Comp. Biochem. Physiol.Vol.99A, No. 1/2, pp. 223-228, 1991 Printed in Great Britain
0300-9629/91 $3.00+ 0.00 © 1991PergamonPress plc
THE ASSOCIATION OF HEPATIC APOPROTEIN A N D LIPID METABOLISM IN HAMSTERS A N D RATS G. L. Liu, L. M. FAN and R. N. I~DINGER Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. Telephone (502) 588-6991 (Received 10 August 1990) Almtract--1. The hamster liver but not that of the rat, secretes VLDL containing only apoprotein B~00. Apoprotein B,~ was identified in mesenteric lymph of hamsters and therefore plasma apoprotein B4s is of intestinal origin. 2. Male hamster livers secrete less free cholesterol but similar cholesterol ester than male rats resulting in a higher CE/FC ratio in hamsters. 3. Hepatic VLDL from male hamsters contain more apo B and E while that from femalescontains more TG and apo A-II/C. 4. Hamsters fed high-C diets secrete more hepatic VLDL-apoprotein B, -free and -cholesterol ester, and biliary cholesterol.
INTRODUCTION
The liver of mammals is unique in that it has the capacity to both assemble and degrade lipoproteins. Since lipoproteins transport cholesterol to and from the liver, their metabolism by this organ is critical for cholesterol homeostasis. Further, this organ also secretes cholesterol and its metabolic end-product bile acids into bile. The role of apoproteins in the regulation of hepatic lipid metabolism is also important, since apoprotein B is recognized by the low density lipoprotein receptor of the liver and levels of LDL apoprotein B are better predictors of atherosclerosis than even LDL-cholesterol. A better understanding of both lipid and apoprotein metabolism by the liver of lower animals is therefore important and may allow better strategies for treatment of human atherosclerosis and cholesterol gallstones. Since it is difficult to assess many aspects of this metabolism in man, a greater reliance has been placed on other mammalian species for such studies. However, hepatic disposal of cholesterol differs substantially within mammalian species. Cholesterol metabolism in the hamster differs from that of the rat and more closely resembles that of man (Spady and Dietschy, 1983). The hamster, like man, for example, carries more cholesterol in low density lipoprotein and demonstrates much lower rates of whole body and hepatic cholesterol synthesis than rat (Turley et al., 1983; Spady et al., 1986). While more is known about cholesterol metabolism in the hamster, little information is available regarding the origin and composition of apoproteins in the hamster. Conversely, considerable knowledge has been garnered in this respect for the rat (Chapman, 1986; Wu and Windmueller, 1979). We therefore compared both lipid and apoprotein origin and composition in the hamster and rat. We utilized the in situ liver perfusion system to investigate selective components of hepatic lipoprotein production, as well as biliary lipid secretion in both sexes
of the hamster during control and high cholesterol feeding. We also utilized the mesenteric lymph fistula to assess intestinal lipoprotein origin and composition in both hamster and rat. METHODS AND MATERIALS
Animal and diets Adult Golden Syrian hamsters (Mesocricetus auratus) and Sprague--Dawley rats (Rattus rattus) were purchased from Charles River, OH. They were cared for according to the Guides for the Care and Use of Laboratory Animals of the National Research Council, U.S. Department of Health, Education and Welfare (Guide for the Care and Use of Laboratory Animals, DHEW Publications No. [NIH] 85-23, Revised 1985) and the University of Louisville Institutional Animal Care and Use Committee. The animals were kept in individual cages and observed over 12 hr light/dark cycles. Purina rodent chow and water were given to animals ad libitum. For the cholesterol feeding group 2% (w/w) cholesterol rodent diet was obtained from the same commercial source. The body weights of the animals used in the study were 113.88+4.9 for hamsters and 224.3 +9.7 for rats. Liver perfusion Liver perfusion was performed in situ in a recirculation system for 2--4hr, essentially using the technique described by Miller (Miller, 1973) for rat liver. The animals were anesthetized with pentobarbital, administered intraperitoneally at 50 mg/kg body weight. A mid line abdominal incision was made and then the cystic bile duct was tied off. The common bile duct was cannulated with PE 10 tubing and a 21 gauge angiocatheter was put into the portal vein. The liver was immediately flushed with oxygenated Krebs-Ringer bicarbonate. The abdominal inferior vena cava was cut and another angiocatheter inserted into the thoracic inferior vena cava through the right atrium after thoracotomy. The abdominal vena cava was then ligated above the right kidney and animal with liver placed into a thermostated plexiglass cabinet with temperature kept as 38°C. The perfusion medium consisted of Krebs-Ringer bicarbonate containing 100 mg/dl glucose, 3 g bovine serum albumin and expired (Red Cross Blood) human red cells at
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Fig. 1. Species comparison. Plasma and hepatic VLDL apoprotein in hamster and rat in 4-30% SDA-PAGE. Lane 1, molecular weight marker; Lane 2, hamster hepatic VLDL; Lane 3, rat hepatic VLDL; Lane 4, hamster plasma VLDL; Lane 5, rat plasma VLDL. 22% hematocrit. The perfusate was gassed with oxygencarbon dioxide (95:5) using a silastic tubing lung described by Hamilton (Hamilton et al., 1974). The pH of the perfusate was maintained at 7.35-7.45 and the flow rate was kept at 1 ml/min/g liver. The liver's viability and performance was monitored to ensure maintenance of normal colour and consistent steady bile production and oxygen consumption. Instability of these parameters resulted in termination of the experiment. At the conclusion of the experiment, the liver was weighed and the perfusate collected for separation of lipoprotein. Mesenteric lymph collection The technique used for collecting mesenteric lymph was the same as described by Warshaw (Warshaw, 1972). Animals were anesthetized with pentobarbital as described for liver preparation. After exposure of the mesenteric lymph duct, it was nicked and PE 10 tubing filled with normal saline inserted and fixed with a drop of tissue glue. Lymph was then collected in a test tube containing EDTA and sodium azide at 1 and 0.5 mg, respectively, as final concentrations. The collection time was no longer than 4 hr. Lipoprotein isolation and characterization The plasma, perfusate and mesentric lymph were similarly processed to isolate the d < 1.006 fraction (Tso et al., 1983). All samples were spun by ultracentrifugation for 18hr at 40,000rpm at 10°C in a T70.1 rotor (Beckman Instruments, Inc., CA). The d < 1.006 lipoprotein at the top was aspirated and washed once under the same conditions. The lipoproteins were delipidated with methanolether (Osborne, 1986) and resolubized in a sample buffer containing SDS and mercaptoethanol. Samples were then heated at 90°C for 5 min and applied to a discontinuous
SDS polyacrylamide slab gel (Laemmli, 1970). The separating gel was a linear gradient from 4-30% polyacrylamide. Electrophoresis was performed using a Bio-Rad mini vertical slab gel apparatus. Gels were stained with 0.05% Coomassie brilliant blue and scanned with a Beckman gel scanner (model CDC 200). The apolipoprotein bands were identified by comparison to Pharmacia molecular weight standards (phosphorylase b 94,000, bovine serum albumin 67,000, ovalbumin 43,000, carbonic anhydrase 30,000 and a-lactalbumin 14,000). Analytical techniques Lipids were extracted by the method of Bligh (Bligh and Dyer, 1957) and total cholesterol and free cholesterol were determined enzymatically according to Sale et al. (1984). Triglycerides were measured as described by Mendez et al. (1975) and phospholipids by the method of Bartlett (1959). Lipoprotein-protein was assayed by the Lowry technique (Lowry et al., 1951). Bile acid was determined using the enzymatic method described by Talalay (Talalay, 1960). Statistical analysis All data were expressed as mean __+SE and statistical significant difference determined using the Student's t-test for comparisons between species, sexes and diets. RESULTS (A) Species comparison Figure 1 shows the a p o p r o t e i n profile in b o t h p l a s m a a n d hepatic V L D L o f h a m s t e r a n d rat as separated by 4 - 3 0 % gradient S D A - P A G E . H a m s t e r s secreted only a p o p r o t e i n B100 from the liver, while rats secreted b o t h a p o p r o t e i n B100 a n d a p o p r o t e i n
Lipoprotein metabolism in hamsters and rats Table 1. Apolipoproteindistributionin hamsters Percentage B100 B48 A-IV E Hepatic 19.2t:~ --35.3~" N=9 +3.1 --_ 1.7 Intestinal 1.6 9.2 21.9 14.2 N=8 + 0.8 +__0.9 + 6.8 __+1.1 *P < 0.05, ~P < 0.01, ~Mean_ SE. B48. Since plasma of hamsters also contained apoprotein B48, such apoprotein must have originated from other source(s). This source was established as shown in Table 1 by comparing hepatic and intestinal apoprotein composition. The intestine was shown to be the source of apo B48 because hepatic VLDL apoproteins again carried no B48 or A-IV apoproteins, while lipoproteins originating from the d < 1.006 fraction of intestinal lymph contained both apoprotein B48 and A-IV. In hamsters, there was a higher percentage of apoprotein A-IV and A-I and a lower percentage of apoprotein E in d < 1.006 fraction of intestinal lymph compared to hepatic VLDL apoproteins. In fact, intestinal lymph composition resembled that of human chylomicrons from the thoracic duct (Green et al., 1979). The accumulation rates of total triglycerides, free and esterified cholesterol and the cholesterol ester to free cholesterol ratio in perfusate of hamster and rat livers, are shown in Fig. 2. The hamster liver secreted less free cholesterol than did the rat liver. Since cholesterol esterification was the same in both species, the cholesterol ester to free cholesterol ratio was much higher in the hamster. The triglyceride secretion of all lipoproteins secreted by the liver into the perfusate was similar in the two species. (B) Sex comparison Figure 3 compares the apoprotein distribution within hepatic VLDL in male and female hamsters. Table 2 shows that in the female, VLDL contained less apoprotein B100 than the male as determined by densitometry measurements, while apoprotein A-II and C were higher in the female. Table 3 shows the lipid composition of hepatic VLDL in male and female hamsters. No significant differences were evi-
~g/gj
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FC p
0300-9629/91 $3.00+ 0.00 © 1991PergamonPress plc
THE ASSOCIATION OF HEPATIC APOPROTEIN A N D LIPID METABOLISM IN HAMSTERS A N D RATS G. L. Liu, L. M. FAN and R. N. I~DINGER Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. Telephone (502) 588-6991 (Received 10 August 1990) Almtract--1. The hamster liver but not that of the rat, secretes VLDL containing only apoprotein B~00. Apoprotein B,~ was identified in mesenteric lymph of hamsters and therefore plasma apoprotein B4s is of intestinal origin. 2. Male hamster livers secrete less free cholesterol but similar cholesterol ester than male rats resulting in a higher CE/FC ratio in hamsters. 3. Hepatic VLDL from male hamsters contain more apo B and E while that from femalescontains more TG and apo A-II/C. 4. Hamsters fed high-C diets secrete more hepatic VLDL-apoprotein B, -free and -cholesterol ester, and biliary cholesterol.
INTRODUCTION
The liver of mammals is unique in that it has the capacity to both assemble and degrade lipoproteins. Since lipoproteins transport cholesterol to and from the liver, their metabolism by this organ is critical for cholesterol homeostasis. Further, this organ also secretes cholesterol and its metabolic end-product bile acids into bile. The role of apoproteins in the regulation of hepatic lipid metabolism is also important, since apoprotein B is recognized by the low density lipoprotein receptor of the liver and levels of LDL apoprotein B are better predictors of atherosclerosis than even LDL-cholesterol. A better understanding of both lipid and apoprotein metabolism by the liver of lower animals is therefore important and may allow better strategies for treatment of human atherosclerosis and cholesterol gallstones. Since it is difficult to assess many aspects of this metabolism in man, a greater reliance has been placed on other mammalian species for such studies. However, hepatic disposal of cholesterol differs substantially within mammalian species. Cholesterol metabolism in the hamster differs from that of the rat and more closely resembles that of man (Spady and Dietschy, 1983). The hamster, like man, for example, carries more cholesterol in low density lipoprotein and demonstrates much lower rates of whole body and hepatic cholesterol synthesis than rat (Turley et al., 1983; Spady et al., 1986). While more is known about cholesterol metabolism in the hamster, little information is available regarding the origin and composition of apoproteins in the hamster. Conversely, considerable knowledge has been garnered in this respect for the rat (Chapman, 1986; Wu and Windmueller, 1979). We therefore compared both lipid and apoprotein origin and composition in the hamster and rat. We utilized the in situ liver perfusion system to investigate selective components of hepatic lipoprotein production, as well as biliary lipid secretion in both sexes
of the hamster during control and high cholesterol feeding. We also utilized the mesenteric lymph fistula to assess intestinal lipoprotein origin and composition in both hamster and rat. METHODS AND MATERIALS
Animal and diets Adult Golden Syrian hamsters (Mesocricetus auratus) and Sprague--Dawley rats (Rattus rattus) were purchased from Charles River, OH. They were cared for according to the Guides for the Care and Use of Laboratory Animals of the National Research Council, U.S. Department of Health, Education and Welfare (Guide for the Care and Use of Laboratory Animals, DHEW Publications No. [NIH] 85-23, Revised 1985) and the University of Louisville Institutional Animal Care and Use Committee. The animals were kept in individual cages and observed over 12 hr light/dark cycles. Purina rodent chow and water were given to animals ad libitum. For the cholesterol feeding group 2% (w/w) cholesterol rodent diet was obtained from the same commercial source. The body weights of the animals used in the study were 113.88+4.9 for hamsters and 224.3 +9.7 for rats. Liver perfusion Liver perfusion was performed in situ in a recirculation system for 2--4hr, essentially using the technique described by Miller (Miller, 1973) for rat liver. The animals were anesthetized with pentobarbital, administered intraperitoneally at 50 mg/kg body weight. A mid line abdominal incision was made and then the cystic bile duct was tied off. The common bile duct was cannulated with PE 10 tubing and a 21 gauge angiocatheter was put into the portal vein. The liver was immediately flushed with oxygenated Krebs-Ringer bicarbonate. The abdominal inferior vena cava was cut and another angiocatheter inserted into the thoracic inferior vena cava through the right atrium after thoracotomy. The abdominal vena cava was then ligated above the right kidney and animal with liver placed into a thermostated plexiglass cabinet with temperature kept as 38°C. The perfusion medium consisted of Krebs-Ringer bicarbonate containing 100 mg/dl glucose, 3 g bovine serum albumin and expired (Red Cross Blood) human red cells at
223
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Fig. 1. Species comparison. Plasma and hepatic VLDL apoprotein in hamster and rat in 4-30% SDA-PAGE. Lane 1, molecular weight marker; Lane 2, hamster hepatic VLDL; Lane 3, rat hepatic VLDL; Lane 4, hamster plasma VLDL; Lane 5, rat plasma VLDL. 22% hematocrit. The perfusate was gassed with oxygencarbon dioxide (95:5) using a silastic tubing lung described by Hamilton (Hamilton et al., 1974). The pH of the perfusate was maintained at 7.35-7.45 and the flow rate was kept at 1 ml/min/g liver. The liver's viability and performance was monitored to ensure maintenance of normal colour and consistent steady bile production and oxygen consumption. Instability of these parameters resulted in termination of the experiment. At the conclusion of the experiment, the liver was weighed and the perfusate collected for separation of lipoprotein. Mesenteric lymph collection The technique used for collecting mesenteric lymph was the same as described by Warshaw (Warshaw, 1972). Animals were anesthetized with pentobarbital as described for liver preparation. After exposure of the mesenteric lymph duct, it was nicked and PE 10 tubing filled with normal saline inserted and fixed with a drop of tissue glue. Lymph was then collected in a test tube containing EDTA and sodium azide at 1 and 0.5 mg, respectively, as final concentrations. The collection time was no longer than 4 hr. Lipoprotein isolation and characterization The plasma, perfusate and mesentric lymph were similarly processed to isolate the d < 1.006 fraction (Tso et al., 1983). All samples were spun by ultracentrifugation for 18hr at 40,000rpm at 10°C in a T70.1 rotor (Beckman Instruments, Inc., CA). The d < 1.006 lipoprotein at the top was aspirated and washed once under the same conditions. The lipoproteins were delipidated with methanolether (Osborne, 1986) and resolubized in a sample buffer containing SDS and mercaptoethanol. Samples were then heated at 90°C for 5 min and applied to a discontinuous
SDS polyacrylamide slab gel (Laemmli, 1970). The separating gel was a linear gradient from 4-30% polyacrylamide. Electrophoresis was performed using a Bio-Rad mini vertical slab gel apparatus. Gels were stained with 0.05% Coomassie brilliant blue and scanned with a Beckman gel scanner (model CDC 200). The apolipoprotein bands were identified by comparison to Pharmacia molecular weight standards (phosphorylase b 94,000, bovine serum albumin 67,000, ovalbumin 43,000, carbonic anhydrase 30,000 and a-lactalbumin 14,000). Analytical techniques Lipids were extracted by the method of Bligh (Bligh and Dyer, 1957) and total cholesterol and free cholesterol were determined enzymatically according to Sale et al. (1984). Triglycerides were measured as described by Mendez et al. (1975) and phospholipids by the method of Bartlett (1959). Lipoprotein-protein was assayed by the Lowry technique (Lowry et al., 1951). Bile acid was determined using the enzymatic method described by Talalay (Talalay, 1960). Statistical analysis All data were expressed as mean __+SE and statistical significant difference determined using the Student's t-test for comparisons between species, sexes and diets. RESULTS (A) Species comparison Figure 1 shows the a p o p r o t e i n profile in b o t h p l a s m a a n d hepatic V L D L o f h a m s t e r a n d rat as separated by 4 - 3 0 % gradient S D A - P A G E . H a m s t e r s secreted only a p o p r o t e i n B100 from the liver, while rats secreted b o t h a p o p r o t e i n B100 a n d a p o p r o t e i n
Lipoprotein metabolism in hamsters and rats Table 1. Apolipoproteindistributionin hamsters Percentage B100 B48 A-IV E Hepatic 19.2t:~ --35.3~" N=9 +3.1 --_ 1.7 Intestinal 1.6 9.2 21.9 14.2 N=8 + 0.8 +__0.9 + 6.8 __+1.1 *P < 0.05, ~P < 0.01, ~Mean_ SE. B48. Since plasma of hamsters also contained apoprotein B48, such apoprotein must have originated from other source(s). This source was established as shown in Table 1 by comparing hepatic and intestinal apoprotein composition. The intestine was shown to be the source of apo B48 because hepatic VLDL apoproteins again carried no B48 or A-IV apoproteins, while lipoproteins originating from the d < 1.006 fraction of intestinal lymph contained both apoprotein B48 and A-IV. In hamsters, there was a higher percentage of apoprotein A-IV and A-I and a lower percentage of apoprotein E in d < 1.006 fraction of intestinal lymph compared to hepatic VLDL apoproteins. In fact, intestinal lymph composition resembled that of human chylomicrons from the thoracic duct (Green et al., 1979). The accumulation rates of total triglycerides, free and esterified cholesterol and the cholesterol ester to free cholesterol ratio in perfusate of hamster and rat livers, are shown in Fig. 2. The hamster liver secreted less free cholesterol than did the rat liver. Since cholesterol esterification was the same in both species, the cholesterol ester to free cholesterol ratio was much higher in the hamster. The triglyceride secretion of all lipoproteins secreted by the liver into the perfusate was similar in the two species. (B) Sex comparison Figure 3 compares the apoprotein distribution within hepatic VLDL in male and female hamsters. Table 2 shows that in the female, VLDL contained less apoprotein B100 than the male as determined by densitometry measurements, while apoprotein A-II and C were higher in the female. Table 3 shows the lipid composition of hepatic VLDL in male and female hamsters. No significant differences were evi-
~g/gj
::oF
FC p
