33
Atherosclerosis, 32 (1979) 33-42 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
HYPERLIPOPROTEINEMIA INDUCED PITUITARY TUMOR IN THE RAT
A. CHRISTINE NESTRUCK, MICHEL CHRkTIEN Institut de Recherches H2 W 1 R7 (Canada)
CHRISTINA
BY A TRANSPLANTABLE
GIANOULAKIS,
JEAN DAVIGNON
and
Cliniques de Montre’al, 110 avenue des Pins, ouest, Montre’al, P. QuB.,
(Received 8 May, 1978) (Revised, received 6 September, 1978) (Accepted 15 September, 1978)
Summary The MtT-F4 tumor, a transplantable pituitary tumor of rats, induces significant hyperlipidemia in male Fisher 344 rats. The increasive hypercholesterolemia was accompanied by hypertriglyceridemia only in the first month of tumor implantation. Clofibrate feeding inhibited the development of hypercholesterolemia and maintained normal serum triglyceride levels. In contrast to the changes in lipoprotein cholesterol distribution and profile found in experimental hyperlipidemia induced by high fat and cholesterol feeding, the hypercholesterolemic tumor-bearing rats showed no accumulation of cholesterol in the very low density and intermediate density lipoproteins, and no appearance of a new class of lipoprotein, B-VLDL. An HDLc-like lipoprotein appeared as hypercholesterolemia developed. Increased amounts of cholesterol were deposited in the aorta. The effects are attributed to the lipolytic hormones secreted by the tumor and antagonism to their action by clofibrate. Key words:
Atherogenesis
- Clofibrate - HDL,
- Hypercholesterolemia
Introduction The rat, a widely used model in lipid and lipoprotein metabolic studies, is very resistant to the development of hypercholesterolemia and atherosclerosis. Early studies [ 1,2] demonstrated that hypercholesterolemia sufficient to This work was supported by grants from the Medical Research Council of Canada (MA-6119) and the Quebec Heart Foundation. A.C.N. holds a Research Scholarship from the Cons4 de la Recherche en Sante du Quebec and C.G. is a Medical Research Council of Canada post-doctoral fellow.
34
induce experimental atherosclerosis in the rat required diets containing saturated fat, bile salts, cholesterol and the thyrotoxic agent propylthiouracil. More recently, the changes in lipoprotein and apoprotein profiles of these rats have been characterized [3] and shown to be similar to those in animals in which hypercholesterolemia can be induced by cholesterol feeding alone. We recently reported that implantation of a transplantable pituitary tumor (MtTF4) in chow-fed male Fisher 344 rats induced hyperlipidemia [4] and hypertension [5]. Thirty-one days after implantation, plasma free fatty acid, triglyceride and cholesterol levels were significantly raised. The tumor, which secretes large quantities of prolactin, growth hormone and ACTH [4,6], also induces a fatty liver [4,7] and a significantly higher lipolytic response to ACTH by isolated adipocytes [ 81. Tumor-bearing rats fed a clofibrate-supplemented chow diet developed a lesser degree of hyperlipidemia [4]. It has been suggested that the hyperlipidemia and fatty liver of the tumor-bearing rats are consequences of chronic lipolysis and mobilization of lipid from peripheral depots. The purpose of this study was to characterize the hyperlipoproteinemia induced by the tumor and to extend the period of tumor implantation to two months, since preliminary results [9] after one month of implantation had shown differences in the lipoprotein profile from that associated with experimental hypercholesterolemias dependent on high cholesterol diet. The early inhibitory effect of clofibrate feeding on the development of the hyperlipidemia was also investigated after 2 months of tumor implantation. Methods Male Fisher 344 rats were purchased from Canadian Breeding Laboratories. The MtT-F4 tumors were provided by the Mason Research Institute of Boston, MA, and were taken through at least 10 successive transplantations before being used for the present experiments. The tumors were transplanted subcutaneously under ether anesthesia in the posterior half of the dorsal area. A sham implantation was performed in control rats. The animals were divided into 4 groups: controls, with sham implantation (S); rats with MtT-F4 tumor implantation (T); shams fed clofibrate (SCF); and rats with MtT-F4 tumor that were fed clofibrate (TCF). Rats in all groups weighed 100 f 5 g at the start of the experiments. Groups S and T were fed Purina laboratory chow, while groups SCF and TCF received the same chow containing 0.25% clofibrate (ethyl-p-chlorophenoxyisobutyrate). The clofibrate-supplemented diet was prepared by Ayerst Laboratories, Montreal. Daily caloric intake was similar for all rats, and clofibrate intake was calculated to be approximately 30 mg/kg per day. All rats were fasted for 24 h prior to killing by decapitation at 31 or 60 days after implantation. Trunk blood was collected and allowed to clot. The adrenals, liver and tumor were removed, dissected free from adhering connective tissue and weighed. After centrifugation the serum was kept at 4°C for determination of triglyceride and cholesterol and for the isolation of lipoproteins. Serum cholesterol [lo] and triglyceride [ 111 were measured with a Tech-
35
nicon Autoanalyser or individual rat serum samples at 31 days or on pooled serum at 60 days. Lipoproteins were isolated from pooled serum (4 rats/group) containing sodium azide (0.1%) by sequential ultracentrifugation at increasing densities [ 121. All centrifugations were carried out in a Beckman 50 Ti rotor at 47,000 rpm for various times: at density = 1.006, very low density lipoproteins (VLDL), 1.02 (intermediate fraction) and 1.063 for 16 h and at density 1.21, high density lipoproteins (HDL) for 40 h. The fractions were dialyzed against 5 mM ammonium bicarbonate buffer and protein content was determined [13]. The lipids of the various fractions were extracted with chloroform-methanol (2 : 1, v/v) [ 141. Aliquots of the chloroform phase were evaporated to dryness under nitrogen and taken up in isopropanol for tri[lo] determinations. Phospholipids were meaglyceride [ 111 and cholesterol sured according to the method of Bartlett [15]. Aliquots of the isolated lipoprotein fractions were also subjected to agar-agarose electrophoresis [16]. After delipidation [ 171, the lipoprotein apoproteins were subjected to electrophoresis on 10% polyacrylamide gels containing 7 M urea [ 181 with buffers as described by Kane [ 191 or in the SDS system of Weber and Osborne [20] for molecular weight determinations. In the 60-day studies, immediately after the rat was killed the aorta from the aortic arch to iliac bifurcation was dissected free of connective tissue, blotted and weighed. The aortas from half the rats in each group were fixed and stained with hematoxylin-eosin [21]. The aortas from the remaining rats were quickfrozen in liquid nitrogen and stored at -30°C. The frozen aortas were later cut into small fragments and saponification and lipid extraction procedures were applied [22]. Total cholesterol was then determined [lo]. Liver slices were taken at random, fixed and stained with Oil Red 0 [ 231. The results reported here are from studies of one 31-day tumor implantation with 4-5 rats per treatment group and from two separate 60-day studies, also with 4-5 rats per treatment group. For statistical analysis, the Newman-Keuls test was used [ 241. Results Organ weights The body weight of the sham-operated rats increased throughout the postoperative period. Growth of the rats with an MtT-F4 tumor was retarded: their body weight was significantly lower than that of the controls at both 31 and 60 days after implantation, Liver and adrenal weights were, however, significantly higher than in the controls (Table 1). Clofibrate treatment partially prevented the tumor effect on liver weight without affecting the tumor growth or adrenal hypertrophy. At 31 days the body weight retardation of this group was the same as in the tumor group and at 60 days was slightly more severe (Table 1). Serum lipids Table 2 presents the effects of the tumor on serum lipids. Both sham groups showed normal lipid levels throughout this study. Thirty-one days after implantation a significant hypertriglyceridemia and hypercholesterolemia had been induced by the tumor. Previous studies had shown a progressive increase in
36 TABLE 1 EFFECTS OF MtT-F4 TUMOR f CLOFIBRATE
ON BODY AND ORGAN WEIGHTS
Values are means + SEM. Time a
Tumor (g) 31
60
31
60
31
60
31
60
13.6 k1.97 13.7 t1.89
231 t2.a 230 to.9 199 * t3.7 188 * t4.3
261 56.5 210 f2.7 217 * t4.3 200 *.***
5.8 to.13 5.4 to.12 9.3 * to.43 1.5 *.** to.26
7.5 il.05 1.7 r0.19 20.2 * il.32 14.6 *. ** il.17
40 +1.7 39 t1.2 124 * t8.0 114 * +I.7
38 +1.6 39 t1.6 445 * 221.3 466 * t26.8
Sb SCF T TCF
1.4 to.15 1.2 +0.16
Adrenals (me)
Liver (5)
Body (g)
k5.6
a Days after sham or tumor implantation. b S = sham-implanted rats: SCF = sham-implanted, clofibrate-fed; planted. clofibrate-fed (10 rats/group). * Significant difference from S or SCF, P < 0.001. ** Significant difference from T, P < 0.001. * * * Significant difference from T. P < 0.05.
T = tumor-implanted:
TCF = tumor-im-
these two plasma lipid concentrations up to 41 days [4]. By 60 days of tumor implantation, however, the serum triglyceride level had reverted to normal levels despite a severe remaining hypercholesterolemia (Table 2). This decline in plasma triglyceride was confirmed in other studies at 60 days of tumor implantation. The clofibrate-fed tumor-bearing rats showed little change in serum triglyceride and only moderate hypercholesterolemia at 31 days. By 60 days, however, the serum cholesterol levels were as high as in the tumor group: about 5 times the control level (>300 mg/dl). Phospholipids were also increased in both tumor groups at 60 days. The ratio of free : esterified cholesterol was unchanged.
TABLE 2 TIME EFFECT OF MtT-F4 TUMOR Time
TG 31 a
f CLOFIBRATE
TC
ON SERUM LIDIDS PL
FC/TC (%)
6ob
31
60
31
60
31
60
38 39 33 69
41 48 219 * 132 **
68 65 330 * 306 **
ND ND ND ND
100 98 380 * 347 **
ND ND ND ND
21 20 21 21
(mgld) S SCF T TCF
33 31 130 * 48
TG = triglyceride; TC = total cholesterol: PL = phospholipids; FC = free cholesterol: ND = not determined; S. SCF, T. TCF: see legend,Table 1. a Mean of 6 individual samples/group, 31 days after sham or tumor implantation. b Mean of two experiments, pooled serum samples (4-5 rats/group), 60 days after implantation. * Significantly different from S. P < 0.001. ** Significantly different from SCF. P < 0.01.
37
Lipoproteins The lipid and protein distributions in the ultracentrifugal fractions 31 and 60 days after implantation are shown in Table 3. Clofibrate-fed sham-implanted rats showed no change in the distribution of serum lipids or lipoprotein protein at any time in the study. In all determinations more than 90% of the serum triglyceride was recovered in the lipoprotein fractions. For serum cholesterol, the recovery was >80% except in the two tumor groups at 60 days, where the serum values were >300 mg/dl and only 60% of the serum level was accounted for. At 31 days, there was very little change in the distribution of the plasma lipids or lipoprotein protein in either tumor-bearing group and no accumulation of cholesterol in the d < 1.02 fractions, in spite of mean serum cholesterol levels of 132-219 mg/dl. All groups throughout the study had less than 10 mg/ dl in the d < 1.006 lipoproteins, with the exception of the tumor-bearing group at 31 days. The elevated levels of serum cholesterol and phospholipid were predominantly carried in the d > 1.02 lipoproteins in both tumor groups. At 31 days, the d < 1.006 fraction in both tumor groups had a higher lipid : protein ratio because of the increase in all lipid components. This lipid enrichment was slightly decreased at 60 days. The 1.02-1.063 lipoproteins in both tumor groups were enriched in phospholipid and to a lesser extent protein at the TABLE 3 DISTRIBUTION
OF LIPID AND PROTEIN IN THE ULTRACENTRIFUGAL
Density a
1.006
Time b
31
FRACTIONS
1.0061.02
1.021.063
1.0061.063
1.063-1.21
60 c
31
31
60
31
60
(mg/dI) TG TC PL P
_d -
25.1 6.2 5.5 7.4
-
SCF
TG TC PL P
27.9 5.4 2.1 4.9
T
TG TC PL P TG TC PL P
S
TCF
-
-
-
7.1 8.8 3.2 5.0
-
1.1 39.2 28.1 53.0
32.1 5.2 6.7 8.0
1.3 1.1 0.9 0.6
3.5 4.8 4.5 6.0
4.7 5.1 1.6 2.1
0.6 25.1 20.1 45.4
1.1 29.8 25.2 56.1
136.6 28.8 30.1 15.4
15.8 4.5 2.4 2.5
2.9 3.0 3.5 1.6
3.2 36.0 31.7 31.7
6.2 33.0 29.2 23.1
3.1 106.3 120.5 174.3
6.3 165.2 210.4 249.5
45.8 9.0 13.3 5.8
49.5 9.0 2.6 6.8
2.3 2.8 1.8 1.0
2.8 13.9 14.3 15.4
5.7 30.1 31.8 21.1
1.8 103.3 113.2 171.1
6.0 142.3 205.4 249.1
S. SCF, T. TCF: see legend Table 1: TG, TC. PL: see legend Table 2; P = protein. a Sequential ultracentrifugal density cuts. Intermediate density fraction 1.006-1.02 day experiment. b Days after sham or tumor implantation. c Mean of two separate serum pools. d sample lost.
isolated only in 31-
38
expense of triglyceride at 31 days, and these changes were enhanced at 60 days. The d 1.063-1.23. lipoproteins of both tumor groups were enriched in phospholipid, but not cholesterol or triglyceride, both at 31 and 60 days. Agar-agarose electrophoresis of the isolated density fractions revealed changes in the tumor-bearing animals at 31 days (Fig. 1, top-half). In contrast to the controls which showed in the d < 1.006 fraction a pre-fl band and virtually no material at the origin, both tumor groups showed increased trailing and material at the origin (tumor >> tumor + clofibrate). Little stainable material was found in the d 1.006-1.02 fraction in any group. The d 1.02-1.063
Fig. 1. Ag-garose electrophoretograms of the sequential ultracentrifugal fractions for sham and tumorbearing rats at 31 days (above); tumor and tumor plus clofibrate at 60 days (below). 0 indicates the origin. and /3 and (Y the migration of lipoprotein bands. Slides are stained with Oil Red 0.
39
TABLE 4 CHOLESTEROL
CONTENT
OF THE AORTA
AT 60 DAYS
Values are mean t SD. Mg/mg wet weight S SCF T TCF
1.76 1.77 2.61 2.53
* + f +
0.08 0.03 0.26 * 0.26 *
* Significantly different from S or SCF, P < 0.001.
fraction displayed a single P-migrating band in the control and tumor + clofibrate groups, but in the MtT-F4 group, a second band with a-mobility was observed (Fig. 1). Furthermore in the d 1.063-1.21 fractions, both tumor groups showed a band with P-mobility in addition to the a-migrating band characteristic of this fraction. At 60 days (Fig. 1, lower half), both tumor groups showed a single pre-fl band in the VLDL. Clofibrate feeding appeared to prevent the build-up of a-migrating proteins in the d 1.006-1.063 fraction, whereas the tumor group showed a less prominent P-migrating band. In the d 1.063-1.21 fraction both tumor groups showed the characteristic a-migrating band as well as a P-band. On analytical SDS polyacrylamide gel electrophoresis, in the d 1.063-1.21 fractions, all groups showed bands with molecular weights of 25,000 (AI), 33,000 (apo E), and 44,000 (apo A IV) in addition to low molecular weight bands < 10,000 (apo CII, CIII). Additionally, both tumor groups showed a peptide with molecular weight = 19,500. For basic polyacrylamide gel electrophoresis the apoproteins of the d 1.006-1.063 fraction of the control groups were almost insoluble in the solvent (7 M urea, 40 mM Tris, pH 8.9). Only l2% of the lipoprotein was soluble and only bands in the apo CII-CIII area were found. In contrast, 50-60% of the d 1.006-1.063 lipoprotein-protein of the two tumor groups was soluble in this solvent. These soluble peptides showed banding in the apo-AI, -E, -CII and -CIII areas [ 251. Tissue studies The cholesterol content of the aorta was measured in all groups at 60 days. Both tumor groups showed significantly higher values than the controls (Table 4). Histology of liver sections taken from the 4 groups to be killed showed significant fatty infiltration of the parenchyma in the tumor group with tumor >> tumor + clofibrate. No structural modifications, however, were seen in the aortic sections. Discussion A hypercholesterolemia develops following high fat and cholesterol feeding in the rat [ 261, pig [ 271, or dog [28] and the propylthiouracil-supplemented atherogenic diet in the rat [ 31. The diet-induced hypercholesterolemia is accompanied by cholesterol enrichment of the very low density lipoproteins
40
and the finding of a cholesterol-enriched B-VLDL in the intermediate density range. In our study, although the tumor induced an elevated serum cholesterol as high as that reported after cholesterol feeding in rats [3,26], the very low density lipoproteins had an essentially normal complement of cholesterol and there was no accumulation of lipoprotein or cholesterol in the d 1.006-1.02 range. For both tumor groups, the significantly increased serum cholesterol was found predominantly in the d > 1.063 lipoproteins. The appearance of a second band with cr-migration on the d 1.02-1.063 fraction, possibly an HDLc-type particle [ 3,27-28 1, is similar to electrophoretograms following cholesterol feeding in various species. This band was not apparent in the clofibrate-fed tumor group at 31 days, when serum cholesterol had risen to 132 mg/dl, but did become visible at 60 days when cholesterol levels were equal to those in the tumor groups not clofibrate-fed (>300 mg/dl). This a-migrating lipoprotein was also associated with significant amounts of urea-soluble apoproteins. In comparison with the hyperlipidemia induced by cholesterol feeding in the rat, these differences in lipoprotein distribution and composition in relation to an almost equal degree of hypercholesterolemia may reflect different methods of induction: an endogenous versus an exogenous stimulation of lipid metabolism. The hyperlipidemia of the tumor-bearing rats is probably the result of the high concentration of lipolytic hormones secreted by the tumor. Both ACTH and GH are lipolytic in the rat [29,30 1, although increased production of corticosteroids may also play a role [ 31-341, The importance of adrenal glands and hypersecretion of corticosteroids in the induction of hyperlipidemia is indicated by the observation that adrenalectomized rats bearing the MtT-F4 tumor did not develop a fatty liver [35]. The lipolytic response of isolated adipose tissue cells [8] and the accumulation of cyclic AMP following stimulation by ACTH were significantly higher in cells derived from rats bearing the tumor [36]. It is well established that the availability of fatty acid to the liver regulates triglyceride synthesis and VLDL secretion [37-401. In the normal rat, the further conversion of VLDL &protein and cholesterol ester to LDL appears to be only a minor process [41,42]. However, in this study, it is possible that the continued stimulation of VLDL production enhanced this conversion process especially in the early stages of tumor implantation. However, in the long term, after two months of tumor implantation, the severe widespread fatty infiltration of the liver suggests that the synthetic and/or secretory mechanisms for triglyceride export (VLDL production) were insufficient and resulted in a reversal of the hypertriglyceridemia, decreased numbers of VLDL lipoproteins and decreased numbers of fraction. P-migrating lipoproteins in the d 1.006-1.063 Clofibrate is known to have several actions on lipid metabolism and extrahepatic tissues [43-441, including an antilipolytic effect on human and rat adipose tissue mediated by the adenylate cyclase system [45]. In this study, clofibrate appears to have protected the organism against the enhanced lipolysis induced by the tumor: at 31 and 60 days, the serum triglyceride levels were only slightly increased, the P-migrating lipoproteins of the 1.006-1.063 fraction remained prominent and the liver showed a lesser degree of fatty infiltration. Thus, possibly by slowing the degree of extrahepatic lipolysis, the hepatic
41
handling and disposal mechanisms of endogenous free fatty acids were maintained. However, in the long term, clofibrate did not prevent hypercholesterolemia or cholesterol deposition in the aorta. In summary, rats bearing the MtT-F4 tumor present a developing hypercholesterolemia which is different from that resulting from cholesterol feeding. The absence of B-VLDL with its significant cholesterol-carrying function is possibly due to different disposal mechanisms for chylomicron and hepatic VLDL lipid. The finding of an HDLc-like lipoprotein and its associated ureasoluble apoproteins is possibly analogous to the HDLc previously reported in various species. In the present study, this lipoprotein was associated with serum cholesterol levels >200 mg/dl and ultimately with increased cholesterol deposition in the aorta. Acknowledgements The authors wish to thank Ayerst Laboratories of Montreal for a generous supply of the clofibrate-supplemented diet; Dr. S.A. Bencosme, Department of Pathology, Queen’s University, Kingston, Ontario for performing the histology and Margaret Bergseth for excellent technical assistance. References 1 Malinow, M.R., Hojman, D. and Pellegrino, A., Different methods for the experimental production of generalized atherosclerosis in the rat, Acta Cardiol., 9 (1954) 480. 2 Wissler, R.W.. Eilert, M.L., Schroeder, M.A. and Cohen, L., Production of lipomatous and atheromatous arterial lesions in the albino rat, Arch. Path., 57 (1954) 333. 3 Mahley, R.W. and Holcombe, K.S., Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat. J. Lipid Res., 18 (1977) 314. 4 Gianoulakis. C.. Passe&i. L., Lis, M., Davignon, J. and Chretien, M., Effect of clofibrate treatment on chronic hyperlipidemia induced by an ACTH producing tumor, Canad. J. Physiol. Pharmacol.. 55 (1977) 220. 5 Kubo. S.. Ganten, D., Ganten, U.. Nowaczynski, W. and Genes& J.. Plasma steroids in rats with experimental ectopic pituitary tumor and arterial hypertension, Endocrinology, 94 (19741459. 6 Bates, R.W.. Milkovic. S. and Garrison, M.M., Concentration of prolactin, growth hormone and ACTH in blood and tumor of rats with transplantable mammotropic pituitary tumors, Endocrinology, 71 (1962) 943. 7 Milcovic, 8. Ganison. M.M. and Bates, R.W.. Study of the hormonal control of body and organ size in rats with mammotropic tumor, Endocrinology, 75 (1964) 670. 8 Gianoulakis, C., Lis, M., Davignon, J. and Chretien, M., Development of hyperlipidemia associated with increased lipolytic response of isolated adipose tissue cells following prolonged stimulation by an ectopic pituitary tumor, Harm. Metab. Res., In press. 9 Gianoulakis, C.. Nestruck, A.C., Lis, M., Davignon, J. and Chretien, M., New model for experimental hyperlipidemia. In: Abstracts of the 59th Annual Meeting of the Endocrine Society, 1977, p. 339. 10 Block, W.D., Jarret. K.J. and Levine, L.B.. An improved automated determination of serum cholcsterol with a single color reagent, Clin. Chem., 12 (1966) 681. 11 Kraml, N. and Cosyns. L., A semiautomated determination of serum triglycerides, Clin. Biochem., 2 (19691 373. 12 Havel, R.J.. Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest., 34 (1955) 1346. 13 Lowry, O.H., Rosebrough, N.J., Farr. A.L. and Randall, R.J.. Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (19511 265. 14 Folch, J., Lees, N. and Sloane-Stanley, G.H., A simple method for the isolation and Purification of total lipides from animal tissues, J. Biol. Chem.. 226 (19571 497. 15 Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem.. 234 (19591466. 16 Noble, R.P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (19681 693.
42 17
Scanu,
A.M. and Ed&t&i.
C., Solubllity
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weight
peptides of serum very low density and high density lipoproteins, Anal. Biochem.. 44 (1971) 576. 18 Reisfeld. R.A. and Small, P.A., Electrophoretic heterogeneity of polypeptide chains of specific antibodies. Science, 152 (1966) 1253. 19 Kane, J.P. A rapid electrophoretic technique for identification of subunit species of apoproteins in 20
serum lipoproteins. Anal. Biochem.. 53 (1973) 350. Weber, K. and Osbom, M., The reliability of molecular polyacrylamide
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Hlstochemical and Histopathology Methods, Charles Thompson, S.W.. In: Selected Springfield. IL, 1966, p. 763. Lelorier, J., Tremblay, M.. De Champlain, J.. Gattereau, A. and Davignon, J.. Effect of dopamine of diet-induced hyperlipidemla and atherosclerosis in the rat, Canad. J. Physiol. 54 (1976) 83. Thompson, S.W., In: Selected Histochemical and Histopathological Methods, Charles Springfield. IL, 1966. p. 330.
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24 Zar. H.J.. In: Biostatistical Analysis, Prentice Hall, Englewood Cliffs. NJ, 1974, p. 151. 25 Bar-On, H., Roheim, P.S. and Eder, H.A.. Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes, J. Clln. Invest., 57 (1976) 714. 26 Lasser. N.L., Roheim, P.S., Edelstein, D. and Eder, H.A., Serum lipoproteins of normal and cholesterol-fed rats, J. Lipid Res., 14 (1973) 1. 27 Mahley. R.W., Weisgraber. K.H., Innerarlty. T.. Brewer, Jr., B.H. and Assmann, G.. Swine lipoproteins and atherosclerosis, changes in the plasma lipoproteins and apoproteins induced by cholesterol feeding, Biochemistry, 14 (1975) 2817. R.W.. Weisgraber, K.H. and Innerarity. T., Canine lipoproteins and atherosclerosis. Part 2 28 Mahley. (Characterization of the plasma lipoproteins associated with atherogenic and nonatherogenic hyperlipidemla), Circulat. Res.. 35 (1974) 722. 29 Hollenberg, C.H., Vast, A. and Patten, R.L.. Regulation of adipose mass -Control of fat cell development and lipid content, Rec. Progr. Horm. Res.. 26 (1970) 403. 30 Ozegovic, B. and Milkovic. S.. Inhibitory effect of prolactin on the development of fatty liver induced by ACTH in the rat, Proc. Sot. Exp. Biol. Med., 149 (1975) 262. 31 Rudman, D. and Di Girolamo, M., Effects of adrenal cortical steroids on lipid metabolism. In: Human Adrenal Cortex, Harper and Row. New York, NY, 1972. p. 241. 32 Kyner, J.L., Levy, R.I.. Soelder. J.D.. Gleason. R.E. and Fredickson. D.S., The short term effect of cortisone acetate upon fasting triglyceride and cholesterol in normal subjects and offspring of diabetic couples, Metabolism, 21 (1972) 329. 33 34 35
36
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Stem. M.P., Kolterman. O.G.. Fries. J.F., McDeritt. H.O. and Reaven, G.M.. Adrenocortical steroid treamtent of rheumatic diseases - Effects on lipid metabolism, Arch. Intern. Med., 132 (1973) 97. Goodman, M.H., Permissive effects of hormones on lipolysis, Endocrinology. 86 (1970) 1064. Ozegovic. B., Rode, B. and Mllkovic. S., The role of the adrenal gland in the lipid accumulation process ln the liver of rats bearing an ACTH and prolactin producing tumor, Endocrlnologie. 66 (1975) 128. Gianoulakis, C., Lis. M. and Chretien, M.. Lipolytic response, cyclic AMP and cyclic GMP accumulation in rat adipocytes stimulated with ACTH - Effect of MtT-F4 tumor and clofibrate treatment, Submitted for publication. Ruderman, N.B., Richards, K.V., Valles de Bourges. V. and Jones, A.L.. Regulation of production and release of lipoprotein by the perfused rat liver, J. Lipid Res.. 9 (1968) 613. Heimberg, M.. Weinstein, I. and Kohout, M., The effect of glucagon. dibutyryl cyclic adenosine 3’.5’monophosphate, and concentration of free fatty acids on hepatic lipid metabolism, J. Biol. Chem.. 244 (1969) 5131. Topping, D.L. and Mayes, P.A., The immediate effects of insulin and fructose on the metabolism of the perfused liver - Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism, Biochem.. J., 126 (1972) 295. Buckley, J.T., Delahunty. T.J. and Rubinstein, D.. The relationship of protein synthesis to the secretion of the lipid moiety of low density lipoprotein by the liver, Canad. J. Biochem., 46 (1968) 341. Faegerman. 0.. Sata. T., Kane, J.P. and Havel. R.J., Metabolism of apoprotein beta of plasma very low density lipoproteins in the rat, J. Clin. Invest., 56: 1396-1403. Faegerman. 0. and Havel, R.J., Metabolism of cholesteryl esters of rat very low density lipoproteins, J. Clin. Invest., 55 (1975) 1210. Nikkilii, G.A., Effect of drugs on plasma triglyceride metabolism. In: W.L. Holmes, R. Paoletti and D. Kritchevsky (Eds.). Pharmacological Control of Lipid Metabolism, Plenum. New York, NY, 1971. p. 113. Havel, R.J. and Kane, J.P.. Drugs and lipid metabolism, Ann. Rev. Pharmacol., 13 (1973) 287. D’Costa. M.A. and Angel, A., Inhibition of hormone stimulated lipolysis by clofibrate - A possible mechanism for its hypolipidemic action, J. Clin. Invest., 55 (1975) 138.
Atherosclerosis, 32 (1979) 33-42 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
HYPERLIPOPROTEINEMIA INDUCED PITUITARY TUMOR IN THE RAT
A. CHRISTINE NESTRUCK, MICHEL CHRkTIEN Institut de Recherches H2 W 1 R7 (Canada)
CHRISTINA
BY A TRANSPLANTABLE
GIANOULAKIS,
JEAN DAVIGNON
and
Cliniques de Montre’al, 110 avenue des Pins, ouest, Montre’al, P. QuB.,
(Received 8 May, 1978) (Revised, received 6 September, 1978) (Accepted 15 September, 1978)
Summary The MtT-F4 tumor, a transplantable pituitary tumor of rats, induces significant hyperlipidemia in male Fisher 344 rats. The increasive hypercholesterolemia was accompanied by hypertriglyceridemia only in the first month of tumor implantation. Clofibrate feeding inhibited the development of hypercholesterolemia and maintained normal serum triglyceride levels. In contrast to the changes in lipoprotein cholesterol distribution and profile found in experimental hyperlipidemia induced by high fat and cholesterol feeding, the hypercholesterolemic tumor-bearing rats showed no accumulation of cholesterol in the very low density and intermediate density lipoproteins, and no appearance of a new class of lipoprotein, B-VLDL. An HDLc-like lipoprotein appeared as hypercholesterolemia developed. Increased amounts of cholesterol were deposited in the aorta. The effects are attributed to the lipolytic hormones secreted by the tumor and antagonism to their action by clofibrate. Key words:
Atherogenesis
- Clofibrate - HDL,
- Hypercholesterolemia
Introduction The rat, a widely used model in lipid and lipoprotein metabolic studies, is very resistant to the development of hypercholesterolemia and atherosclerosis. Early studies [ 1,2] demonstrated that hypercholesterolemia sufficient to This work was supported by grants from the Medical Research Council of Canada (MA-6119) and the Quebec Heart Foundation. A.C.N. holds a Research Scholarship from the Cons4 de la Recherche en Sante du Quebec and C.G. is a Medical Research Council of Canada post-doctoral fellow.
34
induce experimental atherosclerosis in the rat required diets containing saturated fat, bile salts, cholesterol and the thyrotoxic agent propylthiouracil. More recently, the changes in lipoprotein and apoprotein profiles of these rats have been characterized [3] and shown to be similar to those in animals in which hypercholesterolemia can be induced by cholesterol feeding alone. We recently reported that implantation of a transplantable pituitary tumor (MtTF4) in chow-fed male Fisher 344 rats induced hyperlipidemia [4] and hypertension [5]. Thirty-one days after implantation, plasma free fatty acid, triglyceride and cholesterol levels were significantly raised. The tumor, which secretes large quantities of prolactin, growth hormone and ACTH [4,6], also induces a fatty liver [4,7] and a significantly higher lipolytic response to ACTH by isolated adipocytes [ 81. Tumor-bearing rats fed a clofibrate-supplemented chow diet developed a lesser degree of hyperlipidemia [4]. It has been suggested that the hyperlipidemia and fatty liver of the tumor-bearing rats are consequences of chronic lipolysis and mobilization of lipid from peripheral depots. The purpose of this study was to characterize the hyperlipoproteinemia induced by the tumor and to extend the period of tumor implantation to two months, since preliminary results [9] after one month of implantation had shown differences in the lipoprotein profile from that associated with experimental hypercholesterolemias dependent on high cholesterol diet. The early inhibitory effect of clofibrate feeding on the development of the hyperlipidemia was also investigated after 2 months of tumor implantation. Methods Male Fisher 344 rats were purchased from Canadian Breeding Laboratories. The MtT-F4 tumors were provided by the Mason Research Institute of Boston, MA, and were taken through at least 10 successive transplantations before being used for the present experiments. The tumors were transplanted subcutaneously under ether anesthesia in the posterior half of the dorsal area. A sham implantation was performed in control rats. The animals were divided into 4 groups: controls, with sham implantation (S); rats with MtT-F4 tumor implantation (T); shams fed clofibrate (SCF); and rats with MtT-F4 tumor that were fed clofibrate (TCF). Rats in all groups weighed 100 f 5 g at the start of the experiments. Groups S and T were fed Purina laboratory chow, while groups SCF and TCF received the same chow containing 0.25% clofibrate (ethyl-p-chlorophenoxyisobutyrate). The clofibrate-supplemented diet was prepared by Ayerst Laboratories, Montreal. Daily caloric intake was similar for all rats, and clofibrate intake was calculated to be approximately 30 mg/kg per day. All rats were fasted for 24 h prior to killing by decapitation at 31 or 60 days after implantation. Trunk blood was collected and allowed to clot. The adrenals, liver and tumor were removed, dissected free from adhering connective tissue and weighed. After centrifugation the serum was kept at 4°C for determination of triglyceride and cholesterol and for the isolation of lipoproteins. Serum cholesterol [lo] and triglyceride [ 111 were measured with a Tech-
35
nicon Autoanalyser or individual rat serum samples at 31 days or on pooled serum at 60 days. Lipoproteins were isolated from pooled serum (4 rats/group) containing sodium azide (0.1%) by sequential ultracentrifugation at increasing densities [ 121. All centrifugations were carried out in a Beckman 50 Ti rotor at 47,000 rpm for various times: at density = 1.006, very low density lipoproteins (VLDL), 1.02 (intermediate fraction) and 1.063 for 16 h and at density 1.21, high density lipoproteins (HDL) for 40 h. The fractions were dialyzed against 5 mM ammonium bicarbonate buffer and protein content was determined [13]. The lipids of the various fractions were extracted with chloroform-methanol (2 : 1, v/v) [ 141. Aliquots of the chloroform phase were evaporated to dryness under nitrogen and taken up in isopropanol for tri[lo] determinations. Phospholipids were meaglyceride [ 111 and cholesterol sured according to the method of Bartlett [15]. Aliquots of the isolated lipoprotein fractions were also subjected to agar-agarose electrophoresis [16]. After delipidation [ 171, the lipoprotein apoproteins were subjected to electrophoresis on 10% polyacrylamide gels containing 7 M urea [ 181 with buffers as described by Kane [ 191 or in the SDS system of Weber and Osborne [20] for molecular weight determinations. In the 60-day studies, immediately after the rat was killed the aorta from the aortic arch to iliac bifurcation was dissected free of connective tissue, blotted and weighed. The aortas from half the rats in each group were fixed and stained with hematoxylin-eosin [21]. The aortas from the remaining rats were quickfrozen in liquid nitrogen and stored at -30°C. The frozen aortas were later cut into small fragments and saponification and lipid extraction procedures were applied [22]. Total cholesterol was then determined [lo]. Liver slices were taken at random, fixed and stained with Oil Red 0 [ 231. The results reported here are from studies of one 31-day tumor implantation with 4-5 rats per treatment group and from two separate 60-day studies, also with 4-5 rats per treatment group. For statistical analysis, the Newman-Keuls test was used [ 241. Results Organ weights The body weight of the sham-operated rats increased throughout the postoperative period. Growth of the rats with an MtT-F4 tumor was retarded: their body weight was significantly lower than that of the controls at both 31 and 60 days after implantation, Liver and adrenal weights were, however, significantly higher than in the controls (Table 1). Clofibrate treatment partially prevented the tumor effect on liver weight without affecting the tumor growth or adrenal hypertrophy. At 31 days the body weight retardation of this group was the same as in the tumor group and at 60 days was slightly more severe (Table 1). Serum lipids Table 2 presents the effects of the tumor on serum lipids. Both sham groups showed normal lipid levels throughout this study. Thirty-one days after implantation a significant hypertriglyceridemia and hypercholesterolemia had been induced by the tumor. Previous studies had shown a progressive increase in
36 TABLE 1 EFFECTS OF MtT-F4 TUMOR f CLOFIBRATE
ON BODY AND ORGAN WEIGHTS
Values are means + SEM. Time a
Tumor (g) 31
60
31
60
31
60
31
60
13.6 k1.97 13.7 t1.89
231 t2.a 230 to.9 199 * t3.7 188 * t4.3
261 56.5 210 f2.7 217 * t4.3 200 *.***
5.8 to.13 5.4 to.12 9.3 * to.43 1.5 *.** to.26
7.5 il.05 1.7 r0.19 20.2 * il.32 14.6 *. ** il.17
40 +1.7 39 t1.2 124 * t8.0 114 * +I.7
38 +1.6 39 t1.6 445 * 221.3 466 * t26.8
Sb SCF T TCF
1.4 to.15 1.2 +0.16
Adrenals (me)
Liver (5)
Body (g)
k5.6
a Days after sham or tumor implantation. b S = sham-implanted rats: SCF = sham-implanted, clofibrate-fed; planted. clofibrate-fed (10 rats/group). * Significant difference from S or SCF, P < 0.001. ** Significant difference from T, P < 0.001. * * * Significant difference from T. P < 0.05.
T = tumor-implanted:
TCF = tumor-im-
these two plasma lipid concentrations up to 41 days [4]. By 60 days of tumor implantation, however, the serum triglyceride level had reverted to normal levels despite a severe remaining hypercholesterolemia (Table 2). This decline in plasma triglyceride was confirmed in other studies at 60 days of tumor implantation. The clofibrate-fed tumor-bearing rats showed little change in serum triglyceride and only moderate hypercholesterolemia at 31 days. By 60 days, however, the serum cholesterol levels were as high as in the tumor group: about 5 times the control level (>300 mg/dl). Phospholipids were also increased in both tumor groups at 60 days. The ratio of free : esterified cholesterol was unchanged.
TABLE 2 TIME EFFECT OF MtT-F4 TUMOR Time
TG 31 a
f CLOFIBRATE
TC
ON SERUM LIDIDS PL
FC/TC (%)
6ob
31
60
31
60
31
60
38 39 33 69
41 48 219 * 132 **
68 65 330 * 306 **
ND ND ND ND
100 98 380 * 347 **
ND ND ND ND
21 20 21 21
(mgld) S SCF T TCF
33 31 130 * 48
TG = triglyceride; TC = total cholesterol: PL = phospholipids; FC = free cholesterol: ND = not determined; S. SCF, T. TCF: see legend,Table 1. a Mean of 6 individual samples/group, 31 days after sham or tumor implantation. b Mean of two experiments, pooled serum samples (4-5 rats/group), 60 days after implantation. * Significantly different from S. P < 0.001. ** Significantly different from SCF. P < 0.01.
37
Lipoproteins The lipid and protein distributions in the ultracentrifugal fractions 31 and 60 days after implantation are shown in Table 3. Clofibrate-fed sham-implanted rats showed no change in the distribution of serum lipids or lipoprotein protein at any time in the study. In all determinations more than 90% of the serum triglyceride was recovered in the lipoprotein fractions. For serum cholesterol, the recovery was >80% except in the two tumor groups at 60 days, where the serum values were >300 mg/dl and only 60% of the serum level was accounted for. At 31 days, there was very little change in the distribution of the plasma lipids or lipoprotein protein in either tumor-bearing group and no accumulation of cholesterol in the d < 1.02 fractions, in spite of mean serum cholesterol levels of 132-219 mg/dl. All groups throughout the study had less than 10 mg/ dl in the d < 1.006 lipoproteins, with the exception of the tumor-bearing group at 31 days. The elevated levels of serum cholesterol and phospholipid were predominantly carried in the d > 1.02 lipoproteins in both tumor groups. At 31 days, the d < 1.006 fraction in both tumor groups had a higher lipid : protein ratio because of the increase in all lipid components. This lipid enrichment was slightly decreased at 60 days. The 1.02-1.063 lipoproteins in both tumor groups were enriched in phospholipid and to a lesser extent protein at the TABLE 3 DISTRIBUTION
OF LIPID AND PROTEIN IN THE ULTRACENTRIFUGAL
Density a
1.006
Time b
31
FRACTIONS
1.0061.02
1.021.063
1.0061.063
1.063-1.21
60 c
31
31
60
31
60
(mg/dI) TG TC PL P
_d -
25.1 6.2 5.5 7.4
-
SCF
TG TC PL P
27.9 5.4 2.1 4.9
T
TG TC PL P TG TC PL P
S
TCF
-
-
-
7.1 8.8 3.2 5.0
-
1.1 39.2 28.1 53.0
32.1 5.2 6.7 8.0
1.3 1.1 0.9 0.6
3.5 4.8 4.5 6.0
4.7 5.1 1.6 2.1
0.6 25.1 20.1 45.4
1.1 29.8 25.2 56.1
136.6 28.8 30.1 15.4
15.8 4.5 2.4 2.5
2.9 3.0 3.5 1.6
3.2 36.0 31.7 31.7
6.2 33.0 29.2 23.1
3.1 106.3 120.5 174.3
6.3 165.2 210.4 249.5
45.8 9.0 13.3 5.8
49.5 9.0 2.6 6.8
2.3 2.8 1.8 1.0
2.8 13.9 14.3 15.4
5.7 30.1 31.8 21.1
1.8 103.3 113.2 171.1
6.0 142.3 205.4 249.1
S. SCF, T. TCF: see legend Table 1: TG, TC. PL: see legend Table 2; P = protein. a Sequential ultracentrifugal density cuts. Intermediate density fraction 1.006-1.02 day experiment. b Days after sham or tumor implantation. c Mean of two separate serum pools. d sample lost.
isolated only in 31-
38
expense of triglyceride at 31 days, and these changes were enhanced at 60 days. The d 1.063-1.23. lipoproteins of both tumor groups were enriched in phospholipid, but not cholesterol or triglyceride, both at 31 and 60 days. Agar-agarose electrophoresis of the isolated density fractions revealed changes in the tumor-bearing animals at 31 days (Fig. 1, top-half). In contrast to the controls which showed in the d < 1.006 fraction a pre-fl band and virtually no material at the origin, both tumor groups showed increased trailing and material at the origin (tumor >> tumor + clofibrate). Little stainable material was found in the d 1.006-1.02 fraction in any group. The d 1.02-1.063
Fig. 1. Ag-garose electrophoretograms of the sequential ultracentrifugal fractions for sham and tumorbearing rats at 31 days (above); tumor and tumor plus clofibrate at 60 days (below). 0 indicates the origin. and /3 and (Y the migration of lipoprotein bands. Slides are stained with Oil Red 0.
39
TABLE 4 CHOLESTEROL
CONTENT
OF THE AORTA
AT 60 DAYS
Values are mean t SD. Mg/mg wet weight S SCF T TCF
1.76 1.77 2.61 2.53
* + f +
0.08 0.03 0.26 * 0.26 *
* Significantly different from S or SCF, P < 0.001.
fraction displayed a single P-migrating band in the control and tumor + clofibrate groups, but in the MtT-F4 group, a second band with a-mobility was observed (Fig. 1). Furthermore in the d 1.063-1.21 fractions, both tumor groups showed a band with P-mobility in addition to the a-migrating band characteristic of this fraction. At 60 days (Fig. 1, lower half), both tumor groups showed a single pre-fl band in the VLDL. Clofibrate feeding appeared to prevent the build-up of a-migrating proteins in the d 1.006-1.063 fraction, whereas the tumor group showed a less prominent P-migrating band. In the d 1.063-1.21 fraction both tumor groups showed the characteristic a-migrating band as well as a P-band. On analytical SDS polyacrylamide gel electrophoresis, in the d 1.063-1.21 fractions, all groups showed bands with molecular weights of 25,000 (AI), 33,000 (apo E), and 44,000 (apo A IV) in addition to low molecular weight bands < 10,000 (apo CII, CIII). Additionally, both tumor groups showed a peptide with molecular weight = 19,500. For basic polyacrylamide gel electrophoresis the apoproteins of the d 1.006-1.063 fraction of the control groups were almost insoluble in the solvent (7 M urea, 40 mM Tris, pH 8.9). Only l2% of the lipoprotein was soluble and only bands in the apo CII-CIII area were found. In contrast, 50-60% of the d 1.006-1.063 lipoprotein-protein of the two tumor groups was soluble in this solvent. These soluble peptides showed banding in the apo-AI, -E, -CII and -CIII areas [ 251. Tissue studies The cholesterol content of the aorta was measured in all groups at 60 days. Both tumor groups showed significantly higher values than the controls (Table 4). Histology of liver sections taken from the 4 groups to be killed showed significant fatty infiltration of the parenchyma in the tumor group with tumor >> tumor + clofibrate. No structural modifications, however, were seen in the aortic sections. Discussion A hypercholesterolemia develops following high fat and cholesterol feeding in the rat [ 261, pig [ 271, or dog [28] and the propylthiouracil-supplemented atherogenic diet in the rat [ 31. The diet-induced hypercholesterolemia is accompanied by cholesterol enrichment of the very low density lipoproteins
40
and the finding of a cholesterol-enriched B-VLDL in the intermediate density range. In our study, although the tumor induced an elevated serum cholesterol as high as that reported after cholesterol feeding in rats [3,26], the very low density lipoproteins had an essentially normal complement of cholesterol and there was no accumulation of lipoprotein or cholesterol in the d 1.006-1.02 range. For both tumor groups, the significantly increased serum cholesterol was found predominantly in the d > 1.063 lipoproteins. The appearance of a second band with cr-migration on the d 1.02-1.063 fraction, possibly an HDLc-type particle [ 3,27-28 1, is similar to electrophoretograms following cholesterol feeding in various species. This band was not apparent in the clofibrate-fed tumor group at 31 days, when serum cholesterol had risen to 132 mg/dl, but did become visible at 60 days when cholesterol levels were equal to those in the tumor groups not clofibrate-fed (>300 mg/dl). This a-migrating lipoprotein was also associated with significant amounts of urea-soluble apoproteins. In comparison with the hyperlipidemia induced by cholesterol feeding in the rat, these differences in lipoprotein distribution and composition in relation to an almost equal degree of hypercholesterolemia may reflect different methods of induction: an endogenous versus an exogenous stimulation of lipid metabolism. The hyperlipidemia of the tumor-bearing rats is probably the result of the high concentration of lipolytic hormones secreted by the tumor. Both ACTH and GH are lipolytic in the rat [29,30 1, although increased production of corticosteroids may also play a role [ 31-341, The importance of adrenal glands and hypersecretion of corticosteroids in the induction of hyperlipidemia is indicated by the observation that adrenalectomized rats bearing the MtT-F4 tumor did not develop a fatty liver [35]. The lipolytic response of isolated adipose tissue cells [8] and the accumulation of cyclic AMP following stimulation by ACTH were significantly higher in cells derived from rats bearing the tumor [36]. It is well established that the availability of fatty acid to the liver regulates triglyceride synthesis and VLDL secretion [37-401. In the normal rat, the further conversion of VLDL &protein and cholesterol ester to LDL appears to be only a minor process [41,42]. However, in this study, it is possible that the continued stimulation of VLDL production enhanced this conversion process especially in the early stages of tumor implantation. However, in the long term, after two months of tumor implantation, the severe widespread fatty infiltration of the liver suggests that the synthetic and/or secretory mechanisms for triglyceride export (VLDL production) were insufficient and resulted in a reversal of the hypertriglyceridemia, decreased numbers of VLDL lipoproteins and decreased numbers of fraction. P-migrating lipoproteins in the d 1.006-1.063 Clofibrate is known to have several actions on lipid metabolism and extrahepatic tissues [43-441, including an antilipolytic effect on human and rat adipose tissue mediated by the adenylate cyclase system [45]. In this study, clofibrate appears to have protected the organism against the enhanced lipolysis induced by the tumor: at 31 and 60 days, the serum triglyceride levels were only slightly increased, the P-migrating lipoproteins of the 1.006-1.063 fraction remained prominent and the liver showed a lesser degree of fatty infiltration. Thus, possibly by slowing the degree of extrahepatic lipolysis, the hepatic
41
handling and disposal mechanisms of endogenous free fatty acids were maintained. However, in the long term, clofibrate did not prevent hypercholesterolemia or cholesterol deposition in the aorta. In summary, rats bearing the MtT-F4 tumor present a developing hypercholesterolemia which is different from that resulting from cholesterol feeding. The absence of B-VLDL with its significant cholesterol-carrying function is possibly due to different disposal mechanisms for chylomicron and hepatic VLDL lipid. The finding of an HDLc-like lipoprotein and its associated ureasoluble apoproteins is possibly analogous to the HDLc previously reported in various species. In the present study, this lipoprotein was associated with serum cholesterol levels >200 mg/dl and ultimately with increased cholesterol deposition in the aorta. Acknowledgements The authors wish to thank Ayerst Laboratories of Montreal for a generous supply of the clofibrate-supplemented diet; Dr. S.A. Bencosme, Department of Pathology, Queen’s University, Kingston, Ontario for performing the histology and Margaret Bergseth for excellent technical assistance. References 1 Malinow, M.R., Hojman, D. and Pellegrino, A., Different methods for the experimental production of generalized atherosclerosis in the rat, Acta Cardiol., 9 (1954) 480. 2 Wissler, R.W.. Eilert, M.L., Schroeder, M.A. and Cohen, L., Production of lipomatous and atheromatous arterial lesions in the albino rat, Arch. Path., 57 (1954) 333. 3 Mahley, R.W. and Holcombe, K.S., Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat. J. Lipid Res., 18 (1977) 314. 4 Gianoulakis. C.. Passe&i. L., Lis, M., Davignon, J. and Chretien, M., Effect of clofibrate treatment on chronic hyperlipidemia induced by an ACTH producing tumor, Canad. J. Physiol. Pharmacol.. 55 (1977) 220. 5 Kubo. S.. Ganten, D., Ganten, U.. Nowaczynski, W. and Genes& J.. Plasma steroids in rats with experimental ectopic pituitary tumor and arterial hypertension, Endocrinology, 94 (19741459. 6 Bates, R.W.. Milkovic. S. and Garrison, M.M., Concentration of prolactin, growth hormone and ACTH in blood and tumor of rats with transplantable mammotropic pituitary tumors, Endocrinology, 71 (1962) 943. 7 Milcovic, 8. Ganison. M.M. and Bates, R.W.. Study of the hormonal control of body and organ size in rats with mammotropic tumor, Endocrinology, 75 (1964) 670. 8 Gianoulakis, C., Lis, M., Davignon, J. and Chretien, M., Development of hyperlipidemia associated with increased lipolytic response of isolated adipose tissue cells following prolonged stimulation by an ectopic pituitary tumor, Harm. Metab. Res., In press. 9 Gianoulakis, C.. Nestruck, A.C., Lis, M., Davignon, J. and Chretien, M., New model for experimental hyperlipidemia. In: Abstracts of the 59th Annual Meeting of the Endocrine Society, 1977, p. 339. 10 Block, W.D., Jarret. K.J. and Levine, L.B.. An improved automated determination of serum cholcsterol with a single color reagent, Clin. Chem., 12 (1966) 681. 11 Kraml, N. and Cosyns. L., A semiautomated determination of serum triglycerides, Clin. Biochem., 2 (19691 373. 12 Havel, R.J.. Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest., 34 (1955) 1346. 13 Lowry, O.H., Rosebrough, N.J., Farr. A.L. and Randall, R.J.. Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (19511 265. 14 Folch, J., Lees, N. and Sloane-Stanley, G.H., A simple method for the isolation and Purification of total lipides from animal tissues, J. Biol. Chem.. 226 (19571 497. 15 Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem.. 234 (19591466. 16 Noble, R.P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (19681 693.
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