ABSTRACT
Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.
Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Islets of Langerhans , Mice , Adult , Animals , Humans , Glucagon/genetics , Glucagon/metabolism , Diabetes Mellitus, Type 2/metabolism , Islets of Langerhans/metabolism , Insulin/metabolism , Pancreas/metabolism , MafB Transcription Factor/genetics , MafB Transcription Factor/metabolismABSTRACT
Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.
Subject(s)
Brain/growth & development , Encephalitis/genetics , Endogenous Retroviruses/genetics , Gene Deletion , Tripartite Motif-Containing Protein 28/genetics , Animals , Brain/immunology , Brain/virology , CRISPR-Cas Systems , Cells, Cultured , Encephalitis/immunology , Encephalitis/virology , Endogenous Retroviruses/immunology , Epigenesis, Genetic , Gene Expression Regulation , Histones/metabolism , Mice , Transcriptional ActivationABSTRACT
Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.
Subject(s)
GTPase-Activating Proteins/metabolism , Organogenesis , Pancreas/embryology , Animals , Cell Polarity , Epithelial Cells/metabolism , Laminin/metabolism , Mice , Mice, Knockout , Pancreas/cytology , Pancreas/metabolism , Pancreas, Exocrine/cytology , Pancreas, Exocrine/embryology , Pancreas, Exocrine/metabolism , Stem Cells/metabolism , rho-Associated Kinases/metabolismABSTRACT
Cellulose nanofibrils (CNFs) were obtained by applying a chemical pretreatment consisting of autoclaving the pulp fibers in sodium hydroxide, combined with 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation. Three levels of sodium hypochlorite were applied (2.5, 3.8, and 6.0 mmol/g) to obtain CNF qualities (CNF_2.5, CNF_3.8, and CNF_6.0) with varying content of carboxyl groups, that is, 1036, 1285, and 1593 µmol/g cellulose. The cytotoxicity and skin irritation potential (indirect tests) of the CNFs were determined according to standardized in vitro testing for medical devices. We here demonstrate that autoclaving (121 °C, 20 min), which was used to sterilize the gels, caused a modification of the CNF characteristics. This was confirmed by a reduction in the viscosity of the gels, a morphological change of the nanofibrils, by an increase of the ultraviolet-visible absorbance maxima at 250 nm, reduction of the absolute zeta potential, and by an increase in aldehyde content and reducing sugars after autoclaving. Fourier-transform infrared spectroscopy and wide-angle X-ray scattering complemented an extensive characterization of the CNF gels, before and after autoclaving. The antibacterial properties of autoclaved carboxylated CNFs were demonstrated in vitro (bacterial survival and swimming assays) on Pseudomonas aeruginosa and Staphylococcus aureus. Importantly, a mouse in vivo surgical-site infection model on S. aureus revealed that CNF_3.8 showed pronounced antibacterial effect and performed as good as the antiseptic Prontosan wound gel.
Subject(s)
Nanofibers , Animals , Anti-Bacterial Agents/pharmacology , Cellulose , Mice , Staphylococcus aureus , WoodABSTRACT
INTRODUCTION: Our previous studies have shown that amyloid ß peptide (Aß) is subject to complement-mediated clearance from the peripheral circulation, and that this mechanism is deficient in Alzheimer's disease. The mechanism should be enhanced by Aß antibodies that form immune complexes (ICs) with Aß, and therefore may be relevant to current Aß immunotherapy approaches. METHODS: Multidisciplinary methods were employed to demonstrate enhanced complement-mediated capture of Aß antibody immune complexes compared with Aß alone in both erythrocytes and THP1-derived macrophages. RESULTS: Aß antibodies dramatically increased complement activation and opsonization of Aß, followed by commensurately enhanced Aß capture by human erythrocytes and macrophages. These in vitro findings were consistent with enhanced peripheral clearance of intravenously administered Aß antibody immune complexes in nonhuman primates. DISCUSSION: Together with our previous results, showing significant Alzheimer's disease deficits in peripheral Aß clearance, the present findings strongly suggest that peripheral mechanisms should not be ignored as contributors to the effects of Aß immunotherapy.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides/immunology , Antibodies/blood , Complement System Proteins/metabolism , Erythrocytes/metabolism , Immunotherapy/methods , Alzheimer Disease/immunology , Alzheimer Disease/pathology , Alzheimer Disease/therapy , Amyloid beta-Peptides/administration & dosage , Amyloid beta-Peptides/metabolism , Animals , Cell Adhesion/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Female , Humans , Immunologic Factors , Macaca fascicularis , Macrophages/metabolism , Male , Phagocytosis , THP-1 Cells/metabolism , THP-1 Cells/pathologyABSTRACT
INTRODUCTION: Genome-wide association studies consistently show that single nucleotide polymorphisms (SNPs) in the complement receptor 1 (CR1) gene modestly but significantly alter Alzheimer's disease (AD) risk. Follow-up research has assumed that CR1 is expressed in the human brain despite a paucity of evidence for its function there. Alternatively, erythrocytes contain >80% of the body's CR1, where, in primates, it is known to bind circulating pathogens. METHODS: Multidisciplinary methods were employed. RESULTS: Conventional Western blots and quantitative polymerase chain reaction failed to detect CR1 in the human brain. Brain immunohistochemistry revealed only vascular CR1. By contrast, erythrocyte CR1 immunoreactivity was readily observed and was significantly deficient in AD, as was CR1-mediated erythrocyte capture of circulating amyloid ß peptide. CR1 SNPs associated with decreased erythrocyte CR1 increased AD risk, whereas a CR1 SNP associated with increased erythrocyte CR1 decreased AD risk. DISCUSSION: SNP effects on erythrocyte CR1 likely underlie the association of CR1 polymorphisms with AD risk.
Subject(s)
Amyloid beta-Peptides/metabolism , Polymorphism, Single Nucleotide , Receptors, Complement 3b/genetics , Receptors, Complement 3b/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Apolipoproteins E/genetics , Erythrocytes/metabolism , Female , Genetic Predisposition to Disease , Humans , Male , Microglia/metabolism , Neocortex/metabolism , Prospective Studies , Protein Isoforms , Receptors, Complement 3b/chemistryABSTRACT
Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that ß cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in ß cells inhibits ß cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in ß cells expressing constitutively active Cdc42 partially restores both delamination and ß cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis.
Subject(s)
Actins/metabolism , Cell Differentiation , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Signal Transduction , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , cdc42 GTP-Binding Protein/metabolism , Animals , Animals, Newborn , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Epithelium/metabolism , Humans , Hyperglycemia/metabolism , Hyperglycemia/pathology , Intercellular Junctions/metabolism , Intercellular Junctions/pathology , Mice , Rats , Time-Lapse ImagingABSTRACT
INTRODUCTION: Although amyloid ß peptide (Aß) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role. METHODS: Multidisciplinary methods were used to demonstrate immune adherence capture of Aß by erythrocytes and its deficiency in Alzheimer's disease (AD). RESULTS: Aß was shown to be subject to immune adherence at every step in the pathway. Aß dose-dependently activated serum complement. Complement-opsonized Aß was captured by erythrocytes via CR1. Erythrocytes, Aß, and hepatic Kupffer cells were colocalized in the human liver. Significant deficits in erythrocyte Aß levels were found in AD and mild cognitive impairment patients. DISCUSSION: CR1 polymorphisms elevate AD risk, and >80% of human CR1 is vested in erythrocytes to subserve immune adherence. The present results suggest that this pathway is pathophysiologically relevant in AD.
Subject(s)
Alzheimer Disease/blood , Amyloid beta-Peptides/metabolism , Cognitive Dysfunction/blood , Erythrocytes/metabolism , Peptide Fragments/metabolism , Receptors, Complement/physiology , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/pharmacology , Animals , Case-Control Studies , Cognitive Dysfunction/pathology , Cognitive Dysfunction/physiopathology , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Female , Humans , Liver/metabolism , Liver/pathology , Liver/ultrastructure , Macaca fascicularis/blood , Male , Mental Status and Dementia Tests , Microscopy, Electron , Middle Aged , Peptide Fragments/pharmacology , Protein Binding/drug effects , Receptors, Complement/geneticsABSTRACT
A persistent and nonresolving inflammatory response to accumulating Aß peptide species is a cardinal feature in the development of Alzheimer's disease (AD). In response to accumulating Aß peptide species, microglia, the innate immune cells of the brain, generate a toxic inflammatory response that accelerates synaptic and neuronal injury. Many proinflammatory signaling pathways are linked to progression of neurodegeneration. However, endogenous anti-inflammatory pathways capable of suppressing Aß-induced inflammation represent a relatively unexplored area. Here we report that signaling through the prostaglandin-E2 (PGE2) EP4 receptor potently suppresses microglial inflammatory responses to Aß42 peptides. In cultured microglial cells, EP4 stimulation attenuated levels of Aß42-induced inflammatory factors and potentiated phagocytosis of Aß42. Microarray analysis demonstrated that EP4 stimulation broadly opposed Aß42-driven gene expression changes in microglia, with enrichment for targets of IRF1, IRF7, and NF-κB transcription factors. In vivo, conditional deletion of microglial EP4 in APPSwe-PS1ΔE9 (APP-PS1) mice conversely increased inflammatory gene expression, oxidative protein modification, and Aß deposition in brain at early stages of pathology, but not at later stages, suggesting an early anti-inflammatory function of microglial EP4 signaling in the APP-PS1 model. Finally, EP4 receptor levels decreased significantly in human cortex with progression from normal to AD states, suggesting that early loss of this beneficial signaling system in preclinical AD development may contribute to subsequent progression of pathology.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Inflammation/metabolism , Microglia/metabolism , Peptide Fragments/pharmacology , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Signal Transduction/physiology , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Animals , Cell Survival/drug effects , Cell Survival/physiology , Humans , Inflammation/pathology , Methyl Ethers/pharmacology , Microglia/drug effects , Receptors, Prostaglandin E, EP4 Subtype/agonists , Signal Transduction/drug effectsABSTRACT
Lack or dysfunction of insulin producing ß cells results in the development of type 1 and type 2 diabetes mellitus, respectively. Insulin secretion is controlled by metabolic stimuli (glucose, fatty acids), but also by monoamine neurotransmitters, like dopamine, serotonin, and norepinephrine. Intracellular monoamine levels are controlled by monoamine oxidases (Mao) A and B. Here we show that MaoA and MaoB are expressed in mouse islet ß cells and that inhibition of Mao activity reduces insulin secretion in response to metabolic stimuli. Moreover, analysis of MaoA and MaoB protein expression in mouse and human type 2 diabetic islets shows a significant reduction of MaoB in type 2 diabetic ß cells suggesting that loss of Mao contributes to ß cell dysfunction. MaoB expression was also reduced in ß cells of MafA-deficient mice, a mouse model for ß cell dysfunction, and biochemical studies showed that MafA directly binds to and activates MaoA and MaoB transcriptional control sequences. Taken together, our results show that MaoA and MaoB expression in pancreatic islets is required for physiological insulin secretion and lost in type 2 diabetic mouse and human ß cells. These findings demonstrate that regulation of monoamine levels by Mao activity in ß cells is pivotal for physiological insulin secretion and that loss of MaoB expression may contribute to the ß cell dysfunction in type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Maf Transcription Factors, Large/biosynthesis , Monoamine Oxidase/metabolism , Animals , Cells, Cultured , Humans , Insulin Secretion , Mice , Mice, Inbred C57BL , Mice, Transgenic , Transcriptional ActivationABSTRACT
Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP(1-4) receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2(lox/lox) mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2(lox/lox) and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.
Subject(s)
Brain/metabolism , Inflammation/metabolism , Macrophages/metabolism , Microglia/metabolism , Receptors, Prostaglandin E, EP2 Subtype/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Brain/drug effects , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Inflammation/chemically induced , Inflammation/genetics , Lipopolysaccharides , Macrophages/drug effects , Mice , Mice, Transgenic , Microglia/drug effects , Receptors, Prostaglandin E, EP2 Subtype/geneticsABSTRACT
Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP-Binding Cassette Transporters/antagonists & inhibitors , Cytochrome P-450 Enzyme Inhibitors , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Pharmaceutical Preparations/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Cells, Cultured , Cryopreservation , Drug Interactions , Female , Hepatocytes/drug effects , Hepatocytes/enzymology , Hepatocytes/metabolism , Humans , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Pharmaceutical Preparations/chemistry , Substrate SpecificityABSTRACT
Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Hepatocytes/metabolism , Pharmaceutical Preparations/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/biosynthesis , Animals , Cells, Cultured , Cryopreservation , Female , Fluorescent Antibody Technique , Hepatocytes/drug effects , Humans , Male , Metabolic Clearance Rate , Rats , Rats, Sprague-Dawley , Species Specificity , Tissue DistributionABSTRACT
Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bile/metabolism , Hepatocytes/metabolism , Models, Biological , Pharmaceutical Preparations/metabolism , Animals , Blood Proteins/metabolism , Cells, Cultured , Chromatography, Liquid , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/drug effects , Hepatocytes/enzymology , Male , Metabolic Clearance Rate , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Predictive Value of Tests , Protein Binding , Rats , Rats, Sprague-Dawley , Substrate Specificity , Tandem Mass Spectrometry , Time FactorsABSTRACT
Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na(+)-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved hepatocytes.
Subject(s)
Cryopreservation , Hepatocytes/metabolism , Organic Anion Transporters, Sodium-Dependent/metabolism , Organic Anion Transporters/metabolism , Organic Cation Transport Proteins/metabolism , Pharmaceutical Preparations/metabolism , Symporters/metabolism , Animals , Biological Transport , Blood Proteins/metabolism , Blotting, Western , Cell Culture Techniques , Cells, Cultured , Fluorescent Antibody Technique , Hepatocytes/drug effects , Humans , Metabolic Clearance Rate , Metabolic Detoxication, Phase I , Metabolic Detoxication, Phase II , Microsomes, Liver/metabolism , Pharmaceutical Preparations/blood , Predictive Value of Tests , Protein Binding , Rats , Species SpecificityABSTRACT
Biomass-derived oligo- and polysaccharides may act as elicitors, i.e., bioactive molecules that trigger plant immune responses. This is particularly important to increase the resistance of plants to abiotic and biotic stresses. In this study, cellulose nanofibrils (CNF) gels were obtained by TEMPO-mediated oxidation of unbleached and bleached kraft pulps. The molecular structures were characterized with ESI and MALDI MS. Analysis of the fine sequences was achieved by MS and MS/MS of the water-soluble oligosaccharides obtained by acid hydrolysis of the CNF gels. The analysis revealed the presence of two families: one corresponding to homoglucuronic acid sequences and the other composed by alternating glucose and glucuronic acid units. The CNF gels, alone or with the addition of the water-soluble oligosaccharides, were tested on Chili pepper (Capsicum annuum). Based on the characterization of the gene expression with Next Generation Sequencing (NGS) of the C. annuum's total messenger RNA, the differences in growth of the C. annuum seeds correlated well with the downregulation of the pathways regulating photosynthesis. A downregulation of the response to abiotic factors was detected, suggesting that these gels would improve the resistance of the C. annuum plants to abiotic stress due to, e.g., water deprivation and cold temperatures.
Subject(s)
Capsicum , Cellulose , Gene Expression Regulation, Plant , Nanofibers , Oligosaccharides , Cellulose/chemistry , Oligosaccharides/chemistry , Nanofibers/chemistry , Capsicum/chemistry , Capsicum/genetics , Gene Expression Regulation, Plant/drug effectsABSTRACT
OBJECTIVE: There is significant evidence for a central role of inflammation in the development of Alzheimer disease (AD). Epidemiological studies indicate that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX-1 and COX-2, these findings suggest that downstream prostaglandin signaling pathways function in the preclinical development of AD. Here, we investigate the function of prostaglandin E(2) (PGE(2) ) signaling through its EP3 receptor in the neuroinflammatory response to Aß peptide. METHODS: The function of PGE(2) signaling through its EP3 receptor was examined in vivo in a model of subacute neuroinflammation induced by administration of Aß(42) peptides. Our findings were then confirmed in young adult APPSwe-PS1ΔE9 transgenic mice. RESULTS: Deletion of the PGE(2) EP3 receptor in a model of Aß(42) peptide-induced neuroinflammation reduced proinflammatory gene expression, cytokine production, and oxidative stress. In the APPSwe-PS1ΔE9 model of familial AD, deletion of the EP3 receptor blocked induction of proinflammatory gene and protein expression and lipid peroxidation. In addition, levels of Aß peptides were significantly decreased, as were ß-secretase and ß C-terminal fragment levels, suggesting that generation of Aß peptides may be increased as a result of proinflammatory EP3 signaling. Finally, deletion of EP3 receptor significantly reversed the decline in presynaptic proteins seen in APPSwe-PS1ΔE9 mice. INTERPRETATION: Our findings identify the PGE(2) EP3 receptor as a novel proinflammatory, proamyloidogenic, and synaptotoxic signaling pathway, and suggest a role for COX-PGE(2) -EP3 signaling in the development of AD.
Subject(s)
Alzheimer Disease/pathology , Brain/metabolism , Dinoprostone/metabolism , Encephalitis/metabolism , Gene Expression Regulation/genetics , Signal Transduction/physiology , Age Factors , Alzheimer Disease/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Animals, Newborn , Aspartic Acid Endopeptidases/metabolism , Brain/drug effects , Brain/pathology , Calcium-Binding Proteins , Cells, Cultured , Cognitive Dysfunction/pathology , DNA-Binding Proteins/metabolism , Disease Models, Animal , Encephalitis/chemically induced , Female , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins , Microtubule-Associated Proteins/metabolism , Mutation/genetics , Neurons/drug effects , Neurons/metabolism , Oxidative Stress/drug effects , Oxidative Stress/genetics , Peptide Fragments/pharmacology , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/metabolism , Receptors, Prostaglandin E, EP3 Subtype/deficiency , Signal Transduction/drug effects , Signal Transduction/genetics , Synaptosomal-Associated Protein 25/metabolism , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
The beneficial effects of growth hormone (GH) on memory and learning have previously been confirmed in both humans and in animal models. An important role of GABAB receptors for multiple forms of learning and memory has also been reported. In this study, we examined the effect of GH on the density and functionality of the metabotropic GABAB receptors in the rat brain. Male Sprague-Dawley rats (n = 24) divided into 3 groups were injected twice daily with recombinant human GH (0.07 or 0.7 IU/kg) for 7 days. The effects of the hormone were determined by quantitative autoradiography and by GABAB stimulated [(35)S]-GTPγS binding using the selective GABAB receptor agonist baclofen. The results demonstrate moderate but significant alterations in both receptor density and functionality in a number of brain regions. For example, a dose-dependent upregulation of GABAB receptors was found in the cingulate cortex, primary motor cortex and caudate putamen, whereas attenuation in the receptor density was encountered in, for example, the medial geniculate nucleus. Although the GH-induced effects on the GABAB receptor in brain areas associated with cognition were fairly pronounced, they were significant and we propose that the physiological responses observed after GH administration at least partly can be mediated through a mechanism involving GABAB receptors.
Subject(s)
Brain/drug effects , Brain/metabolism , Human Growth Hormone/pharmacology , Receptors, GABA-B/metabolism , Animals , Baclofen/pharmacology , Dose-Response Relationship, Drug , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Male , Rats , Recombinant Proteins/pharmacology , Sulfur RadioisotopesABSTRACT
Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs.
Subject(s)
Endoderm/embryology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Animals , Cell Differentiation , Cell Proliferation , Embryonic Development , Endoderm/cytology , Endoderm/metabolism , Gene Expression Regulation, Developmental , Gestational Age , Homeodomain Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Morphogenesis/genetics , Morphogenesis/physiology , Pancreas/blood supply , Pancreas/cytology , Pancreas/embryology , Pancreas/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Lysosphingolipid/deficiency , Receptors, Lysosphingolipid/genetics , Sphingosine-1-Phosphate Receptors , Trans-Activators/metabolismABSTRACT
Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in ß-cell stimulus-secretion coupling. We show that pancreatic ß-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in ß-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated ß-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).