Diabetes Metab J.  2022 Nov;46(6):829-842. 10.4093/dmj.2021.0274.

Peroxisomal Fitness: A Potential Protective Mechanism of Fenofibrate against High Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice

• Jiang S
• Uddin MJ
• Yu X
• Piao L
• Dorotea D
• Oh GT
• Ha H

• KMID: 2536142
• DOI: http://doi.org/10.4093/dmj.2021.0274

Abstract

Background
Non-alcoholic fatty liver disease (NAFLD) has been increasing in association with the epidemic of obesity and diabetes. Peroxisomes are single membrane-enclosed organelles that play a role in the metabolism of lipid and reactive oxygen species. The present study examined the role of peroxisomes in high-fat diet (HFD)-induced NAFLD using fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist.
Methods
Eight-week-old male C57BL/6J mice were fed either a normal diet or HFD for 12 weeks, and fenofibrate (50 mg/kg/day) was orally administered along with the initiation of HFD.
Results
HFD-induced liver injury as measured by increased alanine aminotransferase, inflammation, oxidative stress, and lipid accumulation was effectively prevented by fenofibrate. Fenofibrate significantly increased the expression of peroxisomal genes and proteins involved in peroxisomal biogenesis and function. HFD-induced attenuation of peroxisomal fatty acid oxidation was also significantly restored by fenofibrate, demonstrating the functional significance of peroxisomal fatty acid oxidation. In Ppara deficient mice, fenofibrate failed to maintain peroxisomal biogenesis and function in HFD-induced liver injury.
Conclusion
The present data highlight the importance of PPARα-mediated peroxisomal fitness in the protective effect of fenofibrate against NAFLD.

Keyword

Fenofibrate; Non-alcoholic fatty liver disease; Peroxisomal disorders; PPAR alpha

Figure

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  Fig. 1. Fenofibrate (FF) ameliorates high-fat diet (HFD)-induced liver dysfunction in wild-type (WT) mice. (A) Liver sections were immunofluorescence (IF) stained for adipose differentiation-related protein (ADFP; red) and 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear counterstaining (blue). Original magnification, 200×; scale bar, 100 μm. (A, B, C, D, E) Liver sections were also stained with anti-F4/80, 8-hydroxyguanine (8-oxo-dG), nitrotyrosine (NT), and 4-hydroxynonena (4-HNE) antibodies and were quantified. Original magnification, 100×; scale bar, 200 μm, n=4. (F) Interleukin 1β (Il1b), Il6, F4/80, and monocyte chemoattractant protein 1 (Mcp1) were measured by real-time polymerase chain reaction, and the results were normalized to the 18S rRNA levels. (G, H) The protein levels of phospho-nuclear factor kappa B (p-NF-κB) and total-NF-κB (t-NF-κB) were measured by Western blotting. (I) Plasma alanine aminotransferase (ALT) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Data are expressed as the mean±standard error of 6 mice/group. ND, normal diet. aP<0.05 vs. ND mice, bP<0.05 vs. HFD mice.

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  Fig. 2. Fenofibrate (FF) improves liver peroxisomal function in high-fat diet (HFD)-fed wild-type (WT) mice. (A) Peroxisome-related genes were analyzed by real-time polymerase chain reaction, and the results were normalized to 18S rRNA levels. (B, C, D) Liver sections were used for immunofluorescence (IF) staining of ATP binding cassette subfamily D member 3 (ABCD3; red), catalase (green), and 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) counterstaining (blue) and were quantified. Original magnification, 200×; scale bar, 200 μm. (E, F) Protein levels of acyl-CoA oxidase 1 (ACOX1) were measured by Western blotting, and the results were normalized to β-actin levels. (G) Peroxisomal fatty acid oxidation (FAO) was measured in liver tissue. Data are expressed as the mean±standard error of 6 mice/group. ND, normal diet; Pex, peroxisomal biogenesis factor; DPM, disintegration per minute. aP<0.05 vs. ND mice, bP<0.05 vs. HFD mice.

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  Fig. 3. Role of peroxisome proliferator-activated receptor α (PPARα) in maintaining liver homeostasis in Ppara-/- mice. (A, B, C) Liver morphology was detected by H&E staining. Original magnification, 100×; scale bar, 200 μm. Liver sections were immunofluorescence (IF) stained for adipose differentiation-related protein (ADFP; red) and 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear counterstaining (blue). Original magnification, 200×; scale bar, 200 μm. Liver sections were also stained with anti-4-hydroxynonena (4-HNE) and collagen 1 (COL1) antibody and the positive area were quantified. Original magnification, 100×; scale bar, 200 μm. (D, E, F) Interleukin 1β (Il1b), Il6, and F4/80 were measured by real-time polymerase chain reaction, and the results were normalized to the 18S rRNA levels. (G) Plasma alanine aminotransferase (ALT) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Data are expressed as the mean±standard error of 6 mice/group. ND, normal diet; WT, wild-type; HFD, high-fat diet; ADRP, adipose differentiation-related protein. aP<0.05 vs. WT mice with ND, bP<0.05 vs. Ppara-/- mice with ND, cP<0.05 vs. WT mice with HFD.

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  Fig. 4. Fenofibrate fails to maintain peroxisomal biogenesis in Ppara-/- mice. (A) The expression of peroxisome-related genes was decreased in Ppara-/- mice compared to wild-type (WT) mice. (B) Peroxisome-related genes in Ppara-/- mice were analyzed by real-time polymerase chain reaction, and the results were normalized to 18S rRNA levels. Data are expressed as the mean±standard error of 6 mice/group. PPAR, peroxisome proliferator-activated receptor; Pex, peroxisomal biogenesis factor; Abcd, ATP binding cassette subfamily D member; Acox1, acyl-CoA oxidase 1; ND, normal diet; HFD, high-fat diet; FF, fenofibrate. aP<0.05 vs. WT mice or Ppara-/- mice with ND.

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  Fig. 5. Fenofibrate fails to improve peroxisomal function in Ppara-/- mice. (A, B, C) Liver sections were immunofluorescence stained for ATP binding cassette subfamily D member 3 (ABCD3; red), catalase (green), and 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear counterstaining (blue) and intensities were quantified. Original magnification, 200×; scale bar, 200 μm. (D) Peroxisomal fatty acid oxidation (FAO) was measured in liver tissue. Data are expressed as the mean±standard error of 6 mice/group. PPAR, peroxisome proliferator-activated receptor; ND, normal diet; HFD, high-fat diet; FF, fenofibrate; DPM, disintegration per minute. aP<0.05 vs. Ppara-/- mice with ND.

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  Fig. 6. Suggested model of fenofibrate-mediated peroxisomal fitness against high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). HFD or obesity decreases peroxisomal biogenesis and function and increases liver injury, including oxidative stress, inflammation, and fibrosis, due to inhibition of the peroxisome proliferator-activated receptor α (PPARα) pathway. Subsequently, it results in decreased hepatic fatty acid oxidation, increased lipid accumulation, and the induction of liver dysfunction, which leads to the development of NAFLD. Fenofibrate maintains peroxisomal biogenesis and function through activation of the PPARα pathway. It also attenuates liver injury and increases hepatic fatty acid oxidation. Thus, fenofibrate may mediate protective effects against NAFLD by maintaining peroxisomal biogenesis and function.

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