Hepatic Fgf21 Expression Is Repressed after Simvastatin Treatment in Mice

Abstract

Fibroblast growth factor 21 (Fgf21) is a hormone with emerging beneficial roles in glucose and lipid homeostasis. The interest in Fgf21 as a potential antidiabetic drug and the factors that regulate its production and secretion is growing. Statins are the most widely prescribed drug for the treatment of dyslipidemia. However, the function of statins is not limited to the lowering of cholesterol as they are associated with pleiotropic actions such as antioxidant, anti-inflammatory and cytoprotective effects. The recently described effect of statins on mitochondrial function and the induction of Fgf21 by mitochondrial stress prompted us to investigate the effect of statin treatment on Fgf21 expression in the liver. To this end, C57BL6J male mice and primary mouse hepatocytes were treated with simvastatin, and Fgf21 expression was subsequently assessed by immunoblotting and quantitative real-time PCR. Hepatic Fgf21 protein and mRNA and circulating levels of FGF21significantly decreased in mice that had received simvastatin in their food (0.1% w/w) for 1 week. This effect was also observed with simvastatin doses as low as 0.01% w/w for 1 week or following 2 intraperitoneal injections within a single day. The reduction in Fgf21 mRNA levels was further verified in primary mouse hepatocytes, indicating that the effect of simvastatin is cell autonomous. In conclusion, simvastatin treatment reduced the circulating and hepatic Fgf21 levels and this effect warrants further investigation with reference to its role in metabolism.

Fig 1. Expression of Fgf21 in liver and serum after simvastatin treatment in mice.

A. Immunoblotting analysis of Fgf21 in the livers of 3-month old male mice treated with simvastatin (0.1% w/w in chow) for 1 week. Immunoblots for Ampkα and elf2a and Coomassie blue staining of the gel were used as loading controls. B. Relative Fgf21 protein levels as assessed by immunoblotting after normalization to elf2a levels. Data are presented as the mean ± SEM. n = 5 per treatment. *P<0.05. C, D, E, F, G, H. mRNA levels of Fgf21(C), Hmgcr (D), Pcsk9 (E), Acox1 (F), Cyp4a10 (G), Cyp7a1 (H) in the livers of 3-month old male mice treated with simvastatin (0.1% w/w in chow) for 1 week. Relative mRNA levels were assessed by qRT-PCR. Data are presented as the mean ±SEM. n = 7–8 per treatment. *P<0.05. I. Assessment of serum Fgf21 levels by ELISA in 3-month old male mice treated with vehicle or simvastatin. Data are presented as the mean ±SEM. n = 7–8 per treatment. *P<0.05.

Fig 2. Body weights, food consumption and hepatic Fgf21 mRNA levels in 1-month old male mice following the administration of increasing doses of simvastatin for 1 week.

A. Body weights of mice before and after exposure to simvastatin expressed as the % of initial weight. *P<0.05 compared with baseline weight. B. Cumulative food intake for the 1-week treatment with simvastatin. *P<0.05 compared with vehicle treatment (0% simvastatin w/w). C. Fgf21 hepatic mRNA levels as assessed by qRT-PCR. *P<0.05 compared with the 0% dose. a, b, c, d, e denote 0%, 0.01%, 0.05%, 0.1% and 0.5% w/w simvastatin in chow, respectively. For panels A, B, C the data are presented as the mean ± SEM. n = 6 per treatment with the exception of the 0.5% dose (n = 3; 5 mice received the treatment and 2 died after the treatment).

Fig 3. Fgf21 mRNA levels after intraperitoneal administration of simvastatin.

Fgf21 (A) and Pcsk9 (B) mRNA levels in the liver of mice administered vehicle (control) or simvastatin intraperitoneally twice (20 and 12 hours before sacrifice). Data are presented as the mean ± SEM, n = 10 per treatment. *P<0.05 compared with control.

Fig 4. mRNA levels of Fgf21 and relevant genes in mouse primary hepatocytes after simvastatin treatment.

mRNA levels of Fgf21 (A), Pcsk9 (B), Acox1 (C), Cyp4a10 (D) in primary hepatocytes treated with vehicle or simvastatin (3 different doses) for 12 hours. Data are presented as the means ±SEM from 3 individual experiments, each of which included 3 technical replicates. *P<0.05 (compared with vehicle treatment).

Fig 5. mRNA levels of Fgf21 in primary hepatocytes and HepG2 cells after overexpression of Srebp-2 or miR-33.

Srebp-2 (A) and Fgf21 (B) mRNA levels in primary hepatocytes after overexpression of Srebp-2. Data are presented as the means ±SEM from 3 individual experiments, each of which included 3 technical replicates. *P<0.05 (compared with empty vector transfection). LDLR (C) and FGF21 (D) mRNA levels in HepG2 cells after overexpression of Srebp-2. Data are presented as the means ±SEM from 6 individual experiments, each of which included 3 technical replicates. *P<0.05 (compared with empty vector transfection). ABCA1 (E) and FGF21 (F) mRNA levels in HepG2 cells after overexpression of miR-33. Data are presented as the means ±SEM from 3 individual experiments, each of which included 3 technical replicates. *P<0.05 (compared with empty vector transfection).