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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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 4

apoB ASO–treated livers have evidence of increased autophagy of ER, with no change in ER stress levels.

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apoB ASO–treated livers have evidence of increased autophagy of ER, with...
(A) Liver homogenates from control ASO–, apoB ASO–, and MTP ASO–treated mice were separated by SDS-PAGE and immunoblotted for phoso-eIF2α, GRP78, and actin. Representative immunoblots are shown. cDNA from the same ASO-treated livers was digested by PstI. The upper band represents the spliced form of X-box–binding protein 1 (XBP1). N = 8–12 per group. (B) Primary hepatocytes were stained with ER-Tracker Red for 30 minutes. Images were taken with a Zeiss Axiovert 200M microscope (original magnification, ×200). N = 2 hepatocyte isolations per group; N = 5 images per group. (C) Representative autolysosomes from apoB ASO–treated livers (see Figure 1G) (original magnification, ×20,000) showing lamellae-like structures. Scale bar: 100 nm. (D) Representative images of liver sections that were incubated with anti-LC3 Ab (red) and anti-calnexin Ab (green) and then stained with DAPI (blue). Images were taken with a Nikon A1RPM microscope (original magnification, ×600). N = 3 livers per group; 5 images per liver. Scale bar: 20 μm. (E) Primary hepatocytes isolated from control ASO– and apoB ASO–treated mice were incubated with anti-LC3 Ab (red) and anti-calnexin Ab (green) and then stained for DAPI (blue). Images were visualized using a Nikon Ti Eclipse inverted confocal microscope and a 60×/1.49 NA oil lens. N = 3 hepatocyte isolations per group; 3 images per isolation. Scale bars: 10 μm.