Defective HDL particle uptake in ob/ob hepatocytes causes decreased recycling, degradation, and selective lipid uptake
David L. Silver, … , Nan Wang, Alan R. Tall
David L. Silver, … , Nan Wang, Alan R. Tall
Published January 15, 2000
Citation Information: J Clin Invest. 2000;105(2):151-159. https://doi.org/10.1172/JCI8087.
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Article

Defective HDL particle uptake in ob/ob hepatocytes causes decreased recycling, degradation, and selective lipid uptake

Abstract

Levels of plasma HDL are determined in part by catabolism in the liver. However, it is unclear how the hepatic catabolism of holo-HDL is regulated or mediated. Recently, we found that ob/ob mice have defective liver catabolism of HDL apoproteins in vivo that can be reversed by low-dose leptin treatment. Here we examined HDL catabolism and trafficking at the cellular level using isolated hepatocytes. We demonstrate that ob/ob hepatocytes have reduced binding, association, degradation, and resecretion of HDL apoproteins and 50% less selective lipid uptake relative to wild-type hepatocytes. In addition, HDL apoproteins were found to colocalize with transferrin in the general endosomal recycling compartment (ERC) in wild-type hepatocytes. However, the localization to the ERC was markedly reduced in ob/ob hepatocytes. Filipin staining of cellular cholesterol revealed decreased cholesterol in the ERC in ob/ob hepatocytes. Defects in HDL cell association and cholesterol distribution were reversed by leptin administration. The findings show a major defect in HDL uptake and recycling in ob/ob hepatocytes and suggest that HDL recycling through the ERC plays a role in the determination of plasma HDL protein and cholesterol levels.

Authors

David L. Silver, Nan Wang, Alan R. Tall

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Analysis of binding, association, and degradation of HDL and LDL with ob...
Analysis of binding, association, and degradation of HDL and LDL with ob/ob and wild-type hepatocytes. (a) Binding at 4°C for 1 hour of apoE-free human 125I-HDL or 125I-LDL with freshly isolated hepatocytes. (b) Association of apoE-free human 125I-HDL or 125I-LDL at 37°C for 1 hour with hepatocytes. (c) Degradation of apoE-free human 125I-HDL was determined by following a pulse-chase scheme (see Methods) in the presence or absence of 50 μM chloroquine. Degradation was calculated as the amount of TCA-soluble counts in the media at the end of the chase period (ob/ob versus WT, *P < 0.05, **P < 0.0001). (d) Degradation of 125I-LDL was determined as in c. All experiments used 5 μg of HDL or LDL and were performed in triplicate a total of 3 times, with similar results. Results were reported as mean nanogram of HDL or LDL per milligram of cell protein per hour ± SD, after subtraction of background counts measured in the presence of 100-fold excess unlabeled HDL or LDL. WT, wild-type.