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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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FoxO6 subcellular distribution, DNA binding activity, and interaction with CRM-1. A: Immunohistochemistry. HepG2 cells pretransduced with 50 plaque forming units (pfu)/cell of FoxO6 vector (a and b) or FoxO1 vector (c and d) were serum-starved for 6 h, followed by incubation in the absence or presence of insulin (100 nmol/L) for 30 min. Cells were immunostained using rabbit anti-FoxO6 (a, b) or anti-FoxO1 (c, d) antibodies, followed by anti-rabbit IgG conjugated with fluorescein isothiocyanate (green). Bar = 25 μm. B: Immunoblot. HepG2 cells were transduced with 50 pfu/cell of FoxO6 vector in the absence or presence of Adv-Akt-CA vector (50 pfu/cell) expressing Akt-CA. After 24-h incubation, cells were harvested for the preparation of nuclear and cytoplasmic fractions, which were subjected to anti-FoxO6 immunoblot assay. C: Chromatin immunoprecipitation (ChIP) analysis of liver. C57BL/6J male mice (aged 10 weeks) were fasted for 16 h, followed by an injection of insulin (2 units/kg i.p.; n = 3) or phosphate-buffered saline (PBS; n = 3). Mice were killed 20 min after insulin injection, and aliquots of liver tissue (20 mg) underwent ChIP assay using rabbit anti-FoxO6 antibody for determining FoxO6 association with G6Pase promoter DNA. D: The amount of G6Pase promoter DNA bound by FoxO6 relative to input control DNA was determined in the liver of PBS- and insulin-treated mice. In addition, HepG2 cells were cotransduced with FoxO1 and FoxO6 vectors at a fixed dose (50 pfu/cell). *P < 0.005 vs. control by ANOVA. After a 24-h incubation, cells were collected for the preparation of total protein lysates, which were subjected to immunoprecipitation using anti–14-3-3 (E), anti-FoxO1 (F), anti–CRM-1 (G), or anti-FoxO6 antibody (H). The control antibody was anti–β-galactosidase IgG. The immunoprecipitates were analyzed for the presence of FoxO1, FoxO6, 14-3-3, or CRM-1. IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; bp, base pair. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 4: FoxO6 subcellular distribution, DNA binding activity, and interaction with CRM-1. A: Immunohistochemistry. HepG2 cells pretransduced with 50 plaque forming units (pfu)/cell of FoxO6 vector (a and b) or FoxO1 vector (c and d) were serum-starved for 6 h, followed by incubation in the absence or presence of insulin (100 nmol/L) for 30 min. Cells were immunostained using rabbit anti-FoxO6 (a, b) or anti-FoxO1 (c, d) antibodies, followed by anti-rabbit IgG conjugated with fluorescein isothiocyanate (green). Bar = 25 μm. B: Immunoblot. HepG2 cells were transduced with 50 pfu/cell of FoxO6 vector in the absence or presence of Adv-Akt-CA vector (50 pfu/cell) expressing Akt-CA. After 24-h incubation, cells were harvested for the preparation of nuclear and cytoplasmic fractions, which were subjected to anti-FoxO6 immunoblot assay. C: Chromatin immunoprecipitation (ChIP) analysis of liver. C57BL/6J male mice (aged 10 weeks) were fasted for 16 h, followed by an injection of insulin (2 units/kg i.p.; n = 3) or phosphate-buffered saline (PBS; n = 3). Mice were killed 20 min after insulin injection, and aliquots of liver tissue (20 mg) underwent ChIP assay using rabbit anti-FoxO6 antibody for determining FoxO6 association with G6Pase promoter DNA. D: The amount of G6Pase promoter DNA bound by FoxO6 relative to input control DNA was determined in the liver of PBS- and insulin-treated mice. In addition, HepG2 cells were cotransduced with FoxO1 and FoxO6 vectors at a fixed dose (50 pfu/cell). *P < 0.005 vs. control by ANOVA. After a 24-h incubation, cells were collected for the preparation of total protein lysates, which were subjected to immunoprecipitation using anti–14-3-3 (E), anti-FoxO1 (F), anti–CRM-1 (G), or anti-FoxO6 antibody (H). The control antibody was anti–β-galactosidase IgG. The immunoprecipitates were analyzed for the presence of FoxO1, FoxO6, 14-3-3, or CRM-1. IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; bp, base pair. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: FoxO6, although phosphorylated in response to insulin, did not undergo insulin-dependent nuclear exclusion in HepG2 cells, as determined by immunohistochemistry (Fig. 4A). In contrast, FoxO1 was translocated from the nucleus to the cytoplasm in the presence of insulin. To consolidate these findings, we determined FoxO6 subcellular distribution in the absence or presence of constitutively active Akt (Akt-CA), which has been shown to phosphorylate its targets independently of insulin (13). FoxO6 remained predominantly in the nucleus regardless of Akt-CA in HepG2 cells (Fig. 4B). Using the same assay, we previously showed that Akt-CA stimulates FoxO1 phosphorylation and promotes its trafficking from the nucleus to cytoplasm (7). It follows that insulin inhibits FoxO6 activity by a distinct mechanism that is different from other members of the FoxO family.


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)

FoxO6 subcellular distribution, DNA binding activity, and interaction with CRM-1. A: Immunohistochemistry. HepG2 cells pretransduced with 50 plaque forming units (pfu)/cell of FoxO6 vector (a and b) or FoxO1 vector (c and d) were serum-starved for 6 h, followed by incubation in the absence or presence of insulin (100 nmol/L) for 30 min. Cells were immunostained using rabbit anti-FoxO6 (a, b) or anti-FoxO1 (c, d) antibodies, followed by anti-rabbit IgG conjugated with fluorescein isothiocyanate (green). Bar = 25 μm. B: Immunoblot. HepG2 cells were transduced with 50 pfu/cell of FoxO6 vector in the absence or presence of Adv-Akt-CA vector (50 pfu/cell) expressing Akt-CA. After 24-h incubation, cells were harvested for the preparation of nuclear and cytoplasmic fractions, which were subjected to anti-FoxO6 immunoblot assay. C: Chromatin immunoprecipitation (ChIP) analysis of liver. C57BL/6J male mice (aged 10 weeks) were fasted for 16 h, followed by an injection of insulin (2 units/kg i.p.; n = 3) or phosphate-buffered saline (PBS; n = 3). Mice were killed 20 min after insulin injection, and aliquots of liver tissue (20 mg) underwent ChIP assay using rabbit anti-FoxO6 antibody for determining FoxO6 association with G6Pase promoter DNA. D: The amount of G6Pase promoter DNA bound by FoxO6 relative to input control DNA was determined in the liver of PBS- and insulin-treated mice. In addition, HepG2 cells were cotransduced with FoxO1 and FoxO6 vectors at a fixed dose (50 pfu/cell). *P < 0.005 vs. control by ANOVA. After a 24-h incubation, cells were collected for the preparation of total protein lysates, which were subjected to immunoprecipitation using anti–14-3-3 (E), anti-FoxO1 (F), anti–CRM-1 (G), or anti-FoxO6 antibody (H). The control antibody was anti–β-galactosidase IgG. The immunoprecipitates were analyzed for the presence of FoxO1, FoxO6, 14-3-3, or CRM-1. IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; bp, base pair. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 4: FoxO6 subcellular distribution, DNA binding activity, and interaction with CRM-1. A: Immunohistochemistry. HepG2 cells pretransduced with 50 plaque forming units (pfu)/cell of FoxO6 vector (a and b) or FoxO1 vector (c and d) were serum-starved for 6 h, followed by incubation in the absence or presence of insulin (100 nmol/L) for 30 min. Cells were immunostained using rabbit anti-FoxO6 (a, b) or anti-FoxO1 (c, d) antibodies, followed by anti-rabbit IgG conjugated with fluorescein isothiocyanate (green). Bar = 25 μm. B: Immunoblot. HepG2 cells were transduced with 50 pfu/cell of FoxO6 vector in the absence or presence of Adv-Akt-CA vector (50 pfu/cell) expressing Akt-CA. After 24-h incubation, cells were harvested for the preparation of nuclear and cytoplasmic fractions, which were subjected to anti-FoxO6 immunoblot assay. C: Chromatin immunoprecipitation (ChIP) analysis of liver. C57BL/6J male mice (aged 10 weeks) were fasted for 16 h, followed by an injection of insulin (2 units/kg i.p.; n = 3) or phosphate-buffered saline (PBS; n = 3). Mice were killed 20 min after insulin injection, and aliquots of liver tissue (20 mg) underwent ChIP assay using rabbit anti-FoxO6 antibody for determining FoxO6 association with G6Pase promoter DNA. D: The amount of G6Pase promoter DNA bound by FoxO6 relative to input control DNA was determined in the liver of PBS- and insulin-treated mice. In addition, HepG2 cells were cotransduced with FoxO1 and FoxO6 vectors at a fixed dose (50 pfu/cell). *P < 0.005 vs. control by ANOVA. After a 24-h incubation, cells were collected for the preparation of total protein lysates, which were subjected to immunoprecipitation using anti–14-3-3 (E), anti-FoxO1 (F), anti–CRM-1 (G), or anti-FoxO6 antibody (H). The control antibody was anti–β-galactosidase IgG. The immunoprecipitates were analyzed for the presence of FoxO1, FoxO6, 14-3-3, or CRM-1. IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; bp, base pair. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: FoxO6, although phosphorylated in response to insulin, did not undergo insulin-dependent nuclear exclusion in HepG2 cells, as determined by immunohistochemistry (Fig. 4A). In contrast, FoxO1 was translocated from the nucleus to the cytoplasm in the presence of insulin. To consolidate these findings, we determined FoxO6 subcellular distribution in the absence or presence of constitutively active Akt (Akt-CA), which has been shown to phosphorylate its targets independently of insulin (13). FoxO6 remained predominantly in the nucleus regardless of Akt-CA in HepG2 cells (Fig. 4B). Using the same assay, we previously showed that Akt-CA stimulates FoxO1 phosphorylation and promotes its trafficking from the nucleus to cytoplasm (7). It follows that insulin inhibits FoxO6 activity by a distinct mechanism that is different from other members of the FoxO family.

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