Research
Abstract
Insulin-mediated suppression of white adipose tissue (WAT) lipolysis is an important anabolic function that is dysregulated in states of overnutrition. However, the mechanism of short-term high-fat diet (HFD)-induced WAT insulin resistance is poorly understood. Based on our recent studies we hypothesize that a short-term HFD causes WAT insulin resistance through increases in plasma membrane (PM) sn-1,2-diacylglycerols (DAG), which promotes protein kinase C-ε (PKCε) activation to impair insulin signaling by phosphorylating insulin receptor (Insr) Thr1160. To test this hypothesis, we assessed WAT insulin action in 7-day HFD-fed versus regular chow diet-fed rats during a hyperinsulinemic-euglycemic clamp. HFD feeding caused WAT insulin resistance, reflected by reductions in both insulin-mediated WAT glucose uptake and suppression of WAT lipolysis. These changes were specifically associated with increased PM sn-1,2-diacylglycerol (DAG) content, increased PKCε activation and impaired insulin-stimulated InsrY1162 phosphorylation. In order to examine the role of InsrT1160 phosphorylation in mediating lipid-induced WAT insulin resistance, we examined these same parameters in short-term HFD-fed InsrT1150A knockin mice (mouse homolog for human Thr1160). Similar to the rat study HFD feeding induced WAT insulin resistance in WT control mice but failed to induce WAT insulin resistance in InsrT1150A mice. Taken together these data demonstrate that the PM sn-1,2-DAG - PKCε - InsrT1160 phosphorylation pathway plays an important role in mediating lipid-induced WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in WAT.
Authors
Kun Lyu, Dongyan Zhang, Joongyu D. Song, Xiruo Li, Rachel J. Perry, Varman T. Samuel, Gerald I. Shulman
Abstract
Reestablishing an appropriate balance between T effector cells (Teff) and T regulatory cells (Treg) is essential for correcting autoimmunity. Multiple Sclerosis (MS) is an immune-mediated chronic central nervous system (CNS) disease characterized by neuroinflammation, demyelination, and neuronal degeneration, in which the Teff:Treg balance is skewed toward pathogenic Teff cells, Th1 and Th17 cells. Signal transducer and activator of transcription 3 (STAT3) is a key regulator of Teff:Treg balance. Using the structure-based design, we have developed a novel small-molecule prodrug LLL12b that specifically inhibits STAT3 and suppresses Th17 differentiation and expansion. Moreover, LLL12b regulates the fate decision between Th17 and Tregs in an inflammatory environment, shifting Th17:Treg balance toward Tregs and favoring the resolution of inflammation. Therapeutic administration of LLL12b after disease onset significantly suppresses disease progression in adoptively transferred, chronic, and relapsing-remitting experimental autoimmune encephalomyelitis. Disease relapses were also significantly suppressed by LLL12b given during the remission phase. Additionally, LLL12b shifts Th17:Treg balance of CD4 T cells from MS patients toward Tregs and increases Teff sensitivity to Treg-mediated suppression. These data suggest selective inhibition of STAT3 by the novel small molecule LLL12b recalibrates the effector and regulatory arms of CD4 T responses, representing a potentially clinically translatable therapeutic strategy for MS.
Authors
Saba I. Aqel, Xiaozhi Yang, Emma E. Kraus, Jinhua Song, Marissa F. Farinas, Erin Y. Zhao, Wei Pei, Amy E. Lovett-Racke, Michael K. Racke, Chenglong Li, Yuhong Yang
Abstract
Glucagon regulates glucose and lipid metabolism and also promotes weight loss. Thus, therapeutics stimulating glucagon-receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating Fibroblast Growth Factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon’s weight-loss effects. FGF21 signaling requires an obligate co-receptor (B-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb (Klb∆CNS) exhibit a partial reduction in body weight with chronic GCGR-agonism (via IUB288) compared to controls (p<0.0001), supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist (1153) also display partial weight loss (p<0.0001). Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR-agonism mediates part of its weight loss properties through central KLB and has implications for future treatments against obesity and metabolic syndrome.
Authors
Shelly R. Nason, Jessica P. Antipenko, Natalie Presedo, Stephen E. Cunningham, Tanya H. Pierre, Teayoun Kim, Jodi R. Paul, Cassie L. Holleman, Martin E. Young, Karen L. Gamble, Brian Finan, Richard DiMarchi, Chad S. Hunter, Alexei Kharitonenkov, Kirk M. Habegger
Abstract
Background: Loss-of-function variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including epileptic encephalopathy and cardiac arrhythmia. β1 subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming a subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Methods: We investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase.Results: We show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA-seq, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null cardiac tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. Conclusion: We propose that human disease variants resulting in SCN1B loss-of-function cause transcriptional dysregulation that contributes to altered excitability. These results provide important new insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multi-functionality of sodium channel β1 subunits.
Authors
Alexandra A. Bouza, Nnamdi Edokobi, Samantha L. Hodges, Alexa M. Pinsky, James Offord, Lin Piao, Yan-Ting Zhao, Anatoli N. Lopatin, Luis F. Lopez-Santiago, Lori L. Isom
Abstract
2'3'-cGAMP is known as a non-classical 2nd messenger and small immune modulator that possesses potent anti-tumor and antiviral activities through stimulating STING-mediated signaling pathway. However, its function in regulating type 2 immune responses remains unknown. We sought to determine a role of STING activation by 2'3'-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2'3'-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperresponsiveness (AHR) induced by IL-33 and a fungal allergen, A. flavus. Mechanistically, upon the respiratory delivery, 2'3'-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING-IRF3-IFN-I signaling axis to induce the production of inhibitory factors containing IFNα, which blocked the IL-33-mediated activation of group 2 innate lymphoid cells (ILC2) in vivo. We further demonstrated that 2'3'-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 in vitro. Taken together, our findings suggest that STING activation by 2'3'-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2'3'-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.
Authors
Li She, Gema D. Barrera, Liping Yan, Hamad Hazzaa Alanazi, Edward G. Brooks, Peter H. Dube, Yilun Sun, Hong Zan, Daniel P. Chupp, Nu Zhang, Xin Zhang, Yong Liu, Xiao-Dong Li
Abstract
Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca+2. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl- secretion by greater than 80%. Cinacalcet also reduced Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion following activation of CaSR, and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed-loop models of cholera and Traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3 (NHE3)-mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas including cholera, Traveler’s diarrhea and VIPoma.
Authors
Apurva A. Oak, Parth D. Chhetri, Amber Rivera, Alan S. Verkman, Onur Cil
Abstract
Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contributes to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single surface markers, such as CD15, CD44, CD133, and alpha-6 integrin. It remains unknown how these single surface marker-defined GSC populations compare to each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost, and previously undetectable ones materialized. GSCs that highly expressed all four surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings.
Authors
Luciano Galdieri, Arijita Jash, Olga Malkova, Diane D. Mao, Patrick A. DeSouza, Yunli E. Chu, Amber Salter, Jian L. Campian, Kristen M. Naegle, Cameron W. Brennan, Hiroaki Wakimoto, Stephen T. Oh, Albert H. Kim, Milan G. Chheda
Abstract
Antiretroviral therapies (ART) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which re-seed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time, and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.
Authors
Eva M. Stevenson, Adam R. Ward, Ronald Truong, Allison S. Thomas, Szu-Han Huang, Thomas R. Dilling, Sandra Terry, John K. Bui, Talia M. Mota, Ali Danesh, Guinevere Q. Lee, Andrea Gramatica, Pragya Khadka, Winiffer D. Conce Alberto, Rajesh T. Gandhi, Deborah K. McMahon, Christina M. Lalama, Ronald J. Bosch, Bernard J. Macatangay, Joshua C. Cyktor, Joseph J. Eron, John W. Mellors, R. Brad Jones
Abstract
Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2 and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK up-regulated the expression of genes involved in OS cell metabolism and down-regulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux towards lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of peroxisome proliferator-activated receptor-γ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.
Authors
Toru Hirozane, Mari Masuda, Teppei Sugano, Tetsuya Sekita, Naoko Goto, Toru Aoyama, Takato Sakagami, Yuko Uno, Hideki Moriyama, Masaaki Sawa, Naofumi Asano, Masaya Nakamura, Morio Matsumoto, Robert Nakayama, Tadashi Kondo, Akira Kawai, Eisuke Kobayashi, Tesshi Yamada
Abstract
Infantile hemangioma is a vascular tumor characterized by the rapid growth of disorganized blood vessels followed by slow spontaneous involution. The underlying molecular mechanisms that regulate hemangioma proliferation and involution still are not well elucidated. Our previous studies reported that NOGOB receptor (NGBR), a transmembrane protein, is required for the translocation of prenylated RAS from the cytosol to the plasma membrane and promotes RAS activation. Here, we show that NGBR is highly expressed in the proliferating phase of infantile hemangioma, but its expression decreases in the involuting phase, suggesting that NGBR may be involved in regulating the growth of proliferating hemangioma. Moreover, we demonstrated that NGBR knockdown in hemangioma stem cells (HemSCs) attenuates growth factors-stimulated RAS activation and diminishes the migration and proliferation of HemSCs, which is consistent with the effects of RAS knockdown in HemSCs. In vivo differentiation assay further showed that NGBR knockdown inhibits blood vessel formation and adipocyte differentiation of HemSCs in immunodeficient mice. Our data suggest that NGBR serves as a RAS modulator in controlling the growth and differentiation of HemSCs.
Authors
Wenquan Hu, Zhong Liu, Valerie Salato, Paula E. North, Joyce Bischoff, Suresh N. Kumar, Zhi Fang, Sujith Rajan, M. Mahmood Hussain, Qing R. Miao
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