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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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Related in: MedlinePlus

Effect of FoxO6 gain-of-function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 9), which were intravenously injected with Adv-glucose-6-phosphate (G6P)-Luc vector (0.5 × 1011 plaque forming units [pfu]/kg) that is premixed with Adv-FoxO6-CA or Adv- vector (1.5 × 1011 pfu/kg). A: Mice were injected 5 days after vector administration with a dose of d-luciferin (200 μg/g i.p.), followed by whole-body imaging. B: The mean radiance of mice, defined as the light unit (photons/s/cm2/sr [steradian]), was compared between FoxO6-CA and control groups. Mice were killed after 14 days of hepatic FoxO6-CA production. C: Liver tissues were subjected to luciferase activity assay for determining hepatic luciferase activity. Aliquots of liver tissues (20 mg) were analyzed for the determination of PEPCK mRNA (D), PEPCK protein (E), G6Pase mRNA (F), and FoxO6 mRNA levels (G). In parallel, two groups of CD1 mice (n = 5) were identically treated with FoxO6-CA or control vector, without the inclusion of the luciferase vector, for determining the effect of FoxO6-CA on glucose metabolism. H: Blood glucose profiles of the pyruvate tolerance test (PTT). Mice were fasted for 16 h, followed by an injection of pyruvate (2 g/kg i.p.). Blood glucose levels were measured before and after pyruvate infusion. Data were obtained after 8 days of hepatic FoxO6-CA production. I: Fasting blood glucose levels. J: Fasting plasma insulin levels. Mice were fasted for 16 h, followed by determination of fasting blood glucose levels. In addition, aliquots of blood (20 μL) were collected from individual mice for the determination of fasting plasma insulin levels. Data were obtained on day 5 after vector administration. K: Blood glucose profiles of glucose tolerance test (GTT). Mice were fasted for 16 h, followed by a glucose injection (2 g/kg i.p.). Blood glucose levels were measured before and after glucose infusion. Data were obtained after 5 days of hepatic FoxO6-CA production. L: Body weight. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Effect of FoxO6 gain-of-function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 9), which were intravenously injected with Adv-glucose-6-phosphate (G6P)-Luc vector (0.5 × 1011 plaque forming units [pfu]/kg) that is premixed with Adv-FoxO6-CA or Adv- vector (1.5 × 1011 pfu/kg). A: Mice were injected 5 days after vector administration with a dose of d-luciferin (200 μg/g i.p.), followed by whole-body imaging. B: The mean radiance of mice, defined as the light unit (photons/s/cm2/sr [steradian]), was compared between FoxO6-CA and control groups. Mice were killed after 14 days of hepatic FoxO6-CA production. C: Liver tissues were subjected to luciferase activity assay for determining hepatic luciferase activity. Aliquots of liver tissues (20 mg) were analyzed for the determination of PEPCK mRNA (D), PEPCK protein (E), G6Pase mRNA (F), and FoxO6 mRNA levels (G). In parallel, two groups of CD1 mice (n = 5) were identically treated with FoxO6-CA or control vector, without the inclusion of the luciferase vector, for determining the effect of FoxO6-CA on glucose metabolism. H: Blood glucose profiles of the pyruvate tolerance test (PTT). Mice were fasted for 16 h, followed by an injection of pyruvate (2 g/kg i.p.). Blood glucose levels were measured before and after pyruvate infusion. Data were obtained after 8 days of hepatic FoxO6-CA production. I: Fasting blood glucose levels. J: Fasting plasma insulin levels. Mice were fasted for 16 h, followed by determination of fasting blood glucose levels. In addition, aliquots of blood (20 μL) were collected from individual mice for the determination of fasting plasma insulin levels. Data were obtained on day 5 after vector administration. K: Blood glucose profiles of glucose tolerance test (GTT). Mice were fasted for 16 h, followed by a glucose injection (2 g/kg i.p.). Blood glucose levels were measured before and after glucose infusion. Data were obtained after 5 days of hepatic FoxO6-CA production. L: Body weight. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To provide in vivo evidence that FoxO6 targets the G6Pase promoter for trans-activation, we transferred adenoviral vectors encoding the G6Pase promoter-directed luciferase reporter system along with the Adv-FoxO6-CA or Adv- vector into CD1 mice (n = 10 per group), followed by whole-body imaging (Fig. 3A). This assay detected a significant induction of hepatic luciferase activity, defined by the luminescent radiance in the liver with FoxO6-CA production (Fig. 3B). This result was confirmed by determination of luciferase activity in the liver protein extracts of FoxO6-CA vector-treated mice (Fig. 3C). Consistent with its role in gluconeogenesis, FoxO6-CA augmented hepatic PEPCK mRNA (Fig. 3D) and PEPCK protein (Fig. 3E), as well as G6Pase mRNA expression (Fig. 3F), correlating with increased FoxO6 production in the liver (Fig. 3G). This effect contributed to augmented gluconeogenesis, as proven by significantly higher blood glucose levels in FoxO6-CA vector-treated mice after an intraperitoneal dose of pyruvate solution (Fig. 3H). Mice with hepatic FoxO6-CA production were associated with elevated fasting blood glucose (Fig. 3I) and plasma insulin levels (Fig. 3J), accompanied by impaired glucose tolerance (Fig. 3K). No differences were seen in body weight between FoxO6-CA and control groups (Fig. 3L). These data support the idea that FoxO6 targets the G6Pase gene for trans-activation, contributing to the induction of hepatic gluconeogenesis. As a control, we determined the potential effect of FoxO6 on hepatic expression of other members in the FoxO family. No significant differences in hepatic FoxO1, FoxO3, and FoxO4 mRNA levels were detected in control versus FoxO6-CA groups (Supplementary Fig. 6), precluding the possibility that the observed induction of hepatic gluconeogenesis was secondary to altered production of other FoxO proteins in FoxO6-CA mice.


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 gain-of-function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 9), which were intravenously injected with Adv-glucose-6-phosphate (G6P)-Luc vector (0.5 × 1011 plaque forming units [pfu]/kg) that is premixed with Adv-FoxO6-CA or Adv- vector (1.5 × 1011 pfu/kg). A: Mice were injected 5 days after vector administration with a dose of d-luciferin (200 μg/g i.p.), followed by whole-body imaging. B: The mean radiance of mice, defined as the light unit (photons/s/cm2/sr [steradian]), was compared between FoxO6-CA and control groups. Mice were killed after 14 days of hepatic FoxO6-CA production. C: Liver tissues were subjected to luciferase activity assay for determining hepatic luciferase activity. Aliquots of liver tissues (20 mg) were analyzed for the determination of PEPCK mRNA (D), PEPCK protein (E), G6Pase mRNA (F), and FoxO6 mRNA levels (G). In parallel, two groups of CD1 mice (n = 5) were identically treated with FoxO6-CA or control vector, without the inclusion of the luciferase vector, for determining the effect of FoxO6-CA on glucose metabolism. H: Blood glucose profiles of the pyruvate tolerance test (PTT). Mice were fasted for 16 h, followed by an injection of pyruvate (2 g/kg i.p.). Blood glucose levels were measured before and after pyruvate infusion. Data were obtained after 8 days of hepatic FoxO6-CA production. I: Fasting blood glucose levels. J: Fasting plasma insulin levels. Mice were fasted for 16 h, followed by determination of fasting blood glucose levels. In addition, aliquots of blood (20 μL) were collected from individual mice for the determination of fasting plasma insulin levels. Data were obtained on day 5 after vector administration. K: Blood glucose profiles of glucose tolerance test (GTT). Mice were fasted for 16 h, followed by a glucose injection (2 g/kg i.p.). Blood glucose levels were measured before and after glucose infusion. Data were obtained after 5 days of hepatic FoxO6-CA production. L: Body weight. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Effect of FoxO6 gain-of-function on gluconeogenesis. CD1 male mice (aged 10 weeks) were stratified by body weight and randomly assigned to two groups (n = 9), which were intravenously injected with Adv-glucose-6-phosphate (G6P)-Luc vector (0.5 × 1011 plaque forming units [pfu]/kg) that is premixed with Adv-FoxO6-CA or Adv- vector (1.5 × 1011 pfu/kg). A: Mice were injected 5 days after vector administration with a dose of d-luciferin (200 μg/g i.p.), followed by whole-body imaging. B: The mean radiance of mice, defined as the light unit (photons/s/cm2/sr [steradian]), was compared between FoxO6-CA and control groups. Mice were killed after 14 days of hepatic FoxO6-CA production. C: Liver tissues were subjected to luciferase activity assay for determining hepatic luciferase activity. Aliquots of liver tissues (20 mg) were analyzed for the determination of PEPCK mRNA (D), PEPCK protein (E), G6Pase mRNA (F), and FoxO6 mRNA levels (G). In parallel, two groups of CD1 mice (n = 5) were identically treated with FoxO6-CA or control vector, without the inclusion of the luciferase vector, for determining the effect of FoxO6-CA on glucose metabolism. H: Blood glucose profiles of the pyruvate tolerance test (PTT). Mice were fasted for 16 h, followed by an injection of pyruvate (2 g/kg i.p.). Blood glucose levels were measured before and after pyruvate infusion. Data were obtained after 8 days of hepatic FoxO6-CA production. I: Fasting blood glucose levels. J: Fasting plasma insulin levels. Mice were fasted for 16 h, followed by determination of fasting blood glucose levels. In addition, aliquots of blood (20 μL) were collected from individual mice for the determination of fasting plasma insulin levels. Data were obtained on day 5 after vector administration. K: Blood glucose profiles of glucose tolerance test (GTT). Mice were fasted for 16 h, followed by a glucose injection (2 g/kg i.p.). Blood glucose levels were measured before and after glucose infusion. Data were obtained after 5 days of hepatic FoxO6-CA production. L: Body weight. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To provide in vivo evidence that FoxO6 targets the G6Pase promoter for trans-activation, we transferred adenoviral vectors encoding the G6Pase promoter-directed luciferase reporter system along with the Adv-FoxO6-CA or Adv- vector into CD1 mice (n = 10 per group), followed by whole-body imaging (Fig. 3A). This assay detected a significant induction of hepatic luciferase activity, defined by the luminescent radiance in the liver with FoxO6-CA production (Fig. 3B). This result was confirmed by determination of luciferase activity in the liver protein extracts of FoxO6-CA vector-treated mice (Fig. 3C). Consistent with its role in gluconeogenesis, FoxO6-CA augmented hepatic PEPCK mRNA (Fig. 3D) and PEPCK protein (Fig. 3E), as well as G6Pase mRNA expression (Fig. 3F), correlating with increased FoxO6 production in the liver (Fig. 3G). This effect contributed to augmented gluconeogenesis, as proven by significantly higher blood glucose levels in FoxO6-CA vector-treated mice after an intraperitoneal dose of pyruvate solution (Fig. 3H). Mice with hepatic FoxO6-CA production were associated with elevated fasting blood glucose (Fig. 3I) and plasma insulin levels (Fig. 3J), accompanied by impaired glucose tolerance (Fig. 3K). No differences were seen in body weight between FoxO6-CA and control groups (Fig. 3L). These data support the idea that FoxO6 targets the G6Pase gene for trans-activation, contributing to the induction of hepatic gluconeogenesis. As a control, we determined the potential effect of FoxO6 on hepatic expression of other members in the FoxO family. No significant differences in hepatic FoxO1, FoxO3, and FoxO4 mRNA levels were detected in control versus FoxO6-CA groups (Supplementary Fig. 6), precluding the possibility that the observed induction of hepatic gluconeogenesis was secondary to altered production of other FoxO proteins in FoxO6-CA mice.

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