Bax inhibition protects against free fatty acid-induced lysosomal permeabilization

Abstract

Lysosomal permeabilization is a key feature of hepatocyte lipotoxicity, yet the mechanisms mediating this critical cellular event are unclear. This study examined the mechanisms involved in free fatty acid (FFA)-induced lysosomal permeabilization and the role of Bax, a Bcl-2 family member, in this event. Exposure of liver cells to palmitate induced Bax activation and translocation to lysosomes. Studies to suppress Bax activation either by pharmacological approaches or small interfering-RNA-mediated inhibition of Bax expression showed that lysosomal permeabilization is Bax dependent. In addition, palmitate treatment resulted in a significant decrease in Bcl-X(L), a Bax antagonist. Moreover, forced Bcl-X(L) expression blocked lysosomal permeabilization. Lysosomal permeabilization by FFA was ceramide and caspase independent. Finally, paradigms that inhibit lysosomal permeabilization also reduced apoptosis. In conclusion, these data strongly support a regulatory role for Bax in FFA-mediated lysosomal permeabilization and subsequent cell death.

Fig. 1

Bax, a Bcl-2 family member, is activated upstream of cathepsin B (Cat B) and localizes in lysosomes during palmitate treatment. Isolated hepatocytes from C57BL/6 male mice were incubated with 0.2 mM palmitate in media with serum albumin as a carrier in the presence or absence of the Cat B selective inhibitor E-64 for 3 h. Cells were then fixed and subjected to immunofluorescence detection of the active “apoptotic” conformation of Bax by using a monoclonal antibody (MAb 6A7; BD Biosciences) that selectively binds to an NH₂-terminal epitope that is exposed only after Bax activation. A: cells were then imaged by using an inverted laser-scanning confocal microscope. Palmitate treatment resulted in significant Bax activation regardless of the presence or absence of the Cat B inhibitor (inh). B: to assess the cellular localization of active Bax, HepG2 cells were loaded with LysoTracker Red (DND-99, Molecular Probes, Eugene, OR), a fluorescent dye that loads predominantly into lysosomes, for 30 min at 37°C. Cells were then fixed, subjected to immunofluorescence detection of active Bax, and imaged by confocal microscopy as described above. Following palmitate treatment, active Bax fluorescence overlaps in part with LysoTraker Red, suggesting colocalization. FFA, free fatty acid.

Fig. 2

Genetic inhibition of Bax prevents FFA-induced lysosomal permeabilization. Bax expression in HepG2 cells was silenced by small interfering-RNA (siRNA) as described in Materials and Methods. After transfection of HepG2 cells with Bax siRNA, an immunoblot for Bax and β-actin was performed (A). Following 48 h of transfection with the Bax siRNA constructs, cells were loaded with LysoTracker Red (LTR) for 30 min at 37°C and incubated with 0.2 mM palmitate in media with BSA for 24 h. B: cells were then imaged by confocal microscopy. C: cells were scored as either having a predominantly punctuate or diffuse fluorescent pattern. Bax inhibition significantly reduced palmitate-initiated lysosomal permeabilization (*P < 0.01 vs. palmitate-treated cells).

Fig. 3

Bax inhibition reduces palmitate-induced apoptosis. Bax expression in HepG2 cells was silenced by siRNA as described in Materials and Methods. After transfection of HepG2 cells with Bax siRNA, cells were incubated with 0.2 mM palmitate in media with BSA for 24 h. A: apoptosis was quantified by using the DNA-binding dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy. Bax inhibition significantly reduced palmitate-associated liver cell apoptosis (*P < 0.001 vs. palmitate-treated cells).

Fig. 4

Pharmacological Bax inhibition attenuates FFA-induced ctsb-green fluorescent protein (ctsb-GFP) release from lysosomes into the cytosol. McNtcp.24 cells were transfected with ctsb-GFP as described in Materials and Methods. After 48 h cells were pretreated with 20 μM of the selective Bax inhibitor CN 196805 (EMD Biosciences) for 30 min followed by incubation with or without 0.2 mM palmitate in media with serum albumin for 24 h. Subsequently, lysosomal permeabilization was assessed by confocal microscopy of ctsb-GFP (A). B: cells were scored as either having a predominantly punctuate or diffuse fluorescent pattern. Bax inhibition significantly reduced palmitate-associated ctsb-GFP release from lysosomes into the cytosol (*P < 0.01 vs. palmitate-treated cells).

Fig. 5

Palmitate treatment decreases expression of the Bax antagonist Bcl-X_L. HepG2 cells were incubated in the absence or presence of palmitate for up to 6 h. Whole cell lysates containing 40 μg of protein were subjected to SDS-PAGE and immunoblot using a rabbit polyclonal anti-human Bax antibody and a mouse monoclonal anti-human Bcl-X_L antibody. Representative results of at least 3 different experiments are depicted. Palmitate treatment resulted in time-dependent decrease in Bcl-X_L protein expression, whereas no changes was observed for total Bax.

Fig. 6

Bcl-X_L overexpression attenuates palmitate-induced lysosomal permeabilization. A: immunoblot analysis was performed on whole cell lysates from stably transfected clones and the parent cell line. Primary antibody was a mouse monoclonal anti-human Bcl-X_L antibody. β-Actin was used as a protein loading control. Immunoreactivity bands were visualized using a goat anti-mouse or anti-rabbit human IgG peroxidase-conjugated antibody, enhanced by chemiluminescence techniques. Expression of Bcl-X_L was significantly enhanced in transfected cells compared with the control (parent) cell line. Next, control cells and cells overexpressing Bcl-X_L were treated in the absence or presence of palmitate for 24 h. B: cells were then imaged by confocal microscopy. C: cells were scored as either having a predominantly punctuate or diffuse fluorescent pattern. Bcl-X_L overexpression resulted in a significant reduced palmitate-associated lysosomal permeabilization (*P < 0.01 vs. palmitate-treated cells).

Fig. 7

Bcl-X_L-overexpressing cells are protected from palmitate-induced apoptosis. McNtcp.24 cells were stablytransfected with Bcl-X_L as described in Materials and Methods. Stable transformants and nontransfected control cells were incubated with or without 0.2 mM palmitate in media with BSA for 24 h. A: apoptosis was quantified by using the DNA-binding dye DAPI and fluorescence microscopy. Bcl-X_L overexpression significantly reduced palmitate-associated liver cell apoptosis (*P < 0.01 vs. palmitate-treated cells).

Fig. 8

Palmitate-induced lysosomal permeabilization is independent of caspase activation and de novo ceramide synthesis. HepG2 cells were loaded with LTR for 30 min at 37°C and incubated with 0.2 mM palmitate in media with serum albumin in the absence or presence of either the pan-caspase inhibitor z-VAD-fmk (20 μM) or 50 μM of a ceramide synthase inhibitor (fumonisin B₁) for 24 h. A: cells were then imaged by confocal microscopy. B: cells were scored as either having a predominantly punctuate or diffuse fluorescent pattern. Inhibition of caspase activation as well as de novo ceramide synthesis failed to affect palmitate-induce lysosomal permeabilization.