The origin and fate of chylomicrons and VLDL.
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Animals↗
Biomedical subjects
R J Havel
Publications and source records attributed to R J Havel.
Apoprotein content of plasma lipoproteins of the rat separated by gel chromatography or ultracentrifugation.
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Animals↗
Evidence that a separate particle containing B-apoprotein is present in high-density lipoproteins from perfused rat liver.
High-density lipoproteins (HDL) (1.075 less than d less than 1.175) from perfusates of rat liver, unlike those of blood plasma, contain protein with the properties of B-apolipoprotein. This protein remains near the origin upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, has beta electrophoretic mobility in agarose gel, is insoluble in tetramethylurea, and precipitates with antisera to the B-apoprotein isolated from low-density lipoprotein. B-apolipoprotein in HDL from perfusates binds to concanavalin-A Sepharose and can thus be separated from the characteristic HDL, the chemical and physical properties of which are otherwise preserved. These observations suggest that in addition to the discoidal lipoproteins, another particle that contains B-apoprotein exists in HDL of perfusates.
Animals↗
Metabolism of cholesteryl esters of very low density lipoproteins in the guinea pig.
Very low density lipoproteins from guinea pig plasma, endogenously labeled with 3H in both the esterified and free cholesterol moieties, were obtained from serum collected 20 hr after the intravenous injection of 3H-cholesterol into donor animals. When these lipoproteins were injected into recipient guinea pigs, the esterified 3H-cholesterol was rapidly cleared from the plasma; 24% was in the liver in 5 min and 54% in 15 min. A smaller fraction of the esterified cholesterol appeared in other plasma lipoprotein fractions, with 3H in the low density lipoproteins reaching a peak of 9%-18% of the injected esterified 3H-cholesterol between 30 and 60 min after the injection. The results indicate that most of the esterified cholesterol in very low density lipoproteins of guinea pig plasma is removed directly by the liver and a minor fraction is transferred to low density lipoproteins. The pattern of labeling of cholesteryl esters of high density lipoproteins in these experiments suggests that their low concentration in the guinea pig is accompanied by a rapid turnover rate.
Animals↗
Classifications of the hyperlipidemias.
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Apolipoproteins↗
The Prader-Willi syndrome. Regulation of fat transport.
Studies of fat mobilization and transport are reported in six patients with the Prader-Willi syndrome. Two patients had carbohydrate intolerance. One of these had a low and the other an augmented insulin response to glucose challenge. Following challenge with glucose, three of the four nondiabetics had normal insulin responses or increased responses consistent with their obesity; the other nondiabetic had insulinopenia. Measurements of the effects of norepinephrine, insulin, glucose, and 5-methylpyrazole-3-carboxylic acid on plasma levels of FFA, glycerol, and ketones provide no evidence for abnormal regulation of mobilization of fat from adipose tissue. Measurements of plasma lipids and postheparin lipolytic activity are consistent with normal uptake of fat into adipose tissue, and normal fatty acid composition of adipose tissue gives no evidence for abnormal lipogenesis.
Adipose Tissue↗
The significance of lipoprotein lipase in rat skeletal muscles.
Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.
Adipose Tissue↗
Lipoprotein lipase.
The activity of lipoprotein lipase in the vascular bed represents the major pathway by which triglyceride fatty acid is cleared from the plasma and made available to the epripheral tissues. Recent studies on the properties of the enzyme, both solubilized and membrane-bound have provided new information on the regulation of its activity with the major triglyceride-rich lipoprotein substrates. A key role for this enzyme in the regulation of plasma triglyceride levels is indicated from studies of lipase levels in human adipose tissue and in blood plasma obtained after injection of heparin.
Adipose Tissue↗
Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.
Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.
Animals↗
Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia.
Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.
Abetalipoproteinemia↗
Radioimmunoassay of apolipoprotein A-I of rat serum.
A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.
Animals↗
New protein in human blood plasma, rich in proline, with lipid-binding properties.
A protein that binds to a lecithin-stabilized triglyceride emulsion has been separated from plasma after removal of major lipoprotein classes by ultracentrifugation at density 1.21 g/ml. This protein, rich in proline, has been purified to electrophoretic and immunochemical homogeneity by subsequent gel and ion-exchange chromatography. In native plasma and after purification, it exists as a large particle exceeding 10(6) daltons, but a single component with a molecular weight of about 74,000 is found upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Although the purified protein contains very little bound lipid and is not present in the major lipoprotein classes from post-absorptive individuals, it is present in chylomicrons. Its concentration in plasma varies from 12 to 41 mg/dl and is significantly correlated with that of cholesterol in lipoproteins of very low and low density but not in those of high denisty.
Amino Acids↗
Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.
Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.
Animals↗
Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver.
Rat livers were perfused for 6 h without added plasma proteins using washed erythrocytes and buffer in a recirculating system. An inhibitor to the enzyme lecithin-cholesterol acyltransferase (5,5'-dithionitrobenzoic acid) was added in some experiments to prevent modification of substrate-lipids contained in secreted lipoproteins. The inhibitor did not detectably alter hepatic ultrastructure or gas exchange, but it inhibited the secreted lecithin-cholesterol acyltransferase by more than 85%. Very low density lipoproteins in perfusate were unaltered but the high density lipoproteins obtained from livers perfused with the inhibitor appeared disk-shaped in negative stain by electron microscopy with a mean edge thickness of 46 +/- 5 A and a mean diameter of 190 +/- 25 A. The high density lipoproteins were composed predominantly of polar lipids and protein with only small amounts of cholesteryl esters and triglycerides. The major apoprotein of these discoidal fractions had the same electrophoretic mobility as the arginine-rich apoprotein, whereas plasma high density lipoproteins contained mainly the A-I approtein. In all these respects the discoidal perfusate high density lipoproteins closely resemble those found in human plasma which is deficient in lecithin-cholesterol acyltransferase. Perfusate high density lipoproteins obtained in the absence of the enzyme inhibitor more closely resembled plasma high density lipoproteins in chemical composition (content of cholesteryl esters and apoproteins) and in electron microscopic appearance. Purified lecithin-cholesterol acyltransferase synthesized cholesteryl esters at a substantially faster rate from substrate lipids of perfusate high density lipoproteins than those from plasma. The discoidal high density lipoproteins were the best substrate for this reaction. Thin sections of plasma high density lipoproteins indicated a spherical particle whereas discoidal high density lipoproteins stained with the characteristic trilaminar image of membranes. These observations suggest that the liver secretes disk-shaped lipid bilayer particles which represent both the nascent form of high density lipoproteins and preferred substrate for lecithin-cholesterol acyltransferase.
Animals↗
Mathematical evaluation of methods for estimation of the concentration of the major lipid components of human serum lipoproteins.
The concentration of total cholesterol and triglycerides in the three major lipoprotein classes of human serum was measured in 136 men, randomly selected from an industrial population, by a quantitative method of lipoprotein electrophoresis on agarose gel and of fractions separated by preparative ultracentrifugation. Correlation coefficients for the two estimates were 0.98 for triglycerides in very low-density lipoproteins, 0.93 for total cholesterol in low-density lipoproteins, and 0.75 for total cholesterol in high-density lipoproteins. Data obtained form the analyses of the ultracentrifugal fractions were used to develop regression equations that predict the concentrations of total cholesterol and triglycerides in the lipoprotein classes from their concentrations in whole serum. These equations take into account the inverse curvilinear relationship between total cholesterol in high-density lipoproteins and serum tiriglyceride concentration. When applied to a separate sample of 530 men, the predicted values for triglycerides in very low-density lipoproteins and total cholesterol in low-density lipoproteins correlated as well with ultracentrifugal values as did the electrophoretic estimates. However, for total cholesterol in high-density lipoproteins, the electrophoretic method was superior. Similar regression equations were developed from ultracentrifugal lipoprotein analyses in 158 women from the same industrial population. Although the concentration of total cholesterol in the low-density lipoproteins estimated by both electrophoresis and the regression equations agreed closely in most cases with the ultracentrifugal values, errors exceeded 10% with sufficient frequency to limit the value of the estimates for this purpose. In both men and women, the ratio of total cholesterol to triglycerides in high-density lipoproteins was a hyperbolic function of serum triglyceride concentration, suggesting that cholesteryl esters in the core of this lipoprotein are progressively replaced by triglycerides as the concentration of triglycerides in very low-density lipoproteins increases. This altered composition of nonpolar lipids accounts, at least in part, for the reduction of cholesterol in high-density lipoproteins in hyperlipemic individuals.
Adolescent↗
Lipoproteins and lipid transport.
Continued advances in the delineation of pathways of lipid transport in lipoproteins now provide substantial information on all phases of plasma triglyceride transport. Analysis of certain genetic human disorders, together with studies in experimental animals, has begun to show how lipoproteins transport cholesterol as esters of long chain fatty acids. Both triglycerides and cholesterol are esters of long chain fatty acids. Both triglycerides and cholesteryl esters are transported in the "core" of lipoproteins, but the polar lipids and the apoproteins at the aqueous interface critically determine the interactions with enzymes and cellular receptors that control this complex transport system. Differences in pathways as well as in rates of lipid transport appear to underlie the large interspecies variations in lipoprotein concentrations.
Animals↗
Metabolism of cholesteryl esters of rat very low density lipoproteins.
Rat very low density lipoproteins (d smaller than 1.006), biologically labeled in esterified and free cholesterol, were obtained form serum 6 h after intravenous injection of particulate (3-H) cholesterol. When injected into recipient animals, the esterified cholesterol was cleared form plasma with a half-life of 5 min. After 15 min, 71% of the injected esterified (3-H) cholesterol had been taken up by the liver, where it was rapidly hydrolyzed. After 60 min only 3.3% of the amount injected had been transferred, via lipoproteins of intermediate density, to the low density lipoproteins of plasma (d 1.019-1.063). Both uptake in the liver and transfer to low density lipoproteins occurred without change of distribution of 3-H in the various cholesteryl esters. 3-H appearing in esterified cholesterol of high density lipoproteins (d greater than 1.063) was derived from esterification, presumably by lecithin: cholesterol acyltransferase, of simultaneously injected free (3-H) cholesterol. Content of free (3-H) cholesterol in the very low density lipoproteins used for injection could be reduced substantially by incubation with erythrocytes. This procedure, however, increased the rate of clearance of the lipoproteins after injection into recipient rats. These studies show that hepatic removal is the major catabolic pathway for cholesteryl esters of rat very low density lipoproteins and that transfer to low density lipoproteins occurs to only a minor extent.
Animals↗
Characterization of remnants produced during the metabolism of triglyceride-rich lipoproteins of blood plasma and intestinal lymph in the rat.
The metabolism of intravenously injected large and small chylomicrons from intestinal lymph and of very low density lipoproteins from blood plasma was studied in functionally eviscerated "supradiaphragmetic" rats. For studies with lymph lipoproteins, recipient animals were injected with 4-amino-pyrazolopyrimidine 18 h before injection of lipoprotein to prevent secretion of very low density lipoproteins into their blood plasma. In all cases, most of the triglycerides (labeled with 14C) were rapidly metabolized, whereas cholesteryl esters (labeled with 3H) persisted in the blood. Most of the cholesteryl esters remained in smaller "remnant" lipoproteins, less dense that 1.006, which retained an apparently spherical shape, as determined by electron microscopy of negatively stained preparations. Whereas the diameters and chemical compositions of large chylomicrons were substantially different from those of small chylomicrons and very low density lipoproteins, all remnants were similar in these respects. Average remnant diameters were 400-600 A and remnants were enriched in cholesteryl esters and in protein insoluble in tetramethylurea. In addition to triglycerides, remnants were depleted of phospholiarticle size, the composition of remnants, like that of their precursors, was consistent with the "pseudomicellar" model of lipoproteins, in which a core of nonpolar lipids is covered by a monolayer of polar lipids and protein. These results domonstrate the fundamental similarity of the initial step in the metabolism of triglyceride-rich lipoproteins from intestinal mucosa and liver and show that loss of triglycerides from the core of the particles is accompanied by removal of polar components from the surface.
Animals↗