Inhibition of apolipoprotein B synthesis stimulates endoplasmic reticulum autophagy that prevents steatosis
Donna M. Conlon, … , Jing Liu, Henry N. Ginsberg
Donna M. Conlon, … , Jing Liu, Henry N. Ginsberg
Published September 6, 2016
Citation Information: J Clin Invest. 2016;126(10):3852-3867. https://doi.org/10.1172/JCI86028.
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Research Article Metabolism

Inhibition of apolipoprotein B synthesis stimulates endoplasmic reticulum autophagy that prevents steatosis

Abstract

Inhibition of VLDL secretion reduces plasma levels of atherogenic apolipoprotein B (apoB) lipoproteins but can also cause hepatic steatosis. Approaches targeting apoB synthesis, which lies upstream of VLDL secretion, have potential to effectively reduce dyslipidemia but can also lead to hepatic accumulation of unsecreted triglycerides (TG). Here, we found that treating mice with apoB antisense oligonucleotides (ASOs) for 6 weeks decreased VLDL secretion and plasma cholesterol without causing steatosis. The absence of steatosis was linked to an increase in ER stress in the first 3 weeks of ASO treatment, followed by development of ER autophagy at the end of 6 weeks of treatment. The latter resulted in increased fatty acid (FA) oxidation that was inhibited by both chloroquine and 3-methyl adenine, consistent with trafficking of ER TG through the autophagic pathway before oxidation. These findings support the concept that inhibition of apoB synthesis traps lipids that have been transferred to the ER by microsomal TG transfer protein (MTP), inducing ER stress. ER stress then triggers ER autophagy and subsequent lysosomal lipolysis of TG, followed by mitochondrial oxidation of released FA, leading to prevention of steatosis. The identification of this pathway indicates that inhibition of VLDL secretion remains a viable target for therapies aiming to reduce circulating levels of atherogenic apoB lipoproteins.

Authors

Donna M. Conlon, Tiffany Thomas, Tatyana Fedotova, Antonio Hernandez-Ono, Gilbert Di Paolo, Robin B. Chan, Kelly Ruggles, Sarah Gibeley, Jing Liu, Henry N. Ginsberg

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Figure 6

Treatment with apoB ASO for 3 weeks results in increased steatosis and increased ER stress but no change in autophagy.

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Treatment with apoB ASO for 3 weeks results in increased steatosis and i...
Triton WR1339 and 35S-methionine were injected i.v. into apobec-1–KO mice that were treated for 3 weeks with control, MTP, or apoB ASO, and blood samples were obtained over the next 120 minutes. (A) TG levels in plasma were measured enzymatically. (B) apoB was isolated by 4% SDS-PAGE of the 120-minute plasma sample, and bands were cut and counted. N = 5 mice per group. (C) Liver lipid was extracted from mice treated with control ASO, MTP ASO, or apoB ASO for 3 weeks. Liver TG was measured enzymatically and normalized to liver protein. N = 13–14 per group. (B and C) *P < 0.05 for both MTP ASO– and apoB ASO– versus control ASO–treated mice, by ANOVA. Values represent the mean ± SD. (D) Liver sections (5-μm) were stained for neutral lipid using oil red O. Representative images are shown (original magnification, ×400). N = 4 livers per group with 5 images per liver. (E) Liver homogenate was separated by SDS-PAGE and immunoblotted for p-eIF2α, GRP78, and actin. Representative blots are shown. N = 6 livers per group. (F) Representative immunoblots of LC3-I and LC3-II with or without lysosomal inhibition. Graph shows quantification by ImageJ densitometry of immunoblots of the nontreated LC3-II bands. N = 6 per group. (G) Livers from apobec-1–KO mice treated with each ASO for 3 weeks were incubated with anti-LC3 Ab (red) and then stained with DAPI (blue). Images were taken with a NikonA1RMP confocal microscope (original magnification, ×600). N = 3 mice per group; 5 images per mouse. Scale bar: 20 μm. (H) Primary hepatocytes isolated from mice treated for 3 weeks with either control ASO or apoB ASO were incubated with anti-LC3 Ab (red) and stained with DAPI (blue). N = 3 hepatocyte isolations per group; 3 images per isolation. Scale bars: 10 μm.