Impact of global PTP1B deficiency on the gut barrier permeability during NASH in mice.

OBJECTIVE:Non-alcoholic steatohepatitis (NASH) is characterized by a robust pro-inflammatory component at both hepatic and systemic levels together with a disease-specific gut microbiome signature. Protein tyrosine phosphatase 1 B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory signaling cascades. In this study, we have explored the role of PTP1B in the composition of gut microbiota and gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. METHODS:Gut features and barrier permeability were characterized in wild-type (PTP1B WT) and PTP1B-deficient knockout (PTP1B KO) mice fed a chow or methionine/choline-deficient (MCD) diet for 4 weeks. The impact of inflammation was studied in intestinal epithelial and enteroendocrine cells. The secretion of GLP-1 was evaluated in primary colonic cultures and plasma of mice. RESULTS:We found that a shift in the gut microbiota shape, disruption of gut barrier function, higher levels of serum bile acids, and decreased circulating glucagon-like peptide (GLP)-1 are features during NASH. Surprisingly, despite the pro-inflammatory phenotype of global PTP1B-deficient mice, they were partly protected against the alterations in gut microbiota composition during NASH and presented better gut barrier integrity and less permeability under this pathological condition. These effects concurred with higher colonic mucosal inflammation, decreased serum bile acids, and protection against the decrease in circulating GLP-1 levels during NASH compared with their WT counterparts together with increased expression of GLP-2-sensitive genes in the gut. At the molecular level, stimulation of enteroendocrine STC-1 cells with a pro-inflammatory conditioned medium (CM) from lipopolysaccharide (LPS)-stimulated macrophages triggered pro-inflammatory signaling cascades that were further exacerbated by a PTP1B inhibitor. Likewise, the pro-inflammatory CM induced GLP-1 secretion in primary colonic cultures, an effect augmented by PTP1B inhibition. CONCLUSION:Altogether our results have unraveled a potential role of PTP1B in the gut-liver axis during NASH, likely mediated by increased sensitivity to GLPs, with potential therapeutic value

A Genomic Approach for the Identification and Classification of Genes Involved in Cell Wall Formation and its Regulation in Saccharomyces Cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation

Efecto delas vesiculas intracelulares hepaticas en la inflamacion y resistencia a la insulina en la enfermedad del hígado graso no alcoholico

Comunicación presentada en el 45º Congreso anual Asosciacion Española de Estudio del Hígado, celebrado en Madrid (España) del 12 al 14 de febrero de 2020.[Introducción]: La proteína tirosina-fosfatasa 1B (PTP1B) ha sido ampliamente estudiada por su papel como regulador negativo del receptor de insulina, así como de otros receptores de tipo tirosina-quinasa implicados en cascadas inflamatorias. Por lo tanto PTP1B juega papeles distintos sobre células de tipo noinmune y sobre células inmunes, inhibiendo en estas últimas la señalización de tipo pro-inflamatorio. La esteatoheptitis no alcohólica (de sus siglas en inglés NASH) se caracteriza por presentar un fuerte componente inflamatorio tanto a nivel hepático como sistémico, así como un perfil de microbiota intestinal específico. En este trabajo nuestro objetivo ha sido descifrar el papel de PTP1B en el eje intestino-hígado en un modelo preclínico murino de NASH inducido por una dieta deficiente en metionina y colina (MCD). [Métodos]: Se determinaron las características intestinales así como la función de barrera intestinal en animales de fenotipo salvaje y en ratones con una deficiencia sistémica para PTP1B alimentados con dieta estándar o MCD durante 4 semanas. Se estudió el efecto de un ambiente pro-inflamatorio en células enteroendocrinas y de epitelio intestinal. Se analizó la secreción del péptido similar a glucagón (GLP) tipo 1 en cultivos primarios de colon de ratón. Resultados: Observamos cambios en la conformación de la microbiota intestinal, aumento de los niveles séricos de ácidos biliares, disrupción de la función de barrera intestinal y disminución de los niveles circulantes de GLP1 en el modelo murino de NASH. Sorprendentemente, a pesar del fenotipo proinflamatorio del ratón deficiente para PTP1B, éste presentaba una mejor integridad de la barrera intestinal tanto a nivel de la proteína de unión estrecha ZO-1, como de los niveles séricos de endotoxemia y de la permeabilidad al compuesto FITC-dextrano. Además, estos efectos concurren con una protección contra la caída de los niveles de GLP-1 circulante en los ratones deficientes para PTP1B comparados con los ratones control, junto con un incremento en la expresión de genes diana de GLP-2 en el colon. Por otro lado, la inhibición de PTP1B en células enteroendocrinas STC-1 provocó una señalización pro-inflamatoria exacerbada cuando se sometió a dichas células a un daño pro-inflamatorio con medio condicionado (MC) procedente de macrófagos estimulados con lipopolisacárido (LPS). Del mismo modo, el MC pro-inflamatorio indujo la secreción de GLP-1 en cultivos primarios de colon, un efecto que se veía aumentado por la inhibición de PTP1B. [Conclusión]: Nuestros resultados en conjunto muestran un papel potencial de PTP1B en el eje intestinohígado durante el NASH, un efecto que probablemente está mediado por un aumento de la sensibilidad a GLPs, lo cual podría tener un valor terapéu co potencial.Peer reviewe

Efecto de la deficiencia sistémica de la proteína tirosina fosfatasa 1B (PTP1B) sobre la microbiota intestinal y la permeabilidad de barrera durante la esteatohepatitis no alcohólica

Resumen del trabajo presentado al 45º Congreso anual Asociación Española de Estudio del Hígado (AEEH), celebrado en Madrid del 12 al 14 de febrero de 2020.Peer reviewe

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp. Funct. Genomics 2:124–142

Abstract Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of b1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect b1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation

Impact of global PTP1B deficiency on the gut barrier permeability during NASH in mice.

Objective: Non-alcoholic steatohepatitis (NASH) is characterized by a robust pro-inflammatory component at both hepatic and systemic levels together with a disease-specific gut microbiome signature. Protein tyrosine phosphatase 1B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory signaling cascades. In this study we have explored the role of PTP1B in the composition of gut microbiota and gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. Methods: Gut features and barrier permeability were characterized in wild-type (PTP1B WT) and PTP1B-deficient (PTP1B KO) mice fed a chow or methionine/choline-deficient (MCD) diet during 4 weeks. The impact of inflammation was studied in intestinal epithelial and enteroendocrine cells. Secretion of GLP-1 was evaluated in primary colonic cultures and plasma of mice. Results: We found that a shift in the gut microbiota shape, disruption of gut barrier function, higher levels of serum bile acids and decreased circulating glucagon-like peptide (GLP)-1 are features during NASH. Surprisingly, despite the pro-inflammatory phenotype of global PTP1B deficient mice, they were partly protected against the alterations in gut microbiota composition during NASH and presented better gut barrier integrity and less permeability under this pathological condition. These effects concurred with higher colonic mucosal inflammation, decreased serum bile acids, protection against the drop of circulating GLP-1 levels during NASH in comparison to their wild-type counterparts together with increased expression of GLP-2-sensitive genes in the gut. At the molecular level, stimulation of enteroendocrine STC-1 cells with a pro-inflammatory conditioned medium (CM) from lipopolysaccharide (LPS)- stimulated macrophages triggered pro-inflammatory signaling cascades that were further exacerbated by a PTP1B inhibitor. Likewise, the pro-inflammatory CM induced GLP-1 secretion in primary colonic cultures, an effect augmented by PTP1B inhibition. Conclusion: Altogether our results have unraveled a potential role of PTP1B in the gut-liver axis during NASH, likely mediated by increased sensitivity to GLPs, with potential therapeutic value.Wellcome Trust MR