Heparin decreases the degradation rate of hepatic lipase in Fu5AH rat hepatoma cells. A model for hepatic lipase efflux from hepatocytes.
The mechanism for the stimulation of hepatic lipase secretion by heparin was studied in cultured Fu5AH rat hepatoma cells. Quantitative immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radioactive enzyme; hepatic lipase protein mass was quantitated by ELISA. Addition of heparin to the medium resulted in a 2-fold increase in lipase secretion rate, whereas cell-surface-associated and intracellular lipase decreased by 76 and 20%, respectively. Rates of synthesis of hepatic lipase measured by incorporation of Trans 35S-label into enzyme protein were not different in control or heparin-treated dishes. In pulse-chase studies, it was estimated that the degradation rate constants for control and heparin-treated cultures were 0.51 +/- 0.09 and 0.14 +/- 0.13 h-1 for control and heparin-treated cultures, respectively. 52% of the synthesized enzyme was degraded in control cultures; addition of heparin to the culture medium reduced this figure to 11% of the synthetic rate. Equilibrium binding data of highly purified 125I-hepatic lipase to Fu5AH cells at 4 degrees C demonstrate the presence of a class of high-affinity binding sites. At 37 degrees C, cell-surface-bound 125I-hepatic lipase is internalized and either degraded or recycled to the medium. The half-intracellular residence times of hepatic lipase were 55 and 31 min in control and heparin-treated cultures, respectively. Radioactivity incorporated in the 55.4 kDa high-mannose-containing lipase and the mature 57.6 kDa species was measured as a means of locating the enzyme in the secretory pathway before or beyond the medial Golgi. The disappearance of the 55.4 kDa species from the cell is similar in control and heparin-treated cultures with half-intracellular residence times of 29 and 25 min, respectively. In contrast, the amount of radiolabeled 57.6 kDa species in control cells remained constant from 15 min to 2 h, whereas it decreased by 79% in heparin-treated cells. The above data demonstrate that the increase in hepatic lipase secretion is due to a decreased degradation rate with no change in synthetic rate and that heparin primarily affected the residence time of hepatic lipase in the medial Golgi-plasma membrane region.
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Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853.
Melford
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Sensel
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Bensadoun
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D000818:Animals; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000494:pharmacology*; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D051381:Rats; D014407:Tumor Cells, Cultured
D007457:Iodine Radioisotopes; D006493:Heparin; D008049:Lipase