RESUMEN
The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Descubrimiento de Drogas , Hipoglucemiantes/farmacología , Pirroles/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Relación Dosis-Respuesta a Droga , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Ratones , Estructura Molecular , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.
Asunto(s)
Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Fenilpropionatos/química , Fenilpropionatos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Disponibilidad Biológica , Glucemia/análisis , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diseño de Fármacos , Células HEK293 , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacocinética , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéutico , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Ratones Endogámicos C57BL , Fenilpropionatos/farmacocinética , Fenilpropionatos/uso terapéutico , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic ß-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant ß-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Péptido 1 Similar al Glucagón/agonistas , Islotes Pancreáticos/citología , Receptores de la Hormona Gastrointestinal/agonistas , Animales , Línea Celular , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Secreción de Insulina , Cariotipificación , Ratones , Ratones Noqueados , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
The identification of fatty acids as ligands for the G-protein coupled free fatty acid (FFA) receptor family over 10 years ago led to intensive chemistry efforts to find small-molecule ligands for this class of receptors. Identification of potent, selective modulators of the FFA receptors and their utility in medicine has proven challenging, in part due to their complex pharmacology. Nevertheless, ligands have been identified that are sufficient for exploring the therapeutic potential of this class of receptors in rodents and, in the case of FFA1, FFA2, FFA4, and GPR84, also in humans. Expression profiling, the phenotyping of FFA receptor knockout mice, and the results of studies exploring the effects of these ligands in rodents have uncovered a number of indications where engagement of one or a combination of FFA receptors might provide some clinical benefit in areas including diabetes, inflammatory bowel syndrome, Alzheimer's, pain, and cancer. In this chapter, we will review the clinical potential of modulating FFA receptors based on preclinical and in some cases clinical studies with synthetic ligands. In particular, key aspects and challenges associated with small-molecule ligand identification and FFA receptor pharmacology will be addressed with a view of the hurdles that need to be overcome to fully understand the potential of the receptors as therapeutic targets.
Asunto(s)
Ácidos Grasos no Esterificados/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Ligandos , Enfermedades Metabólicas/tratamiento farmacológico , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
GPR40 (FFAR1) and GPR120 (FFAR4) are G-protein-coupled receptors (GPCRs) that are activated by long chain fatty acids (LCFAs). GPR40 is expressed at high levels in islets and mediates the ability of LCFAs to potentiate glucose-stimulated insulin secretion (GSIS). GPR120 is expressed at high levels in colon, adipose, and pituitary, and at more modest levels in pancreatic islets. The role of GPR120 in islets has not been explored extensively. Here, we confirm that saturated (e.g. palmitic acid) and unsaturated (e.g. docosahexaenoic acid (DHA)) LCFAs engage GPR120 and demonstrate that palmitate- and DHA-potentiated glucagon secretion are greatly reduced in isolated GPR120 KO islets. Remarkably, LCFA potentiated glucagon secretion is similarly reduced in GPR40 KO islets. Compensatory changes in mRNA expression of GPR120 in GPR40 KO islets, and vice versa, do not explain that LCFA potentiated glucagon secretion seemingly involves both receptors. LCFA-potentiated GSIS remains intact in GPR120 KO islets. Consistent with previous reports, GPR120 KO mice are hyperglycemic and glucose intolerant; however, our KO mice display evidence of a hyperactive counter-regulatory response rather than insulin resistance during insulin tolerance tests. An arginine stimulation test and a glucagon challenge confirmed both increases in glucagon secretion and liver glucagon sensitivity in GPR120 KO mice relative to WT mice. Our findings demonstrate that GPR120 is a nutrient sensor that is activated endogenously by both saturated and unsaturated long chain fatty acids and that an altered glucagon axis likely contributes to the impaired glucose homeostasis observed in GPR120 KO mice.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Femenino , Humanos , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring ß cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into ß cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that ß cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the ß cell surface and contributes to the increased insulin secretion brought about by ß cell-to-ß cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 ß cell proliferation and insulin content. These results extend the known parallels between the synaptic and ß cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of ß cell function.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Comunicación Celular/fisiología , Proliferación Celular , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Células HEK293 , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Obesidad/metabolismo , Péptido YY/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Energía/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Derivación Gástrica , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Hipotálamo , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/fisiopatología , Péptido YY/fisiología , Pérdida de PesoRESUMEN
Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Secretoras de Insulina/fisiología , Vesículas Sinápticas/fisiología , Factores de Transcripción/fisiología , Animales , Western Blotting , Brefeldino A/farmacología , Proteínas de Unión al ADN/genética , Inmunohistoquímica , Insulina/fisiología , Microscopía Confocal , Células PC12 , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/química , ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción/genética , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/fisiologíaRESUMEN
The composition of the beta-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of beta-cells and neurons substantially overlap. beta-Cells secrete gamma-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the beta-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in beta-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe beta-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 beta-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the beta-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Empalme Alternativo , Animales , Western Blotting , Encéfalo/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Glicoproteínas/genética , Glicoproteínas/metabolismo , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Humanos , Secreción de Insulina , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Neuropéptidos/genética , Neuropéptidos/metabolismo , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.
Asunto(s)
Moléculas de Adhesión Celular/deficiencia , Epitelio Corneal/anomalías , Receptores de Superficie Celular/deficiencia , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Forma de la Célula/genética , Forma de la Célula/fisiología , Cartilla de ADN/genética , Epitelio Corneal/citología , Epitelio Corneal/embriología , Epitelio Corneal/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/deficiencia , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Fenotipo , Embarazo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.
Asunto(s)
Aloxano/farmacología , Glutamato Descarboxilasa/sangre , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Estreptozocina/farmacología , Aloxano/administración & dosificación , Animales , Biomarcadores/sangre , Péptido C/sangre , Muerte Celular , Relación Dosis-Respuesta a Droga , Semivida , Inyecciones Intraperitoneales , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatectomía , Ratas , Ratas Wistar , Estreptozocina/administración & dosificaciónRESUMEN
We have discovered a novel series of isothiazole-based phenylpropanoic acids as GPR120 agonists. Extensive structure-activity relationship studies led to the discovery of a potent GPR120 agonist 4x, which displayed good EC50 values in both calcium and ß-arrestin assays. It also presented good pharmaceutical properties and a favorable PK profile. Moreover, it demonstrated in vivo antidiabetic activity in C57BL/6 DIO mice. Studies in WT and knockout DIO mice showed that it improved glucose handling during an OGTT via GPR120. Overall, 4x possessed promising antidiabetic effect and good safety profile to be a development candidate.
RESUMEN
BACKGROUND: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies. METHODS: A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies. RESULTS: The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples. CONCLUSIONS: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.
Asunto(s)
Glutamato Descarboxilasa/sangre , Fragmentos de Péptidos/sangre , Autoanticuerpos , Autoantígenos/sangre , Diabetes Mellitus/sangre , Diabetes Mellitus/inmunología , Glutamato Descarboxilasa/inmunología , Humanos , Técnicas de Inmunoadsorción , Células Secretoras de Insulina/metabolismo , Fragmentos de Péptidos/inmunología , Plasma/química , Proteínas Recombinantes/sangre , Suero/química , Extractos de Tejidos/químicaRESUMEN
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function. Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions. Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
Asunto(s)
Comunicación Celular/fisiología , Técnicas de Cocultivo/métodos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Línea Celular , Citoplasma/metabolismo , Células HEK293 , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Mapas de Interacción de ProteínasRESUMEN
Neuroligin-2 is a transmembrane, cell-surface protein originally identified as an inhibitory synapse-associated protein in the central nervous system. Neuroligin-2 is also present on the pancreatic beta-cell surface, and there it engages in transcellular interactions that drive functional maturation of the insulin secretory machinery; these are necessary for normal insulin secretion. The effects of neuroligin-2 deficiency on brain and neuronal function and morphology and on behavior and coordination have been extensively characterized using neuroligin-2 knockout mice. The effects of absent neuroligin-2 expression on islet development and function, however, are unknown. Here, to help test whether neuroligin-2 is necessary for normal islet development, we characterized islet morphology in mice lacking neuroligin-2. To test whether-as predicted by our earlier co-culture studies-absence of neuroligin-2 impairs beta cell function, we compared glucose-stimulated insulin secretion by islets from mutant and wild-type mice. Our results show that while islets from neuroligin-2-deficient mice do not to appear to differ architecturally from wild-type islets, they are smaller, fewer in number, and contain beta cells with lower insulin content. Evaluation of transcript levels suggests that upregulation of neuroligin-1 helps compensate for loss of neuroligin-2. Surprisingly, under both basal and stimulating glucose levels, isolated islets from the knockout mice secreted more of their intracellular insulin content. Rat islets with shRNA-mediated neuroligin-2 knockdown also exhibited increased insulin secretion. Neurexin transcript levels were lower in the knockout mice and, consistent with our prior finding that neurexin is a key constituent of the insulin granule docking machinery, insulin granule docking was reduced. These results indicate that neuroligin-2 is not necessary for the formation of pancreatic islets but that neuroligin-2 influences islet size and number. Neuroligin-2-perhaps through its effects on the expression and/or activity of its binding partner neurexin-promotes insulin granule docking, a known constraint on insulin secretion.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/deficiencia , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patología , Proteínas del Tejido Nervioso/deficiencia , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , RatasRESUMEN
The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer.