Dexamethasone-induced Krüppel-like factor 9 expression promotes hepatic gluconeogenesis and hyperglycemia

Abstract

Chronic glucocorticoid therapy has serious side effects, including diabetes and fatty liver. However, the molecular mechanisms responsible for steroid-induced diabetes remain largely enigmatic. Here, we show that hepatic Krüppel-like factor 9 (Klf9) gene expression is induced by dexamethasone and fasting. The overexpression of Klf9 in primary hepatocytes strongly stimulated Pgc1a gene expression through direct binding to its promoter, thereby activating the gluconeogenic program. However, Klf9 mutation abolished the stimulatory effect of dexamethasone on cellular glucose output. Adenovirus-mediated overexpression of KLF9 in the mouse liver markedly increased blood glucose levels and impaired glucose tolerance. Conversely, both global Klf9-mutant mice and liver-specific Klf9-deleted mice displayed fasting hypoglycemia. Moreover, the knockdown of Klf9 in the liver in diabetic mouse models, including ob/ob and db/db mice, markedly lowered fasting blood glucose levels. Notably, hepatic Klf9 deficiency in mice alleviated hyperglycemia induced by chronic dexamethasone treatment. These results suggest a critical role for KLF9 in the regulation of hepatic glucose metabolism and identify hepatic induction of KLF9 as a mechanism underlying glucocorticoid therapy-induced diabetes.

Keywords: Diabetes; Endocrinology; Gluconeogenesis; Glucose metabolism; Metabolism.

Figure 1. Hepatic Klf9 expression is induced by fasting and Dex.

(A) Quantitative PCR analysis of hepatic Klf9, Pgc1a, Pck1, and G6pc in C57BL/6J mice 12 hours after injection with saline or Dex (1 mg/kg) (n = 5/group). (B) Representative Western blot analysis of hepatic KLF9 and PGC1α in the mice described in A. (C) Quantitative PCR analysis of hepatic Klf9 and Pgc1a in mice under ad libitum–fed, 24 hour–fasted or 12 hour–refed conditions (n = 5/group). (D) Representative Western blot analysis of hepatic KLF9 and PGC1α in mice described in C. (E) Quantitative PCR analysis of Klf9, Pgc1a, Pck1, and G6pc in mouse primary hepatocytes treated with 100 nM Dex and/or 10 μM of the GR antagonist RU486 for 12 hours. (F) ChIP assays performed as described in Methods showing that fasting leads to the binding of endogenous GR proteins to GRE1/2 on the Klf9 promoter region, but not the distal region, which lacks GREs (negative control), as indicated. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test (A), 1-way ANOVA (C, E).

Figure 2. KLF9 activates the gluconeogenic program in primary hepatocytes through PGC1α.

(A) Quantitative PCR analysis showing mRNA levels of Pgc1a, G6pc, Pck1, and Glut2 in mouse primary hepatocytes infected with Ad-GFP or Ad-Klf9. Cells were harvested for further analysis 48 hours after infection. (B) Western blot analysis of KLF9 and PGC1α in primary hepatocytes treated as described in A. (C) Glucose output assay showing the effects of Klf9 overexpression on glucose production in primary hepatocytes as described in A. (D) ChIP assay performed as described in Methods, showing that both fasting (top panel) and Dex treatment (bottom panel) promote endogenous KLF9 binding to the proximal region of the Pgc1a gene promoter, but not the distal region (as a negative control). (E) Glucose output assay showing glucose production in primary hepatocytes isolated from global Klf9-mutant and WT C57BL/6 mice treated with Dex (100 nM) or saline for 12 hours. (F) Quantitative PCR analysis (left) of Pgc1a, G6pc, Pck1, and Glut2 in the primary hepatocytes described in E. (G) Representative Western blot analysis of hepatic KLF9 and PGC1α in the primary hepatocytes described in E. (H) Quantitative PCR analysis of G6pc and Pck1 in primary hepatocytes infected with the indicated adenoviruses. (I) Glucose output assay showing glucose production in the primary hepatocytes described in H. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test (A, C), 2-way ANOVA (E, F), or 1-way ANOVA (H, I).

Figure 3. Hepatic overexpression of Klf9 elevates blood glucose and impairs glucose tolerance in C57BL/6J mice.

(A) Quantitative PCR analysis of Klf9, Pgc1a, G6pc, Pck1, and Glut2 in the livers of C57BL/6J mice infected with Ad-GFP or Ad-Klf9 (n = 6/group). Seven days after infection, the 6 hour–fasted mice were sacrificed for further analysis. (B) Representative Western blot analysis of the hepatic KLF9 and PGC1α in mice treated as in A. (C) Blood glucose of 6 hour–fasted C57BL/6J mice treated as in A on day 7 after adenovirus injection. (D, E) Blood glucose during the GTT (D) and PTT (E) of C57BL/6J mice treated as in A (n = 6/group). Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test (A, C-E).

Figure 4. Liver-specific Klf9-deficient mice display decreased fasting blood glucose and enhanced glucose tolerance.

(A) Quantitative PCR (left panel) and Western blot (right panel) of Klf9 and other gluconeogenic genes/proteins in the livers of 6 hour–fasted Klf9^alb–/– mice and control littermates (Klf9^fl/fl) at 8 to 9 weeks of age (n = 5/group). (B) Blood glucose in 6 hour–fasted Klf9^fl/fl mice or Klf9^alb–/– mice (n = 8/group). (C, D) Blood glucose during GTT (C) and PTT (D) of Klf9^fl/fl mice or Klf9^alb–/– mice (n = 6/group). (E) Blood glucose in 6 hour–fasted Klf9^fl/fl mice and Klf9^alb–/– mice injected with Ad-GFP or Ad-Pgc1a (n = 5/group). (F) Quantitative PCR analysis of hepatic Pgc1a and G6pc mRNA levels in mice treated as in E (n = 5/group). (G) Representative Western blot analysis of hepatic PGC1α protein levels in mice treated as in E. Data are represented as mean ± SEM. *P < 0.05; ***P < 0.001, 2-tailed Student’s t test (A-D) or 2-way ANOVA (E, F).

Figure 5. Liver-specific Klf9-deficient mice display fasting-induced hepatic steatosis.

(A) Representative gross morphology and H&E staining of livers from Klf9^fl/fl mice and Klf9^alb–/– mice fasted for 24 hours. (B–D) Biochemical analysis showing hepatic TG (B), serum TG (C), and FFA (D) content in ad libitum–fed or 24 hour–fasted Klf9^fl/fl mice and Klf9^alb–/– mice (n = 5/group). (E) Quantitative PCR analysis of Pgc1a, Ppara, Mcad, Cpt1a, Cyp4a10 and Cyp4a14 in the livers of 24 hour–fasted Klf9^fl/fl mice and Klf9^alb–/– mice (n = 5/group). Scale bars: 20 μm. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 1-way ANOVA (B-D) or 2-tailed Student’s t test (E).

Figure 6. Hepatic Klf9 deficiency alleviates hyperglycemia induced by chronic Dex treatment.

(A)Blood glucose in the 16 hour–fasted Klf9^fl/fl mice and Klf9^alb–/– mice (n = 6/group) treated with saline or Dex (1 mg/kg) every other day for 2 months (n = 6/group). (B, C) Blood glucose during GTT (B) and PTT (C) of Klf9^fl/fl mice and Klf9^alb–/– mice treated as in A. (D) Quantitative PCR analysis of Pgc1a, G6pc, and Glut2 in livers of Klf9^fl/fl mice and Klf9^alb–/– mice treated as in A (n = 6/group). (E) Representative Western blot analysis of hepatic KLF9 and PGC1α in mice treated as in A. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 2-way ANOVA (A-D).

Figure 7. Hepatic Klf9 knockdown decreases blood glucose in db/db mice.

(A) Quantitative PCR analysis of hepatic Klf9 and Pgc1a in db/db or db/m mice (db/db, n = 10 mice; db/m, n = 6 mice). (B) Representative Western blot analysis of hepatic KLF9 and PGC1α in db/db or db/m mice. (C) Quantitative PCR analysis of hepatic Pgc1a, G6pc, Pck1, and Glut2 in db/db mice injected with Ad-shCtrl or Ad-shKlf9 (n = 6/group). Seven days after infection, 6 hour–fasted mice were sacrificed for further analysis. (D) Representative Western blot analysis of KLF9 and PGC1α in livers of db/db mice treated as in C. (E) Blood glucose of 6 hour–fasted db/db or db/m mice treated as in C (n = 6/group). (F–G) Blood glucose during GTT (F) and PTT (G) in db/db mice infected with Ad-shCtrl or Ad-shKlf9 or db/m mice (n = 6/group). (H) Proposed model of GC induction of hepatic gluconeogenesis and hyperglycemia. GCs are diffused into hepatocytes, where GCs bind to GRs in the cytosol. The GC/GR complex translocates into the nucleus to activate Klf9 gene transcription, which in turn promotes Pgc1a expression, thereby activating the hepatic gluconeogenic program and leading to hyperglycemia. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test (A, C) or 1-way ANOVA (E-G)