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
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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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Leptin restores the defect in HDL uptake by ob/ob primary hepatocytes an...
Leptin restores the defect in HDL uptake by ob/ob primary hepatocytes and the altered localization pattern of HDL apoproteins in primary ob/ob hepatocytes. (a) ob/ob mice were injected twice daily with 1 μg/g body weight leptin for the times indicated. Control ob/ob mice (0 hour time point) were injected twice daily with saline and pair-fed to the leptin-treated mice. After treatments, hepatocytes were isolated and incubated with either apoE-free human 125I-HDL or Alexa-HDL (see Figure 6) at 37°C for 1 hour. Cells were then washed, and total cell radioactivity was counted. All experiments used 5 μg of HDL and were performed a total of 2–3 times in triplicate with similar results. Results were reported as mean nanogram of HDL per milligram of cell protein per hour ± SD, after subtraction of background counts measured in the presence of 100-fold excess unlabeled HDL (*0 and 24 hours versus 48 and 72 hours; P < 0.005). (b) Primary hepatocytes from a were incubated with Alexa-HDL at 37°C for 1 hour. Cells were then washed, fixed, and examined using confocal microscopy. Apoproteins in hepatocytes from leptin-treated ob/ob mice were localized in a similar pattern as seen in wild-type mice, i.e., juxtanuclear location. All experiments used 5 μg of HDL and were performed a total of 3 times with similar results.