Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2

Abstract

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase-AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2-/- but not of Bmal1-/- mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2-/- mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2-/- genes.

Fig 1. AKT exhibits cell-autonomous circadian phosphorylation rhythms.

(A) Immunoblot analysis of protein samples from 3T3-L1 cells collected at 3-hour intervals at the indicated times. The samples for CTs 39 to 45 were run twice on 2 separate gels to enable normalization between same antibodies on different blots and quantification of the entire series as one sequence (see Materials and methods). (B) Intensity quantification of pAKT/AKT, PER2, and pNR1D1 protein levels. Values were normalized to the maximum for each blot (mean ± SEM, n = 3 to 4 technical replicates, each consists of a mixture of n = 3 biological replicates). (C) Periodogram derived from B, based on JTK_CYCLE test. CT, circadian time, time since dexamethasone shock; arrow marks the position of the specific band. The molecular mass is marked in kDa. Numerical values for panels B and C can be found in S1 Raw Data. kDa, kilodalton.

Fig 2. Ultradian rhythms of pAKT in Per1,2^−/− mice under free-running conditions.

(A) Immunoblot analyses of protein samples extracted from livers of WT or Per1,2^−/− mice housed in constant dark regimen. Next to each blot an intensity quantification of pAKT/AKT. Values were normalized to the maximum for each blot (mean ± SEM, n = 3 to 4 mice per time point). (B) Periodogram derived from A, showing a dominant period of about 24 hours for WT and about 16 hours for Per1,2^−/− (JTK_CYCLE test). The molecular mass is marked in kDa. Numerical values for panels A and B can be found in S1 Raw Data. CT, circadian time; kDa, kilodalton; WT, wild-type.

Fig 3. Liver gene expression cycles with ultradian periodicity in Per1,2^−/− mice under free-running conditions.

(A) Heatmap of expression profiles of genes that were rhythmic (q < 0.2, JTK_CYCLE analysis) in WT with an about 24-hour period and their corresponding profiles in Per1,2^−/− mice. Data are presented as z-scores of the average expression in each CT. (B) Venn diagrams representing the overlap between 24-hour rhythmic genes in WT or Per1,2^−/− mice. (C) Periodograms of the transcriptome in WT or Per1,2^−/− mice. (D) Venn diagrams representing the overlap between 24-hour rhythmic genes in WT or 16-hour rhythmic genes in Per1,2^−/− mice. (E) Heatmap of expression profiles of the rhythmic genes in Per1,2^−/− mice with an about 16-hour period and their corresponding profiles in WT. Data are presented as z-scores of the average expression in each CT. (F) Histogram representing the distribution of phases of 16-hour rhythmic genes in Per1,2^−/− mice. (G) Enrichment analysis of rhythmic genes of 16-hour rhythmic genes in Per1,2^−/− mice based on ChEA dataset (left) and MSigDB C3:TFT collection (right) (p < 0.1, overrepresentation test). (H) Daily profiles of selected 16-hour rhythmic genes in Per1,2^−/− mice. (I) GO cellular compartment enrichment analysis of 16-hour rhythmic genes in Per1,2^−/− mice (p < 0.05, overrepresentation test). (J) Heatmap of expression profiles of mitochondria related genes that were rhythmic with about 16-hour period in Per1,2^−/− mice. Data are presented as z-scores of the average expression in each CT. (K) Graphic representation of mitochondrial electron transport chain complexes, highlighting related genes that were rhythmic with about 16-hour period in Per1,2^−/− mice. See also S1 and S2 Tables. CT, circadian time; GO, Gene Ontology; WT, wild-type.

Fig 4. Phosphorylation of AKT is not required for circadian clock rhythmicity.

(A) Bioluminescence recording from 3T3-L1 stably expressing a Per2:luciferase reporter (top) and TTFs prepared from PER2::LUC mice (bottom). Cells were treated with 0.2 μM of either MK-2206 (MK), GDC-0491 (GDC), or DMSO control. Data shown as mean of n = 3 per condition. (B) Analysis of the effect of MK and GDC treatment on the clock phase (as determined by the time of the first peak in A) and period (the time between the first and the second peaks) (mean ± SEM; *p < 0.05, Student t test). (C) Immunoblot analyses of protein samples from the indicated cells treated with 2 doses of MK or GDC (0.05 and 0.5 μM, + and ++, respectively) for 4 hours prior to sample collection. Arrow marks the position of the specific band. The molecular mass is marked in kDa. Numerical values for panels A and B can be found in S1 Raw Data. kDa, kilodalton; TTF, tail tip fibroblast.