132
Biochimica et Biophysics Acta, 380 (1975) 132-140 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56539
LIPOPROTEIN LIPASE IN RAT LUNG THE EFFECT OF FASTING
MARGIT HAMOSH and PAUL HAMOSH Department of Anatomy and Department of Physiology and Biophysics, University School of Medicine, Washington, D.C. 20007 (U.S.A.)
Georgetown
(Received May 2’7th, 1974)
Summary We measured lipoprotein lipase activity in dried defatted preparations of rat lung using doubly labeled chylomicron triglyceride as substrate. The enzyme activity was linear for the first hour of incubation at 37”C, had a pH optimum of 8.1 and was completely inhibited by 0.5 M NaCI. Lungs from fed rats hydrolyzed chylomicron triglyceride at a rate of 13.00 pmoles/g per h; the activity rate was unchanged by fasting 8-72 h. Heparin infusion into isolated lungs caused immediate release of lipoprotein lipase to the venous effluent. The activity released was equivalent to about 10% of total lung lipoprotein lipase activity in both fed and fasted rats. Since the ability to remove blood triglyceride is directly related to the level of lipoprotein lipase activity, these findings indicate that the lung is one of the few tissues able to remove efficiently blood triglyceride during fasting.
Introduction The alveoli in the lung are lined with a surface active material (surfactant) consisting mainly of dip~mitoyll~ithin [l] , The main sources of palmitic acid for surfactant synthesis in lung are blood free fatty acids [ 2,3 J and triglyceride fatty acids [4]. Uptake of blood triglyceride fatty acids by most tissues is catalyzed and regulated by lipoprotein lipase [ 51, an enzyme that hydrolyzes blood triglycerides in or near the capillary wall [6] . Enzyme activity in several tissues is greatly affected by the nutritional state of the animal [7,8] . Little is known about the characteristics of lipoprotein lipase or the factors that regulate the enzyme activity in lung [ 9,101. This paper describes a study of lipoprotein lipase in lung and the effect of fasting on enzyme activity.
133
Methods Adult female Charles River (Charles River Breeding Labs, Inc., Wilmington, Mass.) rats weighing 160-200 g were used in this study. The animals were fed ad libitum Purina Laboratory Chow (Ralston Purina Co., St. Louis, MO.) or were fasted for 8,16,24,48 and 72 h; all groups had free access to water. Preparation of tissue for Lipoprotein lipase assay. Dried defatted tissue preparations were made and assayed for lipoprotein lipase activity by methods similar to those described by Robinson [ll] . The rats were killed by decapitation, the lungs were rapidly excised, weighed, rinsed in ice-cold saline, minced and homogenized in 5 ml of cold 0.025 M NHs-HCI buffer (pH 8.1) containing heparin (1 unit/ml) with the aid of a Tekmar Tissumizer (model SDT 182N, Tekmar Co., Cincinnati, Ohio). The.homogenates were poured into 100 ml of cold acetone. The tissue constituents insoluble in acetone were collected by filtering through Whatman No. 1 paper on a Buchner funnel. In order to increase the mass of precipitate 80 mg of bovine plasma albumin in 0.5 ml of 0.85% NaCl (pH 8.1) were added to the lung homogenate-acetone mixture before filtering. The tissue residue was washed with 100 ml of cold acetone, 150 ml of acetone at room temperature and finally with 150 ml of diethyl ether at room temperature. The dried defatted preparations were further dried overnight at 4°C in vacuum. The next morning the preparations were scraped off the filter paper and dissolved in cold 0.025 M NH3-HCI buffer, pH 8.1, containing heparin (1 unit/ml), by a 15-s homogenization with the tissumizer. The enzyme.suspensions were kept on ice until assayed within 30-60 min. Lipoprotein lipase assay. Lipoprotein lipase activity in the dried defatted tissue preparation was measured by the amount of chylomicron triglyceride hydrolyzed to glycerol and free fatty acids at pH 8.1. Each assay tube (Falcon, polypropylene, Division of BioQuest, Oxnard, Calif.) contained 62.5 ~1 of 1 M Tris buffer, 125 ~1 of 16% bovine plasma albumin (Fraction V, Metrix, Armour Pharmaceutical Co., Chicago, Ill., Lot G-36912), 1 pmole of double labeled chylomicron triglyceride in 75 ,ul of rat serum, 50-200 ~1 of enzyme suspension and water in a final volume of 0.5 ml. The chylomicrons and serum were incubated together for 30 min at 37°C before being added to the assay mixture. The assay mixture was incubated at 37°C in a Dubnoff shaking bath for 5-120 min. At the end of the incubation the lipid was extracted in hexane by a modification [12] of the procedure of Dole and Meinertz [ 131. The chylomicron triglyceride was labeled with [” H] glycerol and ’ 4 C-labeled fatty acids so that the decrease in glyceride could be measured by the change in ratio of 3 H to ’ 4 C in the hexane extract of the assay medium. Lipoprotein lipase activity is expressed as fimoles triglyceride hydrolyzed/g of tissue (wet weight) per h. Doubly labeled chylomicrons were isolated from thoracic duct chyle collected from rats tube-fed corn oil containing [l-l 4 C] palmitic acid (56 mCi/mmole, Lot No. 594D-62), and trioleyl[ 2-3 H] glycerol (2 Ci/mmole, Lot No. 733864) (International Chemical and Nuclear Corp., Irvine, Calif.) as described previously [ 14,151. The presence of partial glycerides in the hexane extracts was analyzed by thin-layer chromatography as described elsewhere
WI.
Radioacti~ty
in the total lipid extracts and in the thin-layer chromato-
134
graphy samples was measured [ 161 in a liquid scintillation spectrometer (Packard Tricarb model 314-EX, Packard Instrument Co., Inc., Downers Grove, Ill.) with a scintillation fluid consisting of 4.2% Liqui~uor (New England Nuclear, Pilot Chemicals Division, Boston, Mass., Cat. No. NEF-903). The triglyceride content of chylomicrons was measured by the method of Rapport and Alonzo [ 171. Perfusion of isolated rat lung. Rats were anesthetized with sodium pentobarbital (150 mg/kg), the chest was opened and the pulmonary artery canulated through and incision in the right ventricle. The left atrium and ventricle were widely transected; the thoracic content was then removed and suspended by the trachea in a chamber at 37°C and saturated with water. The rate of perfusion was constant at 1 ml/min; the perfusate was collected by gravity. The lungs were perfused with 4% albumin in Krebs-Ringer bicarbonate buffer, pH 7.4. Infusion of heparin (100 pg/ml) started after 5 min and continued for 15 min. Venous effluent was collected at 2-min intervals into ice-chilled tubes for the determination of lipoprotein lipase activity. Results Dried defatted prep~ations of rat lung readily hydrolyzed ~hylomi~ron triglyceride to free fatty acids and glycerol (Figs 1 and 2 and Table II). There was no accumulation of partial glycerides even during the initial stage of the reaction. The lipolytic activity was proportional to the amount of acetone powder present in the assay system in a concentration range equivalent to 10-50 mg
TISSUE
(mg)
Fig. 1. Hydrolysis of chylomicron triglyceride as a function of tissue concentration. The tubes contained dried defatted lung acetone powder equivalent to 10-80 mg wet weight tissue; incubation was for 15 min at 37°C in the reaction mixture described in Methods. The results are means of three experiments.
135
v
I
I
I
L
30
60
90
INCUBATION
I
120
(mln)
Fig. 2. Time curve of chylomicron triglyceride hydrolysis by dried defatted preparations of rat lung. Each tube contained the equivalent of 20 mg lung homogenate; incubation was at 3’l°C in the reaction mixture described in Methods. The results are means of tdree ekperiments.
tissue wet weight (Fig. 1). The reaction rate was constant for the first 60 min of incubation and leveled off thereafter (Fig. 2). The pH optimum of the reaction was 7.7-8.1 (Fig. 3), similar to that of lipoprotein lipase from other tissues [ 8,11,14] . Since the level of .lipoprotein lipase activity in a variety of tissues is markedly affected by the nutritional state of the animal [E&7,8] we determined the activity of the enzyme in lungs of rats fasted from 8 to 72 h. Fasting up to 72 h caused no change in the wet and dry weight of rat lung compared to lungs of fed rats (Table I). Fasting 8-72 h caused no change in the level of lipoprotein lipase activity in rat lung (Table II). Similar to tissue preparations from fed rats, there was no accumulation of partial glycerides during the initial stage of chylomicron triglyceride hydrolysis by acetone powders prepared from lungs of fasted rats. 0.5 M NaCl completely inhibited lipolysis, indicating that the activity in the six preparations tested is that of lipoprotein lipase [ 5,141. The level of lipoprotein lipase activity in fresh tissue homogenate was similar to that in the acetone powder preparations (Table III); there was no difference in the activity rate in lungs from fed or 8-16-h-fasted rats. Failure to “activate” the chylomicron triglyceride by incubation with serum led to a 70-80% decrease in activity in the three tissue preparations tested (Table III). Similar results were obtained with acetone powders of lungs from fed or fasted rats. The residual 20-30s activity is probably due to the presence of chylomicron apoprotein. We have made the same observation with acetone powder preparations of adipose tissue and mammary gland. 0.5 M NaCl markedly inhibited the lipolytic activity of fresh homogenates. 12-14% of the lipoprotein lipase was released from tissue slices into heparin-containing medium during 30 min incubation at 37°C. There was no release of lipolytic activity when heparin was omitted from the medium. A
136
Fig. 3. Effect of pH on hydrolysis of chylomicron triglyceride by dried defatted preparations of rat lung. Each tube contained the equivalent of 20 mg rat lung homogenate in the reaction mixture as described in Methods. In the pH range of 5.0-6.5 the buffer was Trismaleate and in the range of 6.54.0 Tris-HCI; both buffers were present in a final concentration of 0.125 M. The mixture was incubated at 37’C for 30 min. The results are means of three experiments.
similar percentage of tissue lipase was released from lungs of both fed and fasted rats. The activity was markedly inhibited by 0.5 M NaCl. Release of lipoprotein lipase from isolated perfused lungs. The venous effluent collected from lungs perfused with buffered albumin solution contained only traces of lipoprotein lipase activity. Infusion of heparin (100 pg/ml) caused a rapid increase in the amount of lipase released to the venous effluent. The amount of lipoprotein lipase released within 2-3 min of heparin
TABLE
I
EFFECT
OF FASTING
ON WET AND DRY WEIGHT
OF RAT
LUNG
Values are mean + S.E. Nutritional state
Fed Fasted. Fasted. Fasted, Fasted, Fasted.
8 16 24 48 72
h h h h h
No. of animals
Body weight (9)
Lung weight
Lung weight
(g)
(g/l00 g body weight)
Lung dry weight/% of wet weight*
7 4 5 6 6 5
196 203 209 157 185 197
1.07 1.13 1.10 0.875 0.99 1.10
0.54 0.55 0.53 0.55 0.53 0.52
17.14 17.20 18.81 17.85 16.45 17.94
* Dry weight was determined
f 8.00 i: 5.50 f 3.20 + 4.16 f 6.16 5 11.00 in defatted
+ * * k f +
0.061 0.043 0.016 0.025 0.038 0.620
lung preparations.
f 0.030 f 0.043 i 0.010 + 0.018 + 0.025 zk0.034
* f + + + ?
0.40 0.64 0.45 0.50 0.83 0.47
137 TABLE
II
HYDROLYSIS OF CHYLOMICRON EFFECT OF FASTING
TRIGLYCERIDE
BY LIPOPROTEIN
LIPASE
OF RAT
LUNG
-
Nutritional state
No. of animals
Chylomicron triglyceride* hydrolyzed (units/g wet wt***)
Chylomicron triglyceride* hydrolyzed (units/100 mg dry wt* * *)
Effect of 0.5 M NaCl (W inhibition)
Fed Fasted, 8 h Fasted, 16 h Fasted, 24 h Fasted, 48 h Fasted. 72 h
I**
13.00 15.47 11.85 15.10 11.00 12.15
6.70 8.82 6.30 5.70 7.50 7.60
99.0 95.5 92.0 98.5 99.0 99.8
4 5 6 6 5
?r 0.80 r 0.98 + 1.88 + 1.78 k 1.61 ?r.1.08
f f f f ? ?
0.45 0.50 1.00 0.72 0.41 0.51
* Values are means f S.E. ** The activity rate for each tissue is the result of five separate determinations. *** lunit = 1 pm01 triglyceride hydrolysed/h.
infusion increased from 0.025 to 0.38. units/min in lungs from fed rats and from 0.017 to 0.41 units/min in lungs from 36-h-fasted rats. The amount of activity released during the first 5-7 min of heoarin infusion was equivalent to about k-10% of the total activity present in the lungs of fed or fasted rats. Discussion The possible role of blood triglyceride fatty acids as a reservoir of substrate for lung metabolism has been suggested by Felts [4] who reported the TABLE
III
CHARACTERISTICS
OF LIPOPROTEIN
LIPASE
ACTIVITY
OF RAT
LUNG-EFFECT
OF FASTING
Lungs were homogenized in 0.025 M NH3-HCl buffer as described under Methods. Aliquots of fresh homogenate equivalent to 20-40 mg tissue were tested for lipoprotein lipase as described under Methods. 500 mg lung slices were incubated for 30 min at 37’C in 2 ml KrebvRinger bicarbonate buffer. pH 7.4, containing 25 fig/ml heparin. After 30 min. 0.5 ml akquots of the medium were tested for the presence of lipoprotein lipase activity (incubation with 1 pmole serum-activated chylomicron triglyceride at pH 8.1 and 37’C for 30 and 60 min). Aliquots were incubated also in the complete system in the presence of 0.5 M NaCl. There was no release of lipoprotein lipase when heparin was omitted from the incubation medium. The results are mean * S.E. There were four to five animals in each group. The activity rate for each tissue is the result of four separate determinations. Tissue preparation
Assay system
Lipoprotein Fed
lipase activity
Fasted 8h
Homogenate
Slices*
Complete Serum omitted + 0.5 M NaCl Complete + 0.5 M NaCl
* Release of lipoprotein
(units/g)
16 h
12.30 f 1.00 10.70 11.50 f 0.35 3.69 f 0.39 2.85 3.96 f 0.60 1.23 f 0.02 0.62 0.80 f 0.06 Lipoprotein lipase activity (units/O.5 g per 30 min) 0.83 f 0.01 0.67 0.76 f 0.025 0.12 f 0.013 0.067 0.098 * 0.004
lipase during incubation
with heparin.
* 1.00 f 1.00 + 0.01 * 0.02 k 0.003
138
uptake of very-low-density lipoprotein triglyceride fatty acids by rabbit lung slices. Bragdon and Gordon (181 have shown uptake of triglyceride by the lung after intravenous administration of chylomicrons to rats. Studies by Jones and Have1 (191 indicate that chylomicron triglycerides are hydrolyzed during uptake in vivo by the lung. The uptake of blood triglyceride is directly related to the level of lipoprotein lipase in a number of tissues [ 5,6]. Although early reports [20] indicated the absence of lipoprotein lipase from the lung, more recent investigations [9,10] indicate that the enzyme is present in lung. The lung contains also a lipase 1211, active in the breakdown of tissue lipid and release of free fatty acids to the blood, and it seems that earlier reports [ 221 on lipolytic activity in the lung were unable to differentiate between the two activities. We think therefore that by using dried, defatted tissue powders we were able to study lipoprotein lipase activity without interference by other lipases 1231. Indeed the present study shows that lung lipoprotein lipase has characteristics similar to those described in other tissues [8,11,14], i.e. pH optimum of 7.7-8.1, marked inhibition by high concentrations of NaCl (Fig. 3 and Tables II and III) or by omission of serum from the reaction mixture (Table III). Hydrolysis of triglyceride to glycerol and free fatty acids without the accumulation of 2-monoglyceride is due to the presence of monoglyceride lipase in rat lung 1241. The monoglyceride lipase activity was reported to be 3-4 times higher than that of triglyceride lipase in the lung [ 24]. There is no change in the level of lipoprotein lipase activity in lung during fasting 8-72 h (Table II). Our data agree well with those of Felts [4], who found no marked difference in the uptake of triglyceride fatty acids by lung slices from fed or fasted rabbits; and with the in vivo studies of Bragdon and Gordon [18] who TABLE EFFECT
IV OF
HEPARIN
ON RELEASE
OF
LIPOPROTEIN
LIPASE
FROM
PERFUSED
RAT
LUNG
Isolated lungs were perfused with 4% atbumin in KrebsRinger bicarbonate buffer (pH 7.4). Heparin infusion (100 &@/ml perfusate) was started after 5 min and continued for 15 min. Lipoprotein lipase activity in the venous effluent was determined by incubating the sample with 1 &nnole chylomieron trigfyeeride at pH 8.1 and 37’C. Heparin (100 pg/mlf was added for the in vitro incubation of the effluent samplescollected prior to heparin infusion. The activity is expressed in units: 1 unit = 1 /.&mole triglyceride hydrolyzed per h. The activity was completely inhibited by 0.5 M NaCl. There were three tissues in each group. Time (mW
Heparin infusion
Lipoprotein (units/min) Fed
Iipase activity
36 h fast ---
lx-2 24 46 68 8-10 1*12 12-14 14-16 16-18 1 S-20
0.018 0.025 0.13 0.38 0.25 0.13 0.15 0.12 0.06 0.06
released
0.012 0.017 0.077 0.41 0.28 0.107 0.064 0.066 0.05 0.04
to the venous f!ffIuent
139
found no change in the amount of labeled chylomicron triglyceride taken up by the lung in fed or fasted rats. Lung slices from fed or fasted rats (Table III) release identical amounts of lipoprotein lipase into heparin-containing medium. Infusion of heparin (Table IV) into isolated rat lung causes the immediate release of lipoprotein lipase to the venous effluent. The amount released is equivalent to about 8-10% of total tissue lipoprotein lipase activity and is identical in lungs from fed or fasted rats. These results indicate that during fasting there is no change in the amount of lipoprotein lipase located in or near the capillary wall in the lung. Our previous studies with rat adipose tissue ,[6,25] and those of Borensztajn and Robinson with rat heart [26] have shown that this fraction of lipoprotein lipase, which is readily released during heparin infusion, is active in the hydrolysis of circulating lipoprotein triglyceride. The level of lipoprotein lipase activity depends on the physiological and nutritional state of the animal and varies from tissue to tissue. Thus in adipose tissue the activity decreases sharply during fasting [7], diabetes [27], or lactation [ 141 whereas the activity in’heart increases 2-3-fold during fasting [ ES]; in the mammary gland activity is present only during lactation and virtually disappears several hours after removal of the pups [ 141. Our finding that lipoprotein lipase activity in the lung is unchanged during fasting, indicates that this tissue has the ability to remove efficiently blood triglyceride during food deprivation. Surfactant is essential for survival and our results suggest that during fasting, triglycerides might be the source of fatty acids for the synthesis of surfactant. Since lipoprotein lipase activity reflects the ability of a tissue to remove triglyceride from the blood, the changes in enzyme activity would channel blood triglyceride from storage in adipose tissue to the heart and lung during fasting. Acknowledgments The authors are grateful to Mr Michael Blunda for excellent assistance. This work was supported in part by ‘grants from the Washington Heart Association and Council for Tobacco Research Inc., U.S.A. Some of the data in this work were presented at the spring meeting of the American Physiological Society, Atlantic City, April, 1974. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Clements, J.A., Nellenbogen. J. and Trahan. H.J. (1970) Science 169, 603-604 Young, S.L. and Tiemey, D.F. (1972) Am. J. Physiol. 222, 1539-1544 Darrah. H.K. and Hedley-Whyte. J. (1973) J. Appl. Physiol. 34, 205-213 Felts, J.M. (1964) Health Phys. 10.973-979 Robinson, D.S. (1970) Compr. Biochem. 18. 61-116 Scow, R.O.. Hamosh. M.. Blanchette-Mackie. J.E. and Evans, A.J. (1972) Lipids 7.497-505 Bezman. A., Felts, J.M. and Have& R.J. (1962) J. Lipid Res. 3, 427-431 Borensztain, J., Otway, S. and Robinson, D.S. (1970) J. Lipid Res. 11.102-110 Radomsky, M.W. and Orme. T. (1971) Am. J. Physiol. 220.1852-1856 Abe. K. and Yoshimura, K. (1972) J. Physiol. Sot. Japan 34.81-82 Salaman. M.R. and Robinson, D.S. (1966) Biochem. J. 99,640-647 Chernick, S.S. and Novack, M. (1970) Diabetes 19. 563-570 Dole, V.P. and Meinertz. H. (1960) J. Biol. Chem. 235, 2595-2599 Hamosh. M.. Claw. T.R.. Chernick. S.S. and Scow, R.O. (1970) Biochim. Biophys. Acta 210, 473482
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Hamosh,
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Rapport,
M.M.
18
Bragdon,
J.H.
19
Jones,
X.L.
and
20
Kom.
E.D.
(1955)
21
Armstrong.
22
Heineman.
M. and
23
Hamosh,
24
Wang.
25
Hamosh,
26
Borensztajn,
27
Schatz,
28
Robinson,
and and
H.Y., H.O. M. and
M.C.
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Y.
and
and
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Alonzo,
Havel,
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Evans.
(1967)
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R.H.
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D.S.
242,
4919-4924
88-95 217,193-198
Invest. 213.
37,
574-578
824-828
l-14 L.F.
201.
Biophys.
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32,
Proc.
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EXP.
231,283-289
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607-610
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Biochimica et Biophysics Acta, 380 (1975) 132-140 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56539
LIPOPROTEIN LIPASE IN RAT LUNG THE EFFECT OF FASTING
MARGIT HAMOSH and PAUL HAMOSH Department of Anatomy and Department of Physiology and Biophysics, University School of Medicine, Washington, D.C. 20007 (U.S.A.)
Georgetown
(Received May 2’7th, 1974)
Summary We measured lipoprotein lipase activity in dried defatted preparations of rat lung using doubly labeled chylomicron triglyceride as substrate. The enzyme activity was linear for the first hour of incubation at 37”C, had a pH optimum of 8.1 and was completely inhibited by 0.5 M NaCI. Lungs from fed rats hydrolyzed chylomicron triglyceride at a rate of 13.00 pmoles/g per h; the activity rate was unchanged by fasting 8-72 h. Heparin infusion into isolated lungs caused immediate release of lipoprotein lipase to the venous effluent. The activity released was equivalent to about 10% of total lung lipoprotein lipase activity in both fed and fasted rats. Since the ability to remove blood triglyceride is directly related to the level of lipoprotein lipase activity, these findings indicate that the lung is one of the few tissues able to remove efficiently blood triglyceride during fasting.
Introduction The alveoli in the lung are lined with a surface active material (surfactant) consisting mainly of dip~mitoyll~ithin [l] , The main sources of palmitic acid for surfactant synthesis in lung are blood free fatty acids [ 2,3 J and triglyceride fatty acids [4]. Uptake of blood triglyceride fatty acids by most tissues is catalyzed and regulated by lipoprotein lipase [ 51, an enzyme that hydrolyzes blood triglycerides in or near the capillary wall [6] . Enzyme activity in several tissues is greatly affected by the nutritional state of the animal [7,8] . Little is known about the characteristics of lipoprotein lipase or the factors that regulate the enzyme activity in lung [ 9,101. This paper describes a study of lipoprotein lipase in lung and the effect of fasting on enzyme activity.
133
Methods Adult female Charles River (Charles River Breeding Labs, Inc., Wilmington, Mass.) rats weighing 160-200 g were used in this study. The animals were fed ad libitum Purina Laboratory Chow (Ralston Purina Co., St. Louis, MO.) or were fasted for 8,16,24,48 and 72 h; all groups had free access to water. Preparation of tissue for Lipoprotein lipase assay. Dried defatted tissue preparations were made and assayed for lipoprotein lipase activity by methods similar to those described by Robinson [ll] . The rats were killed by decapitation, the lungs were rapidly excised, weighed, rinsed in ice-cold saline, minced and homogenized in 5 ml of cold 0.025 M NHs-HCI buffer (pH 8.1) containing heparin (1 unit/ml) with the aid of a Tekmar Tissumizer (model SDT 182N, Tekmar Co., Cincinnati, Ohio). The.homogenates were poured into 100 ml of cold acetone. The tissue constituents insoluble in acetone were collected by filtering through Whatman No. 1 paper on a Buchner funnel. In order to increase the mass of precipitate 80 mg of bovine plasma albumin in 0.5 ml of 0.85% NaCl (pH 8.1) were added to the lung homogenate-acetone mixture before filtering. The tissue residue was washed with 100 ml of cold acetone, 150 ml of acetone at room temperature and finally with 150 ml of diethyl ether at room temperature. The dried defatted preparations were further dried overnight at 4°C in vacuum. The next morning the preparations were scraped off the filter paper and dissolved in cold 0.025 M NH3-HCI buffer, pH 8.1, containing heparin (1 unit/ml), by a 15-s homogenization with the tissumizer. The enzyme.suspensions were kept on ice until assayed within 30-60 min. Lipoprotein lipase assay. Lipoprotein lipase activity in the dried defatted tissue preparation was measured by the amount of chylomicron triglyceride hydrolyzed to glycerol and free fatty acids at pH 8.1. Each assay tube (Falcon, polypropylene, Division of BioQuest, Oxnard, Calif.) contained 62.5 ~1 of 1 M Tris buffer, 125 ~1 of 16% bovine plasma albumin (Fraction V, Metrix, Armour Pharmaceutical Co., Chicago, Ill., Lot G-36912), 1 pmole of double labeled chylomicron triglyceride in 75 ,ul of rat serum, 50-200 ~1 of enzyme suspension and water in a final volume of 0.5 ml. The chylomicrons and serum were incubated together for 30 min at 37°C before being added to the assay mixture. The assay mixture was incubated at 37°C in a Dubnoff shaking bath for 5-120 min. At the end of the incubation the lipid was extracted in hexane by a modification [12] of the procedure of Dole and Meinertz [ 131. The chylomicron triglyceride was labeled with [” H] glycerol and ’ 4 C-labeled fatty acids so that the decrease in glyceride could be measured by the change in ratio of 3 H to ’ 4 C in the hexane extract of the assay medium. Lipoprotein lipase activity is expressed as fimoles triglyceride hydrolyzed/g of tissue (wet weight) per h. Doubly labeled chylomicrons were isolated from thoracic duct chyle collected from rats tube-fed corn oil containing [l-l 4 C] palmitic acid (56 mCi/mmole, Lot No. 594D-62), and trioleyl[ 2-3 H] glycerol (2 Ci/mmole, Lot No. 733864) (International Chemical and Nuclear Corp., Irvine, Calif.) as described previously [ 14,151. The presence of partial glycerides in the hexane extracts was analyzed by thin-layer chromatography as described elsewhere
WI.
Radioacti~ty
in the total lipid extracts and in the thin-layer chromato-
134
graphy samples was measured [ 161 in a liquid scintillation spectrometer (Packard Tricarb model 314-EX, Packard Instrument Co., Inc., Downers Grove, Ill.) with a scintillation fluid consisting of 4.2% Liqui~uor (New England Nuclear, Pilot Chemicals Division, Boston, Mass., Cat. No. NEF-903). The triglyceride content of chylomicrons was measured by the method of Rapport and Alonzo [ 171. Perfusion of isolated rat lung. Rats were anesthetized with sodium pentobarbital (150 mg/kg), the chest was opened and the pulmonary artery canulated through and incision in the right ventricle. The left atrium and ventricle were widely transected; the thoracic content was then removed and suspended by the trachea in a chamber at 37°C and saturated with water. The rate of perfusion was constant at 1 ml/min; the perfusate was collected by gravity. The lungs were perfused with 4% albumin in Krebs-Ringer bicarbonate buffer, pH 7.4. Infusion of heparin (100 pg/ml) started after 5 min and continued for 15 min. Venous effluent was collected at 2-min intervals into ice-chilled tubes for the determination of lipoprotein lipase activity. Results Dried defatted prep~ations of rat lung readily hydrolyzed ~hylomi~ron triglyceride to free fatty acids and glycerol (Figs 1 and 2 and Table II). There was no accumulation of partial glycerides even during the initial stage of the reaction. The lipolytic activity was proportional to the amount of acetone powder present in the assay system in a concentration range equivalent to 10-50 mg
TISSUE
(mg)
Fig. 1. Hydrolysis of chylomicron triglyceride as a function of tissue concentration. The tubes contained dried defatted lung acetone powder equivalent to 10-80 mg wet weight tissue; incubation was for 15 min at 37°C in the reaction mixture described in Methods. The results are means of three experiments.
135
v
I
I
I
L
30
60
90
INCUBATION
I
120
(mln)
Fig. 2. Time curve of chylomicron triglyceride hydrolysis by dried defatted preparations of rat lung. Each tube contained the equivalent of 20 mg lung homogenate; incubation was at 3’l°C in the reaction mixture described in Methods. The results are means of tdree ekperiments.
tissue wet weight (Fig. 1). The reaction rate was constant for the first 60 min of incubation and leveled off thereafter (Fig. 2). The pH optimum of the reaction was 7.7-8.1 (Fig. 3), similar to that of lipoprotein lipase from other tissues [ 8,11,14] . Since the level of .lipoprotein lipase activity in a variety of tissues is markedly affected by the nutritional state of the animal [E&7,8] we determined the activity of the enzyme in lungs of rats fasted from 8 to 72 h. Fasting up to 72 h caused no change in the wet and dry weight of rat lung compared to lungs of fed rats (Table I). Fasting 8-72 h caused no change in the level of lipoprotein lipase activity in rat lung (Table II). Similar to tissue preparations from fed rats, there was no accumulation of partial glycerides during the initial stage of chylomicron triglyceride hydrolysis by acetone powders prepared from lungs of fasted rats. 0.5 M NaCl completely inhibited lipolysis, indicating that the activity in the six preparations tested is that of lipoprotein lipase [ 5,141. The level of lipoprotein lipase activity in fresh tissue homogenate was similar to that in the acetone powder preparations (Table III); there was no difference in the activity rate in lungs from fed or 8-16-h-fasted rats. Failure to “activate” the chylomicron triglyceride by incubation with serum led to a 70-80% decrease in activity in the three tissue preparations tested (Table III). Similar results were obtained with acetone powders of lungs from fed or fasted rats. The residual 20-30s activity is probably due to the presence of chylomicron apoprotein. We have made the same observation with acetone powder preparations of adipose tissue and mammary gland. 0.5 M NaCl markedly inhibited the lipolytic activity of fresh homogenates. 12-14% of the lipoprotein lipase was released from tissue slices into heparin-containing medium during 30 min incubation at 37°C. There was no release of lipolytic activity when heparin was omitted from the medium. A
136
Fig. 3. Effect of pH on hydrolysis of chylomicron triglyceride by dried defatted preparations of rat lung. Each tube contained the equivalent of 20 mg rat lung homogenate in the reaction mixture as described in Methods. In the pH range of 5.0-6.5 the buffer was Trismaleate and in the range of 6.54.0 Tris-HCI; both buffers were present in a final concentration of 0.125 M. The mixture was incubated at 37’C for 30 min. The results are means of three experiments.
similar percentage of tissue lipase was released from lungs of both fed and fasted rats. The activity was markedly inhibited by 0.5 M NaCl. Release of lipoprotein lipase from isolated perfused lungs. The venous effluent collected from lungs perfused with buffered albumin solution contained only traces of lipoprotein lipase activity. Infusion of heparin (100 pg/ml) caused a rapid increase in the amount of lipase released to the venous effluent. The amount of lipoprotein lipase released within 2-3 min of heparin
TABLE
I
EFFECT
OF FASTING
ON WET AND DRY WEIGHT
OF RAT
LUNG
Values are mean + S.E. Nutritional state
Fed Fasted. Fasted. Fasted, Fasted, Fasted.
8 16 24 48 72
h h h h h
No. of animals
Body weight (9)
Lung weight
Lung weight
(g)
(g/l00 g body weight)
Lung dry weight/% of wet weight*
7 4 5 6 6 5
196 203 209 157 185 197
1.07 1.13 1.10 0.875 0.99 1.10
0.54 0.55 0.53 0.55 0.53 0.52
17.14 17.20 18.81 17.85 16.45 17.94
* Dry weight was determined
f 8.00 i: 5.50 f 3.20 + 4.16 f 6.16 5 11.00 in defatted
+ * * k f +
0.061 0.043 0.016 0.025 0.038 0.620
lung preparations.
f 0.030 f 0.043 i 0.010 + 0.018 + 0.025 zk0.034
* f + + + ?
0.40 0.64 0.45 0.50 0.83 0.47
137 TABLE
II
HYDROLYSIS OF CHYLOMICRON EFFECT OF FASTING
TRIGLYCERIDE
BY LIPOPROTEIN
LIPASE
OF RAT
LUNG
-
Nutritional state
No. of animals
Chylomicron triglyceride* hydrolyzed (units/g wet wt***)
Chylomicron triglyceride* hydrolyzed (units/100 mg dry wt* * *)
Effect of 0.5 M NaCl (W inhibition)
Fed Fasted, 8 h Fasted, 16 h Fasted, 24 h Fasted, 48 h Fasted. 72 h
I**
13.00 15.47 11.85 15.10 11.00 12.15
6.70 8.82 6.30 5.70 7.50 7.60
99.0 95.5 92.0 98.5 99.0 99.8
4 5 6 6 5
?r 0.80 r 0.98 + 1.88 + 1.78 k 1.61 ?r.1.08
f f f f ? ?
0.45 0.50 1.00 0.72 0.41 0.51
* Values are means f S.E. ** The activity rate for each tissue is the result of five separate determinations. *** lunit = 1 pm01 triglyceride hydrolysed/h.
infusion increased from 0.025 to 0.38. units/min in lungs from fed rats and from 0.017 to 0.41 units/min in lungs from 36-h-fasted rats. The amount of activity released during the first 5-7 min of heoarin infusion was equivalent to about k-10% of the total activity present in the lungs of fed or fasted rats. Discussion The possible role of blood triglyceride fatty acids as a reservoir of substrate for lung metabolism has been suggested by Felts [4] who reported the TABLE
III
CHARACTERISTICS
OF LIPOPROTEIN
LIPASE
ACTIVITY
OF RAT
LUNG-EFFECT
OF FASTING
Lungs were homogenized in 0.025 M NH3-HCl buffer as described under Methods. Aliquots of fresh homogenate equivalent to 20-40 mg tissue were tested for lipoprotein lipase as described under Methods. 500 mg lung slices were incubated for 30 min at 37’C in 2 ml KrebvRinger bicarbonate buffer. pH 7.4, containing 25 fig/ml heparin. After 30 min. 0.5 ml akquots of the medium were tested for the presence of lipoprotein lipase activity (incubation with 1 pmole serum-activated chylomicron triglyceride at pH 8.1 and 37’C for 30 and 60 min). Aliquots were incubated also in the complete system in the presence of 0.5 M NaCl. There was no release of lipoprotein lipase when heparin was omitted from the incubation medium. The results are mean * S.E. There were four to five animals in each group. The activity rate for each tissue is the result of four separate determinations. Tissue preparation
Assay system
Lipoprotein Fed
lipase activity
Fasted 8h
Homogenate
Slices*
Complete Serum omitted + 0.5 M NaCl Complete + 0.5 M NaCl
* Release of lipoprotein
(units/g)
16 h
12.30 f 1.00 10.70 11.50 f 0.35 3.69 f 0.39 2.85 3.96 f 0.60 1.23 f 0.02 0.62 0.80 f 0.06 Lipoprotein lipase activity (units/O.5 g per 30 min) 0.83 f 0.01 0.67 0.76 f 0.025 0.12 f 0.013 0.067 0.098 * 0.004
lipase during incubation
with heparin.
* 1.00 f 1.00 + 0.01 * 0.02 k 0.003
138
uptake of very-low-density lipoprotein triglyceride fatty acids by rabbit lung slices. Bragdon and Gordon (181 have shown uptake of triglyceride by the lung after intravenous administration of chylomicrons to rats. Studies by Jones and Have1 (191 indicate that chylomicron triglycerides are hydrolyzed during uptake in vivo by the lung. The uptake of blood triglyceride is directly related to the level of lipoprotein lipase in a number of tissues [ 5,6]. Although early reports [20] indicated the absence of lipoprotein lipase from the lung, more recent investigations [9,10] indicate that the enzyme is present in lung. The lung contains also a lipase 1211, active in the breakdown of tissue lipid and release of free fatty acids to the blood, and it seems that earlier reports [ 221 on lipolytic activity in the lung were unable to differentiate between the two activities. We think therefore that by using dried, defatted tissue powders we were able to study lipoprotein lipase activity without interference by other lipases 1231. Indeed the present study shows that lung lipoprotein lipase has characteristics similar to those described in other tissues [8,11,14], i.e. pH optimum of 7.7-8.1, marked inhibition by high concentrations of NaCl (Fig. 3 and Tables II and III) or by omission of serum from the reaction mixture (Table III). Hydrolysis of triglyceride to glycerol and free fatty acids without the accumulation of 2-monoglyceride is due to the presence of monoglyceride lipase in rat lung 1241. The monoglyceride lipase activity was reported to be 3-4 times higher than that of triglyceride lipase in the lung [ 24]. There is no change in the level of lipoprotein lipase activity in lung during fasting 8-72 h (Table II). Our data agree well with those of Felts [4], who found no marked difference in the uptake of triglyceride fatty acids by lung slices from fed or fasted rabbits; and with the in vivo studies of Bragdon and Gordon [18] who TABLE EFFECT
IV OF
HEPARIN
ON RELEASE
OF
LIPOPROTEIN
LIPASE
FROM
PERFUSED
RAT
LUNG
Isolated lungs were perfused with 4% atbumin in KrebsRinger bicarbonate buffer (pH 7.4). Heparin infusion (100 &@/ml perfusate) was started after 5 min and continued for 15 min. Lipoprotein lipase activity in the venous effluent was determined by incubating the sample with 1 &nnole chylomieron trigfyeeride at pH 8.1 and 37’C. Heparin (100 pg/mlf was added for the in vitro incubation of the effluent samplescollected prior to heparin infusion. The activity is expressed in units: 1 unit = 1 /.&mole triglyceride hydrolyzed per h. The activity was completely inhibited by 0.5 M NaCl. There were three tissues in each group. Time (mW
Heparin infusion
Lipoprotein (units/min) Fed
Iipase activity
36 h fast ---
lx-2 24 46 68 8-10 1*12 12-14 14-16 16-18 1 S-20
0.018 0.025 0.13 0.38 0.25 0.13 0.15 0.12 0.06 0.06
released
0.012 0.017 0.077 0.41 0.28 0.107 0.064 0.066 0.05 0.04
to the venous f!ffIuent
139
found no change in the amount of labeled chylomicron triglyceride taken up by the lung in fed or fasted rats. Lung slices from fed or fasted rats (Table III) release identical amounts of lipoprotein lipase into heparin-containing medium. Infusion of heparin (Table IV) into isolated rat lung causes the immediate release of lipoprotein lipase to the venous effluent. The amount released is equivalent to about 8-10% of total tissue lipoprotein lipase activity and is identical in lungs from fed or fasted rats. These results indicate that during fasting there is no change in the amount of lipoprotein lipase located in or near the capillary wall in the lung. Our previous studies with rat adipose tissue ,[6,25] and those of Borensztajn and Robinson with rat heart [26] have shown that this fraction of lipoprotein lipase, which is readily released during heparin infusion, is active in the hydrolysis of circulating lipoprotein triglyceride. The level of lipoprotein lipase activity depends on the physiological and nutritional state of the animal and varies from tissue to tissue. Thus in adipose tissue the activity decreases sharply during fasting [7], diabetes [27], or lactation [ 141 whereas the activity in’heart increases 2-3-fold during fasting [ ES]; in the mammary gland activity is present only during lactation and virtually disappears several hours after removal of the pups [ 141. Our finding that lipoprotein lipase activity in the lung is unchanged during fasting, indicates that this tissue has the ability to remove efficiently blood triglyceride during food deprivation. Surfactant is essential for survival and our results suggest that during fasting, triglycerides might be the source of fatty acids for the synthesis of surfactant. Since lipoprotein lipase activity reflects the ability of a tissue to remove triglyceride from the blood, the changes in enzyme activity would channel blood triglyceride from storage in adipose tissue to the heart and lung during fasting. Acknowledgments The authors are grateful to Mr Michael Blunda for excellent assistance. This work was supported in part by ‘grants from the Washington Heart Association and Council for Tobacco Research Inc., U.S.A. Some of the data in this work were presented at the spring meeting of the American Physiological Society, Atlantic City, April, 1974. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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