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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

Insulin regulation of FoxO6 transcriptional activity. Human primary hepatocytes were cultured in the absence or presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (Dex, 100 μmol/L), with and without the inclusion of insulin (100 nmol/L). After 24-h incubation, cells were subjected to real-time quantitative (q) RT-PCR assay for determination of FoxO6 mRNA levels (A), G6Pase mRNA levels (B), and PEPCK mRNA levels (C). In addition, HepG2 cells were transduced with control and FoxO6 vectors at a fixed dose (100 plaque-forming units [pfu]/cell). After 24-h incubation, cells were replenished with glucose-free glutamine-containing Dulbecco’s modified Eagle’s medium that was supplemented with 1 mmol/L pyruvate. After 6-h incubation, conditioned medium was used for determination of glucose. Cells were subjected to real-time qRT-PCR analysis and anti-FoxO6 immunoblot assay using anti-actin antibody as control. D: G6Pase mRNA levels. E: Hepatic G6Pase activity. F: FoxO6 mRNA levels. G: Glucose levels in the medium. AU, arbitrary unit. H: The mouse G6Pase promoter contains three tandem copies of the insulin-responsive element (IRE). This 1.2-kb G6Pase promoter was cloned in the luciferase reporter expression vector pG6P-Luc. I: FoxO6 binds to the G6Pase promoter in HepG2 cells. HepG2 cells were transfected with pG6P-Luc in the presence of FoxO6 vector at 50 pfu/cell in duplicate. After 24-h incubation, cells were subjected to chromatin immunoprecipitation (ChIP) assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Immunoprecipitates were subjected to PCR analysis using a pair of primers flanking the IRE sequence (−471/−122 nucleotide [nt]) in the G6Pase promoter. As a negative control, the immunoprecipitates were analyzed using a pair of off-target primers flanking the G6Pase coding region (684–1,074 nt). As a positive control, aliquots of input DNA samples (3 μL) were used in PCR analysis. J: FoxO6 binds to the G6Pase promoter in the liver. CD-1 mice (n = 4) were killed after a 16-h fast. Liver tissues were subjected to ChIP assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Furthermore, HepG2 cells were transfected with 1 μg of the plasmid encoding the G6Pase promoter-directed luciferase reporter system in the presence of FoxO6 (K) or its constitutively active allele FoxO6-CA (L) production. Adv-FoxO6, Adv-FoxO6-CA, and control Adv- vectors were used at a fixed dose (50 pfu/cell). Cells were incubated for 24 h in the presence or absence of insulin (100 nmol/L), followed by luciferase assay. To normalize the transfection efficiency, 1 μg of pCA35-LacZ encoding β-galactosidase was included in each transfection. The promoter activity, defined as the ratio between luciferase and β-galactosidase activities, was determined. Data were from 4 to 6 independent experiments. *P < 0.05 and **P < 0.001 vs. control by ANOVA; NS, not significant. bp, base pairs; MW, molecular weight. (A high-quality color representation of this figure is available in the online issue.)
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Figure 2: Insulin regulation of FoxO6 transcriptional activity. Human primary hepatocytes were cultured in the absence or presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (Dex, 100 μmol/L), with and without the inclusion of insulin (100 nmol/L). After 24-h incubation, cells were subjected to real-time quantitative (q) RT-PCR assay for determination of FoxO6 mRNA levels (A), G6Pase mRNA levels (B), and PEPCK mRNA levels (C). In addition, HepG2 cells were transduced with control and FoxO6 vectors at a fixed dose (100 plaque-forming units [pfu]/cell). After 24-h incubation, cells were replenished with glucose-free glutamine-containing Dulbecco’s modified Eagle’s medium that was supplemented with 1 mmol/L pyruvate. After 6-h incubation, conditioned medium was used for determination of glucose. Cells were subjected to real-time qRT-PCR analysis and anti-FoxO6 immunoblot assay using anti-actin antibody as control. D: G6Pase mRNA levels. E: Hepatic G6Pase activity. F: FoxO6 mRNA levels. G: Glucose levels in the medium. AU, arbitrary unit. H: The mouse G6Pase promoter contains three tandem copies of the insulin-responsive element (IRE). This 1.2-kb G6Pase promoter was cloned in the luciferase reporter expression vector pG6P-Luc. I: FoxO6 binds to the G6Pase promoter in HepG2 cells. HepG2 cells were transfected with pG6P-Luc in the presence of FoxO6 vector at 50 pfu/cell in duplicate. After 24-h incubation, cells were subjected to chromatin immunoprecipitation (ChIP) assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Immunoprecipitates were subjected to PCR analysis using a pair of primers flanking the IRE sequence (−471/−122 nucleotide [nt]) in the G6Pase promoter. As a negative control, the immunoprecipitates were analyzed using a pair of off-target primers flanking the G6Pase coding region (684–1,074 nt). As a positive control, aliquots of input DNA samples (3 μL) were used in PCR analysis. J: FoxO6 binds to the G6Pase promoter in the liver. CD-1 mice (n = 4) were killed after a 16-h fast. Liver tissues were subjected to ChIP assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Furthermore, HepG2 cells were transfected with 1 μg of the plasmid encoding the G6Pase promoter-directed luciferase reporter system in the presence of FoxO6 (K) or its constitutively active allele FoxO6-CA (L) production. Adv-FoxO6, Adv-FoxO6-CA, and control Adv- vectors were used at a fixed dose (50 pfu/cell). Cells were incubated for 24 h in the presence or absence of insulin (100 nmol/L), followed by luciferase assay. To normalize the transfection efficiency, 1 μg of pCA35-LacZ encoding β-galactosidase was included in each transfection. The promoter activity, defined as the ratio between luciferase and β-galactosidase activities, was determined. Data were from 4 to 6 independent experiments. *P < 0.05 and **P < 0.001 vs. control by ANOVA; NS, not significant. bp, base pairs; MW, molecular weight. (A high-quality color representation of this figure is available in the online issue.)

Mentions: To address the above hypothesis, we studied hepatic regulation of FoxO6 expression by insulin in cultured human primary hepatocytes. Hepatic FoxO6 expression was upregulated by cAMP/dexamethasone (Fig. 2A), correlating with the induction of G6Pase (Fig. 2B) and PEPCK (Fig. 2C), two key enzymes in gluconeogenesis. This effect was reversed by insulin, suggesting that hepatic FoxO6 activity is induced by glucagon (via cAMP) and inhibited by insulin.


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)

Insulin regulation of FoxO6 transcriptional activity. Human primary hepatocytes were cultured in the absence or presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (Dex, 100 μmol/L), with and without the inclusion of insulin (100 nmol/L). After 24-h incubation, cells were subjected to real-time quantitative (q) RT-PCR assay for determination of FoxO6 mRNA levels (A), G6Pase mRNA levels (B), and PEPCK mRNA levels (C). In addition, HepG2 cells were transduced with control and FoxO6 vectors at a fixed dose (100 plaque-forming units [pfu]/cell). After 24-h incubation, cells were replenished with glucose-free glutamine-containing Dulbecco’s modified Eagle’s medium that was supplemented with 1 mmol/L pyruvate. After 6-h incubation, conditioned medium was used for determination of glucose. Cells were subjected to real-time qRT-PCR analysis and anti-FoxO6 immunoblot assay using anti-actin antibody as control. D: G6Pase mRNA levels. E: Hepatic G6Pase activity. F: FoxO6 mRNA levels. G: Glucose levels in the medium. AU, arbitrary unit. H: The mouse G6Pase promoter contains three tandem copies of the insulin-responsive element (IRE). This 1.2-kb G6Pase promoter was cloned in the luciferase reporter expression vector pG6P-Luc. I: FoxO6 binds to the G6Pase promoter in HepG2 cells. HepG2 cells were transfected with pG6P-Luc in the presence of FoxO6 vector at 50 pfu/cell in duplicate. After 24-h incubation, cells were subjected to chromatin immunoprecipitation (ChIP) assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Immunoprecipitates were subjected to PCR analysis using a pair of primers flanking the IRE sequence (−471/−122 nucleotide [nt]) in the G6Pase promoter. As a negative control, the immunoprecipitates were analyzed using a pair of off-target primers flanking the G6Pase coding region (684–1,074 nt). As a positive control, aliquots of input DNA samples (3 μL) were used in PCR analysis. J: FoxO6 binds to the G6Pase promoter in the liver. CD-1 mice (n = 4) were killed after a 16-h fast. Liver tissues were subjected to ChIP assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Furthermore, HepG2 cells were transfected with 1 μg of the plasmid encoding the G6Pase promoter-directed luciferase reporter system in the presence of FoxO6 (K) or its constitutively active allele FoxO6-CA (L) production. Adv-FoxO6, Adv-FoxO6-CA, and control Adv- vectors were used at a fixed dose (50 pfu/cell). Cells were incubated for 24 h in the presence or absence of insulin (100 nmol/L), followed by luciferase assay. To normalize the transfection efficiency, 1 μg of pCA35-LacZ encoding β-galactosidase was included in each transfection. The promoter activity, defined as the ratio between luciferase and β-galactosidase activities, was determined. Data were from 4 to 6 independent experiments. *P < 0.05 and **P < 0.001 vs. control by ANOVA; NS, not significant. bp, base pairs; MW, molecular weight. (A high-quality color representation of this figure is available in the online issue.)
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Figure 2: Insulin regulation of FoxO6 transcriptional activity. Human primary hepatocytes were cultured in the absence or presence of 8-cpt-cAMP (cAMP analog, 500 μmol/L) and dexamethasone (Dex, 100 μmol/L), with and without the inclusion of insulin (100 nmol/L). After 24-h incubation, cells were subjected to real-time quantitative (q) RT-PCR assay for determination of FoxO6 mRNA levels (A), G6Pase mRNA levels (B), and PEPCK mRNA levels (C). In addition, HepG2 cells were transduced with control and FoxO6 vectors at a fixed dose (100 plaque-forming units [pfu]/cell). After 24-h incubation, cells were replenished with glucose-free glutamine-containing Dulbecco’s modified Eagle’s medium that was supplemented with 1 mmol/L pyruvate. After 6-h incubation, conditioned medium was used for determination of glucose. Cells were subjected to real-time qRT-PCR analysis and anti-FoxO6 immunoblot assay using anti-actin antibody as control. D: G6Pase mRNA levels. E: Hepatic G6Pase activity. F: FoxO6 mRNA levels. G: Glucose levels in the medium. AU, arbitrary unit. H: The mouse G6Pase promoter contains three tandem copies of the insulin-responsive element (IRE). This 1.2-kb G6Pase promoter was cloned in the luciferase reporter expression vector pG6P-Luc. I: FoxO6 binds to the G6Pase promoter in HepG2 cells. HepG2 cells were transfected with pG6P-Luc in the presence of FoxO6 vector at 50 pfu/cell in duplicate. After 24-h incubation, cells were subjected to chromatin immunoprecipitation (ChIP) assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Immunoprecipitates were subjected to PCR analysis using a pair of primers flanking the IRE sequence (−471/−122 nucleotide [nt]) in the G6Pase promoter. As a negative control, the immunoprecipitates were analyzed using a pair of off-target primers flanking the G6Pase coding region (684–1,074 nt). As a positive control, aliquots of input DNA samples (3 μL) were used in PCR analysis. J: FoxO6 binds to the G6Pase promoter in the liver. CD-1 mice (n = 4) were killed after a 16-h fast. Liver tissues were subjected to ChIP assay using rabbit preimmune IgG (lanes 1 and 2) or anti-FoxO6 antibody (lanes 3 and 4). Furthermore, HepG2 cells were transfected with 1 μg of the plasmid encoding the G6Pase promoter-directed luciferase reporter system in the presence of FoxO6 (K) or its constitutively active allele FoxO6-CA (L) production. Adv-FoxO6, Adv-FoxO6-CA, and control Adv- vectors were used at a fixed dose (50 pfu/cell). Cells were incubated for 24 h in the presence or absence of insulin (100 nmol/L), followed by luciferase assay. To normalize the transfection efficiency, 1 μg of pCA35-LacZ encoding β-galactosidase was included in each transfection. The promoter activity, defined as the ratio between luciferase and β-galactosidase activities, was determined. Data were from 4 to 6 independent experiments. *P < 0.05 and **P < 0.001 vs. control by ANOVA; NS, not significant. bp, base pairs; MW, molecular weight. (A high-quality color representation of this figure is available in the online issue.)
Mentions: To address the above hypothesis, we studied hepatic regulation of FoxO6 expression by insulin in cultured human primary hepatocytes. Hepatic FoxO6 expression was upregulated by cAMP/dexamethasone (Fig. 2A), correlating with the induction of G6Pase (Fig. 2B) and PEPCK (Fig. 2C), two key enzymes in gluconeogenesis. This effect was reversed by insulin, suggesting that hepatic FoxO6 activity is induced by glucagon (via cAMP) and inhibited by insulin.

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