Dual roles of circadian clock proteins in development and adult homeostasis

The skeletal system comprises bone and cartilage made of mesenchymal stem cell-derived osteocytes and chondrocytes, respectively. The balance between bone/cartilage resorption and formation is a tightly regulated, timed process¹⁰. Xu et al. showed that bone resorption in fully developed osteoclasts is higher in the dark (active) phase of the day, and is compromised in embryonic osteoclast-specific Bmal1 KO mice¹¹. Bone growth is also under clock control, since embryonic Bmal1 KO in mouse chondrocytes and fibroblast-like synoviocytes leads to impaired joint development, including abnormal cartilage formation and altered cellular composition within the synovium¹². Disrupted circadian rhythms, in turn, can alter cartilage breakdown or bone remodeling patterns, leading to diseases such as osteoarthritis or osteoporosis. In humans, chronic circadian disruption due to rotating work schedules (shift work) compromises bone health, fueling bone fracture risk^13,14.

BMAL1 is essential for early skeletal development due to its roles in regulating mesenchymal stem cell differentiation, BMP/TGFβ signaling, and metalloproteinase activity. Lack of BMAL1 during development disrupts normal bone formation and joint integrity, leading to skeletal abnormalities. For example, Samsa et al.'s global embryonic Bmal1 KO mice exhibit reduced mass and length of both trabecular and cortical bone¹⁵. The low bone mass phenotype of Bmal1⁻/⁻ mice is absent at birth, but is detectable by 8 weeks (young adulthood in mice), and worsens with age, showing severe cortical/trabecular deficits and reduced osteoblast/osteocyte activity by 7–9 months. This likely stems from the reduced ability of mesenchymal stem cells to differentiate into osteoblasts, observed in vitro, leading to the low levels of active osteoblasts and osteocytes observed in vivo.

Beyond impaired bone formation, Bmal1 deficiency also affects joint integrity. Bunger et al. reported that global embryonic Bmal1 KO mice develop abnormal joint phenotypes, characterized by cartilage degeneration and increased ossification and calcification in the joints, which are only apparent as they age, not during development¹⁶. Liang et al. further showed that BMAL1 suppresses BMP/TGFβ signaling, and its absence activates this pathway, leading to increased ossification and calcification in tendons and ligaments¹⁷. These phenotypes were seen as early as birth. Since these effects occurred in mice housed under 12 h:12 h light-dark cycles, without assessing time-of-day variation, it is uncertain whether BMAL1's regulation of BMP/TGFβ reflects circadian-dependent or independent roles in skeletal formation¹⁷. Additionally, cartilage degeneration in global Bmal1-deficient mice can be explained by increased metalloproteinase activity, which accelerates extracellular matrix breakdown, contributing to joint degradation^18–20.

Tissue-specific embryonic Bmal1 KOs match the cartilage degeneration phenotypes of global KOs. Liao et al. found that articular-cartilage embryonic Bmal1 KO mice have reduced chondrocyte proliferation and cartilage matrix synthesis, as well as elevated cartilage apoptosis. The phenotype worsened over time, causing no cartilage changes at 2 months, mild degeneration by 6 months, and severe osteoarthritis-like damage with elevated catabolic markers by 12 months²¹. Dudek et al. found that cartilage-specific embryonic Bmal1 ablation led to more severe, progressively worsening lesions in the articular cartilage than those seen in global embryonic Bmal1-KO²². This was attributed to desynchrony between the chondrocyte peripheral clock and the central hypothalamic pacemaker, rather than to global loss of rhythmicity. Similarly, Hand et al. found that deleting Bmal1 in mesenchymal cells during embryogenesis caused articular cartilage cells to lose their rhythmicity and led to disruptions in joint architecture in adult mice, causing arthritis¹².

Adult-life Bmal1 KOs show conflicting bone phenotypes, however. Osteoblast-specific Bmal1 ablation in adult mice still leads to BMP/TGFβ pathway activation, consistent with BMAL1 binding and repressing the Bmp2 promoter (Fig. 2a), but only causes decreased mass in cortical bone, not trabecular bone²³. This contrasts with observations from Samsa et al. in global embryonic Bmal1 KOs, in which both trabecular and cortical bone show reduced mass¹⁵. This discrepancy may be due to compensatory pathways in the adult KO that are absent or impaired in the global embryonic KO. The higher turnover rate of trabecular bone may also explain why osteoblast numbers increase there but remain unchanged in cortical bone, which remodels more slowly¹⁰. Finally, since BMP/TGFβ activation is observed in both embryonic global and adult tissue-specific KOs, it is likely that BMAL1's role in suppressing this pathway persists into adulthood but affects bone homeostasis differently depending on when it is lost. Yang et al. also found that global Bmal1 KO in adult mice does not increase calcification of cartilage in the Achilles' tendon, unlike global embryonic KOs²⁴. This suggests that BMAL1's role in suppressing tendon ossification is developmentally programmed rather than continuously required in adulthood.

BMAL1's role in the skeletal system thus appears to shift over time. While embryonic deletion results in widespread skeletal defects, adult-specific KOs show more nuanced, site-dependent effects. This suggests BMAL1 enacts context-dependent inhibition of pathways regulating bone formation, such as BMP/TGFβ signaling (Fig. 2a). The diurnal regulation of bone resorption is clearly dependent on the circadian clock, whereas suppression of BMP/TGFβ signaling and prevention of ectopic ossification may reflect non-circadian roles of BMAL1. Overall, BMAL1 serves as both a developmental regulator and a homeostatic gatekeeper, integrating circadian control with skeletal health to maintain structural integrity throughout life.

The circadian clock is essential for maintaining glucose homeostasis by anticipating daily energy needs. Without BMAL1, insulin secretion and glucose metabolism become impaired. Embryonic Bmal1 KO models develop insulin deficiency, hyperglycemia, and glucose intolerance as adults, indicating that BMAL1 is critical for the function and metabolic set point programming of pancreatic β-cells. In contrast, the effects of Bmal1 loss during adulthood are more variable or compensable: some studies report minimal impact, while others show a role in β-cell function and adaptation to metabolic stress. This suggests that BMAL1 is indispensable for metabolic programming during early development, but its necessity in adulthood depends on tissue-specific adaptations and external conditions.

Global embryonic Bmal1 KO models show defective glucose-stimulated insulin secretion as adults^25–28. This may be due to upregulation of UCP2, a mitochondrial uncoupling protein that decreases ATP production, in β-cells, leading to reduced insulin secretion and hypo-insulinemic diabetes²⁷ (Fig. 2b). In global embryonic Bmal1 KO mice, gluconeogenesis is completely abolished, yet corticosterone and glucagon responses to insulin-induced hypoglycemia remain unaffected²⁸. Marcheva et al. found that global embryonic Bmal1 KO and Clock-mutant mice develop hypo-insulinemic diabetes during adult life²⁵. These mice present impaired glucose tolerance and hyperglycemia due to insufficient insulin release, which disrupts their metabolic homeostasis. Their pancreatic islets show impaired stimulus-coupled insulin secretion, attributed to disrupted transcriptional rhythms, including in exocytosis networks. Specifically, Perelis et al. found that CLOCK and BMAL1 bind pancreas-specific enhancers to impart circadian expression to genes for late secretory machinery, including vesicle transport and insulin exocytosis, in β-cells²⁹ (Fig. 2b). Bmal1 loss thus dysregulates genes controlling insulin secretion, in addition to vesicular transport and MAPK signaling pathways²⁵, resulting in decreased insulin release²⁹.

Tissue-specific embryonic Bmal1 KO supports results from global embryonic KO. Pancreas-specific embryonic Bmal1 KOs develop hyperglycemia and hypo-insulinemia as adults¹². Additionally, they show reduced insulin release in islets stimulated by glucose or specific chemicals^25,26. Beyond pancreatic dysfunction, BMAL1 loss disrupts insulin sensitivity in peripheral tissues. SIRT1 expression rhythms dampen in muscle-specific embryonic Bmal1 KOs, impairing circadian insulin signaling. BMAL1 binds to E-box elements in the Sirt1 promoter, promoting its transcription (Fig. 2b). Without BMAL1, SIRT1 levels drop, leading to reduced insulin-stimulated glucose uptake and insulin resistance in skeletal muscle³⁰, manifest from early developmental stages. However, glucose tolerance remains unchanged in muscle-specific Bmal1 KOs, suggesting that other tissues like adipose compensate to stabilize glucose uptake³¹. Liver-specific embryonic Bmal1 KO mice also develop insulin resistance, and during adulthood show decreased ability to adapt to different nutrient conditions, such as a high-fat diet, attributed to elevated mitochondrial oxidative damage³².

The effects of adult-life Bmal1 KO on glucose metabolism vary, depending on the knockout tissue and the timing of metabolic assessments. In adult muscle-specific Bmal1 KOs, insulin resistance develops in part due to GLUT4 downregulation, which impairs glucose uptake^31,33 (Fig. 2b). Perelis et al. found that β-cell-specific Bmal1 KO during adulthood still causes insulin deficiency and glucose intolerance²⁹. However, Yang et al. reported no significant metabolic changes following global Bmal1 knockout in adult mice²⁴. A key difference between these studies is the timing of metabolic tests—Yang et al. conducted tests 8 months after inducible adult KO, while Perelis et al. tested within 10–14 days. Evidence suggests that acute glucose intolerance and diabetic phenotypes, as observed upon Bmal1 loss, can be compensated by age-dependent mechanisms over time³⁴. Acute and long-term metabolic studies are thus needed to resolve these discrepancies.

These findings underscore BMAL1's dual role: it is indispensable for metabolic programming during development, while in adulthood, its impact on glucose homeostasis is shaped by tissue-specific adaptations, compensatory responses, and external factors such as diet and circadian misalignment. Adult β cell-specific or global KOs can thus show from minimal to moderate metabolic changes^24,29. BMAL1's role in glucose metabolism thus appears to involve both developmental programming—such as regulating islet mass and secretory capacity—and lifelong circadian control, such as imparting rhythmic transcription of genes regulating glucose uptake in muscle and insulin exocytosis and signaling in β cells. Whether developmental effects arise from circadian or non-circadian roles demands further investigation.

Blood pressure (BP) shows distinct time-of-day variation. In healthy individuals, BP has the steepest increase during a "morning surge" at the onset of waking, remains stable through the daytime with a peak in the late afternoon³⁵, and then "dips" during nighttime sleeping, decreasing 10-20% relative to daytime BP⁴. Moreover, heart infarctions tend to occur earlier in the day rather than later at night³⁶, highlighting the importance of studying BP in the context of circadian rhythms. BP rhythms reflect regulation by both the central nervous system and peripheral tissue clocks, especially those in the kidney, adrenal glands, nervous system, heart, and vasculature.

Global embryonic Bmal1 KO results in a general hypotensive phenotype in adult mice³⁷. This is likely due to impaired vascular smooth muscle function, as embryonic Bmal1 KO in smooth muscle cells suppresses the maximal contractile responses of blood vessels, lowering BP³⁸. Another reason could be disruption of the renin-angiotensin-aldosterone system (RAAS), which follows a circadian rhythm to regulate BP fluctuations³⁹. Kidney-renin-cell-specific Bmal1 KO during embryogenesis disrupts the rhythmic release of renin, which normally increases BP, causing a hypotensive phenotype in adult mice⁴⁰. Thus, the renal circadian clock is essential to maintain BP homeostasis by ensuring proper RAAS responses to physiological demands. Furthermore, embryonic Bmal1 KO in distal nephron and collecting ducts lowers baseline BP in male mice, suggesting that BP regulation relies on circadian control of sodium transport in aldosterone-sensitive regions of the kidney^41,42.

In contrast, adult nephron-specific Bmal1 KO mice show no changes in glomerular filtration rate, renal function, or BP regulation^43,44. Whereas postnatal cardiomyocyte-specific Bmal1 KO mice do exhibit enhanced BP-induced cardiac remodeling, potentially mediated by PI3K/AKT signaling⁴⁵. This suggests that BMAL1 plays a role in adaptive cardiovascular responses, rather than baseline BP regulation, during adulthood.

In sum, BMAL1 plays distinct roles in BP regulation depending on the developmental stage. Embryonic Bmal1 loss leads to a hypotensive phenotype in adult mice, likely due to circadian control of vascular contractility and the RAAS. This phenotype does not occur when BMAL1 is disrupted in adulthood, suggesting that while BMAL1 remains important for homeostasis, its role in BP maintenance is more nuanced and context-dependent. These findings highlight BMAL1's dual role: during development, it is essential for establishing BP homeostasis, whereas in adulthood, it modulates physiological responses to maintain BP stability rather than acting as a primary regulator. BMAL1's mechanisms for BP regulation thus involve both developmental programming—such as vascular and renal programming—and circadian homeostatic control, such as establishing rhythms in adaptive cardiovascular responses. Remodeling responses in cardiomyocyte-specific knockouts may represent a hybrid mechanism, with basal structural changes modulated by circadian gating under stress.

Circadian rhythms influence airway health and asthma development. In patients with asthma, symptoms often worsen at night or during sleep. Scheer et al. showed that this is driven by the endogenous circadian clock⁴⁶. Using a forced desynchrony protocol to isolate circadian effects from behavioral and environmental factors, they demonstrated that airway resistance increased and pulmonary function decreased during the biological night, even in the absence of sleep or external cues, evidencing circadian control over respiratory function. Understanding circadian mechanisms in asthma pathogenesis is therefore essential. Ehlers et al. studied the effects of Bmal1 KO in both embryonic and adult models of airway pathology⁴⁷. They found that postnatal BMAL1 loss alone is sufficient to compromise the immune system's ability to fight viral infections in the lung and cause asthma. This finding established BMAL1 as a key regulator of antiviral defense during both early development and adulthood. Given that immune impairment occurs even when Bmal1 is disrupted postnatally, the study argues against developmental effects as the primary cause. Instead, it argues that BMAL1 actively regulates immune function throughout life. Further studies will be needed to delineate the specific pathways downstream of BMAL1 that modulate airway immune responses. Dissecting the temporal regulation of immune signaling, epithelial barrier integrity, and cytokine production may in turn potentiate the development of novel time-dependent therapeutic strategies for asthma.

BMAL1 coordinates sleep and regulates detoxification and waste clearance in the brain, which are linked to Alzheimer's and Parkinson's syndromes. Global embryonic Bmal1 KO weakens the brain over time, making neurons more vulnerable to stress and aging in mice⁴⁸. These mice show increased oxidative damage, inflammation, and loss of neural connections, especially in memory-related brain regions. Though neurons do not die immediately, they become fragile and prone to degeneration, especially under stress. Without BMAL1, these protective processes are compromised, increasing the risk of neurodegenerative disease.

Global Bmal1 KO in adults causes rapid and direct neuron loss, particularly in dopamine-producing brain regions linked to movement⁴⁹. Unlike embryonic loss, this kills neurons within a month, even without external triggers. The damage is not just due to circadian rhythm disruption: light-induced circadian desynchrony did not cause TH+ neuron loss, whereas neuron-restricted (Nestin-Cre) and TH-specific Bmal1 disruption produced cell-autonomous degeneration even when behavioral rhythms were intact. BMAL1 also plays an active role in protecting neurons by supporting energy production and stress resistance, while its loss triggers gene changes linked to Parkinson's disease⁴⁹. This shows that BMAL1 is essential for both long-term brain health and immediate neuron survival.

Neuron-specific BMAL1 KO in adults leads to oxidative stress, synapse loss, and dopaminergic neuron degeneration, independent of disruptions in behavioral rhythms⁴⁹. In contrast, astrocyte- or microglia-specific BMAL1 deletion does not induce dopaminergic neuron loss, though BMAL1's role in these glial cells influences redox balance and gliosis, which are relevant to Alzheimer's and Parkinson's pathology.

Thus, BMAL1's neuroprotective roles are not restricted to a developmental window. Increased oxidative stress and synapse loss occur with both developmental and adult-specific deletion, though whether early-life BMAL1 loss increases long-term vulnerability remains unknown. These effects likely reflect circadian and non-circadian functions. Oxidative stress and synapse loss occur without behavioral rhythm disruption⁴⁹, whereas processes like glymphatic clearance and detoxification are linked to circadian cycles^7,48. Redox cycling and proteostasis likely integrate both mechanisms.

REV-ERBs are nuclear receptors and important transcriptional repressors in the circadian clock. They play key roles in liver metabolism by preventing mistimed feeding responses and controlling energy and lipid metabolism⁵⁰. In particular, REV-ERBβ binds and suppresses transcription of lipogenic genes, including those regulated by SREBP, a transcription factor that promotes cholesterol and fatty acid synthesis. Thus, REV-ERBβ acts as a metabolic repressor that limits the activation of the mTOR pathway and fine-tunes lipid production in the liver^51,52. REV-ERBα similarly represses SREBP activity in hepatocytes while also playing a broader role in metabolic regulation, such as the modulation of bile acid synthesis and inhibition of TGFβ signaling, which affects brown adipose tissue differentiation^52,53. Single REV-ERBα or REV-ERBβ KOs are viable but present developmental abnormalities when either gene is lost embryonically, or disrupted homeostasis when lost in adulthood. In contrast, double REV-ERBα and REV-ERBβ KO during embryogenesis can be lethal in global knockouts or sublethal in inducible models, depending on the tissue⁵⁴.

Global embryonic Rev-erbα KO in mice disturbs SREBP activity, leading to systemic metabolic dysfunction during adulthood. This includes high lipid accumulation, causing a metabolic dysfunction-associated steatotic liver disease (MASLD) phenotype, reduced brown fat, hyperglycemia without insulin resistance, muscular atrophy, and metabolic syndrome^53–57. REV-ERBα can also suppress TGFβ signaling. Without REV-ERBα as the repressor, increased TGFβ signaling inhibits brown adipose tissue differentiation, leading to reduced burning of excess fat⁵³. Dysregulated bile accumulation is also seen, leading to oxidative stress, inflammation, and activation of the fibrosis pathway, which worsens MASLD⁵².

Unlike global embryonic REV-ERBα KOs, adult hepatocyte-specific KOs show enhanced lipid accumulation in the liver without increased body weight⁵⁸. Similarly, adult hepatocyte-specific REV-ERBα and REV-ERBβ double KOs (hepDKO) show increased hepatic triglyceride levels⁵⁴. This is thought to be mediated by dysregulated SREBP signaling, since loss of REV-ERBs in hepatocytes can unleash SREBP activity, causing overproduction of triglycerides⁵¹. Indeed, blocking SREBP signaling, via knockout of SREBP cleavage–activating protein in hepDKO mice, prevents diet-induced hepatic triglyceride accumulation⁵¹, restoring lipid homeostasis⁵⁹. HepDKO mice also show lower circulating free fatty acid levels⁵⁴, suggesting a more active uptake and utilization of fatty acids for energy through increased oxidative metabolism.

Overall, while global embryonic REV-ERBα loss causes severe systemic metabolic dysfunction, hepatocyte-specific loss of REV-ERBα or of both REV-ERBα and REV-ERBβ during adulthood causes more localized and comparatively milder effects. This indicates that REV-ERBα is critical for metabolic programming during early development. In contrast, in adulthood, the phenotype cannot be explained simply by compensation from REV-ERBβ, since deleting both paralogs in hepDKO still results in marked hepatic triglyceride accumulation^54,58. REV-ERBs' roles in hepatic metabolism involve both rhythmic and constitutive repression of metabolic pathways. Feeding phase-specific lipid accumulation is circadian rhythm-dependent, whereas basal suppression of SREBP activity and bile acid synthesis appears non-circadian. Mitochondrial oxidative metabolism control may involve both mechanisms, with REV-ERBs regulating both baseline respiratory activity and its alignment with circadian cues.

Cardiomyocyte-specific deletion of REV-ERBα and REV-ERBβ during embryogenesis is sublethal, causing postnatal heart failure and dilated cardiomyopathy⁵⁴. The double knockout disrupts myocardial metabolic rhythms, impairing fatty acid oxidation during the light phase. This prompts a compensatory reliance on glucose metabolism in the dark (active) phase. Such a shift in fuel substrate utilization occurs whether REV-ERBs are ablated in cardiomyocytes during embryonic development or during adulthood⁶⁰. This indicates that REV-ERBs play continuous roles in cardiac energy metabolism beyond development. Further investigation will be needed to determine if these roles depend on circadian or non-circadian mechanisms. Light-phase restricted effects on substrate utilization reflect direct circadian regulation, whereas cardiac structural remodeling in embryonic knockouts may be secondary to altered substrate utilization.