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A Bensadoun

Publications and source records attributed to A Bensadoun.

At least 37 records · Page 2Linked to original sources
PubMed1997

Hepatic lipase is abundant on both hepatocyte and endothelial cell surfaces in the liver.

The cellular location of hepatic lipase was investigated in transgenic rabbits that expressed human hepatic lipase in the liver. The binding of monoclonal antibodies to human hepatic lipase, as detected by either fluorescence-tagged or gold-conjugated secondary antibodies, showed that hepatic lipase was concentrated at the surfaces of hepatic sinusoids. This distribution was the same as observed in the human liver. At the ultrastructural level, immunogold labeling of the space of Disse showed hepatic lipase on both lumenal and sublumenal surfaces of rabbit liver sinusoidal endothelial cells. An equivalent amount of hepatic lipase also was found on the external surfaces of hepatocyte microvilli in the space of Disse, as well as in the interhepatocyte spaces. The distribution suggests that a majority of the hepatic lipase produced by the liver is associated with hepatocyte surfaces, consistent with the functions of this enzyme in lipoprotein metabolism.

Animals↗
PubMed1996

Genetics and molecular biology of hepatic lipase.

Hepatic lipase is emerging as a major factor in the control of HDL metabolism. Hepatic lipase plays a central role in the hydrolysis of HDL2 triglycerides and phospholipids and in the concomitant apolipoprotein A-I efflux from this density class. New data suggest that allelic variation at the hepatic lipase gene locus accounts for 25% of the total variability in HDL cholesterol. Recent information suggests that this lipase may enhance the uptake of lipoproteins by cell surface receptors.

Animals↗
PubMed1996

A compound heterozygote for hepatic lipase gene mutations Leu334-->Phe and Thr383-->Met: correlation between hepatic lipase activity and phenotypic expression.

We have characterized the molecular basis for familial hepatic lipase (HL) deficiency in a Finnish family. In the propositus, the HL deficiency results from compound heterozygosity for two rare HL gene mutations, a previously unknown missense mutation designated L334F and the previously reported T383M mutation. These mutations were introduced into human HL cDNA by site-directed mutagenesis and the constructs expressed in COS-1 cells. In the homogenate of COS-1 cell transfected with the L334F mutant cDNA, a high amount of inactive protein accumulated. In the media of L334F transfected cells, 30% of the wild type activity and 80% of wild type mass were detected. The lysates of COS-1 cells transfected with the T383M mutant cDNA contained 39% of wild type HL activity and 34% of wild type HL mass. In the media of COS-1 cells transfected with the T383M cDNA construct, 50% of wild type HL mass but only 6% of wild type activity was present. The single amino acid substitutions present in L334F and T383M are therefore sufficient to severely affect the HL enzyme. These defects explain the HL-deficient phenotype of the individual carrying the two mutations. The lipoprotein phenotype associated with compound heterozygosity for L334F and T383M mutations is characterized by a slight increase in the buoyant low density lipoprotein (LDL) fraction and an increase in the light high density lipoprotein (HDL) fractions, HDL2a and HDL2b. These results demonstrate that lipoprotein changes occurring in HL deficiency are difficult to identify and support the hypothesis that HL is important in HDL remodeling and metabolism in vivo.

Adult↗
PubMed1995

Heparan sulfate proteoglycans are primarily responsible for the maintenance of enzyme activity, binding, and degradation of lipoprotein lipase in Chinese hamster ovary cells.

Various aspects of lipoprotein lipase (LPL) metabolism, including cell surface binding, degradation, and enzymatic activity, were compared between Chinese hamster ovary (CHO) cells and two distinct proteoglycan-deficient CHO cell lines. The contribution of low density lipoprotein receptor-related protein in binding LPL was also analyzed by the use of a 39-kDa receptor-associated protein expressed as a glutathione S-transferase fusion protein (GST-RAP). Equilibrium binding data with 125I-LPL revealed the presence of a class of high affinity binding sites with a KD of 7.8 nM in CHO cells, whereas no high affinity binding was observed for proteoglycan-deficient cells. The high affinity binding of LPL in CHO cells appeared to be concentrated in cell surface projections and was not effectively inhibited by GST-RAP. Moreover, degradation of endogenous and exogenous LPL was significantly greater in control CHO cells than in proteoglycan-deficient cells. Degradation of LPL in CHO cells was not affected by GST-RAP, suggesting that proteoglycans and not low density lipoprotein receptor-related protein are responsible for the majority of binding and degradation of LPL in these cells. Our data also show that proteoglycan binding is not essential for the assembly of active LPL homodimers, although proteoglycan binding controls the distribution of LPL activity. Furthermore, LPL produced by CHO cells was more stable than LPL produced by proteoglycan-deficient cells.

Animals↗
PubMed1994

Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase.

Lipoprotein lipase (LPL)-binding heparan sulfate proteoglycans (HSPGs) were isolated from cell extracts and conditioned media of cultured adipocytes treated with phosphatidylinositol-specific phospholipase C (PIPLC). The methodology employed included anion exchange chromatography, affinity chromatography on LPL Affi-Prep 10 and hydrophobic chromatography. HSPGs were resolved into two distinct fractions on the Octyl-Sepharose CL-4B matrix. Treatment of the eluted fractions with heparinase and heparitinase yielded core proteins of 48.4 and 39 kDa. The 39-kDa core protein is anchored to the cell surface by a glycosyl phosphatidylinositol anchor as evidenced by 1) release of the HSPG with the 39-kDa core protein into media by PIPLC treatment and 2) biosynthetic incorporation of [3H]ethanolamine and [32P]orthophosphate into the PIPLC-releasable 39-kDa core protein. PIPLC released 23% of the total heparin-releasable LPL. A similar percentage (24.5%) of the total heparan sulfate chains was released by PIPLC. Over 96% of the total adipocyte heparan sulfate chains bound to LPL Affi-Prep 10 column. The heterogeneity of core proteins of HSPGs with affinity for LPL may provide a structural basis for the multiple fates of LPL on the surface of adipocytes, i.e. internalization, degradation, or recycling to the cell surface and translocation into the medium.

Adipocytes↗
PubMed1994

Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the administration of heparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 and HDL2) as well as dense HDL3. A reduction in the quantity of intermediate density lipoproteins (IDL) was also observed. These results demonstrate that HL functions in the metabolism of HDL and IDL, thereby playing a key role in plasma cholesterol homeostasis.

Animals↗
PubMed1994

Enhanced binding and uptake of remnant lipoproteins by hepatic lipase-secreting hepatoma cells in culture.

Rat hepatoma McA-RH7777 cells transfected with a human hepatic lipase (HL) cDNA synthesized and secreted 50-80 ng of human HL/mg of cell protein at 4 h, approximately 50% of which was bound to cell-surface heparan sulfate proteoglycans (HSPG). The newly synthesized HL possessed enzymatic activity. When rabbit beta-very low density lipoproteins (beta-VLDL) and canine chylomicrons or chylomicron remnants were incubated with HL-secreting cells, remnant binding and uptake were enhanced 3-fold compared with nontransfected cells. Furthermore, fluorescence microscopy showed enhanced uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled beta-VLDL by the HL-transfected cells. When 125I-beta-VLDL were added to conditioned medium from HL-secreting cells, the HL in the media enhanced the binding and uptake of the remnant lipoproteins by nontransfected cells about 3-fold. Likewise, surface-bound HL (without HL in the medium) also was able to mediate the enhanced binding of the remnants. This HL-enhanced binding was shown to be mediated by an interaction with cell-surface HSPG. Heparinase treatment to remove cell-surface HSPG or chlorate treatment to prevent HSPG sulfation of the HL-secreting cells abolished all the HL-mediated enhanced binding and uptake. Furthermore, heparinase pretreatment of nontransfected cells prevented the enhanced binding and uptake of beta-VLDL incubated with conditioned medium from HL-secreting cells. As binding was not enhanced in the absence of HSPG, an HL-HSPG initial interaction appears essential. Addition of apolipoprotein (apo) E to the beta-VLDL did not facilitate HL-mediated binding and uptake; in fact, beta-VLDL from apoE-null mice demonstrated a similar degree of enhanced binding as did rabbit beta-VLDL with or without added apoE. On the other hand, beta-VLDL from transgenic mice overexpressing binding-defective apoE(Arg142-->Cys) did not display any enhanced binding and uptake by the HL-secreting cells, and it appears that the apoE(Arg142-->Cys) actually inhibited the HL-mediated interaction. This mutant form of apoE is associated with a dominant mode of expression of type III hyperlipoproteinemia in contrast to the more commonly occurring recessive disorder. Impaired HL interaction with the apoE(Arg142-->Cys) beta-VLDL may contribute to remnant lipoprotein accumulation in the plasma of patients with this mutant form of apoE. Thus, HL contributes to the enhanced cell association of specific types of remnant lipoproteins by initiating their binding to cell-surface HSPG.

Animals↗
PubMed1994

Role of hepatic lipase in the uptake and processing of chylomicron remnants in rat liver.

The participation of hepatic lipase in the initial uptake and processing of chylomicron remnants was studied in the isolated, perfused rat liver. Hepatic lipase activity was either reduced by preperfusion of livers with heparin or inhibited with specific rat hepatic lipase antibodies. [3H]palmitate-labeled chylomicron remnants were recirculated through control and treated livers for 15 min; the livers were then flushed, homogenized, and endosome-rich fractions were isolated. Depletion of hepatic lipase activity by both methods reduced the uptake of chylomicron remnants and hydrolysis of their component triglycerides by perfused rat livers, but at the same time significantly increased the rate of endocytosis of those chylomicron remnants taken up. We conclude that hepatic lipase facilitates, but is not essential for, the initial uptake of chylomicron remnants by rat liver. Furthermore, endocytosis of chylomicron remnants does not require binding to hepatic lipase or the associated hydrolysis of remnant lipids.

Animals↗
PubMed1993

Identification of the 5' regulatory elements of avian lipoprotein lipase gene: synergistic effect of multiple factors.

The organization of cis-acting regulatory elements of the chicken lipoprotein lipase gene was investigated in 5.4 kb of 5' flanking sequences. Various lengths of 5' flanking sequence were linked to the bacterial chloramphenicol acyltransferase (CAT) gene and transfected into primary cultures of chicken adipocytes by DEAE-dextran transfection method. Negative elements are present between -1947 and -139 of the 5' flanking sequence. Removal of these sequences revealed the presence of positive elements located within 138 bp upstream of the major transcription start site. Sequence analysis showed that the region from the major transcription start site to -138 contains an inverted GC box (ACCACGCCCC), a CCAAT element and two direct repeats of the octamer motif, ATTTGCAT. DNase I footprinting assays using a probe extending from -175 to +191, identified three sites protected by nuclear factors. Site I (-126 to -123), a C-rich sequence, GCCC, was identified only on the coding strand. Site II covered the sequence from -95 to -68 and includes the GC box. Site III, from -53 to -26, contained two octamer repeats. Site I is the 5' portion of a 10 bp sequence (CCCTCCCCCC; -126/-116) which is perfectly conserved in the avian and the human promoter. Single or multiple copies of a 37 bp DNA fragment (-138/-102) containing the 10 bp conserved sequence were cloned into LPLCAT-51, upstream or downstream of the major transcription start site and in both orientations; transfection and CAT activity assays with these constructs indicate that the -138/-102 fragment has an enhancer like activity. Additional 5' and internal deletions of LPLCAT-138 suggest that the factors binding to the C-rich element, the GC box and the two octamer repeats have a synergistic effect on promoter activity.

Adipocytes↗
PubMed1993

Site-directed mutagenesis of a putative heparin binding domain of avian lipoprotein lipase.

Lipoprotein lipase (LPL) binds to heparin and heparan sulfate proteoglycans. We have employed site-directed mutagenesis to dissect one of the proposed heparin binding domains of avian LPL, which contains the sequence Arg-Lys-Asn-Arg (amino acids 281-284). Various single, double, and triple mutants of chicken LPL were constructed in order to alter the positive charge of this region. The mutant and wild-type cDNAs were subcloned into an expression vector, pRc/CMV, and expressed in Chinese hamster ovary cells. In general, the LPL mutants with a decrease in regional positive charge showed a decrease in affinity for heparin and heparan sulfate proteoglycans. The greatest effect was seen with the triple mutant, LPL 5G, in which all of the positively charged amino acids were altered to neutral residues. On a heparin-Sepharose column, LPL 5G eluted at 0.96 M NaCl compared with 1.35 M for wild-type LPL. This mutant also had the lowest specific activity with 1.5 mu eq fatty acid/micrograms/h for the cell-associated pool and with no detectable activity in the media. Wild-type cells, however, produced a lipase with a specific activity of 12.4 and 13.1 mu eq fatty acid/micrograms/h for cell-associated and media lipase pools, respectively. LPL 5G also showed a decrease in affinity for the heparan sulfate proteoglycans on the cell surface of Chinese hamster ovary cells. In conclusion, the region of avian LPL between Arg281 and Arg284 does appear to be involved in heparin-binding; however, additional regions must be involved since binding was not completely abolished. In addition, specific activity of the cell-associated and secreted LPL is correlated to affinity of the enzyme for heparan sulfate chains.

Amino Acid Sequence↗
PubMed1993

Lipoprotein lipase synthesis and secretion: effects of concentration and type of fatty acids in adipocyte cell culture.

Fatty acids (FA) were added to differentiating chick adipocytes to study their effects on the synthesis and secretion of avian lipoprotein lipase (LPL). Oleate (18:1n-9), eicosapentaenoate (EPA, 20:5n-3), and linoleate (18:2n-6) were complexed to fatty acid-free bovine serum albumin (BSA) and separately added to cells in RMPI-1640 media containing 0.5% delipidated hen serum. LPL secretion in the presence of 10 U/ml heparin was used as a means of estimating LPL synthesis. FA from 50 microM to 165 microM depressed LPL secretion for cells exposed to 20:5n-3 and 18:2n-6. The decrease in adipocyte LPL secretion was only observed with chronic administration of FA. In other experiments, the rate of LPL synthesis was estimated by incorporation of radiolabel (Tran 35S-label) in cells exposed to 50 microM FA. Total radioactivity incorporated into LPL, expressed as a percentage of total trichloroacetic acid (TCA)-precipitable radioactivity, was not affected in cells fed 18:1n-9 but was significantly decreased for cells fed 20:5n-3 (45%) or 18:2n-6 (67%) relative to controls given equimolar BSA (33 microM). Abundance of LPL message in similarly treated cells also decreased for cells incubated with 20:5n-3 or 18:2n-6 (35%) relative to controls and 18:1n-9-treated cells. A decrease in adipocyte LPL secretion was also observed with administration of lipoprotein (d < 1.006 g/ml) enriched in n-3 and n-6 fatty acids. LPL secretion of cells incubated with n-3 enriched lipoprotein at 50 microM and 75 microM triglyceride fatty acid equivalents was significantly greater than that of cells incubated with n-6-enriched lipoprotein. Interestingly, at the same concentrations of triglyceride fatty acids, lipoproteins enriched with n-9 fatty acids had no effect on LPL secretion relative to controls. These studies document that in cultured avian adipocytes, LPL secretion, synthesis, and level of message are decreased by chronic administration of n-3 and n-6 fatty acids. In contrast, in adipocytes supplemented with oleic acid there was no effect on LPL synthesis and secretion.

Adipose Tissue↗
PubMed1993

Hepatic lipase treatment of chylomicron remnants increases exposure of apolipoprotein E.

The consequences of hepatic lipase treatment of chylomicron remnants were studied. Rats were fed corn oil to induce production and secretion of chylomicrons and were then injected with polyclonal antiserum raised against hepatic lipase to specifically and quantitatively inhibit hepatic lipase activity in vivo. A fraction enriched in chylomicron remnants was isolated from rat plasma by a brief centrifugation step that preferentially isolates triglyceride-rich apolipoprotein (apo) B-48-containing lipoproteins. The chylomicron remnants were then treated with hepatic lipase in vitro, or incubated under identical conditions in the absence of enzyme (control incubations). Hepatic lipase-treated and control chylomicron remnants were isolated by a second brief centrifugation step using discontinuous salt gradients. Control lipoproteins were collected from one discrete band at d < 1.02 g/ml. Hepatic lipase-treated chylomicron remnants formed two discrete bands and were collected at two densities: d < 1.02 g/ml and 1.02 < d < 1.04 g/ml. The buoyant (d < 1.02 g/ml) subfraction of hepatic lipase-treated chylomicron remnants was depleted of 62% of the total phospholipid when compared to control d < 1.02 g/ml lipoproteins. The dense (1.02 < d < 1.04 g/ml) subfraction of hepatic lipase-treated chylomicron remnants was depleted of 65% of particle phospholipid content and 90% of particle triglyceride content when compared to control d < 1.02 g/ml lipoproteins. The dense (1.02 < d < 1.04 g/ml) subfraction of hepatic lipase-treated chylomicron remnants showed 5- to 7-fold greater immunoreactivity of apoE when compared to control lipoproteins in competitive displacement immunoassays. These data suggest that extensive hydrolysis of chylomicron remnant phospholipid and triglyceride leads to the formation of a dense remnant particle that contains highly exposed apoE. This increased exposure of apoE may be the key to the previously observed increased degradation of chylomicron remnants treated with hepatic lipase because more exposed apoE may bind better to cell surface lipoprotein receptors. Furthermore, the data imply that hepatic lipase cleaves chylomicron remnant phospholipid and triglyceride in a sequential fashion; hydrolytic intermediates depleted only of phospholipid precede the formation of a smaller dense remnant particle depleted of phospholipid and triglyceride.

Animals↗
PubMed1992

The structure and complete nucleotide sequence of the avian lipoprotein lipase gene.

The entire gene for chicken lipoprotein lipase (LPL) has been isolated and characterized by primer extension and sequence analysis. The gene is 17 kilobase pairs long and comprises 10 exons and 9 introns. As determined by primer extension analysis the start sites of transcription map 176, 204 and 218 nucleotides upstream of the initiator methionine codon. The 1947 base pairs of 5' flanking sequence contains several putative regulatory elements including two adjacent Oct I binding elements, four glucocorticoid regulatory elements and a sequence very homologous to the previously described fat specific element at--1402 nt. The first intron is very large (6433 bp) and contains four consensus SpI binding-site sequences. Five polyadenylation signals are found in the 3' untranslated region, the last three of which give predicted mRNA species identical in size to those determined by Northern blot. The 5' flanking sequences of the LPL, pancreatic lipase and hepatic lipase genes do not show homology, however. This may account for the homologous amino acid sequences but dissimilar gene expression of these enzymes.

Amino Acid Sequence↗
PubMed1991

Stearyl-CoA desaturase activity in cultured rat hepatocytes.

Stearyl-CoA desaturase activity was measured as a function of time in culture in rat primary hepatocytes up to 90 h of culture. A decrease to half of the original activity occurred within 40 h of culture. Activity remained constant thereafter. Stearyl-CoA desaturase activity increased with insulin molarity in the medium and remained constant as dexamethasone or triiodothyronine concentrations were increased in the medium. When fructose replaced glucose in the medium, the desaturase activity was enhanced. No stimulating effect of cholesterol on the enzyme activity was observed and no inhibiting effect of griseofulvin was shown. Finally, starvation (48 h) of the donor rat followed by 60% sucrose refeeding (48 h) did not enhance delta 9-desaturase activity in the cultured hepatocytes compared with ad libitum nutritional status.

Animals↗
PubMed1991

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation.

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.

Adipose Tissue↗
PubMed1991

Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase.

After adipocytes were labeled with Na2[35SO4], immunoadsorbed with immobilized antilipoprotein lipase, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, a labeled band was identified at 59,700 daltons, the molecular mass of chicken lipoprotein lipase (LPL). Excess unlabeled LPL prevented the immunoadsorption of this labeled species, hence the labeled species was determined to be LPL. Digestion of LPL with endo-beta-N-acetylglucosaminidase H (Endo H) caused a shift in mobility of LPL in SDS-PAGE with no loss of radioactivity, whereas digestion with glycopeptidase F resulted in removal of 99% of the radioactivity. Adipocytes cultured with Trans35S-label and tunicamycin produced an LPL species of 52,000 daltons, but tunicamycin abolished the incorporation of 35SO4 into LPL. This established that 35SO4 was incorporated into an N-linked oligosaccharide of LPL. Endo H digestion of pulse-chase labeled LPL revealed the presence of two complex and one high mannose-type N-linked oligosaccharides. A single 35SO4-labeled tryptic peptide was isolated by reverse phase chromatography. The amino acid sequence of the peptide established that the 35SO4 oligosaccharide is conjugated at Asn-45. Behavior of the 35SO4-labeled oligosaccharide on concanavalin A-agarose, sequential exoglycosidase digestion, and chemical analysis of the 35SO4 oligosaccharide confirms that this moiety is of the complex type. Sequential exoglycosidase digestion, thin layer chromatography of the released monosaccharides, and the use of glycosylation inhibitors established that the sulfated sugar is a core N-acetylglucosamine (GlcNAc). The data show that chicken LPL contains two complex and one high mannose N-linked oligosaccharides and that 35SO4 is incorporated into LPL on a GlcNAc residue of a complex oligosaccharide located at Asn-45.

Adipose Tissue↗
PubMed1991

Lipoprotein lipase.

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Amino Acid Sequence↗