Cytochalasin-stimulated steroidogenesis from high density lipoproteins

The cytochalasins stimulate steroid secretion of Y-1 adrenal tumor cells two-to threefold. The order of potencies is cytochalasin E is greater than D is greater than B, but the maximum response is the the same and always less than with ACTH. Like that with ACTH, the stimulation has a rapid onset, is easily reversible, is inhibited by cucloheximide and aminoglutethimide, and occurs at a stage before pregnenolone. Although the cytochalasin, like ACTH, produce cell rounding, it is shown that this morphological change is not necessarily coupled to steridogenesis. Unlike ACTH, cytochalasin B does not measurably increase cellular levels of cAMP at concentrations that lead to maximal steroidogenesis. The cytochalasin B-induced stimulation of steroidogenesis, unlike the short-term ACTH effect, fails to occur in the absence of serum. This lack of response can be corrected by even low concentrations of human high density lipoproteins (HDL) but not by low density lipoproteins (LDL). We, therefore, propose that cytochalasin B enhances the availability of cholesterol bound to HDL for steroidogenesis by Y-1 adrenal cells.     

Effects of guanidine hydrochloride on human plasma high density lipoproteins.

Denaturation of human plasma high density lipoproteins during ultracentrifugation in guanidine-HCl is characterized by: dissociation of apoA-I, in the range of 2-3 M guanidine-HCl, and dissociation of apoA-I and apoA-II in 5-6 M guanidine-HCl. Denaturation of high density lipoprotein species, during a sequence of timed exposure to guanidine-HCl followed first by removal of the denaturant by dialysis and then by ultracentrifugation, is characterized by:dissociation of lipid-poor apoA-I, which follows a time course similar to denaturation-related changes in reported spectroscopic parameters; and apparent formation of lipoprotein aggregation products depleted in apoA-I and relatively enriched in apoA-II. These studies indicate differential properties of the major apoproteins in stabilizing high density lipoprotein structure and characterize a mode of lipoprotein transformation and degradation which apparently results from apoprotein dissociation coupled with aggregation of denatured lipoprote species.     

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

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

alpha-cyclodextrin extracts diacylglycerol from insect high density lipoproteins

alpha-Cyclodextrins are water-soluble cyclic hexamers of glucose units with hydrophobic cavities capable of solubilizing lipophiles. Incubating alpha-cyclodextrin with high density lipophorin from Manduca sexta or Bombyx mori resulted in a cloudy, turbid solution. Centrifugation separated a pale yellowish precipitate. Thin-layer chromatography analysis of the lipid extract of the precipitate showed that the major lipid was diacylglycerol, while KBr density gradient analysis of the supernatant demonstrated the presence of a lipid-depleted very high density lipophorin. Transfer of diacylglycerol from lipophorin to cyclodextrin was specific to alpha-cyclodextrin and was not observed with beta- or gamma-cyclodextrins. pH had no effect on diacylglycerol transfer to alpha-cyclodextrin. However, the transfer was strongly dependent on the concentration of alpha-cyclodextrin and temperature. Increasing the concentration of alpha-cyclodextrin in the incubation mixture was associated with the formation of increasingly higher density lipophorins. Thus, at 20, 30, and 40 mm alpha-cyclodextrin, the density of B. mori lipophorin increased from 1.107 g/ml to 1.123, 1. 148, and 1.181 g/ml, respectively. At concentrations greater than 40 mm, alpha-cyclodextrin had no further effect on the density of lipophorin. alpha-Cyclodextrin removed at most 83;-87% of the diacylglycerol present in lipophorin. Temperature played an important role in altering the amount of diacylglycerols transferred to alpha-cyclodextrin. At 30 mm alpha-cyclodextrin, the amount of diacylglycerol transferred at different temperatures was 50% at 4 degrees C, 41% at 15 degrees C, 20% at 28 degrees C, and less than 3% at 37 degrees C. We propose that diacylglycerol transfers to alpha-cyclodextrin via an aqueous diffusion pathway and that the driving force for the transfer is the formation of an insoluble alpha-cyclodextrin-diacylglycerol complex.     

Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose-response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.     

Human plasma lipid transfer protein catalyzes the speciation of high density lipoproteins

The role of purified plasma lipid transfer protein complexes in determining the particle size distribution of human plasma high density lipoproteins (HDL) was examined in vitro. Incubation of HDL2 or HDL3, isolated from normolipemic subjects with very low density lipoproteins (VLDL) or VLDL-remnants and lipid transfer protein complex had little or no effect on HDL particle size. In contrast, HDL isolated from patients with hypertriglyceridemia, designated HDL3D, showed speciation of particle size distribution when incubated with VLDL-remnants and the transfer protein. Incubation of HDL3D with VLDL-remnants and lipid transfer complex resulted in the production of two particles of radius 4.3 and 3.7 nm; incubation with VLDL or in the absence of the transfer protein did not result in a redistribution of particle size. We suggest that the action of lipid transfer protein complex on triacylglycerol-rich lipoprotein remnants and HDL accounts for the low levels of HDL-cholesterol observed in subjects with severe hypertriglyceridemia.     

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of nascent high density lipoproteins containing arginine-rich protein in human plasma.

A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism.     

In vivo transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins in man.

The fate of cholesteryl esters in high density lipoprotein (HDL) was studied to determine whether the transfer of esterified cholesterol from HDL to other plasma lipoproteins occurred to a significant extent in man. HDL cholesteryl ester, labelled in vitro with [3H] cholesterol, was injected into human subjects. Labelling of cholesteryl esters in very low density (VLDL) occurred rapidly and by 3 h, the esterified cholesterol in VLDL reached peak specific radioactivity. The removal rate of cholesteryl esters from HDL appeared to be exponential and of the order of 0.2/h; calculation of the apparent flux was about 150 mg/h which approximates reported values for total cholesterol esterification in human plasma in vivo. The rapid rate of labelling of VLDL from HDL suggests that the transfer of HDL cholesteryl esters to VLDL may represent a significant pathway for the disposal of HDL cholesterol.     

Immunochemical study of the plasma low and high density lipoproteins in Tangier disease

Major disturbances of the lipoproteins in Tangier serum have been investigated using electrophoretic and immunochemical techniques. Previously described anomalies concerning the striking deficiency in HDL and the very low levels of apo A-I and apo A-II in Tangier patients are illustrated and explained. Anomalies concerning the fast LDL of Tangier serum are attributed to different forms of apo B not previously described. These data are strengthened by the features of a 2-dimensional electrophoresis method elaborated in the laboratory which allows apoproteins to separate in the second dimension. These apoproteins are obtained by the delipidation of the lipoproteins fractionated in a first polyacrylamide discontinuous gel. This method clearly shows the distribution of apoproteins in the first lipoprotein track and is in perfect accordance with the new concept of lipoprotein particles.     

Apolipoprotein A-II content of human plasma high density lipoproteins measured by radioimmunoassay.

A double antibody radioimmunoassay for human ApoA-II is reported. ApoA-II isolated from human plasma high density lipoprotein (HDL) by column chromatography migrated as a single band on polyacrylamide disc gel electrophoresis, had the appropriate amino acid composition, and provoked the production of monospecific antisera. (125)I-ApoA-II (iodinated by lactoperoxidase, purified by Sephadex G-75 chromatography) migrated with "cold" ApoA-II as a single band on disc gel electrophoresis in SDS. Its specific radioactivity was 5-12 mCi/ micro g. In assays, (0.05 M barbital buffer, 0.01% Triton X-100, pH 8.6) over 90% of (125)I-ApoA-II was bound by excess first antibody and over 95% was displaced by excess "cold" ApoA-II. Low density lipoprotein, very low density lipoprotein, ApoA-I, ApoC-II, and ApoC-III displaced no counts. Intraassay and interassay coefficients of variation for lipoprotein or plasma samples were 7 +/- 4 and 11 +/- 6%, respectively. As little as 1.0 ng of ApoA-II was detectable with a precision of 10%. ApoA-II made up 20-25% of the proteins of HDL (d 1.083-1.19), HDL(2) (d 1.083-1.124), and HDL(3) (d 1.124-1.19) on column chromatography. The ApoA-II contents of these HDL fractions were also 20-25% by radioimmunoassay. Similar results were obtained whether assays were carried out on intact or delipidated HDL samples. Thus, in contrast with ApoA-I (only 10% of which is detectable), all of the ApoA-II contents of intact HDL are detected with accuracy by this assay. Plasma levels of ApoA-II in young normolipemic subjects were approximately 40 mg/dl (n = 29). In these subjects, over 98% of ApoA-II was found in the d 1.063-1.21 density fractions.     

Dynamics of lipid-protein interactions. Interaction of apolipoprotein A-II from human plasma high density lipoproteins with dimyristoylphosphatidylcholine

ApoA-II and dimyristoylphosphatidylcholine (DMPC) spontaneously associate to give three different complexes whose structures are determined by the initial reactant concentration and by the reaction temperature with respect to Tc (23.9 degrees C), the gel to liquid crystalline transition temperature of DMPC. At an initial lipid to protein ratio of 45/1, a single complex (2.29 x 10(5) daltons) is quantitatively formed at all temperatures between Tc - 4 degrees C and Tc + 6 degrees C. When the 45/1 complex is mixed with DMPC liposomes there is lipid exchange but no net transfer of lipid, so that the structure of the complex remains unaltered. At an initial molar ratio of 100 to 300:1, the reaction scheme is more complex. At 24 degrees C a 240/1 complex (1.5 x 10(6) daltons) is formed from a precursor 75/1 complex (3.43 x 10(5) daltons) if excess (approximately 300 mol/mol) lipid is present. The 75/1 complex exhibits lipid exchange in the presence of added DMPC liposomes at 24 degrees C, and both the 75/1 and the 240/1 complex can be converted to smaller protein-rich complexes in the presence of added apoA-II. These results suggest that the initial lipid/protein ratio and the physical state of a lipid or lipid . protein complex determines the composition and structure of the resulting complex and support the view that lipid-protein interactions are stronger than protein-protein or lipid-lipid interactions.     

Association of lysolecithin acyltransferase with the high density lipoproteins and its activation by the low density lipoproteins in normal human plasma.

The lysolecithin acyltransferase of human plasma is shown to be associated with the high-density lipoprotein fraction. Although the low density lipoproteins do not have intrinsic enzyme activity, their presence activated the enzyme 3--7-fold. This activation is not affected by heat-treatment of the low density lipoproteins, but is abolished by the addition of heparin.