Supplemental Information
S1a. Insulin-dependent coactivation of FOXO1 by PGC-1 on the glucose-6-Pase
promoter. Immortalized hepatocytes were treated and transfected as in a except that a
glucose-6-Pase promoter/luciferase plasmid was used.
S1b. FOXO1 coactivation by PGC-1 on the glucose-6-Pase promoter depends on
FOXO1 DNA binding sites. Immortalized hepatocytes were transfected as in b, with the
addition of the glucose-6-Pase promoter/luciferase plasmids containing mutations of the
FOXO1 DNA binding sites. Error bars indicate SEM of four independent experiments
made in duplicate. Glucose-6-phosphatase promoter linked to luciferase was previously
described 5
S2. PGC-1 physically interacts with FOXO1 in vitro S2a, Mapping of the FOXO1
binding site on PGC-1. FOXO1, SRC-1 and HNF4 proteins were radiolabelled with
[35S]-methionine using an in vitro translation kit. Binding assays were performed as
described in the Methods sections using various deletions of PGC-1, fused to GST.
After binding reactions, proteins were separated by SDS-PAGE and detected by
autoradiography. S2b, Mapping of the PGC-1 binding site on FOXO1. Generation of
FOXO1 fusion protein, radiolabelling of PGC-1, HNF4 and FOXO1, and binding
assays are described in Methods.
S3. Insulin-dependent decrease of PGC-1-induced glucose production is reversed by a
constitutively active form of FOXO1. Fao rat hepatocytes were infected with
adenoviruses expressing GFP, PGC-1 and various alleles of FOXO1 for 24 hours. Cells
were changed to a serum-free medium containing 0.5% BSA and treated for 12 hours
with vehicle or 10 nM insulin, 1M forskolin and 1 M dexamethasone. After 12 hours
incubation, cells were washed three times with phosphate-buffered saline and incubated
in the gluconeogenic medium (glucose and phenol red-free DMEM supplemented with
20 mM sodium lactate and 2 mM sodium pyruvate.) for 3 hours. Glucose concentrations
were measured with a colorimetric glucose assay kit (Sigma). The readings were then
normalized to total protein content determined by the BCA method (Pierce). Error bars
indicate SEM of three independent experiments made in duplicate.