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FoxO6 integrates insulin signaling with gluconeogenesis in the liver.

Kim DH, Perdomo G, Zhang T, Slusher S, Lee S, Phillips BE, Fan Y, Giannoukakis N, Gramignoli R, Strom S, Ringquist S, Dong HH - Diabetes (2011)

Bottom Line: This effect stems from inept insulin suppression of hepatic gluconeogenesis.FoxO6 stimulates gluconeogenesis, which is counteracted by insulin.Insulin inhibits FoxO6 activity via a distinct mechanism by inducing its phosphorylation and disabling its transcriptional activity, without altering its subcellular distribution in hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. dongh@pitt.edu

ABSTRACT

Objective: Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. This effect stems from inept insulin suppression of hepatic gluconeogenesis. To understand the underlying mechanisms, we studied the ability of forkhead box O6 (FoxO6) to mediate insulin action on hepatic gluconeogenesis and its contribution to glucose metabolism.

Research design and methods: We characterized FoxO6 in glucose metabolism in cultured hepatocytes and in rodent models of dietary obesity, insulin resistance, or insulin-deficient diabetes. We determined the effect of FoxO6 on hepatic gluconeogenesis in genetically modified mice with FoxO6 gain- versus loss-of-function and in diabetic db/db mice with selective FoxO6 ablation in the liver.

Results: FoxO6 integrates insulin signaling to hepatic gluconeogenesis. In mice, elevated FoxO6 activity in the liver augments gluconeogenesis, raising fasting blood glucose levels, and hepatic FoxO6 depletion suppresses gluconeogenesis, resulting in fasting hypoglycemia. FoxO6 stimulates gluconeogenesis, which is counteracted by insulin. Insulin inhibits FoxO6 activity via a distinct mechanism by inducing its phosphorylation and disabling its transcriptional activity, without altering its subcellular distribution in hepatocytes. FoxO6 becomes deregulated in the insulin-resistant liver, accounting for its unbridled activity in promoting gluconeogenesis and correlating with the pathogenesis of fasting hyperglycemia in diabetes. These metabolic abnormalities, along with fasting hyperglycemia, are reversible by selective inhibition of hepatic FoxO6 activity in diabetic mice.

Conclusions: Our data uncover a FoxO6-dependent pathway by which the liver orchestrates insulin regulation of gluconeogenesis, providing the proof-of-concept that selective FoxO6 inhibition is beneficial for curbing excessive hepatic glucose production and improving glycemic control in diabetes.

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Effect of FoxO6 loss of function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 10), which were intravenously injected with a predefined dose (1.5 × 1011 plaque forming units [pfu]/kg) of Adv-FoxO6-siRNA vector expressing FoxO6-specific siRNA under the U6 promoter or control Adv-Sc-siRNA vector encoding scrambled siRNA under the U6 promoter. A: Blood glucose levels. B: Hepatic FoxO6 mRNA levels. C: Hepatic FoxO6 protein levels. D: Hepatic G6Pase activity. E: Hepatic G6Pase mRNA levels. F: Hepatic PEPCK mRNA levels. G: Hepatic PEPCK protein levels. H: Hepatic FoxO1 mRNA levels. I: Body weight. J: Plasma insulin levels. K: Blood glucose profiles of pyruvate tolerance tests (PTT). Blood glucose and plasma insulin levels were determined after a 16-h fast at day 10 after vector administration. PTT was performed at day 14. Mice were killed after a 16-h fast after 15 days of hepatic FoxO6-siRNA expression. Liver tissues were subjected to real-time quantitative RT-PCR analysis and G6Pase activity assay. L: Glucose production in FoxO6-deficient hepatocytes. Mouse primary hepatocytes (2 × 105 cells/well in 12-well microplates) were treated with Adv-FoxO6-siRNA or Adv-Sc-siRNA vector at the dose of 100 pfu/cell in the presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (100 μmol/L). Each condition was run in six replicates. After 24-h incubation, the amount of glucose released from hepatocytes into culture medium was determined between FoxO6-siRNA and control Sc-siRNA groups. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant.
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Figure 7: Effect of FoxO6 loss of function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 10), which were intravenously injected with a predefined dose (1.5 × 1011 plaque forming units [pfu]/kg) of Adv-FoxO6-siRNA vector expressing FoxO6-specific siRNA under the U6 promoter or control Adv-Sc-siRNA vector encoding scrambled siRNA under the U6 promoter. A: Blood glucose levels. B: Hepatic FoxO6 mRNA levels. C: Hepatic FoxO6 protein levels. D: Hepatic G6Pase activity. E: Hepatic G6Pase mRNA levels. F: Hepatic PEPCK mRNA levels. G: Hepatic PEPCK protein levels. H: Hepatic FoxO1 mRNA levels. I: Body weight. J: Plasma insulin levels. K: Blood glucose profiles of pyruvate tolerance tests (PTT). Blood glucose and plasma insulin levels were determined after a 16-h fast at day 10 after vector administration. PTT was performed at day 14. Mice were killed after a 16-h fast after 15 days of hepatic FoxO6-siRNA expression. Liver tissues were subjected to real-time quantitative RT-PCR analysis and G6Pase activity assay. L: Glucose production in FoxO6-deficient hepatocytes. Mouse primary hepatocytes (2 × 105 cells/well in 12-well microplates) were treated with Adv-FoxO6-siRNA or Adv-Sc-siRNA vector at the dose of 100 pfu/cell in the presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (100 μmol/L). Each condition was run in six replicates. After 24-h incubation, the amount of glucose released from hepatocytes into culture medium was determined between FoxO6-siRNA and control Sc-siRNA groups. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant.

Mentions: To further illustrate the importance of FoxO6 in gluconeogenesis, we used the siRNA-mediated gene-silencing approach to knock down FoxO6 expression in the liver. This approach generated a liver-specific FoxO6-deficient model. The FoxO6-deficient mice (n = 10) exhibited significantly reduced fasting blood glucose levels (Fig. 7A). Hypoglycemia (blood glucose <50 mg/dL) developed in ∼50% after a 24-h fast, indicating that FoxO6 loss-of-function was associated with impaired ability to maintain fasting euglycemia during prolonged fasting. This effect correlated with near depletion of hepatic FoxO6 expression (Fig. 7B and C) and significant reduction in hepatic G6Pase activity (Fig. 7D), G6Pase mRNA (Fig. 7E), PEPCK mRNA (Fig. 7F), and PEPCK protein (Fig. 7G) expression in FoxO6-siRNA vector-treated mice. Hepatic FoxO1 expression remained unchanged in FoxO6-siRNA vector-treated mice (Fig. 7H), confirming the specificity of FoxO6-siRNA for selective FoxO6 knockdown in the liver. Likewise, no significant impact of FoxO6-siRNA on hepatic FoxO3 and FoxO4 expression was seen in FoxO6-siRNA vector-treated mice versus the control group (Supplementary Fig. 11). Furthermore, no differences in body weight (Fig. 7I) and liver enzyme levels (Supplementary Fig. 11) were detected, ruling out the possibility that the reduction in blood glucose levels was the result of hepatotoxicity in FoxO6-siRNA vector-treated mice. Mice with hepatic FoxO6 depletion displayed lower fasting insulin levels (Fig. 7J), secondary to reduced hepatic gluconeogenesis. This was validated by pyruvate tolerance test. FoxO6-deficient mice exhibited significantly lower blood glucose levels after the intraperitoneal pyruvate injection (Fig. 7K), suggesting that FoxO6 depletion was associated with diminished hepatic gluconeogenesis.


FoxO6 integrates insulin signaling with gluconeogenesis in the liver.

Kim DH, Perdomo G, Zhang T, Slusher S, Lee S, Phillips BE, Fan Y, Giannoukakis N, Gramignoli R, Strom S, Ringquist S, Dong HH - Diabetes (2011)

Effect of FoxO6 loss of function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 10), which were intravenously injected with a predefined dose (1.5 × 1011 plaque forming units [pfu]/kg) of Adv-FoxO6-siRNA vector expressing FoxO6-specific siRNA under the U6 promoter or control Adv-Sc-siRNA vector encoding scrambled siRNA under the U6 promoter. A: Blood glucose levels. B: Hepatic FoxO6 mRNA levels. C: Hepatic FoxO6 protein levels. D: Hepatic G6Pase activity. E: Hepatic G6Pase mRNA levels. F: Hepatic PEPCK mRNA levels. G: Hepatic PEPCK protein levels. H: Hepatic FoxO1 mRNA levels. I: Body weight. J: Plasma insulin levels. K: Blood glucose profiles of pyruvate tolerance tests (PTT). Blood glucose and plasma insulin levels were determined after a 16-h fast at day 10 after vector administration. PTT was performed at day 14. Mice were killed after a 16-h fast after 15 days of hepatic FoxO6-siRNA expression. Liver tissues were subjected to real-time quantitative RT-PCR analysis and G6Pase activity assay. L: Glucose production in FoxO6-deficient hepatocytes. Mouse primary hepatocytes (2 × 105 cells/well in 12-well microplates) were treated with Adv-FoxO6-siRNA or Adv-Sc-siRNA vector at the dose of 100 pfu/cell in the presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (100 μmol/L). Each condition was run in six replicates. After 24-h incubation, the amount of glucose released from hepatocytes into culture medium was determined between FoxO6-siRNA and control Sc-siRNA groups. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant.
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Figure 7: Effect of FoxO6 loss of function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 10), which were intravenously injected with a predefined dose (1.5 × 1011 plaque forming units [pfu]/kg) of Adv-FoxO6-siRNA vector expressing FoxO6-specific siRNA under the U6 promoter or control Adv-Sc-siRNA vector encoding scrambled siRNA under the U6 promoter. A: Blood glucose levels. B: Hepatic FoxO6 mRNA levels. C: Hepatic FoxO6 protein levels. D: Hepatic G6Pase activity. E: Hepatic G6Pase mRNA levels. F: Hepatic PEPCK mRNA levels. G: Hepatic PEPCK protein levels. H: Hepatic FoxO1 mRNA levels. I: Body weight. J: Plasma insulin levels. K: Blood glucose profiles of pyruvate tolerance tests (PTT). Blood glucose and plasma insulin levels were determined after a 16-h fast at day 10 after vector administration. PTT was performed at day 14. Mice were killed after a 16-h fast after 15 days of hepatic FoxO6-siRNA expression. Liver tissues were subjected to real-time quantitative RT-PCR analysis and G6Pase activity assay. L: Glucose production in FoxO6-deficient hepatocytes. Mouse primary hepatocytes (2 × 105 cells/well in 12-well microplates) were treated with Adv-FoxO6-siRNA or Adv-Sc-siRNA vector at the dose of 100 pfu/cell in the presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (100 μmol/L). Each condition was run in six replicates. After 24-h incubation, the amount of glucose released from hepatocytes into culture medium was determined between FoxO6-siRNA and control Sc-siRNA groups. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant.
Mentions: To further illustrate the importance of FoxO6 in gluconeogenesis, we used the siRNA-mediated gene-silencing approach to knock down FoxO6 expression in the liver. This approach generated a liver-specific FoxO6-deficient model. The FoxO6-deficient mice (n = 10) exhibited significantly reduced fasting blood glucose levels (Fig. 7A). Hypoglycemia (blood glucose <50 mg/dL) developed in ∼50% after a 24-h fast, indicating that FoxO6 loss-of-function was associated with impaired ability to maintain fasting euglycemia during prolonged fasting. This effect correlated with near depletion of hepatic FoxO6 expression (Fig. 7B and C) and significant reduction in hepatic G6Pase activity (Fig. 7D), G6Pase mRNA (Fig. 7E), PEPCK mRNA (Fig. 7F), and PEPCK protein (Fig. 7G) expression in FoxO6-siRNA vector-treated mice. Hepatic FoxO1 expression remained unchanged in FoxO6-siRNA vector-treated mice (Fig. 7H), confirming the specificity of FoxO6-siRNA for selective FoxO6 knockdown in the liver. Likewise, no significant impact of FoxO6-siRNA on hepatic FoxO3 and FoxO4 expression was seen in FoxO6-siRNA vector-treated mice versus the control group (Supplementary Fig. 11). Furthermore, no differences in body weight (Fig. 7I) and liver enzyme levels (Supplementary Fig. 11) were detected, ruling out the possibility that the reduction in blood glucose levels was the result of hepatotoxicity in FoxO6-siRNA vector-treated mice. Mice with hepatic FoxO6 depletion displayed lower fasting insulin levels (Fig. 7J), secondary to reduced hepatic gluconeogenesis. This was validated by pyruvate tolerance test. FoxO6-deficient mice exhibited significantly lower blood glucose levels after the intraperitoneal pyruvate injection (Fig. 7K), suggesting that FoxO6 depletion was associated with diminished hepatic gluconeogenesis.

Bottom Line: This effect stems from inept insulin suppression of hepatic gluconeogenesis.FoxO6 stimulates gluconeogenesis, which is counteracted by insulin.Insulin inhibits FoxO6 activity via a distinct mechanism by inducing its phosphorylation and disabling its transcriptional activity, without altering its subcellular distribution in hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. dongh@pitt.edu

ABSTRACT

Objective: Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. This effect stems from inept insulin suppression of hepatic gluconeogenesis. To understand the underlying mechanisms, we studied the ability of forkhead box O6 (FoxO6) to mediate insulin action on hepatic gluconeogenesis and its contribution to glucose metabolism.

Research design and methods: We characterized FoxO6 in glucose metabolism in cultured hepatocytes and in rodent models of dietary obesity, insulin resistance, or insulin-deficient diabetes. We determined the effect of FoxO6 on hepatic gluconeogenesis in genetically modified mice with FoxO6 gain- versus loss-of-function and in diabetic db/db mice with selective FoxO6 ablation in the liver.

Results: FoxO6 integrates insulin signaling to hepatic gluconeogenesis. In mice, elevated FoxO6 activity in the liver augments gluconeogenesis, raising fasting blood glucose levels, and hepatic FoxO6 depletion suppresses gluconeogenesis, resulting in fasting hypoglycemia. FoxO6 stimulates gluconeogenesis, which is counteracted by insulin. Insulin inhibits FoxO6 activity via a distinct mechanism by inducing its phosphorylation and disabling its transcriptional activity, without altering its subcellular distribution in hepatocytes. FoxO6 becomes deregulated in the insulin-resistant liver, accounting for its unbridled activity in promoting gluconeogenesis and correlating with the pathogenesis of fasting hyperglycemia in diabetes. These metabolic abnormalities, along with fasting hyperglycemia, are reversible by selective inhibition of hepatic FoxO6 activity in diabetic mice.

Conclusions: Our data uncover a FoxO6-dependent pathway by which the liver orchestrates insulin regulation of gluconeogenesis, providing the proof-of-concept that selective FoxO6 inhibition is beneficial for curbing excessive hepatic glucose production and improving glycemic control in diabetes.

Show MeSH
Related in: MedlinePlus