ORP8 acts as a lipophagy receptor to mediate lipid droplet turnover

Abstract

Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on LDs and is phosphorylated by AMPK, thereby enhancing its affinity for LC3/GABARAPs. Deletion of ORP8 or interruption of ORP8-LC3/GABARAP interaction results in accumulation of LDs and increased intracellular triglyceride. Overexpression of ORP8 alleviates LD and triglyceride deposition in the liver of ob/ob mice, and Osbpl8-/- mice exhibit liver lipid clearance defects. Our results suggest that ORP8 is a lipophagy receptor that plays a key role in cellular lipid metabolism.

Keywords: ORP8; autophagy; lipid; lipophagy.

Figure 1.

The localization of ORP8 on LDs. (A) Live-cell images of mCherry-ORP8-expressing HeLa cells treated with 200 µmol/L OA or serum-starved for 6 h. Cells were stained with Bodipy. (B) Endogenous ORP8 and PLIN2 proteins in the subcellular fractions of HeLa cells were analyzed by Western blot. Cells were treated with 200 µmol/L OA for 12 h. WCL, whole cell lysate; PNS, post-nuclear supernatant; Cyto, cytosol; MEM, cellular membranes. (C) 3D reconstruction image of LDs coated with mCherry-ORP8. (D) APEX-EM image of HeLa cells expressing HA-APEX2-ORP8. The cells were treated with 200 µmol/L OA for 6 h. The arrow indicates APEX2-ORP8 signal on LDs. (E) APEX-EM image of the HA-APEX2-ORP8 on the surface of a purified LD. LDs were purified from HeLa cells expressing HA-APEX2-ORP8 which were pre-treated with 200 µmol/L OA for 12 h. Purified LDs were embedded in 6% gelatin, and performed the APEX-EM analysis. (F and G) Confocal images of HeLa cells expressing the indicated mCherry-ORP8 truncated mutants. Cells were treated with 200 µmol/L OA for 6 h. OA, oleate acid; SS, serum starvation. Scale bars, 5 µm (A, F, G); 0.5 µm (C–E).

Figure 2.

ORP8 promotes turnover of LDs. (A) Maximum intensity projection (MIP) confocal images of HeLa cells treated with ORP8 or ORP5 siRNA for 48 h and serum starvation for 24 h. The cells were stained with Bodipy. (B) PLIN2 in WT and ORP8-KO HeLa cells treated with or without serum starvation or 200 µmol/L OA for 24 h was analyzed by Western blot. (C) TG level in cells treated with or without serum starvation or 200 µmol/L OA for 24 h. Quantitative data are derived from three independent experiments. (D) MIP images of HeLa cells expressing GFP-2A or GFP-2A-ORP8 with or without serum starvation for 24 h. The cells were stained with LipidTox. (E) WT or ORP8-KO HeLa cells were pre-treated with 200 µmol/L OA overnight and cultured in serum-free medium containing 6 μmol/L TrC for the indicated times. Cell lysates were then analyzed by Western blot using the indicated antibodies. (F and G) PLIN2 expression (F) and TG levels (G) in WT or the indicated ORP8 or/and ATGL-KO HeLa cells with or without serum starvation for 24 h. Results are reported as means ± SEM of three replicates (G). (H) Bodipy staining of ATGL-KO HeLa cells pre-loaded with 200 µmol/L OA overnight and cultured with 6 μmol/L TrC and ORP8 siRNA or ORP5 siRNA for 48 h. (I and J) PLIN2 expression (I) and TG levels (J) in ATGL-KO cells treated as (H). Quantitative data are derived from three independent experiments (J). OA, oleate acid; SS, serum starvation; TrC, triacsin C. Scale bars, 10 μm.

Figure 3.

ORP8 is required for lipophagy. (A) WT and ATG7-KO MEFs stained with Bodipy. Cells were pre-loaded with OA overnight and treated with or without ORP8 siRNA for 48 h in regular medium. (B) Quantification of LD number per cell in (A). n = 30 cells. (C) WT or ORP8-KO HeLa cells stably expressing mCherry-GFP-livedrop were serum-starved for 24 h and imaged by confocal microscopy. (D) Quantification of the number of mCherry⁺ GFP^- puncta per cell in (C). n = 30 cells. (E) Representative FACS scatterplots of GFP and mCherry fluorescence in WT or ORP8-KO cells. Cells expressing mCherry-GFP-livedrop were serum-starved for 24 h. (F) Confocal images of GFP-LC3-expressing HeLa cells transfected with mCherry-ORP8 or mCherry-ORP5. The cells were serum-starved for 24 h. (G) Co-localization of mCherry-ORP8, GFP-LC3 and PLIN2-BFP in serum-starved HeLa cells for 24 h. (H) The indicated proteins in HeLa cells and HEK293 cells with or without 50 μmol/L CQ treatment for 24 h were analyzed by Western blot (upper panel). Quantification of the relative protein level of ORP8 (lower panel). Quantitative data are from three independent experiments. (I) The indicated proteins in WT and ORP8-KO HeLa cells cultured in Earle’s balanced salt solution (EBSS) for 12 h with or without 50 μmol/L CQ for 6 h were analyzed by Western blot. OA, oleate acid; SS, serum starvation; CQ, chloroquine. Scale bars, 10 μm (A, C, F), 2 μm (G).

Figure 4.

Characterization of the interaction of ORP8-LC3. (A) The indicated proteins in the subcellular fraction of HeLa cells were analyzed by Western blot. The cells were treated with 200 μmol/L OA for 12 h. (B) GFP-LC3 membranes from EBSS-treated HEK 293 cells stably expressing GFP-LC3 were pulled down by GFP-TRAP beads. Then the beads were incubated with equivalent Lipi-blue stained LDs purified from WT HeLa cells or ORP8-KO HeLa cells. The beads were then imaged by confocal microscope. Note the binding of LDs (red) from WT but not ORP8-KO cells to beads. In order to better show the attachment of LDs to the beads, we used red to mark Lipi-Blue. Scale bar, 10 μm. (C) Purified GST-LC3B was incubated with HEK293T cell lysates overexpressing HA-ORP8 or HA-ORP5, pulled down by GSH beads, and the bound HA-ORP8 and HA-ORP5 were analyzed by Western blot using anti-HA antibody. (D) Purified GST-tagged ATG8 family proteins were incubated with HEK293T cell lysates overexpressing HA-ORP8, pulled down by GSH beads, and the bound HA-ORP8 was analyzed by Western blot. (E) Co-immunoprecipitation of LC3B with ORP8 from HeLa cells. The cells were serum-starved for 24 h, or treated with 200 μmol/L OA or 50 μmol/L CQ for 6 h. (F) Purified GST-LC3B was incubated with HEK293T cell lysates overexpressing WT ORP8 or ORP8 mutants, pulled down by GSH beads, and the bound HA-ORP8 was analyzed by Western blot. (G) Representative MIP confocal images of WT and ORP8-KO cells with or without overexpression of HA-ORP8 WT, HA-ORP8-LIRs-6A or HA-ORP8-H514A/H515A. Cells were serum-starved for 24 h and stained by Bodipy. Scale bars, 10 μm. (H) Quantification of the number of LDs per cell in (G). n = 30 cells. OA, oleate acid; SS, serum starvation; CQ, chloroquine; GABA, GABARAP; LIR, LC3-interacting region.

Figure 5.

AMPK regulates ORP8 activity in lipophagy. (A) Phosphorylation of ORP8 in HeLa cells. Cells were treated with 200 μmol/L OA for 6 h or 2 mmol/L acadesine (AICAR) for 1 h, or were serum-starved for 24 h with or without 20 μmol/L compound C for 6 h. ORP8 was immunoprecipitated by anti-ORP8 and analyzed using an anti-phosphoserine/threonine antibody. (B) Phosphorylation of ORP8 in WT or AMPK α1/α2-DKO MEF cells. Cells were treated with or without serum starvation for 24 h. (C) Co-immunoprecipitation of Myc-AMPK with HA-ORP8 from transfected HEK293T cells. Cells were treated with or without 20 μmol/L compound C for 6 h or were serum- starved for 24 h. (D) In vitro kinase assay of AMPK using purified active AMPK complex (0.2 μg) with purified ORP8 (0.5 μg) as substrate. (E) Phosphorylation of ORP8 mutants in transfected HEK293T cells. Cells were glucose- and serum-starved for 6 h. Immunoprecipitated ORP8 was analyzed by Western blot using a pan phosphoserine/threonine antibody. (F) Phosphorylation of HA-tagged ORP8 and ORP8 mutants in HEK293T cells. Cells were glucose- and serum-starved for 6 h in the presence or absence of 20 μmol/L Compound C. HA-tagged ORP8 was immunoprecipitated with anti-HA and analyzed by Western blot using a specific antibody against ORP8 phosphorylated at Thr54. (G and H) Protein pull-down assay of ORP8 mutants and ORP5 from transfected HEK293T cell lysates after incubation with purified GST-LC3B (G and H) or GST-GABARAP (H). The cells were glucose- and serum-starved for 6 h. (I) Confocal images of ORP8-KO cells transfected with HA-tagged ORP8 or ORP8-T54A/S65A. The cells were serum-starved for 24 h and stained by Bodipy. (J and K) Quantification of the number (J) and total area (K) of LDs per cell in (I). n = 30 cells. Scale bar, 10 μm. (L) FACS scatterplots of GFP and mCherry fluorescence in mCherry-GFP-livedrop expressing ORP8-KO cells. Cells were transfected with or without HA-ORP8-WT or HA-ORP8-T54A/S65A and were serum-starved for 24 h. OA, oleate acid; CC, compound C; SS, serum starvation.

Figure 6.

ORP8 alleviates lipid deposition in mice. (A) Expression of ORP8 protein in the liver of fasting ob/ob mice. (B) TG levels in the liver of ob/ob mice. Mice were intraperitoneally injected with HA-tagged rAAV-vector, rAAV-ORP8-WT, rAAV-ORP8-T54/S65A, rAAV-ORP8-LIRs-6A or rAAV-ORP5. n = 5 mice. (C) Oil red O and HE staining of liver tissue of mice treated in (B). Scale bar, 100 μm. (D) Quantification of Oil red O staining area in (C). n = 5 views from 5 mice. (E) Oil red O staining of liver tissue of WT and Osbpl8^−/− mice after 24 h of fasting or no fasting. Scale bar, 100 μm. (F) Quantification of Oil red O staining area in (E). n = 9 views from 3 mice. (G) TG levels in the liver of WT and Osbpl8^−/− mice after 24 h of fasting or no fasting. n = 3 mice.