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 2

No consistent differences in hepatic DNL, FA oxidation, FA uptake, or FA secretion between apoB ASO– and MTP ASO–treated mice.

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No consistent differences in hepatic DNL, FA oxidation, FA uptake, or FA...
(A) mRNA levels of genes related to lipid synthesis. N = 4–5 per group. (B) Mice were injected i.p. with 3H2O, and the amount of newly synthesized liver lipids was measured after 1 hour. N = 6–8 per group. (C) mRNA levels of genes related to hepatic uptake of plasma FA. N = 4 per group. Groups with different letters above the bars are significantly different at P < 0.05, by ANOVA. FATP2, fatty acid transport protein 2; FATP5, fatty acid transport protein 5; FABP, fatty acid binding protein; Lipc, hepatic lipase; Lpl, Lipoprotein lipase. (D) Mice were injected i.v. with 14C OA, and the disappearance of the labeled FA from plasma was measured over a 5-minute period. N = 3–6 per group. (E) The amount of labeled FA uptake by the liver was measured after 5 minutes in the same mice. N = 3–6 per group. (F) mRNA levels of genes related to FA oxidation. N = 4–5 per group. PPARA, peroxisome proliferator activated receptor α; MCAD, Medium-chain acyl-CoA dehydrogenase; Cpt1, Carnitine palmitoyltransferase 1; Acox, acyl-coenzyme A oxidase. (G) Primary hepatocytes from control ASO– and apoB ASO–treated mice were labeled for 2 hours with 14C OA, and then the amount of 14CO2 and 14C ASMs released into the media was measured and normalized to cell protein levels. N = 12 wells from 4 mice per group. (H) An enzymatic assay was used to measure total plasma ketones in plasma taken from mice treated for 6 weeks with ASO. N = 6–10 per group. (I) Primary hepatocytes from control ASO– and apoB ASO–treated mice were labeled with 14C OA for 16 hours and then chased with unlabeled media for 4 hours. Lipid was extracted from the chase media and separated by TLC. The amount of 14C FA was counted and normalized to total cell protein as a measure of direct FA secretion. N = 9 wells from 3 mice per group. All values represent the mean ± SD. (A and F) *P < 0.05 versus control ASO–treated mice, by ANOVA.