Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis

Masanori Hiramitsu, Yasuhito Shimada, Junya Kuroyanagi, Takashi Inoue, Takao Katagiri, Liqing Zang, Yuhei Nishimura, Norihiro Nishimura, Toshio Tanaka, Masanori Hiramitsu, Yasuhito Shimada, Junya Kuroyanagi, Takashi Inoue, Takao Katagiri, Liqing Zang, Yuhei Nishimura, Norihiro Nishimura, Toshio Tanaka

Abstract

Lemon (Citrus limon) contains various bioactive flavonoids, and prevents obesity and obesity-associated metabolic diseases. We focused on eriocitrin (eriodictyol 7-rutinoside), a powerful antioxidative flavonoid in lemon with lipid-lowering effects in a rat model of high-fat diet. To investigate the mechanism of action of eriocitrin, we conducted feeding experiments on zebrafish with diet-induced obesity. Oral administration of eriocitrin (32 mg/kg/day for 28 days) improved dyslipidaemia and decreased lipid droplets in the liver. DNA microarray analysis revealed that eriocitrin increased mRNA of mitochondrial biogenesis genes, such as mitochondria transcription factor, nuclear respiratory factor 1, cytochrome c oxidase subunit 4, and ATP synthase. In HepG2 cells, eriocitrin also induced the corresponding orthologues, and reduced lipid accumulation under conditions of lipid loading. Eriocitrin increased mitochondrial size and mtDNA content, which resulted in ATP production in HepG2 cells and zebrafish. In summary, dietary eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.

Figures

Figure 1. Assessment of body weight and…
Figure 1. Assessment of body weight and length, plasma TG, and hepatic steatosis in zebrafish overfed with eriocitrin.
(a) Average body weight; (b) average body length; and (c) BMI in each group during 4 weeks feeding. Each group contained 10 fish. All values are mean ± SEM. *P < 0.05, **P < 0.01 versus vehicle in the NF group. (d) Plasma TG levels in each group. Four weeks' administration of eriocitrin reduced plasma TG in the OF group. Values are mean ± SEM; n = 10, *P < 0.05. (e) Fasting blood glucose in each group. Values are mean ± SEM; n = 10. (f) Oil Red O staining of liver sections. Eriocitrin reduced the number of lipid droplets (red) compared with the OF group. Erio: eriocitrin.
Figure 2. Analysis of DNA microarray data.
Figure 2. Analysis of DNA microarray data.
(a) Clustering analysis of DNA microarrays and (b) the average expression levels of clusters 7 and 10. (c) GSEA plots showed that expression of a mitochondrial gene module was more enriched in the OF + Erio group compared with the OF group.
Figure 3. qRT-PCR of genes related to…
Figure 3. qRT-PCR of genes related to lipid metabolism and mitochondrial functions, and eriocitrin reduced lipid accumulation in HepG2 cells.
To confirm the DNA microarray analyses, qRT-PCR was conducted. Genes related to lipid metabolism in zebrafish (a) and HepG2 cells (b). Genes related to mitochondrial biogenesis and respiratory function in zebrafish (c) and HepG2 cells (d). All values are means ± SEM; n = 5, *P < 0.05, **P < 0.01. (e) Oil Red O staining of HepG2. (f) Absorbance of Oil Red O during lipid accumulation. Eriocitrin reduced lipid accumulation in palmitate-stimulated HepG2. All values are means ± SEM; n = 8, *P < 0.05.
Figure 4. Eriocitrin increased mitochondrial biogenesis and…
Figure 4. Eriocitrin increased mitochondrial biogenesis and ATP production.
(a) Eriocitrin (10 μM) increased mitochondrial size (red) of HepG2 cells using MitoTracker Red CMXRos staining. Blue colour represents the nucleus (Hoechst 33342). (b) Quantitative analysis of mitochondrial staining. (c) Quantification of mtDNA was accomplished by calculating the ratio of CYTB to nuclear PK and expressing it as mtDNA copy number per cell. (d), (e) ATP quantification with eriocitrin administration. Upon 72 h administration of eriocitrin, there were increased intracellular ATP in HepG2 cells (d) and systemic ATP of 7 dpf zebrafish (e). All values are mean ± SEM; n = 8, *P < 0.05, **P < 0.01.

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