Lipid metabolism.
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ATP-Binding Cassette Transporters↗
Biomedical subjects
M M van Greevenbroek
Publications and source records attributed to M M van Greevenbroek.
Identification of TNFRSF1B as a novel modifier gene in familial combined hyperlipidemia.
Familial combined hyperlipidemia (FCHL) is the most commonly inherited hyperlipidemia in man, with a frequency of +/-1% in the general population and approximately 10% in myocardial infarction survivors. A genomic scan in 18 Dutch FCHL families resulted in the identification of several loci with evidence for linkage. One of these regions, 1p36.2, contains TNFRSF1B which encodes one of the tumor necrosis factor receptors. An intron 4 polymorphic CA-repeat was used to confirm linkage to FCHL. Linear regression analysis using 79 independent sib pairs showed linkage with a quantitative FCHL discriminant function (P = 0.032), and, borderline, with apolipoprotein B levels (P = 0.064). Furthermore, in a case-control study, association was demonstrated since the overall CA-repeat genotype distribution was significantly different among 40 unrelated FCHL patients and 48 unrelated healthy spouse controls (P = 0.029). This difference was due to a significant increase in allele CA271 homozygotes in the FCHL patients (P = 0.019). Mutation analysis of exon 6 in 73 FCHL family members demonstrated the presence of a single nucleotide polymorphism with two alleles, coding for methionine (196M) and arginine (196R). Complete linkage disequilibrium between CA267, CA271 and CA273 and this polymorphism was detected. In 85 hyperlipidemic FCHL subjects, an association was demonstrated between soluble TNFRSF1B plasma concentrations and the CA271-196M haplotype. In conclusion, TNFRSF1B was found to be associated with susceptibility to FCHL. Our data suggest that an as yet unknown disease-associated mutation, linked to alleles 196M and CA271, plays a role in the pathophysiology of FCHL.
Adult↗
Soluble receptors for tumor necrosis factor-alpha (TNF-R p55 and TNF-R p75) in familial combined hyperlipidemia.
We investigated the potential role of the 75 kD receptor for tumor necrosis factor-alpha (TNF-alpha) (TNFRSF1B, located on chromosome 1 band p36.2) as a modifier gene in familial combined hyperlipidemia (FCH), based on previous linkage and association data. Age-corrected values for the soluble (s) extracellular domain of TNF-R p75 were lower in 156 well-characterized hyperlipidemic (HL) FCH relatives than in 168 normolipidemic (NL) relatives (P<0.01). Plasma concentrations of the soluble domain of the 55 kD receptor (sTNF-R p55, the other TNF-alpha receptor) did not differ between HL and NL relatives. In conditional logistic regression analysis, plasma sTNF-R p75 concentration was the only non-lipid variable that contributed significantly to prediction of affected FCH status (regression coefficient=-0.413, P=0.01). The present findings have potentially important diagnostic and therapeutic implications in FCH.
Adult↗
Caco-2 cells secrete two independent classes of lipoproteins with distinct density: effect of the ratio of unsaturated to saturated fatty acid.
Polarized Caco-2 cells can synthesize two distinct density classes of lipoproteins, i.e. chylomicron/VLDL (d<1.006 g/ml) or IDL/LDL density (1.009<d<1.068 g/ml). When saturated fatty acid in the incubation medium is replaced with unsaturated fatty acid, this results in an increase in the basolateral secretion of triglycerides from 18.6+/-3.6 nmol/filter (with 0.5 mmol/l 16:0) to 21.4+/-6.2, 27. 5+/-4.8 and 28.9+/-5.3 nmol/filter when 10, 20 or 30% of 16:0 were substituted by 18:1. The secretion of IDL/LDL-sized lipoproteins diminished and chylomicron/VLDL secretion increased in proportion to the increase of unsaturated fatty acid in the medium. To gain insight into the relationship between these lipoprotein classes, we determined their secretion at several time intervals (0-4, 4-8 and 22-26 h) after incubation with a fatty acid mixture containing 16:0 and 18:1 in a 9:1 molar ratio (total fatty acid concentration was 0.5 mmol/l). Chylomicron/VLDL secretion was detectable immediately upon the start of the incubation and persisted during all intervals. In contrast, IDL/LDL density lipoproteins were first detectable in the 4-8 h time interval and their secretion was highest in the final phase of the incubation (22-26 h). We conclude that Caco-2 cells secrete two distinct density classes of lipoproteins that show no precursor-product relation.
Caco-2 Cells↗
Genome scan for adiposity in Dutch dyslipidemic families reveals novel quantitative trait loci for leptin, body mass index and soluble tumor necrosis factor receptor superfamily 1A.
OBJECTIVE: To search for novel genes contributing to adiposity in familial combined hyperlipidemia (FCH), a disorder characterized by abdominal obesity, hyperlipidemia and insulin resistance, using a 10cM genome-wide scan. DESIGN: Plasma leptin and soluble tumor necrosis factor receptor superfamily members 1A and 1B (sTNFRSF1A and sTNFRSF1B, also known as sTNFR1 and sTNFR2) were analyzed as unadjusted and adjusted quantitative phenotypes of adiposity, in addition to body mass index (BMI), in multipoint and single-point analyses. In the second stage of analysis, an important chromosome 1 positional candidate gene, the leptin receptor (LEPR), was studied. SUBJECTS: Eighteen Dutch pedigrees with familial combined hyperlipidemia (FCH) (n= 198) were analyzed to search for chromosomal regions harboring genes contributing to adiposity. RESULTS: Multipoint analysis of the genome scan data identified linkage (log of odds, LOD, 3.4) of leptin levels to a chromosomal region defined by D1S3728 and D1S1665, flanking the leptin receptor (LEPR) gene by approximately 9 and 3 cM, respectively. The LOD score decreased to 1.8 with age- and gender-adjusted leptin levels. Notably, BMI also mapped to this region with an LOD score of 1.2 (adjusted BMI: LOD 0.5). Two polymorphic DNA markers in LEPR and their haplotypes revealed linkage to unadjusted and adjusted BMI and leptin, and an association with leptin levels was found as well. In addition, the marker D8S1110 showed linkage (LOD 2.8) with unadjusted plasma concentrations of soluble TNFRSF1A. BMI gave a LOD score of 0.6. Moreover, a chromosome 10 q-ter locus, AFM198ZB, showed linkage with adjusted BMI (LOD 3.3). CONCLUSION: These data provide evidence that a human chromosome 1 locus, harboring the LEPR gene, contributes to plasma leptin concentrations, adiposity and body weight in humans affected with this insulin resistant dyslipidemic syndrome. Novel loci on chromosome 8 and 10 qter need further study.
Adult↗
Chylomicron synthesis by intestinal cells in vitro and in vivo.
Synthesis and secretion of chylomicrons by the intestine is essential to transport dietary fats in the circulation and to deliver these fats to the appropriate peripheral tissues. The assembly of chylomicrons within the enterocyte and the subsequent secretion of these lipoprotein particles into the lymph is a complex, multi-step process that includes absorption of lipids by the enterocytes, cellular lipid (re)synthesis and translocation of cellular lipid pools, synthesis and post-translational modification of various apolipoproteins and, finally, the assembly of lipid and lipoprotein components into a chylomicron. The key process in chylomicron synthesis is the intracellular association of apolipoprotein (apo)B48, the structural protein of chylomicrons, with lipids.
Animals↗
Lipoprotein secretion by intestinal Caco-2 cells is affected differently by trans and cis unsaturated fatty acids: effect of carbon chain length and position of the double bond.
The effects of trans fatty acids on intestinal lipoprotein secretion were determined in polarized Caco-2 cells. Palmitic acid (16:0), palmitoleic acid (c-16:1delta9), and palmitelaidic acid (t-16:1delta9), as well as stearic acid (18:0), oleic acid (c-18:1delta9), c-vaccenic acid (c-18:1delta11), elaidic acid (t-18:1delta9), and t-vaccenic acid (t-18:1delta11) were studied. Compared with 18:0 (control), c- and t-18:1delta9 increased triacylglycerol secretion (2.7- and 3.6-fold, respectively) as well as apolipoprotein (apo) B-48 and apo B-100 secretion (both 1.6-fold compared with 18:0); c- and t-18:1delta11 caused a modest 1.7-fold increase in triacylglycerol secretion with no significant effect on secretion of apo B. Thus, the position of the double bond in the 18:1 isomers, but not its geometrical configuration, affected lipoprotein secretion by Caco-2 cells. In contrast, the effects of the geometrical isomers (cis and trans) of C16 fatty acids were not comparable: t-16:1delta9 did not affect triacylglycerol and apo B secretion (compared with 16:0, as control) whereas c-16:1delta9 was a potent stimulator of secretion of triacylglycerol (2.4-fold higher than 16:0), apo B-48 (1.3-fold higher than 16:0), and apo B-100 (1.5-fold higher than 16:0). We conclude that the carbon chain length of fatty acids, as well as the position of double bonds and their stereochemical configuration, are important determinants of the unique effects of various species of dietary trans fatty acids on lipoprotein secretion and composition in Caco-2 cells.
Caco-2 Cells↗
Participation of the microsomal triglyceride transfer protein in lipoprotein assembly in Caco-2 cells: interaction with saturated and unsaturated dietary fatty acids.
This study was designed to gain insight into the role of microsomal triglyceride transfer protein (MTP) in the association of apolipoprotein (apo) B with lipid during intestinal lipoprotein assembly. The MTP-inhibiting compound BMS-200150 (Jamil et al. 1996. Proc. Natl. Acad. Sci. USA 93: 11991-11995) was used to inhibit the lipid transfer activity of MTP in Caco-2 cells. MTP inhibition reduced the number of apoB-containing lipoproteins that were secreted from the cells. Secretion of apoB-100 appeared to be more sensitive to BMS-200150 than apoB-48 secretion, which appeared to be relatively insensitive. BMS-200150 caused a decrease in the triglyceride content of the secreted lipoproteins, compared with control incubations without MTP inhibition. This indicated that, in Caco-2 cells, MTP is not only involved in the first step of lipoprotein synthesis, i.e., the rescue of apoB from intracellular degradation through early lipidation of the protein, but also in further steps involving the association of lipoproteins with triglycerides. When 0.5 mM oleic acid (18:1) was used to stimulate cellular lipid synthesis, secreted lipoproteins were predominantly of chylomicron/VLDL density and their secretion could be efficiently inhibited with BMS-200150. With 0.5 mm palmitic acid (16:0), lipoproteins of distinct densities (i.e., chylomicron/VLDL and IDL/LDL) were secreted by Caco-2 cells, as reported before (van Greevenbroek et al. 1995. J. Lipid Res. 36: 13-24). Secretion of the lipoproteins at chylomicron/VLDL density was strongly reduced by inhibition of MTP activity by BMS-200150, whereas the IDL/LDL density lipoproteins were relatively insensitive. In conclusion, specific inhibition of MTP activity in Caco-2 cells with BMS-200150 resulted in reduced secretion of apoB-containing lipoproteins (predominantly apoB-100) by Caco-2 cells and furthermore reduced the triglyceride content of these lipoproteins. MTP inhibition preferentially reduced the secretion of triglyceride-rich lipoproteins (d < 1.006 g/ml).
Apolipoproteins B↗
Abnormal postprandial apolipoprotein B-48 and triglyceride responses in normolipidemic women with greater than 70% stenotic coronary artery disease: a case-control study.
Because remnants of triglyceride-rich lipoproteins (TRLP) are potentially atherogenic, the postprandial lipoprotein metabolism was studied in 12 normocholesterolemic, normotriglyceridemic women, aged 60 +/- 2 years, with angiographically proven coronary artery disease (CAD+; cholesterol 5.7 +/- 0.1 (S.E.) mmol/l, triglyceride 1.35 +/- 0.10 mmol/l) and in 12 individually matched controls, aged 59 +/- 2 years, without angiographical abnormalities (CAD-; cholesterol 5.1 +/- 0.2 mmol/l and triglyceride 1.16 +/- 0.13 mmol/l). Following an oral retinyl palmitate-fat load, the CAD+ women showed a significantly higher triglyceride response in the chylomicron, or Sf > 1000, fraction (P < 0.05 vs. controls). Total plasma apolipoprotein (apo) B and retinyl palmitate concentrations were similar in both groups. Fasting apo B-48 levels in the d < 1.006 g/ml fraction were significantly higher in CAD+ cases (0.25 +/- 0.03 integrated optical density (iod) units) than CAD- controls (0.15 +/- 0.03; P < 0.05). Furthermore, after the fat load, a greater absolute and incremental apo B-48 response in the intermediate density lipoprotein (IDL) fraction (d = 1.006-1.019 g/ml) was observed in CAD+ cases (incremental area under the curve (Delta-AUC)8: 0.40 +/- 0.12 h.iod) than CAD- controls (0.01 +/- 0.06 h.iod; P = 0.01). Post-heparin hepatic lipase (HL) activities were higher in the CAD+ group: 422 +/- 22 mU/l vs 288 +/- 20 mU/ml in the CAD- group (P < 0.001) while lipoprotein lipase (LPL) activities were identical. The results provide evidence that the metabolism of intestinal TRLP is significantly different in normolipidemic women with angiographically proven CAD compared with individually matched controls without coronary disease. Fasting apo B-48 levels in d< 1.006 g/ml fractions represent a potentially useful marker in women at risk for CAD.
Analysis of Variance↗
Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells.
We studied the effects of addition of physiological concentrations (0.5 mM) of fatty acids i.e., palmitic (16:0), stearic (18:0), oleic (18:1) and linoleic acid (18:2) on lipoprotein secretion by polarized Caco-2 cells. With saturated fatty acids, secreted lipoproteins were at IDL/LDL density, 1.009 < d < 1.068 g/ml. The numbers of secreted lipoproteins, expressed as secreted apolipoprotein (apo) B, were comparable to control without fatty acid (palmitic acid, 551 +/- 185; stearic acid, 629 +/- 304 and control, 504 +/- 176 ng apo B/4.7 cm2 filter). With unsaturated fatty acids, apo B containing lipoproteins were secreted at chylomicron/VLDL density (d < 1.006 g/ml). Oleic acid caused a two-fold higher secretion of apo B than control (1058 +/- 87 vs. 504 +/- 176 ng/4.7 cm2 filter, P < 0.001). The increase in apo B secretion was attributed to a specific increase in apo B-48. Unsaturated fatty acid caused a two-fold higher secretion of triglyceride than saturated fatty acids but incorporation of newly synthesized lipid into the secreted lipoproteins, measured by incorporation of a fatty acid marker, was 10- to 20-fold higher, indicating preferential translocation of unsaturated triglycerides into lipoproteins. Mixtures rich in either polyunsaturated, monounsaturated, or saturated fatty acids, resembling nutritional fat and oils, were capable of a two-fold stimulation of secretion of apo B containing triglyceride-rich lipoproteins. The triglyceride/apo B ratio in the basolateral medium was higher with the monounsaturated 'olive oil' mixture (12 250 +/- 2000 mol/mol) than with the polyunsaturated 'corn oil' mixture (7830 +/- 2480 mol/mol) and incorporation of newly synthesized lipid into the secreted lipoproteins was 1.5-fold higher as well. In conclusion, unsaturated fatty acids were most potent in stimulating the secretion of apo B by specifically increasing apo B-48 secretion. Unsaturated triglycerides, that contain mainly oleic acid, were more efficiently incorporated into lipoproteins than saturated triglycerides, suggesting preferential translocation by microsomal triglyceride transfer protein.
Apolipoprotein B-100↗
Palmitic acid and linoleic acid metabolism in Caco-2 cells: different triglyceride synthesis and lipoprotein secretion.
Polarized monolayers of intestinal Caco-2 cells were used to study the effects of saturated palmitic acid (16:0) and polyunsaturated linoleic acid (18:2) on triglyceride synthesis and lipoprotein secretion. Monolayers were incubated for 24 h, at the apical or lumenal side, with palmitic acid (16:0) or linoleic acid (18:2) in physiological concentrations. Incubation with 1.0 mM 16:0 or 18:2 resulted in differences in the composition and amount of secreted lipoproteins. Radiolabeled lipids in the lipoproteins secreted during incubation with 18:2 were found in the chylomicron/VLDL (very low density lipoprotein) density whereas with 16:0 the secreted lipoproteins were in the intermediate density/low density lipoprotein (IDL/LDL) density range. More triglyceride was secreted into the (basolateral) medium during incubation with 1.0 mM 18:2 (41 +/- 12% of total triglyceride synthesized) than with 1.0 mM 16:0 (18 +/- 3% of total). The biochemical findings correlate with conspicuous morphological changes in the cells in the presence of 16:0, but not 18:2. Increasing concentrations of 16:0 (0.1-1.0 mM) caused gradual accumulation of intracellular membrane. Microvilli became strongly reduced in number. With 1.0 mM palmitic acid we found an increased incorporation of [1-14C]palmitic acid into phosphatidic acid (14.8% of total incorporation into phospholipid with 16:0 vs. < 0.5% with 18:2) and diacylglycerol (12.5% with 16:0 vs. < 0.5% with 18:2) and diacylglycerol (12.5% with 16:0 vs. 0.5% with 18:2) and the amount of intracellular phospholipid doubled. The morphological changes were completely reversed after 24 h with 1.0 mM 18:2. We conclude from our results that, compared to 18:2, 16:0 is not efficiently incorporated into triglycerides. 16:0 is incorporated into cellular phospholipids in a greater proportion than 18:2, causing accumulation of intracellular phospholipid and the precursors phosphatidic acid and diacylglycerol. Different processing of 18:2 and 16:0 by Caco-2 cells resulted in profound differences in triglyceride synthesis and lipoprotein composition and secretion.
Carbon Radioisotopes↗
Structural and functional changes of surfactant protein A induced by ozone.
The major surfactant protein SP-A is thought to play an important role in the regulation of the structure and the metabolism of pulmonary surfactant. We investigated whether this protein may be a target in ozone toxicity. Several properties were studied that are probably relevant to the physiological functions of SP-A. In vitro exposure of human and canine SP-A to ozone led to decreases in 1) self-association of SP-A, 2) SP-A-mediated lipid aggregation, and 3) binding of SP-A to immobilized mannose. SP-A was neither degraded nor cross-linked by ozone exposure. Exposure of canine SP-A to ozone led to an increase in the apparent molecular weight of monomeric SP-A. Human SP-A did not show this change. Oxidation of methionine and tryptophan residues in canine SP-A was detected following ozone exposure. Reactions of ozone with other amino acid residues were not observed. The impairments of structure and properties of SP-A may contribute to the toxic action of ozone in the lungs.
Animals↗
Genetic dissection of familial combined hyperlipidemia.
Familial combined hyperlipidemia (FCHL) is the most common genetic hyperlipidemia in man. FCHL is characterized by familial clustering of hyperlipidemia and clinical manifestations of premature coronary heart disease, i.e., before the age of 60. Although FCHL was delineated about 25 years ago, at present the FCHL phenotype and its complex genetics are not fully understood. Initially, the familial aggregation of high plasma total cholesterol and triglyceride levels, with a bimodal distribution of triglycerides, was taken as evidence of a dominant mode of inheritance. However, it is now clear that the genetics of FCHL is more complex, and it has been suggested that FCHL is heterogeneous. Several approaches can be taken to identify genes contributing to the disease phenotype in complex genetic disorders either by studying the disease in the human situation or by using animal models. Recent reports have shown that a combination of genetic linkage studies, association studies, and differential gene expression studies provides a useful tool for the genetic dissection of complex diseases. Therefore, the genetic strategies that will be used to dissect the genetic background of FCHL are reviewed.
Animals↗