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1.
Int J Obes Relat Metab Disord ; 25 Suppl 1: S19-21, 2001 May.
Article in English | MEDLINE | ID: mdl-11466580

ABSTRACT

Polymorphisms in a number of candidate genes have been reported to be associated with obesity. We have determined the incidence of the following polymorphisms in the following candidate genes in a group of 388 morbid obese patients (mean body mass index (BMI) 52+/-8.01) who underwent gastric banding surgery: lipoprotein lipase (LpL) t-93 g and N291S; peroxisome proliferator receptor gamma (PPARgamma), P12A, P115Q and c1431t; peroxisome proliferator receptor alpha (PPARalpha) L162V; beta-adrenergic receptor 2 (beta-AR 2), Q27E; beta-adrenergic receptor 3 (beta-AR 3) W64R; uncoupling protein 1 (ucp-1), a-3826g, ucp-2, 45 bp insertion. Only for the ucp2 polymorphism did we find a statistically significant association with obesity. The beta-AR 3 W64R and ucp-1 a-3826g polymorphisms influenced the rate of the development of obesity and may act synergistically.


Subject(s)
Body Weight/genetics , Carrier Proteins/genetics , Membrane Transport Proteins , Mitochondrial Proteins , Obesity, Morbid/genetics , Polymorphism, Genetic , Adult , Body Mass Index , Female , Humans , Ion Channels , Male , Proteins , Uncoupling Protein 2
2.
Atherosclerosis ; 141(2): 273-85, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9862176

ABSTRACT

Selective uptake of high-density lipoprotein- (HDL-) associated cholesteryl esters (CE), i.e. lipid uptake independent from particle uptake, delivers CE to the liver and steroidogenic tissues in vivo. In vitro, besides hepatocytes and steroidogenic cells many other cell types selectively take up HDL CE. Hepatic lipase (HL) stimulates the internalisation of apoprotein (apo) B-containing lipoproteins by hepatocytes independent from lipolysis. In this study the role of HL in the hepatic metabolism of apo A-I-containing lipoproteins, i.e. HDL, was investigated. HDL3 (d = 1.125-1.21 g/ml) was radiolabeled in its protein (125I) and in its CE moiety ([3H]cholesteryl oleyl ether, ([3H]CEt)). HL originated from tissue culture media of hepatoma cells and from post-heparin plasma. Human Hep 3B hepatoma cells incubated in medium containing radiolabeled HDL3. In the absence of HL, the rate of apparent HDL3 particle uptake according to the lipid tracer ([3H]CEt) was in most cases in approximately 10-fold excess on that due to the protein label (125I), indicating selective CE uptake from HDL3. Addition of HL to these incubations increased the cellular uptake of [3H]CEt and of 125I from HDL3 and quantitatively the most prominent effect was an up to approximately 2.5-fold stimulation of apparent selective CE uptake ([3H]CEt-125I). This increase in selective CE uptake was observed in the presence of tetrahydrolipstatin, an inhibitor of the catalytically active site of HL, suggesting that this HL effect is independent from lipolysis. HL binds to cell surface heparan sulfate proteoglycans. To explore the role of these molecules for the HL effect on selective CE uptake, hepatoma cells were depleted of proteoglycans or Chinese hamster ovary (CHO) cells deficient in proteoglycan synthesis were used. Proteoglycan-deficiency reduced the HL-mediated increase in selective uptake by more than 80%. To investigate if low-density lipoprotein (LDL) receptors or the LDL receptor-related protein (LRP) are involved in the HL effect on selective CE uptake, murine embryonic fibroblasts (MEF) were used which are deficient in these receptors; alternatively, monensin, an inhibitor of endocytosis was present in the medium of Hep 3B cells during the uptake assay for labeled HDL3. These experiments yielded no evidence for a role of LDL receptors or LRP in the HL-mediated increase in selective CE uptake. In summary, HL mediates an increase in HDL3 selective CE uptake by human Hep 3B hepatoma cells. This HL effect is independent from lipolysis and independent from LRP and LDL receptors. However this HL effect is susceptible to cell surface proteoglycan deficiency. The potential physiologic implication is that HL modifies HDL selective CE uptake by the liver in vivo and such an effect could play a role in reverse cholesterol transport.


Subject(s)
Cholesterol Esters/metabolism , Lipase/physiology , Lipoproteins, HDL/metabolism , Liver/metabolism , Animals , CHO Cells , Carcinoma, Hepatocellular , Cricetinae , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Heparan Sulfate Proteoglycans/metabolism , Heparin Lyase/pharmacology , Humans , Lactones/pharmacology , Lipase/pharmacology , Lipoproteins, HDL3 , Liver/enzymology , Mice , Monensin/pharmacology , Orlistat , Receptors, LDL/deficiency , Receptors, LDL/physiology , Tumor Cells, Cultured
3.
Hum Mutat ; 12(3): 217, 1998.
Article in English | MEDLINE | ID: mdl-10660334

ABSTRACT

Familial chylomicronemia is an autosomal recessive disease characterised by fasting triglyceridemia and an absence of lipoprotein lipase (LpL) activity in post-heparin plasma. The disease is a result of mutation in either the lipoprotein lipase (Lpl) gene or in the apoCII gene which codes for an essential co-factor. To date, over 80 mutations in the LpL gene have been reported. The proband, a 30 month old female, presented with fasting triglycerides of 3192 mg/dl, and no detectable LpL mass or activity in post-heparin plasma. Sequencing of all of the exons and exon/intron boundaries of the LpL gene showed that she was a compound heterozygote with G-A transitions in codon 188 (G188E:GGG to GAG) generating an avall restriction site and in codon 259 (S259G:AGT to GGT) generating a bssKI site. Restriction digests confirmed the mutations and determined the incidence within the family. The father (55%LPL activity), paternal aunt (82%) and paternal grandmother (29%) were all heterozygous for the S259G mutation whilst her sister (55%), mother (73%) and maternal grandfather (45%) were heterozygous for the G188E mutation. The maternal grandmother (114%) was unaffected.


Subject(s)
Genetic Carrier Screening , Hyperlipoproteinemia Type I/genetics , Lipoprotein Lipase/genetics , Point Mutation/genetics , Female , Humans , Infant
4.
J Lipid Res ; 37(5): 926-36, 1996 May.
Article in English | MEDLINE | ID: mdl-8725146

ABSTRACT

The uptake of triglyceride-rich lipoproteins has been described as being mediated by apolipoprotein E and lipoprotein lipase (LpL). Proteoglycans, the LDL-receptor, and the LDL receptor-related protein (LRP) are the cellular acceptors. In addition to LpL, hepatic lipase (HL) has been shown to bind to LRP. In this study, the role of HL in lipoprotein uptake was investigated. Human chylomicrons and rabbit beta-VLDL were used as ligands for human hepatoma cells, primary human hepalocytes, normal and proteoglycan-deficient Chinese hamster ovary (CHO) cells, and normal and LDL receptor-deficient human fibroblasts. We show that HL induces stimulation of the uptake of chylomicrons and beta-VLDL into the different cell lines. HL is known to bind to heparan sulfate, and experiments on normal and proteoglycan-deficient CHO cells showed that cell surface proteoglycans are essential for HL-mediated uptake of lipoproteins. To exclude LDL receptor-mediated uptake. we performed experiments on LDL receptor-deficient fibroblasts that demonstrated that the LDL receptor was not important for the HL-mediated uptake of lipoproteins. Crosslinking experiments confirmed the binding of HL to LRP on the cell surface. To identify the region of HL involved in the interaction with LRP, we used a C-terminal fragment of LpL, known to inhibit LpL-mediated uptake. HL-mediated lipoprotein uptake was suppressed by this fragment. Our experiments indicate that HL, like LpL, can mediate the uptake of lipoproteins into cells, most probably via a C-terminal binding site. The uptake, initiated by proteoglycan binding, is mediated by LRP.


Subject(s)
Chylomicrons/metabolism , Lipase/physiology , Lipoproteins, VLDL/metabolism , Liver/enzymology , Receptors, Immunologic/physiology , Receptors, LDL , Animals , CHO Cells , Cells, Cultured , Cricetinae , Fibroblasts/metabolism , Humans , Lipoproteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-1 , Membrane Proteins/metabolism , Protein Binding , Proteoglycans/metabolism , Rabbits , Tumor Cells, Cultured
5.
J Cell Biol ; 111(1): 19-29, 1990 Jul.
Article in English | MEDLINE | ID: mdl-1973169

ABSTRACT

We have identified a new coat protein in clathrin-coated vesicles from bovine brain by urea-SDS gel electrophoresis. The protein was purified from Tris-solubilized coat proteins either by combination of hydroxyapatite chromatography and gel filtration or more rapidly in a single step by immunoaffinity chromatography. The purified protein binds to clathrin triskelia and thereby promotes clathrin assembly into regular 50-100-nm cages. We propose for the new protein the name auxilin (Latin auxilium, meaning support). Auxilin migrates as a 110-kD polypeptide in standard type SDS-PAGE, but in the presence of 6 M urea shifts to a position corresponding to 126 kD. Gel filtration in 6 M guanidinium hydrochloride gives a molecular weight of approximately 86,000. The native protein is monomeric in 0.5 M Tris. Antigenic reactivity and two-dimensional peptide maps gave no evidence of gross similarities between auxilin and any of the other known coated vesicle-associated proteins. Since the structural organization of auxilin does not resemble that of the ubiquitous heterotetrameric HA1 and HA2 adaptor complexes, that are believed to connect clathrin to receptors, it is unlikely that it functions as an adaptor. Immunoblotting did not reveal the presence of auxilin in tissues other than brain. If auxilin and AP 180 are indeed both confined to neuronal cells, as the immunochemical evidence suggests, it might be inferred that both serve to adapt clathrin-coated vesicles to an as yet undisclosed function unique to this cell type.


Subject(s)
Brain Chemistry , Clathrin/analysis , Coated Pits, Cell-Membrane/analysis , Endosomes/analysis , Membrane Proteins/analysis , Nerve Tissue Proteins/analysis , Animals , Antibodies , Brain/ultrastructure , Cattle , Chromatography, Affinity , Clathrin/isolation & purification , Clathrin/metabolism , Coated Pits, Cell-Membrane/ultrastructure , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Microscopy, Electron , Molecular Weight , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Peptide Mapping , Protein Binding , Protein Conformation
6.
J Biol Chem ; 264(33): 20089-93, 1989 Nov 25.
Article in English | MEDLINE | ID: mdl-2573598

ABSTRACT

The HA2 adaptor complex, comprising alpha-, beta-, 50-kDa, and 16-kDa subunits, was partially dissociated into its constituents with 3 M urea, and the beta-subunit was purified from the mixture by hydroxylapatite and affinity chromatography. The renatured beta-subunit behaves hydrodynamically as a single polypeptide of Mr approximately 128,000. In a sedimentation assay the purified beta-polypeptide co-sediments with pre-formed clathrin cages. The beta-polypeptide, however, will not induce assembly of clathrin triskelia. Our results support the conjecture that the beta-type subunits (beta and beta') of the HA2 and HA2 adaptor complexes serve to attach the HA-2 adaptor complex to clathrin (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929), while the other subunits may determine the specificity of binding to docking proteins and receptors on cytoplasmic membrane surfaces.


Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Endosomes/metabolism , Animals , Binding Sites , Binding, Competitive , Carrier Proteins/isolation & purification , Cattle , Chromatography , Durapatite , Electrophoresis, Polyacrylamide Gel , Hydroxyapatites , Kinetics , Macromolecular Substances , Molecular Weight , Protein Binding
8.
EMBO J ; 7(4): 919-29, 1988 Apr.
Article in English | MEDLINE | ID: mdl-3402440

ABSTRACT

We have established by peptide mapping and immunochemical analysis of purified clathrin assembly protein preparations from bovine brain, that the cluster of components of mol. wt 100-120 kd fall into four classes, which we term alpha, beta, beta' and gamma. The beta and beta' proteins are immunologically related and generate a series of common tryptic peptides. The same criteria reveal no such homologies between the alpha, beta(beta') and gamma polypeptides. The so-called HA-II assembly protein group contains equimolar amounts of alpha and beta class polypeptides, which are shown to interact with each other. In the HA-I group assembly protein complex gamma and beta' class polypeptides form a stoichiometric complex. Immunofluorescence microscopy reveals that the HA-I complex is specifically associated with clathrin-coated membranes in the Golgi region of cultured cells, whereas the HA-II complex appears to be restricted to coated pits on the plasma membrane. The data lead to the tentative conclusion that the clathrin assembly proteins are involved in the recognition of the intracellular targets by uncoated vesicles.


Subject(s)
Brain/metabolism , Cell Membrane/metabolism , Clathrin/genetics , Golgi Apparatus/metabolism , Animals , Cattle , Clathrin/biosynthesis , Clathrin/isolation & purification , Electrophoresis, Polyacrylamide Gel , Macromolecular Substances , Molecular Weight , Peptide Fragments/analysis , Protein Processing, Post-Translational
9.
EMBO J ; 5(12): 3143-9, 1986 Dec 01.
Article in English | MEDLINE | ID: mdl-3816757

ABSTRACT

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.


Subject(s)
Brain Chemistry , Clathrin/isolation & purification , Animals , Cattle , Clathrin/metabolism , Macromolecular Substances , Microscopy, Electron , Molecular Weight , Peptide Fragments/analysis , Trypsin
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