RESUMEN
Loss of functional ß-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces ß-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates ß-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated ß-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in ß-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of ß-cell proliferation, suggesting several unique targets for expansion of functional ß-cell mass.
Asunto(s)
Proliferación Celular , Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/fisiología , Islotes Pancreáticos/citología , Proteínas del Tejido Nervioso/fisiología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Animales , Animales Recién Nacidos , Inmunoprecipitación de Cromatina , Proteínas de Homeodominio/genética , Masculino , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Ratas , Ratas Wistar , Enzimas Ubiquitina-Conjugadoras/metabolismo , Regulación hacia ArribaRESUMEN
Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.
Asunto(s)
Antiinflamatorios/farmacología , Bencimidazoles/farmacología , Benzotiazoles/farmacología , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacología , Inflamación/tratamiento farmacológico , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Tiazoles/química , Células 3T3-L1 , Animales , Antiinflamatorios/síntesis química , Apoptosis/efectos de los fármacos , Bencimidazoles/síntesis química , Benzotiazoles/síntesis química , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Perfilación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Hidrocortisona/síntesis química , Técnicas para Inmunoenzimas , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Metabolómica , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno/efectos de los fármacos , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Enhanced leukocytic infiltration into pancreatic islets contributes to inflammation-based diminutions in functional ß-cell mass. Insulitis (aka islet inflammation), which can be present in both T1DM and T2DM, is one factor influencing pancreatic ß-cell death and dysfunction. IL-1ß, an inflammatory mediator in both T1DM and T2DM, acutely (within 1h) induced expression of the CCL20 gene in rat and human islets and clonal ß-cell lines. Transcriptional induction of CCL20 required the p65 subunit of NF-κB to replace the p50 subunit at two functional κB sites within the CCL20 proximal gene promoter. The NF-κB p50 subunit prevents CCL20 gene expression during unstimulated conditions and overexpression of p50 reduces CCL20, but enhances cyclooxygenase-2 (COX-2), transcript accumulation after exposure to IL-1ß. We also identified differential recruitment of specific co-activator molecules to the CCL20 gene promoter, when compared with the CCL2 and COX2 genes, revealing distinct transcriptional requirements for individual NF-κB responsive genes. Moreover, IL-1ß, TNF-α and IFN-γ individually increased the expression of CCR6, the receptor for CCL20, on the surface of human neutrophils. We further found that the chemokine CCL20 is elevated in serum from both genetically obese db/db mice and in C57BL6/J mice fed a high-fat diet. Taken together, these results are consistent with a possible activation of the CCL20-CCR6 axis in diseases with inflammatory components. Thus, interfering with this signaling pathway, either at the level of NF-κB-mediated chemokine production, or downstream receptor activation, could be a potential therapeutic target to offset inflammation-associated tissue dysfunction in obesity and diabetes.
Asunto(s)
Quimiocina CCL20/genética , Diabetes Mellitus/genética , Inflamación/genética , Obesidad/genética , Factor de Transcripción ReIA/genética , Animales , Quimiocina CCL20/biosíntesis , Quimiocina CCL20/metabolismo , Diabetes Mellitus/patología , Humanos , Inmunidad Innata/genética , Inflamación/patología , Resistencia a la Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ratones , Ratones Obesos , FN-kappa B/genética , Obesidad/metabolismo , Obesidad/fisiopatología , Ratas , Receptores CCR6/genética , Transducción de Señal/genética , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/metabolismoRESUMEN
Proinflammatory cytokines impact islet ß-cell mass and function by altering the transcriptional activity within pancreatic ß-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1ß, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1ß. Nitric oxide production, which is markedly elevated in pancreatic ß-cells exposed to IL-1ß, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1ß-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1ß were dependent on NF-κB transcriptional activity. We conclude that IL-1ß-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating ß-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet ß-cell function and mass.
Asunto(s)
Quimiocinas/metabolismo , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , ARN Mensajero/metabolismo , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocinas/genética , Espectroscopía de Resonancia por Spin del Electrón , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Immunoblotting , Insulina/genética , Secreción de Insulina , Insulinoma , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Consumo de Oxígeno , Neoplasias Pancreáticas , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Ratas Zucker , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Ribosómica S9 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Células Tumorales CultivadasRESUMEN
The CXCL10 gene encodes a peptide that chemoattracts a variety of leukocytes associated with type 1 and type 2 diabetes. The present study was undertaken to determine the molecular mechanisms required for expression of the CXCL10 gene in response to IL-1ß and IFN-γ using rat islets and ß cell lines. IL-1ß induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1ß plus IFN-γ was synergistic. Small interfering RNA-mediated suppression of NF-κB p65 markedly inhibited the ability of cytokines to induce the expression of the CXCL10 gene, whereas targeting STAT1 only diminished the synergy provided by IFN-γ. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-γ-controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr(701). We further discovered that, although the Tyr(701) phosphorylation site is inducible (within 15 min of IFN-γ exposure), the Ser(727) site within STAT1 is constitutively phosphorylated. Thus, we generated single-mutant STAT1 Y701F and double-mutant STAT1 Y701F/S727A adenoviruses. Using these recombinant adenoviruses, we determined that overexpression of either the single- or double-mutant STAT1 decreased the IFN-γ-mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser(727) phosphorylation was neither contingent on a functional Y701 site in ß cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1ß and IFN-γ to induce expression of the CXCL10 gene requires NF-κB, STAT1 phosphorylated at Tyr(701), recruitment of coactivators, and acetylation of histones H3 and H4.
Asunto(s)
Quimiocina CXCL10/genética , Regulación de la Expresión Génica/inmunología , Histonas/metabolismo , Interferón gamma/fisiología , Interleucina-1beta/fisiología , FN-kappa B/fisiología , Factor de Transcripción STAT1/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Células Cultivadas , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL10/metabolismo , Histonas/genética , Humanos , Interferón gamma/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Fosforilación/genética , Fosforilación/inmunología , Ratas , Ratas Wistar , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Tirosina/genética , Tirosina/metabolismoRESUMEN
Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet ß-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of ß-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 ß-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Isocitratos/metabolismo , Ácido Pirúvico/metabolismo , Canales de Potasio Shab/biosíntesis , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Masculino , Modelos Biológicos , Péptidos/farmacología , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/genética , Venenos de Araña/farmacologíaRESUMEN
Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing ß-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1ß markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and ß-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1ß to promote transcription. In addition, IL-1ß induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1ß induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1ß-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic ß-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.
Asunto(s)
Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Animales , Células Cultivadas , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , Ratas , Ratas Wistar , Factor de Transcripción STAT1/genética , Transcripción Genética/efectos de los fármacosRESUMEN
A surface contact model of ring type traveling wave ultrasonic motors is proposed in this paper in order to describe the circular three-dimensional contact and friction problem between the stator and rotor accurately. Differing from previous contact models of traveling wave ultrasonic motors, the flexibility of the rotor, which results in the irregular shape of contact area and inhomogeneous distribution of contact pressure along radial direction, is taken into account in the proposed model. The normal contact between the stator and rotor is analyzed with finite element method and the tangential force transmission on the contact surface is investigated with analytical method. The contact pressures and the tangential stresses on the contact surface are obtained. Mechanical performances of the motor are also studied based upon the proposed model. Finally, torque-speed curves of a prototype motor are measured and compared with the calculated results of the proposed model, and good agreements are obtained. In addition, the proposed surface contact model is compared with previous line contact models. It is found that the calculated results of the surface contact model are closer to measured results than those of line contact models. The comparison results verify the accuracy of the proposed surface contact model.
RESUMEN
A coupled linear ultrasonic motor (LUSM) based on an eccentric constraint was proposed. Two pieces of oblique piezoelectric ceramics were arranged at each end of the elastomer, and the polarization direction of the ceramics was vertically upward. Using the tilting characteristics of the piezoelectric ceramics, the two ends of the fixed piezoelectric ceramics formed an eccentric restraint on the motor, providing conditions for the motor to generate coupled modes. When the elastomer of the motor generated the coupling vibration, the motion trajectories of the driving feet ends were oblique straight lines, and the oblique straight-line motion trajectories of the upper and lower driving feet ends were in opposite directions, driving the upper and lower sliders to run simultaneously. The stator parameters were optimized by using ANSYS to obtain larger amplitudes for the ends of the driving feet in both X and Z directions. The structure and operation principle of the motor are explained in detail. A prototype was fabricated to study the arrangement scheme with fixed constraints at the ends of the motor. The frequency-velocity characteristics, voltage-velocity characteristics, and mechanical characteristics of the motor were tested. The no-load speed and maximum output power were measured to be 45.9 mm/s and 3.24 mW.
RESUMEN
Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.
Asunto(s)
Glucosa/metabolismo , Glutamina/metabolismo , Insulina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Citosol/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Modelos Biológicos , NADP/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A contact model of ring type traveling wave ultrasonic motors is proposed in this paper in order to investigate the dynamic contact and friction drive mechanism between the stator and rotor. Differing from previous contact models of ring type traveling wave ultrasonic motors, the Dahl friction model is adopted and the stator teeth are taken into account in the proposed model. The normal stress, the tangential stress, and the torque-speed characteristics of the motor are evaluated in detail based on the proposed model. Finally, the torque-speed values of the prototype motor are measured and compared with the calculation ones. The results show that the calculation values are in good agreement with the experimental values, which validates the proposed model. Moreover, the proposed contact model is compared with previous contact models that adopt Coulomb friction law and meanwhile ignore stator teeth by assuming teeth surfaces to be continuous. The comparison results show that the proposed model is more accurate than previous models.
Asunto(s)
Ultrasonido/instrumentación , Suministros de Energía Eléctrica , Diseño de Equipo , Fricción , Rotación , TorqueRESUMEN
Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.
Asunto(s)
Ciclo del Ácido Cítrico/fisiología , Glucosa/farmacología , Glutamina/farmacología , Secreción de Insulina/efectos de los fármacos , Animales , Células Cultivadas , Glucosa/metabolismo , Glutamina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Compuestos de Fenilurea/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Sulfonamidas/farmacología , Sumoilación/efectos de los fármacosRESUMEN
OBJECTIVE: The expression of the interleukin-1 receptor type I (IL-1R) is enriched in pancreatic islet ß-cells, signifying that ligands activating this pathway are important for the health and function of the insulin-secreting cell. Using isolated mouse, rat, and human islets, we identified the cytokine IL-1α as a highly inducible gene in response to IL-1R activation. In addition, IL-1α is elevated in mouse and rat models of obesity and Type 2 diabetes. Since less is known about the biology of IL-1α relative to IL-1ß in pancreatic tissue, our objective was to investigate the contribution of IL-1α to pancreatic ß-cell function and overall glucose homeostasis in vivo. METHODS: We generated a novel mouse line with conditional IL-1α alleles and subsequently produced mice with either pancreatic- or myeloid lineage-specific deletion of IL-1α. RESULTS: Using this in vivo approach, we discovered that pancreatic (IL-1αPdx1-/-), but not myeloid-cell, expression of IL-1α (IL-1αLysM-/-) was required for the maintenance of whole body glucose homeostasis in both male and female mice. Moreover, pancreatic deletion of IL-1α led to impaired glucose tolerance with no change in insulin sensitivity. This observation was consistent with our finding that glucose-stimulated insulin secretion was reduced in islets isolated from IL-1αPdx1-/- mice. Alternatively, IL-1αLysM-/- mice (male and female) did not have any detectable changes in glucose tolerance, respiratory quotient, physical activity, or food intake when compared with littermate controls. CONCLUSIONS: Taken together, we conclude that there is an important physiological role for pancreatic IL-1α to promote glucose homeostasis by supporting glucose-stimulated insulin secretion and islet ß-cell mass in vivo.
Asunto(s)
Glucosa/metabolismo , Homeostasis , Secreción de Insulina/fisiología , Interleucina-1alfa/metabolismo , Células Mieloides/metabolismo , Páncreas/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Intolerancia a la Glucosa/metabolismo , Proteínas de Homeodominio , Inflamación , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratas , Receptores de Citocinas , Receptores Tipo I de Interleucina-1/metabolismo , TransactivadoresRESUMEN
The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/efectos de los fármacos , Mitocondrias/enzimología , Animales , Biguanidas/farmacología , Relación Dosis-Respuesta a Droga , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Immunoblotting , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Hepatic GK (glucokinase) plays a key role in maintaining glucose homoeostasis. Many stimuli regulate GK activity by controlling its gene transcription. We hypothesized that endogenous lipophilic molecules modulate hepatic Gck expression. Lipophilic molecules were extracted from rat livers, saponified and re-constituted as an LE (lipophilic extract). LE synergized with insulin to induce primary hepatocyte, but not beta-cell, Gck expression in an SREBP-1c (sterol-regulatory-element-binding protein-1c)-independent manner. The dramatic induction of Gck mRNA resulted in a significant increase in GK activity. Subsequently, the active molecules were identified as retinol and retinal by MS after the purification of the active LE fractions. Retinoids synergized with insulin to induce Gck expression by the activation of both RAR [RA (retinoic acid) receptor] and RXR (retinoid X receptor). Inhibition of RAR activation completely abolished the effect of retinal. The hepatic GK specific activity and Gck mRNA levels of Zucker lean rats fed with a VAD [VA (vitamin A)-deficient] diet were significantly lower than those of rats fed with VAS (VA-sufficient) diet. Additionally, the hepatic Gck mRNA expression of Sprague-Dawley rats fed with a VAD diet was lower than that of rats fed with VA-marginal, -adequate or -supplemented diets. The reduced expression of Gck mRNA was increased after an intraperitoneal dose of RA in VAD rats. Furthermore, an intravenous injection of RA rapidly raised hepatic Gck expression in rats fed with a VAS control diet. Understanding the underlying mechanism that mediates the synergy may be helpful for developing a treatment strategy for patients with diabetes.
Asunto(s)
Glucoquinasa/biosíntesis , Insulina/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Retinoides/farmacología , Animales , Células Cultivadas , Fraccionamiento Químico , Inducción Enzimática/efectos de los fármacos , Glucoquinasa/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/metabolismo , Retinaldehído/farmacología , Receptor alfa de Ácido Retinoico , Receptores X Retinoide/metabolismo , Retinoides/administración & dosificación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Extractos de Tejidos/metabolismo , Vitamina A/farmacología , Deficiencia de Vitamina A/metabolismoRESUMEN
We have developed a single-modal linear motor which contains two kinds of PZT ceramics. The linear motor works by exciting the transverse vibration mode of the PZT ceramic on the upper surface of stator elastomer and the shear vibration mode of PZT ceramics at two ends simultaneously. The operation principle of the motor is analyzed in detail. And the rule of the driving foot amplitude varies with the size of the end protruding structure has been further researched by FEM analysis. The prototype is manufactured, and its operating characteristics are experimentally studied and analyzed. The no-load velocity and the maximum output force are tested to be 169.4 mm/s and 1.1 N, respectively. The results show that extra arrangement of PZT ceramics which works on shear vibration mode can efficiently improve the output of motor.
RESUMEN
A key event in the development of both major forms of diabetes is the loss of functional pancreatic islet ß-cell mass. Strategies aimed at enhancing ß-cell regeneration have long been pursued, but methods for reliably inducing human ß-cell proliferation with full retention of key functions such as glucose-stimulated insulin secretion (GSIS) are still very limited. We have previously reported that overexpression of the homeobox transcription factor NKX6.1 stimulates ß-cell proliferation, while also enhancing GSIS and providing protection against ß-cell cytotoxicity through induction of the VGF prohormone. We developed an NKX6.1 pathway screen by stably transfecting 832/13 rat insulinoma cells with a VGF promoter-luciferase reporter construct, using the resultant cell line to screen a 630,000 compound chemical library. We isolated three compounds with consistent effects to stimulate human islet cell proliferation, but not expression of NKX6.1 or VGF, suggesting an alternative mechanism of action. Further studies of the most potent of these compounds, GNF-9228, revealed that it selectively activates human ß-cell relative to α-cell proliferation and has no effect on δ-cell replication. In addition, pre-treatment, but not short term exposure of human islets to GNF-9228 enhances GSIS. GNF-9228 also protects 832/13 insulinoma cells against ER stress- and inflammatory cytokine-induced cytotoxicity. GNF-9228 stimulates proliferation via a mechanism distinct from recently emergent DYRK1A inhibitors, as it is unaffected by DYRK1A overexpression and does not activate NFAT translocation. In conclusion, we have identified a small molecule with pleiotropic positive effects on islet biology, including stimulation of human ß-cell proliferation and insulin secretion, and protection against multiple agents of cytotoxic stress.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/patología , Glucosa/farmacología , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/patología , Insulinoma/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas , Quinasas DyrKRESUMEN
Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Neuropéptidos/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Humanos , Immunoblotting , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Masculino , Neuropéptidos/genética , Neuropéptidos/metabolismo , Proteína Oncogénica v-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Transfección , Factor Trefoil-3RESUMEN
Carbohydrate metabolism in pregnancy reflects the balance between counterregulatory hormones, which induce insulin resistance, and lactogenic hormones, which stimulate beta-cell proliferation and insulin production. Here we explored the interactions of prolactin (PRL) and glucocorticoids in the regulation of beta-cell gene expression, fatty acid oxidation, and glucose-stimulated insulin secretion (GSIS). In rat insulinoma cells, rat PRL caused 30-50% (P < 0.001) reductions in Forkhead box O (FoxO)-1, peroxisome proliferator activator receptor (PPAR)-gamma coactivator-1alpha (PGC-1alpha), PPARalpha, and carnitine palmitoyltransferase 1 (CPT-1) mRNAs and increased Glut-2 mRNA and GSIS; conversely, dexamethasone (DEX) up-regulated FoxO1, PGC1alpha, PPARalpha, CPT-1, and uncoupling protein 2 (UCP-2) mRNAs in insulinoma cells and inhibited GSIS. Hydrocortisone had similar effects. The effects of DEX were attenuated by coincubation of cells with PRL. In primary rat islets, PRL reduced FoxO1, PPARalpha, and CPT-1 mRNAs, whereas DEX increased FoxO1, PGC1alpha, and UCP-2 mRNAs. The effects of PRL on gene expression were mimicked by constitutive overexpression of signal transducer and activator of transcription-5b. PRL induced signal transducer and activator of transcription-5 binding to a consensus sequence in the rat FoxO1 promoter, reduced nuclear FoxO1 protein levels, and induced its phosphorylation and cytoplasmic redistribution. DEX increased beta-cell fatty acid oxidation and reduced fatty acid esterification; these effects were attenuated by PRL. Thus, lactogens and glucocorticoids have opposing effects on a number of beta-cell genes including FoxO1, PGC1alpha, PPARalpha, CPT-1, and UCP-2 and differentially regulate beta-cell Glut-2 expression, fatty acid oxidation, and GSIS. These observations suggest new mechanisms by which lactogens may preserve beta-cell mass and function and maternal glucose tolerance despite the doubling of maternal cortisol concentrations in late gestation.
Asunto(s)
Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Ácidos Grasos/metabolismo , Glucocorticoides/farmacología , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Embarazo/metabolismo , Prolactina/farmacología , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Dexametasona/farmacología , Interacciones Farmacológicas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Oxidación-Reducción/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Factor de Transcripción STAT5/metabolismoRESUMEN
Type 1 diabetes mellitus (T1DM) results from immune cell-mediated reductions in function and mass of the insulin-producing ß-cells within the pancreatic islets. While the initial trigger(s) that initiates the autoimmune process is unknown, there is a leukocytic infiltration that precedes islet ß-cell death and dysfunction. Herein, we demonstrate that genes encoding the chemokines CXCL9, 10, and 11 are primary response genes in pancreatic ß-cells and are also elevated as part of the inflammatory response in mouse, rat, and human islets. We further established that STAT1 participates in the transcriptional control of these genes in response to the pro-inflammatory cytokines IL-1ß and IFN-γ. STAT1 is phosphorylated within five minutes after ß-cell exposure to IFN-γ, with subsequent occupancy at proximal and distal response elements within the Cxcl9 and Cxcl11 gene promoters. This increase in STAT1 binding is coupled to the rapid appearance of chemokine transcript. Moreover, circulating levels of chemokines that activate CXCR3 are elevated in non-obese diabetic (NOD) mice, consistent with clinical findings in human diabetes. We also report herein that mice with genetic deletion of CXCR3 (receptor for ligands CXCL9, 10, and 11) exhibit a delay in diabetes development after being injected with multiple low doses of streptozotocin. Therefore, we conclude that production of CXCL9, 10, and 11 from islet ß-cells controls leukocyte migration and activity into pancreatic tissue, which ultimately influences islet ß-cell mass and function. © 2016 BioFactors, 42(6):703-715, 2016.