Increased Production of HDL ApoA-I in Homozygous Familial Defective ApoB-100
Abstract—Familial defective apolipoprotein (apo) B-100 (FDB) is a frequent cause of hypercholesterolemia. Hypercholesterolemia in homozygous FDB is less severe than in homozygotes for familial hypercholesterolemia. Recently, we showed decreased low density lipoprotein (LDL) apoB-100 fractional catabolism and decreased production of LDL due to an enhanced removal of apoE-containing precursors in a patient with homozygous FDB. The effects of defective apoB-100 on high density lipoprotein (HDL) metabolism are unknown. We studied HDL apoA-I metabolism in this FDB patient and in 6 control subjects by using 2H3-l-leucine as a tracer. ApoA-I levels were normal in all study subjects. However, the fractional catabolic rate and the production rate of apoA-I were increased, by 79% and 70%, respectively, in FDB; the fractional catabolic rate of apoA-I in FDB was 0.34 day−1 compared with 0.19±0.03 day−1 in normal controls. The production rate of apoA-I in FDB was 18.4 mg · kg−1 · d−1 compared with 10.8±2.3 mg · kg−1 · d−1 in controls. Thus, we have shown for the first time that defective apoB-100 may influence HDL kinetics. The increase in total HDL turnover might enhance reverse cholesterol transport and could contribute to the seemingly benign clinical course of FDB compared with that of familial hypercholesterolemia.
- Received March 18, 1999.
- Accepted March 31, 2000.
Familial defective apoB-100 (FDB) is one of the most common monogenetic abnormalities of lipoprotein metabolism. This disorder results from a glutamine-for-arginine substitution at position 3500 of apoB-100, which leads to defective binding of apoB-100 to the LDL receptor and the accumulation of LDL in the plasma (reviewed in References 1 and 2 ).
Recently, we identified a homozygous FDB patient3 4 and studied the in vivo kinetics of apoB-100–containing lipoproteins in this subject by using a stable isotope tracer technique.5 Our investigation revealed an increased removal of VLDL apoE, whereas the VLDL apoB-100 residence time was prolonged. The LDL apoB-100 production was reduced (7.4 versus 15 mg · kg−1 · d−1 in normals), and the residence time of LDL was increased (8.3 versus 2.3 days in normals).5 These findings are in agreement with data from heterozygous FDB subjects.6 They indicate that the metabolism of apoB-100–containing lipoproteins is distinct in FDB compared with that in familial hypercholesterolemia (FH) due to LDL receptor deficiency.
In FH, HDL metabolism has also been found to be abnormal. Schaefer et al7 demonstrated decreased production and increased fractional catabolism of apoA-I in individuals with FH, suggesting a “cross-talk” between LDL and HDL metabolism. On the background of these observations, we decided to examine whether HDL metabolism might be altered in FDB as well. We therefore investigated the kinetics of HDL apoA-I in a homozygous FDB subject by using the stable-isotope tracer technique.
We studied the homozygous FDB patient described previously.3 4 The patient is a 54-year-old white male without any symptoms of disease. He had multiple xanthelasmas and a discrete arcus corneae. His apoE phenotype was 3/3. Routine blood biochemistry was normal except for a slight increase in γ-glutamyl transferase (36 U/L; upper limit of normal, 27 U/L). The patient did not take any lipid-lowering drugs in the 6 weeks before and during the study. Six healthy, normolipidemic male subjects served as controls. All but one (No. 6 was apoE 2/4) had an apoE 3/3 phenotype and normal body-weight.
Sequencing of ApoA-I
DNA of the homozygous FDB patient was extracted from white blood cells by using blood polymerase chain reaction (PCR) DNA isolation cartridges (Diagen GmbH). Oligonucleotide primers were synthesized (BIG) to allow amplification of part of the promoter and of individual exons of the apoA-I gene: apoA-I promoter 5′: 5′-AGA GCT GAT CCT TGA ACT CTT AAG-3′ (77–100); apoA-I promoter 3′: 5′-GAC ACC TAC CCG TCA GGA AGA GCA-3′ (475–499); apoA-I exon 2, 5′: 5′-TCA CCT GGC TGC AAT GAG T-3′ (343–362); apoA-I exon 2, 3′: 5′-ACG GGG ATT TAG GGA GAA AG-3′ (530–549); apoA-I exon 3, 5′: 5′-CTG GCC TGA TCT GGG TCT C-3′ (644–662); apoA-I exon 3, 3′: 5′-CCA GTC TGG CTT CAA CAT CA-3′ (898–908); apoA-I exon 4, 5′: 5′-GGC TCA CCC CTG ATA GGC-3′ (1359–1376); and apoA-I exon 4, 3′: 5′-GCA CGG AGT TGT TGA GAT CC-3′ (2179–2198). Typical PCR conditions were as follows: 50 ng of DNA, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 1 μmol/L of each primer, and 0.5 U Taq polymerase (Roche) in a 50-μL reaction. Amplification was performed by using an initial denaturing step at 95°C for 3 minutes and then 30 cycles at 95°C for 45 seconds, 60°C for 1 minute, and 72°C for 1 minute, followed by a final elongation step at 72°C for 10 minutes. Purified PCR products were directly sequenced with the dideoxy chain-termination method (BigDye sequencing kit, Perkin-Elmer) on an ABI 377 sequencer.
The in vivo turnover study followed a protocol that has recently been described.7 The protocol had been approved by the Internal Review Boards of the Philips University Marburg and of the Albert Ludwigs University Freiburg. Informed, written consent was obtained from each study subject. Three days before the study all subjects received a standardized isocaloric diet. The stable-isotope turnover study was performed under fasting conditions in all subjects, starting at 8 am. Two plastic indwelling catheters were placed intravenously on contralateral arm veins: one was used for the tracer infusion and the other was used for the frequent blood sampling during the study. [2H3]l-leucine (l-leucine-methyl-D3 99, Cambridge Isotope Laboratories) was administered as a priming bolus of 1.34 mg/kg, immediately followed by a constant infusion of 22 μg · kg−1 · min−1 for up to 12 hours. Blood samples (9 mL) were drawn into tubes containing EDTA at a final concentration of 1 g/L before the tracer injection; after 10, 20, 30, 40, 60, 90, and 120 minutes; and then hourly for up to 12 hours. The blood was kept on ice and the plasma was immediately separated by centrifugation (3000g, 20 minutes at 4°C). NaN3 and aprotinin were added to the plasma at final concentrations of 0.5 g/L and 200 000 KIU/L, respectively.
Lipoproteins and Apolipoproteins
VLDL (d<1.006 g/mL), LDL (d=1.019 to 1.063 g/mL), and HDL (d=1.063 to 1.21 g/mL) were isolated by sequential preparative ultracentrifugation from 5 mL of plasma (50.3 Ti rotor, L8-55M centrifuge; Beckman Instruments). VLDL apoB-100 and HDL apoA-I were isolated by preparative gradient (T=5% to 15%) SDS–polyacrylamide gel electrophoresis. Cholesterol, triglycerides, and phospholipids were measured by using enzymatic reagents provided by Wako Chemicals. ApoA-I and A-II were quantified turbidimetrically with the use of antibodies from Rolf Greiner.
Determination of Isotopic Enrichment
Samples were prepared for analysis by gas chromatography–mass spectrometry (GC-MS) as described previously. Apolipoprotein bands were excised from the gels and hydrolyzed in 6 mol/l HCl at 110°C for 24 hours under N2. The protein hydrolysates were lyophilized in a Speed-Vac evaporator (Savant Instruments). Free amino acids were purified from plasma or protein hydrolysates by cation exchange chromatography (AG-50W-X6, Bio-Rad Laboratories) and then derivatized to n-heptafluorobutyrylisobutyl esters and analyzed by GC-MS in the chemical ionization and selected ion monitoring mode as reported recently.8 The ions monitored were 363.1 m/z (mass-to-charge ratio) for unlabeled l-leucine and 366.1 m/z for radiolabeled [2H3]l-leucine as parent ions (first MS) and 280.1 m/z for the daughter ions of both types of leucine (second MS). Tracer enrichment was calculated as the tracer-to-tracee ratio according to Cobelli et al.9
A monoexponential function was fitted to the tracer-to-tracee curves of VLDL apoB-100 and HDL apoA-I with the use of saam 30 software as previously described. The function was defined as Et=Ep[1 −e−k(t−d)], where Et is the tracer-to-tracee ratio at time t of HDL apoA-I and Ep is the tracer-to-tracee ratio at plateau of the precursor pool of interest (VLDL apoB-100)10 ; d is the delay time and k is the fractional catabolic rate.
Results and Discussion
Lipids and lipoprotein profiles in the FDB and in 6 normal control subjects are shown in Table 1⇓. In the FDB patient, total cholesterol was higher compared with that in controls, but his total apoA-I was normal. Although HDL cholesterol, HDL phospholipids, and HDL apoA-II were significantly lower in FDB compared with controls, there was no significant difference in the composition of HDL (Table 2⇓). In addition, sequencing revealed no mutation either in the promoter or in the coding sequence of the apoA-I gene of the homozygous FDB patient. The kinetic parameters of HDL apoA-I metabolism in the FDB homozygote and in the 6 control subjects are shown in Table 3⇓. The FDB subject revealed a clearly higher fractional catabolic rate (+79%, P<0.0001) and production rate (+70%, P<0.0005) of apoA-I compared with those values in normal subjects.
Defective apoB-100 leads to significant changes in the in vivo kinetics of apoB-containing lipoproteins. We and others have shown that both the production rate of LDL apoB and its fractional catabolic rate are reduced compared with normal,2 5 6 but the metabolic situation in FDB is completely different from that in FH. In FH, delayed fractional catabolism of LDL apoB occurs together with an increased production of LDL apoB.11 12 13 Moreover, in homozygous FH, in an earlier report we found dramatic changes in HDL apoA-I kinetics, characterized by a decrease in apoA-I production and an increased fractional catabolic rate of apoA-I.7 In FH, coordinated regulation of apoB and apoA-I kinetics was thus suggested, and we were interested to search for a similar effect in FDB.
This is the first study to examine the metabolism of HDL apoA-I in FDB. The single homozygous individual presented in this study may not be completely representative of the entire disorder. However, owing to the potential influence of normal apoB-100 in heterozygous FDB, the study of a homozygous individual may provide more valid information about the metabolism in FDB than would study of several individuals heterozygous for the disease.
Despite normal apoA-I levels, we found an increased fractional catabolic rate of apoA-I in FDB and a marked increase in the total production rate of apoA-I. These changes may indicate that reverse cholesterol transport is upregulated in FDB. Increased production of apoA-I is found in subjects with the longevity syndrome.14 In addition, an increased fractional catabolic rate of apoA-I might be beneficial, since it is known that hypoalphalipoproteinemia due to increased fractional catabolism of apoA-I appears to be less atherogenic compared with the inborn abnormalities of apoA-I metabolism associated with decreased apoA-I production.15 The increased production of apoA-I may thus protect our FDB homozygote from atherosclerosis.
The metabolic basis for the increase in apoA-I production and fractional catabolism in FDB is currently unclear. Alterations of the apoA-I gene in our homozygous FDB patient and major alterations of the composition of HDL have been excluded as possible reasons for our findings. A speculative possibility is that the increase in the fractional catabolism of apoA-I in FDB is due to an increase in the catabolic rate of apoE-containing HDL. ApoE-containing HDLs are known to be catabolized by the LDL receptor,16 and LDL receptor activity appears increased in FDB due to the low influx of LDL cholesterol into the liver.4 5
Brown et al17 demonstrated an increase in apoA-I levels when they transferred human LDL receptors by using a recombinant adenovirus into LDL receptor–deficient rabbits, a finding that, at first sight, appears inconsistent with an increased apoA-I fractional catabolic rate in FDB due to the upregulation of LDL receptors. However, in the study by Brown et al, the greatest increase of apoA-I was found in pre-β-HDL, a fraction considered to contain mainly nascent HDL. This suggests that the increase in HDL was due to an increase in apoA-I production rather than decreased catabolism of apoA-I. Such interpretation of their data would then completely align with our findings in FDB, wherein we observed an increased apoA-I production together with an increased fractional catabolic rate. A mechanism linking the upregulation of LDL receptors and stimulation of apoA-I production is also not readily apparent at this time. The existence of such a mechanism, however, is well in agreement with studies by Mitchell et al,18 as well as with the studies of our group. In rats treated with drugs that upregulate the LDL receptor, Mitchell et al18 demonstrated an increase in apoA-I mRNA and a decrease in hepatic apoB mRNA. Consistently in subjects treated with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin, we have found an increased production rate of apoA-I.19 Finally, we recently obtained evidence of increased production of apoA-I during administration of lifibrol to individuals with moderate hyperlipidemia.20 Lifibrol is a novel hypolipidemic compound that stimulates LDL receptors without substantially affecting cholesterol biosynthesis.21 22
In conclusion, the finding of a significantly increased HDL metabolism in FDB is in clear contrast to the situation in FH. In FH there is an increased fractional catabolic rate of apoA-I combined with a decrease in apoA-I production. With this combination, there are major metabolic differences between FDB and FH, with respect to not only the kinetics of apoB-containing lipoproteins but also in the kinetics of apoA-I. These differences might contribute to the rather benign clinical course of FDB compared with homozygous FH.
Innerarity TL, Weisgraber KH, Arnold KS, Mahley RW, Krauss RM, Vega GL, Grundy SM. Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding. Proc Natl Acad Sci U S A. 1987;84:6919–6923.
Innerarity TL, Mahley RW, Weisgraber KH, Bersot TP, Krauss RM, Vega GL, Grundy SM, Friedl W, Davignon J, McCarthy BJ. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia. J Lipid Res. 1990;31:1337–1349.
März W, Ruzicka C, Pohl T, Usadel KH, Gross W. Familial defective apolipoprotein B-100: mild hypercholesterolaemia without atherosclerosis in a homozygous patient. Lancet. 1992;340:1362. Letter.
März W, Baumstark MW, Scharnagl H, Ruzicka V, Buxbaum S, Herwig J, Pohl T, Russ A, Schaaf L, Berg A, Böhles H-J, Usadel KH, Gross W. Accumulation of “small dense” low density lipoproteins (LDL) in a homozygous patient with familial defective apolipoprotein B-100: results from heterogenous interaction of LDL subfractions with the LDL receptor. J Clin Invest. 1993;92:2922–2933.
Schaefer JR, Scharnagl H, Baumstark MW, Schweer H, Zech LA, Seyberth H, Winkler K, Steinmetz A, März W. Homozygous familial defective apolipoprotein B-100: enhanced removal of apolipoprotein E–containing very low density lipoproteins and decreased production of low density lipoproteins. Arterioscler Thromb Vasc Biol. 1997;17:348–353.
Pietzsch J, Wiedemann B, Julius U, Nitzsche S, Gehrisch S, Bergmann S, Leonhardt W, Jaross W, Hanefeld M. Increased clearance of low density lipoprotein precursors in patients with heterozygous familial defective apolipoprotein B-100: a stable isotope approach. J Lipid Res. 1996;37:2074–2087.
Schaefer JR, Rader DJ, Ikewaki K, Fairwell T, Zech LA, Kindt MR, Davignon J, Gregg, RE, Brewer HB Jr. In vivo metabolism of apolipoprotein A-I in a patient with homozygous familial hypercholesterolemia. Arterioscler Thromb. 1992;12:843–848.
Cobelli C, Toffolo G, Foster DM. Tracer-to-tracee ratio for analysis of stable isotope tracer data: link with radioactive formalism. Am J Physiol. 1992;262:E968–E975.
Ikewaki K, Rader DJ, Schaefer JR, Fairwell T, Zech LA, Brewer HB, Jr. Evaluation of apoA-I kinetics in humans using simultaneous endogenous stable isotope and exogenous radiotracer methods. J Lipid Res. 1993;34:2207–2215.
Bilheimer DW, Goldstein JL, Grundy SM, Brown MS. Reduction in cholesterol and low density lipoprotein synthesis after portocaval shunt surgery in a patient with homozygous familial hypercholesterolemia. J Clin Invest. 1975;56:1420–1430.
Bilheimer DW, Stone NJ, Grundy SM. Metabolic studies in familial hypercholesterolemia: evidence for a gene-dosage effect in vivo. J Clin Invest. 1979;64:524–533.
Shepherd J, Packard CJ. Lipoprotein metabolism in familial hypercholesterolemia. Arteriosclerosis. 1989;9:139–142.
Rader DJ, Schaefer JR, Lohse P, Ikewaki K, Thomas F, Harris WA, Zech LA, Dujovne CA, Brewer HB Jr. Increased production of apolipoprotein A-I associated with elevated plasma levels of high-density lipoproteins, apolipoprotein A-I, and lipoprotein A-I in a patient with familial hyperalphalipoproteinemia. Metabolism. 1993;42:1429–1434.
Mahley RW, Innerarity TL, Weisgraber KH, Oh SY. Altered metabolism (in vivo and in vitro) of plasma lipoproteins after selective chemical modification of lysine residues of the apolipoproteins. J Clin Invest. 1979;64:743–750.
Brown DR, Brousseau ME, Shamburek RD, Tally GD, Meyn S, Demosky SJ Jr, Santamarina-Fojo S, Brewer HB Jr, Hoeg JM. Adenoviral delivery of low-density lipoprotein receptors to hyperlipidemic rabbits: receptor expression modulates high-density lipoproteins. Metabolism. 1996;45:1447–1457.
Schaefer JR, Schweer H, Ikewaki K, Stracke H, Seyberth HJ, Kaffarnik H, Maisch B, Steinmetz A. Metabolic basis of high density lipoproteins and apolipoprotein A-I increase by HMG-CoA reductase inhibition in healthy subjects and a patient with coronary artery disease. Atherosclerosis. 1999;144:177–184.
Winkler K, Schaefer JR, Klima B, Nuber C, Friedrich I, Köster W, Gierens H, Scharnagl H, Soufi M, Wieland H, März W. HDL steady state levels are not affected, but HDL apoA-I turnover is enhanced by lifibrol in patients with hypercholesterolemia and mixed hyperlipidemia. Atherosclerosis. 2000;150:113–120.
Scharnagl H, Wieland H, März W. Lifibrol: first member of a new class of lipid lowering drugs? Expert Opin Investig Drugs. 1997;6:583–591.
Scharnagl H, Schliack H, Loser R, Nauck M, Gierens H, Jeck N, Wieland H, Grob W, März W. The effects of lifibrol (K12.148) on the cholesterol metabolism of cultured cell evidence for sterol-independent stimulation of the LDL receptor pathway. Atherosclerosis. In press.