Plasma metabolism of apolipoprotein A-IV in humans

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.     

Lipoprotein affinity of human apolipoprotein A-IV during cholesterol esterification

We have studied the lipoprotein distribution of human apo A-IV during cholesterol esterification by the action of endogenous lecithin-cholesterol acyltransferase. Using immunologic and radiotracer techniques at 4 degrees C, apo A-IV was found in two discrete monomeric and dimeric populations, unassociated with plasma lipoproteins. With incubation at 37 degrees C, apo A-IV initially associated with the high density lipoprotein-3 fraction, but thereafter dissociated from its surface, and reappeared as unbound protein and in association with a complex in the low density lipoprotein size range. Inclusion of LCAT inhibitors in the incubations abolished these changes. We conclude that the changes in lipoprotein distribution of human apo A-IV closely parallel the formation and exchange of plasma cholesteryl esters.     

Congruence of individual responsiveness to dietary cholesterol and to saturated fat in humans

Previous experiments have shown that differences between humans in the response of serum cholesterol to dietary cholesterol are at least partly reproducible and stable over a prolonged period. In this study it was investigated whether enhanced sensitivity to dietary cholesterol and saturated fat go together. The subjects had also participated in three or four experiments dealing with the reproducibility of the effect on blood cholesterol of either adding cholesterol to the diet in normal subjects (NORM-EGG group; n = 23) or of cessation of egg consumption in subjects with a high habitual egg intake (HAB-EGG group; n = 24). In the present experiment the NORM-EGG subjects were fed a mixed natural diet providing 21% of energy as polyunsaturated and 11% as saturated fat (P/S2 ratio, 1.9) for 3 weeks, and one providing 5% of energy as polyunsaturated fat and 23% as saturated fat (P/S ratio, 0.2) for the next 3 weeks. The HAB-EGG group was fed the same diets in reverse order. The serum cholesterol concentrations were higher on the low P/S diet than on the high P/S diet (on average 23% in normal subjects and 16% in habitual egg eaters). The correlation coefficient between each subject's serum cholesterol response to fatty acids and his or her average response to dietary cholesterol in the dietary cholesterol experiments was 0.62 for the normal subjects (P less than 0.01) and 0.15 for the HAB-EGG group. We conclude that modest differences in responsiveness of serum cholesterol to dietary saturated fat do exist in humans, and that, in people of normal cholesterol intake, responsiveness to dietary cholesterol and to saturated fat tend to go together.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

Effect of ursodeoxycholic acid on cholesterol absorption and metabolism in humans

Qualitative and quantitative changes in intraluminal bile acid composition may alter cholesterol absorption and synthesis and LDL receptor expression. In a randomized crossover design outpatient study, 12 adults aged 24-36 years took 15 mg/kg/day ursodeoxycholic acid (UDCA) or no bile acid supplement (control) for 20 days while being fed a controlled diet (AHA Step II). A liquid meal of defined composition was then given and luminal samples collected. Cholesterol absorption and cholesterol fractional synthetic rate (FSR) were assessed by stable isotopic methods. With UDCA treatment, bile was enriched significantly (P < 0.0001) to 40.6 +/- 2.6% (mean +/- SEM) compared with 2.2 +/- 2.6% for controls. Regardless, plasma total, HDL, and LDL cholesterol were unchanged with UDCA treatment. Intraluminal cholesterol solubilized in the aqueous phase during the entire collection was decreased (P = 0.012) in UDCA-treated subjects (101.0 +/- 7.2 mg/ml/120 min) compared with controls (132.5 +/- 7.2 mg/ml/120 min.). Percent micellar cholesterol was increased in UDCA-treated versus controls after meal ingestion. No changes were found in cholesterol absorption, FSR, or LDL receptor mRNA with UDCA treatment compared with controls. Thus, despite marked enrichment in luminal bile with UDCA and decreased cholesterol solubilization, no differences in cholesterol absorption or metabolism are found when diet and genetic differences in absorption are carefully controlled.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Pancreatic triglyceride lipase deficiency minimally affects dietary fat absorption but dramatically decreases dietary cholesterol absorption in mice

This study generated pancreatic triglyceride lipase (PTL)-null mice to test the hypothesis that PTL-mediated hydrolysis of dietary triglyceride is necessary for efficient dietary cholesterol absorption. The PTL-/- mice grew normally and displayed similar body weight as their PTL+/+ littermates. Plasma lipid levels between animals of various PTL genotypes were similar when they were maintained on either a basal low fat diet or a western-type high fat/high cholesterol diet. Although the lack of a functional PTL delayed fat absorption during the initial hour of feeding a bolus load of olive oil containing [3H]triolein and [14C]cholesterol, the rate of [3H]triolein absorption was similar between PTL+/+ and PTL-/- mice after the initial 1-h period. Importantly, comparison of fecal fat content revealed similar overall fat absorption efficiency between PTL+/+ and PTL-/- mice. In contrast, the PTL-/- mice displayed significant decrease in both the rate and the amount of cholesterol absorbed after a single meal. The plasma appearance of [14C]cholesterol was found to be 75% lower (p < 0.0005) in PTL-/- mice compared with PTL+/+ mice after 4 h. The total amount of [14C]cholesterol excreted in the feces was 45% higher (p < 0.0004) in PTL-/- mice compared with PTL+/+ mice over a 24-h period. These results indicate that the delayed fat digestion due to PTL deficiency results in a significant reduction in cholesterol absorption, although other enzymes in the digestive tract may compensate for the lack of PTL in PTL-/- mice in fat digestion and absorption.     

Effect of dietary cholesterol and alloxan-diabetes on tissue cholesterol and apolipoprotein E mRNA levels in the rabbit

Alloxan-diabetic rabbits develop hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. To determine whether alterations in apolipoprotein composition of plasma lipoproteins were due to changes in apolipoprotein gene expression, we measured the steady state apoE mRNA levels in several tissues from both control and diabetic rabbits. Control rabbits were fed either chow or chow plus 1.5% cholesterol (chow-fed or cholesterol-fed groups) and diabetic rabbits were fed either chow or chow plus 0.5% cholesterol for dietary periods of 5, 21, and 42 days. The tissues examined were liver, small intestine, brain, adrenals, and aorta. ApoE mRNA levels were measured by Northern and dot blot analysis with a human apoE cDNA probe. In control rabbits fed either chow or cholesterol diets for up to 42 days, the steady state apoE mRNA levels remained relatively constant in all of the tissues examined. In contrast, in alloxan-diabetic rabbits fed a 0.5% cholesterol diet, apoE mRNA was reduced in liver, brain, and adrenals (46 +/- 19%, 56 +/- 5%, and 39 +/- 18% of chow-fed control, respectively), but showed little change in the aorta (91 +/- 22% of chow-fed control). Despite a similar increase in plasma cholesterol, the cholesterol content of the liver and adrenals of cholesterol-fed diabetic rabbits were 20% and 50%, respectively, of that of the liver and adrenals in cholesterol-fed control rabbits. The result that apoE mRNA levels and tissue cholesterol content are altered in the diabetic cholesterol-fed rabbit suggests that insulin deficiency in the rabbit may influence apoE gene expression and tissue cholesterol homeostasis.     

The value of apolipoprotein E knockout mice for studying the effects of dietary fat and cholesterol on atherogenesis

The ability of the apolipoprotein E-deficient mouse to develop spontaneous atherosclerosis, which resembles the human process, is an excellent model in which to assess the impact of dietary factors. This review discusses the role of several nutrients in the development of atherosclerosis and the mechanisms through which they act.     

Metabolism of apolipoprotein A-IV in rat

The metabolism of apolipoprotein A-IV (apo-IV) has been investigated in the rat. In this animal species, apoA-IV is a major protein constituent of plasma HDL and lymph chylomicron. The apolipoprotein is also present in the lipoprotein-deficient fraction (LDF) of plasma and lymph. In vivo studies with the radioiodinated protein showed the apoA-IV does not exchange freely between HDL and LDF and that LDF apoA-IV had a faster catabolism than HDL apoA-IV. ApoA-IV in chylomicrons is a direct precursor of apoA-IV in plasma HDL but not of that in LDF. On the other hand lymph LDF apoA-IV is an important precursor of plasma LDF apoA-IV. Transfer of apoA-IV from plasma to lymph is negligible, and since most of apoA-IV in lymph is present in LDF, we speculate that LDF apoA-IV is the major apoA-IV secretory product of the intestine. Studies aimed at identifying the site of catabolism of apoA-IV utilizing either radioiodinated or [14C]sucrose labelled apoA-IV, gave results consistent with the view that the liver plays a major role. When tested, human apoA-IV behaved in vivo in rat as the autologous protein. These findings, together with others previously published (Ghiselli, G. et al. (1987) J. Lipid Res. 27, 813-827), support the conclusion that the plasma metabolism of apoA-IV is remarkably similar in rat and human. We speculate that in mammals the rapid plasma catabolism of apoA-IV is mediated by an efficient uptake by the liver.     

Radioimmunoassay of rat apolipoprotein A-IV

We have developed a specific and sensitive radioimmunoassay for rat apolipoprotein A-IV (apoA-IV). The protocol includes treatment of the samples for 1 h at 60 degrees C with 0.7% Tween 20. Under these conditions, linear logit-log plots have been obtained for apoA-IV in lymph and plasma lipoprotein fractions as well as for purified apoA-IV. The sensitivity of the assay is to 20 ng. Absolute mass values obtained with the assay were validated by comparison with values obtained with an independent method of colorimetric reading of apoA-IV separated by polyacrylamide gel electrophoresis from plasma high density lipoproteins. The concentration of apoA-IV in fasting plasma averaged 10.2 mg/dl and in the mesenteric duct lymph 15.8 and 12.6 mg/dl during the fasting and the fat absorption states, respectively.     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Effects of dietary cholesterol and hypothyroidism on rat apolipoprotein mRNA metabolism

The effects of dietary cholesterol and hypothyroidism on the mRNA levels of rat apolipoproteins A-I, A-IV, and E were measured in extracts of rat liver and rat intestine by hybridization to specific cDNA. Four groups, each comprised of six rats, were fed diets consisting of normal laboratory rat chow and either no supplements (control); 5% lard, 1% cholesterol, and 0.3% taurocholic acid (CF); 5% lard, 1% cholesterol, 0.3% taurocholic acid, and 0.1% propylthiouracil (CF-PTU); or 0.1% propylthiouracil (PTU) for 32 days. At the conclusion of the diets, serum cholesterol, triiodothyronine, and thyroxine levels were measured. The average serum cholesterol concentrations for the four groups were 50.4 +/- 3.7, 75.6 +/- 15.3, 135.3 +/- 41.5, and 73.3 +/- 16.4 mg/dl, respectively. The presence of propylthiouracil in the diets significantly lowered triiodothyronine and thyroxine levels in the serum. The mRNA levels for apolipoproteins A-I and A-IV in rat liver decreased significantly after the feeding of the CF-PTU diet (31 +/- 4% and 32 +/- 3% of normal, respectively) and the PTU diet (34 +/- 8% and 43 +/- 12% of normal, respectively), but showed little change after the CF diet (88 +/- 16% and 108 +/- 15% of normal, respectively). The effects of dietary propylthiouracil on the hepatic mRNA levels for apolipoproteins A-I and A-IV imply a role for thyroid hormones in regulating the mRNA levels for these apolipoproteins in rat liver. ApoE mRNA levels in the rat liver decreased slightly after the CF-PTU diet (74 +/- 12% of normal) and after the PTU diet (73 +/- 10% of normal).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary cholesterol on plasma cholesterol concentration in subjects following reduced fat,high fibre diet.

One hundred and sixty eight subjects participated in a randomised crossover study to determine whether halving or doubling the present dietary cholesterol intake from eggs had any influence on blood cholesterol concentration in people following current dietary recommendations. During the first eight weeks all participants were advised to follow a reduced fat diet (26% total energy for hyperlipidaemic patients, 35% total energy for normolipidaemic volunteers) with an increased ratio of polyunsaturated to saturated fatty acids. This background diet was continued throughout the 16 week experimental period, during which participants ate either two or seven eggs a week. A small but significant increase in total cholesterol was seen after four weeks in the group eating seven eggs a week compared with that in the group eating two eggs a week, but this was no longer apparent after eight weeks. Previous studies suggesting that dietary cholesterol has a greater effect on the serum cholesterol concentration either have been carried out against a background of a higher fat intake or have contrasted extreme cholesterol intakes. A further reduction in dietary cholesterol seems to be unnecessary in those people who have already reduced their intake of saturated fat and increased the ratio of polyunsaturated to saturated fatty acids and fibre rich carbohydrate.     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.