360

Based on the plasma kinetics of chylomicron triglyceride, retinyl palmitateand apolipoprotein Bog following a lipid meal, chylomicron clearance wasfound to be substantially delayed compared with normolipidemic controls . Infact, when intestinal production was reduced following fasting, accumulationof chylomicron remnants in plasma persisted. Consistent with involvement ofthe LDL receptor in chylomicron clearance, binding and uptake of chylomi-cron remnants by fibroblasts of FH subjects was found to be substantially lessthan cells from controls .

This study identifies that in addition to LDL, chylomicron metabolismis severely impaired in FH . The data suggests that the LDL-receptor issignificantly involved in the clearance of postprandial lipoproteins in man .More importantly, this study raises the possibility that in FH, and in otherdisorders where LDL-receptor expression is reduced, atherogenesis might bea postprandial disease .

HDL (high density lipoprotein) and LDL (low densitylipoprotein) receptors regulation in human adrenal cortex

G. Martin, A. Pilon l , C . Alberta , M. Valle3 , D.W. Hum3 , J .C. Fruchart 12 ,J . Auwerx l , V. Clavey 12 .13 . Staels 1 . t U3251nserm, Dept d'Atherosclerose,Institut Pasteur, BP 245, 59019 Lille ; 2 Fac. de Pharmacie, Univ. de Lille II,59006 Lille, France; 3 Lab. Endocrinol. Mol., CH, Univ. Laval, 2705 BdLaurier, Quebec, GIV4G2, Canada

In rodents cholesterol for adrenal steroidogenesis is mainly derived from HDLvia the HDL receptor, Scavenger Receptor BI (SR-BI) . In the present studySR-BI gene expression and regulation by steroidogenic stimuli was analyzedin the human adrenal gland and compared to LDL receptor gene expression .Northern blot, RT PCR and sequence analysis demonstrated the presence ofhigh levels of SR-BI mRNA in fetal and adult human adrenal cortex . SR-BImRNA was detected in human primary adrenocortical and adrenocorticalcarcinoma NCI-H295 cells, indicating its production by the steroid-producingcells . Treatment of primary adrenal cells with adrenocorticotropic hormone(ACTH) or 8Br-cAMP, a stimulator of glucocorticoid synthesis via the proteinkinase A (PKA) second messenger signal transduction pathway, resulted ina pronounced increase of SR-BI mRNA levels . In NCI-H295 cells differentPKA activators coordinately increased SR-BI and LDL receptor mRNA lev-els . The induction of SR-BI and LDL receptor by cAMP was independentof ongoing protein synthesis and occured at the transcriptional level . Theincrease of SR-BI expression after cAMP was associated with an increasedbinding of HDL3 to and uptake of cholesteryl esters by NCI-H295 cells .In contrast to PKA activators, treatment of NCI-H295 cells with phorbol12-myristate 13-acetate (PMA), which activates the protein kinase C pathwaymediating angiotensin-II induction of mineralocorticoid synthesis, resulted ina time-dependent, transcriptional induction of SR-BI mRNA levels, whereasLDL receptor expression decreased . In conclusion, both the SR-BI and LDLreceptor genes are highly expressed in the human adrenal cortex and coor-dinately regulated by glucocorticoid synthesis activators . In contrast, SR-BIand LDL receptor expression are differentially regulated by activation ofmineralocorticoid synthesis, suggesting that the HDL receptor pathway maybe an important contributor of cholesterol to the human adrenal gland formineralcorticoid synthesis .

4 .P.304*

4.P.305 A reliable flow cytometric assay for functional low densitylipoprotein receptors in human lymphocytes

M. Nagano, T. Iwasaki, H. Hattori, T. Egashira, T. Okada' . Physiobioloy Div.,R&D Center, BML Inc., Saitama; 'Dept. of Pediatr., Nihon Univ. Sch. ofMed., Tokyo, Japan

A simple, specific flow cytometric method to measure the low density lipopro-tein receptor (LDL-R) activity in lymphocytes has been developed for thediagnosis of familial hypercholesterolemia (FH), which is caused by mutationof the LDL-R gene . The LDL-R activity in 94 patients with clinically diag-nosed for FH was measured to evaluate the specificity . In 94 FH heterozygouspatients, the LDL-R activity was 61 ± 16% (mean ± SD) compared to thatof control subjects (100 f 10%), but in 10 patients showed normal activity(>80%o) . Subsequently, the mutations of LDL-R gene were screened by DGGEtechnique and determined the nucleotide sequence by sequencing . 21 differentmutations were identified in 94 FH patients. The mutations of the LDL-Rgene are classified on the basis of the phenotypic behavior of the mutantproteins . According to this classification, the LDL-R activities were 56 ± 11 %(n = 37) in FH patients with receptor-negative type mutations involving Fs98,Fs109, Fs395, W512X, Fs542, 1845 + 2 T-*C, and Fs658 ; 60 ± 15% (n =38) in transport-defective type mutations involving C74S, El 19K, 0200-206,

Thursday 9 October 1997: PostersLipoprotein receptors

C317S, C317R, M351-354/OALN, F382L, A410T, D412H, L547V, P664L .E693K, and M753-759 ; 75 ± 20% (n = 19) in internalization-defective typemutation of K790X. The normal receptor activity was observed in patientswith P664L and K790X. The specificity for the flow cytometric procedure inthis study was 89% . These studies suggested that a flow cytometric procedureof the LDL-R activity might be specific and reliable for the diagnosis of FH .

4 .P.306

4.R307

II th International Symposium on Atherosclerosis, Paris, October 1997

Lipoproteins regulate expression of the class B macrophagescavenger receptor, CD36

A.C . Nicholson, J . Han, M . Febbraio, D .P. Hajjar. Cornell University MedicalCollege, New York NY USA

Uptake of oxidized low density lipoprotein (OxLDL) by macrophages is akey event in the initiation and development of atherosclerotic lesions . Twomacrophage receptors, CD36 (a class B scavenger receptor) and the class Ascavenger receptor have been identified as the major receptors which bind andinternalize OxLDL . Monocyte/macrophage expression of CD36 is dependentthe differentiation state as well as exposure to cytokines and growth factors .The purpose of this study was to determine the role of lipoproteins in mod-ulating CD36 expression . We studied the effect, of native LDL and modifiedLDL (acetylated LDL [AcLDL] and OxLDL) on the expression of CD36 inJ774 cells, a murine macrophage cell line . Exposure to lipoproteins resultedin a marked induction of CD36 mRNA expression (4-8 fold) . Maximuminduction was observed 2 hr. after treatment with AcLDL and at 4 hr. withLDL and OxLDL. Increased expression of CD36 mRNA persisted through24 hr. with each treatment group . Induction of CD36 mRNA expression wasparalleled by an increase in CD36 protein as determined by Western blot, withthe greatest induction by OxLDL (4-fold) . In the presence of actinomycinD, treatment of macrophages with LDL, AcLDL or OxLDL did not affectCD36 mRNA stability. Peritoneal macrophages were then isolated from micefollowing intraperitoneal injection of lipoproteins . Macrophage expressionof CD36 mRNA was significantly increased by LDL, AcLDL or OxLDLrelative to mice infused with PBS, with OxLDL causing the greatest induction(8-fold). This is the first demonstration that lipids augment functional expres-sion of CD36, and imply that lipoproteins can further contribute to foam celldevelopment in atherosclerosis by upregulating a major OxLDL receptor .(NIH grants K01-RR00085 and T32-HL-07423-17)

Modified very low density lipoprotein receptor lacking thefirst repeat in the ligand binding domain binds fl-very lowdensity lipoprotein but not low density lipoprotein

D. Norman, D.D . Patel, X.-M . Sun, B .L . Knight, A .K . Soutar. MRCLipoprotein Team, Clinical Sciences Centre, Hammersmith Hospital, London,UK

The low density lipoprotein receptor (LDLR) and the very low densitylipoprotein receptor (VLDLR) share very similar domain structures, but theLDLR binds both low density lipoprotein (LDL) and lipoproteins contain-ing apolipoprotein E (apoE), while the VLDLR binds only apoE-containinglipoproteins . Although these receptors have highly homologous protein se-quences, one major structural difference is that the ligand-binding domain ofthe VLDLR contains 8 cysteine-rich repeats, compared with 7 in the LDLR .By PCR-based mutagenesis we have constructed a modified VLDLR cDNAfrom which the sequence coding for the first, additional repeat has beenremoved. Expression of the modified and normal VLDLR in stable trans-fectants of LDLR-deficient CHO cells was detected by immunoblotting withpolyclonal anti-peptide antibodies . The modified VLDLR was not detected byan antiserum to the first cysteine-rich repeat of the VLDLR, but was detectedby one to the epidermal growth factor precursor-like domain . Ligand blotanalysis of cell extracts and assay of uptake and degradation of 125 I-labelledlipoproteins by transfected cells showed that the modified VLDLR bindsrabbit ,B-VLDL as well as the normal VLDLR, but still does not bind humanLDL. These results show that the inability of the VLDLR to bind LDL is notbecause of the extra cysteine-rich repeat in its ligand-binding domain, andsuggest that LDL binding by the LDLR must depend on specific amino acidresidues in other repeats of the ligand binding domain that are not present inthe VLDLR.