Abstract
Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the US population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, we studied flux through pathways of hepatic oxidative metabolism and gluconeogenesis using 5 simultaneous stable isotope tracers in ketotic (24-hour-fasted) individuals with a wide range of hepatic triglyceride content levels (0%–52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic acid (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when β-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as calculated hepatic oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is a consequence not of acetyl-CoA production per se, but rather of how acetyl-CoA is further metabolized in liver.
Authors
Justin A. Fletcher, Stanisław Deja, Santhosh Satapati, Xiaorong Fu, Shawn C. Burgess, Jeffrey D. Browning
Abstract
In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN-Tg mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography revealed that SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN-Tg mdx mice by hemodynamic pressure-volume methods highlighted enhanced systolic performance compared with mdx controls. SSPN restored cardiac sarcolemma stability, the primary defect in DMD disease; reduced fibrotic response; and improved contractile function. We demonstrate that SSPN ameliorated more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin, which mitigate disease severity, and partially restored responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest that SSPN overexpression in DMD mouse models positively affects skeletal, pulmonary, and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
Authors
Michelle S. Parvatiyar, Alexandra J. Brownstein, Rosemeire M. Kanashiro-Takeuchi, Judd R. Collado, Karissa M. Dieseldorff Jones, Jay Gopal, Katherine G. Hammond, Jamie L. Marshall, Abel Ferrel, Aaron M. Beedle, Jeffrey S. Chamberlain, Jose Renato Pinto, Rachelle H. Crosbie
Abstract
Induction of a potent CD4 and CD8 T cell response against tumor-specific and tumor-associated antigens is critical for eliminating tumor cells. Recent vaccination strategies have been hampered by an inefficacious and low-amplitude immune response. In this article, we describe a self-adjuvanted chimeric protein vaccine platform to address these challenges, characterized by a multidomain construction incorporating (a) a cell-penetrating peptide allowing internalization of several multiantigenic MHC-restricted peptides within (b) the multiantigenic domain and (c) a TLR2/4 agonist domain. Functionality of the resulting chimeric protein is based on the combined effect of the above-mentioned 3 different domains for simultaneous activation of antigen-presenting cells and antigen cross-presentation, leading to an efficacious multiantigenic and multiallelic cellular immune response. Helper and cytotoxic T cell responses were observed against model-, neo-, and self-antigens and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a nonhuman primate model. This therapeutic vaccine approach, which we believe to be newly engineered, is promising for the treatment of poorly infiltrated tumors that do not respond to currently marketed immunotherapies.
Authors
Elodie Belnoue, Jean-François Mayol, Susanna Carboni, Wilma Di Berardino Besson, Eloise Dupuychaffray, Annika Nelde, Stefan Stevanovic, Marie-Laure Santiago-Raber, Paul R. Walker, Madiha Derouazi
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. Recently, we created a mouse model with spontaneous and sustained AF caused by a mutation in the NaV1.5 channel (F1759A) that enhances persistent Na+ current, thereby enabling the investigation of molecular mechanisms that cause AF and the identification of potentially novel treatment strategies. The mice have regional heterogeneity of action potential duration of the atria similar to observations in patients with AF. In these mice, we found that the initiation and persistence of the rotational reentrant AF arrhythmias, known as spiral waves or rotors, were dependent upon action potential duration heterogeneity. The centers of the rotors were localized to regions of greatest heterogeneity of the action potential duration. Pharmacologically attenuating the action potential duration heterogeneity reduced both spontaneous and pacing-induced AF. Computer-based simulations also demonstrated that the action potential duration heterogeneity is required to generate rotors that manifest as AF. Taken together, these findings suggest that action potential duration heterogeneity in mice and humans is one mechanism by which AF is initiated and that reducing action potential duration heterogeneity can lessen the burden of AF.
Authors
Uma Mahesh R. Avula, Jeffrey Abrams, Alexander Katchman, Sergey Zakharov, Sergey Mironov, Joseph Bayne, Daniel Roybal, Anirudh Gorti, Lin Yang, Vivek Iyer, Marc Waase, Deepak Saluja, Edward J. Ciaccio, Hasan Garan, Andrew R. Marks, Steven O. Marx, Elaine Y. Wan
Glycolytic inhibitor 2-deoxyglucose prevents cortical hyperexcitability after traumatic brain injury
Abstract
Traumatic brain injury (TBI) causes cortical dysfunction and can lead to posttraumatic epilepsy. Multiple studies demonstrate that GABAergic inhibitory network function is compromised following TBI, which may contribute to hyperexcitability and motor, behavioral, and cognitive deficits. Preserving the function of GABAergic interneurons, therefore, is a rational therapeutic strategy to preserve cortical function after TBI and prevent long-term clinical complications. Here, we explored an approach based on the ketogenic diet, a neuroprotective and anticonvulsant dietary therapy that results in reduced glycolysis and increased ketosis. Utilizing a pharmacologic inhibitor of glycolysis (2-deoxyglucose, or 2-DG), we found that acute in vitro application of 2-DG decreased the excitability of excitatory neurons, but not inhibitory interneurons, in cortical slices from naive mice. Employing the controlled cortical impact (CCI) model of TBI in mice, we found that in vitro 2-DG treatment rapidly attenuated epileptiform activity seen in acute cortical slices 3–5 weeks after TBI. One week of in vivo 2-DG treatment immediately after TBI prevented the development of epileptiform activity, restored excitatory and inhibitory synaptic activity, and attenuated the loss of parvalbumin-expressing inhibitory interneurons. In summary, 2-DG may have therapeutic potential to restore network function following TBI.
Authors
Jenny B. Koenig, David Cantu, Cho Low, Mary Sommer, Farzad Noubary, Danielle Croker, Michael Whalen, Dong Kong, Chris G. Dulla
Abstract
Preneoplastic lesions carry many of the antigenic targets found in cancer cells but often exhibit prolonged dormancy. Understanding how the host response to premalignancy is maintained and altered during malignant transformation is needed to prevent cancer. To understand the immune microenvironment in precursor monoclonal gammopathy of undetermined significance (MGUS) and myeloma, we analyzed bone marrow immune cells from 12 healthy donors and 26 patients with MGUS/myeloma by mass cytometry and concurrently profiled transcriptomes of 42,606 single immune cells from these bone marrow samples. Compared with age-matched healthy donors, memory T cells from both MGUS and myeloma patients exhibited greater terminal effector differentiation. However, memory T cells in MGUS show greater enrichment of stem-like TCF1/7hi cells. Clusters of T cells with stem-like and tissue residence genes were also found to be enriched in MGUS by single-cell transcriptome analysis. Early changes in both NK and myeloid cells were also observed in MGUS. Enrichment of stem-like T cells correlated with a distinct genomic profile of myeloid cells and levels of Dickkopf-1 in bone marrow plasma. These data describe the landscape of changes in both innate and adaptive immunity in premalignancy and suggest that attrition of the bone marrow–resident T cell compartment because of loss of stem-like cells may underlie loss of immune surveillance in myeloma.
Authors
Jithendra Kini Bailur, Samuel S. McCachren, Deon B. Doxie, Mahesh Shrestha, Katherine Pendleton, Ajay K. Nooka, Natalia Neparidze, Terri L. Parker, Noffar Bar, Jonathan L. Kaufman, Craig C. Hofmeister, Lawrence H. Boise, Sagar Lonial, Melissa L. Kemp, Kavita M. Dhodapkar, Madhav V. Dhodapkar
Abstract
T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce IL-21 promote germinal center (GC) B cell responses, leading to Ig production. Here we investigate the role of IL-21 and Tfh cells in hypertension. In response to angiotensin II–induced (Ang II–induced) hypertension, T cell IL-21 production was increased, and Il21–/– mice developed blunted hypertension, attenuated vascular end-organ damage, and decreased IL-17A and IFN-γ production. Tfh-like cells and GC B cells accumulated in the aorta, and plasma IgG1 was increased in hypertensive WT but not Il21–/– mice. Furthermore, Tfh cell–deficient mice developed blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL-21 neutralization reduced BP and reversed endothelial dysfunction and vascular inflammation. Moreover, recombinant IL-21 impaired endothelium-dependent relaxation ex vivo and decreased NO production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL-21 correlated with systolic BP and IL-17A production. These data suggest that IL-21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.
Authors
Bethany L. Dale, Arvind K. Pandey, Yuhan Chen, Charles D. Smart, Fanny Laroumanie, Mingfang Ao, Liang Xiao, Anna E. Dikalova, Sergey I. Dikalov, Fernando Elijovich, Jason D. Foss, Natalia R. Barbaro, Justin P. Van Beusecum, Serpil M. Deger, Aseel Alsouqi, Hana A. Itani, Allison E. Norlander, Matthew R. Alexander, Shilin Zhao, T. Alp Ikizler, Holly M. Scott Algood, Meena S. Madhur
Abstract
Right ventricular (RV) dysfunction is highly prevalent across cardiopulmonary diseases and independently predicts death in both heart failure (HF) and pulmonary hypertension (PH). Progression towards RV failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting current insufficient understanding of RVF molecular pathophysiology. To identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced-HF patients, with and without RVF. Using an integrated systems genomic and functional biology approach, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers, and confirmed the ventricular specificity of Wipi1, Hspb6, and Map4 transcriptional changes in adult murine models of pressure overload–induced RV versus left ventricular failure. We uncovered a shift towards noncanonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited noncanonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and noncanonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential target for RV-directed HF therapy.
Authors
Christos Tzimas, Christoph D. Rau, Petra E. Buergisser, Gaston Jean-Louis Jr., Katherine Lee, Jeffrey Chukwuneke, Wen Dun, Yibin Wang, Emily J. Tsai
Abstract
Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+-sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxyvitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia, a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium; however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a nonfunctional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when coexpressed with the CaSR. Moreover, the phospholipase C inhibitor U73122 prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.
Authors
Justin J. Lee, Xiong Liu, Debbie O’Neill, Megan R. Beggs, Petra Weissgerber, Veit Flockerzi, Xing-Zhen Chen, Henrik Dimke, R. Todd Alexander
Abstract
Potassium (K+) secretion by kidney tubule cells is central to electrolyte homeostasis in mammals. In the K+-secreting principal cells of the distal nephron, electrogenic Na+ transport by the epithelial sodium channel (ENaC) generates the electrical driving force for K+ transport across the apical membrane. Regulation of this process is attributable in part to aldosterone, which stimulates the gene transcription of the ENaC-regulatory kinase, SGK1. However, a wide range of evidence supports the conclusion that an unidentified aldosterone-independent pathway exists. We show here that in principal cells, K+ itself acts through the type 2 mTOR complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion. The effect depends on changes in K+ concentration on the blood side of the cells, and requires basolateral membrane K+-channel activity. However, it does not depend on changes in aldosterone, or on enhanced distal delivery of Na+ from upstream nephron segments. These data strongly support the idea that K+ is sensed directly by principal cells to stimulate its own secretion by activating the mTORC2/SGK1 signaling module, and stimulate ENaC. We propose that this local effect acts in concert with aldosterone and increased Na+ delivery from upstream nephron segments to sustain K+ homeostasis.
Authors
Mads Vaarby Sørensen, Bidisha Saha, Iben Skov Jensen, Peng Wu, Niklas Ayasse, Catherine E. Gleason, Samuel Levi Svendsen, Wen-Hui Wang, David Pearce
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