The end of the experiments, frogs were decapitated by guillotine, and blood was collected. Plasma was separated from blood cells by centrifugation at 3000g for 15 min at 4 and stored at -35 for later use. The gastrointestinal tract was removed and weighed, and its length was measured after the small intestine lumen was washed with ice-cold frog Ringer’s solution to remove digesta. The middle part of the intestine was used for morphological studies. Proximal and distal parts were mixed, and then used for RNA preparation, and ChIP and enzyme assays. For enzyme assays, after the lumen was rinsed with frog Ringer’s solution, small intestine was homogenized in 4 volumes of 10 mM potassium phosphate (K2HPO4/KH2PO4) buffer, pH 7.0, 1 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine HCl in a Potter lvehjem homogenizer. Homogenate was stored at -84 until required. All housing and experimental procedures were GGTI298 web conducted in accordance with the code of ethics on the Animal Welfare Committee of Shizuoka University.Histologywith a semidry transfer system for 2 h at 1.2 mA/cm2. The membranes were incubated in a 10 skimmed milk in Tris-buffered saline (TBS), overnight at 4 and then probed with primary antibody against PCNA (1:1000) in 1 skimmed milk in TBS for 1 h. After washing with TBS containing 0.1 Tween 20 three times, blots were then incubated with horseradish peroxidase conjugated anti-mouse IgG (1:3000) for 0.5 h. Signals were developed by chemiluminescence (ECL reagent, GE Healthare, Piscataway, NJ). -Tubulin level was used to normalize protein expression. Protein expression was quantified using a lumino-image analyzer (LAS-4000, Fuji Film, Tokyo, Japan).Measurements of plasma biochemical parametersThe plasma concentration of glucose was determined by the mutarotase-glucose oxidase method [43] using a kit (Glucose CII-testWako), triglycerides by the glycerol3-phosphate oxidase method [44] using a kit (Triglyceride E-testWako), PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27607577 cholesterol by the cholesterol oxidase method [45] using a kit (Cholesterol E-testWako), and free fatty acids by the acyl-CoA synthetase and acyl-CoA oxidase method [46] using a kit (NEFA C-testWako) according to the manufacturer’s directions.Enzyme assaysPieces (5 mm in length) of intestine were fixed in 4 paraformaldehyde in phosphate buffered saline (PBS) overnight at room temperature. The fixed tissues were dehydrated through a graded ethanol series and embedded in Paraplast Plus (McCormick Scientific, St. Louis, MO, USA). Samples were transversely sectioned at 4 m thickness using a microtome (Yamato Koki, Saitama, Japan). The specimens were stained with Mayer’s hematoxylin and eosin, dehydrated with ethanol, and then mounted in Entellan (Merck, Darmstadt, Germany), according to Nakakura et al. [42]. Stained sections were observed under a light microscope (BX61, Olympus, Tokyo, Japan). The intestinal diameter, the outer diameter of the mucosa/submucosa layer, the circumference of the epithelial layer, the width of the muscularis externa, the number of PAS-positive goblet cells (panel E in Additional file 1: Figure S1) and the number of troughs in a villus-trough unit were measured and using Image J (http://imagej.nih.gov/ij/), and calculated from three randomly selected sections per animal.ImmunoblottingIntestinal homogenates (60 g) were run on 10 sodium dodecyl sulfate olyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranesIntestinal alkaline phosphata.
