Oat Beta-Glucan as a Metabolic Regulator in Early Stage of Colorectal Cancer-A Model Study on Azoxymethane-Treated Rats.

Factors that reduce the risk of developing colorectal cancer include biologically active substances. In our previous research, we demonstrated the anti-inflammatory, immunomodulatory, and antioxidant effects of oat beta-glucans in gastrointestinal disease models. The aim of this study was to investigate the effect of an 8-week consumption of a diet supplemented with low-molar-mass oat beta-glucan in two doses on the antioxidant potential, inflammatory parameters, and colonic metabolomic profile in azoxymethane(AOM)-induced early-stage colorectal cancer in the large intestine wall of rats. The results showed a statistically significant effect of AOM leading to the development of neoplastic changes in the colon. Consumption of beta-glucans induced changes in colonic antioxidant potential parameters, including an increase in total antioxidant status, a decrease in the superoxide dismutase (SOD) activity, and a reduction in thiobarbituric acid reactive substance (TBARS) concentration. In addition, beta-glucans decreased the levels of pro-inflammatory interleukins (IL-1α, IL-1ß, IL-12) and C-reactive protein (CRP) while increasing the concentration of IL-10. Metabolomic studies confirmed the efficacy of oat beta-glucans in the AOM-induced early-stage colon cancer model by increasing the levels of metabolites involved in metabolic pathways, such as amino acids, purine, biotin, and folate. In conclusion, these results suggest a wide range of mechanisms involved in altering colonic metabolism during the early stage of carcinogenesis and a strong influence of low-molar-mass oat beta-glucan, administered as dietary supplement, in modulating these mechanisms.

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell-cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

Interleukin-6 mimics insulin-dependent cellular distribution of some cytoskeletal proteins and Glut4 transporter without effect on glucose uptake in 3T3-L1 adipocytes.

Interleukin (IL)-6, a known proinflammatory cytokine, is released in both visceral adipose tissue and contracting skeletal muscle. In this study, we used microRNA profiling as a screening method to identify miRNA species modified by IL-6 treatment in mouse 3T3-L1 adipocytes. miRNA microarray analysis and qRT-PCR revealed increased expression of miR-146b-3p in adipocytes exposed to IL-6 (1 ng/ml) during 8-day differentiation. On the basis of ontological analysis of potential targets, selected proteins associated with cytoskeleton and transport were examined in the context of adipocyte response to insulin, using immunofluorescence and confocal microscopy. We concluded that IL-6: (i) does not affect insulin action on actin cellular distribution; (ii) modulates the effect of insulin on myosin light chain kinase (Mylk) distribution by preventing its shift toward cytoplasm; (iii) mimics the effect of insulin on dynein distribution by increasing its near-nuclear accumulation; (iv) mimics the effect of insulin on glucose transporter Glut4 distribution, especially by increasing its near-nuclear accumulation; (v) supports insulin action on early endosome marker Rab4A near-nuclear accumulation. Moreover, as IL-6 did not disturb insulin-dependent glucose uptake, our results do not confirm the IL-6-induced impairment of insulin action observed in some in vitro studies, suggesting that the effect of IL-6 is dose dependent.

The influence of eicosapentaenoic acid and docosahexaenoic acid on expression of genes connected with metabolism and secretory functions of ageing 3T3-L1 adipocytes.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are n-3 long chain polyunsaturated fatty acids. The purpose of our study was to evaluate the influence of EPA and DHA on expression of genes connected with metabolism and secretory functions of ageing adipocytes. Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48h in the presence of EPA, or DHA. Both fatty acids increased the expression of Pparg, FATP1, FATP4 and ATGL genes, but only in young 3T3-L1 adipocytes. Moreover, in young, mature and old cells DHA elevated the expression of CPT1 gene. In addition, EPA and DHA enhanced the expression of leptin, adiponectin and apelin genes only in young cells. Investigated fatty acids changed mRNA levels of IL6 and MCP1 in young, mature and old cells. EPA increased the expression of these two genes, whereas DHA decreased it. Furthermore, EPA and DHA treatment changed the expression of IRS1 and GLUT4 genes involved in insulin signalling pathway, but their effects were opposite. Expression of these genes was decreased by EPA and increased by DHA in young, mature and old cells. In summary, the investigated fatty acids are able to affect the expression of genes associated with lipid metabolism, secretory functions and insulin resistance in ageing 3T3-L1 adipocytes, but their impact is age-dependant. Young cells seem to be more sensitive to EPA and DHA than mature and old ones. Furthermore, the effect of these two fatty acids is not always identical, and therefore requires further investigation.

Lymphocytic, cytokine and transcriptomic profiles in peripheral blood of dogs with atopic dermatitis.

BACKGROUND: Canine atopic dermatitis (cAD) is a common chronic and pruritic skin disease in dogs. The development of cAD involves complex interactions between environmental antigens, genetic predisposition and a number of disparate cell types. The aim of the present study was to perform comprehensive analyses of peripheral blood of AD dogs in relation to healthy subjects in order to determine the changes which would be characteristic for cAD. RESULTS: The number of cells in specific subpopulations of lymphocytes was analyzed by flow cytometry, concentration of chosen pro- and anti-inflammatory cytokines (IL-4, IL-10, IL-13, TNF-α, TGF-ß1) was determined by ELISA; and microarray analysis was performed on RNA samples isolated from peripheral blood nuclear cells of AD and healthy dogs. The number of Th cells (CD3(+)CD4(+)) in AD and healthy dogs was similar, whereas the percentage of Tc (CD3(+)CD8(+)) and Treg (CD4(+)CD25(+) Foxp3(+)) cells increased significantly in AD dogs. Increased concentrations of IL-13 and TNF-α, and decreased levels of IL-10 and TGF-ß1 was observed in AD dogs. The level of IL-4 was similar in both groups of animals. Results of the microarray experiment revealed differentially expressed genes involved in transcriptional regulation (e.g., transcription factors: SMAD2, RORA) or signal transduction pathways (e.g., VEGF, SHB21, PROC) taking part in T lymphocytes lineages differentiation and cytokines synthesis. CONCLUSIONS: Results obtained indicate that CD8(+) T cells, beside CD4(+) T lymphocytes, contribute to the development of the allergic response. Increased IL-13 concentration in AD dogs suggests that this cytokine may play more important role than IL-4 in mediating changes induced by allergic inflammation. Furthermore, observed increase in Treg cells in parallel with high concentrations of TNF-α and low levels of IL-10 and TGF-ß1 in the peripheral blood of AD dogs point at the functional insufficiency of Treg cells in patients with AD.

The influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation.

BACKGROUND: EPA and DHA have been reported to have anti-obesity and anti-inflammatory properties. Recent studies revealed that these positive actions of n-3 PUFA at least partially are connected with their influence on metabolism and secretory functions of the adipose tissue. However, their impact on old adipocytes is still poorly understood. Therefore the aim of the present study was to evaluate the influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation. METHODS: Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 µM EPA, or 50 µM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits. RESULTS: The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted. CONCLUSIONS: In summary, age is an important determinant of fat accumulation in adipocytes and affects adipokines secretion by these cells. Moreover, the impact of investigated fatty acids: EPA and DHA on fat cells varies depending on the stage of maturation, and seems to be stronger in young cells than in mature and old ones. Docosahexaenoic acid exerts an anti-inflammatory action; however, on the basis of the obtained data it was not possible to determine whether eicosapentaenoic acid shows anti- or pro-inflammatory properties.

Cytokine and Lymphocyte Profiles in Dogs with Atopic Dermatitis after Allergen-Specific Immunotherapy.

Canine atopic dermatitis (cAD) is a chronic and recurrent inflammatory and pruritic skin disease in dogs. Currently, allergen-specific immunotherapy (ASIT) is the only identified disease-modifying intervention for allergic diseases. It decreases the symptoms triggered by allergens and prevents recurrence of the disease in the long-term. The aim of our research was to determine how immunotherapy changes the proportion of lymphocyte subsets in dog peripheral blood and the levels of cytokines secreted by these cells during therapy. ASIT was applied for 6 months. Blood samples for further analyses were collected from patients in the third and sixth month of immunotherapy. Six out of seven dogs receiving ASIT showed a positive effect. A reduction in cytokine levels (IL-13, TNF-α) in peripheral blood of cAD patients and changes in the number of specific T cell subpopulations-reduction of Tc cells (CD8+) and increase of activated T cells (CD3+CD25+)-confirmed the beneficial effect of the applied ASIT. In addition, a significantly higher percentage of Treg cells (CD4+CD25+FOXP3+) was noted in cAD patients before treatment compared to healthy dogs. After 3 months of therapy, the percentage of Tregs significantly decreased, and after 6 months, it increased significantly again.