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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

Characterization of FoxO6 and its hepatic expression under physiologic and pathologic conditions. A: FoxO6 is divergent from FoxO1. FoxO6 contains 640 amino acid (aa) residues and two Akt/PKB phosphorylation sites, lacking NES. NLS, nuclear localization signal. B: FoxO6 is ubiquitously expressed. C57BL/6J male mice (aged 10 weeks) were killed under fed conditions for collecting tissues (20 mg), which were subjected to RT-PCR analysis using FoxO6 and β-actin primers. Data were representative of three independent assays from three mice. C57BL/6J male mice (aged 10 weeks, n = 3) were killed under fed conditions or after a 16-h fast. C: Total liver RNA was subjected to real-time quantitative (q) RT-PCR assay using FoxO6 and β-actin primers. Total liver proteins were separated into nuclear (D) and cytoplasmic (E) fractions, which were analyzed by immunoblot assay using anti-FoxO6 antibody. Furthermore, C57BL/6J male mice (aged 6 weeks) were rendered obese after 8 weeks of high-fat feeding. Mice in groups fed regular chow (n = 6, body wt 26.5 ± 2.1 g) and a high-fat diet (n = 6, 51.4 ± 4.9 g) were killed after a 16-h fast. F: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Total liver proteins were separated into cytoplasmic (G) and nuclear (H) fractions, which were analyzed by anti-FoxO6 immunoblot assay. Likewise, male diabetic db/db (n = 8, aged 6 months; blood glucose levels, 62.8 ± 3.9 mg/dL) vs. male age-matched control db/+ mice (n = 8, 310 ± 28 mg/dL) were killed after a 16-h fast, and liver tissue was processed for the preparation of cytoplasmic and nuclear fractions. I: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Aliquots of cytoplasmic (J) and nuclear (K) proteins (20 μg) were subjected to semiquantitative immunoblot analysis for FoxO6. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality color representation of this figure is available in the online issue.)
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Figure 1: Characterization of FoxO6 and its hepatic expression under physiologic and pathologic conditions. A: FoxO6 is divergent from FoxO1. FoxO6 contains 640 amino acid (aa) residues and two Akt/PKB phosphorylation sites, lacking NES. NLS, nuclear localization signal. B: FoxO6 is ubiquitously expressed. C57BL/6J male mice (aged 10 weeks) were killed under fed conditions for collecting tissues (20 mg), which were subjected to RT-PCR analysis using FoxO6 and β-actin primers. Data were representative of three independent assays from three mice. C57BL/6J male mice (aged 10 weeks, n = 3) were killed under fed conditions or after a 16-h fast. C: Total liver RNA was subjected to real-time quantitative (q) RT-PCR assay using FoxO6 and β-actin primers. Total liver proteins were separated into nuclear (D) and cytoplasmic (E) fractions, which were analyzed by immunoblot assay using anti-FoxO6 antibody. Furthermore, C57BL/6J male mice (aged 6 weeks) were rendered obese after 8 weeks of high-fat feeding. Mice in groups fed regular chow (n = 6, body wt 26.5 ± 2.1 g) and a high-fat diet (n = 6, 51.4 ± 4.9 g) were killed after a 16-h fast. F: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Total liver proteins were separated into cytoplasmic (G) and nuclear (H) fractions, which were analyzed by anti-FoxO6 immunoblot assay. Likewise, male diabetic db/db (n = 8, aged 6 months; blood glucose levels, 62.8 ± 3.9 mg/dL) vs. male age-matched control db/+ mice (n = 8, 310 ± 28 mg/dL) were killed after a 16-h fast, and liver tissue was processed for the preparation of cytoplasmic and nuclear fractions. I: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Aliquots of cytoplasmic (J) and nuclear (K) proteins (20 μg) were subjected to semiquantitative immunoblot analysis for FoxO6. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality color representation of this figure is available in the online issue.)

Mentions: FoxO6 consists of an amino DNA-binding domain and a carboxyl trans-activation domain, a structural feature that is characteristic of the FoxO family. However, FoxO6 differs from other members of FoxO family in fundamental ways (Fig. 1A):1) FoxO6 has the lowest degree of homology (<30%) in amino acid sequence with other members of the FoxO family. 2) FoxO6 contains only two consensus Akt/PKB phosphorylation sites (Thr26 and Ser184) within its amino DNA-binding domain. In contrast, other members of FoxO family contain three highly conserved phosphorylation sites (Thr24, Ser256, and Ser319 in FoxO1).3) FoxO6 lacks the nuclear export signal (NES), a motif that is conserved in other members of the FoxO family.We determined FoxO6 tissue distribution, demonstrating that FoxO6 was ubiquitously expressed in mice (Fig. 1B). Such a broad tissue distribution of FoxO6 presages a wide spectrum of FoxO6 function in different organs. However, because of scant data on FoxO6 in the literature, little is known about its role in metabolism in response to nutritional cues.


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)

Characterization of FoxO6 and its hepatic expression under physiologic and pathologic conditions. A: FoxO6 is divergent from FoxO1. FoxO6 contains 640 amino acid (aa) residues and two Akt/PKB phosphorylation sites, lacking NES. NLS, nuclear localization signal. B: FoxO6 is ubiquitously expressed. C57BL/6J male mice (aged 10 weeks) were killed under fed conditions for collecting tissues (20 mg), which were subjected to RT-PCR analysis using FoxO6 and β-actin primers. Data were representative of three independent assays from three mice. C57BL/6J male mice (aged 10 weeks, n = 3) were killed under fed conditions or after a 16-h fast. C: Total liver RNA was subjected to real-time quantitative (q) RT-PCR assay using FoxO6 and β-actin primers. Total liver proteins were separated into nuclear (D) and cytoplasmic (E) fractions, which were analyzed by immunoblot assay using anti-FoxO6 antibody. Furthermore, C57BL/6J male mice (aged 6 weeks) were rendered obese after 8 weeks of high-fat feeding. Mice in groups fed regular chow (n = 6, body wt 26.5 ± 2.1 g) and a high-fat diet (n = 6, 51.4 ± 4.9 g) were killed after a 16-h fast. F: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Total liver proteins were separated into cytoplasmic (G) and nuclear (H) fractions, which were analyzed by anti-FoxO6 immunoblot assay. Likewise, male diabetic db/db (n = 8, aged 6 months; blood glucose levels, 62.8 ± 3.9 mg/dL) vs. male age-matched control db/+ mice (n = 8, 310 ± 28 mg/dL) were killed after a 16-h fast, and liver tissue was processed for the preparation of cytoplasmic and nuclear fractions. I: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Aliquots of cytoplasmic (J) and nuclear (K) proteins (20 μg) were subjected to semiquantitative immunoblot analysis for FoxO6. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality color representation of this figure is available in the online issue.)
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Figure 1: Characterization of FoxO6 and its hepatic expression under physiologic and pathologic conditions. A: FoxO6 is divergent from FoxO1. FoxO6 contains 640 amino acid (aa) residues and two Akt/PKB phosphorylation sites, lacking NES. NLS, nuclear localization signal. B: FoxO6 is ubiquitously expressed. C57BL/6J male mice (aged 10 weeks) were killed under fed conditions for collecting tissues (20 mg), which were subjected to RT-PCR analysis using FoxO6 and β-actin primers. Data were representative of three independent assays from three mice. C57BL/6J male mice (aged 10 weeks, n = 3) were killed under fed conditions or after a 16-h fast. C: Total liver RNA was subjected to real-time quantitative (q) RT-PCR assay using FoxO6 and β-actin primers. Total liver proteins were separated into nuclear (D) and cytoplasmic (E) fractions, which were analyzed by immunoblot assay using anti-FoxO6 antibody. Furthermore, C57BL/6J male mice (aged 6 weeks) were rendered obese after 8 weeks of high-fat feeding. Mice in groups fed regular chow (n = 6, body wt 26.5 ± 2.1 g) and a high-fat diet (n = 6, 51.4 ± 4.9 g) were killed after a 16-h fast. F: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Total liver proteins were separated into cytoplasmic (G) and nuclear (H) fractions, which were analyzed by anti-FoxO6 immunoblot assay. Likewise, male diabetic db/db (n = 8, aged 6 months; blood glucose levels, 62.8 ± 3.9 mg/dL) vs. male age-matched control db/+ mice (n = 8, 310 ± 28 mg/dL) were killed after a 16-h fast, and liver tissue was processed for the preparation of cytoplasmic and nuclear fractions. I: Total liver RNA was subjected to real-time qRT-PCR assay for determining hepatic FoxO6 mRNA levels. Aliquots of cytoplasmic (J) and nuclear (K) proteins (20 μg) were subjected to semiquantitative immunoblot analysis for FoxO6. *P < 0.05 and **P < 0.005 vs. control by ANOVA; NS, not significant. (A high-quality color representation of this figure is available in the online issue.)
Mentions: FoxO6 consists of an amino DNA-binding domain and a carboxyl trans-activation domain, a structural feature that is characteristic of the FoxO family. However, FoxO6 differs from other members of FoxO family in fundamental ways (Fig. 1A):1) FoxO6 has the lowest degree of homology (<30%) in amino acid sequence with other members of the FoxO family. 2) FoxO6 contains only two consensus Akt/PKB phosphorylation sites (Thr26 and Ser184) within its amino DNA-binding domain. In contrast, other members of FoxO family contain three highly conserved phosphorylation sites (Thr24, Ser256, and Ser319 in FoxO1).3) FoxO6 lacks the nuclear export signal (NES), a motif that is conserved in other members of the FoxO family.We determined FoxO6 tissue distribution, demonstrating that FoxO6 was ubiquitously expressed in mice (Fig. 1B). Such a broad tissue distribution of FoxO6 presages a wide spectrum of FoxO6 function in different organs. However, because of scant data on FoxO6 in the literature, little is known about its role in metabolism in response to nutritional cues.

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