Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver.

Abstract

A B S T R A C T Rat livers were perfused for 6 h without added plasma proteins using washed erythrocytes and buffer in a recirculating system. An inhibitor to the enzyme lecithin-cholesterol acyltransferase (5,5'-dithionitrobenzoic acid) was added in some experiments to prevent modification of substrate-lipids contained in secreted lipoproteins. The inhibitor did not detectably alter hepatic ultrastructure or gas exchange, but it inhibited the secreted lecithin-cholesterol acyltransferase by more than 85%. Very low density lipoproteins in perfusate were unaltered but the high density lipoproteins obtained from livers perfused with the inhibitor appeared disk-shaped in negative stain by electron microscopy with a mean edge thickness of 46±5 A and a mean diameter of 190±25 A. The high density lipoproteins were composed predominantly of polar lipids and protein with only small amounts of cholesteryl esters and triglycerides. The major apoprotein of these discoidal fractions had the same electrophoretic mobility as the arginine-rich apoprotein, whereas plasma high density lipoproteins contained mainly the A-I apoprotein. In all these respects the discoidal perfusate high density lipoproteins closely resemble those found in human plasma which is deficient in lecithin-cholesterol acyltransferase. Perfusate high density lipoproteins obtained in the absence of the enzyme inhibitor more closely resembled plasma high density lipoproteins in Portions of this work have appeared in an abstract and in a review (1, 2).Dr. Hamilton is the recipient of a Research Career Development Award. Dr. Williams was the recipient of a Postdoctoral Fellowship from the National Heart and Lung Institute. Dr. Fielding was an Established Investigator of the American Heart Association.Received for publication 30 January 1976 and in revised form 12 April 1976. chemical composition (content of cholesteryl esters and apoproteins) and in electron microscopic appearance. Purified lecithin-cholesterol acyltransferase synthesized cholesteryl esters at a substantially faster rate from substrate lipids of perfusate high density lipoproteins than those from plasma. The discoidal high density lipoproteins were the best substrate for this reaction. Thin sections of plasma high density lipoproteins indicated a spherical particle whereas discoidal high density lipoproteins stained with the characteristic trilaminar image of membranes. These observations suggest that the liver secretes disk-shaped lipid bilayer particles which represent both the nascent form of high density lipoproteins and preferred substrate for lecithin-cholesterol acyltransferase. INTRODUCTIONThe plasma lipoproteins are usually separated into four major physically defined groups although it is recognized that heterogeneity exists within each. This heterogeneity is further complicated by movements of both lipids and apoproteins between particles of the different groups. One approach to obtain a clearer understanding of the physiological significance of the different plasma lipoproteins h...

Interaction of microtubule proteins with phospholipid vesicles.

“…2 b). This stacking pattern resembles that obtained from has- cent high density lipoproteins (HDL) (15). At higher concentrations the vesicles appear to fuse into multilamellar sheets (Fig.…”

Hepatic Secretion of Lipoproteins in the Rat and the Effect of Experimental Nephrosis

“…The high triglyceride content of the nascent HDL that we reported earlier (3) has been confirmed. It may be related to the presence of apo B in this fraction that has also been found by Hamilton et al (19). Those investigators have shown that the apo B-containing portion of nascent HDL can be separated from the rest of this fraction by concanavalin-A affinity chromatography (20).…”

Isolated hepatocytes from hypercholesterolemic rats secrete discoidal lipoproteins

“…A similar mechanism was proposed for the accumulation of discoidal HDL in the sera of cholesterol-fed guinea pigs [6]. It is significant to note that nascent discoidal HDL with a FC:PL molar ratio of 0.65 isolated from perfused rat livers is, in contrast, a good substrate for LCAT [5]. The lipoproteins secreted into fraction 1 are spherical despite their high free cholesterol-to-phospholipid molar ratio.…”

“…De novo synthesis of discoidal HDL by perfused livers from normal and hypercholesterolemic African green monkeys was shown in [23]. The tendency of the discoidal lipoproteins to stack into rouleau formations, (fig.2b,c) is characteristic of negatively stained preparations of these abnormal lipoproteins obtained from animal [5,7] or human studies [ 1,2].…”

“…Abbreviations: LCAT, lecithin-cholesterol acyl-transferase; VLDL, very low density lipoproteins; HDL, high density lipoproteins; DTNB, 5,5'dithionitrobenzoic acid;EDTA,ethylenediaminetetraacetic acid;FC,free (unesterified) Isolated hepatocytes from hypercholesterolemic rats were prepared as in [ 111 with extensive cell washing (5-6 washes) to remove particulate fat. The hepatocytes from 2 livers were incubated at 37'C as a suspension for up to 24 h in 600 ml of Waymouth's MB 752/l medium supplemented with 17.5% heatinactivated, delipoproteinated horse serum, 10 000 U penicillin/ml and 0.05 mM streptomycin sulphate.…”