The glucagon-like peptide-1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β cell and α cell function and relative volumes, and islet cell apoptosis and replication in nondiabetic nonhuman primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an l-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated, and the remnant pancreas (head-body) was harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by about 2-fold. β, α, and δ cell relative volumes in exenatide-treated baboons were significantly increased compared with saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β, α, and δ cells and produces a robust increase in insulin sensitivity in nonhuman primates.
Teresa Vanessa Fiorentino, Francesca Casiraghi, Alberto M. Davalli, Giovanna Finzi, Stefano La Rosa, Paul B. Higgins, Gregory A. Abrahamian, Alessandro Marando, Fausto Sessa, Carla Perego, Rodolfo Guardado-Mendoza, Subhash Kamath, Andrea Ricotti, Paolo Fiorina, Giuseppe Daniele, Ana M. Paez, Francesco Andreozzi, Raul A. Bastarracea, Anthony G. Comuzzie, Amalia Gastaldelli, Alberto O. Chavez, Eliana S. Di Cairano, Patrice Frost, Livio Luzi, Edward J. Dick, Glenn A. Halff, Ralph A. DeFronzo, Franco Folli
Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, β-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid β-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid β-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.
Yazmin I. Rovira Gonzalez, Adam L. Moyer, Nicolas J. LeTexier, August D. Bratti, Siyuan Feng, Congshan Sun, Ting Liu, Jyothi Mula, Pankhuri Jha, Shama R. Iyer, Richard Lovering, Brian O’Rourke, Hye Lim Noh, Sujin Suk, Jason K. Kim, George K. Essien Umanah, Kathryn R. Wagner
miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with WT mice, the allergen-challenged Mrc1–/– mice showed increased airway hyperresponsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pretransduced with adeno-associated virus–miR-511-3p (AAV–miR-511-3p). Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miR pulldown assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p–transduced macrophages and in bronchoalveolar lavage fluids of AAV–miR-511-3p–infected Mrc1–/– mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by coimmunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen–induced AHR and lung inflammation. These findings suggest a potentially novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.
Danh C. Do, Jie Mu, Xia Ke, Karan Sachdeva, Zili Qin, Mei Wan, Faoud T. Ishmael, Peisong Gao
Depletion of epithelial cells after lung injury prompts proliferation and epithelial mesenchymal transition (EMT) of progenitor cells, and this repopulates the lost epithelial layer. To investigate the cell proliferative function of human oncoprotein MDM2, we generated mouse models targeting human MDM2 expression in either lung Club or alveolar cells after doxycycline treatment. We report that MDM2 expression in lung Club or alveolar cells activates DNA replication specifically in lung progenitor cells only after chemical- or radiation-induced lung injury, irrespective of their p53 status. Activation of DNA replication by MDM2 triggered by injury leads to proliferation of lung progenitor cells and restoration of the lost epithelial layers. Mouse lung with no Mdm2 allele loses its ability to replicate DNA, whereas loss of 1 Mdm2 allele compromises this function, demonstrating the requirement of endogenous MDM2. We show that the p53-independent ability of MDM2 to activate Akt signaling is essential for initiating DNA replication in lung progenitor cells. Furthermore, MDM2 activates the Notch signaling pathway and expression of EMT markers, indicative of epithelial regeneration. This is the first report to our knowledge demonstrating a direct p53-independent participation of MDM2 in progenitor cell proliferation and epithelial repair after lung injury, distinct from a p53-degrading antiapoptotic effect preventing injury.
Shilpa Singh, Catherine A. Vaughan, Christopher Rabender, Ross Mikkelsen, Sumitra Deb, Swati Palit Deb
Epicardial adipose tissue (EAT) is the visceral fat depot of the heart. Inflammation of EAT is thought to contribute to coronary artery disease (CAD). Therefore, we hypothesized that the EAT of patients with CAD would have increased inflammatory gene expression compared with controls without CAD. Cardiac surgery patients with (n = 13) or without CAD (n = 13) were consented, and samples of EAT and subcutaneous adipose tissue (SAT) were obtained. Transcriptomic analysis was performed using Affymetrix Human Gene 1.0 ST arrays. Differential expression was defined as a 1.5-fold change (ANOVA P < 0.05). Six hundred ninety-three genes were differentially expressed between SAT and EAT in controls and 805 in cases. Expression of 326 genes was different between EAT of cases and controls; expression of 14 genes was increased in cases, while 312 were increased in controls. Quantitative reverse transcription PCR confirmed that there was no difference in expression of CCL2, CCR2, TNF-α, IL-6, IL-8, and PAI1 between groups. Immunohistochemistry showed more macrophages in EAT than SAT, but there was no difference in their number or activation state between groups. In contrast to prior studies, we did not find increased inflammatory gene expression in the EAT of patients with CAD. We conclude that the specific adipose tissue depot, rather than CAD status, is responsible for the majority of differential gene expression.
Timothy P. Fitzgibbons, Nancy Lee, Khanh-Van Tran, Sara Nicoloro, Mark Kelly, Stanley K.C. Tam, Michael P. Czech
Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti–PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1–expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.
Amber J. Giles, Shuyu Hao, Michelle Padget, Hua Song, Wei Zhang, John Lynes, Victoria Sanchez, Yang Liu, Jinkyu Jung, Xiaoyu Cao, Rika Fujii, Randy Jensen, David Gillespie, Jeffrey Schlom, Mark R. Gilbert, Edjah K. Nduom, Chunzhang Yang, John H. Lee, Patrick Soon-Shiong, James W. Hodge, Deric M. Park
Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
Jonathan Merola, Melanie Reschke, Richard W. Pierce, Lingfeng Qin, Susann Spindler, Tania Baltazar, Thomas D. Manes, Francesc Lopez-Giraldez, Guangxin Li, Laura G. Bracaglia, Catherine Xie, Nancy Kirkiles-Smith, W. Mark Saltzman, Gregory T. Tietjen, George Tellides, Jordan S. Pober
Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene–related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an “itch cocktail” augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.
Joel Castro, Andrea M. Harrington, TinaMarie Lieu, Sonia Garcia-Caraballo, Jessica Maddern, Gudrun Schober, Tracey O’Donnell, Luke Grundy, Amanda L. Lumsden, Paul Miller, Andre Ghetti, Martin S. Steinhoff, Daniel P. Poole, Xinzhong Dong, Lin Chang, Nigel W. Bunnett, Stuart M. Brierley
Chromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.
Giovanni A. Carosso, Leandros Boukas, Jonathan J. Augustin, Ha Nam Nguyen, Briana L. Winer, Gabrielle H. Cannon, Johanna D. Robertson, Li Zhang, Kasper D. Hansen, Loyal A. Goff, Hans T. Bjornsson
Dendritic cells (DCs) are crucial to balance protective immunity and autoimmune inflammatory processes. Expression of CD83 is a well-established marker for mature DCs, although its physiological role is still not completely understood. Using a DC-specific CD83–conditional KO (CD83ΔDC) mouse, we provide new insights into the function of CD83 within this cell type. Interestingly, CD83-deficient DCs produced drastically increased IL-2 levels and displayed higher expression of the costimulatory molecules CD25 and OX40L, which causes superior induction of antigen-specific T cell responses and compromises Treg suppressive functions. This also directly translates into accelerated immune responses in vivo. Upon Salmonella typhimurium and Listeria monocytogenes infection, CD83ΔDC mice cleared both pathogens more efficiently, and CD83-deficient DCs expressed increased IL-12 levels after bacterial encounter. Using the experimental autoimmune encephalomyelitis model, autoimmune inflammation was dramatically aggravated in CD83ΔDC mice while resolution of inflammation was strongly reduced. This phenotype was associated with increased cell influx into the CNS accompanied by elevated Th17 cell numbers. Concomitantly, CD83ΔDC mice had reduced Treg numbers in peripheral lymphoid organs. In summary, we show that CD83 ablation on DCs results in enhanced immune responses by dysregulating tolerance mechanisms and thereby impairing resolution of inflammation, which also demonstrates high clinical relevance.
Andreas B. Wild, Lena Krzyzak, Katrin Peckert, Lena Stich, Christine Kuhnt, Alina Butterhof, Christine Seitz, Jochen Mattner, Niklas Grüner, Maximilian Gänsbauer, Martin Purtak, Didier Soulat, Thomas H. Winkler, Lars Nitschke, Elisabeth Zinser, Alexander Steinkasserer
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