Maternal Calorie Restriction Induces a Transcriptional Cytoprotective Response in Embryonic Liver Partially Dependent on Nrf2

Background: Calorie restriction is known to enhance Nrf2 signaling and longevity in adult mice, partially by reducing reactive oxygen species, but calorie restriction during pregnancy leads to intrauterine growth retardation. The latter is associated with fetal reprogramming leading to increased incidence of obesity, metabolic syndrome and diabetes in adult life. Transcription factor Nrf2 is a central regulator of the antioxidant response and its crosstalk with metabolic pathways is emerging. We hypothesized that the Nrf2 pathway is induced in embryos during calorie restriction in pregnant mothers. Methods: From gestational day 10 up to day 16, 50% of the necessary mouse diet was provided to Nrf2 heterozygous pregnant females with fathers being of the same genotype. Embryos were harvested at the end of gestational day 16 and fetal liver was used for qRT-PCR and assessment of oxidative stress (OS). Results: Intrauterine calorie restriction led to upregulation of mRNA expression of antioxidant genes (Nqo1, Gsta1, Gsta4) and of genes related to integrated stress response (Chac1, Ddit3) in WT embryos. The expression of a key gluconeogenic (G6pase) and two lipogenic genes (Acacb, Fasn) was repressed in calorie-restricted embryos. In Nrf2 knockout embryos, the induction of Nqo1 and Gsta1 genes was abrogated while that of Gsta4 was preserved, indicating an at least partially Nrf2-dependent induction of antioxidant genes after in utero calorie restriction. Measures of OS showed no difference (superoxide radical and malondialdehyde) or a small decrease (thiobarbituric reactive substances) in calorie-restricted WT embryos. Conclusions: Calorie restriction during pregnancy elicits the transcriptional induction of cytoprotective/antioxidant genes in the fetal liver, which is at least partially Nrf2-dependent, with a physiological significance that warrants further investigation

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.

<p><b>A.</b> Body weights of mice before and after exposure to simvastatin expressed as the % of initial weight. *P<0.05 compared with baseline weight. <b>B.</b> Cumulative food intake for the 1-week treatment with simvastatin. *P<0.05 compared with vehicle treatment (0% simvastatin w/w). <b>C.</b> 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).</p

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

<p>Srebp-2 (<b>A</b>) and Fgf21 (<b>B</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 (<b>C</b>) and FGF21 (<b>D</b>) 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 (<b>E</b>) and FGF21 (<b>F</b>) 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).</p

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

<p>mRNA levels of Fgf21 (<b>A</b>), Pcsk9 (<b>B</b>), Acox1 (<b>C</b>), Cyp4a10 (<b>D</b>) 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).</p

Fgf21 mRNA levels after intraperitoneal administration of simvastatin.

<p>Fgf21 (<b>A</b>) and Pcsk9 (<b>B</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.</p

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

<p><b>A.</b> 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>B.</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. <b>C, D, E, F, G, H</b>. 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. <b>I.</b> 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.</p

Primer sequences used for qRT-PCR.

<p>Primer sequences used for qRT-PCR.</p