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. 2025 Apr 28;57(1):2497112. doi: 10.1080/07853890.2025.2497112

PPAR-mediated reduction of lipid accumulation in hepatocytes involves the autophagy-lysosome-mitochondrion axis

Federica Cetti a,*, Alice Ossoli a,*, Carola Garavaglia a, Lorenzo Da Dalt b, Giuseppe Danilo Norata b, Monica Gomaraschi a,✉
PMCID: PMC12039397  PMID: 40289698

Abstract

Background and aim

Lipid accumulation in hepatocytes is reduced by the activation of the peroxisome proliferator-activated receptor (PPAR) α, which is associated with increased lysosomal acid lipase (LAL) activity, transcription factor EB (TFEB) expression, and mitochondrial β-oxidation.

Aim of the study was to assess whether the three isoforms of PPAR, i.e. α, δ and γ, share the same ability to reduce lipid accumulation in hepatocytes and to clarify the involvement of autophagy activation, lysosomal hydrolysis, and mitochondrial β-oxidation in lipid clearance induced by PPARs.

Methods

HepG2 cells were treated with oleate/palmitate (O/P) to induce lipid accumulation and exposed to the PPARα agonist fenofibric acid, the γ agonist pioglitazone, the δ agonist seladelpar, or the dual α/γ agonist saroglitazar.

Results

The treatment of HepG2 cells with fenofibric acid, pioglitazone, seladelpar, or saroglitazar halved lipid accumulation induced by O/P. PPAR agonists increased TFEB, p62, and LC3 expression and rescued LAL impairment induced by O/P. Moreover, PPAR agonists significantly increased mitochondrial mass and the expression of genes involved in mitochondrial dynamics and fatty acid catabolism. Interestingly, PPAR agonists lost their ability to reduce lipid accumulation when autophagic flux, LAL activity, or fatty acid transport in the mitochondria were blocked by specific inhibitors.

Conclusion

All PPAR agonists were able to promote the clearance of lipids in cells loaded with long-chain fatty acids. The key role of acid hydrolysis to generate fatty acids, which can be then catabolized in the mitochondria, and the ability of the PPAR system to sustain each phase of this clearing process were elucidated.

Keywords: Peroxisome proliferator-activated receptors, hepatocytes, lipid accumulation, lysosomal acid lipase

1. Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) or metabolic dysfunction-associated fatty liver disease (MAFLD), the new nomenclature for nonalcoholic fatty liver disease (NAFLD), is the most frequent chronic liver disease in Western countries and it is characterized by hepatic steatosis in the absence of excessive alcohol consumption, infections, or autoimmune diseases [1]. Lipid accumulation in the form of cytosolic lipid droplets (LD) occurs when free fatty acid (FFA) levels exceed the ability of hepatocytes to either promote their catabolism through β-oxidation in the mitochondria or increase very low-density lipoprotein production; they are therefore stored in LD following the conversion into triglycerides (TG) in order to prevent their pro-oxidant and pro-inflammatory activities [2]. Strategies to increase energetic metabolism for the treatment of MASLD are heavily investigated, and agonists of peroxisome proliferator-activated receptors (PPAR) are among the most studied candidates.

The PPAR family consists of three isoforms (α, δ and γ), which control lipid and glucose metabolism and are expressed in tissues with high metabolic activity. Endogenous ligands are n-3 polyunsaturated fatty acids and eicosanoids [3]. PPARα is primarily expressed in liver, heart, kidneys, and brown adipose tissue. PPARδ is expressed ubiquitously, while PPARγ is mainly expressed in white adipose tissue and to a lower amount in the liver, in both hepatocytes and Kupffer cells. PPARs act as transcription factors with both overlapping and distinct (even contrasting) effects [4,5]. For this reason, dual and pan agonists are under development to maximize the effects of PPAR system activation and/or dampen unintended responses. In addition, PPARs can exert anti-inflammatory activities through transrepression. PPARα and δ mainly induce the expression of genes involved in peroxisomal and mitochondrial β-oxidation; indeed, fibrates, which are PPARα agonists, are used to treat hypertriglyceridemia. PPARγ regulates adipocyte differentiation, lipogenesis, and fatty acid uptake. Since their activation increases insulin sensitivity, thiazolidinediones are used for the treatment of type 2 diabetes.

In searching for other players involved in the development and progression of MASLD, we and others have shown that MASLD is frequently associated with an acquired deficiency of lysosomal acid lipase (LAL) activity [6,7]. LAL is responsible for the lysosomal hydrolysis of TG and cholesteryl esters, which come from the receptor-mediated internalization of circulating lipoproteins [8]. On the contrary, the hydrolysis of lipids within LD was classically ascribed to cytosolic neutral esterases; however, a role for the lysosome in the catabolism of LD through the activation of autophagy is now recognized, as well [9]. In this context, an impaired LAL activity could favour and worsen lipid accumulation in the hepatocytes. Recently, we showed that the activation of PPARα by the synthetic agonist WY14643 rescued LAL deficiency and decreased lipid accumulation in a cell model of steatosis through the activation of transcription factor EB (TFEB) and mitochondrial β-oxidation [7]. Since TFEB is a master regulator of autophagy and lysosomal biogenesis [10], these results support the hypothesis that PPARα could reduce lipid accumulation in the hepatocytes by promoting LD catabolism in the lysosomes and the consequent oxidation of generated fatty acids in the mitochondria. Aim of this study was to assess whether the three isoforms of PPAR, i.e. α, δ and γ, share the same ability to reduce lipid accumulation in hepatocytes by using specific or dual agonists already approved for clinical use or under clinical development. Then, we sought to clarify the direct involvement of autophagy activation, lysosomal hydrolysis by LAL and mitochondrial fatty acid β-oxidation in lipid clearance induced by PPAR activation. An in vitro model of MASLD based on the incubation of HepG2 cells with a mixture of fatty acids was used [11]. Even if a cancer-derived cell line could display different metabolic features if compared to primary hepatocytes, HepG2 cells express PPARα, δ and γ [12,13,14]. We previously showed that HepG2 cells loaded with fatty acids fully recapitulate the in vivo conditions observed in MASLD patients, i.e. progressive reduction of LAL activity, but not of LAL protein levels, with lipid accumulation [7].

2. Materials and methods

2.1. Cells and treatments

The human hepatoma cell line HepG2 was obtained from ATCC (code HB-8065, ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 2 mM L-glutamine, 0.1 U/ml penicillin and 0.1 µg/ml streptomycin. To induce lipid accumulation, cells were incubated with 1 mM long-chain oleate and palmitate (O/P, at 2:1 ratio, Merck, Germany) up to 48 h in complete growth medium. O/P mixture was prepared and used as previously described [7]. Incubation with O/P was carried out in the presence or in absence of the following PPAR agonists: the α agonist fenofibric acid at 50 µM (FA), the γ agonist pioglitazone at 20 µM (PGZ), the δ agonist seladelpar at 20 µM (SEL) and the dual α/γ agonist saroglitazar at 20 µM (SAR) (MedChemExpress, USA). Experiments were replicated in the presence of specific inhibitors of autophagy, LAL activity and mitochondrial β-oxidation. In particular, cells were pre-treated for 6h with (i) the inhibitor of the autophagosome-lysosome fusion CA-5f at 20 μM [12](MedChemExpress, USA), or (ii) the LAL inhibitor lalistat 2 at 100 µM [15] (Merck, Germany), or (iii) the inhibitor of carnitine palmitoyltransferase-1 (CPT-1) etomoxir at 10 μM (MedChemExpress, USA). At the indicated concentrations, all the agonists and inhibitors did not affect cell viability, assessed by the MTS assay (Promega Corp, USA).

2.2. Lipid accumulation

Intracellular lipid accumulation was assessed by Oil-Red-O (ORO) staining. Briefly, cells were washed with PBS and fixed with formalin 10% (Sigma-Aldrich, USA) for 1h at room temperature. After fixation, cells were washed with water, incubated with 1,2-propanediol (Sigma-Aldrich, USA) for 5 min at room temperature, and washed again with water. Cells were stained with ORO solution (BioOptica, Italy) for 20 min at room temperature, and unbound staining was removed with PBS. Images were captured with a bright-field stereo microscope (Nikon Europe, The Netherlands) and analyzed with ImageJ software (NIH, USA).

2.3. Gene expression analysis

Gene expression was assessed by real time-PCR. RNA was extracted with Trizol reagent and cDNA was synthetized by the iScript cDNA Synthesis kit (Bio-Rad Laboratories Inc., USA). Amplification was performed in a MiniOpticon System with the iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc., USA). β-actin was used as housekeeping gene for data normalization. The sequences of the qPCR primers are listed in Supplementary Table 1.

2.4. Western blotting

Cells were homogenized in lysis buffer (20 mmol/L Tris pH 6.8 containing 4% SDS, 20% glycerol, 1 mmol/L EDTA, 0.5 mmol/L DTT and a protease inhibitor cocktail) and protein concentration was assessed by the microBCA assay (ThermoFisher Scientific, USA). After separation by SDS-PAGE, proteins were transferred on a nitrocellulose membrane, followed by incubation with primary antibodies (Supplementary Table 2), and then with HRP–conjugated secondary antibodies (Dako Cytomation, Denmark). Bands were visualized by enhanced chemiluminescence (GE Biosciences, Sweden). Tubulin expression was used as loading control (Merck, Germany).

2.5. LAL activity

LAL activity was measured by fluorescence, using 4-methylumbelliferone palmitate (4-MUP, Cayman Chemical, USA), cardiolipin (Avanti Polar Lipids, USA) and Lalistat 2 (Merck, Germany), according to the method of Hamilton et al. [15], as previously described [7]. HepG2 were lysed in 0.15 M acetate buffer pH 4.0 with 1% Triton X-100 (assay buffer), incubated with H2O or with Lalistat 2 for 30 min at 37 °C, and then with cardiolipin and 4-MUP in assay buffer for 1h at 37 °C. Generated 4-methylumbelliferone (4-MU) was detected by fluorescence using the Synergy H1 Multi-Mode microplate reader (BioTek, USA), setting excitation at 320 nm and emission at 460 nm. LAL activity was calculated by subtracting the activity in the inhibited reaction (with Lalistat 2) from total activity (with H2O) and expressed as nmol of 4-MU generated in 1h/µg of protein.

2.6. Mitochondrial mass

Cells were dissociated using trypsin (Euroclone, Italy), washed with PBS and then stained for mitochondrial mass quantification according to the manufacturer’s guidelines using MitoTracker Green (20 nM) for 15 min at 4 °C. To quantify total mitochondrial mass, the mean fluorescence intensity (MFI) for FITC was detected by flow cytometry analysis (Fortessa, Becton Dickinson, USA).

2.7. ROS production

Cells were washed with PBS and loaded with 3 µM 2′,7′-dichlorofluorescein (Life Technologies, USA) for 30 min. ROS production was measured by fluorescence using a Synergy H1 multi-mode reader (BioTek, USA) and normalized by the protein concentration of the total cell lysate.

2.8. Statistical analysis

Data are expressed as mean ± SD, if not otherwise stated. Comparisons between groups were performed by two-sided one-way ANOVA for independent samples with the Student-Newman-Keuls test or by non-parametric Kruskal-Wallis test, according to data distribution. P values <0.05 were considered as statistically significant. Statistical analysis was performed with SigmaPlot 12.5 (Systat Software, USA).

3. Results

3.1. PPAR agonists halved lipid accumulation induced by O/P

The incubation of HepG2 cells with the mixture of oleate and palmitate (O/P) for 48h induced a significant increase of lipid content; indeed, a 23.7 ± 2.3 fold increase of the ORO-positive area was observed in O/P-treated cells compared to untreated cells (p < 0.001, Figure 1). The presence of PPAR agonists in O/P treated cells almost halved lipid accumulation (Figure 1). Indeed, PPAR agonists similarly reduced ORO-positive area by 39.6 ± 21.7% (fenofibric acid) to 50.5 ± 13.7% (saroglitazar) compared to O/P only treated cells.

Figure 1.

Figure 1.

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Effect of PPAR agonists on lipid accumulation induced by oleate and palmitate.

HepG2 cells were treated with oleate and palmitate (O/P) for 48h in the presence or absence of the PPAR agonists fenofibric acid (FA), pioglitazone (PGZ), seladelpar (SEL) or saroglitazar (SAR). Lipid accumulation was assessed as percentage of ORO-positive area and expressed as fold of untreated cells (Panel B). Data are mean ± SD, n = 3. *p < 0.05 vs O/P. Representative images are shown in Panel A (scale bar 100 μm).

3.2. PPAR agonists activated autophagy and increased LAL activity

To evaluate the role of autophagy and lysosomal hydrolysis in the reduction of lipid accumulation induced by PPAR agonists, the expression of transcription factor EB (TFEB), the master regulator of both autophagy and lysosomal biogenesis, was evaluated. Compared to cells incubated with O/P only, TFEB mRNA levels significantly increased when PPAR agonists were added to the medium (Figure 2A). This resulted in the activation of autophagy as shown by the significant increase of p62 and LC3 mRNA levels, which are involved in autophagosome formation (Figure 2B and 2C). LAL mRNA levels tended to increase in cells treated with PPAR agonists as well (Figure 2D), but most importantly LAL activity was significantly affected. Indeed, consistent with our previous data [7], lipid loading with O/P impaired LAL activity compared to untreated cells (−18.6 ± 9.3%, p = 0.012); this impairment was completely rescued following the treatment with the PPAR agonists (Figure 2E).

Figure 2.

Figure 2.

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Effect of PPAR agonists on markers of autophagy and LAL activity.

Levels of mRNA for transcription factor EB (TFEB, Panel A), p62 (Panel B), LC3 (Panel C) and LAL (Panel D) were assessed by real-time PCR in cells treated with oleate and palmitate (O/P) for 48h, in the presence or absence of the PPAR agonists fenofibric acid (FA), pioglitazone (PGZ), seladelpar (SEL) or saroglitazar (SAR). Data are expressed as fold of untreated cells, mean ± SD, n = 5. *p < 0.05 vs O/P. Panel E, LAL activity measured by fluorescence in cells treated with O/P for 48h, in the presence or absence of PPAR agonists. Data are expressed as fold of untreated cells, mean ± SD, n = 6. *p < 0.05 vs O/P.

3.3. PPAR agonists stimulated mitochondrial β-oxidation

The involvement of mitochondria in the reduction of lipid accumulation induced by PPAR agonists was also evaluated. Firstly, PPAR agonists significantly increased mitochondrial mass, as assessed by fluorescence using the MitoTracker Green probe (Figure 3A); while O/P almost halved mitochondrial mass, in the presence of PPAR agonists fluorescence intensity increased between 1.98 ± 0.51 and 2.50 ± 0.51 fold if compared to untreated cells. In addition, the expression of a panel of genes involved in mitochondrial dynamics and in fatty acid catabolism was upregulated in the presence of PPAR agonists with a similar profile among the different agonists (Figure 3C-E). The latest observation, suggesting an efficient fatty acid catabolism for energy generation, could also contribute to explaining the reduction of oxidative stress, measured as levels of reactive oxygen species (ROS), which was more pronounced with pioglitazone and seladelpar (Figure 3B). Indeed, an excess of free fatty acids, especially of saturated ones, would trigger the production of cytosolic and mitochondrial ROS [16].

Figure 3.

Figure 3.

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Effect of PPAR agonists on markers of mitochondrial β-oxidation.

HepG2 cells were treated with oleate and palmitate (O/P) for 48h in the presence or absence of the PPAR agonists fenofibric acid (FA), pioglitazone (PGZ), seladelpar (SEL) or saroglitazar (SAR). Panel A, Mitochondrial mass estimated by fluorescence of MitoTracker Green. Panel B, Levels of reactive oxygen species (ROS) were evaluated by fluorescence using 2’,7’-dichlorofluorescein. Data are expressed as fold of untreated cells, mean ± SD, n = 3. *p < 0.05 vs O/P. Panel C, Heatmap of the real-time PCR analysis of genes involved in mitochondrial dynamics and fatty acid catabolism by β-oxidation. Color mapping is shown at the bottom. MNF, mitofusin; OPA1, optic atrophy 1; DRP1, dynamin–related protein 1; FIS1, mitochondrial fission protein 1; CPT, carnitine palmitoyltransferase; ECHS1, short-chain enoyl-CoA hydratase; HADHB, 3-ketoacyl-CoA thiolase subunit B; ACADVL, very long-chain specific acyl-CoA dehydrogenase; ACADL, acyl-CoA dehydrogenase, long-chain; ACADM, acyl-CoA dehydrogenase, medium-chain. Results for HADHB and ACADL are also reported as bar charts in Panel D and E, respectively. Data are expressed as fold of untreated cells, mean ± SD, n = 3. *p < 0.05 vs O/P; #p < 0.05 vs untreated cells.

3.4. Inhibition of autophagic flux, LAL activity or mitochondrial β-oxidation halted lipid clearance induced by PPAR agonists

To stress the relevance of the autophagy-driven lysosomal hydrolysis and the consequent increase of mitochondrial oxidation of fatty acids delivered following PPAR agonists incubation, experiments were replicated in the presence of specific inhibitors of the different pathways (Figure 4, Supplementary Figure 1). Firstly, to block autophagic flux upstream of the lysosomes, cells were pre-treated with CA-5f, an irreversible inhibitor of autophagosome fusion with the lysosomes, before the loading with O/P. The presence of CA-5f did not affect the extent of lipid accumulation; indeed, the ORO-positive area after incubation with O/P increased by 13.9 ± 2.0 fold in cells pre-treated with CA-5f and by 12.0 ± 1.7 fold in cells that were not pre-treated with the inhibitor (p = 0.212). Most importantly, when autophagic flux has been blocked by CA-5f, PPAR agonists completely lost their ability to reduce lipid accumulation induced by the incubation with O/P (p = 0.164, Figure 4A).

Figure 4.

Figure 4.

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Effect of selective inhibitors on lipid accumulation induced by oleate and palmitate.

Lipid accumulation assessed as ORO-positive area in cells treated with oleate and palmitate (O/P) for 48h in the presence or absence of the PPAR agonists fenofibric acid (FA), pioglitazone (PGZ), seladelpar (SEL) or saroglitazar (SAR). Cells were pre-treated with CA-5f (Panel A), lalistat 2 (Panel B) or etomoxir (Panel C). Data are expressed as fold of untreated cells, mean ± SD, n = 3. *p < 0.05 vs O/P. Representative images are shown in Panel D (scale bar 100 μm).

Then, the relevance of lipid hydrolysis by LAL was tested by using lalistat 2, a specific inhibitor of LAL. In untreated cells, LAL activity was reduced by 93.5 ± 8.0% in the presence of lalistat 2. Lipid accumulation induced by O/P was not affected by the presence of lalistat 2: the ORO-positive area increased by 9.4 ± 1.3 fold in lalistat-treated cells and by 9.2 ± 1.3 fold when LAL was not inhibited (p = 0.758). As for CA-5f, when LAL activity was inhibited, the PPAR-mediated reduction of lipid accumulation induced by O/P was completely lost despite the increase in TFEB and LAL expression (p = 0.149, Figure 4B and Supplementary Figure 1).

Finally, the irreversible inhibitor of CPT-1 etomoxir was used to block fatty acid transport in the mitochondria. The increase of the ORO-positive area induced by O/P was comparable in cells pre-treated or not with etomoxir (9.7 ± 2.8 fold and 9.7 ± 1.9 fold, respectively; p = 0.991). Again, PPAR agonists completely lost their ability to reduce lipid accumulation induced by the incubation with O/P when CPT-1 was blocked despite the increased expression of genes involved in fatty acid catabolism (p = 0.998, Figure 4C and Supplementary Figure 1).

4. Discussion

Using an in vitro model of lipid accumulation in hepatocytes and clinically relevant PPAR agonists, we showed that the activation of all PPAR isoforms resulted in a significant reduction of lipid content in cells loaded with long-chain fatty acids. Furthermore, we elucidated (i) the role of acid hydrolysis in the clearance of lipid droplets to generate FFA which can be then catabolized in the mitochondria, and (ii) the ability of the PPAR system to sustain each phase of this clearing process.

The central role of PPARα in fatty acid metabolism, especially in the liver, is well established, being one of the most studied targets for the prevention and correction of MASLD [17]. We previously showed that lipid accumulation impaired LAL activity and that PPARα activation by WY14643 significantly reduced cell lipid content by restoring LAL activity and promoting mitochondrial fatty acid oxidation [7]. To increase the translational potential of these findings, in the present study the hypotriglyceridemic drug fenofibrate was used, which shared the effect of WY14643. The relatively elevated concentration of fenofibrate needed to observe a significant reduction of lipid accumulation is not unexpected since its EC50 is almost 5-fold higher than that of WY14643 [18]. These results are in line with the improved LAL activity observed in MASLD and dyslipidemic patients after treatment with fenofibrate [7,19].

We next investigated whether the activation of the other PPAR isoforms, i.e. γ and δ, could also impact lipid accumulation. To this aim, pioglitazone and seladelpar were used and they both resulted as effective as PPARα agonists in reducing lipid accumulation. PPARγ is highly expressed in adipose tissue, while its levels are relatively low in the liver; however, an increased hepatic expression was shown in patients with MASLD [5]. Pioglitazone is a PPARγ agonist approved for the treatment of type 2 diabetes which is also recommended for fatty liver disease [20,21). Extra-hepatic activities of pioglitazone are thought to be responsible for the beneficial effect on liver steatosis, but our results indicate that direct effects on hepatocytes could be involved as well. It has been postulated that pioglitazone could act as a weak PPARα agonist [22]; however, transactivation assays in transfected COS-7 cells showed that pioglitazone acts as a full agonist of PPARγ with an EC50 of 479 nM, while the EC50 for PPARα was 4.8 µM with a low efficacy of activation (25% of the reference full agonist) [23]. In addition, we cannot rule out that pioglitazone could also act through ‘non genomic’ mechanisms, as previously shown for mitochondrial pyruvate carrier or long-chain acyl-CoA synthetase 4 inhibition [24]. PPARδ is well expressed in the liver and its modulation of gene expression largely resembles that of PPARα [25]. Seladelpar is a potent and selective PPARδ agonist [26] whose efficacy in preclinical models of NASH has been demonstrated [27]; however, its clinical development was halted in phase 2b, since differences in the primary endpoint (i.e. change of liver fat content at week 12) were not appreciated and atypical histological findings were observed [28]. Recently, seladelpar met the primary and key secondary endpoints in a Phase 3 study for the treatment of primary biliary cholangitis (PBC) [29].

Due to the complementary effects of the three PPAR isoforms, drugs that can activate more than one PPAR isoform were developed to obtain a superior clinical efficacy with a better safety profile [5]. In the present work, we tested saroglitazar, a dual α/γ agonist authorized only in India for the treatment of patients with diabetic dyslipidemia and NASH [30]. A significant reduction of lipid accumulation was induced by saroglitazar in our experimental model, which is in line with its predominant activity as PPARα agonist coupled to a moderate PPARγ affinity. In preliminary experiments, elafibranor (an α/δ agonist) and the pan-agonist lanifibranor were also tested; however, due to their toxicity in our experimental setting, their concentration had to be reduced resulting in no significant effect on lipid accumulation.

Regarding the mechanisms responsible for the PPAR-mediate reduction of lipid accumulation, we showed that the activation of autophagy to route lipids from cytosolic droplets to the lysosome for hydrolysis by LAL is a key step leading to the generation of fatty acids, which can then be catabolized in the mitochondria. Indeed, when this pathway was blocked by the inhibition of the autophagosome-lysosome fusion or of LAL [15,31], the reduction of lipid accumulation induced by PPAR activation was completely lost. In this context, PPAR-mediated induction of TFEB is instrumental due to its key role as autophagy regulator and in lysosomal biogenesis [10]. Autophagy contributes to cellular homeostasis by regulating the turnover and degradation of macromolecules. In patients with MASLD, the homeostasis is lost and results in the accumulation of lipid droplets and cytotoxic proteins (Mallory-Denk bodies) in hepatocytes, thus contributing to disease progression. Accordingly, the pharmacological activation of autophagy reduced fatty liver in preclinical models of MASLD [32]. Historically, neutral cytosolic esterases were considered the main responsible for the hydrolysis of lipids stored in LDs; however, the role of acidic hydrolysis in the lysosomes through autophagy activation is emerging [9,33]. In our experimental model, neutral hydrolysis in the cytosol proved not only marginal for PPAR-mediated lipid reduction but did not show any compensative potential when the acidic pathway was blocked. Finally, fatty acids generated by LAL can be routed to the mitochondria for β-oxidation. The ability of PPARs to stimulate fatty acid catabolism is well known [4,5] and confirmed by our results. Indeed, PPARs increased mitochondrial mass and affected the signature of genes involved in mitochondrial plasticity, acyl-carnitine synthesis and β-oxidation. Moreover, the inhibition of acyl-carnitine synthesis, the rate limiting step of the entire catabolic process [34], resulted in a complete loss of PPAR-mediated reduction of lipid accumulation.

Our results were obtained in an in vitro model mainly based on gene expression and the use of chemical modulators. Nevertheless, they support the potential of the entire PPAR system in hepatic lipid clearance and the key role of autophagy-driven acid hydrolysis of lipid droplets, which would deserve additional validation in experimental models of higher complexity.

As stated above, several PPAR agonists have been tested in patients with fatty liver disease. Since the three isoforms together can address the entire biology of metabolic-associated steatohepatitis (MASH), PPAR pan-agonists have been developed and tested [35]. Indeed, only the pan agonist lanifibranor is currently in phase III for patients with MASH. Even if other therapeutic approaches, as thyroid hormone receptor β agonists, fibroblast growth factor 21 analogues and glucagon-like peptide-1 receptor agonists, seem more effective, PPAR agonists could still find their way: they improved histological MASH, liver enzymes and glucose homeostasis, representing a possible therapeutic option for non-obese patients with MASH and type 2 diabetes [36].

Supplementary Material

Supplementary filedocx
IANN_A_2497112_SM2067.docx (427.6KB, docx)

Funding Statement

This work was partially supported by: Università degli Studi di Milano (SEED 2019-1229 to MG), Italian Ministry of Health – Ricerca Finalizzata (RF-2021-12374481 to MG and RF-2019-12370896 to GDN), Progetti di Rilevante Interesse Nazionale (PRIN 2022 7KTSAT to GDN), Nanokos (European Commission Ref EUROPEAID/173691/DD/ACT/XK to GDN), PNRR Missione 6 (PNRR-MAD-2022-12375913 to GDN) and European Atherosclerosis Society (Research Grant 2023 to LDD).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data are available upon reasonable request to the corresponding author.

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Supplementary Materials

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Data Availability Statement

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