The circadian clock orchestrates adipocyte development and lipid remodeling, with its disruption leading to the development of obesity and insulin resistance. Here we demonstrate that the flavonoid compound naringenin displays clock modulatory activity via RORα that suppresses adipocyte lipid storage while promoting browning. In adipogenic progenitors, naringenin activates RORα with induction of clock gene expression to promote circadian clock oscillation with protective effect against cytokine-induced dampening. The clock-enhancing properties of naringenin suppressed lipogenesis in mature adipocytes together with induction of browning characteristics. The inhibitory effect of naringenin on lipogenesis was dependent on clock modulation as it was abolished in RORα-deficient adipocytes. We further show that naringenin administration in vivo up-regulated RORα expression with clock gene induction together with browning of subcutaneous beige fat depot, resulting reduced fat mass and body weight. Naringenin treatment in vivo also lowered plasma glucose and free fatty acid levels, with markedly enhanced insulin signaling in adipose depots and skeletal muscle. Collectively, our findings uncover a new clock-activating mechanism of action in mediating the metabolic benefits of naringenin, suggesting its potential as a natural supplement for anti-obesity and metabolic disease interventions.

Introduction and Objective: Obesity and diabetes are global health threats. A BMI-increasing variant in CREBRF [rs373863828, R457Q] protects against diabetes despite increasing obesity in humans. The mechanisms by which CREBRF and its R457Q variant mediate these effects are unknown. Since beta cell failure is the common pathway to diabetes, we tested the hypothesis that CREBRF is required for functional beta cell mass (BCM). Methods: We generated mice with global deletion of Crebrf (gCrebrfKO). We then evaluated the impact of CREBRF on mechanistic determinants of functional BCM in mice in vivo, islets ex vivo, and INS-1 832/13 cells. Results: In contrast to human carriers of the CREBRF variant, gCrebrfKO mice have impaired fasting glucose and glucose tolerance despite lower total body and fat mass compared to wild-type. BCM, size, and insulin content per beta cell were decreased in gCrebrfKO mice. In addition, ex vivo glucose-stimulated insulin secretion was reduced in gCrebrfKO islets. Gene expression analysis of islets revealed differential expression of genes involved in proliferation, cell cycle progression, cytoskeleton, and secretion. The cell autonomous effect of Crebrf was confirmed by demonstrating reduced proliferation in INS-1 cells following knockdown. Conclusion: These data suggest that CREBRF is critical in maintaining functional BCM, and that the R457Q variant may be a gain-rather than loss-of-function with respect to energy and glucose homeostasis. gCrebrfKO mice exhibit a dramatic decrease in serum insulin, and our results confirm reduction of beta cell size, insulin content, and stimulated insulin secretion in gCrebrfKO islets. We also show the cell autonomous effect of Crebrf by demonstrating reduced proliferation in a beta cell line following its knockdown. CREBRF is required for glucose homeostasis despite lower body weight primarily through effects on functional BCM and regulation of key genes in beta cell proliferation and cell cycle progression. Disclosure M.G. Rossillo: None. P.E. Mattila: None. Z. Ross: None. A. Wood: None. G. Schoiswohl: None. R. Liu: None. F. Shah: None. B. Chuan: None. M.K. Basantani: None. D. Gupta: None. D.E. Weeks: None. S.T. McGarvey: None. L. Heinsberg: None. M. Jurczak: None. V. Yechoor: None. E.E. Kershaw: Consultant; Ended; Novo Nordisk. Consultant; Current; NodThera, Sparrow Pharmaceuticals. Research Support; Current; Pfizer Inc., Arrowhead Pharmaceuticals, Inc. Funding American Diabetes Association (1-17-PMF-02), National Institutes of Health (T32DK007052), National Institutes of Health (K01DK115543), National Institutes of Health (R21KD121266), Winters Foundation Award HHMI Pittsburgh Foundation (MR2018-98421), National Institutes of Health (HL093093)

How cellular metabolism facilitates tissue-resident macrophage maintenance remains elusive. Here we show that visceral adipose tissue (VAT)-resident macrophages, unlike monocyte-derived macrophages, are enriched with mitochondrial-specific antioxidant enzymes restraining inflammation and promoting VAT homeostasis and insulin sensitivity. Additionally, VAT resident macrophages express high levels of plasminogen activator inhibitor type 2, encoded by SerpinB2, which is involved in the blood coagulation cascade. SerpinB2 promotes adipose resident macrophage survival by regulating mitochondrial oxidative phosphorylation and preventing the release of pro-apoptotic cytochrome c from the mitochondria into the cytoplasm via antioxidant glutathione production. Chronic inflammation, such as obesity, diminishes SerpinB2 expression in VAT macrophages in patients and mice, leading to the decline of this macrophage subset. Mechanistically, interferon-γ elevation in diabetes induces Ikaros, a transcriptional suppressor, which binds to the SerpinB2 promoter and decreases SerpinB2 expression. Congruently, selective depletion of the IFN-γ receptor in myeloid cells or supplementation of macrophage-specific SerpinB2 deficient mice with N-acetylcysteine, a glutathione precursor, restores VAT resident macrophage survival, decreases adipocyte size, and improves glucose tolerance and insulin sensitivity. Our data thus reveal an unexpected function of SerpinB2 in the regulation of mitochondrial function and survival of tissue-resident macrophages.

The circadian clock maintains temporal control of metabolic processes and exerts a key role in adipocyte development. Discovery of clock-modulatory compounds may provide new avenues for metabolic disease therapy. Here we report the identification of flavonoid compounds, Quercetin and Fisetin, as clock-activating molecules with direct inhibitory action on adipogenesis and adipocyte lipid metabolism. Quercetin and Fisetin displayed robust RORα agonism that promoted clock oscillation with induction of clock genes. Treating preadipocytes with these compounds blocked their adipogenic differentiation. In mature adipocytes, Quercetin and Fisetin suppressed lipid accumulation by inhibiting lipogenic enzymes. Furthermore, activation of RORα by a synthetic agonist or ectopic expression were sufficient to inhibit adipogenesis. In mice treated with Quercetin or Fisetin, RORα was markedly induced in adipose depots with strong suppression of the adipogenic and lipogenic programs. While quercetin significantly attenuated lipid storage in adipose tissue in vivo accompanied with lowering of free fatty acids and improved insulin sensitivity, fisetin displayed a less robust effect with differential regulation of lipolytic pathway. Collectively, these findings uncovered the clock-activating properties of quercetin and fisetin that prevent adipocyte maturation and hypertrophy to limit adipose tissue expansion. These actions contribute, at least in part, to their beneficial effects on metabolic disorders.

Introduction and Objective: Literature supports preserving β-cell mass and function to slow diabetes progression. To do so, β-cells withstand stressors including inflammation and increased insulin demand. Mitochondria emerge as an area of focus, as respiration drives ATP generation required for glucose-stimulated insulin secretion (GSIS), and damaged mitochondria lead to ROS generation. Mitochondria recycle via mitophagy, and defects have been linked to β-cell death. We isolated RIMOC1 (RAB7A-Interacting MON1-CCZ1 Complex Subunit 1) as a protein highly expressed in β-cells. While its function is unknown, human GEO datasets highlight a negative correlation with glucose load and HgbA1c. Considering this, our hypothesis is that RIMOC1 preserves mitophagy and mitochondrial function in β-cells, and a loss of RIMOC1 function in β-cells would lead to β-cell failure. Methods: We generated a stable INS2 RIMOC1 KO cell line and an INS1-Cre RIMOC1 KO mouse model using CRISPR-Cas9. Mitochondrial membrane potential was quantified via TMRE, mitophagy via mtKeima probe, and mitochondrial respiration via Seahorse. Ongoing in vivo experiments include glucose and insulin tolerance testing (GTT/ITT), and ex vitro GSIS experiments. Results: Human GEO datasets show that RIMOC1 expression is positively correlated with β-cell maturity and negatively correlated with glucose load and HgbA1c. Our in vitro data supports this, demonstrating RIMOC1 KO leads to decreased insulin mRNA expression, decreased insulin content, diminished TMRE staining, altered mitophagy, impaired mitochondrial respiration, and increased β-cell cycle inhibition. Early in vivo data shows significant impairment of glucose tolerance in oral GTT vs IP GTT. Conclusion: RIMOC1 is enriched in β-cells and has a significant role in maintaining euglycemia and mitochondrial function, and ongoing studies will further explore β-cell specific RIMOC1 KO. This research highlights RIMOC1 as a promising drug target for preserving β-cell function for the prevention or treatment of diabetes. D. Filingeri: None. R. Liu: None. V. Negi: None. J. Lee: None. A. Kumar: None. V. Mandi: None. M. Moulik: None. V. Yechoor: None. NIH-T32 Fellowship Grant (5-T32-DK007052)

BACKGROUND TEAD1, the mammalian Hippo pathway-regulated transcription factor, plays a critical and non-redundant role in maintaining cardiomyocyte (CM) homeostasis. However, the specific cellular pathways regulated by TEAD1 in CMs remain poorly defined. We hypothesized that TEAD1 has an essential, cell-autonomous role in the CM oxidative stress response by directly regulating the transcription of NRF2, the master regulator of oxidative stress response. METHODS AND RESULTS Tamoxifen-induced conditional CM-specific TEAD1 deletion in adult mice leads to acute heart failure (HF) and altered expression of antioxidant genes. In silico analysis of publicly available RNA-seq data from human hearts with end-stage dilated (DCM) and ischemic (ICM) cardiomyopathy revealed significant downregulation of TEAD1 transcript levels and a positive correlation between TEAD1 and NRF2 gene expression. ChIP-seq and ATAC-seq in adult mouse hearts confirmed TEAD1 occupancy at promoter/enhancer elements within open chromatin regions of multiple antioxidant genes, including NRF2 and its targets. Ex vivo and in vitro TEAD1 knockout in primary neonatal and adult murine CMs, as well as in H9C2 cells, resulted in significantly increased cellular and mitochondrial ROS le, accompanied by a marked decrease in NRF2 expression and promoter-luciferase activity, under both basal and oxidative stress conditions. Mosaic, conditional deletion of TEAD1 in ∼40–50% of murine heart CMs provided a novel in vivo model for studying TEAD1-regulated pathways in the heart, independent of the confounding effects of HF. This model demonstrated reduced NRF2 expression and heightened oxidative stress in neonatal and adult TEAD1 mosaic knockout hearts. Notably, 8OHdG staining identified oxidative DNA damage in TEAD1-deficient CMs compared to TEAD1-expressing CMs within the mosaic knockout hearts. Upon in vivo AngII infusion, TEAD1 mosaic knockout hearts showed a significant increase in oxidative stress markers and an impaired NRF2 response. Overexpression of human TEAD1 restored NRF2 activity and mitigated ROS accumulation in TEAD1 knockout CMs in vitro. Furthermore, TEAD1 deletion in human iPSC-derived CMs resulted in increased oxidative stress and downregulation of NRF2 expression and functional activity, confirming the requirement of TEAD1 in NRF2-mediated oxidative stress response in human CMs. Collectively, these findings establish that TEAD1 is essential for NRF2 expression and activity under both basal and AngII-induced conditions and plays a crucial role in the oxidative stress response in CMs. CONCLUSIONS TEAD1 is a cell-autonomous, direct transcriptional regulator of NRF2 and the cardiomyocyte (CM) oxidative stress response. Its gene expression, which directly correlates with NRF2 transcript levels in the human myocardium, is significantly downregulated in human end-stage heart failure, potentially compromising the oxidative stress response in the failing heart. ### Competing Interest Statement The authors have declared no competing interest.

Long-term exposure to nonstandard work schedules can result in circadian misalignment, which has been linked to a series of maladies. To test whether modulating light patterns reduces shiftwork-induced rest/activity disruptions, 30 male C57BL/6 J mice individually housed in cages outfitted with running wheels were exposed to 6 simulated shiftwork light interventions. Mice experiencing high light levels during shiftwork exhibited a significant decrease in activity compared to low light levels during shiftwork and a conventional 12 L:12D condition, indicating circadian misalignment. In contrast, mice experiencing shiftwork in darkness combined with either modulated evening light pulses or circadian blind, vision-permissive light showed similar levels of rest/activity compared to a 12 L:12D condition, with phasor analysis indicating that their 24-h circadian rest/activity patterns were not misaligned. The results show that exposure to light that permits visibility but is below activation of the circadian system during shiftwork can prevent circadian misalignment.

Human cytomegalovirus (HCMV) infects up to 80% of the world’s population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hr after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon 6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV’s established roles in these processes.

Proliferative quiescence in β-cells is required to maintain functional competence. While this presents a significant hurdle in regenerative therapy for diabetes, the molecular underpinnings of this reciprocal relationship remain unclear. Here, we demonstrate that TEAD1, the transcription effector of the mammalian-Hippo pathway, drives developmental stage-specific β-cell proliferative capacity in conjunction with its functional maturation. TEAD1 promotes adult β-cell mature identity by direct transcriptional control of a network of critical β-cell transcription factors, including, Pdx1, Nkx6.1, and MafA, while its regulation of Cdkn2a maintains proliferative quiescence. Consequently, mice with either constitutive or inducible genetic deletion of TEAD1 in β-cells developed overt diabetes due to a severe loss of secretory function despite induction of proliferation. Furthermore, we show that TEAD1 has a similar regulatory role in human β-cells. Consistent with this function in β-cells, variants in TEAD1 have been associated with c-HOMA-B in American Indians. We propose that TEAD1 is an essential intrinsic molecular switch coordinating adult β-cell proliferative quiescence with mature identity and its differential modulation may be necessary to overcome the challenge of inducing proliferation with functional competence in human beta cells.

Foam cells in atheroma are engorged with lipid droplets (LDs) that contain esters of regulatory lipids whose metabolism remains poorly understood. LD-associated hydrolase (LDAH) has a lipase structure and high affinity for LDs of foam cells. Using knockout and transgenic mice of both sexes, here we show that LDAH inhibits atherosclerosis development and promotes stable lesion architectures. Broad and targeted lipidomic analyzes of primary macrophages and comparative lipid profiling of atheroma identified a broad impact of LDAH on esterified sterols, including natural liver X receptor (LXR) sterol ligands. Transcriptomic analyzes coupled with rescue experiments show that LDAH modulates the expression of prototypical LXR targets and leads macrophages to a less inflammatory phenotype with a profibrotic gene signature. These studies underscore the role of LDs as reservoirs and metabolic hubs of bioactive lipids, and suggest that LDAH favorably modulates macrophage activation and protects against atherosclerosis via lipolytic mobilization of regulatory sterols.

Cytokine-induced β-cell apoptosis is a major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding its underlying mechanisms, few drugs have been translated to protect β-cells in T1D. Epigenetic modulators such as bromodomain-containing BET (bromo- and extra-terminal) proteins are important regulators of immune responses. Pre-clinical studies have demonstrated a protective effect of BET inhibitors in an NOD (non-obese diabetes) mouse model of T1D. However, the effect of BET protein inhibition on β-cell function in response to cytokines is unknown. Here, we demonstrate that I-BET, a BET protein inhibitor, protected β-cells from cytokine-induced dysfunction and death. In vivo administration of I-BET to mice exposed to low-dose STZ (streptozotocin), a model of T1D, significantly reduced β-cell apoptosis, suggesting a cytoprotective function. Mechanistically, I-BET treatment inhibited cytokine-induced NF-kB signaling and enhanced FOXO1-mediated anti-oxidant response in β-cells. RNA-Seq analysis revealed that I-BET treatment also suppressed pathways involved in apoptosis while maintaining the expression of genes critical for β-cell function, such as Pdx1 and Ins1. Taken together, this study demonstrates that I-BET is effective in protecting β-cells from cytokine-induced dysfunction and apoptosis, and targeting BET proteins could have potential therapeutic value in preserving β-cell functional mass in T1D.

Preclinical and clinical studies suggest that lipid-induced hepatic insulin resistance is a primary defect that predisposes to dysfunction in islets, implicating a perturbed liver-pancreas axis underlying the comorbidity of T2DM and MASLD. To investigate this hypothesis, we developed a human biomimetic microphysiological system (MPS) coupling our vascularized liver acinus MPS (vLAMPS) with pancreatic islet MPS (PANIS) enabling MASLD progression and islet dysfunction to be assessed. The modular design of this system (vLAMPS-PANIS) allows intra-organ and inter-organ dysregulation to be deconvoluted. When compared to normal fasting (NF) conditions, under early metabolic syndrome (EMS) conditions, the standalone vLAMPS exhibited characteristics of early stage MASLD, while no significant differences were observed in the standalone PANIS. In contrast, with EMS, the coupled vLAMPS-PANIS exhibited a perturbed islet-specific secretome and a significantly dysregulated glucose stimulated insulin secretion response implicating direct signaling from the dysregulated liver acinus to the islets. Correlations between several pairs of a vLAMPS-derived and a PANIS-derived factors were significantly altered under EMS, as compared to NF conditions, mechanistically connecting MASLD and T2DM associated hepatic-factors with islet-derived GLP-1 synthesis and regulation. Since vLAMPS-PANIS is compatible with patient-specific iPSCs, this platform represents an important step towards addressing patient heterogeneity, identifying disease mechanisms, and advancing precision medicine. A coupled liver-islet MPS was used to investigate the comorbidity between MASLD and T2DM. This study demonstrated that secreted factors from the MASLD liver disrupt islet function and suggest potential underlying mechanisms of disease progression along the liver-islet axis.

Background: The pathogenic mechanisms underlying HFpEF (Heart Failure with preserved Ejection Fraction), which accounts for 50% of heart failure, remain unclear. Elevated endoplasmic reticulum (ER) stress has been implicated in HFpEF. Circadian disruption (CD) as seen in shift workers has been associated with an excess risk for chronic age-related disorders, including cardiovascular disease. Currently, the role of CD in HFpEF is not known. Hypothesis: The dual 'hits' of chronic metabolic stress and circadian disruption lead to increased ER stress, impaired unfolded protein response (UPR), and adverse cardiac remodeling resulting in HFpEF. Methods: High fat diet (HFD) fed C57BL6 male mice were subjected to chronic shifted light-dark (LD) cycles mimicking shift work (Shift), with appropriate controls. Metabolic (glucose tolerance, insulin sensitivity, body fat) and cardiac function parameters (systolic and diastolic function by ECHO, MRI, and terminal PV-loops) were collected. Cardiac tissue was analyzed using qPCR, western blot, bulk RNA-seq, spatial transcriptomics, proteomics, and histology. For in vitro studies, Bmal1 was deleted in H9C2 cells using CRISPR/Cas9. Results: ‘HS’ mice (HFD & Shift) had the most insulin resistance, glucose intolerance, and obesity, compared to ‘HR’ (HFD & Regular LD), ‘CS’ (Normal Chow & Shift) and ‘CR’ (Chow and Regular LD) groups. ECHO and MRI revealed preserved EF in all groups, with diastolic impairment in both HS and HR (↑MV E/E’, ↓MV E/A, and ↓LV diastolic strain) groups, HS>HR. Only ‘HS’ mice showed ↓cardiac compliance (↑β in EDPVR) and ↑lung weight suggestive of HF consistent with HFpEF. Gene and protein expression revealed dysregulation of numerous UPR pathway molecules (Atf6, Chop, Bip, Perk, and others) in HS mice. Mechanistically, Bmal1 deletion in CMs resulted in similar dysregulation of UPR genes. HS mouse hearts showed increased fibrosis, and spatial transcriptomics displayed enrichment of ATF4-related pathways in CMs and activated fibroblasts. Conclusions: The dual hit of metabolic and circadian stress induces HFpEF, which cannot be recapitulated by only one stressor. Our results identify previously unrecognized roles of UPR perturbation in driving HFpEF under circadian and metabolic stress.

Background: COVID-19 pandemic has caused more than 6 million deaths worldwide. Co-morbid conditions such as Type 2 Diabetes (T2D) have increased mortality in COVID-19. With limited translatability of in vitro and small animal models to human disease, human organ-on-a-chip models are an attractive platform to model in vivo disease conditions and test potential therapeutics. Methods: T2D or non-diabetic patient-derived macrophages and human liver sinusoidal endothelial cells were seeded, along with normal hepatocytes and stellate cells in the liver-on-a-chip (LAMPS - liver acinus micro physiological system), perfused with media mimicking non-diabetic fasting or T2D (high levels of glucose, fatty acids, insulin, glucagon) states. The macrophages and endothelial cells were transduced to overexpress the SARS-CoV2-S (spike) protein with appropriate controls before their incorporation into LAMPS. Cytokine concentrations in the efflux served as a read-out of the effects of S-protein expression in the different experimental conditions (non-diabetic-LAMPS, T2D-LAMPS), including incubation with tocilizumab, an FDA-approved drug for severe COVID-19. Findings: S-protein expression in the non-diabetic LAMPS led to increased cytokines, but in the T2D-LAMPS, this was significantly amplified both in the number and magnitude of key pro-inflammatory cytokines (IL6, CCL3, IL1β, IL2, TNFα, etc.) involved in cytokine storm syndrome (CSS), mimicking severe COVID-19 infection in T2D patients. Compared to vehicle control, tocilizumab (IL6-receptor antagonist) decreased the pro-inflammatory cytokine secretion in T2D-COVID-19-LAMPS but not in non-diabetic-COVID-19-LAMPS. Interpretation: macrophages and endothelial cells play a synergistic role in the pathophysiology of the hyper-inflammatory response seen with COVID-19 and T2D. The effect of Tocilizumab was consistent with large clinical trials that demonstrated Tocilizumab's efficacy only in critically ill patients with severe disease, providing confirmatory evidence that the T2D-COVID-19-LAMPS is a robust platform to model human in vivo pathophysiology of COVID-19 in T2D and for screening potential therapeutics.

TEAD1 and the mammalian Hippo pathway regulate cellular proliferation and function, though their regulatory function in β cells remains poorly characterized. In this study, we demonstrate that while β cell-specific TEAD1 deletion results in a cell-autonomous increase of β cell proliferation, β cell-specific deletion of its canonical coactivators, YAP and TAZ, does not affect proliferation, suggesting the involvement of other cofactors. Using an improved split-GFP system and yeast two-hybrid platform, we identify VGLL4 and MENIN as TEAD1 corepressors in β cells. We show that VGLL4 and MENIN bind to TEAD1 and repress the expression of target genes, including FZD7 and CCN2, which leads to an inhibition of β cell proliferation. In conclusion, we demonstrate that TEAD1 plays a critical role in β cell proliferation and identify VGLL4 and MENIN as TEAD1 corepressors in β cells. We propose that these could be targeted to augment proliferation in β cells for reversing diabetes.

Introduction: Adult human cardiomyocytes (CMs) have poor capacity for proliferation and functional recovery after heart attack, thus leading to the high morbidity and mortality of cardiovascular diseases. Thus, there is great need to understand the underlying mechanisms of adult CM turnover and maturation. While screening for modulators of TEAD1, the downstream transcriptional effector of Hippo pathway, my lab identified a novel factor—C5ORF51/RIMOC1—as a potential cardiomyogenic modulator. C5ORF51 (hereinafter referred to as C5x) is a previously uncharacterized protein of nearly unknown function, which appears to play critical roles in the regulation of CM homeostasis and heart size. Hypothesis: C5x is a novel cardiomyogenic regulator which determines CM turnover, maturation, and function. Methods and Results: (1) Ex vivo C5x deletion in mouse neonatal CMs leads to significantly increased percentages of cells in S-G2-M phases, suggesting that C5x mediates CM cell cycle arrest in a cell-autonomous manner; (2) In vivo CM-specific C5x-knockout mouse model shows significantly enhanced CM proliferation in adult mice as well as increased CM endowment and mononuclear diploid populations, indicating that C5x has an essential role in cell replication and division in vivo; (3) With age, C5x-CM-knockout mice develop systolic dysfunction, massive cardiomegaly, late-onset dilated cardiomyopathy, and premature mortality, demonstrating a critical role of C5x in maintaining normal cardiac function; (4) RNA sequencing analysis further reveals that C5x loss-of-function activates fetal gene expression while its gain-of-function suppresses fetal gene expression, suggesting C5x as a gatekeeper of the fetal gene program. Conclusions: C5x is required for CM cell cycle arrest, and its loss results in fetal gene activation leading to a primitive cell state and immature CM expansion.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here, we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays antiadipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the reactivation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Last, Nobiletin inhibited the differentiation of primary preadipocytes, and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.

The COVID-pandemic has contributed to more than 5 million deaths worldwide in the last two years. Co-morbid conditions such as Type 2 Diabetes (T2D) , HTN, obesity, and CKD have been associated with increased mortality with COVID-19. In a large meta-analysis, the relative risk of mortality was 1.54 for patients with T2D and COVID-19. Thus, there is an imperative need to develop a platform for rapid and reliable drug screening/selection against COVID-related morbidity/mortality in T2D patients. With limited translatability of in vitro and small animal models to humans, human organ-on-a-chip models are an attractive platform to model in vivo disease conditions and test potential therapeutics. We seeded T2D or nondiabetes patient-derived macrophage and human liver sinusoidal endothelial cells along with normal hepatocytes and kupffer cells in the liver-on-a-chip (LAMPS - Liver Acinus MicroPhysiological System) developed by our group, perfused with media mimicking normal fasting or late metabolic syndrome (LMS - high levels of glucose, fatty acids, insulin, glucagon) states. We transduced both macrophage and endothelial cells to overexpress the SARS-CoV2-S (spike) protein and compared it with a control lentivirus transduction. We found that T2D cells overexpressing S-protein in LMS media (T2D chip) displayed an increased secretion of inflammatory cytokines compared to the nondiabetes chip over days. We then tested the effect of Tocilizumab (IL6-receptor antagonist) in T2D chips. Compared to vehicle control, Tocilizumab significantly decreased the S-protein induced inflammatory cytokine secretion in T2D chips but not in nondiabetes chips, indicating its higher efficacy in severe disease states only. This is consistent with what was observed in large clinical trials providing confirmatory evidence that the LAMPS T2D and nondiabetes chips serve as a relevant in vitro model system to replicate human in vivo pathophysiology of COVID and for screening potential therapeutics. Disclosure V.Negi: None. D.Gavlock: None. J.Lee: None. T.Shun: None. A.Gough: None. M.T.Miedel: None. D.L.Taylor: None. V.Yechoor: n/a.