Lipophagy-derived fatty acids undergo extracellular efflux via lysosomal exocytosis

Abstract

The autophagic degradation of lipid droplets (LDs), termed lipophagy, is a major mechanism that contributes to lipid turnover in numerous cell types. While numerous factors, including nutrient deprivation or overexpression of PNPLA2/ATGL (patatin-like phospholipase domain containing 2) drive lipophagy, the trafficking of fatty acids (FAs) produced from this pathway is largely unknown. Herein, we show that PNPLA2 and nutrient deprivation promoted the extracellular efflux of FAs. Inhibition of autophagy or lysosomal lipid degradation attenuated FA efflux highlighting a critical role for lipophagy in this process. Rather than direct transport of FAs across the lysosomal membrane, lipophagy-derived FA efflux requires lysosomal fusion to the plasma membrane. The lysosomal Ca2+ channel protein MCOLN1/TRPML1 (mucolipin 1) regulates lysosomal-plasma membrane fusion and its overexpression increased, while inhibition blocked FA efflux. In addition, inhibition of autophagy/lipophagy or MCOLN1, or sequestration of extracellular FAs with BSA attenuated the oxidation and re-esterification of lipophagy-derived FAs. Overall, these studies show that the well-established pathway of lysosomal fusion to the plasma membrane is the primary route for the disposal of FAs derived from lipophagy. Moreover, the efflux of FAs and their reuptake or subsequent extracellular trafficking to adjacent cells may play an important role in cell-to-cell lipid exchange and signaling.Abbreviations: ACTB: beta actin; ADRA1A: adrenergic receptor alpha, 1a; ALB: albumin; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; BECN1: beclin 1; BHBA: beta-hydroxybutyrate; BSA: bovine serum albumin; CDH1: e-cadherin; CQ: chloroquine; CTSB: cathepsin B; DGAT: diacylglycerol O-acyltransferase; FA: fatty acid; HFD: high-fat diet; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LIPA/LAL: lysosomal acid lipase A; LLME: Leu-Leu methyl ester hydrobromide; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryo fibroblast; PBS: phosphate-buffered saline; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA2/ATGL patatin-like phospholipase domain containing 2; RUBCN (rubicon autophagy regulator); SM: sphingomyelin; TAG: triacylglycerol; TMEM192: transmembrane protein 192; VLDL: very low density lipoprotein.

Keywords: Fatty acid; MCOLN1/TRPML1; PNPLA2/ATGL; lipid droplets; lipid metabolism; lipophagy.

Figure 1.

Nutrient deprivation and PNPLA2 promote FA efflux. (A–D) Sequestration of media BODIPY C16 in response to fasting and/or BSA in (A) primary mouse hepatocytes, (B) MEFs, (C) Hep3B and (D) HepG2 cells. (E) Effects of nutrient removal on the efflux of ¹⁴C-oleate from hepatocytes with BSA present in the media. (F–G) Effects of Pnpla2 overexpression (AdPnpla2) on FA efflux with BSA present in the media. (H) Effects of silencing Pnpla2 by the administration of shPnpla2 on BODIPY C16 FA efflux in the presence of BSA under fasting conditions in primary mouse hepatocytes. (I) Inhibition of PNPLA2 using 20 μM ATGListat (Astat) on BODIPY C16 FA efflux in the presence of BSA in primary mouse hepatocytes. All experiments were performed at least three times with n = 3, mean±SEM. Statistical differences among groups were determined using one-way ANOVA followed by Dunnett’s post hoc test in A-E, and I; or a two-way ANOVA followed by Turkey’s post hoc test in F-G; or student t-test in G-H. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 were compared to control within groups unless specified otherwise. ^####P < 0.001 were compared to the fasted group

Figure 2.

Lipophagy contributes to PNPLA2 and fasting-initiated FA efflux. (A–E) Media chase ¹⁴C-oleate in response to Pnpla2 overexpression (AdPnpla2) and (A) knockdown of Atg5, (B) inhibition of PIK3C3 (VPS34-IN1, 5 μM) (C) chloroquine (CQ, 5 μM), (D) knockdown of Lipa, or (E) inhibition of LIPA using LAListat1 (10 μM) in mouse hepatocytes. (F) Media BODIPY C16 FA in cells cultured in fed or fasted conditions along with bafilomycin A1 (BafA1, 100 nM) or the above inhibitors. (G) Effect of knockdown of Atg7 on BODIPY C16 FA efflux in primary hepatocytes. The effluxed FAs were measured in the chase media containing 2% FA-free BSA in A-G. (H) Experimental design of FA transfer assay for I-L. (I) FA transfer assay measuring BODIPY C12 FA transfer from donor MEF cells treated with adenoviruses to the GFP-labeled receptor cells. Scale bars: 10 μm. (J) Quantification of the BODIPY-labeled LD in receptor cells from 6 images for I. (K) Effects of Lipa knockdown on fasting-mediated FA transfer in MEF cells. Scale bars: 10 μm. (L) Quantification of the BODIPY-labeled LD in receptor cells from 6 images for K. The receptor cells are outlined. All experiments were repeated at least three times with n = 3, mean±SEM. Statistical differences among groups were determined using two-way ANOVA followed by the Turkey’s post hoc test in A-E, G, J, L; or a one-way ANOVA followed by the Dunnett’s post hoc test in F. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 were compared to control groups (AdCtrl in A-E; Fed in F, G, J, and L). ^#P < 0.05, ^##P < 0.01, ^####P < 0.001 were compared to siCtrl in A, E, G, and L, compared to Vehicle in B-D, compared to Fasted in F, compared to AdCtrl in J

Figure 3.

FA efflux occurs through lysosomal fusion to the plasma membrane. (A) Effect of vacuolin-1 (1 μM) on the efflux of BODIPY C16 FA under fed or fasted conditions in mouse hepatocytes. (B) Media chase ¹⁴C-oleate in response to Pnpla2 overexpression and vacuolin-1 treatment in mouse hepatocytes. (C) Effect of knocking down Mcoln1 (shMcoln1) on the efflux of BODIPY C16 FA under fed or fasted conditions in AML12 cells. The effluxed FAs were measured in the chase media containing 2% FA-free BSA in A–C. (D) Effects of vacuolin-1 on fasting-mediated FA transfer. The receptor cells are outlined. Scale bars: 10 μm. (E) Quantification of the BODIPY-labeled LD in receptor cells from 6 images for D. (F) Effect of transient overexpression of MCOLN1 (TRPML1-YFP) on BODIPY C16 FA efflux in mouse hepatocytes. (G) Workflow for measurement of intracellular FFA using biosensor ADIFAB. (H) Fold-change of intracellular FFA level measured by ADIFAB in AML12 cells. (I) The workflow of lysosome isolation and measurement of lysosomal FFA using ADIFAB. (J) ADIFAB measured the lysosomal FFA level in AML12 cells. (K) Effluxed FFA from in situ liver perfusate (n = 6). All experiments were repeated at least three times with n = 4 unless specified otherwise. Mean±SEM. Statistical differences among groups were determined using two-way ANOVA followed by Turkey’s post hoc test in A-C, F, J, K; or a one-way ANOVA followed by the Dunnett’s post hoc test in E, H. **P < 0.01, ***P < 0.005, ****P < 0.001 were compared to fed or control group. ^#P < 0.05, ^##P < 0.01, ^####P < 0.001 were compared to vehicle (A-B), or shScr (C), or Null (F), or Fasted (H, and J)

Figure 4.

Sphingomyelins regulate FA efflux. (A and B) The lipidomic analysis showed that relative abundances of SM species are upregulated with knockdown Pnpla2 (shPnpla2) in mouse livers (n = 8). (C) SM(d18:1/16:0) reduced Pnpla2 overexpression-mediated FA efflux in mouse hepatocytes. (D) SM(d181/16:0) decreased fasting-induced FA efflux in mouse hepatocytes. (E) SM(d181/16:0) failed to further decrease the reduced FA efflux in AML12 cells treated with shMcoln1 to knockdown Mcoln1. The effluxed FAs were measured in the chase media containing 2% FA-free BSA in C-E. FA efflux assay was repeated at least three times with n = 4. Data presented as mean±SEM, n = 4. Statistical differences among groups were determined using two-way ANOVA followed by Turkey’s post hoc test in B, C, E; or a one-way ANOVA followed by Dunnett’s post hoc test in D. *P < 0.05, ****P < 0.001 were compared to shScr or Vehicle or Fed. ^####P < 0.001 were compared to AdCtrl or Fasted or shMcoln1.

Figure 5.

Blocking FA reuptake decreases intracellular TAG levels. (A) Representative images of LipidTOX stained intracellular LDs under fed or fasted media conditions along with either 2% BSA or CB16.2 (10 μM) in mouse hepatocytes. Scale bars: 20 μm. (B) Quantifications of LD area from 6 images in A. (C) Intracellular TAG levels in mouse hepatocytes under either fed or fasted conditions in the presence or absence of 2% BSA were measured and quantified. (D) Effect of transient overexpression of dsRed2-Dgat1 under indicated media conditions in MEF cells. Representative images of BODIPY C12 FA-labeled LDs. Scale bars: 10 μm. (E) Quantification of LD area from 6 images in C. (F) Effect of DGAT1 and DGAT2 inhibitors on BODIPY C16 FA efflux with BSA present in the chase media in mouse hepatocytes. All experiments were performed at least three times with n = 6. Mean±SEM. Statistical differences among groups were determined using two-way ANOVA followed by Turkey’s post hoc test in B, C, E; or a one-way ANOVA followed by the Dunnett’s post hoc test in F. *P < 0.05, **P < 0.01, ****P < 0.001 were compared to the fed group. ^##P < 0.01, ^###P < 0.005, ^####P < 0.001 was compared to BSA negative group or null group

Figure 6.

Fasting and PNPLA2-mediated FA efflux require reuptake for channeling toward oxidative pathways. (A–B) Fold change of chase ASM level under either overexpression of Pnpla2 along with vacuolin-1 or LAListat1 treatment in mouse hepatocytes. (A) Effect of overexpression of Pnpla2 (AdPnpla2) on ASM. (B) Effect of vacuolin-1 or LAListat1 on ASM while overexpression of Pnpla2 (AdPnpla2). (C) Fold change of chase ASM under fasting conditions in MEF cells. (D) Representative images showing the co-localization of BODIPY C12 FA-labeled LDs with mitochondria. Scale bars: 10 μm. (E) Quantification for D, co-localization was quantified from 6 images using Mander’s overlap analysis. (F) BHBA level was determined from in situ liver perfusates. All experiments were performed at least 3 times with n = 4, mean±SEM. Statistical differences among groups were determined using two-way ANOVA followed by Turkey’s post hoc test in A–C, F; or a one-way ANOVA followed by the Dunnett’s post hoc test in E. *P < 0.05, ***P < 0.005, ****P < 0.001 were compared to AdCtrl or Fed. ^#P < 0.05, ^##P < 0.01, ^###P < 0.005, ^####P < 0.001 were compared to treatment control or fasted group

Figure 7.

Working model. Nutrient deprivation and PNPLA2 activation drive lipophagy and FA generation in lysosomes. FFA are effluxed extracellularly through lysosome fusion with the plasma membrane, which is regulated by the lysosome calcium channel protein MCOLN1. Subsequently, effluxed FA undergo reuptake and then either channeled to mitochondria for oxidation or are re-esterified to TAG and stored in LDs