J. Biosci., Vol. 12, Number 2, June 1987, pp. 137 142. Printed in India. Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats P. VALSALA and P. A. KURUP Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum 695 581, India MS received 29 July 1986; revised 28 April 1987 Abstract. The mechanism of hypercholesterolemia effect of Cu 2+ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu 2+ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu 2+ deficient rats. Keywords. Copper deficiency; hypercholesterolemia; HMG-CoA reductase; lipoprotein lipase; apo B; hepatic bile acids. Introduction Copper deficiency has been reported to produce hypercholesterolemia in rats (Allen and Klevay, 1978) but very little is known about its mechanism. Allen and Klevay (1978) observed that Cu 2+ deficiency had no effect on liver total cholesterol or on biliary steroid excretion in weanling rats, but increased biliary bile acid excretion. Increased incorporation of [ 3 H]-mevalonate into the plasma free and ester cholesterol was observed by these workers. Apart from these, no report seems to be available as to how the cholesterol metabolism is affected in Cu 2+ deficiency. In view of this, the effect of Cu 2+ deficiency was studied on some aspects of cholesterol metabolism using rats as experimental animals. The activity of hepatic hydroxylmethylglutaryl (HMG)-coenzyme A (CoA) reductase, incorporation of [ 14 C]- acetate into hepatic and serum cholesterol, activity of lipoprotein lipase (LPL) of the extrahepatic tissues, concentration of cholesterol in the serum lipoprotein fractions and concentration of hepatic bile acids have been studied. The effect of including cholesterol in the diet on these aspects has also been studied. Materials and methods Male albino rats (Sprague-Dawley strain, weight 40 50 g) were divided into 4 groups of 8 rats each as follows: A. Cholesterol free diet group 1. Cu 2+ deficient rats 2. Control rats Abbreviations used: HMG, Hydroxymethylglutaryl; CoA, coenzyme A; LPL, lipoprotein lipase; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein. 137
138 Valsala and Kurup B. Cholesterol diet group 3. Cu 2+ deficient rats 4. Control rats The cholesterol free diet contained (g/100g): Casein-20, glucose-62, cellulose-5, groundnut oil-8, salt mixture-4, vitamin mixture-1. Cholesterol diet had the following composition (g/100 g): Casein-20, glucose-53, cellulose-5, coconut oil-15, cholesterol-2, salt mixture-4, vitamin mixture-1. Wesson's salt mixture (Oser, 1965) without copper was used. ZnCl 2 and CoCl 2. 6H 2 O were also added to the diet at a concentration of 15 0 and 0 15 mg/kg diet respectively. The Cu 2+ content of the diet was determined by atomic absorption spectrophotometry. Cu SO 4. 5H 2 O (AR. BDH) was added to the diet to give a concentration of 0 8 µg Cu 2+ /g diet in the deficient groups and 5 0 µg/g in the control groups. All chemicals used for the salt mixture were of analytical grade. Composition of the vitamin mixture has been given before (Jayakumari and Kurup, 1979). The rats of groups 2 and 4 were pairfed controls of groups 1 and 3, respectively. The diet consumption was adjusted to be the same in the control and deficient groups. Deionised water after distillation was available to the rats ad libitum. The animals were housed individually in polypropylene cages in rooms maintained at 25 C. The duration of the experiment was 6 months. At the end of this period, the animals were deprived of food overnight, stunned by a blow at the back of the neck and killed by decapitation. Blood and tissues were removed to ice cold containers for various estimations. Cholesterol and triglycerides in the serum and tissue were estimated as described before (Menon and Kurup, 1976). Extraction of liver for bile acids was carried out according to the procedure of Okishio et al. (1957). Bile acids were separated from free fatty acids by thin layer chromatography over silica gel using n hexane: ether : acetic acid (30:6:0 5 v/v/v) as the solvent system and estimated enzymatically using 3 α-hydroxy steroid dehydrogenase (Palmer, 1969). HMG-CoA reductase (EC 1 1 1 34) activity of liver was estimated as described by Rao and Ramakrishnan (1975) by determining the ratio of HMG-CoA: mevalonic acid. In vivo incorporation of [1, 2-14 C] -acetate into cholesterol in the liver and serum was carried out as described before (Thomas et al., 1983). 5 µci of [ 1, 2 14 C] -acetate per 100 g body weight was administered intraperitonially at 9 A.M. and the rats were killed 3 h later. Separation of serum high density lipoprotein (HDL) and low density lipoprotein (LDL) + very low density lipoproteins (VLDL) was carried out using the procedure described by Warnick and Albers (1978). Lipoprotein lipase (EC 3 1 1 3) activity of heart and adipose tissue was estimated according to the procedure of Krauss et al. (1974). Protein in the enzyme extract was determined after trichloroacetic acid precipitation by the method of Lowry et al. (1951). Estimation of Cu 2+ in the plasma and liver was carried out by atomic absorption spectrophotometry. The tissues were first ashed and then dissolved in dilute HCl. Statistical analysis was carried out by Student's 't' test (Bennet and Franklin, 1967). Results and discussion Diet consumption in the cholesterol free and cholesterol fed diet groups were 11 0 ± 1 2 and 10 0 ± 1 0 g, respectively. The intake of Cu 2+ was 8 72 ± 1 4 and 55 6 ± 2 2 µg
Hypercholesterolemic effect of copper deficiency 139 in the Cu 2+ deficient and control rats respectively in the cholesterol free diet group. The corresponding values in the cholesterol fed diet group were 8 14 ± 1 2 and 52 45 ±2 6 µg, respectively. The intake of cholesterol in the cholesterol fed diet group was 0 20±0 01 g. The gain in body weight was significantly lower (140 0 ± 5 2 and 210 0 ± 8 4 in the Cu 2+ deficient and control rats in the cholesterol free diet group and 180 0 ± 5 4 and 253 0 ± 10 1 respectively in the cholesterol fed diet group) and the heart weight was more (0 98 g±0 04 and 0 60 ± 0 02 in the deficient and control rats respectively in cholesterol free diet and 1 01 g±0 03 and 0 75 ± 0 04 respectively in the cholesterol fed diet group) in the copper deficient rats, in agreement with previous reports (Prohaska and Heller, 1982). The concentration of Cu 2+ in the plasma and liver was significantly lower in the Cu 2+ deficient group. (Plasma Cu 2+ (µg/ml):- 1 8 ± 0 05 and 4 12 ± 0 16 in the deficient and control group respectively in cholesterol free diet group and 1 2 ± 0 03 and 3 62 ± 0 14 respectively in the cholesterol fed diet group. Liver Cu 2+ (µg/g):- 6 4 ± 0 10 and 14 6 ± 0 68 in the deficient and control rats respectively in cholesterol free diet group and 5 2 ± 0 15 and 12 8 ± 0 51 respectively in the cholesterol fed diet group). Cu 2+ deficient rats showed hypercholesterolemia, confirming previous reports (Allen and Klevay, 1978), the extent of which was more in the presence of cholesterol in the diet (114 0 ± 4 56 and 56 0 ± 1 68 mg/100ml in the deficient and control rats respectively in cholesterol free diet group and 265 0 ± 10 6 and 112 0 ± 3 36 mg/100 ml respectively in the cholesterol fed diet group). It is significant that the copper deficient rats in the cholesterol free diet group showed almost the same extent of hypercholesterolemia (114 mg/ 100 ml) as that in the control animals of the cholesterol diet group fed adequate copper (112 mg/100 ml). There was also hypertriglyceridemia in the Cu 2+ deficient rats (10 2 ± 0 30 and 8 4 ± 0 32 mg triglyceride glycerol/100 ml in the deficient and control rats respectively in cholesterol free diet group and 22 2 ± 0 66 and 14 0 ± 0 56 respectively in the cholesterol diet group). Thus inclusion of cholesterol in the diet potentiated the hypercholesterolemic and hypertriglyceridemic effect of copper deficiency. Concentration of cholesterol in the liver and aorta There was no significant alteration in the concentration of cholesterol in the liver in Cu 2+ deficient rats in the cholesterol free diet group, but it was more in the cholesterol fed diet group (table 1). Concentration of cholesterol in the aorta was significantly more in the Cu 2+ deficient rats in both groups, the increase being more in the cholesterol fed diet group. Allen and Klevay (1978) also reported no significant alteration in hepatic total cholesterol in rats fed cholesterol free diet, but there is no previous report about the increase in aortic cholesterol in Cu 2+ deficiency. Concentration of cholesterol and apo B in serum lipoproteins HDL cholesterol was lower in the Cu 2+ deficient rats in both cholesterol free and cholesterol fed diet groups, but VLDL + LDL cholesterol was significantly higher (table 1). Thus the increase in serum cholesterol is manifested by increase in VLDL + LDL cholesterol. The increase in LDL + VLDL cholesterol was significantly more in the
140 Valsala and Kurup Table 1. Concentration of cholesterol in serum and tissue and of apo B in lipoproteins. Values are the mean±sem for 8 rats. Group 1 has been compared with group 2 and group 3 with group 4. a P<0 01, b P between 0 01 and 0 05. N,D, Not done. rats fed cholesterol diet. Concentration of apo B was also more in the deficient rats in the cholesterol free diet group. Incorporation of labelled acetate into hepatic and serum cholesterol Incorporation of labelled acetate into hepatic free cholesterol was significantly more in the Cu 2+ deficient rats in the cholesterol free diet group when compared to control rats, while incorporation into ester cholesterol was lower (the incorporation was not studied in the cholesterol diet group). The incorporation of label into the serum free and ester cholesterol was also significantly more in these rats. The observations in the case of serum cholesterol and liver ester cholesterol are in agreement with those of Allen and Klevay (1978). The increase in incorporation of label, into free cholesterol in the liver has not been reported before. Activity of HMG-CoA reductase in the liver was also more in the deficient rats (table 2). No previous reports are available on the activity of hepatic HMG-CoA reductase. This enzyme catalyses the rate limiting step in cholesterol biosynthesis in the tissues and its activity closely correlates with cholesterogenesis in the tissues. The increased activity of the enzyme in the liver in Cu 2+ deficient rats corresponds with increased cholesterogenesis, as indicated by the higher incorporation of label into hepatic cholesterol. Thus chole- Table 2. Activity of HMG-CoA reductase, in vivo incorporation of [l,2-14 C]-acetate into cholesterol (serum and liver) and concentration of bile acids in liver. Same notations as in table 1. *Ratio of HMG-CoA/mevalonate. Lower ratio indicates higher enzyme activity. N D, Not done.
Hypercholesterolemic effect of copper deficiency 141 sterogenesis is increased in Cu 2+ deficient rats. Since the activity of this enzyme is similar in both Cu 2+ deficient rats fed cholesterol free and cholesterol diet, it is evident that Cu 2+ deficiency is able to offset the, depression of cholesterogenesis caused by dietary cholesterol in the cholesterol fed rats. Concentration of bile acids in the liver There was no significant alteration in the total bile acids in the liver in the Cu 2+ deficient rats when compared to control rats in the cholesterol free diet group, but it was significantly decreased in the cholesterol fed diet group (table 2). Activity of lipoprotein lipase in the heart and adipose tissue The enzyme activity was significantly lower in the heart and adipose tissue in the Cu 2+ deficient rats (table 3). This enzyme is concerned with the uptake of circulating triglyceride rich lipoproteins (Chylomicrons and VLDL) by the extrahepatic tissues and the lower activity of this enzyme indicates decreased uptake of circulating triglyceride rich lipoproteins. The increase in the concentration of serum triglycerides in the Cu 2+ deficient rats may be due to the decreased activity of this enzyme. Table 3. Activity of lipoprotein lipase in the heart and adipose tissue. Same notations as in table 1. The liver occupies a key position in cholesterol metabolism. Hepatocytes derive cholesterol from circulating lipoproteins or by de novo synthesis and use it for membrane synthesis, bile acid synthesis and secretion, secretion of free sterol into the bile, lipoprotein formation, and storage of excess sterol as cholesterol ester. In Cu 2+ deficiency there is increased cholesterogenesis in the liver as is evident from the increased activity of HMG-CoA reductase and increased incorporation of label into liver cholesterol. But there is no significant alteration in the utilization of hepatic cholesterol for bile acid synthesis as indicated by the lack of significant alteration in the concentration of bile acids in the Cu 2+ deficient rats fed cholesterol free diet. There is also decreased esterification of the sterol, as is evident from the decreased incorporation of lable into ester cholesterol. Therefore more of this newly synthesized cholesterol may be channelled for the synthesis of lipoproteins and their subsequent release into circulation. The increase in the incorporation of label into serum cholesterol in the Cu 2+ deficient rats may support this view. More apo B is also synthesized in the liver of deficient rats and used to assemble the lipoproteins as is evident from the higher concentration of apo B present in VLDL and LDL fractions.
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