RESUMEN
Bile acids play a major role in the regulation of lipid and energy metabolism. Here we propose the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as a target to prolong postprandial bile acid elevations in plasma. Reducing hepatic clearance of bile acids from plasma by genetic deletion of NTCP moderately increased plasma bile acid levels, reduced diet-induced obesity, attenuated hepatic steatosis, and lowered plasma cholesterol levels. NTCP-G protein-coupled bile acid receptor (TGR5) double knockout mice were equally protected against diet-induced-obesity as NTCP single knockout mice. NTCP knockout mice displayed decreased intestinal fat absorption and a trend towards higher fecal energy output. Furthermore, NTCP deficiency was associated with an increased uncoupled respiration in brown adipose tissue, leading to increased energy expenditure. We conclude that targeting NTCP-mediated bile acid uptake can be a novel approach to treat obesity and obesity-related hepatosteatosis by simultaneously dampening intestinal fat absorption and increasing energy expenditure.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/genética , Obesidad/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Receptores Acoplados a Proteínas G/genética , Simportadores/genética , Aumento de Peso/genética , Tejido Adiposo Pardo/metabolismo , Animales , Ácidos y Sales Biliares/sangre , Glucemia/metabolismo , Peso Corporal , Colesterol/sangre , Grasas de la Dieta/metabolismo , Metabolismo Energético/genética , Hígado Graso/metabolismo , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Absorción Intestinal/genética , Ratones , Ratones Noqueados , Obesidad/metabolismo , Triglicéridos/sangreRESUMEN
Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 µm in DDC-fed mice, 432±280 to 762±288 µm in Atp8b1-G308V mice and from 522±130 to 3625±378 µm in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by â¼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).
Asunto(s)
Colestasis/tratamiento farmacológico , Lipopéptidos/uso terapéutico , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Simportadores/antagonistas & inhibidores , Animales , Ácidos y Sales Biliares/sangre , Colestasis/sangre , Evaluación Preclínica de Medicamentos , Lipopéptidos/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolípidos/metabolismoRESUMEN
Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.
Asunto(s)
Neoplasias Óseas/genética , Condrosarcoma/genética , Glutaratos/metabolismo , Isocitrato Deshidrogenasa/genética , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Condrosarcoma/metabolismo , Condrosarcoma/patología , Cromatografía Liquida , Análisis Mutacional de ADN , Humanos , Isocitrato Deshidrogenasa/metabolismo , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the ß1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of ß1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked ß1 integrin function or knockdown of ß1 switched the migratory behavior of human and mouse E-cadherin-positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor-ß (TGFß) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFß receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in ß1 integrin-deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that ß1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting ß1 integrins may have undesirable effects in TNBC.
Asunto(s)
Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/fisiopatología , Transducción de Señal/fisiología , Neoplasias de la Mama Triple Negativas/fisiopatología , Animales , Western Blotting , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas de Unión al ADN/genética , Citometría de Flujo , Silenciador del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Integrina beta1/genética , Luciferasas , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Imagen de Lapso de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Pez Cebra , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Colitis/inmunología , Colon/inmunología , Mucosa Intestinal/inmunología , Administración Oral , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Colitis/inducido químicamente , Colitis/patología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Memoria Inmunológica , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Bazo/inmunología , Bazo/patologíaRESUMEN
In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits ß-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.