Fasting or low blood sugar induces the expression of genes required for production of glucose through gluconeogenesis. Once glucose levels increase, signaling through the insulin receptor terminates glucose production in the liver and activates glucose uptake in the peripheral tissues, stabilizing blood glucose levels. Dysregulation of this balance results in development of metabolic disorders such as type 2 diabetes. FOXO transcription factors control the expression of genes involved in metabolism, proliferation, and cell survival. FOXO activity is regulated by acetylation. Deacetylation of FOXO transcription factors by sirtuins and now class IIa histone deacetylaces (HDAC) activates the transcription of enzymes required for gluconeogenesis. Recent discovery of the role of class IIa HDAC activity in regulation of glucose and lipid metabolism opens up the possibility for use of class IIa HDAC inhibitors for treatment of metabolic disorders. Evaluation of FOXO target gene expression using real-time PCR analysis will further our understanding of the role of HDACs in regulation of genes involved in metabolism, proliferation, and cell survival.
Glucose homeostasis is regulated through a balance between the activity of the insulin and glucagon signaling pathways. Low blood glucose stimulates the production of glucagon, a 29 amino acid hormone that is processed into functional peptide hormones from the proglucagon precursor. Binding to the glucagon receptor activates a signaling pathway that includes the cAMP dependent protein kinase A (PKA) and the AMP dependent protein kinase family members, AMPK, SIK1, and SIK2 (SIK3 in Drosphila). PKA blocks AMPK, SIK1 and SIK2, allowing nuclear translocation of transcriptional activators of gluconeogenic gene expression. Transcription of gluconeogenic enzymes such as glucose-6-phosphatase and phosphophenol pyruvate carboxykinase is regulated by the transcription factors FOXO1 and HNF4a and initiates glucose production in the liver (see figure, Model for regulation of glucose metabolism by class IIa HDACs) (1-4).
Once glucose levels are sufficient, signaling through the insulin receptor attenuates gluconeogenesis, and initiates glucose uptake. Signaling through the insulin receptor involves activation of AKT kinase by PI3 kinase (PI3K). AKT phosphorylates GSK3, deactivating the kinase, which results in activation of glycogen synthase, inducing energy storage in the form of glycogen. AKT also acts to shut off gluconeogenesis by phosphorylating FOXO1. Phosphorylated FOXO1 is excluded from the nucleus and can no longer activate transcription of gluconeogenic enzymes. PI3K also plays a role in insulin stimulated glucose uptake through activation of atypical PKCs (λ and ζ). Atypical PKCs induce translocation of the insulin sensitive glucose transporter, GLUT4, to the plasma membrane, mediating glucose uptake (1-4).
Blood glucose levels are maintained through the balance between insulin mediated glucose uptake and storage in the form of glycogen, and glucagon mediated gluconeogenesis in the liver. Dysfunction within these pathways results in type 2 diabetes and metabolic syndrome. Further understanding of the mechanisms of regulation of glucose homeostasis will aid in the development of treatment for these disorders.
Back to top Regulation of Gluconeogenesis through Post Translational Modification of FOXO
Post translational modifications alter the function of proteins by influencing stability, localization, and interaction with other proteins or nucleic acids. Acetylation can both activate and suppress transcription. Traditionally acetylation of lysines on histone tails has been associated with transcriptional activation. The acetyl group neutralizes the positive charge of the lysine, relaxing chromatin structure, therefore increasing the accessibility of target genes to transcription factors. Acetylated histones are also binding sites for bromodomain proteins, transcriptional activators. In this setting HDACs are acting to repress transcription by allowing compaction of the chromatin (5).
Direct acetylation of transcription factors, however, has a variety of outcomes in terms of transcriptional activity. Acetylation has been shown to enhance specific DNA binding while inhibiting non-specific DNA binding. Acetylation can form a docking site for recruitment of co-activators. Lysine acetylation can inhibit ubiquitination on the same residue, affecting protein stability and localization. Acetylation can therefore induce repression and activation depending on the setting (5).
The FOXO transcription factors are a subgroup of the forkhead family of transcription factors, characterized by a conserved DNA binding domain. There are more than 100 members of the forkhead family in humans. They have been implicated in regulation of a wide range of cellular processes including differentiation, proliferation, survival, and metabolism. There are 4 FOXO genes in mammals, FOXO 1, 3, 4, and 6. FOXO transcription factors were first linked to longevity in C. elegans (DAF-16) where they were shown to be negatively regulated by the insulin/PI3K/Akt signaling pathway. They were later shown to be inhibited by insulin and growth factors in mammalian cells. FOXO transcription factors have since been implicated in a number of critical cellular processes from apoptosis to oxidative stress response to energy homeostasis and glucose metabolism (6).
FOXO transcription factors are regulated by post-translational modification; namely by phosphorylation and acetylation. Phosphorylation affects FOXO activity by sequestering the protein in the cytoplasm. FOXO 1, 3a, and 4, are phosphorylated by AKT, resulting in 14-3-3 chaperone mediated nuclear exclusion, thereby preventing initiation of transcription. FOXO is also phosphorylated by JNK under conditions of oxidative stress, inducing nuclear translocation (6).
Acetylation affects FOXO transcriptional activity through a number of mechanisms. Lysine 245 and 248 reside in the DNA binding domain. The positive charge of lysine contributes to DNA binding and lysine acetylation decreases DNA binding affinity (7). Lysine 265 resides within the nuclear localization signal and acetylation on this residue may interfere with nuclear import. FOXO is acetylated in response to cell stress by the nuclear hormone receptor coactivators CBP and p300. The nuclear sirtuins deacetylate FOXO in response to the cellular NAD:NADH ratio. The sirtuin family member, SIRT1, has been implicated in regulation of gluconeogenesis and fatty acid beta-oxidation and has been shown to induce nuclear retention of FOXO. Disruption of this class III HDAC in the mouse liver significantly decreases these metabolic processes, however the ultimate role of SIRT1 loss on glucose homeostasis is still unclear (8).
Back to top Class IIa HDACs Regulate FOXO Activity
The HDAC superfamily, in mammals, is made up of 11 proteins sharing a conserved deacetylase domain, that fall into 4 families based on structure, function, localization, and expression pattern. Class I HDACs (1, 2, 3, and 8) are ubiquitously expressed in the nucleus and function in deacetylation of histone substrates. Class III represents the sirtuins. Class IIa HDACs (4, 5, 7, and 9) have large N-terminal extensions with binding sites where the MEF2 transcription factor and the 14-3-3 chaperone bind and regulate HDAC function. The class IIa HDACs are phosphorylated by kinases and then bound by 14-3-3 chaparones that shuttle the complex from the nucleus to the cytoplasm. The function of MEF2 as transcriptional activator or repressor is determined by whether the histone acetyl transferase, p300, or HDAC is bound to the N terminal extension.
The mechanism by which class IIa HDACs repress transcription is not clear. Purified recombinant class IIa HDACs lack catalytic activity and endogenous HDACs copurify in a high molecular weight complex. Recruitment of class I HDACs through their C terminal HDAC domain is a possible mechanism for the observed repressive activity (5).
Recently, class IIa HDACs have been implicated in regulation of FOXO mediated transcription of gluconeogenic genes. These findings provide insight into a possible mode of treatment for metabolic disorders such as type 2 diabetes. In fact, a number of class IIa HDAC inhibitors are currently in development as anti-cancer therapeutics (2, 3).
Mihaylova et al and Wang et al report a role for class IIa HDACs in the regulation of FOXO during glucose and lipid metabolism (2, 3). Both groups report direct regulation of the HDACs by LKB1, SIK1/2, and AMPK. Mihaylova et al deleted LKB1 in the mouse liver and observed decreased phosphorylation of HDAC 4/5/7(2). Treatment with metformin, an AMPK/LKB1 agonist, increased HDAC 4/5/7 phosphorylation, resulting in retention in the cytoplasm. Treatment with glucagon induced HDAC 4/5/7 dephosphorylation and translocation into the nucleus. In the nucleus, the class IIa HDACs recruited the catalytically active class I HDAC 3. HDAC 3 deacetylated the FOXO family of transcription factors resulting in FOXO target gene expression.
Wang et al observed a similar mechanism in Drosophila. During fasting, SIK3, a member of the AMPK family of Ser/Thr kinases, is inactivated. This results in dephosphorylation and nuclear localization of HDAC4 and FOXO deacetylation. During feeding SIK3 is activated by AKT which blocks FOXO activity, promoting lipid storage (3).
Inhibition of class IIa HDACs by Mihaylova et al in a mouse model of type 2 diabetes inhibited hyperglycemia, highlighting the potential for use of class I/II HDAC inhibitors for treatment of metabolic disorders (2).
Identification of class IIa HDACs as regulators of glucose metabolism opens up the world of HDAC inhibitors for potential use in the treatment of metabolic disorders. However, many questions remain. The FOXO family of transcription factors regulates diverse cellular processes from metabolism to apoptosis. What roles do class IIa HDACs and sirtuins play in the regulation of these other pathways? How will HDAC inhibitors affect the other FOXO regulated genes? The transcriptional co-activator CRTC2 mediates an additional mechanism of gluconeogenic gene regulation. What is the purpose of this redundancy? What does this teach us about the function of the FOXO family of transcription factors?
- Karpac, Jason, and Heinrich Jasper, (2011) Metabolic Homeostasis: HDACs Take Center Stage. Cell 145, 497-499.
- Mihaylova, Maria M, et al., (2011) Class IIa Histone Deacetylases Are Hormone-Activated Regulators of FOXO and Mammalian Glucose Homeostasis. Cell 145, 607-621.
- Wang, Biao, et al., (2011) A Hormone-Dependent Module Regulating Energy Balance. Cell 145, 596-606.
- Biddinger, Sudha B., and C. Ronald Kahn, (2006) FROM MICE TO MEN: Insights into the Insulin Resistance Syndromes. Annual Review of Physiology 68, 123-158.
- Haberland, Michael, Rusty L. Montgomery, and Eric N. Olson, (2009) The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 10, 32-42.
- Accili, Domenico, and Karen C. Arden, (2004) FoxOs at the Crossroads of Cellular Metabolism, Differentiation, and Transformation. Cell 117, 421-426.
- Matsuzaki, Hitomi, Hiroaki Daitoku, Mitsutoki Hatta, Hisanori Aoyama, Kenji Yoshimochi, and Akiyoshi Fukamizu, (2005) Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation. Proceedings of the National Academy of Sciences of the United States of America 102, 11278-11283.
- Yang, Xiang-Jiao, and Edward Seto, (2008) Lysine Acetylation: Codified Crosstalk with Other Posttranslational Modifications. Molecular cell 31, 449-461.