Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents
Tsuguhito Ota, … , Constance Gayet, Henry N. Ginsberg
Tsuguhito Ota, … , Constance Gayet, Henry N. Ginsberg
Published December 3, 2007
Citation Information: J Clin Invest. 2008;118(1):316-332. https://doi.org/10.1172/JCI32752.
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Research Article Cell biology

Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents

Abstract

ER stress can cause hepatic insulin resistance and steatosis. Increased VLDL secretion could protect the liver from ER stress–induced steatosis, but the effect of lipid-induced ER stress on the secretion of VLDL is unknown. To determine the effect of lipids on hepatic ER stress and VLDL secretion, we treated McA-RH7777 liver cells with free fatty acids. Prolonged exposure increased cell triglycerides, induced steatosis, and increased ER stress. Effects on apoB100 secretion, which is required for VLDL assembly, were parabolic, with moderate free fatty acid exposure increasing apoB100 secretion, while greater lipid loading inhibited apoB100 secretion. This decreased secretion at higher lipid levels was due to increased protein degradation through both proteasomal and nonproteasomal pathways and was dependent on the induction of ER stress. These findings were supported in vivo, where intravenous infusion of oleic acid (OA) in mice increased ER stress in a duration-dependent manner. apoB secretion was again parabolic, stimulated by moderate, but not prolonged, OA infusion. Inhibition of ER stress was able to restore OA-stimulated apoB secretion after prolonged OA infusion. These results suggest that excessive ER stress in response to increased hepatic lipids may decrease the ability of the liver to secrete triglycerides by limiting apoB secretion, potentially worsening steatosis.

Authors

Tsuguhito Ota, Constance Gayet, Henry N. Ginsberg

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Figure 4

OA or IL induces alternative splicing of XBP1 mRNA and translocation of the alternative form of the protein into the nucleus in McA cells.

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OA or IL induces alternative splicing of XBP1 mRNA and translocation of ...
McA cells were exposed for 3 or 6 hours (A) or for 16 hours (B) to OA (0.4 mM) or IL (500 mg/dl). XBP1 cDNA was amplified by PCR followed by incubation with Pst1. The products of the incubation with Pst1 are shown on the left; the bar graphs show the percentage of total XBP1 mRNA that was resistant to Pst1 and was, therefore, already spliced and activated. After 3 or 6 hours of incubation with OA or IL, most of the XBP1 PCR products were cut by Pst1 (Pst1+), producing a 300-bp amplification product, indicating a predominance of the native, unspliced form of XBP1 mRNA; less than 20% of total XBP1 was detected as the Pst1–, 601-bp amplification product, indicative of spliced XBP1 mRNA. By contrast, after a 16-hour incubation with either OA or IL, a larger proportion of the XBP1 PCR product was Pst1– and kept its full 601-bp length (OA 43% ± 6%; IL 38% ± 4%), indicating partial XBP1 mRNA splicing and presence of ER stress. Complete XBP1 activation and splicing were induced by a 3-hour treatment with tunicamycin (5 μg/ml) as a positive control. Data are mean ± SD (n = 6 for each condition); *P < 0.05, **P < 0.01 versus control incubations (without OA or IL). The nuclear content of XBP1 was not increased after a 6-hour incubation with OA or IL (C) but was increased after incubation with either lipid source for 16 hours (D). Nucleophosmin is shown as a control for the efficiency of the nuclear extraction. ER stress was also induced by a 3-hour treatment with 5 μg/ml tunicamycin as a positive control. Lanes were run on the same gel but were noncontiguous. Data are mean ± SD normalized to cells incubated without OA or IL (n = 3 for each condition); *P < 0.05, **P < 0.01 versus control incubations.