipid formation although the rate of formation was about 40%

322

Fig.

1.

Incorporation

labeled release mean and

of

glycerolipid were i

S.E.M.

medium

into

measured of

three

[1,3the

in

24 h

srparate

glycerolipids,

I,hatidylethanolamine),

TG PA

“Clglycerol

culture old

into

medium monolayers

cellular

as a function

as described

determinations. (triacylglycerol).

(phosphatidic

acid)

neutral

The DG and

under

following

(diacylglycerol), PI,

total

lipid

of incubation

and time.

Methods.

abbreviations PC

phospholip;d Glyccrolipid Each were

point used

(phosphatidylcholine).

and

release

formation

of and

represents

the

to designate

cell

PE

(phos-

phospholipids).

greater than is shown in Fig. 1 for 24 h old hepatocyte monolayers. These results are different from those reported by Sundler et al. [3] who described accumulation of diacylglycerol in similar incubations of isolated rat hepatocytes. The release of labeled triacylglycerol and phospholipid into the cell medium is shown in Fig. 1. More than 90% of the small amount of radioactive glycerolipid appearing in the medium was triacylglycerol and phosphatidylcholine. The time course of phospholipid release was linear throughout the incubation period, although triacylglycerol release showed an initial 30 min lag before becoming linear. This observation suggests that phospholipid and triacylglycerol release are independent processes. Exposure of monolayers to cycloheximide (1 . lo-’ M) for 24 h significantly reduced triacylglycerol but not phospholipid release (Table I). However, exposing monolayers to colchicine (1 . lo-” M) for 24 h decreased the release of both phospholipid as triacylglycerol (Table I). Neither cycloheximide or colchicine reduced cellular phospholipid or triacylglycerol formation. These results suggest that both triacylglycerol and phospholipid release involves the microtubular system but only triacylglycerol release is dependent upon protein synthesis. This also raises the interesting possibility that phospholipid release is a measure of biliary phospholipid output since Gregory et al. [25] has shown that colchicine blocks biliary phospholipid output in perfused livers. The density class of lipids released into the media was studied by differential ultracentrifugation according to Have1 et al. [26]. Aliquots (0.5 ml) of the various density fractions prepared from cell media were removed, the lipids extracted, and radioactivity determined in triacylglycerol and phospholipid. Under these conditions all glycerolipid radioactivity was associated with the

323 TABLE THE

I INFLUENCE

LIPIDS

FORMED

OF

VARIOUS

FROM

AGENTS

ON

[1,3-14C]GLYCEROL

THE

FORMATION

BY HEPATOCYTE

AND RELEASE

2 h after freshly isolated hepatocytes had been plated. drugs were added to produce concentration. After the monolayers had been exposed to the appropriate agents biosynthesis was measured as described under Methods. Each point represents the (nmol glycerol incorporated/mg protein) f S.E.M. of 3-5 separate experiments in tion was done in triplicate. Control values for cellular and medium triacylglycerol 18.34. 1.10, 7.79 and 0.47 respectively. Addition

COllCIl. (M)

“b control ? S.E.M. ~_~ --__

Cell

None Cycloheximide Colchicine Estradiol Ethanol * 1’ < 0.01 ** I’ < 0.05

_ 1 1 1 1

10-S . 10-S 10-S 10-3

level of significance level of significance

100 108+ 135 189 155

___ + 10 9 ? 14 t 13 * i 12 *

the indicated medium for 24 h, glycerolipid % control of the mean which each determinaand phospholipid were

_____

Triacylglycerol

__-

OF GLYCERO-

MONOLAYERS

Phospholipid Mvdla

Celi

100 i 13 392 5* 202 5* 177 2 13 * 44? 15 **

loo? 9 95? 6 137 + 15 179?15’ 190 ? 14 *

____

Media _~..~~_ 100 105 61’ 111 81

? 15 ? 13 8** f 9 + 12

from control. from control.

albumin fraction. When rat serum VLDL * was added as carrier; however, 50% of the triacylglycerol radioactivity was associated with the VLDL fraction. This might indicate an exchange of triacylglycerol from albumin to VLDL. The residual labeled triacylglycerol and all of the released phospholipid remained associated with albumin. Media samples from 15-20 plates on two separate occasions were combined and condensed by pressure dialysis packed in ice and shipped to NIH ** for analysis of lipoprotein and apoprotein content. No rat apolipoprotein (B, A, C3 and arginine rich ***) could be detected by the double diffusion immunologic techniques used and the only protein identified in the media was rat liver albumin. At present, it is not clear why the triacylglycerol released into the medium by hepatocyte monolayers is not bound to VLDL as described by other investigators using isolated hepatocytes [l] and hepatocyte suspensions [ 271. Perhaps, the amount of lipoprotein released into the medium by hepatocyte monolayers is too small to measure by currently available techniques. Figs. 2 and 3 show the effects of increasing concentrations of glycerol and palmitoleate on glycerolipid production and release. Maximum rates of production and release were obtained at 0.5 mM glycerol. Triacylglycerol formation and release into media also was markedly increased by the addition of palmitoleate. Although triacylglycerol synthesis increased through 2.0 mM palmitoleate, release into the medium was maximum at 1.0 mM and was reduced at higher concentrations. Cellular phospholipid synthesis and release was maximum at 1.0 mM palmitoleate and the effect of fatty acid level on phospholipid formation was much less than for triacylglycerol. * Very low density lipoprotein (VLDL) was isolated according to Have1 et al. C251 1.006. * * Kindly performed by Dr. Peter Herbert. Dr. Lloyd Henderson and Robert Heinen. *** Nomenclature defined in article by Eisenberg and Levy [40].

at a

density

of

324

Fig. 2. Effect of increasing medium glycerol concentrations on the formation and release of monolayer glycerolipids. The rate of cellular triacylglycerol (TG) and phospholipid (PL) formation and release into the medium was determined as described under Methods. All determinations were made in 24 h old monolayers that were incubated with the standard [1.3- 14C]glycerol incubation medium for 90 min. Each point represents the mean + S.E.M. of three separate determinations.

Table II shows the comparative effect of several fatty acids (1.0 mM) on the incorporation of [ 1,3-14C]glycerol into cellular triacylglycerol and phospholipids and their release into the medium. “Relative rate” refers to the incorporation of radioactive glycerol into the indicated lipid in comparison with incubations containing all the standard incubation components except fatty acid. The latter is assigned a value of 1.0. The results indicate that triacylglycerol formation and release is influenced by the type of fatty acid present in the incubation medium. Palmitate was the most active in cell triacylglycerol production, although release of triacylglycerol into medium was greater for 16 : 1, 18 : 1 and 18 : 2. In contrast, phospholipid synthesis and release exhibited only modest variation in response to the different fatty acids.

I

I

,‘.I

NH,,,. ,;,I.

Fig. 3. Effect of increasing medium palmitoleate concentrations on the incorporation of [1,3-14C1glycerol into cellular phospholipids (PL) and triacylglycerol (TG) and release of these labeled glycerolipids into the incubation medium. All determinations were done in 24 h old monolayers under conditions described under Methods. Each value is the mean ? S.E.M. of three separate determinations.

325

TABLE

II

THE

EFFECT

RELEASE

OF

OF

THE

ADDITION

GLYCEROLIPIDS

OF

VARIOUS

FORMED

FATTY

FROM

ACIDS

ON

THE

[1,3-‘4CJGLYCEROL

BY

PRODUCTION

AND

HEPATOCYTE

MONO-

LAYERS The

indicated

fatty

Glyccrolipid

acid

formation

Each

point

done

in triplicate.

represents

Fatty

was and

the

Rrlativr

added release

mean

rate

to

value

(nmol

the

was

incubation

mrasured

of

at least

glycerol

in

medium 24

2 separate

incorp/mg

h old

to

give

a final

monolayers

experiments

concentration

as described

in which

each

uf under

1.0

determination

was

protein) _____.._ ____ ~__

acid Cells

_ _. .~~_

Mrdia

Triacvlalvwrol NOYX?

mM.

Methods.

Ph~~sl~h~.~lipid ~_- -___-__

1 .o

Triacylslvcerol

Ph[)sph~~li~)~d

1.0

1.0

16:O

13.8

2.6

9.8

1.4

16:l

10.6

2.1

17.8

2.2

1.0

18:0

5.8

I.9

4.0

1.3

18:l

8.0

1.7

14.9

1.3

18:2 -~

1.0

4.2

28.1 --

1.9

Mixtures of these fatty acids did not increase glycerolipid production or release more effectively than the individual fatty acids at comparable total concentrations. Similar results were obtained when fatty acids were compared at 0.1 and 0.5 mM concentrations. The stimulatory effect of p~mitoleate on glycerolipid production and release and the acceleration of triacylglycerol release in the presence of unsaturated fatty acids has been reported previously in isolated hepatocytes [ 31 and liver perfusion studies [ 81. The incorporation of [14C]glucose into glycerolipids by hepatic monolayers also was studied. Formation of glycerolipid from [ 14C] glucose (5-10 mM) was lower (0.05-0.1 nmol/min/mg) than the rate using 0.5 mM [1,3-‘4C]glycerol (0.2-0.4 nmol/min/mg). The influence of the age of the monolayer preparation on the rate of glucose incorporation into glycerolipids was similar to that CELL.

I

o-t

' 24

0

MFDiA

I

a 72

48

0 t L----J-._..._;i.cl 24

72

Plate Age (hours) Fig.

4.

S.E.M. cell

Effect of

neutral

determined

of

values lipid

monolayer obtained (NL)

as described

age

on

glyceroiipid

in several

and

different

phospholipid

under

Methods.

(PL)

formation experiments. and

media

and The

release.

Each

incorporation

triacyklyceroi

(TG)

point of

represents

the mean

~1,3-14C181ycernl

and Phospholipid

*

into 1PL)

was

326

of glycerol (Fig. 4). Differences in the rates of glycerolipid formation from glucose and glycerol may be related to intracellular pool sizes of substrates or iI~termediates in the glycolytic pathway. One advantage of monolayers over other in vitro techniques used for measuring cellular glycerolipid formation is the maintenance of many basic liver functions for 3-5 days rather than hours. However, changes in the rate of these functions may occur with time. Fig. 4 shows the effect of monolayer age formation decreased on glycerolipid formation and release. Triacylglycerol rapidly within 24 h and then declined slowly during the next 48 h. Phospholipid synthesis decreased slowly and in a linear fashion throughout the 72 h period. The rapid decline in glycerolipid biosynthesis from [ 1,3-‘4C]glycerol during the first 24 h of culture may be explained by the marked decrease in glycerol kinase and sn-glycerol-3-phosphate acyltransferase activities observed during this period (Table III). This marked drop in glycerolipid production (Table I) and glycerol kinase activity (Table III) is reduced by exposing monolayers to cstradiol. Hepatocyte monolayers were exposed to various agents for 24 h prior to measuring the incorporation of [ 14C]glycerol into glycerolipids. Table I shows that monolayers exposed to ethanol (1 mM) and estradiol (10 PM) showed increased capacity to incorporate [ 1,3-‘“C]glycerol into triacyl~lycerol and phospllolipid. The data in Table III (Experiment Bj demonstrates that glycerol kinase but not sn-glycerol-3-phosphate acyltransferase activity was increased in monolayer homogenates of cells exposed to ethanol and estradiol. The results in Table III (Experiment A, B), Table I and Fig. 4 suggest that under these TABLE

III

EFFECT AND

OF

FERASE

AGE

Glycerol layers

kinase under

that

after

the

were

and 0,

monolayer

represents

in triplicate

from

the

OF

AND KINASE

HEPATOCYTE

12,

In 24

were

48

to

homogenates mean

A the

h old.

plated

prepared

of at least

were activity

measured was

B sufficient

indicated and

the

ethanol

in which

and

enzyme each

homogenates

in homogenates e&radio1

concentration.

appropriate

activity

enzyme

were

After

24

nmoI!min/mg

2 h

h of

drug Each

was measured

protein

2 S.E.M.

Glycerol

Giycerolphosphatr

kinase

acvltransferase ___~._

-

-.

.~

A

0

_

12

6.41

? 0.59

0.34

i 0.06

2.85

i 0.26

*

0.17

+ 0.01

**

24

_

1.81

i 0.22

*

0.15

? 0.01

*

48

-

1.34

? 0.22

*

0.12

? 0.01

*

0.20

_+ 0.02

B

24

Control

24

Ethanol

24

Estradiot

* P G 0.01 * *

added

measured.

activity

as

of mono-

(Mt

~~

Experiment

ETHANOL

ACYLTRANS-

plates.

(h)

Expernnent

TO

in monolayer

measured

medium

3 experiments

Addition

ltIt>~