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. 2011 May 13;145(4):607-21.
doi: 10.1016/j.cell.2011.03.043.

Class IIa histone deacetylases are hormone-activated regulators of FOXO and mammalian glucose homeostasis

Maria M Mihaylova  1 , Debbie S Vasquez, Kim Ravnskjaer, Pierre-Damien Denechaud, Ruth T Yu, Jacqueline G Alvarez, Michael Downes, Ronald M Evans, Marc Montminy, Reuben J Shaw
Affiliations

Class IIa histone deacetylases are hormone-activated regulators of FOXO and mammalian glucose homeostasis

Maria M Mihaylova et al. Cell. .

Abstract

Class IIa histone deacetylases (HDACs) are signal-dependent modulators of transcription with established roles in muscle differentiation and neuronal survival. We show here that in liver, class IIa HDACs (HDAC4, 5, and 7) are phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, class IIa HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 recruit HDAC3, which results in the acute transcriptional induction of these genes via deacetylation and activation of FOXO family transcription factors. Loss of class IIa HDACs in murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Finally, suppression of class IIa HDACs in mouse models of type 2 diabetes ameliorates hyperglycemia, suggesting that inhibitors of class I/II HDACs may be potential therapeutics for metabolic syndrome.

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Figures

Figure 1
Figure 1. Class IIa HDACs Are Regulated by LKB1-dependent Kinases and Metformin Treatment in Liver
(A) Clustal alignment of Class IIa HDACs showing sequence conservation on established phosphorylation sites matching the optimal AMPK motif. (B) Primary mouse hepatocytes or mouse livers lysates infected with adenoviruses bearing indicated shRNAs and immunoblotted with indicated antibodies (full description in Supplemental Supporting Text). (C) Lysates of HepG2 or Huh7 cells transfected with indicated siRNA pools and treated with either 2mM Phenformin or vehicle for 1hr and subjected to immunoblotting. (D) Immunoblot of lysates from murine livers from LKB1+/+ or LKB1lox/lox mice deleted for hepatic LKB1 and treated with either 250mg/kg metformin, or saline alone for 1h. See also Figure S1.
Figure 2
Figure 2. Glucagon Induces Dephosphorylation and Nuclear Translocation of Class IIa HDACs in Hepatocytes
(A) Liver lysates from C57Bl/6J mice either fasted for 18h and/or then refed for 4h (left panel). Mice were either fasted for 6h or fed ad libitum (right panel). (B) Primary mouse hepatocytes treated with 100nM glucagon or vehicle for indicated times, lysed and immunoblotted with indicated antibodies. (C) Primary hepatocytes were treated with either 10uM Forskolin, 100nM Glucagon or 100nM Insulin for 1h. Cells were lysed and blotted for indicated proteins. Results are representative of 3 independent experiments for each panel. (D) Primary mouse hepatocytes infected with adenovirus expressing GFP-HDAC5 WT either treated with 100nM glucagon or vehicle (media) for indicated times and analyzed by confocal microscopy. See also Figure S2.
Figure 3
Figure 3. Class IIa HDACs are Required for the Induction of Gluconeogenic Genes and Associate with the G6Pase Locus Following Glucagon
(A) Microarray data analysis on genes induced by forskolin in primary mouse hepatocytes and whose expression is altered in due to depletion of HDAC4 & 5 (HDAC) but not scrambled (scram) control shRNA. Cells were treated with 10uM forskolin or vehicle (DMSO) for 2 or 4h as indicated. Duplicate samples are shown for each condition. Gene expression shown relative to scrambled shRNA cells treated with vehicle for 2h. FOXO regulated targets (Dong et al., 2006; Dansen et al., 2004; Renault et al., 2009; Paik et al., 2009) (#) or CREB regulated targets (Zhang et al., 2005) (*) as indicated. Rate-limiting gluconeogenic enzymes highlighted in red. Representative 15 of the top 50 HDAC4/5-regulated genes shown. (B) qRT-PCR from primary hepatocytes of FOXO target genes whose FSK-induced expression is attenuated following depletion with HDAC4/5/7 shRNAs. Expression relative to cyclophilin. (n=9) *p<0.01 (C) Ad-G6Pase-luc activity (top panel) or CRE-luc activity (bottom panel) in primary hepatocytes expressing indicated adenoviruses and treated with vehicle or 10uM Forskolin for 4h as indicated. Representative of 4 independent experiments. (n=6) *p< 0.007 (D) G6Pase-luc activity in 18h fasted mice expressing Ad-scrambled or Ad-HDAC4/5/7 shRNAs. Results representative of 3 independent experiments and quantified using the Living Image 3.2 program. (n=6) *p<0.002 (E) Endogenous HDAC4 or HDAC5 chromatin immunoprecipitation (ChIP) with primers against indicated regions of the murine G6Pase promoter at the times indicated following 100nM glucagon treatment. (n=4) *p<0.05 Data are shown as mean +/− s.e.m. using Student’s t-test. See also Figure S3.
Figure 4
Figure 4. Class IIa HDACs control FOXO Acetylation
(A) HEK293T cells transfected with MYC- Foxo1 and GFP-HDAC5 as indicated, treated with 10uM forskolin or vehicle for 1h and immunoprecipitated with anti-myc tag antibody. (B) Primary hepatocytes treated for 1h with vehicle or 10uM Forskolin and endogenous Foxo1 and HDAC4 were detected by immunocytochemistry. (C) Immunoblot showing amounts of acetylated FOXO (Ac-Lys259/262/271) from primary hepatocytes transduced with adenoviruses expressing Foxo3 and indicated shRNAs. Total cell lysates were blotted with indicated antibodies. (D) Primary hepatocytes were transduced with adenoviruses expressing Foxo3 or GFP-FOXO1 and indicated shRNA-expressing adenoviruses. Foxo immunoprecipitates were immunoblotted with indicated antibodies. (E) Lysates from mouse livers knockdown with either scrambled or Class IIa HDAC4/5/7 shRNAs were immunoprecipitated for endogenous Foxo1 protein and immunoblotted with indicated antibodies. (F) Primary hepatocytes knocked down for the Class IIa HDACs or control scramble shRNAs and total cell lysates were immunoblotted with indicated antibodies. See also Figure S4.
Figure 5
Figure 5. Class I HDAC3 is Recruited by Class IIa HDACs to Deacetylate Foxo
(A) ChIP analysis on primary hepatoyctes transduced with control scramble or HDAC4/5/7 shRNAs and assessed for HDAC3 association on Foxo binding sites within G6Pase or PCK1 or the housekeeping TFIIB promoter following 1h treatment with 100nM Glucagon. (n= 4) *p<0.05 (B) HEK293T cells transfected with a FLAG-HDAC3 and GFP-HDAC5 as indicated and treated with forskolin or vehicle for 1h and then immunoprecipitated with anti-FLAG tag antibodies. Immunoprecipitates and input cell lysates were blotted with indicated antibodies. (C) In vitro deacetylation assays were performed on recombinant GST-FOXO1, pre-acetylated in vitro with a recombinant fragment of p300. GST-FOXO1 acetylation is detected using the Foxo1 K242/245 acetylation specific antibody. Recombinant HDAC3 or HDAC3 complexed with Ncor was used at varying concentrations. Recombinant SIRT1 used as positive control. (D) ChIP analysis on primary hepatoyctes transduced with control scramble or HDAC4/5/7 shRNAs and assessed for Foxo1 on G6Pase or PCK1 promoters following 1h treatment with 100nM Glucacon. (n= 4) *p<0.05 Data are shown as mean +/− s.e.m. using Student’s t-test. See also Figure S5.
Figure 6
Figure 6. Class IIa HDACs are Required for Glucose Homeostasis
(A) C57Bl/6J mice infected with indicated shRNAs in liver and fasted for 18h and/or then refed for 4h. Livers were processed for histology and stained with hematoxilin and eosin (H&E) or periodic-shiff’s stain (PAS) to detect glycogen. Images were taken at 40x. (B) qRT-PCR for G6Pase expression from livers of ad lib fed C57Bl/6J mice expressing GFP or GFP-HDAC5-AA or HDAC4/5 shRNAs in liver. (n=9) *p<0.01, **p<0.001, ***p<0.0001 (C) Albumin-creERT2 LKB1+/+ or LKB1lox/lox mice were tamoxifen-treated and and subsequently infected with scrambled or HDAC4/5/7 (HDAC) shRNAs. 5 days later, mice were fasted for 18h, and blood glucose was measured. Average blood glucose value shown in red. (n=5) **p<0.001 ***p<0.0001 (D) qRT-PCR for FOXO target genes (Igfbp1, Agxt2l1, Mmd2) or Hdac4 (control) from livers of indicated mice from C. (n=9) *p<0.01, **p<0.001, ***p<0.0001. , (E) Liver lysates from mice in C were immunoblotted with indicated antibodies. Asterisk indicates a non-specific band recognized by the HDAC5 antibody. Data are shown as mean +/− s.e.m. using Student’s t-test. See also Figure S6.
Figure 7
Figure 7. Suppression of Class IIa HDACs Lowers Blood Glucose in Mouse Models of Metabolic Disease
(A) Glucose tolerance test was performed on db/db mice infected with either scramble (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs. (n=5) *p<0.02 (B) Db/db mice knocked down with (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs in liver. 5 days later, mice were fasted for 18h and blood glucose was measured. Average blood glucose value shown in red. (n=5) *p<0.02 (C) Pyruvate tolerance test performed on db/db mice injected with either scramble (scramb) shRNA or HDAC4/5/7 (HDAC) shRNAs. (n=5) *p<0.04 (D) 7 month old B6 mice on a high fat diet (HFD) were treated as in A. (n=5) *p<0.03 (E) Glucose tolerance test was performed on 7 month old B6 mice on HFD as in C. (n=5) *p<0.02 (F) Model for Glucagon dependent regulation of Class IIa HDACs and FOXO. Under fasting conditions, glucagon induces dephosphorylation and nuclear translocation of Class IIa HDACs. Once nuclear, they associate with the G6Pase and PEPCK promoters and bind to HDAC3-Ncor/SMRT and FOXO1/3, resulting in HDAC3-mediated deacetylation and activation of FOXO. Under fed conditions insulin-dependent activation of the LKB1-dependent kinases SIK1/2 stimulates phosphorylation and cytoplasmic shuttling of Class IIa HDACs. Similarly, following metformin treatment, the LKB1-dependent AMPK activation induces Class IIa HDAC phosphorylation and 14-3-3 binding. In response to glucagon, PKA is activated and directly phosphorylates and inactivates AMPK, SIK1, and SIK2 hence resulting in loss of HDAC phosphorylation. Data are shown as mean +/− s.e.m. using Student’s t-test. See also Figure S7.
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