RESUMEN
Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.
Asunto(s)
Ovario , Procesos de Determinación del Sexo , Proteínas WT1 , Animales , Femenino , Masculino , Ratones , Ovario/crecimiento & desarrollo , Procesos de Determinación del Sexo/genética , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Testículo/crecimiento & desarrollo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Isoformas de ProteínasRESUMEN
Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.
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Linfocitos T CD8-positivos , Neoplasias , Animales , Ratones , Antígeno B7-H1 , Diferenciación Celular , Neoplasias/patología , Receptores de QuimiocinaRESUMEN
The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.
Asunto(s)
Sistemas CRISPR-Cas , Cigoto , Ratones , Animales , Sistemas CRISPR-Cas/genética , Intrones/genética , Técnicas de Inactivación de GenesRESUMEN
Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Infecciones por Arterivirus/inmunología , Interferones/metabolismo , Virus Elevador de Lactato Deshidrogenasa/inmunología , Receptores de IgG/metabolismo , Anemia Hemolítica Autoinmune/virología , Animales , Infecciones por Arterivirus/virología , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Ratones Noqueados , FagocitosisRESUMEN
The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/inmunología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Lapatinib/uso terapéutico , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Quinolinas/uso terapéutico , Ratas , Receptor ErbB-2/genética , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Trastuzumab/uso terapéuticoRESUMEN
Here we show, for the first time, spontaneous cortical spreading depolarization (CSD) events - the electrophysiological correlate of the migraine aura - in animals by using the first generated familial hemiplegic migraine type 3 (FHM3) transgenic mouse model. The mutant mice express L263V-mutated α1 subunits in voltage-gated NaV 1.1 sodium channels (Scn1aL263V ). CSDs consistently propagated from visual to motor cortex, recapitulating what has been shown in patients with migraine with aura. This model may be valuable for the preclinical study of migraine with aura and other diseases in which spreading depolarization is a prominent feature.
Asunto(s)
Depresión de Propagación Cortical/fisiología , Trastornos Migrañosos/fisiopatología , Corteza Motora/fisiopatología , Corteza Visual/fisiopatología , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.1/genéticaRESUMEN
Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.
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Adenocarcinoma , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Neoplasias del Colon , Receptores de IgG , Animales , Anticuerpos Monoclonales , Modelos Animales de Enfermedad , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.
Asunto(s)
Anticuerpos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inmunoterapia/métodos , Interleucina-2/uso terapéutico , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Melanoma/terapia , Receptores de IgG/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunización , Masculino , Melanoma/inmunología , Melanoma Experimental , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/inmunología , Receptores de IgG/genética , Receptores Toll-Like/agonistasRESUMEN
Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.
RESUMEN
By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.
RESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Asunto(s)
Secuencia de Bases , Distrofina , Exones , Oligodesoxirribonucleótidos Antisentido , Eliminación de Secuencia , Animales , Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofina/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacologíaRESUMEN
The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-ß-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
Asunto(s)
Proteína Axina/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Vía de Señalización Wnt , Animales , Proteína Axina/genética , Sistemas CRISPR-Cas , Marcación de Gen/métodos , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
AIMS/HYPOTHESIS: Glucocorticoids (GCs) are widely used anti-inflammatory agents that frequently induce side effects, including insulin resistance, diabetes and hypertension. Here, we investigated the contribution of microvascular dysfunction to the development of these adverse effects in healthy men. METHODS: In a randomised, placebo-controlled, dose-response intervention study, 32 healthy normoglycaemic men (age: 21 ± 2 years; BMI: 21.9 ± 1.7 kg/m(2)) were allocated to receive prednisolone 30 mg once daily (n = 12), prednisolone 7.5 mg once daily (n = 12) or placebo (n = 8) for 2 weeks using block randomisation. A central office performed the treatment allocation, and medication was dispersed by the hospital pharmacy that was also blinded. Treatment allocation was kept in concealed envelopes. Participants, study personnel conducting the measures and assessing the outcome were blinded to group assignment. The study was conducted at a university hospital. Primary endpoint was prednisolone-induced changes in microvascular function, which was assessed by capillary microscopy. Insulin sensitivity was determined by hyperinsulinaemic-euglycaemic clamp and postprandial glycaemic excursions by standardised meal tests. RESULTS: Compared with placebo, prednisolone 7.5 mg and 30 mg decreased insulin-stimulated capillary recruitment by 9 ± 4% and 17 ± 3%, respectively (p < 0.01). In addition, prednisolone 7.5 mg and 30 mg reduced insulin sensitivity (M value) by -11.4 ± 4.5 µmol kg(-1) min(-1) and -25.1 ± 4.1 µmol kg(-1) min(-1) (p < 0.001) and increased postprandial glucose levels by 11 ± 5% and 27 ± 9% (p < 0.001), respectively. Only high-dose prednisolone increased systolic blood pressure (6 ± 1.2 mmHg, p = 0.006). Prednisolone-induced changes in insulin-stimulated capillary recruitment were associated with insulin sensitivity (r = +0.76; p < 0.001), postprandial glucose concentrations (r = -0.52; p < 0.03) and systolic blood pressure (r = -0.62; p < 0.001). Prednisolone increased resistin concentrations, which were negatively related to insulin-stimulated capillary recruitment (r = -0.40; p = 0.03). No effects were noted on adiponectin and leptin concentrations. Prednisolone treatment was well tolerated; none of the participants left the study. CONCLUSIONS/INTERPRETATION: Prednisolone-induced impairment of insulin-stimulated capillary recruitment was paralleled by insulin resistance, increased postprandial glucose levels, hypertension and increased circulating resistin concentrations in healthy men. We propose that GC-induced impairments of microvascular function may contribute to the adverse effects of GC treatment on glucose metabolism and blood pressure. TRIAL REGISTRATION: isrctn.org ISRTCN 78149983. FUNDING: The study was funded by the Dutch Top Institute Pharma T1-106.
Asunto(s)
Antiinflamatorios/efectos adversos , Glucemia/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Glucocorticoides/efectos adversos , Adulto , Antiinflamatorios/uso terapéutico , Glucocorticoides/uso terapéutico , Humanos , Masculino , Prednisolona/efectos adversos , Prednisolona/uso terapéutico , Adulto JovenRESUMEN
De novo germline variants in several components of the SWI/SNF-like BAF complex can cause Coffin-Siris syndrome (CSS), Nicolaides-Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD-powered databases to facilitate further genotype-phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype-genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation.
Asunto(s)
Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Cara/anomalías , Estudios de Asociación Genética , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Micrognatismo/diagnóstico , Micrognatismo/genética , Complejos Multiproteicos/genética , Cuello/anomalías , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Exones , Facies , Orden Génico , Humanos , Proteínas Nucleares/genética , Fenotipo , Proteína SMARCB1 , Factores de Transcripción/genéticaRESUMEN
The proline-rich Akt substrate of 40-kDa (PRAS40) has been linked to the regulation of the activity of the mammalian target of rapamycin complex 1 as well as insulin action. Despite these cytosolic functions, PRAS40 was originally identified as nuclear phosphoprotein in Hela cells. This study aimed to detail mechanisms and consequences of the nucleocytosolic trafficking of PRAS40. Sequence analysis identified a potential leucine-rich nuclear export signal (NES) within PRAS40. Incubation of A14 fibroblasts overexpressing human PRAS40 (hPRAS40) resulted in nuclear accumulation of the protein. Furthermore, mutation of the NES mimicked the effects of leptomycin B, a specific inhibitor of nuclear export, on the subcellular localization of hPRAS40. Finally, A14 cells expressing the NES-mutant showed impaired activation of components of the Akt-pathway as well as of the mTORC1 substrate p70 S6 kinase after insulin stimulation. This impaired insulin signaling could be ascribed to reduced protein levels of insulin receptor substrate 1 in cells expressing mutant NES. In conclusion, PRAS40 contains a functional nuclear export signal. Furthermore, enforced nuclear accumulation of PRAS40 impairs insulin action, thereby substantiating the function of this protein in the regulation of insulin sensitivity.
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Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Señales de Exportación Nuclear , Prolina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Núcleo Celular/metabolismo , Humanos , Insulina/metabolismo , Proteínas de la Membrana , Ratones , Mutación , Células 3T3 NIH , Ratas , Proteínas de Saccharomyces cerevisiaeRESUMEN
Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.
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Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Prednisolona/efectos adversos , Prednisolona/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Células 3T3-L1 , Adipoquinas/genética , Animales , Desoxiglucosa/metabolismo , Expresión Génica/efectos de los fármacos , Glucocorticoides/efectos adversos , Glucocorticoides/farmacología , Inmunidad/efectos de los fármacos , Insulina/farmacología , Resistencia a la Insulina , Ratones , Transducción de Señal/efectos de los fármacos , Análisis de Matrices Tisulares , Transcriptoma/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Glucocorticoids (GCs), such as prednisolone (PRED), are widely prescribed anti-inflammatory drugs, but their use may induce glucose intolerance and diabetes. GC-induced beta cell dysfunction contributes to these diabetogenic effects through mechanisms that remain to be elucidated. In this study, we hypothesized that activation of the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress could be one of the underlying mechanisms involved in GC-induced beta cell dysfunction. We report here that PRED did not affect basal insulin release but time-dependently inhibited glucose-stimulated insulin secretion in INS-1E cells. PRED treatment also decreased both PDX1 and insulin expression, leading to a marked reduction in cellular insulin content. These PRED-induced detrimental effects were found to be prevented by prior treatment with the glucocorticoid receptor (GR) antagonist RU486 and associated with activation of two of the three branches of the UPR. Indeed, PRED induced a GR-mediated activation of both ATF6 and IRE1/XBP1 pathways but was found to reduce the phosphorylation of PERK and its downstream substrate eIF2α. These modulations of ER stress pathways were accompanied by upregulation of calpain 10 and increased cleaved caspase 3, indicating that long term exposure to PRED ultimately promotes apoptosis. Taken together, our data suggest that the inhibition of insulin biosynthesis by PRED in the insulin-secreting INS-1E cells results, at least in part, from a GR-mediated impairment in ER homeostasis which may lead to apoptotic cell death.
Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Insulina , Prednisolona , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calpaína/genética , Calpaína/metabolismo , Línea Celular , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mifepristona/farmacología , Fosforilación/efectos de los fármacos , Prednisolona/efectos adversos , Prednisolona/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptores de Glucocorticoides/antagonistas & inhibidores , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/metabolismo , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
OBJECTIVE: Glucocorticoids (GCs) are regarded as diabetogenic because they impair insulin sensitivity and islet-cell function. This study assessed whether treatment with the glucagon-like peptide receptor agonist (GLP-1 RA) exenatide (EXE) could prevent GC-induced glucose intolerance. RESEARCH DESIGN AND METHODS: A randomized, placebo-controlled, double-blind, crossover study in eight healthy men (age: 23.5 [20.0-28.3] years; BMI: 26.4 [24.3-28.0] kg/m(2)) was conducted. Participants received three therapeutic regimens for 2 consecutive days: 1) 80 mg of oral prednisolone (PRED) every day (q.d.) and intravenous (IV) EXE infusion (PRED+EXE); 2) 80 mg of oral PRED q.d. and IV saline infusion (PRED+SAL); and 3) oral placebo-PRED q.d. and intravenous saline infusion (PLB+SAL). On day 1, glucose tolerance was assessed during a meal challenge test. On day 2, participants underwent a clamp procedure to measure insulin secretion and insulin sensitivity. RESULTS: PRED+SAL treatment increased postprandial glucose levels (vs. PLB+SAL, P = 0.012), which was prevented by concomitant EXE (vs. PLB+SAL, P = NS). EXE reduced PRED-induced hyperglucagonemia during the meal challenge (P = 0.018) and decreased gastric emptying (vs. PRED+SAL, P = 0.028; vs. PLB+SAL, P = 0.046). PRED+SAL decreased first-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL, P = 0.017 and P = 0.05, respectively), whereas PRED+EXE improved first- and second-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL; P = 0.017, 0.012, and 0.093, respectively). CONCLUSIONS: The GLP-1 RA EXE prevented PRED-induced glucose intolerance and islet-cell dysfunction in healthy humans. Incretin-based therapies should be explored as a potential strategy to prevent steroid diabetes.