Rats fasted 24 h were killed, and their liver samples removed at 11:00 h. Each experimental group contained 6 rats. Figure 9 Time of treatment, feeding conditions, times of sampling and light – darkness cycle used in the experimental protocol. RFS = restricted feeding schedule. Liver sampling Each animal was deeply anesthetized with Anestesal® (sodium pentobarbital)

at a dose of 1 ml per 2.5 kg of body weight. In one set of experiments the rats were killed by decapitation, and their livers removed and weighed. A fragment (0.3 – 0.5 g) was weighed, then kept at ≈ 65°C for one week and weighed again; the initial #check details randurls[1|1|,|CHEM1|]# water content was calculated as the difference between the initial and final weights. In a different set of experiments, small sections of each liver were rapidly removed and cut into pieces of about 1 mm3 with sharp razors to be fixed for morphometric measurements and histochemical techniques or processed for electron microscopy. Morphometry CX-6258 Small tissues blocks (≈ 1 mm3) for each rat, 6 per group, were immediately fixed in a cold solution of 2.5% glutaraldehyde diluted in 0.15 M cacodylate buffer, pH 7.3. After 60 min, tissues were postfixed for 1 h in 1% osmium tretroxide dissolved

in the same buffer. Then, liver fragments were dehydrated in graded acetone dissolved in deionized water and embedded in epoxy resin. One-micron thick semi-thin sections were obtained by a Leica ultramicrotome equipped with glass knives and stained with toluidine blue. Observations were done in a Nikon Eclipse E600 microscope, and images were obtained with a digital camara Photometrics Cool SNAP. Hepatocytes with a single, clear nucleus

were selected, and their surfaces were measured with the program IPLab V 3.6 for cross-sectional area determination. Histochemical techniques For glycogen staining, liver fragments (6 rats for each experimental group) were immediately placed and kept 48 h in a fixative (freshly prepared 10% w/v formaldehyde in 0.1 M phosphate buffer, pH 7.2), embedded in paraffin, sectioned at 5-μm thickness, and assessed to detect the content of glycogen within the hepatocytes by the periodic acid-Schiff reaction, with diastase addition for non-specific staining (PAS/D). In this method periodate oxidizes the hydroxyl Linifanib (ABT-869) moieties of glucose residues to aldehydes, which in turn react with the Schiff reagent generating a purple-magenta color. Ten representative fields from at least 4 different liver fragments per rat were analyzed by light microscopy (Olympus BX51; Olympus American, Melville, NY) and captured with a digital video camera (Cool Snap Pro, Media Cybernetics, Silver Spring, MD). Each digital image was photographed with the ×10 objective and formatted at fixed pixel density (8 × 10 inches at 150 dpi) using Adobe Photoshop software (v. 5.5). Each digital image was then analyzed using the MetaMorph Imaging Processing and Analysis software (v. 4.