Abstract 25: Inhibition of VLDL Assembly by Apolipoprotein B Antisense Oligonucleotides Stimulates Autophagy
In order to investigate the effects of reduced VLDL secretion on hepatic lipid metabolism, we examined the effects of knockdown of either apolipoproteinB (apoB) or microsomal triglyceride transfer protein (MTP) using antisense oligonucleotides (ASO) for 6 weeks in apobec-1 knockout mice that only synthesize apoB100. Despite a similar decrease VLDL secretion in mice treated with apoB ASO as compared with MTP ASO, there was an increase in liver triglyceride (TG) content only in the MTP ASO-treated mice. There were no differences in either fatty acid (FA) uptake or secretion, or lipid synthesis from de novo lipogenesis, between the two groups to explain the difference in liver TG mass. In primary hepatocytes isolated from mice treated with apoB ASO, FA oxidation measured after a 16 hr steady state label with 14C oleic acid showed a 3 fold increase in FA oxidation versus cells from control ASO-treated mice. A labeled time course study showed that the increase in FA oxidation was delayed, indicating compartmentalization of lipid in the apoB ASO-treated hepatocytes. Electron microsopy (EM) showed swollen endoplasmic reticulum (ER) and increased autophagosomes in the apoB ASO hepatocytes. The latter was confirmed by an increase in immunofluoresent staining for the autophagy marker LC3b. The LC3b protein was perinuclear suggesting that it was present on mature autophagolysosomes. Treatment with lysosomal inhibitors showed that the accumulation of LC3b was not due to a defect in autophagic flux in these hepatocytes. Autophagosomes in the EMs contained large double membrane structures suggestive of ER. In primary hepatocytes from apoB ASO-treated mice, labeling with ER Tracker showed abnormal aggregates of ER. In addition, immunofluorescent staining studies showed that these abnormal ER aggregates, as represented by calnexin staining, co-localized with the LC3b protein. We propose that autophagy of lipid-filled ER, with subsequent lipolysis of TG in the autophagolysosome and later oxidation of released FA, prevent hepatic steatosis in mice treated with apoB ASO.
- © 2012 by American Heart Association, Inc.