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Effects of anti-microtubular agents and cycloheximide on the metabolism of chylomicron cholesteryl esters by hepatocyte suspensions.

ABSTRACT: 1. Post-heparin plasma that promoted rapid hydrolysis of about 90% of the triacylglycerol markedly stimulated the uptake or binding of chylomicron cholesteryl ester by suspended hepatocytes. The net hydrolysis of chyle cholesteryl ester after the uptake by the cells was, however, slower than in vivo. 2. The cholesteryl ester uptake in the presence of post-heparin plasma was larger if the cells had been preincubated for 2h. It was inhibited by the presence of colchicine, vinblastine or cycloheximide during the preincubation, and by mild trypsin treatment of the preincubated cells. 3. The results suggested that the anti-microtubular agents, but not cycloheximide, also inhibited the hydrolysis of chyle cholesteryl ester after uptake or binding to the cells. 4. The uptake of isolated chylomicron remnant particles was more efficient than that of native chyle lipoproteins. It was, however, still stimulated by heparin alone and by post-heparin plasma. The heparin-stimulated uptake was markedly decreased if cycloheximide was present during the preincubation period.

SUBMITTER: Nilsson A

PROVIDER: S-EPMC1164610 | biostudies-other | 1977 Feb

REPOSITORIES: biostudies-other

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Project description:1. The cholesteryl ester of isolated chylomicron-remnant particles was efficiently degraded by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors. 2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached saturation and conformed to a linear double-reciprocal plot. The V(max.) was determined as 80ng of cholesteryl ester/h per mg of protein and the apparent K(m) as 1.4mug of cholesteryl ester per mg of protein. The time course for the uptake and hydrolysis suggested that binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of native chylomicrons were degraded to a much smaller extent and their presence had only a small inhibitory effect on the degradation of chylomicron remnants. Intestinal very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to 50%, but had less influence on the amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake and hydrolysis, whereas d=1.21 infranatant had no effect. 4. The cholesteryl ester hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine. Only 28-36% of the unhydrolysed cholesteryl ester could be released from these cells by trypsin treatment, indicating that the major portion was truly intracellular. The particles that could be released from the cell surface by trypsin and those remaining in the medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant particles. Significant amounts of denser particles were thus not formed during contact with the cell surface. 5. The presence of heparin, as well as preincubation of the cells with heparin, increased the uptake of chylomicron remnants. This effect was most marked in the presence of serum. A much smaller proportion of the other serum lipoproteins was taken up, and this proportion was not increased by heparin.

Project description:Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. For both transport and storage, phospholipids and proteins embrace the CEs to form an amphipathic monolayer that surrounds the CEs. CEs are transported extracellularly in lipoproteins and are stored intracellularly as cytoplasmic lipid droplets. To clarify the molecular phenomena related to the above structures, we conducted atomistic molecular-dynamics simulations for a spherical, approximately high density lipoprotein sized lipid droplet comprised of palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesteryl oleate (CO) molecules. An additional simulation was conducted for a lamellar lipid trilayer consisting of the same lipid constituents. The density profiles showed that COs were located in the core of the spherical droplet. In trilayer simulations, CO molecules were also in the core and formed two denser strata. This is remarkable because the intra- and intermolecular behaviors of the COs were similar to previous findings from bulk COs in the fluid phase. In accordance with previous experimental studies, the solubility of COs in the POPC monolayers was found to be low. The orientation distribution of the sterol moiety with respect to the normal of the system was found to be broad, with mainly isotropic or slightly parallel orientations observed deep in the core of the lipid droplet or the trilayer, respectively. In both systems, the orientation of the sterol moiety changed to perpendicular with respect to the normal close to the phopsholipid monolayers. Of interest, within the POPC monolayers, the intramolecular conformation of the COs varied from the previously proposed horseshoe-like conformation to a more extended one. From a metabolic point of view, the observed solubilization of CEs into the phospholipid monolayers, and the conformation of CEs in the phospholipid monolayers are likely to be important regulatory factors of CE transport and hydrolysis.

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Effects of anti-microtubular agents and cycloheximide on the metabolism of chylomicron cholesteryl esters by hepatocyte suspensions.

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