Distribution of apolipoproteins A-I and B among intestinal lipoproteins

Chylomicrons and very low density lipoproteins (VLDL) are produced by the intestine and these nascent particles are thought to be similar to their counterparts in intestinal lymph. To study the relationship between these lipoproteins within the cell and those secreted into the lamina propria and lymph, we have isolated enterocytes, lamina propria, and mesenteric lymph from rats while fasted and after corn oil feeding. Apolipoprotein A-I and B content were measured by radioimmunoassay in cell, lamina propria, and lymph fractions separated by Sepharose 6B and 10% agarose chromatography, and by KBr isopycnic density centrifugation. ApoA-I in the cell and the underlying lamina propria was found partly in those fractions in which chylomicron and very low density lipoproteins (chylo-VLDL) and high density lipoproteins (HDL) elute, but more abundantly where unassociated 125I-labeled apoA-I was eluted. In the lymph, however, 74% of apoA-I eluted in the HDL region and no peak of free apoA-I was found. ApoB and apoC-III within the enterocyte were found distributed in the position of particles eluting not only with chylomicrons and VLDL, but also in the regions corresponding to LDL and HDL. In the lamina propria and lymph, on the other hand, most of the apoB was found in the region of VLDL and chylomicrons. These results indicate that the patterns in lymph lipoproteins and the lamina propria do not exactly mirror the distribution of apoA-I and B among lipoproteins inside the cell. This may be because intracellular apoproteins may be unassociated with lipoproteins, or they could be associated with lipoproteins in various stages of assembly of protein with lipids. Furthermore, the apoprotein composition of intestinal lipoproteins is altered after secretion from the enterocyte. Finally, not all apoproteins seem to be secreted in association with identifiable lipoprotein particles from the enterocyte.     

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.     

Human apolipoprotein A-I isoprotein metabolism: proapoA-I conversion to mature apoA-I

ProapoA-I (apoA-i+2 isoform) is the major apoA-I isoprotein secreted by the liver and intestine; however, it is a minor isoprotein in plasma and lymph where the major A-I apo-lipoprotein is mature apoA-I (apoA-I0, apoA-I-1, and apoA-I-2 isoforms). In the present report we provide evidence that apoA-I is rapidly and quantitatively converted to mature apoA-I, and the mature apoA-I isoforms are catabolized at equal rates. In these studies, human proapoA-I was isolated from thoracic duct chylomicrons collected during active fat absorption and mature apoA-I was isolated from plasma high density lipoproteins. The isolated lipoproteins were delipidated, fractionated by gel permeation chromatography, and the individual apoA-I isoforms were separated by preparative isoelectrofocusing. The metabolism of apoA-I isoproteins was studied in normal volunteers (N = 6) in a metabolic ward. In the first study proapoA-I and mature apoA-I (apoA-I0 isoform) were injected simultaneously into two normal subjects and the conversion of proapoA-I to mature apoA-I and the decay of radioactivity were followed in plasma and HDL over a 14-day period. ProapoA-I was rapidly and completely converted to mature apoA-I with a fractional rate of conversion of 4.0 pools/day. The average residence times of proapoA-I and mature apoA-I were 0.23 and 6.5 days, respectively. The mature apoA-I derived from proapoA-I had a residence time which was the same as the injected mature apoA-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular apoA-I and apoB distribution in rat intestine is altered by lipid feeding

Intracellular forms of chylomicrons, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) have previously been isolated from the rat intestine. These intracellular particles are likely to be nascent precursors of secreted lipoproteins. To study the distribution of intracellular apolipoprotein among nascent lipoproteins, a method to isolate intracellular lipoproteins was developed and validated. The method consists of suspending isolated enterocytes in hypotonic buffer containing a lipase inhibitor, rupturing cell membranes by nitrogen cavitation, and isolating lipoproteins by sequential ultracentrifugation. ApoB and apoA-I mass are determined by radioimmunoassay and newly synthesized apolipoprotein characterized following [3H]leucine intraduodenal infusion. Intracellular chylomicron, VLDL, low density lipoprotein (LDL), and HDL fractions were isolated and found to contain apoB, and apoA-IV, and apoA-I. In the fasted animal, less than 10% of total intracellular apoB and apoA-I was bound to lipoproteins and 7% of apoB and 35% of apoA-I was contained in the d 1.21 g/ml infranatant. The remainder of intracellular apolipoprotein was in the pellets of centrifugation. Lipid feeding doubled the percentage of intracellular apoA-I bound to lipoproteins and increased the percentage of intracellular apoB bound to lipoproteins by 65%. Following lipid feeding, the most significant increase was in the chylomicron apoB and HDL apoA-I fractions. These data suggest that in the fasting state, 90% of intracellular apoB and apoA-I is not bound to lipoproteins. Lipid feeding shifts intracellular apolipoprotein onto lipoproteins, but most intracellular apolipoprotein remains non-lipoprotein bound. The constant presence of a large non-lipoprotein-bound pool suggests that apolipoprotein synthesis is not the rate limiting step in lipoprotein assembly or secretion.     

Identification of proapoa-I in rat lymph and plasma: metabolic conversion to "mature" apoA-I

Rat apoA-I polymorphism has been analyzed in lymph and plasma. Two major proteins were present and their relative distribution was different in lymph and plasma lipoproteins. The basic protein (pI 5.60) was quantitatively most abundant among plasma lipoproteins and the acidic protein (pI 5.50) was predominant in lymph chylomicrons and lipoproteins. Microsequence amino acid analysis of the two proteins isolated by preparative isoelectrofocusing revealed that pI 5.50 apoA-I was proapoA-I with six additional amino acids (H2N-Ser-Glu-Phe-Trp-Gln-Gln) at the N-terminal end of "mature" apoA-I (pI 5.60 apoA-I). When radioiodinated proapoA-I was injected in rats, a conversion to "mature" apoA-I was observed and the process reached 92% completion in six hours. These data demonstrate the origin of apoA-I polymorphism in vivo.     

Effect of saturated and unsaturated lipid on the composition of mesenteric triglyceride-rich lipoproteins in the rat

The effect of saturated and unsaturated lipids on the composition of mesenteric lymph triglyceride-rich lipoproteins was studied in rats. A short-term steady-state infusion model was developed in mesenteric lymph fistula rats. Micellar solutions of linoleate, oleate, or palmitate were infused intraduodenally. Steady-state conditions of lymph flow, triglyceride, and apoA-I and apoB secretion rates were achieved in hours 3-5 after the start of the infusion. During this steady-state period, triglyceride-rich lipoproteins were prepared and characterized. With lipid infusion there were the expected increases in secretion rates of triglyceride, apoB, and apoA-I both in whole lymph and in the d less than 1.006 g/ml lipoproteins. Compositional analysis of d less than 1.006 g/ml lipoproteins revealed no difference in the ratios of phospholipid or apoA-I (surface) to triglyceride (core) constituents between saturated and unsaturated lipids, suggesting a similar particle size. This was directly verified by agarose gel filtration and electron microscopy carried out at 27 degrees C, which showed no difference in particle size between linoleate and palmitate chylomicrons. When these lipoproteins were prepared at 4 degrees C, palmitate lipoproteins exhibited dramatically changed gel filtration elution profiles, suggesting a shift to smaller or at least distorted particles and questioning earlier results suggesting a smaller size for saturated fat d less than 1.006 g/ml lipoproteins. Despite the similarity of size between saturated and unsaturated chylomicrons, the apoB content of unsaturated linoleate chylomicrons was significantly lower than that of palmitate chylomicrons. This difference was present whether chylomicrons were prepared by centrifugation or by gel filtration. The clearance of palmitate chylomicrons from the circulation of recipient rats was slightly more rapid than that of linoleate chylomicrons. The mechanism for this apparently selective increase in the apoB content of saturated fat chylomicrons is unknown but the present studies suggest that these changes may be of physiologic significance, perhaps relating to the potential atherogenicity of saturated lipids.     

Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Dietary saturated fatty acid content affects lymph lipoproteins: studies in the rat

We examined effects on intestinal absorption of cholesterol and triglycerides and intestinal lipoprotein formation by feeding rats diets in which saturated fatty acids (palmitic plus stearic) comprised 78%, 68%, 48%, or 38% of triglyceride fatty acids. Absorption into lymph of radiolabeled cholesterol was proportional to triglyceride absorption. The rates of absorption of these lipids were related inversely to the % saturated fatty acids fed. The distribution of newly absorbed cholesterol and triglyceride into intestinal lipoproteins differed. With increasing cholesterol absorption more was recovered in very low density lipoproteins in contrast to the appearance preferentially in chylomicrons of larger quantities of fatty acid. Lymph lipid content did not reflect a consistent pattern in relation to the experimental diet fed. The fatty acid composition of triglyceride-rich lymph lipoproteins resembled the diet closely. One-quarter of the intestinal lymph particles from rats fed the highly saturated diets was flattened and polygonal as judged by electron microscopy if cooled to room temperature; whereas with the same diets, particles collected and isolated at 37 degrees C were round. Proportions of A-I and C apolipoproteins in triglyceride-rich intestinal particles varied inversely; apoA-I increased as fat/cholesterol absorption was greater. Diet-induced alterations in plasma lipoproteins and increased circulating triglycerides in this study in rats were unrelated to the variations in intestinal absorption or lymph lipoprotein formation.     

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins

We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.     

The lecithin-cholesterol acyl transferase activity of rat intestinal lymph.

The lecithin-cholesterol acyl transferase (LCAT) activity in rat mesenteric lymph was examined as a possible source of chylomicron cholesteryl ester. Lymph activity was only 2-3% of rat serum activity. Removal of d less than 1.006 lipoproteins increased lymph LCAT activity, but only to 6-8% of that of serum. Relative to total cholesterol in the d greater than 1.08 g/ml fractions, lymph LCAT activity in lymph from fasting rats was less than serum, but in lymph from nonfasting rats the ratio LCAT/HDL-cholesterol reached levels greater than serum, suggesting a contribution of enzyme from the gut. Both LCAT activity and HDL concentration in mesenteric lymph increased during feeding. Subfractions of lymph that inhibited serum LCAT were: chylomicrons, VLDL, chylomicron lipid, VLDL apoprotein, and HDL apoprotein. In the rat, the low LCAT activity of mesenteric lymph was in part due to the low enzyme concentration present, and the activity was apparently lowered further by lipid-rich lipoproteins that inhibited the reaction. Enzyme inhibition due to the apoprotein fractions of lipoproteins is probably minor in the rat in vivo.     

Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV

Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent chylomicrons.     

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.     

Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of dietary lipids on hepatic mRNA levels of proteins regulating plasma lipoproteins in baboons with high and low levels of large high density lipoproteins.

Selective breeding of baboons has produced families with increased plasma levels of large high density lipoproteins (HDL1) and very low (VLDL) and low (LDL) density lipoproteins when the animals consume a diet enriched in cholesterol and saturated fat. High HDL1 baboons have a slower cholesteryl ester transfer, which may account for the accumulation of HDL1, but not of VLDL and LDL. To investigate the mechanism of accumulation of VLDL + LDL in plasma of the high HDL1 phenotype, we selected eight half-sib pairs of baboons, one member of each pair with high HDL1, the other member with little or no HDL1 on the same high cholesterol, saturated fat diet. Baboons were fed a chow diet and four experimental diets consisting of high and low cholesterol with corn oil, and high and low cholesterol with lard, each for 6 weeks, in a crossover design. Plasma lipids and lipoproteins and hepatic mRNA levels were measured on each diet. HDL1 phenotype, type of dietary fat, and dietary cholesterol affected plasma cholesterol and apolipoprotein (apo) B concentrations, whereas dietary fat alone affected plasma triglyceride and apoA-I concentrations. HDL1 phenotype and dietary cholesterol alone did not influence hepatic mRNA levels, whereas dietary lard, compared to corn oil, significantly increased hepatic apoE mRNA levels and decreased hepatic LDL receptor and HMG-CoA synthase mRNA levels. Hepatic apoA-I message was associated with cholesterol concentration in HDL fractions as well as with apoA-I concentrations in the plasma or HDL. However, hepatic apoB message level was not associated with plasma or LDL apoB levels. Total plasma cholesterol, including HDL, was negatively associated with hepatic LDL receptor and HMG-CoA synthase mRNA levels. However, compared with low HDL1 baboons, high HDL1 baboons had higher concentrations of LDL and HDL cholesterol at the same hepatic mRNA levels. These studies suggest that neither overproduction of apoB from the liver nor decreased hepatic LDL receptor levels cause the accumulation of VLDL and LDL in the plasma of high HDL1 baboons. These studies also show that, in spite of high levels of VLDL + LDL and HDL1, the high HDL1 baboons had higher levels of mRNA for LDL receptor and HMG-CoA synthase. This paradoxical relationship needs further study to understand the pathophysiology of VLDL and LDL accumulation in the plasma of animals with the high HDL1 phenotype.     

Dietary supplementation with Pluronic L-81 modifies hepatic secretion of very low density lipoproteins in the rat

Supplementation of high fat/cholesterol-enriched diets with polyoxypropylene-polyoxyethylene copolymers containing 90% hydrophobic constituents has been found to impair enteric secretion of chylomicrons, lower plasma levels of very low density (VLDL) and low density (LDL) lipoprotein cholesterol and prevent diet-induced hypercholesterolemia and atherosclerosis. These agents are known to be absorbed from the gastrointestinal tract and excreted in bile. In order to determine whether dietary supplementation with this group of hydrophobic poloxalenes influences hepatic secretion of triglyceride-rich lipoproteins, groups of rats were maintained for 21-34 days on either standard chow, semisynthetic diet containing 10.0% safflower oil/1.0% cholesterol, or each of the above diets supplemented with the hydrophobic poloxalene Pluronic L-81. At the end of the feeding period, newly secreted hepatic VLDL were isolated from 2-hr recirculating liver perfusates, quantitated, and characterized. Compared to perfusions in chow-fed rats, perfusion experiments in rats fed the high fat/cholesterol-enriched semisynthetic diet revealed a 3.1-fold increased net hepatic VLDL secretion rate; enrichment of secretory VLDL in cholesteryl esters and in C18:2 core lipid fatty acids; and a shift in the size distribution of secretory VLDL towards larger particles. When the 0.5% Pluronic L-81 was included in the high fat/cholesterol-enriched semisynthetic diet, the net hepatic VLDL secretion rate fell significantly and the physicochemical properties of secretory VLDL in these rats were found to resemble those of chow-fed animals. Supplementation of the chow diet with L-81 resulted in a significant fall in the net hepatic VLDL secretion rate from that observed in rats fed chow alone. Compared to rats fed chow alone, perfusate VLDL from rats fed each of the other experimental diets contained markedly lower amounts of both apoB molecular weight variants, as analyzed by gradient gel electrophoresis and densitometric gel scanning. Since previous studies have demonstrated that VLDL are the major cholesterol transport lipoproteins following fat/cholesterol feeding; a precursor-product relationship exists between fat/cholesterol-induced hepatic VLDL and plasma VLDL; such particles are capable of delivering cholesterol to the arterial wall; and dietary supplementation with hydrophobic poloxalenes prevents both the increase in plasma VLDL-cholesterol and diet-induced atherosclerosis, it is possible that dietary supplementation with hydrophobic poloxalenes may influence the atherogenic process through direct and/or indirect effects on hepatic VLDL transport.     

Effect of the apolipoprotein A-IV Q360H polymorphism on postprandial plasma triglyceride clearance

Apolipoprotein (apo)A-IV is synthesized in the small intestine during fat absorption and is incorporated onto the surface of nascent chylomicrons. In circulation, apoA-IV is displaced from the chylomicron surface by high density lipoprotein-associated C and E apolipoproteins; this exchange is critical for activation of lipoprotein lipase and chylomicron remnant clearance. The variant allele A-IV-2 encodes a Q360H polymorphism that increases the lipid affinity of the apoA-IV-2 isoprotein. We hypothesized that this would impede the transfer of C and E apolipoproteins to chylomicrons, and thereby delay the clearance of postprandial triglyceride-rich lipoproteins. We therefore measured triglycerides in plasma, S(f) > 400 chylomicrons, and very low density lipoproteins (VLDL) in 14 subjects heterozygous for the A-IV-2 allele (1/2) and 14 subjects homozygous for the common allele (1/1) who were fed a standard meal containing 50 gm fat per m(2) body surface area. All subjects had the apoE-3/3 genotype. Postprandial triglyceride concentrations in the 1/2 subjects were significantly higher between 2;-5 h in plasma, chylomicrons, and VLDL, and peaked at 3 h versus 2 h for the 1/1 subjects. The area under the triglyceride time curves was greater in the 1/2 subjects (plasma, P = 0.045; chylomicrons, P = 0.027; VLDL, P = 0.063). A post-hoc analysis of the frequency of the apoA-IV T347S polymorphism suggested that it had an effect on triglyceride clearance antagonistic to that of the A-IV-2 allele. We conclude that individuals heterozygous for the A-IV-2 allele display delayed postprandial clearance of triglyceride-rich lipoproteins.     

Effects of triiodothyronine and propylthiouracil on plasma lipoproteins in male rats

Hyperalphalipoproteinemia, characterized by increased plasma concentrations of apoA-I and of HDL lipid and protein, was observed in rats treated with triiodothyronine (T(3)) for 7 days. The increase in the plasma HDL apoproteins was general for apoC, apoE plus A-IV, and apoA-I, as determined by isoelectric focusing. Hypotriglyceridemia, characterized by decreased concentrations of VLDL and apoB, was also observed in the hyperthyroid state. Although in the mildly hypothyroid animals (propylthiouracil-treated), hepatic metabolism of free fatty acid is shifted toward esterification to triglyceride and VLDL formation, as we reported previously, plasma HDL and apoA-I concentrations were not different from control plasma values, while the d 1.006-1.063 g/ml (IDL + LDL) lipoprotein fraction tended to be increased. In general, the proportion of apoE in the (IDL + LDL) fraction of the hypothyroid rat was greater than in controls and hyperthyroid animals, while the proportion of apoE tended to be lower in VLDL from both hypo- and hyperthyroid rats than in VLDL from controls. An enhanced release of apoA-I by perfused livers isolated from rats treated with T(3) was also observed; this enhanced output of apoA-I may explain, in part, the hyperalphalipoproteinemia observed in these rats. The depressed net output of apoA-I in vitro by perfused livers from rats treated with propylthiouracil (PTU) was not expressed in a statistically significant diminished plasma concentration of HDL or apoA-I in the intact animals. Treatment with T(3) also resulted in modification of the content of essential fatty acids in various lipid classes. Linoleic acid residues were significantly reduced and arachidonic acid content was increased in plasma phospholipids and esterified cholesterol in T(3)-treated rats. However, the relative fatty acid composition of unesterified fatty acids and triglyceride fatty acids was not altered by T(3) treatment. PTU treatment had no effect on fatty acid distribution in any of the plasma lipids. Secretion of biliary lipids was increased in perfused livers from T(3)-treated rats, while treatment with PTU did not affect release of lipids in the bile. These observations suggest a regulatory role for thyroid hormones that determine concentration and composition of plasma HDL and other lipoproteins.-Wilcox, H. G., W. G. Keyes, T. A. Hale, R. Frank, D. W. Morgan, and M. Heimberg. Effects of triiodothyronine and propylthiouracil on plasma lipoproteins in male rats.     

An electron microscopic and functional study of very low density lipoproteins in intestinal lymph

Previous studies with fasting rats showed that the intestine produces endogenous very low density lipoproteins (VLDL) which resemble those in the plasma. Intestinal VLDL also were found to be important in lipid transport during absorption of saturated but not of unsaturated fat. These findings depended upon separations of a chylomicron-rich fraction (S(f) > 400) from VLDL (S(f) 20-400) by preparative ultracentrifugation methods based on particle flotation rates. The present studies correlate this method with electron microscopic measurement of lipoprotein particle size. Almost all intestinal lymph lipoprotein particles from fasting rats were less than 750 A in diameter, and could not be distinguished morphologically from plasma VLDL. Cholestyramine administration or bile diversion led to decreased lymph lipid output, correlating with marked reduction in VLDL. This supports the concept that lymph VLDL contain endogenous lipid which is reabsorbed from the intestinal lumen. During exogenous fatty acid absorption, lymph lipoprotein particle sizes were significantly smaller after administration of palmitate than after administration of linoleate, a finding consistent with ultracentrifugal evidence of the importance of VLDL in lipid transport during palmitate absorption. These studies fully confirm and extend earlier observations. Together, they show that the intestine is a source of endogenous VLDL in the fasting animal. In addition, significant quantities of exogenous lipid are transported in VLDL during palmitate absorption, whereas with linoleate absorption nearly all lipid is in chylomicrons. These findings indicate that the small intestine plays a role in lipoprotein metabolism which extends beyond the absorption of dietary fat.