BIOCHEMICAL
MEDICINE
21, 156-161 (1979)
Esterification of cis and trans Fatty Acids by Swine Aortic Smooth Muscle Ceils during Aerobic and Anaerobic Incubations WILLIAM Y. HUANG AND FRED A. KUMMEROW The Harlan E. Moore Heart Research Foundation, Champaign, Illinois and The Burnsides Research Laboratory, University of Illinois, Urbana, Illinois 61801
61820,
Received October 28, 1978
It has been shown that dietary tram fatty acids can be incorporated into tissue and serum lipids (l-7). It was postulated that dietary tram fatty acids might be related to atherosclerosis and other diseases (8). The dietary tram fatty acids have been shown to increase the serum cholesterol level in animals (6,9) as well as in man (10). The accumulation of lipids, especially in the level of triglyceride in hypoxia, has been reported in the cell culture of strain L mouse fibroblasts (11) and intimal cells of swine aorta (12). In rabbits exposed in vivo to hypoxic conditions, there has been found an increase of arterial lipid content and development of atherosclerosis (13-16). Studies on the effect of hypoxia on the fatty acid esterification in the aortic smooth muscle cells have not been reported to date. Furthermore, the incorporation of cis and trans fatty acids into swine aortic smooth muscle cell lipids has not been determined to date. The present studies were designed to investigate the incorporation of labeled oleic acid (cis-9-octadecenoic acid) and elaidic acid (tram-9octadecenoic acid) into lipids in the swine aortic smooth muscle cells under aerobic and anaerobic incubations. METHODS AND MATERIALS
Cell culture techniques. Segments of thoracic aorta were obtained from approximately 6-month-old swine weighing from 100 to 110 kg. Explants of intimal-medial segments of swine aortas were prepared according to the method of Kamio et al. (17). The explants were first grown in %-ml Falcon flasks. Smooth muscle cells usually started growing out of the 156 0006-2!344/79/020156-06$02.00/O Copytight @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
ESTERIFICATION
OF FATTY
ACIDS
157
intimal-medial explants after about 14 days, and the growth appeared to be confluent 3 weeks later. The cells were then released from the flask by trypsinization and transferred to a new 250~ml flask. The explants of intimal-medial segments (smooth muscle cells) were cultivated in the modified Dulbecco-Vogt medium supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic solution. The media were changed every third day. Incubation temperature was 37°C in a moist atmosphere of 95% air-5% CO,. Incubations. When growth appeared to be confluent, the cells were harvested by trypsinization, washed three times, and resuspended in a balanced salt solution. The incubation medium, containing 3.0 ml of Krebs-Ringer bicarbonated buffer (pH 7.4), 5 mM glucose, 0.1 unit insulin, 0.1 mM fatty acid-free bovine serum albumin, 0.5 mM [1J4C]01eic acid (specific activity 55 mCi/mmole), or [l-‘4Clelaidic acid (specific activity 50 mCi/mmole) was added to the smooth muscle cells (cell protein 2.5 mg) in the flask. The flasks were gassed with 95% O,-5% CO, mixture (aerobically) or 95% N,-5% CO, mixture (anaerobically) and stoppered with rubber serum stoppers. The flasks were shaken in a gyrotory water bath shaker at 37” for 4 hr. After incubation, the cells were washed three times by resuspending in a balanced salt solution and centrifuged (600g for 2 min) at 4°C. Extraction and chromatography. The lipids were extracted from the cells according to the method of Folch et al. (18). Radioactive lipids were separated by thin-layer chromatography (19). Separation of the lipids into phospholipids, diglycerides, free fatty acids, triglycerides, and cholesteryl esters was performed by thin-layer chromatography (Instant thin-layer chromatography, polysilicic acid gel-impregnated glass fiber sheets, Gelman Instrument Co., Ann Arbor, Mich.) with petroleum ether:ethyl etheracetic acid (85: 15: 1) as solvent system. Individual phospholipids were separated by two-dimensional thin-layer chromatography using a modification of the method of Parson and Patton (20). Silica gel H plates were developed in the first dimension with chloroform-methanol-15 M ammonium hydroxide-water (130:70: 10:5) and in the second dimension with chloroform-acetone-methanol-glacial acetic acid-water (100:40:20:20: 10). The spots were visualized with iodine vapor. The individual spot corresponding to the known standard was scraped into scintillation vials. Scinti&ztion counting. The vials were filled with 10 ml of Bio-Solv scintillation cocktail (5 g PPO, 30 mg POPQP, 130 ml methanol, and 108 ml Bio-Solv and diluted to 1000 ml with toluene) and assayed for radioactivity in a Packard scintillation counter. Quenching was corrected by the use of an external standard. Protein measurement. Protein determination was carried out by the method of Schaterle and Dallack (21).
158
HUANG AND KUMMEROW
RESULTS
The uptake and esterification of labeled fatty acids by aortic smooth muscle cells under aerobic and anaerobic incubations are shown in Table 1. The uptake of elaidic acid was higher than that of oleic acid in smooth muscle cells both in aerobic and anaerobic conditions. However, most of elaidic acid taken up by the cells was kept in an unesterified form. Incorporation of oleic acid into tissue lipids was significantly higher than that of elaidic acid incubated under both aerobic and anaerobic conditions. As compared to elaidic acid, more oleic acid was incorporated into triglycerides. With labeled elaidic acid as the precursor, the formation of labeled diglycerides was relatively large. An increase in the incorporation of oleic acid into diglycerides, free fatty acids, and triglycerides was found under anaerobic conditions. More elaidic acid was incorporated only into triglycerides when the cells were incubated under anaerobic than aerobic conditions. The incorporation of (l-l40labeled fatty acids into individual phospholipids under aerobic and anaerobic conditions is shown in Table 2. Phosphatidyl choline was found to be the major labeled component under both conditions. Among phospholipid classes, approximately 78% of labeled oleic acid was recovered in phosphatidylcholine but only about 6% of the total activity was accounted for by the incorporation of elaidic acid into phosphatidylcholine. As compared to oleic acid as a precursor, relatively high percentages of the total activities were found in the lysophosphatidylcholine and phosphatidylethanolamine fractions with elaidic acid as a precursor. No significant difference of the incorporation of labeled fatty acids into phospholipid classes between aerobic and anaerobic conditions was observed under these studies. TABLE UPTAKE [lJ4C]E~~t~~~
AND
DURING
ESTERIFICATION ACID BY SWINE AEROBIC
Incubation condition Substrate
Phospholipids DigIycerides Free fatty acids Trilgycerides Cholesteryl esters
AND
I
OF [I-**C]OLEIC AORTIC SMOOTH ANAEROBIC
ACID MUSCLE
INCUBATIONS
Anaerobic
Aerobic Oleic
19 0.78 5.6 36 1.2
acid
2 2 k 2 5
2 0.04 0.4 5 0.1
AND CELLS
Elaidic
10.0 2.3 157 6.6 0.21
2 2 f + f
acid
Oleic acid
Elaidic acid
0.4 0.4 9 0.3 0.02
22 1.1 8.9 47 1.4
10.6 2.2 157 a.4 0.33
It 2 + 2 2
1 0.1 0.5 1 0.1
LI Results expressed as nanomoles of substrate taken up or incorporated milligram protein per 4 hr. Mean t SD for three determinations.
2 2 -t 2 rt
0.3 0.3 2 0.9 0.18
into lipids per
ESTERIFICATION
OF FATTY
TABLE
159
ACIDS
2
SEPARATION OF PHOSPHOLIPIDS SYNTHESIZED IN SWINE AORTK SMOOTH MUSCLE CELLS INCUBATED WITH ( I-WJLABELED FATTY ACIDS UNDER AEROBIC AND ANAEROBIC CONDITIONS
Radioactivity as percentages of total labeled phospholipids Incubation condition
Anaerobic
Aerobic
Substrate
Oleic acid
Elaidic acid
Oleic acid
LPC” SP + PI PS PC PE
0.5 10.9 3.1 78 7.3
1.8 14.7 3.5 69 10.6
0.3 10.3 2.4 79 7.7
2 f * lr +
0.2” 0.4 0.6 1 0.1
5 2 + 2 r
0.1 0.6 0.7 I 0.5
2 2 z It t
0.1 0.1 0.3 1 0.9
Elaidic acid 2.6 14.1 2.5 70 10.5
’ LPC, lysophosphatidylcholine; SP + PI sphingomyelin + phosphatidylinositol; phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. ’ Mean f SD for three determinations.
-c 0.2 t 0.8 -c 0.5 2 2 + 0.3 PS,
DISCUSSION
The present study showed that in swine aortic smooth muscle cells, labeled oleic acid was incorporated preferentially into triglycerides and phospholipids and comparably less into diglycerides and cholesteryl esters. Oleic acid appeared to be more readily utilized for cholesteryl ester synthesis than elaidic acid. The difference in cholesterol esterification between these two fatty acids could be, as observed in the liver microsomes (22), due to a reflection of the enzyme specificity toward the fatty acid substrates. Although the uptake of elaidic acid by the smooth muscle cells was greater than oleic acid, the incorporation of elaidic acid into cell lipids was much lower than that of oleic acid incubated under both aerobic and anaerobic conditions. Most of elaidic acid taken up by the cells was kept in an unesterified form. The high radioactivity in the free fatty acid fraction and low incorporation into complex lipid fractions may in part be due to the relatively low rate of oxidation of elaidic acid. Anderson (23) reported that the oxidation of uniformly [14C]oleic acid was faster than that of uniformly [r4Clelaidic acid and concluded that the difference in oxidation rate between cis and tram fatty acids was due to a slower oxidation of the alkyl chain on the methyl side of the trans double bond. Recently Hsu and Kummerow (24) demonstrated that elaidyl camitine has lower oxygen uptake rate than that of oleyl carnitine in rat heart mitochondria. As compared to the esterification of oleic acid, the incorporation of elaidic acid into triglycerides and phospholipids was relatively small, and its incorporation into diglycerides was relatively high, suggest-
160
HUANG
AND KUMMEROW
ing the existence of substrate specificity for triglyceride and phospholipid synthesis. The significant increase in the incorporation of labeled oleic acid into triglycerides, diglycerides, and free fatty acids was observed in the smooth muscle cells under anaerobic conditions. It was reported that a marked accumulation of triglycerides, and to a lesser extent of free fatty acids, was found in the hypoxic strain L mouse fibroblast cells (11). The results showed that more oleic acid was taken up and incorporated into triglycerides and diglycerides, and more elaidic acid was esterified to triglycerides when the smooth muscle cells were incubated under anaerobic conditions. It has been reported that hypoxia and other injuries can cause increased arterial endothelial permeability (25, 26). it is possible that hypoxia causes decreased fatty acid oxidation with the resultant accumulation of glycerides and free fatty acids. Chiodi (27) and SolerArgilaga et al. (28) reported that fatty acid oxidation was decreased and triglycerides accumulated in hypoxic liver. The impairment of this utilization of free fatty acid under anaerobic conditions results in increased esterification and storage of glycerides. SUMMARY
The incorporation of [ l-14C]01eic acid and [ 1-i4C]elaidic acid into cell lipids of the swine aortic smooth muscle cells under aerobic and anaerobic conditions was investigated. The uptake of elaidic acid was higher than that of oleic acid; however, most elaidic acid taken up by aortic smooth muscle cells was kept in an unesterified form. As compared to elaidic acid, significant amounts of oleic acid were incorporated into phospholipids, triglycerides, and cholesteryl esters under both aerobic and anaerobic conditions. More labeled oleic acid and elaidic acid was esterified into triglycerides under anaerobic than aerobic conditions. Of individual phospholipid fractions studied, both labeled oleic and elaidic acids were incorporated predominantly into phosphatidylcholine fraction. ACKNOWLEDGMENTS The authors are grateful to Ms. Meme Dai for her technical assistance. The work was supported by NIH Grant HL 15504-5 and the Wallace Genetic Foundation.
REFERENCES 1. 2. 3. 4. 5. 6.
Johnston, P. V., Johnson, 0. C., and Kummerow, F. A., J. Nutr. 65, 13-23 (1958). Egwim, P. O., and Sgoutas, D. S., J. Nutr. 101, 307-314 (1971). Takatori, T., Philips, F. C., Shimasaki, H., and Privett, 0. S.,Lipids l&272-280(1976). Egwim, P. O., and Kummerow, F. A., J. Nutr. 102, 783-792 (1972). Egwim, P. 0.. and Sgoutas, D. S., J. Nutr. 101, 315-322 (1971). Weigenberg, B. I., McMillian, G. C., and Ritchie, A. C., Arch. Pat/d. 72, 358-366 (l%l).
ESTERIFICATION
OF FATTY
ACIDS
161
7. Dayton, S., and Hashimoto, S., J. Atheroscler. Res. 8, 555-568 (1968). 8. Sinclair, H. M., Lancet 270, 381-383 (1956). 9. McMiIIian, G. C., Silver, M. D., and Weigenberg, B. I., Arch. Pathol. 76, 106-112 (1963). 10. Anderson, J. T., Grande, F., and Keys, A., J. Nurr. 75, 388-394 (l%l). 11. Gordon, G. B., Barcza, M. A., and Bush, M. E., Amer. J. Pathof. 88, 663 (1977). 12. Briggs, R. G., and Glenn, J. L., Lipids 11, 791-797 (1976). 13. Kjeldsen, K., Wanstrup, J., and Astrup, P., J. Atheroscler. Res. 8, 835-841 (1968). 14. Helin, P., Lorenzen, I., Garbarsch, C., and Matthiessen, M. E., ./. Atheroscler. Res. 9, 295-304 (1969). 15. Helin, P., and Lorenzen, I., Angiology 20, l-12 (1%9). 16. Myasnikov, A. L., Circulation 17, 99-113 (1958). 17. Kamio, A., Huang, W. Y.. Cho, B. H. S., Imai, H., and Kummerow, F. A., Arterial Wall 4, 27-44 (1977). 18. Folch, J. M., Lees, M., and Sloane-Stanely, G. H., J. Biol. Chem. 226, 497-509. 19. Huang, W. Y., Kamio, A., Yeh, S. J. C., and Kummerow, F. A., Artery 3, 439-455 (1977). 20. Parson, J. G., and Patton, S., J. Lipid Res. 8, 6%-699 (1%7). 21. Schaterle, G. R., and Dallack, R. L., Anal. Biochem. 51, 654-655 (1973). 22. Sgoutas, D. S., Biochemistry 9, 1826-1833 (1970). 23. Anderson, R. I., Biochim. Biophys. Acta 144, 18-24 (1%7). 24. Hsu, C. M. L., and Kummerow, F. A., Lipids 12, 486-494 (1977). 25. Constantinides, P., and Robinson, M., Arch. Pathol. 88, 99-105 (1969). 26. Robertson, A. L., Jr., and Khairallah, P. A., Exp. Mol. Puthol. 18, 241-260 (1973). 27. Chiodi, H., Amer. J. Physiol. 218, 92-94 (1970). 28. Soler-Argilaga, C., Infante, R., Polonovski, J., and Caroli, J., Biochim. Biophys. Acta 239, 154-161 (1971).
MEDICINE
21, 156-161 (1979)
Esterification of cis and trans Fatty Acids by Swine Aortic Smooth Muscle Ceils during Aerobic and Anaerobic Incubations WILLIAM Y. HUANG AND FRED A. KUMMEROW The Harlan E. Moore Heart Research Foundation, Champaign, Illinois and The Burnsides Research Laboratory, University of Illinois, Urbana, Illinois 61801
61820,
Received October 28, 1978
It has been shown that dietary tram fatty acids can be incorporated into tissue and serum lipids (l-7). It was postulated that dietary tram fatty acids might be related to atherosclerosis and other diseases (8). The dietary tram fatty acids have been shown to increase the serum cholesterol level in animals (6,9) as well as in man (10). The accumulation of lipids, especially in the level of triglyceride in hypoxia, has been reported in the cell culture of strain L mouse fibroblasts (11) and intimal cells of swine aorta (12). In rabbits exposed in vivo to hypoxic conditions, there has been found an increase of arterial lipid content and development of atherosclerosis (13-16). Studies on the effect of hypoxia on the fatty acid esterification in the aortic smooth muscle cells have not been reported to date. Furthermore, the incorporation of cis and trans fatty acids into swine aortic smooth muscle cell lipids has not been determined to date. The present studies were designed to investigate the incorporation of labeled oleic acid (cis-9-octadecenoic acid) and elaidic acid (tram-9octadecenoic acid) into lipids in the swine aortic smooth muscle cells under aerobic and anaerobic incubations. METHODS AND MATERIALS
Cell culture techniques. Segments of thoracic aorta were obtained from approximately 6-month-old swine weighing from 100 to 110 kg. Explants of intimal-medial segments of swine aortas were prepared according to the method of Kamio et al. (17). The explants were first grown in %-ml Falcon flasks. Smooth muscle cells usually started growing out of the 156 0006-2!344/79/020156-06$02.00/O Copytight @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
ESTERIFICATION
OF FATTY
ACIDS
157
intimal-medial explants after about 14 days, and the growth appeared to be confluent 3 weeks later. The cells were then released from the flask by trypsinization and transferred to a new 250~ml flask. The explants of intimal-medial segments (smooth muscle cells) were cultivated in the modified Dulbecco-Vogt medium supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic solution. The media were changed every third day. Incubation temperature was 37°C in a moist atmosphere of 95% air-5% CO,. Incubations. When growth appeared to be confluent, the cells were harvested by trypsinization, washed three times, and resuspended in a balanced salt solution. The incubation medium, containing 3.0 ml of Krebs-Ringer bicarbonated buffer (pH 7.4), 5 mM glucose, 0.1 unit insulin, 0.1 mM fatty acid-free bovine serum albumin, 0.5 mM [1J4C]01eic acid (specific activity 55 mCi/mmole), or [l-‘4Clelaidic acid (specific activity 50 mCi/mmole) was added to the smooth muscle cells (cell protein 2.5 mg) in the flask. The flasks were gassed with 95% O,-5% CO, mixture (aerobically) or 95% N,-5% CO, mixture (anaerobically) and stoppered with rubber serum stoppers. The flasks were shaken in a gyrotory water bath shaker at 37” for 4 hr. After incubation, the cells were washed three times by resuspending in a balanced salt solution and centrifuged (600g for 2 min) at 4°C. Extraction and chromatography. The lipids were extracted from the cells according to the method of Folch et al. (18). Radioactive lipids were separated by thin-layer chromatography (19). Separation of the lipids into phospholipids, diglycerides, free fatty acids, triglycerides, and cholesteryl esters was performed by thin-layer chromatography (Instant thin-layer chromatography, polysilicic acid gel-impregnated glass fiber sheets, Gelman Instrument Co., Ann Arbor, Mich.) with petroleum ether:ethyl etheracetic acid (85: 15: 1) as solvent system. Individual phospholipids were separated by two-dimensional thin-layer chromatography using a modification of the method of Parson and Patton (20). Silica gel H plates were developed in the first dimension with chloroform-methanol-15 M ammonium hydroxide-water (130:70: 10:5) and in the second dimension with chloroform-acetone-methanol-glacial acetic acid-water (100:40:20:20: 10). The spots were visualized with iodine vapor. The individual spot corresponding to the known standard was scraped into scintillation vials. Scinti&ztion counting. The vials were filled with 10 ml of Bio-Solv scintillation cocktail (5 g PPO, 30 mg POPQP, 130 ml methanol, and 108 ml Bio-Solv and diluted to 1000 ml with toluene) and assayed for radioactivity in a Packard scintillation counter. Quenching was corrected by the use of an external standard. Protein measurement. Protein determination was carried out by the method of Schaterle and Dallack (21).
158
HUANG AND KUMMEROW
RESULTS
The uptake and esterification of labeled fatty acids by aortic smooth muscle cells under aerobic and anaerobic incubations are shown in Table 1. The uptake of elaidic acid was higher than that of oleic acid in smooth muscle cells both in aerobic and anaerobic conditions. However, most of elaidic acid taken up by the cells was kept in an unesterified form. Incorporation of oleic acid into tissue lipids was significantly higher than that of elaidic acid incubated under both aerobic and anaerobic conditions. As compared to elaidic acid, more oleic acid was incorporated into triglycerides. With labeled elaidic acid as the precursor, the formation of labeled diglycerides was relatively large. An increase in the incorporation of oleic acid into diglycerides, free fatty acids, and triglycerides was found under anaerobic conditions. More elaidic acid was incorporated only into triglycerides when the cells were incubated under anaerobic than aerobic conditions. The incorporation of (l-l40labeled fatty acids into individual phospholipids under aerobic and anaerobic conditions is shown in Table 2. Phosphatidyl choline was found to be the major labeled component under both conditions. Among phospholipid classes, approximately 78% of labeled oleic acid was recovered in phosphatidylcholine but only about 6% of the total activity was accounted for by the incorporation of elaidic acid into phosphatidylcholine. As compared to oleic acid as a precursor, relatively high percentages of the total activities were found in the lysophosphatidylcholine and phosphatidylethanolamine fractions with elaidic acid as a precursor. No significant difference of the incorporation of labeled fatty acids into phospholipid classes between aerobic and anaerobic conditions was observed under these studies. TABLE UPTAKE [lJ4C]E~~t~~~
AND
DURING
ESTERIFICATION ACID BY SWINE AEROBIC
Incubation condition Substrate
Phospholipids DigIycerides Free fatty acids Trilgycerides Cholesteryl esters
AND
I
OF [I-**C]OLEIC AORTIC SMOOTH ANAEROBIC
ACID MUSCLE
INCUBATIONS
Anaerobic
Aerobic Oleic
19 0.78 5.6 36 1.2
acid
2 2 k 2 5
2 0.04 0.4 5 0.1
AND CELLS
Elaidic
10.0 2.3 157 6.6 0.21
2 2 f + f
acid
Oleic acid
Elaidic acid
0.4 0.4 9 0.3 0.02
22 1.1 8.9 47 1.4
10.6 2.2 157 a.4 0.33
It 2 + 2 2
1 0.1 0.5 1 0.1
LI Results expressed as nanomoles of substrate taken up or incorporated milligram protein per 4 hr. Mean t SD for three determinations.
2 2 -t 2 rt
0.3 0.3 2 0.9 0.18
into lipids per
ESTERIFICATION
OF FATTY
TABLE
159
ACIDS
2
SEPARATION OF PHOSPHOLIPIDS SYNTHESIZED IN SWINE AORTK SMOOTH MUSCLE CELLS INCUBATED WITH ( I-WJLABELED FATTY ACIDS UNDER AEROBIC AND ANAEROBIC CONDITIONS
Radioactivity as percentages of total labeled phospholipids Incubation condition
Anaerobic
Aerobic
Substrate
Oleic acid
Elaidic acid
Oleic acid
LPC” SP + PI PS PC PE
0.5 10.9 3.1 78 7.3
1.8 14.7 3.5 69 10.6
0.3 10.3 2.4 79 7.7
2 f * lr +
0.2” 0.4 0.6 1 0.1
5 2 + 2 r
0.1 0.6 0.7 I 0.5
2 2 z It t
0.1 0.1 0.3 1 0.9
Elaidic acid 2.6 14.1 2.5 70 10.5
’ LPC, lysophosphatidylcholine; SP + PI sphingomyelin + phosphatidylinositol; phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. ’ Mean f SD for three determinations.
-c 0.2 t 0.8 -c 0.5 2 2 + 0.3 PS,
DISCUSSION
The present study showed that in swine aortic smooth muscle cells, labeled oleic acid was incorporated preferentially into triglycerides and phospholipids and comparably less into diglycerides and cholesteryl esters. Oleic acid appeared to be more readily utilized for cholesteryl ester synthesis than elaidic acid. The difference in cholesterol esterification between these two fatty acids could be, as observed in the liver microsomes (22), due to a reflection of the enzyme specificity toward the fatty acid substrates. Although the uptake of elaidic acid by the smooth muscle cells was greater than oleic acid, the incorporation of elaidic acid into cell lipids was much lower than that of oleic acid incubated under both aerobic and anaerobic conditions. Most of elaidic acid taken up by the cells was kept in an unesterified form. The high radioactivity in the free fatty acid fraction and low incorporation into complex lipid fractions may in part be due to the relatively low rate of oxidation of elaidic acid. Anderson (23) reported that the oxidation of uniformly [14C]oleic acid was faster than that of uniformly [r4Clelaidic acid and concluded that the difference in oxidation rate between cis and tram fatty acids was due to a slower oxidation of the alkyl chain on the methyl side of the trans double bond. Recently Hsu and Kummerow (24) demonstrated that elaidyl camitine has lower oxygen uptake rate than that of oleyl carnitine in rat heart mitochondria. As compared to the esterification of oleic acid, the incorporation of elaidic acid into triglycerides and phospholipids was relatively small, and its incorporation into diglycerides was relatively high, suggest-
160
HUANG
AND KUMMEROW
ing the existence of substrate specificity for triglyceride and phospholipid synthesis. The significant increase in the incorporation of labeled oleic acid into triglycerides, diglycerides, and free fatty acids was observed in the smooth muscle cells under anaerobic conditions. It was reported that a marked accumulation of triglycerides, and to a lesser extent of free fatty acids, was found in the hypoxic strain L mouse fibroblast cells (11). The results showed that more oleic acid was taken up and incorporated into triglycerides and diglycerides, and more elaidic acid was esterified to triglycerides when the smooth muscle cells were incubated under anaerobic conditions. It has been reported that hypoxia and other injuries can cause increased arterial endothelial permeability (25, 26). it is possible that hypoxia causes decreased fatty acid oxidation with the resultant accumulation of glycerides and free fatty acids. Chiodi (27) and SolerArgilaga et al. (28) reported that fatty acid oxidation was decreased and triglycerides accumulated in hypoxic liver. The impairment of this utilization of free fatty acid under anaerobic conditions results in increased esterification and storage of glycerides. SUMMARY
The incorporation of [ l-14C]01eic acid and [ 1-i4C]elaidic acid into cell lipids of the swine aortic smooth muscle cells under aerobic and anaerobic conditions was investigated. The uptake of elaidic acid was higher than that of oleic acid; however, most elaidic acid taken up by aortic smooth muscle cells was kept in an unesterified form. As compared to elaidic acid, significant amounts of oleic acid were incorporated into phospholipids, triglycerides, and cholesteryl esters under both aerobic and anaerobic conditions. More labeled oleic acid and elaidic acid was esterified into triglycerides under anaerobic than aerobic conditions. Of individual phospholipid fractions studied, both labeled oleic and elaidic acids were incorporated predominantly into phosphatidylcholine fraction. ACKNOWLEDGMENTS The authors are grateful to Ms. Meme Dai for her technical assistance. The work was supported by NIH Grant HL 15504-5 and the Wallace Genetic Foundation.
REFERENCES 1. 2. 3. 4. 5. 6.
Johnston, P. V., Johnson, 0. C., and Kummerow, F. A., J. Nutr. 65, 13-23 (1958). Egwim, P. O., and Sgoutas, D. S., J. Nutr. 101, 307-314 (1971). Takatori, T., Philips, F. C., Shimasaki, H., and Privett, 0. S.,Lipids l&272-280(1976). Egwim, P. O., and Kummerow, F. A., J. Nutr. 102, 783-792 (1972). Egwim, P. 0.. and Sgoutas, D. S., J. Nutr. 101, 315-322 (1971). Weigenberg, B. I., McMillian, G. C., and Ritchie, A. C., Arch. Pat/d. 72, 358-366 (l%l).
ESTERIFICATION
OF FATTY
ACIDS
161
7. Dayton, S., and Hashimoto, S., J. Atheroscler. Res. 8, 555-568 (1968). 8. Sinclair, H. M., Lancet 270, 381-383 (1956). 9. McMiIIian, G. C., Silver, M. D., and Weigenberg, B. I., Arch. Pathol. 76, 106-112 (1963). 10. Anderson, J. T., Grande, F., and Keys, A., J. Nurr. 75, 388-394 (l%l). 11. Gordon, G. B., Barcza, M. A., and Bush, M. E., Amer. J. Pathof. 88, 663 (1977). 12. Briggs, R. G., and Glenn, J. L., Lipids 11, 791-797 (1976). 13. Kjeldsen, K., Wanstrup, J., and Astrup, P., J. Atheroscler. Res. 8, 835-841 (1968). 14. Helin, P., Lorenzen, I., Garbarsch, C., and Matthiessen, M. E., ./. Atheroscler. Res. 9, 295-304 (1969). 15. Helin, P., and Lorenzen, I., Angiology 20, l-12 (1%9). 16. Myasnikov, A. L., Circulation 17, 99-113 (1958). 17. Kamio, A., Huang, W. Y.. Cho, B. H. S., Imai, H., and Kummerow, F. A., Arterial Wall 4, 27-44 (1977). 18. Folch, J. M., Lees, M., and Sloane-Stanely, G. H., J. Biol. Chem. 226, 497-509. 19. Huang, W. Y., Kamio, A., Yeh, S. J. C., and Kummerow, F. A., Artery 3, 439-455 (1977). 20. Parson, J. G., and Patton, S., J. Lipid Res. 8, 6%-699 (1%7). 21. Schaterle, G. R., and Dallack, R. L., Anal. Biochem. 51, 654-655 (1973). 22. Sgoutas, D. S., Biochemistry 9, 1826-1833 (1970). 23. Anderson, R. I., Biochim. Biophys. Acta 144, 18-24 (1%7). 24. Hsu, C. M. L., and Kummerow, F. A., Lipids 12, 486-494 (1977). 25. Constantinides, P., and Robinson, M., Arch. Pathol. 88, 99-105 (1969). 26. Robertson, A. L., Jr., and Khairallah, P. A., Exp. Mol. Puthol. 18, 241-260 (1973). 27. Chiodi, H., Amer. J. Physiol. 218, 92-94 (1970). 28. Soler-Argilaga, C., Infante, R., Polonovski, J., and Caroli, J., Biochim. Biophys. Acta 239, 154-161 (1971).
