The Krüppel-like zinc finger transcription factor Gli-similar 3 (GLIS3) plays a critical role in the regulation of pancreatic beta cells, with global Glis3-knockout mice suffering from severe hyperglycemia and dying by post-natal day 11. In addition, GLIS3 has been shown to directly regulate the early endocrine marker Ngn3, as well as Ins2 gene expression in mature beta cells. We hypothesize that GLIS3 regulates several other genes critical to beta cell function, in addition to Ins2, by directly binding to regulatory regions. We therefore generated a pancreas-specific Glis3 deletion mouse model (Glis3 Δ panc) using a Pdx1-driven Cre mouse line. Roughly 20% of these mice develop hyperglycemia by 8 weeks and lose most of their insulin expression. However, this did not appear to be due to loss of the beta cells themselves, as no change in cell death was observed. Indeed, presumptive beta cells appeared to persist as PDX1+/INS−/MAFA−/GLUT2− cells. Islet RNA-seq analysis combined with GLIS3 ChIP-seq analysis revealed apparent direct regulation of a variety of diabetes-related genes, including Slc2a2 and Mafa. GLIS3 binding near these genes coincided with binding for other islet-enriched transcription factors, indicating these are distinct regulatory hubs. Our data indicate that GLIS3 regulates not only insulin expression, but also several additional genes critical for beta cell function.
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David W Scoville, Kristin Lichti-Kaiser, Sara A Grimm and Anton M Jetten
Jessica L Pierce, Ke-Hong Ding, Jianrui Xu, Anuj K Sharma, Kanglun Yu, Natalia del Mazo Arbona, Zuleika Rodríguez-Santos, Paul J Bernard, Wendy B Bollag, Maribeth H Johnson, Mark W Hamrick, Dana L Begun, Xing-Ming Shi, Carlos M Isales and Meghan E McGee-Lawrence
Excess fat within bone marrow is associated with lower bone density. Metabolic stressors such as chronic caloric restriction (CR) can exacerbate marrow adiposity, and increased glucocorticoid signaling and adrenergic signaling are implicated in this phenotype. The current study tested the role of glucocorticoid signaling in CR-induced stress by conditionally deleting the glucocorticoid receptor (Nr3c1; hereafter abbreviated as GR) in bone marrow osteoprogenitors (Osx1-Cre) of mice subjected to CR and ad libitum diets. Conditional knockout of the GR (GR-CKO) reduced cortical and trabecular bone mass as compared to WT mice under both ad libitum feeding and CR conditions. No interaction was detected between genotype and diet, suggesting that the GR is not required for CR-induced skeletal changes. The lower bone mass in GR-CKO mice, and the further decrease in bone by CR, resulted from suppressed bone formation. Interestingly, treatment with the β-adrenergic receptor antagonist propranolol mildly but selectively improved metrics of cortical bone mass in GR-CKO mice during CR, suggesting interaction between adrenergic and glucocorticoid signaling pathways that affects cortical bone. GR-CKO mice dramatically increased marrow fat under both ad libitum and CR-fed conditions, and surprisingly propranolol treatment was unable to rescue CR-induced marrow fat in either WT or GR-CKO mice. Additionally, serum corticosterone levels were selectively elevated in GR-CKO mice with CR, suggesting the possibility of bone–hypothalamus–pituitary–adrenal crosstalk during metabolic stress. This work highlights the complexities of glucocorticoid and β-adrenergic signaling in stress-induced changes in bone mass, and the importance of GR function in suppressing marrow adipogenesis while maintaining healthy bone mass.
Anna E Bollag, Tianyang Guo, Ke-Hong Ding, Vivek Choudhary, Xunsheng Chen, Qing Zhong, Jianrui Xu, Kanglun Yu, Mohamed E Awad, Mohammed Elsalanty, Maribeth H Johnson, Meghan E McGee-Lawrence, Wendy B Bollag and Carlos M Isales
Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than three-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (NRF2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated NRF2 and induced anti-oxidant NRF2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters were monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.
Kasper Faarkrog Høyer, Christoffer Laustsen, Steffen Ringgaard, Haiyun Qi, Christian Østergaard Mariager, Thomas Svava Nielsen, Ulrik Kræmer Sundekilde, Jonas T Treebak, Niels Jessen and Hans Stødkilde-Jørgensen
Hyperpolarized [1-13C]pyruvate magnetic resonance (MR) spectroscopy has the unique ability to detect real-time metabolic changes in vivo owing to its high sensitivity compared with thermal MR and high specificity compared with other metabolic imaging methods. The aim of this study was to explore the potential of hyperpolarized MR spectroscopy for quantification of liver pyruvate metabolism during a hyperinsulinemic–isoglycemic clamp in mice. Hyperpolarized [1-13C]pyruvate was used for in vivo MR spectroscopy of liver pyruvate metabolism in mice. Mice were divided into two groups: (i) non-stimulated 5-h fasted mice and (ii) hyperinsulinemic-isoglycemic clamped mice. During clamp conditions, insulin and donor blood were administered at a constant rate, whereas glucose was infused to maintain isoglycemia. When steady state was reached, insulin-stimulated mice were rapidly infused with hyperpolarized [1-13C]pyruvate for real-time tracking of the dynamic distribution of metabolic derivatives from pyruvate, such as [1-13C]lactate, [1-13C]alanine and [13C]bicarbonate. Isotopomer analysis of plasma glucose confirmed 13C-incorporation from [1-13C]pyruvate into glucose was increased in fasted mice compared with insulin-stimulated mice, demonstrating an increased gluconeogenesis in fasted mice. The AUC ratios for [1-13C]alanine/[1-13C]pyruvate (38.2%), [1-13C]lactate/[1-13C]pyruvate (41.8%) and [13C]bicarbonate/[1-13C]pyruvate (169%) all increased significantly during insulin stimulation. Hyperpolarized [1-13C]pyruvate can be used for in vivo MR spectroscopy of liver pyruvate metabolism during hyperinsulinemic-isoglycemic clamp conditions. Under these conditions, insulin decreased gluconeogenesis and increased [1-13C]alanine, [1-13C]lactate and [13C]bicarbonate after a [1-13C]pyruvate bolus. This application of in vivo spectroscopy has the potential to identify impairments in specific metabolic pathways in the liver associated with obesity, insulin resistance and nonalcoholic fatty liver disease.
Aran Son, Namju Kang, Sue Young Oh, Ki Woo Kim, Shmuel Muallem, Yu-Mi Yang and Dong Min Shin
The receptor activator of nuclear factor-kappa B ligand (RANKL) induces osteoclastogenesis by induction of Ca2+ oscillation, calcineurin activation and translocation into the nucleus of nuclear factor of activated T cells type c1 (NFATc1). Homer proteins are scaffold proteins. They regulate Ca2+ signaling by modulating the activity of multiple Ca2+ signaling proteins. Homers 2 and 3, but not Homer1, also independently affect the interaction between NFATc1 and calcineurin. However, to date, whether and how the Homers are involved in osteoclastogenesis remains unknown. In the present study, we investigated Homer2 and Homer3 roles in Ca2+ signaling and NFATc1 function during osteoclast differentiation. Deletion of Homer2/Homer3 (Homer2/3) markedly decreased the bone density of the tibia, resulting in bone erosion. RANKL-induced osteoclast differentiation is greatly facilitated in Homer2/3 DKO bone marrow-derived monocytes/macrophages (BMMs) due to increased NFATc1 expression and nuclear translocation. However, these findings did not alter RANKL-induced Ca2+ oscillations. Of note, RANKL treatment inhibited Homer proteins interaction with NFATc1, but it was restored by cyclosporine A treatment to inhibit calcineurin. Finally, RANKL treatment of Homer2/3 DKO BMMs significantly increased the formation of multinucleated cells. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation. Specifically, we found that Homer2 and Homer3 regulate NFATc1 function through its interaction with calcineurin to regulate RANKL-induced osteoclastogenesis and bone metabolism.
Wu Luo, Lan Huang, Jingying Wang, Fei Zhuang, Zheng Xu, Haimin Yin, Yuanyuan Qian, Guang Liang, Chao Zheng and Yi Wang
Emerging evidence implicates elevated activity of STAT3 transcription factor in driving the development and progression of diabetic cardiomyopathy (DCM). We hypothesized that the fibrosis-promoting and hypertrophic actions of STAT3 are linked to the activation by epidermal growth factor receptor (EGFR). We tested this hypothesis by challenging cultured cardiomyocytes to high-concentration glucose and heart tissues of streptozotocin (STZ)-induced type 1 diabetic mice. Our results indicated that, in diabetic mice, the blockade of STAT3 or EGFR using selective inhibitors S3I-201 and erlotinib, respectively, abrogated the increased activating STAT3 phosphorylation and the induction of genes regulating fibrosis and hypertrophy in myocardial tissue. S3I-201 and erlotinib significantly reduced myocardial structural and functional deficits in diabetic mice. In cultured cardiomyocytes, high-concentration glucose induced EGFR-mediated STAT3 phosphorylation. We further showed that blockade of STAT3 or EGFR using selective inhibitors and siRNAs significantly reduced the increased expression of genes known to promote fibrosis and hypertrophy in cardiomyocytes. These results provide novel evidence that the EGFR-STAT3 signaling axis likely plays a crucial role in the development and progression of DCM.
Xiaoning Li, Junhua Xiao, Yating Fan, Kan Yang, Kai Li, Xin Wang, Yanhua Lu and Yuxun Zhou
Gonadotropin-releasing hormone (GnRH) is the ultimate signal by which the neuroendocrine system controls the puberty onset and fertility in mammals. The pulsatile release of GnRH is regulated by numerous extracellular and intracellular factors, including miRNAs. Here, we report a novel regulation mechanism mediated by miR-29 family. We found that the absence of miR-29s resulted in elevated expression of Gnrh1 in GT1-7 cells. Through in silico and wet analysis, we identified Tbx21, a target gene of miR-29, as the main effector. As a transcription activator, TBX21 stimulates the expression of Gnrh1 directly by binding to its promoter region, and indirectly by activating the expression of Dlx1, another transcription activator of Gnrh1. Stereotactic brain infusion of miR-29 inhibitor into the hypothalamus caused earlier puberty onset in prepubertal female mice than that of intact controls. The female mice with ectopic expression of Tbx21 in the hypothalamus were affected in both puberty onset and fertility, as they had higher level of serum LH and FSH, larger litter size but steeper decline of fertility compared with those of controls. Our results revealed that miR-29-3p and its target Tbx21 played a role in regulating the mammalian puberty onset and reproduction by modulating the Gnrh1 expression.
Kelly De Sousa, Alaa B Abdellatif, Rami M El Zein and Maria-Christina Zennaro
Primary aldosteronism (PA) is the most common form and an under-diagnosed cause of secondary arterial hypertension, accounting for up to 10% of hypertensive cases and associated to increased cardiovascular risk. PA is caused by autonomous overproduction of aldosterone by the adrenal cortex. It is mainly caused by a unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. Excess aldosterone leads to arterial hypertension with suppressed renin, frequently associated to hypokalemia. Mutations in genes coding for ion channels and ATPases have been identified in APA, explaining the pathophysiology of increased aldosterone production. Different inherited genetic abnormalities led to the distinction of four forms of familial hyperaldosteronism (type I to IV) and other genetic defects very likely remain to be identified. Somatic mutations are identified in APA, but also in aldosterone-producing cell clusters (APCCs) in normal adrenals, in image-negative unilateral hyperplasia, in transitional lesions and in APCC from adrenals with bilateral adrenal hyperplasia (BAH). Whether these structures are precursors of APA or whether somatic mutations occur in a proliferative adrenal cortex, is still a matter of debate. This review will summarize our knowledge on the molecular mechanisms responsible for PA and the recent discovery of new genes related to early-onset and familial forms of the disease. We will also address new issues concerning genomic and proteomic changes in adrenals with APA and discuss adrenal pathophysiology in relation to aldosterone-producing structures in the adrenal cortex.
Bo He, Yinxian Wen, Shuwei Hu, Guihua Wang, Wen Hu, Jacques Magdalou, Liaobin Chen and Hui Wang
We previously showed that prenatal caffeine exposure (PCE) induces intrauterine growth retardation (IUGR) and high susceptibility to nonalcoholic fatty liver disease in offspring rats, and the underlying mechanisms are associated with fetal overexposure to maternal glucocorticoids. Herein, we aimed to verify whether PCE disrupts liver development before and after birth and explore its possible programming mechanism. In vivo, reduced fetal weights and increased IUGR rates were accompanied by fetal liver developmental dysfunction in PCE rats. Increased fetal serum corticosterone and decreased insulin-like growth factor 1 (IGF1) levels were observed. Both male and female fetal livers exhibited increased glucocorticoid function-related gene (Gr/C/ebpα) expression and inhibited IGF1 signaling pathway (Igf1/Igf1r/Akt2) expression. At PW6, the levels of serum corticosterone and glucocorticoid function-related genes in PCE offspring livers were decreased, while serum IGF1 and liver IGF1 signaling pathway expression were increased, accompanied by obvious catch-up growth and enhanced liver function. Furthermore, in PCE adult offspring under chronic stress, serum corticosterone and liver Gr/C/ebpα expression levels were elevated, while the serum IGF1 and liver IGF1 signaling pathway levels were decreased. In vitro, cortisol (not caffeine) upregulated GR and C/EBPα expression and downregulated IGF1R expression. The IGF1R expression downregulated by cortisol was partially reversed by GR or C/EBPα knockdown. In conclusion, PCE-induced liver developmental dysfunction in fetal rats and catch-up growth in IUGR offspring. The mechanisms may be closely associated with GR/C/EBPα upregulation and IGF1/IGF1R signaling pathway downregulation in the fetal liver, caused by intrauterine programming of the liver glucocorticoid–IGF1 axis induced by glucocorticoid overexposure.
Ioannis Simitsidellis, Arantza Esnal-Zuffiaure, Olympia Kelepouri, Elisabeth O’Flaherty, Douglas A Gibson and Philippa T K Saunders
Selective androgen receptor modulators (SARMs) have been proposed as therapeutics for women suffering from breast cancer, muscle wasting or urinary incontinence. The androgen receptor (AR) is expressed in the uterus but the impact of SARMs on the function of this organ is unknown. We used a mouse model to compare the impact of SARMs (GTx-007/Andarine®, GTx-024/Enobosarm®), Danazol (a synthetic androstane steroid) and dihydrotestosterone (DHT) on tissue architecture, cell proliferation and gene expression. Ovariectomised mice were treated daily for 7 days with compound or vehicle control (VC). Uterine morphometric characteristics were quantified using high-throughput image analysis (StrataQuest; TissueGnostics), protein and gene expression were evaluated by immunohistochemistry and RT-qPCR, respectively. Treatment with GTx-024, Danazol or DHT induced significant increases in body weight, uterine weight and the surface area of the endometrial stromal and epithelial compartments compared to VC. Treatment with GTx-007 had no impact on these parameters. GTx-024, Danazol and DHT all significantly increased the percentage of Ki67-positive cells in the stroma, but only GTx-024 had an impact on epithelial cell proliferation. GTx-007 significantly increased uterine expression of Wnt4 and Wnt7a, whereas GTx-024 and Danazol decreased their expression. In summary, the impact of GTx-024 and Danazol on uterine cells mirrored that of DHT, whereas GTx-007 had minimal impact on the tested parameters. This study has identified endpoints that have revealed differences in the effects of SARMs on uterine tissue and provides a template for preclinical studies comparing the impact of compounds targeting the AR on endometrial function.