Rat Very Low Density Lipoproteins Research Articles

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Utilization of triacylglycerol‐rich lipoproteins by the working rat heart: routes of uptake and metabolic fates

Very-low-density lipoprotein (VLDL) and chylomicrons (CMs) transport triacylglycerol (TAG) to peripheral tissues. Lipoprotein-TAG may gain access to target cells by lipoprotein lipase (LPL) hydrolysis or via receptor-mediated uptake; the principal routes of entry of VLDL and CM into heart are unknown, and different routes of entry may result in different metabolic fates. To examine this, isolated working rat hearts were perfused with rat VLDL and CMs, dual-labelled with [3H]TAG and [14C]cholesterol. Uptake and utilization of CM-TAG were significantly greater than VLDL-TAG, but both were decreased significantly (more than halved) by tetrahydrolipstatin (THL, an inhibitor of lipoprotein lipase). By contrast, uptake of VLDL-cholesterol was much higher than CM-cholesterol (P < 0.01), and suramin (a lipoprotein receptor antagonist) decreased cholesterol uptake of both forms. CM-TAG oxidation rate was more than 4-fold higher than VLDL-TAG oxidation. However, suramin decreased TAG oxidation from both VLDL and CM without affecting TAG uptake or total utilization, suggesting that the TAG gaining access through receptor-mediated pathways is preferentially 'channelled' towards oxidation. Most (79%) CM-TAG was oxidized whilst the proportion of VLDL-TAG oxidized was only about half (49%). In the presence of suramin, there was a significant increase in esterification (incorporation of assimilated [3H]TAG into myocardial tissue [3H]lipids, mainly TAG) of assimilated TAG from both VLDL and CMs, again suggesting that receptor-mediated TAG uptake is directed towards oxidation rather than esterification. The importance of this relatively small pool of TAG is indicated by the fact that cardiac mechanical function declined markedly when lipoprotein receptors were inhibited. These results suggest that CMs, most fatty acids of which gain access into cardiomyocytes through LPL-mediated hydrolysis, are the major supplier of TAG for hearts to oxidize; however, the metabolic fate of VLDL was split evenly between oxidation and deposition as myocardial tissue lipid. Most importantly, VLDL may play a regulatory role in heart lipid metabolism through a lipoprotein receptor-mediated mechanism.

Utilisation of Fatty Acid and Triacylglycerol by Rat Macrophages: the Effect of Endotoxin

Background/Aims: Plasma very-low-density lipoprotein (VLDL) concentrations are increased during sepsis/endotoxaemia and VLDL may be substrates for the activated immune system. Lipid substrate utilisation by quiescent and activated macrophages was therefore examined. Methods: Rat R2 macrophages were incubated with oleate or rat VLDL, with or without lipopolysaccharide (LPS) stimulation. The metabolic fate of the lipids was examined. Results: Macrophages utilised both oleate and (control) VLDL-triacylglycerol (TAG) to a similar extent. Most was deposited as cellular lipid; about 10% of oleate was oxidised compared to 25% of cVLDL-TAG. LPS significantly increased oleate oxidation and its deposition as cellular lipid (mostly as TAG) at 48hrs but abolished both oxidation and storage of VLDL-TAG. VLDL produced during endotoxaemia (eVLDL) was assimilated by the unstimulated macrophage to a greater extent than oleate or cVLDL-TAG and selectively stored as cellular lipids (no oxidation); LPS decreased eVLDL utilisation. VLDL-TAG utilisation was decreased by the lipoprotein lipase (LPL) inhibitor terahydrolipstatin. LPL activity was greater in oleate than in VLDL incubations, but was increased by incubation with eVLDL. LPS had no effect (oleate, cVLDL) or increased (eVLDL) LPL activity. Conclusion: VLDL represented an efficient substrate for the macrophage. However under conditions of sepsis/endotoxaemia eVLDL utilisation was limited and directed away from oxidation, suggesting that the increased plasma TAG (eVLDL) concentrations resulting from sepsis is not a respiratory fuel for the macrophage but may supply cellular lipid.

Anomalous change of viscosity and conductivity in blood plasma lipoproteins in the physiological temperature range

Study on temperature dependencies of viscosity (η) and conductivity (σ) of blood lipoproteins [high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL)] and A-I apolipoprotein (human and rats) has revealed the presence of the anomalous region at temperature 35–38±0.5°C (Tc). Transition width is 2°C. Viscous flow enthalpy, activation energy (ΔH), and transition enthalpy (ΔHtrans.) as well as thermal coefficients Δη/ΔT and Δσ/ΔT on either side of Tc have been calculated. Transition heat is very low in human HDL, VLDL and apoA-I, and in LDL it is higher by a factor of 4–5. Some mechanisms of the cortisol interaction with HDL and apoA-I have been studied by infrared (IR) spectroscopy and conductometry. The hormone has been found to strengthen the tangle → α-helixes and tangle → β-structures transitions and increase the ordering of lipids. Therewith, ΔHtrans. rises markedly (13 and more times), and at the same time the anomalous region is shifted by 1–2°C in apoA-I. The anomalous change of viscosity and conductivity in the physiological temperature range for all lipoprotein fractions and apoA-I seems to be due to the structural phase transition in both proteins and lipids. In view of the heat capacity jump and a low value of ΔHtrans. in human HDL, one may assume the phospholipids of these particles to exhibit the orientation transition of smectic A↔C type, which is assigned to the second type of phase transition. The structural transition into apoA-I is likely to contain the elements of phase transition of the first and second types. In human and rat VLDL, the smectic → cholesteric phase transition seems to occur. Enthalpy of viscous flow and structural transition in VLDL is higher for rats than for human. The pH shift of the medium to the neutral region (pH 6.1) results in shifting the anomalous region by 1–2°C. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 81: 348–369, 2001

Perfused heart studies to investigate lipid metabolism.

The isolated perfused heart preparation is an invaluable model for investigating metabolism in a variety of physiological and pathological states. It avoids confounding systemic factors (e.g. endocrine, metabolic and work load changes) and permits simultaneous measurement of mechanical function. The ability to measure arteriovenous concentration differences across the myocardium and the coronary flow rate, together with the use of radiolabelled substrates, permits assessment of substrate assimilation and disposition of most potential energetic substrates. In the case of lipids, metabolism of non-esterified fatty acids has been extensively investigated in the perfused rat heart, but fatty acids may also be derived from circulating triacylglycerols (TAG) in lipoproteins [chylomicrons, very-low-density-lipoprotein (VLDL)]. TAG requires initial hydrolysis by the endothelial enzyme lipoprotein lipase and hence an intact heart preparation is vital to maintain tissue structural integrity. Chylomicron-TAG utilization and fate (oxidation, tissue-lipid deposition) in isolated working hearts has been studied using chylomicrons obtained from thoracic-duct catheters. However, lack of availability of sufficient quantities of VLDL has hindered examination of their cardiac utilization; the recent development of a technique to produce large quantities of radio-labelled rat VLDL has facilitated these studies and established that VLDL-TAG is an important metabolic substrate for working heart. Results relating to myocardial utilization of VLDL-TAG under varying physiological (lactation) and pathological (endotoxinaemia) conditions will be presented. The putative role of VLDL as a regulator of cardiac lipid metabolism will also be discussed.

Dietary regulation of the very low density lipoprotein receptor in mouse heart and fat.

The very low density lipoprotein (VLDL) receptor is a member of the LDL receptor family. As opposed to the LDL receptor, the VLDL receptor is expressed primarily in muscle and adipose tissue. Although the VLDL receptor is capable of binding lipoproteins, its functional role is still unclear. Previous studies found that VLDL receptor expression is unaffected by fasting in the rat. The current studies examined whether VLDL receptor expression is altered with fasting in the mouse. Balb/c mice were fasted for periods up to 48 hours, killed, hearts and epididymal fat obtained, and total membranes prepared. To detect the VLDL receptor a portion of the rat VLDL receptor was expressed as a bacterial fusion protein, purified and used to immunize rabbits. The antibodies raised specifically recognized intact VLDL receptor. When cardiac membranes were immunoblotted, VLDL receptor expression increased progressively with fasting, doubling at 36 hours. In contrast, VLDL receptor expression decreased progressively with fasting in membranes from epididymal fat, being reduced 70% by 48 hours. Thus, VLDL receptor expression appears to be regulated in mouse heart and fat by nutritional perturbation, supporting a potential role for the VLDL receptor in the delivery of triglycerides/fatty acids as fuel.

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.

Serum lipids and lipoprotein composition in spontaneously diabetic BB Wistar rats.

Serum lipid and lipoprotein composition in spontaneously diabetic BB Wistar rats, nondiabetic littermates, and control Wistar rats was studied to elucidate diabetes-related abnormalities of lipoprotein composition. Serum total triglycerides and pre-beta-lipoprotein concentrations of insulin-treated spontaneously diabetic BB and nondiabetic littermate rats were significantly higher than those of control Wistar rats. Serum cholesterol and HDL cholesterol concentrations of spontaneously diabetic BB and nondiabetic littermate rats did not differ from controls. Concentrations of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) of spontaneously diabetic BB and nondiabetic littermate rats were higher than those of normal rats. With sodium dodecylsulfate-polyacrylamide gel electrophoresis it was observed that the spontaneously diabetic BB and nondiabetic littermate rat VLDL contained higher percentages of apoE relative to total apoC when compared with control Wistar rats. With isoelectric focusing, apoC-II relative percentages in VLDL and HDL of both spontaneously diabetic BB and nondiabetic littermate rats were higher than apoC-II proportions in VLDL and HDL of controls. Apolipoprotein A-I of the control rat HDL showed four isoforms that focused at pI 5.8 (17.3%), 5.75 (30.6%), 5.65 (31.8%), and 5.55 (20.5%); however, the spontaneously diabetic BB and nondiabetic littermate rat HDL apoA-I was mainly represented by two isoforms that focused at pI 5.8 and 5.75. VLDL of both diabetic and nondiabetic BB rats contained higher levels of acidic apoE isoforms compared to their counterparts in control Wistar rats. Although HDL cholesterol concentrations of spontaneously diabetic BB rats remained normal, protein concentrations were higher resulting in a low cholesterol/protein ratio in HDL suggesting that the cholesterol-carrying capacity of spontaneously diabetic BB rat HDL could be less than normal and may be due to an abnormal apoA-I composition. Quantitative alterations of lipid and lipoprotein composition appear in the BB Wistar rat when compared to the Wistar rat, but some of the changes are more pronounced in the spontaneously diabetic BB Wistar rat.

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins.

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.

Very low density lipoproteins promote triglyceride accumulation in macrophages.

Incubation of mouse peritoneal macrophages with very low density lipoproteins (VLDL) from normal rats or rhesus monkeys markedly increased the levels of intracellular triglycerides by 10- to 56-fold and was accompanied by the production of oil red O positive vacuoles. The stimulation of triglyceride accumulation in macrophages was time- and concentration-dependent and was specific for VLDL. Three possible mechanisms for the VLDL-stimulated triglyceride accumulation in macrophages were explored: receptor-mediated uptake, action of lipoprotein lipase, and phagocytosis. Macrophage uptake and degradation of 125I-monkey and rat VLDL demonstrated saturable and nonsaturable components. Uptake of 125I-VLDL could be inhibited by unlabeled normal VLDL, although hyperlipemic VLDL was more effective. HDL did not compete to a significant extent. Heparin released lipoprotein lipase-like activity from peritoneal macrophages. Addition of heparin with VLDL resulted in a greater, more rapid elevation in intracellular triglycerides, which was partially inhibited by albumin. Free fatty acid and Intralipid also produced triglyceride accumulation in macrophages. The data showed that all three of the mechanisms examined could contribute to the metabolism of VLDL by macrophages and cause the production of triglyceride-rich cells with a "foamy" appearance, although the evidence suggested that the action of lipoprotein lipase was probably the most important in this process.

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity.

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.

Uptake and degradation of rat and human very low density (remnant) apolipoprotein by parenchymal and non-parenchymal rat liver cells

1. 1. The relative contribution of parenchymal and non-parenchymal cells to the in vivo hepatic uptake of serum apolipoproteins was measured 30 min after intravenous injection of radioiodinated rat very low density lipoprotein (VLDL) remnants, rat and human VLDL, low density lipoprotein (LDL) and high density lipoprotein (HDL). Using rat VLDL, VLDL-remnants, LDL and HDL, respectively, the non-parenchymal cells contain 4.7, 4.9, 6.1 and 5.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For human VLDL, LDL and HDL these values are 5.1, 12.0 and 5.9, respectively. 2. 2. The abilities of homogenates of human liver, rat liver parenchymal cells and rat liver non-parenchymal cells to hydrolyze human and rat iodinated VLDL apoprotein were determined by measuring the amount of trichloroacetic acid-soluble (non-iodide) radioactivity liberated upon incubation at the optimal pH of 4.2. Non-parenchymal cells possess a 8–21-fold higher maximal capacity to degrade VLDL apoprotein per mg of cell protein than parenchymal cells. This factor is 5–6 for VLDL-remnant apoprotein degradation measured at low (suboptimal) apolipoprotein concentrations. 3. 3. It is concluded that, in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake and possibly degradation of VLDL-(remnant) apoprotein.

The catabolism of human and rat very low density lipoproteins by perfused rat hearts

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d < 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019–1.063 and 1.063–1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019–1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019–1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.

Synthesis of VLDL by isolated rat hepatocytes in suspension

Very low density lipoprotein (VLDL) synthesis was studied using suspensions of isolated rat hepatocytes prepared and incubated as described previously (1). These hepatocytes synthesized and secreted VLDL over a 24-hr period, in quantities permitting its isolation, without using carrier, for determining absolute synthesis rates and analysing the peptide pattern. The mean secretion of triglyceride, cholesterol and rat VLDL protein was 410 ± 46.6, 36.6 ± 0.1 and 34.9 ± 5.4 (mean ± SEM, n = 5) μg/g hepatocyte/hr over 24 hr, during which incorporation of 3H-valine into VLDL protein approached linearity. Suitable polyacrylamide gel electrophoresis showed the hepatocytes secreted the peptides found in circulating rat VLDL but with a different proportion of the fast to the slowly migrating ones.

Studies on the metabolism of rat serum very low density apolipoprotein.

There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules.

Studies on the radioiodination of very low density lipoprotein obtained from different mammalian species

Very low density lipoprotein (VLDL) isolated from human, pig and rat serum showed marked differences in labelling patterns when radioiodinated under similar conditions, with greater amounts of 125 I-labelled iodine being incorporated into the lipid moieties of pig and rat VLDL than into human VLDL. The effect (on their biological behaviour) of introducing increasing amounts of iodine into the lipoproteins was tested for both human and rat VLDL with preparations containing iodine:protein (I/P) ratios varying from < 0.5 to 12. After injection into rats and analysis of disappearance curves, only minor differences in catabolic rates were observed between different preparations. Thus, rat VLDL, with I/P ratios varying from < 0.5–12 showed fast and slow exponential components varying in half life ( t 1 2 ) between 40–63 min and 10–11 h, respectively, while human VLDL with comparable I/P ratios, had a fast component varying from ( t 1 2 ) 50–78 min and a slower component ( t 1 2 ) of 10.5–12.5 h The plasma disappearance of human VLDL, labelled by the lactoperoxidase method, was also characterised by two exponential components with mean half lives of 67 min and 11 h, respectively, suggesting similarity in the biological behaviour of human VLDL labelled both enzymatically and with iodine monochloride. The results also indicated similarity in the catabolism of rat and human VLDL by the rat, suggesting that this animal is a suitable model for studying VLDL metabolism.

The dissociation in vitro of the alpha- and beta-lipoprotein components of human and rat very low density lipoproteins.

Heparin precipitates of human and rat serum lipoproteins, dissolved, dialyzed, and analyzed by paper electrophoresis, were found to migrate as β-lipoproteins. The pre-β-lipoproteins disappeared. Using specific anti-human β- and α-lipoprotein sera it was shown that the heparin precipitate of human very low density lipoproteins (VLDL) contained β-lipoproteins, whereas the supernatant contained α-lipoproteins. This indicates that these components are dissociated by the heparin precipitation procedure. Following partial delipidation of the heparin precipitate, a procedure which exposes the antigenic site of the α-lipoprotein of the VLDL, it can be demonstrated that the α-lipoprotein component is completely removed by the heparin precipitation procedure. Partially delipidated rat VLDL showed four immuno-reactive components when reacted against rabbit anti-rat VLDL serum, but only one, corresponding to the β-lipoprotein, when reacted with rabbit antiserum to heparin precipitate.Chemical analysis of the lipid components of the heparin precipitate and supernatant of human and rat VLDL reveals that over 90% of the triglycerides are precipitated by the heparin and that the supernatant fraction contains lipoproteins having a phospholipid to protein ratio higher than any of the other lipoproteins.Upon reincubation of the dissolved and dialyzed human serum heparin precipitate and its supernatant a pre-β band reappeared on paper electrophoresis. The recombined pre-β-lipoprotein was isolated and found to react with anti-β-, but not with anti-α-lipoprotein serum. This is a characteristic of native pre-β-lipoproteins. Evidence has also been obtained that the high density lipoproteins combine only with the β-lipoproteins originally present in the VLDL.