Effect of the ACAT Inhibitor CL 277,082 on Apolipoprotein B48 Transport in Mesenteric Lymph and on Plasma Clearance of Chylomicrons and Remnants
Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) activity previously have been found to decrease the absorption of cholesterol and to be effective antiatherosclerotic agents. Effects on chylomicron (CM) transport could contribute to these effects. No previous study has examined the effect of inhibition of ACAT activity on the intestinal lymph output of apolipoprotein (apo) B48 or on the clearance from plasma of lymph CM. In this study, we selected 2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-(hepthyl)urea (CL 277,082) to inhibit intestinal ACAT activity and measured its effects on the output of lipids and apo B48 in intestinal lymph. Compared with control untreated rats, treatment with CL 277,082 decreased the lymph outputs of apo B48 and triglyceride. Associated with the effects on transport, the lymph CM were smaller in diameter in rats treated with CL 277,082. The unesterified cholesterol content of lymph CM was markedly increased and the cholesteryl ester (CE) content was decreased. The contents of triglyceride were decreased and phospholipid was increased. Labeled CM were prepared by feeding donor rats with a test meal containing 3H-cholesterol and 14C–fatty acid. Traced by the CE label in lymph CM in both control rats and rats treated with CL 277,082, the remnants derived after intravenous injection of CM from rats treated with CL 277,082 were cleared significantly more slowly than CM from untreated rats. Moreover, less CE label was recovered in the livers of both groups of rats after injection of CM from rats treated with CL 277,082. Recovery in the spleen was significantly higher in recipient rats injected with CM from rats treated with CL 277,082 when compared with injections of CM obtained from untreated rats. We conclude that the metabolism of CM is affected by treatment with CL 277,082, partly due to the changes in lymph CM composition and partly due to other effects on the recipient rat.
- Received October 30, 1995.
- Revision received May 16, 1996.
Chylomicrons are triacylglycerol-rich lipoproteins formed in the intestine during lipid absorption. The intestine is also a site of synthesis of cholesterol. Absorbed exogenous cholesterol and newly synthesized cholesterol are transported in CM from the intestine to plasma. Esterification by intestinal ACAT accounts for the CE in CM. A number of pharmacological agents affect the delivery of cholesterol by means of the intestine. Various drugs are available to prevent the esterification of cholesterol by ACAT, to inhibit the uptake or absorption of cholesterol from the intestinal lumen, or to inhibit new synthesis of cholesterol.
In this study we measured the composition and size of mesenteric lymph CM collected from rats treated with CL 277,082 (an inhibitor of ACAT activity), cholestyramine (to inhibit or restrict absorption of cholesterol from the intestinal lumen), or pravastatin, which inhibits cholesterol synthesis in the liver and intestine.1 The clearance of lymph CM from rats treated with CL 277,082 was also studied.
Male Wistar rats weighing ≈300 g were obtained from the Animal Resources Centre (Murdoch, Western Australia). Eight groups of rats were studied, with one for each drug studied plus untreated control groups with subgroups in which added cholesterol was a variable. In all groups, intestinal lymph was collected through a plastic tube surgically implanted in the principal mesenteric lymph duct. After surgery the rats were placed in individual cages to recover from anesthesia and kept hydrated by a steady intragastric injection of 0.15 mol/L NaCl at 2.0 mL/h through a gastrostomy tube placed at the time of lymph cannulation. Tap water was freely available. Details of these procedures have been given previously.2
Rats were treated for 5 days with CL 277,082 mixed with the diet in the proportion of 0.5% by weight. The diet was fed in broad-rimmed containers designed to prevent food spillage. In other groups, rats were placed on a diet containing 2% cholestyramine for 2 weeks. Other groups of rats had ad libitum access for 14 days to drinking water containing 0.04% pravastatin sodium, and after lymph duct cannulation these groups of rats were infused through the gastrostomy cannula with 0.15 mol/L NaCl containing 0.04% pravastatin sodium.
Preparation of CM
On the first postoperative day, lymph was collected for 4 hours while rats received a steady intestinal infusion through the gastrostomy tube of Intralipid (Vitrum AB) at a TG concentration of 125 mg/h, either with or without 2% cholesterol. Beginning 2 hours after the start of Intralipid infusion, lymph was collected for 4 hours into vessels containing EDTA, gentamicin, reduced glutathione, aprotinin, and phenyl methyl-sulfonyl fluoride (0.3 g/mL in dimethylsulfoxide) to final concentrations of 1 mg/mL, 0.1 mg/mL, 1.6 mmol/L, 10 kallikrein units/mL and 0.15% (wt/vol), respectively. Cells were removed by low-speed centrifugation, and solid KBr was then added (1.96 g/14 mL) to increase the density to 1.1 g/mL. After degassing in vacuo, 14 mL of lymph was injected under discontinuous density gradients consisting of 6-mL solutions with densities 1.065, 1.040, 1.020, and 1.006 g/mL.3 The gradients were then centrifuged at 20°C in the SW28 rotor of the Beckman L8-M ultracentrifuge for 15 hours at 28 000 rpm. The triglyceride-rich lymph lipoproteins containing CM were removed from the top 0.5 cm of the tube.
For clearance studies, male rats weighing 250 to 300 g were prepared surgically with arterial and venous cannulas as described earlier.2 CM labeled with trace amounts of radioactive triglyceride and CE were prepared and injected to measure plasma clearance exactly as described previously.2 Labeled CM were prepared by addition of 10 μCi of [1-14C] palmitic acid and 20 μCi [7(n)-3H]cholesterol at 2 hours after the start of Intralipid (without added cholesterol) infusion. Lymph was collected for 4 hours into vessels containing EDTA (1 mg/mL) and reduced glutathione (1.6 mmol/L). CM were isolated under the same conditions as before except the gradients were centrifuged for 75 minutes. CM that floated to the surface were analyzed and injected into rats within 1 day. Oxidation was prevented by the addition of reduced glutathione (50 μg/mL) and storage under argon.
Preparation of Intralipid-Containing Cholesterol
Intralipid has the composition of soybean oil (20% wt/vol), egg phospholipid (1.2% wt/vol), and glycerol (2.25% wt/vol) in an emulsion with particles ranging in diameter from 100 to 800 nm.4 To prepare Intralipid with 2% cholesterol, aliquots containing 196 mg of TG were dispensed into vials with 4 mg cholesterol and sonicated in 8.5 mL of 0.15 mol/L NaCl, 10 mmol/L N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid], pH 7.4, at 55° to 56°C (monitored by a thermocouple in the vessel) with the atmosphere above the mixture purged with nitrogen to prevent lipid oxidation. Sonication was for 20 minutes with the use of a 1-cm probe at a continuous output of 90 to 110 W with a Vibra-Cell high-intensity ultrasonic processor (Sonics and Materials Inc). The lipid mixture was then centrifuged at 3000 rpm for 1 hour to remove titanium debris.
Quantitation of Apo B48
The quantitation of apo B48 was determined as recently published.2 In summary, lymph CM were partially delipidated by mixing 0.5 mL of sample with 3 mL of diethyl ether. Aliquots of the partially delipidated material of 50 μL were mixed with 50 μL of reducing buffer (0.01 mol/L Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 0.04 mol/L dithiothreitol) and 0.02% (wt/vol) bromophenol blue and applied to a 4% stacking gel that overlayed a 5% to 25% vertical slab of sodium dodecyl sulfate polyacrylamide gel 1.5 mm in thickness. A set of standards containing known amounts of pure apo B48 protein in duplicate was applied to each gel for quantitation, as shown in Fig 1⇓. Apo B48 was assayed by the Lowry procedure with the use of bovine serum albumin as standard, corrected by 13% to account for greater chromogenicity of apo B48.5 The gels were imaged with an Epson Scanner 4000, and the image was analyzed by a computerized Scan Analysis program (Biosoft).
The lipids extracted from CM with chloroform-methanol (2:1 vol/vol) were separated by thin-layer chromatography on 0.2-mm layers of silica gel in the solvent system petroleum ether 40° to 60°C/diethyl ether/formic acid (90/10/1 by volume). The TG, CE, and cholesterol bands were scraped from the plate for assay of TG by the chromotropic acid method,6 and free and esterified cholesterol were assayed by the o-phthaldialdehyde procedure.7 Protein was assayed by the procedure of Lowry et al8 with extraction of turbidity due to lipids with chloroform. Phospholipid was measured directly on CM samples.9
Statistical comparisons were made on the lipid radioactivity data of the plasma clearances by repeated-measures ANOVA with SPSS software. Other data were compared by one-way ANOVA or two-way ANOVA where appropriate.
The ACAT inhibitor CL 277,082 was a gift from American Cyanamid Co, Pearl River, NY. Cholestyramine was obtained from Astra Pharmaceuticals, North Ryde, New South Wales. Pravastatin sodium (SQ31000) was a gift from the Squibb Institute for Medical Research, Princeton, NJ.
Effects of CL 277,082, Pravastatin, and Cholestyramine on CM Composition and Size and Transport of TG and Apo B48
Table 1⇓ shows that the contents of TG were slightly decreased, whereas PL were increased in lymph CM from rats treated with CL 277,082. Table 1⇓ also shows that compared with CM from other groups, the lymph CM from rats treated with CL 277,082 were significantly increased in contents of FC (P<.001 by one-way ANOVA), whereas CE contents were significantly decreased irrespective of cholesterol in the infused Intralipid. The mean lipid and protein compositions of the lymph CM were used to calculate the diameters of the average CM particles, as described by Miller and Small.10 Lymph CM from rats treated with CL 277,082 were 21% and 30% smaller in diameter than CM from control rats without or with cholesterol, consistent with the changes in composition (Table 1⇓).
Table 1⇑ also shows that the outputs of TG and apo B48 in lymph were both decreased in rats treated with CL 277,082. The reduction in lymph TG transport in rats treated with CL 277,082 was by 46% in rats infused with Intralipid without cholesterol and by 71% with cholesterol in the infusate. The corresponding reductions in lymph apo B48 transport in rats treated with CL 277,082 were by 40% and 60%, respectively. The reduction in lymph apo B48 transport in treated rats was consistent with a decrease in CM particle number.
As shown in Table 1⇑, CM from rats treated with cholestyramine and pravastatin showed no significant changes in composition when infused with Intralipid either with or without cholesterol. However, the transport of lymph TG was significantly reduced by 44% in rats treated with cholestyramine when cholesterol was present in the infusate. Cholestyramine and pravastatin had no significant effects on the transport of apo B48.
Table 2⇓ summarizes the results of two-way ANOVA of the data of Table 1⇑. All parameters were significantly affected by the effects caused by the different drug treatments. The CM contents of CE, PL, and FC were affected by the presence of cholesterol in the perfusate, whereas other parameters were unaffected. The changes in CM composition are obvious from Table 3⇓, showing the calculated ratios for FC/CE and FC/total protein were markedly increased in lymph CM from CL 277,082–treated rats. To confirm that the treatment was inhibiting the esterification of exogenous cholesterol, labeled [3H]-cholesterol was injected into the duodenum during lymph collection. Fig 2⇓ shows that the [3H]-cholesterol distributed significantly more into the FC fraction of lymph CM from CL 277,082–treated rats and significantly less in the CE fraction compared with control rats.
Effect of CL 277,082 on Plasma Clearance of CM
Fig 3⇓ shows the clearances from plasma after injection of radiolabeled lymph CM into control rats and rats treated with CL 277,082. In each case, lymph CM were obtained from either control untreated rats or from rats treated with CL 277,082. Fig 3A⇓ and Fig 3C⇓ show the effect of treatment of the recipient rat with CL 277,082 on the clearance of CM obtained from untreated donor rats. Treatment slowed the clearance of the CE label, whereas the clearance of the TG label was faster in the treated recipients. Fig 3B⇓ and Fig 3D⇓ show that the clearances of labels were faster overall when chylomicrons were obtained from donor rats treated with CL 277,082. However the slower relative clearance of the CE label and the faster relative clearance of the TG label due to treatment of the recipients persisted. The plasma contents of cholesterol (P<.02) and triglyceride (P<.01) were both significantly reduced in rats treated with CL 277,082 (Fig 4⇓).
The plasma clearance data of Fig 3⇑ were compared by repeated-measures ANOVA. To simplify data presentation, the curves were fitted to biexponentials, and the average fractional clearance rates were calculated as described by Matthews et al11 and converted to half-lives by division into 0.693. The results of the clearance studies are summarized in Table 4⇓, which shows that the half-life of TG label was shorter in the plasma of treated rats (Fig 3C and 3D⇑⇑, P<.02 by repeated-measures ANOVA), whereas the half-life of the CE label after injection of CM derived from either control or treated donors was significantly longer (P<.01) in rats treated with CL 277,082 (Fig 3A and 3B⇑⇑). Further, the half-lives of CE label after injection of CM obtained from treated donor rats were significantly shorter in both control and treated recipient rats (P<.05 by repeated-measures ANOVA).
The recoveries in the livers and spleens of the TG and CE labels from CM injected in control and treated groups of rats are also shown in Table 4⇑. Data were analyzed by two-way ANOVA. The recoveries in the liver of TG label after injection of CM from treated donors were significantly greater (P<.001) in both control rats and rats treated with CL 277,082. Significantly less (P<.02) TG label was found in the livers of rats treated with CL 277,082 after injection of CM obtained from either control or treated donor rats. The recoveries in the liver of CE after injection of CM from control or treated donors were significantly less (P<.05) in recipient rats treated with CL 277,082. The recoveries of TG and CE labels by the spleen were significantly greater (P<.001) in control and treated recipient rats when the injected CM were obtained from donor rats treated with CL 277,082.
Effects of Treatment With CL 277,082 on the Apolipoproteins Associated With Lymph CM
When compared with control CM (lanes 3, 4, and 5), Fig 5⇓ shows that the apolipoproteins associated with lymph CM from rats treated with CL 277,082 (lanes 6, 7, and 8) contained less apo B48. This finding was consistent with the evidence of decreased apo B48 transport presented in Table 1⇑. The contents of other apolipoproteins in the CM from treated rats also were reduced, consistent with the reduction in CM transport shown by the data of Table 1⇑.
The effects of ACAT inhibitors as inhibitors of cholesterol absorption,12 13 14 15 16 17 as hypocholesterolemic agents,18 19 20 21 22 23 and as antiatherosclerotic agents24 25 26 have been the subject of many recent studies. Inhibition of the activity of ACAT in tissues such as intestine, liver, and artery wall has clear implications in the treatment of hypercholesterolemia and atherosclerosis. Our present study shows two further consequences of inhibition of ACAT activity. First, treatment with CL 277,082 altered the composition of lymph CM with subsequent affects on CM clearance from the plasma. Second, CL 277,082 decreased the outputs of apo B48 and triglyceride in lymph (Table 1⇑).
The lymph CM from rats treated with CL 277,082 contained significantly more FC and significantly less CE (Table⇑s 1 through 3 and Fig 2⇑). Lymph CM from treated rats were significantly smaller in diameter, reflecting the decreased TG contents and increased phospholipid and protein contents of these particles (Table 1⇑). Plasma TG and cholesterol contents were decreased when compared with control rats, consistent with published studies.12 13 14 15 16 17
In the present study, CL 277,082 decreased both apo B48 transport and TG transport in lymph, perhaps indicating that CL 277,082 inhibits CM formation in the enterocyte. A decrease in apo B48 transport was found with CL 277,082 but not with pravastatin (Table 1⇑ and Fig 5⇑). Therefore, synthesis of apo B48 in the intestine may be regulated by the availability of CE, as found by Cianflone et al27 in HepG2 cells for the production of apo B100.
Another possibility is that inhibition of ACAT activity, by increasing cellular FC, decreased CM formation. Alternatively, CL 277,082 may affect the transcription of apo B. Apo B48 production by the intestine is normally rather constant. For example, large changes in lipid transport by the intestine are not accompanied by corresponding changes in apo B48 transport.2 However, bile diversion decreased intestinal apo B48 output,28 whereas treatment with ethinyl estradiol decreased rat lymph apo B48 transport by ≈50% and inhibited TG transport in postprandial intestinal lymph.29
In rats treated with Triton WR1339 to block TG removal from plasma, pravastatin decreased the secretion of TG into the plasma of fed rats but not fasted rats,30 which suggests that the TG output in CM was decreased. However, this interpretation cannot be supported by our direct measurements of TG output in CM of rats treated with pravastatin. Neither pravastatin nor cholestyramine affected lymph CM composition or size. The output of lymph TG was significantly decreased when rats were treated with cholestyramine without an accompanying reduction in the output of apo B48. This observation warrants further investigation and possibly indicates an effect of cholestyramine on the size distribution of CM.
After injection of labeled lymph CM obtained from rats treated with CL 277,082, the plasma clearance of radiolabeled CE, a tracer for the remnants derived from these particles, was slow in control rats and in rats treated with CL 277,082 (Table 4⇑). The uptake of remnant particles from these CM was significantly less by the liver and significantly more by the spleen (Fig 3⇑ and Table 4⇑). The slow remnant clearance in rats treated with CL 277,082 occurred despite the lower plasma cholesterol content (Fig 4⇑) and therefore was not explained by an increase in pool size. The slow clearance was possibly related to a lower expression of receptors or to an effect of treatment with CL 277,082 on the interaction of remnant particles with LDL receptors or other apo E receptors. Inhibition of ACAT activity would be expected to decrease the expression of LDL receptors because of the increased contents of cell unesterified cholesterol. However, this explanation is not consistent with the decrease in plasma cholesterol concentration, and additional studies including measurements of the expression of LDL receptors and other apo E receptors are needed to resolve the question.
In contrast to the slow clearance of CE label, the clearance of the TG label was faster from the plasma of rats treated with CL 277,082 (Fig 3C and 3D⇑⇑) when labeled CM were obtained from either control or donor rats treated with CL 277,082. The faster TG clearance was consistent with the low plasma TG content in rats treated with CL 277,082 (Fig 4⇑) and suggests that lipase activities may be increased.
Many pharmacological agents are known to inhibit the activity of ACAT, some with greater potency and some with different tissue selectivities than CL 277,082. Measurements with other agents are needed to determine whether the effects on CM transport are specific to CL 277,082 or represent a general response to inhibition of ACAT activity. The present studies provide novel data relating to the effects of CL 277,082 on lymph CM metabolism and apo B48 transport. The reduction in transport of lymph apo B48 suggests that CL 277,082 affects not only cholesterol esterification but possibly also gene transcription or assembly of CM in the enterocyte.
Selected Abbreviations and Acronyms
|ACAT||=||acyl CoA:cholesterol acyltransferase|
This work was supported by grants from the National Health and Medical Research Council of Australia. The ACAT inhibitor CL 277,082 was a gift from the American Cyanamid Company, Pearl River, NY. Pravastatin was a gift from the Squibb Institute for Medical Research, Princeton, NJ.
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