Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin–sensitizing

Histological analyses tissues were fixed in formalin

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Histological analyses Tissues were fixed in formalin for at least 48 h, embedded in paraffin and H&E stained. After staining, each sample was imaged in triplicate. For determination of hepatic fibrosis, trichrome staining was used to visualize collagen. This was then quantified using the color segmentation function of Image J software (NIH). Western blotting, inflammation and real time quantitative PCR Tissues were dissected rapidly, snap frozen in liquid nitrogen and stored at −80 °C until subsequent analyses. Blotting for total and phosphorylated Akt, Jnk, Ampk and Acc (antibodies all from Cell Signaling) were performed as previously described 42 . Phosphorylated FoxO1 Ser 253, was normalized to Gapdh (Cell Signaling). For Pkc– ε phosphorylation, an antibody directed against Ser729 was used (Abcam). For membrane– associated Pkc activation, tissues were homogenized in 300 μl of buffer I (20 mM Tris·HCl, pH 7.4, 1 mM EDTA, 0.25 mM EGTA, 250 mM sucrose and protease inhibitor mixture) and centrifuged at 100,000 × g for 1 h (4 °C). The supernatants containing the cytosolic fraction Fullerton et al. Page 7 Nat Med . Author manuscript; available in PMC 2016 July 28. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscript
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were removed to new tubes. Pellets were then resuspended in 300 μl of buffer II (250 mM Tris·HCl, pH 7.4, 1 mM EDTA, 0.25 mM EGTA, 2% Triton X 100, protease inhibitor mixture) and centrifuged at 100,000 × g for 1 h (4 °C) to obtain the plasma membrane fraction. Gapdh and caveolin–1 were used as markers of the cytosolic and membrane preparation purity, respectively. Pkc activation is expressed as a ratio of membrane (normalized to caveolin–1) to cytosolic (normalized to Gapdh) localization, which was assessed from the same membrane to eliminate exposure bias. Hepatic tissues were prepared as previously described 43 and pro–inflammatory cytokines were determined using commercially available kits. Total RNA isolation, cDNA synthesis and quantitative real time PCR were performed as described previously 42 . Metabolic studies For glucose, insulin and metformin tolerance tests, mice were injected with D–glucose (2 g/kg and 1 g/kg for chow and HFD, respectively), human insulin (0.6 U/kg and 1 U/kg for chow and HFD, respectively) or 50 and 200 mg/kg metformin 37 via IP injection and blood glucose monitored at the indicated times by a small cut in the tail vein. Whole body adiposity was assessed by computed tomography and respiratory exchange ratio was determined using Columbus Laboratory Animal Monitoring System, as previously described 42 . Hyperinsulinemic–euglycemic clamps were performed as previously described 42 . Briefly, 5 days post–cannulation, only mice that lost < 8% of their weight were clamped. Mice, fasted 6 h were infused with a basal infusate containing D–[3– 3 H]–glucose (7.5 μCi/h, 0.12 ml/h) for 1 h to determine basal glucose disposal. An insulin infusate (10 mU/kg/min insulin in 0.9% saline) containing D–[3– 3 H]–glucose (7.5 μCi/h, 0.12 ml/h) was then initiated and blood glucose monitored and titrated with 50% dextrose infused at a variable rate to achieve and maintain euglycemia. For rates of glucose uptake, [
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  • Winter '19
  • Robert S Kiss
  • Insulin resistance, Fatty acid metabolism

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