Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease

doi:10.1172/jci.insight.93626

. 2018 Mar 22;3(6):e93626.

doi: 10.1172/jci.insight.93626.

Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease

Maria E Moreno-Fernandez^{1

2}, Daniel A Giles^{1

2

3}, Traci E Stankiewicz^{1

2}, Rachel Sheridan^{1

4}, Rebekah Karns^{1

5}, Monica Cappelletti^{1

2}, Kristin Lampe^{1

2}, Rajib Mukherjee^{1

2}, Christian Sina⁶, Anthony Sallese^{1

7}, James P Bridges^{1

7}, Simon P Hogan^{1

8}, Bruce J Aronow^{1

9}, Kasper Hoebe^{1

2}, Senad Divanovic^{1

2}

Affiliations

¹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
² Division of Immunobiology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, Ohio, USA.
³ Immunology Graduate Program, CCHMC, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
⁴ Division of Pathology, CCHMC, Cincinnati, Ohio, USA.
⁵ Division of Gastroenterology, Hepatology, and Nutrition, CCHMC, Cincinnati, Ohio, USA.
⁶ Molecular Gastroenterology, University Hospital Schleswig-Holstein, Campus Lübeck, Germany.
⁷ Division of Neonatology and Pulmonary Biology.
⁸ Division of Allergy and Immunology, and.
⁹ Division of Biomedical Informatics, CCHMC, Cincinnati, Ohio, USA.

PMID: 29563328
PMCID: PMC5926941
DOI: 10.1172/jci.insight.93626

Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease

Maria E Moreno-Fernandez et al. JCI Insight. 2018.

. 2018 Mar 22;3(6):e93626.

doi: 10.1172/jci.insight.93626.

Authors

Affiliations

¹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
² Division of Immunobiology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, Ohio, USA.
³ Immunology Graduate Program, CCHMC, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
⁴ Division of Pathology, CCHMC, Cincinnati, Ohio, USA.
⁵ Division of Gastroenterology, Hepatology, and Nutrition, CCHMC, Cincinnati, Ohio, USA.
⁶ Molecular Gastroenterology, University Hospital Schleswig-Holstein, Campus Lübeck, Germany.
⁷ Division of Neonatology and Pulmonary Biology.
⁸ Division of Allergy and Immunology, and.
⁹ Division of Biomedical Informatics, CCHMC, Cincinnati, Ohio, USA.

PMID: 29563328
PMCID: PMC5926941
DOI: 10.1172/jci.insight.93626

Abstract

Nonalcoholic fatty liver disease (NAFLD), a metabolic predisposition for development of hepatocellular carcinoma (HCC), represents a disease spectrum ranging from steatosis to steatohepatitis to cirrhosis. Acox1, a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, regulates metabolism, spontaneous hepatic steatosis, and hepatocellular damage over time. However, it is unknown whether Acox1 modulates inflammation relevant to NAFLD pathogenesis or if Acox1-associated metabolic and inflammatory derangements uncover and accelerate potential for NAFLD progression. Here, we show that mice with a point mutation in Acox1 (Acox1Lampe1) exhibited altered cellular metabolism, modified T cell polarization, and exacerbated immune cell inflammatory potential. Further, in context of a brief obesogenic diet stress, NAFLD progression associated with Acox1 mutation resulted in significantly accelerated and exacerbated hepatocellular damage via induction of profound histological changes in hepatocytes, hepatic inflammation, and robust upregulation of gene expression associated with HCC development. Collectively, these data demonstrate that β-oxidation links metabolism and immune responsiveness and that a better understanding of peroxisomal β-oxidation may allow for discovery of mechanisms central for NAFLD progression.

Keywords: Cytokines; Fatty acid oxidation; Inflammation; Metabolism; Obesity.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

**Figure 1. Acox1^Lampe1 mutation promotes proinflammatory immune responsiveness.**
(A and B) Twelve-week-old, chow diet–fed, Acox1^Lampe1 mice and WT littermate controls were challenged i.p. with biotinylated capture Ab (20 μg/mouse) 3 hours prior to i.v. saline (NS) or LPS challenge (25 μg/mouse; ultrapure *E. coli* 0111:B4). Twenty-four hours later, serum was collected, and IL-6, TNF-α, IFN-γ, and IL-17A levels were quantified by IVCCA ELISA. (C) Supernatant IL-6 and IL-1β levels from BM-derived DCs (BMDC), of chow diet–fed, Acox1^Lampe1 mice and WT littermate controls stimulated for 24 hours with saline (NS) or LPS (100 ng/ml). (D) Splenic naive CD4⁺ T cells from chow diet–fed WT mice, isolated by negative selection using magnetic beads, were cultured under Th17 and Th1 polarizing conditions with or without peroxisomal β-oxidation inhibitor (thioridizine, 1 μM) and the frequency of IL-17A– and IFN-γexpressing cells were quantified by flow cytometry. (E and F) Splenic naive CD4⁺ T cells from 12-week-old, chow diet–fed, Acox1^Lampe1 mice and WT littermate controls, isolated by negative selection using magnetic beads, were cultured under Th17 and Th1 polarizing conditions, and the frequency of IL-17A– and IFN-γ–expressing cells was quantified by flow cytometry. (G) Frequency of total splenic Th1 (CD3⁺CD4⁺IFN-γ⁺IL-17A^–), Th17 (CD3⁺CD4⁺IL-17A⁺IFN-γ^–), and Treg (CD3⁺CD4⁺FOXP3⁺) in chow diet–fed Acox1^Lampe1 mice and WT littermate controls. Data represent means ± SEM. (A, B, and **E–G**) Unpaired Student’s t test; *P < 0.05, **P < 0.01. (C) One-way ANOVA followed by Tukey’s correction; *P < 0.05. White bars denote WT mice; black bars denote Acox1^Lampe1 mice. (A, B and D) Data combined from 2 independent experiments, n = 3–8/condition. (C and E) Data combined from 2 independent experiments, n = 7/condition. (F and G) A single experiment, n = 3–4/condition.

**Figure 2. Acox1^Lampe1 mutation modulates whole body metabolism and brown adipose tissue activity.**
(**A–D**) Analysis of factors contributing to differential weight gain. Twelve-week-old, chow diet–fed, Acox1^Lampe1 mice and WT littermate controls were placed in metabolic chambers, and (A) food intake, (B) total daily locomotor activity, (C) total daily energy expenditure (EE), and (D) total daily oxygen consumption were quantified. (**E–G**) Analysis of brown adipose tissue (BAT) function. Twelve-week-old, chow diet–fed, Acox1^Lampe1 mice and WT littermate controls were utilized for analysis of (E) BAT/body mass ratio, (F) BAT *Ucp1* mRNA expression levels, and (G) BAT mitochondrial oxygen consumption rate (OCR). Data represent means ± SEM. (**A–G**) Unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. (**A–F**) White bars denote WT mice; black bars denote Acox1^Lampe1 mice. (C) Shaded regions denote night cycle; clear regions denote day cycle. (G) White squares denote WT mice; black squares denote Acox1^Lampe1 mice. (A) Representative of 3 individual experiments, n = 4/condition. (**B–G**) Data combined from 2 independent experiments, n = 3-6/condition.

**Figure 3. Acox1^Lampe1 mutation alters hepatic mitochondrial activity, hepatic inflammation, and hepatocellular damage.**
(A) Representative liver histology (H&E staining; 20×) of chow diet–fed Acox1^Lampe1 mice and WT littermate controls. E15; 2 weeks of age; and 12 weeks of age were analyzed. Representative hepatic H&E staining. (**B–H**) Characterization of liver phenotype and function in 12-week-old, chow diet–fed, Acox1^Lampe1 mice and WT littermate controls. (B) Liver/body mass ratio. (C) Hepatic triglyceride (TG) levels. (D) Hepatic mitochondrial OCR. (E) Hepatic 4-hydroxynonenal (4-HNE) levels. (F) Serum alanine transaminase (ALT) levels. (G) Total hepatic immune (CD45⁺) cell infiltration determined by flow cytometry. (H) Hepatic chemokine mRNA expression of *Cxcl10* and *Ccl22*. Data represent means ± SEM. (**B–H**) Unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. White bars denote WT mice; black bars denote Acox1^Lampe1 mice. (**B–D**) Representative of 3 individual experiments, n = 3/condition. (E, G, H) A single experiment, n = 3/condition. (F) Data combined from 2 independent experiments, n = 5–12/condition.

**Figure 4. Short-term obesogenic-diet challenge accelerates and exacerbates Acox1^Lampe1 mutation–driven hepatocellular damage and systemic inflammation.**
Eight-week-old Acox1^Lampe1 mice and WT littermate controls were fed a HFHCD or chow diet for 4 weeks. (A) Weight gain. (B) Liver/body mass ratio. (C) Serum ALT levels. (D) Representative liver histology (H&E staining; 20x). (E) Total hepatic immune (CD45⁺) cell infiltration determined by flow cytometry. (F) Hepatic chemokine mRNA expression of *Ccl2*, *Ccl3*, *Ccl4*, *Ccl22*, and *Cxcl10*. (G) Hepatic TG levels. (H) Hepatic lipid content measured by thin layer chromatography and visualized by primuline staining. Cholesterol ester (CE), triglyceride (TG), diacylglycerol (DAG), free cholesterol (FC), monoacylglycerol (MAG), phospholipids (polar). Data represent means ± SEM. (A, E and F) Unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.01. (B, C, and G) One-way ANOVA followed by Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.01. (A) White squares denote WT mice fed chow diet; white circles denote WT mice fed HFHCD; black squares denote Acox1^Lampe1 mice fed chow diet; black circles denote Acox1^Lampe1 mice fed HFHCD. (B, C, and **E–G**) White bars denote WT mice fed HFHCD; black bars denote Acox1^Lampe1 mice fed HFHCD. (A) Representative of 2 individual experiments, n = 3/condition. (B, C, F and G) Data combined from 2 independent experiments, n = 6–9/condition. (E and H) A single experiment, n = 3/condition.

**Figure 5. Acox1^Lampe1 mutation in hematopoietic cells contributes to exacerbated hepatocellular damage and systemic inflammation during short-term obesogenic-diet challenge.**
Eight- ton 10-week-old WT mice were lethally irradiated and subsequently reconstituted with BM from WT or Acox1^Lampe1 mice. Following successful immunological reconstitution, mice were fed a HFHCD for 4 weeks. (A) Schematic representation. (B) Weight gain. (C) Hepatic TG levels. (D) Representative liver histology (H&E staining; 20×). (E) Serum ALT levels. (F) Total hepatic immune (CD45⁺) cell infiltration determined by flow cytometry. (G) Hepatic chemokine mRNA expression of *Ccl2*, *Ccl3*, *Ccl4*, *Ccl22*, and *Cxcl10*. (H) Frequency of hepatic CD45⁺TCRB⁺CD4⁺ T cells producing IFN-γ, TNF-α, and IL-17A. (I) Frequency of hepatic CD45⁺TCRB⁺CD8⁺ T cells producing IFN-γ and TNF-α. (J) Mice were challenged i.p. with biotinylated capture Ab (20 μg/mouse) for 3 hours, and serum IL-6, TNF-α, IFN-γ, and IL-17A levels were quantified by IVCCA ELISA. Data represent means ± SEM. (**B–J**) Unpaired Student’s t test; *P < 0.05, **P < 0.01. A single experiment, n = 5–6/condition.

**Figure 6. Acox1^Lampe1 mutation in hematopoietic cells alters immune responsiveness to secondary inflammatory challenge during short-term obesogenic-diet challenge.**
Eight- to 10-week-old WT mice were lethally irradiated and subsequently reconstituted with BM from WT or Acox1^Lampe1 mice. Following successful immunological reconstitution, mice were fed a HFHCD for 4 weeks and challenged with LPS. (A) Mice were challenged i.p. with biotinylated capture Ab (20 μg/mouse) 3 hours prior to i.v. LPS challenge (25 μg/mouse; ultrapure *E. coli* 0111:B4). Twenty-four hours later, serum was collected, and IL-6, TNF-α, IFN-γ, and IL-17A levels were quantified by IVCCA ELISA. (B) Serum ALT levels. (C) Total hepatic immune (CD45⁺) cell infiltration determined by flow cytometry. (D) Hepatic chemokine mRNA expression of *Ccl2*, *Ccl3*, *Ccl4*, *Ccl22*, and *Cxcl10*. (E) Frequency of hepatic CD45⁺TCRB⁺CD4⁺ T cells producing IFN-γ, TNF-α, and IL-17A. (F) Frequency of hepatic CD45⁺TCRB⁺CD8⁺ T cells producing IFN-γ and TNF-α. Data represent means ± SEM. (**A–F**) Unpaired Student’s t test; *P < 0.05, **P < 0.01. A single experiment, n = 5–6/condition.

**Figure 7. Coupling of Acox1^Lampe1 mutation with obesogenic diet stress amplifies biological pathways associated with HCC development.**
Eight-week-old Acox1^Lampe1 mice and WT littermate controls were fed a HFHCD or chow diet for 4 weeks. (A) Genes upregulated at least 2-fold in the liver of mice fed chow or HFHCD, as determined by RNA-seq. (B and C) Upregulated gene expression pathways determined from RNA-seq analysis. (D and E) Hepatic mRNA expression of genes associated with HCC development: *H19* and *Afp*. Data represent means ± SEM. One-way ANOVA with Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.001. White bars denote WT mice; black bars denote Acox1^Lampe1 mice. (**A–C**) A single experiment, n = 3/condition. (D and E) Data combined from 2 independent experiments, n = 3–6/condition.

**Figure 8. Coupling of Acox1^Lampe1 mutation with obesogenic diet stress accelerates hepatic injury and expression of genes associated with HCC development.**
Eight-week-old Acox1^Lampe1 mice and WT littermate controls were fed a HFHCD or chow diet for 4 weeks (young) and compared with Acox1^Lampe1 were fed chow diet for 59 weeks (old). (**A–E**) Hepatic mRNA expression of genes associated with HCC development; *H19*, *Afp*, *Igfbp1*, *Fabp5*, and *Spp1*. (F) Upper panel: Representative liver histology (H&E staining; 20×) of the temporal evolution (8, 12, 16, 23, and 59 weeks of age) and histopathological changes in chow-fed Acox1^Lampe1 mice. Acox1^Lampe1 mice display steatosis, increasing inflammation, and liver cell injury over time with development of hepatocellular tumors at 59 weeks of age. Lower panel: Representative liver histology (H&E staining; 20×) of 12-week-old (young) Acox1^Lampe1 and WT mice fed a HFHCD. Acox1^Lampe1 mice display steatosis, inflammation, and liver cell injury after 4 weeks of HFHCD feeding. PT depicts portal tract; arrowhead depicts mitotic hepatocytes. Data represent mean ± SEM. One-way ANOVA with Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.001. White bars denote WT mice fed chow diet; gray bars denote old Acox1^Lampe1 mice fed chow diet; black bars denote young Acox1^Lampe1 mice fed HFHCD. A single experiment, n = 3/condition.

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References

1. Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J Clin Invest. 2008;118(9):2992–3002. doi: 10.1172/JCI34260. - DOI - PMC - PubMed
1. Strissel KJ, et al. Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes. 2007;56(12):2910–2918. doi: 10.2337/db07-0767. - DOI - PubMed
1. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112(12):1796–1808. doi: 10.1172/JCI19246. - DOI - PMC - PubMed
1. Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol. 2010;5:145–171. doi: 10.1146/annurev-pathol-121808-102132. - DOI - PubMed
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[1] Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J Clin Invest. 2008;118(9):2992–3002. doi: 10.1172/JCI34260. - DOI - PMC - PubMed

[2] Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J Clin Invest. 2008;118(9):2992–3002. doi: 10.1172/JCI34260. - DOI - PMC - PubMed

[3] Strissel KJ, et al. Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes. 2007;56(12):2910–2918. doi: 10.2337/db07-0767. - DOI - PubMed

[4] Strissel KJ, et al. Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes. 2007;56(12):2910–2918. doi: 10.2337/db07-0767. - DOI - PubMed

[5] Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112(12):1796–1808. doi: 10.1172/JCI19246. - DOI - PMC - PubMed

[6] Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112(12):1796–1808. doi: 10.1172/JCI19246. - DOI - PMC - PubMed

[7] Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol. 2010;5:145–171. doi: 10.1146/annurev-pathol-121808-102132. - DOI - PubMed

[8] Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol. 2010;5:145–171. doi: 10.1146/annurev-pathol-121808-102132. - DOI - PubMed

[9] Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science. 2011;332(6037):1519–1523. doi: 10.1126/science.1204265. - DOI - PMC - PubMed

[10] Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science. 2011;332(6037):1519–1523. doi: 10.1126/science.1204265. - DOI - PMC - PubMed

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Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease

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Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease

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