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 7

Three weeks of apoB ASO treatment does not cause a change in ER autophagy but does show an increase in lipid accumulation in the ER lumen.

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Three weeks of apoB ASO treatment does not cause a change in ER autophag...
(A) Primary hepatocytes were stained with ER-Tracker Red for 30 minutes. N = 2 mice; 3 images per mouse. Scale bar: 10 μm. (B) Liver sections were incubated with anti-LC3 Ab (red) and anti-calnexin Ab (green) and then stained with DAPI (blue). Scale bar: 20 μm. (C) Primary hepatocytes were incubated with anti-LC3 Ab (red) and anti-calnexin (green) and stained with DAPI (blue). N = 3 mice per group; 3 images per mouse. Scale bars: 10 μm. (D) Representative EM images (original magnification, ×6,000) of livers from mice treated for 3 weeks. Scale bars: 5 μm. (E) Insets of representative electron micrographs from D showing perinuclear areas with enlarged smooth ER in apoB ASO–treated mice. (F) Primary hepatocytes from mice treated for 3 or 6 weeks were labeled with 14C OA for 16 hours and chased for 4 hours. 14CO2 and 14C ASM were measured and normalized to cell protein levels. N = 9 wells from 3 mice (3 weeks); N = 15 wells from 5 mice (6 weeks). *P < 0.05, by Student’s t test, for apoB ASO versus control ASO for each time period. (G) Primary hepatocytes were labeled with 14C OA for 2 hours, and chase media were added to the hepatocytes for 12 hours and then changed for another 4 hours. The amount of 14CO2 and 14C ASMs produced during labeling and the 12- to 16-hour chase was measured. N = 9 wells from 3 mice per group. (H and I) Hepatocytes were labeled with 14C OA for 16 hours and then chased for 4 hours with or without 50 μM chloroquine or 5 mM 3-MA, after which 14CO2 and 14C ASMs were measured and normalized to cell protein levels. N = 9 wells from 3 mice per group. All values represent the mean ± SD.