sepharose and Obesity

Exosomes are nanovesicles that participate in cell-to-cell communication and are secreted by a variety of cells including neurons. Recent studies suggest that neuronally-derived exosomes are detectable in plasma and that their contents likely reflect expression of various biomarkers in brain tissues. The receptor for advanced glycation endproducts (RAGE) has been implicated in the pathophysiology of Alzheimer's disease (AD) and is increased in brain regions affected by AD. The goal of our project was to determine whether RAGE is present in plasma exosomes, and specifically exosomes derived from neurons. Exosomes were isolated from plasma samples (n = 8) by precipitation (ExoQuick) and ultracentrifugation methods. Neuronally-derived exosomes were isolated using a biotin-tagged L1 Cell Adhesion Molecule (L1CAM) specific antibody and streptavidin-tagged agarose resin. RAGE expression was measured by Western blots and ELISA. Western Blotting showed that RAGE is present in L1CAM-positive exosomes isolated using both methods. Mean (SD) exosomal RAGE levels were 164 (60) pg/ml by ExoQuick and were highly correlated with plasma sRAGE levels (r = 0.87, p = 0.005), which were approximately 7.5-fold higher than exosomal levels. Weak to moderate correlations were found between exosomal RAGE and age, BMI, and cognitive function. These results show for the first time that RAGE is present in neuronally-derived plasma exosomes, and suggest that exosomal RAGE may be a novel biomarker that reflects pathophysiological processes in the brain.

Topics: Age Factors; Aged; Antigens, Neoplasm; Bacterial Proteins; Biotinylation; Body Mass Index; Brain; Cell Separation; Exosomes; Female; Gene Expression; Humans; Male; Mitogen-Activated Protein Kinases; Neural Cell Adhesion Molecule L1; Neurons; Obesity; Protein Binding; Sepharose

Gut microbiota are deeply associated with the prevalence of obesity. Agarose is hydrolyzed easily to yield oligosaccharides, designated as agaro-oligosaccharides (AGO). This study evaluated the effects of AGO on obese phenotype and gut microbial composition in mice. Mice were administered AGO in drinking water (AGO-receiving mice). 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Serum bile acids were ascertained using a LC-MS/MS system. Compared to the control group, AGO administration significantly reduced epididymal adipose tissue weights and serum non-esterified fatty acid concentrations, but the cecal content weights were increased. Data from the serum bile acid profile show that concentrations of primary bile acids (cholic acid and chenodeoxycholic acid), but not those of secondary bile acids (deoxycholic acid, lithocholic acid, and ursodeoxycholic acid), tended to increase in AGO-receiving mice. 16S rRNA gene sequencing analyses showed that the relative abundances of 15 taxa differed significantly in AGO-receiving mice. Of these, the relative abundances of Rikenellaceae and Lachnospiraceae were found to be positively correlated with epididymal adipose tissue weight. The relative abundances of Bacteroides and Ruminococcus were correlated negatively with epididymal adipose tissue weight. Although the definitive role of gut microbes of AGO-received mice is still unknown, our data demonstrate the possibility that AGO administration affects the gut microbial composition and inhibits obesity in mice.

Topics: Adipose Tissue; Animals; Bacteria; Bacterial Load; Bile Acids and Salts; Cecum; Fatty Acids, Nonesterified; Gastrointestinal Microbiome; Male; Mice; Mice, Inbred C57BL; Obesity; Oligosaccharides; Organ Size; RNA, Ribosomal, 16S; Sepharose; Sequence Analysis, RNA; Water

High-fat diet (HFD)-induced alteration in the gut microbial composition, known as dysbiosis, is increasingly recognized as a major risk factor for various diseases, including colon cancer. This report describes a comprehensive investigation of the effect of agaro-oligosaccharides (AGO) on HFD-induced gut dysbiosis, including alterations in short-chain fatty acid contents and bile acid metabolism in mice. C57BL/6N mice were fed a control diet or HFD, with or without AGO. Terminal restriction fragment-length polymorphism (T-RFLP) analysis produced their fecal microbiota profiles. Profiles of cecal organic acids and serum bile acids were determined, respectively, using HPLC and liquid chromatography-tandem mass spectrometry systems. T-RFLP analyses showed that an HFD changed the gut microbiota significantly. Changes in the microbiota composition induced by an HFD were characterized by a decrease in the order Lactobacillales and by an increase in the Clostridium subcluster XIVa. These changes of the microbiota community generated by HFD treatment were suppressed by AGO supplementation. As supported by the data of the proportion of Lactobacillales order, the concentration of lactic acid increased in the HFD + AGO group. Data from the serum bile acid profile showed that the level of deoxycholic acid, a carcinogenic secondary bile acid produced by gut bacteria, was increased in HFD-receiving mice. The upregulation tended to be suppressed by AGO supplementation. Finally, results show that AGO supplementation suppressed the azoxymethane-induced generation of aberrant crypt foci in the colon derived from HFD-treated mice. Our results suggest that oral intake of AGO prevents HFD-induced gut dysbiosis, thereby inhibiting colon carcinogenesis.

Topics: Animals; Bile Acids and Salts; Clostridium; Colonic Neoplasms; Diet, High-Fat; Dietary Fiber; Dysbiosis; Endotoxins; Fatty Acids; Feces; Lactobacillales; Male; Mice; Mice, Inbred C57BL; Microbiota; Obesity; Oligosaccharides; Sepharose

Hepatic insulin receptor levels in 6-week-old obese (fa/fa) rats were about 2-fold lower than those from lean (Fa/-) rats, which agrees with their insulin-resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese (fa/fa) rats were similar to those of lean (Fa/-) animals but the cytosolic levels were reduced to half, due to a decrease in the 39-kD)a catalytic subunit. Marked increases in activity, due to rises in the 44-kDa and 39-kDa catalytic subunits, were seen in the 16000 x g sediments (M1) from insulin-resistant rats, with moderate changes in the 100000xg sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin-resistant rats.

Topics: Animals; Casein Kinase II; Chromatography, Affinity; Female; HSP70 Heat-Shock Proteins; Insulin Resistance; Lectins; Liver; Membrane Proteins; Obesity; Protein Serine-Threonine Kinases; Rats; Rats, Zucker; Receptor, Insulin; Sepharose; Subcellular Fractions