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C Vannier

Publications and source records attributed to C Vannier.

At least 37 records · Page 2Linked to original sources
PubMed1989

Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport.

The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.

Animals↗
PubMed1989

Coupling growth arrest and adipocyte differentiation.

The complete differentiation program of preadipose cells can be divided into early and late events. The expression of early markers takes place at growth arrest (G1/S boundary), whereas that of late markers, leading to terminal differentiation, takes place after a limited number of mitoses of early marker-containing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine and results in the formation of triacylglycerol-filled, nondividing cells. The events of adipose cell differentiation which take place in vitro allow a better understanding of the development of adipose tissue in vivo.

Adipose Tissue↗
PubMed1988

Short-term stimulation by insulin of lipoprotein lipase secretion in adipose cells.

The spontaneous secretion of lipoprotein lipase has been examined in adipose cells of mouse Ob17, Ob17SA and 3T3-F442A clonal lines as well as in rat adipose cells in primary culture. Striking differences are observed both in serum-free and serum-supplemented media, rat adipose cells and 3T3-F442A cells being the most active. Insulin from 10(-11) M to 10(-9) M was able to modulate the rate of LPL secretion from 2- to 4-fold. The stimulatory effect of insulin on this process occurred within 30 min in cells treated or not with cycloheximide. It is concluded that insulin is able to modulate the rate of LPL secretion independently of the synthesis of new enzyme molecules on a short-term basis and within a physiological range of concentrations.

Adipose Tissue↗
PubMed1987

Differentiation of human adipocyte precursors in a chemically defined serum-free medium.

Stromal-vascular cells from the inguinal fat tissue of human (age range 1.5 month-27 years), were able to undergo adipose conversion when cultured in a medium containing insulin, transferrin and triiodothyronine. Between 10 and 20 per cent of the cells changed their morphology and accumulated lipid droplets within 10 to 15 days. In most cultures, differentiated cells were present in clusters. These clustered cells were shown by indirect immunofluorescence to contain lipoprotein lipase (located in the Golgi region) and by histochemistry to contain glycerol-3-phosphate dehydrogenase. The occurrence of both enzymes was assessed directly by determining enzyme activities and the synthesis of triacylglycerol was demonstrated by incorporation of [U-14C]glucose into lipids. Foreskin fibroblasts did not display any of these phenotypes. The development of a serum-free, chemically defined medium for the differentiation of diploid adipocyte precursors from human should be of interest for the characterization of factors involved in the stimulation or inhibition of the differentiation process.

Adipose Tissue↗
PubMed1986

Reconstitution of brush border membrane proteins in phosphatidylcholine vesicles. Biochemical and functional characterization.

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.

Alanine↗
PubMed1986

A continuous flow method for the study of lipoprotein lipase secretion in adipose cells.

The secretion of lipoprotein lipase has been examined in Ob17 adipose cells. No spontaneous secretion is detected. The activity of the heparin-releasable enzyme shows a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevents any significant determination of enzyme secretion in heparin-containing media. Thus, a perifusion system, with which the rate of enzyme inactivation is minimal and systematic, has been devised and used. The data show that the secretion of a pool of pre-existing lipoprotein lipase molecules is followed by the secretion of newly synthesized enzyme molecules. The results are discussed with respect to the significance of the determinations of the heparin-releasable enzyme in most studies as well as with respect to the intracellular localization of lipoprotein lipase in Ob17 cells.

Adipose Tissue↗
PubMed1986

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme.

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.

Adipose Tissue↗
PubMed1986

Intracellular localization of lipoprotein lipase in adipose cells.

Subcellular localization of lipoprotein lipase has been examined in differentiated Ob17 adipose cells. No patent activity is detectable in carefully homogenized cells. All latent activity can be unmasked by disrupting membrane structures with neutral detergents. The sequestration of lipoprotein lipase in closed membrane structures is supported by experiments of immunotitration with anti-lipoprotein lipase antibodies and by experiments showing a full protection of the masked activity against proteolytic attack by trypsin. The intracellular distribution of lipoprotein lipase investigated by immunofluorescence staining and by isopycnic centrifugation indicates that a large proportion of the enzyme is located in the Golgi apparatus, in which the activation of the enzyme is likely to take place (C. Vannier et al. (1985) J. Biol. Chem. 260, 4424-4431). Altogether, the results are in favor of a localization of lipoprotein lipase in adipose cells as being typical of that of a secretory protein and underline the absence of lipoprotein lipase in the cell cytoplasm.

Adipose Tissue↗
PubMed1985

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms.

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.

Animals↗
PubMed1985

Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of the enzyme.

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competition for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m-chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.

Adipose Tissue↗
PubMed1985

[Lipoprotein lipase and adipocyte differentiation].

Some hormonal factors, possibly involved in the proliferation and differentiation of adipose precursor cells in vivo, have been characterized in vitro using different preadipocyte cell lines established from rodent adipose tissue. The process of adipose conversion has also been studied using these cell lines; in this process, stem cells (adipoblasts) were committed at any cell division during the growth phase. At confluence, committed cells (preadipocytes) underwent a limited number of mitoses and differentiated into adipose cells, whereas the uncommitted cells remained as stem cells in the cell population. This stochastic model could be extended to the development of rat adipose tissue in vivo. The study of adipose conversion showed the early emergence of lipoprotein lipase (LPL) and monoglyceride lipase (MGL). LPL activity appeared in the cells before any triglyceride accumulation. In contrast, this accumulation seemed dependent upon the emergence of glycerol-3-phosphate dehydrogenase. In vitro experiments clearly established that LPL-containing (differentiating) cells underwent postconfluent mitoses. This limited proliferation was in agreement with previous data obtained in vivo and indicates that only triglyceride-containing (mature) cells could not divide.

Adipose Tissue↗
PubMed1985

Adipose conversion of ob17 cells and hormone-related events.

The ob17 preadipocyte clonal line has been established from the adipocyte fraction of the epididymal fat pads of adult C57 BL/6J ob/ob mice. In vivo, injection of ouabain-resistant mutant cells (ob 17OR11 cell line) into athymic mice is followed by the formation of fat pads containing ouabain-resistant mature fat cells. In vitro, ob17 cells develop after confluence biochemical and morphological characteristics of adipocytes. The adipose conversion process is best represented by a stochastic model in which a pool of stem cells (adipoblasts) give rise to clusters of adipose cells and to additional stem cells that remain in the population. The role of the different factors involved in such conversion is discussed; (1) factors that enhance the number of susceptible cells (ACF or ACF-like compounds), (2) factors without which no adipose conversion takes place (triiodothyronine, growth hormone and other factors still to be characterized), (3) factors that enhance the expression of the differentiation program (insulin). The early emergence of lipoprotein lipase occurs normally in insulin-depleted medium. The separation of ob17 cells by isopycnic centrifugation shows that lipoprotein lipase is present at high levels in early differentiating cells which are still devoid of late markers, ie glycerol-3-phosphate dehydrogenase and triglycerides. These results are discussed with respect to the determination of cellularity during development of adipose tissue in vivo.

Adipose Tissue↗
PubMed1981

Crossed-immunoelectrophoretic study on human renal brush border membrane vesicles.

The human kidney brush border membrane proteins were studied by crossed-immunoelectrophoresis. An antiserum against membrane vesicles was raised in rabbits and used in establishing a reference immunoelectrophoregram with the antigens released by Triton X-100. Among the precipitates observed, the following hydrolases were identified by zymogram staining: Microvillus aminopeptidase (EC 3..4.11.2), gamma-glutamyltransferase (EC 2.3.2.2), maltase (EC3.2.1.20) and trehalase (EC 3.2.1.28). Depletion of the antiserum with sealed, right-side-out vesicles was performed. No precipitates could be seen when the Triton X-100 extract was electrophoresed in a gel containing the depleted antibody. It is therefore suggested that the precipitation of membrane components by the complete antibody is mainly due to externally-located determinants and that the precipitates of the reference pattern correspond to membrane components pointing, at least in part, towards the tubular lumen. Evidence was also noted for a differential removal of antibodies directed against the different antigens. Such an observation could not be explained by the antigen accessibility nor by its amount in the membrane. Parallel crossed-immunoelectrophoresis of Triton X-100 and papain extracts gave rise to an "identity" pattern for only some antigens, particularly for microvillus aminopeptidase and maltase. It is thus strongly suggested that the papain-released form of these enzymes bears nearly all the antigenicity of the whole molecule.

Animals↗
PubMed1980

Purification by affinity chromatography and characterization of a neutral alpha-glucosidase from horse kidney.

A horse kidney neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was purified about 580-fold with a yield of 33% by an affinity chromatography technique using the p-aminophenyl-beta-D-maltoside, a substrate derivative, as ligand. The purified enzyme, homogeneous in polyacrylamide gel electrophoresis, was a glycoprotein with a molecular weight of 280 000 as calculated by gel filtration and its isoelectric focusing points was found to be pH 4.1. The purified enzyme was able to hydrolyze various substrates having (alpha-1,2), (alpha-1,3), (alpha-1,4), and (alpha-1,6) glucosidic linkages. The V/Km ratio shows that the (alpha-1,4) linkages are the best substrates. The pKm of the purified enzyme determined at different pH values indicated that two ionizable groups with pK values 5.2 and 6.9 could be essential in the active site. Enzyme modification with cardodiimide abolished the maltase activity. The turanose, a substrate analogue, protected the enzyme against this inactivation.

Animals↗
PubMed1979

Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.

Glucan 1,4-alpha-Glucosidase↗
PubMed1978

Glucose transport by horse kidney brush borders. I.--Transport properties of brush border membrane closed vesicles.

Brush border membranes isolated from horse kidney cortex as closed right-side out vesicles show selective permeability when analyzed on sucrose and dextran gradients. These vesicles can actively accumulate D-glucose. The preservation of the glucose transport system is demonstrated by the following features: (a) the uptake and release rates of D-glucose are higher in the presence of a sodium gradient, showing that D-glucose transport is a sodium-dependent process; (b) this transport, specific for the D-isomer, is inhibited by phlorizin; (c) the D-glucose transport system is saturable; (d) no inhibition of D-glucose transport is found with C-mannose; (e) the D-glucose uptake is sensitive to osmotic variations.

Animals↗