In vivo contribution of LCAT to apolipoprotein B lipoprotein cholesteryl esters in LDL receptor and apolipoprotein E knockout mice

Previous studies have indicated that LCAT may play a role in the generation of cholesteryl esters (CE) in plasma apolipoprotein B (apoB) lipoproteins. The purpose of the present study was to examine the quantitative importance of LCAT on apoB lipoprotein CE fatty acid (CEFA) composition. LCAT(-/-) mice were crossed into the LDL receptor (LDLr)(-/-) and apoE(-/-) background to retard the clearance and increase the concentration of apoB lipoprotein in plasma. Plasma free cholesterol was significantly elevated but total and esterified cholesterol concentrations were not significantly affected by removal of functioning LCAT in either the LDLr(-/-) or apoE(-/-) mice consuming a chow diet. However, when functional LCAT was removed from LDLr(-/-) mice, the CEFA ratio (saturated + monounsaturated/polyunsaturated) of plasma LDL increased 7-fold because of a 2-fold increase in saturated and monounsaturated CE, a 40% reduction in cholesteryl linoleate, and a complete absence of long chain (>18 carbon) polyunsaturated CE (20:4, 20:5n-3, and 22:6n-3), from 29.3% to 0%. Removal of functional LCAT from apoE(-/-) mice resulted in only a 1.6-fold increase in the CEFA ratio, due primarily to a complete elimination of long chain CE (7.7% to 0%).Our results demonstrate that LCAT contributes significantly to the CEFA pool of apoB lipoprotein and is the only source of plasma long chain polyunsaturated CE in these mice.     

Role of apolipoprotein E and B gene variation in determining response of lipid, lipoprotein, and apolipoprotein levels to increased dietary cholesterol.

A large segment of the population is modifying its dietary cholesterol intake to achieve a healthier life-style. However, all individuals do not respond equally. We have investigated the effects that that two physiologically important polymorphisms in the apolipoprotein (apo) E and B genes have on the responses of plasma lipid, lipoprotein, and apolipoprotein levels to a high-cholesterol diet. Over a 6-wk period, individuals were prescribed two diets, one consisting of 300 mg dietary cholesterol/d for 3 wk and one consisting of 1,700 mg dietary cholesterol/d for 3 wk. Total cholesterol, low-density-lipoprotein cholesterol (LDL-C), and apo B levels were significantly increased on the high-cholesterol diet. Average total cholesterol (numbers in parentheses are SDs) went from 167.6 (23.4) mg/dl on the low-cholesterol diet to 190.8 (36.2) mg/dl on the high-cholesterol diet; LDL-C went from 99.9 (24.8) mg/dl to 119.2 (33.4) mg/dl, and apo B went from 74.9 (24.5) mg/dl to 86.8 (29.5) mg/dl. In 71 individuals, the frequencies of the apo epsilon 2, epsilon 3, and epsilon 4 alleles were .09, .84, and .07, respectively. The frequency of the longer, apo B signal peptide allele (5'beta SP27) was .68. Apo epsilon 2/3 individuals had significantly lower LDL-C levels than did epsilon 3/3 homozygotes, on both the low-cholesterol diet (LDL-C lower by 21 mg/dl) and the high-cholesterol diet (LDL-C lower by 27 mg/dl). Average triglyceride levels were significantly different among apo B signal peptide genotypes, with the 5'beta SP27/37 homozygotes having the lowest levels (70 mg/dl). When individuals were switched from the low-cholesterol diet to the high-cholesterol diet, in no case were the average responses in lipid levels significantly different among apo E or B genotypes. Therefore, these gene loci do not have a major effect on the response of lipid levels to increased dietary cholesterol.     

Rate of gastric emptying influences dietary cholesterol absorption efficiency in selected inbred strains of mice

This study compared the physiological process of cholesterol absorption in different strains of inbred mice with the goal of identifying novel mechanism(s) by which cholesterol absorption can be controlled. The rate and amount of cholesterol absorption were evaluated based on [14C]cholesterol appearance in plasma after feeding a meal containing [14C]cholesterol and by the percentage of [14C]-cholesterol absorbed over a 24 h period. Results showed that the rate of [14C]cholesterol appearance in plasma was slower in 129P3/J mice than in SJL/J mice. However, more dietary cholesterol was absorbed over a 24 h period by 129P3/J mice than by SJL/J mice. In both strains of mice, cholesterol delivered with medium-chain triglyceride was absorbed less efficiently than cholesterol delivered with olive oil. The strain- and vehicle-dependent differences in cholesterol absorption efficiency correlated negatively with stomach-emptying rates. Furthermore, inhibition of gastric emptying with nitric oxide synthase inhibitor increased cholesterol absorption efficiency in SJL/J mice. These results document that stomach-emptying rate contributes directly to the rate of dietary cholesterol absorption, which is inversely correlated with the total amount of cholesterol absorbed from a single meal. Additionally, genetic factor(s) that influence gastric emptying may be an important determinant of cholesterol absorption efficiency.     

Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.     

Plasma lipoprotein profiles and arylesterase activities in two inbred strains of rabbits with high or low response of plasma cholesterol to dietary cholesterol

• 1.1. Cholesterol feeding for 4 weeks of female and male rabbits of two inbred strains increased plasma cholesterol concentrations by about 11 and 48 mmole/I in the hypo- and hyperresponsive strain, respectively.
• 2.2. On the low-cholesterol pre-experimental diet, the hyporesponsive animals had significantly higher plasma HDL (high density protein) cholesterol levels than hyperresponders.
• 3.3. In both strains, cholesterol feeding caused elevations of cholesterol in all lipoprotein classes, the difference between the hypo- and hyperresponsive strains in essence only being observed in the VLDL (very low density lipoprotein) fraction.
• 4.4. Basal plasma total arylesterase activity was significantly higher in the hypo- than in the hyperresponsive rabbits.
• 5.5. Dietary cholesterol caused an increase in plasma esterase activity in both strains.
• 6.6. We suggest that in rabbits a low plasma arylesterase activity and a low concentration of HDL cholesterol are associated with an increased sensitivity to dietary cholesterol.

     

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.     

Effect of apolipoprotein A-IV genotype and dietary fat on cholesterol absorption in humans

We investigated the effect of the A-IV-2 allele, which encodes a Q360H substitution in apolipoprotein (apo) A-IV, and dietary fat on cholesterol absorption in humans. In three separate studies we compared fractional intestinal cholesterol absorption between groups of subjects heterozygous for the A-IV-2 allele (1/2) and homozygous for the common allele (1/1) receiving high cholesterol ( approximately 800 mg/day) diets with different fatty acid compositions. All subjects had the apoE 3/3 genotype. There was no difference in cholesterol absorption between the two genotype groups receiving a high saturated fat diet (33% of total energy as fat; 18% saturated, 3% polyunsaturated, 12% monounsaturated) or a low fat diet (22% of total energy as fat; 7% saturated, 7% polyunsaturated, 8% monounsaturated) diet. However, on a high polyunsaturated fat diet (32% of total energy as fat; 7% saturated, 13% polyunsaturated, 12% monounsaturated) mean fractional cholesterol absorption was 56. 7% +/- 1.9 in 1/1 subjects versus 47.5% +/- 2.1 in 1/2 subjects (P = 0.004). A post hoc analysis of the effect of the apoA-IV T347S polymorphism across all diets revealed a Q360H x T347S interaction on cholesterol absorption, and suggested that the A-IV-2 allele lowers cholesterol only in subjects with the 347 T/T genotype.We conclude that a complex interaction between apoA-IV genotype and dietary fatty acid composition modulates fractional intestinal cholesterol absorption in humans.     

Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.     

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

Retention of apolipoprotein B and cholesterol by perfused heart during lipolysis of very-low-density lipoprotein

The fate and mechanism of removal of apolipoproteins and lipids of human very-low-density lipoproteins were determined in the perfused rat heart. Approx. 50% of the VLDL triacylglycerol was hydrolyzed during a 2 h perfusion. Phospholipid phosphorus, apolipoproteins C-II, C-III and E were quantitatively recovered in the medium. However, there was a loss of unesterified (17 +/- 6%) and esterified (19 +/- 8%) cholesterol from the perfusion medium. Apolipoprotein B was retained by the heart, as determined by the loss of immunoassayable apolipoprotein B (30 +/- 5%) or the uptake of 125I-labelled apolipoprotein of VLDL (9 +/- 2%) from the perfusion medium. The discrepancy in the two methods for estimating apolipoprotein removal was shown to be due to the modification of apolipoprotein B-containing lipoproteins, which was such that they were no longer precipitated with antibodies to apolipoprotein B. The labelled apolipoprotein B, retained by the heart, could be partially released by perfusion of the heart with buffer containing heparin (14 +/- 2%) or trypsin (50 +/- 2%). Labelled apolipoprotein uptake by the heart was reduced by 90% when lipoprotein lipase was first released by heparin or when VLDL was treated with 1,2-cyclohexanedione to modify arginine residues of apolipoproteins. Very little extensive degradation of the apoprotein to low molecular weight material occurred during the 2 h perfusion, since 95% of the tissue label was precipitated by trichloroacetic acid. It is concluded that there is retention of apolipoprotein B, cholesteryl ester and cholesterol by the perfused heart during catabolism of VLDL. The data are consistent with the concept that the retention of apolipoprotein B requires membrane-bound lipoprotein lipase or an interaction with the cell surfaces that is modified by heparin. The overall process also involves arginine residues of apolipoproteins. At least 50% of the labelled apolipoprotein retained in the tissue is associated with lipoprotein lipase and other cell surface sites, while the remainder may be taken up by the cells.