Animal models are widely used in the initial screening of antihyperlipidemic agents and atherosclerosis research. Whole-animal studies are often combined with various cellular assays to screen the effectiveness of these drugs. However, several of the most commonly used animal models have significant limitations. Typically, hypercholesterolemia is induced in several species by ingestion of a high-fat and cholesterol (CHO) diet (1). This necessitates a lag-time before evaluation of the new antihyperlipidemic agent (1); thus studies may become very expensive and time consuming. Genetically hyperlipidemic animals, such as the Watanabe heritable hyperlipidemic rabbit, also are used in research. This animal model has a deficiency in low-density-lipoprotein receptors, a condition that simulates familial hyperlipidemia (2). However, this model has limited applicability because familial hyperlipidemia represents only a small percentage of patients with lipid abnormalities (3).
A new rapid, convenient, and low-cost animal model is being developed by using poloxamer 407 (P-407). Poloxamer 407 (Pluronic F-127) is a hydrophilic, nonionic surface active agent with virtually no toxicity (4-6) and an oral 50% lethal dose (LD50) in rabbits and mice ≥15 and 1.8 g/kg, respectively (7). P-407 also exhibits the property of reverse thermal gelation (6). At reduced temperatures, the surfactant exists as a mobile viscous liquid and then forms a semisolid gel on warming. One 300-mg intraperitoneal (i.p.) injection of P-407 causes the surfactant solution to gel in vivo and produce a very protracted (>96 h) hyperlipidemia (8). In fact, the maximal plasma P-407 concentration after i.p. injection of rats is ∼13 h after injection (9). Maximal plasma CHO and triglyceride concentrations are observed at ∼48 h after a single i.p. injection of P-407 and remain above normal levels for ≥96 h after injection (8). Others have used the nonionic surfactant Triton WR-1339 to produce hyperlipidemia in a variety of animal species (10-11). However, Triton WR-1339, unlike P-407, causes hemolysis and so could not be used in a long-term study. Moreover, because P-407 forms a thermally reversible gel after injection, the time course for hyperlipidemia should presumably be extended compared with the viscous liquid Triton WR-1339 (9).
Previously we reported that the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors lovastatin and pravastatin were able to reduce the total plasma CHO concentrations in P-407-induced hyperlipidemic rats (8,12). This suggested that HMG-CoA reductase may play a role in hypercholesterolemia and may be affected directly or indirectly by the surfactant. Thus the purpose of our investigation was to determine the effect of P-407 on HMG-CoA reductase in rats. Our first objective was to determine whether P-407 directly affected the enzymatic activity of HMG-CoA reductase in vitro. An additional aim was to determine the activity of HMG-CoA reductase in microsomal fractions of liver at various times after i.p. injection of P-407. Our last objective was to determine the time course of hepatic CHO after administration of the surfactant to rats.
MATERIALS AND METHODS
Poloxamer 407, N.F. (Pluronic F-127; BASF Corporation, Parsippany, NJ, U.S.A.) solution for intraperitoneal injection was prepared by combining the agent with double deionized water for a final concentration of 30% wt/wt. The solution was refrigerated overnight to facilitate dissolution of the P-407 by the cold method of incorporation (13). Tritiated mevalonolactone (30 Ci/mmol) was purchased from Amersham (Arlington Heights, IL, U.S.A.). The substrate, [14C]3-hydroxy-3-methyl-glutaryl coenzyme A (59.9 mCi/mmol) was purchased from Amersham. Anion exchange resin (AG 1-X8; formate form) was purchased from Bio-Rad (Richmond, CA, U.S.A.). Diagnostic enzyme kits for determination of total plasma CHO were obtained from Sigma (St. Louis, MO, U.S.A.).
Animals used in all experiments were male Sprague-Dawley rats weighing 225-250 g (Harlan Laboratories, Indianapolis, IN, U.S.A.) and were housed in individual cages for 2 weeks before the experiments. Rats were fasted overnight in the experiments that determined whether injection of P-407 resulted in changes in plasma CHO. All other rats were allowed access to rat chow and water ad libitum. Fasted and fed rats used in these studies were all housed under a 12-h reverse light-dark schedule (0600-1800 dark) for 2 weeks before experimentation. The 12-h reverse light-cycling schedule was used so that HMG-CoA reductase activity was maximal at 1000 h (14). At the time of the experiment, the rats weighed 325-350 g. All animals were treated in accordance with the institution's guide for the care and use of laboratory animals, and the treatment protocol was approved by the Animal Care Committee at the University of Illinois at Chicago.
Plasma cholesterol after P-407 administration
This study examined the plasma CHO concentration after i..p. injection of P-407. After the overnight fast, each of six rats was individually injected with 1 ml (≈1 g) of the P-407 solution (30% wt/wt). At 0, 1, 2, 4, 6, 8, 12, and 24 h after the injection, a 1-ml blood sample was obtained from each rat by the tail-clip method, as reported previously (12). The samples were collected in heparinized collection tubes. Samples were centrifuged immediately after collection, and the plasma separated and frozen at −70°C until analysis.
HMG-CoA reductase study
This experiment was conducted to determine the time course of enzymatic activity of hepatic microsomal HMG-CoA reductase after administration of P-407. At the time of the experiment, 1 ml of a 30% wt/wt solution of P-407 was injected i.p. into each of 42 rats at 2, 4, 6, 15, 24, 48, and 72 h before the animals were killed. An additional group of 42 rats served as sham-injected controls and were administered normal saline at the same times before the experiment as treatment rats. Because there is a significant circadian rhythm in HMG-CoA reductase activity (15), all animals were killed at 10:00 a.m. Therefore, preinjection of poloxamer 407 had to be made at different times before death.
At the time of death (10:00 a.m.), each rat was decapitated and killed by exsanguination. The abdominal cavity was opened and the liver excised and immediately placed into 25 ml of a chilled buffer containing potassium phosphate (0.04 M; pH 7.2), KCl (0.05 M), sucrose (0.1 M), ethylenediamine tetraacetic acid (0.03 M), and aprotinin (500 KI units/ml). No kinase or phosphatase inhibitors were included in the homogenization medium. With this buffer, we assumed that the enzyme was in a dephosphorylated state, thus maximizing the enzyme activity. All livers were then homogenized in glass/glass homogenizers, and the homogenates centrifuged at 16,000 g for 15 min at 4°C. The supernatant was removed and recentrifuged under the same conditions a second time. The second 16,000 g supernatant was then spun at 100,000 g for 70 min at 4°C. Pelleted microsomes were then resuspended in 1.5 ml of buffer and homogenized in a glass/glass homogenizer. A protein assay (Micro BCA; Pierce, Rockford, IL, U.S.A.) was performed on the microsomal preparation to monitor protein recovery and to equate the protein content of enzyme assays. Last, dithiothreitol (10 mM) was added and the preparation appropriately aliquoted and frozen at −70°C.
HMG-CoA reductase assay
The reductase activity of the microsomal homogenates was assayed in 0.25 ml by the method of Edwards et al. (16). In summary, the substrate ([14C]HMG-CoA) is added to the microsomal fraction containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and incubated at 37°C for 20 min. The reaction was stopped with KOH, and then 5N HCl was added to lactonize the mevalonic acid. Reaction mixtures were centrifuged, and the supernatants layered onto 2 g of the anion-exchange resin and eluted with 2.0 ml of deionized water. The first 0.5-ml fraction was discarded, and the next 1.5-ml fraction collected and counted for both tritium and carbon 14 in 10 ml of scintillation fluid (Aquasol; NEN, Boston, MA, U.S.A.). Tritiated mevalonolactone (0.05 μCi) was added to each assay mixture to determine the percentage recovery of [14C]mevalonate after elution from the anion-exchange columns. Under the conditions described previously, the enzyme activity increased linearly up to 300 μg of microsomal protein per reaction mixture and was linear with respect to time up to 30 min.
Effect of P-407 on HMG-CoA reductase activity in vitro
This study was conducted to assess the effect of poloxamer 407 on the activity of HMG-CoA reductase in vitro. Rats that had received a 1-ml i.p. injection of normal saline 24 h before death were used for isolation of hepatic microsomes. After isolation of hepatic microsomes as described, the activity of the HMG-CoA reductase was determined in a 0.04 M potassium phosphate buffer (pH 7.2) with and without P-407. The 150-μl aliquots of microsomal homogenate assayed for enzyme activity contained increasing amounts of P-407 (0-5 mM). The amount of microsomal protein assayed at each P-407 concentration was 1.5 mg. All activity measurements were performed in duplicate and were normalized to the activity of the HMG-CoA reductase determined in the phosphate buffer alone.
Hepatic cholesterol concentrations
This study was conducted to determine whether the concentration of CHO in hepatic tissue was changed after an i.p. injection of P-407. Fifty-four rats were injected with normal saline (three rats/time point) or a 30% wt/wt solution of P-407 (six rats/time point) at 2, 4, 8, 15, 24, and 48 h before death. At the time of death, the rats were killed by decapitation, and a representative section of hepatic tissue was obtained and weighed (∼250 mg). The liver specimens were homogenized with a 3:1 ethanol/ether mixture as described by Entenman (17). The samples were then evaporated at 90°C to dryness. Residues were solubilized with 1.5 ml of 2-propanol and a 20-μl aliquot assayed for CHO as described, by using a standard enzymatic CHO determination kit. Previous pilot studies demonstrated linearity of CHO quantitation in 2-propanol and recoveries near 100% by using the CHO assay kit.
All plasma and tissue CHO concentrations were expressed as the mean concentration ± standard error of the mean (SEM). The mean values of the activity of HMG-CoA reductase determined in hepatic microsomal fractions obtained from P-407- and saline-injected rats were tested for a statistical difference at each time point by using Student's t test (18). Students's t test also was used to determine whether the mean value of hepatic CHO at each time point after injection of P-407 was significantly different from the mean value of hepatic CHO determined in control animals. Activities of homogenates that contained varying concentrations of P-407 were expressed as the mean value ± SEM of the percentage of the enzyme's activity when assayed in phosphate buffer only.
Accumulation of cholesterol in plasma after P-407 administration
Cholesterol accumulation in the plasma of fasted rats after injection of P-407 is shown in Fig. 1. Concentrations of plasma CHO were significantly (p < 0.05) elevated as soon as 1 h after injection of the surfactant. The plasma concentration of CHO was 449 ± 57 mg/dl at 24 h after administration, with the fastest rate of accumulation occurring from 1 to 12 h after injection (≈16.6 mg/dl/h). Plasma CHO reached a maximum concentration (663 ± 91 mg/dl) at 48 h after injection and returned to approximately normal values at ∼96 h after injection.
Effect of P-407 on HMG-CoA reductase in vitro
As illustrated in Fig. 2, P-407 did not appear to significantly affect the activity of HMG-CoA reductase in vitro over the P-407 concentration range of 0-5 mM compared with control samples (buffer only; no P-407). Values ranged from ∼92 ± 2.9% (0.5 mM) to 98 ± 14.8% (5 mM) of control activity.
Effect of P-407 on HMG-CoA reductase activity in vivo
Figure 3 illustrates the enzymatic activity of hepatic microsomal HMG-CoA reductase at various times after injection of 1 ml (≈300 mg) of P-407 or normal saline (1 ml) in reverse light-cycled rats. The enzymatic activity of HMG-CoA reductase, as assessed by the formation of mevalonic acid, reached a maximum of 262 ± 42.6 pmol/min/mg at ∼15 h after administration of the surfactant, with a subsequent decline to control activity (94.1 ± 8.7 pmol/min/mg) at ∼40 h after injection. At 48 h after injection of P-407, the activity of HMG-CoA reductase decreased below control values to a mean activity of 9.4 ± 1.2 pmol/min/mg. Sham-injected control rats had enzyme activities that ranged from 70.1 ± 9.02 (2 h) to 97.5 ± 12.2 pmol/min/mg (72 h), with a mean value of 94.1 ± 8.7 pmol/min/mg.
Effect of P-407 on hepatic cholesterol concentrations
As shown in Fig. 4, the CHO concentration in hepatic tissue after injection of P-407 was significantly (p < 0.01) elevated at 2 h (3.26 ± 0.19 mg/g) and 4 h (3.75 ± 0.38 mg/g) and significantly (p < 0.01) reduced at 15 h (1.56 ± 0.19 mg/g) compared with tissue CHO concentrations determined in control animals (2.65 ± 0.18 mg/g). However, the hepatic CHO content appeared to return to control values by ∼24 h (2.61 ± 0.08 mg/g) after injection of P-407. The hepatic CHO concentration in P-407-injected rats was not significantly different at either 24 or 48 h after injection of the surfactant compared with controls.
Our investigation demonstrated that injection of P-407 in rats affects the enzymatic activity of hepatic microsomal HMG-CoA reductase. The effect of the surfactant on the enzymatic activity of HMG-CoA reductase appeared to be mediated by an indirect pathway, because our data demonstrated no significant reduction in the activity of the enzyme when incubated with P-407 in vitro over a wide range of surfactant concentrations. Presumably, increased CHO in the plasma after an i.p. injection of P-407 may occur as a result of an increase in synthesis, a decrease in elimination, or a redistribution from other tissues such as the liver. Because we demonstrated both an increase in HMG-CoA reductase activity and a depletion of hepatic CHO at 15 h after injection, our data suggest that the reduction in hepatic CHO may have contributed to the specific event(s) that released the inhibition of HMG-CoA reductase synthesis. Kuroda et al. (19) and Goldfarb (20) both demonstrated with rats that injection of the nonionic surfactant Triton WR-1339 caused a depletion of hepatic CHO that preceded stimulation of HMG-CoA reductase activity and cholesterolgenesis. However, the time course of the changes was much longer in Kuroda's study (19) than in the study by Goldfarb (20). Kuroda demonstrated that within 3 h after intravenous injection of Triton WR-1339 in rats, serum CHO levels doubled, and liver CHO levels decreased up to 35% (19). At 6-9 h after i.v. injection of Triton WR-1339 in rats, Kuroda et al. demonstrated that hepatic HMG-CoA reductase activity was increased fourfold to fivefold over baseline activity (19).
Our data demonstrated an approximate threefold increase in HMG-CoA reductase activity and a 40% decrease in liver CHO concentrations at 15 h after an injection of P-407. Because liver CHO concentrations were significantly (p < 0.01) increased relative to control tissue at 2 and 4 h after injection of P-407 in rats, it suggests that the initial increase in plasma CHO (1 h after injection) was caused by cholesterolgenesis or decreased removal or both. Presumably at later times (8-24 h after injection), increased plasma CHO was caused by both increased CHO synthesis (Fig. 3) and redistribution of hepatic CHO into the plasma compartment (Fig. 4). Hepatic CHO depletion or redistribution into plasma may first be observable at 8 h (Fig. 4) because of the route of P-407 administration. Kuroda et al. (19) observed hepatic CHO depletion (35%) at 3 h after intravenous administration of Triton WR-1339, whereas we observed a 40% reduction in hepatic CHO 15 h after an intraperitoneal injection of P-407. Additional work in our laboratory demonstrated that maximal P-407 plasma concentrations are obtained at 12-15 h after intraperitoneal injection of P-407 (9). It is possible that the maximal depletion in liver CHO after i.p. injection of P-407 occurs when the plasma concentration of P-407 reaches a maximum.
At 24 h after injection of P-407, liver CHO concentrations returned to normal values, although the activity of HMG-CoA reductase was still increased. This suggests that from 24 to ∼40 h after injection of P-407, de novo CHO synthesis or decreased removal of CHO as bile acids or both were responsible for increased plasma CHO. Our data suggest that P-407 caused hypercholesterolemia in rats by both stimulation of CHO synthesis and redistribution of hepatic CHO into the plasma compartment. Further experimentation is required to determine whether injection of P-407 inhibits the activity of cholesterol 7α-hydroxylase, which would decrease CHO removal by inhibiting bile acid synthesis (21). Previous work in our laboratory indicated that an increase in CHO synthesis is probably the primary mechanism responsible for increased plasma CHO concentrations because oral administration (75 mg/kg) of lovastatin to rats 24 h before, at the time of, and 24 h after an i.p. injection of P-407 completely abolished the hypercholesterolemic response (8). It should also be noted that the time at which the maximal reduction in hepatic CHO occurred coincided with the time point (15 h) observed for the maximal increase in HMG-CoA reductase activity (Figs. 3 and 4). This is not altogether unexpected, because total hepatic CHO content has been shown to be inversely related to the activity of HMG-CoA reductase assayed in hepatic microsomes of rats (21). With feedback control of HMG-CoA reductase activity by hepatic CHO, the increased activity of HMG-CoA reductase from 8 to 24 h (hepatic CHO below normal) was a mixed effect of both indirect stimulation by P-407 and feedback enzyme activation caused by depleted hepatic CHO stores. The apparent inhibition of HMG-CoA reductase activity from ≈40 to 72 h (Fig. 3), when the concentration of hepatic CHO was normal, suggests a mechanism different from hepatic CHO content regulation. The possibility exists that HMG-CoA synthase, which ultimately synthesizes HMG-CoA from acetate, may directly or indirectly be activated similar to HMG-CoA reductase (21). Increased synthesis of HMG-CoA may lead to deactivation of HMG-CoA reductase. Last, it is possible that P-407 also affects the activity of other cholesterolgenic enzymes.
Comparison of the activity-time profile for HMG-CoA reductase (Fig. 3) with the hepatic CHO content-time plot (Fig. 4) reveals a dissociation between hepatic CHO and enzyme activity. Specifically, hepatic microsomal HMG-CoA reductase activity increased during a time when hepatic CHO increased (0-4 h) and decreased during a time when hepatic CHO content was determined to be normal (>24 h). Thus this may suggest that there are additional regulatory mechanisms that control HMG-CoA reductase activity after administration of P-407 other than eventual CHO formation. One such possibility may involve covalent modification of HMG-CoA reductase when plasma CHO was increasing and the activity of HMG-CoA reductase was below normal, although homogenization and assay conditions were such as to minimize these effects. It is assumed that enzyme activity may reflect hepatic HMG-CoA reductase content.
The increase in plasma CHO caused by increased CHO synthesis soon after injection of a nonionic surfactant is supported by the work of others. Although it was shown in vitro by Anderson and Dietschy (22) that CHO synthesis significantly increased in six organs in addition to the liver after 3 days of Triton WR 1339 infusion to rats, the liver was suggested to be the organ primarily responsible for the newly synthesized CHO because of its relative mass and potential for cholesterolgenesis. This conclusion is also supported by the observation that hepatectomy followed by Triton WR 1339 injection did not cause an increase in plasma CHO concentration (23). The chemical similarity of the nonionic surface-active agents Triton WR-1339 and P-407 caused by the polyoxyethylene ether bonds (24,25) may suggest a common mechanism(s) for induction of hypercholesterolemia in rats.
In conclusion, we demonstrated that i.p. injection of P-407 to rats results in time-dependent changes in plasma and hepatic CHO and the activity of HMG-CoA reductase assayed in hepatic microsomes. Although the initial activation in HMG-CoA reductase activity appears to be mediated by an indirect effect of P-407, the model may prove beneficial for dose-response studies aimed at determining the potency of new HMG-CoA reductase-inhibitor drugs. In addition, the P-407 model produces a temporary and reversible hyperlipidemic state with well-characterized alterations in plasma and liver total CHO concentrations and the activity of HMG-CoA reductase. This experimentally induced hypercholesterolemia in rats may provide a convenient rapid, and cost-effective animal model for the study of atherosclerosis after extended treatment with P-407.
Acknowledgment: This study was supported by the Parenteral Drug Association for a research grant awarded to T.P.J. and W.K.P. and a research grant from the American Heart Association, Kansas affiliate, awarded to T.P.J.
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