Leptin-dependent fat accumulation triggers autophagy in metabolic dysfunction-associated steatohepatitis model

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by hepatic accumulation of fat, mediated by metabolic dysregulation, in the absence of alcohol consumption (1). Metabolic syndrome, which characterizes MASLD, accounts for a prevalence of over 25 percent in Western countries and correlates with an increasing prevalence of MASLD (2). MASLD increases the risk of inflammation and, further, the development of metabolic dysfunction-associated steatohepatitis (MASH) (1). MASH is characterized by hepatic lipid accumulation and intralobular inflammation followed by fibrosis. Patients with MASH have a risk for progressing towards fibrosis, cirrhosis and hepatocellular carcinoma (2).

Autophagy is a cellular catabolic mechanism that can reduce cellular stress by degrading cellular components such as misfolded proteins or damaged organelles. Especially under metabolic stress, autophagy serves as a compensator for the cell by obtaining energy from degraded cellular components (3). Macroautophagy delivers amino acids through the degradation of proteins and recycling of organelles, mobilizes intracellular glycogen storage under metabolic stress, and degrades lipid droplets, leading to increased levels of intracellular triglycerides and free fatty acids (3). Metabolic stress is a strong inducer of autophagy in hepatocytes, whereas the occurrence of high levels of energy substrates, e.g., insulin and/or fatty acids, inhibits autophagy in hepatocytes (3).

The role of autophagy in the pathogenesis of MASLD, MASH and liver fibrosis is controversial and exerts different roles in the cellular population of the liver (4,5). MASLD is characterized by an excessive storage of triglyceride-rich lipid droplets in the cytosol of the hepatocytes (6), which prompts autophagy (4). Autophagy counteracts the accumulation of lipid droplets in the hepatocytes by increased degradation of lipid droplets (lipophagy), thereby preventing the development of MASLD and the progression to MASH and liver fibrosis (4).

Susceptibility to the development of MASLD, such as aging and obesity are associated with a decreased level of autophagy in hepatocytes (6). Therefore, this suggests that autophagy exerts a protective role in the hepatocytes against hepatic steatosis. The activation of the autophagic process also leads to a decrease in intracellular lipid droplets in hepatic stellate cells (HSCs). Thus, promoting the activation of the stellate cells, which further trigger liver fibrosis (5,7).

Adipose tissue secretes various biologically active peptide hormones, called adipokines. They are involved in the regulation of lipid/glucose metabolism, inflammation and fibrogenesis in the liver (8). Adiponectin and leptin are considered the key hormones of adipokines (8).

Leptin binds to the hypothalamic receptors that transduce the signal by the activation of the JAK/STAT and the PI3K signaling pathways. The same signaling pathway is responsible for the proliferation of both HSCs and hepatocellular cancer cells (9,10). Additionally, leptin activates the HSCs through the secretion of tumor growth factor beta (TGF-β) and connective tissue growth factor (CTGF) thereby promoting liver fibrosis (11).

Furthermore, leptin promotes the survival of HSCs by inhibiting TNF-related apoptosis inducing ligand (TRAIL)- and Fas ligand/CD95L (FasL)-induced apoptosis (9). Leptin is responsible for inducing autophagy in hepatoma and breast cancer cells by modulating p53/Forkhead box O3A (FoxO3A) axis (12). However, no correlation between leptin and autophagy has been found in MASLD/MASH yet.

Bursting of the autophagy process could be an effective strategy for the treatment of MASLD and MASH by reducing intracellular lipid accumulation in hepatocytes and cellular stress (4,13).

However, further investigations are needed to verify the HSC-specific inhibition of autophagy as a promising antifibrotic strategy in liver disease.

In this study, we examined the autophagy process in MASLD and MASH mouse models and investigated the effect of monounsaturated fatty acids and caffeine on the metabolism of HSCs and liver cancer cells with emphasis on the link between leptin and autophagy to develop a better understanding of dietary effects in the development of MASLD. We present this article in accordance with the MDAR reporting checklist (available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-17/rc).

Methods

Cell lines

The human hepatoblastoma cell line HepG2 (ACC180, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany), the human hepatocellular carcinoma cell line Hep3B (ACC93, DSMZ) and the human HSC line LX-2, a kindly gift from Scott Friedmann (Icahn School of Medicine at Mount Sinai), were grown in DMEM (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) (11548876, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were routinely tested for mycoplasma.

Mouse strain

Fatty liver Shionogi (FLS) mouse spontaneously develops fatty liver without obesity and is used as an MASLD model in this study (14). The ob/ob mouse strain is a leptin-deficient mouse model for obesity, which develops MASH (15,16). In contrast to the FLS mouse, the ob/ob mouse develops hepatic steatosis due to its excessive ingestion with consecutive obesity, which is mediated by its leptin deficiency (15). Both mouse strains were fed with normal food and water ad libitum. Neither special food nor medications were used to induce MASLD and MASH.

Liver specimens, used in this study, were collected from 24-week-old male mice (FLS, n=6; ob/ob, n=6) at Tottori University (17). No new animal experiments were performed for this study. The original animal study was approved by the ethics committee of Tottori University (protocol No. 11-Y-54). The liver specimens were snap frozen and kept at −80 °C till the isolation of proteins and RNA for the performance of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting.

Substances

Bafilomycin A1 (tlrl-baf1, InvivoGen, Toulouse, France), oleic acid (4954, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and caffeine (205548, Sigma-Aldrich) were dissolved in sterile DMSO (WAK-DMSO-50, WAK-Chemie Medical GmbH, Steinbach, Germany).

RT-qPCR

Total RNA was isolated with the RNeasy Mini Kit (74106, QIAGEN, Hilden, Germany) according to the manufacturer’s protocol from HepG2 and LX-2 cells previously treated for 24 h with 2 mM oleic acid alone or in combination with 100 pM bafilomycin and 2 mM caffeine. Reverse Transcription of mRNA with iScript™ cDNA Synthesis Kit (170-8891, Bio-Rad Laboratories, Hercules, CA, USA) on FlexCycler (Analytik Jena AG, Jena, Deutschland). Qiagen primers for human BECN1 (QT00004221), UVRAG (QT00034328), MAP1LC3B (QT00055069), SQSTM1 (QT00095676), ACTA2 (QT00088102), COL1A1 (QT00088711) and GAPDH (QT01192646) were used with GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) on RT-qPCR thermocycler CFX96™ Real-Time System (Bio-Rad Laboratories). Results were analyzed with the Bio-Rad CFX-Manager (Bio-Rad Laboratories) and normalized with GAPDH mRNA content for each sample. Raw data were further analyzed with Rest2009 (Relative Expression Software Tool V.2.0.13, Qiagen).

Western blot analysis

Whole cell lysates were prepared with Jie’s Buffer (10 mM NaCl, 0.5% NonidetP40, 20 mM Tris-HCL pH 7.4, 5 mM MgCl₂, 1 mM PMSF, Complete Protease Inhibitor and Phosphatase Inhibitor (Sigma-Aldrich Chemie GmbH) from HepG2 and LX-2 cells previously treated for 24 h with 2 mM oleic acid alone or in combination with 100 pM bafilomycin and 2 mM caffeine. Proteins were separated using SDS-Page (NP0342, Life Technologies, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (10600009, GE Healthcare Life Science, Chicago, IL, USA) by semi-dry-blotting with Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories). The membranes were then cut into strips corresponding to the molecular weight of the target proteins, blocked with 4% BSA (23208, Thermo Fisher Scientific) in TBS-Tween20 (0.5%) and incubated with primary antibodies against Beclin1 (ab92389, Abcam, Cambridge, UK), UVRAG (U7508. Sigma-Aldrich, St. Louis, USA), LC3B (ab51520, Abcam), SQSTM1 (ab96706, Abcam), AMPK-α (2532S, Cell Signaling Technology, Danvers, MA, USA), Phospho-AMPK-α (T172) (2525S, Cell Signaling Technology) and β-actin (A5441 Sigma-Aldrich). Bound primary antibodies were detected by secondary horseradish-labeled goat anti-rabbit (A0545, Sigma-Aldrich) and goat anti-mouse (A9917, Sigma-Aldrich) antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The immuno-detection was quantified using Fusion image capture (VILBER LOURMAT Deutschland GmbH, Eberhardzell, Germany) and Bio-1D analysis System (VILBER LORUMAT Deutschland GmbH). The uncropped original blots are shown in Figure S1.

Picrosirius red stain

Collagen fibers of the human HSCs LX-2 were stained with the Picrosirius Red Stain Kit (24901, Polysciences Inc., Warrington, PA, USA). Collagen assumes a red color under light microscopy. Muscle fibers and cytoplasm are identified by yellow colors. Polarized light was used to differentiate between the different types of collagen fibers. Collagen type I (yellow/orange birefringence), collagen type II (green birefringence). The micrographs were acquired by Leica DM5000.

Oil Red O Stain

Lipids of the human HepG2 cells and HSCs LX-2 were stained with Oil Red O Stain (Merck KGaA, Darmstadt, Germany) and hematoxylin. Light microscopy (Leica DM5000) was used to identify the intracellular accumulation of lipid droplets.

Stable transfection

Hep3B cells were stably transfected with an E. coli plasmid encoding red fluorescent protein-green fluorescent protein-MAP1LC3B (RFP-GFP-MAP1LC3B) by incubating them with 20 µg/mL plasmid in serum-free medium along with FUGENE^® HD Transfection Reagent (Promega). Three days post-transfection, the selective agent G-418 (04727894001, Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland) was introduced into the fresh medium. Over the following weeks, transfected cells were distinguished from non-transfected cells by pipette scratching under a fluorescence microscope, and the positively transfected cells were plated into new dishes containing fresh medium supplemented with G-418 (18). These stably transfected cells were subsequently seeded in 6-well plates and allowed to attach for 24 hours before being treated for 48 hours with 2 mM oleic acid dissolved in phenol-red free complete DMEM growth medium, which was supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) (Thermo Fisher Scientific). The fluorescence of the cells was monitored using the IncuCyte^® S3 Live-Cell Analysis System (Sartorius, Göttingen, Germany).

Statistical analysis

Statistical analysis was performed using SPSS 15.0.1 for Windows (SPSS Inc., Chicago, IL, USA). Significance was calculated using the 2-way analysis of variance (ANOVA) with Tukey’s post hoc test/Dunnett’s multiple comparison test for paired samples. A P value of less than 0.05 was considered to indicate statistical significance. Detailed P values for all comparisons are provided in Appendix 1.

Results

Characterization of FLS and ob/ob mice

The expression of autophagic markers was detected in the liver of 24-week-old male FLS (n=6) and ob/ob (n=6) mice. The FLS mouse spontaneously develops hepatic steatosis without obesity, whereby the FLS mouse represents the early stage of MASLD, a non-inflammatory fatty liver (14). The ob/ob mice, owing to their leptin deficiency mediated increased ingestion/hyperphagia, develops obesity resulting in a fatty liver with inflammatory and hepatocyte damaging characteristics consistent with MASH (15,16).

As shown in Table 1, FLS and ob/ob mice differed significantly for body weight, blood sugar, liver weight, fat, triglycerides and fatty acids. The level of alanine aminotransferase (ALT) was significantly higher in ob/ob compared to FLS. These parameters confirmed that ob/ob are affected by non-alcoholic steatohepatitis as previously shown (17).

Leptin-deficient mice show increased autophagic markers expression associated with MASH

ob/ob mice (n=6) showed a significantly higher expression of the autophagic transcripts Becn1, Map1lc3b, Sqstm1 Uvrag and Prkaa1_2 compared to the FLS mice (n=6) (Figure 1A). The detection of the protein level of the autophagy players in ob/ob mice (n=6) evidenced, as shown in Figure 1B, an over-expression of Beclin1, Lc3b-I and Uvrag (P<0.05). Interestingly, FLS (n=6) and ob/ob (n=6) mice differ substantially concerning the expression of Ampk-α, a key regulator of the autophagy process, and its active phosphorylated form P-Ampk-α (Thr172). The ob/ob mice (n=6) showed a reduction of the protein level of the total Ampk-α, which was outbalanced in favor of the active form P-Ampk-α. Instead, FLS mice (n=6) showed no detectable expression of P-Ampk-α even though expressing a higher protein level of the total Ampk-α (Figure 1B).

Figure 1 Detection of the autophagy markers in the liver tissue of FLS and ob/ob mice. (A) Whisker and box plots of the expression of the transcripts for Becn1, Map1lc3b, Sqstm1, Uvrag, Prkaa1_2 and Prkaa2_2 in the liver tissue of ob/ob mice. Values above 2 were considered significantly over-expressed genes. Instead, values below 0.5 were assigned as down-regulated expression of the target genes. The expression was normalized to Gapdh. The expression of the genes in each single ob/ob mouse (n=6) was normalized to the mean expression of the FLS mice (n=6). (B) Detection (Western blot) and densitometric quantification of the protein level of Beclin1, Lc3b-I, Lc3b-II, Uvrag, p62, Ampk-α and P-Ampk-α. Beta-actin was detected as equal loading control and used for further densitometric normalization of the protein level of the target proteins. The error bars of the densitometric graph represent the SEM of experiments performed in triplicates. *, P<0.05 by 2-way ANOVA with Tukey’s test (see Appendix 1 for detailed statistical analysis). ANOVA, analysis of variance; FLS, fatty liver Shionogi; SEM, standard error mean.

These results suggest that the autophagy flux is activated in ob/ob mice. The absence of Leptin gene and the fat accumulation could correlate with the autophagy process in ob/ob mice. Instead, an inactive autophagy flux, as supported by the lower expression of the autophagy markers, could characterize FLS mice.

Effects of monounsaturated fatty acids and caffeine on the expression of autophagy markers

The efficacy of the solo and combined treatment of oleic acid, bafilomycin and caffeine on the expression of the autophagic markers in human hepatoblastoma HepG2 cells was determined by RT-qPCR and by Western blotting.

The oleic acid was administered for 24 h in order to increase the cellular content of unsaturated fatty acids (19) with emphasis on the autophagy process.

Treatment with 2 mM oleic acid, for up to 24 h, did not modulate the expression of autophagic transcripts, besides a significant reduction of PRKAA2_1 in HepG2 cells compared with untreated cells (Figure 2A). Bafilomycin A1, a V-ATPase inhibitor, prevents maturation of autophagic vacuoles by inhibiting the fusion of autophagosomes and lysosomes (20). The administration of 100 pM bafilomycin (24 h) caused a significant down-regulation of MAP1LC3B and PRKAA1_1 transcript in HepG2 cells. Caffeine was included in the treatment because of its ability to burst the cellular metabolism (21). The 24 h treatment of HepG2 cells with 2 mM caffeine also caused a significant down-regulation of MAP1LC3B and UVRAG transcripts. The combination of oleic acid and bafilomycin (24 h) caused no variation of the transcript level of the autophagy markers. The addition of caffeine to oleic acid was responsible for the significant down-regulation of UVRAG (Figure 2A).

Figure 2 Detection of the autophagy markers in liver cancer cells. (A) RT-qPCR detection of BECN1, MAP1LC3B, SQSTM1, UVRAG, TFEB, PRKAA1_1, and PRKAA2_1 in HepG2 cells after the administration of 2 mM OA, 100 pM BAF, 2 mM caffeine and their combination. The target expression was normalized to GAPDH. Data are presented as means ± SEM of treated vs. untreated cells from three independent experiments. (B,C) Western blot detection and densitometric quantification of the protein level of Beclin1, LC3B-I, LC3B-II, UVRAG, p62, AMPK-α and P-AMPK-α. Beta-actin was detected as equal loading control and used for the further densitometric normalization of the protein level of the target proteins. The error bars of the densitometric graph represent the SEM of experiments performed in triplicates. (D) Micrographs of MAP1LC3B-GFP-RFP stably transfected Hep3B cells after the administration of 2 mM OA (scale bar =300 µm). The cells were live monitored for up to 48 h (please refer to the Videos S1,S2). *, P<0.05 by 2-way ANOVA with Dunnett’s test (see Appendix 1 for detailed statistical analysis). ANOVA, analysis of variance; OA, oleic acid; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SEM, standard error mean.

The protein level of the autophagy markers was investigated in HepG2 cells after 24 h administration of 2 mM oleic acid, 100 pM bafilomycin and 2 mM caffeine (Figure 2B,2C). Oleic acid caused a down-regulation of Beclin1, LC3B-I, LC3B-II, UVRAG and p62 and an over-expression of AMPK-α and its phosphorylated active form P-AMPK-α (Thr172). Bafilomycin caused as well a reduction of the protein level of Beclin1, LC3B-I, LC3B-II, UVRAG, and p62, and an over-expression of AMPK-α, whereas the level of P-AMPK-α was slightly down-regulated. Caffeine showed similar effects of bafilomycin. The addition of bafilomycin did not alter the effect of oleic acid responsible for the down-regulation of Beclin1, LC3B-I, LC3B-II, UVRAG and p62 and an over-expression of AMPK-α and its phosphorylated active form P-AMPK-α (Thr172). Instead, the addition of caffeine to the oleic acid caused an increase of Beclin1 and UVRAG but not of P-AMPK-α (Figure 2B,2C).

The administration of oleic acid caused a reduction in the protein level of the autophagy proteins being responsible for the synthesis of autophagosome vesicles. Additionally, it induced an increase in P-AMPK-α protein level, an energy sensor that regulates cellular metabolism and in particular serves as the autophagy promoter. Thus, confirming that oleic acid is responsible for the activation of autophagy. Instead, a down-regulation of both autophagy markers and P-AMPK-α was observed after treatment with bafilomycin, the autophagy inhibitor, and caffeine, thus suggesting that both substances impair autophagy.

Real-time monitoring of autophagy process in Hep3B liver cancer cells

After detecting the expression of autophagy associated marker proteins and genes indicative of an enhanced autophagic activity under treatment with oleic acid, we investigated to what extent oleic acid induced functionally active autophagy by real-time monitoring of stably GFP-RFP-MAP1LC3B transfected Hep3B cells.

The stably transfected cells expressed ectopic double labeled LC3B which allows to monitor autophagosome maturation and terminal degradation activity of autolysosomes, thereby offering qualitative assessment of autophagic activity (18,20,22). Through the fusion of the autophagosome and the lysosome, the GFP-RFP-labeled LC3B enters the acidic milieu where the green fluorescent acid-sensitive GFP is degraded while the red fluorescent acid-stable RFP retains its fluorescent function (18,22).

The 48 h treatment with 2 mM oleic acid caused a decrease in the GFP and the stabilization of the RFP in Hep3B cells demonstrating a functional active autophagy maturation process and the final fusion of the autophagosome vesicles with the lysosomes (Figure 2D and Videos S1,S2).

Accumulation of lipid droplets in HepG2 cells

An increase in the concentration of monounsaturated fatty acids could prompt the accumulation of lipid droplets in hepatic cells. In order to analyze the involvement of autophagy in such process, HepG2 cells were treated for 24 h with 2 mM oleic acid, the inhibitor of autophagy bafilomycin and the metabolic promoter caffeine. As shown in Figure 3, the 24 h administration of 2 mM oleic acid caused a slight increase in lipid droplets in HepG2 cells. However, the increase of lipid droplets was lower than in cells treated with TGF-β, included as positive control. Interestingly, the administration of 100 pM bafilomycin, as negative regulator of autophagy, caused a strong accumulation of lipid droplets. Its combined administration with oleic acid showed a similar accumulation of lipid droplets in comparison with the treatment with the single substance. The treatment with 2 mM caffeine evidenced an increase in lipid droplets in HepG2 cells when administered for 24 h in combination with 2 mM oleic acid (Figure 3). Once again, fatty acid administration prompted autophagy, thereby impeding the accumulation of lipid droplets. In contrast, lipid droplets accumulated extensively after treatment with the autophagy inhibitor bafilomycin or the metabolism inducer caffeine.

Figure 3 Autophagosome maturation monitoring in Hep3B treated with oleic acid. Intracellular lipid accumulation in HepG2 cells treated for 24 h with 500 ng/mL of rhTGF-β, 2 mM oleic acid, 100 pM bafilomycin, 2 mM caffeine and their combination. The lipids were stained with Oil Red O. Magnification: 100× (A,C) and 200× (B,D).

Expression of lipid droplets in HSCs

Occurrence of lipid droplets correlates negatively with the activation status of stellate cells. The administration of 5 ng/mL of TGF-β to HSCs was used as positive control of their active status (23).

Treatment with 2 mM oleic acid for 24 h increased the number of lipid droplets in the HSCs (Figure 4A). The administration of 100 pM bafilomycin caused a slight increase in the number of lipid droplets comparable to the TGF-β treated cells (transactivation positive control). The administration of caffeine, instead, evidenced a strong accumulation of lipid droplets. The combination of both oleic acid and bafilomycin or caffeine sustained the accumulation of lipid droplets similar to oleic acid alone.

Figure 4 Detection of lipid accumulation and stellate cells activation. (A) Intracellular lipid accumulation in LX-2 cells treated for 24 h with 500 ng/mL of rhTGF-β, 2 mM OA, 100 pM bafilomycin, 2 mM caffeine and their combination. The lipids were stained with Oil Red O. The magnification is 200×. (B) RT-qPCR detection of ACTA2 and COL1A1 in LX-2 cells treated for 24 h with 2 mM OA, 100 pM bafilomycin, 2 mM caffeine and their combination. The target expression was normalized to GAPDH. Shown are means ± SEM of treated vs. untreated of experiments performed in triplicates. (C) Picrosirius red staining of the collagen fibers of LX-2 cells treated for 24 h with 500 ng/mL of rhTGF-β, 2 mM OA, 100 pM bafilomycin, 2 mM caffeine and their combination. The micrographs were acquired by polarized light. The magnification is 200×. *, P<0.05 by 2-way ANOVA with Dunnett’s test (see Appendix 1 for detailed statistical analysis). OA, oleic acid; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SEM, standard error mean.

Monitoring of HSC activation status

Stellate cells are strictly involved in the MASH pathogenesis of the liver. For this reason, the trans-activation status of HSCs LX-2 was monitored after the treatment for 24 h with 2 mM oleic acid, 100 pM bafilomycin, 2 mM caffeine and their combination by detecting the expression of stellate cell activation genes (ACTA2, COL1A1) and staining of collagen fibers (24) associated with stellate cell activation.

The anti-fibrogenic effects of oleic acid have been previously described in HSCs (25). Here, the HSCs LX-2 treated for 24 h with 2 mM oleic acid showed a significantly increased expression of COL1A1 transcript and a stable expression of ACTA2 (Figure 4B). Treatment with 100 pM bafilomycin (24 h) caused no alteration of COL1A1 and ACTA2 transcripts and neutralized the over-expression of COL1A1 determined by oleic acid (Figure 4B). Caffeine caused a significant down-regulation of ACTA2 transcript. Additionally, its combined administration with oleic acid caused a significant reduction of both ACTA2 and COL1A1 (Figure 4B).

Furthermore, we investigated the production of collagen by the HSCs under different treatment modalities. The protein level of collagen was detected in LX-2 cells under polarized light after staining with picrosirius red. The HSCs treated with TGF-ß were included as positive control (23). Neither after solo treatment with oleic acid, bafilomycin, caffeine nor after their combination was a change of collagen distribution, which was unchanged in comparison to untreated cells as shown by the overall red staining of the collagen fibers (Figure 4C).

Despite an over-expression of the transcripts of the genes deputed to the transactivation of the stellate cells, no increase in the inducible collagen could be observed. Thus, highlighting that the stellate cells probably do not trans-activate after an increase in the amount of fatty unsaturated acids.

Autophagy status in HSCs

We further investigated the possible implication of autophagy after accumulation of monounsaturated fatty acids by analysis of autophagic marker genes and proteins.

Transcript analysis showed significantly increased expression of BECN1 and MAP1LC3B and a significant reduction of PRKAA1_1 and PRKAA2_1 transcripts in LX-2 cells treated for 24 h with 2 mM oleic acid (Figure 5A). The oleic acid caused a downregulation of the protein level of all analyzed autophagy markers; interestingly the protein level of P-AMPK-α was not affected and kept stable (Figure 5B). The administration of 100 pM bafilomycin caused no significant alterations of the expression of autophagic transcripts (Figure 5A). In addition, bafilomycin reduced the expression of Beclin1, UVRAG and p62 proteins and suppressed AMPK-α and P-AMPK-α protein levels (Figure 5B).

Figure 5 Detection of the autophagy markers in LX-2 cells. (A) RT-qPCR detection of BECN1, MAP1LC3B, SQSTM1, UVRAG, TFEB, PRKAA1_1 and PRKAA2_1 in LX-2 cells after the administration of 2 mM OA, 100 pM bafilomycin, 2 mM caffeine and their combination. The target expression was normalized to GAPDH. Data are presented as means ± SEM of treated vs. untreated cells from three independent experiments. (B) Western blot detection and densitometric quantification of the protein level of Beclin1, LC3B-I, LC3B-II, UVRAG, p62, AMPK-α and P-AMPK-α. Beta-actin was detected as equal loading control and used for the further densitometric normalization of the protein level of the target proteins. The error bars of the densitometric graph represent the SEM of experiments performed in triplicates. *, P<0.05 by 2-way ANOVA with Dunnett’s test (see Appendix 1 for detailed statistical analysis). ANOVA, analysis of variance; OA, oleic acid; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SEM, standard error mean.

The combined administration of bafilomycin and oleic acid caused a significant reduction of BECN1, SQSTM1 and UVRAG transcripts in LX-2 cells (Figure 5A); furthermore, a reduction of the protein level of Beclin1, LC3B-I and p62 was observed. AMPK-α showed a higher expression compared to the untreated cells but P-AMPK-α was almost not detectable (Figure 5B).

Caffeine-treated LX-2 cells were characterized by an unchanged expression of autophagic transcripts (Figure 5A) and a reduction of Beclin1 and p62 proteins. P-AMPK-α protein level was not detectable despite AMPK-α being increased (Figure 5B). Combined treatment of caffeine and oleic acid showed a stable expression of all autophagic transcripts (Figure 5A). Beclin1, p62 and UVRAG proteins were decreased, while AMPK-α was increased. No protein band of P-AMPK was detectable (Figure 5B).

The lipidated active form LC3B-II was not detectable in both untreated and treated cells.

To summarize, oleic acid could be responsible for the autophagy process in LX-2 cells because of the down-regulation of autophagy proteins and detectable level of P-AMPK-α. Instead, caffeine caused a reduction of autophagy markers, similar to the administration of bafilomycin. Both compounds were able to reduce the transcript and protein levels of autophagy genes alone and in combination with oleic acid. In particular, the protein level of P-AMPK-α, the autophagy sensor kinase, was not detectable after bafilomycin and caffeine, thus assuming an inhibition of autophagy.

Discussion

Autophagy is involved in the pathogenesis of MASLD, with peculiar mechanisms that differ and characterize the cellular population of the liver parenchyma. The induction of autophagy leads to activation of the HSCs and thus exerts a pro-fibrotic property (5). In the hepatocytes, in turn, autophagy reduces the lipid accumulation, thereby preventing the development and progression of hepatic steatosis (14). Furthermore, the role of autophagy in hepatocellular carcinoma cells must be considered separately. In hepatocellular carcinoma cells, the role of autophagy depends on the stage of tumorigenesis. Impaired autophagy has been linked to liver cancer tumorigenesis and cancer progression (26).

This study highlighted that the administration of monounsaturated fatty acids promoted autophagy mechanism in liver cancer cells, whereas caffeine hampered the autophagy process in these cells. Oleic acid, a monounsaturated fatty acid, increased in hepatoblastoma cells HepG2, P-AMPK-α, which retained even after the combined administration of bafilomycin and caffeine. The detection of reduced autophagy associated marker proteins accompanied by increased activity of the autophagy key regulator P-AMPK-α, indicated an increased autophagic activity. Oleic acid-mediated autophagy could be further confirmed by real-time autophagy monitoring of GFP-RFP-MAP1LC3B stably transfected hepatocellular carcinoma cells Hep3B, thus proving a complete functional process in such cells.

The administration of caffeine attenuated the expression of the autophagic genes MAP1LC3B and UVRAG and all investigated autophagy associated proteins. The energy sensor P-AMPK-α was not detectable. Thus, indicating that caffeine was able to hamper autophagy in liver cancer cells. In contrast, Quan and colleagues have found that the treatment of HepG2 cells with caffeine leads to AMPK-α activation with an associated decrease in intracellular lipid accumulation but without the assessment of the autophagy status (27). However, the observed decrease in lipid droplets could be attributed to autophagy.

The effect of unsaturated fatty acids on MASLD progression and hepatocellular carcinogenesis requires further investigation to better understand the role of dietary interventions in preventing MASLD and liver tumors.

The HSCs are responsible for the accumulation of components of the extracellular matrix and the associated fibrosis of the liver, which triggers MASLD (5). The administration of oleic acid decreased the transcript level of PRKAA1_1 and PRKAA1_2 followed by the downregulation of all autophagic proteins but not P-AMPK-α. Additionally, it could be observed that the over-expression of COL1A1, a marker of transactivation, has been previously correlated with autophagy activation (28). Thus, supporting a rising autophagy process, which has been further confirmed by the positive stain of more lipid droplets.

The administration of caffeine led to a suppression of P-AMPK-α and of the autophagy-associated proteins accompanied by absent stellate cell activation, indicating that caffeine inhibited autophagy and HSC activation.

The addition of caffeine was even able to counteract the oleic acid-mediated induction of P-AMPK-α and the expression of COL1A1 and ACTA2, both markers for stellate cell activation that strictly correlate with autophagy (28). Further investigation of autophagy is necessary to understand the role of this cell mechanism in liver tumor metabolism.

Caffeine could serve as an approach to hamper the pro-fibrotic properties of autophagy in stellate cells through suppression of stellate cell activation as shown in our study and furthermore, could simultaneously raise beneficial effects in hepatocytes through induction of autophagy with consecutive degradation of lipid droplets as shown by data from several reports, thereby preventing MASLD within the metabolic syndrome (29-31). Recent studies have also demonstrated the beneficial effects of caffeine against chronic liver diseases. In particular, caffeine counteracts progression of liver fibrosis by promoting apoptosis in HSCs and suppresses activation of HSCs independently of cAMP by antagonizing adenosine receptors (32,33). However, the induction of apoptosis by caffeine was accompanied by endoplasmic reticulum stress mediated autophagy (33).

Focusing on the MASH mouse model, an increased expression of P-Ampk-α associated with autophagy was observed in leptin-deficient ob/ob mice. The absence of leptin impedes PI3K/Akt/mTOR signaling (34) that cannot counteracts Ampk-α and suppress autophagy (3). Furthermore, obesity has been linked to the occurrence of ER-stress (35). The endoplasmic reticulum serves as a membrane resource for autophagosome formation and experimental induction of ER-stress has also been observed as an inducer of autophagy (18). Even though previous findings showed that Ampk-α activity does not ameliorate the condition of ob/ob mice (36), the current results sustain that ob/ob mice are characterized by Ampk-α-mediated autophagy. Increased Ampk-α activity is actually considered beneficial in metabolic syndrome for overcoming insulin resistance and normalizing fat metabolism (37). FLS mice showed, instead, an absent P-Ampk-α (6/6). Emerging data of several reports have highlighted the beneficial effects of increased AMPK-α activity and induction of autophagy in MASLD (6,37). This study highlighted that the monounsaturated fatty acid oleic acid causes increased autophagy activity associated with active P-AMPK-α, suggesting that oleic acid could have beneficial effects on MASLD by overcoming dysregulation of fat metabolism. Olive oil is composed by 75% of oleic acid and it has already been shown that olive oil rich diet ameliorates hepatic steatosis in rats (19,38). However, an autophagic process independent of the activity of AMPK-α (22) could not be excluded in the current study.

Conclusions

In conclusion, our findings show that the autophagy mechanism is significantly activated in liver cancer cells and HSCs treated with oleic acid and hampered under treatment with caffeine.

Caffeine could represent a new therapeutic approach to selectively inhibit autophagy-mediated activation of stellate cells thereby hampering pro-fibrotic stellate cell activation and simultaneously inducing autophagy with preventive effect against fatty degeneration in hepatocytes.

The absence of leptin is associated with obesity-mediated MASH and induction of autophagy in liver tissue.

Recently, autophagy has been connected to a lipid-related degradation mechanism called lipophagy. Here, the autophagy markers exert a substantial role during the formation of the lipid vesicles and their final degradation (39). However, the exact link between autophagy and lipophagy and the potential of modulating these catabolic processes in MASH still needs to be further clarified.

Acknowledgments

We are thankful to Professor Tamotsu Yoshimori from Osaka University for providing the GFP-mRFP-MAP1LC3B plasmid distributed by Addgene (Cambridge).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-17/rc

Data Sharing Statement: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-17/dss

Peer Review File: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-17/prf

Funding: The study was supported by Anneliese Pohl and Kempkes Foundation.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-17/coif). P.D.F. serves as an unpaid editorial board member of Translational Gastroenterology and Hepatology from August 2024 to December 2026. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. No new experiments have been performed on animals for this study. The mouse specimens have been gently provided by Kinya Okamoto and Tomomitsu Matono (Tottori University), who have previously published the animal study approved by the ethics committee of Tottori University (protocol No. 11-Y-54).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Diehl AM, Day C. Cause, Pathogenesis, and Treatment of Nonalcoholic Steatohepatitis. N Engl J Med 2017;377:2063-72. [Crossref] [PubMed]
2. Perumpail BJ, Khan MA, Yoo ER, et al. Clinical epidemiology and disease burden of nonalcoholic fatty liver disease. World J Gastroenterol 2017;23:8263-76. [Crossref] [PubMed]
3. Galluzzi L, Pietrocola F, Levine B, et al. Metabolic control of autophagy. Cell 2014;159:1263-76. [Crossref] [PubMed]
4. Singh R, Kaushik S, Wang Y, et al. Autophagy regulates lipid metabolism. Nature 2009;458:1131-5. [Crossref] [PubMed]
5. Hernández-Gea V, Ghiassi-Nejad Z, Rozenfeld R, et al. Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues. Gastroenterology 2012;142:938-46. [Crossref] [PubMed]
6. Czaja MJ. Function of Autophagy in Nonalcoholic Fatty Liver Disease. Dig Dis Sci 2016;61:1304-13. [Crossref] [PubMed]
7. Thoen LF, Guimarães EL, Dollé L, et al. A role for autophagy during hepatic stellate cell activation. J Hepatol 2011;55:1353-60. [Crossref] [PubMed]
8. Parker R. The role of adipose tissue in fatty liver diseases. Liver Research 2018;2:35-42.
9. Saxena NK, Titus MA, Ding X, et al. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J 2004;18:1612-4. [Crossref] [PubMed]
10. Chen C, Chang YC, Liu CL, et al. Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway. Endocr Relat Cancer 2007;14:513-29. [Crossref] [PubMed]
11. Wang J, Leclercq I, Brymora JM, et al. Kupffer cells mediate leptin-induced liver fibrosis. Gastroenterology 2009;137:713-23. [Crossref] [PubMed]
12. Nepal S, Kim MJ, Hong JT, et al. Autophagy induction by leptin contributes to suppression of apoptosis in cancer cells and xenograft model: involvement of p53/FoxO3A axis. Oncotarget 2015;6:7166-81. [Crossref] [PubMed]
13. Lin CW, Zhang H, Li M, et al. Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice. J Hepatol 2013;58:993-9. [Crossref] [PubMed]
14. Soga M, Kishimoto Y. The FLS mouse: a new inbred strain with spontaneous fatty liver. Lab Anim Sci 1999;49:269-75.
15. Lindström P. The physiology of obese-hyperglycemic mice ob/ob mice. ScientificWorldJournal 2007;7:666-85. [Crossref] [PubMed]
16. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver disease and steatohepatitis research. Int J Exp Pathol 2006;87:1-16. [Crossref] [PubMed]
17. Okamoto K, Koda M, Okamoto T, et al. A Series of microRNA in the Chromosome 14q32.2 Maternally Imprinted Region Related to Progression of Non-Alcoholic Fatty Liver Disease in a Mouse Model. PLoS One 2016;11:e0154676. [Crossref] [PubMed]
18. Di Fazio P, Waldegger P, Jabari S, et al. Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer. Oncotarget 2016;7:28998-9010. [Crossref] [PubMed]
19. Hussein O, Grosovski M, Lasri E, et al. Monounsaturated fat decreases hepatic lipid content in non-alcoholic fatty liver disease in rats. World J Gastroenterol 2007;13:361-8. [Crossref] [PubMed]
20. Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1). Autophagy 2021;17:1-382.
21. Zhang SJ, Li YF, Wang GE, et al. Caffeine ameliorates high energy diet-induced hepatic steatosis: sirtuin 3 acts as a bridge in the lipid metabolism pathway. Food Funct 2015;6:2578-87. [Crossref] [PubMed]
22. Matrood S, de Prisco N, Wissniowski TT, et al. Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death. Neuroendocrinology 2021;111:965-85. [Crossref] [PubMed]
23. Hellerbrand C, Stefanovic B, Giordano F, et al. The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo. J Hepatol 1999;30:77-87. [Crossref] [PubMed]
24. Lattouf R, Younes R, Lutomski D, et al. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem 2014;62:751-8. [Crossref] [PubMed]
25. Hu S, Bae M, Park YK, et al. n-3 PUFAs inhibit TGFβ1-induced profibrogenic gene expression by ameliorating the repression of PPARγ in hepatic stellate cells. J Nutr Biochem 2020;85:108452. [Crossref] [PubMed]
26. Di Fazio P, Matrood S. Targeting autophagy in liver cancer. Transl Gastroenterol Hepatol 2018;3:39. [Crossref] [PubMed]
27. Quan HY, Kim DY, Chung SH. Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells. BMB Rep 2013;46:207-12. [Crossref] [PubMed]
28. Bernard M, Dieudé M, Yang B, et al. Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF. Autophagy 2014;10:2193-207. [Crossref] [PubMed]
29. Birerdinc A, Stepanova M, Pawloski L, et al. Caffeine is protective in patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2012;35:76-82. [Crossref] [PubMed]
30. Molloy JW, Calcagno CJ, Williams CD, et al. Association of coffee and caffeine consumption with fatty liver disease, nonalcoholic steatohepatitis, and degree of hepatic fibrosis. Hepatology 2012;55:429-36. [Crossref] [PubMed]
31. Sinha RA, Farah BL, Singh BK, et al. Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice. Hepatology 2014;59:1366-80. [Crossref] [PubMed]
32. Yamaguchi M, Saito SY, Nishiyama R, et al. Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors. Biol Pharm Bull 2017;40:658-64. [Crossref] [PubMed]
33. Li Y, Chen Y, Huang H, et al. Autophagy mediated by endoplasmic reticulum stress enhances the caffeine-induced apoptosis of hepatic stellate cells. Int J Mol Med 2017;40:1405-14. [Crossref] [PubMed]
34. Donato J Jr, Frazão R, Elias CF. The PI3K signaling pathway mediates the biological effects of leptin. Arq Bras Endocrinol Metabol 2010;54:591-602. [Crossref] [PubMed]
35. Gregor MF, Hotamisligil GS. Thematic review series: Adipocyte Biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease. J Lipid Res 2007;48:1905-14. [Crossref] [PubMed]
36. Zachariah Tom R, Garcia-Roves PM, Sjögren RJ, et al. Effects of AMPK activation on insulin sensitivity and metabolism in leptin-deficient ob/ob mice. Diabetes 2014;63:1560-71. [Crossref] [PubMed]
37. Ruderman NB, Carling D, Prentki M, et al. AMPK, insulin resistance, and the metabolic syndrome. J Clin Invest 2013;123:2764-72. [Crossref] [PubMed]
38. Hernández R, Martínez-Lara E, Cañuelo A, et al. Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: involvement of perisinusoidal stellate cells. World J Gastroenterol 2005;11:7480-5. [Crossref] [PubMed]
39. Filali-Mouncef Y, Hunter C, Roccio F, et al. The ménage à trois of autophagy, lipid droplets and liver disease. Autophagy 2022;18:50-72. [Crossref] [PubMed]