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Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.
OBJECTIVES - Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy.
DESIGN - Gastric colonisation with Altered Schaedler's flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection.
RESULTS - Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice.
CONCLUSIONS - rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk.
Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.
In vivo animal experiments are essential to current prostate cancer research, and are particularly critical to studying interactions between tumor cells and their microenvironment. Numerous pre-clinical animal models of prostate cancer are currently available, including transgenic mouse models and human prostate cancer xenograft mouse models. In contrast to transgenic mouse models producing more heterogeneous cohorts of tumors, xenograft mouse models provide more controlled approaches. This unit describes the detailed procedures necessary to establish several distinct pre-clinical mouse models of human prostate cancer, including an orthotopic prostate xenograft model, an orthotopic bone metastasis model, an experimental metastasis model of intra-cardiac injection, and a vossicle model of tumor-bone interaction.
Extensive apoptosis of leukocytes during sepsis and endotoxic shock constitutes an important mechanism linked to the excessive mortality associated with these disorders. Caspase inhibitors confer protection from endotoxin-induced lymphocyte apoptosis and improve survival, but it is not clear which caspases mediate lipopolysaccharide (LPS)-induced lymphocyte apoptosis and mortality. We report here that the apoptotic executioner caspase-7 was activated in the splenocytes of LPS-injected mice, suggesting a role for caspase-7 in lymphocyte apoptosis. Indeed, caspase-7-deficient mice were resistant to LPS-induced lymphocyte apoptosis and were markedly protected from LPS-induced lethality independently of the excessive production of serum cytokines. These results reveal for the first time a nonredundant role for caspase-7 in vivo and identify caspase-7 inhibition as a component of the mechanism by which caspase inhibitors protect from endotoxin-induced mortality.
During hematopoiesis, myeloid cell leukemia-1 (MCL-1) mediates the survival of bone marrow progenitors and lymphocytes. However, its requirement during myeloid cell differentiation, development, and effector function is less clear. Lineage-specific deletion of MCL-1 in myeloid precursors results in neutropenia due to death during differentiation. The loss of mature neutrophils induced by Mcl-1 deletion was not rescued by genetic deletion of proapoptotic Bim and Puma or by exogenous cytokine treatment. However, blockade of intrinsic apoptosis by lineage-specific deletion of both multidomain proapoptotics Bax and Bak was capable of rescuing the neutropenia associated with Mcl-1 deletion. In the monocytic lineage, despite efficient Mcl-1 deletion, monocytes and macrophages undergo normal development. During the phagocytosis of extracellular bacteria, macrophages concomitantly increase the expression of both MCL-1 and BIM. However, Mcl-1-deficient macrophages exhibit increased sensitivity to death during bacterial phagocytosis that can be abolished by codeletion of Bim. These data suggest that MCL-1 may be necessary to antagonize BIM during macrophage effector responses. Thus, MCL-1 plays selective roles in myeloid development, being required for neutrophil development and setting the threshold for apoptosis during a macrophage effector response.
Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet the effects of bacterial eradication on carcinogenesis remain unclear. Animal models provide important insights into factors that are involved in gastric carcinogenesis, and we previously utilized such a model to demonstrate that an in vivo-adapted H. pylori strain, 7.13, rapidly and reproducibly induces inflammation-mediated gastric carcinoma. In the current study, we used this bacterial strain as a prototype to define the role of targeted antimicrobial therapy in gastric carcinogenesis. Mongolian gerbils were infected with H. pylori for 4 or 8 weeks, treated with antimicrobial agents or vehicle, and then euthanized at 8 weeks after the completion of therapy. All infected gerbils developed gastritis; however, inflammation was significantly attenuated in animals receiving antimicrobial therapy. Gastric dysplasia or cancer developed in >60% of the gerbils that remained persistently colonized with H. pylori, but in none of the animals treated with antibiotics following 4 weeks of infection. Infection with H. pylori for 8 weeks prior to therapy resulted in an attenuation, but not complete prevention, of pre-malignant and malignant lesions. Similarly, antibiotic therapy initiated at 4, but not 8, weeks after H. pylori challenge significantly reduced expression of the Th1 pro-inflammatory cytokine interferon-gamma within colonized gastric mucosa. These results indicate that treatment of H. pylori in this model decreases the incidence and severity of lesions with carcinogenic potential. The effectiveness of eradication is dependent upon the timing of intervention, providing insights into mechanisms that may regulate the development of malignancies arising within the context of inflammatory states.
Cytokine genes undergo progressive changes in chromatin organization when naïve CD4+ T helper (Th) cells differentiate into committed Th1 and Th2 lineages. Here, we analyzed nuclear matrix attachment regions (MARs) in the Ifng gene by DNA array technique in unactivated and activated CD4+ Th cells. This approach was combined with analysis of spatial organization of the Ifng gene by chromosome conformation capture approach to assess the relationship between the gene conformation and matrix attachment organization in functionally different cell subsets. We report that the Ifng gene in unactivated cells displays a linear conformation, but in T-cell receptor-activated cells, it adopts a loop conformation. The selective MARs support the spatial gene organization and characteristically define the Ifng gene in functionally different cell subsets. The pattern of interaction of the Ifng gene with the nuclear matrix dynamically changes in a lineage-specific manner in parallel with the changes in Ifng gene conformation. The data suggest that such structural dynamics provide the means for transcriptional regulation of the Ifng gene in the course of activation and differentiation of CD4+Th cells.
The allergic inflammation occurring in asthma is believed to be accompanied by the production of free radicals. To investigate the role of free radicals and the cells affected we turned to a murine model of allergic inflammation produced by sensitization to ovalbumin with subsequent aerosol challenge. We examined oxidant stress by measuring and localizing the sensitive and specific marker of lipid peroxidation, the F2-isoprostanes. F2-isoprostanes in whole lung increased from 0.30 +/- 0.08 ng/lung at baseline to a peak of 0.061 +/- 0.09 ng/lung on the ninth day of daily aerosol allergen challenge. Increased immunoreactivity to 15-F2t-IsoP (8-iso-PGF2alpha) or to isoketal protein adducts was found in epithelial cells 24 h after the first aerosol challenge and at 5 days in macrophages. Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased immunoreactivity after ovalbumin challenge. Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes while supplemental vitamin E suppressed their formation. Similar changes in immunoreactivity to F2-isoprostanes were seen. Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant. Our findings indicate that allergic airway inflammation in mice is associated with an increase in oxidant stress, which is most striking in airway epithelial cells and macrophages. Oxidant stress plays a role in the production of airway responsiveness.
Type I or insulin dependent diabetes mellitus develops in the non-obese diabetic (NOD) mouse as a consequence of T cell mediated autoimmune attack on pancreatic beta cells. B lymphocytes are required for disease progression in NOD and loss of tolerance in the B cell compartment is one of the earliest manifestation of the autoimmune process. To understand how the fate and function of B lymphocytes may be regulated in the context of an organ specific autoimmune disease, the B cell co-receptor CD72 (Lyb-2) was examined in NOD mice. Mab that recognize a,b, and d alleles of CD72 reacted poorly with NOD B cells while western blots of B cell extracts show that CD72 is abundant in NOD B cells. Nucleotide sequencing of CD72 cDNA confirms that an uncommon allele, CD72c, is expressed in NOD. Functional studies using monoclonal antibodies indicate that the CD72c allele of NOD can serve as a positive regulator of B cell responses both as a single signal and in synergy with BCR or IL-4 stimulation. Since CD72c differs principally in the extra cellular or ligand binding portion of the molecule, interactions with its natural ligand in vivo may contribute to functional differences in mouse strains that express this allele. NOD and lupus prone strains share the CD72c allele and its functions may contribute to overlapping features of organ specific and systemic autoimmune disorders.