Abstract
Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.
Highlights
Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied
The following two types of labeled HDL preparations were used (i) partially purified HDL3 consisting of plasma of d > 1.125 g/ml (3H-CE-1.125B)and (ii) fully purified HDL3 prepared from 1.125B by centrifugation and freed of apoE by heparinSepharose chromatography.In separate experiments employing eight different cell preparations and five different labeled HDL preparations,with concentrations of HDL cholesterol ranging from 4.8 to 100 pg/ml, from 1.5 to 17.5% of the added counts were converted to steroid products (Table I)
Under these conditions, where 80% or more of thesubstrate cholesterol is derived from HDL [5, 14], Sandoz 58-035 had no effect on steroid hormone production (Fig. 5 B ) indicating that extracellular cholesterolneed not pass through intracellular storage pools before being utilizedfor steroidogenic substrate
Summary
Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Since cholesteryl ethers cannot be hydrolyzed, they do not provide a suitable probe for followingfurther the metabolism ' T h e abbreviations used are: HDL, high densitylipoprotein; ACTH, adrenocorticotrophin;AG, aminoglutethimide; 1.125B, serum of density greater than 1.125 g/ml; CE, cholesteryl ester; LDL, low density lipoprotein; TLC, thinlayer chromatography; LPP, lipoprotein poor media. Materials-Heparin-Sepharose was obtained from Pharmacia LKB Biotechnology Inc. lZ5Iand [3H](1,2)-cholesteryI-oleatewere purchased from Du Pont-New England Nuclear. [3HlCholesteryloleate was chromatographed on TLC plates prior to use
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