CETP expression increases HDL cholesteryl ester tissue uptake and plasma fractional catabolic rate in transgenic mice

114

Friday 28 May 1999 Poster presentation: Enz3,mes and transJer proteins #zvolved in intravascular modeling of lipoproteins

of free radical activity in tissues. Inhibition of LDL oxidation suggests that the enzyme may also play an important role in the pathogenesis of atherosclerosis. ESTRADIOL TRANSFER BETWEEN LIPOPROTEINS A. H/Sekerstedt, M. Jauhiainen, H. Adlercreutz, M.J. Tikkanen. Department of Medicine. Helsinki University Central Hospital: National Public Health Institute, Helsinki, Finland The aim of the study was to explore the molecular mechanisms underlying the cardioprotective effects of estrogens We focused on the association of estradiol with lipoproteins and the possible transfer nlechanisms between lipoproteins. 171~,-estradiol ( E ) is a relatively hydrophilic molecule and therefore unlikely to bind to lipoproteins in the free form. It has been suggested that E2 may form lipophilic derivatives, E2 fatty acid esters, in human blood. On this basis we explored the esterification and incorporation of E2 into lipoproteins. Following incubation of plasma with 3H-E2, we isolated lipoproteins by sequential ultracentrifugation and purified them by gel filtration on Sephadex G-25 to remove free, unattached 3H-E2. Most of the radioactivity coincided with the protein peak of LDL or HDL. The LDL and HDL fractions were extracted and further subjected to gel chromatography on Sephadex LH20 demonstrating that most of the radioactivity was recovered in the ester fraction. The experiment was repeated in the presence of dithio-2-nitrobenzoic acid (DTNB), the inhibitor of lecithin: cholesterol acyl transferase (LCAT). Almost no radioactivity was detected in lipoproteins in the presence of DTNB. suggesting that LCAT was catalyzing the esterification of E2, and that this was required for incorporation of E2 into lipoproteins. However, as LCAT is associated only with HDL but not with other lipoproteins, our finding of 3H-E2-esters also in LDL-fraction raised the question how E2esters might be transferred from HDL to LDL. Therefore we investigated the possible role of cholesterol ester transfer protein (CETP) in the transfer of 3H-E2 between these lipoproteins. We incubated non-radioactive LDL with HDL containing 3H-E~-esters (prepared as above) with and without CETP in phosphate buffered saline (PBS). After incubation, the lipoproteins were again separated by ultracentrifugation. The transfer of E2-esters from HDL to LDL could then be assessed by measuring the radioactivity of both lipoproteins. The results show that CETP accelerated the transfer of E2-esters from HDL to LDL but was not the only factor involved. This finding was further strengthened by demonstrating that addition of increasing amounts of CETP enhanced the transfer in a dose-dependent fashion. In conclusion, we were able to demonstrate that 3H-E2 was esterified and incorporated into both HDL and LDL. In addition, we present preliminary evidence that CETP is involved in the E2-ester transfer from HDL to LDL. Further studies are needed to determine to what extent these in vitro findings reflect the process occurring in oivo. LCAT INDUCED ACCUMULATION OF ESTRADIOL INTO HDL H. Helisten, O. Ylikorkala, A. Tiitinen, H. Adlercreutz, M.J. Tikkanen.

Department of Medicine. Helsinki University Central Hospital; Department of Gynecolog3'; Folkhalsan Research Center and Department of Clinical Chemistr)., Helsinki, Finland We explored the possibility that estrogen esters might be formed and incorporated into lipoproteins where they could, in theory, act as antioxidants. Ovarian as well as exogenous estrogens are believed to decrease the risk of coronary heart disease in women. Oxidative modification of LDL and HDL may have a role in the pathogenesis of atherosclerosis. Accordingly, estrogen could oppose oxidation of LDL and HDL. The most important estrogen, 171~estradiol (E2), has a ring structure somewhat similar to that of cholesterol. Most of the cholesterol in lipoproteins is present in esterified form. Lecithin: cholesterol acyltransferase (LCAT) is known to be the esterifying enzyme. Recent reports indicate that the fatty acid esters of E2 are present in human tissues. These lipophilic molecules may be incorporated into lipoproteins in contrast to unesterified E2. Human ovarian follicular fluid was chosen for this experiment because of its high E2 concentration and the presence of one lipoprotein, HDL, in this material. To investigate E2 ester formation, follicular fluid was incubated with tritiated estradiol ([3H]E2) for 4 and 24 hours at +37°C with and without the LCAT inhibitor 5.5-dithiobis 2-nitrobentzoic acid (DTNB). After incubation HDL was isolated by ultra--centrifugation and purified by gel filtration to remove unbound estrogens and other molecules not associated

with HDL. Next the HDL-containing fraction was extracted and subjected to gel chromatography to separate E2 esters from unesterified E2. The radioactivity was measured from in the ester- and free E2-fractions to find out whether [3H]E2 had been esterified and incorporated into HDL. The radioactivity present in the HDL farction consisted mainly of E2 esters. However, addition of DTNB abolished the 3H-label completely from HDL. Hence it may be concluded that E2 esterification occurred during incubation in follicular fluid, and such E2 esters were incorporated into HDL. The almost complete suppression of E2 esterification in the presence of DTNB indicates that this reaction was catalyzed by LCAT. While we were able to demonstrate E2 esterification and incorporation into HDL in our experimental system, it is not clear to what extent this occurs in uiuo and what the physiologic role of this might be. LIPOPROTEIN LIPASE CYS z39- > TRP: A NEW LOSS OF FUNCTION MUTATION ASSOCIATED WITH TYPE I HYPERLIPOPROTEINEMIA M.M. Hoffmann 1 S. Matthaei 2, H. Wieland 1, W. M/irz 1, ;Department of Internal Medicine; Division of Clinical Chemistr3.'; Albert-L,~dwigsUniuersio; Freiburg i. Br.; 2Department of Internal Medicine; Eberhard-Karls-Uniuersio,. Tfibingen. Germany Lipoprotein lipase (LPL) is the major enzyme responsible for the hydrolysis of triglyceride-rich lipoproteins in plasma. Genetic deficiency of LPL causes type I hyperlipoproteinemia syndrome, which is characterised by the presence of chylomicrons in fasting plasma and a marked increase in plasma triglyceride levels. Several types of mutations occurring predominantly in exons 4, 5, and 6 of the LPL gene have been shown to cause LPL deficiency: missense, nonsense, deletion, insertion, and splicing mutations. The index case, a 47-year-old German woman, presented with severe type I hyperlipoproteinemia (triglycerides 1233 mg/dll. The apolipoprotein CII concentration was normal (6.9 mg/dl) and apoCII was able to activate LPL in an in uitro assay. In post hepann plasma no LPL mass and activity was detectable whereas hepatic lipase activity was normal. Direct sequencing of all ten exons of the LPL gene lead to the detection of a hithero unknown mutation in exon 6, Cys "~3'~- > Trp. The patient was homozygous for this mutation. The mutation prevents the formation of the second disulfide bridge of LPL, which is part of the lid covering the catalytic center. There is one other missense mutation, disturbing the formation of a disulphide bridge, Cys418Tyr (Henderson et al. BBRC; 227: 189-1941, leading to the secretion of inactive LPL into the plasma. Recently. the role of disulfide bridging in the stability of LPL was determined in vitro by generating variants with individually substituted cysteine pairs (Lo et al. BBRC; 206:266-271 ). In contrast to our observation the authors found that LPL, in which the formation of the Cys216-Cys239 disulfide bridge was disturbed, was secreted (22% of wild type mass), whereby the mutant was of course inactive. It is not surprising that the Cys239Trp mutation lead to complete absense of protein in plasma because the possibility of wrong cysteine bridging within the LPL is very probable if one cysteine is missing. Consistently, Busca et al (JBC; 271: 2139-2146) could show that misfolded LPL is rapidly degraded within the ceils. CETP EXPRESSION INCREASES HDL CHOLESTERYL ESTER TISSUE UPTAKE AND PLASMA FRACTIONAL CATABOLIC RATE IN TRANSGENIC MICE L.M. Harada 1, RM. Cazita t, J.A. Berti 2, E.C.R. Quint~o 1, A.R. Tall 3, H.C.F Oliveira 2. l Lipids Laborato~. , LIM-IO. UnioersiO, of Sdo Paulo

Medical School. Sdo Paulo: 2Department of Ph.vsiology and Biophysics, Institute of Biology, State University of Campinas (UNICAMP). Campinas. SP. Brazil; 3Department of Medicine. Columbia UniuersiO" New York. USA The role of cholesteryl ester transfer protein (CETP) in the tissue uptake of HDL cholesteryl ester tissue uptake and plasma removal rate was investigated in transgenic (Tg) mice. Male and female heterozygous human CETP expressing mice (line 5203), aged 14-16 weeks, and nontransgenic (non-Tg) C57BL/6J control mice were used. Human HDL labeled with 3H-cholesteryl oleoyl ether (3H-HDL) was adminsitered intraperitoneally ( 1,000,000 dpm/animal) into mice, and blood samples were drawn from the tail at 0.5, 1, 2, 3, 4, 6, 8 and 24 h for radioactivity determination. Twenty four hour after the 3H-HDL injection, 12 organs were excised (abdominal muscle, small intestine, liver, lungs, spleen, heart, kidneys, adrenals, testis or ovaries, adipose tissue, skeletal muscle and brain) and radioactivity was measured in the tissue lipid extracts. The 3H-HDL plasma fractional

71st EAS Congress and Satellite Symposia

Fridat: 28 Mat, 1999 Poster presentation." Enzymes and tran,~er proteins involved in intrauascular modeling of lipoproteins

I 15

calabolic rates (FCR) were calculated from the plasma radioactivity curves. Male and female CETP Tg mice exibited an increase of 79% (p < 0.0001 ) and 38% (p < 0.005), respectively, in 3H-HDL FCR as compared to nonTg control mice. ~H-HDL tissue uptake was significantly increased in liver, adrenals and adipose tissue by 47% (p < 0.005), 50% (p < 0.005) and 42% (p < 0.05) respectively in male CETP Tg mice and 32% (p < 0.005), 24% (0.0001) and 97% (p < 0.0001 ) respectively in female CETP Tg mice. Taken together these results indicate that the decrease o f plasma HDL concentration brought about by CETP is due to stimulation of cholesteryl ester uptake by the liver, adrenals and adipose tissue, and favors the reverse cholesterol transport system. Supported by FAPESP, S~o Paulo, Brazil

transfer from HDL to LDL. Hyperthyroid animals had twice as much plasma CETP activity as compared to their controls, while in hypothyroid animals plasma CETP activity did not change. Tissue uptake of HDL 3Hcholesteryl ether (Cet) was examined in eleven tissues. Hyperthyroid mice exibited a significant increase in skeletal muscle HDL-CEt uptake (p = 0.016) and decrease in small intestine and spleen (p < 0,05). Hypothyroid animals showed a decrease in small intestine HDL-Cet uptake (p = 0.03). In conclusion, the decrease in HDL levels found in hyperthyroid animals can be explained by an increase in HDL plasma removal rate, muscle uptake and CETP activity. However, hypothyroid status did not change HDL metabolism and CETP activity. Financial support: FAPESE FINEP/PRONEX, FAEP/UNICAMP

INFLUENCE OF PLASMA LIPASES AND LIPID TRANSFER PROTEINS ON THE PHOSPHOLIPIDS (PL) AND FREE CHOLESTEROL (FC) TRANSFER FROM LIPID EMULSION TO HDL

CARBAMAZEPINE INFLUENCES CONCENTRATIONS OF 27BUT NOT OF 24S-HYDROXYCHOLESTEROL: EVIDENCE FOR CYTOCHROME-P450-MEDIATED METABOLISM OF 27HYDROXYCHOLESTEROL

V.S. Nunes 1, RM. Cazita '1, L.M. Harada ~. E.C.R. Quintfio 1,

S. Locatelli, S. Brfimswig, D. Liitjohann, K. von Bergmann, H.K. Berthold. Department of Clinical Pharmacology. UniversiO, of Bonn.

H.C.E Oliveira 2. t Lilfids Lab.. LIM-IO. University of S~o Paulo Medical &.hool." "Dept. Pt~vsialogv and Biopto,sic, State University of Campinas (UNICAMP). Sdzo Pavia. SP. Brazil Plasma lipases and lipid transfer proteins activities may be involved in HDL generation and speciation. In this study, we examined the influence of plasma lipases and lipid transfer proteins on the ~H-FC and t4C-PL transfer from lipid emulsion to human, rat and mouse lipoproteins. Human plasma (control. post-heparin and post-heparin + lipasc inhibitor) and rat plasma /control and post-heparin) were incubated with 3H-FC/14C-PL-emulsion during 30 minutes at 37"C and lipoproteins were separated by FPLC. The table shows the pcrcent transfer of 3H-FC and 14C-PL from the emulsion to human HDL Po~t-heparm~PII~

t. o m r o /

lIt-F("

14C-PL

~II-F("

14('-PL

PIt * LIpase inhibitor 3H-FC I'I('-PL

I~

31

18

50 a'h

15

39

(7 2~1

(IS 471

(12 3()1

(35 6M)

18-33]

123--.18)

~,lanrJ-WhJlney Icsl. a P}I • o m l r o | , p - 0 0 0 1 ; b P}I ~ P[-I + h p a s c inhibltt)r, p < 1) 1)1

The results of control rat plasma were similar to post-heparin human plasma. The effect of CETP on PL transfer was analysed in human plasma Icontrol and and with CETP monoclonal antibody) and mouse plasma icontrol and CETP transgenic) incubated with laC-PL-emulsion. The presence of CETP inhibitor reduced the PL transfer to human HDL by approximately 50%. p < 0.05. Otherwise no difference was observed on the PL transfer in CETP transgenic mouse comparing to control. In conclusion, human and rat plasma lipases stimulated the PL transfer to HDL but did not influence the FC transfer. CETP influenced the PL transfer to human HDL. since its inhibition reduced the transfer. However, comparing control and CETP transgenic mouse plasma, no effect of CETP on the PL transfer was observed. THYROID HORMONES INCREASE HDL CATABOLISM AND PLASMA CHOLESTERYL ESTER TRANSFER PROTEIN (CETP) ACTIVITY IN CETP TRANSGENIC MICE H.C.E Oliveira 1, J.A. Berti 1, M.E.C. Amaral 1. A.C. Boschero 1, L.M. Harada 2, A.R. Tall 3. ~Department qfPhysiotoKv and biophysics. State

Sigmund-Freud-Str. 25. D-53105 Bonn. Federal Republic of Germam, Introduction: The antiepileptic drug carbamazepine (CBZ), a cytochromeP450-enzyme inductor, is known to influence lipoprotein metabolism, mainly by increasing total cholesterol (Chol) and LDL-cholesterol. Oxysterols are formed during cholesterol catabolism by cytochrome-P450-dependent hydroxylation pathways. 24S-hydroxycholesterol (24S-OH-Chol), an oxysterol mainly produced in the brain, is of importance for cholesterol transport through the blood-brain barrier whereas 27-hydroxycholesterol (27-OHCholL mainly synthezised in extrahepatic tissues, plays a role in removal o f cholesterol from macrophages. The latter mechanism may be of importance for development of atheroscterosis. Materials and Methods: In a prospective study we investigated whether CBZ administration to healthy young male volunteers influences the concentration of the two oxysterols in serum. Twenty healthy young men (mean age of 32 + 4 years) were treated with CBZ (2-3 x 400 mg per day) for 7.5 to 9 weeks. CBZ serum concentrations ranged from 4.8 to 9.2 p.g/ml (median 6.6 ~.tg/ml). Total cholesterol and LDL-C were measured enzymatically; 24SOH-Chol and 27-OH-Chol were measured using a highly sensitive isotope dilution method with gas-chromatography mass spectrometry. Cholesterol and oxysterol concentrations under drug treatment were compared with baseline measurements. Results: Total and LDL-cholesterol significantly increased from 1914-31 to 218±32 mg/dl and 1275:29 to 1525:33 mg/d~ respectively (means:SD; p < 0.0011. A significant decrease was observed for 27-OH-Chol from 1725:40 to 1335:25 ng/ml (-21%; p < 0.001), whereas 24S-OH-Chol was not altered during treatment with CBZ (72± 18 to 735:13 ng/ml), Conclusions: The results indicate that the concentration of 27-OH-Chol but not of 24S-OH-Chol is reduced by CBZ treatment. The reduction of 27OH-Chol is probably related to an increased catabolism by 7ct-hydroxylation, indicating increased bile acid synthesis induced by CBZ. Thus, 24S-OHChol is not catabolized by CBZ-induced cytochrome-P450 enzymes, and CBZ might not affect cholesterol metabolism in the central nervous system. CHARACTERISATION OF FUNCTIONAL RESIDUES IN THE INTERFACIAL RECOGNITION DOMAIN OF LECITHIN CHOLESTEROL ACYLTRANSFERASE B. Vanloo 1, E Peelman 1, C. Labeur 1, J. Vandekerckhove2, J. Tavernier2, M. Rosseneul, I Laborato~. for Lipoprotein Chemist~. Universi O, of Gent.

UniuersiO, of Campinas," 2Lipid laboratoo,, S•o Paulo UniversiO' A4edieal School, SP, Brazil: JDept of Medicine. Columbia UniversiO,. NE USA

B-9000 Gent. 2Flanders InteruniuersiO' Institute for Biotechnology. Fac. Medicine. Department of Biochemistry, University of Gent. B-9000 Gent. Belgium

Thyroid dysfunction produces multiple alterations on the plasma lipoprotein levels, including HDL. CETP is an important factor that modulates HDL plasma levels. Thus, the effects o f thyroid hormone on the HDL metabolism and CETP expression was investigated in hypo and hyperthyroid CETP transgenic mice. Transgenic mice were divided in 4 groups: hypo and hyperthyroid and their respective controls. The plasma FPLC lipoprotein profile from hyperthyroid mice showed significant increase in the VLDL fraction and decrease in HDL fraction. In the hypothyroid animals an increase in LDL fraction was observed (p < 0.01). 3H-cholesteryl ether-HDL kinetic studies showed that plasma fractional catabolic rate was approximately 2 fold greater in hyperthyroid animals than in controls while it did not change in hypothyroid animals. CETP activity was measured as cholesteryl ester

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high density (HDL) and low density lipoproteins (LDL). Threading alignment o f LCAT with lipases, suggest that residues 50-74 form an Interracial Recognition Site, and this hypothesis was tested by sitedirected mutagenesis. Phospholipase activity was measured on a fluorescent phospholipid analogue (bis-pyrene-PC), either monomeric or incorporated into an ether-DPPC/bis-pyrene-PC/apoA-I rHDL. Acyltransferase activity was determined on rHDL consisting of PLPC/cholesterol/apoA-I and on 3Hlabeled LDL. The (A56-68) deletion mutant had no activity on any substrate. Substitution of W61 with E Y, L or G, suggested that an aromatic residue is required for full enzymatic activity. Moreover, the pbospholipase activity of the W61F and W61Ymutants was twofold higher on the monomerie

71st EAS Congress and Satellite Symposia