RESUMEN
Paired box 4 (Pax4) is a key transcription factor involved in the embryonic development of the pancreatic islets of Langerhans. Consisting of a conserved paired box domain and a homeodomain, this transcription factor plays an essential role in early endocrine progenitor cells, where it is necessary for cell-fate commitment towards the insulin-secreting ß cell lineage. Knockout of Pax4 in animal models leads to the absence of ß cells, which is accompanied by a significant increase in glucagon-producing α cells, and typically results in lethality within days after birth. Mutations in Pax4 that cause an impaired Pax4 function are associated with diabetes pathogenesis in humans. In adulthood, Pax4 expression is limited to a distinct subset of ß cells that possess the ability to proliferate in response to heightened metabolic needs. Upregulation of Pax4 expression is known to promote ß cell survival and proliferation. Additionally, ectopic expression of Pax4 in pancreatic islet α cells or δ cells has been found to generate functional ß-like cells that can improve blood glucose regulation in experimental diabetes models. Therefore, Pax4 represents a promising therapeutic target for the protection and regeneration of ß cells in the treatment of diabetes. The purpose of this review is to provide a thorough and up-to-date overview of the role of Pax4 in pancreatic ß cells and its potential as a therapeutic target for diabetes.
Asunto(s)
Diabetes Mellitus , Islotes Pancreáticos , Animales , Humanos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Islotes Pancreáticos/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica , Diabetes Mellitus/genéticaRESUMEN
BACKGROUND/OBJECTIVES: NISCH-STAB1 is a newly identified locus correlated to human waist-hip ratio (WHR), which is a risk indicator of developing obesity-associated diabetes. Our previous studies have shown that Nisch mutant male mice increased glucose tolerance in chow-fed conditions. Thus we hypothesized that Nisch mutant mice will have changes in insulin resistance, adipocytes, hepatic steatosis when mice are fed with high-fat diet (HFD). METHODS: Insulin resistance was assessed in Nisch mutant mice and WT mice fed with high-fat diet (60% by kCal) or chow diet. Whole-body energy metabolism was examined using an indirect calorimeter. Adipose depots including inguinal white adipose tissue (WAT), perigonadal WAT, retroperitoneal WAT, and mesenteric WAT were extracted. Area and eqdiameter of each adipocyte were determined, and insulin signaling was examined as well. Paired samples of subcutaneous and omental visceral adipose tissue were obtained from 400 individuals (267 women, 133 men), and examined the expression of Nischarin. RESULTS: We found that insulin signaling was impaired in major insulin-sensitive tissues of Nisch mutant female mice. When mice were fed with HFD for 15 weeks, the Nisch mutant female mice not only developed severe insulin resistance and decreased glucose tolerance compared with wild-type control mice, but also accumulated more white fat, had larger adipocytes and developed severe hepatic steatosis than wild-type control mice. To link our animal studies to human diseases, we further analyzed Nischarin expression in the paired human samples of visceral and subcutaneous adipose tissue from Caucasians. In humans, we found that Nischarin expression is attenuated in adipose tissue with obesity. More importantly, we found that Nischarin mRNA inversely correlated with parameters of obesity, fat distribution, lipid and glucose metabolism. CONCLUSIONS: Taken together, our data revealed sexual dimorphism of Nischarin in body fat distribution, insulin resistance, and glucose tolerance in mice.
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Adipocitos/patología , Resistencia a la Insulina/fisiología , Grasa Intraabdominal/patología , Metabolismo de los Lípidos/fisiología , Mutación , Obesidad/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Distribución de la Grasa Corporal , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Humanos , Receptores de Imidazolina/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/fisiología , Caracteres SexualesRESUMEN
The transcription factor Pax4 plays a critical role in the determination of α- versus ß-cell lineage during endocrine pancreas development. In this study, we explored whether Pax4 gene transfer into α-cells could convert them into functional ß-cells and thus provide therapeutic benefits for insulin-deficient diabetes. We found that Pax4 delivered by adenoviral vector, Ad5.Pax4, induced insulin expression and reduced glucagon expression in αTC1.9 cells. More importantly, these cells exhibited glucose-stimulated insulin secretion, a key feature of functional ß-cells. When injected into streptozotocin-induced diabetic mice, Pax4-treated αTC1.9 cells significantly reduced blood glucose, and the mice showed better glucose tolerance, supporting that Pax4 gene transfer into αTC1.9 cells resulted in the formation of functional ß-cells. Furthermore, treatment of primary human islets with Ad5.Pax4 resulted in significantly improved ß-cell function. Detection of glucagon(+)/Pax4(+)/Insulin(+) cells argued for Pax4-induced α-to-ß cell transitioning. This was further supported by quantification of glucagon and insulin bi-hormonal cells, which was significantly higher in Pax4-treated islets than in controls. Finally, direct administration of Ad5.Pax4 into the pancreas of insulin-deficient mice ameliorated hyperglycemia. Taken together, our data demonstrate that manipulating Pax4 gene expression represents a viable therapeutic strategy for the treatment of insulin deficient diabetes.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Terapia Genética/métodos , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Factores de Transcripción Paired Box/genética , Animales , Diferenciación Celular , Células Cultivadas , Dependovirus/genética , Diabetes Mellitus Experimental/metabolismo , Vectores Genéticos/administración & dosificación , Glucagón/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patología , RatonesRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase-related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.
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Proteínas Tirosina Quinasas/metabolismo , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/patología , Adulto , Animales , Bleomicina , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/efectos de los fármacos , Miofibroblastos/enzimología , Miofibroblastos/patología , Proteínas Tirosina Quinasas/deficiencia , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Glucagon-like peptide 1 (GLP-1) and glucagon share the same precursor molecule proglucagon, but each arises from a distinct posttranslational process in a tissue-specific manner. Recently, it has been shown that GLP-1 is co-expressed with glucagon in pancreatic islet cells. This study was aimed to investigate the progressive changes of GLP-1 versus glucagon production in pancreatic islets during the course of diabetes development. METHODS: Both type 1 (non-obese diabetes mice) and type 2 (db/db mice) diabetes models were employed in this study. The mice were monitored closely for their diabetes progression and were sacrificed at different stages according to their blood glucose levels. GLP-1 and glucagon expression in the pancreatic islets was examined using immunohistochemistry assays. Quantitative analysis was performed to evaluate the significance of the changes. RESULTS: The ratio of GLP-1-expressing cells to glucagon-expressing cells in the islets showed significant, progressive increase with the development of diabetes in db/db mice. The increase of GLP-1 expression was in agreement with the upregulation of PC1/3 expression in these cells. Interestingly, intra-islet GLP-1 expression was not significantly changed during the development of type 1 diabetes in non-obese diabetes mice. CONCLUSIONS: The study demonstrated that GLP-1 was progressively upregulated in pancreatic islets during type 2 diabetes development. In addition, the data suggest clear differences in intra-islet GLP-1 production between type 1 and type 2 diabetes developments. These differences may have an effect on the clinical and pathophysiological processes of these diseases and may be a target for therapeutic approaches.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Islotes Pancreáticos/metabolismo , Estado Prediabético/metabolismo , Algoritmos , Animales , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Progresión de la Enfermedad , Femenino , Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/patología , Inmunohistoquímica , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Estado Prediabético/patología , Estado Prediabético/fisiopatología , Proproteína Convertasa 1/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND AND AIM: The relationships between the X-ray repair cross-complementing 1 (XRCC1) Arg399Gln polymorphism (rs25487, G > A) and responses to platinum-based chemotherapy of gastric and colorectal cancer patients are controversial. Therefore, we performed a meta-analysis to assess the relationships. METHODS: We retrieved the relevant articles from MEDLINE and EMBASE databases. Fourteen studies with 1618 gastric and colorectal cancer patients were included. Primary outcomes included response rate (RR), progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) or hazard ratio with 95% confidence interval (CI) were estimated. All analyses were performed using the Stata software version 11.0 and Review Manager (v5.0). RESULTS: In the dominant model, the A allele of XRCC1 Arg399Gln polymorphism was associated with reduced RR to platinum-based chemotherapy in all gastric and colorectal cancer patients (A/G + A/Aâ vsâ G/G OR, 0.73; 95% CI, 0.55-0.96) and in Asians (OR, 0.62; 95% CI, 0.44-0.89) but not in Caucasians (OR, 0.92; 95% CI, 0.60-1.42). In addition, stratified analysis for different types of cancers indicated a marginally significant decrease of RR in colorectal cancer patients (OR, 0.68; 95% CI, 0.46-1.00) but not in gastric cancer patients (OR, 0.78; 95% CI, 0.53-1.15). However, we did not observe a significant association between XRCC1 Arg399Gln polymorphism and hazard for PFS and OS for gastric and colorectal cancer patients in all tested models. CONCLUSIONS: XRCC1 Arg399Gln polymorphism may be a valuable genetic marker for platinum-based chemotherapy of gastric and colorectal cancer patients, and more well-designed studies with large samples are needed to confirm our findings.
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Cisplatino/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Bases de Datos Bibliográficas , Compuestos Organoplatinos/uso terapéutico , Farmacogenética , Polimorfismo Genético/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Alelos , Pueblo Asiatico/genética , Neoplasias Colorrectales/mortalidad , Intervalos de Confianza , Humanos , Oportunidad Relativa , Oxaliplatino , Pronóstico , Neoplasias Gástricas/mortalidad , Tasa de Supervivencia , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Aim: Type II diabetes (T2D) stems from insulin resistance, with ß-cell dysfunction as a hallmark in its progression. Studies reveal that ß cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for ß differentiation and survival, thus may be a potential enhancer of ß-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on ß cells were examined. Results: PAX4 gene delivery significantly improved ß-cell survival, and increased ß-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves ß-cell function, thus could provide therapeutic benefits for T2D treatment.
Type II diabetes (T2D) results from insulin resistance, with ß-cell dysfunction playing a pivotal role in its progression. Deficits in ß-cell mass and function have been attributed primarily to ß-cell death through apoptosis; however, recent studies suggest ß-cell failure can also arise from ß-cell dedifferentiation that is, ß cells undergo a loss of mature identity, adopting either progenitor-like or glucagon-producing α cell states during T2D development. Therefore, a strategy preventing ß-cell dedifferentiation while promoting its survival is beneficial for T2D treatment. In this study, we explored whether PAX4, a critical transcription factor for ß differentiation and survival, could alleviate ß-cell dysfunction in human islets derived from T2D patients. To accomplish that, human PAX4 cDNA was delivered into human islets isolated from T2D donors by an adenoviral vector-based vector, Ad5.Pax4 and its effects on ß-cell function were evaluated. The results showed PAX4 expression significantly improved ß-cell survival and increased ß-cell composition in the T2D islets. Notably, PAX4-treated T2D islets exhibited significantly higher basal insulin secretion and glucose-stimulated insulin secretion than control-treated islets. The data demonstrate that PAX4 gene delivery into T2D human islets enhances ß-cell mass and function, and thus may offer therapeutic benefits in the treatment of T2D.
Asunto(s)
Diabetes Mellitus Tipo 2 , Proteínas de Homeodominio , Células Secretoras de Insulina , Insulina , Factores de Transcripción Paired Box , Humanos , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción Paired Box/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/metabolismo , Secreción de Insulina , Técnicas de Transferencia de Gen , Supervivencia Celular , Islotes Pancreáticos/metabolismo , Terapia Genética/métodosRESUMEN
Photocatalytic CO2 reduction to generate high value-added and renewable chemicals is of great potential in facilitating the realization of closed-loop and carbon-neutral hydrogen economy. Stabilizing and accelerating the formation of COCO* intermediate is crucial to achieve high-selectivity ethane production. Herein, a novel 3D/2D NiSe2/g-C3N4 heterostructure that mesoscale hedgehog nickel selenide (NiSe2) grown on the ultrathin g-C3N4 nanosheets were synthesized via a successively high temperature calcination process and in-situ thermal injection method for the first time. The optimum 2.7 % NiSe2/g-C3N4 heterostructure achieved moderate C2H6 generation rate of 46.1 µmol·g-1·h-1 and selectivity of 97.5 % without any additional photosensitizers and sacrificial agents under light illumination. Based on the results of the theoretical calculations and experiments, the improvement of photocatalytic CO2 to C2H6 production and selectivity should be ascribed to the increased visible light absorption ability, unique 3D/2D heterostructures with promoted adsorption of CO2 molecules on the Ni active sites, the type II heterojunction with improved charge transfer dynamics and lowered interfacial transfer resistance, as well as the formation of COCO* key intermediate. This work provides an inspiration to construct efficient photocatalysts for the direct transformation of CO2 to multicarbon products (C2+).
RESUMEN
Oculocerebrorenal syndrome of Lowe (OCRL) gene product is a phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P(2)] 5-phosphatase, and mutations of OCRL cause Lowe syndrome and Dent disease, both of which are frequently associated with hypercalciuria. Transient receptor potential, vanilloid subfamily, subtype 6 (TRPV6) is an intestinal epithelial Ca(2+) channel mediating active Ca(2+) absorption. Hyperabsorption of Ca(2+) was found in patients of Dent disease with increased Ca(2+) excretion. In this study, we tested whether TRPV6 is regulated by OCRL and, if so, to what extent it is altered by Dent-causing OCRL mutations using Xenopus laevis oocyte expression system. Exogenous OCRL decreased TRPV6-mediated Ca(2+) uptake by regulating the function and trafficking of TRPV6 through different domains of OCRL. The PI(4,5)P(2) 5-phosphatase domain suppressed the TRPV6-mediated Ca(2+) transport likely through regulating the PI(4,5)P(2) level needed for TRPV6 function without affecting TRPV6 protein abundance of TRPV6 at the cell surface. The forward trafficking of TRPV6 was decreased by OCRL. The Rab binding domain in OCRL was involved in regulating the trafficking of TRPV6. Knocking down endogenous X. laevis OCRL by antisense approach increased TRPV6-mediated Ca(2+) transport and TRPV6 forward trafficking. All seven Dent-causing OCRL mutations examined exhibited alleviation of the inhibitory effect on TRPV6-mediated Ca(2+) transport together with decreased overall PI(4,5)P(2) 5-phosphatase activity. In conclusion, OCRL suppresses TRPV6 via two separate mechanisms. The disruption of PI(4,5)P(2) 5-phosphatase activity by Dent-causing mutations of OCRL may lead to increased intestinal Ca(2+) absorption and, in turn, hypercalciuria.
Asunto(s)
Calcio/metabolismo , Enfermedad de Dent/metabolismo , Mucosa Intestinal/metabolismo , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Enfermedad de Dent/enzimología , Enfermedad de Dent/genética , Femenino , Técnicas de Silenciamiento del Gen/métodos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Síndrome Oculocerebrorrenal/enzimología , Síndrome Oculocerebrorrenal/genética , Fosfatidato Fosfatasa/genética , Fosfatidato Fosfatasa/fisiología , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica/genética , Transporte de Proteínas/genética , Ratas , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Xenopus laevisRESUMEN
Myosin VI is an actin-based motor protein that is enriched at the postsynaptic density and appears to interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs) via synapse associated protein 97 (SAP97). Here, we find that a Flag epitope-tagged dominant negative construct that inhibits the interaction between SAP97 and myosin VI (Flag-myoVI-DN) causes a dramatic reduction in the number of synapses and the surface expression of AMPARs in cultured hippocampal neurons. Furthermore, we find that Flag-myoVI-DN also prevents the rapid delivery of AMPARs to synapses that can be induced by the transient activation of N-methyl-d-aspartate receptors. The Flag-myoVI-DN induced decrease in surface AMPARs is not because of reduced AMPAR subunit protein synthesis. Using whole-cell recording, we show that Flag-myoVI-DN also prevents the activity-induced increase in miniature excitatory postsynaptic current frequency that is normally associated with recruitment of AMPARs to the cell surface at synaptic sites that lack these receptors (i.e. 'silent' synapses). Together, these results indicate that myosin VI/SAP97 plays an important role in trafficking and activity-dependent recruitment of AMPARs to synapses.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hipocampo/citología , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Aminoquinolinas/farmacología , Animales , Animales Recién Nacidos , Carcinoma/patología , Células Cultivadas , Homólogo 1 de la Proteína Discs Large , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/genética , Cadenas Pesadas de Miosina/genética , Neuronas/citología , Técnicas de Placa-Clamp/métodos , Cloruro de Potasio/farmacología , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/fisiología , Ratas , Sinapsis/efectos de los fármacos , Transfección/métodosRESUMEN
Insulating interlayer between nanoparticles and polymer matrix is crucial for suppressing the dielectric loss of polymer composites. In this study, titanium carbide (TiC) particles were surface modified by polydopamine (PDA), and the obtained PDA@TiC powders were used to reinforce thermoplastic polyurethane (TPU). The results indicate that the PDA@TiC were homogenously dispersed in the matrix compared with the pristine TiC, and that the PDA@TiC/TPU composites show improved dielectric and mechanical properties, i.e., much lower dissipation factors and obviously enhanced dielectric breakdown strength, as well as higher tensile strength and elongation at break as compared to the raw TiC/TPU. The nanoscale PDA interlayer contributes to the dielectric and mechanical enhancements because it not only serves as an insulating shell that prevents TiC particles from direct contacting and suppresses the loss and leakage current to very low levels, but also enhances the interfacial interactions thereby leading to improved mechanical strength and toughness. The prepared flexible PDA@TiC/TPU with high permittivity but low loss will find potential applications in electronic and electrical applications.
RESUMEN
The transcription factor Pax4 plays an essential role in the development of insulin-producing ß cells in pancreatic islets. Ectopic Pax4 expression not only promotes ß cell survival but also induces α-to-ß cell transdifferentiation. This dual functionality of Pax4 makes it an appealing therapeutic target for the treatment of insulin-deficient type 1 diabetes (T1D). In this study, we demonstrated that Pax4 gene delivery by an adenoviral vector, Ad5.Pax4, improved ß cell function of mouse and human islets by promoting islet cell survival and α-to-ß cell transdifferentiation, as assessed by multiple viability assays and lineage-tracing analysis. We then explored the therapeutic benefits of Pax4 gene delivery in the context of islet transplantation using T1D mouse models. Both mouse-to-mouse and human-to-mouse islet transplantation, via either kidney capsule or portal vein, were examined. In all settings, Ad5.Pax4-treated donor islets (mouse or human) showed substantially better therapeutic outcomes. These results suggest that Pax4 gene delivery into donor islets may be considered as an adjunct therapy for islet transplantation, which can either improve the therapeutic outcome of islet transplantation using the same amount of donor islets or allow the use of fewer donor islets to achieve normoglycemia.
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Transdiferenciación Celular , Técnicas de Transferencia de Gen , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Trasplante de Islotes Pancreáticos , Factores de Transcripción Paired Box/genética , Animales , Linaje de la Célula , Supervivencia Celular , Diabetes Mellitus Tipo 1/terapia , Femenino , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Factores de Transcripción Paired Box/metabolismo , Resultado del TratamientoRESUMEN
Testosterone (T) affects ß-cell function in men and women. T is a prohormone that undergoes intracrine conversion in target tissues to the potent androgen dihydrotestosterone (DHT) via the enzyme 5α-reductase (5α-R) or to the active estrogen 17ß-estradiol (E2) via the aromatase enzyme. Using male and female human pancreas sections, we show that the 5α-R type 1 isoform (SRD5A1) and aromatase are expressed in male and female ß-cells. We show that cultured male and female human islets exposed to T produce DHT and downstream metabolites. In these islets, exposure to the 5α-R inhibitors finasteride and dutasteride inhibited T conversion into DHT. We did not detect T conversion into E2 from female islets. However, we detected T conversion into E2 in islets from two out of four male donors. In these donors, exposure to the aromatase inhibitor anastrozole inhibited E2 production. Notably, in cultured male and female islets, T enhanced glucose-stimulated insulin secretion (GSIS). In these islets, exposure to 5α-R inhibitors or the aromatase inhibitor both inhibited T enhancement of GSIS. In conclusion, male and female human islets convert T into DHT and E2 via the intracrine activities of SRD5A1 and aromatase. This process is necessary for T enhancement of GSIS.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Aromatasa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Testosterona/farmacología , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Aromatasa/genética , Células Cultivadas , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/metabolismo , MasculinoRESUMEN
BACKGROUND: A major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection. METHODS: A recombinant Ad5 vector was genetically modified to display a peptide sequence ("rH17d'"), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d' and Ad5.HVR5-rH17d' were constructed by engineering the rH17d' peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively. RESULTS: In vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d', whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d' or Ad5.HVR5-rH17d' showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d' and His6 vectors following their intratumoral injection was similar. CONCLUSIONS: Displaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism.
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Adenovirus Humanos/genética , Activación de Complemento/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Neoplasias/terapia , Adenovirus Humanos/inmunología , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Terapia Genética/efectos adversos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Humanos , Inmunidad Humoral/inmunología , Inyecciones Intralesiones , Hígado/citología , Hígado/inmunología , Masculino , Ratones , Neoplasias/inmunología , Péptidos/genética , Transducción GenéticaRESUMEN
Glucagon-like peptide 1 (GLP-1) is known to suppress glucagon secretion, but the mechanism by which GLP-1 exerts this effect is unclear. In this study, we demonstrated GLP-1 receptor (GLP-1R) expression in α-cells using both antibody-dependent and antibody-independent strategies. A novel α-cell-specific GLP-1R knockout (αGLP-1R-/-) mouse model was created and used to investigate its effects on glucagon secretion and glucose metabolism. Male and female αGLP-1R-/- mice both showed higher nonfasting glucagon levels than their wild-type littermates, whereas insulin and GLP-1 levels remained similar. Female αGLP-1R-/- mice exhibited mild glucose intolerance after an intraperitoneal glucose administration and showed increased glucagon secretion in response to a glucose injection compared with the wild-type animals. Furthermore, using isolated islets, we confirmed that αGLP-1R deletion did not interfere with ß-cell function but affected glucagon secretion in a glucose-dependent bidirectional manner: the αGLP-1R-/- islets failed to inhibit glucagon secretion at high glucose and failed to stimulate glucagon secretion at very low glucose condition. More interestingly, the same phenomenon was recapitulated in vivo under hypoglycemic and postprandial (fed) conditions. Taken together, this study demonstrates that GLP-1 (via GLP-1R in α-cells) plays a bidirectional role, either stimulatory or inhibitory, in glucagon secretion depending on glucose levels.
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Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glucosa/metabolismo , Animales , Femenino , Citometría de Flujo , Receptor del Péptido 1 Similar al Glucagón/genética , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Metallic nanoparticles (NPs) can be used for the diagnosis, imaging, and therapy of tumors and cardiovascular disease. However, targeted delivery of NPs to specific cells remains a major limitation for clinical realization of these potential treatment options. Herein, a novel strategy for the specific coupling of NPs to a targeted adenoviral (Ad) platform to deliver NPs to specific cells is defined. Genetic manipulation of the gene-therapy vector is combined with a specific chemical coupling strategy. In particular, a high-affinity interaction between a sequence of six-histidine amino acid residues genetically incorporated into Ad capsid proteins and nickel(II) nitrilotriacetic acid on the surface of gold NPs is employed. The selective self-assembly of gold NPs and Ad vectors into multifunctional platforms does not negatively affect the targeting of Ad to specific cells. This opens the possibility of using Ad vectors for targeted NP delivery, thereby providing a new type of combinatorial approach for the treatment of diseases that involves both nanotechnology and gene therapy.
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Adenoviridae/genética , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/química , Proteínas de la Cápside/genética , Antígeno Carcinoembrionario/genética , Línea Celular , Expresión Génica , Terapia Genética , Vectores Genéticos , Oro , Células HeLa , Humanos , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Transmisión , Nanotecnología , Espectrofotometría AtómicaRESUMEN
Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.
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Adenoviridae/genética , Proliferación Celular , Viroterapia Oncolítica , Neoplasias Ováricas/terapia , Replicación Viral , Animales , Femenino , Terapia Genética , Vectores Genéticos , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/genética , Transducción Genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite the many potential advantages of Ad vectors for vaccine application, the full utility of current Ad vaccines may be limited by the host anti-vector immune response. Direct incorporation of antigens into the adenovirus capsid offers a new and exciting approach for vaccination strategies; this strategy exploits the inherent antigenicity of the Ad vector. Critical to exploiting Ad in this new context is the placement of antigenic epitopes within the major Ad capsid protein, hexon. In our current study we illustrate that we have the capability to place a range of antigenic epitopes within Ad5 capsid protein hexon hypervariable regions (HVRs) 2 or 5, thus producing viable Ad virions. Our data define the maximal incorporation size at HVR2 or HVR5 as it relates to identical antigenic epitopes. In addition, this data suggests that Ad5 HVR5 is more permissive to a range of insertions. Most importantly, repeated administration of our hexon-modified viruses resulted in a secondary anti-antigen response, whereas minimal secondary effect was present after administration of Ad5 control. Our study describes antigen placement and optimization within the context of the capsid incorporation approach of Ad vaccine employment, thereby broadening this new methodology.
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Adenoviridae/genética , Adenoviridae/inmunología , Antígenos/inmunología , Proteínas de la Cápside/genética , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Vacunas/administración & dosificación , Animales , Antígenos/genética , Proteínas de la Cápside/inmunología , Línea Celular , Epítopos/genética , Epítopos/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas/genética , Vacunas/inmunologíaRESUMEN
Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector. To achieve highly efficient and specific gene delivery, it is often necessary to re-direct Ad5 tropism. Because the capsid protein fiber plays an essential role in directing Ad5 infection, our laboratory attempted to re-target Ad5 through fiber modification. We have developed two strategies in this regard. One is a bi-specific adaptor protein strategy, in which the adaptor protein is designed to bind both the Ad5 fiber and an alternative cell-surface receptor. Another is genetic modification, in which alternative targeting motifs are genetically incorporated into the fiber knob domain so that the Ad5 vectors can infect cells through the alternative receptors. In this chapter, we will focus on the genetic fiber modification strategy and provide a detailed protocol for generation of fiber-modified Ad5 vectors. A series of techniques/procedures used in our laboratory will be described, which include the generation of fiber-modified Ad5 genome by homologous recombination in a bacterial system, rescuing the modified Ad5 viruses, virus amplification and purification, and virus titration.
Asunto(s)
Adenoviridae/genética , Proteínas de la Cápside/genética , Terapia Genética , Vectores Genéticos , Células Cultivadas , Genoma Viral , Humanos , Transducción GenéticaRESUMEN
INTRODUCTION: The etiology of diabetes is mainly attributed to insulin deficiency due to the lack of ß cells (type 1), or to insulin resistance that eventually results in ß cell dysfunction (type 2). Therefore, an ultimate cure for diabetes requires the ability to replace the lost insulin-secreting ß cells. Strategies for regenerating ß cells are under extensive investigation. AREAS COVERED: Herein, the authors first summarize the mechanisms underlying embryonic ß cell development and spontaneous adult ß cell regeneration, which forms the basis for developing ß cell regeneration strategies. Then the rationale and progress of each ß cell regeneration strategy is reviewed. Current ß cell regeneration strategies can be classified into two main categories: in vitro ß cell regeneration using pluripotent stem cells and in vivo reprogramming of non-ß cells into ß cells. Each has its own advantages and disadvantages. EXPERT OPINION: Regenerating ß cells has shown its potential as a cure for the treatment of insulin-deficient diabetes. Much progress has been made, and ß cell regeneration therapy is getting closer to a clinical reality. Nevertheless, more hurdles need to be overcome before any of the strategies suggested can be fully translated from bench to bedside.