Biochimica et Biophysica Acta. 1089 (1991) 83-87
83
1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 0167478191001328 BBAEXP 92247
Recombinant human interleukin-1 suppresses lipoprotein lipase activity, but not expression of lipoprotein lipase mRNA in mesenchymal rat heart cell cultures G. F r i e d m a n ~, V. B a r a k 2 T. C h a j e k - S h a u l i, j. E t i e n n e 3 A.J. T r e v e s 2, O. Stein 4 a n d Y. Stein t t Lipid Research Laboratoo', Department of Medicine B. 2 Department of Oncololo', Hadas,mh University Hospital, Jerusalem (IsraeO. ~ Laboratoire de Biochimie. Facult~ de Medicine. St. Antoine. Paris (France) and ~ Department of Experimental Medicine and Cam'er Research. Hebrew Universio,-Hadazsah Medwal School. Jermalem (Israel)
(Received 13 December 1990)
Key words: Tumor necrosis factor; Lipoprotein lipase mRNA; Cytokine: Interleukin-I; Lipoprotein lipase activity; (Rat heart cell)
The effect of human recombinant interleukin-I (IL-I) on the regulation of lipoprotein lipase (LPL) was studied in rat heart mesenchymal cell cultures. A time-dependent reduction in enzyme activity occurred with a 30% fall after I h. The suppression of enzyme activity was accompanied by a commensurate reduction in enzyme mass. The reduction in LPL activity was most prominent in the heparin releasable pool; IL-I treatment resulted in a 7.2-L~-fold decrease in the functional compartment and a 2.5-2.8-fold decrease in residual cellular activity. The effect of IL-! could he prevented by the addition of the IL-I inhibitor. However, in contradistinction to the effect of tumor necrosis factor O'NF), there was no change in LPL mRNA in cultures treated with IL-I. The present results show that the regulation of LPL in mescoehymal heart cell cultures by IL-I occurs posttranscriptionully, as has been shown in 3T3 cells |5]. The more pronounced effect on LPL activity in the functional pool suggests that IL-I treatment might have influenced also the processing a n d / o r transport of the enzyme to the cell sudace.
Introduction Regulation of lipoprotein lipase (LPL) by cytokines such as tumor necrosis factor (TNF) and interleukin-I (IL-I) has been studied extensively in an adipocyte derived cell line (3T3-L1) [1-4]. Both cytokines were shown to reduce enzyme activity and enzyme synthesis in a time related manner. However, while the effect of TNF could be related to a reduction of LPL mRNA, IL-1 caused inhibition of enzyme synthesis but did not interfere with gene transcription [5]. Observations derived from the study of a cell line were not always corroborated in primary cultures of cells. Thus, no effect of TNF on LPL activity could be demonstrated when the cytokine was added to human adipocytcs [6]
Abbreviations: rH-IL-I, recombinant human interleukin-l; TNF. tumor necrosis factor; LPL, lipoprotein lipase. Correspondence: Y. Stein. Lipid Research Laboratory. Department of Medicine B. Hadassah University Hospital. P.O. Box 12000. Jerusalem 91120, Israel.
or rat adipocytes in culture [7]. A similar discrepancy was observed when the effect of cytokines was compared in J774 macrophages and primary peritoneal macrophages. Neither TNF nor IL-I affected LPL activity in the cell line [8], but exposure of peritoneal macrophages to rTNF did result in suppression of LPL mass and activity [9]. Therefore, it seemed of interest to determine whether the different level of regulation by the two cytokines described in a cell line occurs also in primary culture. The cultures used consisted mainly of mesenchymal cells, derived from new-born rat hearts [10], which were shown to respond to TNF by a very pronounced reduction in LPL synthesis [11]. Materials and Methods
Cell cultures. F~ heart cell cultures which consisted mainly of non-beating mesenchymal cells were prepared from 2-4-day-old rat hearts, as described [10]. Cultures were used between 6-8 days after their isolation. Determination of ;ipoprotein lipase activity. The enzyme activity was determined on aliquots of medium and homogenates of cells which had been released from
84 the petri dish with a rubber policeman in 1 ml 0.025 M NH~/NH4CI buffer (pH 8.1) containing 1 unit/ml of heparin. The assay system consisted of 0.1 ml of medium, or 0.1 ml of cell homogenate (50-70/~g protein) and 0.1 ml of substrate containing labeled triolein, prepared according to the method of Nilsson-Ehle and Schotz [12]. lncubatioi s were carried out at 37°C for 45 min. The reaction was stopped by addition of methanol/chloroform/heptane (1.4: 1.25: 1, v/v) and the extraction of fatty acids was performed according to the methods of Belfrage and Vaughan [13] as modified by Nilsson-Ehle and Schotz [12]. Enzyme activity was calculated according to the formula of Nilsson-Ehle and Sehotz [12] and was expressed per mg cell protein per h. To measure the effect of 1 M NaCI on the lipolytic activity, cell homogenates or medium samples were preincubated in the presence of I M NaCI for 10 rain at 27 o C. For determination of heparin releasable lipoprotein lipase activity, the medium was collected at the end of the incubation and cultures were incubated for 3 rain at room temperature with 0.5 mi F~0 medium containing 20% serum and 5 U/ml of heparin. 0.1 ml of the medium was assayed for lipoprotein lipase activity. Partial purification of lipoprotein lipase. Partial purification of lipoprotein lipase from heart cell cultures was performed by heparin sepharose column chromatography as described previously [11]. Briefly, heart cell cultures were washed extensively, and homogenized in phosphate-buffered saline (PBS). The cell homogenate was extracted with cold diethyl ether and the resultant powder was dissolved in 10 mM Tris-HCl buffer (pH 7.4), 0.1% Triton X-100, 20% glycerol and sonicated. The sonic.ate was centrifuged for 30 min at 4 ° C at 12000 rpm in an SS-34 rotor. The supernatant was collected, applied to a beparin-Sepharose column, and eluted at 4 ° C with 2 M NaCI as described [11]. Gel electrophoresis and immunobiotting. Electrophoresis was carried out at 50 mA/slab gel for 3 h at 23°C. Gels were calibrated with molecular weight standards (Sigma). lmmunoblotting of lipoprotein lipase was performed as described [11,14]. Briefly, proteins were transferred from the sodi,~m dodecyl sulfate (SDS) gels onto nitrocellulose paper in a t:dnsblot cell apparatus (Bio-Rad). Electroblotting was carried out at 400 mA for 4 h at 23°C. After incubation of the nitrocellulose paper with goat antiserum to lipoprotein lipase for 3 h and subsequently with rabbit antigoat antiserum (BioYeda, Israel), diluted 1:50 for 1 h, the paper was washed extensively and incubated with 1"51-labeled protein A (specific activity 30/zCi/mg, Amersham, U.K.), followed by washing and air drying. Immunoreactive proteins on nitrocellulose paper were visualized by exposure to Agfa (Curix RP~) film for 4-7 days. Nucleic acid probes. Hybridization probes were prepared using plasmid pBSLPL which includes a 213 bp fragment of genomic sequences from exon 5 of the
mouse LPL gene. This fragment was isolated from a 1.3 kb Xbal fragment of LPL cDNA subcloned into pBS vector (Stratagene). Sequence analysis confirmed that the clones were identical to those published [15]. Sense and antisense probes were prepared by linearizing the constructs with an appropriate restriction endonuclease and synthesizing [ ' S ] U T P labeled RNA transcripts (Stratagene RNA Transcription Kit). Following probe preparation, the labeled transcripts were separated by gel electrophoresis, blotted onto a nylon membrane (Biotrans Nylon Membrane, ICN) and autoradiographed. Only probes which consisted of a single band with the expected size on autoradiography were used. A cloned ,8-tubulin cDNA insert [16] was used as the control probe. DNA probes were labeled by random hexanucleotide priming by using 3zP-labeled nucleotide triphosphates [17]. RNA preparation and blot hybridization analysis. Total cellular RNA was extracted from cultured rat heart cells with guanidine thiocyanate and sedimented through a cesium chloride cushion according to Chirgwin et al. [18]. The integrity of the RNA preparation was verified by denaturing gel electrophoresis [19]. The RNA was transferred to a nylon-based membrane (Biotrans Nylon Membrane, ICN) and hybridized at 55 °C with the indicated probe in 50% formamide, 50 mM phosphate buffer, 5 x SSC, 0.1% SDS, 1 mM EDTA, 0.4 mg/ml ssDNA and 2.5 x Denhardt's. Post hybridization washing was performed under stringent conditions which include 2 × SSC and 0.5% SDS at room temperature followed by 2 x SSC and 0.1% SDS at 65°C. Dot blots analysis was performed with a template manifold apparatus (Sehleicher & SehueU, Keene NH), as described [20]. The LPL mRNA signals were compared between dots based on densitometry normalized relative to the ~8-tubulin signal [21]. Analytical procedures. Protein was determined according to the methods of Lowry et al. [22], using bovine serum albumin as standard. Radioactivity was determined in a //-scintillation spectrometer (Tri-carb 2660). The scintillation fluid used was 20% Triton X100/0.005% 1,4-bis(2-(5-phenyl-oxazolyl)benzene) (POPOP)/0.4%, 2,5-diphenyl-oxazole (PPO) in toluene. Materials. Recombinant human interleukin-l/] (riLl) was obtained from Cistron Technology, Pine Brook, N.Y. Recombinant human tumor necrosis factor (rTNF), specific activity 1.0- 107 U/mg) was obtained from Cetus, Emeryville, CA. Partially purified preparation of an IL-1 inhibitor which inhibited specifically IL-1 activity, was isolated from supernatants of cultured myelomonocytic cell line (M20) as described [23,24]. Glycerol tri[9,10(n)-~H]oleate, specific activity 1 Ci/mmol was obtained from Amersham International, U.K. [a-3:P]dCTP, specific activity 3000 Ci/mmol was obtained from New England Nuclear, U.S.A. Heparin,
85
120
thrombolique was obtained from Organon, Oss, The Netherlands. Culture media and sera were obtained from Gibco, Grand Island, N.Y.
,,
Results and Discussion
~, o e01-
To study the role of human rlL-I on the activity of LPL in cultured rat mesenchymal heart cells, various concentrations of the cytokine were added to complete culture medium. As seen in Fig. 1, a 48~ decrease in cellular enzyme activity was seen after 24 h of incubation with 10 u n i t s / m i of rIL-I and almost maximal suppression occurred at a concentration of 200 u n i t s / m l of incubation medium, rIL-I had no effect on the LPL activity when added directly to the enzyme assay. The time course of the reduction of cellular lipoprotein lipase activity was studied in the presence of 100 u n i t s / m l of rIL-1. As seen in Fig. 2, a 3 0 ~ decrease in cellular LPL activity occurred at 1 h, and only 25~ of the original enzyme activity remained after 24 h of incubation. This decrease in enzyme activity was not due to a leak or secretion of the enzyme into the culture medium, since the lipoprotein lipase activity in the culture medium was 102 + 12 nmol free fatty a c i d s / h per mg cell protein in the control dishes and 57 + 9 nmol free fatty a c i d s / h per mg cell protein after treatment with rIL-1. To evaluate whether the decrease in cellular lipolytic activity in the rat heart cell cultures treated with rIL-I was the result of non:;pecific inhibition of cellular pro-
>,
120 100
a0 ..J
l
0 . - - 20[
}
0
i
0
I
I
z
I
i
,
i
6
".I
2, I
Hours
Fig. 2. Suppression of cellular lipoprotein lipase activity by rlL*! as a function of time. Conditions as in legend to Fig. I. Incubation was carried out with 100 units/ml of rlL-I. Cellular lipoprotein lipase activity in Ihe control dishes incubated in the absence of rlL-1 was 1001 :t:73 nmol of fatty acid relea:,~d/h per mg cell protein. Values are means ± S.E. of triplicate dishes, from two experiments,
rein synthesis, incorporation of [~Hlleucine into cellular proteins was studied. Rat heart cell cultures were exposed to rIL-1, 100 u n i t s / m l for 24 h, and labeled leucine was added for the last 20 min of incubation. Protein synthesis was not affected by the exposure to the cytokine (33.6 =1: 1.1; 36.8 + 7.4 d p m . 1 0 - 3 / r a g cell protein in control and IL-I treated cells, respectively). Lipoprotein lipase in the cultured mesenchymal rat heart cells is located in an intracellular and in a cell surface related functional compartment from which it can be released by heparin [25]. In the experiments presented in Table l, rat heart cell cultures were exposed to 100 u n i t s / m l of rlL-I for 24 h. Thereafter, heparin was added for 3 rain to release the cell surface
!
\
tJ
c. 5
0 601-
TABLE I Effect of rlL*l on the functional (heparin releasable) LPL activity in mesenchymal rat heart cell cultures
t ~)
Conditions: as in legend to Fig. I. The cells were cultured for 7 days ¢1 o CL
20 0
I
0
I
I
I
i
,
,
!
"..t" i
20 AO 60 80 200 riLl (units/ml )
Fig, 1. Effect of increasing concentrations of rlL-I in culture medium on cellular lipoprotein lipase activity of mesenchymal rat he,art cell cultures. The cells were cultured in F)o medium supplemented with 10¢$ fetal bovine serum and 10~ horse serum for 7 days. The medium was changed every 3 days. and the last medium change was 48 h prior to the start of the experiment. On day 7, riL-! was added to the serum containing culture medium for 24 h. At the end of incubation, the medium was collected and cellular lipoprotein lipase activity was determined on aliquots of cell homogenates. Cellular LPL activity in the control dishes incubated in the absence of rlL-1 was 1016±54 nmol of fatty acid released/h per mg cell protein. Values ~ ,-~ + S.E. of triplicate dishes from three experiment,,
in complete culture medium. On day 7. cuhures were exposed to 1OO U / m l of IL-I or to medium alone for 24 h. For the determination of heparin releasable lipoprotcin lipase, the cultures wen= incubated for 3 min with 0.5 ml F,o containing 20~[ serum and 5 unils/ml of heparin, and both medium and cells were a.~ayed for enzyme activity as described under Materials and Methods. Resuhs are means±S.E, of
triplicate dishes. Experiment No.
Additions to medium
!
none H-1 none IL-I
II
LPL activity, (nmolfatty acid released/ h per mg cell protein) cellular releasable total 608+46 317+ 5 925+31 244+14 44+12 288+18 703 + 16 310:t:! 1 1013± 21 250±17 37± 8 287±15
86
A
TABI,E II
Neutrah:atton ,] I1,-I Htdu(ed suppression of cellular hpoprotem hpaye acttrt(v tn mt~ien
83
1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 0167478191001328 BBAEXP 92247
Recombinant human interleukin-1 suppresses lipoprotein lipase activity, but not expression of lipoprotein lipase mRNA in mesenchymal rat heart cell cultures G. F r i e d m a n ~, V. B a r a k 2 T. C h a j e k - S h a u l i, j. E t i e n n e 3 A.J. T r e v e s 2, O. Stein 4 a n d Y. Stein t t Lipid Research Laboratoo', Department of Medicine B. 2 Department of Oncololo', Hadas,mh University Hospital, Jerusalem (IsraeO. ~ Laboratoire de Biochimie. Facult~ de Medicine. St. Antoine. Paris (France) and ~ Department of Experimental Medicine and Cam'er Research. Hebrew Universio,-Hadazsah Medwal School. Jermalem (Israel)
(Received 13 December 1990)
Key words: Tumor necrosis factor; Lipoprotein lipase mRNA; Cytokine: Interleukin-I; Lipoprotein lipase activity; (Rat heart cell)
The effect of human recombinant interleukin-I (IL-I) on the regulation of lipoprotein lipase (LPL) was studied in rat heart mesenchymal cell cultures. A time-dependent reduction in enzyme activity occurred with a 30% fall after I h. The suppression of enzyme activity was accompanied by a commensurate reduction in enzyme mass. The reduction in LPL activity was most prominent in the heparin releasable pool; IL-I treatment resulted in a 7.2-L~-fold decrease in the functional compartment and a 2.5-2.8-fold decrease in residual cellular activity. The effect of IL-! could he prevented by the addition of the IL-I inhibitor. However, in contradistinction to the effect of tumor necrosis factor O'NF), there was no change in LPL mRNA in cultures treated with IL-I. The present results show that the regulation of LPL in mescoehymal heart cell cultures by IL-I occurs posttranscriptionully, as has been shown in 3T3 cells |5]. The more pronounced effect on LPL activity in the functional pool suggests that IL-I treatment might have influenced also the processing a n d / o r transport of the enzyme to the cell sudace.
Introduction Regulation of lipoprotein lipase (LPL) by cytokines such as tumor necrosis factor (TNF) and interleukin-I (IL-I) has been studied extensively in an adipocyte derived cell line (3T3-L1) [1-4]. Both cytokines were shown to reduce enzyme activity and enzyme synthesis in a time related manner. However, while the effect of TNF could be related to a reduction of LPL mRNA, IL-1 caused inhibition of enzyme synthesis but did not interfere with gene transcription [5]. Observations derived from the study of a cell line were not always corroborated in primary cultures of cells. Thus, no effect of TNF on LPL activity could be demonstrated when the cytokine was added to human adipocytcs [6]
Abbreviations: rH-IL-I, recombinant human interleukin-l; TNF. tumor necrosis factor; LPL, lipoprotein lipase. Correspondence: Y. Stein. Lipid Research Laboratory. Department of Medicine B. Hadassah University Hospital. P.O. Box 12000. Jerusalem 91120, Israel.
or rat adipocytes in culture [7]. A similar discrepancy was observed when the effect of cytokines was compared in J774 macrophages and primary peritoneal macrophages. Neither TNF nor IL-I affected LPL activity in the cell line [8], but exposure of peritoneal macrophages to rTNF did result in suppression of LPL mass and activity [9]. Therefore, it seemed of interest to determine whether the different level of regulation by the two cytokines described in a cell line occurs also in primary culture. The cultures used consisted mainly of mesenchymal cells, derived from new-born rat hearts [10], which were shown to respond to TNF by a very pronounced reduction in LPL synthesis [11]. Materials and Methods
Cell cultures. F~ heart cell cultures which consisted mainly of non-beating mesenchymal cells were prepared from 2-4-day-old rat hearts, as described [10]. Cultures were used between 6-8 days after their isolation. Determination of ;ipoprotein lipase activity. The enzyme activity was determined on aliquots of medium and homogenates of cells which had been released from
84 the petri dish with a rubber policeman in 1 ml 0.025 M NH~/NH4CI buffer (pH 8.1) containing 1 unit/ml of heparin. The assay system consisted of 0.1 ml of medium, or 0.1 ml of cell homogenate (50-70/~g protein) and 0.1 ml of substrate containing labeled triolein, prepared according to the method of Nilsson-Ehle and Schotz [12]. lncubatioi s were carried out at 37°C for 45 min. The reaction was stopped by addition of methanol/chloroform/heptane (1.4: 1.25: 1, v/v) and the extraction of fatty acids was performed according to the methods of Belfrage and Vaughan [13] as modified by Nilsson-Ehle and Schotz [12]. Enzyme activity was calculated according to the formula of Nilsson-Ehle and Sehotz [12] and was expressed per mg cell protein per h. To measure the effect of 1 M NaCI on the lipolytic activity, cell homogenates or medium samples were preincubated in the presence of I M NaCI for 10 rain at 27 o C. For determination of heparin releasable lipoprotein lipase activity, the medium was collected at the end of the incubation and cultures were incubated for 3 rain at room temperature with 0.5 mi F~0 medium containing 20% serum and 5 U/ml of heparin. 0.1 ml of the medium was assayed for lipoprotein lipase activity. Partial purification of lipoprotein lipase. Partial purification of lipoprotein lipase from heart cell cultures was performed by heparin sepharose column chromatography as described previously [11]. Briefly, heart cell cultures were washed extensively, and homogenized in phosphate-buffered saline (PBS). The cell homogenate was extracted with cold diethyl ether and the resultant powder was dissolved in 10 mM Tris-HCl buffer (pH 7.4), 0.1% Triton X-100, 20% glycerol and sonicated. The sonic.ate was centrifuged for 30 min at 4 ° C at 12000 rpm in an SS-34 rotor. The supernatant was collected, applied to a beparin-Sepharose column, and eluted at 4 ° C with 2 M NaCI as described [11]. Gel electrophoresis and immunobiotting. Electrophoresis was carried out at 50 mA/slab gel for 3 h at 23°C. Gels were calibrated with molecular weight standards (Sigma). lmmunoblotting of lipoprotein lipase was performed as described [11,14]. Briefly, proteins were transferred from the sodi,~m dodecyl sulfate (SDS) gels onto nitrocellulose paper in a t:dnsblot cell apparatus (Bio-Rad). Electroblotting was carried out at 400 mA for 4 h at 23°C. After incubation of the nitrocellulose paper with goat antiserum to lipoprotein lipase for 3 h and subsequently with rabbit antigoat antiserum (BioYeda, Israel), diluted 1:50 for 1 h, the paper was washed extensively and incubated with 1"51-labeled protein A (specific activity 30/zCi/mg, Amersham, U.K.), followed by washing and air drying. Immunoreactive proteins on nitrocellulose paper were visualized by exposure to Agfa (Curix RP~) film for 4-7 days. Nucleic acid probes. Hybridization probes were prepared using plasmid pBSLPL which includes a 213 bp fragment of genomic sequences from exon 5 of the
mouse LPL gene. This fragment was isolated from a 1.3 kb Xbal fragment of LPL cDNA subcloned into pBS vector (Stratagene). Sequence analysis confirmed that the clones were identical to those published [15]. Sense and antisense probes were prepared by linearizing the constructs with an appropriate restriction endonuclease and synthesizing [ ' S ] U T P labeled RNA transcripts (Stratagene RNA Transcription Kit). Following probe preparation, the labeled transcripts were separated by gel electrophoresis, blotted onto a nylon membrane (Biotrans Nylon Membrane, ICN) and autoradiographed. Only probes which consisted of a single band with the expected size on autoradiography were used. A cloned ,8-tubulin cDNA insert [16] was used as the control probe. DNA probes were labeled by random hexanucleotide priming by using 3zP-labeled nucleotide triphosphates [17]. RNA preparation and blot hybridization analysis. Total cellular RNA was extracted from cultured rat heart cells with guanidine thiocyanate and sedimented through a cesium chloride cushion according to Chirgwin et al. [18]. The integrity of the RNA preparation was verified by denaturing gel electrophoresis [19]. The RNA was transferred to a nylon-based membrane (Biotrans Nylon Membrane, ICN) and hybridized at 55 °C with the indicated probe in 50% formamide, 50 mM phosphate buffer, 5 x SSC, 0.1% SDS, 1 mM EDTA, 0.4 mg/ml ssDNA and 2.5 x Denhardt's. Post hybridization washing was performed under stringent conditions which include 2 × SSC and 0.5% SDS at room temperature followed by 2 x SSC and 0.1% SDS at 65°C. Dot blots analysis was performed with a template manifold apparatus (Sehleicher & SehueU, Keene NH), as described [20]. The LPL mRNA signals were compared between dots based on densitometry normalized relative to the ~8-tubulin signal [21]. Analytical procedures. Protein was determined according to the methods of Lowry et al. [22], using bovine serum albumin as standard. Radioactivity was determined in a //-scintillation spectrometer (Tri-carb 2660). The scintillation fluid used was 20% Triton X100/0.005% 1,4-bis(2-(5-phenyl-oxazolyl)benzene) (POPOP)/0.4%, 2,5-diphenyl-oxazole (PPO) in toluene. Materials. Recombinant human interleukin-l/] (riLl) was obtained from Cistron Technology, Pine Brook, N.Y. Recombinant human tumor necrosis factor (rTNF), specific activity 1.0- 107 U/mg) was obtained from Cetus, Emeryville, CA. Partially purified preparation of an IL-1 inhibitor which inhibited specifically IL-1 activity, was isolated from supernatants of cultured myelomonocytic cell line (M20) as described [23,24]. Glycerol tri[9,10(n)-~H]oleate, specific activity 1 Ci/mmol was obtained from Amersham International, U.K. [a-3:P]dCTP, specific activity 3000 Ci/mmol was obtained from New England Nuclear, U.S.A. Heparin,
85
120
thrombolique was obtained from Organon, Oss, The Netherlands. Culture media and sera were obtained from Gibco, Grand Island, N.Y.
,,
Results and Discussion
~, o e01-
To study the role of human rlL-I on the activity of LPL in cultured rat mesenchymal heart cells, various concentrations of the cytokine were added to complete culture medium. As seen in Fig. 1, a 48~ decrease in cellular enzyme activity was seen after 24 h of incubation with 10 u n i t s / m i of rIL-I and almost maximal suppression occurred at a concentration of 200 u n i t s / m l of incubation medium, rIL-I had no effect on the LPL activity when added directly to the enzyme assay. The time course of the reduction of cellular lipoprotein lipase activity was studied in the presence of 100 u n i t s / m l of rIL-1. As seen in Fig. 2, a 3 0 ~ decrease in cellular LPL activity occurred at 1 h, and only 25~ of the original enzyme activity remained after 24 h of incubation. This decrease in enzyme activity was not due to a leak or secretion of the enzyme into the culture medium, since the lipoprotein lipase activity in the culture medium was 102 + 12 nmol free fatty a c i d s / h per mg cell protein in the control dishes and 57 + 9 nmol free fatty a c i d s / h per mg cell protein after treatment with rIL-1. To evaluate whether the decrease in cellular lipolytic activity in the rat heart cell cultures treated with rIL-I was the result of non:;pecific inhibition of cellular pro-
>,
120 100
a0 ..J
l
0 . - - 20[
}
0
i
0
I
I
z
I
i
,
i
6
".I
2, I
Hours
Fig. 2. Suppression of cellular lipoprotein lipase activity by rlL*! as a function of time. Conditions as in legend to Fig. I. Incubation was carried out with 100 units/ml of rlL-I. Cellular lipoprotein lipase activity in Ihe control dishes incubated in the absence of rlL-1 was 1001 :t:73 nmol of fatty acid relea:,~d/h per mg cell protein. Values are means ± S.E. of triplicate dishes, from two experiments,
rein synthesis, incorporation of [~Hlleucine into cellular proteins was studied. Rat heart cell cultures were exposed to rIL-1, 100 u n i t s / m l for 24 h, and labeled leucine was added for the last 20 min of incubation. Protein synthesis was not affected by the exposure to the cytokine (33.6 =1: 1.1; 36.8 + 7.4 d p m . 1 0 - 3 / r a g cell protein in control and IL-I treated cells, respectively). Lipoprotein lipase in the cultured mesenchymal rat heart cells is located in an intracellular and in a cell surface related functional compartment from which it can be released by heparin [25]. In the experiments presented in Table l, rat heart cell cultures were exposed to 100 u n i t s / m l of rlL-I for 24 h. Thereafter, heparin was added for 3 rain to release the cell surface
!
\
tJ
c. 5
0 601-
TABLE I Effect of rlL*l on the functional (heparin releasable) LPL activity in mesenchymal rat heart cell cultures
t ~)
Conditions: as in legend to Fig. I. The cells were cultured for 7 days ¢1 o CL
20 0
I
0
I
I
I
i
,
,
!
"..t" i
20 AO 60 80 200 riLl (units/ml )
Fig, 1. Effect of increasing concentrations of rlL-I in culture medium on cellular lipoprotein lipase activity of mesenchymal rat he,art cell cultures. The cells were cultured in F)o medium supplemented with 10¢$ fetal bovine serum and 10~ horse serum for 7 days. The medium was changed every 3 days. and the last medium change was 48 h prior to the start of the experiment. On day 7, riL-! was added to the serum containing culture medium for 24 h. At the end of incubation, the medium was collected and cellular lipoprotein lipase activity was determined on aliquots of cell homogenates. Cellular LPL activity in the control dishes incubated in the absence of rlL-1 was 1016±54 nmol of fatty acid released/h per mg cell protein. Values ~ ,-~ + S.E. of triplicate dishes from three experiment,,
in complete culture medium. On day 7. cuhures were exposed to 1OO U / m l of IL-I or to medium alone for 24 h. For the determination of heparin releasable lipoprotcin lipase, the cultures wen= incubated for 3 min with 0.5 ml F,o containing 20~[ serum and 5 unils/ml of heparin, and both medium and cells were a.~ayed for enzyme activity as described under Materials and Methods. Resuhs are means±S.E, of
triplicate dishes. Experiment No.
Additions to medium
!
none H-1 none IL-I
II
LPL activity, (nmolfatty acid released/ h per mg cell protein) cellular releasable total 608+46 317+ 5 925+31 244+14 44+12 288+18 703 + 16 310:t:! 1 1013± 21 250±17 37± 8 287±15
86
A
TABI,E II
Neutrah:atton ,] I1,-I Htdu(ed suppression of cellular hpoprotem hpaye acttrt(v tn mt~ien
