Binding and intracellular trafficking of lipoprotein lipase and triacylglycerol-rich lipoproteins by liver cells.
ABSTRACT The cellular mechanisms and pathways by which lipoprotein lipase (LPL) enhances the binding and uptake of lipoproteins remains unknown. Confocal and immunoelectron microscopy demonstrated that primary binding of bovine LPL (bLPL) occurs at the microvilli surface of HepG2 cells and hepatocytes. Internalized bLPL was associated with endocytic vesicles and multivesicular bodies. Quantitative immunofluorescence indicated that the presence of bLPL caused a marked increase in the cell-surface binding of DiI-conjugated triacylglycerol-rich lipoproteins (DiI-TRL). Confocal microscopy showed that when DiI-TRL was incubated with bLPL at 4 degrees C, the distributions of bound LPL and DiI-TRL were totally coincident, and covered the apical surface of both HepG2 cells and hepatocytes. When incubated separately, the time-courses of the internalization of fluorescence associated with DiI-TRL and bLPL were different: DiI-TRL was quickly internalized by both HepG2 cells and hepatocytes, and reached a plateau at 30 min, whereas intracellular LPL increased continuously, but more slowly in the same period. In the presence of bLPL, DiI-TRL was internalized progressively by HepG2 and by cultured hepatocytes for up to 1 h and no saturation was reached. At this time the intensity of labeling of bLPL was lower than of DiI-TRL and a higher number of DiI spots did not colocalize with bLPL immunofluorescence, suggesting that the ligands follow a different pathway after internalization. The data suggest that when lipoprotein lipase (LPL) is associated with the lipoproteins it directs them to specific endocytic pathways. A hypothetical model of the intracellular pathways followed by triacylglycerol-rich lipoproteins and LPL after internalization is proposed.
Article: [Lipoprotein receptors].Duodecim; lääketieteellinen aikakauskirja 02/1981; 97(18):1534-45.
- [show abstract] [hide abstract]
ABSTRACT: The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane-intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma; there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes.The Journal of Cell Biology 04/1985; 100(3):975-80. · 10.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism. A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1. The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits. Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d < 1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL. However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor. CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment. Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C. Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor. Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect. Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor. These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL. The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.Journal of Biological Chemistry 06/1995; 270(26):15747-54. · 4.65 Impact Factor