Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression

Abstract

The AKT signaling pathway is important for circadian rhythms in mammals and flies ( Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1^-/- mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1^-/- aorta, compared with control aorta, follows a distinct pattern. In the Akt1^-/- aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms.

Keywords: Akt; amplitude; aorta; circadian rhythms; gene expression.

Figure 1

Circadian gene expression in Akt1^−/− hearts. Mice were sacrificed at the given times in complete darkness. Negative regulators (A) and positive regulators (B) of circadian rhythms were quantified by qPCR and gene expression normalized to Gapdh expression and CT0 (0700 h). Black bars, Akt1^+/−. White bars, Akt1^−/−. Horizontal black and white bar shows subjective dark and light phases of circadian time. Data are represented by mean ± SEM. n = 3; ***p < 0.001, *p < 0.05.

Figure 2

Circadian gene expression in Akt1^−/− livers. Mice were sacrificed at the given times in complete darkness. Negative regulators (A) and positive regulators (B) of circadian rhythms were quantified by qPCR and gene expression normalized to Gapdh expression and CT0 (0700 h). Black bars, Akt1^+/−. White bars, Akt1^−/−. Horizontal black and white bar shows subjective dark and light phases of circadian time. Data are represented by mean ± SEM. n = 3; ***p < 0.001, *p < 0.05.

Figure 3

Akt1 affects timing and amplitude of circadian gene expression in the aorta. Mice were sacrificed at the given times in complete darkness. Gene expression was normalized to Gapdh expression and CT0 (0700 h). (A) Negative regulators show larger amplitude and later peak of expression. (B) Positive regulators generally show smaller amplitude and earlier peak of expression. Clock in the Akt1^−/− aortae was arrhythmic using JTK_CYCLE analysis. Black bars, Akt1^+/−. White bars, Akt1^−/−. Horizontal black and white bar shows subjective dark and light phases of circadian time. Data represented as mean ± SEM. n = 3; ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 4

Altered circadian gene expression in Akt1^−/− mouse lung endothelial cells. Expression at the indicated times post shock was normalized to Gapdh expression and the first time point at 12 hours post shock. (A) Negative regulators have higher amplitude of rhythms and earlier peak expression in Akt1^−/− cells. (B) Positive regulators in Akt1^−/− cells have lower amplitude rhythms than Akt1^+/+ transcripts and earlier peak expression. Neither the negative regulators nor Dbp in Akt1^−/− nor Clock in either cell type are significantly rhythmic according to JTK_CYCLE (Suppl. Table S4). Data are represented as mean ± SEM. A Lowess curve is fitted to the data. Akt1^+/+, closed circles and solid line. Akt1^−/−, open circles and dotted line. n = 5; *p < 0.05, **p < 0.01, ***p < 0.001.