Nighttime light exposure enhances Rev-erbα-targeting microRNAs and contributes to hepatic steatosis

Abstract

Objective: The exposure to artificial light at night (ALAN) disrupts the biological rhythms and has been associated with the development of metabolic syndrome. MicroRNAs (miRNAs) display a critical role in fine-tuning the circadian system and energy metabolism. In this study, we aimed to assess whether altered miRNAs expression in the liver underlies metabolic disorders caused by disrupted biological rhythms.

Results: We found that C3H/HePas mice exposed to ALAN developed obesity, and hepatic steatosis, which was paralleled by decreased expression of Rev-erbα and up-regulation of its lipogenic targets ACL and FAS in liver. Furthermore, the expression of Rev-erbα-targeting miRNAs, miR-140-5p, 185-5p, 326-5p and 328-5p were increased in this group. Consistently, overexpression of these miRNAs in primary hepatocytes reduced Rev-erbα expression at the mRNA and protein levels. Importantly, overexpression of Rev-erbα-targeting miRNAs increased mRNA levels of Acly and Fasn.

Conclusion: Thus, altered miRNAs profile is an important mechanism underlying the disruption of the peripheral clock caused by exposure to ALAN, which could lead to hepatic steatosis.

Keywords: Clock genes; De novo lipogenesis; Exposure to artificial light at night; Hepatic steatosis; microRNAs.

Figure 1.. Exposure to artificial light at night (ALAN) disrupts the biological rhythms.

Mice were kept on standard light/dark cycles (lights on at 6hr/lights off at 18hr) or exposed to artificial light at night during 8 weeks. Then (A) Locomotor activity during 24 hours; (B) Average of locomotor activity measured in beam breaks; and (C) Food intake was evaluated. Data are expressed as the mean ± S.E.M., n= 7–9 mice. Two-way ANOVA with post hoc Tukey test correction was used. *p<0.05 vs ZT0–12 or 6–18 hr; #p<0.05 vs L/D group. The (D) Urinary 6-sulfatoxymelatonin was also measured. Data are expressed as the mean ± S.E.M., n= 7–9 mice Student t-test was used. #p<0.05 vs L/D group.

Figure 2.. Exposure to ALAN leads to the development of obesity and insulin resistance.

Mice were kept on standard light/dark cycles (L/D) or exposed to artificial light at night (L/L) for eight weeks. After this period, the (A) Body, and (B) Perigonadal and (C) Retroperitoneal fat pad weights were measured (n=7–9), as well as the (D) Diameter of adipocytes from the perigonadal adipose tissue (n=4). We also performed (E) ip Insulin Tolerance Test on these mice and calculated the (F) glucose decay constant (KiTT) (n=7–9). Data are expressed as the mean ± S.E.M. The student’s t-test was used to analyze the biometric parameters and diameter of adipocytes. For the KiTT, two-way ANOVA with post hoc Tukey test correction was used. *p<0.05 vs ZT2 or 8hr; #p<0.05 vs L/D group.

Figure 3.. Exposure to ALAN leads to hepatic steatosis and altered expression of lipogenic proteins.

(A) H&E-stained liver sections (n=4), Hepatic (B) Triglycerides, and (C) Cholesterol levels (n=7–9); (D) Representative Western Blots and (E) Densitometric determination from proteins involved in de novo lipogenesis (n= 4–6). Data are expressed as the mean ± S.E.M. For the analysis of hepatic lipid levels, the Student’s t-test was used. #p<0.05 vs L/D group. Two-way ANOVA with post hoc Tukey test correction was used to analyze the protein expression. *p<0.05 vs ZT2 or 8hr; #p<0.05 vs L/D group.

Figure 4.. Exposure to ALAN leads to disruption of the expression of clock genes in the liver.

After eight weeks exposed or not to ALAN, the expression of Bmal1, Reverbα, Per1 and Per2 was evaluated in both groups. Data are expressed as the mean ± S.E.M., n= 5–9. Two-way ANOVA with post hoc Tukey test correction was used. *p<0.05 vs ZT2 or 8hr; #p<0.05 vs L/D group.

Figure 5.. Exposure to ALAN leads to altered expression of several Rev-erbα–targeting miRNAs in liver.

(A) Expression of altered miRNAs candidates in the liver from both groups, which were selected according to their scores. Data are expressed as the mean ± S.E.M., n= 7–9 mice. Two-way ANOVA with post hoc Tukey test correction was used. *p<0.05 vs ZT2 or 8hr; #p<0.05 vs L/D group. Effects of the miRNAs mimics upon (B) Rev-erbα gene and (C) REV-ERBα protein expression in primary hepatocyte culture. The values are mean ± SEM. n=4 (2 experiments/2 mice each); *p<0.05, **p<0.01, ***p<0.0005 NC (negative control) vs. miRNA mimic. Student’s t-test.

Figure 6.. miRs-185–5p and 140–5p bind to the Rev-ERBα 3’UTR and regulate the expression of lipogenic genes.

(A) Normalized bioluminescence from HEK293 cells co-transfected with the candidate miRNAs mimics or negative control (NC), and the vector containing the full-length WT Rev-erbα 3’UTR clone (n=1 experiment in triplicates). Diagram with the putative binding sites to (B) miR-185–5p and (C) miR140–5p; (D) Luciferase activity of the wild-type and mutant constructs of the Rev-erbα 3’UTR in the HEK293 cells co-transfected with the vector and the miRNA mimics or negative control. The plotted values correspond to the ratio of Renilla luciferase signal to firefly luciferase activity in the same sample. n= 1 experiments in triplicates; (E) Expression of Rev-erbα target genes in primary hepatocyte culture transfected with miR-185–5p, miR-140–5p, miR-326–5p and miR-328–5p mimic or negative control; n=4 (2 mice/2 experiments). The values are Mean ± SEM. *p<0.05; **p<0.01, ****p<0.0001 NC vs. miRNA mimic. Student’s t-test.