Normolipidemic Human Subjects Research Articles

HDL-Free Cholesterol Influx into Macrophages and Transfer to LDL Correlate with HDL-Free Cholesterol Content

High-density lipoprotein (HDL)-free cholesterol (FC) transfers to other lipoproteins and cells, the former by a spontaneous mechanism and the latter by both spontaneous and receptor-mediated mechanisms. Macrophages are an important cell type in all stages of atherosclerotic cardiovascular disease (ASCVD), and the magnitude of FC efflux from macrophages to HDL, a metric of HDL function, inversely associates with several metrics of ASCVD. Very high plasma HDL concentrations are associated with increased all cause and ASCVD mortality, suggesting that the reverse process, FC influx from HDL into macrophages, is atherogenic. We hypothesize that HDL-FC is a metric of dysfunctional HDL, and when combined with HDL particle number (HDL-P), is an ASCVD risk factor. The magnitude of FC influx from HDL to macrophages is expected to be a function of HDL-P and HDL-FC content. Here we show that plasma HDL-FC content varies 2-fold among normolipidemic human subjects and linearly correlates with low-density lipoprotein (LDL)-FC content. The influx of HDL-FC into macrophages and transfer to LDL increase linearly with HDL-FC. As expected, influx of HDL-FC into macrophages and transfer to LDL are positively correlated. These data support the hypothesis that high HDL FC content is a marker for dysfunctional HDL, resulting in greater influx into macrophages and greater HDL-FC transfer to LDL. HDL-FC transfer to LDL is a valid surrogate for influx into macrophages. This study of HDL composition and function of normolipidemic subjects provides the basis for further investigation and establishment of HDL-FC content as an ASCVD risk factor.

The roles of different pathways in the release of cholesterol from macrophages

Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins. We determined the effect of enriching cells with free cholesterol (FC) on the release of FC to human serum. Loading Fu5AH cells with FC had no effect on fractional efflux, whereas enriching mouse peritoneal macrophages (MPMs) resulted in a doubling of fractional efflux. Efflux from cholesterol-normal MPM and Fu5AH cells to 15 human sera correlated well with HDL parameters. However, these relationships were reduced or lost with cholesterol-loaded MPMs. Using macrophages from scavenger receptor class B type I (SR-BI)-, ABCA1-, and ABCG1-knockout mice, together with inhibitors of SR-BI- and ABCA1-mediated efflux, we were able to quantitate efflux upon loading macrophages with excess cholesterol and to establish the contributions of the various efflux pathways in cholesterol-normal and -enriched cells. The removal of ABCA1 had essentially no effect on the total efflux when cell cholesterol levels were normal. However, in cholesterol-enriched cells, the removal of ABCA1 reduced efflux by 50%. Approximately 20% of the efflux stimulated by FC-loading MPM is attributable to ABCG1. The SR-BI contribution to efflux was small. Another pathway that is present in all cells is aqueous diffusion. Our studies demonstrate that this mechanism is one of the major contributors to efflux, particularly in cholesterol-normal cells.

Phosphatidylinositol increases HDL-C levels in humans

Studies have shown that phosphatidylinositol (PI) can stimulate reverse cholesterol transport by enhancing the flux of cholesterol into HDL and by promoting the transport of high density lipoprotein-cholesterol (HDL-C) to the liver and bile. The goal of this study was to determine the safety and therapeutic value of PI after oral administration to normolipidemic human subjects. We performed a randomized 2 week study in 16 normolipidemic subjects. Subjects received either 2.8 or 5.6 g of PI, with or without food. PI was well tolerated by all subjects. PI significantly affected the levels of HDL-C and triglyceride in the plasma of subjects receiving PI with food. The lower dose showed a 13% increase in HDL-C, whereas the high dose showed an increase of 18% over the 2 week period. Both low- and high-dose groups showed significant increases in plasma apolipoprotein A-I. The high dose of PI also decreased plasma triglycerides by 36% in the fed subjects. These data suggest that after only 2 weeks, PI may have a comparable therapeutic value to niacin, with negligible side effects.

Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover

Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, involving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measuring VLDL-TG kinetics in normolipidemic human subjects. When administered as a bolus simultaneously, both tracers provided identical VLDL-TG FCRs when the data were analyzed by a compartmental model that accounted for hepatic lipid tracer recycling, but not by non-compartmental analysis. The model-derived FCR was greater than that determined using a non-compartmental approach, and was 2- to 3-fold higher than that usually reported by using a bolus of radioactive [3H]glycerol. When palmitate tracer was given as a constant infusion, VLDL-TG turnover appeared 5-fold slower, because tracer recycling through hepatic lipid pools could not be resolved with the infusion protocol. We conclude that accounting for tracer recycling, particularly the contribution of hepatic glycerolipid pools, is essential to accurately measure VLDL-TG kinetics, and that bolus injection of stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling analysis offers a reliable approach for measuring VLDL-TG kinetics.—Patterson, B. W., B. Mittendorfer, N. Elias, R. Satyanarayana, and S. Klein. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover.

Effect of the fat composition of a single meal on the composition and cytotoxic potencies of lipolytically-releasable free fatty acids in postprandial plasma

Ingestion of a meal increases plasma levels of triglyceride (TG)-rich lipoproteins through the secretion of intestine-derived chylomcirons and liver-derived very low density lipoproteins (VLDL). We have determined the effects of the fat composition of a single meal on the composition of TG in TG-rich lipoproteins (VLDL+chylomicrons) and circulating and lipolytically-releasable free fatty acids (FFA) in postprandial (PP) plasma and on the cytotoxic potencies of the lipolytically-released FFA to cultured arterial wall cells. PP lipemia was induced by feeding fasted normolipidemic human subjects with a meal rich in saturated fat (SF) and another meal rich in polyunsaturated fat (PUF), or vice versa; each meal provided 65% of energy as fat, and polyunsaturated to saturated fatty acid ratios (P/S) of the SF and PUF in the meals were 0.40 and 2.49, respectively. The mean P/S of TG in TG-rich lipoproteins (1.43) and circulating FFA (1.46) in 4 h PP plasma of PUF were significantly higher than those in PP plasma of SF (0.44 and 0.59, respectively) in fasting plasma (0.52 and 0.53, respectively). In vitro lipolysis of fasting and PP serum by purified bovine milk lipoprotein lipase (LpL) resulted in a marked (8.8–12.3-fold) increase in the serum FFA level. The P/S of serum FFA in postlipolysis fasting and PP serum were consistently higher than that of FFA or that of TG associated with TG-rich lipoproteins in prelipolysis fasting and PP serum, indicating that polyunsaturated TG in VLDL and/or chylomicrons is more susceptible than saturated TG to lipolysis. When postlipolysis serum was interacted with cultured endothelial cells and mouse peritoneal macrophages (MPM), the lipolytically-released FFA in PP serum of SF and PUF disrupted the barrier function of endothelial cells and were cytotocxic to cultured MPM; FFA in postlipolysis fasting serum was not cytotoxic. FFA in postlipolysis PP serum of PUF were consistently more potent than that in postlipolysis PP serum of SF. Further study showed that all long-chain monounsaturated FFA and polyunsaturated FFA, but not saturated FFA, incorporated into lipoproteins (LDL) were cytotoxic to cultured MPM. In conclusion, despite the generally well-accepted belief that SF is more atherogenic than PUF, the present study provides in vitro evidence that the lipolytic remnant products of TG-rich lipoproteins produced after a meal rich in PUF are more injurious to arterial wall cells than those produced after a meal rich in SF.

Chimpanzee lipoprotein(A): Relationship between apolipoprotein(A) isoform size and the density profile of lipoprotein(A) in animals with different heterozygous apo(A) phenotypes.

In a previous study [C. Doucet et al., J. Lipid Res 35:263-270, 1994], we have shown that plasma lipoprotein (a) [Lp(a)] levels were significantly elevated in a population of unrelated chimpanzees as compared to those in normolipidemic human subjects. Nonetheless, the inverse correlation between Lp(a) levels and apolipoprotein (a) [apo(a)] isoforms typical of man was maintained in the chimpanzee. In the present study, we describe the density profiles of apo B- and apo A1-containing lipoproteins and of Lp(a) in chimpanzee plasmas heterozygous for apo(a) isoforms after fractionation by single spin ultracentrifugation in an isopycnic gradient. The distribution of apo(a) isoforms in the density gradient was also examined by SDS-agarose gel electrophoresis and immunoblotting using chemiluminescence detection. In all double-band phenotypes examined, the smallest isoform was present along the entire length of the density gradient. The density distribution of the second isoform varied according to the size difference between the respective isoforms. Two isoforms close in size (difference in apparent molecular mass = 60 kDa) were present together in every gradient subfraction. On the contrary, when the two isoforms displayed distinct molecular mass (maximal difference in apparent molecular mass = 340 kDa), then the largest was principally present in the densest fractions of the gradient (d > 1.1 mg/ml). These observations suggest that Lp(a) particles with small apo(a) isoforms are more susceptible to interact with other lipoproteins than are Lp(a) particles with large isoforms.

Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects

Plasma cholesterol level is controlled by various factors. In the present study, high plasma activity of cholesterol esterase was found to correlate with plasma total cholesterol and low density lipoprotein (LDL) cholesterol levels in normolipidemic human subjects. However, the cholesterol esterase is not elevated in plasma of patients with familial hypercholesterolemia. These observations suggest that cholesterol esterase level is not determined by plasma cholesterol level, but elevated cholesterol esterase may be causative in increasing plasma cholesterol and LDL. Additional experiments further demonstrated that cholesterol esterase can convert the larger and less-atherogenic LDL to the smaller and more atherogenic LDL subspecies in vitro. These results suggest that plasma cholesterol esterase contributes to the formation and accumulation of atherogenic lipoproteins, and thus is a major risk factor for premature atherosclerosis in normal human subjects.

Effects of increasing amounts of dietary cholesterol on postprandial lipemia and lipoproteins in human subjects.

Our aim was to determine the effects of increasing amounts of dietary cholesterol (0-710 mg) on the postprandial plasma lipid responses and lipoprotein changes in normolipidemic human subjects. Ten subjects were fed five different test meals in a random order: one meal did not contain fat or cholesterol while the four others contained a fixed amount of lipids (45 g) and 0, 140, 280, and 710 mg cholesterol, respectively. Fasting and post-meal blood samples were obtained for 7 h. Large and small triglyceride-rich lipoproteins (TRL), low density (LDL), and high density (HDL) lipoproteins were isolated. Compared to the no-fat, no-cholesterol meal, the fat-enriched meals raised (P < 0.05) plasma triglycerides, phospholipids, and free cholesterol and lowered cholesteryl esters postprandially. The meals containing zero or 140 mg cholesterol generally elicited comparable postprandial plasma and lipoprotein lipid responses. The meals providing 280 or 710 mg cholesterol significantly increased postprandial plasma phospholipids and large TRL triglycerides and decreased plasma esterified cholesterol. The lipid composition of the large TRLs and the concentrations of the small TRL lipid components were not altered postprandially by cholesterol intake. On the other hand, LDL free cholesterol increased after 3 h, LDL cholesteryl esters dropped after 3 and 7 h, HDL cholesteryl esters dropped after 3 h, and HDL phospholipids increased 7 h after ingesting meals highly enriched in cholesterol. Blood insulin, apoA-I and apoB were not altered postprandially by cholesterol intake. Thus, the data show that ingesting more than 140 mg cholesterol per meal significantly alters the postprandial lipoprotein response in healthy subjects.

Exchanging dietary protein for carbohydrate in normolipidemic human subjects lowers LDL-C

Exchanging dietary protein for carbohydrate in normolipidemic human subjects lowers LDL-C

Rapid in vivo transport and catabolism of human apolipoprotein A-IV-1 and slower catabolism of the apoA-IV-2 isoprotein.

Apolipoprotein (apo) A-IV is a polymorphic, intestinally derived apolipoprotein that is genetically linked to and similar in structure to apoA-I, the major apolipoprotein in high density lipoproteins (HDL). ApoA-IV plays a potentially important role in lipoprotein metabolism and reverse cholesterol transport, but its in vivo metabolism is poorly understood. In order to gain insight into factors modulating apoA-IV metabolism in humans, the in vivo kinetics of the two major human apoA-IV isoproteins apoA-IV-1 and apoA-IV-2 were investigated in normolipidemic human subjects. 131I-apoA-IV-1 and 125I-apoA-IV-2 were reassociated with autologous plasma and injected into study subjects. Analysis of the kinetic data revealed a rapid mean fractional catabolic rate (FCR) for apoA-IV-1 of 2.42 +/- 0.11 d-1. The mean production, or transport, rate of apoA-IV-1 was 16.3 +/- 1.4 mg/kg per d. Plasma apoA-IV concentrations were highly correlated with apoA-IV production rate (r = 0.84, P < 0.001) and not correlated with apoA-IV fractional catabolic rate (r = 0.25, P = NS). The mean FCR of apoA-IV-2 was 2.21 +/- 0.10 d-1. In the ten subjects in whom 131I-apoA-IV-1 and 125I-apoA-IV-2 were simultaneously injected, the FCR of apoA-IV-2 was significantly slower by paired t test (P = 0.003). The FCR of apoA-IV-2 in an apoA-IV-2/2 homozygote was only 1.49 d-1, substantially slower than in all other subjects. We conclude that: (a) apoA-IV is a rapidly catabolized apolipoprotein in humans, with a fractional catabolic rate more than 10 times greater than that of apoA-I; (b) apoA-IV has a high absolute transport rate similar to that of apoA-I; (c) plasma levels of apoA-IV are primarily determined by apoA-IV production rate in normolipidemic subjects; and (d) the fractional catabolic rate of the common variant apoA-IV-2 is slower than that of the wild-type apoA-IV-1.

In vivo metabolism of apolipoprotein A-I on high density lipoprotein particles LpA-I and LpA-I,A-II.

Apolipoprotein (apo) A-I is the major protein in high density lipoproteins (HDL) and is found in two major subclasses of lipoproteins, those containing apolipoprotein A-II (termed LpA-I,A-II) and those without apoA-II (termed LpA-I). The in vivo kinetics of apoA-I on LpA-I and LpA-I,A-II were investigated in normolipidemic human subjects. In the first series of studies, radiolabeled apoA-I and apoA-II were reassociated with autologous plasma lipoproteins and injected into normal subjects. LpA-I and LpA-I,A-II were isolated from plasma at selected time points by immunoaffinity chromatography. By 24 h after injection, only 52.8 +/- 1.0% of the apoA-I in LpA-I remained, whereas 66.9 +/- 2.7% of apoA-I in LpA-I,A-II remained (P less than 0.01). In the second series of studies, purified apoA-I was labeled with either 131I or 125I and reassociated with autologous plasma. Isolated LpA-I and LpA-I,A-II particles differentially labeled with 131I-labeled apoA-I and 125I-labeled apoA-I, respectively, were simultaneously injected into study subjects. The plasma residence time of apoA-I injected on LpA-I (mean 4.39 days) was substantially shorter than that of apoA-I injected on LpA-I,A-II (mean 5.17 days), with a mean difference in residence times of 0.79 +/- 0.08 days (P less than 0.001). These data demonstrate that apoA-I injected on LpA-I is catabolized more rapidly than apoA-I injected on LpA-I,A-II. The results are consistent with the concept that LpA-I and LpA-I,A-II have divergent metabolic pathways.

Diet-induced alteration in the activity of plasma lipid transfer protein in normolipidemic human subjects

Studies were performed to investigate the effect of diets rich in oleic or linoleic acids on the activity of plasma cholesteryl ester transfer protein (CETP) in normolipidemic subjects. Previous to the test diets, all subjects consumed a baseline diet rich in saturated fatty acids (“sat-diet”) for 17 days. The test diets, rich in either monounsaturated fatty acids (“mono-diet”) or rich in polyunsaturated fatty acids (“poly-diet”), were given for 5 weeks to 52 normolipidemic healthy volunteers. The activity of CETP was measured, using a method independent of endogenous plasma lipoproteins, as the rate of exchange of radioactive cholesteryl oleate between labelled LDL and unlabelled HDL. The “mono-diet” induced a statistically significant decrease in CETP activity (from 115 ± 20 to 102 ± 19 units/ml plasma, P < 0.01), while the small decrease on the “poly-diet” (from 111 ± 23 to 107 ± 22 units/ml plasma) did not reach significancy. The percentual decrease in CETP activity induced by the “mono-diet” was higher than that induced by the “poly-diet” as was also found for the decrease in LDL cholesterol. In both diet groups a positive correlation was found between changes in CETP activity and changes in plasma total or (VLDL + LDL) cholesterol. The results suggest that high levels of dietary monounsaturated fatty acids may result in decreased plasma CETP activity, as well as LDL cholesterol levels. The mechanisms of these effects, and their possible interrelations, remain to be established.

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL ( d 1.006–1.019 g/ml) and LDL ( d 1.019–1.063 g/ml) were prepared by centrifugation from the plasma of apo E- 3 3 or 4 3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50–150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [ 14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 μg protein/10 μg IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B, E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with K d values of 2.50, 0.93 μg protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2–3 molecules /particle) and enrichment of IDL with apo E-3 (to 4–5 molecules /particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.

Enhanced metabolism of normolipidemic human plasma very low density lipoprotein in cultured cells by exogenous apolipoprotein E-3.

In this investigation in cultured human fibroblasts, an attempt was made to determine the optimal metabolism of apolipoprotein (apo) B-100 lipoproteins from normolipidemic human subjects. We supplemented culture systems containing 125I-lipoproteins with exogenous recombinant or plasmatic apo E-3. Very low density lipoprotein (VLDL) fractions I, II, and III, and low density lipoproteins (LDL) were prepared from one E 4/3 and four E 3/3 subjects. Without added apo E-3, cellular metabolism (binding, cell association, and degradation) of VLDL-I, II, and III was negligible. Exogenous apo E-3 caused a many-fold enhancement of the metabolism of the three VLDL fractions, but LDL was not affected. The effects of apo E-3 were specific, not observed with apo E-2, and not observed on receptor-negative cells. Exogenous apo E-3 also enhanced down-regulation of cellular sterol synthesis by the VLDLs, but not LDL, indicating increased particle catabolism by the cells. The optimal concentrations of exogenous apo E-3 were 4 to 6 micrograms protein/15 micrograms VLDL-protein, when most of the added apo E-3 became associated with the VLDL particles. Apo E-3 failed to associate with LDL. These results demonstrate that availability and association of adequate amounts of apo E-3 are crucial for optimal cellular metabolism of apo B-100 lipoproteins along the VLDL----LDL cascade.