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. 2014 Aug 4;5:4535. doi: 10.1038/ncomms5535

Figure 2. Liver-specific SIK2-knockout mice display normal glycaemia.

Figure 2

(a) Diagram of the generation of liver-specific SIK2-knockout (liver SIK2 KO) mice. Exon 5 was flanked by loxP sites and a hygromycin resistance cassette flanked by FRT sites was inserted upstream from the 5′ loxP site. The hygromycin resistance cassette was excised by the expression of the FLP recombinase in vivo. Liver disruption of exon 5 flanked by loxP sites was achieved by crossing SIK2-floxed mice with Alfp–Cre transgenic mice. (b) Immunoblotting of SIK2 in liver, vastus lateralis muscle, inguinal white adipose tissue (WAT), brown adipose tissue (BAT) and heart from WT, SIK2-floxed (Lox) and liver SIK2-deficient (CRE) mice, demonstrating that the deletion was effective and liver specific. (c) Kinase activity of SIK1, 2 and 3 in liver extracts generated from randomly fed control (white bars) and liver SIK2-KO (black bars) mice. SIK1, 2 and 3 were immunoprecipitated with respective isoform-specific antibodies from liver extracts in triplicate and subjected to in vitro kinase assay. Data is presented as mean±s.d., n=3. (d) Blood glucose levels of control (white bars) and liver SIK2-KO (black bars) male mice (10-week-old) fed ad libitum (fed), fasted for 18 h (fasted) or refed overnight with a high-carbohydrate diet following a 24 h fast (refed). n=6–12 for each group. (e) Evaluation of blood glucose levels during an oral glucose tolerance test (3 g kg−1) in control and liver SIK2-KO mice (n=6 for each group). Values are presented as mean±s.e.m. (f) Relative Ppargc1a, Pck1, Srebp-1c and Fasn mRNA levels measured by quantitative reverse transcription–PCR in the liver of control (white bars) and liver SIK2-KO (black bars) mice fasted for 18 h or refed overnight with a high-carbohydrate diet following an 24 h fast (refed). n=6 for each group. All values are presented as mean±s.e.m. (g) Immunoblotting of SIK2, PEPCK and GAPDH in the liver of control and liver SIK2-KO mice fasted for 18 h or refed overnight with a high-carbohydrate diet following an 24 h fast (refed). Data are representative of nine mice per group.