Exp. Geront. Vol. 1I. pp. 57-58. Pergamon Press 1976. Printed in Great Britain.
CHOLESTEROL U P T A K E IN VITRO BY Y O U N G A N D OLD RAT AORTAS SAROJ MALHOTRA a n d DAVID KR1TCHEVSKY The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104, U.S.A. (Received 18 June 1975)
Abstract--In general, aging appears to reduce an organism's ability to metabolize lipid (Kritehevsky, 1972). Cholesterol synthesis (Bloch et aL, 1946), degradation (Yamamoto and Yamamura, 1971) and turnover (Hruza and Wachtlova, 1969) are all reduced in aging rats. Fatty acid synthesis and oxidation by rat epididymal fat are also reduced (Benjamin et al., 1961). Aortic metabolism does not appear to follow this trend. Zemplenyi and Grafnetter (1959) and Dury (1961) measured lipolysis by aortic tissue and found lipolytic activity higher in old than in young rats. Cholesterol ester synthetase and hydrolase activities are also higher in old than in young rats (Kritchevksy et al., 1973b). We would like to report the results of preliminary experiments to determine the uptake of cholesterol in vitro by aortas from young and old rats. METHODS YOUNG (45 DAYS) and old (24 month) male Wistar rats were given single feedings of [1,2-3Hlcholesterol (3 laCi of cholesterol/10 g of body weight) in propylene glycol and killed 48 hr later. The sera of several rats from each age group were pooled and the specific activity and cholesterol content were determined (Kritchevsky and Malhotra, 1970; Kritehevsky et al., 1973a). Aortas from other young and old rats were excised and rinsed in oxygenated Tyrode's buffer, pH 7.4. These aortas were suspended in 3 ml of Tyrode's buffer in a 25 ml Erlenmeyer flask and 1,5 ml of radioactive serum was added. The mixture was then incubated with shaking at 37°C for 3 hr. The aortas were removed from the incubation mixture, rinsed in several changes of buffer, saponified and the total cholesterol content and specific activity were determined. RESULTS The characteristics of the pooled radioactive rat sera are given in Table 1. Pools of y o u n g a n d old labeled sera were incubated with aortas of y o u n g a n d old rats. The results are presented in Table 2. The a m o u n t of radioactive cholesterol t a k e n u p by old aorta from old serum was 14.5 lag. The a m o u n t of radioactive cholesterol f o u n d in the aorta TABLE. 1 CHARACTERISTICS OF LABELED RAT SERAt Cholesterol Serum
Ng/ml
dpm
Specific activity (dpm/~g )
Old*.l Old* 2
463 453
7760 5731
16.8 12~7
Young* 1 Young* 2
377 251
2327 11184
6.2 44.6
*Radioactive. i'Serum labeled by feeding [l,2-aH]cholesterol to rats (3 IxCi/10g). Old rats--24 months; young rats--45 days. could represent either true uptake (i.e. a d d i t i o n to the aortic cholesterol pool) or exchange with cholesterol present in the aorta. Thus, ca. 1 1 . 6 ~ of the cholesterol of the aorta was exchangeable or labile. By similar calculations 3.1 ~ of the serum cholesterol was exchanged or depleted. 57
58
SAROJ MALHOTRA AND DAVID KRITCHEVSKY TABLE 2, UPTAKE OF CHOLESTEROLBY RAT AORTA in vitrot Cholesterol Serum
Aorta
Incorporation Total (~g)
dpm
of
[14C] (~g)
Per c e n t incorporation
Per cent [14C] loss from serum
Old* 1 Young* 1
Old Old
]40 ].40
244 86
14.5 13.9
ll.6 ii.0
3.1 3.7
Old* 2 Young* 2
Young Young
128 128
120 92
9.4 2.1
7.9 7.1
2.1 0.8
* Labeled serum. ]~Aorta incubated with labeled serum, 3 hr at 37°C.
The data (Table 2) show that old aorta interchanged or took up c a . 11 ~ of its original content of cholesterol irrespective of the source of serum. Young aorta, on the other hand, took up 78 %oless cholesterol from young than it did from old serum. DISCUSSION The cholesterol content of serum from young rats was 18--45 ~ lower than that from old rats. The aortic cholesterol levels were similar. Hruza and Wachtlova (1969) found the cholesterol content of aortas of young and old Sprague-Dawley rats were also similar. Our data indicate that old rat aortas take up or exchange more cholesterol from serum than do young aortas. The level of uptake of cholesterol by old aortas seems to be similar regardless of the source of the serum in which it has been incubated. Young aortas, on the other hand, take up more cholesterol from old than from young serum. The uptake of cholesterol by aortas depends upon factors in the serum as well as in the tissue. The serum factor may be the increasing concentration of cholesterol or the 13 lipoprotein concentration. In the aorta, the factors affecting cholesterol uptake may be the ratio of free to esterified cholesterol (which generally decreases with age) or the phospholipid composition. Stein and Stein (1972) found that aortas of 18-24-month-old rats contained 205 ~ more sphingomyelin than did aortas of 1-month-old rats. The effect of serum factors, aortic chemistry and the question of true uptake versus exchange are all under investigation. Acknowledgements--This work was supported, in part, by USPHS grants (HL-03299 and HL-05209) and a Research Career Award (HL-0734) from the National Heart and Lung Institute.
REFERENCES BENJAMIN,W., GELLHORN,A., WAGNER,M. and KUNDEL,H. (1961) Am. J. Physiol. 201, 540. BLOCH, K., BOREK, E. and RITTENBERG, D. (1946) J. biol. Chem. 162, 441. DLrRy, A. (1961)J. Geront. 16, 114. HRUZA, Z. and WACHTLOVA, M. (1969) Exp. Geront. 4, 245. KRITCHEVSKY, D. (1972) Mech. Aging Dev. 1, 275. KRITCHEVSKY, D., DAVIDSON, L. M., KIM, H. K. and MALHOTRA, S. (1973a) Clinica chim. Acta 46, 63. KRtrCHEVSKY, D., GENZANO, J. C. and KOTHARI, H. V. (1973b) 3Iech. Aging Dev. 2, 345. KRXTCHEVSKY, D. and MALHOTRA, S. (1970) J. Chromat. 52, 498. STEIN, Y. and STEIN, O. (1972) Exposds an. Biochem. Mdd. 31, 97. YAMAMOTO, M. and YAMAMURA,Y. (1971) Atherosclerosis 13, 365. ZEMPLENYL T. and GRAFNETTER, D. (1959) Gerontologia 3, 55.
CHOLESTEROL U P T A K E IN VITRO BY Y O U N G A N D OLD RAT AORTAS SAROJ MALHOTRA a n d DAVID KR1TCHEVSKY The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104, U.S.A. (Received 18 June 1975)
Abstract--In general, aging appears to reduce an organism's ability to metabolize lipid (Kritehevsky, 1972). Cholesterol synthesis (Bloch et aL, 1946), degradation (Yamamoto and Yamamura, 1971) and turnover (Hruza and Wachtlova, 1969) are all reduced in aging rats. Fatty acid synthesis and oxidation by rat epididymal fat are also reduced (Benjamin et al., 1961). Aortic metabolism does not appear to follow this trend. Zemplenyi and Grafnetter (1959) and Dury (1961) measured lipolysis by aortic tissue and found lipolytic activity higher in old than in young rats. Cholesterol ester synthetase and hydrolase activities are also higher in old than in young rats (Kritchevksy et al., 1973b). We would like to report the results of preliminary experiments to determine the uptake of cholesterol in vitro by aortas from young and old rats. METHODS YOUNG (45 DAYS) and old (24 month) male Wistar rats were given single feedings of [1,2-3Hlcholesterol (3 laCi of cholesterol/10 g of body weight) in propylene glycol and killed 48 hr later. The sera of several rats from each age group were pooled and the specific activity and cholesterol content were determined (Kritchevsky and Malhotra, 1970; Kritehevsky et al., 1973a). Aortas from other young and old rats were excised and rinsed in oxygenated Tyrode's buffer, pH 7.4. These aortas were suspended in 3 ml of Tyrode's buffer in a 25 ml Erlenmeyer flask and 1,5 ml of radioactive serum was added. The mixture was then incubated with shaking at 37°C for 3 hr. The aortas were removed from the incubation mixture, rinsed in several changes of buffer, saponified and the total cholesterol content and specific activity were determined. RESULTS The characteristics of the pooled radioactive rat sera are given in Table 1. Pools of y o u n g a n d old labeled sera were incubated with aortas of y o u n g a n d old rats. The results are presented in Table 2. The a m o u n t of radioactive cholesterol t a k e n u p by old aorta from old serum was 14.5 lag. The a m o u n t of radioactive cholesterol f o u n d in the aorta TABLE. 1 CHARACTERISTICS OF LABELED RAT SERAt Cholesterol Serum
Ng/ml
dpm
Specific activity (dpm/~g )
Old*.l Old* 2
463 453
7760 5731
16.8 12~7
Young* 1 Young* 2
377 251
2327 11184
6.2 44.6
*Radioactive. i'Serum labeled by feeding [l,2-aH]cholesterol to rats (3 IxCi/10g). Old rats--24 months; young rats--45 days. could represent either true uptake (i.e. a d d i t i o n to the aortic cholesterol pool) or exchange with cholesterol present in the aorta. Thus, ca. 1 1 . 6 ~ of the cholesterol of the aorta was exchangeable or labile. By similar calculations 3.1 ~ of the serum cholesterol was exchanged or depleted. 57
58
SAROJ MALHOTRA AND DAVID KRITCHEVSKY TABLE 2, UPTAKE OF CHOLESTEROLBY RAT AORTA in vitrot Cholesterol Serum
Aorta
Incorporation Total (~g)
dpm
of
[14C] (~g)
Per c e n t incorporation
Per cent [14C] loss from serum
Old* 1 Young* 1
Old Old
]40 ].40
244 86
14.5 13.9
ll.6 ii.0
3.1 3.7
Old* 2 Young* 2
Young Young
128 128
120 92
9.4 2.1
7.9 7.1
2.1 0.8
* Labeled serum. ]~Aorta incubated with labeled serum, 3 hr at 37°C.
The data (Table 2) show that old aorta interchanged or took up c a . 11 ~ of its original content of cholesterol irrespective of the source of serum. Young aorta, on the other hand, took up 78 %oless cholesterol from young than it did from old serum. DISCUSSION The cholesterol content of serum from young rats was 18--45 ~ lower than that from old rats. The aortic cholesterol levels were similar. Hruza and Wachtlova (1969) found the cholesterol content of aortas of young and old Sprague-Dawley rats were also similar. Our data indicate that old rat aortas take up or exchange more cholesterol from serum than do young aortas. The level of uptake of cholesterol by old aortas seems to be similar regardless of the source of the serum in which it has been incubated. Young aortas, on the other hand, take up more cholesterol from old than from young serum. The uptake of cholesterol by aortas depends upon factors in the serum as well as in the tissue. The serum factor may be the increasing concentration of cholesterol or the 13 lipoprotein concentration. In the aorta, the factors affecting cholesterol uptake may be the ratio of free to esterified cholesterol (which generally decreases with age) or the phospholipid composition. Stein and Stein (1972) found that aortas of 18-24-month-old rats contained 205 ~ more sphingomyelin than did aortas of 1-month-old rats. The effect of serum factors, aortic chemistry and the question of true uptake versus exchange are all under investigation. Acknowledgements--This work was supported, in part, by USPHS grants (HL-03299 and HL-05209) and a Research Career Award (HL-0734) from the National Heart and Lung Institute.
REFERENCES BENJAMIN,W., GELLHORN,A., WAGNER,M. and KUNDEL,H. (1961) Am. J. Physiol. 201, 540. BLOCH, K., BOREK, E. and RITTENBERG, D. (1946) J. biol. Chem. 162, 441. DLrRy, A. (1961)J. Geront. 16, 114. HRUZA, Z. and WACHTLOVA, M. (1969) Exp. Geront. 4, 245. KRITCHEVSKY, D. (1972) Mech. Aging Dev. 1, 275. KRITCHEVSKY, D., DAVIDSON, L. M., KIM, H. K. and MALHOTRA, S. (1973a) Clinica chim. Acta 46, 63. KRtrCHEVSKY, D., GENZANO, J. C. and KOTHARI, H. V. (1973b) 3Iech. Aging Dev. 2, 345. KRXTCHEVSKY, D. and MALHOTRA, S. (1970) J. Chromat. 52, 498. STEIN, Y. and STEIN, O. (1972) Exposds an. Biochem. Mdd. 31, 97. YAMAMOTO, M. and YAMAMURA,Y. (1971) Atherosclerosis 13, 365. ZEMPLENYL T. and GRAFNETTER, D. (1959) Gerontologia 3, 55.
