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

['Rona Aviram', 'Department Of Biomolecular Sciences', 'Weizmann Institute Of Science', 'Rehovot', 'Vaishnavi Dandavate', 'Gal Manella', 'Marina Golik', 'Gad Asher']

Date: 2022-01 

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.

Indeed, previous studies that examined daily changes in AKT phosphorylation in mice fed ad libitum found elevated levels of pAKT at nighttime, when the animals normally ingest food [ 8 ]. Furthermore, time-restricted feeding protocols (i.e., daytime or nighttime) showed that feeding rhythms are sufficient to generate cycles of AKT phosphorylation [ 8 , 9 ]. Thus, rhythmic activation of AKT can be achieved in response to external signals such as food ingestion; however, it is still unknown whether they can be driven through intrinsic mechanisms, such as the circadian clock.

The PI3K–AKT signaling pathway relays environmental information of nutritional/metabolic state to regulate cell size and proliferation [ 5 , 6 ]. This signaling cascade relies on the phosphorylation of phosphatidylinositol (PI) to generate PIP, PIP2, or PIP3 (PIs with 1, 2, or 3 phosphorylated residues, respectively), which subsequently facilitate phosphorylation and activation of downstream targets. A principal effector is the serine/threonine protein kinase AKT. Binding to either PIP2 or PIP3 leads to phosphorylation of multiple sites of AKT, out of which the Serine residue 473 (pAKT) is required for its maximal activity and conventionally serves as readout for its activation [ 7 ]. Once AKT is activated, it phosphorylates dozens of target proteins that convey the signal to regulate gene expression and other key cellular functions. Overall, this pathway is widely known to be activated in response to feeding-related signals [ 6 , 7 ].

In mammals, the circadian clock is present in almost every cell of the body and functions based on a network of transcription–translation feedback loops [ 1 , 2 ]. The heterodimer of BMAL1 and CLOCK (or its paralog NPAS2) drives the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes. In turn, PERIOD and CRYPTOCHROME proteins accumulate and repress the transcription of their own genes. An additional essential feedback loop involves the expression of the nuclear receptors NR1D1/2 and ROR, which regulate Bmal1 transcription. These so termed “core clock components” not only interact with one another, but also orchestrate a myriad of cellular metabolic processes [ 3 , 4 ].

(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.

Next, we examined the reciprocal relationship, namely, whether phosphorylation of AKT affects the circadian clock’s rhythmicity. Pharmacological inhibition of AKT (either directly by MK-2206 or via upstream inhibition of PI3K by GDC-0941) strongly inhibited AKT phosphorylation ( S5A Fig ). However, we did not observe any overt effects on the phase or period of circadian reporters; Per2:luciferase in 3T3-L1 and in tail tip fibroblasts (TTFs) from PER2::LUC mice, and overall, the clock function was unperturbed ( Fig 4A and 4B ). In agreement with this, the levels of several core clock proteins remained unchanged upon pAKT inhibition, across different drugs, doses, and in opposing administration times ( Fig 4C , S5B Fig ).

Next, we analyzed another clock mutant mouse model, namely Bmal1 −/− mice [ 12 , 23 ] and examined both liver pAKT levels and liver gene expression by RNA-seq, in constant dark for 2 consecutive days. Unlike Per1,2 −/− mice, in Bmal1 −/− mice we did not observe significant rhythms in neither pAKT levels nor in gene expression (aside of 12 genes that showed 12-hour rhythms) ( S4 Fig , S3 Table ). We, therefore, concluded that the observed about 16-hour ultradian rhythms in liver pAKT levels and gene expression specifically emerge in the absence of Per1/2 and not Bmal1.

Next, we examined the annotated biological functions of the rhythmic genes in the clock mutant mice. Comparison to mouse ChEA dataset (compilation of about 200 published ChIP-seqs [ 18 ]) showed enrichment of targets for several transcription factors in mouse tissues ( Fig 3G , S2 Table ). Among them were ESR1 and PPAR gamma, which have been shown to work upstream of AKT, via modulation of insulin signaling and PTEN regulation [ 19 – 21 ]. Importantly, we observed enrichment for targets of FOXO1, a canonical downstream effector of the AKT pathway, whose FOXO4 isoform was also identified in the MSigDB C3:TFT collection [ 22 ], a transcription factor targets dataset ( Fig 3G , S2 Table ). An additional analysis of upstream regulators predicted the involvement of prominent AKT-related factors, such as RICTOR, PTEN, insulin, and again FOXO1, consistent with the rhythmicity of some notable genes related to this signaling pathway ( S2 Table ). Profiles of representative genes are presented in Fig 3H .

Roughly half of the knockout 16-hour rhythmic genes were de novo oscillations (i.e., were not rhythmic in wild type), whereas the other half showed about 24-hour rhythmicity in wild-type mice ( Fig 3D ). Within the 48-hour window of collection, 3 peaks could be observed, with a bimodal phase distribution ( Fig 3E and 3F ). Their median amplitude was slightly lower than that of the wild-type 24-hour rhythmic genes ( S3E Fig , S1 Table ).

Our observation that pAKT exhibits ultradian rhythms in liver of Per1,2 −/− mice prompted us to examine periods other than 24 hours. Strikingly, we detected rhythmicity with an about 16-hour period, which was essentially the only other period detected in this method. Similar conclusions were drawn across additional data filtration threshold, significance cutoffs, as well as other widely used rhythmicity tests (i.e., harmonic regression [ 16 ] and RAIN [ 17 ]) ( S3A–S3D Fig , S1 Table ).

(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.

(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.

Our observations in cell culture prompted us to examine whether AKT phosphorylation rhythms are also present in vivo in mice. Analysis of pAKT in liver of wild-type mice under free-running conditions (i.e., in constant dark) over the course of 2 days showed about 24-hour rhythms ( Fig 2A and 2B ), consistent with the animals’ rhythmic feeding behavior ( S2A and S2B Fig ). To eliminate any circadian effects that might mask or interfere with the endogenous pAKT rhythms, we tested clock mutant Per1,2 −/− mice [ 12 ] housed in constant dark and fed ad libitum. As expected, Per1,2 −/− mice showed no rhythmicity of clock proteins (as demonstrated by absence of BMAL1 and pNR1D1 rhythmicity) or feeding behavior ( S2A and S2B Fig ) (see also [ 12 , 13 ]). Unexpectedly, under these conditions, we detected ultradian rhythms in pAKT levels, with a period of about 16 hours ( Fig 2A and 2B ). This suggested that presence of ultradian pAKT oscillations, in vivo, in the absence of environmental rhythms and the circadian clock.

AKT phosphorylation was previously shown to be cell cycle regulated, with elevated activation of AKT at the G2 phase [ 11 ]. If the rhythms observed herein are driven by the cell cycle, it entails that (i) the cells in the culture are dividing; and (ii) the divisions are synchronized in coordination with AKT phosphorylation (i.e., doubling time and prevalence of G2 cells correspond to pAKT rhythmicity). The 3T3-L1 cells indeed proliferate but with a doubling time of 33 hours, beyond the observed period for pAKT ( S1A Fig ). This, together with propidium iodide staining ( S1B Fig ), demonstrated that these divisions are not synchronized and do not oscillate in coordination with AKT phosphorylation.

(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.

Discussion

In this study, we found that the protein kinase AKT exhibits ultradian phosphorylation rhythms in Per1,2−/− mouse livers that are associated with ultradian gene expression. These oscillations are intriguing as they emerge in the absence of rhythmic environmental cues (e.g., light or food intake) or known endogenous mechanisms of rhythmicity (e.g., circadian clock). Notably, these ultradian rhythms were not detected in another clock mutant mouse model, namely Bmal1−/− mice, suggesting that they are not common to all clock mutant animals and raising the possibility that Bmal1 might be implicated in these rhythms.

The molecular nature underlying this rhythmicity remains open for investigation, and at this stage, we cannot determine whether AKT is a core component of an alternative oscillator or a rhythmic output of such, nor establish a causal relation between pAKT rhythmicity and rhythmic gene expression. Negative feedback loops are a common design principle underlying oscillatory behavior [24], and in the AKT signaling pathway, at least 2 different negative feedback loops have been identified [6,25]. It is conceivable that members within this complex network, which function under certain constraints [26,27], may generate a feedback loop that drives about 16-hour rhythms. Among others, these may include lipid species such as polyphosphoinositides, primarily PIP3, which facilitate phosphorylation and activation of AKT, or proteins such as mTORC2 and RICTOR, as well as downstream AKT effectors such as phosphorylated S6K, S6, and mTOR [6,25]. Another interesting candidate is GSK3β, a target of AKT, which was recently suggested to function also as a cryptochrome-independent component of a cytosolic oscillator [28]. Furthermore, our bioinformatic analysis hints toward some transcription factor complexes, whose relevant transcripts exhibit about 16-hour rhythmicity. For instance, aside from FOXO, which functions downstream to AKT activation, other transcriptional regulators of metabolism such as PPAR and HNF might be of relevance. Future studies utilizing different AKT mutants, or kinase activity modifiers, might shed light on the molecular mechanisms underlying the ultradian rhythms observed herein. Due to the complex signaling network comprising of multiple components and feedback loops, exploring these options pose a considerable challenge. It is noteworthy that deciphering the circadian core clock machinery was achieved only decades after the groundbreaking discovery of clock mutants that differ in their circadian period length (i.e., the Drosophila period gene) [29–31].

If indeed pAKT is a mere output of an alternative oscillator, our finding that inhibition of AKT phosphorylation does not affect the circadian clock should be interpreted cautiously as it does not rule out a possible interaction between the oscillators. Indeed, several studies showed that other components of the PI3K–AKT pathway are implicated in the circadian clock function. Specifically, TOR was shown to affect the clock’s rhythmicity in different models such as Arabidopsis [32], flies (together with AKT mutants) [33], and mammals [34,35]. While neither of the pathway’s components seem to be an integral part of the mammalian clock machinery (i.e., not core clock components), they appear to be significant for determination of parameters such as period and amplitude. Furthermore, given that AKT responds to known circadian clock timing cues such as serum and oxygen [36,37], it is tempting to speculate that, while it is not required for sustaining rhythmicity, AKT phosphorylation could play a role in conveying external signals (e.g., nutritional or metabolic cues) to the circadian clock.

Over the past years, evidence from different in vitro models, from red blood cells to clock component–deficient cells, suggest the presence of circadian rhythms in the absence of the canonical clock [38–40]. For instance, it was recently shown that ex vivo liver slices and skin fibroblasts cultivated from Bmal1 null mice exhibit about 24-hour oscillations based on several omics screens [41]. Overall, evidence point toward the involvement of metabolic cycles, redox cycles, and posttranslational processes, yet the underlying molecular mechanism(s) are largely unknown [24,39,42,43].

It is notable that these works identified about 24-hour rhythms while our study shows shorter rhythms. It appears that the period of pAKT oscillations is significantly shorter than 24 hours in livers of Per1,2−/− mice and is within the range of 16 hours. Period estimation can be achieved through the use of different algorithms for rhythm detection (e.g., JTK_CYCLE and cosinor fitting), yet sampling resolution is critical for determining the exact period. The JTK_CYCLE methodology analyzes rhythmicity only in discrete period lengths along the sampling interval (i.e., in our case, 4-hour intervals). Consequently, it cannot distinguish between 16 hours and, for instance, 17 hours or 15 hours, but indicates that 16 hours is more likely than 12 hours or 20 hours. In addition, analysis of pAKT/AKT levels in Per1/2 null mice using a cosinor fitting approach showed as well a period of about 16 hours (period estimate of 16.02 with 95% confidence interval of 14.88 to 17.47). It is noteworthy that a previous analysis of pAKT in liver of clock mutant Cry1,2−/− mice showed that pAKT cycles with comparable peak times to our study [8], further supporting our findings.

Interestingly, about 16-hour rhythms in locomotor activity were reported in Cry1,2−/− mice [28] as well as Per1,2−/− animals under specific experimental settings [44], yet we did not detect any rhythmicity in feeding or locomotor activity of Per1,2−/− mice in constant dark. Ultradian rhythms of gene expression were reported in mammals in vivo in harmonics of 24 hours [14]. Specifically, about 12-hour rhythms were shown in relation to endoplasmic reticulum (ER) function and the unfolded protein response [45,46]. Remarkably, these about 12-hour rhythms persisted in Bmal1 null mice under free-running conditions [45]. The extent of about 12-hour rhythms in our Bmal1 null mice was much lower, as only few genes showed statistically significant about 12-hour rhythms, which might stem from differences in the methods and cutoffs used for rhythm detection. Finally, ultradian rhythms were reported in other organisms among other yeast [47] and Neurospora crassa [48], albeit with a much shorter period, namely about 5 hours and about 7 hours, respectively. These reports, together with our findings, support the presence of a wide range of rhythms that are shorter than 24 hours. Future studies are expected to shed more mechanistic insight and uncover potential biological implications that are related to these ultradian rhythms.

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