Fig. 4.

Insulin signaling is increased in the liver of HFD-fed LysM-GRK2^+/− mice, accompanied by decreased steatosis and inflammation. Following 12 weeks of HFD, control (LysM-GRK2^+/+) and LysM-GRK2^+/− mice were injected vehicle (NaCl 0.9%) or insulin (1 IU/kg body weight) and after 10 min tissue lysates from liver were subjected to western blot (WB) and probed with antibodies against total and phosphorylated AKT (Ser473). Representative immunoblots and densitometric analysis (a) of four mice per group are shown. Results are expressed as % of stimulation over vehicle-treated mice (basal). b Liver sections from HFD-fed mice from both genotypes were stained with haematoxylin and eosin (10×magnification). c Liver TG were extracted, measured and expressed as equivalent triolein concentration (mg/ml). Data are means±SEM of 6 animals per genotype. Statistical significance was analyzed by unpaired two-tailed t test *p < 0.05 d Immunohistochemical analysis of liver sections stained with F4/80 antibody and counterstained with haematoxylin (magnification×20). Representative photomicrographs are shown. Positively stained area was quantified using Image J Software in at least three different randomly chosen fields per mouse. Results are expressed as % change over control (HFD-fed LysM-GRK2^+/+) mice and are means ± SEM of 6 animals per genotype. Statistical significance was analyzed by Mann Whitney test *p < 0.05. Real-time quantitative polymerase chain reaction (qPCR) was used to measure the hepatic expression of the F4/80 gene (e) and genes encoding TNF- , IL-1beta, IL-6, CCL2, CCR2 (f) and NRLP3 and COX-2 (Ptgs2) (g). Results were normalized against Ywhaz and Gapdh mRNAs. Values are represented as fold change over control mice and are means ± SEM of 5–6 animals per genotype. Statistical significance was analyzed by unpaired two-tailed t test (e) or by one-way ANOVA followed by Bonferroni post-hoc test (f, g) (*p < 0.05; **p < 0.05)