Fig 1: The activation of SIK1 mediated by phanginin A was LKB1-dependent. (A–B) Primary mouse hepatocytes were pretreated with LKB1 siRNA for 48 h and then treated with 5 µM of phanginin A and then the LKB1 protein and SIK1 phosphorylation levels (A) (n = 4) and gluconeogenesis (B) (n = 5) were examined. (C–D) Primary mouse hepatocytes were pretreated with SIK2 and SIK3 siRNA for 24 h and then treated with or without 5 µM of phanginin A, and the SIK mRNA (C) and gluconeogenesis (D) were tested (n = 5). (E) Primary mouse hepatocytes were pretreated with LKB1 siRNA for 48 h and then treated with 5 µM of phanginin A and then the LKB1 protein and AMPK phosphorylation levels were examined (n = 4). (F–G) Primary mouse hepatocytes were pretreated with AMPK siRNA for 48 h and then treated with or without 5 µM of phanginin A and the AMPK protein level (F) (n = 3) and gluconeogenesis (G) (n = 4) were studied. All of the results are presented as the mean ± SEM. **P < 0.01 vs individual controls.
Fig 2: Phanginin A increased PDE4 activity and inhibited the cAMP pathway by activating SIK1. (A–D) Primary mouse hepatocytes were pretreated with 0.5 µM of pan-SIK inhibitor HG-9-91-01 for 30 min and cotreated with 5 or 10 µM of phanginin A and then the PDE4 activity (A), cAMP concentration (B), PKA activity (C), and CREB phosphorylation (D) were detected. *P < 0.05 and **P < 0.01 vs controls under basal conditions. #P < 0.05 and ##P < 0.01 vs controls under HG-9-91-01 conditions. (E–J) Primary mouse hepatocytes were pretreated with SIK1 siRNA for 48 h and then treated with 5 µM or 10 µM of phanginin A and then the PDE4 activity (E), cAMP concentration (F), and PKA activity (G) were examined. Primary mouse hepatocytes were treated with 5 µM of phanginin A after SIK1 interference and then the CREB phosphorylation (H), mRNA expression of G6P (I), and PEPCK (J) were evaluated. *P < 0.05 and **P < 0.01 vs controls under negative control conditions. ##P < 0.01 vs controls under siSIK1 conditions. All of the results are presented as the mean ± SEM (n = 5).
Fig 3: SIK1 overexpression inhibited gluconeogenesis and increased PDE4 activity in primary mouse hepatocytes. Primary mouse hepatocytes were overexpressed with SIK1 via transient plasmid transfections for 24 h and then the mRNA expression of SIKs (A) and Western blotting analysis of SIK1 (B) were conducted, and the PDE4 activity (C), cAMP levels (D), mRNA expression of gluconeogenic genes (E), and gluconeogenesis (F) were examined. All of the results are presented as the mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 vs the vector control group.
Fig 4: Chronic treatment of phanginin A improved metabolic disorders in ob/ob mice. Male ob/ob mice were treated with phanginin A (100 mg/kg, once daily, p. o.), metformin (250 mg/kg, once daily, p. o.), or vehicle for 26 days. (A–B) Random (A) and fasting blood glucose levels (B) were detected on days 4, 8, 12, 16, 20, 23, and 26. (C) Oral glucose tolerance tests were conducted on day 23. (D) HbA1c was determined on day 26. (E–F) Food intake accumulation (E) and body weight (F) were regularly measured during treatment. (G–H) Triglyceride and total cholesterol in the serum (G) and liver (H) were detected. (I) SIK1 phosphorylation in the liver was analyzed by Western blotting and quantified as the relative optical density. (J) Hepatic PDE4 activity and (K) cAMP concentration were examined after treatment. (L) CREB phosphorylation in the liver was analyzed by Western blotting. (M) Hepatic gluconeogenesis gene expression in ob/ob mice were evaluated. All of the results are presented as the mean ± SEM (n = 8). *P < 0.05 and **P < 0.01 vs the vehicle group.
Fig 5: Phanginin A inhibits gluconeogenesis by activating SIK1 in primary mouse hepatocytes. (A–B) Primary hepatocytes were incubated with 5 µM phanginin A for different times (A) or with different doses of phanginin A for 2 h (B). SIK1 phosphorylation at Thr182 was detected. met = metformin. (C) Primary hepatocytes were pretreated with or without 0.5 µM of HG-9-91-01 for 30 min and cotreated with 5 µM of phanginin A or 500 µM of metformin for 2 h and then SIK1 phosphorylation was detected. (D) Primary hepatocytes were pretreated with or without 0.5 µM of HG-9-91-01 for 30 min and cotreated with 5 µM or 10 µM of phanginin A or 500 µM of metformin for 4 h and then gluconeogenesis was detected. (E–G) Primary hepatocytes were pretreated with SIK1 siRNA for 48 h followed with or without phanginin A treatment, and the mRNA expression of SIK1-3 (E), SIK1 protein, (F) and gluconeogenesis (G) were detected. (H) The effect of phanginin A on gluconeogenesis in SIK1-knockdown hepatocytes under 10 nM glucagon-induced conditions. All of the results are presented as the mean ± SEM (n = 5 for all of the groups, except for n = 4 in Figure H). *P < 0.05 and **P < 0.01 vs basal controls. ##P < 0.01 vs HG-9-91-01 controls or controls under glucagon-induced conditions.
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