Journal:

Article Title: Insulin Enhances Post-translational Processing of Nascent SREBP-1c by Promoting Its Phosphorylation and Association with COPII Vesicles *

doi: 10.1074/jbc.M805746200

Figure Lengend Snippet: Effects of PKB on SREBP-1c phosphorylation and activation by insulin. A, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without Akt inhibitor II (10 μm) or III (10 μm) for 30 min before adding insulin. Microsomal membranes (500 μg) were immunoprecipitated (IP) with anti-FLAG (full-length SREBP-1c) and immunoblotted for phosphoserine antibody or FLAG antibody (upper left panel). Hepatocytes infected with Ad-His-SREBP-1cFLAG were preincubated with or without Akt inhibitor II or III for 30 min and then treated with insulin for 1 h. Nuclear protein extracts (50 μg) were separated by SDS-PAGE and immunoblotted for anti-His (nSREBP-1c) or anti-histone H1 antibodies (upper right panel). Hepatocytes were treated with or without insulin (100 nm) in the presence and absence of Akt inhibitor II (10 μm) or III (10 μm), and cell extracts were prepared. An equal amount of protein (50 μg) from control and each treatment was analyzed by Western blotting for phosphorylated GSK-3β-specific antibodies. The blots were reprobed with anti-GSK-3β antibodies to assess the levels of total GSK-3β (lower panel). B, hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins were immunoprecipitated (500 μg) with anti-SREBP-1c or preimmune IgG, fractionated by SDS-PAGE, and immunoblotted for Sec23 (left panel). Hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins (500 μg) were immunoprecipitated with anti-Sec23 or preimmune IgG, fractionated by SDS-PAGE, and immunoreacted with anti-SREBP-1c antibodies (right panel). C, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes and assayed by GST-Sar1 pulldown assay as described under “Materials and Methods.” D, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes for 1 h at 37°C and an ATP-regenerating system containing 50 μCi of [γ-32P]ATP. Membranes were reisolated, solubilized, fractionated by SDS-PAGE, and subjected to autoradiography as described under “Materials and Methods.” E, recombinant SREBP-1c, recombinant active PKB, or recombinant SREBP-1c and active PKB were incubated in vitro in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE, and autoradiographed. Alternatively, proteins were detected by immunoblotting using either SREBP-1 antibody or Akt antibody (left panel). Recombinant wild-type GSK-3β or its nonphosphorylatable mutant (S9A) and active PKB were incubated in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE and autoradiographed or immunoblotted using GSK-3β or Akt antibody (right panel).

Article Snippet: Akt inhibitor II (SH-5) and Akt inhibitor III (SH-6) were procured from Invitrogen.

Techniques: Activation Assay, Infection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Recombinant, In Vitro, Mutagenesis

Journal: Basic Research in Cardiology

Article Title: Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

doi: 10.1007/s00395-010-0133-0

Figure Lengend Snippet: Experimental protocols. All groups were subjected to 30 min of regional ischemia (RI) followed by 120 min of reperfusion. Hearts receiving no other treatments constitute the control group (Ctr). a The IPC group was exposed to 3× 5 min global ischemia (GI), with 5 min intermittent reperfusion periods prior to RI. To test the temporal cytoprotective efficacy of IPC effluent in recipient hearts, freshly collected effluent was administered for 10 min prior to RI (Eff Pre ) or for 10 min at onset of ischemic reperfusion (Eff Rep ). Furthermore, to mimic ischemic postconditioning, the IPC effluent was administered for 3× 30 s with 30 s intermittent KHB perfusion at immediate reperfusion (Eff Post ). Ischemic postconditioning (IPost) was achieved by 3× 30 s of global ischemia (GI) at early ischemic reperfusion. The PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) were administered for 10 min at immediate onset of ischemic reperfusion in conjunction with 10 min of pre- or post-ischemic treatment with IPC effluent (Eff Pre + WI/SH-6, Eff Rep + WI/SH-6 or Eff Post + WI). b The IPC effluent was fractionated in respect to charge and size. Effluent collected during IPC was first separated into a hydrophilic (HPhil) and a hydrophobic (HPhob) fraction. All total effluent fractions were administered for either 10 min prior to ischemia (HPhil Pre and HPhob Pre ) or for 10 min at reperfusion (HPhob Rep and HPhob Post ). The hydrophobic fraction was further fractionated by a 10 or 30 kDa size filter resulting in four hydrophobic fractions with molecules below 10/30 kDa (HPhob Pre < 10 and HPhob Rep < 30) or above 10/30 kDa (HPhob Pre > 10 and HPhob Rep > 30). Arrows indicate time points for tissue sampling. Solid lines KH buffer perfusion

Article Snippet: Furthermore, to explore if the cardioprotective properties mediated by the coronary IPC effluent were dependent on PI3K/Akt-dependent signaling at reperfusion, the PI3K-inhibitor Wortmannin (WI; 1 μM) (Tocris Bioscience, UK) and the Akt-inhibitor SH-6 (10 μM) (Enzo Life Sciences, New York, USA) were administered for 10 min at ischemic reperfusion (Eff Pre + WI/SH-6 and Eff Rep + WI/SH-6 and Eff Post + WI; Fig. a).

Techniques: Sampling

Journal: Basic Research in Cardiology

Article Title: Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

doi: 10.1007/s00395-010-0133-0

Figure Lengend Snippet: Phosphorylation status of myocardial Akt in recipient hearts exposed to treatment with IPC effluent. a Representative immunoblots of Akt phosphorylation (Ser 473 ) showing the effects of treatment with IPC effluent (Eff Pre ) in recipient hearts (tissue harvested at end of treatment), as compared to hearts exposed to the standard IPC protocol (IPC) and baseline hearts ( C B ). c Representative immunoblots of Akt phosphorylation (Ser 473 ) in pre-ischemic IPC effluent-treated hearts that was also reperfused for 15 min (Eff Pre+Rep ) and reperfusion treatment with IPC effluent (Eff Rep ) in recipient hearts as compared to ischemic reperfused control hearts (Ctr Rep ) (tissues harvested at 15 min of reperfusion). Administering the PI3K-inhibitor WI (1 μM) and the Akt-inhibitor SH-6 (10 μM) for 10 min at reperfusion abrogated Akt phosphorylation in IPC effluent-treated hearts. Total Akt and GADPH indicates equal loading. b , d Densitometric analysis of total and phosphorylated Akt immunoblots expressed in arbitrary units (AU). p-Akt expressed as a ratio of total Akt with C B = 1. Bars represent mean ± SEM. N ≥ 3 in each group. * P < 0.05 versus C B , † P < 0.05 versus IPC, § P < 0.05 versus Eff Pre+Rep , ¤ P < 0.05 versus Eff Rep , # P < 0.05 versus Ctr Rep

Article Snippet: Furthermore, to explore if the cardioprotective properties mediated by the coronary IPC effluent were dependent on PI3K/Akt-dependent signaling at reperfusion, the PI3K-inhibitor Wortmannin (WI; 1 μM) (Tocris Bioscience, UK) and the Akt-inhibitor SH-6 (10 μM) (Enzo Life Sciences, New York, USA) were administered for 10 min at ischemic reperfusion (Eff Pre + WI/SH-6 and Eff Rep + WI/SH-6 and Eff Post + WI; Fig. a).

Techniques: Western Blot

Journal: Basic Research in Cardiology

Article Title: Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

doi: 10.1007/s00395-010-0133-0

Figure Lengend Snippet: The effect of inhibiting PI3K and Akt upon reperfusion in IPC effluent-treated recipient hearts. When administering the PI3K-inhibitor WI and the Akt-inhibitor SH-6 at the onset of reperfusion in IPC effluent pre-ischemic (Eff Pre ) or post-ischemic (Eff Rep and Eff Post ) treated hearts, the cardioprotective effect of the effluent was completely abolished. Ctr control; Eff Pre 10 min effluent administration prior to RI; Eff Rep 10 min effluent administration after RI; Eff Post effluent administration 3× 30 s at start of reperfusion; WI 10 min Wortmannin (1 μM) at reperfusion; SH-6 10 min SH-6 (10 μM) at reperfusion. Bars represent mean ± SEM. N ≥ 6 in each group. * P < 0.05 versus Ctr

Article Snippet: Furthermore, to explore if the cardioprotective properties mediated by the coronary IPC effluent were dependent on PI3K/Akt-dependent signaling at reperfusion, the PI3K-inhibitor Wortmannin (WI; 1 μM) (Tocris Bioscience, UK) and the Akt-inhibitor SH-6 (10 μM) (Enzo Life Sciences, New York, USA) were administered for 10 min at ischemic reperfusion (Eff Pre + WI/SH-6 and Eff Rep + WI/SH-6 and Eff Post + WI; Fig. a).

Techniques: