RESUMEN
ß-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, ß-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether ß-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-ß-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for ß-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.
Asunto(s)
Diabetes Mellitus Tipo 2 , Dieta Alta en Grasa , Animales , Femenino , Masculino , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Peso Corporal/genética , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/genética , Glucosa/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismoRESUMEN
The ß-cells of the islets of Langerhans are the sole producers of insulin in the human body. In response to rising glucose levels, insulin-containing vesicles inside ß-cells fuse with the plasma membrane and release their cargo. However, the mechanisms regulating this process are only partly understood. Previous evidence indicated reductions in α-catenin elevate insulin release, while reductions in ß-catenin decrease insulin release. α- and ß-catenin contribute to cellular regulation in a range of ways but one is as members of the adherens junction complex. Therefore, we investigated the effects of adherens junctions on insulin release. We show in INS-1E ß-cells knockdown of either E- or N-cadherin had only small effects on insulin secretion, but simultaneous knockdown of both cadherins resulted in a significant increase in basal insulin release to the same level as glucose-stimulated release. This double knockdown also significantly attenuated levels of p120 catenin, a cadherin-binding partner involved in regulating cadherin turnover. Conversely, reducing p120 catenin levels with siRNA destabilized both E- and N-cadherin, and this was also associated with an increase in levels of insulin secreted from INS-1E cells. Furthermore, there were also changes in these cells consistent with higher insulin release, namely reductions in levels of F-actin and increased intracellular free Ca2+ levels in response to KCl-induced membrane depolarization. Taken together, these data provide evidence that adherens junctions play important roles in retaining a pool of insulin secretory vesicles within the cell and establish a role for p120 catenin in regulating this process.
Asunto(s)
Uniones Adherentes , Cateninas , Células Secretoras de Insulina , Insulina , Vesículas Secretoras , Uniones Adherentes/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Cateninas/genética , Cateninas/metabolismo , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Vesículas Secretoras/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Catenina deltaRESUMEN
The presence of adherens junctions and the associated protein ß-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in ß-cells. Evidence indicates that modulation of ß-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from ß-cells but the role of ß-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of ß-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of ß-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of ß-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on ß-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of ß-catenin Ser552 and the effects of these on insulin secretion in ß-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both ß-cell models and mouse islets. Together this identifies phosphorylation of ß-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of ß-catenin to modulation of insulin secretory capacity of ß-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in ß-cells.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/genética , Células Secretoras de Insulina/metabolismo , Insulina/genética , Islotes Pancreáticos/metabolismo , beta Catenina/genética , Quinasas p21 Activadas/genética , Actinas/genética , Actinas/metabolismo , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Animales , Línea Celular Transformada , Disulfuros/farmacología , Regulación de la Expresión Génica , Péptido 1 Similar al Glucagón/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hidrazonas/farmacología , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Isoxazoles/farmacología , Masculino , Ratones , Naftoles/farmacología , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Transducción de Señal , Técnicas de Cultivo de Tejidos , beta Catenina/metabolismo , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismoRESUMEN
AIMS/HYPOTHESIS: The minor A allele of rs373863828 (CREBRF p.Arg457Gln) is associated with increased BMI, but reduced risk of type 2 and gestational diabetes in Polynesian (Pacific peoples and Aotearoa New Zealand Maori) populations. This study investigates the effect of the A allele on insulin release and sensitivity in overweight/obese men without diabetes. METHODS: A mixed meal tolerance test was completed by 172 men (56 with the A allele) of Maori or Pacific ancestry, and 44 (24 with the A allele) had a frequently sampled IVGTT and hyperinsulinaemic-euglycaemic clamp. Mixed linear models with covariates age, ancestry and BMI were used to analyse the association between the A allele of rs373863828 and markers of insulin release and blood glucose regulation. RESULTS: The A allele of rs373863828 is associated with a greater increase in plasma insulin 30 min following a meal challenge without affecting the elevation in plasma glucose or incretins glucagon-like polypeptide-1 or gastric inhibitory polypeptide. Consistent with this point, following an i.v. infusion of a glucose bolus, participants with an A allele had higher early (p < 0.05 at 2 and 4 min) plasma insulin and C-peptide concentrations for a similar elevation in blood glucose as those homozygous for the major (G) allele. Despite increased plasma insulin, rs373863828 genotype was not associated with a significant difference (p > 0.05) in insulin sensitivity index or glucose disposal during hyperinsulinaemic-euglycaemic clamp. CONCLUSIONS/INTERPRETATION: rs373863828-A allele associates with increased glucose-stimulated insulin release without affecting insulin sensitivity, suggesting that CREBRF p.Arg457Gln may increase insulin release to reduce the risk of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Insulina , Alelos , Glucemia , Diabetes Mellitus Tipo 2/genética , Humanos , Insulina/genética , Masculino , Nativos de Hawái y Otras Islas del Pacífico , Proteínas Supresoras de Tumor/genéticaRESUMEN
KEY POINTS: Loss of ß-catenin impairs in vivo and isolated muscle exercise/contraction-stimulated glucose uptake. ß-Catenin is required for exercise-induced skeletal muscle actin cytoskeleton remodelling. ß-Catenin675 phosphorylation during exercise may be intensity dependent. ABSTRACT: The conserved structural protein ß-catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin-stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction-stimulated glucose uptake. Here we investigated whether ß-catenin is involved in regulating exercise/contraction-stimulated glucose uptake. We report that the muscle-specific deletion of ß-catenin induced in adult mice (BCAT-mKO) impairs both exercise- and contraction (isolated muscle)-induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of ß-cateninS675 , and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1 muscle-specific knockout mice did not induce muscle ß-cateninS675 phosphorylation, suggesting exercise intensity-dependent regulation of ß-cateninS675 . Introduction of a non-phosphorylatable S675A mutant of ß-catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise-induced increases in cross-sectional phalloidin staining (F-actin marker) of gastrocnemius muscle was impaired in muscle from BCAT-mKO mice. Collectively our findings suggest that ß-catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.
Asunto(s)
Glucosa , beta Catenina , Animales , Estudios Transversales , Transportador de Glucosa de Tipo 4 , Insulina/metabolismo , Ratones , Contracción Muscular , Músculo Esquelético/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.
Asunto(s)
Resistencia a Antineoplásicos , Melanoma/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Neoplasias Cutáneas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Humanos , Isoenzimas , Melanoma/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Triazinas/uso terapéutico , Regulación hacia Arriba , Vemurafenib/uso terapéuticoRESUMEN
The recent finding that ß-catenin levels play an important rate-limiting role in processes regulating insulin secretion lead us to investigate whether its binding partner α-catenin also plays a role in this process. We find that levels of both α-E-catenin and α-N-catenin are rapidly up-regulated as levels of glucose are increased in rat clonal ß-cell models INS-1E and INS-832/3. Lowering in levels of either α-catenin isoform using siRNA resulted in significant increases in glucose stimulated insulin secretion (GSIS) and this effect was attenuated when ß-catenin levels were lowered indicating these proteins have opposing effects on insulin release. This effect of α-catenin knockdown on GSIS was not due to increases in insulin expression but was associated with increases in calcium influx into cells. Moreover, simultaneous depletion of α-E catenin and α-N catenin decreased the actin polymerisation to a similar degree as latrunculin treatment and inhibition of ARP 2/3 mediated actin branching with CK666 attenuated the α-catenin depletion effect on GSIS. This suggests α-catenin mediated actin remodelling may be involved in the regulation of insulin secretion. Together this indicates that α-catenin and ß-catenin can play opposing roles in regulating insulin secretion, with some degree of functional redundancy in roles of α-E-catenin and α-N-catenin. The finding that, at least in ß-cell models, the levels of each can be regulated in the longer term by glucose also provides a potential mechanism by which sustained changes in glucose levels might impact on the magnitude of GSIS.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Edulcorantes/farmacología , alfa Catenina/metabolismo , Animales , Células Cultivadas , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Isoformas de Proteínas , Ratas , alfa Catenina/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Excessive adipose tissue macrophage accumulation in obesity has been implicated in mediating inflammatory responses that impair glucose homeostasis and promote insulin resistance. Colony-stimulating factor 1 (CSF1) controls macrophage differentiation, and here we sought to determine the effect of a CSF1 receptor inhibitor, PLX3397, on adipose tissue macrophage levels and understand the impact on glucose homeostasis in mice. METHODS: A Ten-week-old mice were fed a chow or high-fat diet for 10 weeks and then treated with PLX3397 via oral gavage (50 mg/kg) every second day for 3 weeks, with subsequent monitoring of glucose tolerance, insulin sensitivity and assessment of adipose tissue immune cells. RESULTS: PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization. CONCLUSIONS: Our results indicate that macrophage infiltration of adipose tissue induced by a high-fat diet may not be the trigger for impairments in whole body glucose homeostasis, and that anti-CSF1 therapies are not likely to be useful as treatments for insulin resistance.
Asunto(s)
Tejido Adiposo , Aminopiridinas/farmacología , Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Macrófagos/efectos de los fármacos , Pirroles/farmacología , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Animales , Dieta Alta en Grasa , Homeostasis/efectos de los fármacos , Ratones , Obesidad , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidoresRESUMEN
BACKGROUND: The CREBRF missense variant (p.Arg457Gln) is paradoxically associated with lower risk of type 2 diabetes, yet higher body mass index (BMI). Here we sought to determine whether this CREBRF variant might be associated with adult height. METHODS: Linear regression was used to analyse the association of the CREBRF minor (A) allele with height in 2286 Maori and Pacific adults living in Aotearoa/New Zealand. A potential type 2 diabetes index event was corrected to account for a bias that may be the cause of paradoxical association between the CREBRF diabetes-protective allele and higher BMI and height. RESULTS: The CREBRF protective allele was associated with increased adult height (ß = 1.25 cm, P = 3.9 × 10-6), with the effect being more pronounced in males. The lower odds of diabetes remained similar when analyses were adjusted for height (OR = 0.67-0.65). We found no evidence of a diabetes index event bias to explain the paradoxical effect of CREBRF with either BMI or height and diabetes. The orthologous CREBRF p.Arg457Gln variant was created in knock-in mice to independently assess the effect of the variant, and length was found to be greater in male mice at 8 weeks of age. CONCLUSION: These data taken together indicate that CREBRF p.Arg457Gln is associated with taller stature in Maori and Pacific adults.
Asunto(s)
Estatura/genética , Nativos de Hawái y Otras Islas del Pacífico/genética , Proteínas Supresoras de Tumor/genética , Adulto , Alelos , Animales , Estudios de Cohortes , Femenino , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación Missense/genética , Nueva ZelandaRESUMEN
A novel peptide stapling method effected by a double thiol-ene reaction between two cysteine residues and a divinyl diester to access stapled peptides with enhanced cell permeability is reported. This diverse chemical tool kit provides facile access to stapled peptides with varying bridge lengths. Stapled Axin mimetics were synthesised by using this stapling method resulting in improved α-helicity relative to the unstapled peptide. Cell penetrating stapled analogues of the SIGK peptide that targets the protein-protein interaction hotspot of Gßγ proteins were also synthesised that exhibited a moderate increase in α-helicity and were cell permeable. This chemoselective peptide stapling method is highly amenable as a facile method to easily modify synthetic α-helical peptides to target intracellular proteins.
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Cisteína/química , Ésteres/química , Péptidos/química , Compuestos de Sulfhidrilo/química , Estructura Secundaria de ProteínaRESUMEN
Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.
Asunto(s)
Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Sulfonamidas/química , Triazinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Trasplante HeterólogoRESUMEN
GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for ß-catenin in this process. In 3T3-L1 adipocytes in which ß-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit ß-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that ß-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.
Asunto(s)
Adipocitos/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Animales , Línea Celular , Membrana Celular/metabolismo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas de la Membrana/metabolismo , RatonesRESUMEN
AIMS/HYPOTHESIS: The A (minor) allele of CREBRF rs373863828 has been associated with increased BMI and reduced risk of type 2 diabetes in the Samoan populations of Samoa and American Samoa. Our aim was to test rs373863828 for associations with BMI and the odds of type 2 diabetes, gout and chronic kidney disease (CKD) in Maori and Pacific (Polynesian) people living in Aotearoa/New Zealand. METHODS: Linear and logistic regression models were used to analyse the association of the A allele of CREBRF rs373863828 with BMI, log-transformed BMI, waist circumference, type 2 diabetes, gout and CKD in 2286 adults. The primary analyses were adjusted for age, sex, the first four genome-wide principal components and (where appropriate) BMI, waist circumference and type 2 diabetes. The primary analysis was conducted in ancestrally defined groups and association effects were combined using meta-analysis. RESULTS: For the A allele of rs373863828, the effect size was 0.038 (95% CI 0.022, 0.055, p = 4.8 × 10-6) for log-transformed BMI, with OR 0.59 (95% CI 0.47, 0.73, p = 1.9 × 10-6) for type 2 diabetes. There was no evidence for an association of genotype with variance in BMI (p = 0.13), and nor was there evidence for associations with serum urate (ß = 0.012 mmol/l, pcorrected = 0.10), gout (OR 1.00, p = 0.98) or CKD (OR 0.91, p = 0.59). CONCLUSIONS/INTERPRETATION: Our results in New Zealand Polynesian adults replicate, with very similar effect sizes, the association of the A allele of rs373863828 with higher BMI but lower odds of type 2 diabetes among Samoan adults living in Samoa and American Samoa.
Asunto(s)
Índice de Masa Corporal , Diabetes Mellitus Tipo 2/prevención & control , Nativos de Hawái y Otras Islas del Pacífico/genética , Obesidad/genética , Polimorfismo de Nucleótido Simple , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/etnología , Diabetes Mellitus Tipo 2/genética , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Nueva Zelanda/epidemiología , Obesidad/diagnóstico , Obesidad/etnología , Fenotipo , Polinesia/etnología , Factores Protectores , Factores de RiesgoRESUMEN
BACKGROUND: While it has been challenging to establish prostate cancer patient-derived xenografts (PDXs), with a take rate of 10-40% and long latency time, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer PDXs using a variety of immune deficient mice. In 2014, the Movember Foundation launched a Global Action Plan 1 (GAP1) project to support an international collaborative prostate cancer PDX program involving eleven groups. Between these Movember consortium members, a total of 98 authenticated human prostate cancer PDXs were available for characterization. Eighty three of these were derived directly from patient material, and 15 were derived as variants of patient-derived material via serial passage in androgen deprived hosts. A major goal of the Movember GAP1 PDX project was to provide the prostate cancer research community with a summary of both the basic characteristics of the 98 available authenticated serially transplantable human prostate cancer PDX models and the appropriate contact information for collaborations. Herein, we report a summary of these PDX models. METHODS: PDX models were established in immunocompromised mice via subcutaneous or subrenal-capsule implantation. Dual-label species (ie, human vs mouse) specific centromere and telomere Fluorescence In Situ Hybridization (FISH) and immuno-histochemical (IHC) staining of tissue microarrays (TMAs) containing replicates of the PDX models were used for characterization of expression of a number of phenotypic markers important for prostate cancer including AR (assessed by IHC and FISH), Ki67, vimentin, RB1, P-Akt, chromogranin A (CgA), p53, ERG, PTEN, PSMA, and epithelial cytokeratins. RESULTS: Within this series of PDX models, the full spectrum of clinical disease stages is represented, including androgen-sensitive and castration-resistant primary and metastatic prostate adenocarcinomas as well as prostate carcinomas with neuroendocrine differentiation. The annotated clinical characteristics of these PDXs were correlated with their marker expression profile. CONCLUSION: Our results demonstrate the clinical relevance of this series of PDXs as a platform for both basic science studies and therapeutic discovery/drug development. The present report provides the prostate cancer community with a summary of the basic characteristics and a contact information for collaborations using these models.
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Xenoinjertos , Trasplante de Neoplasias/métodos , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Biomarcadores de Tumor/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Neoplasias de la Próstata/metabolismoRESUMEN
The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC50 values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein-ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.
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Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Dominio Catalítico , Línea Celular Tumoral , Pruebas de Enzimas , Humanos , Proteínas Inmediatas-Precoces/química , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The phosphatidylinositol 3-kinase (PI3K) pathway is involved in many cellular functions including cell growth, metabolism, and transformation. Hyperactivation of this pathway contributes to tumorigenesis, therefore, PI3K is a major target for anticancer drug discovery. Since the PI3Kα isoform is implicated mostly in cancer, we conducted a high-throughput screening (HTS) campaign using a 3-step PI3K homogenous time-resolved fluorescence assay against this isoform bearing the H1047R mutation. A total of 288,000 synthetic and natural product-derived compounds were screened and of which, we identified 124 initial hits that were further selected by considering the predicted binding mode, relationship to known pan-assay interference compounds and previous descriptions as a lipid kinase inhibitor. A total of 24 compounds were then tested for concentration-dependent responses. These hit compounds provide novel scaffolds that can potentially be optimized to create novel PI3K inhibitors.
Asunto(s)
Isoenzimas/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Isoenzimas/genética , Isoenzimas/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismoRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are major regulators of many cellular functions, and hyperactivation of PI3K cell signalling pathways is a major target for anticancer drug discovery. PI3Kα is the isoform most implicated in cancer, and our aim is to selectively inhibit this isoform, which may be more beneficial than concurrent inhibition of all Class I PI3Ks. We have used structure-guided design to merge high-selectivity and high-affinity characteristics found in existing compounds. Molecular docking, including the prediction of water-mediated interactions, was used to model interactions between the ligands and the PI3Kα affinity pocket. Inhibition was tested using lipid kinase assays, and active compounds were tested for effects on PI3K cell signalling. The first-generation compounds synthesized had IC50 (half maximal inhibitory concentration) values >4â µM for PI3Kα yet were selective for PI3Kα over the other Class I isoforms (ß, δ and γ). The second-generation compounds explored were predicted to better engage the affinity pocket through direct and water-mediated interactions with the enzyme, and the IC50 values decreased by â¼30-fold. Cell signalling analysis showed that some of the new PI3Kα inhibitors were more active in the H1047R mutant bearing cell lines SK-OV-3 and T47D, compared with the E545K mutant harbouring MCF-7 cell line. In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Antineoplásicos/química , Antineoplásicos/farmacología , Dominio Catalítico , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Conformación ProteicaRESUMEN
The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic ß-cells are only partly understood. Here we investigate the involvement of ß-catenin in these processes. Reducing ß-catenin levels using siRNA knockdown attenuated GSIS in a range of ß-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both ß-cell models and isolated islets by short-term exposure to the ß-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases ß-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that ß-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of ß-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating ß-catenin from the plasma membrane pool to the nucleus. We show that ß-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that ß-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that ß-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretoras/metabolismo , beta Catenina/metabolismo , Citoesqueleto de Actina/genética , Animales , Línea Celular , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Ratones , Vesículas Secretoras/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , beta Catenina/genéticaRESUMEN
As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Hidrazonas/síntesis química , Hidrazonas/farmacología , Hidrazonas/toxicidad , Ratones , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/toxicidad , Pirazoles/síntesis química , Pirazoles/toxicidad , Piridinas/síntesis química , Piridinas/toxicidad , SolubilidadRESUMEN
Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.