ABSTRACT
In the aftermath of the COVID-19 pandemic, opportunities to modulate biological pathways common to the lifecycles of viruses need to be carefully considered. N-linked glycosylation in humans is mediated exclusively by the oligosaccharyltransferase complex and is frequently hijacked by viruses to facilitate infection. As such, STT3A/B, the catalytic domain of the OST complex, became an intriguing drug target with broad-spectrum antiviral potential. However, due to the critical role N-linked glycosylation plays in a number of fundamental human processes, the toxicological ramifications of STT3A/B inhibition required attention commensurate to that given to antiviral efficacy. Herein, we describe how known STT3A/B inhibitor NGI-1 inspired the discovery of superior tool compounds which were evaluated in in vitro efficacy and translational safety (e.g., CNS, cardiovascular, liver) studies. The described learnings will appeal to those interested in the therapeutic utility of modulating N-linked glycosylation as well as the broader scientific community.
ABSTRACT
Drug development (target identification, advancing drug leads to candidates for preclinical and clinical studies) can be facilitated by genetic and genomic knowledge. Here, we review the contribution of population genomics to target identification, the value of bulk and single cell gene expression analysis for understanding the biological relevance of a drug target, and genome-wide CRISPR editing for the prioritization of drug targets. In genomics, we discuss the different scope of genome-wide association studies using genotyping arrays, versus exome and whole genome sequencing. In transcriptomics, we discuss the information from drug perturbation and the selection of biomarkers. For CRISPR screens, we discuss target discovery, mechanism of action and the concept of gene to drug mapping. Harnessing genetic support increases the probability of drug developability and approval.
Subject(s)
Drug Development , Genomics , Animals , CRISPR-Cas Systems , Drug Development/methods , Gene Editing , Gene Expression Profiling , Genome-Wide Association Study , Genomics/methods , Genotype , Humans , Pharmacogenomic Testing/methods , Single-Cell Analysis , Transcriptome , Whole Genome SequencingABSTRACT
Induced pluripotent stem (iPS) cells offer the exciting opportunity for modeling neurological disorders in vitro in the context of a human genetic background. While significant progress has been made in advancing the use of iPS cell-based disease models, there remains an unmet need to characterize the electrophysiological profile of individual neurons with sufficient throughput to enable statistically robust assessment of disease phenotypes and pharmacological modulation. Here, we describe the Optopatch platform technology that utilizes optogenetics to both stimulate and record action potentials (APs) from human iPS cell-derived excitatory neurons with similar information content to manual patch clamp electrophysiology, but with ~ 3 orders of magnitude greater throughput. Cortical excitatory neurons were produced using the NGN2 transcriptional programming approach and cultured in the presence of rodent glial cells. Characterization of the neuronal preparations using immunocytochemistry and qRT-PCR assays reveals an enrichment of neuronal and glutamatergic markers as well as select ion channels. We demonstrate the scale of our intrinsic cellular excitability assay using pharmacological assessment with select ion channel modulators quinidine and retigabine, by measuring changes in both spike timing and waveform properties. The Optopatch platform in human iPS cell-derived cortical excitatory neurons has the potential for detailed phenotype and pharmacology evaluation, which can serve as the basis of cellular disease model exploration for drug discovery and phenotypic screening efforts.
Subject(s)
Cell Differentiation/physiology , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Neurons/cytology , Action Potentials/physiology , Cells, Cultured , Electrophysiological Phenomena/physiology , Humans , Optogenetics/methodsABSTRACT
Knockout mice studies implicate the mammalian short-chain fatty acid (SCFA) receptors, FFAR2 and FFAR3- in colitis, arthritis and asthma. However, the correlation with human biology is uncertain. Here, we detected FFAR2 and FFAR3 expression in human monocytes via immunohistochemistry. Upon treatment with acetate SCFA or FFAR2- and FFAR3-specific synthetic agonists, human monocytes displayed elevated p38 phosphorylation and attenuated C5, CCL1, CCL2, GM-CSF, IL-1α, IL-1ß and ICAM-1 inflammatory cytokine expression. Acetate and FFAR2 agonist treatment also repressed Akt and ERK2 signalling. Surprisingly, mouse monocytes displayed a distinct response to acetate treatment, elevating GM-CSF, IL-1α, and IL-1ß cytokine expression. This effect persisted in FFAR2/3-knockout mouse monocytes and was not reproduced by synthetic agonists, suggesting a FFAR2/3 independent mechanism in mice. Collectively, we show that SCFAs act via FFAR2/3 to modulate human monocyte inflammatory responses- a pathway that is absent in mouse monocytes.
ABSTRACT
Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.
Subject(s)
Insulin/agonists , Peptides/pharmacology , Protein Engineering , Proteins/agonists , Animals , CHO Cells , Cricetulus , Dose-Response Relationship, Drug , Humans , Mice , Models, Molecular , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistry , Structure-Activity RelationshipABSTRACT
Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.
Subject(s)
Amides/chemistry , Insulin/chemistry , Peptides/chemistry , Proteins/chemistry , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Amides/chemical synthesis , Amides/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , CHO Cells , Cricetulus , Cyclic AMP/metabolism , Gene Expression/drug effects , Humans , Insulin/chemical synthesis , Insulin/pharmacology , Kinetics , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Peptides/chemical synthesis , Peptides/pharmacology , Protein Binding , Protein Domains , Proteins/chemical synthesis , Proteins/pharmacology , Receptors, G-Protein-Coupled/chemistry , Receptors, Peptide/chemistry , Solid-Phase Synthesis Techniques , Static Electricity , Structure-Activity RelationshipABSTRACT
Free fatty acid receptor 2 (FFA2) is expressed on enteroendocrine L cells that release glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) when activated by short-chain fatty acids (SCFAs). Functionally GLP-1 and PYY inhibit gut transit, increase glucose tolerance, and suppress appetite; thus, FFA2 has therapeutic potential for type 2 diabetes and obesity. However, FFA2-selective agonists have not been characterized in vivo. Compound 1 (Cpd 1), a potent FFA2 agonist, was tested for its activity on the following: GLP-1 release, modulation of intestinal mucosal ion transport and transit in wild-type (WT) and FFA2(-/-) tissue, and food intake and glucose tolerance in lean and diet-induced obese (DIO) mice. Cpd 1 stimulated GLP-1 secretion in vivo, but this effect was only detected with dipeptidyl peptidase IV inhibition, while mucosal responses were PYY, not GLP-1, mediated. Gut transit was faster in FFA2(-/-) mice, while Cpd 1 slowed WT transit and reduced food intake and body weight in DIO mice. Cpd 1 decreased glucose tolerance and suppressed plasma insulin in lean and DIO mice, despite FFA2(-/-) mice displaying impaired glucose tolerance. These results suggest that FFA2 inhibits intestinal functions and suppresses food intake via PYY pathways, with limited GLP-1 contribution. Thus, FFA2 may be an effective therapeutic target for obesity but not for type 2 diabetes.
Subject(s)
Eating/drug effects , Gastrointestinal Transit/drug effects , Glucose Intolerance/metabolism , Intestines/drug effects , Peptide YY/metabolism , Receptors, Cell Surface/agonists , Animals , Appetite/drug effects , Cells, Cultured , Eating/physiology , Gastrointestinal Transit/physiology , Glucagon-Like Peptide 1/metabolism , Intestinal Mucosa/metabolism , Mice , Mice, Obese , Obesity/metabolismABSTRACT
The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.
Subject(s)
Colon/metabolism , Eating/drug effects , Eating/physiology , Enteroendocrine Cells/metabolism , Peptide Hormones/metabolism , Peptide Hormones/pharmacology , Animals , Female , Glucagon-Like Peptide 1/metabolism , Humans , Male , Mice , Mice, Knockout , Peptide YY/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolismABSTRACT
UDP sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor P2Y14 (GPR105) was found to bind extracellular UDP and UDP sugars. Little is known about the physiological functions of this G protein-coupled receptor. To study its physiological role, we used a gene-deficient mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung, and uterus. Among other phenotypical differences, knock-out mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets, highlighting P2Y14 as a new modulator of proper insulin secretion.
Subject(s)
Insulin/metabolism , Muscle, Smooth/physiology , Receptors, Purinergic P2Y/physiology , Animals , Base Sequence , DNA Primers , Female , Gastric Emptying , Glucose Intolerance , Insulin Secretion , Islets of Langerhans/metabolism , Mice , Mice, Knockout , Polymerase Chain Reaction , Receptors, Purinergic P2Y/geneticsABSTRACT
GPR81, which exhibits a high degree of homology with GPR109a, has been recently identified as a lactate receptor. Similar to GPR109a, the activation of GPR81 by lactate suppresses lipolysis, suggesting that GPR81 may be a potential drug target for treating dyslipidemia. In addition, the fact that GPR81 is expressed only in adipocytes, whereas GPR109a is expressed in various tissues and cells, including Langerhans cells, which are considered responsible for flushing, indicates that targeting GPR81 could lead to the development of antidyslipidemia agents with a reduced risk of this side effect. However, the pharmacological role of GPR81 remains largely unclear, mainly because of the lack of potent and selective surrogate GPR81 agonists suitable for in vivo studies. In the present study, we showed that lactate-induced suppression of lipolysis in explants of white adipose tissue (WAT) depends on the presence of GPR81. We also performed high-throughput screening (HTS) and identified four novel chemical clusters as GPR81 agonists. Chemical optimization of aminothiazole derivatives led to the discovery of a lead compound with improved potency. The compound inhibited lipolysis in differentiated 3T3-L1 adipocytes. Finally, intraperitoneal administration of this compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. This is the first description of a 50nM GPR81 selective agonist with in vivo efficacy, without the side effect, i.e., flushing. These results suggest that GPR81 is an attractive drug target for treating dyslipidemia without the risk of flushing.
Subject(s)
Adipocytes/drug effects , Adipose Tissue, White/drug effects , Flushing/prevention & control , Hypolipidemic Agents/pharmacology , Lipolysis/drug effects , Receptors, G-Protein-Coupled/agonists , Thiazoles/pharmacology , 3T3-L1 Cells , Adipocytes/metabolism , Adipose Tissue, White/metabolism , Animals , Dose-Response Relationship, Drug , Drug Discovery , High-Throughput Screening Assays , Hypolipidemic Agents/administration & dosage , Hypolipidemic Agents/chemical synthesis , Injections, Intraperitoneal , Lactic Acid/pharmacology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Structure-Activity Relationship , Thiazoles/administration & dosage , Thiazoles/chemical synthesis , TransfectionABSTRACT
BACKGROUND: Gastrointestinal (GI) disorders are commonly associated with chronic conditions such as diabetes, obesity, and hypertension. Direct consequences are obstipation or diarrhea as opposite aspects of the irritable bowel syndrome, and more indirectly, alteration of appetite, feeling of fullness, flatulence, bloatedness, and eventually leading to altered absorption of nutrients. Moreover, GI retention and passage times have been recognized as important factors in determining the release site and hence the bioavailability of orally administered drugs. To facilitate the understanding of physiological and pathological processes involved, it is necessary to monitor the gut motility in animal models. Here, we describe a method for studying the GI transit time using technetium-labeled activated charcoal diethylenetriaminepentaacetic acid (99mTc-Ch-DTPA) detected by single-photon emission computed tomography (SPECT). METHODS: Tc-DTPA was adsorbed onto activated charcoal and administered orally to trypan blue-tainted (n = 4) 129SvEv mice (50 to 80 MBq/animal, n = 11). The exact distribution and movement of radioactivity in the gastrointestinal tract was measured at intervals of 1, 3, 6, 12, and 22 h by SPECT-CT. In addition, in order to validate the imaging of GI transient time, loperamide (0.25 mg/animal, n = 3) was used to delay the GI transit. RESULTS: The transit time measured as the peak radioactivity occurring in the rectum was 6 to 7 h after gavaging of 99mTc-Ch-DTPA. After 1 h, the bolus had passed into the small intestine and entered the cecum and the colon. At 6 and 8 h, the cecum, the ascending, transverse, and descending colon, and the rectum showed significant labeling. Several pellets were stored in the rectum for defecation. After 22 h, little activity remained in the stomach and none was detected in the transverse colon or other GI locations. In contrast, 6 h after administration of loperamide, only the cecum and part of the transverse colon were labeled. After 22 h, both structures retained significant amount of label. This delay has been verified by non-radiolabeled dye trypan blue GI measurements (n = 4). CONCLUSION: Here, we present the first non-invasive study of mouse GI transit time, allowing clear differentiation between vehicle- and loperamide-treated animals. This technique is useful for the investigation of GI motility in mice.
ABSTRACT
BACKGROUND: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. RESULTS: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115, and Gpr116. We show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111, or Gpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. CONCLUSIONS: The aGPCR cluster Gpr110, Gpr111, Gpr115, and Gpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115.
Subject(s)
Evolution, Molecular , Genetic Loci/genetics , Multigene Family/genetics , Receptors, G-Protein-Coupled/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , DNA Primers/genetics , Epithelium/metabolism , Galactosides , Gene Expression Profiling , Humans , Indoles , Kidney/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Skin/metabolism , Species SpecificityABSTRACT
Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.
Subject(s)
Fatty Acids, Volatile/pharmacology , Glucagon-Like Peptide 1/metabolism , Receptors, G-Protein-Coupled/physiology , Animals , Calcium/metabolism , Colon/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Mice , Mice, Inbred C57BL , Signal TransductionABSTRACT
Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.
Subject(s)
Embryo, Mammalian/metabolism , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Receptors, G-Protein-Coupled/genetics , Animals , Cardiovascular Abnormalities/embryology , Cardiovascular Abnormalities/genetics , Cardiovascular Abnormalities/metabolism , Embryo, Mammalian/embryology , Female , Genotype , Humans , Immunohistochemistry , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Myocardium/pathology , Placenta/metabolism , Pregnancy , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
A recessive mutation named Justy was found that abolishes B lymphopoiesis but does not impair other major aspects of hematopoiesis. Transplantation experiments showed that homozygosity for Justy prevented hematopoietic progenitors from generating B cells but did not affect the ability of bone marrow stroma to support B lymphopoiesis. In bone marrow from mutant mice, common lymphoid progenitors and pre-pro-B cells appeared normal, but cells at subsequent stages of B lymphopoiesis were dramatically reduced in number. Under culture conditions that promoted B lymphopoiesis, mutant pre-pro-B cells remained alive and began expressing the B cell marker CD19 but failed to proliferate. In contrast, these cells were able to generate myeloid or T/NK precursors. Genetic and molecular analysis demonstrated that Justy is a point mutation within the Gon4-like (Gon4l) gene, which encodes a protein with homology to transcriptional regulators. This mutation was found to disrupt Gon4l pre-mRNA splicing and dramatically reduce expression of wild-type Gon4l RNA and protein. Consistent with a role for Gon4l in transcriptional regulation, the levels of RNA encoding C/EBPalpha and PU.1 were abnormally high in mutant B cell progenitors. Our findings indicate that the Gon4l protein is required for B lymphopoiesis and may function to regulate gene expression during this process.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Lymphopoiesis/genetics , Mutation/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Base Sequence , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , DNA-Binding Proteins , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Male , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/metabolism , Protein Biosynthesis , RNA Splicing/genetics , Sequence Homology, Amino Acid , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.
Subject(s)
Cell Size , Neuroglia/cytology , Osmosis , Receptors, Purinergic/metabolism , Retina/cytology , Signal Transduction/physiology , 5'-Nucleotidase/deficiency , Adenine/analogs & derivatives , Adenine/pharmacology , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Animals , Barium Compounds/metabolism , Calcium/metabolism , Chlorides/metabolism , Cyclic AMP/metabolism , Drug Combinations , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Inositol 1,4,5-Trisphosphate Receptors/deficiency , Mice , Mice, Knockout , Neuroglia/drug effects , Neurons/drug effects , Neurons/metabolism , Osmolar Concentration , Potassium Channel Blockers/pharmacology , Purinergic Agonists , Purinergic Antagonists , Pyrimidine Nucleotides/pharmacology , Quaternary Ammonium Compounds/pharmacology , Receptors, Purinergic/deficiency , Signal Transduction/drug effects , Signal Transduction/genetics , Thionucleotides/pharmacology , Time Factors , Valerates/pharmacology , Xanthines/pharmacologyABSTRACT
Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.
Subject(s)
Calcium/metabolism , EF Hand Motifs/genetics , Hemorrhage , Membrane Glycoproteins/metabolism , Mutation , Platelet Activation , Thrombocytopenia , Animals , Bone Marrow/pathology , Calcium Channels/metabolism , Fibrosis/pathology , Hemorrhage/genetics , Hemorrhage/metabolism , Megakaryocytes/cytology , Megakaryocytes/metabolism , Membrane Glycoproteins/genetics , Mice , Mice, Inbred Strains , Platelet Activation/genetics , Platelet Membrane Glycoproteins/metabolism , Signal Transduction/physiology , Splenomegaly/metabolism , Stromal Interaction Molecule 1 , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thrombocytopenia/genetics , Thrombocytopenia/metabolismABSTRACT
We sought to identify associations of basal metabolic rate (BMR) with morphological traits in laboratory mice. In order to expand the body mass (BM) range at the intra-strain level, and to minimize relevant genetic variation, we used male and female wild type mice (C3HeB/FeJ) and previously unpublished ENU-induced dwarf mutant littermates (David mice), covering a body mass range from 13.5 g through 32.3 g. BMR was measured at 30 degrees C, mice were killed by means of CO(2 )overdose, and body composition (fat mass and lean mass) was subsequently analyzed by dual X-ray absorptiometry (DEXA), after which mice were dissected into 12 (males) and 10 (females) components, respectively. Across the 44 individuals, 43% of the variation in the basal rates of metabolism was associated with BM. The latter explained 47% to 98% of the variability in morphology of the different tissues. Our results demonstrate that sex is a major determinant of body composition and BMR in mice: when adjusted for BM, females contained many larger organs, more fat mass, and less lean mass compared to males. This could be associated with a higher mass adjusted BMR in females. Once the dominant effects of sex and BM on BMR and tissue mass were removed, and after accounting for multiple comparisons, no further significant association between individual variation in BMR and tissue mass emerged.
Subject(s)
Basal Metabolism/physiology , Body Composition/physiology , Body Mass Index , Dwarfism/physiopathology , Mice, Mutant Strains/physiology , Animals , Basal Metabolism/genetics , Body Composition/genetics , Body Size/genetics , Body Size/physiology , Dwarfism/genetics , Female , Male , Mice , Mice, Inbred C3H , Organ Size/genetics , Organ Size/physiology , PhenotypeABSTRACT
Peroxisome proliferator-activated receptor (PPAR)gamma is a key transcription factor facilitating fat deposition in adipose tissue through its proadipogenic and lipogenic actions. Human patients with dominant-negative mutations in PPARgamma display lipodystrophy and extreme insulin resistance. For this reason it was completely unexpected that mice harboring an equivalent mutation (P465L) in PPARgamma developed normal amounts of adipose tissue and were insulin sensitive. This finding raised important doubts about the interspecies translatability of PPARgamma-related findings, bringing into question the relevance of other PPARgamma murine models. Here, we demonstrate that when expressed on a hyperphagic ob/ob background, the P465L PPARgamma mutant grossly exacerbates the insulin resistance and metabolic disturbances associated with leptin deficiency, yet reduces whole-body adiposity and adipocyte size. In mouse, coexistence of the P465L PPARgamma mutation and the leptin-deficient state creates a mismatch between insufficient adipose tissue expandability and excessive energy availability, unmasking the deleterious effects of PPARgamma mutations on carbohydrate metabolism and replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPARgamma mutations. Thus, adipose tissue expandability is identified as an important factor for the development of insulin resistance in the context of positive energy balance.
Subject(s)
Leptin/deficiency , PPAR gamma/physiology , Adipose Tissue/pathology , Animals , Blood Glucose/metabolism , Gene Expression Profiling , Genes, Lethal , Homozygote , Insulin/blood , Insulin Resistance/genetics , Leptin/genetics , Lipid Metabolism/genetics , Mice , Mice, Obese , PPAR gamma/geneticsABSTRACT
Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.