UCP2 induction by KB228.

<p>Chow-fed (n = 7/7, 6 months of age) and HFD-fed (n = 9/9, 6 months of age) C57/Bl6J male mice underwent vehicle or KB228 treatment. 2 hours post-treatment livers and gastrocnemius muscles were removed and homogenized. (A, B) In liver homogenates from chow-fed (A) or HFD-fed (B) mice UCP2 mRNA and protein levels were assayed in RT-qPCR reactions and Western blotting. (C, D) In skeletal muscle homogenates from chow-fed (C) or HFD-fed (D) mice UCP2 mRNA levels were measured in RT-qPCR reactions. (E) Differentiated C2C12 myoblasts were treated with 3 µM of KB228 for 8 hours then UCP2 expression was determined in RT-qPCR reactions. Error is given as SD on panel E. * and *** indicate statistically significant difference between vehicle and GPi-treated groups at p<0.05, or p<0.001, respectively.</p

KB228 induced mTORC2 in mice.

<p>Chow-fed (n = 7/7, 6 months of age) and HFD-fed (n = 9/9, 6 months of age) C57/Bl6J male mice underwent vehicle or KB228 treatment (90 mg/kg). 2 hours post-treatment livers were removed and homogenized. From liver homogenates of chow-fed (A) and HFD-fed (B) mice Akt and phospho-Akt (⁴⁷³Ser) levels were determined by Western blotting.</p

Other GPi-s also induce UCP2 expression in HepG2 cells.

<p>(A) Three GPi-s TH, NV50 and NV76 were tested on HepG2 cells. (B–C) HepG2 cells (n = 3) kept under normoglycemic conditions were treated with the inhibitors at the indicated concentrations for 8 hours, then (B) glycogen content and (C) UCP-2 expression was determined as described in Materials and Methods. Error is given as SD throughout the figure. * and *** indicate statistically significant difference between vehicle and GPi-treated groups at p<0.05, or p<0.001, respectively. VEH – vehicle, other abbreviations are in the text.</p

Primers used in RT-qPCR reactions.

<p>Primers used in RT-qPCR reactions.</p

Synthesis of KB228.

<p><i>N</i>-(3,5-dimethyl-benzoyl)-<i>N’</i>-(β-D-glucopyranosyl)urea (KB228), a GP inhibitor was prepared as described in the Materials and Methods section.</p

Effect of propofol on tau phosphatases in mouse cortical tissue 0.5 h and 2 h following the administration of propofol under normothermic conditions.

<p>Cortical protein extracts were separated by SDS-PAGE and levels of phosphatases were determined using antibodies directed at the following proteins: (<i>1</i>) PP1 catalytic subunit, (<i>2</i>) PP2B catalytic subunit, and (<i>3</i>) PP2A catalytic subunit. Relative immunoreactive band intensities are expressed as a percent of control (Ctl; intralipid) and are displayed for each epitope. For each condition, 1 representative datum is displayed. (<i>4</i>) PP2A activity was measured with the PP2A Immunoprecipitation Phosphatase BioAssay Kit from US Biological and values expressed as percentage of control. All data are expressed as mean ± SD. * and ** denote <i>P</i><0.05 and <i>P</i><0.01 vs. ctl, respectively; Ctl (n = 6), 0.5 h (n = 6), and 2 h (n = 7); ANOVA with Newman-Keuls <i>post hoc</i> test.</p

Effect of propofol on tau phosphorylation in Tau-SH-SY5Y cells.

<p>Cells were harvested after 1 h incubation at 37°C (n = 4) or 30°C (n = 3) in the absence of propofol (<i>A</i>), or following 1 h exposure to 10% intralipid in DMEM (Ctl, n = 3) or propofol (Prop, n = 5) at 3 µg/ml (16.8 µM), both at 37°C (<i>B</i>). Cell lysate protein extracts were separated by SDS-PAGE and the level of tau phosphorylation was determined using antibodies directed at the AT8 (<i>A1, B1</i>), CP13 (<i>B2</i>), or PHF-1 (<i>B3</i>) phosphoepitopes, or total tau (<i>A2, B4</i>). Relative immunoreactive band intensities are expressed as a percent of control and are displayed for each phosphoepitope and total tau. Tau phosphoepitopes are normalized on total tau. For each condition, 2 representative data are displayed. Data are expressed as mean ± SD, with *, **, and *** denoting <i>P</i><0.05, <i>P</i><0.01 and <i>P</i><0.001 vs. ctl, respectively with unpaired <i>t</i>-test.</p

Regional anatomical localization of tau phosphorylation following 30 min of anesthesia with propofol.

<p>Fluorescence photomicrographs of hippocampal sagittal sections are shown with AT8 (Green, <i>A,B,C</i>), Total Tau (Red, <i>D,E,F</i>), or merged with DAPI (Green-Red-Blue, <i>G,H,I</i>), for the following conditions: Control (Intralipid, <i>A,D,G</i>), Hypothermia (<i>B,E,H</i>), and Normothermia (<i>C,F,G</i>). All images were taken at 5x magnification.</p

Tau phosphorylation in mouse hippocampal tissue 30 min following the administration of propofol under hypothermic (<i>A</i>) or normothermic (<i>B</i>) conditions.

<p>Hippocampal protein extracts were separated by SDS-PAGE and levels of tau phosphorylation were determined using antibodies directed at the AT8 (Ser²⁰²/Thr²⁰⁵;<i>A1,B1</i>), CP13 (Ser²⁰²;<i>A2,B2</i>), and PHF-1 (Ser³⁹⁶/Ser⁴⁰⁴;<i>A3,B3</i>) phosphoepitopes, or total tau (<i>A4, B4</i>). Relative immunoreactive band intensities are expressed as a percent of control (Ctl; intralipid) and are displayed for each phosphoepitope and total tau. For each condition, 2 representative data are displayed with Ctl (n = 4), and Prop (n = 5). Data are expressed as mean ± SD. *** denotes <i>P</i><0.001, ** denotes <i>P</i><0.01, and * denotes <i>P</i><0.05 vs. Ctl with unpaired <i>t</i>-test.</p

Tau phosphorylation in mouse cortical tissue 30 min and 2 h following the administration of propofol under normothermic conditions.

<p>Cortical protein extracts were separated by SDS-PAGE and levels of tau phosphorylation were determined using antibodies directed at the AT8 (<i>1</i>), PHF-1 (<i>2</i>), pS262 (<i>3</i>), MC6 (<i>4</i>), and pS422 (<i>5</i>) phophoepitopes, or Total Tau (<i>6</i>). Relative immunoreactive band intensities are expressed as a percent of control (Ctl; intralipid) and are displayed for each phosphoepitope and total tau. For each condition, 1 representative datum is displayed with Ctl (n = 6), 0.5 h (n = 6), and 2 h (n = 7). Data are expressed as mean ± SD. *, and ** and denote <i>P</i><0.05, and <i>P</i><0.01 vs. Ctl, respectively; ANOVA with Newman-Keuls <i>post hoc</i> test.</p