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Biomedical subjects

T L Innerarity

Publications and source records attributed to T L Innerarity.

At least 109 records · Page 6Linked to original sources
PubMed1981

Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man. Apo-B,E and apo-E receptors.

We have reported previously that canine livers possess two distinct lipoprotein receptors, an apoprotein (apo)-B,E receptor capable of binding the apo-B-containing low density lipoproteins (LDL) and the apo-E-containing cholesterol-induced high density lipoproteins (HDLc), and an apo-E receptor capable of binding apo-E HDLc but not LDL. Both the apo-B,E and apo-E receptors were found on the liver membranes obtained from immature growing dogs, but only the apo-E receptors were detected on th hepatic membranes of adult dogs. In this study, the expression of the apo-B,E receptors, as determined by canine LDL binding to the hepatic membranes, was found to be highly dependent on the age of the dog and decreased linearly with increasing age. Approximately 30 ng of LDL protein per milligram of membrane protein were bound via the apo-B,E receptors to the hepatic membranes of 7- to 8-wk-old immature dogs as compared with no detectable LDL binding in the hepatic membranes of adult dogs (greater than 1--1.5 yr of age). Results obtained by in vivo turnover studies of canine 125I-LDL correlated with the in vitro findings. In addition to a decrease in the expression of the hepatic apo-B,E receptors with age, these receptors were regulated, i.e., cholesterol feeding suppressed these receptors in immature dogs and prolonged fasting induced their expression in adult dogs. Previously, it was shown that the apo-B,E receptors were induced in adult livers following treatment with the hypocholesterolemic drug cholestyramine. In striking contrast, the apo-E receptors, as determined by apo-E HDLc binding, remained relatively constant for all ages of dogs studied (10--12 ng/mg). Moreover, the expression of the apo-E receptors was not strictly regulated by the metabolic perturbations that regulated the apo-B,E receptors. Similar results concerning the presence of apo-B,E and apo-E receptors were obtained in swine and in man. The hepatic membranes of adult swine bound only apo-E HDLc (apo-E receptors), whereas the membranes from fetal swine livers bound both LDL and apo-E HDLc (apo B,E and apo-E receptors). Furthermore, the membranes from adult human liver revealed the presence of the apo-E receptors as evidenced by the binding of 12--14 ng of HDLc protein per milligram of membrane protein and less than 1 ng of LDL protein per milligram. The membranes from the human liver also bound human chylomicron remnants and a subfraction of human HDL containing apo-E. These data suggest the importance of the E apoprotein and the apo-E receptors in mediating lipoprotein clearance, including chylomicron remnants, by the liver of adult dogs, swine, and man.

Adult↗
PubMed1980

Immunoregulatory plasma lipoproteins. Role of apoprotein E and apoprotein B.

Plasma lipoproteins containing either the B (apo-B) or E (apo-E) apoproteins, e.g. apo-B low density lipoproteins (LDL) isolated from normolipemic humans and apo-E-containing cholesterol-induced high density lipoproteins (apo-E HDLc) isolated from cholesterol-fed dogs, suppress phytohemagglutinin (PHA)-induced lymphocyte activation and inhibit early events such as mitogen-enhanced 45Ca2+ accumulation and late events such as enhanced DNA synthesis. On a molar basis, apo-E HDLc are 3.5 times more effective than apo-B LDL in inhibiting 45Ca2+ accumulation by 50% and 3.8 times more effective than apo-B LDL in suppressing DNA synthesis by 50%. Both lipoproteins bind to the lymphocyte surface, and in competitive binding assays apo-E HDLc and apo-B LDL bind to the same receptors. These receptors, termed immunoregulatory receptors, comprise a homogeneous class of binding sites which do not act cooperatively. The equilibrium dissociation constant (Kd) for apo-B LDL is 2.0 X 10(-7) M at 4 degrees C and 37 degrees C; the Kd for apo-E HDLc is 9.3 X 10(-8) M at 4 degrees C and 7.3 X 10(-8) M at 37 degrees C. At saturation, 16,000 and 20,000 LDL particles are bound/cell at 4 degrees C and 37 degrees C, respectively. The corresponding values for apo-E HDLc are 6,700 at 4 degrees C and 5,500 at 37 degrees C. The increased effectiveness of apo-E HDLc in supp]ressing the PHA-induced Ca2+ accumulation and DNA synthesis is due to the multiple receptor binding of apo-E HDLc. At 37 degrees C, each apo-E HDLc particle occupies multiple receptors at a ratio of 3.6:1 relative to apo-B LDL. The enhanced affinity of apo-E HDLc most likely results from the multiple interactions of this lipoprotein with the receptors.

Apolipoproteins↗
PubMed1980

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.

Animals↗
PubMed1980

Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein by a high affinity receptor. Identity with the chylomicron remnant transport process.

The canine lipoproteins containing only the E apoprotein (apo-E HDLc), when perfused through a rat liver, display high affinity, receptor-mediated uptake, and saturation kinetics. These data indicate that the hepatic uptake of apo-E HDLc proceeds via a finite number of binding sites. The kinetic values determined for apo-E HDLc binding and uptake during a single-pass perfusion through a rate liver are: Km = 4.99 micrograms of protein . ml-1 and Vmax = 6.42 microgram of protein . g-1 . min-1. On a molar basis, the hepatic extraction of apo-E HDLc is almost equivalent to the high hepatic extraction of a class of lipoproteins that is most avidly removed from systemic circulation--the rat chylomicron remnants. Competition experiments between apo-E HDLc and rat chylomicron remnants indicate that the hepatic uptake mechanism for the two macromolecules is identical. Given that the recognition of apo-E HDLc is mediated by the E apoprotein and that the kinetic uptake properties of chylomicron remnants and apo-E HDLc are almost identical, it is probable that the E apoprotein is the major determinant responsible for hepatic chylomicron remnant recognition.

Animals↗
PubMed1980

Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine beta-very low density lipoproteins.

The synthesis and accumulation of cholesteryl esters by monolayers of mouse peritoneal macrophages was stimulated 20- to 160-fold by incubation with beta-migrating very low density lipoproteins (beta-VLDL, density less than 1.006 g/ml) isolated from the plasma of cholesterol-fed dogs. Three other cholesterol-rich lipoprotein fractions obtained from the plasma of the same hypercholesterolemic dogs, including low density lipoprotein (LDL), cholesterol-induced high density lipoprotein (HDLc), and apo-E HDLc, had little to no stimulatory effect. Plasma VLDL (density less than 1.006 g/ml) from normal dogs did not increase cholesteryl ester formation in macrophages. The enhancement in cholesteryl ester synthesis and accumulation by hypercholesterolemic canine beta-VLDL was due to the presence of a high affinity binding site on the macrophage cell surface that mediated the uptake and lysosomal degradation of the beta-VLDL. Competition studies with fucoidin and dextran sulfate indicated that the receptor for canine beta-VLDL was different from that previously described for human acetylated low density lipoprotein (acetyl-LDL). Prior incubation of macrophage monolayers with either unlabeled canine beta-VLDL or human acetyl-LDL, both of which raised the cellular content of cholesteryl esters, reduced the ability of the cells to degrade 125I-labeled beta-VLDL, suggesting that the receptor for beta-VLDL is subject to regulation. The current findings indicate: 1) that macrophages possess a high affinity receptor that recognizes one of the four cholesterol-rich lipoproteins present in the plasma of cholesterol-fed dogs, beta-VLDL, and 2) that the receptor-mediated ingestion of beta-VLDL leads to cholesteryl ester deposition in these cells.

Animals↗
PubMed1980

Inhibition of receptor-mediated clearance of lysine and arginine-modified lipoproteins from the plasma of rats and monkeys.

Reductive methylation of at least 30% of the lysine residues or 1,2-cyclohexanedione modification of 45% of the arginine residues prevented low density lipoproteins (LDL) from binding to cell surface receptors of fibroblasts in vitro, without significantly altering other physical or chemical properties of the LDL. When rat or human LDL with more than 30% of the lysine residues methylated were injected intravenously into rats, the clearance of these lipoproteins from the plasma was slowed considerably. The half-life of the reductively methylated LDL was approximately twice that obtained for control (unmodified) LDL, and the value for the fractional catabolic rate was approximately half that of the control. Furthermore, when human LDL modified by reductive methylation were injected into rhesus monkeys, the rate of clearance was similarly retarded, and the value for the fractional catabolic rate was reduced by approximately 50% as compared with the value for control LDL. A dual isotope labeling technique ((125)I and (131)I) was used to compare the disappearance of the control and modified LDL in the same animal. It was demonstrated that not only modification of lysine residues but also modification of the arginine residues with 1,2-cyclohexanedione retarded the plasma clearance of the rat LDL. However, the cyclohexanedione modification was spontaneously reversible at 37 degrees C, whereas reductive methylation of the lysine residues was stable. It is concluded that the selective chemical modification of lysine or arginine residues of LDL interferes with the normal uptake of these lipoproteins in vivo as well as by fibroblasts in vitro. These data provide an estimation of the level of receptor-mediated clearance of LDL from the plasma, a value that may be as high as 50% in rats and monkeys.

Animals↗
PubMed1980

Cholesteryl ester synthesis in macrophages: stimulation by beta-very low density lipoproteins from cholesterol-fed animals of several species.

Animals fed cholesterol accumulate several types of cholesterol-rich lipoproteins in their plasma and ultimately develop cholesteryl ester deposition in tissue macrophages. Previous studies in the cholesterol-fed dog have shown that one class of cholesterol-rich lipoproteins. beta-migrating very low density lipoproteins (beta-VLDL, density < 1.006 g/ml), possesses a unique ability to produce cellular cholesteryl ester accumulation when incubated with mouse peritoneal macrophages in vitro. This accumulation results from the receptor-mediated uptake of beta-VLDL with subsequent lysosomal hydrolysis of the lipoprotein and re-esterification of the liberated cholesterol. In the current studies, we demonstrate that beta-VLDL obtained from cholesterol-fed animals of several other species, including monkeys, rabbits, and rats, also causes cholesteryl ester accumulation in monolayers of mouse peritoneal macrophages, as monitored by an increase in the rate at which the cells incorporate exogenous [14C]oleate into cholesteryl [14C]oleate. Like canine beta-VLDL, the beta-VLDL from these three other species were effective at low concentrations and exhibited saturation kinetics, suggesting that they, too, entered macrophages by receptor-mediated endocytosis. Very low density lipoprotein (VLDL) from normal animals and low density lipoprotein (LDL) from normal and cholesterol-fed monkeys, rats, and rabbits did not stimulate cholesteryl ester synthesis in mouse peritoneal macrophages. In addition to their effects on mouse macrophages, the beta-VLDL from cholesterol-fed dogs and rabbits stimulated cholesteryl ester synthesis in cultured human monocytes. The current findings suggest that beta-VLDL from cholesterol-fed animals has the general property of stimulating cholesteryl ester synthesis and accumulation in macrophages.

Animals↗
PubMed1979

Accelerated clearance of low-density and high-density lipoproteins and retarded clearance of E apoprotein-containing lipoproteins from the plasma of rats after modification of lysine residues.

Selective chemical modification of lysine residues of lipoproteins by acetoacetylation dramatically altered the metabolism of the lipoproteins without significantly altering other physical or chemical properties. Modification of 30-60% of the total lysine residues of iodinated rat or human low-density lipoproteins ((125)I-LDL) resulted in a rapid removal of these acetoacetylated lipoproteins from the plasma of rats. Within minutes after intravenous injection into intact rats, greater than 80% of the total injected dose disappeared from the plasma. The rapidly cleared acetoacetylated LDL appeared in the liver, and within 6-30 min as much as 50-80% of the total injected dose of modified LDL could be accounted for in the liver. Furthermore, it was possible to demonstrate in the isolated perfused rat liver that the Kupffer cells were responsible for the lipoprotein uptake. Human high-density lipoproteins (HDL(3)) were also rapidly removed from the plasma after acetoacetylation. In striking contrast, acetoacetylation (30-60%) of two E apoprotein-containing lipoproteins (rat HDL(1) and dog HDL(c)) retarded their removal from the plasma. The accelerated removal of modified LDL and HDL(3), in contrast to the retarded removal of modified HDL(1) and HDL(c), suggests that the recognition and removal process is specific for a property acquired by only certain lipoproteins after acetoacetylation. Moreover, these results suggest that lysine residues of the E apoprotein may play a functional role in the recognition process for the normal clearance of HDL(1) and HDL(c), a process that is interfered with after acetoacetylation.

Acetylation↗
PubMed1979

Rate and equilibrium constants for binding of apo-E HDLc (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: evidence for multiple receptor binding of apo-E HDLc.

Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDL(c)) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDL(c) have a much greater binding activity than LDL. Equilibrium and kinetic binding studies were conducted at 4 degrees C to determine the mechanism for this enhanced receptor binding activity. Based on the data, the binding of both LDL and apo-E HDL(c) appears to be a simple bimolecular receptor interaction, and no heterogeneity of binding sites or cooperative effects among the receptor sites were observed. Equilibrium dissociation constants determined by Scatchard analysis of the equilibrium binding data for apo-E HDL(c) (K(d) = 0.12 x 10(-9) M) and LDL (K(d) = 2.8 x 10(-9) M) revealed a 23-fold greater affinity of HDL(c) for the receptors. Association and dissociation rate constants for the lipoprotein-receptor complex were determined from the time course of binding at various lipoprotein concentrations. The equilibrium dissociation constants calculated from these kinetic data confirmed that apo-E HDL(c) had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDL(c) bound more rapidly than LDL with rates of association of 18.0 x 10(4) and 5.5 x 10(4) M(-1) sec(-1), respectively. The rate of dissociation of the apo-E HDL(c)-receptor complex (1.7 x 10(-5) sec(-1)) was slower than that of the LDL receptor complex (6.3 x 10(-5) sec(-1)). An additional important difference between the binding of apo-E HDL(c) and LDL was that 4 times (3.6 +/- 0.4) as many LDL particles as HDL(c) particles were required for saturation of the receptors at maximal binding. These data indicate that each HDL(c) particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding.

Apolipoproteins↗
PubMed1979

Altered metabolism (in vivo and in vitro) of plasma lipoproteins after selective chemical modification of lysine residues of the apoproteins.

Chemical modification of lysine residues by acetoacetylation of the apoproteins of iodinated canine and human low density lipoproteins (LDL) and canine high density lipoproteins (HDL) resulted in a marked acceleration in the rate of removal of these lipoproteins from the plasma after intravenous injection into dogs. Clearance of the lipoproteins from the plasma correlated with their rapid appearance in the liver. Acetoacetylated canine (125)I-LDL (30-60% of the lysine residues modified) were essentially completely removed from the plasma within an hour, and > 75% of the activity cleared within 5 min. Reversal of the acetoacetylation of the lysine residues of the LDL restored to these lipoproteins a rate of clearance essentially identical to that of control LDL. Identical results were obtained with modified human LDL injected into dogs. At 10 min, when congruent with 90% of the acetoacetylated human (125)I-LDL had been removed from the plasma, 90% of the total injected activity could be accounted for in the liver. Furthermore, it was possible to demonstrate an enhancement in uptake and degradation of acetoacetylated LDL by canine peritoneal macrophages in vitro. The mechanism(s) responsible for the enhanced removal of the LDL and HDL in vivo and in vitro remains to be determined. By contrast, however, acetoacetylation of canine (125)I-apoE HDL(c) did not accelerate their rate of removal from the plasma but, in fact, retarded their clearance. Control (native) apoE HDL(c) were removed from the plasma (64% within 20 min) and rapidly appeared in the liver (39% at 20 min). At the same time point, only 45% of the acetoacetylated apoE HDL(c) were cleared from the plasma and <10% appeared in the liver. Acetoacetylation of the apoE HDL(c) did not enhance their uptake or degradation by macrophages. The rapid clearance from the plasma of the native apoE HDL(c) in normal and hypercholesterolemic dogs suggests that the liver may be a normal site for the removal of the cholesteryl ester-rich apoE HDL(c). The retardation in removal after acetoacetylation of apoE HDL(c) indicates that the uptake process may be mediated by a lysine-dependent recognition system.

Acetoacetates↗
PubMed1978

Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

The low density lipoprotein (LDL) cell surface receptors on human fibroblasts grown in culture bind specific plasma lipoproteins, initiating a series of events which regulate intracellular cholesterol metabolism. Specificity for the interaction with the receptors resides with the protein moieties of the lipoproteins, specifically with the B and E apoproteins of LDL and certain high density lipoproteins (HDLc HDLl), respectively. It was previously established that the amino acid arginine is a functionally significant residue in or near the recognition sites on the B and E apoproteins and that modification of this residue abolishes the ability of these apolipoproteins to bind to the receptor. The present study indicates that lysine residues are also involved in the lipoprotein-receptor interaction. Chemical modification of 15% of the lysine residues of LDL by carbamylation with cyanate or 20% by acetoacetylation with diketene prevents the LDL from competitively displacing unmodified 125I-LDL from the high affinity receptor sites or from binding directly to the receptor. Moreover, quantitative reversal of the aceto-acetylation of the lysine residues of LDL by hydroxylamine treatment regenerates the lysyl residues and reestablishes greater than 90% of the original binding activity of the LDL. The reversibility of this reaction establishes that the loss of binding activity which follows lysine modification is not due to an irreversible alteration of the LDL or HDLc but is probably due to an alteration of a property of the recognition site associated with specific lysine residues. While acetoacetylation and carbamylation neutralize the positive charge on the epsilon-amino group of lysine, reductive methylation selectively modifies lysine residues of LDL and HDLc without altering the positive charge, yet abolishes their ability to bind to the receptor. Preservation of the charge but loss of binding activity following reductive methylation of the lipoproteins suggests that the specificity of the recognition site does not reside simply with the presence of positive charges but depends on other more specific properties of the site determined by the presence of a limited number of the lysine (and arginine) residues. The precise role of lysine remains to be defined, but its function may be to establish and maintain the conformation of the recognition site or the alignment of reactive residues, or both, or to chemically react, through its epsilon-amino group, with the receptor (hydrogen bond formation would be such a possibility).

Amino Acids↗
PubMed1978

Alterations in human high-density lipoproteins, with or without increased plasma-cholesterol, induced by diets high in cholesterol.

High-density lipoproteins (d=1.095--1.21) (H.D.L.( were isolated from six healthy men and women who added 4 to 6 eggs per day to their diet for 4 weeks and from five individuals who gradually increased their egg consumption to 3 per day over an 18-week period. Pre-diet and post-diet H.D.L.-binding activities for the cell-surface receptors of fibroblasts were compared by determining the quantity of 125I-labelled low-density lipoprotein which was competitively displaced by H.D.L. in binding, internalisation, and degradation assays. Irrespective of whether plasmacholesterol changed during the course of the diet, the binding activity of the post-diet H.D.L. was enhanced 2.6-fold to 4-fold compared with pre-diet activity. Furthermore, the increased binding activity in the H.D.L. could be accounted for by a minor, but potent, H.D.L. subfraction precipitated by heparin/manganese. Both binding activity and heparin precipitability appeared to correlate with an increase in arginine-rich apoprotein (apo-E) in the active H.D.L. subfraction. These data show that consumption of large numbers of eggs, whether or not it leads to an increase in plasma-cholesterol, does alter the properties of human H.D.L.

Adult↗