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.


GCN5 switches substrate in a cAMP- and CITED2-dependent manner.(a) Immunoblot analysis of the effects of FLAG-CITED2 expression or pCPT-cAMP treatment (for 1 h) in AML12 cells on the HAT activity of immunoprecipitated Myc epitope-tagged GCN5 assayed in vitro with histone H3 as substrate. (b) Effects of haemagglutinin epitope (HA)-tagged CITED2 expression in AML12 cells on the acetyltransferase activity of immunoprecipitated FLAG-GCN5 assayed in vitro with histone H3 and a His6-tagged NH2-terminal fragment of PGC-1α as substrates. (c) Interaction of GCN5 with PGC-1α or histone H3 was assessed by PLA in primary hepatocytes expressing either Myc-GCN5 with FLAG–PGC-1α (top) or FLAG-GCN5 alone (bottom) and exposed (or not) to pCPT-cAMP for 1 h. PLA signals (red dots) represent proximity (<40 nm) of GCN5 and either PGC-1α (top) or histone H3 (bottom). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole. Scale bars, 10 μm. All data are representative of at least three independent experiments. Adenoviral vectors were used for these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: GCN5 switches substrate in a cAMP- and CITED2-dependent manner.(a) Immunoblot analysis of the effects of FLAG-CITED2 expression or pCPT-cAMP treatment (for 1 h) in AML12 cells on the HAT activity of immunoprecipitated Myc epitope-tagged GCN5 assayed in vitro with histone H3 as substrate. (b) Effects of haemagglutinin epitope (HA)-tagged CITED2 expression in AML12 cells on the acetyltransferase activity of immunoprecipitated FLAG-GCN5 assayed in vitro with histone H3 and a His6-tagged NH2-terminal fragment of PGC-1α as substrates. (c) Interaction of GCN5 with PGC-1α or histone H3 was assessed by PLA in primary hepatocytes expressing either Myc-GCN5 with FLAG–PGC-1α (top) or FLAG-GCN5 alone (bottom) and exposed (or not) to pCPT-cAMP for 1 h. PLA signals (red dots) represent proximity (<40 nm) of GCN5 and either PGC-1α (top) or histone H3 (bottom). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole. Scale bars, 10 μm. All data are representative of at least three independent experiments. Adenoviral vectors were used for these experiments.

Mentions: Given that glucagon-induced GCN5-CITED2 interaction is mediated by the cAMP-PKA pathway20, we tested whether GCN5 activity is regulated by cAMP, CITED2 or both in hepatocytes. The HAT activity of GCN5 measured in vitro with histone H3 as a substrate was increased by treatment of AML12 cells with pCPT-cAMP, and this effect was enhanced by CITED2 overexpression in an additive manner (Fig. 5a). These data thus indicated that the HAT activity of GCN5 was increased in the presence of cAMP and CITED2. We next examined the effect of CITED2 on the balance between the HAT and PGC-1α acetyltransferase activities of GCN5 measured in vitro with histone H3 and an NH2-terminal fragment of PGC-1α as substrates. In this setting, GCN5 activity was reciprocally regulated by CITED2: HAT activity was increased, whereas PGC-1α acetyltransferase activity and autoacetylation activity were decreased (Fig. 5b). These results indicated that CITED2 binds to GCN5 and promotes a substrate switch from PGC-1α to histone H3 as measured in vitro. To examine whether this switch also occurs in hepatocytes, we investigated the interaction of GCN5 with PGC-1α or histone H3 in the nucleus with an in situ proximity ligation assay (PLA)25. Exposure of primary hepatocytes to pCPT-cAMP disrupted GCN5–PGC-1α interaction and promoted GCN5–histone H3 interaction (Fig. 5c). Together with our results showing that GCN5 recruitment to and H3K9ac marking of gluconeogenic gene promoters are dependent on cAMP and CITED2 (Fig. 4a,b and Supplementary Fig. 3a), these findings supported the notion that a cAMP- and CITED2-dependent substrate switch of GCN5 integrates epigenetic changes and co-activator activity in gluconeogenic gene induction.


The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch
GCN5 switches substrate in a cAMP- and CITED2-dependent manner.(a) Immunoblot analysis of the effects of FLAG-CITED2 expression or pCPT-cAMP treatment (for 1 h) in AML12 cells on the HAT activity of immunoprecipitated Myc epitope-tagged GCN5 assayed in vitro with histone H3 as substrate. (b) Effects of haemagglutinin epitope (HA)-tagged CITED2 expression in AML12 cells on the acetyltransferase activity of immunoprecipitated FLAG-GCN5 assayed in vitro with histone H3 and a His6-tagged NH2-terminal fragment of PGC-1α as substrates. (c) Interaction of GCN5 with PGC-1α or histone H3 was assessed by PLA in primary hepatocytes expressing either Myc-GCN5 with FLAG–PGC-1α (top) or FLAG-GCN5 alone (bottom) and exposed (or not) to pCPT-cAMP for 1 h. PLA signals (red dots) represent proximity (<40 nm) of GCN5 and either PGC-1α (top) or histone H3 (bottom). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole. Scale bars, 10 μm. All data are representative of at least three independent experiments. Adenoviral vectors were used for these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: GCN5 switches substrate in a cAMP- and CITED2-dependent manner.(a) Immunoblot analysis of the effects of FLAG-CITED2 expression or pCPT-cAMP treatment (for 1 h) in AML12 cells on the HAT activity of immunoprecipitated Myc epitope-tagged GCN5 assayed in vitro with histone H3 as substrate. (b) Effects of haemagglutinin epitope (HA)-tagged CITED2 expression in AML12 cells on the acetyltransferase activity of immunoprecipitated FLAG-GCN5 assayed in vitro with histone H3 and a His6-tagged NH2-terminal fragment of PGC-1α as substrates. (c) Interaction of GCN5 with PGC-1α or histone H3 was assessed by PLA in primary hepatocytes expressing either Myc-GCN5 with FLAG–PGC-1α (top) or FLAG-GCN5 alone (bottom) and exposed (or not) to pCPT-cAMP for 1 h. PLA signals (red dots) represent proximity (<40 nm) of GCN5 and either PGC-1α (top) or histone H3 (bottom). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole. Scale bars, 10 μm. All data are representative of at least three independent experiments. Adenoviral vectors were used for these experiments.
Mentions: Given that glucagon-induced GCN5-CITED2 interaction is mediated by the cAMP-PKA pathway20, we tested whether GCN5 activity is regulated by cAMP, CITED2 or both in hepatocytes. The HAT activity of GCN5 measured in vitro with histone H3 as a substrate was increased by treatment of AML12 cells with pCPT-cAMP, and this effect was enhanced by CITED2 overexpression in an additive manner (Fig. 5a). These data thus indicated that the HAT activity of GCN5 was increased in the presence of cAMP and CITED2. We next examined the effect of CITED2 on the balance between the HAT and PGC-1α acetyltransferase activities of GCN5 measured in vitro with histone H3 and an NH2-terminal fragment of PGC-1α as substrates. In this setting, GCN5 activity was reciprocally regulated by CITED2: HAT activity was increased, whereas PGC-1α acetyltransferase activity and autoacetylation activity were decreased (Fig. 5b). These results indicated that CITED2 binds to GCN5 and promotes a substrate switch from PGC-1α to histone H3 as measured in vitro. To examine whether this switch also occurs in hepatocytes, we investigated the interaction of GCN5 with PGC-1α or histone H3 in the nucleus with an in situ proximity ligation assay (PLA)25. Exposure of primary hepatocytes to pCPT-cAMP disrupted GCN5–PGC-1α interaction and promoted GCN5–histone H3 interaction (Fig. 5c). Together with our results showing that GCN5 recruitment to and H3K9ac marking of gluconeogenic gene promoters are dependent on cAMP and CITED2 (Fig. 4a,b and Supplementary Fig. 3a), these findings supported the notion that a cAMP- and CITED2-dependent substrate switch of GCN5 integrates epigenetic changes and co-activator activity in gluconeogenic gene induction.

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.