@Copyright 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/34024107 $02.00
The Effect of Dietary Copper on Rat Plasma Apolipoprotein B, E Plasma Levels, and Apolipoprotein Gene Expression in Liver and Intestine A . M A Z U R , *,1
F. NASSIR, 1, E. GUEUX, 1 P. CARDOT, 1
J. BELLANGER, 2 M. LAMAND,2 AND Y. RAYSS1GUIER2 1Laboratoire des/Vlaladies/v16taboliques; and 2Unit6 des Maladies Nutritionnelles, INRA Theix, 63122 St Gen~s Champanelle, France Received June 8, 1991; Accepted July 29, 1991
ABSTRACT The plasma levels of apo B and apo E, and the level of hepatic and intestinal mRNA coding for these apolipoproteins were investigated in weanling male rats pair-fed for 6 wk with a control or copperdeficient diet. Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats. Copper deficiency significantly increased triglyceride levels and decreased cholesterol levels in the liver. No major differences in the levels of hepatic and intestinal apo B and apo E mRNA occurred between control and copper-deficient rats. These data imply that hypertriglyceridemia dn hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in the gene expression at the mRNA level in the liver and intestine of the apolipoproteins studied. Index Entries: Copper deficiency; hyperlipidemia; apolipoprotein B; apolipoprotein E; mRNA. *Author to whom all correspondence and reprint orders should be addressed.
Biological TraceElement Research
] 07
Vol. 34, 1992
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Mazur et al.
INTRODUCTION Copper deficiency has been demonstrated to induce hypertriglyceridemia and hypercholesterolemia in rats, and cause changes in lipoprotein concentration and composition; however, the mechanisms responsible for these observations are not fully understood (1,2). Available evidence suggests that hypercholesterolemia in copper-deficient rats may be the result of an increased rate of hepatic release of cholesterol into the plasma (3) and that an impaired receptor-mediated binding of lipoproteins does not contribute to the hypercholesterolemia (4). Copper deficiency increases hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity (5), and decreases lipoprotein lipase, hepatic lipase (6), and lecithin:cholesterol acyltransferase (LCAT) activities (7), which could contribute to an elevated plasma cholesterol and triglyceride plasma levels. However, the possible contribution of apolipoproteins to the observed hyperlipemia in copper-deficient rats remains unclear. Regulation of apo B and apo E metabolism constitutes a major mechanism by which dietary agents may alter plasma lipids. Apo B is an apolipoprotein synthesized in enterocytes and hepatocytes as an obligate component of triglyceride-rich lipoproteins, and serves as recognition marker for the uptake of low-density lipoprotein (LDL) by the LDL (B, E receptor) (8). Apo E is a constituent of various plasma lipoproteins, and is synthesized in most tissues with the liver being the major site of production. Apo E plays an important role in the clearance of triglyceride-rich lipoproteins and in the redistribution of the cholesterol among tissues by its participation in the reverse transport of cholesterol (8). It is well known that the type of dietary carbohydrates plays a role in the expression of copper deficiency (9). The use of sucrose or fructorse as a carbohydrate source in the diet increases the severity of the signs of copper deficiency as compared to those exhibited when either starch or glucose was used as a dietary carbohydrate (9). On the other hand, diets rich in fructose are known to stimulate triglyceride synthesis and secretion from the liver (10). Investigations of apolipoprotein mRNA levels can provide information on factors controlling apolipoprotein and lipoprotein metabolism. Thus, in the present investigations, the rats were fed a diet containing sucrose with or without the addition of copper. We have focused our attention on the effects of copper deficiency on the plasma levels of apo B and apo E, and their mRNAs in the liver and in the intestine.
/V TERIALS AND METHODS Weanling male Wistar rats (IFFA-Credo, L'Arbresle, France) weighing 60 + 2 g (mean + SEM) were divided at random into copper deficient and control group (12 rats/group). The Institute's guide for the care and Biological Trace Element Research
Vol. 34, 1992
Apo B and Apo E in Copper Deficiency
109
use of laboratory animals was used. Rats were housed in wire-bottomed cages in a temperature-controlled room (22~ with a dark period from 20.00 to 08.00 h and pair-fed the appropriate diets for 6 wk. The semipurified diets contained (g/kg) casein 200, sucrose 705, corn oil 50, mineral mixture 35, and vitamin mixture 10, as described previously (11). Distilled water was provided ad libitum. The copper contents of diet were 0.6 mg/kg (deficient) and 7.3 mg/kg (control). Nonfasted animals were sacrificed after being anesthetized with sodium pentobarbital (40 mg/kg body wt) ip. Blood was collected into heparinized tubes, and plasma was obtained by low-speed centrifugation (2000g). The liver was excised, and portions from the right lobe were immediately plunged into liquid nitrogen, and then stored frozen at - 70~ until the lipid and RNA extractions were performed. Triglycerides, cholesterol, and phospholipids were determined in plasma by enzymatic procedures as described previously (12). Liver samples were homogenized, lipids were extracted with chloroform/ methanol (2/1, v/v) and triglyceride, and cholesterol and phospholipid contents were measured in the lipid residue (12,13). Copper in plasma and diets was determined with a Perkin Elmer 400 atomic absorption spectrophotometer. Plasma apo B and apo E levels were estimated by rocket immunoelectrophoresis (14) using antirat apo B and apo E rabbit immunosera. Serial dilutions of rat LDL or of pooled rat serum were used as standards. Hemoglobin was estimated by cyanmethemoglobin method (Test-Combination Hemoglobin, Boehringer, Mannheim, FRG). Total cellular RNA was isolated from liver tissue using the guanidium/phenol/chloroform method according to Chomczynski and Sacchi (15). RNA was quantitated by measuring the absorbance at 260 nm. Its integrity was systematically assessed by agarose-gel electrophoresis and visualization of 18 S and 28 S ribosomal RNAs by ethidium bromide staining. Aliquots of total RNA were subjected to quantification of mRNA content by dot-blot analysis on nylon filters. Hybridization of immobilized RNA to cDNA probes for apo E, apo B, and ~-actin labeled with [o~-32P]dATP and washing conditions have been previously described (16,17). The filters were blotted dry, and autoradiography was performed with intensifying screens at -70~ Quantification of the relative amounts of specific mRNA was performed by densitometric analysis of the hybridization signal using a laser densitometer (Ultrascan XL, LKB, Sweden). Relative abundance of the mRNA apolipoprotein was calculated with respect to the mRNA ~-actin content. Student's t-test was used to assess differences between copper-deficient and control groups.
RESULTS Copper-deficient rats had lower body weights and higher relative liver and heart weights than control rats (Table 1), but mortality was absent. Reduced plasma copper concentrations and blood hemoglobin Biological Trace Element Research
VoL 34, 1992
110
Mazur et ai.
Table 1 Body and Organ Weights, and Biochemical Data of Control and Copper-Deficient Rats I Control Body wt (g) Relative liver wt (g/100 g) Relative heart wt (g/100 g) Plasma copper (~mol/L) Blood hemoglobin (g/100 mL) Plasma triglycerides (raM) Plasma total cholesterol (raM) Plasma phospholipids (mM) Plasma apo B (AU) 2 Plasma apo E (AU) 2 Liver triglycerides (mg/g wet wt) Liver total cholesterol (mg/g wet wt) Liver phospholipids (mg/g wet wt)
253 3.70 0.32 17.81 12.2 0.50 1.85 1.37 100 100 10.5 4.4 35.8
_+ 5 + 0.08 _+ 0.01 _+ 0.52 + 0.2 _+ 0.10 + 0.08 + 0.05 + 6 ___ 3 + 0.5 _+ 0.2 _+ 0.7
Copper deficient 207 6.43 0.71 0.67 7.0 0.96 2.40 2.01 210 250 15.1 3.5 32.9
+ 9*** + 0.20*** _+ 0.05*** _+ 0.07*** + 0.4*** ___ 0.20* ___ 0.13"* + 0.09*** + 8*** + 8*** + 1.4" _+ 0.1"* + 1.2
1Mean + SEM; *P K .05; **P ~ .01; ***P ~ .001 as compared with the respective control value. 2Results of plasma apo B and apo E are expressed as arbitrary units (AU) taking the mean value of the control rats as 100.
were found (Table 1). Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats (Table 1). Copper deficiency significantly increased the triglyceride level and decreased the cholesterol level in the liver (Table 1). The hepatic apo B and apo E m R N A were slightly decreased in copperdeficient rats compared to control rats, but the difference was not statistically significant (Table 2). Dietary copper did not affect jejunal and ileal apo B mRNA, even if slight increases in m R N A levels were observed in deficient rats (Table 2).
DISCUSSION Several m e a s u r e m e n t s are indicators of dietary copper deficiency in addition to the reduced plasma copper concentration, such as decreased body weights, increased heart and liver to body weight ratios, and lower blood hemoglobin levels (9). Similar to previous findings (9), we observed both hypercholesterolemia and hypertriglyceridemia. All these traits indicate that the animals on the copper-deficient diet were indeed copper deficient. The increase in plasma triglycerides suggests that corresponding increase in plasma triglyceride-rich lipoproteins occurs. Conversely, the increase in plasma cholesterol suggests an increase in highdensity lipoprotein (HDL) cholesterol (2), since plasma cholesterol in the rat is mainly transported by HDL. The increase in plasma apo B observed Biological Trace Element Research
Vol. 34, 1992
Apo B and Apo E in Copper Deficiency
111
Table 2 Apolipoprotein B and E mRNA Levels in Control and Copper-Deficient Rats ~ Control
Copper deficient
Liver Apo B mRNA Apo E mRNA
100 + 14 100 + 11
77 +__ 15 78 +_ 12
Intestine Apo B rnRNA:jejunum Apo B mRNA:ileum
100 _+ 25 109 +__8
155 +_ 14 128 _+ 17
1Results are mean +_ SEM of six samples. Results are expressed as apo B/actin or apo E/actin mRNA ratios, and are normalized taking the mean value of the control liver or of the control j e j u n u m as 100.
in the present study is consistent with the elevation of triglyceride-rich lipoproteins in copper-deficient rats (2). It has been shown that the hypercholesterolemia observed in copper-deficient rats is mainly the result of a selective increase in the concentration of HDL1 subfraction (18). The increase in plasma apo E is also consistent with the selective increase in the concentration of HDL1, which is characterized by an elevated apo E content compared to HDL2 particles. Our results indicate that apo B and apo E mRNA levels in the liver of copper-deficient rats were slightly decreased (about 22%) as compared to control rats, but these differences were not significant. A recent study indicates that the apo A-I mRNA was also decreased by 25% in the liver of copper-deficient rats (19). Whether these decreases reflect a general reduction in all liver mRNA is unknown. The liver plays a central role in lipoprotein metabolism. Triglycerides synthesized in the liver, mainly from dietary fatty acids and carbohydrates, are transported to peripheral tissues as the very low density lipoproteins (VLDL). Apo B and apo E are the major apolipoproteins of VLDL, and are important for regulating synthesis, secretion, and catabolism of these particles (8). Copper deficiency is associated with a significant increase in plasma triglycerides and apo B, as well as with a significant increase in hepatic triglyceride concentration. Previous studies have consistently shown a decreased hepatic cholesterol content in copper-deficient rats (9). Similarly, our studies found significantly reduced levels of total hepatic cholesterol. However, previous investigations did not observe the increased levels of hepatic triglycerides in copper-deficient rats that had been fasted (20). Higher triglyceride to protein ratios in VLDL from copper-deficient rats than from control rats have been also observed (21). The results concerning apo B gene expression in the liver is of interest, since secretion of VLDL is dependent upon apo B (8). Apo E also has a control role in cholesterol metabolism (8); however, hypercholesterolemia and decreased hepatic Biological Trace Element Research
Vol. 34, 1992
1 12
/Vlazur et ai.
content of cholesterol were not associated with changes in apo E mRNA in the liver. Little is known about the influence of copper on intestinal production of lipoproteins. A recent study showed that copper deficiency impairs the intestinal transport of cholesterol (6), which involves transport into the lymphatic system via chylomicrons, and it is also well known that apo B is essential for the transport of triglycerides out of the intestine (8). However, our results indicate that apo B mRNA levels in the intestine were not significantly modified by copper deficiency. Finally, we have shown that the increase in plasma triglyceride and cholesterol levels observed in copper deficiency is associated with a marked increase in plasma apo B and apo E levels. However, our data do not show major differences in apo B and apo E mRNA levels between control and copper-deficient rats. These data imply that hypertriglyceridemia and hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in mRNA levels in the tissues measured. Thus, plasma apo B and apo E levels may not be determined by factors that regulate the level of apo mRNA. It has been established that the type of dietary carbohydrate used lays a major role in the expression and severity of copper deficiency, and it has been suggested that certain metabolic pathways of fructose may be responsible for the severity of copper deficiency (22). Recent results support a stimulatory effect of dietary sucrose on apo B and apo E gene expression in rat liver (23). Thus, it would be of interest to evaluate the effect of changing the type of dietary carbohydrate on the effect of copper deficiency on apo B and apo E gene expression in rat liver.
ACKNOWLEDGMENT This work was supported by a grant from Fondation Fran~aise pour la Nutrition. We thank D. Bayle, A. Bellanger, and C. Lab for their excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5.
L. M. Klevay, Am. J. Clin. Nutr. 26, 1060 (1973). K. Y. Lei, J. Nutr. 113, 2178 (1983). M. S. J. Shao and K. Y. Lei, J. Nutr. 110, 859 (1980). C. A. Hassel, T. P. Carr, and K. Y. Lei, Nutr. Res. 10, 903 (1990). N. Y. Yount, D. J. McNamara, A. A1-Othman, and K. Y. Lei, J. Nutr. Biochem,. 1, 21 (1990). 6. S. I. Koo, C. C. Lee, and J. E. Norvell, Proc. Soc. Exp. Biol. Med. 188, 410 (1988). 7. B. W. C. Lau and L. M. Klevay, ]. Nutr. 111, 1698 (1981): 8. R. W. Mahley, T. L. Innerarity, S. C. Rail, and K. H. Weisgraber, J. Lipid Res. 25, 1277 (1984).
Biological Trace Element Research
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Apo B and Apo E in Copper Deficiency
113
9. S. Reiser, R. J. Ferretti, M. Fields, and J. C. Smith, Jr. Am. J. Clin. Nutr. 38, 214 (1983). 10. P. A. Mayes and M. E. Laker, Prog. Biochem. Pharmacol. 21, 33 (1986). 11. Y Rayssiguier, E. Gueux, and D. Weiser, J. Nutr. 111, 1876 (1981). 12. A. Mazur, C. R6m6sy, E. Gueux, M. A. Levrat, and C. Demign6, J. Nutr. 120, 1037 (1990). 13. G. R. Bartlett, ]. Biol. Chem. 234, 466 (1959). 14. C. B. Laurell, Anal. Biochem. 15, 45 (1966). 15. P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987). 16. M. Mangeney, P. Cardot, S. Lyonnet, C. Coupe, R. Benarous, A. Munnich, J. Girard, J. Chambaz, and G. B6r6ziat, Eur. J. Biochem. 181, 225 (1989). 17. A. Ribeiro, M. Mangeney, P. Cardot, C. Loriette, Y. Rayssiguier, J. Chambaz, and G. B6r6ziat, Eur. J. Biochem. 196, 499 (1991). 18. S. C. Croswell and K. Y. Lei, J. Nutr. 115, 473 (1985). 19. D. B. Ray, C. C. Lee, and S. I. Koo, FASEB J. 3, A1062 (1990). 20. P. W. Harvey and K. G. D. Allen, Nutr. Res. 5, 511 (1985). 21. A. A. AI-Othman, F. Rosenstein, and K. Y. Lei, FASEB J. 4, A393 (1990). 22. M. Fields and Ch. G. Lewis in Metal Ions in Biology and Medicine, Ph. Collery, L. A. Poirier, M. Monfait, and J. C. Etienne, eds., John Libbey Eurotext, Paris, 1990, pp. 80-83. 23. A. Ribeiro, M. Mangeney, P. Cardot, C. Loriette, J. Chambaz, Y. Rayssiguier, and G. Ber6ziat. Diab~te & Mdtabolisme (in press).
Biological Trace Element Research
Vol. 34, 1992
The Effect of Dietary Copper on Rat Plasma Apolipoprotein B, E Plasma Levels, and Apolipoprotein Gene Expression in Liver and Intestine A . M A Z U R , *,1
F. NASSIR, 1, E. GUEUX, 1 P. CARDOT, 1
J. BELLANGER, 2 M. LAMAND,2 AND Y. RAYSS1GUIER2 1Laboratoire des/Vlaladies/v16taboliques; and 2Unit6 des Maladies Nutritionnelles, INRA Theix, 63122 St Gen~s Champanelle, France Received June 8, 1991; Accepted July 29, 1991
ABSTRACT The plasma levels of apo B and apo E, and the level of hepatic and intestinal mRNA coding for these apolipoproteins were investigated in weanling male rats pair-fed for 6 wk with a control or copperdeficient diet. Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats. Copper deficiency significantly increased triglyceride levels and decreased cholesterol levels in the liver. No major differences in the levels of hepatic and intestinal apo B and apo E mRNA occurred between control and copper-deficient rats. These data imply that hypertriglyceridemia dn hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in the gene expression at the mRNA level in the liver and intestine of the apolipoproteins studied. Index Entries: Copper deficiency; hyperlipidemia; apolipoprotein B; apolipoprotein E; mRNA. *Author to whom all correspondence and reprint orders should be addressed.
Biological TraceElement Research
] 07
Vol. 34, 1992
108
Mazur et al.
INTRODUCTION Copper deficiency has been demonstrated to induce hypertriglyceridemia and hypercholesterolemia in rats, and cause changes in lipoprotein concentration and composition; however, the mechanisms responsible for these observations are not fully understood (1,2). Available evidence suggests that hypercholesterolemia in copper-deficient rats may be the result of an increased rate of hepatic release of cholesterol into the plasma (3) and that an impaired receptor-mediated binding of lipoproteins does not contribute to the hypercholesterolemia (4). Copper deficiency increases hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity (5), and decreases lipoprotein lipase, hepatic lipase (6), and lecithin:cholesterol acyltransferase (LCAT) activities (7), which could contribute to an elevated plasma cholesterol and triglyceride plasma levels. However, the possible contribution of apolipoproteins to the observed hyperlipemia in copper-deficient rats remains unclear. Regulation of apo B and apo E metabolism constitutes a major mechanism by which dietary agents may alter plasma lipids. Apo B is an apolipoprotein synthesized in enterocytes and hepatocytes as an obligate component of triglyceride-rich lipoproteins, and serves as recognition marker for the uptake of low-density lipoprotein (LDL) by the LDL (B, E receptor) (8). Apo E is a constituent of various plasma lipoproteins, and is synthesized in most tissues with the liver being the major site of production. Apo E plays an important role in the clearance of triglyceride-rich lipoproteins and in the redistribution of the cholesterol among tissues by its participation in the reverse transport of cholesterol (8). It is well known that the type of dietary carbohydrates plays a role in the expression of copper deficiency (9). The use of sucrose or fructorse as a carbohydrate source in the diet increases the severity of the signs of copper deficiency as compared to those exhibited when either starch or glucose was used as a dietary carbohydrate (9). On the other hand, diets rich in fructose are known to stimulate triglyceride synthesis and secretion from the liver (10). Investigations of apolipoprotein mRNA levels can provide information on factors controlling apolipoprotein and lipoprotein metabolism. Thus, in the present investigations, the rats were fed a diet containing sucrose with or without the addition of copper. We have focused our attention on the effects of copper deficiency on the plasma levels of apo B and apo E, and their mRNAs in the liver and in the intestine.
/V TERIALS AND METHODS Weanling male Wistar rats (IFFA-Credo, L'Arbresle, France) weighing 60 + 2 g (mean + SEM) were divided at random into copper deficient and control group (12 rats/group). The Institute's guide for the care and Biological Trace Element Research
Vol. 34, 1992
Apo B and Apo E in Copper Deficiency
109
use of laboratory animals was used. Rats were housed in wire-bottomed cages in a temperature-controlled room (22~ with a dark period from 20.00 to 08.00 h and pair-fed the appropriate diets for 6 wk. The semipurified diets contained (g/kg) casein 200, sucrose 705, corn oil 50, mineral mixture 35, and vitamin mixture 10, as described previously (11). Distilled water was provided ad libitum. The copper contents of diet were 0.6 mg/kg (deficient) and 7.3 mg/kg (control). Nonfasted animals were sacrificed after being anesthetized with sodium pentobarbital (40 mg/kg body wt) ip. Blood was collected into heparinized tubes, and plasma was obtained by low-speed centrifugation (2000g). The liver was excised, and portions from the right lobe were immediately plunged into liquid nitrogen, and then stored frozen at - 70~ until the lipid and RNA extractions were performed. Triglycerides, cholesterol, and phospholipids were determined in plasma by enzymatic procedures as described previously (12). Liver samples were homogenized, lipids were extracted with chloroform/ methanol (2/1, v/v) and triglyceride, and cholesterol and phospholipid contents were measured in the lipid residue (12,13). Copper in plasma and diets was determined with a Perkin Elmer 400 atomic absorption spectrophotometer. Plasma apo B and apo E levels were estimated by rocket immunoelectrophoresis (14) using antirat apo B and apo E rabbit immunosera. Serial dilutions of rat LDL or of pooled rat serum were used as standards. Hemoglobin was estimated by cyanmethemoglobin method (Test-Combination Hemoglobin, Boehringer, Mannheim, FRG). Total cellular RNA was isolated from liver tissue using the guanidium/phenol/chloroform method according to Chomczynski and Sacchi (15). RNA was quantitated by measuring the absorbance at 260 nm. Its integrity was systematically assessed by agarose-gel electrophoresis and visualization of 18 S and 28 S ribosomal RNAs by ethidium bromide staining. Aliquots of total RNA were subjected to quantification of mRNA content by dot-blot analysis on nylon filters. Hybridization of immobilized RNA to cDNA probes for apo E, apo B, and ~-actin labeled with [o~-32P]dATP and washing conditions have been previously described (16,17). The filters were blotted dry, and autoradiography was performed with intensifying screens at -70~ Quantification of the relative amounts of specific mRNA was performed by densitometric analysis of the hybridization signal using a laser densitometer (Ultrascan XL, LKB, Sweden). Relative abundance of the mRNA apolipoprotein was calculated with respect to the mRNA ~-actin content. Student's t-test was used to assess differences between copper-deficient and control groups.
RESULTS Copper-deficient rats had lower body weights and higher relative liver and heart weights than control rats (Table 1), but mortality was absent. Reduced plasma copper concentrations and blood hemoglobin Biological Trace Element Research
VoL 34, 1992
110
Mazur et ai.
Table 1 Body and Organ Weights, and Biochemical Data of Control and Copper-Deficient Rats I Control Body wt (g) Relative liver wt (g/100 g) Relative heart wt (g/100 g) Plasma copper (~mol/L) Blood hemoglobin (g/100 mL) Plasma triglycerides (raM) Plasma total cholesterol (raM) Plasma phospholipids (mM) Plasma apo B (AU) 2 Plasma apo E (AU) 2 Liver triglycerides (mg/g wet wt) Liver total cholesterol (mg/g wet wt) Liver phospholipids (mg/g wet wt)
253 3.70 0.32 17.81 12.2 0.50 1.85 1.37 100 100 10.5 4.4 35.8
_+ 5 + 0.08 _+ 0.01 _+ 0.52 + 0.2 _+ 0.10 + 0.08 + 0.05 + 6 ___ 3 + 0.5 _+ 0.2 _+ 0.7
Copper deficient 207 6.43 0.71 0.67 7.0 0.96 2.40 2.01 210 250 15.1 3.5 32.9
+ 9*** + 0.20*** _+ 0.05*** _+ 0.07*** + 0.4*** ___ 0.20* ___ 0.13"* + 0.09*** + 8*** + 8*** + 1.4" _+ 0.1"* + 1.2
1Mean + SEM; *P K .05; **P ~ .01; ***P ~ .001 as compared with the respective control value. 2Results of plasma apo B and apo E are expressed as arbitrary units (AU) taking the mean value of the control rats as 100.
were found (Table 1). Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats (Table 1). Copper deficiency significantly increased the triglyceride level and decreased the cholesterol level in the liver (Table 1). The hepatic apo B and apo E m R N A were slightly decreased in copperdeficient rats compared to control rats, but the difference was not statistically significant (Table 2). Dietary copper did not affect jejunal and ileal apo B mRNA, even if slight increases in m R N A levels were observed in deficient rats (Table 2).
DISCUSSION Several m e a s u r e m e n t s are indicators of dietary copper deficiency in addition to the reduced plasma copper concentration, such as decreased body weights, increased heart and liver to body weight ratios, and lower blood hemoglobin levels (9). Similar to previous findings (9), we observed both hypercholesterolemia and hypertriglyceridemia. All these traits indicate that the animals on the copper-deficient diet were indeed copper deficient. The increase in plasma triglycerides suggests that corresponding increase in plasma triglyceride-rich lipoproteins occurs. Conversely, the increase in plasma cholesterol suggests an increase in highdensity lipoprotein (HDL) cholesterol (2), since plasma cholesterol in the rat is mainly transported by HDL. The increase in plasma apo B observed Biological Trace Element Research
Vol. 34, 1992
Apo B and Apo E in Copper Deficiency
111
Table 2 Apolipoprotein B and E mRNA Levels in Control and Copper-Deficient Rats ~ Control
Copper deficient
Liver Apo B mRNA Apo E mRNA
100 + 14 100 + 11
77 +__ 15 78 +_ 12
Intestine Apo B rnRNA:jejunum Apo B mRNA:ileum
100 _+ 25 109 +__8
155 +_ 14 128 _+ 17
1Results are mean +_ SEM of six samples. Results are expressed as apo B/actin or apo E/actin mRNA ratios, and are normalized taking the mean value of the control liver or of the control j e j u n u m as 100.
in the present study is consistent with the elevation of triglyceride-rich lipoproteins in copper-deficient rats (2). It has been shown that the hypercholesterolemia observed in copper-deficient rats is mainly the result of a selective increase in the concentration of HDL1 subfraction (18). The increase in plasma apo E is also consistent with the selective increase in the concentration of HDL1, which is characterized by an elevated apo E content compared to HDL2 particles. Our results indicate that apo B and apo E mRNA levels in the liver of copper-deficient rats were slightly decreased (about 22%) as compared to control rats, but these differences were not significant. A recent study indicates that the apo A-I mRNA was also decreased by 25% in the liver of copper-deficient rats (19). Whether these decreases reflect a general reduction in all liver mRNA is unknown. The liver plays a central role in lipoprotein metabolism. Triglycerides synthesized in the liver, mainly from dietary fatty acids and carbohydrates, are transported to peripheral tissues as the very low density lipoproteins (VLDL). Apo B and apo E are the major apolipoproteins of VLDL, and are important for regulating synthesis, secretion, and catabolism of these particles (8). Copper deficiency is associated with a significant increase in plasma triglycerides and apo B, as well as with a significant increase in hepatic triglyceride concentration. Previous studies have consistently shown a decreased hepatic cholesterol content in copper-deficient rats (9). Similarly, our studies found significantly reduced levels of total hepatic cholesterol. However, previous investigations did not observe the increased levels of hepatic triglycerides in copper-deficient rats that had been fasted (20). Higher triglyceride to protein ratios in VLDL from copper-deficient rats than from control rats have been also observed (21). The results concerning apo B gene expression in the liver is of interest, since secretion of VLDL is dependent upon apo B (8). Apo E also has a control role in cholesterol metabolism (8); however, hypercholesterolemia and decreased hepatic Biological Trace Element Research
Vol. 34, 1992
1 12
/Vlazur et ai.
content of cholesterol were not associated with changes in apo E mRNA in the liver. Little is known about the influence of copper on intestinal production of lipoproteins. A recent study showed that copper deficiency impairs the intestinal transport of cholesterol (6), which involves transport into the lymphatic system via chylomicrons, and it is also well known that apo B is essential for the transport of triglycerides out of the intestine (8). However, our results indicate that apo B mRNA levels in the intestine were not significantly modified by copper deficiency. Finally, we have shown that the increase in plasma triglyceride and cholesterol levels observed in copper deficiency is associated with a marked increase in plasma apo B and apo E levels. However, our data do not show major differences in apo B and apo E mRNA levels between control and copper-deficient rats. These data imply that hypertriglyceridemia and hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in mRNA levels in the tissues measured. Thus, plasma apo B and apo E levels may not be determined by factors that regulate the level of apo mRNA. It has been established that the type of dietary carbohydrate used lays a major role in the expression and severity of copper deficiency, and it has been suggested that certain metabolic pathways of fructose may be responsible for the severity of copper deficiency (22). Recent results support a stimulatory effect of dietary sucrose on apo B and apo E gene expression in rat liver (23). Thus, it would be of interest to evaluate the effect of changing the type of dietary carbohydrate on the effect of copper deficiency on apo B and apo E gene expression in rat liver.
ACKNOWLEDGMENT This work was supported by a grant from Fondation Fran~aise pour la Nutrition. We thank D. Bayle, A. Bellanger, and C. Lab for their excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5.
L. M. Klevay, Am. J. Clin. Nutr. 26, 1060 (1973). K. Y. Lei, J. Nutr. 113, 2178 (1983). M. S. J. Shao and K. Y. Lei, J. Nutr. 110, 859 (1980). C. A. Hassel, T. P. Carr, and K. Y. Lei, Nutr. Res. 10, 903 (1990). N. Y. Yount, D. J. McNamara, A. A1-Othman, and K. Y. Lei, J. Nutr. Biochem,. 1, 21 (1990). 6. S. I. Koo, C. C. Lee, and J. E. Norvell, Proc. Soc. Exp. Biol. Med. 188, 410 (1988). 7. B. W. C. Lau and L. M. Klevay, ]. Nutr. 111, 1698 (1981): 8. R. W. Mahley, T. L. Innerarity, S. C. Rail, and K. H. Weisgraber, J. Lipid Res. 25, 1277 (1984).
Biological Trace Element Research
Vol. 34, 1992
Apo B and Apo E in Copper Deficiency
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Biological Trace Element Research
Vol. 34, 1992
