Figure 6. Glucose production impairment in SRC-1 KO hepatocytes is rescued by C/EBPα re-expression.

A) C/EBPα controls PC and FOXO1 gene expression but not other important known genes of the gluconeogenic pathway. Primary hepatocytes were treated with a C/EBPα adenovirus or empty virus (GFP) and used 48 hours later for measurement of gluconeogenic gene mRNA by qPCR.

B) In the mouse liver, C/EBPα and SRC-1 bind to the PC promoter in the region containing C/EBPα responsive elements specifically upon fasting. ChIP assays were performed with 100 bp amplicons flanking the region containing the C/EBPα motifs of the PC promoter. qPCR (normalized to input) was used to assess SRC-1 and C/EBPα occupancy of the PC promoter upon chromatin immunoprecipitation, using SRC-1 and C/EBPα specific antibodies.

C) The combination of C/EBPα and SRC-1 controls the PC promoter in a synergistic manner. Hela cells were transfected in the presence of the proximal promoter of the PC gene in presence of C/EBPα, SRC-1 or both expression vectors.

D) SRC-1 KO mice placed on a high protein diet are hypoglycemic. Blood glucose levels were determined in mice in the fed state (n = 5 mice), using a hand-held glucometer.

E) In SRC-1 KO mice placed on a high protein diet, PC and C/EBPα gene expression are decreased. PC and C/EBPα mRNA levels were measured by qPCR in the liver of KO and WT animals in the fed state (n=5 mice per group).

F) Acute overexpression of C/EBPα reactivates glucose production in primary hepatocytes from SRC-1 KO mice. Primary hepatocytes from SRC-1 KO mice and WT littermates were treated with C/EBPα or GFP adenoviruses and cultured for an additional 48 hours prior to measurement of glucose production over a period of 6 hours in the presence of forskolin and dexamethasone.

G) Re-expression of C/EBPα in liver rescued the glucose production in SRC-1 KO mice. Adenoviruses (GFP and C/EBPα) were injected via the tail vein of each mouse (n=5-6 for each group). Blood glucose was checked after 7 days of infection using a hand-held glucometer.

H) SRC-1 LiKO mice are hypoglycemic upon fasting. Blood glucose levels were determined in mice (n = 5 mice) in the fasted state (24h), using a hand-held glucometer. I) In SRC-1 liKO mice placed on fasting conditions, PC and C/EBPα gene expression are decreased. PC and C/EBPα mRNA levels were measured by qPCR in the liver of KO and WT animals (n=3-4 mice per group) upon fasting (24h).

Data are represented as mean ± SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05; two asterisks indicate p < 0.01 and three asterisks indicate p < 0.005.

“See also Fig.S5”