Abstract
The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CTP:phosphocholine cytidylyltransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.
Highlights
The abundance of nuclear lipid droplets (LDs) in hepatocytes led us to hypothesize that they may be related to the synthesis of very lowdensity lipoprotein (VLDL)
We found that microsome triglyceride transfer protein (MTP) inhibitors (MTPi), BAY 13-995212, and CP-34608613, suppressed the oleic acid (OA)-induced increase of nuclear LDs, but not that of cytoplasmic LDs in Huh[7] (Fig. 1a)
The MTP overexpression increased nuclear LDs significantly above the control level (Fig. 1b), suggesting that a disproportionate increase of MTP, which enhances the production of apolipoprotein B (ApoB)-free lumenal LDs7,14, may enhance nuclear LD formation
Summary
MTP activity is essential for nuclear LD formation. Incubation with 0.4 mM oleic acid (OA) increases both nuclear and cytoplasmic LDs in hepatocarcinoma cell lines[6]. Much less ApoB was in the OA/TM-treated microsome than in the control, indicating that NR-lumenal LDs in the OA/TM-treated cell are largely ApoB-free lumenal LDs (Fig. 2d) This conclusion was supported by the lack of ApoB labeling in the nucleus of OA- or OA/TM-treated Huh[7] despite the presence of abundant nuclear LDs (Supplementary Fig. 2d). The latter structure was observed by immunofluorescence microscopy as ApoE-positive NR-lumenal LDs bound with smaller nucleoplasmic LDs labeled for CCTα and perilipin-3 (Fig. 3b, Supplementary Fig. 3a) These results indicated that NR-lumenal LDs turn into nucleoplasmic LDs by disintegration of the surrounding NR membrane, and that LDs newly exposed to the nucleoplasm might fuse with existing nucleoplasmic LDs. To study whether and how NR-lumenal LDs and nucleoplasmic LDs grow in situ, LDs metabolically labeled with BODIPYC12 were bleached with a laser and the fluorescence recovery was examined. LD formation in hepatocytes is drastically different from that reported in yeast[19]
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