We applied a protocol GLUT2 site described previously [14], viz., soon after overnight incubation in
We made use of a protocol described previously [14], viz., immediately after overnight incubation in insulin-containing medium as described above, hepatocytes had been incubated for three hours in similar but insulin-free Williams E medium, followed by six hours 100nmoll insulin, 1 or 10mmoll metformin, 100nmoll ICAP. Immediately after incubation, cells had been sonicated in homogenizing buffer for protein studies or placed into Trizol reagent (Invitrogen) for mRNA research. All experimental procedures involving human materials had been authorized by the Institutional Evaluation Board of your University of South Florida College of Medicine, and also the James A. Haley Veterans Administration Health-related Center Investigation and Improvement Committee, Tampa, Fl, and carried out in accordance with all the Declaration of Helsinki and Superior Clinical Practice. Tissue Preparation As described [14], hepatocytes have been homogenized in ice-cold buffer containing 0.25moll sucrose, 20mmoll TrisHCl (pH, 7.5), 2mmoll EGTA, 2mmoll EDTA, 1mmoll phenlysulfonlyfluoride (PMSF), 20gml leupeptin, 10gml aprotinin, 2mmoll Na4P2O7, 2mmoll Na3VO4, 2mmoll NaF, and 1moll microcystin, then supplemented with 1 TritonX-100, 0.6 Nonidet and 150mmoll NaCl, and cleared by low-speed centrifugation.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptaPKC, Akt, and AMPK Assays As described [114,17], aPKCs were immunoprecipitated from lysates with rabbit polyclonal antiserum (Santa Cruz Biotechnologies, Santa Cruz, California, USA) which recognizes C-termini of PKC- and PKC- (PKC- will be the human homolog of mouse PKC- with 98 homology; human and mouse muscle contain mostly PKC- and tiny PKC-; mouse and human liver contain substantial amounts of both PKC- and PKC- [23]). Immunoprecipitates had been collected on Sepharose-AG beads (Santa Cruz Biotechnologies) and incubated for eight min at 30 in 100l buffer containing 50mmoll TrisHCl (pH,7.five), 100moll Na3VO4, 100moll Na4 P2O4, 1mmoll NaF, 100moll PMSF, 4g phosphatidylserine (Sigma, St. Louis, Missouri, USA), 50moll [-32P]ATP (NEN Life Science Products, Beverly, Massachussetts, USA), 5mmoll MgCl2, and, as substrate, 40moll serine analogue in the PKC- pseudocIAP-2 custom synthesis substrate (Millipore, Bedford, Massachussetts, USA). Just after incubation, 32P-labeled substrate was trapped on P-81 filter paper and counted. aPKC activation was also assessed by immunoblotting for phosphorylation of the auto(trans)phosphorylation web site, thr-555560 in PKC-, essential for, and reflective of, activation [23]. As described [14], for assays of recombinant PKC- and PKC- (50ngassay; Biovision, Mountain, California, USA), 10fmoll phosphatidylinositol-3,4,5-(PO4)3 (PIP3; Matreya, Pleasant Gap, Pennsylvania, USA) was added to activate and define aPKC activity. Activation of AMPK was assessed by measurement of immunoprecipitable AMPK activity as described [3,14], and by immunoblotting for phosphorylation of each threonine-172AMPK plus the AMPK substrate, serine-79-acetyl-CoA carboxylase (ACC). Western Analyses Western analyses have been conducted as described [114,17], working with: anti-phospho-serine-473Akt and glyceraldehyde-phosphate dehydrogenase (GAPDH) antisera (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); anti-phospho-threonine-560555-PKC-PKC- antiserum (Invitrogen, Carlsbad, CA, USA); mouse monoclonal anti-PKC- antibodies (Transduction Labs, Bedford, Massachussets, USA); and anti-phospho-threonine-172-Diabetologia. Author manuscript; offered in PMC 2014 April 02.Sajan et al.PageAMPK; and anti-phospho-serine-79-ACC antisera.