Mary. N. Teruel

Progenitor cells must proliferate to expand the cell population, yet terminal differentiation requires this proliferative state to end. How signaling controls the duration of this proliferative window remains poorly understood. Using adipogenesis and live single-cell imaging of differentiation, cell-cycle, and ERK-activity reporters, we show that YAP and TAZ coordinate progenitor expansion with differentiation commitment by regulating ERK dynamics. YAP/TAZ maintain cells in a fluctuating high-ERK state that promotes proliferation while actively keeping the differentiation driver PPARG below the threshold for irreversible commitment. Crucially, this differentiation block is not explained by proliferation alone: inhibiting CDK4/6 or AKT suppressed proliferation without restoring differentiation, whereas MEK–ERK inhibition restored differentiation even when YAP/TAZ activity remained high. As YAP/TAZ activity decreases, dampened ERK fluctuations trigger PPARG activation. These findings support a self-limiting model in which YAP/TAZ-driven progenitor expansion progressively increases cell density and contact-dependent Hippo signaling, reducing YAP/TAZ activity and terminating the proliferative phase. Consequently, transient YAP/TAZ activation expands the progenitor pool while preserving subsequent differentiation, whereas sustained activation suppresses commitment. Together, these findings identify YAP/TAZ-controlled ERK dynamics as the nexus coordinating progenitor expansion with terminal differentiation and suggest that slower density-dependent Hippo feedback may set the duration of this proliferative window to regulate differentiated cell-number output.

Mitogens trigger cell-cycle entry by activating E2F at the restriction point, which is followed by B-MYB/FOXM1 activation and progression to mitosis. How mitogens control continued cycling and cell-cycle exit after the restriction point is not well-understood. By developing an E2F and B-MYB/FOXM1 dual transcriptional biosensor system, we show that S/G2 phase duration is set by timed mitogen-regulated B-MYB/FOXM1 activation, while E2F activity gradually declines before mitosis. As a striking consequence, rapid B-MYB/FOXM1 activation shortens S/G2, delivering high mitotic E2F activity to daughter cells which keeps them cycling. Delayed B-MYB/FOXM1 activation prolongs S/G2, depleting mitotic E2F which drives daughters to quiescence. When S/G2 is further prolonged, partially activated B-MYB/FOXM1 frequently reverts, triggering mitotic bypass and polyploid quiescence. Thus, B-MYB/FOXM1 governs a tri-directional “G2 restriction point” where cells commit to continued cycling through early B-MYB/FOXM1 activation; cell-cycle exit through delayed B-MYB/FOXM1 activation; or mitotic bypass by B-MYB/FOXM1 inactivation.

Aims/hypothesis: Disruption of the circadian glucocorticoid rhythm occurs in human settings including chronic stress, sleep restriction, circadian misalignment, ageing and autonomous cortisol secretion; in mild autonomous cortisol secretion (MACS) and Cushing’s syndrome, loss of the normal cortisol trough is clinically informative, and flatter diurnal cortisol profiles are associated with cardiometabolic disease. We previously showed that flattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinemia without hyper or hypo-glycaemia, implying that glucocorticoid rhythms may directly regulate the relationship between circulating glucose and systemic insulin output. Here we tested the hypothesis that beta cell glucocorticoid receptor (GR) signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis during glucocorticoid rhythm flattening, and that this reflects glucocorticoid-dependent reprogramming of beta cell stimulus-secretion coupling. Methods: Glucocorticoid rhythms were flattened in male C57BL/6J mice by subcutaneous corticosterone pellet implantation, which elevates trough levels and reduces peak amplitude while preserving the daily mean hormone concentration. Fasting plasma insulin and blood glucose were measured longitudinally and compared with placebo-implanted controls and high-fat diet–fed mice. Beta cell secretory function was assessed by static and dynamic glucose-stimulated insulin secretion in isolated islets, and beta cell excitability by GCaMP6f Ca²⁺ imaging in islets from Ins1-Cre;GCaMP6f mice. To test the requirement for beta cell GR in mature beta cells while avoiding developmental effects of constitutive GR deletion, we generated adult-inducible beta cell–specific GR knockout mice (MIP-CreERT;Nr3c1fl/fl; βGRKO). Combined beta cell and hepatic GR knockout mice (double-GRKO) were used to examine an additional extra-pancreatic contribution to systemic insulin availability. Glucose tolerance and insulin sensitivity were assessed by intraperitoneal glucose and insulin tolerance tests. As a secondary question, a possible contribution of altered insulin clearance was examined from plasma C-peptide:insulin ratios and hepatic insulin-degrading enzyme (IDE) abundance. Results: Glucocorticoid rhythm flattening produced sustained hyperinsulinaemia with maintained euglycaemia, distinct from the delayed hyperinsulinaemia and hyperglycaemia observed in high-fat diet–fed mice. Islets from glucocorticoid-flattened mice exhibited increased insulin secretion at subthreshold (3 mmol/l) glucose, enhanced secretory responses to stimulatory glucose and increased Ca²⁺ responses, indicating a lowered glucose threshold for beta cell activation that persisted ex vivo. Beta cell–specific deletion of GR markedly attenuated the hyperinsulinaemic response to glucocorticoid flattening (insulin AUC reduced ∼40% vs controls; p < 0.001) and produced progressive hyperglycaemia and impaired glucose tolerance, despite unchanged or improved insulin sensitivity. A reduced plasma C-peptide:insulin molar ratio (p = 0.007) and decreased hepatic IDE abundance (p = 0.032) indicated that reduced insulin clearance contributes additionally to the elevated circulating insulin, and combined beta cell and hepatic GR deletion lowered circulating insulin further than beta cell GR deletion alone. The absence of hypoglycaemia despite persistent hyperinsulinaemia is consistent with concurrent insulin resistance. Conclusions/interpretation: Beta cell GR signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis when glucocorticoid rhythmicity is disrupted, acting through glucocorticoid-dependent lowering of the glucose threshold for insulin secretion; reduced insulin clearance contributes additionally to the rise in circulating insulin. These findings identify beta cell GR signalling as a key determinant of glucose homeostasis during disrupted glucocorticoid rhythmicity. Clinically, the work is most relevant not simply to nonspecific chronic stress, but to human states in which the cortisol rhythm is measurably flattened or the nocturnal trough is lost, including MACS, Cushing’s syndrome, sleep restriction, shift work/circadian misalignment and ageing. RESEARCH IN CONTEXT: What is already known about this subject?: Flattened or disrupted glucocorticoid rhythmicity in humans is observed most directly in MACS and Cushing’s syndrome, where loss of the late-night cortisol nadir is clinically informative, and more broadly as flatter salivary cortisol slopes or elevated evening cortisol in ageing, sleep restriction and circadian misalignment; these patterns are associated with type 2 diabetes, cardiovascular disease and mortality. Flattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinaemia without hypoglycaemia, indicating that circulating insulin can be elevated independently of glucose. Hepatic insulin clearance, mediated in part by insulin-degrading enzyme and CEACAM1, is a major determinant of circulating insulin levels. What is the key question?: How does disruption of glucocorticoid rhythmicity increase circulating insulin while maintaining glycaemic control, and is beta cell glucocorticoid receptor signalling required for this adaptive response? What are the new findings?: Glucocorticoid rhythm flattening lowers the glucose threshold for beta cell activation through enhanced Ca²⁺ excitability, an effect that persists in isolated islets and indicates in vivo reprogramming of beta cell function. Beta cell–specific deletion of the glucocorticoid receptor blunts the hyperinsulinaemic response to glucocorticoid flattening and produces hyperglycaemia and impaired glucose tolerance despite unchanged or improved insulin sensitivity. Reduced insulin clearance, associated with decreased hepatic insulin-degrading enzyme abundance, contributes additionally to the elevated circulating insulin, but is not required for maintenance of glucose homeostasis. How might this impact on clinical practice in the foreseeable future?: Identifying beta cell glucocorticoid receptor signalling as a requirement for glucose homeostasis during disrupted glucocorticoid rhythmicity may inform strategies for understanding hyperinsulinaemia and steroid-associated metabolic dysfunction in human conditions marked by loss of the cortisol trough or flatter diurnal cortisol profiles, particularly MACS, Cushing’s syndrome, shift work/circadian misalignment and ageing.

Hippo-YAP signaling orchestrates transient proliferation during tissue repair and is therefore an attractive target in regenerative medicine. However, it is unclear how YAP integrates mitogen and contact signals to start and stop proliferation. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during early G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio in concert with inducing mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity, which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Thus, critical for regeneration without cancer initiation, robust proliferation responses result from a YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is reversed by delayed suppression of receptor signaling after contact inhibition of YAP.

Terminal cell differentiation, a process vital for tissue development and regeneration where progenitor cells acquire specialized functions and permanently exit the cell cycle, remains poorly understood at the molecular level. Using live-cell imaging and adipogenesis as a model, we show that the initial stage involves a variable number of cell divisions, driven by redundant CDK4/6 or CDK2 activation. Afterwards, a delayed decrease in cyclin D1 and an increase in p27 levels reduce CDK4/6 and CDK2 activity. This results in G1 lengthening and the induction of PPARG, the master regulator of adipogenesis. PPARG then induces p21, and later p18, ultimately causing irreversible inactivation of CDK4/6 and CDK2, and thus, permanent cell cycle exit. However, contrary to expectation, CDK inactivation alone is not sufficient to trigger differentiation commitment; ERK inactivation is also necessary. Our study reveals that the coordinated activation and subsequent delayed inactivation of CDK4/6, CDK2, and ERK are crucial for irreversible cell cycle exit and differentiation commitment in terminal cell differentiation.

SUMMARY: Terminal cell differentiation, a process vital for tissue development and regeneration where progenitor cells acquire specialized functions and permanently exit the cell cycle, is still poorly understood at the molecular level. Using live-cell imaging and adipogenesis as a model, we demonstrate that the initial stage involves a variable number of cell divisions driven by redundant CDK4/6 or CDK2 activation.. Subsequently, a delayed decrease in cyclin D1 and an increase in p27 levels leads to the attenuation of CDK4/6 and CDK2 activity. This results in G1 lengthening and the induction of PPARG, the master regulator of adipogenesis. PPARG then induces p21, and later p18, culminating in the irreversible inactivation of CDK4/6 and CDK2, and thus, permanent cell cycle exit. However, contrary to expectation, CDK inactivation alone is not sufficient to trigger commitment to differentiation and functional specialization; ERK inactivation is also required. Our study establishes that the coordinated activation and subsequent delayed inactivation of CDK4/6, CDK2, and ERK are crucial determinants for irreversible cell cycle exit and differentiation commitment in terminal cell differentiation.

Chronic stress, modeled by flattened circadian glucocorticoid (GC) oscillations, is increasingly implicated in obesity. We investigated the underlying mechanisms by disrupting GC rhythms in mice, elevating normally low GC levels during the rest period. This disruption induced substantial obesity, comparable to a 60% high-fat diet (HFD), with additive effects on fat mass, suggesting distinct mechanisms driving adiposity. Despite similar adiposity, GC-flattening and HFD produced profoundly different metabolic outcomes. HFD led to hepatic steatosis and elevated fasting glucose/fatty acid levels, reflecting typical diet-induced dysfunction. In contrast, GC-flattening maintained low fasting glucose/fatty acid levels and prevented hepatic lipid accumulation, with increased adiposity driven by a shift of glucose uptake from muscle to fat and suppression of lipolysis. This reveals a previously unrecognized mechanism of obesity development where excess fat accumulation occurs independently of the mechanisms driving metabolic dysfunction observed in diet-induced obesity. The dissociation between obesity and metabolic dysfunction in GC-flattening challenges the view that increased adiposity and persistent hyperinsulinemia inevitably leads to fatty liver disease. Understanding this pathway opens avenues for novel therapeutic interventions targeting stress-related metabolic disorders.

How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given the fluctuating signaling environment in which cells exist and that critical transcription factors are often not unique to a differentiation process. Here, we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after up-regulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of adipogenesis in mouse preadipocyte cells, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty acid-loaded FABP4 increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state.