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The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch

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ABSTRACT

Hepatic gluconeogenesis during fasting results from gluconeogenic gene activation via the glucagon–cAMP–protein kinase A (PKA) pathway, a process whose dysregulation underlies fasting hyperglycemia in diabetes. Such transcriptional activation requires epigenetic changes at promoters by mechanisms that have remained unclear. Here we show that GCN5 functions both as a histone acetyltransferase (HAT) to activate fasting gluconeogenesis and as an acetyltransferase for the transcriptional co-activator PGC-1α to inhibit gluconeogenesis in the fed state. During fasting, PKA phosphorylates GCN5 in a manner dependent on the transcriptional coregulator CITED2, thereby increasing its acetyltransferase activity for histone and attenuating that for PGC-1α. This substrate switch concomitantly promotes both epigenetic changes associated with transcriptional activation and PGC-1α–mediated coactivation, thereby triggering gluconeogenesis. The GCN5-CITED2-PKA signalling module and associated GCN5 substrate switch thus serve as a key driver of gluconeogenesis. Disruption of this module ameliorates hyperglycemia in obese diabetic animals, offering a potential therapeutic strategy for such conditions.

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Suppression of hepatic GCN5 phosphorylation at Ser275 ameliorates diabetes.(a) Analysis of GCN5 phosphorylated at Ser275 in the liver of db/db or db/m (control) mice or of C57BL/6J mice fed NC or a HFD. All mice were deprived of food for 16 h before analysis. Liver extracts were subjected to IP with antibodies to Ser275-phosphorylated GCN5 followed by immunoblot analysis with antibodies to GCN5. (b,c) Effects of expression of GCN5(WT), GCN5(S275A) or GCN5(S275D) in the liver of db/db mice on plasma glucose concentration under fed or fasted (24 h) conditions (b) as well as on hepatic expression of G6pc and Pck1 under the fasted (24 h) condition (c). (d) Effect of CITED2 depletion on GCN5 phosphorylation at Ser275 in the liver of db/db mice deprived of food for 24 h. All quantitative data are means±s.e.m. (n=7 (b,c)). *P<0.05, **P<0.01 (ANOVA with Bonferroni's post hoc test). Data in a,d are representative of at least three independent experiments. Adenoviral vectors were used for these experiments. ANOVA, analysis of variance; NC, normal chow.
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f9: Suppression of hepatic GCN5 phosphorylation at Ser275 ameliorates diabetes.(a) Analysis of GCN5 phosphorylated at Ser275 in the liver of db/db or db/m (control) mice or of C57BL/6J mice fed NC or a HFD. All mice were deprived of food for 16 h before analysis. Liver extracts were subjected to IP with antibodies to Ser275-phosphorylated GCN5 followed by immunoblot analysis with antibodies to GCN5. (b,c) Effects of expression of GCN5(WT), GCN5(S275A) or GCN5(S275D) in the liver of db/db mice on plasma glucose concentration under fed or fasted (24 h) conditions (b) as well as on hepatic expression of G6pc and Pck1 under the fasted (24 h) condition (c). (d) Effect of CITED2 depletion on GCN5 phosphorylation at Ser275 in the liver of db/db mice deprived of food for 24 h. All quantitative data are means±s.e.m. (n=7 (b,c)). *P<0.05, **P<0.01 (ANOVA with Bonferroni's post hoc test). Data in a,d are representative of at least three independent experiments. Adenoviral vectors were used for these experiments. ANOVA, analysis of variance; NC, normal chow.

Mentions: We also examined the phosphorylation level of GCN5 at Ser275 in the liver of diabetic mice. Phosphorylation of GCN5 (Fig. 9a) as well as the amount of GCN5 protein (Fig. 1a–c) were increased in the liver of db/db mice and of C57BL/6J mice fed a HFD. The expression of CITED2 (ref. 20), but not that of PKA (Supplementary Fig. 7a), was also increased in the liver of diabetic mice. These changes in GCN5 and CITED2 are consistent with enhancement of gluconeogenesis and prompted us to address whether GCN5 phosphorylation at Ser275 contributes to hyperglycemia through promotion of hepatic gluconeogenesis in these animals. Hepatic expression of GCN5(S275D) increased, whereas that of GCN5(S275A) or GCN5(WT) decreased, blood glucose levels and gluconeogenic gene expression in the liver of fasted db/db mice (Fig. 9b,c and Supplementary Fig. 7b). These effects are consistent with those observed in hepatocytes (Fig. 8a–c) or in the liver of lean mice (Fig. 8e,f), and they indicate that inhibition of GCN5 phosphorylation at Ser275 suppresses gluconeogenesis and therefore ameliorates hyperglycemia, whereas enhanced phosphorylation of GCN5 at Ser275 promotes gluconeogenesis and exacerbates hyperglycemia, in diabetic animals. We have previously shown that depletion of CITED2 in the liver of db/db mice suppresses gluconeogenesis through enhancement of GCN5-dependent acetylation and the consequent inactivation of PGC-1α and thereby ameliorates diabetes20. In this setting, phosphorylation of GCN5 at Ser275 was also inhibited (Fig. 9d), indicating that disruption of the GCN5-CITED2-PKA signalling module by CITED2 depletion attenuates gluconeogenesis and ameliorates hyperglycemia through suppression of GCN5 phosphorylation at Ser275 and consequent enhanced GCN5-dependent acetylation and inhibition of PGC-1α as well as reduced GCN5-dependent acetylation of histone H3K9.


The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch
Suppression of hepatic GCN5 phosphorylation at Ser275 ameliorates diabetes.(a) Analysis of GCN5 phosphorylated at Ser275 in the liver of db/db or db/m (control) mice or of C57BL/6J mice fed NC or a HFD. All mice were deprived of food for 16 h before analysis. Liver extracts were subjected to IP with antibodies to Ser275-phosphorylated GCN5 followed by immunoblot analysis with antibodies to GCN5. (b,c) Effects of expression of GCN5(WT), GCN5(S275A) or GCN5(S275D) in the liver of db/db mice on plasma glucose concentration under fed or fasted (24 h) conditions (b) as well as on hepatic expression of G6pc and Pck1 under the fasted (24 h) condition (c). (d) Effect of CITED2 depletion on GCN5 phosphorylation at Ser275 in the liver of db/db mice deprived of food for 24 h. All quantitative data are means±s.e.m. (n=7 (b,c)). *P<0.05, **P<0.01 (ANOVA with Bonferroni's post hoc test). Data in a,d are representative of at least three independent experiments. Adenoviral vectors were used for these experiments. ANOVA, analysis of variance; NC, normal chow.
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f9: Suppression of hepatic GCN5 phosphorylation at Ser275 ameliorates diabetes.(a) Analysis of GCN5 phosphorylated at Ser275 in the liver of db/db or db/m (control) mice or of C57BL/6J mice fed NC or a HFD. All mice were deprived of food for 16 h before analysis. Liver extracts were subjected to IP with antibodies to Ser275-phosphorylated GCN5 followed by immunoblot analysis with antibodies to GCN5. (b,c) Effects of expression of GCN5(WT), GCN5(S275A) or GCN5(S275D) in the liver of db/db mice on plasma glucose concentration under fed or fasted (24 h) conditions (b) as well as on hepatic expression of G6pc and Pck1 under the fasted (24 h) condition (c). (d) Effect of CITED2 depletion on GCN5 phosphorylation at Ser275 in the liver of db/db mice deprived of food for 24 h. All quantitative data are means±s.e.m. (n=7 (b,c)). *P<0.05, **P<0.01 (ANOVA with Bonferroni's post hoc test). Data in a,d are representative of at least three independent experiments. Adenoviral vectors were used for these experiments. ANOVA, analysis of variance; NC, normal chow.
Mentions: We also examined the phosphorylation level of GCN5 at Ser275 in the liver of diabetic mice. Phosphorylation of GCN5 (Fig. 9a) as well as the amount of GCN5 protein (Fig. 1a–c) were increased in the liver of db/db mice and of C57BL/6J mice fed a HFD. The expression of CITED2 (ref. 20), but not that of PKA (Supplementary Fig. 7a), was also increased in the liver of diabetic mice. These changes in GCN5 and CITED2 are consistent with enhancement of gluconeogenesis and prompted us to address whether GCN5 phosphorylation at Ser275 contributes to hyperglycemia through promotion of hepatic gluconeogenesis in these animals. Hepatic expression of GCN5(S275D) increased, whereas that of GCN5(S275A) or GCN5(WT) decreased, blood glucose levels and gluconeogenic gene expression in the liver of fasted db/db mice (Fig. 9b,c and Supplementary Fig. 7b). These effects are consistent with those observed in hepatocytes (Fig. 8a–c) or in the liver of lean mice (Fig. 8e,f), and they indicate that inhibition of GCN5 phosphorylation at Ser275 suppresses gluconeogenesis and therefore ameliorates hyperglycemia, whereas enhanced phosphorylation of GCN5 at Ser275 promotes gluconeogenesis and exacerbates hyperglycemia, in diabetic animals. We have previously shown that depletion of CITED2 in the liver of db/db mice suppresses gluconeogenesis through enhancement of GCN5-dependent acetylation and the consequent inactivation of PGC-1α and thereby ameliorates diabetes20. In this setting, phosphorylation of GCN5 at Ser275 was also inhibited (Fig. 9d), indicating that disruption of the GCN5-CITED2-PKA signalling module by CITED2 depletion attenuates gluconeogenesis and ameliorates hyperglycemia through suppression of GCN5 phosphorylation at Ser275 and consequent enhanced GCN5-dependent acetylation and inhibition of PGC-1α as well as reduced GCN5-dependent acetylation of histone H3K9.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatic gluconeogenesis during fasting results from gluconeogenic gene activation via the glucagon&ndash;cAMP&ndash;protein kinase A (PKA) pathway, a process whose dysregulation underlies fasting hyperglycemia in diabetes. Such transcriptional activation requires epigenetic changes at promoters by mechanisms that have remained unclear. Here we show that GCN5 functions both as a histone acetyltransferase (HAT) to activate fasting gluconeogenesis and as an acetyltransferase for the transcriptional co-activator PGC-1&alpha; to inhibit gluconeogenesis in the fed state. During fasting, PKA phosphorylates GCN5 in a manner dependent on the transcriptional coregulator CITED2, thereby increasing its acetyltransferase activity for histone and attenuating that for PGC-1&alpha;. This substrate switch concomitantly promotes both epigenetic changes associated with transcriptional activation and PGC-1&alpha;&ndash;mediated coactivation, thereby triggering gluconeogenesis. The GCN5-CITED2-PKA signalling module and associated GCN5 substrate switch thus serve as a key driver of gluconeogenesis. Disruption of this module ameliorates hyperglycemia in obese diabetic animals, offering a potential therapeutic strategy for such conditions.

No MeSH data available.


Related in: MedlinePlus