RESUMEN
Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. Here, we report that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1α induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1α can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Dieta Alta en Grasa , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Resistencia a la Insulina , Obesidad/metabolismo , Oxígeno/metabolismo , Translocador 2 del Nucleótido Adenina/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Ácidos Grasos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inflamación/metabolismo , Ácido Láctico/metabolismo , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismoRESUMEN
1. Leukotriene B4 (LTB4) is a proinflammatory mediator important in the progression of a number of inflammatory diseases. Preclinical models can explore the role of LTB4 in pathophysiology using tool compounds, such as CP-105696, that modulate its activity. To support preclinical pharmacology studies, micro-sampling techniques and mathematical modeling were used to determine the pharmacokinetics of CP-105696 in mice within the context of systemic inflammation induced by a high-fat diet (HFD). 2. Following oral administration of doses > 35 mg/kg, CP-105696 kinetics can be described by a one-compartment model with first order absorption. The compound's half-life is 44-62 h with an apparent volume of distribution of 0.51-0.72 L/kg. Exposures in animals fed an HFD are within 2-fold of those fed a normal chow diet. Daily dosing at 100 mg/kg was not tolerated and resulted in a >20% weight loss in the mice. 3. CP-105696's long half-life has the potential to support a twice weekly dosing schedule. Given that most chronic inflammatory diseases will require long-term therapies, these results are useful in determining the optimal dosing schedules for preclinical studies using CP-105696.
Asunto(s)
Benzopiranos/farmacocinética , Ácidos Carboxílicos/farmacocinética , Leucotrieno B4/antagonistas & inhibidores , Administración Oral , Animales , Dieta Alta en Grasa , Semivida , Inflamación , Ratones , Modelos Biológicos , NeutrófilosRESUMEN
Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) and loss (Lrp6(-)) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6(-/-) MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6(-/-) embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1.
Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Tubo Neural/embriología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alelos , Animales , Polaridad Celular , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Células 3T3 NIH , Cresta Neural/metabolismo , Tubo Neural/fisiología , Neurulación/genética , Embarazo , Proteínas Wnt/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
It is generally accepted that longer microsatellites mutate more frequently in defective DNA mismatch repair (MMR) than shorter microsatellites. Indeed, we have previously observed that the A10 microsatellite of transforming growth factor beta type II receptor (TGFBR2) frameshifts -1 bp at a faster rate than the A8 microsatellite of activin type II receptor (ACVR2), although both genes become frameshift-mutated in >80% of MMR-defective colorectal cancers. To experimentally determine the effect of microsatellite length upon frameshift mutation in gene-specific sequence contexts, we altered the microsatellite length within TGFBR2 exon 3 and ACVR2 exon 10, generating A7, A10 and A13 constructs. These constructs were cloned 1 bp out of frame of EGFP, allowing a -1 bp frameshift to drive EGFP expression, and stably transfected into MMR-deficient cells. Subsequent non-fluorescent cells were sorted, cultured for 7-35 days and harvested for EGFP analysis and DNA sequencing. Longer microsatellites within TGFBR2 and ACVR2 showed significantly higher mutation rates than shorter ones, with TGFBR2 A13, A10 and A7 frameshifts measured at 22.38x10(-4), 2.17x10(-4) and 0.13x10(-4), respectively. Surprisingly, shorter ACVR2 constructs showed three times higher mutation rates at A7 and A10 lengths than identical length TGFBR2 constructs but comparably lower at the A13 length, suggesting influences from both microsatellite length as well as the sequence context. Furthermore, the TGFBR2 A13 construct mutated into 33% A11 sequences (-2 bp) in addition to expected A12 (-1 bp), indicating that this construct undergoes continual subsequent frameshift mutation. These data demonstrate experimentally that both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA MMR.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Mutación del Sistema de Lectura , Repeticiones de Microsatélite , Receptores de Activinas Tipo II/genética , Secuencia de Bases/genética , Línea Celular Tumoral , Reparación de la Incompatibilidad de ADN/genética , Mutación del Sistema de Lectura/genética , Proteínas Fluorescentes Verdes , Humanos , Repeticiones de Microsatélite/genética , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.
Asunto(s)
Receptores de Activinas Tipo II/genética , Reparación de la Incompatibilidad de ADN , Mutación del Sistema de Lectura , Nucleótidos/genética , Receptores de Activinas Tipo II/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Trastornos por Deficiencias en la Reparación del ADN/genética , Trastornos por Deficiencias en la Reparación del ADN/metabolismo , Exones , Citometría de Flujo/métodos , Humanos , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Tasa de Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
BACKGROUND AND OBJECTIVES: Colorectal tumors are often observed with tumor infiltrating lymphocytes, presumably as a host-immune response, and patterns may segregate by types of genomic instability. Microsatellite unstable (MSI) colorectal cancers contain a pronounced lymphocyte reaction that can pathologically identify these tumors. Colorectal tumors with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) have not been examined for lymphocyte patterns. METHODS: We evaluated a 108-person cohort with 24 adenomas and 84 colorectal cancers for MSI and EMAST. Immunohistochemical detection of CD4+ and CD8+ T cell infiltration were performed. Prognostic relevance was assessed by survival analysis. RESULTS: CD8+ T cell infiltration in the tumor cell nest (p = 0.013) and tumor stroma (p = 0.004) were more prominent in moderately and poorly differentiated adenocarcinoma than in adenoma and well-differentiated adenocarcinoma. CD8+ T cells in the tumor cell nest (p = 0.002) and tumor stroma (p = 0.009) were at higher density in tumors with ulcerating features compared to tumors with a sessile or polypoid appearance. MSI-H tumors showed a higher density of CD8+ T cell infiltrations in tumor cell nests (p = 0.003) and tumor stroma (p = 0.001). EMAST-positive tumors showed a higher density of CD8+ T cell infiltrations than EMAST-negative tumors both in tumor cell nest (p = 0.027) and in tumor stroma (p = 0.003). These changes were not observed with CD4+ T lymphocytes. There was no difference in cancer patient survival based on density of CD8+ cells. CONCLUSIONS: CD8+ T lymphocytes, but not CD4+ cells, were increased in tumor cell nests and the tumor stroma in both MSI and EMAST tumors, and showed higher infiltration in ulcerated tumors. CD8+ T lymphocyte infiltration is associated with both EMAST and MSI patterns, and increases with histological advancement.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Linfocitos Infiltrantes de Tumor/patología , Inestabilidad de Microsatélites , Repeticiones de Microsatélite/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/genética , Adenoma/patología , Relación CD4-CD8 , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Estudios de Cohortes , Humanos , Estudios RetrospectivosRESUMEN
BACKGROUND & AIMS: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) occurs during microsatellite instability (MSI) that is not associated with major defects in DNA mismatch repair (MMR) but rather the reduced (heterogenous) expression of the MMR protein hMSH3; it occurs in sporadic colorectal tumors. We examined the timing of development of EMAST during progression of colorectal neoplasias and looked for correlations between EMAST and clinical and pathology features of tumors. METHODS: We evaluated tumor samples from a cohort of patients that had 24 adenomas and 84 colorectal cancers. EMAST were analyzed after DNA microdissection of matched normal and tumor samples using the polymorphic tetranucleotide microsatellite markers MYCL1, D9S242, D20S85, D8S321, and D20S82; data were compared with clinical and pathology findings. Traditional MSI analysis was performed and hMSH3 expression was measured. RESULTS: Moderately differentiated adenocarcinomas and poorly differentiated adenocarcinomas had higher frequencies of EMAST (56.9% and 40.0%, respectively) than well-differentiated adenocarcinomas (12.5%) or adenomas (33.3%) (P = .040). In endoscopic analysis, ulcerated tumors had a higher frequency of EMAST (52.3%) than flat (44.0%) or protruded tumors (20.0%) (P = .049). In quantification, all tumors with >3 tetranucleotide defects lost MSH3 (>75% of cells); nuclear heterogeneity of hMSH3 occurred more frequently in EMAST-positive (40.0%) than in EMAST-negative tumors (13.2%) (P = .010). CONCLUSIONS: EMAST is acquired during progression of adenoma and well-differentiated carcinomas to moderately and poorly differentiated carcinomas; it correlates with nuclear heterogeneity for hMSH3. Loss of hMSH3 corresponds with multiple tetranucleotide frameshifts. The association between EMAST and ulcerated tumors might result from increased inflammation.
Asunto(s)
Neoplasias Colorrectales/genética , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Repeticiones de Microsatélite/genética , Disparidad de Par Base , Colonoscopía , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteína 3 Homóloga de MutS , Reacción en Cadena de la PolimerasaRESUMEN
TGF-beta utilizes receptor-activated SMAD signaling to mediate growth suppression; however, non-SMAD signaling that modulates the TGF-beta response in epithelial cells become apparent when the SMAD signaling is abrogated, a common occurrence in pancreatic cancers. Here, we examined whether TGF-beta utilized NF-kappaB to downregulate PTEN, a gene that is rarely mutated in pancreatic cancers. SMAD4-null BxPc3 and CAPAN-1 pancreatic cancer cells were treated with TGF-beta (10 ng/ml) and lysed, and cellular proteins were analyzed by Western blots using p-IkappaB, p65, and PTEN antibodies. PTEN promoter and NF-kappaB activities were assessed by PTEN-luc and p-NF-luc constructs, respectively. Dominant negative p-IkappaB-alpha-M (NF-kappaB superrepressor) was used to block activation of NF-kappaB. Cell motility was assessed by Boyden chamber migration assay. TGF-beta induced IkappaB-alpha phosphorylation followed by NF-kappaB p65 subunit nuclear translocation and increased NF-kappaB activity. IkappaB-alpha-M blocked TGF-beta-induced NF-kappaB activity, reversed downregulated PTEN promoter activity and PTEN expression, and prevented augmentation of cell motility induced by TGF-beta. SMAD4 restoration, but not knockdown of SMAD2 and/or 3, reversed TGF-beta-induced NF-kappaB activity. Thus TGF-beta suppresses PTEN in pancreatic cancer cells through NF-kappaB activation and enhances cell motility and invasiveness in a SMAD4-independent manner that can be counteracted when TGF-beta-SMAD signaling is restored. The TGF-beta/NF-kappaB/PTEN cascade may be a critical pathway for pancreatic cancer cells to proliferate and metastasize.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Fosfohidrolasa PTEN/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adenocarcinoma/fisiopatología , Carcinoma Ductal Pancreático/fisiopatología , División Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Genes Reporteros/fisiología , Humanos , Proteínas I-kappa B/metabolismo , Inhibidor NF-kappaB alfa , Fosfohidrolasa PTEN/genética , Fosforilación/fisiología , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Factor de Transcripción ReIA/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The Wnt/ß-catenin signaling pathway is aberrantly activated in colorectal (CRC) and many other cancers, and novel strategies for effectively targeting it may be needed due to its complexity. In this report, SM08502, a novel small molecule in clinical development for the treatment of solid tumors, was shown to reduce Wnt pathway signaling and gene expression through potent inhibition of CDC-like kinase (CLK) activity. SM08502 inhibited serine and arginine rich splicing factor (SRSF) phosphorylation and disrupted spliceosome activity, which was associated with inhibition of Wnt pathway-related gene and protein expression. Additionally, SM08502 induced the generation of splicing variants of Wnt pathway genes, suggesting that its mechanism for inhibition of gene expression includes effects on alternative splicing. Orally administered SM08502 significantly inhibited growth of gastrointestinal tumors and decreased SRSF phosphorylation and Wnt pathway gene expression in xenograft mouse models. These data implicate CLKs in the regulation of Wnt signaling and represent a novel strategy for inhibiting Wnt pathway gene expression in cancers. SM08502 is a first-in-class CLK inhibitor being investigated in a Phase 1 clinical trial for subjects with advanced solid tumors (NCT03355066).
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factores de Empalme Serina-Arginina/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Vía de Señalización Wnt/efectos de los fármacos , Empalme Alternativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Concentración 50 Inhibidora , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas , Neoplasias Gástricas/patología , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and ß cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased ß cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered ß cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.
Asunto(s)
Glucemia/metabolismo , Quimiocina CX3CL1/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Animales , Glucemia/genética , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Quimiocina CX3CL1/genética , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Fragmentos Fc de Inmunoglobulinas/genética , Secreción de Insulina/genética , Células Secretoras de Insulina/patología , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
BACKGROUND: Menopause is associated with significant hormonal changes that result in increased total body fat and abdominal fat, amplifying the risk for metabolic syndrome and diseases such as diabetes, cardiovascular disease and cancer in postmenopausal women. Intermittent fasting regimens hold significant health benefit promise for obese humans, however, regimens that include extreme daytime calorie restriction or daytime fasting are generally associated with hunger and irritability, hampering long-term compliance and adoption in the clinical setting. Time-restricted feeding (TRF), a regimen allowing eating only during a specific period in the normal circadian feeding cycle, without calorie restriction, may increase compliance and provide a more clinically viable method for reducing the detrimental metabolic consequences associated with obesity. METHODS: We tested TRF as an intervention in a mouse model of postmenopausal obesity. Metabolic parameters were measured using Clinical Laboratory Animal Monitoring System (CLAMS) and we carried out glucose tolerance tests. We also stained liver sections with oil red O to examine steatosis and measured gene expression related to gluconeogenesis. RESULTS: Preexisting metabolic disease was significantly attenuated during 7 weeks of TRF. Despite having access to the same high fat diet (HFD) as ad libitum fed (ALF) mice, TRF mice experienced rapid weight loss followed by a delayed improvement in insulin resistance and a reduced severity of hepatic steatosis by having access to the HFD for only 8h during their normal nocturnal feeding period. The lower respiratory exchange ratio in the TRF group compared with the ALF group early in the dark phase suggested that fat was the predominant fuel source in the TRF group and correlated with gene expression analyses that suggested a switch from gluconeogenesis to ketogenesis. In addition, TRF mice were more physically active than ALF fed mice. CONCLUSIONS: Our data support further analysis of TRF as a clinically viable form of intermittent fasting to improve metabolic health due to obesity.
Asunto(s)
Hígado Graso/dietoterapia , Conducta Alimentaria/fisiología , Resistencia a la Insulina , Obesidad/etiología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Ingestión de Energía , Femenino , Perfilación de la Expresión Génica , Gluconeogénesis/genética , Ratones , Ratones Endogámicos C57BL , PosmenopausiaRESUMEN
Insulin resistance results from several pathophysiologic mechanisms, including chronic tissue inflammation and defective insulin signaling. We found that liver, muscle and adipose tissue exhibit higher levels of the chemotactic eicosanoid LTB4 in obese high-fat diet (HFD)-fed mice. Inhibition of the LTB4 receptor Ltb4r1, through either genetic or pharmacologic loss of function, led to an anti-inflammatory phenotype with protection from insulin resistance and hepatic steatosis. In vitro treatment with LTB4 directly enhanced macrophage chemotaxis, stimulated inflammatory pathways, reduced insulin-stimulated glucose uptake in L6 myocytes, and impaired insulin-mediated suppression of hepatic glucose output in primary mouse hepatocytes. This was accompanied by lower insulin-stimulated Akt phosphorylation and higher Irs-1/2 serine phosphorylation, and all of these events were dependent on Gαi and Jnk1, two downstream mediators of Ltb4r1 signaling. These observations elucidate a novel role of the LTB4-Ltb4r1 signaling pathway in hepatocyte and myocyte insulin resistance, and they show that in vivo inhibition of Ltb4r1 leads to robust insulin-sensitizing effects.
Asunto(s)
Hepatocitos/inmunología , Resistencia a la Insulina/inmunología , Leucotrieno B4/inmunología , Macrófagos/inmunología , Fibras Musculares Esqueléticas/inmunología , Obesidad/inmunología , Receptores de Leucotrieno B4/inmunología , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Hígado Graso/inmunología , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Inflamación/inmunología , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Ratones Obesos , Fibras Musculares Esqueléticas/metabolismo , Obesidad/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Leucotrieno B4/antagonistas & inhibidores , Receptores de Leucotrieno B4/genética , Transducción de SeñalRESUMEN
OBJECTIVE: Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ). METHODS: To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1 (flx/flx) aP2Cre (+) (ATKO) and Sirt1 (flx/flx) aP2Cre (-) (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue. RESULTS: On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity. CONCLUSION: Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.
RESUMEN
The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance.
Asunto(s)
Gluconeogénesis/fisiología , Hígado/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Hígado Graso/metabolismo , Hígado Graso/patología , Interleucina-6/metabolismo , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Hígado/patología , Macrófagos/patología , Masculino , Ratones , Ratones Obesos , Obesidad/etiología , Obesidad/patología , Receptores CCR2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Aging is associated with impairment of T cell function. We demonstrate here that age-associated declines in T cell signaling are due to the inability to form effective immune synapses at the site of the T cell receptor and antigen interaction. On the basis of our previous research with vitamin E (VE), we hypothesized that VE supplementation of old CD4(+) T cells enhances effective immune synapse formation through increased translocation of signaling proteins. Using confocal microscopy, we found that when exposed to antigen-presenting cells, CD4(+) T cells from old mice have a lower percentage of effective immune synapses compared to those from young mice. Furthermore, we show that in vitro and in vivo VE supplementation increases the percentage of old CD4(+) T cells capable of forming a functional immune synapse. Further studies are under way to determine the mechanisms of age and VE-induced enhancement of effective immune synapse formation.
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Envejecimiento/inmunología , Linfocitos T CD4-Positivos/inmunología , Sinapsis/fisiología , Vitamina E/administración & dosificación , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/ultraestructura , Linfocitos T CD4-Positivos/ultraestructura , Ratones , Transducción de SeñalRESUMEN
To determine the effect of age on susceptibility to azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and its underlying mechanism, young and old mice were injected with AOM weekly for 4 or 5 weeks and euthanized 5 or 6 weeks later. Given the same (12 or 15) mg/kg body weight dose of AOM, old mice had significantly more ACF than young mice. However, given the same total dose of AOM (to avoid confounding effect of higher dose to heavier old mice), at a low total dose (1.5 mg) there was no age difference, but at higher total doses (1.8 and 2.2 mg) young mice had significantly more ACF than old mice. These results indicate that the age-related susceptibility to AOM differs depending on whether administration of the carcinogen is based on weight or total dose. These age differences are not due to variations in cyclooxygenase-2 expression, cell proliferation, or AOM hydroxylase activity.
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Azoximetano/toxicidad , Carcinógenos/toxicidad , Colon/efectos de los fármacos , Neoplasias del Colon/química , Factores de Edad , Animales , Azoximetano/administración & dosificación , Carcinógenos/administración & dosificación , Colon/patología , Neoplasias del Colon/patología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inmunohistoquímica , Isoenzimas/análisis , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Prostaglandina-Endoperóxido Sintasas/análisis , Pérdida de PesoRESUMEN
It is well known that the ω-3 fatty acids (ω-3-FAs; also known as n-3 fatty acids) can exert potent anti-inflammatory effects. Commonly consumed as fish products, dietary supplements and pharmaceuticals, ω-3-FAs have a number of health benefits ascribed to them, including reduced plasma triglyceride levels, amelioration of atherosclerosis and increased insulin sensitivity. We reported that Gpr120 is the functional receptor for these fatty acids and that ω-3-FAs produce robust anti-inflammatory, insulin-sensitizing effects, both in vivo and in vitro, in a Gpr120-dependent manner. Indeed, genetic variants that predispose to obesity and diabetes have been described in the gene encoding GPR120 in humans (FFAR4). However, the amount of fish oils that would have to be consumed to sustain chronic agonism of Gpr120 is too high to be practical, and, thus, a high-affinity small-molecule Gpr120 agonist would be of potential clinical benefit. Accordingly, Gpr120 is a widely studied drug discovery target within the pharmaceutical industry. Gpr40 is another lipid-sensing G protein-coupled receptor, and it has been difficult to identify compounds with a high degree of selectivity for Gpr120 over Gpr40 (ref. 11). Here we report that a selective high-affinity, orally available, small-molecule Gpr120 agonist (cpdA) exerts potent anti-inflammatory effects on macrophages in vitro and in obese mice in vivo. Gpr120 agonist treatment of high-fat diet-fed obese mice causes improved glucose tolerance, decreased hyperinsulinemia, increased insulin sensitivity and decreased hepatic steatosis. This suggests that Gpr120 agonists could become new insulin-sensitizing drugs for the treatment of type 2 diabetes and other human insulin-resistant states in the future.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ácidos Grasos Omega-3/metabolismo , Resistencia a la Insulina/fisiología , Receptores Acoplados a Proteínas G/agonistas , Animales , Arginasa/biosíntesis , Linfocitos B Reguladores/inmunología , Secuencia de Bases , Diabetes Mellitus Tipo 2/genética , Ácidos Docosahexaenoicos/farmacología , Hígado Graso/tratamiento farmacológico , Hiperinsulinismo/tratamiento farmacológico , Inflamación , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Obesidad/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1's promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency. AIM: To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency. METHODS: We measured -4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1â»/â», hMSH6â»/â», and hMSH3â»/â» CRC cells. We observed the subcellular location of hMSH3 with oxidative stress. RESULTS: D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1â»/â» and hMSH3â»/â» cells, respectively, than in hMSH6â»/â» or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10â»4 mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10â»4 mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H2O2 to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H2O2 treatment. CONCLUSION: EMAST is dependent upon the MMR background, with hMSH3â»/â» more prone to frameshift mutations than hMSH6â»/â», opposite to frameshift mutations observed for mononucleotide repeats. hMSH3â»/â» mimics complete MMR failure (hMLH1â»/â») in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3's subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.
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Núcleo Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Repeticiones de Microsatélite/genética , Estrés Oxidativo , Transporte Activo de Núcleo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Reparación de la Incompatibilidad de ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Mutación del Sistema de Lectura/genética , Técnicas de Silenciamiento del Gen , Sitios Genéticos/genética , Humanos , Proteína 3 Homóloga de MutS , Estrés Oxidativo/genéticaRESUMEN
Several studies indicate that the DNA mismatch repair (MMR) system may trigger cytotoxicity upon 5-fluorouracil (5-FU) recognition, but signaling pathways regulated by MMR in response to 5-FU are unknown. We hypothesize that recognition of 5-FU in DNA by MMR proteins trigger specific signaling cascades that results in slowing of the cell cycle and cell death. Whole human genome cDNA microarrays were used to examine relative signaling responses induced in MMR-proficient cells after 5-FU (5 µM) treatment for 24 hours. Analysis revealed 43 pathways differentially affected by 5-FU compared to control (P 1.4-fold) and downregulated cdc25C, cyclins B1 and B2, histone H2A, H2B, and H3 (< -1.4-fold) over control. Cell cycle analysis revealed a G1/S arrest by 5-FU that was congruent with increased cyclin E and decreased cdc25C protein expression. Importantly, with knockdown of hMLH1 and hMSH2, we observed that decreased histone H3 expression by 5-FU was dependent on hMLH1. Additionally, 5-FU treatment dramatically decreased levels of several histone H3 modifications. Our data suggest that 5-FU induces a G1/S arrest by regulating cyclin E and cdc25C expression, and MMR recognition of 5-FU in DNA may modulate cyclin E to affect the cell cycle. Furthermore, MMR recognition of 5-FU reduces histone H3 levels that could be related to DNA access by proteins and/or cell death during the G1/S phase of the cell cycle.
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Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Ciclina E/metabolismo , Reparación de la Incompatibilidad de ADN , Fluorouracilo/farmacología , Histonas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Neoplasias Colorrectales/metabolismo , Ciclina E/biosíntesis , Ciclina E/genética , Daño del ADN , Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Histonas/biosíntesis , Histonas/genética , Humanos , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Mesalazine (5-aminosalicylic acid, 5-ASA) has chemopreventive properties in colitis-associated cancer. In vitro, it improves replication fidelity at (CA)13 microsatellites independent of mismatch repair proficiency. Therefore, 5-ASA might be advantageous in patients with hereditary nonpolyposis colorectal cancer. At this point, however, it is uncertain whether this improvement of replication fidelity is specific for (CA)13 repetitive sequences. Here, we tested the effect of 5-ASA on replication fidelity in mononucleotide, dinucleotide, and tetranucleotide repeats. EXPERIMENTAL DESIGN: HCT116 and HCT116+chr3 cells were transfected with pIREShyg2-EGFP reporter plasmids harboring the following microsatellites: A10, G10, (CA)13, (CA)26, (AAAG)17, poly-A tracts, and their flanking sequences of transforming growth factor beta receptor II (TGFBR2; A10) and activin type II receptor (ACVR2; A8). Stably transfected single-cell clones were selected, characterized by Southern blotting, sorted into six-well plates, and cultured with or without 5-ASA. Frameshift mutations that shift the enhanced green fluorescence protein into its proper reading frame were quantified by flow cytometry. RESULTS: In HCT116, 5-ASA reduced the mutant fraction at (CA)13 by 48.3%, at A10 by 35.6-43.6%, at G10 by 74.9-83.6%, and at (AAAG)17 by 37.6-44.4%. Similar results were observed in hMLH1-proficient HCT116+chr3 cells. Moreover, the presence of 5-ASA significantly reduced mutations in TGFBR2 (A10) and ACVR2 (A8) by 39.9% and 46.2%, respectively. CONCLUSIONS: 5-ASA increases replication fidelity in mononucleotide, dinucleotide, and tetranucleotide repeats and reduces mutations in tumor suppressor genes TGFBR2 and ACVR2, a finding that may provoke in vivo studies for the prevention of colorectal cancer in hereditary nonpolyposis colorectal cancer.