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The morphological transformation of adipogenic progenitors into mature adipocytes requires dissolution of cytoskeleton driven by progressive loss of MRTF-SRF activity. Circadian clock confers temporal control in adipogenic differentiation, and MRTF-SRF inhibits beige adipocyte development. Here we identify that key components of the actin cytoskeleton-MRTF/SRF signaling cascade are direct transcriptional targets of circadian clock, and a clock-MRTF/SRF regulatory axis suppresses beige adipocyte thermogenic recruitment. Genetic loss- or gain-of-functions of the core clock regulator, Brain and Muscle Arnt-like 1 (Bmal1), altered actin cytoskeleton organization, cell shape and SRF activity. Genes involved in actin dynamic-MRTF-SRF pathway display diurnal expression profiles in beige adipose tissue, with identification of Bmal1 direct transcriptional regulation. Using beige adipocyte-selective genetic models together with pharmacological approaches, we demonstrate that Bmal1 inhibits beige adipogenesis and thermogenic capacity via the MRTF-SRF pathway. Selective ablation of Bmal1 induced beigeing, whereas its targeted overexpression attenuated the beige thermogenic program resulting in obesity. Collectively, our findings define a novel circadian clock control in actin cytoskeleton-MRTF-SRF signaling that suppresses beige adipogenesis to maintain metabolic homeostasis.

The circadian clock confers temporal regulation in metabolism, and its disruption leads to obesity and insulin resistance. In the current study, we identify that the opposing circadian clock regulators, transcription activator Bmal1 and its repressor Rev-erbα, exert concerted control of the actin cytoskeleton-MRTF/SRF pathway to drive beige adipocyte development, and that this clock regulatory axis is required for beige fat thermogenic capacity. Key components of the MRTF/SRF signaling display circadian oscillations in beige fat, and cistromic analyses revealed Bmal1 and Rev-erbα chromatin occupancy of genes involved in MRTF/SRF regulation. Genetic loss- or gain-of-functions of Bmal1 and Rev-erbα in adipogenic precursors markedly altered actin cytoskeleton organization. Bmal1 silencing inhibits F-actin formation and MRTF/SRF activity whereas its forced expression augments actin cytoskeleton. Notably, Bmal1 and Rev-erbα transcriptional control of the MRTF/SRF signaling modulates beige fat thermogenic capacity in vivo. Prrx1-Cre-mediated subcutaneous beige fat-selective Bmal1 ablation induced browning with augmented mitochondrial metabolism, resulting in resistance to obesity that improved insulin sensitivity. Conversely, overexpression of Bmal1 in beige depot suppressed the thermogenic program with adipose expansion and impaired glucose tolerance. In contrast, Rev-erbα gain-of-function phenocopied that of Bmal1 ablation, leading to browning of beige fat, resistance to obesity and insulin sensitivity. Mechanistically, we show that genetic loss of Bmal1 enhanced beige precursor differentiation and thermogenic induction, whereas Rev-erbα overexpression similarly promoted beige adipogenesis. Collectively, these findings uncover a novel concerted circadian clock control in beige adipocyte development that determines thermogenic capacity via the cytoskeleton-MRTF/SRF signaling cascade. Disclosure X.Xiong: None. W.Li: None. R.Liu: None. V.Yechoor: n/a. K.Ma: None. Funding NIH 1R01DK112794 to KM and DK097160 to VY

β-cell mass is significantly reduced especially in the late stage of diabetes. Hence, increasing β-cell proliferation to increase β-cell mass remains a desirable goal for diabetes treatment. The mechanisms that regulate β cell proliferation and concurrent maintenance of mature function is not well understood. Hippo-TEAD pathways regulated proliferation in many tissues. Our data show that TEAD1 (Hippo) pathway activities were significantly reduced in mature β cell. TEAD1 knockout results in the cell-autonomous increase of β cell proliferation, while loss of TAZ has not change in β cell proliferation, suggesting that TEAD1 corepressors, rather than coactivators, play a dominant role in β-cells. Improved Split-GFP system and yeast two-hybrid platform were also adapted to screen TEAD1 cofactors in β cells. Our study showed that MENIN can bind to TEAD1 directly and the pocket area of TEAD1 is critical for this binding. VGLL4 and MENIN work as TEAD1 corepressors to inhibit the expression of target genes in β-cells, including FZD7 and CCN2, which leads to a restriction of β cell proliferation. We also found that TEAD1 can regulate TEAD1, and this auto-inhibition contributes to TEAD1 pathway inhibition with β cell maturity. Our study sheds light on the mechanisms involved in β cell maintenance of mature function and proliferation and offer the mechanistic basis for modulating TEAD pathway to increase proliferation in β cells. Disclosure F.Li: None. R.Liu: None. V.Negi: None. P.Yang: None. J.K.Lee: None. M.Moulik: None. V.Yechoor: n/a.

The transcriptional factor, Tead1, mediates the transcriptional output of the Hippo pathway. Tead1 activates the transcription of downstream genes including many cell cycle promoting genes that lead to cell proliferation in many tissues, but its role in β-cells is unknown. To test if Tead1 is required for β-cell proliferation and function, we deleted Tead1 in β-cell. Deletion of Tead1 in β-cells was confirmed in these β-cell Tead1 KO (β-Tead1-/-) , by a significant decrease in transcript and protein from whole islet lysates. The β-Tead1-/- mice had normal body weight but developed significantly higher fasting and fed glucose levels starting at 5 weeks of age and progressed to frank diabetes by 8 weeks, with fasting blood glucose >300 mg/dl, accompanied by hypoinsulinemia, as compared to floxed control mice. 8wk β-Tead1-/- mice display severe glucose intolerance due to an abrogation of GSIS during GTT, with a loss of first phase insulin secretion. Pancreatic insulin content was decreased by ∼50%, due to a decrease in expression of mature β-cell genes including, Pdx1, Nkx6.1 and MafA, all of which, we show to be direct transcriptional targets of Tead1. Tead1 also binds to the proximal promoter at the Ink4a locus to regulate p16/p19, critical cell cycle inhibitors, and this loss of activation in Tead1-deficient β-cells leads to an enhanced entry into cell cycle. Furthermore, to test if Tead1 has a similar regulatory role in human β-cells, we deleted TEAD1 knockout in human iPS cell-derived β-cells using CRISPR/Cas9 system. Tead1 deficient iPS cell-derived β-cells displayed a significant decrease in vitro GSIS. Our results demonstrate Tead1 to be a transcriptional switch required for β-cells to maintain mature function and remain in proliferative quiescence. This novel regulatory pathway could be targeted for β-cell replacement therapy to achieve β-cell proliferation without a loss of mature function. Disclosure J. Lee: None. V. Yechoor: n/a. R. Liu: None. F. Li: None. V. Negi: None. R. Jagannathan: None. M. Huising: Consultant; Crinetics Pharmaceuticals, Inc., Research Support; Crinetics Pharmaceuticals, Inc. K. Ma: None. B. Shih: None. M. Moulik: None. Funding VA-MERIT Grant (I01BX002678)

Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that Tead1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function, and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, although its role in cardiomyocytes is unknown. Here, we show that Tead1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle, and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss of function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that Tead1 regulation of OXPHOS in cardiomyocytes is cell autonomous. Taken together, our data demonstrate that Tead1 is a crucial transcriptional node that is a cell-autonomous regulator, a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis. NEW & NOTEWORTHY Mitochondrial dysfunction constitutes an important aspect of heart failure etiopathogenesis and progression. However, the molecular mechanisms are still largely unknown. Growing lines of evidence indicate that Hippo-Tead pathway plays a role in cellular bioenergetics. This study reveals the novel role of Tead1, the downstream transcriptional effector of Hippo pathway, as a novel regulator of mitochondrial oxidative phosphorylation and in vivo cardiomyocyte energy metabolism, thus providing a potential therapeutic target for modulating mitochondrial function and enhancing cytoprotection of cardiomyocytes.

The adipogenic progenitor transformation into mature adipocytes requires dissolution of intracellular actin cytoskeleton, and the cytoskeleton-MRTF/SRF signaling blocks adipogenesis. The circadian clock confers temporal control in metabolism, with tissue-intrinsic clock circuits contributing to metabolic homeostasis. In the current study, we identify a novel clock-MRTF/SRF regulatory axis that suppresses beige adipogenesis that is required for whole-body glucose metabolism. Key components of the cytoskeleton-MRTF/SRF signaling cascade display circadian oscillations in beige fat depot mediated by Bmal1 transcriptional activity. Genetic loss- or gain-of-functions of Bmal1 in adipogenic precursors markedly altered actin cytoskeleton organization, with silencing of Bmal1 inhibiting F-actin formation and MRTF/SRF activity whereas its forced expression augmenting actin architecture. Furthermore, we show that Bmal1 circadian control of the MRTF/SRF pathway drives beige thermogenic capacity in vivo. Prrx1-Cre-mediated beige fat-selective Bmal1 ablation induced beige depot browning with augmented mitochondrial metabolism, resulting in improved whole-body insulin sensitivity and resistance to obesity. Conversely, beige fat-specific overexpression of Bmal1 suppressed beige thermogenic program leading to impaired glucose tolerance and adipose expansion. Mechanistically, we show that Bmal1 deficiency enhanced beige precursor differentiation and thermogenic induction. Collectively, our findings uncover a temporal regulation of the cytoskeleton-MRTF/SRF signaling that modulates beige thermogenic capacity to maintain metabolic homeostasis. Disclosure X. Xiong: None. R. Liu: None. V. Yechoor: None. P. Saha: None. K. Ma: None. Funding National Institutes of Health (DK112794, DK097160-01); American Heart Association (17GRNT33370012)

Cytokine induced β-cell apoptosis is the major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding 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 NOD (non-obese diabetes) mouse model of T1D. However, the role of BET proteins in 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 and preserved β-cell mass, suggesting a cytoprotective function of I-BET. Furthermore, human islets treated with I-BET displayed better glucose stimulated insulin secretion compared to controls, when exposed to cytokines. Mechanistically, RNA-Seq analysis revealed I-BET treatment suppressed pathways involved in apoptosis, including NF-kB signaling, 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 may have potential therapeutic values in T1D.

Diabetes is one of the most prevalent disease in the United States, affecting 9% of the population, which is expected to increase by 54% by 2030. It occurs due to the body’s inability to make or respond to insulin, leading to hyperglycemia and significant risk of chronic complications. Pre-clinical investigations have reported the beneficial effect of an anti-cancer drug, I-BET (inhibitor of bromodomain and extra-terminal protein) on β-cell function but the underlying mechanisms remain unknown. As apoptosis of β-cells is a characteristic event in both T1D and T2D, we were interested in determining the effect of I-BET762 on inflammation induced β-cell apoptosis. In the present study, we found that I-BET was able to reduce the inflammation induced apoptosis in insulinoma cell line (Ins1); as determined by Annexin V-PI staining using flow cytometry (Figure 1) and cleaved caspase-3 quantitation using western blot. In addition, we also found that I-BET was able to reduce hyperglycemia in vivo in the low dose STZ (streptozotocin) model of T1D. This anti-apoptotic effect of I-BET is mediated via a reduction in the cytokine-induced activation of NF-kB and STAT1 signaling. In conclusion, our results indicate that inhibition of bromodomain proteins could be a therapeutic strategy in all forms of diabetes by protecting β-cells from cytokine-induced apoptosis. Disclosure V. Negi: None. J.K. Lee: None. R. Liu: None. R. Jagannathan: None. F. Li: None. P. Yang: None. M. Perez-Garcia: None. M. Moulik,: None. V. Yechoor: None. Funding National Institutes of Health (R01DK097160)

SummaryThe low proliferative rate of adult β-cells presents a challenge for diabetes therapy to increase β-cell numbers while maintaining mature function. Although proliferative quiescence in β-cells is required to maintain functional competence, exemplified by glucose stimulated insulin secretion, the molecular underpinnings of this reciprocal relationship remain enigmatic. 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. We propose that Tead1 is an essential intrinsic molecular switch coordinating adult β-cell proliferative quiescence with mature identity and functional competence to maintain glucose homeostasis.

The low proliferative rate of adult β-cells presents a challenge for diabetes therapy to increase β-cell numbers while maintaining mature function. Although proliferative quiescence in β-cells is required to maintain functional competence, exemplified by glucose stimulated insulin secretion, the molecular underpinnings of this reciprocal relationship remain enigmatic. 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. We propose that Tead1 is an essential intrinsic molecular switch coordinating adult β-cell proliferative quiescence with mature identity and functional competence to maintain glucose homeostasis.

Background: Gene expression is determined by structural interactions in between transcription factors, cofactors and enhancer elements, as well as enhancer-promoter interactions (1). Both YY1 and CTCF are essential, zinc finger proteins that bind hypo-methylated DNA sequences, form homodimers, and thus facilitate DNA loop formation (1). ​However, YY1 preferentially occupies interacting enhancers and promoters, whereas CTCF preferentially occupies sites distal from these regulatory elements, forming larger loops and participating in insulation (1). A sequencing study of spontaneous functional insulinomas in a Chinese cohort identified a somatic a hotspot mutation in YY1 (c.C1115G/p.T372R) in 30% of the cases, associated with increased YY1 activity (2). YY1 is a critical transcription factor involved in the regulation of proliferation and metabolism (2). Hypothesis: YY1 loss-of-function alters energy source preference in pancreatic β-cells. Methods: YY1 stable loss-of-function in mouse insulinoma cell lines was achieved by shRNA lentiviral transduction. Mitochondrial membrane potential (MMP) was measured via flow cytometry of aggregated mitochondria to monomeric mitochondria ratio. Mitostress and complex-substrate controlled respiration were measured by Seahorse analyzer. Mitochondrial copy number was assessed by mitochondrial to nuclear DNA ratio. Quantitative qPCR and Western blotting were used to assess mitochondrial gene and protein expression. Results: Our data indicated that YY1 deficient β-cells showed increased MMP and maximal respiration. No significant differences were found in basal respiration, ATP production, proton leak, non-mitochondrial oxygen consumption or coupling efficiency. We also found that YY1 deficient β-cells exhibited reduced glycolytic capacity and decreased ETC complex IV activity, with concurrent increased complex I and II activity. In addition, YY1 deficient β-cells exhibited elevated mitochondrial copy number​ and increased quantitative mRNA of mitochondrial gene expression, which could be correlated with increased PGC1-α expression. Conclusions: YY1 is critical in the metabolic regulation of β-cells, particularly in the facilitation of glycolytic metabolism. YY1 activating mutations in functional spontaneous insulinoma cells can lead to a proliferation dysregulation accompanied by a metabolic switch that favors glycolysis, while the opposite occurs in YY1 deficient β-cells. References: (1) Weintraub AS et al, Cell 2017 Dec 14; 171:1573–1588 (2) Cao Y et al, Nat Commun 2013 Dec 10; 4:2810

The SRF/MRTF and upstream signaling cascade play key roles in actin cytoskeleton organization and myocyte development. To date, how this signaling axis may function in brown adipocyte lineage commitment and maturation has not been delineated. Here we report that MRTF-SRF signaling exerts inhibitory actions on brown adipogenesis, and suppressing this negative regulation promotes brown adipocyte lineage development. During brown adipogenic differentiation, protein expressions of SRF, MRTFA/B and its transcription targets were down-regulated, and MRTFA/B shuttled from nucleus to cytoplasm. Silencing of SRF or MRTF-A/MRTF-B enhanced two distinct stages of brown adipocyte development, mesenchymal stem cell determination to brown adipocytes and terminal differentiation of brown adipogenic progenitors. We further demonstrate that the MRTF-SRF axis exerts transcriptional regulations of the TGF-β and BMP signaling pathway, critical developmental cues for brown adipocyte development. TGF-β signaling activity was significantly attenuated, whereas that of the BMP pathway augmented by inhibition of SRF or MRTF-A/MRTF-B, leading to enhanced brown adipocyte differentiation. Our study demonstrates the MRTF-SRF transcriptional cascade as a negative regulator of brown adipogenesis, through its transcriptional control of the TGF-β/BMP signaling pathways.

Introduction: Mammalian cardiomyocyte (CM) proliferation peaks in the embryonic and neonatal periods. TEAD1, a key transcription factor regulated by the Hippo pathway, is critical for early embryonic CM proliferation. But mid gestation lethality of Tead1 germline deletion precluded the study of its role in CMs at later developmental stages. We recently generated Tead1 floxed (Tead1 F/F ) mice which allows the study of TEAD1 function in CMs at later stages. The objective of this study was to determine requirement of TEAD1 for neonatal CM proliferation. Hypothesis: TEAD1 remains critical for CM proliferation in late embryonic and early neonatal periods through transcriptional regulation of cell cycle promoting genes. Methods and Results: We observed that TEAD1 cardiac expression peaks in the perinatal period. Using Myh6-Cre deletor mice, we knocked out Tead1 in CMs at E10.5 (referred as cKO). cKO pups were born in expected Mendelian frequency, but survived only till day of life (DOL) 9. Systolic dysfunction was evident by ECHO in DOL1 cKO pups and progressed to frank heart failure (HF) by DOL9. Histological exam showed decreased myocardial mass with increased intercellular fibrosis. Ventricles of DOL1 cKO pups demonstrated increased expression of Acta1, Nppa, and Nppb, consistent with HF but showed decreased expression of Myh7, suggesting an impairment in the typical fetal gene program activated in HF. Myocardial immunostaining showed reduction in Ki67 (G1/S/G2/M phase marker) (Fig 1) and PH3-S10 (M phase marker) positive CMs by 82% and 46% respectively in DOL1 cKO hearts, indicating significantly reduced CM proliferation. The expression of essential cell cycle proteins showed a significant decrease in the levels of G1/S regulating proteins, CDK4, CDK6, ppRB S807/811 and S/G2 and G2/M regulating proteins, pWEE1 S642 and Cyclin B1 in cKO hearts (Fig 2). Similar results in ex vivo and in vitro Tead1 knockout models in CMs using neonatal Tead1 F/F CMs and HL1 cells validated the cell autonomous regulation of CM cell cycle by TEAD1. Conclusions: TEAD1 is required for embryonic and neonatal CM proliferation and its loss at mid gestation leads to neonatal HF associated with impaired fetal gene program activation and decreased expression of cell cycle promoting genes.

Islet transplantation for type 1 diabetes is limited by a shortage of donor islets and requirement for immunosuppression. We approached this problem by inducing in vivo islet neogenesis in non-obese diabetic (NOD) diabetic mice, a model of autoimmune diabetes. We demonstrate that gene therapy with helper-dependent adenovirus carrying neurogenin3 (Ngn3), an islet lineage-defining transcription factor, and betacellulin (Btc), an islet growth factor, leads to the induction of periportal insulin-positive cell clusters in the liver, which are rapidly destroyed. To specifically accord protection to these 'neo-islets' from cytokine-mediated destruction, we overexpressed suppressor of cytokine signaling 1 (SOCS1) gene, using a rat insulin promoter in combination with Ngn3 and Btc. With this approach, about half of diabetic mice attained euglycemia sustained for over 4 months, regain glucose tolerance and appropriate glucose-stimulated insulin secretion. Histological analysis revealed periportal islet hormone-expressing 'neo-islets' in treated mouse livers. Despite evidence of persistent 'insulitis' with activated T cells, these 'neo-islets' persist to maintain euglycemia. This therapy does not affect diabetogenicity of splenocytes, as they retain the ability to transfer diabetes. This study thus provides a proof-of-concept for engineering in vivo islet neogenesis with targeted resistance to cytokine-mediated destruction to provide a long-term reversal of diabetes in NOD mice.

Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM-specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell-derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.

Bile acid receptor FXR (farnesoid X receptor) is a key regulator of hepatic bile acid, glucose and lipid homeostasis through regulation of numerous genes involved in the process of bile acid, triglyceride and glucose metabolism. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains and acts primarily as a co-repressor of many nuclear receptors. Here, we demonstrated that DAX1 is co-localized with FXR in the nucleus and acted as a negative regulator of FXR through a physical interaction with FXR. Our study showed that over-expression of DAX1 down-regulated the expression of FXR target genes, whereas knockdown of DAX1 led to their up-regulation. Furthermore, three LXXLL motifs in the N-terminus of DAX1 were required for the full repression of FXR transactivation. In addition, our study characterized that DAX1 suppresses FXR transactivation via competing with co-activators such as SRC-1 and PGC-1α. In conclusion, DAX1 acts as a co-repressor to negatively modulate FXR transactivity.

The epididymis and efferent ducts play major roles in sperm maturation, transport, concentration and storage by reabsorbing water, ions and proteins produced from seminiferous tubules. Gpr48-null male mice demonstrate reproductive tract defects and infertility. In the present study, we found that estrogen receptor alpha (ERalpha) was dramatically reduced in the epididymis and efferent ducts in Gpr48-null male mice. We further revealed that ERalpha could be upregulated by Gpr48 activation via the cAMP/PKA signaling pathway. Moreover, we identified a cAMP responsive element (Cre) motif located at -1307 to -1300 bp in the ERalpha promoter that is able to interact with Cre binding protein (Creb). In conclusion, Gpr48 participates in the development of the male epididymis and efferent ducts through regulation of ERalpha expression via the cAMP/PKA signaling pathway.

Circadian disruption is the bane of modern existence and its deleterious effects on health; in particular, diabetes and metabolic syndrome have been well recognized in shift workers. Recent human studies strongly implicate a 'dose-dependent' relationship between circadian disruption and diabetes. Genetic and environmental disruption of the circadian clock in rodents leads to diabetes secondary to β-cell failure. Deletion of Bmal1, a non-redundant core clock gene, leads to defects in β-cell stimulus-secretion coupling, decreased glucose-stimulated ATP production, uncoupling of OXPHOS and impaired glucose-stimulated insulin secretion. Both genetic and environmental circadian disruptions are sufficient to induce oxidative stress and this is mediated by a disruption of the direct transcriptional control of the core molecular clock and Bmal1 on Nrf2, the master antioxidant transcription factor in the β-cell. In addition, circadian disruption also leads to a dysregulation of the unfolded protein response and leads to endoplasmic reticulum stress in β-cells. Both the oxidative and endoplasmic reticulum (ER) stress contribute to an impairment of mitochondrial function and β-cell failure. Understanding the basis of the circadian control of these adaptive stress responses offers hope to target them for pharmacological modulation to prevent and mitigate the deleterious metabolic consequences of circadian disruption.

Brown adipose tissue is a major thermogenic organ that plays a key role in maintenance of body temperature and whole-body energy homeostasis. Rev-erbα, a ligand-dependent nuclear receptor and transcription repressor of the molecular clock, has been implicated in the regulation of adipogenesis. However, whether Rev-erbα participates in brown fat formation is not known. Here we show that Rev-erbα is a key regulator of brown adipose tissue development by promoting brown adipogenesis. Genetic ablation of Rev-erbα in mice severely impairs embryonic and neonatal brown fat formation accompanied by loss of brown identity. This defect is due to a cell-autonomous function of Rev-erbα in brown adipocyte lineage commitment and terminal differentiation, as demonstrated by genetic loss- and gain-of-function studies in mesenchymal precursors and brown preadipocytes. Moreover, pharmacological activation of Rev-erbα activity promotes, whereas its inhibition suppresses brown adipocyte differentiation. Mechanistic investigations reveal that Rev-erbα represses key components of the TGF-β cascade, an inhibitory pathway of brown fat development. Collectively, our findings delineate a novel role of Rev-erbα in driving brown adipocyte development and provide experimental evidence that pharmacological interventions of Rev-erbα may offer new avenues for the treatment of obesity and related metabolic disorders.

DAX1 (for dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1; also called NROB1) mutations are responsible for adrenal failure and hypogonadotropic hypogonadism in patients with adrenal hypoplasia congenita (AHC), through a loss of trans-repression of SF-1 (for steroidogenic factor-1)-mediated StAR (for steroidogenic acute regulatory protein) and LHbeta transcriptional activities and a reduction of GnRH expression. The correlation of clinical features with genetic and functional alterations of the gene was investigated in detail in AHC patients.The present study aimed at identifying DAX1 mutations in Chinese AHC patients and investigating the functional defects of detected novel mutations.Nine patients with AHC were recruited from eight families. DAX1 mutations were screened, and the transcriptional activities of the identified mutations were assessed in vitro.DAX1 mutations were detected in all nine patients enrolled in the study, with eight different mutations. Among the latter, seven are novel mutations, including two missense (L262P and C368F), one nonsense (Q222X), and four frame-shift (637delC, 652_653delAC, 973delC, and 774_775insCC) mutations. The functional studies showed that the mutant DAX1 was impaired by nuclear localization, loss of trans-repression of StAR and LHbeta transcriptional activities, and reduction of GnRH expression.These findings provide insight into the molecular events by which DAX1 mutations influence the hypothalamus-pituitary-gonadal and hypothalamus-pituitary-adrenal axis and lead to AHC and hypogonadotropic hypogonadism.