Chronic Inhibition Research Articles

NNOS-induced tyrosine nitration of TRKB impairs BDNF signaling and restrains neuronal plasticity.

Nitric oxide (NO) has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that NO induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase Cγ1 (PLCγ1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP-2 and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the visual cortex, and promoted a shift in ocular dominance upon monocular deprivation - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.

Nitric oxide and salt resistance in Dahl rats: no role of inducible NO synthase.

Inducible NO synthase (NOS II) was proposed to play an important role in salt resistance of Dahl salt-resistant (SR/Jr) rats. Its chronic inhibition by specific inhibitors was accompanied by blood pressure (BP) elevation in animals subjected to high salt intake. The aim of our study was to evaluate 1) whether such inhibitors affect BP and/or its particular components (sympathetic tone and NO-dependent vasodilation) only under the conditions of high salt intake, and 2) whether similar BP effects are elicited after systemic or intracerebroventricular (icv) application of these inhibitors. Wistar rats fed Altromin diet (0.45 % NaCl) and SR/Jr rats fed either a low-salt (LS, 0.3 % NaCl) or a high-salt (HS, 4 % NaCl) diet were studied. Aminoguanidine (AMG) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) were used as NOS II inhibitors. BP and its responses to acute blockade of renin-angiotensin system (captopril), sympathetic nervous system (pentolinium) and NO synthase (L-NAME) were measured in conscious cannulated rats. There were no significant changes of BP or its components in either Wistar rats or SR/Jr rats subjected to chronic inhibition of NOS II by peroral aminoguanidine administration (50 mg/kg/day for 4 weeks). This was true for SR/Jr rats fed either LS or HS diets. Furthermore, we have studied BP effects of chronic icv administration of both NOS II inhibitors in SR/Jr rats fed HS diet, but we failed to find any BP changes elicited by such treatment. In conclusion, inducible NO synthase does not participate in the resistance of SR/Jr rats to hypertensive effects of excess salt intake.

The Alzheimer’s disease-linked protease BACE1 modulates neuronal IL-6 signaling through shedding of the receptor gp130

BackgroundThe protease BACE1 is a major drug target for Alzheimer’s disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates.MethodsTo identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors.ResultsBesides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal.ConclusionBACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.Graphical abstract

Chronic unilateral inhibition of GABA synthesis in the amygdala increases specificity of conditioned fear in a discriminative fear conditioning paradigm in rats

Neural activity in the amygdala is critical for fear learning. In anxiety disorder patients, bilateral hyperactivity of the amygdala can be observed. This hyperactivation is often associated with the facilitation of fear learning and/or over-generalization of conditioned fear. In contrast, hypoactivity of the amygdala, e.g. by pharmacological interventions, attenuates or blocks fear learning. To date, little is known about how neural excitability of the amygdala affects specificity or generalization of fear. Therefore, the present study utilized chronic inhibition of GABA synthesis in the amygdala to increase excitability and investigated the effect on the specificity of fear learning. In rats, unilateral cannulas aiming at the amygdala were implanted. The cannulas were connected to subcutaneously implanted osmotic mini pumps that delivered either the GABA synthesis inhibitor L-allylglycine or its inactive enantiomer D-allylglycine. Following one week of chronic GABA synthesis manipulation, the rats were submitted to a discriminative fear conditioning protocol. In addition, anxiety-like behavior in the light-dark box was measured. Our data show that chronic unilateral L-AG infusions into the amygdala improve the specificity of learned fear, support safety learning, and reduce fear generalization and anxiety. This data demonstrates that moderately increased amygdala excitability can be beneficial for the specificity of fear learning and highlights the potential application for therapeutic interventions.

4-EA-NBOMe, an amphetamine derivative, alters glutamatergic synaptic transmission through 5-HT1A receptors on cortical neurons from Sprague[sbnd]Dawley rat and pyramidal neurons from C57BL/6 mouse

New psychoactive substances (NPSs) are compounds designed to mimic illegal recreational drugs. In particular, there are difficulties in legal restrictions because there is no fast NPS detection method to suppress the initial spread of NPS with criminal records; thus, they expose the public to serious health threats, including toxicity and dependence. However, the effects of NPSs on the brain and the related cellular mechanisms are well unknown. One of the recently emerging drugs is 4-ethylamphetamine-NBOMe (4-EA-NBOMe), a member of the 2 C phenylalanine family with a similar structure to methamphetamine (methA). In this study, we tested the effect of methA analogs on the glutamatergic synaptic transmission on primary cultured cortical neurons of SpragueDawley (SD) rats and C57BL/6 mice, and also layer 2/3 pyramidal neurons of the medial prefrontal cortex (mPFC) of C57BL/6 mice. We found that acute treatment with 4-EA-NBOMe inhibits spontaneous excitatory postsynaptic currents (EPSCs) and that withdrawal after chronic inhibition by 4-EA-NBOMe augments glutamatergic synaptic transmission. These modifications of synaptic responses are mediated by 5-HT1A receptors. These findings suggest that 4-EA-NBOMe directly affects the central nervous system by changing the efficacy of glutamatergic synaptic transmission.

Differential inhibition of T-cell receptor and STAT5 signaling pathways determines the immunomodulatory effects of dasatinib in chronic phase chronic myeloid leukemia.

Dasatinib is a multi-kinase inhibitor with activity against the SRC kinase LCK, which plays a critical role in T-cell receptor signaling. Dasatinib, initially developed as an immunosuppressive agent, is by contrast, also noted to result in enhanced tumor immunity in a subset of patients. We studied the impact of dasatinib in chronic myeloid leukemia patients and compared it with patients taking other tyrosine kinase inhibitors (TKI) and healthy controls. We found that patients on dasatinib showed inhibition of both T-cell receptor (TCR) and STAT5 signaling pathways, and reduced expression of Teffector pro-inflammatory cytokines. In addition, dasatinib induced selective depletion of regulatory T cells (Tregs) and effector Tregs, particularly in patients with clonal expansion of effector CD8+ T cells, who demonstrated greater and preferential inhibition of Treg TCR intracellular signaling. In addition, we show that dasatinib selectively reduces Treg STAT5 phosphorylation via reduction of IL-2, in relation with the marked reduction of plasma IL-2 levels in patients taking dasatinib. Finally, patients on other TKI had significantly increased TCR signaling in TIM3+ cells compared to patients taking dasatinib, suggesting that chronic SRC kinase inhibition by dasatinib may play a role in preventing TIM-3-mediated T-cell exhaustion and preserve anti-tumor immunity. These data provide further insight into the selective immunomodulatory effects of dasatinib and its potential use for pharmacologic control of immunotherapies.

PS-B10-5: EFFECTS OF CHRONIC ACETYLCHOLINESTERASE INHIBITION WITH PYRIDOSTIGMINE ON CARDIOVASCULAR SYSTEM AND AUTONOMIC BALANCE IN SPONTANEOUSLY HYPERTENSIVE RATS

Objective: The imbalance in autonomic nervous system, characterized by increased sympathetic and attenuated parasympathetic activity, plays an important role in the pathogenesis of hypertension. The spontaneously hypertensive rats (SHR) which are one of the most studied animal models of essential hypertension, are characterized by autonomic nervous system imbalance. Our study was aimed to determine the effect of chronic inhibition of acetylcholinesterase with pyridostigmine on cardiovascular parameters and sympatho-vagal balance in SHR and Wistar-Kyoto (WKY) rats. Design and method: Adult male SHR and WKY rats were treated with pyridostigmine (25 mg/kg/day) in drinking solution for 2 weeks. The rats were implanted with telemetric transducers to measure blood pressure (BP) and heart rate (HR). Autonomic balance was evaluated by power spectral analysis of systolic blood pressure variability (SBPV) and heart rate variability (HRV). Sympatho-vagal balance was also evaluated pharmacologically by administration of methylatropine (antagonist of muscarinic acetylcholine receptors) and propranolol (antagonist of beta-adrenergic receptors) in a separate set of rats. Results: SHR have elevated BP, increased vascular sympathetic activity (low frequency component of SBPV), and reduced HRV compared to WKY. Pyridostigmine significantly reduced HR in rats of both strains (during both dark and light phase) and BP during the dark (active) phase compared to untreated controls. Acetylcholinesterase activity in plasma was significantly reduced by pyridostigmine treatment in both strains (SHR 53 ± 3%, WKY 46 ± 3%). Sympatho-vagal balance examination revealed reduced cardiac vagal tone in SHR as evidenced by blunted HR response to the muscarinic receptor blocker methylatropine and increased cardiac sympathetic tone as evidenced by elevated HR response to the beta-blocker propranolol. Pyridostigmine treatment increased cardiac vagal tone and decreased cardiac sympathetic tone in SHR to the levels of untreated WKY. Moreover, pyridostigmine increased HRV and reduced low frequency component of SBPV in SHR indicating the restoration of autonomic balance. The reduction of sympathetic tone in pyridostigmine-treated rats was also evident from the reduction of plasma levels of noradrenaline. The potentiation of cardiac parasympathetic activity by pyridostigmine treatment was confirmed by the attenuation of restraint stress-induced tachycardia in both strains. Conclusions: Chronic stimulation of peripheral cholinergic system with pyridostigmine leads to the improvement of autonomic nervous system balance in SHR by increasing cardiac vagal tone and reducing sympathetic tone. Thus, chronic treatment with acetylcholinesterase inhibitors seems to be a promising therapeutic strategy for the restoration of autonomic balance in cardiovascular diseases.

Chronic Inhibition of Aggressive Behavior Induces Behavioral Change in Mice.

Suppression of anger is more common than its expression among Asian individuals. Emotional suppression is considered an unhealthy emotional regulation. Most studies on emotional suppression have concluded that suppression adversely affects social outcomes, with approximately 5% of the world's population suffering from emotional disorders. However, anger suppression has not received academic attention, and details of the effects of chronic anger suppression on the central nervous system remain unclear. In this study, we performed the resident-intruder test to investigate the effect of chronic suppression of aggressive behavior in mice using a behavioral test battery and to clarify whether suppression of this aggressive behavior is stressful for mice. Mice chronically inhibited aggressive behavior and lost weight. Mice with inhibited aggressive behavior showed a reduced percentage of immobility time during the tail suspension test as well as no changes in activity, anxiety-like behavior, muscle strength, or temperature sensitivity. This study provides scientific evidence for the effects of chronic aggressive behavior inhibition on the body and central nervous system.

Role of natural mTOR inhibitors in treatment of diabetes mellitus.

Diabetes mellitus is one of the most common and complex problems in today's society and is responsible for many socio-economic problems. Type 1 diabetes is due to a defect in insulin secretion caused by a destruction of pancreatic β cells. In contrast, the pathogenesis of type 2 diabetes is associated with the development of insulin resistance in the liver and peripheral tissues, a decrease in β-cell mass, and a defect in insulin secretion. Various factors such as inflammation, stress, obesity, and lifestyle are involved in diabetes. Long-term or chronic increase in glucose in these patients is the leading causes of secondary disorders such as micro- and macro-angiopathy, weakness of the antioxidant defense system, and metabolic disorders and altered lipid profile. The above conditions lead to short-term and long-term complications. These complications cause damage to the physical and physiological function of diverse organs of the body and threaten human health. Late complications of diabetes, including nephropathy, retinopathy, cardiovascular complications, neuropathy, hypertension, and weight gain, are common, and more research has been done on them. Numerous drugs such as meglitinides, biguanides, and thiazolidinedione have been proposed to reduce high blood sugar, but due to the lack of complete cure of this disease with the use of existing drugs, the tendency to use alternative and traditional therapies has increased. In the meantime, the role of herbs with hypoglycemic properties in the treatment of diabetic patients cannot be ignored. The consumption of herbs by people with diabetes has become widespread even in Western countries. The use of herbs could be considered when conventional therapies cannot control the disease, and the patient needs to be prescribed insulin. The mammalian target of rapamycin, mTOR, plays a significant role in regulating cell growth, cellular metabolic status in response to nutrients, many extracellular cues, and growth factors. Impaired insulin secretion can lead to altered mTOR signaling. The mTOR pathway has shown different behaviors depending on the situation. It has been shown that mTOR can regulate the adaptation of β cells to blood sugar, and chronic inhibition of the mTOR pathway can also induce diabetes. Here, we have reviewed recent findings on the role of mTOR in major metabolic organs, such as the liver, pancreas, brain, and adipose tissue and muscle, and discussed its potential as a diabetes-related drug target.

Loss of FoxOs in muscle increases strength and mitochondrial function during aging.

Muscle mitochondrial decline is associated with aging-related muscle weakness and insulin resistance. FoxO transcription factors are targets of insulin action and deletion of FoxOs improves mitochondrial function in diabetes. However, disruptions in proteostasis and autophagy are hallmarks of aging and the effect of chronic inhibition of FoxOs in aged muscle is unknown. This study investigatedthe role of FoxOs in regulating muscle strength and mitochondrial function with age. We measured muscle strength, cross-sectional area, muscle fibre-type, markers of protein synthesis/degradation, central nuclei, glucose/insulin tolerance, and mitochondrial bioenergetics in 4.5-month (Young) and 22-24-month-old (Aged) muscle-specific FoxO1/3/4 triple KO (TKO) and littermate control (Ctrl) mice. Lean mass was increased in Aged TKO compared with both Aged Ctrl and younger groups by 26-33% (P<0.01). Muscle strength, measured by max force of tibialis anterior (TA) contraction, was 20% lower in Aged Ctrl compared with Young Ctrls (P<0.01) but was not decreased in Aged TKOs. Increased muscle strength in Young and Aged TKO was associated with 18-48% increased muscle weights compared with Ctrls (P<0.01). Muscle cross-sectional analysis of TA, soleus, and plantaris revealed increases in fibre size distribution and a 2.5-10-fold increase in central nuclei in Young and Aged TKO mice, without histologic signs of muscle damage. Age-dependent increases in Gadd45a and Ube4a expression as well accumulation of K48 polyubiquitinated proteins were observed in quad and TA but were prevented by FoxO deletion. Young and Aged TKO muscle showed minimal changes in autophagy flux and no accumulation of autophagosomes compared with Ctrl groups. Increased strength in Young and Aged TKO was associated with a 10-20% increase in muscle mitochondrial respiration using glutamate/malate/succinate compared with controls (P<0.05). OXPHOS subunit expression and complex I activity were decreased 16-34% in Aged Ctrl compared with Young Ctrl but were prevented in Aged TKO. Both Aged Ctrl and Aged TKO showed impairedglucose tolerance by 33% compared to young groups (P<0.05) indicating improved strength and mitochondrial respiration are not due to improved glycemia. FoxO deletion increases muscle strength even during aging. Deletion of FoxOs maintains muscle strength in part by mild suppression of atrophic pathways, including inhibition of Gadd45a and Ube4a expression, without accumulation of autophagosomes in muscle. Deletion of FoxOs also improved mitochondrial function by maintenance of OXPHOS in both young and aged TKO.

Abstract 9396: Calmodulin-Dependent Protein Kinase II Activation Promotes Kidney Mesangial Expansion in Streptozotocin-Induced Diabetic Mice

Background: Calmodulin-dependent protein kinase II (CaMKII) is upregulated in diabetes mellitus (DM), leading to the overproduction of collagen in the myocardium. We hypothesized that CaMKII also stimulates the extracellular matrix production in the kidneys leading to mesangial matrix expansion, a hallmark of diabetic nephropathy and a major cause of renal failure. To test this hypothesis, we developed an automated process of mesangial matrix measurement. Methods: We induced Type 1 diabetes (DM1) in wild type female FVB mice via an intraperitoneal injection of Streptozotocin (STZ, 200 mg/kg ip) and studied the dynamics of mesangial matrix fractional area (MMFA) over 10 weeks (W) in mice with: DM , DM/KN93 , 6 W after STZ, followed by 4 W treatment with a CaMKII inhibitor KN93 (70 μg/kg/day, via mini pump), and Control (C) (vehicle only) mice. Both kidneys were retrieved. MMFA measurements were performed on 50 glomeruli from each animal on trichrome-stained paraffin sections using a trainable segmentation WEKA (Waikato Environment of Knowledge Analysis) plugin for Fiji, as well as on the electron micrographs of 4-5 whole glomeruli/animal. Results: Both DM and DM/KN93 mice were severely hyperglycemic (517 and 538 mg/dL compared to 161 mg/dL in C). In DM mice, the kidney weight increased by 76% and the MMFA significantly increased by 81% compared to C (9.4 ± 1% vs 5.2 ± 2.8%). In DM/KN93 mice, MMFA was similar to that in controls (6.2 ± 2.3%). The electron microscopy study has confirmed these MMFA changes. Other features of human diabetic nephropathy were not seen and the fractional mesangial cell area and the fractional podocyte area were relatively unchanged in DM and DM/KN93 mice. Conclusions: Our DM1 mouse model reproduces the key features of early human diabetic nephropathy. KN93 prevents DM-induced extracellular matrix production and kidney mesangial matrix expansion, suggesting that chronic inhibition of CaMKII could be a potential treatment for the early diabetic nephropathy. Reliable automated measurement of the MM area can be achieved after training by the WEKA plugin.

Abstract 15087: Chronic Inhibition of NADPH Oxidase Improves Coronary Endothelial Function in Mice With Diabetes

Introduction:: Endothelial dysfunction is the leading cause for diabetes (DM) related coronary heart disease. Diabetes is associated with NADPH Oxidase (Nox) overproduction and related ROS amplification in endothelial cells and coronary vasculature, which may contribute to coronary endothelial dysfunction. Small conductance calcium-activated-potassium (SK) channels also play a key role in diabetes-induced coronary endothelial dysfunction . Hypothesis: Chronic inhibition of Nox may improve coronary endothelial function/coronary arteriolar relaxation, and SK activator-induced vasodilation in mice with or without diabetes. Methods: Wild type (control), non-diabetic (ND) mice and genetically modified, obesity/type-2 diabetic mice (BKS.Cg- Dock7 m +/+ Lepr db /J) with or with treatment with Nox inhibitor apocynin (n = 4-6/group) were used in the study. In the Nox inhibitor treated group, apocynin (30mg/kg/day, i.p) was applied to the diabetic mice for 4 weeks. Small coronary arteries (70-120μM) isolated for harvested hearts were pre-contracted and examined in the presence or absence of the vasodilatory agents, SNP, ADP, and NS309, respectively. Results: After 4-week treatment with apocynin, endothelium-NO-dependent relaxation responses to ADP, and endothelium-dependent, SK channel activator NS309 in the diabetic mice were significantly increased when compared with that in untreated diabetic mice, respectively (# p&lt;0.05 vs ND Figure ). There were no significant differences in responses to the endothelium-independent vasodilator SNP (P&gt;0.05) among the three groups. Conclusions: Chronic inhibition of Nox appears to improve coronary endothelial function/coronary relaxation and SK channel activity in mice with diabetes.

Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model.

The neuromuscular junction (NMJ) mediates neural control of skeletal muscle fibers. Neurotrophic signaling, specifically brain derived neurotrophic factor (BDNF) acting through its high-affinity tropomyosin related kinase B (TrkB) receptor is known to improve neuromuscular transmission. BDNF/TrkB signaling also maintains the integrity of antero- and retrograde communication between the motor neuron soma, its distal axons and pre-synaptic terminals and influences neuromuscular transmission. In this study, we employed a novel rat chemogenetic mutation (TrkB F616), in which a 1-naphthylmethyl phosphoprotein phosphatase 1 (1NMPP1) sensitive knock-in allele allowed specific, rapid and sustained inhibition of TrkB kinase activity. In adult female and male TrkB F616 rats, treatment with either 1NMPP1 (TrkB kinase inhibition) or DMSO (vehicle) was administered in drinking water for 14 days. To assess the extent of neuromuscular transmission failure (NMTF), diaphragm muscle isometric force evoked by nerve stimulation at 40 Hz (330 ms duration trains repeated each s) was compared to isometric forces evoked by superimposed direct muscle stimulation (every 15 s). Chronic TrkB kinase inhibition (1NMPP1 group) markedly worsened NMTF compared to vehicle controls. Acute BDNF treatment did not rescue NMTF in the 1NMPP1 group. Chronic TrkB kinase inhibition did not affect the apposition of pre-synaptic terminals (labeled with synaptophysin) and post-synaptic endplates (labeled with α-Bungarotoxin) at diaphragm NMJs. We conclude that inhibition of BDNF/TrkB signaling in TrkB F616 rats disrupts diaphragm neuromuscular transmission in a similar manner to TrkB F616A mice, likely via a pre-synaptic mechanism independent of axonal branch point failure.

Microglia shield the murine brain from damage mediated by the cytokines IL-6 and IFN-α

Sustained production of elevated levels of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is detrimental and directly contributes to the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Using transgenic mice with CNS-targeted production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN), we have recently demonstrated that microglia are prominent target and effector cells and mount stimulus-specific responses to these cytokines. In order to further clarify the phenotype and function of these cells, we treated GFAP-IL6 and GFAP-IFN mice with the CSF1R inhibitor PLX5622 to deplete microglia. We examined their ability to recover from acute microglia depletion, as well as the impact of chronic microglia depletion on the progression of disease. Following acute depletion in the brains of GFAP-IL6 mice, microglia repopulation was enhanced, while in GFAP-IFN mice, microglia did not repopulate the brain. Furthermore, chronic CSF1R inhibition was detrimental to the brain of GFAP-IL6 and GFAP-IFN mice and gave rise to severe CNS calcification which strongly correlated with the absence of microglia. In addition, PLX5622-treated GFAP-IFN mice had markedly reduced survival. Our findings provide evidence for novel microglia functions to protect against IFN-α-mediated neurotoxicity and neuronal dysregulation, as well as restrain calcification as a result of both IL-6- and IFN-α-induced neuroinflammation. Taken together, we demonstrate that CSF1R inhibition may be an undesirable target for therapeutic treatment of neuroinflammatory diseases that are driven by elevated IL-6 and IFN-α production.

ER-851, a Novel Selective Inhibitor of AXL, Overcomes Resistance to Antimitotic Drugs.

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Chronic reduction of store operated Ca2+ entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1)hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.

Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells

The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.

Abstract P225: Dahl Salt-sensitive And Salt-resistant Rats Exhibit Highly Disparate Blood Pressure Responses To Lifetime High Fat Diet Feeding

In an effort to understand obesity associated hypertension, we and others are using Dahl salt-sensitive (SS) rats fed a high fat diet as an experimental model. One reason for choosing this rat strain is that it is well-known that SS rats exhibit accelerated hypertension development not just in response to high salt intake but to numerous other stimuli as well, when compared to Dahl salt resistant (SR) rats. However, it is not known whether this general dichotomous sensitivity to hypertension development in SS and SR rats also extends to the impact of high fat feeding. Therefore, in the present study we tested the hypothesis that high fat feeding would cause a smaller increase in blood pressure in SR rats versus SS rats in both females and males (n=8 each). To explore possible strain differences in the mechanisms known to contribute to obesity associated hypertension, we also compared the activity of the renin angiotensin and sympathetic nervous systems in SS and SR rats. Rats were fed a high fat diet (60% kcal fat) from weaning. Blood pressure was measured by telemetry starting at 12 weeks of age. At 12 weeks, mean arterial pressures (MAP, mmHg) were 109±8 and 158±6 in male SR and SS rats, and 108±1 and 150±5 in female SR and SS rats, respectively. By 18 weeks MAP were 109±7 and 175±4 in male SR and SS rats, and 108±1 and 180±4 in female SR and SS rats, respectively. In males, plasma renin was lower, and aldosterone the same, in SS rats than SR rats. However, SS rats showed larger falls in blood pressure during week-long ACE inhibitor treatment and to acute sympathoinhibition with hexamethonium than did SR rats. In females, plasma renin and aldosterone both were higher in SS rats than SR rats. Female SS rats also showed larger falls in blood pressure to chronic ACE inhibition and acute ganglion blockade than did SR rats. These results confirm that both male and female SR rats are highly resistant to obesity associated hypertension, and that less robust activation by high fat diet of the renin angiotensin and sympathetic nervous systems may be a causative factor.

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice

Background & AimsNon-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues.MethodsBile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL.ResultsAfter BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology.ConclusionsA reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion.Lay summaryNovel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

Abstract EC103: A Novel Nuclear Role For REDD1 In Cardiac Insulin Sensitivity And Resistance

Insulin resistance (IR) is a hallmark of type II diabetes (TIID) and causes cardiac dysfunction independent of hypertension and coronary artery disease, or diabetic cardiomyopathy (DC). Impaired metabolism and energetics are a major cause of DC, however the molecular mechanisms underlying these defects remain ill-defined. Our unbiased RNA sequencing (RNA-Seq) studies identified ‘regulated in development and DNA damage (REDD)1’ as a potential critical regulator of cardiac insulin sensitivity, as a 1.5-fold increase in REDD1 expression was observed in the hearts of mice treated with acute insulin (1 hour (h)) versus vehicle (n=3). REDD1 is an insulin-sensitive, negative regulator of mTORC1 and global REDD1 deletion leads to whole body IR. Interestingly, cardiac REDD1 is also upregulated in several models of IR. Thus, we hypothesized that while REDD1 is critical for cardiac insulin sensitivity, sustained elevated levels contribute to IR via chronic inhibition of mTORC1. Our findings confirm that acute insulin stimulation or chronic high fat diet (HFD) (2 to 16 weeks) induce cardiac REDD1 expression in vivo (1.7-fold±0.1, n=6). In neonatal rat ventricular myocytes (NRVM) or Hap1 cells, insulin (1 h) or palmitate (24 h) also induce REDD1 expression (1.3-fold±0.1, n=3 or 1.6-fold±0.1, n=9, respectively), which indeed inhibits mTORC1. Notably, insulin treatment following HFD or palmitate, does not further enhance REDD1. In addition, subcellular fractionation shows that REDD1 is uniquely detected in nuclear and chromatin fractions and increases with insulin (1.5-fold±0.1, n=6). We confirmed REDD1 nuclear localization via microscopy and chromatin binding via chromatin immunoprecipitation-deep sequencing (ChIP-Seq) (n=5, pooled). In addition, we determined that REDD1 primarily binds the transcription start sites of metabolic genes and increases with insulin, corresponding to reduced gene expression. Overall, our findings suggest that insulin-inducible cardiac REDD1 exists as part of negative feedback loops that prevent the overactivation of mTORC1 and metabolic gene expression, contributing to insulin sensitivity. Further, sustained fat-induced REDD1 expression, may drive IR via chronic inhibition of mTORC1 and metabolic gene expression.