Rabbit Lipoproteins Research Articles

Role of Hridayarnava Rasa on Inflammatory Responses in Rabbits with High Fat Diet Induced Atherosclerosis

Background: Atherosclerotic plaque formation is a chain of events that begins with fatty streak accumulation followed by monocytes infiltration and lipid core formation. Monocytes/macrophages play an important role in the initiation and progression of atherosclerosis. The role of inflammation and atherosclerosis provides a mechanistic framework for understanding the clinical benefits of lipid-lowering therapies in high-fat diet (HFD) induced atherosclerosis rabbits. Identifying triggers for inflammation and uncovering the details of inflammatory pathways may ultimately present new therapeutic targets. H. Rasa maintains the heart by providing cardioprotective activity along with changes in certain inflammatory markers in atherosclerosis. Objectives: The primary objective of the study is to evaluate the role of oxidized low-density lipoprotein cholesterol (LDLc) in the inflammatory response and how this inflammation triggers the level of white blood cells. The secondary objective is how this Hridayarnava Rasa, an Ayurvedic formulation inhibits the oxidation of LDLc and protects cells from inflammation in HFD-induced atherosclerosis model rabbits. Materials and Methods: Newzealand white rabbits of 24 were randomly divided into 6 groups of 4 animals each. Group I rabbits fed with standard pellet diet; group II rabbits fed with HFD; group III, IV, and V were fed with HFD and different doses of H. Rasa and group VI rabbits were fed with HFD plus Atorvastatin. Results: Total leucocytes, lymphocytes, monocytes, LDLc: high-density lipoprotein cholesterol (HDLc) ratio and total cholesterol (TC): HDLc ratio were increased in group II, III, IV, and VI of 30, 60, and 90 days when compared to group I. The levels of total leukocytes, lymphocytes, monocytes, LDLc: HDLc ratio, and TC: HDLc ratio were significantly reduced in group IV and V of 30, 60, and 90 days when compared to group II. In the present study, treatment with H. Rasa (group V) (i.e., 41.07 mg/kg. b. wt/p. o) was shown to be most effective over 90 days. Conclusion: These results suggest that HFD accelerates the development of atherosclerosis by increasing the inflammatory markers such as oxidized LDL (oxLDLc) and leukocyte counts in a time-dependent manner and H. Rasa protects the aorta by preventing the oxidative damage of LDLc which inturn maintains the inflammatory markers and provided the anti-inflammatory responses and protects the aorta from atherosclerotic plaque formation in a dose-and time-dependent manner. Limitation of the Study: This study focused on the characteristics of the rabbit lipoprotein pathway and pathophysiology of atherosclerotic lesions via inflammatory markers. This paper primarily determines how H. Rasa protects the aorta from the formation of atherosclerotic plaques caused by oxidative low-density lipoprotein. Further studies will need to focus specifically on the inflammatory pathways and the role of H. Rasa.

Clearance of Carbonyl-Modified Low-Density Lipoproteins in Rabbits

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITC. Rabbit or human blood plasma LDL were isolated using differential ultracentrifugation in a density gradient; after labeling by biotinylation or by FITC, LDL were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native (control) and dicarbonyl-modified biotinylated or FITC-labeled LDL were injected into the ear vein of rabbits, and blood samples were taken at certain time intervals. The content of biotinylated LDL in blood plasma was determined by an enzyme immunoassay method; FITC-labeled LDL was determined from the fluorescence spectra. It has been found that glyoxal- and methylglyoxal-modified rabbit and human LDL circulate in the bloodstream of rabbits for almost the same period as native (unmodified) LDL. In contrast to this, MDA-modified rabbit and human LDL were very quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from human blood plasma is not associated with red blood cells or endothelial cells. We found that using the Oxidized LDL ELISA kits (Mercodia, Sweden) it was possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreased during therapy with evolocumab, the hypocholesterolemic inhibitor of PCSK9 (proprotein convertase of subtilisin/kexin type 9), which activates LDL reutilization in the liver cells. These results explain the extremely rapid clearance of MDA-modified LDL in our experiments by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

Top-down and bottom-up lipidomic analysis of rabbit lipoproteins under different metabolic conditions using flow field-flow fractionation, nanoflow liquid chromatography and mass spectrometry

This study demonstrated the performances of top-down and bottom-up approaches in lipidomic analysis of lipoproteins from rabbits raised under different metabolic conditions: healthy controls, carrageenan-induced inflammation, dehydration, high cholesterol (HC) diet, and highest cholesterol diet with inflammation (HCI). In the bottom-up approach, the high density lipoproteins (HDL) and the low density lipoproteins (LDL) were size-sorted and collected on a semi-preparative scale using a multiplexed hollow fiber flow field-flow fractionation (MxHF5), followed by nanoflow liquid chromatography-ESI-MS/MS (nLC-ESI-MS/MS) analysis of the lipids extracted from each lipoprotein fraction. In the top-down method, size-fractionated lipoproteins were directly infused to MS for quantitative analysis of targeted lipids using chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry (cAF4-ESI-MS/MS) in selected reaction monitoring (SRM) mode. The comprehensive bottom-up analysis yielded 122 and 104 lipids from HDL and LDL, respectively. Rabbits within the HC and HCI groups had lipid patterns that contrasted most substantially from those of controls, suggesting that HC diet significantly alters the lipid composition of lipoproteins. Among the identified lipids, 20 lipid species that exhibited large differences (>10-fold) were selected as targets for the top-down quantitative analysis in order to compare the results with those from the bottom-up method. Statistical comparison of the results from the two methods revealed that the results were not significantly different for most of the selected species, except for those species with only small differences in concentration between groups. The current study demonstrated that top-down lipid analysis using cAF4-ESI-MS/MS is a powerful high-speed analytical platform for targeted lipidomic analysis that does not require the extraction of lipids from blood samples.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.

Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. Rabbits are widely used for the study of human atherosclerosis. Because rabbits have a unique feature of lipoprotein metabolism (like humans but unlike rodents) and are sensitive to a cholesterol diet, rabbit models have not only provided many insights into the pathogenesis and development of human atherosclerosis but also made a great contribution to translational research. In fact, rabbit was the first animal model used for studying human atherosclerosis, more than a century ago. Currently, three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (1) cholesterol-fed rabbits, (2) Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors, and (3) genetically modified (transgenic and knock-out) rabbits. Despite their importance, compared with the mouse, the most widely used laboratory animal model nowadays, the use of rabbit models is still limited. In this review, we focus on the features of rabbit lipoprotein metabolism and pathology of atherosclerotic lesions that make it the optimal model for human atherosclerotic disease, especially for the translational medicine. For the sake of clarity, the review is not an attempt to be completely inclusive, but instead attempts to summarize substantial information concisely and provide a guideline for experiments using rabbits.

Human Apolipoprotein A-II Protects Against Diet-Induced Atherosclerosis in Transgenic Rabbits

Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, β-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than β-very-low-density lipoproteins of non-Tg rabbits. These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.

Anion‐exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl‐ligated gel with a perchlorate‐containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion-exchange high-performance liquid chromatography using a column filled with nonporous, diethylaminoethyl-ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high-density lipoprotein, low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein and chylomicrons) were eluted with step-wise increases in a concentration of NaClO₄. The post-column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein-cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within-day and between-day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high-fat diet increased lipoprotein-cholesterol concentrations by 6- to 77-fold. The method described here was nevertheless able to quantitate levels of lipoprotein-cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models.

Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits

Rabbits on a 1% cholesterol diet received injections of vehicle with or without D-4F or L-4F. After 1 month, the percent of aorta with atherosclerotic lesions was 24 +/- 15% (vehicle), 10 +/- 6% (D-4F) (P < 0.01 vs. vehicle), and 13 +/- 9% (L-4F) (P < 0.05 vs. vehicle). Inflammatory indexes for HDL and LDL were determined by measuring monocyte chemotactic activity after adding rabbit lipoproteins to human endothelial cells. HDL-inflammatory index (HII) and LDL-inflammatory index (LII), respectively, were 1.39 +/- 0.24; 1.35 +/- 0.29 (vehicle), 0.67 +/- 0.26; 0.63 +/- 0.38 (D-4F) (P < 0.001 vs. vehicle), and 0.67 +/- 0.2; 0.68 +/- 0.32 (L-4F) (P < 0.01 vs. vehicle). Serum amyloid A (SAA) levels were 95 +/- 39, 8 +/- 22, and 7 +/- 19 mug/ml, respectively, for vehicle, D-4F, and L-4F (P < 0.001 vs. vehicle). There was no correlation between lesion area and total plasma or HDL-cholesterol levels. In contrast, there was a positive correlation with HII, LII, and SAA (P = 0.002, P = 0.0026, P = 0.0079, respectively). HII correlated closely with SAA levels (r = 0.6616; r(2) = 0.4377, P < 0.0001). Thus, HII, LII, and SAA are better predictors of lesion area than are total plasma or HDL-cholesterol levels in cholesterol-fed rabbits.

The effect of acetaminophen on oxidative modification of low-density lipoproteins in hypercholesterolemic rabbits.

Oxidative modification of low-density lipoproteins (LDL) contributes to the pathology of atherosclerosis. Antioxidants may protect LDL against oxidative modification. Acetaminophen, a widely used analgesic and antipyretic agent, has significant antioxidant properties. However, there is little evidence to suggest that acetaminophen acts as an antioxidant for LDL oxidation in vivo. In this study, we investigated the in vivo effect of acetaminophen on LDL oxidation in hypercholesterolemic rabbits. The oxidative modification of LDL was identified by conjugated dienes and thiobarbituric acid-reactive substances (TBARS). In the cholesterol group which rabbits were fed a diet contained 1% g cholesterol for 8 weeks, TBARS contents and conjugated diene levels in the plasma and isolated LDL samples significantly increased compared with the control rabbits (p<0.05). However, in the cholesterol + acetaminophen group, the TBARS contents and conjugated diene levels were significantly lower than that of the cholesterol group (p<0.05). The results from in vitro studies also demonstrated that the LDL isolated from serum was oxidized by Cu++ ions and this oxidation reduced in the presence of acetaminophen. The reduced oxidative modification of LDL by acetaminophen may be of therapeutic value in preventing the development and progression of atherosclerosis.

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine

Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.

Preparation and investigation of 99m technetium-labeled low-density lipoproteins in rabbits with experimentally induced hypercholesterolemia.

Low-density lipoproteins (LDL) were radiolabeled in atherosclerosis studies. The aim was to investigate the biodistribution and uptake of 99mTc-labeled LDL by atherosclerotic plaques in experimentally induced hyperlipidemia. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate human LDL aliquots, to prepare radioactive-labeled lipoproteins and in rabbits with induced hyperlipidemia. Preparative density gradient centrifugation was applied for the simultaneous isolation of the major lipoprotein density classes, which form discrete bands of lipoproteins in the preparative tubes. The cholesterol and protein levels in the lipoprotein fractions were determined. LDL was subsequently dialysed against physiological solution and sterilized and apolipoprotein fragments and aggregates were eliminated by passage through a 0.22-micron filter. LDL was radiolabeled with 99mTc by using sodium dithionite as a reducing agent. Radiochemical purity and in vitro stability were controlled by paper chromatography in acetone. The labelling efficiency was 85-90% for human LDL. Two months after the start of cholesterol feeding, the total cholesterol in the blood serum had increased approximately 33-fold in comparison with the basal cholesterol content of hypercholesterolemic rabbits. Investigation of LDL was performed by Schlieren analysis after adjustment of the density of serum and underlayering by salt solution in a spinning ultracentrifugation capillary band-forming cell. Quantitative results were obtained by measuring the Schlieren areas between the sample curves and the reference baseline curve by means of computerized numerical and graphic techniques. In this manner we measured the concentrations of human LDL and analyzed rabbit LDL levels in induced hyperlipidemia. Gamma scintillation camera scanning of the rabbits was performed. Overnight fasted rabbits were injected in the marginal ear vein with 99mTc-labeled human LDL (4-10 mCi, 0.5-1.5 mg protein). The initial scintigram showing a typical blood-pool scan, gradually changing with time to an image of specific organ uptake of radioactivity by the liver, kidneys and brain and in the bladder. Gamma camera in vivo scintigraphy on rabbits revealed visible signals corresponding to atherosclerotic plaques in the aorta and carotid arteries. Our results show that 99mTc-LDL can be used to assess the organ distribution pattern of LDL in the rabbit, and to detect and localize areas of arterial atherosclerotic lesions.

Influence on hazelnut oil administration on peroxidation status of erythrocytes and apolipoprotein B 100-containing lipoproteins in rabbits fed on a high cholesterol diet.

Hazelnut oil (HO) is rich in monounsaturated fatty acids (MUFA). The effect of a high cholesterol (HC) diet with and without HO on lipids and lipid peroxide levels in plasma, apolipoprotein B 100-containing lipoproteins (VLDL + LDL), and erythrocytes as well as hematological data was investigated in rabbits. A HC diet caused significant increases in lipid peroxide levels in plasma and apo B-containing lipoproteins together with histopathological atherosclerotic findings in aorta. In addition, this diet resulted in hemolytic anemia associated with increased endogenous diene conjugate (DC) levels, but H(2)O(2)-induced malondialdehyde (MDA) levels remained unchanged in erythrocytes. HO supplementation reduced lipid peroxide levels in plasma and apolipoprotein B 100-containing lipoproteins as well as aortic atherosclerotic lesions in rabbits fed an HC diet without any decreasing effect on lipid levels. In addition, HO was found to reduce hemolytic anemia together with significant decreases in DC and H(2)O(2)-induced MDA levels.

Regulation of PUFA metabolism: pharmacological and toxicological aspects

Levels of the long-chain polyunsaturated fatty acids (LCP) of the n-6 and n-3 series in animal plasma and cells are directly or indirectly dependent upon the intakes of either their precursors, the short-chain polyunsaturated fatty acids (SCP), linoleic (LA, 18:2 n-6) and alpha linolenic acid (ALA, 18:3 n-3), respectively, and/or of the preformed products (arachidonic, 20:4 n-6) and eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). We report here that pharmacological agents and cytotoxic compounds significantly affect the production of LCP from SCP in cultured cells. Using labelled substrates and radio HPLC separations, we observed that the potent hypocholesterolemic agent, simvastatin, activates the formation of AA from LA, mainly acting at the Δ5 desaturation step, and increases also the mRNA levels, in cultured monocytic cells (THP-1). Elevation of AA occurs also in plasma lipids of hyperlipemic patients treated with statins (but not with fibrates). Conversely, oxysterols (mainly 7-β-oxysterol), which are detected in circulating lipoproteins of rabbits on a hypercholesterolemic diet, potently inhibit the synthesis of AA from LA in hepatocytic cell lines (Hep-G2). At the same time plasma levels o AA are reduced vs controls, in spite of an identical intake of LA. Finally, on the basis of previous work showing reduced levels of LCP, mainly DHA, in the milk of cigarette-smoking mothers, we have observed that the incubation of human mammary gland cells with sera exposed to cigarette smoke results in marked inhibition of the production of DHA from ALA. The products in smoke responsible for this effect, are being identified through mass spectrometric techniques. In conclusion, pharmacological agents and toxic compounds, such as oxysterols and smoke products affect key steps in the synthesis of the LCP, major bioregulators in mammalian cells.

Studies on effects of dietary fatty acids as related to their position on triglycerides.

This article reviews published literature on how the stereospecific structure of dietary triglycerides may affect lipid metabolism in humans. Animal studies have shown enhanced absorption of fatty acids in the sn-2 position of dietary triglycerides. Increasing the level of the saturated fatty acid palmitic acid in the sn-2 position (e.g., by interesterification of the fat to randomize the positions of the fatty acids along the glycerol backbone) has been shown in rabbits to increase the atherogenic potential of the fat without impacting levels of blood lipids and lipoproteins. In contrast, enhancing the level of stearic acid in the sn-2 position has not been found to affect either atherogenic potential or levels of blood lipids and lipoproteins in rabbits. Fatty acids other than palmitic and stearic have not been studied systematically with respect to possible positional effects. A limited number of human studies have shown no significant effects of interesterified fats on blood lipid parameters. However, it is unknown whether modifying the stereospecific structure of dietary triglycerides would affect atherogenicity or other long-term health conditions in humans. It is possible that incorporation of palmitic acid into the sn-2 position of milk fat is beneficial to the human infant (as a source of energy for growth and development) but not to human adults. Additional research is needed to determine whether processes like interesterification, which can be used to alter physical parameters of dietary fats (e.g., melting characteristics), may result in favorable or unfavorable long-term effects in humans.

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.

L-Carnitine effects on chemical composition of plasma lipoproteins of rabbits fed with normal and high cholesterol diets.

L-Carnitine plays an important role in the mitochondrial uptake of long-chain fatty acids in mammals. It has recently been shown that this compound has a marked hypo-cholesterolemic effect when used in conjunction with lipid-rich diets. The aim of this study was to investigate the effects of L-carnitine on the fatty acid composition of plasma lipoproteins in rabbits fed with different diets. Four different groups were investigated: group I (standard diet), group II (standard diet supplemented with L-carnitine at 80 mg/kg), group III (standard diet supplemented with 0.5% cholesterol), and group IV (standard diet supplemented with 0.5% cholesterol plus L-carnitine at 80 mg/kg). The feeding period was 126 d. Total plasma cholesterol was indistinguishable in groups I and II, but increased nearly 40-fold in group III. This increment was reduced by 50% in group IV. Correspondingly, total cholesterol content in lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) separated by agarose gel chromatography was the same for groups I and II, while for animals fed a cholesterol-rich diet (III) total cholesterol in VLDL + LDL increased nearly 100-fold when compared with groups I and II but, again, the increment was reduced by 50% in group IV. In contrast, total cholesterol in HDL increased only fivefold for both groups III and IV when compared with groups I and II, indicating no effects of L-carnitine on this parameter. The reduction of total cholesterol in VLDL + LDL particles in animals fed a cholesterol-rich diet plus L-carnitine was associated with a marked decrease in the ratio of cholesteryl ester to free cholesterol and a dramatic increase in their phospholipid content; opposite effects were observed for HDL. L-Carnitine induced a marked decrease in the saturated to unsaturated C16 + C18 fatty acid ratio in cholesteryl esters associated with VLDL and LDL from animals fed with both normal and cholesterol-rich diets. The opposite effect (a large increase in the saturated to unsaturated fatty acid ratio) was observed for both cholesteryl esters and phospholipids associated with HDL in animals fed with both diets. The results suggested that the hypocholesterolemic effects of L-carnitine could be associated with increased systemic breakdown of cholesteryl esters, a probable increase in reverse cholesterol transport, and the stabilization of a phospholipid-based structure of VLDL + LDL particles.

Lipoprotein cholesterol uptake mediates up-regulation of bile-acid synthesis by increasing cholesterol 7α-hydroxylase but not sterol 27-hydroxylase gene expression in cultured rat hepatocytes

Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7α-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different lipoproteins in regulating both enzymes are not well established. We studied the effect of different rabbit lipoproteins on cholesterol 7α-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. β-Migrating very-low-density lipoprotein (βVLDL) and intermediate-density lipoprotein (IDL) caused a significant increase in the intracellular cholesteryl ester content of cells (2.3- and 2-fold, respectively) at a concentration of 200 μg of cholesterol/ml, whereas high-density lipoprotein (HDL, 50% v/v), containing no apolipoprotein E (apo E), showed no effect after a 24-h incubation. βVLDL and IDL increased bile-acid synthesis (1.9- and 1.6-fold, respectively) by up-regulation of cholesterol 7α-hydroxylase activity (1.7- and 1.5-fold, respectively). Dose- and time-dependent changes in cholesterol 7α-hydroxylase mRNA levels and gene expression underlie the increase in enzyme activity. Incubation of cells with HDL showed no effect. Sterol 27-hydroxylase gene expression was not affected by any of the lipoproteins added. Transient-expression experiments in hepatocytes, transfected with a promoter-reporter construct containing the proximal 348 nucleotides of the rat cholesterol 7α-hydroxylase promoter, showed an enhanced gene transcription (2-fold) with βVLDL, indicating that a sequence important for a cholesterol-induced transcriptional response is located in this part of the cholesterol 7α-hydroxylase gene. The extent of stimulation of cholesterol 7α-hydroxylase is associated with the apo E content of the lipoprotein particle, which is important in the uptake of lipoprotein cholesterol. We conclude that physiological concentrations of cholesterol in apo E-containing lipoproteins increase bile-acid synthesis by stimulating cholesterol 7α-hydroxylase gene transcription, whereas HDL has no effect and sterol 27-hydroxylase is not affected.

Lipoprotein cholesterol uptake mediates up-regulation of bile-acid synthesis by increasing cholesterol 7α-hydroxylase but not sterol 27-hydroxylase gene expression in cultured rat hepatocytes

Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7alpha-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different lipoproteins in regulating both enzymes are not well established. We studied the effect of different rabbit lipoproteins on cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. beta-Migrating very-low-density lipoprotein (betaVLDL) and intermediate-density lipoprotein (IDL) caused a significant increase in the intracellular cholesteryl ester content of cells (2. 3- and 2-fold, respectively) at a concentration of 200 microgram of cholesterol/ml, whereas high-density lipoprotein (HDL, 50% v/v), containing no apolipoprotein E (apo E), showed no effect after a 24-h incubation. betaVLDL and IDL increased bile-acid synthesis (1. 9- and 1.6-fold, respectively) by up-regulation of cholesterol 7alpha-hydroxylase activity (1.7- and 1.5-fold, respectively). Dose- and time-dependent changes in cholesterol 7alpha-hydroxylase mRNA levels and gene expression underlie the increase in enzyme activity. Incubation of cells with HDL showed no effect. Sterol 27-hydroxylase gene expression was not affected by any of the lipoproteins added. Transient-expression experiments in hepatocytes, transfected with a promoter-reporter construct containing the proximal 348 nucleotides of the rat cholesterol 7alpha-hydroxylase promoter, showed an enhanced gene transcription (2-fold) with betaVLDL, indicating that a sequence important for a cholesterol-induced transcriptional response is located in this part of the cholesterol 7alpha-hydroxylase gene. The extent of stimulation of cholesterol 7alpha-hydroxylase is associated with the apo E content of the lipoprotein particle, which is important in the uptake of lipoprotein cholesterol. We conclude that physiological concentrations of cholesterol in apo E-containing lipoproteins increase bile-acid synthesis by stimulating cholesterol 7alpha-hydroxylase gene transcription, whereas HDL has no effect and sterol 27-hydroxylase is not affected.

Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions.

The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.

Regulation of Plasma and Hepatic Lipids by Dietary Fatty Acids: Effects of Oleic, Elaidic and Palmitic Acids.

This study compares the effects of dietary oleic acid (OA), palmitic acid (PA), and trans fatty acids (TFA) on lipid metabolism in rabbits. Animals were fed semipurified diets in which a fifth of the fat intake (6% total energy) was either OA, PA, or TFA in the presence and absence of dietary cholesterol (0.2% w/w). After 10-12 weeks of feeding the cholesterol-free diets, there were no significant differences in plasma total cholesterol or triacylglycerol concentrations between groups. However, there was an increase in the cholesterol concentration of the lower density lipoproteins in rabbits fed the TFA diet compared with their OA- and PA-fed counterparts. The addition of dietary cholesterol had no effect on plasma lipids, however hepatic cholesterol concentrations were lower in animals fed the PA diet compared with those fed the OA diet (p<0.05), mainly due to a reduction in cholesteryl ester concentration (p<0.05). Despite these changes, hepatic low density lipoprotein (LDL)- receptor activity was not affected by diet. Palmitic and linoleic acids comprised similar proportions in the OA and TFA diets, however the proportion of hepatic phospholipid fatty acids as palmitic acid was lower (p<0.0001) while linoleic acid was higher (p<0.01) in animals fed TFA compared with those fed OA. These findings indicate that in rabbits fed dietary cholesterol (0.2%) TFA do not negatively impact on plasma lipids compared with either OA or PA. Also, increased esterification of TFA and linoleic acid to phospholipids at the expense of palmitic acid in animals fed TFA suggests that feeding TFA may result in cellular membranes with altered physical characteristics.

Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins of rabbits, rats and mice

A fast, accurate and precise high-performance liquid chromatographic assay method has been developed for plasma lipoproteins of experimental animals, rabbits, rats and mice. The method includes complete separation of high, low and very low density lipoproteins from one another within 20 min by a DEAE–glucomannan gel using stepwise elution, and determination by postcolumn reaction with an enzymatic cholesterol reagent as the total cholesterol level. The relative standard deviation of each lipoprotein assay was highly reproducible, being less than 2.0 and 2.4% for repeatability and intermediate precision, respectively. The method was successfully applied to the assays of plasma lipoproteins in three species of normolipidemic and hyperlipidemic animals.