Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Abstract

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa^-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

Keywords: hepatic stellate cell (HSC); lipase; lipid droplet; lipolysis; retinoid; vitamin A.

Figure 1.

The lysosomal lipase inhibitor lalistat increased the levels of neutral lipids in rat HSCs. A and B, isolated rat HSCs were incubated from day 1 to day 7 in medium with 10% FBS (A) or 10% delipidated FBS (delip) (B) containing vehicle (DMSO) or 100 μm lalistat (lalist). C, isolated rat HSCs were incubated from day 1 to day 4 in medium with delipidated FBS and 10 μm DGAT1 inhibitor T863 additionally containing vehicle (DMSO; T863; white bars) or 100 μm lalistat (T863+lalist; gray bars). D, isolated rat HSCs were incubated from day 1 to day 7 in medium with 10% FBS containing vehicle (DMSO) or 10 μm pyrazole-methanone compounds E4, F2, or H4. Subsequently, neutral lipids were determined by HPLC-MS. The values were normalized to the amount of cholesterol in the sample and expressed relative to the level of the respective lipids present in the control cells incubated with FBS at day 7 (A, B, and D) or to the level of the respective lipids present in the cells at day 1 (C). Data are the means ± S.E. of 6 experiments performed in duplicate (A) or the means ± S.D. of 3 (B and D) or 4 (C) experiments performed in duplicate. *, p < 0.05, t test versus control.

Figure 2.

Lalistat did not affect the degradation on newly synthesized TAG species in rat HSCs. Primary rat HSCs were incubated on day 1 with 25 μm D4-palmitate for 48 h. At day 3, part of the cells were harvested (pulse), and the remaining HSCs were chased for 48 h with normal medium with vehicle (DMSO; control), 100 μm lalistat (lalist), or 40 μm orlistat (orlist) and harvested at day 5 (chase). A, single or double deuterium-labeled (gray and dark gray bars) and non-labeled (white bars) TAG fragments with two palmitoyl chains (16:0,16:0,x) were quantitated and expressed as the percentage of all TAG species. B shows breakdown of TAG species from panel A, as the levels of the indicated TAG fragments at day 5, after the chase, were expressed relative to the level of the same TAG species at the beginning of the chase at day 3. Data are the means ± S.D. of five experiments performed in duplicate. *, p < 0.05 paired t test versus control.

Figure 3.

Effect of lalistat and orlistat on LD morphology and on the activation marker α-SMA in rat HSCs. A, confocal images of HSCs isolated from rats incubated from day 1 to day 7 with vehicle (day 7), 100 μm lalistat, or 40 μm orlistat. Lipid droplets were stained with LD540 dye (green) and anti α-SMA antibody (red), and nuclei were stained with Hoechst (blue). In the first panel the red channel was omitted for better visibility of the LDs. Shown are representative pictures from four experiments. B–D, images were analyzed with CellProfiler v2.1.1. B, LD size was expressed in diameter (nm). C, LD numbers were expressed as a ratio of scored lipid droplets and scored nuclei per image. D, LDs distribution is expressed as percentage of LDs with the specified size (in μm). Image analysis was based on at least 50 cells and 3000 lipid droplets per condition. Data are the means ± S.D. *, p < 0.05 t test versus control.

Figure 4.

Lalistat increased colocalization of LD and the lysosomal compartment in rat HSCs. A, confocal images of HSCs isolated from rats incubated from day 1 to day 4 in medium with 10% FBS in the presence of vehicle (control) or 100 μm lalistat. Live HSCs were stained with Lysotracker Red DND99 (red) and Bodipy (LD dye, green) for 30 min before imaging. Blue is the UV autofluorescence signal from REs. In the boxes higher magnification of the separate channels and the merge are shown. The white bar is 10 μm. B, UV-positive LDs were counted and classified manually. Classifications were (i) not in contact with Lysotracker red structures, (ii) in contact or surrounded by Lysotracker red structures (Lysotr.-associated; white bars) or completely colocalized with Lysotracker red structures (Lysotr.-colocalized; gray bars). Image analysis was based on 40 cells from 4 independent experiments. Data are the means ± S.E. *, p < 0.05, t test versus control.

Figure 5.

LAL/Lipa had retinyl esterase activity in vitro. REH was assayed with RP presented in liposomes as substrate in a buffer with pH 7, pH 4, and pH 4 containing 10 μm lalistat or pH4 containing 10 mm MgCl₂ in homogenates of primary rat HSCs cultured for 4 days (16 μg of protein) (A), freshly isolated rat hepatocytes (400 μg of protein) (B), whole mouse livers (400 μg of protein) (C), or cultured CHO cells 1 day after transfection with an empty expression vector or with an expression vector containing rat Lipa cDNA (40 μg of protein) (D). In B, cholesterol esterase activity (CEH) was assayed with D7-cholesterol oleate as substrate presented in liposomes together with RP. Free retinol, RP, D7-cholesterol, and D7-CE levels were quantitated by HPLC-MS in the MRM mode. Retinol and D7-cholesterol are expressed relative to the levels of their respective esters present at the start of the incubation. Data are the means ± S.D. of three experiments performed in duplicate.

Figure 6.

Retinyl esters did not accumulate in livers of LAL-deficient mice. Livers were obtained from either WT and Lipa^−/− mice at an age of 7 weeks (wk) or between 4 and 5 months (20 weeks). A and B, CEs (panel A) and REs (panel B) were quantitated by HPLC-MS in the MRM mode. The following REs were analyzed: RP (gray bars), retinyl stearate (RS; dark gray bars), and retinyl oleate (RO; white bars). C and D, the relative mRNA expression of LRAT (a marker for quiescent HSCs; panel C) and α-SMA (HSC activation marker; panel D) was measured by qPCR. Data are the means ± S.D. of 5 mice (20 weeks) or 4 mice (7 weeks), assayed in duplicate. *, p < 0.05 t test versus WT.

Figure 7.

Effect of lalistat on neutral lipid levels in mouse HSCs. Isolated mouse HSCs were incubated from day 1 to day 7 in medium with 10% FBS containing vehicle (DMSO; control) and 100 μm lalistat (Lalist). Subsequently, neutral lipids were determined by HPLC-MS. The values were normalized to the amount of cholesterol in the sample and expressed relative to the level of the respective lipids present in the control cells at day 7. Data are the means ± S.E. of six experiments performed in duplicate. *, p < 0.05 t test versus control.

Figure 8.

Expression of Lipa, activation of autophagy, and re-esterification of retinol in rat HSCs. A, relative mRNA expression of lysosomal acidic lipase LAL/Lipa in quiescent (day 1; white bar) and activated (day 7; gray bar) rat HSCs was determined by qPCR. B, immunoblot probed with LC3B antibody of total proteins (10 μg) of quiescent (day 1) and activated (day 7) rat HSCs and the human stellate cell line LX2 incubated for 4 h with vehicle (control), the autophagy inducer rapamycin (200 nm), or rapamycin in the presence of 20 μm chloroquine (CQ) to prevent breakdown of LC3B in the lysosome. LC3B-I is the non-activated form, and LC3B-II is the activated form containing the lipid phosphatidylethanolamine. Shown is a representative blot from three experiments. C and D, primary rat HSCs were either harvested at day 1 or incubated in medium with 10% FBS and 25 μm D4-palmitate-containing vehicle (DMSO; control) or with 100 μm lalistat (Lalist) for 3 days (day 1 to day 4; C) or incubated with medium with 10% FBS containing vehicle (DMSO; control) or with 100 μm lalistat (Lalist) for 6 days (day 1 to day 7; D). D4-labeled and non-labeled REs were quantitated by MRM. D4-labeled retinyl palmitate (D4-Palm) and non-labeled RP were expressed relative to the total RP levels at day 1 (C), or specific RE species were expressed as a percentage of total REs (D). Data are the means ± S.D. of three experiments performed in duplicate. *, p < 0.05 paired t test versus day 1 (A) or versus control (C and D).

Figure 9.

Effect of alterations in neutral lipid metabolism on activation of rat and mouse HSCs. Relative gene expression of α-SMA (HSC activation marker) was measured by qPCR. HSCs isolated from rats or mice were incubated from day 1 to day 7 with vehicle (DMSO; control), 100 μm lalistat (lalist), 40 μm orlistat (orlist), 10 μm pyrazole-methanone compounds E4, F2, or H4, or 50 μm etomoxir. Relative mRNA levels of α-SMA were expressed relative to the levels in the control-treated cells (n = 5 or n = 3 in the case of E4, F2, and H4). Data are the means ± S.D. *, p < 0.05 t test versus control.