Fenofibrate, a PPARα agonist, reduces hepatic fat accumulation through the upregulation of TFEB-mediated lipophagy

doi:10.1016/j.metabol.2021.154798

Metabolism

Volume 120, July 2021, 154798

https://doi.org/10.1016/j.metabol.2021.154798 Get rights and content

Highlights

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Activation of PPARα with fenofibrate induces lipophagy and improves hepatic steatosis.
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Fenofibrate treatment mobilizes intracellular Ca²⁺ via lysosomal calcium channel mucolipin 1.
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Fenofibrate treatment upregulates the CaMKKβ-AMPK pathway and calcineurin and subsequently, increases TFEB activation.

Abstract

Background

Recent studies have shown that dysregulation of autophagy is involved in the development of nonalcoholic fatty liver disease (NAFLD). Transcription factors E3 (TFE3) and EB (TFEB) are master regulators of the transcriptional response of basic cellular processes such as lysosomal biogenesis and autophagy. Here, we investigated the role of fenofibrate, a PPARα agonist, in promotion of intracellular lipid clearance by upregulation of TFEB/TFE3.

Methods

We investigated whether the effects of fenofibrate on livers were dependent on TFEB in high fat diet (HFD)-fed mice and in vivo Tfeb knockdown mice. These mice were analyzed for characteristics of obesity and diabetes; the effects of fenofibrate on hepatic fat content, glucose sensitivity, insulin resistance, and autophagy functional dependence on TFEB were investigated. HepG2, Hep3B, TSC2^+/+ and tsc2^−/− MEFs, tfeb wild type- and tfeb knockout-HeLa cells were used for in vitro experiments.

Results

Fenofibrate treatment activated autophagy and TFEB/TFE3 and reduced hepatic fat accumulation in an mTOR-independent manner. Knockdown of TFEB offset the effects of fenofibrate on autophagy and hepatic fat accumulation. In addition, fenofibrate treatment induced lysosomal Ca²⁺ release through mucolipin 1, activated calcineurin and the CaMKKβ-AMPK-ULK1 pathway, subsequently promoted TFEB and TFE3 dephosphorylation and nuclear translocation. Treatment with calcium chelator or knockdown of mucolipin 1 in hepatocytes offset the effects of fenofibrate treatment on autophagy and hepatic fat accumulation.

Conclusion

Activation of PPARα ameliorates hepatic fat accumulation via activation of TFEB and lipophagy induction. Lysosomal calcium signaling appears to play a critical role in this process. In addition, activation of TFEB by modulating nuclear receptors including PPARα with currently available drugs or new molecules might be a therapeutic target for treatment of NAFLD and other cardiometabolic diseases.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide, and the development of NAFLD is associated with obesity, insulin resistance, and type 2 diabetes [1]. Accumulating evidence suggests that dysregulation of lipophagy, a lysosome-dependent selective lipid droplet catabolic process, is implicated in the pathogenesis of NAFLD [2]. In a 2009 study, chemical and genetic inhibition of autophagy increased liver fat content and attenuated subsequent mitochondrial β-oxidation. In addition, a hepatocyte-specific Atg7 knockout mouse showed hepatomegaly with increased triglyceride accumulation after starvation when compared to control mice [3]. Furthermore, a subsequent study demonstrated that chaperone-mediated autophagy was also involved in hepatic autophagy. In this process, the lipid droplet-coating protein perilipin-2 appeared to be selectively recognized by heat shock protein 70 for selective degradation within the lysosome [4].

Transcription factors E3 (TFE3) and EB (TFEB) are master regulators in the transcriptional response of basic cellular processes such as lysosomal biogenesis and autophagy [5]. Subcellular localization of TFEB and TFE3 is controlled by their phosphorylation status. Under nutrient-rich conditions, TFEB and TFE3 are located in the cytoplasm in a phosphorylated form after mammalian target of rapamycin complex 1 (mTORC1) activation. However, nutrient starvation promotes mTORC1 inhibition and subsequent dephosphorylation of TFEB and TFE3 [6]. Dephosphorylated TFEB and TFE3 translocate to the nucleus, thereby inducing the expression of several autophagy- and lysosome-related genes [7]. Recently, TFEB has received a great deal of attention due to its essential role in the regulation of lipid metabolism. TFEB exerts global transcriptional control on lipid catabolism via peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and PPARα. In addition, viral overexpression of TFEB in the liver prevented weight gain and metabolic syndrome in both diet-induced and genetic mouse models of obesity [3].

Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor PPAR family [8] and is highly expressed in the liver, kidney, brain, and heart, where it regulates fatty acid uptake, mitochondrial fatty acid oxidation, peroxisomal fatty acid oxidation, and ketogenesis [9]. In addition, activation of PPARα plays a regulatory role in inflammatory pathways through inhibition of inflammatory gene expression [10]. Furthermore, PPARα positively regulates fatty acid β-oxidation and lipolysis by upregulating the expression of numerous genes involved in lipid metabolism and therefore leads to lower fat accumulation in NAFLD [11]. However, no clear mechanism for the effect of PPARα activation on NAFLD improvement has been fully elucidated. [11]. A recent study showed that activation of PPARα by gemfibrozil and retinoic acid leads to lysosomal biogenesis in brain cells via PPARα-mediated activation of TFEB [8]. Based on the critical role of TFEB in lipid metabolism and the decrease in liver fat with PPARα activation, we investigated whether PPARα activation could alleviate NAFLD by modulating TFEB/TFE3-mediated autophagy responses.

Section snippets

GFP-mRFP plasmid

The pGFP-LC3-mRFP-LC3B plasmid was transfected into HepG2 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Gibco) according to the manufacturer's instructions. After 24 h of transfection, cells were treated with 100 μM fenofibrate for 18 h as indicated. After two washes, sections were counterstained with 1 μg/mL DAPI (blue) for 5–10 min. Cells were finally mounted on glass slides using mounting medium (Dako, Hamburg, Germany), examined under a confocal microscope ZEISS

Activation of PPARα increases autophagy and TFEB/TFE3 nuclear translocation in hepatocytes

TFEB is a known master regulator of lysosomal biogenesis and autophagy. We first examined whether the effects of fenofibrate on autophagy processes and TFEB/TFE3 expression were PPARα-dependent or not. Co-treatment with the PPARα antagonist GW6471 offset the effect of PPARα agonists including fenofibrate and Wy14643 on activation of autophagy and TFEB/TFE3 (Supplementary Fig. 1A and B).

Next, we evaluated whether fenofibrate could induce autophagy and TFEB/TFE3 expression in

Discussion

In the present study, we propose a novel mechanism for PPARα-mediated decreases in liver fat that includes activation of TFEB and TFE3, master regulators of lysosomal biogenesis and autophagy, and lipophagy through subsequent lysosomal degradation of lipid droplets. To promote nuclear translocation and activation of TFEB and TFE3, PPARα activation mobilizes intracellular Ca²⁺ from lysosomes. The calcium-dependent phosphatase calcineurin is then responsible for dephosphorylation and subsequent

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant [Grant number 2016R1D1A1B03935944] funded by the Korean government (MEST) and the Korean Diabetes Association (2018F-3).

Data availability

All data related to this manuscript and Supplementary Information are available from the corresponding author upon reasonable request.

CRediT authorship contribution statement

JY: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Validation, Roles/writing-original draft. IKJ, KJA and HYC: Methodology, Project administration, Resource. YCH: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Resource, Supervision, Validation, Writing-review & editing. All authors approved the final version of the manuscript.

Declaration of competing interest

The authors declare that they have no conflicts of interest to this work.

Acknowledgments

We thank Myung-Shik Lee (Yonsei University College of Medicine) for providing the HeLa and stable Tfeb-GFP-transfected HeLa, CRISPR/Cas9 Tfeb-knockout HeLa cells, Su Han Bae (Yonsei University College of Medicine) for providing the tsc2^+/+ and tsc2^−/− MEFs.

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Basic ScienceFenofibrate, a PPARα agonist, reduces hepatic fat accumulation through the upregulation of TFEB-mediated lipophagy