lipoprotein lipase activity in cultured macrophage cell line j7742 and its increase in variants...

58 Biochimica et Biophysica Acta. 762 (1983) 58-66 Elsevier Biomedical Press

BBA 11116

L I P O P R O T E I N LIPASE ACTIVITY IN CULTURED M A C R O P H A G E CELL LINE J7742 AND ITS INCREASE IN VARIANTS D E F I C I E N T IN ADENYLATE CYCLASE AND CYCLIC A M P - D E P E N D E N T P R O T E I N KINASE

RAPHAEL N. MELMED, GIDEON FRIEDMAN, TOVA CHAJEK-SHAUL, OLGA STEIN and YECHEZKIEL STEIN *

Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital and Department of Experimental Medicine and Cancer Research, Hebrew University .Hadassah Medical School, P. 0., Box 12000, Jerusalem (Israel)

(Received July 14th, 1982) (Revised manuscript received October 8th, 1982)

Key words: Lipoprotein lipase," Adenyl cyclase," Protein kinase," Enzyme deficiency," (Macrophage)

Three macrophage cell lines, J7742, CT 2 and JTI-I I were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCI. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT 2 line which is adenylate cyclase deficient while that in the J 7 H 1 line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30-40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50-70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic A M P in the regulation of lipoprotein lipase activity use was made of dibutyryi cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J7742 line but only dibutyryl cAMP was active in the CT 2 line (adenylate cyclase deficient). In the JTl-I l (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein Upase.

Introduction

Lipoprotein lipase is the key enzyme in the metabolism of plasma chylomicrons and very low density lipoproteins and acts principally at the luminal surface of vascular endothelium. Synthe- sis, secretion and regulation of lipoprotein lipase

* To whom correspondence should be addressed. Abbreviations: IBMX, methyl isobutylxanthine; SV40, simian virus 40.

0167-4889/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

have been studied in cultured cells such as rat heart cells [1-4], preadipocytes [5,6] and clones of the 3T3 line, which are capable of conversion to adipocytes [7,8]. In the cultured rat heart cells production of lipoprotein lipase was shown to be stimulated by corticosteroids [3], while in isolated fat pads [9] and in cultured 3T3-L1 cells and in adipocytes it was stimulated by insulin [8,10]. Re- duction of lipoprotein lipase activity in adipocytes in vitro was observed during activation of intracellular neutral triacylglycerol hydrolase by the cyclic AMP-protein kinase system [11] and after

59

addition of adrenaline and theophylline to the incubation medium [9,12,13]. However,. addition of cyclic AMP-dependent protein kinase to lipoprotein lipase isolated from adipose tissue did not result in deactivation of the enzyme [14]. Recently, secretion of lipoprotein lipase by a macrophage line, J774, has been reported [15] and these cells were shown to also contain a cholesterol esterase, which could be activated by addition of cAMP-dependent protein kinase to the enzyme present in the 40 000 x g supernatant fraction [16]. The avail- ability in our laboratory of the two variant macrophage cell lines derived from J7742 [17], one deficient in adenylate cyclase, CT 2, and the other in cyclic AMP-dependent protein kinase, J7H 1, permitted us to investigate the possible role of cyclic AMP in the regulation of lipoprotein lipase in the intact cell.

Materials and Methods

Cells. The J7742 variant [17] of the mouse lymphosarcoma-derived macrophage cell line originally isolated by Ralph and Nakoinz [18] was kindly given us by Drs. R.D. Berlin and J. Oliver, University of Connecticut Health Center. The two variant lines derived from this cell, one deficient in adenylate cyclase, CT 2 and the other in cyclic AMP-dependent protein kinase, J7H~ were very generously provided by Drs. Ora Rosen and B. Bloom, Albert Einstein College of Medicine, Yeshiva University. These variant cells have been previously defined with respect to their biochemi- cal defects and behavioral characteristics [19,20].

Unless otherwise stated the cells were grown in Modified Eagle's Medium supplemented with glutamine and glucose and 20% horse serum (heat inactivated). Gentamycin and ampicillin were added to this medium.

Determination of lipoprotein lipase activity. The enzyme activity was determined on aliquots of medium and homogenates of cells which had been released from the petri dish with a rubber police- man in 0.5-1 ml 0.025 M NH3/NH4C1 buffer (pH 8.1) containing 1 U / m l heparin (Evans Medi- cal Co., Liverpool, U.K.). The assay system con- sisted of 0.1 ml cell homogenate (40-80 /~g protein) or 0.1 medium and 0.1 ml substrate, prepared according to Ni l s son- Ehle and Schotz [21]. In-

cubations were carried out at 37°C for 60 min. The reaction was stopped by addition of m e t h a n o l / c h l o r o f o r m / h e p t a n e (1.4 : 1.25 : 1, v / v / v ) and the extraction of fatty acids were performed according to Belfrage and Vaughan [22] as modified by Nilsson-Ehle and Schotz [21]. En- zyme activity was calculated according to the for- mula of Nilsson-Ehle and Schotz [21] and was expressed as nmol free fatty acid released/mg cell protein per h.

To measure the effect of 1 M NaC1 and the serum dependence of the lipolytic activity, cell homogenate and medium were preincubated in the presence of 1 M NaC1 for 10 min at 27°C, or with substrate in which serum was replaced by 4% bovine serum albumin.

Determination of cyclic AMP. Cyclic AMP was determined by a sensitive competitive protein binding assay [ 19].

Protein synthesis. Protein synthesis was assayed in each of the three cell lines under the following identical conditions. 10 /~Ci [3H]leucine [50.4 Ci /mmol, New England Nuclear] was added to a 30-ml suspension of each line (1.5.106 cells/ml) in full medium. The suspension was then rapidly divided into four 7 ml aliquots to which were added one of the following reagents, dibutyryl cAMP 0.29 mM, IBMX 1.0 mM or cholera toxin 1 /~g/ml (final concentration). All three lines had been exposed to ganglioside GM 1 the day before the experiment to ensure cholera toxin binding.

The suspensions were incubated at 37°C in a 5% CO 2, 95% air atmosphere and duplicate 1 ml ~amples taken at hourly intervals for 3 h. [3H]Leucine incorporation into trichloroacetic acid-precipitable protein was then determined in a standard manner.

Preparation and labeling of chylomicrons. Mesenteric lymph duct chylomicrons were isolated after intraduodenal infusion of Intralipid [23] and were labeled with 25 ~tCi of [1-14C]palmitic acid complexed to albumin. Labeling of the chylomicrons with [3H]cholestery! linoleyl ether was carried out as described before [24]. To determine the hydrolysis of chylomicron triacylglycerol and the uptake of cholesteryl linoleyl ether by t h e macrophage cell lines, labeled chylomicrons were added to the culture medium, containing 4% bovine serum albumin instead of

60

horse serum, to give 100 ~g triacylglycerol/ml. At the end of 3 h incubation the medium was collected, and an aliquot was taken for radioactivity determination and the rest was extracted according to Bligh and Dyer [25]. The cell layer was washed [24] and incubated at 37 ° for 5 min with 0.05% trypsin in Versene buffer [26]. Following inactivation of the trypsin by addition of serum- containing medium, the cells were separated by centrifugation and were washed twice by suspend- ing in 4 ml phosphate-buffered saline and re- centrifuging. The pellet was removed and used for the determination of radioactivity, after extraction of the lipids according to Folch et al. [27], and for protein determination according to Lowry et al. [28] using bovine albumin as standard. The lipid composition of the labeled media before and after incubation with cell cultures was carried out after extraction of lipids [25] and separation by silica gel thin-layer chromatography, using TLC ready plastic sheets F1500 (Schleicher and Schtill, Dessel, F.R.G.), and hexane/diethyl ether/acetic acid (83 : 16 : 1, v / v / v ) as solvent, as described before [24]. 3H and 14C radioactivities were determined in a /3 scintillation spectrometer (TRI-CARB2660). The percentage of lipolysis of chylomicron triacylglycerol was calculated from the amount of the label found in medium fatty acid plus the label in cells divided by the total radioactivity added to the medium.

Reagents. Trypsin, N 6, O2"-dibutyryladenosine 3',5'-cyclic monophosphoric acid (dibutyryl cAMP) and IBMX were purchased from Sigma, St. Louis, MO. Cholera toxin was purchased from Makor Chemicals Ltd., Jerusalem, Israel.

Materials. [1-t4C]palmitic acid (spec. act. 59 Ci /mol) and glycerol tri[9,10(n)- 3 H]oleate (spec. act. 544 Ci /mol) were obtained from the Ra- diochemical Centre, Amersham, U.K. Culture media were obtained from GIBCO, Grand Island, New York. Heparin, Thrombolique was obtained from Organon, Oss, Holland.

Ganglioside GMt was a gift from Prof. S. Gatt, Department of Biochemistry, Hebrew University- Hadassah Medical School, Jerusalem, Israel. Bovine serum albumin fraction V was obtained from Sigma.

Results

Three macrophage cell lines were compared with respect to synthesis and secretion of lipoprotein lipase. In all cell lines about 80% of all lipase activity was found in the medium. The lipolytic activity both in the cells and in the medium, when compared per mg cell protein, was highest in the mutant CT 2 line. Cultivation in serum-free medium enhanced the secretion of lipolytic activity in the wild type and in the J7H~ cell line by 45-50%, but no increase was seen in the CT 2 line. The experiments shown in Table I were carried out on cells cultured for 24 h. We then studied the effect of time in culture on the synthesis and release of lipolytic activity. The highest activity, 507 nmol fatty acid released/mg cell protein per h, was seen with the CT 2 mutant after 24 h. During the next 48 h more than a 6-fold increase in enzyme activity was seen with the CT 2 mutant and it was more than 3-fold with the J7H~ line, 233 at 24 h; 748 nmol fatty acid released/mg cell protein per h after 3 days. The lipolytic activity in the cells and medium of J7742 increased only by 50% from 186

TABLE I

COMPARISON OF SECRETION OF LIPOPROTEIN LIPASE BY THREE MACROPHAGE LINES CULTURED WITH OR WITHOUT SERUM

The cells were cultured for 24 h in modified Eagle's medium containing either 20% horse serum or without horse serum. The medium was collected, the cell layer washed with phosphate- buffered 0.15 M NaC1 and scraped into NH 3 buffer. De- termination of lipolytic activity was carried out on aliquots of medium and cell homogenates. Values are means of duplicate dishes in two separate experiments. Lipoprotein lipase activity is expressed as nmol fatty acid released/mg cell protein per h.

Cell Serum in line medium

(%)

Lipoprotein lipase activity

Expt. I Expt. I1

Cells Medium Cells Medium

J7742 20 78 258 70 250 None 84 375 91 391

CT 2 20 401 1206 395 1215 None 410 1129 416 1140

J7H I 20 93 346 89 325 None 120 527 132 590

TABLE II

C H A R A C T E R I Z A T I O N OF LIPOLYTIC ACTIVITY IN CELLS A N D M E D I U M OF T H R E E M A C R O P H A G E LINES

The cells were cultured for 48 h in modified Eagle's medium containing 20% horse serum and for an additional 24 h in serum-free medium. Assay of lipolytic activity was carried out on samples of the serum-free medium and cell homogenates. Effect of 1 M NaC1 was determined after preincubation of the enzyme-containing samples for 10 min at 27°C prior to addition of substrate. Values are the mean of duplicate dishes and duplicate determinations of enzyme activity. Lipoprotein lipase activity is expressed as nmol fatty acid re leased/mg cell protein per h.

Cell Assay Lipoprotein lipase line medium activity

Cells Medium

J7742 Complete 117 397 Without serum 10 31 With 1 M NaC1 6 18

CT 2 Complete 796 3 609 Without serum 26 58 With 1 M NaCI 19 26

J7H 1 Complete 306 1 057 Without serum 19 26 With 1 M NaC1 21 24

61

TABLE IlI

EFFECT OF HEPARIN ON LIPOPROTEIN LIPASE RE- LEASE FROM THREE M A C R O P H A G E LINES

The cells were cultures in modified Eagle's medium containing 20% horse serum without (A and B) or with heparin, 5 U / m l (C). After 24 h all media (A, B and C) were collected and lipoprotein lipase was determined in the medium (Medium 24 h) and in cells of A and C. To dishes A and B, fresh medium without (A) or with (B) heparin was added and the cells were incubated for 3 min. Lipoprotein lipase was determined on this medium (Medium 3 min) and on the cells, n.d. = non-detectable. Lipoprotein lipase activity is expressed as nmol fatty acid released/rag cell protein per h.

Cell Heparin in medium Lipoprotein lipase line activity

Cells Medium

24 h 3 min

J7742 None (A) 29 107 n.d. 5 U / m l , 3 min (B) 19 104 17 5 U / m l , 2 4 h (C) 18 219 - -

CT 2 None (A) 404 1 381 n.d. 5 U / m l , 3 min (B) 184 1 336 268 5 U / m l , 2 4 h (C) 162 4535 - -

J7Hj None (A) 125 717 n.d. 5 U / m l , 3 min (B) 84 704 75 5 U / m l , 24h (C) 86 1264 - -

to 269 nmol fatty acid released/mg cell protein per h after 3 days. To determine that the lipolytic activity which increased with the time in culture in the cells and in the medium of the three lines of macrophages is lipoprotein lipase, the cells were cultured for 72 h, the last 24 h in serum-free medium. Omission of serum (donor of apoprotein C-II) from the assay system resulted in detection of only 1-8% of the lipolytic activity measured in the complete assay system. Moreover, addition of 1 M NaCI resulted in 95-99% inhibition of enzyme activity (Table II). The same degree of inhibition by 1 M NaC1 and dependence on the presence of serum was seen with all three cell lines. These criteria permitted us to attribute the lipolytic activity detected in the three cell lines to lipoprotein lipase. Yet another characteristic of lipoprotein lipase is its releasability by heparin and this parameter was also compared in the three cell lines. Exposure of the cells to heparin (5

U / m l ) for 3 min only resulted in a 40-50% decrease in enzyme activity in the cells and the appearance of the missing activity in the medium (Table III). Cultivation of the cells in the presence of heparin for 24 h resulted in a 2- -3- fo ld increase in the lipoprotein lipase activity in the medium and a 50% decrease in cellular activity in all three lines studied.

The shortest time interval studied in this series of experiments was 24 h and in view of the known lability of the enzyme in the medium [15], it seemed important to determine whether the difference in enzyme activity in the medium of the three lines studied was due to a difference in rates of secretion or rates of degradation. As seen in Fig. 1, lipoprotein lipase activity in the medium increased as a function of time of incubation up to 8 h, at which time the level of lipoprotein lipase activity was similar to, or even higher than, that in the medium collected after 48 h of incubation. To

62

/ 300f ~ J 7 7 4 z •

E ~ p , • /

/I " ' , • /

,0o- [ , , I o.~ / ._c. '~ 0 / t r ~-5~U"-o 2

I 4 8

"o _ ~ 4000 CTa •

~ , E /q,, ~ 2GO0 / ', *

'-; ~ Iooo / :,,= o / ,~ /

t.J

~o ~ ISoo~ ~' J T H I ~ / ~ /

I ' ~ - ' o / ', •

V' "/ / r~O 0 L ' - ' / t I I I

..a ~e h 0 2 4 8

Time ( h o u r s )

Fig. l. Secretion of lipoprotein lipase by macrophage lines J7742, CT 2 (adenylate cyclase deficient) and J7H l (protein Kinase deficient). 5 ml of cell suspension in medium containing 20% serum (6.105 cells/plate) was plated in 60 mM plastic dishes. The cells were incubated at 37°C for 48 h and then the old medium was replaced with fresh serum containing medium (arrow). Duplicate 100 ~1 samples were then removed for assay of lipoprotein lipase at 2, 4 and 8 h. Lipoprotein lipase activity was determined on the old (©) and fresh (@) media.

validate the finding that the presence of more enzyme activity in the CT 2 line is not due to a slower rate of enzyme degradation, the g/2 of the enzymic activity in the medium was determined in the absence of cells. It is evident that the loss of enzymatic activity in the media of the three cell lines was quite similar and the g/: ranged between 30 and 40 min (Fig. 2).

The lipoprotein lipase activity towards its na- tive substrate, i.e., chylomicron triacylglycerol, of the three macrophage lines was also compared. The chylomicrons had been labeled biosyntheti- cally with [14C]palmitic acid and more than 85% was recovered in triacylglycerol. The chylomicrons had also been labeled with a nondegradable ana- log of cholesteryl ester, [3H]cholesteryl linoleyl ether. The hydrolysis of chylomicron triacylglycerol was 13.6, 84.9 and 62.2% in line J7742,

~ 40°0 L s 2oooI- ~ ' " '

,,. ',z.

eo Cn • • t lh'31-$

C

~, ~ 1oo g~

>, &0

20

E i

Incubation time (min)

Fig. 2. Half-life of lipoprotein lipase in serum containing media of macrophage lines. Freshly harvested medium from each of the three cell lines was incubated at 37°C in a water bath. Duplicate 100 ~tl samples were taken from each at the time intervals shown and the lipoprotein lipase activity determined. The line represents a linear regression analysis of the decaying activity. @, CT2, adenylate cyclase deficient, y = -0 .010176x + 3.471. A, protein kinase deficient, y = - 0.00955x + 3.03. II, J7742, y = - 0.00767x + 2.394.

CT z and J7H], respectively. The highest uptake of cholesteryl linoleyl ether, 6.8% of medium label, occurred also in the CT 2 cells, as compared to 0.9 and 1.3% in J7742 and J7H~, respectively. The apparent K m values determined on lipoprotein lipase released into complete growth medium of the J7742, CT 2 and J7Hi cells towards chylomicrons were similar, 1.3, 1.4 and 1.5 mM, respectiyely.

The established difference between the three cell lines studied is that the CT 2 has a low activity of adenylate cyclase non-responsive to agonist stimulation, whilst the J7H] has defective cAMP- dependent protein kinase activity. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity, the three cell lines were incubated in the presence of agents known to modulate cellular cyclic AMP levels and the activity of lipoprotein lipase was determined as a function of time. As seen in Fig. 3, addition of IBMX and cholera toxin alone or together with ganglioside GM l to the culture medium, resulted in a marked decrease of lipoprotein lipase activity by 2 h in the wild type (J7742). These agents did not

63

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- 1 0 0 1 i I i 0 2 4 8

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Fig. 3. Effect of modulation of cyclic AMP on lip•protein lipase activity secreted into the medium. A comparable number of cells from each of the three lines (8-105 cells/plate) were grown overnight on 60 mm plastic dishes containing 5 ml modified Eagle's medium with 20% horse serum. At the start of the experiment, the old medium was replaced by fresh medium and the reagents added to the following final concentrations: dibutyryl cAMP 0.29 mM, IBMX 0.1 mM, cholera toxin 1 /~g/ml. To two of the plates of each series at the time of cell plating a sterile solution of ganglioside GM 1 was added to a final concentration of 1 nM. To both plates regular fresh medium was exchanged for the GMl-enriched medium at the start of the experiment. Cholera toxin (1 ttg/ml) was added to one of the plates and the second containing the GM t primed cells was run as an additional control. The plates were incubated at 37°C in a 5% CO 2, 95% air atmosphere and duplicate 100 t~l samples taken from each plate at the timed intervals shown above. Lip•protein lipase secretion in the plate contaimng only GMt primed cells was identical to that of the untreated controls and is therefore not shown. 100% values of lip•protein lipase in the media were 138, 890 and 268 nmol fatty acid released/mg cell protein per h for the J7742, CT 2 (adenylate cyclase deficient) and J7H I (protein kinase deficient) cell lines, respectively. O . . . . . . O, dibutyryl cAMP; • . . . . . A, IBMX; zx-- - - --,'., CT; • e, C T + G M v

a f fec t the e n z y m e ac t iv i ty in the C T 2 va r i an t l ine

o v e r 8 h o f i n c u b a t i o n . H o w e v e r , as in the wi ld

type , a d d i t i o n o f d ibu ty ry l c A M P to C T z cells

r e su l t ed in an 80% dec rease in l i p • p r o t e i n l ipase

ac t iv i ty w i t h i n 8 h. T h e J 7 H 1 var ian t , de f i c i en t in

c A M P - d e p e n d e n t p r o t e i n k inase r e s p o n d e d to the

a d d i t i o n o f cho l e r a tox in wi th an in i t ia l i nc rease in

l i p • p r o t e i n l ipase ac t iv i ty , b u t this e f fec t was shor t - l i ved and was fo l l owed by a dec rea se in

e n z y m e act ivi ty , wh ich fell to 50% at 8 h. O v e r the

first 4 h o f i n c u b a t i o n d i b u t y r y l c A M P or I B M X

TABLE IV

EFFECT OF MODULATION OF CYCLIC AMP ON LIPO- PROTEIN LIPASE ACTIVITY IN THE CELLS OF THREE MACROPHAGE LINES

The conditions were as in the legend to Fig. 3. The cellular lipoprotein lipase activity was determined at the end of the experiment, i.e., 8 h after the addition of fresh medium containing the reagents. Lipoprotein lipase activity is expressed as nmol fatty acid released/mg cell protein per h.

Additions Lip•protein lipase to medium activity in cells

J7742 CT 2 J7H 1

None - 31 394 61 Dibutyryl cAMP 0.29 mM 11 88 50 IBMX 0.1 mM 17 334 47 CT 1 ~g/ml 28 336 27 CT+GM~ 1 #g/ml 14 338 31

d id n o t c h a n g e e n z y m e ac t iv i ty a p p r e c i a b l y in the

p r o t e i n k inase de f i c i en t cells, b u t it was dec reased

3 0 - 4 0 % at the 8 h in terval . T h e d a t a in Fig. 3 are

o f e n z y m e ac t iv i ty d e t e r m i n e d in the m e d i u m .

Ce l lu l a r l i p • p r o t e i n l ipase ac t iv i ty was a lso

d e t e r m i n e d at the end of the 8 h i n c u b a t i o n per iod .

As seen in T a b l e IV, ce l lu la r l i p • p r o t e i n l ipase

ac t iv i ty in the wi ld type and in C T 2 was a f fec ted in

the same m a n n e r as the m e d i u m act ivi ty , i.e., tha t

o n l y a d d i t i o n of d ibu ty ry l c A M P to the C T 2 l ine

resu l ted in a dec rease of e n z y m e act ivi ty . In the

J7H~ var ian t , r e d u c t i o n of e n z y m e ac t iv i ty in the

cel ls on ly o c c u r r e d in the p r e sence of cho l e r a

toxin . S imi la r resul ts were o b t a i n e d in a s e c o n d

e x p e r i m e n t (da ta n o t shown) .

Basal levels o f cycl ic A M P a n d r e spons iveness

o f the three cell l ines to cho l e r a tox in was

d e t e r m i n e d . Cel ls g r o w n in 20% s e r u m - c o n t a i n i n g

m e d i u m were h a r v e s t e d at ze ro t ime and 40 m i n

a f t e r the a d d i t i o n o f cho l e r a tox in (1 / x g / m l ) . T h e

levels o f cycl ic A M P at ze ro t ime were 8.0 a n d 56

p m o l / 5 . 1 0 6 cel ls in J7742 and J7H~ respect ive ly .

F o l l o w i n g cho l e r a tox in the cycl ic A M P levels rose

to 22 and 175 p m o l / 5 , l06 cells in J7742 and

J7H1, respec t ive ly .

Cyc l i c A M P levels in C T 2 were u n m e a s u r a b l e

b o t h b e f o r e and af te r cho le ra tox in (less t han 0.5

pmol ) . These resul ts a re s imi la r to those p r ev ious ly

d e s c r i b e d [ 19].

64

Incorporation of [3H]leucine into total cellular protein was determined after incubation of the J7742 cells for 2 h. In controls and in cells supplemented with dibutyryl cAMP, the protein-bound radioactivity was 22000 dpm/1 .5 .106 cells, in cells treated with cholera toxin or IBMX it was 23000 dpm/1.5 • l0 6 cells.

We therefore conclude that the reduction of lipoprotein lipase activity was not due to a general inhibition of cellular protein synthesis.

Discussion

The studies reported here were aimed principally at confirming the production and secretion of lipoprotein lipase by the three macrophage lines, characterizing the secretory behavior of these cells with respect to lipoprotein lipase, and at defining the regulatory role of cyclic AMP in this process. To this end we have used two variant macrophage cell lines, derived from the well-defined J7742 line, one (CT2) deficient in adenylate cyclase activity, the other (J7H~) deficient in cyclic AMP-dependent protein kinase. Production of lipoprotein lipase by the J774 line was previously described [15] and we here show that the lipolytic activity produced by the two variant lines fulfills the criteria of lipoprotein lipase by virtue of its inhibition by 1 M NaC1 and dependence on the presence of apolipoprotein C-II (added to the assay system in the form of serum). The lipoprotein lipase activity produced by the three cell lines is found mainly in the medium and to a lesser extent in the cells. In this respect the macrophage cell lines resemble adipose tissue [10] and preadipocytes in culture [6-8] and differ from rat heart cells in culture, which produce lipoprotein lipase but release the enzyme into the medium only in the presence of heparin [1-4]. Prolonged exposure of the cultured macrophages to heparin results in a considerable enhancement of enzyme production and release, as was also previously shown in the heart cell cultures [2] and J774 macrophages [15]. It seems plausible, therefore, that the membrane associated lipoprotein lipase could fulfill a regulatory function with respect to enzyme production and that the rapid release of the enzyme in the presence of heparin interferes with such a regulatory process. Previ- ously we have demonstrated that in the rat heart

cell cultures the presence of lipoprotein lipase is required for the uptake of chylomicron cholesteryl ester [24]. Similar findings were obtained with the three macrophage lines, as the ability to internalize cholesterol linoleyl ether, the nondegradable ana- log of cholesteryl ester, was related to the lipoprotein lipase activity of the cell line. One prominent finding of the present study was that under identical conditions of culture, the highest enzymic activity, both in the medium and in the cells, was consistently found in the adenylate cyclase deficient line, the lowest in the J774 cells, while the protein kinase deficient variant was intermediate. This finding prompted a more in-depth analysis of the possible role of cyclic AMP in the regulation of lipoprotein lipase activity.

In the J774 macrophage, increase in cyclic AMP whether by addition of dibutyryl cAMP, IBMX inhibition of phosphodiesterase or cholera toxin activation of endogenous adenylate cyclase activity, is associated with profound inhibition of lipoprotein lipase activity in both medium and cells in the absence of demonstrable inhibition of general cell protein synthetic activity. In the adenylate cyclase deficient variant only dibutyryl cAMP is effective in reducing lipoprotein lipase activity as in this cell the adenylate cyclase fails to respond to cholera toxin and endogenous production of cyclic AMP is so low that IBMX has a trivial effect. Pretreatment of the J774 cells with GM~ ganglioside, the natural receptor for cholera toxin, results with time in the intercalation of the added ganglioside into the cell plasma membrane [29]. This leads to enhanced cholera toxin binding and adenylate cyclase activation and consequently produces a more profound inhibition of lipoprotein lipase activity. Pretreatment of the adenylate cyclase deficient variant with GM~ predictably fails to produce an effect on the lipoprotein lipase activity in cell or medium. In the protein kinase deficient variant, whilst the agents used to enhance cellular cyclic AMP levels all eventually produce some inhibition of lipoprotein lipase production on prolonged incubation, it is clear from studying the kinetics of lipoprotein lipase inhibition in this cell that the effects are partial and considerably delayed. This obtunded response to cyclic AMP is in keeping with the in vitro demonstration of a defective but not totally absent cyclic AMP-depen-

dent protein kinase [19]. In these cells the cAMP- dependent protein kinase has one-tenth of the cyclic AMP binding affinity and a K m ten-times greater than in the wild type, J774 [19]. The use of these variant cells thus provides substantial con- firmation of the importance of cyclic AMP as a physiological regulator of lipoprotein lipase production and secretion in these macrophage lines.

Lipoprotein lipase production and regulation in the three macrophage lines studied is strikingly similar to that of plasminogen activator in both these and other transformed or tumor cells. A study of plasminogen activator secretion by the same three macrophage lines used in this study shows that as with lipoprotein lipase, the adenylate cyclase deficient variant produces considerably more than either of the other two cells [19]. Simi- larly, the inhibition of plasminogen activator production by adenylate cyclase agonists or cyclic AMP analogues was similar to that seen in this study for lipoprotein lipase. It may be noted that plasminogen activator is also considered to be shed from the cell surface [30] and that the major cell-associated activity resides in the plasma membrane fraction [31-33]. An additional characteristic of plasminogen activator secretion by transformed or tumor cells as described, for example, with SV40-transformed 3T3 fibroblasts [34], is an apparent increase of plasminogen activator secretion with time, independent of cell density. This pattern was clearly seen in all three macrophage lines where lipoprotein lipase activity continued to rise progressively over 3 day incubation period. That this continuing production of lipoprotein lipase is not associated specifically with the per- sistent growth of the cells in culture was shown here by the enhanced production of lipoprotein lipase by the macrophages in serum-free medium where growth is totally inhibited. This impressive coincidence in many aspects of the distribution and regulation of both plasminogen activator and lipoprotein lipase strongly suggests that these two important tissue hydrolases are regulated in a highly coordinate fashion and therefore presuma- bly may fulfill an integrated biological function. There is, however, clearly a great deal more that needs to be known about each enzyme to under- stand fully the implication of this observation.

The mechanism by which cyclic nucleotides

65

might regulate lipoprotein lipase activity has not been directly addressed in this study. Since lipoprotein lipase activity isolated from adipose tissue was not affected by in vitro addition of cellular protein kinase, it was concluded that the adenylate cyclase-protein kinase system does not play a di- rect role in the deactivation of lipoprotein lipase [14]. It was therefore proposed that the fall in lipoprotein lipase activity in adipocytes exposed to adrenergic stimulation or to addition of dibutyryl cAMP is related to the activation of the so-called intracellular hormone sensitive lipase [9-14]. Thus, the decrease of lipoprotein lipase activity was considered to be due to an increase in free fatty acid released by hydrolysis of intracellular triacylglycerol [13] and repression of lipoprotein iipase synthesis by fatty acids or to a reduction in enzyme synthesis because of the shift of cellular ATP to esterification of the free fatty acid [11]. The latter possibility is not substantiated by our data as no evidence of decrease in cellular protein synthesis was found when the cells were exposed to either dibutyryl cAMP, IBMX or cholera toxin.

Acknowledgements

We would like to acknowledge the excellent technical assistance of Mrs. Aviva Schneider, Mrs. Rivka Ishai-Michaeli, Mrs. E. Bardach and Mrs. D. Harel. This work was supported in part by a grant No. 1 RO1 HL28454 from the National Institutes of Health, a grant from the Leukemia Research Foundation, Inc., Chicago, U.S.A. and the Stanley Thomas Johnson Foundation, Berne, Switzerland, to R.N.M., from the Joint Research Fund of the Hebrew University and Hadassah to G.F. and from the United States-Israel Binational Science Foundation No. BSF 1880/79 to T.C.-S.

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lipoprotein lipase activity in cultured macrophage cell line j7742 and its increase in variants...

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