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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of GCN5 at Ser275 promotes gluconeogenesis.(a) Effects of forced expression of GCN5(WT) or GCN5(S275A) together with CITED2 on gluconeogenic gene expression in primary hepatocytes exposed (or not) to pCPT-cAMP (6 h). (b,c) Effects of forced expression of GCN5(S275D) on gluconeogenic gene expression (b) and glucose production (c) in primary hepatocytes exposed (or not) to pCPT-cAMP (16 h). (d) qRT-PCR analysis of G6pc and Pck1 expression in primary mouse hepatocytes infected with adenoviruses encoding CITED2 shRNA or GCN5(S275D) and exposed (or not) to pCPT-cAMP for 6 h. (e,f) Effects of GCN5(S275D) expression in the liver of C57BL/6J mice on gluconeogenic gene expression (e) and plasma glycemia (f) under the fasted (24 h) condition. All data are means±s.e.m. (n=3 (a–d) or 7 (e,f)). *P<0.05, **P<0.01 versus control or as indicated (ANOVA with Bonferroni's post hoc test (a–d) or unpaired Student's t-test (e,f)). Adenoviral vectors were used for these experiments. RT–PCR, PCR with reverse transcription. ANOVA, analysis of variance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121418&req=5

f8: Phosphorylation of GCN5 at Ser275 promotes gluconeogenesis.(a) Effects of forced expression of GCN5(WT) or GCN5(S275A) together with CITED2 on gluconeogenic gene expression in primary hepatocytes exposed (or not) to pCPT-cAMP (6 h). (b,c) Effects of forced expression of GCN5(S275D) on gluconeogenic gene expression (b) and glucose production (c) in primary hepatocytes exposed (or not) to pCPT-cAMP (16 h). (d) qRT-PCR analysis of G6pc and Pck1 expression in primary mouse hepatocytes infected with adenoviruses encoding CITED2 shRNA or GCN5(S275D) and exposed (or not) to pCPT-cAMP for 6 h. (e,f) Effects of GCN5(S275D) expression in the liver of C57BL/6J mice on gluconeogenic gene expression (e) and plasma glycemia (f) under the fasted (24 h) condition. All data are means±s.e.m. (n=3 (a–d) or 7 (e,f)). *P<0.05, **P<0.01 versus control or as indicated (ANOVA with Bonferroni's post hoc test (a–d) or unpaired Student's t-test (e,f)). Adenoviral vectors were used for these experiments. RT–PCR, PCR with reverse transcription. ANOVA, analysis of variance.

Mentions: We next investigated the effects of these GCN5 mutants on gluconeogenesis in vitro. In primary hepatocytes overexpressing CITED2, pCPT-cAMP-induced gluconeogenic gene expression was enhanced to a lesser extent by GCN5(S275A) than by GCN5(WT) (Fig. 8a and Supplementary Fig. 6a). On the other hand, expression of the S275D mutant without CITED2 overexpression enhanced cAMP-induced gluconeogenic gene expression (Fig. 8b and Supplementary Fig. 6b)—in contrast to the effect of GCN5(WT) (Fig. 3a)—resulting in enhanced glucose production (Fig. 8c). The selective gluconeogenic effect of GCN5(S275D) was associated with enhanced cAMP-induced acetylation of H3K9 and H3K27 at gluconeogenic gene promoters, but not at Gapdh or Hmgcr promoters (Supplementary Fig. 6c). GCN5(S275D) also rescued, at least in part, the inhibition of gluconeogenic gene expression by CITED2 depletion in pCPT-cAMP-stimulated hepatocytes (Fig. 8d and Supplementary Fig. 6d), suggesting that GCN5 phosphorylated at Ser275 functions as a gluconeogenic HAT downstream of CITED2. These results indicated that phosphorylation of GCN5 at Ser275 by PKA in the GCN5-CITED2-PKA signalling module drives the substrate switch of GCN5 and thereby promotes gluconeogenesis in vitro.


The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch
Phosphorylation of GCN5 at Ser275 promotes gluconeogenesis.(a) Effects of forced expression of GCN5(WT) or GCN5(S275A) together with CITED2 on gluconeogenic gene expression in primary hepatocytes exposed (or not) to pCPT-cAMP (6 h). (b,c) Effects of forced expression of GCN5(S275D) on gluconeogenic gene expression (b) and glucose production (c) in primary hepatocytes exposed (or not) to pCPT-cAMP (16 h). (d) qRT-PCR analysis of G6pc and Pck1 expression in primary mouse hepatocytes infected with adenoviruses encoding CITED2 shRNA or GCN5(S275D) and exposed (or not) to pCPT-cAMP for 6 h. (e,f) Effects of GCN5(S275D) expression in the liver of C57BL/6J mice on gluconeogenic gene expression (e) and plasma glycemia (f) under the fasted (24 h) condition. All data are means±s.e.m. (n=3 (a–d) or 7 (e,f)). *P<0.05, **P<0.01 versus control or as indicated (ANOVA with Bonferroni's post hoc test (a–d) or unpaired Student's t-test (e,f)). Adenoviral vectors were used for these experiments. RT–PCR, PCR with reverse transcription. ANOVA, analysis of variance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121418&req=5

f8: Phosphorylation of GCN5 at Ser275 promotes gluconeogenesis.(a) Effects of forced expression of GCN5(WT) or GCN5(S275A) together with CITED2 on gluconeogenic gene expression in primary hepatocytes exposed (or not) to pCPT-cAMP (6 h). (b,c) Effects of forced expression of GCN5(S275D) on gluconeogenic gene expression (b) and glucose production (c) in primary hepatocytes exposed (or not) to pCPT-cAMP (16 h). (d) qRT-PCR analysis of G6pc and Pck1 expression in primary mouse hepatocytes infected with adenoviruses encoding CITED2 shRNA or GCN5(S275D) and exposed (or not) to pCPT-cAMP for 6 h. (e,f) Effects of GCN5(S275D) expression in the liver of C57BL/6J mice on gluconeogenic gene expression (e) and plasma glycemia (f) under the fasted (24 h) condition. All data are means±s.e.m. (n=3 (a–d) or 7 (e,f)). *P<0.05, **P<0.01 versus control or as indicated (ANOVA with Bonferroni's post hoc test (a–d) or unpaired Student's t-test (e,f)). Adenoviral vectors were used for these experiments. RT–PCR, PCR with reverse transcription. ANOVA, analysis of variance.
Mentions: We next investigated the effects of these GCN5 mutants on gluconeogenesis in vitro. In primary hepatocytes overexpressing CITED2, pCPT-cAMP-induced gluconeogenic gene expression was enhanced to a lesser extent by GCN5(S275A) than by GCN5(WT) (Fig. 8a and Supplementary Fig. 6a). On the other hand, expression of the S275D mutant without CITED2 overexpression enhanced cAMP-induced gluconeogenic gene expression (Fig. 8b and Supplementary Fig. 6b)—in contrast to the effect of GCN5(WT) (Fig. 3a)—resulting in enhanced glucose production (Fig. 8c). The selective gluconeogenic effect of GCN5(S275D) was associated with enhanced cAMP-induced acetylation of H3K9 and H3K27 at gluconeogenic gene promoters, but not at Gapdh or Hmgcr promoters (Supplementary Fig. 6c). GCN5(S275D) also rescued, at least in part, the inhibition of gluconeogenic gene expression by CITED2 depletion in pCPT-cAMP-stimulated hepatocytes (Fig. 8d and Supplementary Fig. 6d), suggesting that GCN5 phosphorylated at Ser275 functions as a gluconeogenic HAT downstream of CITED2. These results indicated that phosphorylation of GCN5 at Ser275 by PKA in the GCN5-CITED2-PKA signalling module drives the substrate switch of GCN5 and thereby promotes gluconeogenesis in vitro.

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