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Cholesteryl ester accumulation in ehrlich cells induced by saturated fats
301
Biochimica et Biophysics Acta, 388 (1975) 301-304 o Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA Report BBA 51180
CHOLESTERYL ESTER ACCUMULATION INDUCED BY SATURATED FATS
DOUGLAS E. BRENNEMAN, ARTHUR A. SPECTOR Departments (U.S.A.) (Received
of Biochemistry
RICHARD
IN EHRLICH
MCGEE, VIESTURS
and Medicine,
CELLS
A. LIEPKALNS
and
University of Iowa, Iowa City, Iowa 52242
February 28th, 1975)
Summary The cholesteryl ester content of Erhlich cells was increased in tumors grown in mice fed saturated fat diets (coconut oil or tristearin) as compared with a polyunsaturated fat diet (sunflower oil). Cholesteryl esters containing monoenoic fatty acids were the predominant species that accumulated in the cells grown on saturated fat. The increase in cholesteryl esters was not accompanied by corresponding increases in the cell content of phospholipids, triacylglycerols,unesterified cholesterol or proteins. This experimental system may be useful for obtaining basic information about intracellular cholesteryl ester accumulation, a process that occurs in atherosclerosis.
Ehrlich ascites tumor cells grow in the peritoneal cavity of mice, suspended in an ascites plasma that is rich in lipids [ 11. The cells can utilize free fatty acids [Z] triacylglycerols [3] and cholesterol [4] from the ascites plasma. Bailey and Dunbar reported that the fatty acid composition of Ehrlich cells is altered when the tumor-bearing mice are fed a fat deficient diet [ 51. Subsequently, we have observed that the fatty acid composition of both the Erhlich cells and the ascites plasma can be modified extensively by changing the type of fat to the tumor-bearing mouse. Therefore, it was of interest to determine if these changes in fatty acid composition are accompanied by alterations in the lipid composition of either the tumor cells or the ascites plasma. Ehrlich ascites tumors were grown in male CBA mice fed diets containing 16% coconut oil, 16% sunflower oil or 4% tristearin. The coconut and sunflower oil diets were purchased from Teklad Test Diets, Madison, Wise., U.S.A. In addition to 16% of the respective oil, each diet contained 26% casein, 10% corn starch, 43% sucrose, 4% mineral mix and 1% vitamin mix. Teklad Test
302
Diets also supplied us with the above mixture containing no fat, and we prepared the third diet by adding 4% tristearin to the fat-free mixture. Mice were placed on these diets 4 weeks prior to tumor inoculation and then maintained on the diets during tumor growth. Tumor cells were separated from the ascites plasma and washed [6]. Aliquots were taken for cell counts and protein determination [ 71. Lipids were extracted from the cells and tumor plasma with chloroform/methanol [8], and the chloroform extract assayed for cholesterol [ 91, cholesteryl ester [lo] , triacylglycerols [ 91 and phospholipids [ 111. Chloroform-soluble lipid was also fractionated by silicic acid chromatography [ 121, saponified and methylated [ 61. The fatty acid compositions of the fractions were determined by gas-liquid chromatography using 10% Silar on Gas-Chrom Q. Peak areas were measured using an Infotronics digital integrator. TABLE I LIPID COMPOSITION
OF EHRLICH CELLS
Dietary fat
Days of growth
n
Lipid content (fig/lo* ceils f SE.) Unesterified Cholesteryl Triacylglycerols cholesterol esters
SunQower oil coconut oil Tristearin Sunflower oil Cocnut oil Triatearin
14 14 14 17 17 17
6 7 6 3 9 7
247 336 391 112 561 433
* 2 f f f *
10 30* 15** 36 27** 35**
368 395 417 392 403 373
t 7 f 18 +_24 f 24 f 16 * 18
2850 2530 2160 2500 3260 2110
k * k f f +
Phospholipids 2730 3220 3180 2900 2600 2680
290 220 260 750 160 190
f f f f + *
130 200 230 200 250 130
* Difference from sunflower oiI diet is P < 0.05. ** Difference from sunfIower oil diet is P < 0.001.
As shown in Table I, there was a significant increase in the cholesteryl ester content of Ehrlich cells grown in mice fed either coconut oil or tristearin as compared with sunflower oil. The increase in cholesteryl esters was observed in cells grown for either 14 or 17 days. Furthermore, cholesteryl ester accumulated between days 14 and 17 with the saturated fat diets whereas it decreased with the polyunsaturated sunflower oil diet. No significant increase in unesterified cholestrol, triacylglycerols or phospholipids occurred in the cells grown in animals fed the saturated fat diets. Likewise, the cell protein content was unaffected by the dietary fat composition. The saturated fat-induced increase in cell cholesteryl esters was not accompanied by any systematic changes in the ascites plasma lipids (Table II). TABLE II LIPID COMPOSITION Dietary fat Sunfiower oil Coconut oil Triatearin Sunfiower oil Coconut oil Tristearin
OF THE EHRLICH
ASCITES PLASMA Triacylglycerols
Phospholipid
BrOWth
Lipid content (mg/dI)* Cholesteryl Unesterified esters cholesterol
14 14 14 17 17 17
55 54 49 54 53 37
330 390 350 270 270 105
110 150 130 110 95 73
Days of
* Each value is the average of 2
47 61 47 39 48 21 samples,
each containing plasma from 34
different animals.
303
TABLE
III
CHOLESTERYL Fatty acids
14:o 16:0 18:0 16:l 18:l 20:1*** 18:2 20:4 22:4 22:6 Othem
ESTER
ACYL
GROUP
Percentage composition (%)* Ehrhch Sunflower* * Coconut 09
Oil
1.4 13.2 8.6 2.2 21.3 2.6 22.6 8.0 6.6 4.0 10.7
4.8 14.4 7.7 5.2 32.2 5.1 6.6 1.0 2.3 2.8 11.4
COMPOSITION
Tristearin 2.2 13.4 7.6 4.6 36.8 6.0 6.6 6.6 1.8 3.0 12.0
Ascites plasma Sunfiower Coconut Oil
Oil
0.4 15.2 18.0 0.7 14.1 0.4 26.2 11.2 5.5 4.4 4.0
0.8 16.0 15.4 2.6 30.6 1.2 10.1 12.0 2.5 4.0 5.7
Tristearin 0.8 15.4 14.4 2.5 34.1 1.7 9.2 10.6 1.9 4.0 5.4
* Average of 2 determinations of pooled samples from the 14 days tumors. * * Dietary fat of the animals in which the tumor was grown. *** This fraction also contained smalI amounts of 18:3.
Table III shows that both the cell and ascites plasma cholesterol esters from mice fed the saturated fat diets contained greater percentages of monoenoic fatty acids (16: 1,18: 1 and 20: 1). There were corresponding decreases in the polyunsaturated fatty acids, especially 18:2 and 22:4. Diet-induced changes in the fatty acid composition of the polar lipid and acylglycerol fractions of the cells and ascites plasma also occurred, and they were qualitatively similar to those observed in the cholesterol esters. There is increasing interest in the possible use of diets high in polyunsaturated fats for the prevention of atherosclerosis. An early event in the atherosclerotic process is the intracellular accumulation of cholesteryl esters [ 131. Based on the present results, we believe that the Ehlich cell may be a useful model system in which to study the regulation of intracellular cholesteryl ester accumulation. This system will make possible detailed studies of cholesterol metabolism in isolated, intact cells in which the cholesteryl ester content is responsive to changes in fatty acid saturation. In the present study, the accumulation of cholesteryl esters was associated with a shift in their acyl group composition to contain more monoenoic and less polyenoic fatty acid. A similar shift in acyl group composition has been observed when cholesteryl esters accumulate in atherosclerotic aortas [ 141. Furthermore, the results with this model system suggest that an increase in polyunsaturated fatty acids can lower the tissue cholesteryl ester content without necessarily lowering the extracellular fluid cholesterol content. These studies were supported by a research grant from the National Heart and Lung Institute (HL-14781). V.A. Liepkalns was supported by a postdoctoral fellowship from the Iowa and American Heart Associations. References 1 Spector, A.A. and Brenneman, D.E. (1973) in Tumor Lipids: Biochemistry and Metabolism Wood. R., ed.). pp. l-13. American Oil Chemists’ Society, Champaign, III. 2 Spector, A.A. (1971) Progr. Biochem. Pharm. 6. 130-176. 3 Brenneman, D.E. and Spector, A.A. (1974) J. Lipid Res. 15.309-316.
304
4 5 6 7 8 9 10 11 12 13 14
Brenneman, D.E.. McGee, R. and Spector, A.A. (1974) Cancer Res. 34,2606-2611. Bailey, J.M. and Dunbar, L.M. (1971) Cancer Res. 31.9147. McGee, R. and Spector, A.A. (1974) Cancer Res. 34.3355-3362. Lees, M.B. and Paxman, S. (1972) Anal. Biochem. 47, 184-192. Folch. J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226.497-509. Manual of Laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, (1974) Vol. 1, DHEW Publication No. (NIH) 75-628. U.S. Government Printing Office, Washington, D.C. Shoenhebner, R. and Sperry, W.M. (1934) J. Biol. Chem. 106,745-760. Raheja, R.K., Kaur, L., Singb. A. and Bhatia, S. (1973) J. Lipid Res. 14,695-697. Christie, W.W. (1973) in Lipid Analysis pp. 158-160. Pergamon Press, Oxford. Smith, E.B., Evans, P.H. and Downman. M.D. (1967) J. Atheroscler. Res. 7, 171-186. Lang, P.D. and Insull, Jr, W. (1970) J. Clin. Invest. 49.1479-1488.
Biochimica et Biophysics Acta, 388 (1975) 301-304 o Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA Report BBA 51180
CHOLESTERYL ESTER ACCUMULATION INDUCED BY SATURATED FATS
DOUGLAS E. BRENNEMAN, ARTHUR A. SPECTOR Departments (U.S.A.) (Received
of Biochemistry
RICHARD
IN EHRLICH
MCGEE, VIESTURS
and Medicine,
CELLS
A. LIEPKALNS
and
University of Iowa, Iowa City, Iowa 52242
February 28th, 1975)
Summary The cholesteryl ester content of Erhlich cells was increased in tumors grown in mice fed saturated fat diets (coconut oil or tristearin) as compared with a polyunsaturated fat diet (sunflower oil). Cholesteryl esters containing monoenoic fatty acids were the predominant species that accumulated in the cells grown on saturated fat. The increase in cholesteryl esters was not accompanied by corresponding increases in the cell content of phospholipids, triacylglycerols,unesterified cholesterol or proteins. This experimental system may be useful for obtaining basic information about intracellular cholesteryl ester accumulation, a process that occurs in atherosclerosis.
Ehrlich ascites tumor cells grow in the peritoneal cavity of mice, suspended in an ascites plasma that is rich in lipids [ 11. The cells can utilize free fatty acids [Z] triacylglycerols [3] and cholesterol [4] from the ascites plasma. Bailey and Dunbar reported that the fatty acid composition of Ehrlich cells is altered when the tumor-bearing mice are fed a fat deficient diet [ 51. Subsequently, we have observed that the fatty acid composition of both the Erhlich cells and the ascites plasma can be modified extensively by changing the type of fat to the tumor-bearing mouse. Therefore, it was of interest to determine if these changes in fatty acid composition are accompanied by alterations in the lipid composition of either the tumor cells or the ascites plasma. Ehrlich ascites tumors were grown in male CBA mice fed diets containing 16% coconut oil, 16% sunflower oil or 4% tristearin. The coconut and sunflower oil diets were purchased from Teklad Test Diets, Madison, Wise., U.S.A. In addition to 16% of the respective oil, each diet contained 26% casein, 10% corn starch, 43% sucrose, 4% mineral mix and 1% vitamin mix. Teklad Test
302
Diets also supplied us with the above mixture containing no fat, and we prepared the third diet by adding 4% tristearin to the fat-free mixture. Mice were placed on these diets 4 weeks prior to tumor inoculation and then maintained on the diets during tumor growth. Tumor cells were separated from the ascites plasma and washed [6]. Aliquots were taken for cell counts and protein determination [ 71. Lipids were extracted from the cells and tumor plasma with chloroform/methanol [8], and the chloroform extract assayed for cholesterol [ 91, cholesteryl ester [lo] , triacylglycerols [ 91 and phospholipids [ 111. Chloroform-soluble lipid was also fractionated by silicic acid chromatography [ 121, saponified and methylated [ 61. The fatty acid compositions of the fractions were determined by gas-liquid chromatography using 10% Silar on Gas-Chrom Q. Peak areas were measured using an Infotronics digital integrator. TABLE I LIPID COMPOSITION
OF EHRLICH CELLS
Dietary fat
Days of growth
n
Lipid content (fig/lo* ceils f SE.) Unesterified Cholesteryl Triacylglycerols cholesterol esters
SunQower oil coconut oil Tristearin Sunflower oil Cocnut oil Triatearin
14 14 14 17 17 17
6 7 6 3 9 7
247 336 391 112 561 433
* 2 f f f *
10 30* 15** 36 27** 35**
368 395 417 392 403 373
t 7 f 18 +_24 f 24 f 16 * 18
2850 2530 2160 2500 3260 2110
k * k f f +
Phospholipids 2730 3220 3180 2900 2600 2680
290 220 260 750 160 190
f f f f + *
130 200 230 200 250 130
* Difference from sunflower oiI diet is P < 0.05. ** Difference from sunfIower oil diet is P < 0.001.
As shown in Table I, there was a significant increase in the cholesteryl ester content of Ehrlich cells grown in mice fed either coconut oil or tristearin as compared with sunflower oil. The increase in cholesteryl esters was observed in cells grown for either 14 or 17 days. Furthermore, cholesteryl ester accumulated between days 14 and 17 with the saturated fat diets whereas it decreased with the polyunsaturated sunflower oil diet. No significant increase in unesterified cholestrol, triacylglycerols or phospholipids occurred in the cells grown in animals fed the saturated fat diets. Likewise, the cell protein content was unaffected by the dietary fat composition. The saturated fat-induced increase in cell cholesteryl esters was not accompanied by any systematic changes in the ascites plasma lipids (Table II). TABLE II LIPID COMPOSITION Dietary fat Sunfiower oil Coconut oil Triatearin Sunfiower oil Coconut oil Tristearin
OF THE EHRLICH
ASCITES PLASMA Triacylglycerols
Phospholipid
BrOWth
Lipid content (mg/dI)* Cholesteryl Unesterified esters cholesterol
14 14 14 17 17 17
55 54 49 54 53 37
330 390 350 270 270 105
110 150 130 110 95 73
Days of
* Each value is the average of 2
47 61 47 39 48 21 samples,
each containing plasma from 34
different animals.
303
TABLE
III
CHOLESTERYL Fatty acids
14:o 16:0 18:0 16:l 18:l 20:1*** 18:2 20:4 22:4 22:6 Othem
ESTER
ACYL
GROUP
Percentage composition (%)* Ehrhch Sunflower* * Coconut 09
Oil
1.4 13.2 8.6 2.2 21.3 2.6 22.6 8.0 6.6 4.0 10.7
4.8 14.4 7.7 5.2 32.2 5.1 6.6 1.0 2.3 2.8 11.4
COMPOSITION
Tristearin 2.2 13.4 7.6 4.6 36.8 6.0 6.6 6.6 1.8 3.0 12.0
Ascites plasma Sunfiower Coconut Oil
Oil
0.4 15.2 18.0 0.7 14.1 0.4 26.2 11.2 5.5 4.4 4.0
0.8 16.0 15.4 2.6 30.6 1.2 10.1 12.0 2.5 4.0 5.7
Tristearin 0.8 15.4 14.4 2.5 34.1 1.7 9.2 10.6 1.9 4.0 5.4
* Average of 2 determinations of pooled samples from the 14 days tumors. * * Dietary fat of the animals in which the tumor was grown. *** This fraction also contained smalI amounts of 18:3.
Table III shows that both the cell and ascites plasma cholesterol esters from mice fed the saturated fat diets contained greater percentages of monoenoic fatty acids (16: 1,18: 1 and 20: 1). There were corresponding decreases in the polyunsaturated fatty acids, especially 18:2 and 22:4. Diet-induced changes in the fatty acid composition of the polar lipid and acylglycerol fractions of the cells and ascites plasma also occurred, and they were qualitatively similar to those observed in the cholesterol esters. There is increasing interest in the possible use of diets high in polyunsaturated fats for the prevention of atherosclerosis. An early event in the atherosclerotic process is the intracellular accumulation of cholesteryl esters [ 131. Based on the present results, we believe that the Ehlich cell may be a useful model system in which to study the regulation of intracellular cholesteryl ester accumulation. This system will make possible detailed studies of cholesterol metabolism in isolated, intact cells in which the cholesteryl ester content is responsive to changes in fatty acid saturation. In the present study, the accumulation of cholesteryl esters was associated with a shift in their acyl group composition to contain more monoenoic and less polyenoic fatty acid. A similar shift in acyl group composition has been observed when cholesteryl esters accumulate in atherosclerotic aortas [ 141. Furthermore, the results with this model system suggest that an increase in polyunsaturated fatty acids can lower the tissue cholesteryl ester content without necessarily lowering the extracellular fluid cholesterol content. These studies were supported by a research grant from the National Heart and Lung Institute (HL-14781). V.A. Liepkalns was supported by a postdoctoral fellowship from the Iowa and American Heart Associations. References 1 Spector, A.A. and Brenneman, D.E. (1973) in Tumor Lipids: Biochemistry and Metabolism Wood. R., ed.). pp. l-13. American Oil Chemists’ Society, Champaign, III. 2 Spector, A.A. (1971) Progr. Biochem. Pharm. 6. 130-176. 3 Brenneman, D.E. and Spector, A.A. (1974) J. Lipid Res. 15.309-316.
304
4 5 6 7 8 9 10 11 12 13 14
Brenneman, D.E.. McGee, R. and Spector, A.A. (1974) Cancer Res. 34,2606-2611. Bailey, J.M. and Dunbar, L.M. (1971) Cancer Res. 31.9147. McGee, R. and Spector, A.A. (1974) Cancer Res. 34.3355-3362. Lees, M.B. and Paxman, S. (1972) Anal. Biochem. 47, 184-192. Folch. J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226.497-509. Manual of Laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, (1974) Vol. 1, DHEW Publication No. (NIH) 75-628. U.S. Government Printing Office, Washington, D.C. Shoenhebner, R. and Sperry, W.M. (1934) J. Biol. Chem. 106,745-760. Raheja, R.K., Kaur, L., Singb. A. and Bhatia, S. (1973) J. Lipid Res. 14,695-697. Christie, W.W. (1973) in Lipid Analysis pp. 158-160. Pergamon Press, Oxford. Smith, E.B., Evans, P.H. and Downman. M.D. (1967) J. Atheroscler. Res. 7, 171-186. Lang, P.D. and Insull, Jr, W. (1970) J. Clin. Invest. 49.1479-1488.