Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.
Lydie Naulé, Alessandra Mancini, Sidney A. Pereira, Brandon M. Gassaway, John R. Lydeard, John C. Magnotto, Han Kyeol Kim, Joy Liang, Cynara Matos, Steven P. Gygi, Florian T. Merkle, Rona S. Carroll, Ana Paula Abreu, Ursula B. Kaiser
Metabolic crosstalk from skeletal muscle to multiple organs is important for maintaining homeostasis, and its dysregulation can lead to various diseases. Chronic glucocorticoid administration often induces muscle atrophy and metabolic disorders such as diabetes and central obesity; however, the detailed underlying mechanism remains unclear. We previously reported that the deletion of glucocorticoid receptor (GR) in skeletal muscle increases muscle mass and reduces fat mass through muscle–liver–fat communication under physiological conditions. In this study, we show that muscle GR signaling plays a crucial role in accelerating obesity through the induction of hyperinsulinemia. Fat accumulation in liver and adipose tissue, muscle atrophy, hyperglycemia, and hyperinsulinemia induced by chronic corticosterone (CORT) treatment improved in muscle-specific GR knockout (GRmKO) mice. Such CORT-induced fat accumulation was alleviated by suppressing insulin production (streptozotocin injection), indicating that hyperinsulinemia enhanced by muscle GR signaling promotes obesity. Strikingly, glucose intolerance and obesity in ob/ob mice without CORT treatment were also improved in GRmKO mice, indicating that muscle GR signaling contributes to obesity-related metabolic changes, regardless of systemic glucocorticoid levels. Thus, this study provides new insight for the treatment of obesity and diabetes by targeting muscle GR signaling.
Hiroki Yamazaki, Masaaki Uehara, Noritada Yoshikawa, Akiko Kuribara-Souta, Motohisa Yamamoto, Yasuko Hirakawa, Yasuaki Kabe, Makoto Suematsu, Hirotoshi Tanaka
Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified L-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor (LepR)-expressing neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1–mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.
Gyujin Park, Kazuya Fukasawa, Tetsuhiro Horie, Yusuke Masuo, Yuka Inaba, Takanori Tatsuno, Takanori Yamada, Kazuya Tokumura, Sayuki Iwahashi, Takashi Iezaki, Katsuyuki Kaneda, Yukio Kato, Yasuhito Ishigaki, Michihiro Mieda, Tomohiro Tanaka, Kazuma Ogawa, Hiroki Ochi, Shingo Sato, Yun-Bo Shi, Hiroshi Inoue, Hojoon Lee, Eiichi Hinoi
Short-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being unclear. We aimed to investigate the role of gut microbiota in the induction of metabolic benefits of dietary butyrate. We performed antibiotic-induced microbiota depletion of the gut and fecal microbiota transplantation (FMT) in APOE*3-Leiden.CETP mice, a well-established translational model for developing human-like metabolic syndrome, and revealed that dietary butyrate reduced appetite and ameliorated high-fat diet–induced (HFD-induced) weight gain dependent on the presence of gut microbiota. FMT from butyrate-treated lean donor mice, but not butyrate-treated obese donor mice, into gut microbiota–depleted recipient mice reduced food intake, attenuated HFD-induced weight gain, and improved insulin resistance. 16S rRNA and metagenomic sequencing on cecal bacterial DNA of recipient mice implied that these effects were accompanied by the selective proliferation of Lachnospiraceae bacterium 28-4 in the gut as induced by butyrate. Collectively, our findings reveal a crucial role of gut microbiota in the beneficial metabolic effects of dietary butyrate as strongly associated with the abundance of Lachnospiraceae bacterium 28-4.
Zhuang Li, Enchen Zhou, Cong Liu, Hope Wicks, Sena Yildiz, Farhana Razack, Zhixiong Ying, Sander Kooijman, Debby P.Y. Koonen, Marieke Heijink, Sarantos Kostidis, Martin Giera, Ingrid M.J.G. Sanders, Ed J. Kuijper, Wiep Klaas Smits, Ko Willems van Dijk, Patrick C.N. Rensen, Yanan Wang
The molecular clock machinery regulates several homeostatic rhythms, including glucose metabolism. We previously demonstrated that Roux-en Y Gastric Bypass (RYGB) has a weight-independent effect on glucose homeostasis, and transiently reduces food intake. In this study we investigate the effects of RYGB on diurnal eating behavior as well as its effects on the molecular clock, and its requirement for the metabolic effects of this bariatric procedure in obese mice. We find that RYGB reverses the high fat diet-induced disruption in diurnal eating pattern during the early post-surgery phase of food reduction. “Dark-cycle” pair-feeding experiments improved glucose tolerance to the level of bypass-operated animals during the physiologic “fasting” phase (Zeitgeber ZT2), but not the “feeding” phase (ZT14). Using a clock gene reporter mouse model (mPer2Luc), we reveal that RYGB induces a liver-specific phase shift in peripheral clock oscillation with no changes to the central clock activity within the suprachiasmatic nucleus (SCN). In addition, we show that weight loss effects are attenuated in obese ClockΔ19 mutant mice post-RYGB that also fail to improve glucose metabolism after surgery, specifically hepatic glucose production. We conclude that RYGB reprograms the peripheral clock within the liver early after surgery to alter diurnal eating behavior and regulate hepatic glucose flux.
Yuanchao Ye, Marwa Abu El Haija, Reine Obeid, Hussein Herz, Liping Tian, Benjamin Linden, Yi Chu, Deng Fu Guo, Daniel C. Levine, Jonathan Cedernaes, Kamal Rahmouni, Joseph Bass, Mohamad Mokadem
The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier. Previously we showed that mineralocorticoid receptor (MR) activation caused GEnGlx damage and albuminuria. Here we investigated whether MR antagonism could limit albuminuria in diabetes and studied the site of action. Streptozotocin-induced diabetic Wistar rats developed albuminuria, increased glomerular albumin permeability (Ps’alb) and increased glomerular matrix metalloproteinase (MMP) activity with corresponding GEnGlx loss. MR antagonism prevented albuminuria progression, restored Ps’alb, preserved GEnGlx and reduced MMP activity. Enzymatic degradation of the GEnGlx negated the benefits of MR antagonism, confirming their dependence on GEnGlx integrity. Exposing human glomerular endothelial cells (GEnC) to diabetic conditions in vitro increased MMPs and caused glycocalyx damage. Amelioration of these effects confirmed a direct effect of MR antagonism on GEnC. To confirm relevance to human disease, we used a novel confocal imaging method to show loss of GEnGlx in renal biopsy specimens from patients with diabetic nephropathy (DN). In addition, DN patients randomised to receive an MR antagonist had reduced urinary MMP2 activity and albuminuria compared with placebo and baseline levels. Taken together our work suggests MR antagonists reduce MMP activity and thereby preserve GEnGlx resulting in reduced glomerular permeability and albuminuria in diabetes.
Michael Crompton, Joanne K. Ferguson, RainaD. Ramnath, Karen L. Onions, Anna S. Ogier, Monica Gamez, Colin J. Down, Laura J. Skinner, Kitty H.F. Wong, Lauren Kari Dixon, Judit Sutak, Steven J. Harper, Paola Pontrelli, Loreto Gesualdo, Hiddo L. Heerspink, Robert D. Toto, Gavin I. Welsh, Rebecca R. Foster, Simon C. Satchell, Matthew J. Butler
The main estrogen, estradiol (E2), exerts several beneficial vascular actions through estrogen receptor (ER)α in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we reported the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, that preserves smooth muscle cells (SMCs), E2, E3 and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4 action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its action independently of ERα membrane initiated signaling by contrast to E2. Consistently, RNAseq analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differ from those of E2. Thus, whereas acceleration of endothelial healing by estrogens was viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.
Morgane Davezac, Rana Zahreddine, Melissa Buscato, Natalia F. Smirnova, Chanaelle Febrissy, Henrik Laurell, Silveric Gilardi-Bresson, Marine Adlanmerini, Philippe Liere, Gilles Flouriot, Rachida Guennoun, Muriel Laffargue, Jean-Michel Foidart, Françoise Lenfant, Jean-François Arnal, Raphaël Métivier, Coralie Fontaine
The G protein–coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor–associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.
Adelaide Bernard, Irene Ojeda Naharros, Xinyu Yue, Francois Mifsud, Abbey Blake, Florence Bourgain-Guglielmetti, Jordi Ciprin, Sumei Zhang, Erin McDaid, Kellan Kim, Maxence V. Nachury, Jeremy F. Reiter, Christian Vaisse
Pathogenic SOX2 variants typically cause severe ocular defects within a SOX2-disorder spectrum that includes hypogonadotropic hypogonadism (HH). We examined exome sequencing data from a large, well-phenotyped cohort of patients (n=1453) with Idiopathic Hypogonadotropic Hypogonadism (IHH) for pathogenic SOX2 variants to investigate the underlying pathogenic SOX2 spectrum and its associated phenotypes. We identified eight IHH individuals harboring heterozygous pathogenic SOX2 variants with variable ocular phenotypes. These variant proteins were tested in vitro to determine whether a causal relationship between IHH and SOX2 exists. We found that Sox2 is highly expressed in the hypothalamus of adult mice and colocalizes with KISS1 expression in the anteroventral periventricular nucleus of adult female mice. In vitro, shRNA suppression of mouse SOX2 protein in Kiss-expressing cell lines increases the levels of human kisspeptin luciferase transcription (hKISS-luc), while SOX2 overexpression represses hKiss-luc transcription. Further, four of the identified SOX2 variants prevented this SOX2-mediated repression of hKiss-luc. Together these data suggest that pathogenic SOX2 variants contribute to both anosmic and normosmic forms of IHH attesting to hypothalamic defects in the SOX2-disorder spectrum. Our study describes novel mechanisms contributing to SOX2-related disease and highlights the necessity of SOX2 screening in IHH genetic evaluation irrespective of associated ocular defects.
Jessica Cassin, Maria I. Stamou, Kimberly W. Keefe, Kaitlin Sung, Celine Bojo, Karen J. Tonsfeldt, Rebecca A. Rojas, Vanessa Ferreira Lopes, Lacey Plummer, Kathryn B. Salnikov, David L. Keefe Jr., Metin Ozata, Myron Genel, Neoklis A. Georgopoulos, Janet E. Hall, William F. Crowley Jr., Stephanie B. Seminara, Pamela L. Mellon, Ravikumar Balasubramanian
CD4+ cytotoxic T lymphocytes (CTLs) were recently implicated in immune-mediated inflammation and fibrosis progression of Graves’ orbitopathy (GO). However, little is known about therapeutic targeting CD4+ CTLs. Herein, we studied the effect of rapamycin, an approved mTORC1 inhibitor, in GO mouse model, in vitro and in refractory GO patients. In the adenovirus-induced model, rapamycin significantly decreased the incidence of orbitopathy. This was accompanied by reduction of CD4+ CTLs, and improvement of inflammation, adipogenesis and fibrosis in orbits. CD4+CTLs from active GO patients showed upregulation of mTOR pathway, while rapamycin decreased their proportions and cytotoxic function. Low-dose rapamycin treatment substantially improved diplopia and clinical activity score in steroid-refractory GO patients. Single-cell RNA sequencing revealed that eye motility improvement was closely related to suppression of inflammation and chemotaxis in CD4+ CTLs. In conclusion, rapamycin is a promising treatment for CD4+ CTL-mediated inflammation and fibrosis in GO.
Meng Zhang, Kelvin K.L. Chong, Zi-yi Chen, Hui Guo, Yu-feng Liu, Yong-yong Kang, Yang-jun Li, Ting-ting Shi, Kenneth Ka Hei Lai, Ming-qian He, Kai Ye, George J. Kahaly, Bing-yin Shi, Yue Wang
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