Introduction

Circadian rhythms are integral biological processes that regulate the timing of various physiological functions within all living organisms, ensuring that internal systems remain synchronized with the external light-dark cycle [1]. Circadian regulation is widely implicated in homeostasis and influences processes such as the sleep-wake cycle, metabolic regulation, immune function, and cellular growth [2–4]. Central to the circadian system is the suprachiasmatic nucleus (SCN), the master pacemaker that coordinates the timing of peripheral clocks located in various tissues, including the brain, liver, and heart [5–7]. These peripheral oscillators, although capable of operating autonomously, are synchronized by signals from the SCN to ensure coordinated physiological functions across the body.

A key player in the molecular regulation of circadian rhythms is basic helix-loop-helix ARNT-like protein 1 (BMAL1), a core clock protein that, together with circadian locomotor output cycles kaput (CLOCK), regulates the expression of downstream clock genes through transcriptional-translational feedback loops. Beyond its canonical role in circadian timing, BMAL1 has been implicated in the modulation of multiple cellular processes, including metabolism, cellular stress responses, and tissue repair [8–10]. Recent studies have suggested an interaction between BMAL1 and the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, a central regulator of cell survival, growth, and metabolic adaptation. Although PI3K/AKT signaling has been investigated across diverse biological and pathological contexts, the present review specifically focuses on its circadian regulation by BMAL1 in the context of ischemic injury and vascular repair, where precise temporal control of survival and metabolic pathways is critical for neurovascular resilience [11–14].

This review focuses on the relationship between BMAL1 and the PI3K/AKT signaling pathway, exploring how circadian regulation by BMAL1 modulates PI3K/AKT activity in different physiological and pathological conditions. In ischemic injury and vascular repair settings, BMAL1 has been associated with changes in PI3K/AKT activation and downstream signaling that may support cellular resilience and tissue recovery, in a context- and timing-dependent manner. Understanding this complex interaction may offer novel insights into therapeutic strategies targeting circadian rhythms and signaling pathways, particularly in the context of diseases linked to circadian disruption.

This review was conducted as a focused narrative synthesis of the literature. Relevant experimental and translational studies were identified through searches of PubMed using combinations of keywords including “BMAL1”, “circadian rhythm”, “PI3K/AKT”, “ischemia”, “stroke”, and/or “vascular repair”. Priority was given to peer-reviewed studies providing mechanistic, temporal, or cell-type-specific insights into BMAL1-PI3K/AKT crosstalk in ischemic and neurovascular contexts.

Circadian rhythms

Circadian rhythms are essential biological phenomena that regulate the timing of various physiological processes in virtually all living organisms. These 24-h cycles help synchronize internal processes with the Earth’s light-dark cycle, optimizing physiological and behavioral responses. Circadian regulation influences key biological functions such as the sleep-wake cycle, metabolic processes, hormone secretion, immune responses, and body temperature regulation [2]. This timing mechanism is fundamental to homeostasis and helps keep physiological systems aligned with external environmental cues [3].

In mammals, the circadian system is structured as a hierarchical network, with central and peripheral oscillators working together to regulate the timing of various physiological processes [15]. At the core of this system lies the SCN in the anterior hypothalamus, which functions as the master pacemaker of circadian rhythms (Figure 1). It synchronizes internal biological clocks across the body and coordinates various functions, including sleep, neuroendocrine activity, and body temperature regulation [16]. The SCN is essential for aligning internal biological clocks with external environmental cues, making it the primary driver of circadian timing [17]. Its synchronization with the environment is largely mediated by light, the most powerful external cue, or zeitgeber. Light is detected by intrinsically photosensitive retinal ganglion cells containing melanopsin, which relay signals via the retinohypothalamic tract to the SCN. This initiates a cascade of molecular and cellular events that reset the SCN’s internal clock, ensuring that the organism’s circadian rhythms remain synchronized with the day-night cycle [18, 19].

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Figure 1. Schematic representation of the central and peripheral circadian clocks in mammals. Light signals are detected by the retina and transmitted to the SCN of the hypothalamus, which functions as the central clock. The SCN synchronizes circadian rhythms throughout the body via neural and hormonal outputs. SCN: suprachiasmatic nucleus. Created in BioRender. Beker, M. Ç. (2026) https://BioRender.com/lgdta09.

Molecular mechanisms of circadian regulation

The molecular regulation of the circadian rhythm is regulated through a series of intricate transcription-translation feedback loops (TTFLs) involving key transcription factors. Central to this process is the heterodimerization of CLOCK and BMAL1, also known as MOP3 or Arnt3, which drives the transcription of various clock-regulated genes. These genes include the negative arm components, cryptochrome (CRY) and period (PER), which play essential roles in feedback inhibition [24–27].

In the canonical model, the CLOCK-BMAL1 complex activates the transcription of CRY and PER genes, which then form a complex in the cytoplasm (Figure 2). After a delay, the CRY-PER complex translocates to the nucleus, where it inhibits or attenuates the transcriptional activity of CLOCK-BMAL1 by binding to the E-box elements in the promoters of target genes, thus completing the feedback loop [2, 24, 26, 28]. However, more recent findings reveal a nuanced regulation. CRY proteins (CRY1 and CRY2) directly interact with the CLOCK-BMAL1 complex, blocking its transcriptional activity even in the absence of PER proteins, revealing an additional mode of repression. In the presence of PER, the CRY-PER complex can further enhance repression by displacing CLOCK-BMAL1 from the E-box, resulting in transcriptional silencing [27, 29, 30].

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Figure 2. Molecular architecture of the mammalian circadian transcription-translation feedback loop. The core clock machinery consists of transcriptional activators BMAL1 and CLOCK, which form a heterodimer that binds to E-box elements in target gene promoters, initiating the transcription of period (PER) and cryptochrome (CRY) genes. The translated PER and CRY proteins accumulate in the cytoplasm, form complexes, and translocate back into the nucleus to inhibit BMAL1-CLOCK activity, thereby generating a negative feedback loop. In a secondary regulatory loop, BMAL1-CLOCK also drives the expression of REV-ERB genes via E-box elements. REV-ERB proteins subsequently repress BMAL1 transcription by binding to RORE sites, creating a stabilizing feedback mechanism that maintains rhythmic oscillations in gene expression and cellular physiology. BMAL1: basic helix-loop-helix ARNT-like protein 1; CLOCK: circadian locomotor output cycles kaput; REV-ERB: nuclear receptor subfamily 1 group D member (NR1D1/2); RORE: retinoic acid receptor-related orphan receptor response element. Adapted from [31]. © 2015 Chen and Yang. Distributed under CC BY 4.0.

At the molecular level, the stability of PER and CRY proteins is tightly regulated by E3 ubiquitin ligase complexes, specifically the Skp1-Cullin-F-box complex, which involves beta-transducin repeat containing protein (β-TrCP) and F-box and leucine-rich repeat protein 3 (FBXL3) [25, 27]. This regulatory mechanism helps regulate the degradation of PER and CRY proteins by the 26S proteasome after phosphorylation by kinases like casein kinase 1 epsilon and delta (CK1ε/δ) and AMP-activated protein kinase (AMPK), thus controlling the duration of their repression [2, 27]. When levels of PER and CRY decline, the inhibition on CLOCK-BMAL1 is increased, allowing the cycle to restart, thus ensuring the continuation of the circadian rhythm. Additionally, nuclear receptor subfamily 1 group D member 1 (NR1D1; REV-ERBα) and nuclear receptor subfamily 1 group D member 2 (NR1D2; REV-ERBβ), as well as the retinoic acid receptor-related orphan receptors (RORs), play important roles in modulating BMAL1 expression. REV-ERBs act as transcriptional repressors of BMAL1, while RORs positively regulate its expression by binding to ROR response elements (ROREs) in the BMAL1 promoter [32].

Beyond these core mechanisms, the circadian clock is influenced by various additional factors. These include intracellular calcium flux, membrane depolarization, and the activation of cyclic AMP (cAMP) signaling, all of which contribute to the rhythmic activity of the SCN neurons. Specifically, calcium influx and the periodic activation of cAMP play crucial roles in modulating the rhythmic expression of circadian regulators. The activation of calcium/cAMP response element-binding protein (CREB) through phosphorylation leads to the binding of CREB to regulatory elements in the promoters of key clock genes such as PER1, further influencing circadian regulation [33].

The PI3K/AKT pathway

The PI3K/AKT signaling pathway is a critical regulator of cellular functions such as growth, survival, metabolism, and differentiation, impacting a wide variety of processes, including cell proliferation, apoptosis, autophagy, and angiogenesis. The activation of this pathway is often triggered by extracellular stimuli such as growth factors, cytokines, and hormones. These signaling molecules bind to receptor tyrosine kinases (RTKs) or G-protein coupled receptors, which initiate a cascade of intracellular signaling events. For example, epidermal growth factor, platelet-derived growth factor (PDGF), and insulin-like growth factor can activate PI3K through the autophosphorylation of tyrosine residues in the cytoplasmic domains of RTKs [34].

BMAL1 and PI3K/AKT signaling in ischemic conditions and vascular repair

Stroke remains a major global health challenge, ranking among the leading causes of mortality and long-term disability worldwide. Each year, more than 12 million individuals experience a stroke, resulting in approximately 6.5 million deaths and millions of survivors requiring sustained medical and social support [79]. Despite advances in acute care, recombinant tissue plasminogen activator (rt-PA) remains the only FDA-approved thrombolytic therapy, and its narrow therapeutic window restricts its use to a small fraction of patients, underscoring the need for alternative and adjunct therapeutic strategies [80].

Importantly, acute ischemic stroke is not a uniform disease entity, but a heterogeneous clinical syndrome composed of distinct etiological subtypes, including cardioembolic, lacunar, atherothrombotic, and cryptogenic infarctions. These subtypes differ substantially in vascular pathology, risk factor profiles, stroke severity, and clinical outcome, and are increasingly recognized to exhibit distinct molecular and biomarker signatures. Accordingly, etiologically stratified analyses are essential for accurate clinical interpretation and for improving the translational relevance of mechanistic stroke research [81]. In addition to etiological diversity, biological sex contributes to variability in stroke presentation and outcome. Clinical cohort studies indicate that female sex is associated with differences in vascular risk factors, a higher prevalence of cardioembolic stroke, greater stroke severity at admission, and less favorable in-hospital outcomes compared with male patients [82]. These observations highlight that stroke subtype and sex interact to shape disease severity and prognosis, emphasizing the importance of demographic and etiological stratification when evaluating molecular pathways implicated in ischemic injury.

Disruption of BMAL1 expression in ischemic conditions impairs circadian regulation and is associated with diminished PI3K/AKT pathway activity. In myocardial ischemia, for instance, decreased BMAL1 levels correlate with reduced phosphorylation of AKT and ERK, accompanied by increased oxidative stress and apoptosis [83]. Key neurological and neurovascular evidence linking BMAL1 to PI3K/AKT-mTOR signaling in ischemic and vascular contexts is summarized in Table 2. Treatment with hydrogen sulfide (H₂S) restores BMAL1 mRNA levels and rhythmicity, reactivating PI3K, AKT, and 14-3-3γ, which together mitigate oxidative damage and cell death, highlighting the association between BMAL1 and PI3K/AKT signaling to protect cells during ischemic events [83].

In focal cerebral ischemia, temporal fluctuations in BMAL1 expression are associated with changes in AKT activation. At zeitgeber time (ZT) 18 (ZT18), when BMAL1 levels peak, phosphorylation of AKT (T308) together with increased phosphorylation of ERK1/2, mTOR, and ribosomal protein S6—is significantly enhanced, promoting neuronal survival through the regulation of apoptotic factors such as Bcl-2-associated agonist of cell death (Bad) and PRAS40 [84]. Furthermore, under oxygen-glucose deprivation, both BMAL1 and melatonin intensify the phosphorylation of key components in the PI3K/AKT cascade—including PDK1, mTOR, PTEN, GSK3α/β, ribosomal S6 kinase 1 (RSK1), ribosomal protein S6 kinase (rpS6K), and p70 rpS6K (p70S6K)—thus promoting cellular resilience [85]. Notably, inhibition of BMAL1, however, not only reduces these phosphorylation events but also disrupts PI3K/AKT signaling, indicating a bidirectional regulatory relationship between BMAL1 and this pathway (Figure 3).

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Figure 3. Crosstalk between BMAL1 and the PI3K/AKT/mTOR signaling network. Upon activation of PI3K, the conversion of PIP2 to PIP3 facilitates the recruitment and phosphorylation of AKT by PDK1, leading to activation of mTOR signaling through phosphorylation-dependent inhibition of TSC2 and PRAS40. AKT phosphorylates GSK3α/β, resulting in reduced kinase activity, and phosphorylates PRAS40 to relieve its inhibitory constraint on mTOR. Activated mTOR modulates protein synthesis via 4E-BP1 and p70S6K, while AKT suppresses pro-apoptotic mediators such as BAD and TSC1/2. BMAL1 receives input from both AKT-mTOR and ERK cascades, integrating metabolic and proliferative signals to fine-tune circadian transcriptional outputs. In turn, BMAL1 influences the activity of AKT, GSK3β, and ERK, thereby establishing a feedback loop that synchronizes cellular growth, metabolism, and circadian timing. Arrows indicate activation, blunt lines represent inhibition, dashed lines denote indirect effects, and bidirectional arrows indicate reciprocal interactions. PI3K: phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; PIP2: phosphatidylinositol (4,5)-bisphosphate; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; PDK1: 3-phosphoinositide-dependent kinase-1; AMPKα: AMP-activated protein kinase α; PRAS40: proline-rich AKT substrate of 40 kDa; BAD: Bcl-2-associated agonist of cell death; RSK1: ribosomal S6 kinase 1; GSK3: glycogen synthase kinase 3; BMAL1: basic helix-loop-helix ARNT-like protein 1; TSC1: tuberous sclerosis protein 1; 4E-BP1: eukaryotic translation initiation factor 4E-binding protein 1; p70S6K: p70 ribosomal protein S6 kinase; rpS6K: ribosomal protein S6 kinase; RAS: rat sarcoma (small GTPase); RAF: rapidly accelerated fibrosarcoma; MEK: mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK: extracellular signal-regulated kinase; EGR1: early growth response protein 1. Created in BioRender. Beker, M. Ç. (2026) https://BioRender.com/suc1om4.

Recent findings have demonstrated that BMAL1 acts as a master integrator of circadian and metabolic resilience during cerebral ischemia. Lentiviral BMAL1 overexpression in mice exposed to transient middle cerebral artery occlusion markedly reduced infarct size and neuronal DNA fragmentation, whereas BMAL1 knockdown or genetic deletion aggravated ischemic damage [86]. Elevated BMAL1 levels increase phosphorylation of mTOR (Ser2448) in ischemic cortex, linking circadian control to metabolic adaptation. Quantitative proteomics has revealed broad BMAL1-dependent modulation of mitochondrial and metabolic pathways, including oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and subunits of ATP synthase and cytochrome c oxidase (ATP5ME, ATP6V1H, ATP5F1C, ATP5PB, ATP6V1A, COX4I1, COX6B1, COX7A2). This metabolic reprogramming preserves ATP synthesis, stabilizes calcium dynamics, and limits excessive reactive oxygen species (ROS) formation, maintaining neuronal energy balance during ischemia [86].

Complementary in vitro evidence suggests that BMAL1 may indirectly sustain PI3K/AKT-mTOR signaling by stabilizing the cellular redox environment and energy metabolism. In PC12 cells exposed to oxygen-glucose deprivation/reoxygenation, BMAL1 overexpression activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) antioxidant axis, reduced ROS accumulation, and restored the Bcl-2/Bax equilibrium, thereby suppressing apoptosis and inflammatory cytokine release [87]. Given that oxidative stress and mitochondrial dysfunction can inhibit PI3K/AKT signaling, these findings support the possibility that BMAL1-mediated redox regulation contributes to the maintenance of AKT phosphorylation and downstream mTOR activity under ischemic stress, potentially reinforcing a time-dependent interplay between circadian control and metabolic survival pathways.

Endothelial- and hematopoietic-specific deletion of BMAL1 further underscores its critical role in vascular PI3K/AKT regulation [88]. In Bmal1^flox/flox; Tek-Cre mice, total and phosphorylated AKT (Ser473) levels were markedly decreased in retinal vessels despite preserved eNOS expression, leading to impaired NO signaling, increased nitrative stress, and extensive microvascular degeneration. The resulting threefold rise in acellular capillaries and enhanced neointimal hyperplasia were accompanied by reduced progenitor cell proliferation, highlighting the dependence of vascular repair on BMAL1-AKT signaling. Loss of BMAL1 likely disrupts mTORC2 [rapamycin-insensitive companion of mTOR (RICTOR)]-mediated phosphorylation of AKT, thereby destabilizing vascular PI3K/AKT-mTOR activity and compromising endothelial survival (Figure 3).

Beyond ischemic injury, BMAL1 also contributes to vascular remodeling under hemodynamic stress. In regions exposed to disturbed flow, such as the aortic arch or ligated carotid arteries, expression of BMAL1 and CLOCK increases in parallel with changes in eNOS and AKT phosphorylation [89]. BMAL1 directly modulates eNOS Ser1177 phosphorylation, thereby influencing endothelial tone and NO signaling. It is known that PDGF-BB activates RTKs and engages the canonical rat sarcoma (RAS; small GTPase)-rapidly accelerated fibrosarcoma (RAF) kinase MAPK/ERK kinase (MEK) signaling cascade, culminating in ERK phosphorylation. Activated ERK induces transcriptional programs via immediate early genes such as early growth response protein 1 (EGR1), thereby supporting proliferative responses. In this setting, PDGF-BB-induced ERK activity upregulates BMAL1 through a ROS–ERK–EGR1 axis, while BMAL1, in turn, modulates ERK signaling to fine-tune PDGF-BB-driven vascular smooth muscle cell proliferation [90]. Collectively, these findings indicate that BMAL1 integrates circadian and growth factor signaling to preserve vascular function and structural integrity under ischemic and mechanical stress conditions (Figure 3).

Conclusions

Evidence across ischemic brain and heart models, vascular endothelium, and metabolic contexts collectively suggests a coherent mechanism: BMAL1 appears to align cellular energy state and stress defenses with environmental time by tuning PI3K/AKT activity. When BMAL1 levels or phase are preserved, upstream PI3K signaling, AKT phosphorylation (Thr308/Ser473), and downstream nodes (mTORC1/2, PRAS40, TSC2, GSK3β, FOXO) shift toward pro-survival, pro-angiogenic, and pro-metabolic phenotypes. This configuration sustains mitochondrial function, limits ROS, maintains NO bioavailability, and stabilizes blood-brain barrier integrity, thereby improving ischemic resilience and vascular repair. Conversely, BMAL1 loss or circadian misalignment dampens PI3K/AKT flux, amplifies oxidative and nitrative stress, and worsens ischemic and metabolic injury. The synthesis of mechanistic, proteomic, and functional data supports a model in which BMAL1 acts both upstream of and responsive to AKT-mTOR signaling, forming a time-coded feedback circuit that coordinates transcriptional timing with kinase-driven survival programs.

Importantly, circadian robustness is not governed solely by photic entrainment. Accumulating evidence indicates that systemic non-photic stressors, through their effects on redox balance, mitochondrial function, and metabolic homeostasis, can disrupt SCN-peripheral clock coupling [91–93]. Experimental and observational studies conducted under hypomagnetic or extreme environmental conditions highlight the vulnerability of circadian organization under such stressors [93–95] and provide a conceptual framework by which SCN-peripheral desynchrony may weaken BMAL1-dependent temporal gating of PI3K/AKT signaling. Although direct experimental evidence linking non-photic environmental perturbations to PI3K/AKT dysregulation in ischemic disease remains limited, these observations reinforce the central premise of this review that intact SCN-to-periphery coupling is required to preserve time-of-day-dependent coordination between circadian transcriptional programs and kinase-driven survival pathways, particularly under systemic stress.

Beyond these mechanistic insights, emerging evidence from vascular and cerebrovascular biology indicates that circadian clocks modulate endothelial function, inflammatory signaling, and treatment responsiveness, suggesting that the efficacy of neurovascular interventions may vary according to circadian phase [96, 97]. In this context, chronotherapy—defined as aligning therapeutic interventions with endogenous circadian timing—represents a promising but still insufficiently explored direction for future research. Systematic preclinical and translational studies incorporating circadian phase, stroke subtype, and sex-specific effects, as well as cell-type-specific responses across neurons, endothelial cells, and immune cells, will be essential to determine whether temporal optimization of BMAL1-PI3K/AKT-targeted strategies can enhance therapeutic efficacy while minimizing oncogenic or unintended pro-proliferative signaling in off-target tissues.

A limitation of the present review is that the available clinical and experimental literature does not uniformly stratify findings by circadian phase, stroke subtype, or biological sex, which may contribute to heterogeneity across reported outcomes.