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Acetyl-CoA carboxylase and fatty acid synthetase activities in cytoplasmic preparations of porcine arteries
BIOCHEMICAL
MEDICINE
26, 115-120
(1981)
Acetyl-CoA Carboxylase and Fatty Acid Synthetase Activities in Cytoplasmic Preparations of Porcine Arteries B. H. s. CHO Harlan Illinois
E. Moore Heart Research Foundation, 503 South Sixth Street. Champaign, 61820 and Burnsides Research Laboratory, Department of Food Science. University of Illinois, Urbana, Illinois 61801
Received February 1I ) 1981
INTRODUCTION
The fatty acid synthetic system has been thoroughly studied in organs such as liver (1). adipose tissue (2), and mammary gland (3), which have a great capacity for triglyceride synthesis. The enzymes for fatty acid synthesis in these organs are mainly soluble enzymes found in the cell compartment, and synthesis has been demonstrated by Wakil to involve malonyl-CoA (4). Acetyl-CoA is first carboxylated to malonyl-CoA in an ATP-requiring reaction catalyzed by acetyl-CoA carboxylase. Then, the concerted reactions involving acetyl-CoA, malonyl-CoA, and NADPH are catalyzed by fatty acid synthetase to form the coenzyme ester of a long-chain fatty acid. The presence of a mitochondrial system for fatty acid synthesis was demonstrated in the liver (5), but quantitatively it was small in contrast to the capacity of the supematant enzymes (6). In metabolic studies, it has been found that labeled precursor such as [14C]acetate or [‘4C]glucose is incorporated into arterial lipids and the rate of fatty acid synthesis is significantly enhanced in the atherosclerotic aorta (7-9). In normal arteries, newly synthesized fatty acids are esterified to phospholipid and triglyceride; in atherosclerotic aorta they are esterified to cholesterol (10-12). Although studies of lipid metabolism in normal and atherosclerotic aortas are numerous, few data are available concerning activities of the acetyl-CoA carboxylase and fatty acid synthetase enzymes which are directly involved in fatty acid synthesis in the arterial tissue. The present report describes acetyl-CoA carboxylase and fatty acid synthetase activities in cytoplasmic preparations from arterial and liver 115 OOOfG2944/81/040115-06$02.00/O Copyright 0 1981 by Academic Press. Inc. All nghis of reproduction in any form reserved.
116
B. H. S.CHO
tissue of swine which were fed either a normal or a high-fat diet, Since arteries reveal selective susceptibility to atherosclerosis, the activities of these fatty acid-synthesizing enzymes were compared among thoracic aorta, abdominal aorta, and coronary arteries. MATERIALS AND METHODS Crossbred (New Hampshire X Duroc), 6-month-old swine weighing 100 to i 10 kg were used. The swine were raised ad libitum on grain diets with and without butterfat supplement. The grain diet consisted of 87.25% ground yellow corn, 10% solvent extracted defatted soybean meal, and 2.75% premix of multiple minerals and vitamins (13). Butterfat was included into the diet by mixing 20 kg of melted fat and 80 kg of grain diet. The whole length of aortas, right and left coronary arteries, and portions of liver tissue were removed within 30 min of slaughter and kept in ice-cold physiological saline solution during preparation. The tissue preparation, homogenization, and subcellular fractionation of arterial and liver tissues were carried out by the methods described, in detail, previously (14,15). In brief, the crude tissue homogenates were centrifuged to sediment cellular debris at 1500 g for 20 min and the supernatant was centrifuged at 105,000 g for 60 min. The resulting 105,000 g high-speed supernatant is referred to as the cytoplasmic preparation and its protein content was determined by the method of Lowry et al. (16). Acetyl-CoA carboxylase was assayed by a modification of the method of Gregolin et al. (17) and Chang et al. (18). For the assay, 0.5 ml of high-speed supernate preparations were first activated by preincubation with shaking for 30 min at 37”C, in air, with 0.5 ml of a buffer solution (pH, 7.5) containing in micromoles: Tris-HCI, 10.0; MgC&, 5.0; sodium citrate, 2.5; glutathion (Nutritional Biochemicals Co., Cleveland, Ohio), 1.0; bovine serum albumin (fatty acid-free), 0.01. Carboxylation was initiated by adding 0.5 ml of reaction components as a mixture in micromoles; Tris-HCl buffer, pH 7.2, 10.0; MgC&, 5.0; sodium citrate 7.5; GSH, 1.0; ATP (NBC), 2.0; acetyl-CoA (NBC), 0.2; NaH14C03 (AmershamlSearle, Arlington Heights, Ill.) (sp act, 0.1 pCi/ kmole), 20.0. The final reaction volume was 1.5 ml in all incubations and the blank for assay contained no protein. The cofactor mixtures for preincubation and main incubation were prepared fresh the day of assay. Following a lo-min incubation at 37°C in a metabolic shaker, the reactions were stopped with 0.2 ml 6 N HCl, and then centrifuged to remove the denatured protein. Aliquots of 0.5 ml solution were transferred into scintillation vials and evaporated to dryness on a warm plate. After drying, 0.2 ml of distilled water was added to each vial followed by 15 ml of Bray’s scintillation solution (19). Acid-stable 14C activity was then de-
FATTY
ACID-SYNTHESIZING
ENZYMES
IN ARTERIES
117
termined and counting efficiency was estimated by counting the appropriate standards under the same conditions. The results are expressed in picomoles of H’4C0,-incorporated per milligram protein per minute. Fatty acid synthetase was assayed by the methods of Chang et al. (18) and Mersmann et al. (20). One milliliter of incubation mixture contained in micromoles: malonyl-CoA (NBC), 0.2; acetyl-CoA (NBC), 0.1; NADPH (NBC), 0.5; malonyl-CoA-1 ,3-14C (DHOM Products Ltd., North Hollywood, Calif.) (sp act, 50 @i/pmole), 1 x 10e3 (0.05 t&i); phosphate buffer (pH, 6.8), 10.0. To this freshly prepared incubation mixture, 0.5 ml of high-speed supernate preparations was added and the reactions, in a final volume of 1.5 ml, were allowed to proceed for 30 min at 37°C with shaking. The assay blank contained no protein as an enzyme source. At the termination of incubation, the reaction was stopped with 0.2 ml 6 N HCl. About an equal volume of 95% ethanol was added to the reaction mixture and the fatty acids were extracted three times with petroleum ether (bp 3040°C). The combined extracts were dried in a scintillation vial, 15 ml of toluene scintillation solution (0.5% PPO and 0.03% dimethyl-POPOP) was added, and the samples were counted in a Packard Tri-Carb Liquid Scintillation Counter. The results were expressed as picomoles of malonyl-CoA incorporated into fatty acids per milligram protein per minute. RESULTS
AN5 DISCUSSION
The cytoplasmic malonyl-CoA pathway for fatty acid biosynthesis, catalyzed by acetyl-CoA carboxylase and fatty acid synthetase multienzyme complex, has been generally recognized on the main de uwo biosynthetic pathway for fatty acids (6,21). The current study demonstrates the presence of these enzymes in cytoplasmic preparations from the arterial tissues of swine as shown in Tables 1 and 2. Although both acetyl-CoA carboxylase and fatty acid synthetase activities in the arteries are quite low compared to liver tissue, arterial tissues clearly possess all the enzymes necessary for fatty acid biosynthesis. In addition, the discovery of a chain elongation system for fatty acids in the mitochondrial and microsomal fractions from monkey aortas (22) further demonstrates the lipid synthesizing capability of the arterial tissue. A noticeable difference in the fatty acid composition of arterial lipids, particularly cholesteryl ester, between normal and atherosclerotic aortas has been a subject of interest. In normal arterial tissue, the fatty acid composition of arterial cholesteryl ester has been found to be relatively similar to that of cholesteryl ester from blood plasma, i.e., high levels of linoleic acid, indicating the influx of plasma cholesteryl ester (23). However, arteries with fatty streaks, characterized microscopically by
118
B. H. S. CHO TABLE
ACETYL-COA
CARBOXYLASE
Tissue
ACTIVITY LIVER
1
IN CYTOPLASMIC PREPARATIONS TISSUES OF SWINE’.”
Grain diet
Thoracic aorta Abdominal aorta Coronary artery Liver
69.6-t 64.12 68.62 537.4~
FROM ARTERIAL
AND
Butterfat diet
5.3’ 3.9 5.8 23.Sd
55.42 4.2 56.7? 7.1’ 54.0* 4.5’ 407.2-c 29.1d
” For the assay, 0.5 ml of cytoplasmic preparations containing 0.3-0.5 mg of protein was first activated by preincubation for 30 min at 37’C with 0.5 ml of a buffer solution (pH 7.5) containing in micromoles: Tris-HCL, 10.0; MgClt, 5.0; sodium citrate, 2.5; glutathion, 1.O; bovine serum albumin, 0.01. Carboxylation was initiated by adding 0.5 ml of reaction components as a mixture in micromoles: Tris-HCl buffer (pH 7.2) 10.0; MgQ. 5.0; sodium citrate, 7.5; glutathione, 1.0; ATP, 2.0; acetyl-CoA, 0.2; NaH14C0, (sp act 0.1 uCi/pmole), 20.0. The reaction mixtures which contained a final volume of 1.5 ml were then incubated for 10 min at 37°C in a metabolic shaker. b Average picomoles of H’%ZO,- incorporated per milligram of protein per minute + SD in duplicate assays from three animals. ‘.d Means in the same column bearing different superscript letters differ significantly from the thoracic aorta (P < 0.01).
numerous foam cells and intracellular lipids, have been shown high cholesteryl oleate concentrations (24,25). The occurence amount of oleate esterified to cholesterol in fatty streaks could increased cellular lipogenic activities which reflect fatty acid coupled with chain elongation and desaturation of fatty acids. local synthesis of lipid has been attributed to its accumulation TABLE FATTY
ACID
SYNTHETASE
ACTIVITY
Tissue Thoracic aorta Abdominal aorta Coronary artery Liver
2
IN CYTOPLASMIC
LIVER
TISSUES
to contain of a high represent synthesis Increased within the
PREPARATIONS
FBOM ARTERIAL
AND
OF SWINE”.~
Grain diet 91.3* 9.9 86.62 15.5’ 96.72 9.5 475.92 28.4d
Butterfat diet 56.92 46.5 2 62.22 291.1 f
9.3’ 10.9 6.4’ 25.7d
” The incubation medium contained in micromoles: Malonyl-CoA, 0.2; acetyl-CoA, 0.1; NADPH, 0.5; malonyl-CoA-1, 3-14C (sp act, 50 pCi/pmole), 0.001 (0.05 PCi); phosphate buffer (pH 6.8), 10.0 and 0.3-0.5 mg of protein in a final volume of 1.5 ml. Reaction mixtures were incubated for 30 min at 37°C in a metabolic shaker. b Average picomoles of malonyl-CoA incorporated per milligram of protein per minute + SD in duplicate assays from three animals. c.d Means in the same column bearing different superscript letters differ significantly from the thoracic aorta (P < 0.01).
FATTY
ACID-SYNTHESIZING
ENZYMES
IN ARTERIES
119
fatty streaks of humans (9). In atherosclerotic aortas from pigeons and rabbits, an increased rate of fatty acid synthesis and incorporation of synthesized fatty acids into various lipid classes has also been observed (26,27). The findings that significant differences in lipid metabolism do exist between normal and atherosclerotic aortas led to the speculation that the lipogenic activity in arteries might be indicative of atherosclerotic susceptibility. Therefore, in the present study, both acetyl-CoA carboxylase and fatty acid synthetase activities were measured in grossly normal thoracic, abdominal, and coronary arteries which reveal differences in susceptibility to atherosclerosis. No significant differences were found in those enzyme activities among arteries as shown in Tables 1 and 2. Similar findings have been reported in different locations of rabbit aorta (28). Although the aortic arch in the rabbit had a disposition for early and rapidly progressive lesions, there was no difference in the rate of fatty acid synthesis from acetate in arch vs thoracic vs abdominal aorta until actual appearance of lesions. Other studies have investigated possible metabolic differences in aortas from two breeds of pigeons which have great differences in susceptibility to spontaneous atherosclerosis, but no significant differences in rates of fatty synthesis were found (10). Based on current and previous findings, it can be stated that susceptibility is apparently not associated with inherent differences in fatty acid synthesis rate or related enzyme activities of the arteries. SUMMARY Acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS) activities were measured in cytoplasmic preparations from thoracic and abdominal aorta, coronary arteries, and liver tissue of swine. Both ACC and FAS activities were present in all three arterial segments, but their activities were quite low compared to those of liver tissue. Between thoracic aorta, abdominal aorta, and coronary arteries, no significant differences in enzyme activities were found. Inclusion of fat in the diet decreased activities of both regulatory enzymes for fatty acid synthesis in vascular as well as liver tissues, and FAS showed more decrease in its activity than ACC in all tissues examined. ACKNOWLEDGMENT This work was supported by the fund for the Moore Investigator (B.H.S.C.) Harlan E. Moore Charitable Trust.
from the
REFERENCES 1. Brady, R. O., and Grin, S.. J. Viol. Chem. 199, 421 (1952). 2. Martin, D. B., Homing, M. G.. and Vagelos, P. R., J. Bid Chem.
236, 663 (1961).
B. H. S. CHO 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Popjok, G., and Tietz, A., Biochem. J. 60, 147 (1955). Wakil, S. J., J. Amer. Chem. Sot. 80, 6465 (1958). Wakil, S. J.. McLain, L. W., and Warshaw, J. B., J. Bio/. Chem. 235, PC31 (1960). Wakil, S. J., J. Lipid Res. 2, 1 (1961). Whereat, A. F., J. Lipid Res. 7, 671 (1966). Howard, C. F., Jr.. J. Lipid Res. 12, 725 (1971). Chobanian, A. V., and Manzur, F., J. Lipid Res. 13, 201 (1972). Lofland, H. B., Jr., Moury, D. M., Hoffman, C. W., and Clarkson, T. B., J. Lipid Res. 6, 112 (1965). St. Clair, R. W., Lofland, H. B., Jr., and Clarkson, T. B., J. Lipid Res. 9, 739 (1968). Dayton, S., and Hashimoto, S., J. Athero. Res. 8, 555 (1%8). Huang, W. Y., and Kummerow, F. A., Lipids 11. 34 (1976). Cho, B. H. S., and Kummerow, F. A.. Proc. Sot. Exp. Biol. Med. 155, 8 (1977). Cho, B. H. S., and Kummerow, F. A., Biochem. Med. 20, 267 (1978). Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J.. 1. Biol. Chem. 193, 265 (1951). Gregolin, C., Ryder, E., Kleinschmidt. A. K., Warner, R. C., and Lane, M. D.. Proc. Nat. Acad. Sci. USN 56, 148 (1966). Chang, H., Seidman, I., Teebor, G., and Lane, M. D., Biochem. Biophys. Res. Comm. 28, 682 (1967). Bray, G. A., Anal. Biochem. 1, 279 (1960). Mersmann, H. J., Houk, J. M., Phinney. G., Underwood, M. C.. and Brown, L. J.. Amer. J. Physiol. 224, 1123 (1973). Vagelos, P. R., Annu. Rev. Biochem. 33, 139 (1964). Howard, C. F.. Jr., J. Lipid Res. 9, 254 (1968). Smith, E. B., J. Athero. Res. 5, 241 (1965). Geer, J. C., and Guidry, M. A., Exp. Mol. Pathol. 9, 230 (1964). Smith, E. B.. Evans, P. H., and Downham, M. D., J. Athero. Res. 7, 171 (1967). Newman, H. A. I., McCandless, E. L., and Zilversmit, D. B., J. Biol. Chem. 236, 1264 (1961). Whereat, A. F., and Orishimo, M. W.. Exp. Mol. Pathol. 9, 230 (1968). Whereat, A. F., J. Athero. Res. 4, 272 (1964).
MEDICINE
26, 115-120
(1981)
Acetyl-CoA Carboxylase and Fatty Acid Synthetase Activities in Cytoplasmic Preparations of Porcine Arteries B. H. s. CHO Harlan Illinois
E. Moore Heart Research Foundation, 503 South Sixth Street. Champaign, 61820 and Burnsides Research Laboratory, Department of Food Science. University of Illinois, Urbana, Illinois 61801
Received February 1I ) 1981
INTRODUCTION
The fatty acid synthetic system has been thoroughly studied in organs such as liver (1). adipose tissue (2), and mammary gland (3), which have a great capacity for triglyceride synthesis. The enzymes for fatty acid synthesis in these organs are mainly soluble enzymes found in the cell compartment, and synthesis has been demonstrated by Wakil to involve malonyl-CoA (4). Acetyl-CoA is first carboxylated to malonyl-CoA in an ATP-requiring reaction catalyzed by acetyl-CoA carboxylase. Then, the concerted reactions involving acetyl-CoA, malonyl-CoA, and NADPH are catalyzed by fatty acid synthetase to form the coenzyme ester of a long-chain fatty acid. The presence of a mitochondrial system for fatty acid synthesis was demonstrated in the liver (5), but quantitatively it was small in contrast to the capacity of the supematant enzymes (6). In metabolic studies, it has been found that labeled precursor such as [14C]acetate or [‘4C]glucose is incorporated into arterial lipids and the rate of fatty acid synthesis is significantly enhanced in the atherosclerotic aorta (7-9). In normal arteries, newly synthesized fatty acids are esterified to phospholipid and triglyceride; in atherosclerotic aorta they are esterified to cholesterol (10-12). Although studies of lipid metabolism in normal and atherosclerotic aortas are numerous, few data are available concerning activities of the acetyl-CoA carboxylase and fatty acid synthetase enzymes which are directly involved in fatty acid synthesis in the arterial tissue. The present report describes acetyl-CoA carboxylase and fatty acid synthetase activities in cytoplasmic preparations from arterial and liver 115 OOOfG2944/81/040115-06$02.00/O Copyright 0 1981 by Academic Press. Inc. All nghis of reproduction in any form reserved.
116
B. H. S.CHO
tissue of swine which were fed either a normal or a high-fat diet, Since arteries reveal selective susceptibility to atherosclerosis, the activities of these fatty acid-synthesizing enzymes were compared among thoracic aorta, abdominal aorta, and coronary arteries. MATERIALS AND METHODS Crossbred (New Hampshire X Duroc), 6-month-old swine weighing 100 to i 10 kg were used. The swine were raised ad libitum on grain diets with and without butterfat supplement. The grain diet consisted of 87.25% ground yellow corn, 10% solvent extracted defatted soybean meal, and 2.75% premix of multiple minerals and vitamins (13). Butterfat was included into the diet by mixing 20 kg of melted fat and 80 kg of grain diet. The whole length of aortas, right and left coronary arteries, and portions of liver tissue were removed within 30 min of slaughter and kept in ice-cold physiological saline solution during preparation. The tissue preparation, homogenization, and subcellular fractionation of arterial and liver tissues were carried out by the methods described, in detail, previously (14,15). In brief, the crude tissue homogenates were centrifuged to sediment cellular debris at 1500 g for 20 min and the supernatant was centrifuged at 105,000 g for 60 min. The resulting 105,000 g high-speed supernatant is referred to as the cytoplasmic preparation and its protein content was determined by the method of Lowry et al. (16). Acetyl-CoA carboxylase was assayed by a modification of the method of Gregolin et al. (17) and Chang et al. (18). For the assay, 0.5 ml of high-speed supernate preparations were first activated by preincubation with shaking for 30 min at 37”C, in air, with 0.5 ml of a buffer solution (pH, 7.5) containing in micromoles: Tris-HCI, 10.0; MgC&, 5.0; sodium citrate, 2.5; glutathion (Nutritional Biochemicals Co., Cleveland, Ohio), 1.0; bovine serum albumin (fatty acid-free), 0.01. Carboxylation was initiated by adding 0.5 ml of reaction components as a mixture in micromoles; Tris-HCl buffer, pH 7.2, 10.0; MgC&, 5.0; sodium citrate 7.5; GSH, 1.0; ATP (NBC), 2.0; acetyl-CoA (NBC), 0.2; NaH14C03 (AmershamlSearle, Arlington Heights, Ill.) (sp act, 0.1 pCi/ kmole), 20.0. The final reaction volume was 1.5 ml in all incubations and the blank for assay contained no protein. The cofactor mixtures for preincubation and main incubation were prepared fresh the day of assay. Following a lo-min incubation at 37°C in a metabolic shaker, the reactions were stopped with 0.2 ml 6 N HCl, and then centrifuged to remove the denatured protein. Aliquots of 0.5 ml solution were transferred into scintillation vials and evaporated to dryness on a warm plate. After drying, 0.2 ml of distilled water was added to each vial followed by 15 ml of Bray’s scintillation solution (19). Acid-stable 14C activity was then de-
FATTY
ACID-SYNTHESIZING
ENZYMES
IN ARTERIES
117
termined and counting efficiency was estimated by counting the appropriate standards under the same conditions. The results are expressed in picomoles of H’4C0,-incorporated per milligram protein per minute. Fatty acid synthetase was assayed by the methods of Chang et al. (18) and Mersmann et al. (20). One milliliter of incubation mixture contained in micromoles: malonyl-CoA (NBC), 0.2; acetyl-CoA (NBC), 0.1; NADPH (NBC), 0.5; malonyl-CoA-1 ,3-14C (DHOM Products Ltd., North Hollywood, Calif.) (sp act, 50 @i/pmole), 1 x 10e3 (0.05 t&i); phosphate buffer (pH, 6.8), 10.0. To this freshly prepared incubation mixture, 0.5 ml of high-speed supernate preparations was added and the reactions, in a final volume of 1.5 ml, were allowed to proceed for 30 min at 37°C with shaking. The assay blank contained no protein as an enzyme source. At the termination of incubation, the reaction was stopped with 0.2 ml 6 N HCl. About an equal volume of 95% ethanol was added to the reaction mixture and the fatty acids were extracted three times with petroleum ether (bp 3040°C). The combined extracts were dried in a scintillation vial, 15 ml of toluene scintillation solution (0.5% PPO and 0.03% dimethyl-POPOP) was added, and the samples were counted in a Packard Tri-Carb Liquid Scintillation Counter. The results were expressed as picomoles of malonyl-CoA incorporated into fatty acids per milligram protein per minute. RESULTS
AN5 DISCUSSION
The cytoplasmic malonyl-CoA pathway for fatty acid biosynthesis, catalyzed by acetyl-CoA carboxylase and fatty acid synthetase multienzyme complex, has been generally recognized on the main de uwo biosynthetic pathway for fatty acids (6,21). The current study demonstrates the presence of these enzymes in cytoplasmic preparations from the arterial tissues of swine as shown in Tables 1 and 2. Although both acetyl-CoA carboxylase and fatty acid synthetase activities in the arteries are quite low compared to liver tissue, arterial tissues clearly possess all the enzymes necessary for fatty acid biosynthesis. In addition, the discovery of a chain elongation system for fatty acids in the mitochondrial and microsomal fractions from monkey aortas (22) further demonstrates the lipid synthesizing capability of the arterial tissue. A noticeable difference in the fatty acid composition of arterial lipids, particularly cholesteryl ester, between normal and atherosclerotic aortas has been a subject of interest. In normal arterial tissue, the fatty acid composition of arterial cholesteryl ester has been found to be relatively similar to that of cholesteryl ester from blood plasma, i.e., high levels of linoleic acid, indicating the influx of plasma cholesteryl ester (23). However, arteries with fatty streaks, characterized microscopically by
118
B. H. S. CHO TABLE
ACETYL-COA
CARBOXYLASE
Tissue
ACTIVITY LIVER
1
IN CYTOPLASMIC PREPARATIONS TISSUES OF SWINE’.”
Grain diet
Thoracic aorta Abdominal aorta Coronary artery Liver
69.6-t 64.12 68.62 537.4~
FROM ARTERIAL
AND
Butterfat diet
5.3’ 3.9 5.8 23.Sd
55.42 4.2 56.7? 7.1’ 54.0* 4.5’ 407.2-c 29.1d
” For the assay, 0.5 ml of cytoplasmic preparations containing 0.3-0.5 mg of protein was first activated by preincubation for 30 min at 37’C with 0.5 ml of a buffer solution (pH 7.5) containing in micromoles: Tris-HCL, 10.0; MgClt, 5.0; sodium citrate, 2.5; glutathion, 1.O; bovine serum albumin, 0.01. Carboxylation was initiated by adding 0.5 ml of reaction components as a mixture in micromoles: Tris-HCl buffer (pH 7.2) 10.0; MgQ. 5.0; sodium citrate, 7.5; glutathione, 1.0; ATP, 2.0; acetyl-CoA, 0.2; NaH14C0, (sp act 0.1 uCi/pmole), 20.0. The reaction mixtures which contained a final volume of 1.5 ml were then incubated for 10 min at 37°C in a metabolic shaker. b Average picomoles of H’%ZO,- incorporated per milligram of protein per minute + SD in duplicate assays from three animals. ‘.d Means in the same column bearing different superscript letters differ significantly from the thoracic aorta (P < 0.01).
numerous foam cells and intracellular lipids, have been shown high cholesteryl oleate concentrations (24,25). The occurence amount of oleate esterified to cholesterol in fatty streaks could increased cellular lipogenic activities which reflect fatty acid coupled with chain elongation and desaturation of fatty acids. local synthesis of lipid has been attributed to its accumulation TABLE FATTY
ACID
SYNTHETASE
ACTIVITY
Tissue Thoracic aorta Abdominal aorta Coronary artery Liver
2
IN CYTOPLASMIC
LIVER
TISSUES
to contain of a high represent synthesis Increased within the
PREPARATIONS
FBOM ARTERIAL
AND
OF SWINE”.~
Grain diet 91.3* 9.9 86.62 15.5’ 96.72 9.5 475.92 28.4d
Butterfat diet 56.92 46.5 2 62.22 291.1 f
9.3’ 10.9 6.4’ 25.7d
” The incubation medium contained in micromoles: Malonyl-CoA, 0.2; acetyl-CoA, 0.1; NADPH, 0.5; malonyl-CoA-1, 3-14C (sp act, 50 pCi/pmole), 0.001 (0.05 PCi); phosphate buffer (pH 6.8), 10.0 and 0.3-0.5 mg of protein in a final volume of 1.5 ml. Reaction mixtures were incubated for 30 min at 37°C in a metabolic shaker. b Average picomoles of malonyl-CoA incorporated per milligram of protein per minute + SD in duplicate assays from three animals. c.d Means in the same column bearing different superscript letters differ significantly from the thoracic aorta (P < 0.01).
FATTY
ACID-SYNTHESIZING
ENZYMES
IN ARTERIES
119
fatty streaks of humans (9). In atherosclerotic aortas from pigeons and rabbits, an increased rate of fatty acid synthesis and incorporation of synthesized fatty acids into various lipid classes has also been observed (26,27). The findings that significant differences in lipid metabolism do exist between normal and atherosclerotic aortas led to the speculation that the lipogenic activity in arteries might be indicative of atherosclerotic susceptibility. Therefore, in the present study, both acetyl-CoA carboxylase and fatty acid synthetase activities were measured in grossly normal thoracic, abdominal, and coronary arteries which reveal differences in susceptibility to atherosclerosis. No significant differences were found in those enzyme activities among arteries as shown in Tables 1 and 2. Similar findings have been reported in different locations of rabbit aorta (28). Although the aortic arch in the rabbit had a disposition for early and rapidly progressive lesions, there was no difference in the rate of fatty acid synthesis from acetate in arch vs thoracic vs abdominal aorta until actual appearance of lesions. Other studies have investigated possible metabolic differences in aortas from two breeds of pigeons which have great differences in susceptibility to spontaneous atherosclerosis, but no significant differences in rates of fatty synthesis were found (10). Based on current and previous findings, it can be stated that susceptibility is apparently not associated with inherent differences in fatty acid synthesis rate or related enzyme activities of the arteries. SUMMARY Acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS) activities were measured in cytoplasmic preparations from thoracic and abdominal aorta, coronary arteries, and liver tissue of swine. Both ACC and FAS activities were present in all three arterial segments, but their activities were quite low compared to those of liver tissue. Between thoracic aorta, abdominal aorta, and coronary arteries, no significant differences in enzyme activities were found. Inclusion of fat in the diet decreased activities of both regulatory enzymes for fatty acid synthesis in vascular as well as liver tissues, and FAS showed more decrease in its activity than ACC in all tissues examined. ACKNOWLEDGMENT This work was supported by the fund for the Moore Investigator (B.H.S.C.) Harlan E. Moore Charitable Trust.
from the
REFERENCES 1. Brady, R. O., and Grin, S.. J. Viol. Chem. 199, 421 (1952). 2. Martin, D. B., Homing, M. G.. and Vagelos, P. R., J. Bid Chem.
236, 663 (1961).
B. H. S. CHO 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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