Hepatic lipase purification and characterization

Twu, Jer-Shung; Garfinkel, Arlene S.; Schotz, Michael C.

doi:10.1016/0005-2760(84)90201-7

Cited by 55 publications

(18 citation statements)

References 24 publications

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“…Plasma total and HDL cholesterol, triglyceride and glycerol concentrations were determined as previously described ( 15). Plasma hepatic lipase activity, measured only in the SPRET/Pt BSB cross, was measured using a glycerol tri [3H]oleate substrate as previously described (20). Briefly, the substrate and aliquots of preheparin plasma were incubated at 370C for 60 min, and the liberated free fatty acid radioactivity was extracted and counted.…”

Section: Methodsmentioning

confidence: 99%

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

et al. 1995

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We previously described a new mouse model for multigenic obesity, designated BSB. We now report the use of a complete linkage map approach to identify loci contributing to body fat and other traits associated with obesity in this model. Four loci exhibiting linkage with body fat, or with the weights of four different fat depots, residing on mouse chromosomes 6, 7,12, and 15, were identified and confirmed by analysis of additional BSB mice. Each of the four loci differed with respect to their effects on the percent of body fat, specific fat depots and plasma lipoproteins. The loci exhibited allele-specific, non-additive interactions. A locus for hepatic lipase activity was co-incident with the body fat and total cholesterol loci on chromosome 7, providing a possible mechanism linking plasma lipoproteins and obesity. The chromosome 7 locus affecting body fat, total cholesterol and hepatic lipase activity was isolated in congenic strains whose donor strain regions overlap with the chromosome 7 BSB locus. These results provide candidate genes and candidate loci for the analysis of human obesity. (J. Clin. Invest. 1995Invest. . 95:1545Invest. -1552

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Section: Methodsmentioning

confidence: 99%

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

et al. 1995

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“…By contrast, gel filtration techniques have suggested a tetrameric form for rat HL (27,28) and a monomeric form for human HL (29). However, the construction and expression of HL-LPL chimera molecules that were catalytically active (30 -32) suggests that HL, like LPL, may also exists as a homodimer.…”

mentioning

confidence: 98%

“…The only previous analyses of the functional molecular weight of HL have relied on conventional size exclusion chromatography methods (27)(28)(29). A disadvantage of this procedure when used to estimate the molecular weight of a protein is that the elution position depends not only on the molecular weight of the molecule, but also its shape.…”

Section: Ls (Solid Line) Ri (Dashed Line) and Uv (Dotted Line)mentioning

confidence: 99%

Human Hepatic Lipase Subunit Structure Determination

Hill

Davis

Yang

et al. 1996

Journal of Biological Chemistry

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Chinese hamster ovary cells were stably transfected with a human hepatic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, specific activity, and heparin affinity equivalent to HL present in human post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, purified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a differential refractometer, and a light scattering photometer. The polypeptide molecular weight (without carbohydrate contributions) was calculated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies revealed that HL had a molecular mass (with carbohydrate contributions) of 121 kDa. Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation. Purified HL was exposed to various doses of high energy electrons at ؊135°C; lipase activity decreased as a single exponential function of the radiation dose to less than 0.01% remaining activity. The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicate that two HL monomer subunits are required for lipolytic activity, consistent with an HL homodimer. A model for active dimeric hepatic lipase is presented with implications for physiological function.Through its ability to catalyze the hydrolysis of triglycerides and phospholipids, hepatic lipase (HL) 1 influences the metabolism of chylomicron remnants, intermediate density lipoproteins, and high density lipoproteins (1). HL is synthesized by hepatocytes and has been shown to be catalytically active within endosomes (2), on the cell surface (3), and on the surface of the sinusoidal endothelium of the liver (4). In addition, there is evidence to suggest that, independent of its catalytic activity, HL can act as a ligand to facilitate the uptake of remnant lipoproteins through its interaction with cell surface proteoglycans and the low density lipoprotein receptor-related protein (5-7).Although HL appears to have a number of roles in lipoprotein metabolism, it is evident in human HL deficiency that remnant metabolism is primarily affected (8). This observation is consistent with animal studies in which the inhibition of HL activity by the administration of HL antibodies decreased the rate of chylomicron remnant uptake by the liver (9, 10). However, there are several in vitro studies which demonstrate that HL also participates in the remodeling of . This has been clearly dem...

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“…Our findings agree with a recent report (23) indicating that active HL reduced the plasma TC by 55-65%, whereas catalytically inactive HL reduced TC by only 12-20% in LDLr-KO3HL-KO mice on a RCD. Surprisingly, the reduction in plasma PL levels was greater than the decrease in plasma TG levels in mice expressing HL-WT, even though the TG lipase activity of HL-WT is greater than its phospholipase activity (32)(33)(34). The very low TG/PL ratio of HDL may account at least in part for this effect.…”

Section: Discussionmentioning

confidence: 99%

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

Freeman

Amar

Shamburek

et al. 2007

Journal of Lipid Research

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To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL -WT) with mice expressing inactive HL (HL -S145G) in a background lacking endogenous HL and the LDLr (LDLr-KO3HL-KO). HL-WT and HL-S145G reduced (P , 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous 125 I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day 21 : 6.07 6 0.25, LDLr-KO3HL-WT; 4.76 6 0.30, LDLr-KO3HL-S145G; 3.70 6 0.13, LDLr-KO3HL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KO3HL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P , 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoBLp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice. Hepatic lipase (HL) is a multifunctional protein with a complex role in lipoprotein metabolism. It is synthesized and secreted primarily by hepatocytes and functions not only as a lipolytic enzyme that hydrolyzes triglycerides (TGs) and phospholipids (PLs) in apolipoprotein Bcontaining lipoproteins (apoB-Lps) and HDL but also as a ligand or coreceptor that facilitates the cellular uptake of lipoproteins and lipoprotein lipids (1-9). These different functions of HL, which facilitate not only plasma lipoprotein metabolism but also cellular lipid uptake, may have variable effects on atherosclerosis; indeed, human and animal studies appear to support both a proatherogenic and an antiatherogenic role for HL (6,7,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).The mechanisms underlying these variable and sometimes opposing atherosclerosis outcomes are not completely understood. The finding that HL in arterial wall lesions enhances atherosclerosis without altering plasma lipoprotein lipid levels (16, 24) provided partial insight into these discrepancies. Another possible yet not fully investigated explanation for these conflicting data may be the potentially differential effects of the lipolytic versus ligandbinding functions of HL on the plasma concentrations and subclass distributions of the different plasma lipoproteins, which in turn may differentially affect atherosclerosis. Initial...

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Hepatic lipase purification and characterization

Cited by 55 publications

References 24 publications

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

Human Hepatic Lipase Subunit Structure Determination

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

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