RESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuronas/citología , Potenciales de Acción/fisiología , Células Cultivadas , Fenómenos Electrofisiológicos/fisiología , Humanos , Optogenética/métodosRESUMEN
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.
Asunto(s)
Colon/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Células Enteroendocrinas/metabolismo , Hormonas Peptídicas/metabolismo , Hormonas Peptídicas/farmacología , Animales , Femenino , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Péptido YY/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
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.
Asunto(s)
Amidas/química , Insulina/química , Péptidos/química , Proteínas/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Amidas/síntesis química , Amidas/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Insulina/síntesis química , Insulina/farmacología , Cinética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Péptidos/síntesis química , Péptidos/farmacología , Unión Proteica , Dominios Proteicos , Proteínas/síntesis química , Proteínas/farmacología , Receptores Acoplados a Proteínas G/química , Receptores de Péptidos/química , Técnicas de Síntesis en Fase Sólida , Electricidad Estática , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Insulina/metabolismo , Músculo Liso/fisiología , Receptores Purinérgicos P2Y/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Vaciamiento Gástrico , Intolerancia a la Glucosa , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos P2Y/genéticaRESUMEN
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.
Asunto(s)
Antivirales , Proteínas de la Membrana , SARS-CoV-2 , Sialiltransferasas , Antivirales/farmacología , Antivirales/química , Humanos , Animales , SARS-CoV-2/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Sialiltransferasas/antagonistas & inhibidores , Sialiltransferasas/metabolismo , Descubrimiento de Drogas , Tratamiento Farmacológico de COVID-19 , Glicosilación , Ratas , HexosiltransferasasRESUMEN
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.
Asunto(s)
Evolución Molecular , Sitios Genéticos/genética , Familia de Multigenes/genética , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN/genética , Epitelio/metabolismo , Galactósidos , Perfilación de la Expresión Génica , Humanos , Indoles , Riñón/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Piel/metabolismo , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Tamaño de la Célula , Neuroglía/citología , Ósmosis , Receptores Purinérgicos/metabolismo , Retina/citología , Transducción de Señal/fisiología , 5'-Nucleotidasa/deficiencia , Adenina/análogos & derivados , Adenina/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Compuestos de Bario/metabolismo , Calcio/metabolismo , Cloruros/metabolismo , AMP Cíclico/metabolismo , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/deficiencia , Ratones , Ratones Noqueados , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Concentración Osmolar , Bloqueadores de los Canales de Potasio/farmacología , Agonistas Purinérgicos , Antagonistas Purinérgicos , Nucleótidos de Pirimidina/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptores Purinérgicos/deficiencia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tionucleótidos/farmacología , Factores de Tiempo , Valeratos/farmacología , Xantinas/farmacologíaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Motivos EF Hand/genética , Hemorragia , Glicoproteínas de Membrana/metabolismo , Mutación , Activación Plaquetaria , Trombocitopenia , Animales , Médula Ósea/patología , Canales de Calcio/metabolismo , Fibrosis/patología , Hemorragia/genética , Hemorragia/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos , Activación Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Transducción de Señal/fisiología , Esplenomegalia/metabolismo , Molécula de Interacción Estromal 1 , Linfocitos T/citología , Linfocitos T/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismoRESUMEN
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.
Asunto(s)
Desarrollo de Medicamentos , Genómica , Animales , Sistemas CRISPR-Cas , Desarrollo de Medicamentos/métodos , Edición Génica , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Genómica/métodos , Genotipo , Humanos , Pruebas de Farmacogenómica/métodos , Análisis de la Célula Individual , Transcriptoma , Secuenciación Completa del GenomaRESUMEN
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.
Asunto(s)
Leptina/deficiencia , PPAR gamma/fisiología , Tejido Adiposo/patología , Animales , Glucemia/metabolismo , Perfilación de la Expresión Génica , Genes Letales , Homocigoto , Insulina/sangre , Resistencia a la Insulina/genética , Leptina/genética , Metabolismo de los Lípidos/genética , Ratones , Ratones Obesos , PPAR gamma/genéticaRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Etilnitrosourea/toxicidad , Mutación/genética , Receptores Acoplados a Proteínas G/genética , Alquilantes/toxicidad , Animales , Células COS , Chlorocebus aethiops , Análisis Mutacional de ADN/métodos , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Mutagénesis/efectos de los fármacos , Filogenia , Receptor de Melanocortina Tipo 3/genética , Receptor de Melanocortina Tipo 3/fisiología , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/fisiología , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , TransfecciónRESUMEN
Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used anti-breast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a short-term treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMR-based metabolite analysis, we found that TMX-treated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly down-regulates FAS expression and activity as indicated by the accumulation of malonyl-CoA, a known inhibitor of mitochondrial beta-oxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonyl-CoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis.
Asunto(s)
Ácido Graso Sintasas/antagonistas & inhibidores , Ácidos Grasos/biosíntesis , Hígado Graso/inducido químicamente , Hígado/efectos de los fármacos , Tamoxifeno/farmacología , Animales , Peso Corporal/efectos de los fármacos , Colesterol/sangre , Ingestión de Alimentos/efectos de los fármacos , Ácido Graso Sintasas/genética , Hígado Graso/metabolismo , Perfilación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/genética , Hígado/metabolismo , Masculino , Malonil Coenzima A/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
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.
RESUMEN
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.
Asunto(s)
Insulina/agonistas , Péptidos/farmacología , Ingeniería de Proteínas , Proteínas/agonistas , Animales , Células CHO , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Tránsito Gastrointestinal/efectos de los fármacos , Intolerancia a la Glucosa/metabolismo , Intestinos/efectos de los fármacos , Péptido YY/metabolismo , Receptores de Superficie Celular/agonistas , Animales , Apetito/efectos de los fármacos , Células Cultivadas , Ingestión de Alimentos/fisiología , Tránsito Gastrointestinal/fisiología , Péptido 1 Similar al Glucagón/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Obesos , Obesidad/metabolismoRESUMEN
The SMA1-mouse is a novel ethyl-nitroso-urea (ENU)-induced mouse mutant that carries an a-->g missense mutation in exon 5 of the GH gene, which translates to a D167G amino acid exchange in the mature protein. Mice carrying the mutation are characterized by dwarfism, predominantly due to the reduction (sma1/+) or absence (sma1/sma1) of the GH-mediated peripubertal growth spurt, with sma1/+ mice displaying a less pronounced phenotype. All genotypes are viable and fertile, and the mode of inheritance is in accordance with a semidominant Mendelian trait. Adult SMA1 mice accumulate excessive amounts of sc and visceral fat in the presence of elevated plasma ghrelin levels, possibly reflecting altered energy partitioning. Our results suggest impaired storage and/or secretion of pituitary GH in mutants, resulting in reduced pituitary GH and reduced GH-stimulated IGF-1 expression. Generation and identification of the SMA1 mouse exemplifies the power of the combination of random mouse mutagenesis with a highly detailed phenotype-analysis as a successful strategy for the detection and analysis of novel gene-function relationships.
Asunto(s)
Enanismo/genética , Hormona del Crecimiento/genética , Mutación Missense , Obesidad/genética , Hormonas Peptídicas/sangre , Tejido Adiposo , Secuencia de Aminoácidos , Animales , Composición Corporal/genética , Peso Corporal/genética , Exones , Femenino , Fertilidad , Ligamiento Genético , Genotipo , Ghrelina , Hormona del Crecimiento/análisis , Hormona del Crecimiento/química , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Fenotipo , Hipófisis/química , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Adipocitos/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Rubor/prevención & control , Hipolipemiantes/farmacología , Lipólisis/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Tiazoles/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Hipolipemiantes/administración & dosificación , Hipolipemiantes/síntesis química , Inyecciones Intraperitoneales , Ácido Láctico/farmacología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-Actividad , Tiazoles/administración & dosificación , Tiazoles/síntesis química , TransfecciónRESUMEN
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.
RESUMEN
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.
Asunto(s)
Ácidos Grasos Volátiles/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Animales , Calcio/metabolismo , Colon/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
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.