Early-life epigenetic changes along the corticotropin-releasing hormone (CRH) gene influence resilience or vulnerability to heat stress later in life.

Stressful events in early life might lead to stress resilience or vulnerability, depending on an adjustable stress-response set-point, which can be altered during postnatal sensory development and involves epigenetic regulation of corticotropin-releasing hormone (CRH). During the critical developmental period of thermal-control establishment in 3-day-old chicks, heat stress was found to affect both body temperature and expression of CRH in the hypothalamic paraventricular nucleus. Both increased during heat challenge in vulnerable chicks, whereas they decreased in resilient chicks. Our aim was to elucidate the epigenetic mechanism underlying the regulation of stress resilience or vulnerability. Accordingly, DNA CpG methylation (5mC) and hydroxymethylation (5hmC) at the CRH intron, which we found to serve as a repressor element, displayed low 5mc% alongside high 5hmc% in resilient chicks, and high 5mc% with low 5hmc% in vulnerable ones. RE1-silencing transcription factor (REST), which has a binding site on this intron, bound abundantly during acute heat stress and was nearly absent during moderate stress, restricting repression by the repressor element, and thus activating CRH gene transcription. Furthermore, REST assembled into a protein complex with TET3, which bound directly to the CRH gene. Finally, the adjacent histone recruited the histone acetylation enzyme GCN5 to this complex, which increased H3K27ac during harsh, but not moderate heat conditioning. We conclude that an epigenetic mechanism involving both post-translational histone modification and DNA methylation in a regulatory segment of CRH is involved in determining a resilient or vulnerable response to stress later in life.

Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

Changes in Exhaled Carbon Dioxide during the Menstrual Cycle and Menopause.

Introduction: The menstrual cycle (MC) reflects multifaceted hormonal changes influencing women's metabolism, making it a key aspect of women's health. Changes in hormonal levels throughout the MC have been demonstrated to influence various physiological parameters, including exhaled carbon dioxide (CO2). Lumen is a small handheld device that measures metabolic fuel usage via exhaled CO2. This study leverages exhaled CO2 patterns measured by the Lumen device to elucidate metabolic variations during the MC, which may hold significance for fertility management. Additionally, CO2 changes are explored in menopausal women with and without hormonal replacement therapy (HRT). Methods: This retrospective cohort study analyzed exhaled CO2 data from 3,981 Lumen users, including eumenorrheal women and menopausal women with and without HRT. Linear mixed models assessed both CO2 changes of eumenorrheal women during the MC phases and compared between menopausal women with or without HRT. Results: Eumenorrheic women displayed cyclical CO2 patterns during the MC, characterized by elevated levels during the menstrual, estrogenic and ovulation phases and decreased levels during post-ovulation and pre-menstrual phases. Notably, despite variations in cycle length affecting the timing of maximum and minimum CO2 levels within a cycle, the overall pattern remained consistent. Furthermore, CO2 levels in menopausal women without HRT differed significantly from those with HRT, which showed lower levels. Conclusion: This study reveals distinct CO2 patterns across MC phases, providing insights into hormonal influences on metabolic activity. Menopausal women exhibit altered CO2 profiles in relation to the use or absence of HRT. CO2 monitoring emerges as a potential tool for tracking the MC and understanding metabolic changes during menopause.

The Association of Macronutrient Consumption and BMI to Exhaled Carbon Dioxide in Lumen Users: Retrospective Real-World Study.

BACKGROUND: Metabolic flexibility is the ability of the body to rapidly switch between fuel sources based on their accessibility and metabolic requirements. High metabolic flexibility is associated with improved health outcomes and a reduced risk of several metabolic disorders. Metabolic flexibility can be improved through lifestyle changes, such as increasing physical activity and eating a balanced macronutrient diet. Lumen is a small handheld device that measures metabolic fuel usage through exhaled carbon dioxide (CO2), which allows individuals to monitor their metabolic flexibility and make lifestyle changes to enhance it. OBJECTIVE: This retrospective study aims to examine the postprandial CO2 response to meals logged by Lumen users and its relationship with macronutrient intake and BMI. METHODS: We analyzed deidentified data from 2607 Lumen users who logged their meals and measured their exhaled CO2 before and after those meals between May 1, 2023, and October 18, 2023. A linear mixed model was fitted to test the association between macronutrient consumption, BMI, age, and gender to the postprandial CO2 response, followed by a 2-way ANOVA. RESULTS: The model demonstrated significant associations (P<.001) between CO2 response after meals and both BMI and carbohydrate intake (BMI: ß=-0.112, 95% CI -0.156 to -0.069; carbohydrates: ß=0.046, 95% CI 0.034-0.058). In addition, a 2-way ANOVA revealed that higher carbohydrate intake resulted in a higher CO2 response compared to low carbohydrate intake (F2,2569=24.23; P<.001), and users with high BMI showed modest responses to meals compared with low BMI (F2,2569=5.88; P=.003). CONCLUSIONS: In this study, we show that Lumen's CO2 response is influenced both by macronutrient consumption and BMI. The results of this study highlight a distinct pattern of reduced metabolic flexibility in users with obesity, indicating the value of Lumen for assessing postprandial metabolic flexibility.

The Effects of Metabolism Tracker Device (Lumen) Usage on Metabolic Control in Adults with Prediabetes: Pilot Clinical Trial.

INTRODUCTION: Prediabetes is a risk factor for type 2 diabetes mellitus (T2DM). However, it may be reversed via lifestyle changes. Lumen is a novel handheld device that measures exhaled CO2 producing results in agreement with those of indirect calorimetry when assessing metabolic fuel usage. The aim of this study was to examine the effects of following Lumen's personalized, measurement-guided lifestyle intervention program on anthropometric and metabolic variables in adults with prediabetes. METHODS: A 12-week single-arm intervention study was conducted in 27 participants. Body composition and blood markers were measured at the start and end of the study. Each participant took a daily morning (fasted) measurement and received feedback on their metabolic state (i.e., their degree of fat vs. carbohydrate oxidation). Participants were then provided with personalized daily guidelines for their carbohydrate, fat, and protein consumption, along with recommended lifestyle changes. RESULTS: Intention-to-treat analysis revealed a significant decrease in body weight (5.99 kg, p < 0.001), comprising a significant reduction in percentage body fat (2.93%, p < 0.001) and waist circumference (6.23 cm, p < 0.001). Significant reductions were also observed in glycated hemoglobin A1c (0.27%, p < 0.001), triglycerides (0.45 mg/dL, p < 0.001), and systolic blood pressure (0.5 mm Hg, p < 0.05). CONCLUSION: In a 12-week pilot study of participants with prediabetes, Lumen usage significantly improved multiple metabolic parameters, demonstrating its potential to deliver better clinical outcomes for patients with T2DM and metabolic syndrome.

Cross-tolerance: embryonic heat conditioning induces inflammatory resilience by affecting different layers of epigenetic mechanisms regulating IL6 expression later in life.

A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks' IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

Embryonic Heat Conditioning Induces TET-Dependent Cross-Tolerance to Hypothalamic Inflammation Later in Life.

Early life encounters with stress can lead to long-lasting beneficial alterations in the response to various stressors, known as cross-tolerance. Embryonic heat conditioning (EHC) of chicks was previously shown to mediate resilience to heat stress later in life. Here we demonstrate that EHC can induce cross-tolerance with the immune system, attenuating hypothalamic inflammation. Inflammation in EHC chicks was manifested, following lipopolysaccharide (LPS) challenge on day 10 post-hatch, by reduced febrile response and reduced expression of LITAF and NFκB compared to controls, as well as nuclear localization and activation of NFκB in the hypothalamus. Since the cross-tolerance effect was long-lasting, we assumed that epigenetic mechanisms are involved. We focused on the role of ten-eleven translocation (TET) family enzymes, which are the mediators of active CpG demethylation. Here, TET transcription during early life stress was found to be necessary for stress resilience later in life. The expression of the TET family enzymes in the midbrain during conditioning increased in parallel to an elevation in concentration of their cofactor α-ketoglutarate. In-ovo inhibition of TET activity during EHC, by the α-ketoglutarate inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), resulted in reduced total and locus specific CpG demethylation in 10-day-old chicks and reversed both thermal and inflammatory resilience. In addition, EHC attenuated the elevation in expression of the stress markers HSP70, CRHR1, and CRHR2, during heat challenge on day 10 post-hatch. This reduction in expression was reversed by BPTES. Similarly, the EHC-dependent reduction of inflammatory gene expression during LPS challenge was eliminated in BPTES-treated chicks. Thus, TET family enzymes and CpG demethylation are essential for the embryonic induction of stress cross-tolerance in the hypothalamus.

PARP Inhibitor Affects Long-term Heat-stress Response via Changes in DNA Methylation.

Resilience to stress can be obtained by adjusting the stress-response set point during postnatal sensory development. Recent studies have implemented epigenetic mechanisms to play leading roles in improving resilience. We previously found that better resilience to heat stress in chicks can be achieved by conditioning them to moderate heat stress during their critical developmental period of thermal control establishment, 3 days posthatch. Furthermore, the expression level of corticotropin-releasing hormone (CRH) was found to play a direct role in determining future resilience or vulnerability to heat stress by alterations in its DNA-methylation and demethylation pattern. Here we demonstrate how intraperitoneal injection of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) influences the DNA methylation pattern, thereby affecting the long-term heat-stress response. Single PARPi administration, induced a reduction in both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), without affecting body temperature. The accumulated effect of three PARPi doses brought about a long-term decrease in 5mC% and 5hmC%. These changes coincided with a reduction in body temperature in non-conditioned chicks, similar to that occurring in moderately conditioned heat-stress-resilient chicks. The observed changes in DNA methylation can be explained by decreased activity of the enzyme DNA methyltransferase as a result of the PARPi injection. Furthermore, evaluation of the DNA-methylation pattern along the CRH intron showed a reduction in 5mC% as a result of PARPi treatment, alongside a reduction in CRH mRNA expression. Thus, PARPi treatment can affect DNA methylation, which can alter hypothalamic-pituitary-adrenal (HPA) axis anchors such as CRH, thereby potentially enhancing long-term resilience to heat stress.

The balance between stress resilience and vulnerability is regulated by corticotropin-releasing hormone during the critical postnatal period for sensory development.

Determining whether a stressful event will lead to stress-resilience or vulnerability depends probably on an adjustable stress response set point, which is most likely effective during postnatal sensory development and involves the regulation of corticotrophin-releasing hormone (CRH) expression. During the critical period of thermal-control establishment in 3-day-old chicks, heat stress was found to render resilient or sensitized response, depending on the ambient temperature. These two different responses were correlated with the amount of activation of the hypothalamic-pituitary-adrenal (HPA) axis. The expression of CRH mRNA in the hypothalamic paraventricular nucleus was augmented during heat challenge a week after heat conditioning in chicks which were trained to be vulnerable to heat, while it declined in chicks that were trained to be resilient. To study the role of CRH in HPA-axis plasticity, CRH or Crh-antisense were intracranially injected into the third ventricle. CRH caused an elevation of both body temperature and plasma corticosterone level, while Crh-antisense caused an opposite response. Moreover, these effects had long term implications by reversing a week later, heat resilience into vulnerability and vice versa. Chicks that had been injected with CRH followed by exposure to mild heat stress, normally inducing resilience, demonstrated, a week later, an elevation in body temperature, and Crh mRNA level similar to heat vulnerability, while Crh-antisense injected chicks, which were exposed to harsh temperature, responded in heat resilience. These results demonstrate a potential role for CRH in determining the stress resilience/vulnerability balance.