All

experiments were performed according to Dutch law and

All

experiments were performed according to Dutch law and approved by the Ethical Committee for Animal Experiments, University of Groningen, the Netherlands. Age-matched mice (8-10 weeks) were fed either a high-fat diet (HFD) containing 36% fat from lard and 0.03% cholesterol (4031.45, Abdiets, Woerden, the Netherlands) or a regular chow diet (2181, RMH-B Arie Blok, Woerden, the Netherlands) for 12 weeks. All mouse experiments were carried out simultaneously, using diets from the same batch numbers. The mice were fasted for 4 hours before being given an intraperitoneal injection with saline or human recombinant insulin Actrapin (Novo Nordisk Canada, Ontario, Canada) (0.75 U/kg) 15 minutes before sacrifice. Tissues LY2109761 price were isolated and snap-frozen in liquid nitrogen and stored either at −80°C, fixed in 4% paraformaldehyde, or stored in paraffin. Nuclear extracts were prepared from fresh liver tissue according to the manufacturer’s protocol and binding of NF-κB to DNA was determined using the p65 TransAM enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, La Hulpe, Belgium). Lipid extraction was performed as described.30 Mice were fasted 9 hours overnight and given 2 g/kg of a 20% glucose solution orally. Glucose levels were measured with a Target Selective Inhibitor Library research buy One Touch Ultra glucose meter before and 15, 30, 45, 90, and 120 minutes after the glucose administration. Insulin

levels were determined with ALPCO immunoassays (ALPCO Diagnostics, Salem, NH) according to the manufacturer’s instructions. Paraffin-embedded sections of the liver (4 μm) were stained with hematoxylin-eosin (H/E), Masson’s Trichrome staining, or Oil Red O. Frozen-cut liver sections (5 μm) were fixed in 4% paraformaldehyde and stained with antibodies against Cd68 and Cd11b (Abcam, Cambridge, UK). We used a Leica DM 3000 microscope. Scoring of steatosis unless and ballooning of hepatocyte degeneration and inflammatory foci was done by a certified

veterinary pathologist and based on a described method.31 Tissues were homogenized and equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and the immune-complex was visualized using the molecular imager ChemiDoc xrs+ system (Bio-Rad). The level of aspartate transaminase (AST) and alanine transaminase (ALT) plasma was measured according to the manufacturer’s protocol (Spinreact, Santa Coloma, Spain). Equal amounts of liver (300 μg) were incubated with Ac-DEVD-AMC Caspase-3 fluorogenic substrate (BD Pharmingen, San Jose, CA) at 37°C for 60 minutes. The amount of cleaved fluorescent AMC was quantified by a spectrofluometer at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Data were statistically analyzed by performing a nonparametric Mann-Whitney test using GraphPad Prism to compare experimental groups (v. 5.00 for Windows, San Diego, CA). Data were expressed as mean ± standard error of the mean (SEM) and considered significant at P < 0.05.

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