Materials and Methods Recombinant adenoviruses. A -expressing adenovirus was generated through homologous recombination between a linearized transfer vector pad-track and the adenoviral backbone vector pad-easy as described (1). pad- contained the fulllength murine cdna or variant with an NH 2 -terminal Flag-tag. In addition to the transgene the virus encoded the green fluorescent protein (GFP) transcribed from an second independent CMV promoter. GFP expression was used to monitor viral infection efficiency. An adenovirus coding for GFP only (pad-gfp) was used as a control in all experiments. Viruses were purified by the CsCl method and dialyzed against PBS buffer containing 10% glycerol as described (2). Two Hybrid Screening. Yeast two-hybrid screening with a GAL4 PH Akt (aa. 150-480) construct as bait was performed in the yeast strain AH109 (Clontech) according to the manufacturer's instructions. A total of 2x10 6 clones from the pre-adipocyte F422A library was screened. Protein Interaction Studies. GST pull down assays were done with baculovirus expressed GST-Akt (gift of T. Roberts, Dana Farber). For co-immunoprecipitation of epitope-tagged proteins, cells were transfected with Akt fused to hemaglutinin antigen (2ug) and Flag- (2ug) expression vectors by Lipofectamine 2000 according to the manufacturer's instructions. Transfected cells were harvested and lysed in Co-IP buffer [25mM Tris (ph 7.6), 150mM NaCl, 2.5mM MgCl 2, 0.5mM EDTA, 0.5% NP-40, 5mM β-glycerophosphate, 1mM DTT, 5% glycerol, and proteinase inhibitors]. Total cell lysate (500µg) was subjected to immunoprecipitation with immobilized anti-ha monoclonal antibodies (COVANCE). For co-immunoprecipitation studies on endogenous Akt and proteins, total cell lysate from HepG2 cells was incubated with antiserum to
. Immunoprecipitates were separated by SDS-PAGE (12% gels), and proteins were analyzed by Western blot assay using monoclonal antiserum to Akt. RNA interference. Double stranded RNA duplexes corresponding to aa. 34-40 of rat and mouse (5'-CGAGUGAGAGAUGAGCCUG-3') or human (5'- CGAGCUCGAAGUGGGCCCC-3') were purified, annealed, and transfected into human HepG2 hepatocytes. The effect of RNAi on expression and on insulin dependent Akt activation was measured after 24 to 48 hours. Mouse- and rat-specific duplex oligos were used as control oligos in experiments with human HepG2 cells. All RNAi experiments were performed on at least three independent occasions with comparable results. In vitro kinase assay. Human embryonic kidney cells (HEK 293) were co-transfected with an expression vector encoding HA-Akt and either a vector encoding or empty expression vector. One day after transfection, cells were deprived of serum for 16 hours and then treated with 100µM of sodium pervanadate or vehicle for 15 mins. Cells were lysed in lysis buffer [20mM Tris, (ph 7.6), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5m β-glycerophosphate, 1mM sodium pervanadate and proteinase inhibitors), and HA-tagged Akt was immunoprecipitated with monoclonal antibody to HA. Immune complexes were washed, and in vitro kinase assays were performed with recombinant GST-GSRSRRPSYRL polypeptide (2µg per reaction) as substrate. Reactions were incubated in kinase assay buffer [20mM Tris ph7.6, 5mM β- glycerophosphate, 2mM DTT, 0.1mM sodium pervanadate, 10mM MgCl 2, 200µM cold ATP, 1µCi [γ-p 32 ]-ATP] at 30 o C for 30 minutes. Reactions were terminated by addition
of 2x SDS PAGE loading buffer, and phosphorylated substrate was resolved by SDS PAGE (12% gels). Radio-labeled bands were quantified by phosphoimager. Western blot assays. Western blot assays were done as described (3) using specific antibodies to the non-phosporylated or phosphorylated forms of Akt or GSK3, respectively (Cell Signaling, Santa Cruz). Anti- rabbit polyclonal antiserum was generated against GST- polypeptide extending from aa. 1 to 145 of mouse protein. Specificity of the antiserum was verified by Western blot assay with purified recombinant protein. Animal experiments. Male 6-week old C57Bl6 mice were obtained from Harlan (San Diego, CA) and housed in an air-conditioned environment, with a 12-hour light-dark cycle, and were fed a regular unrestricted diet. Animals were anaesthetized with Iso- Flurane and a total of 1x10 9 plaque-forming units of recombinant virus was administered by systemic tail vein injection. In each experiment at least 7 animals received identical treatments. During the course of the experiments animals were fasted for 24 hours with free access to water, and then fasting blood glucose was monitored. Mice were then refed for the following 24h and blood glucose was determined thereafter. This fasting-feeding protocol was maintained for at least 7 consecutive days. All mice were sacrificed for blood and tissue collection at the end of the experiment. Blood samples were collected from the tail vein. Plasma was obtained by centrifugation of collected blood and assayed for insulin. Liver tissue for RNA and protein isolation was immediately frozen in liquid nitrogen and stored at 80 C. Cryomicrotome sections of liver samples were used to assess viral infection efficiency by fluorescence microscopy. For glucose tolerance tests, mice were fasted for 24 hours and then injected with 1 unit glucose per g body weight into the peritoneal cavity. Glucose levels were measured from blood collected from the tail immediately before and 10, 20, 30, 60, and 120 minutes after the injection. Hepatic
glycogen content was determined essentially as described (4), and expressed as mg glycogen per gram wet liver tissue. All study protocols were reviewed and approved by the IACUC of the Salk Institute. Blood metabolites. Blood glucose values were determined from whole blood using an automatic glucose monitor (One Touch Ultra, Lifescan). Plasma insulin levels were determined using a commercial insulin ELISA kit (Crystal Chem. Inc., Chicago). All procedures were performed according to the directions provided by the manufacturers. Quantitative Taqman RT-PCR. Total RNA was extracted from homogenized mice livers using the RNeasy (Qiagen, Valencia) kit including DNase I treatment. RNA was processed for quantitative RT-PCR analysis as previously described (5). Cell culture. Cells were maintained as described (6). Glucose output assay. Rat FAO hepatoma cells were cultured in six-well plates in DMEM with 10% FBS and infected 48 hours after plating with adenoviruses expressing either GFP or. 48 hours after infection, cells were treated with 30nM insulin for 3h where indicated. The medium was then replaced with 2 ml of glucose production buffer consisting of glucose-free DMEM, without phenol red, supplemented with 20mM sodium lactate and 2mM sodium pyruvate. After a 3 hour incubation, 0.4 ml of medium was assayed for glucose concentration by using a colorimetric glucose assay kit (Sigma). Readings were normalized to the total protein content determined from the whole-cell lysate.
Supplementary Figures Fig. S1. Co-immunoprecipitation assay of HA-tagged Akt1 and FLAG-tagged proteins in transfected HEK293 cells. Top, Input of wild-type and truncated proteins. Middle, Western blot of HA-tagged Akt1 immunoprecipitates showing FLAG-tagged recovered. Bottom, input levels of HA-Akt1 in transfected cells. - WT INPUT IgG CO-IP Akt INPUT Fig. S2. Western blot analysis of phospho (Thr 308 ) Akt, total Akt, and protein levels in HEK293 cells transfected with HA-tagged Akt1 expression vector plus Flagtagged vector. Cells were treated either with IGF1 (100nM) or left untreated for 30 minutes. Immunoprecipitates of Akt were prepared with antiserum to HA, and Western blot assays were done with antibody specific to phospho (Thr 308 )as well as anti-ha antibody to visualize total amounts of Akt. Amounts of Flag-tagged are shown. IGF - + + + - + P-T308 Akt
Fig. S3. Effect of RNAi oligos on expression in hepatocytes. Human HepG2 hepatocytes were co-transfected with increasing amounts of RNA duplex oligos (0, 0.1, 0.2, 0.4 µg) plus Flag-tagged expression vector. Flag-tagged and endogenous expression in transfected cells was evaluated after 24 hours by Western blot assay. RNAi FLAG- Fig. S4. Enhanced Akt phosphorylation in response to growth factor stimulation in cells with disrupted expression. Western blot assays of phospho (Thr 308 ) and total Akt levels in control and pervanadate (PV) treated HepG2 cells transfected with wild-type (WT) or mutant (mt) RNAi oligos and HA-tagged Akt expression vector. HepG2 extracts were probed with antiserum specific for phospho (Thr 308 ) (top) or nondiscriminating Akt antiserum (middle) and antiserum (bottom). RNAi mt mt WT PV - + - WT + P- Akt
Fig. S5. Potentiation of GSK-3 phosphorylation in response to growth factor stimulation after RNAi-mediated disruption of. Western blot assay of phospho (Ser 21 ) GSK- 3α and phospho (Ser 9 ) GSK-3β in HepG2 hepatocytes transfected with RNAi oligos and treated with pervanadate (PV) or vehicle as indicated. Effect of co-transfected mouse expression vector (which is not recognized by human RNAi oligos) on GSK-3 phosphorylation shown. mtrb + + RNAi mt mt WT WT WT WT PV - + - + - + P-GSK3 α P-GSK3β GSK3 α 1 2 3 4 5 6 GSK3β Fig. S6. Western blot analysis of protein amounts in whole liver extracts from wild-type and db/db mice using antiserum to. Organs were harvested under refed or fasting conditions. refed fast WT db/db
Fig. S7. expression is induced by counter-regulatory hormones. Quantative PCR analysis of RNA levels in FAO hepatocytes treated with dexamethasone (10-7 M) or forskolin (10µM) for 18 hours. TRB-3 expression in FAO hepatoma cells 3.5 3 2.5 2 1.5 1 0.5 Vehicle DEX FSK
Fig. S8. Western blot analysis of Akt in immunoprecipitates prepared from whole liver extracts of fasted db/db mice using either pre-immune (Pre), polyclonal anti- (α) antiserum, or anti- antiserum blocked with polypeptide (aa. 1-145). Recovery of 60 kd Akt immunoreactive band from each immunoprecipitate is shown. Input levels of and Akt indicated. Akt Pre - α + - Pre-blocked IgG Akt INPUT INPUT 1 2 3 Fig. S9. Comparable expression of in db/db mice and in mice infected with adenovirus. Western blot assay of whole liver extracts using anti- or anti-creb antiserum as control. Livers were collected from db/db, adenovirus, or control GFP adenovirus infected mice. GFP db/db anti- 1 2 3 4 5 6 anti-creb
Fig. S10. Serum insulin levels (ng/ml) in control (GFP) or adenovirus infected mice under refed or fasted conditions, as indicated. 2 GFP 1 0 refed fasted References 1. T. He, et al., Proc. Natl. Acad. Sci. 95, 2509-2514 (1998). 2. T. Becker, et al., Methods Cell Biol, 161-189 (1994). 3. L. F. Michael, H. Asahara, A. Shulman, W. Kraus, M. Montminy, Mol Cell Biol. 20, 1596-1603 (2000). 4. W. Shen, L. M. Scearce, J. E. Brestelli, N. J. Sund, K. H. Kaestner, J Biol Chem 276, 42812-7. (2001). 5. S. Herzig, et al., Nature 413, 179-183 (2001). 6. K. Du, M. Montminy, J Biol Chem 273, 32377-9. (1998).