Figure 2
Impairment of CREBH and Insig-2a signaling augments hepatic de novo lipogenesis at the fasting state in CREBH-KO mice.
Liver tissues that were harvested from WT and CREBH-KO mice subjected to the fasting and refeeding protocol as described in the Methods were used to prepare total RNA and tissue homogenates for the following analyses. (A) Protein mass of the precursor, as well as the active forms of SREBP-1 (SREBP-1-n) and SREBP-2 (SREBP-2-n) were measured by immunoblot analysis. Immunoblots were quantified by densitometry and were normalized to β-actin. (B) Relative mRNA expression of SREBP-1c target genes, FASN, ACC and SCD-1 were determined by qRT-PCR. (C) Hepatic lipogenesis was determined by intraperitoneal injection of 3H2O into fasted-WT and fasted-KO mice (n = 3/group). Incorporation of 3H2O into newly synthesized fatty acids was determined as described in the Methods. (D) McA cells were transfected with siRNA against Insig-2 or control siRNA for 36 hours, cells were then treated with glucose (6mM) and LA (200 μM) for additional 36 hours. Cells were used to prepare total RNA and relative mRNA expression of FASN and ACC were determined by qRT-PCR. (E) Secreted TG and CHOL in the culture media of McA cells treated as in (D) were extracted and determined as described in the Methods. KO represents CREBH-KO. For animal studies, results are shown as mean ± SEM. n = 3–12/group. For in vitro studies, data represent three experiments. *P < 0.05, **P < 0.01 versus controls.
