Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of diabetic patients are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of the IR with a reduction of mRNA expression of the selenoproteins Txnrd3, Sephs2, and Gpx3. Consistently, GPX3 is also decreased in adipose tissue of insulin resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation and elevated IR expression in WAT. Again, IR expression correlates positively with Gpx3 expression, a phenotype which is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduces IR expression in 3T3-L1 preadipocytes and insulin sensitivity. Overall our data identify GPx3 as a novel regulator of IR expression and insulin sensitivity in adipose tissue.
Robert Hauffe, Vanessa Stein, Chantal Chudoba, Tanina Flore, Michaela Rath, Katrin Ritter, Mareike Schell, Kristina Wardelmann, Stefanie Deubel, Johannes F. Kopp, Maria Schwarz, Kai Kappert, Matthias Blüher, Tanja Schwerdtle, Anna P. Kipp, Andre Kleinridders
BACKGROUND. Epidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number, and carcinoma associated mesenchymal stem cells (CA-MSC), and clinical outcomes in non-diabetic patients with advanced stage epithelial ovarian cancer (EOC). METHODS. Thirty-eight patients with confirmed stage IIC(n = 1)/III(n = 25)/IV(n = 12) EOC were treated with either (i) neoadjuvant metformin, debulking surgery and adjuvant chemotherapy + metformin, or (ii) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy + metformin. Metformin treated tumors, compared to historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (i) a greater than 2-fold reduction in ALDH+CD133+ CSC and (ii) a relapse free survival at 18 months of greater than 50%. RESULTS. Metformin was well-tolerated. Median progression-free survival was 18.0 months (95% CI 14.0–21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6–70.5). Median overall survival was 57.9 months (95% CI 28.0 – Not Estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+/CD133+ CSC and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSC which prevented CA-MSC driven chemoresistance in vitro. CONCLUSIONS. Translational studies confirm an impact of metformin on EOC CSC and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better than expected overall survival, supporting the use of metformin in phase-III studies.
Jason R Brown, Daniel K. Chan, Jessica J Shank, Kent A. Griffith, Huihui Fan, Robert Szulawski, Kun Yang, R. Kevin Reynolds, Carolyn Johnston, Karen McLean, Shitanshu Uppal, J. Rebecca Liu, Lourdes Cabrera, Sarah E Taylor, Brian C. Orr, Francesmary Modugno, Pooja Mehta, Michael Bregenzer, Geeta Mehta, Hui Shen, Lan Coffman, Ronald J. Buckanovich
Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type-1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type-1 cytokines IFNg and TNFa induce melanocyte detachment through E-cadherin disruption, and the release of its soluble form, partly due to the matrix metalloproteinase MMP-9. MMP-9, whose levels are increased in vitiligo skin and patients’ sera, is produced by keratinocytes in response to IFNg and TNFa. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising to prevent the depigmentation occurring in vitiligo and during chronic skin inflammation.
Nesrine Boukhedouni, Christina Martins, Anne-Sophie Darrigade, Claire Drullion, Jérôme Rambert, Christine Barrault, Julien Garnier, Clement Jacquemin, Denis Thiolat, Fabienne Lucchese, Franck Morel, Khaled Ezzedine, Alain TAIEB, François-Xavier Bernard, Julien Seneschal, Katia Boniface
Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterized by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a significant role of this imprinted gene in the function and organization of the two main neuromodulatory systems of the lateral hypothalamus (LH), namely, the orexin (OX) and melanin concentrating hormone (MCH) systems. We observe that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScrm+/p–) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutants. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutants reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake and temperature control.
Marta Pace, Matteo Falappa, Andrea Freschi, Edoardo Balzani, Chiara Berteotti, Viviana Lo Martire, Fatemeh Kaveh, Eivind Hovig, Giovanna Zoccoli, Roberto Amici, Matteo Cerri, Alfonso Urbanucci, Valter Tucci
Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop genetically-recipient T cells that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell depleted Lewis rats. The rats were then given Lewis x Dark Agouti (LWxDA) CTTI under the kidney capsule and vascularized DA heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naïve and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with (LWxDA) CTTI rejected the third-party BN hearts (mean survival time 10d; n=5). Controls did not (n=5). CTTI recipients produced antibody against third party BN donor but not against the DA thymus donor demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.
Jean Kwun, Jie Li, Clay Rouse, Jae Berm Park, Alton B. Farris III, Maragatha Kuchibhatla, Joseph W. Turek, Stuart Knechtle, Allan D. Kirk, M. Louise Markert
Pancreatic islets secrete insulin from β cells and glucagon from α cells and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the three-dimensional multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system, allowed the co-registration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.
John T. Walker, Rachana Haliyur, Heather A. Nelson, Matthew Ishahak, Gregory Poffenberger, Radhika Aramandla, Conrad Reihsmann, Joseph R. Luchsinger, Diane C. Saunders, Peng Wang, Adolfo Garcia-Ocana, Rita Bottino, Ashutosh Agarwal, Alvin C. Powers, Marcela Brissova
Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. While PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to bring clarity to the cellular basis of PAR2 pain. We developed a PAR2 conditional mutant mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, is dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that co-express the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discreet population of DRG sensory neurons mediate PAR2-evoked pain.
Shayne N. Hassler, Moeno Kume, Juliet Mwirigi, Ayesha Ahmad, Stephanie Shiers, Andi Wangzhou, Pradipta Ray, Sergei N. Belugin, Dhananjay K. Naik, Michael D. Burton, Josef Vagner, Scott Boitano, Armen N. Akopian, Gregory Dussor, Theodore J. Price
BACKGROUND. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a novel viral pneumonia (COVID-19), which is rapidly spreading in the world. The positive result of nucleic acid test is a golden criterion to confirm SARS-CoV-2 infection, but the detection features remain unclear. METHODS. We performed a retrospective analysis in 5,630 high-risk individuals receiving SARS-CoV-2 nucleic acid tests in Wuhan, China, and investigated their characteristics and diagnosis rates. RESULTS. The overall diagnosis rate was 34.7% (1,952/5,630). Male (P = 0.025) and older age (P = 2.525 × 10–39) were two significant risk factors of SARS-CoV-2 infection. People were generally susceptible, and most cases concentrated in people of 30- to 69-years-old. Besides, we investigated the association between diagnosis rate and the number of testing in 501 subjects. Results revealed a 1.27-fold improvement (35.5%/27.9%) of diagnosis rate from testing once to twice (P = 5.847 × 10–9), and a 1.43-fold improvement (39.9%/27.9%) from testing once to three times (P = 7.797 × 10–14). More than three testing times was not helpful for further improvement. However, this improvement was not observed in subjects with pneumonia (P = 0.097). CONCLUSION. All populations are susceptible to SARS-Cov-2 infection, and male and older age are two significant risk factors. Increasing the number of testing could significantly improve diagnosis rates, except for subjects with pneumonia. It is recommended to test twice in those high-risk individuals whose results are negative for the first time, and to perform three testing times is better if available.
Na Shen, Yaowu Zhu, Xiong Wang, Jing Peng, Weiyong Liu, Feng Wang, Yanjun Lu, Liming Cheng, Ziyong Sun
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. When human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patients’ hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these two hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study with hiPSC-derived cardiomyocytes revealed that fenofibrate could inhibit mitochondria-induced apoptosis in the DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified two promising small molecules with in vivo efficacy.
Congshan Sun, In Young Choi, Yazmin I. Rovira Gonzalez, Peter Andersen, C. Conover Talbot Jr., Shama R. Iyer, Richard M. Lovering, Kathryn R. Wagner, Gabsang Lee
Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol-25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds that of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the anti-inflammatory nuclear receptor, Liver X Receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural (oxy)sterols, 25HC is uniquely induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGFb. CH25H/25HC/LXR is thus an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.
Jennifer H. Madenspacher, Eric D. Morrell, Kymberly M. Gowdy, Jeffrey G. McDonald, Bonne M. Thompson, Ginger W. Muse, Jennifer Martinez, Seddon Y. Thomas, Carmen Mikacenic, Jerry A. Nick, Edward Abraham, Stavros Garantziotis, Renee D. Stapleton, Julie M. Meacham, Mary Jane Thomassen, William J. Janssen, Donald N. Cook, Mark M. Wurfel, Michael B. Fessler
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