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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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Hepatic expression of GCN5 is upregulated in obese diabetic mice via cAMP-PKA signalling.(a,b) qRT-PCR analysis of Gcn5 mRNA and immunoblot (IB) analysis of nuclear GCN5 in the liver of C57BL/6J mice maintained on normal chow (NC) or a HFD (a) or of db/db or db/m (control) mice (b) after deprivation of food for 16 h. RT–PCR data are means±s.e.m. (n=7 (a) or 6 (b)). Histone H1 was examined as a loading control for immunoblot analysis. (c) Immunoblot analysis of GCN5, PCAF, CBP and p300 in the liver of db/db or db/m mice deprived of food for 16 h. Quantitative data are means±s.e.m. (n=3). (d) Primary mouse hepatocytes were incubated in the absence or presence of 100 μM pCPT-cAMP or the PKA inhibitor H89 (20 μM) for the indicated times. Cell lysates were then subjected to immunoblot analysis of PCAF or to IP followed by immunoblot analysis with antibodies to GCN5. Data are representative of at least three independent experiments. Statistical analysis was performed with the unpaired Student's t-test (a–c). *P<0.05 versus NC (a) or db/m mice (b,c). RT–PCR, PCR with reverse transcription.
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f1: Hepatic expression of GCN5 is upregulated in obese diabetic mice via cAMP-PKA signalling.(a,b) qRT-PCR analysis of Gcn5 mRNA and immunoblot (IB) analysis of nuclear GCN5 in the liver of C57BL/6J mice maintained on normal chow (NC) or a HFD (a) or of db/db or db/m (control) mice (b) after deprivation of food for 16 h. RT–PCR data are means±s.e.m. (n=7 (a) or 6 (b)). Histone H1 was examined as a loading control for immunoblot analysis. (c) Immunoblot analysis of GCN5, PCAF, CBP and p300 in the liver of db/db or db/m mice deprived of food for 16 h. Quantitative data are means±s.e.m. (n=3). (d) Primary mouse hepatocytes were incubated in the absence or presence of 100 μM pCPT-cAMP or the PKA inhibitor H89 (20 μM) for the indicated times. Cell lysates were then subjected to immunoblot analysis of PCAF or to IP followed by immunoblot analysis with antibodies to GCN5. Data are representative of at least three independent experiments. Statistical analysis was performed with the unpaired Student's t-test (a–c). *P<0.05 versus NC (a) or db/m mice (b,c). RT–PCR, PCR with reverse transcription.

Mentions: We first investigated hepatic expression of GCN5 as well as of CBP, its paralog p300 (also known as Kat3b), and the GCN5 paralog p300/CBP-associated factor (PCAF, also known as Kat2b) in mice. Among these HATs, we found that the expression of GCN5 was selectively increased in the liver of two mouse models of obesity-associated type 2 diabetes—normal mice fed a high-fat diet (HFD) (Fig. 1a) and db/db mice (Fig. 1b,c)—compared with control mice. The amount of GCN5 in primary cultured mouse hepatocytes was also increased by treatment with a cell-permeable analogue of cAMP (pCPT-cAMP) in a PKA-dependent manner (Fig. 1d), suggesting that glucagon-cAMP-PKA signalling increases the hepatic abundance of GCN5 as well as that of CITED2 (ref. 20). These findings prompted us to examine whether GCN5 regulates gluconeogenesis through histone acetylation in collaboration with CITED2.


The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch
Hepatic expression of GCN5 is upregulated in obese diabetic mice via cAMP-PKA signalling.(a,b) qRT-PCR analysis of Gcn5 mRNA and immunoblot (IB) analysis of nuclear GCN5 in the liver of C57BL/6J mice maintained on normal chow (NC) or a HFD (a) or of db/db or db/m (control) mice (b) after deprivation of food for 16 h. RT–PCR data are means±s.e.m. (n=7 (a) or 6 (b)). Histone H1 was examined as a loading control for immunoblot analysis. (c) Immunoblot analysis of GCN5, PCAF, CBP and p300 in the liver of db/db or db/m mice deprived of food for 16 h. Quantitative data are means±s.e.m. (n=3). (d) Primary mouse hepatocytes were incubated in the absence or presence of 100 μM pCPT-cAMP or the PKA inhibitor H89 (20 μM) for the indicated times. Cell lysates were then subjected to immunoblot analysis of PCAF or to IP followed by immunoblot analysis with antibodies to GCN5. Data are representative of at least three independent experiments. Statistical analysis was performed with the unpaired Student's t-test (a–c). *P<0.05 versus NC (a) or db/m mice (b,c). RT–PCR, PCR with reverse transcription.
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f1: Hepatic expression of GCN5 is upregulated in obese diabetic mice via cAMP-PKA signalling.(a,b) qRT-PCR analysis of Gcn5 mRNA and immunoblot (IB) analysis of nuclear GCN5 in the liver of C57BL/6J mice maintained on normal chow (NC) or a HFD (a) or of db/db or db/m (control) mice (b) after deprivation of food for 16 h. RT–PCR data are means±s.e.m. (n=7 (a) or 6 (b)). Histone H1 was examined as a loading control for immunoblot analysis. (c) Immunoblot analysis of GCN5, PCAF, CBP and p300 in the liver of db/db or db/m mice deprived of food for 16 h. Quantitative data are means±s.e.m. (n=3). (d) Primary mouse hepatocytes were incubated in the absence or presence of 100 μM pCPT-cAMP or the PKA inhibitor H89 (20 μM) for the indicated times. Cell lysates were then subjected to immunoblot analysis of PCAF or to IP followed by immunoblot analysis with antibodies to GCN5. Data are representative of at least three independent experiments. Statistical analysis was performed with the unpaired Student's t-test (a–c). *P<0.05 versus NC (a) or db/m mice (b,c). RT–PCR, PCR with reverse transcription.
Mentions: We first investigated hepatic expression of GCN5 as well as of CBP, its paralog p300 (also known as Kat3b), and the GCN5 paralog p300/CBP-associated factor (PCAF, also known as Kat2b) in mice. Among these HATs, we found that the expression of GCN5 was selectively increased in the liver of two mouse models of obesity-associated type 2 diabetes—normal mice fed a high-fat diet (HFD) (Fig. 1a) and db/db mice (Fig. 1b,c)—compared with control mice. The amount of GCN5 in primary cultured mouse hepatocytes was also increased by treatment with a cell-permeable analogue of cAMP (pCPT-cAMP) in a PKA-dependent manner (Fig. 1d), suggesting that glucagon-cAMP-PKA signalling increases the hepatic abundance of GCN5 as well as that of CITED2 (ref. 20). These findings prompted us to examine whether GCN5 regulates gluconeogenesis through histone acetylation in collaboration with CITED2.

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