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Center for Integrative Genomics, National Centre of Competence in Research "Frontiers in Genetics," University of Lausanne, Lausanne, Switzerland
ABSTRACT I. INTRODUCTION II. TRANSCRIPTIONAL CONTROL BY GLUCOSE A. Introduction B. Transcriptional Regulation of Metabolism by High Glucose Levels 1. Transcriptional control of insulin expression and secretion 2. Insulin-regulated transcription of genes involved in glucose metabolism 3. Glucose*insulin regulated transcription of genes involved in glucose metabolism C. Transcriptional Regulation of Metabolism by Low Glucose Levels III. TRANSCRIPTIONAL CONTROL OF AMINO ACID AND PROTEIN METABOLISM A. Introduction B. Transcriptional Regulation of Glutamine Production and Homeostasis C. Transcriptional Regulation of Urea and Ammonia Homeostasis D. Amino Acid Deprivation and Induction of Gene Expression 1. The search for an amino acid response element 2. TOR as a master switch for catabolism versus anabolism IV. TRANSCRIPTIONAL CONTROL OF LIPID METABOLISM A. Transcriptional Control of Fatty Acid Metabolism 1. Fatty acid synthesis and storage: control by an intricate array of transcription factors A) SREBP-1, A MAJOR TRANSCRIPTION FACTOR INVOLVED IN FATTY ACID SYNTHESIS. B) PPARgamma: A MAJOR REGULATOR OF FATTY ACID STORAGE AND ADIPOGENESIS. C) THE PART PLAYED BY C/EBP IN ADIPOGENESIS AND LIPOGENESIS. D) PPARbeta: A ROLE IN ADIPOGENESIS? E) DIVERSE ARRAYS OF SIGNALS PARTICIPATE IN THE TUNING OF THE ADIPOGENESIS PROCESS. 2. Fatty acid oxidation: regulation by PPARalpha and PPARbeta A) PPARalpha: A MAJOR TRANSCRIPTIONAL REGULATOR OF FATTY ACID OXIDATION. B) PPARbeta AND FATTY ACID OXIDATION: OVERLAPS AND SPECIFICITIES WITH RESPECT TO PPARalpha FUNCTIONS. C) TRANSCRIPTIONAL REGULATION OF CPT-I: A COMPLEX ARRAY OF TRANSCRIPTION FACTORS. B. Transcriptional Control of Cholesterol Homeostasis 1. An outline of cholesterol metabolism and its main regulatory factors 2. SREBP-2 is required for the transcriptional activation of the cholesterol synthesis pathway 3. LXR: a player in the reverse cholesterol pathway 4. FXR and the inhibition of bile acid synthesis C. Intricate Regulation of Cholesterol and Fatty Acid Metabolism 1. Regulation of the lipoprotein system and the particular role of PPARs 2. SREBPs and LXR at the branching point between fatty acid and cholesterol metabolism 3. RXR: a pivotal element of sensor-regulated pathways V. TRANSCRIPTION FACTOR INTERPLAY IN THE FASTING-FEEDING CYCLE A. Fasting-Feeding: Metabolic Adjustment in the WAT B. Fasting-Feeding: Metabolic Adjustment in Muscles C. Fasting-Feeding and Gluconeogenesis: Metabolic Adjustment in the Liver, Kidney, and Small Intestine D. Metabolic Adjustment and Circadian Rhythm VI. TRANSCRIPTION FACTORS AND INSULIN RESISTANCE: A MAJOR FOCUS ON PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS A. Introduction B. Insulin Resistance: A Mixed Defect in Glucose and Lipid Metabolism C. Insulin Resistance: On the Molecular Nature of the Causal Mechanism D. The Thiazolidinediones as a Tool for Understanding the Physiopathogeny of the Metabolic Syndrome 1. Hypothesis for the mechanism involved in PPARgamma-mediated improvement of insulin sensitivity 2. The paradox of PPARgamma and insulin resistance 3. A possible role of PPARalpha and PPARbeta in the metabolic syndrome VII. TRANSCRIPTION FACTORS AS TARGETS FOR THERAPEUTIC APPROACHES OF METABOLIC DISORDERS A. How to Modulate Transcription Factor Activity B. Nuclear Receptors as Targets for New Therapeutic Approaches C. Adverse Effects of Drugs on Energy Metabolism VIII. CONCLUSIONS APPENDIX Appendix A: The Nuclear Receptor Family 1. Nuclear receptors share a common structural and functional organization (Fig. 1A) 2. The three functional classes in the nuclear receptor family (Fig. 1B) Appendix B: Peroxisome Proliferator Activated Receptors Appendix C: Liver X Receptor Appendix D: Farnesol X Receptor Appendix E: Hepatic Nuclear Factor 4 Appendix F: Retinoid X Receptor Appendix G: Sterol Regulatory Element Binding Proteins Appendix H: The Liver-Enriched Transcription Factors 1. Hepatocyte nuclear factors 2. CCAAT/enhancer-binding proteins Appendix I: Insulin Resistance: Definition and Characteristics GRANTS ACKNOWLEDGMENTS REFERENCES
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in orchestrating this association. Finally, we discuss the molecular mechanisms underlying metabolic regulations, which provide new opportunities for treating complex metabolic disorders. |
I. INTRODUCTION |
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It is now commonly accepted that metabolic regulation in complex organisms relies on three main types of control. The first corresponds to the classic allosteric control of the activity of a key enzyme along a metabolic pathway triggered by the binding of an activator, which often is the enzyme substrate itself. The second mechanism involves various posttranslational modifications such as proteolytic cleavage, phosphorylation, glycosylation, sumoylation, and acetylation, which may shift the equilibrium between an inactive and active enzyme within seconds and/or affect protein stability. In these two types of control, subsequent changes in protein-protein interaction may participate in producing the active/nonactive enzymatic complex. The third mechanism is transcriptional regulation, which affects the level of expression of key enzymes and is effective on a longer time scale. It clearly appears that most metabolic regulations benefit from a coordination of these various mechanisms. The purpose of this review is to highlight the recent progress in understanding when and how transcriptional regulation participates in the control of metabolic homeostasis.
Transcriptional control requires specific signals to be transduced to the cell nucleus where defined sets of genes are targeted. Thus understanding the transcriptional control of metabolism relies on three complementary pieces of information: 1) events upstream of transcriptional activity, which define the signals involved and their route to the nucleus; 2) the molecular mechanisms by which transcription factors operate; and 3) events downstream of transcriptional activity, which depend on the groups of genes that are targeted and how further signals are generated to reach the dynamic equilibrium of homeostasis. Virtually all transcription factor families are in one way or another involved in metabolic regulation. However, a few of them have a clear predominant role and seem mainly dedicated to metabolic regulation. For the sake of clarity, the transcription factors most often cited herein are briefly presented. Appendix A presents the nuclear receptor family and is accompanied by Figure 1A, which shows the main characteristics of the transcription factors belonging to this family. The notion of "metabolic sensor" receptors was more particularly developed with respect to these nuclear receptors, as also explained in Appendix A with the accompanying Figure 1B. Appendixes B–F describe the main features of some of these "sensors," which belong to the nuclear receptor family, with the peroxisome proliferator activated receptor (PPAR) in Appendix B, the liver X receptor (LXR) in Appendix C, the farnesol X receptor (FXR) in Appendix D, the hepatocyte nuclear factor 4 (HNF4) in Appendix E, and the retinoid X receptor (RXR) in Appendix F. Appendix G details the amazing characteristics of the sterol response element binding proteins (SREBPs), which play a major role in lipid and cholesterol metabolism. Finally, the heterogeneous family of proteins initially grouped under the name of liver-enriched transcription factors and which comprises the CAAT enhancer binding proteins (C/EBP) is discussed in Appendix H.
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The aim of this review is to summarize recent knowledge concerning the transcriptional control of metabolic homeostasis. Analyses of the main transcriptional controls occurring in the regulation of intermediary metabolism is followed by an integrative approach which illustrates how these regulations can take place during the alternation between fasting and feeding to achieve energy homeostasis. In a pathological context, the disruption of energy homeostasis reflected by the metabolic syndrome highlights the intricate link between glucose and lipid metabolism. The last section discusses transcription factors as targets for treating complex metabolic disorders.
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II. TRANSCRIPTIONAL CONTROL BY GLUCOSE |
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Glycemia is a parameter over which the organism establishes tight control. In humans, blood glucose levels are kept constant in a narrow range from 4 to 7 mM, despite discontinued supply due to the alternation between feeding and fasting. One main danger of prolonged hypoglycemia is acute brain damage. At the other end of the scale, acute hyperglycemia is a serious complication of decompensated diabetes mellitus. The associated ketoacidosis and hyperosmolar hyperglycemic state might be fatal due to dehydration and electrolyte imbalance. Chronic hyperglycemia is a major cause of neuropathy and vasculopathy, as seen in diabetes.
Glucose homeostasis is maintained by a hormonal network in which insulin and glucagon are the main agents. Synthesis and secretion of insulin are stimulated by increased glucose levels, particularly after feeding. Insulin release allows the quick removal of glucose from circulation by stimulating the entry of glucose into peripheral tissues, mainly in muscle and adipose tissue cells. In parallel, insulin increases energy storage by inducing glycogen synthesis in liver and muscle, and fatty acid synthesis in liver and adipose tissue. When insulin levels are low, between meals or upon fasting, the hormone glucagon increases the hepatic production and release of glucose by increasing glycogenolysis and stimulating gluconeogenesis. The pancreas is the chief organ of these dual regulations, as it senses glucose levels and produces insulin and glucagon accordingly. The liver functions as the main "buffer," providing glucose when nutrients are scarce and storing glucose as glycogen when food is abundant. Once the liver glycogen store is full, the adipose tissue converts glucose into triacylglycerol for longer term storage as fat. Muscles mainly consume rather than store energy, although they efficiently accumulate glycogen for their own use. The brain is a particular target organ that can use glucose and/or ketone bodies as an energy source. However, the fact that glucose represents the sole source of energy for some of its cells imposes a tight control over glycemia. In this organ, the entry of glucose in cells is mediated by the Glut3 transporter, which maintains a constant supply of glucose to brain cells until glycemia drops to very low levels close to its Km value, i.e., when approaching 2.2 mM.
The aim of this section, which cannot be exhaustive, is to discuss the main threads of the transcriptional network which result in glucose homeostasis. As is the case for many pathways regulated by nutrients, glucose is both an end product and the nutrient substrate that triggers regulation. Therefore, two opposing situations are considered for simplicity: that of high and that of low glucose levels. For each situation, we will describe the signals that are triggered and their action in transcription.
B. Transcriptional Regulation of Metabolism by High Glucose Levels
High glucose levels influence gene expression either directly or through the stimulation of insulin production by the 
-cells of the pancreas. We first analyze the transcriptional regulation of insulin1 and the regulation of insulin secretion. We then review the mechanisms by which glucose and insulin, independently or together, modulate gene transcription.
1. Transcriptional control of insulin expression and secretion
Pro-insulin is synthesized in the -cells of the pancreatic Langerhans islets and is then cleaved by proconvertases in insulin and peptide C. Insulin is stored in secretory vesicles, and its secretion is directly linked to a mechanism sensing glucose availability via an increase in the intracellular ATP/ADP ratio that correlates with the entry and metabolism of glucose in the -cells (Fig. 2, Ref. 230). The entry of glucose into the -cells requires a glucose transporter, Glut2 in rodents but Glut1 rather than Glut2 in humans (56), whose expression and membrane localization are independent of glucose or insulin signaling. The posttranscriptional control of insulin expression and processing, as well as the control over the secretory mechanism, which is dependent on glucose sensing, are key features of the regulation of insulin signaling. However, the pathologies exhibited by patients in whom the regulation of insulin gene expression is altered emphasize the importance of the transcriptional level of control.
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-cells in the pancreatic islets. The tissue-specific expression of insulin is tightly regulated at the transcriptional level, and the major regulatory elements are located in the 5'-flanking region of the insulin gene. Among the set of transcription factors involved, PDX1 (pancreatic duodenum homeobox) is a key component (214) (Fig. 2). PDX1 is the main determinant in the cell lineage of the developing endocrine pancreas and in combination with other transcription factors confers tissue-specific expression of insulin (reviewed in Ref. 215). PDX1 is also the glucose-sensitive transcription factor of the insulin gene transcription machinery. Indeed, glucose triggers the phosphorylation of PDX1, via the phosphatidylinositol 3-kinase (PI3K) pathway, which induces the nuclear translocation of PDX1 and increases insulin expression (185, 237). Other transcription factors and/or coactivators activated by glucose likely contribute to the PDX1-mediated glucose response of insulin (189).
In addition to HNF3/FOXA2, which positively regulates PDX1 expression (83), other members of the HNF family, HNF1
, HNF1, and HNF4 are expressed in the pancreatic -cells. The maturity-onset diabetes of youth (MODY) has highlighted the importance of this network of transcription factors, acting directly or via a cascade of transcriptional regulation on insulin gene expression, and possibly on insulin secretion (310, 319) (see also Fig. 2 and Appendix H). MODY is characterized by the appearance in children or young adults of a non-insulin-dependent form of diabetes mellitus, inherited as an autosomal dominant trait. Except for MODY2, which is caused by a mutation in the enzyme glucokinase, MODY is due to mutations in genes encoding transcription factors involved in insulin gene expression (see Fig. 2). Alteration of HNF1, which causes MODY3, is the most frequent transcription factor defect leading to MODY, whereas MODY1 is rare and due to mutations in HNF4. MODY4 is characterized by a primary defect in insulin synthesis and secretion due to mutations in PDX1. Finally, two Japanese families with mutations in HNF1 responsible for MODY5 have been reported (reviewed in Ref. 319). The fact that mutations in any of these genes result in altered insulin secretion reveals that each of these transcription factors is crucial for the control of cell specificity and metabolic adjustment of insulin expression.
2. Insulin-regulated transcription of genes involved in glucose metabolism
In the insulin-targeted cells, transduction of the insulin signal from the cell surface to key regulatory factors in the cell nucleus occurs in a very short time frame, allowing the immediate adaptive response of the cells (Fig. 3). In short, circulating insulin interacts with its membrane insulin tyrosine kinase receptor, expressed in most cells in vertebrates. This interaction drives the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. The cascade of phosphorylation events starts with the phosphorylation of insulin receptor substrate 1 and/or 2 (IRS1, IRS2). The successive activation of son-of-sevenless (SOS), Ras, and Raf-1 subsequently activates MEK (mitogen-activated, ERK-activating kinase), which in turn phosphorylates MAPK. Activation of this pathway seems to mostly target cellular growth and proliferation, rather than direct metabolic actions, and will not be discussed further.
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and FOXOs, which directly or indirectly mediate the effects of insulin on the transcription of genes involved in glucose metabolism. There are three highly homologous Akt/PKB members, Akt1/PKB, Akt2/PKB, and Akt3/PKB. Specific gene deletion or gene silencing of Akt1 and Akt2 demonstrates the primary role of Akt2 in insulin signaling and glucose metabolism, while significant redundancy of Akt1 and Akt2 still exists (80, 129). In addition to activating Akt, insulin triggers the tyrosine phosphorylation of the protooncogene Cbl in a PI3K-independent manner. Phosphorylated Cbl stimulates the translocation of the glucose transporter 4 (Glut4) to the cell surface membrane, independently of transcriptional regulation (reviewed in Refs. 176, 265). Together, the biological effects of the transduction cascades are Glut4 translocation to increase glucose uptake in adipose tissue and muscle, the activation of glycogen synthase and thus of glycogen synthesis, and the activation of the S6-kinase that will increase protein synthesis. In addition to or as a result of these signaling cascades in target cells, the expression of more than 150 genes expressed in various tissues is transcriptionally regulated by insulin. The diversity of mechanisms of the insulin-mediated transcriptional regulation is extremely wide, as indicated by the variety of promoter sequences that are responsible for insulin-mediated action. A classification of the insulin response sequences or insulin response elements (IRS/IRE) into seven groups has been proposed (212). Whereas there is still much to learn about the factors that are modified by insulin and bind to these elements, recent results have identified sterol response element binding protein 1c (SREBP-1c) as a major contributor. SREBPs are transcription factors of the helix-loop-helix family highly expressed in the liver. Their three forms, SREBP-1a, SREBP-1c, and SREBP-2, were first explored for their role in cholesterol and lipid homeostasis (see Appendix G). Interestingly, SREBP-1c expression in the liver is upregulated by insulin independently of glucose levels (73, 278). This regulation occurs at the transcriptional level, and detailed functional analyses of the SREBP-1c promoter revealed a complex interplay of transcription factors (24, 33). Whereas SREBP itself and nuclear factor Y act to maintain the basal level of SREBP-1c expression, the LXR (see Appendix C), a nuclear receptor activated by oxidized derivatives of cholesterol, plays a crucial role in its insulin-mediated increased expression. The proposed mechanism by which insulin-increased LXR activity would involve the insulin-dependent production of a ligand for LXR (33) is yet to be demonstrated. In addition to this transcriptional upregulation of SREBP-1c expression, insulin triggers the proteolytic cleavage of SREBP-1c in a PI3K-dependent manner to produce the mature active form of this transcription factor (104).
In turn, SREBP-1c acts as an important mediator of insulin action, at least with respect to the transcriptional regulation of glycolytic and lipogenic genes in the liver. Overexpression of a dominant negative form of SREBP-1c counteracts insulin-mediated induction of the expression of liver pyruvate kinase (L-PK), spot 14 (S14), and fatty acid synthase (FAS), three canonical genes with respect to glucose*insulin2 responsiveness (see below and Ref. 73). The expression of glucokinase (GK) in the liver is also regulated by insulin, independently of extracellular glucose levels. GK phosphorylates glucose in glucose-6-phosphate (G6P), a first reaction required for any further intracellular metabolism of glucose. This regulation requires an intact PI3K pathway (124) and might also occur via SREBP-1c (73, 145). Less is known of insulin-mediated activation of SREBP-1c in the adipose tissue. The overlap of the gene expression profile of 3T3-L1 adipocytes subjected to insulin treatment with that of cells overexpressing the mature form or a dominant negative form of SREBP-1c strengthened the notion of a correlation between insulin-induced gene expression and SREBP-1c activity. It also revealed that, in these cells, the transcription factor CAAT/enhancer binding protein (C/EBP) is responsive to insulin via stimulation of SREBP-1c (169a).
Insulin also negatively regulates transcription, particularly that of genes involved in hepatic glucose production, such as those encoding IRS2, phosphoenolpyruvate carboxykinase (PEPCK), insulin growth factor binding protein-1 (IGFBP-1), and glucose-6-phosphatase (G6Pase) (for review, see Ref. 213). A particular sequence element, often contained in a broader insulin response unit, was identified in the promoter region of these genes as a mediator of negative regulation by insulin. Several transcription factors, such as members of the C/EBP, HNF-3/FOXA, and FOXO families, can bind to this element. Of particular interest are the FOXOs, represented by three members, FOXO1, FOXO3a, and FOXO4 (previously called FKHR, FKHRL, and AFX, respectively), which are phosphorylated by Akt-1 upon insulin-mediated activation of the PI3K pathway (reviewed in Ref. 299). Phosphorylated FOXO has a high affinity for protein 14-3-3, which relocates FOXO from the nucleus to the cytosol. In addition, insulin enhances ubiquitination of phosphorylated FOXO and its further degradation (193). Thus, in a simple mechanistic model, insulin would mediate repression via removal from the nucleus and accelerated degradation of the positive transcriptional regulator FOXO. Whereas the correlation between the activity levels of FOXO, HNF3, and C/EBP in mammals and gene repression by insulin remains unclear, their complex intertwined functions are illustrated in insulin-mediated PDX1 regulation. In the pancreatic -cells, nuclear FOXO1 acts as a repressor of the positive activity of HNF3 on the PDX1 promoter, while insulin signaling relieves this repression by excluding FOXO1 from the nucleus (149).
It is interesting to note that the insulin signaling pathway going through PI3K and Akt activation is also present in Drosophila and in Caenorhabditis elegans. Indeed, the identification of FOXO (as DAF-16) and its involvement in insulin signaling was first described in C. elegans, triggering its characterization in mammals. A unique homolog of FOXO, dFOXO, has now been reported in Drosophila (134, 235) where insulin plays a crucial role in cellular growth. As seen in mammals and C. elegans, Drosophila Akt (dAkt) sequesters dFOXO in the cytoplasm when the insulin pathway is active. This results in an inhibition of dFOXO target gene expression, including that of the insulin receptor gene itself.
In summary, the general pathways followed by insulin to trigger many changes in gene expression are beginning to be understood. However, a lot more needs to be done to decipher the molecular mechanisms of this transcriptional regulation. The initial signal is at the cell membrane, and all subsequent events occur via phosphorylation cascades that mainly go through the PI3K pathway, but also possibly via the phosphorylation of the Cbl protooncogene. Thus it is possible that most of the insulin action on gene expression results from posttranslational modifications of various transcription factors, a process that would account for the pleiotropic effects of this hormone.
3. Glucose*insulin regulated transcription of genes involved in glucose metabolism
As mentioned above, glucose, independently of insulin, can regulate the expression of genes involved in carbohydrate metabolism. Upon entry into the cells, glucose is phosphorylated to G6P by GK in hepatocytes and by hexokinase in all other cells. This step is required for glucose to either undergo glycolysis, be used in the glycogen synthesis pathway, or enter the pentose phosphate pathway. This first metabolic transformation of glucose is also required for generating the signal that acts in transcriptional regulation. Some reports suggest that G6P itself might be the signaling molecule. Alternatively, other metabolites such as xylitol produced by the pentose phosphate pathway or intermediates of the hexosamine biosynthetic pathway might also act in tissue-specific regulations (reviewed in Ref. 305).
The analysis of glucose signaling is, however, often difficult to dissociate from insulin signaling (see Fig. 3). First, the entry of glucose into muscle and adipose tissue cells, which are two main insulin target organs, operates through the translocation of the Glut4 transporter via an insulin-mediated transduction signal. In contrast, the expression of the glucose transporter Glut2 in the liver and pancreas, Glut3 in the brain, and the widely distributed Glut1 are insulin independent, and their translocation is constitutive. Second, in the liver and to a lesser extent in the pancreas, the initial metabolic modification of glucose into G6P by GK is required for transcriptional regulation by glucose and is strongly dependent on insulin. Thus the actions of glucose and insulin are often interdependent and, in this review, we refer to this ambiguity by the use of the associated words glucose*insulin when relevant.
Three genes have been important tools for the analysis of the ability of glucose to direct transcriptional regulation; they encode the L-PK (acting on the glycolytic pathway from glucose to pyruvate), S14 (associated to lipogenesis but with an unclear function), and FAS (a key enzyme in lipogenesis). Analyses of the promoter region of these genes have identified response elements called carbohydrate response elements (ChoRE) or glucose response elements (GlRE), which have similarities. The main common feature is the presence of at least one E-box. The GlRE/ChoRE of L-PK and S14 comprises two E-boxes in tandem, in addition to a binding site for an ancillary factor which is HNF4 in the case of L-PK (57, 175, 276). The glucose*insulin responsiveness of FAS also requires a complex array of promoter elements, a complexity that has generated some controversy. Three glucose*insulin response sites are now proposed. A region between –150/+50 centered around an E-box was the first proposed IRE/GlRE/ChoRE. This sequence efficiently binds SREBP-1c and mainly represents an insulin responsive element. A second element is located at –332, but its role in vivo is unclear. Finally, a third far-upstream element located around –7 kb closely resembles the GlRE/ChoRE found in L-PK and S14 (152, 201, 261).
E-boxes are binding motifs for helix-loop-helix transcription factors and can bind the abundant and ubiquitous upstream stimulatory factor (USF), whose two forms USF1 and USF2 can heterodimerize. However, various studies in vivo (with knock-out animals) and in vitro (electromobility shift assays) aimed at elucidating the link between the ability of USFs to bind to the E-box and glucose responsiveness have failed to prove the concept accurate. An alternate hypothesis involves the negative transcription factor COUP-TFII, which is also able to bind to the L-PK GlRE/ChoRE. The equilibrium resulting from the competition between USFI:USF2 and COUP-TFII would then create the dynamic modulation and glucose-dependent regulation (305). A new factor, initially cloned as WBSCR14 (Williams-Beuren syndrome deleted DNA region, Ref. 51), exhibits a GlRE/ChoRE binding activity that could account for glucose responsiveness. Based on its interaction with the L-PK ChoRE, this helix-loop-helix factor has been renamed the ChoRE binding protein (ChREBP) (329). ChREBP is mainly expressed in liver, kidney, and adipose tissue. A mouse line null mutant for ChREBP provided evidence for a direct and dominant role of ChREBP in the glucose-mediated upregulation of LPK, ACC, and FAS gene transcription, coordinating synthesis of fatty acids and triglycerides in vivo in response to high levels of glucose (118, 123). Under basal conditions, ChREBP is phosphorylated by protein kinase A (PKA) and remains cytosolic. The glucose-dependent activation of ChREBP is a two-step process, with a first dephosphorylation at serine-196 which triggers its nuclear translocation, and a second dephosphorylation in the nucleus at serine-568 and threonine-666, which allows it to bind to DNA. This activation requires GK expression, as demonstrated in GK knock-out mice (53), and the glucose metabolite xylulose 5-phosphate from the pentose phosphate pathway is the proposed functional link between high glucose and ChREBP activation (136). Indeed, xylulose 5-phosphate can activate protein phosphatase 2, triggering ChREBP dephosphorylation in the cytosol as well as in the nucleus (reviewed in Ref. 52). Finally, the transcriptional activity of ChREBP requires its heterodimerization with the bHLH/LZ factor Max-like protein X (Mlx) (183), which would allow the complex to specifically target E-box binding sites in glucose-responsive gene promoters.
Thus we have gained extremely interesting new understanding of glucose*insulin regulation of gene expression in the last few years. Present works are now aimed in part at understanding when and how these factors respond to an altered metabolic environment, such as in obesity, insulin resistance, or type 2 diabetes.
C. Transcriptional Regulation of Metabolism by Low Glucose Levels
The prevalence of diabetes, i.e., a deregulation characterized by high glucose levels due to impaired insulin signaling, demonstrates the preeminent role of insulin over the action of all counteracting hormones. This fact possibly explains why less is known about the hormonal regulation of genes in situations of low glucose availability. In periods of starvation, even between regularly spaced meals, the liver and to a lesser extent the kidney are responsible for the glucose production required for a sufficient supply to the brain. The small intestine also provides glucose upon prolonged starvation. Hormonal controls of this adaptation associate increased glucocorticoids, decreased insulin levels, and, importantly, glucagon secretion by the pancreas in response to low glucose.
Glucagon is processed from proglucagon in the -cells of the pancreatic islets and is secreted in response to low blood glucose levels. In the liver, glucagon interacts with a membrane receptor coupled to GTP-binding proteins, inducing a rise in intracellular cAMP (Fig. 4), which in turn activates PKA. By this mechanism, glucagon counteracts some of the glucose*insulin-mediated responses. For example, increasing cAMP levels in primary hepatocytes decrease SREBP-1c expression via a mechanism requiring de novo protein synthesis (75, 279). Also, the PKA-dependent phosphorylation of ChREBP sequesters it in the cytosol and inhibits its lipogenic activity (140). Modulation of cAMP levels is the major mechanism by which the liver adjusts glycogenolysis and gluconeogenesis, which produce and release hepatic glucose in the blood. Several transcription factors such as the cAMP response element binding protein (CREB), the cAMP response element modulator (CREM), and the activation transcription factor-1 (ATF-1) are positively activated upon phosphorylation. All three belong to the bZIP family of transcription factors and share a conserved phosphorylation box and glutamine-rich transactivation domain (reviewed in Ref. 48). Their PKA-dependent phosphorylation allows the recruitment of the CREB-binding protein (CBP) coactivator, which contributes to the transcriptional activity of the DNA-bound complexes. CREB is a ubiquitously expressed transcription factor that induces the expression of key genes involved in the gluconeogenesis pathway, such as those encoding PEPCK, G6Pase, and pyruvate carboxylase. Accordingly, CREB binding sites have been identified in the PEPCK and G6Pase promoters (81, 112, 223), but not in that of pyruvate carboxylase. Additional mechanisms for cAMP-mediated transcriptional response are required to explain the full range of responsive genes and the specificity of the response in gluconeogenic tissues, i.e., liver, kidney, and small intestine. For example, the gene for the cofactor PGC1 is strongly activated by CREB in the liver (106). As a cofactor, PGC1 was shown to increase the transcriptional activity mediated by both HNF4 and the glucocorticoid receptor bound to the PEPCK promoter (334) (Fig. 4). The occurrence of such an indirect mechanism via PGC1 for the positive glucagon-dependent induction of pyruvate carboxylase is yet to be examined. HNF4 together with C/EBP and C/EBP also constitutively binds to the G6Pase promoter. In this context, glucagon further induces gene transcription via CREB binding to its cognate site and further recruitment of CBP (81). CBP might be of prime importance for cessation of gluconeogenesis upon feeding, as it is also a target of insulin-dependent phosphorylation at Ser-436 (339). This phosphorylation impairs CBP recruitment to CREB, thereby inhibiting CREB target genes, as demonstrated for PGC1 (341).
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(see Appendix H and Fig. 4) in the transcriptional control of gluconeogenesis has been revealed by the phenotype of C/EBP null mice (312). The major metabolic disturbance seen in these mice is a lethal neonatal hypoglycemia. This hypoglycemia is due to the combination of two deficiencies; first, reduced glycogen synthase gene expression is responsible for the absence of a glycogen store; second, the very low levels of liver gluconeogenic enzymes, such as G6Pase, PEPCK, and tyrosine aminotransferase, cause the lack of gluconeogenesis (reviewed in Ref. 253). A tissue-specific deletion of C/EBP in the adult liver confirms that these three genes are under the control of C/EBP in adulthood (165). C/EBP null mice have a high susceptibility to hypoglycemia, but survive. In these mice, there is a glycogen store, but glycogenolysis is impaired. This phenotype correlates with decreased levels of cAMP, which could explain an impaired glucagon responsiveness (44).
Finally, the AMP-activated protein kinase (AMPK) seems to play a major role in metabolic homeostasis. Its activation, upon stress or starvation, is caused by a drop in ATP levels with an increased AMP/ATP ratio. In the liver, activation of AMPK leads to an inhibition of lipogenic pathways and affects the glucose*insulin-dependent activation of FAS, S14, and L-PK. Conversely, the knock-out of the 2-subunit of AMPK triggers a metabolic disturbance associated with high glucose and low insulin levels. This perturbation does not seem to be cell-autonomous, as assessed both in pancreatic islet and muscle cells in vitro, but rather caused by a perturbed autonomous nervous system (307). However, one form of AMPK is expressed in the cell nucleus (reviewed in Ref. 318), and AMPK could therefore act directly on transcriptional regulation by inhibiting the DNA binding activity of ChREBP via phosphorylation (139). This is supported by the fact that a specific short-term overexpression of AMPK in the liver decreased the refeeding-induced transcriptional activation of ChREBP, in parallel with a decreased expression of SREBP-1c (72).
In conclusion, this short presentation highlights some of the best-characterized features of the regulation of glucose homeostasis via the transcription of key genes. However, this summary cannot take into account the specificity of these regulations within each tissue, which is essential for the homeostasis at the level of the whole organism. An effort to integrate some of the pathways described above into the global balance of metabolic regulations will therefore be presented in section VI.
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III. TRANSCRIPTIONAL CONTROL OF AMINO ACID AND PROTEIN METABOLISM |
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In addition to being substrates for the synthesis of specialized products such as neurotransmitters, hemes, nucleotides, and polyamines, amino acids (AA) also play an important role in energy supply. Among the 20 AA involved in protein synthesis, around half can be synthesized de novo and 9 essential AA must come from the diet. However, during growth or other situations of high-energy expenditure, some of the nonessential AA such as arginine, whose synthesis is energy demanding, should rather be provided by food (reviewed in Ref. 241).
In western countries, under normal nutritional and physiological conditions, proteins/AA are often ingested in excess and are neither stored nor excreted but catabolized and used as an important source of energy that fuels the production of glucose and fatty acids. With a western diet, degradation of AA provides 10–15% of the total energy requirement of the organism. During starvation, the use of AA degradation for energy supply is increased. In the liver, carbon skeletons of the gluconeogenic AA (e.g., alanine and serine in the liver and glutamine in the kidney and the gut) are catabolized into pyruvate or into one of the metabolites of the citric cycle, which can then be converted into glucose. In contrast, ketogenic AA (e.g., leucine, isoleucine, phenylalanine) degraded into acetyl-CoA or acetoacetate are precursors of the ketone bodies that can be used as an alternate source of energy, in particular by brain cells. The AA carbon skeleton used as the energy source results from an AA deamination process, which leads to the cytosolic accumulation of toxic free ammonia (NH4+). For further processing, NH4+ is transported as glutamate and/or glutamine to the liver and kidneys (see Fig. 5), where ammonia is freed and processed in the urea cycle. Urea is a diffusible molecule, which is then excreted in the urine.
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B. Transcriptional Regulation of Glutamine Production and Homeostasis
As described above, glutamine has a particular status, and it is by far the most abundant AA in the human organism. In addition to being an important energetic and metabolic substrate, it provides sufficient amino groups for AA and nucleotide synthesis. It is also the main transporter of ammonia in the blood towards the liver, and glutamine generation in the brain is crucial for avoiding highly neurotoxic hyperammonemia. Thus it has a dual importance: during growth for anabolism and under catabolic conditions for limiting ammonia levels in peripheral tissues and blood. Glutamine is required for normal growth and proliferation of cells, particularly of enterocytes. Additionally, glutamine requirements are particularly high at times of severe sepsis, when proliferation of the immune cells is necessary (reviewed in Ref. 208). Conversely, depletion of glutamine due to a high cellular metabolic rate, often associated with high catabolism of AA, also occurs in cancer patients.
Glutamine formation via the ATP-dependent glutamine synthase (GS) occurs in most tissues (Fig. 5), with, in adults, the greatest activity in skeletal muscle, lung, brain, and adipose tissue. Whereas the GS turnover is increased by a high concentration of the end-product glutamine, regulation of GS activity also occurs at the transcriptional level, as best characterized by the responsiveness of GS to glucocorticoids in the lungs and muscles. Two broad regions mediating this response have been identified in the upstream promoter and in the first intron of GS. Detailed studies of GS expression in chicken brains showed that glucocorticoids act by relieving the repression mediated by a silencer element located upstream of the glucocorticoid receptor binding site (5). This glucocorticoid-mediated induction of GS occurs particularly in conditions of trauma or high catabolic rate and results in increased glutamine synthesis at the expense of the AA that purvey the amino groups. It is thus believed that the action of glucocorticoids on glutamine metabolism is responsible for some of the deleterious effects of corticotherapy on muscle physiology, such as muscle atrophy. Transcriptional regulation of GS has also been studied in adipocyte differentiation, where the high expression of GS is controlled through a C/EBP responsive element in the distal 5'-flanking promoter region (96). However, little is known about the physiological significance of GS activity in the adipose tissue.
Conversely, glutamine homeostasis for the whole organism is also largely controlled in the liver, and to a lower extent in the kidneys, by the glutaminase activity which participates in the reverse pathway, allowing the disposal of NH4+ (see Fig. 5) and providing gluconeogenic substrate. The hepatic-type glutaminase expression is increased during starvation, diabetes, and protein-rich diets, when AA degradation is increased. At the molecular level, the transcription of hepatic-type glutaminase during fasting (e.g., in conditions of low insulin/high glucagon) is highly activated by cAMP, as well as by glucocorticoids via a promoter element that has been identified (42). In contrast, the kidney-type glutaminase is mainly responsive to metabolic acidosis, triggered by prolonged starvation or uncontrolled diabetes. The catabolism of glutamine in metabolic acidosis has a dual role, both facets contributing to the restoration of metabolic homeostasis. First, the generation of NH4+ from the conversion of glutamine to glutamate and -ketoglutarate facilitates the excretion of acids; second, further catabolism of -ketoglutarate is linked to increased gluconeogenesis, most notably via the increased activity of PEPCK. Whereas the increased expression of glutaminase is mainly the result of mRNA stabilization via an element located in the 3'-UTR region of its mRNA (160, reviewed in Ref. 156), the expression and response to acidosis of PEPCK in the kidney depends on an HNF1 binding site present in its promoter (28).
Thus, whereas glutamine homeostasis seems to be one physiologically important knot of AA metabolism, most of the molecular mechanisms governing the regulation of these enzymatic activities remain to be analyzed. As glutamine synthesis is required, particularly in conditions of cell proliferation, decreasing glutamine intake has been proposed to control cell growth in cancer. It now appears that depletion of glutamine in cancer patients contributes to a global degradation of their health status. Consequently, a supplementation in glutamine is now on trial in these patients, as well as during severe sepsis for reinforcing the immune system (195).
C. Transcriptional Regulation of Urea and Ammonia Homeostasis
Urea production in the liver, via the activity of the five enzymes of the urea cycle, carbamoyl phosphate synthetase 1 (CPS-1), ornithine transcarbamoylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase, is the main pathway for ammonia detoxification (Fig. 6). There are two main sources of ammonia production: the diet with its protein content, and endogenous protein degradation which occurs when there is a relatively low energy supply. The regulations occurring on a short time scale are mainly allosteric reactions, such as CPS-1 activation in the presence of N-acetylglutamate. However, there is a coordinated regulation of the expression of these five enzymes in response to dietary changes or to metabolic challenges during development and in adulthood. Hints regarding the molecular mechanism of this transcriptional regulation and the nature of the coordinating factor are starting to emerge.
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results in hyperammonemia and hypouremia due to a dramatic reduction of the expression of OTC, consistent with the presence of two functional HNF4 response elements in the promoter of this gene. In contrast, the expression of the four other genes of the urea cycle remains unchanged or is only slightly increased (122). Thus the loss of the HNF4 function in ammonia detoxification might be the cause of the premature death of the mice carrying a liver-specific disruption of HNF4. PPAR, on the other hand, is a negative regulator of the urea cycle, consistent with the PPAR-mediated downregulation of CPS-1, OTC, ASS, and ASL in mice treated with a fibrate (141). While the mechanism by which PPAR exerts this coordinated downregulation of urea enzyme gene expression is not yet known, it has been proposed that HNF4 positively regulates PPAR expression via a binding site for HNF4 in the promoter of this gene (231). Accordingly, the relatively low levels of PPAR in HNF4 null mice could thus explain the normal or higher levels of CPS-1, ASS, and ASL enzymes in these mice.
Severe hyperammonemia has been observed in mice carrying a liver-specific deletion of C/EBP. This phenotype correlates with a dramatic fall in CPS-1 and arginase expression, together with a disturbed intrahepatic lobular distribution of OTC expression (147). Glucocorticoids, which have a strong impact on protein degradation in muscle, are efficient inducers of the urea cycle in the liver, allowing excretion in the form of urea of the excess ammonia produced by protein degradation. Intriguingly, the response of arginase to glucocorticoids via binding of the glucocorticoid receptor to its specific response element requires C/EBP expression and the integrity of the C/EBP binding sites in the 5'-flanking region of the gene (89). The same C/EBP dependency of the glucocorticoid response is also true of CPS-1 (146). The CPS-1 promoter contains a complex regulatory module composed of multiple sites for glucocorticoid receptor, HNF3 and C/EBP family members, as well as for unknown factors that control its specific pattern of expression and regulation. The glucocorticoid response unit itself combines a glucocorticoid response element with sites for HNF3 and C/EBP (41). Whereas these in vitro studies performed in primary hepatocytes in culture attributed a preferential role to C/EBP, C/EBP null mice did not present any perturbation of ureagenesis (40, 147), and it seems reasonable to propose that in vivo C/EBP is the functional partner of the glucocorticoid receptor. With respect to HNF3, an embryonic lethality of HNF3 null mutation preempts the analysis of the role of this factor in vivo, whereas HNF3 and HNF3 null mice do not exhibit any perturbation of ammonia metabolism. Thus the phenotype of liver-specific KO of C/EBP together with the subordination of the glucocorticoid response to C/EBP support the hypothesis that C/EBP is the main coordinator of the expression of urea cycle genes.
The above discussion on the search for a coordinator of the ureagenesis pathway underscores the complex interplay established by transcription factors and pinpoints a general mode of homeostasis regulation, in which equilibrium is obtained via the simultaneous control of opposite pathways. One example here is the glucocorticoids that activate the urea cycle via C/EBPs, but at the same time increase the levels of PPAR, which is an inhibitor of the same cycle. Another example is the regulation of PPAR expression by HNF4, which contributes to keeping a balance between activation and inhibition of urea cycle activity. A third example is that of GS and glutaminase, which have opposite activities but are both upregulated in the liver by glucocorticoids (see sect. IIIB). While somewhat counterintuitive, such a mode of regulation should lead to a fine-tuning that limits the oscillations of feed-back and feed-forward regulations.
D. Amino Acid Deprivation and Induction of Gene Expression
1. The search for an amino acid response element
Two genes have been extensively explored for their ability to respond to amino acid deprivation. C/EBP homologous protein (CHOP) is induced by various stresses. One of these is the unfolded protein response pathway triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates chaperones resident in the ER. CHOP is related to the C/EBP family of nuclear factors with which it forms heterodimers. Interestingly, global AA deprivation or starvation in individual AA induces CHOP expression independently of the ER stress pathway. A short promoter sequence or AA response element (AARE) conveys the AA sensitivity of CHOP. This sequence combines the features of a C/EBP consensus element and a cAMP response element (22). The second gene that is used as a model to explore the transcriptional response to amino acid deprivation is the gene encoding asparagine synthetase (AS). AS catalyzes the glutamine- and ATP-dependent conversion of aspartate to asparagine. AS mRNA accumulates in mammalian cell cultures in response to asparagine starvation. Amazingly, deprivation of a wide range of individual AA also induces this accumulation, therefore suggesting that AS responds to a signal reflecting AA deprivation more broadly. Two discrete response elements, called nutrient sensing response elements NSRE-1 and NSRE-2, were found in a short promoter region of human AS. Both of them are required for AS activation and form a nutrient-sensing response unit that mediates not only the response to AA deprivation, but also the response to glucose deprivation (284 and references therein).
The identity of the factors that bind to these elements and are thus responsible for the response to nutrient deprivation remains disputed. They belong to the activating transcription factor (ATF)/CREB family, which includes members sharing a basic leucine zipper motif and a consensus ATF/CRE DNA binding site "TGACGTCA" (reviewed in Ref. 97). The CRE-binding protein 1 (CRE-BP1 or ATF2) binds to the C/EBP-ATF composite site forming the AARE of CHOP, either as a homodimer or as a heterodimer with an unknown dimerization partner (22). ATF4, but not ATF2, binds as a complex with C/EBP to NSRE-1 of CHOP and is required for the response of AS to nutrient deprivation (283). ATF4 itself is transcriptionally and posttranscriptionally regulated by both AA and glucose deprivation (100, 283), which suggests that ATF4 is an important transcriptional mediator of the nutrient-sensing response. However, the response of AS to glucose deprivation is mediated via the unfolded protein response pathway and may be independent of the response to AA deprivation.
Taken together, the differences between and similarities in the regulation of these two genes underline the existence of at least two related but independent pathways, via ATF2 and ATF4, respectively, which control gene expression in response to AA deprivation (21).
2. TOR as a master switch for catabolism versus anabolism
Studies carried out to decipher the yeast response to AA deprivation have identified the target of rapamycin (TOR) proteins as master switches for protein/AA catabolism versus anabolism (125). TOR belongs to the PI3K-related kinase family and appears to function as a nutrient-sensing check-point by controlling many aspects of mRNA translation. Inhibition of TOR proteins by rapamycin in yeast mimics nutrient deprivation. In fact, TOR modulates the transcription of genes involved in AA biosynthesis and the activity of permeases that allow AA transport into the cells. It also inhibits autophagy in yeast and in mammalian cells, a process that degrades cytoplasmic proteins and organelles for scavenging AA when nutrient levels are low. When a sufficient amount of nutrients is sensed, TOR proteins act as a permissive signal for growth and protein synthesis. A single mammalian homologous TOR protein, alternatively called mTOR, FRAP, or RAFT1, has been cloned in various species with a remarkable level of AA identity, suggesting well-conserved functions of the TOR-dependent regulatory pathways. In mammalian cells, as well as in Drosophila, TOR is not only sensitive to the presence of sufficient levels of AA, but also integrates energy and growth signals through the AMPK and PI3K pathways, respectively. Increased Akt activity, via insulin signaling for example, would trigger the phosphorylation of the tuberous sclerosis complex (TSC) relieving the constitutive inhibition that TSC exerts on mTOR activity (reviewed in Ref. 125). In addition to this regulatory interaction, recent evidence has demonstrated that rapamycin-sensitive mTOR kinase activity requires the direct interaction of the small GTPase Rheb-GTP with the TOR-containing complex TORC1 (177).
The best-known molecular mechanism of TOR action is a posttranscriptional action on the phosphorylation status of the initiation and elongation factors involved in translational control (reviewed in Ref. 240). However, it also regulates the abundance of the components of the translation machinery both at the transcriptional and translational levels. This results, for example, in controlling the translational events that regulate mammalian cell size (71). At the transcriptional level, TOR modulates the expression of numerous enzymes involved in multiple metabolic pathways. In yeast, this transcriptional control is mainly exerted by the cytoplasmic sequestration of transcription factors. TOR controls ribosomal protein (RP) gene transcription by maintaining the corepressor CRF1 in the cytoplasm, thereby allowing the forkhead-like transcription factor FHL1 and its coactivator IFH1 to efficiently activate RP gene transcription. Upon TOR inhibition, phosphorylated CRF1 rapidly translocates to the nucleus inhibiting RP transcription (131, 190).
While TORC1 and forkhead-associated domain-containing forkhead transcription factors are conserved from yeast to humans, little is known about the transcriptional mechanisms involved in multicellular organisms. Gene expression profiling in lymphocyte cell lines demonstrated that rapamycin, which inhibits TOR, upregulates genes involved in AA oxidation, fatty acid oxidation, and nucleotide salvage pathways, while it downregulates genes involved in lipid and protein biosynthesis. Furthermore, it was shown that rapamycin and AA deprivation act on overlapping but not identical sets of genes (227). Glutamine deprivation resulted in a broader overlap with rapamycin in terms of gene expression profiles. This reinforces the notion of a parallel between the high increase in the demand for glutamine when the immune system is challenged and the potent immunosuppressive effect of rapamycin. However, the molecular mechanisms of these transcriptional regulations remain entirely unexplained.
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IV. TRANSCRIPTIONAL CONTROL OF LIPID METABOLISM |
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