The Regulation of FGF21 Gene Expression by Metabolic Factors and Nutrients

Horm Mol Biol Clin Invest 2017; 30(1): 20160016

Review

Anjeza Erickson and Régis Moreau* The regulation of FGF21 gene expression by metabolic factors and nutrients

DOI 10.1515/hmbci-2016-0016 epiphenomenon; and (iii) whether FGF21 may have some Received March 28, 2016; accepted May 8, 2016; previously published adverse effects alongside beneficial outcomes. online June 10, 2016 Keywords: butyric acid; curcumin; lipoic acid; methio- Abstract: Fibroblast growth factor 21 (FGF21) gene expres- nine; retinoic acid. sion is altered by a wide array of physiological, metabolic, and environmental factors. Among dietary factors, high dextrose, low protein, methionine restriction, short-chain Introduction fatty acids (butyric acid and lipoic acid), and all-trans- retinoic acid were repeatedly shown to induce FGF21 The importance of diet and nutrition in the etiology of a expression and circulating levels. These effects are usually number of diseases affecting morbidity and mortality is more pronounced in liver or isolated hepatocytes than in well recognized. However, the exact nature of how diet adipose tissue or isolated fat cells. Although peroxisome impacts health and disease is complex and not fully under- proliferator-activated receptor α (PPARα) is a key media- stood. Nutrients and dietary molecules alter gene expres- tor of hepatic FGF21 expression and function, including sion, modulate protein and metabolite levels in blood and the regulation of gluconeogenesis, ketogenesis, torpor, tissues, modify cellular and metabolic pathways, affect epi- and growth inhibition, there is increasing evidence of genetic phenomena, and modify response to drugs. These PPARα-independent transactivation of the FGF21 gene by nutrient-gene interactions thus influence an individual’s dietary molecules. FGF21 expression is believed to follow response to the environment – including diet – and to the circadian rhythm and be placed under the control of therapy. Nutrients act directly, indirectly, and cooperatively first order clock-controlled transcription factors, retinoic with hormones to influence the rate and extent of transcrip- acid receptor-related orphan receptors (RORs) and nuclear tion. Among nutrients, glucose, fatty acids, sterols, vitamin receptors subfamily 1 group D (REV-ERBs), with FGF21 A, vitamin D, and their metabolites are prominent regula- rhythm being anti-phase to REV-ERBs. Key metabolic hor- tors of gene expression. Chief among hormones, insulin, mones such as glucagon, insulin, and thyroid hormone glucagon, glucocorticoids, and growth and thyroid hor- have presumed or clearly demonstrated roles in regulating mones, are essential regulators of metabolic homeostasis. FGF21 transcription and secretion. The control of the FGF21 Since fibroblast growth factor 21 (FGF21) was discov- gene by glucagon and insulin appears more complex than ered nearly two decades ago [1], its physiological and phar- first anticipated. Some discrepancies are noted and will macological effects have been extensively studied whereas, need continued studies. The complexity in assessing the comparatively, the regulation of FGF21 gene expression significance of FGF21 gene expression resides in the diffi- by diet, nutrients, and dietary bioactive compounds is culty to ascertain (i) when transcription results in local or not well characterized. FGF21 is expressed across several systemic increase of FGF21 protein; (ii) if FGF21 is among tissues, predominantly in liver and adipocyte tissues, the first or second order genes upregulated by physiologi- and, to a lower extent, in the pancreas, skeletal muscle, cal, metabolic, and environmental stimuli, or merely an heart, kidneys and testes [1–3]. Unlike most members of the FGF family, FGF21 lacks the FGF heparin-binding domain, allowing FGF21 to be secreted in the bloodstream *Corresponding author: Régis Moreau, Department of Nutrition and and act peripherally as well as centrally [4]. Circulating Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, levels of FGF21 are increased in response to fasting [5–7], USA, Phone: 402-472-7291, Fax: 402-472-1587, E-mail: [email protected] macronutrient diet composition [6, 8], and physiological Anjeza Erickson: Department of Nutrition and Health Sciences, or environmental stress [9] in individuals with obesity, University of Nebraska-Lincoln, Lincoln, Nebraska, USA type 2 diabetes, and non-alcoholic fatty liver disease [10]. 2 Erickson and Moreau: Nutritional regulation of FGF21 gene expression

The broad range responsiveness of FGF21 expression to catabolism of fatty acids, ketogenesis, and gluconeogenesis. metabolic variations suggests that FGF21 contributes to The FGF21 gene is also upregulated by fasting signals con- metabolic homeostasis in health and disease. Indeed, the veyed by glucagon. The actions of the glucagon receptor on pharmacological administration of recombinant FGF21 glucose and lipid metabolism and on the regulation of body resulted in a plethora of metabolic outcomes including the weight and fat mass are mediated in part by FGF21 [36]. The increase of fat utilization and energy expenditure, the pro- glucagon/FGF21 axis is further discussed below. motion of fat browning in white and brown adipose tissue, Fat cell FGF21 has been shown to be upregulated the improvement of thermogenesis, the stimulation of fatty during adipocyte differentiation [28] and to stimulate acid oxidation, the stimulation of peroxisome proliferator- glucose uptake in adipocytes [11, 13]. ChREBP is upregu- activated receptor α (PPARα) and PPARγ transactivation, lated during adipocyte differentiation and, thus, may glucose tolerance and insulin sensitivity, decreased body be involved in the induction of FGF21 in these cells [37]. weight and lowered cholesterol along with serum and In adipocytes, FGF21 is induced by feeding in a PPARγ- hepatic triglyceride levels [5, 6, 11–21]. This review focuses dependent manner [38]. PPARγ agonists, such as the thia- on the nutritional factors and metabolic hormones that zolidinedione drugs, also induce FGF21 gene expression modulate FGF21 gene expression in rodents and humans, in white adipose tissue and isolated adipocytes [28, 39, and discusses the underlying transcriptional mechanisms. 40]. Supporting the mediating role of FGF21 in the therapeutic effects of thiazolidinedione is the observation that FGF21-null mice are refractory to both the beneficial and adverse effects of this family of drugs [17]. Body of review There is evidence to indicate that the FGF21 gene is autoregulated by circulating FGF21. The administration of Regulation by starvation and overnutrition recombinant FGF21 to streptozotocin-treated mice dose- dependently repressed FGF21 gene expression in the liver Animal studies have shown that FGF21 expression is to a greater extent than streptozotocin alone [41]. In a upregulated by drastically different nutritional states, separate study, FGF21 transcript levels were substantially such as in starvation and overnutrition. In contrast, the lowered in the liver following FGF21 administration [42]. FGF21 serum levels of healthy subjects remain stable over These results suggest the presence of a negative feedback the course of a day despite wide interindividual varia- by FGF21 directed at its own expression. tions (21–5300 pg/mL; mean, 450 pg/mL) [22]. Moreover, serum FGF21 is largely unchanged in humans who fasted for 2 days [22], suggesting that secreted FGF21 remains Regulation by hormones within the tissue of production and acts locally. It is after 7 days of fasting that serum FGF21 notably increases in Glucagon and insulin human subjects [22, 23]. Plasma FGF21 levels are reportedly higher in the obese, in individuals with type 2 diabe- A physiological link was established between hepatic tes, and in subjects with non-alcoholic liver disease, which glucagon signaling, 5′ adenosine monophosphate-acti- correlate with elevated levels of triacylglycerols in the liver vated protein kinase (AMPK), PPARα, and the expres- [24–29]. Overnutrition and refeeding also upregulate FGF21 sion of FGF21 in mice lacking the glucagon receptor [43]. gene expression. This response may be induced by dietary In these mice, glucagon failed to induce liver AMPK carbohydrates as it was shown that carbohydrates induce phosphorylation, PPARα and FGF21 gene expression. A FGF21 gene expression through the transcription factor lipid infusion composed of PPARα natural ligands also carbohydrate response element binding protein (ChREBP). failed to induce liver AMPK phosphorylation, and PPARα In the liver and isolated hepatocytes, FGF21 expression and FGF21 gene expression in glucagon receptor-null is induced by starvation in PPARα-dependent and independ- mice, suggesting that a functional glucagon signaling ent manners [5–7, 30, 31]. As a transcription factor activated is required for the PPARα-mediated upregulation of the by fatty acids, phosphatidylcholine, oleoylethanolamine, FGF21 gene. The use of a glucagon receptor agonist raised and hypolipidemic fibrate drugs, PPARα binds to PPRE in hepatic expression and blood levels of FGF21 in wild-type the 5′-flanking region of the FGF21 gene and induces tran- mice [36], supporting the notion that chronic glucagon scription [5, 7] (Figure 1). Liver FGF21 is also induced by a action is mediated through FGF21 expression. Cyphert high-fat low-carbohydrate diet [6], a condition that, similar et al. [44] found that glucagon stimulates FGF21 secretion to starvation, stimulates the breakdown of stored fat, the via a translational and/or post-translational mechanism Erickson and Moreau: Nutritional regulation of FGF21 gene expression 3

Figure 1: Mapping of the response elements and specific transcription factors documented to interact within the 5′-flanking region of the human, mouse, and rat FGF21 gene. (A) Overall layout of the response elements within the 4600 bp upstream of the transcription start site (TSS). (B) Alignment of human, mouse, and rat FGF21 5′-regulatory sequences shown in red or blue boxes the positions of known transcription factor binding sites. The binding sites were retrieved from the literature and confirmed using MatInspector (Genomatix Software GmbH, Munich, Germany). Segments of the 5′-flanking region that do not harbor any documented transcription binding sites were omitted. The 5′ genomic sequences for human, mouse, and rat FGF21 were retrieved from NCBI [32, 33] and aligned using Clustal Omega [34]. The TSSs were positioned based on the following transcript sequences retrieved from ENSEMBL [35]: ENST00000222157 for hFGF21, ENSMUST00000033099 for mFGF21, and ENSRNOT00000028490 for rFGF21. 4 Erickson and Moreau: Nutritional regulation of FGF21 gene expression mediated by the cyclic adenosine monophosphate and retinoid X receptor (RXR) in hepatocytes was sug- (cAMP)/protein kinase A (PKA) axis as well as a PKA-inde- gested based on the rapid induction of the FGF21 gene by pendent mechanism. The latter mechanism involves the T3 [55]. A distal PPRE site in the 5′ regulatory region of the cAMP activation of exchange protein directly activated by FGF21 gene was required for T3 action. T3 did not induce cAMP (EPAC), a guanine nucleotide exchange factor that FGF21 mRNA in the white adipose tissue of wild-type activates the small guanosine-5′-phosphatase (GTPase) mice, indicating that tissue specificity may result from a Ras-proximate-1 (Rap1) [45, 46]. Downstream of cAMP, the distinct expression profile of the thyroid hormone recep- glucagon/cAMP signaling pathway implicates AMPK and tor subtypes. As abnormal blood lipids can be improved p38 mitogen-activated protein kinase (MAPK). with thyroid hormone receptor β agonists, similarly with The cAMP-stimulated transcription factor cyclic recombinant FGF21, FGF21 may be a physiologically rel- AMP-responsive element-binding protein 3-like protein 3 evant mediator of T3’s lipid-ameliorating properties. (CREBH) is upregulated during fasting [47, 48] and possi- bly by glucagon. Fasting and glucagon increases CREBH acetylation, which is required for the formation of the Regulation by miRNA CREBH-PPARα transcriptional complex [49]. In addition to regulating each other’s transcription in fasted liver [47], Recent studies have found evidence linking microRNAs CREBH and PPARα synergize to activate FGF21 gene expres- (miRNAs) to the regulation of metabolism and metabolic sion through the binding of the CREBH-PPARα complex to disorders [56]. miRNAs are circulating, small, non-coding a highly conserved CRE-peroxisome proliferator-activated RNAs of about 19–23 nucleotides long, which regulate gene response element (PPRE) site in the FGF21 proximal pro- expression post-transcriptionally by binding primarily to moter [50]. Glucocorticoid signaling is involved in the regu- the 3′-untranslated region (3′UTR) of mRNAs and target- lation of CREBH [48]. In the fasting state, the adrenal gland ing mRNAs for translational repression and degradation. secretes glucocorticoids, which contribute to upregulate Blood-circulating miRNAs function as signaling molecules gluconeogenesis and stimulate hepatic glucose output [51]. by altering gene expression not only in cells and tissues Glucagon and insulin synergized in primary rat hepato- where they are produced but also in distant organs. Their cytes to induce FGF21 mRNA and FGF21 secretion, whereas critical role in gene regulation suggests that miRNAs can glucagon alone in the absence of insulin lowered FGF21 be markers of health and disease, and potential therapeu- mRNAs [44]. In contrast, Uebanso et al. [52] found that tic targets. The discovery of miRNAs able to regulate FGF21 glucagon and insulin function in an opposite manner in expression and impact the metabolic effects of FGF21 has rat primary hepatocytes: glucagon induced FGF21 mRNA been of special interest in recent years. whereas insulin inhibited gene expression. Insulin, through FGF21 is a well-known regulator of pancreatic insulin protein kinase B (Akt), reportedly induced FGF21 expression production. Treatment of diabetic mice and rats with in cultured skeletal muscle cells [53]. Of note, glucagon has FGF21 increased insulin secretion and content in pancre- been shown to enhance the action of insulin on Akt in hepato- atic islets, improved glucose clearance, and promoted the cytes [54]. Hence, although glucagon and insulin have oppo- survival of islets and insulin-secreting INS-1E pancreatic site broad effects on metabolism, Cyphert et al. [44] showed β-cells against glucolipotoxicity and cytokine-induced that these hormones stimulate FGF21 secretion either alone apoptosis [57]. A recent study investigated miR-577 as a [53] or in combination [44]. Overall, the regulation of FGF21 novel mechanism of improving pancreatic β-cell survival mRNA levels by glucagon and insulin appears more complex and function in diabetic patients [58], in part because than first anticipated. Either glucagon or insulin has the miR-577 was predicted to target FGF21 [59]. Chen et al. [58] potential to upregulate FGF21 gene expression. observed that the blood of diabetic children contained elevated levels of miR-577. They showed that FGF21 3′UTR was a direct target of miR-577 in mouse embryonic pancreatic Thyroid hormone T3 β-cells as the suppression of miR-577 with anti-miR-577 increased FGF21 mRNA and protein expression in these The intraperitoneal injection of thyroid hormone tri-iodo- β-cells. Additional evidence that miR-577 targets FGF21 was thyronine (T3) in mice has been shown to induce hepatic provided when the activation of the extracellular signal- FGF21 gene expression in a dose-dependent manner [55]. regulated kinase 1/2 (ERK1/2) and Akt signaling pathways The induction was abolished in PPARα-null mice, sug- was blocked by miR-577, and when the glucose-stimulated gesting that PPARα mediates the effects of T3. A direct FGF21-induced production of insulin was repressed by miR- interaction among thyroid hormone receptor β, PPARα, 577. Hence, their work points to anti-miR-577 as a means Erickson and Moreau: Nutritional regulation of FGF21 gene expression 5 to protect and restore the insulinotropic action of FGF21. absence of a conserved CHOP binding site in the 5′ flank- There are other predicted targets of miR-577 that belong to ing region (–1497/+5), therefore suggesting that CHOP the FGF family, including FGF5 and FGF23 [59]. stabilizes FGF21 transcripts rather than induces FGF21 Another miRNA that has been shown to play an gene expression [74]. Alternatively, the inositol-requiring important role in metabolic disorders is microRNA-212 enzyme 1 α (IRE1α)/X-box binding protein 1 (XBP1) branch (miR-212). miR-212 modulates alcoholic liver disease [60] of the UPR was implicated in FGF21 upregulation by ER and cardiac insulin resistance during overnutrition [61]. stressors tunicamycin and thapsigargin [76]. ER stress This miRNA is primarily expressed in the central nervous response elements (ERSE, 5′-CCATT…N(n)…CCACG) system and, to a lower extent, in adipose, liver, and many to which XBP1 can bind were identified in the human other tissues. Xiao et al. [62] reported on the beneficial (–150/–128), mouse (–142/–108), and rat (–319/–285) FGF21 effects of miR-212 downregulation through exercise in the promoters [76]. The upregulation of FGF21 expression by prevention of non-alcoholic fatty liver disease (NAFLD). the UPR program is viewed as a countermeasure to ER The evidence that miR-212 targets FGF21 derives primar- stress as FGF21 acts subsequently to suppress the eukary- ily from the observation that synthetic miR-212 mimics otic initiation factor 2α (eIF2α)/ATF4/CHOP pathway [76]. decreased FGF21 expression whereas the miR-212 inhibitor increased FGF21 protein levels and decreased lipid synthesis in HepG2 cells. The silencing of FGF21 increased Regulation by nutrients intracellular lipid droplets in HepG2 cells and hindered the anti-lipogenic effect of the miR-212 inhibitor in these Carbohydrates cells. In animals, hepatic miR-212 was increased in mice fed a high-fat diet, and negatively regulated FGF21. Col- FGF21 hepatic expression and circulating FGF21 levels are lectively, it was concluded that FGF21 is a target of miR-212 influenced by dietary macronutrients. High-carbohydrate and anti-miR-212 therapeutic approaches could be useful diets have been shown to induce FGF21 expression. Liver for NAFLD patients [62]. Other predicted targets of miR-212 FGF21 mRNA levels were upregulated in mice fed a lipo- that belong to the FGF family of proteins include FGF1 genic high-carbohydrate liquid diet rich in dextrose, but [63], FGF2, FGF5, FGF9, FGF11, FGF12, and FGF23 [64]. not by a high-fat obesogenic diet, although both diets induced hepatosteatosis [77]. The stimulatory role of dextrose (27.5 mM) on FGF21 gene expression had been Regulation by endoplasmic reticulum (ER) shown earlier in isolated rat hepatocytes [78]. Hepatic stress FGF21 gene expression is induced not only by glucose but also by fructose, sucrose, and to a lesser extent, saccha- The ER plays a prominent role in cellular homeostasis by rin (an artificial non-caloric sweetener) when provided ad orchestrating the synthesis, folding, maturation, and dis- libitum to mice in the drinking water for 24 h [79]. Glucose, tribution of over a third of all proteins in the cell [65, 66]. fructose, and sucrose, but not saccharin, raised plasma A plethora of studies have linked ER stress with metabolic FGF21 levels in these mice. Similar results were observed disturbances and diseases such as insulin resistance, in human subjects infused with dextrose to reproduce a type 2 diabetes, and obesity [67–69]. New evidence has state of hyperglycemia [79]. ChREBP was implicated in indicated that FGF21 gene expression is induced under the upregulation of hepatic FGF21 gene expression in conditions causing cellular stress, and the upregulation is response to sucrose intake in mice. ChREBP binds to the mediated by activating transcription factor 4 (ATF4) and ChoRE site in the human FGF21 promoter [80]. A typical CCAAT enhancer binding-protein homologous protein ChoRE comprising two E-boxes separated by a 5-bp space (CHOP) [9, 70–74]. For the mechanism, the phosphoryla- exists in the mouse FGF21 promoter [81]. In humans, the tion of the α subunit of the eukaryotic initiation factor ChoRE comprises two imperfect E-boxes separated by (eIF2) by protein kinase R-like endoplasmic reticulum 8 bp at –380 to –366 [80]. A negative feedback loop of kinase (PERK) prompts ATF4 mRNA to be translated. ATF4 liver-derived FGF21 acting centrally in the hypothalamus translocates to the nucleus and induces transcription of to repress sweet-seeking behavior and reduce meal size the unfolded protein response (UPR) target genes. These has been proposed [79]. Counteracting the stimulation by genes include CHOP, a proapoptotic transcription factor sugars, the addition of a soybean oil-based emulsion to a that promotes cell death when stress conditions persist high-carbohydrate dextrose-rich diet led to the suppres- [75]. While conserved ATF4-binding sites are apparent in sion of FGF21 gene expression in mouse tissues and a low- the FGF21 promoter [70], current evidence indicates an ering of plasma FGF21 levels [77]. 6 Erickson and Moreau: Nutritional regulation of FGF21 gene expression

Proteins (low-protein diet, methionine- or leucine- diet for 5 or 8 weeks [88]. In this study, mice on the high- restricted diet) fat diet gained more weight, consumed more food, had higher epididymal white adipose fat and blood triacylglyc- FGF21 hepatic expression and circulating FGF21 are erols than control mice. The sustained feeding of the high- influenced by the level of dietary protein. Serum FGF21 fat diet for 8 weeks led to an upregulation of FGF21 mRNA increases on low-protein diets that typically are high in in the liver and white and brown adipose tissue. In con- carbohydrates. Singly restricted amino acids, such as trast, Hao et al. [77] did not observe a significant change methionine [82–85] or leucine [70], have been shown in hepatic FGF21 mRNA levels in mice fed a high-fat vs. to increase hepatic expression and circulating levels low-fat diet for 16 weeks. The apparent inconsistencies of FGF21. Serum FGF21 also increases in the context of suggest that high dietary fat is only one factor implicated ketogenic diets that are high in fat, very low in carbo- in FGF21 upregulation and warrant continued investiga- hydrate, and moderate to low in protein. Since FGF21 is tion. The upregulation of FGF21 gene expression in the induced by both high and low carbohydrate diets, the adipose tissue by feeding was consistent with the study of view is that FGF21 is not regulated by carbohydrates Dutchak et al. [15] that showed a meal-dependent increase alone but also by the reduction in protein [86]. Feeding in FGF21 mRNA in white adipose tissue 4 h after the initia- a low-protein (5% casein) isoenergetic diet (vs. 15%–20% tion of a meal during the dark phase. These results illus- casein in the control diet) either short-term (8 h) in rats trate the upregulation of FGF21 in liver and adipose tissue or longer-term (5–11 days) in mice led to an upregulation in the context of overnutrition and obesity. Supplementa- of FGF21 gene expression in the liver and an increase tion of the high-fat diet with long-chain polyunsaturated of FGF21 levels in plasma [87]. In humans, restricting n-3 fatty acids (PUFA), among which are eicosapentaenoic dietary protein for 28 days caused plasma FGF21 to rise acid (EPA) and docosahexaenoic acid (DHA), caused a [86]. Mechanistically, the PERK/eIF2α/ATF4 branch of comparatively modest induction of hepatic and adipose the UPR was implicated in FGF21 upregulation by a low- FGF21 gene expression above that of mice fed the control protein diet [86] and methionine restriction [84]. Dietary high-carbohydrate low-fat diet [88]. Mice fed a PUFA- methionine restriction resulted in the upregulation of supplemented high-fat diet gained less weight than mice hepatic ATF4 expression. Subsequently, ATF4 bound to fed the high-fat diet, and stored less fat in the epididymal cAMP response element (CRE). The 5′ flanking region pads and interscapular depot. Their blood triacylglycerols of human FGF21 has two functional ATF4 binding sites were also lower than in mice fed the high-fat diet devoid associated with amino acid response elements (AARE) of PUFA. From these studies, it is concluded that although [9, 70]. Disruption of these sites abolished ER stress- PUFA ameliorated the lipid profile, the mechanism did mediated induction of FGF21. Since leucine deprivation not overtly implicate FGF21. induces the kinase general control non-derepressible protein 2 (GCN2), the involvement of GCN2 (an upstream regulator of ATF4) in FGF21 expression has been pro- Short-chain fatty acids posed. Evidence shows that hepatic FGF21 mRNA levels Butyric acid is a short-chain fatty acid produced in large are markedly decreased in GCN2-null mice fed a low- quantities by bacterial fermentation of dietary fiber in the protein diet [86]; however, low dietary protein is still large intestine. Many of butyrate’s mechanisms of action capable of FGF21 induction, suggesting that there might implicate the regulation of gene expression since butyric be alternative mechanisms independent of GCN2 that link acid has histone deacetylase inhibitor (HDAC) properties. low protein and FGF21 upregulation. These alternative Li et al. [89] showed that butyrate induced FGF21 gene mechanisms include PPARα, which when knocked out in expression in the liver of dietary obese mice and in human mouse liver elicited low serum FGF21 concentrations and hepatocellular carcinoma HepG2 cells. The peritoneal weak FGF21 induction by a low-protein diet [86]. injection of butyrate (500 mg/kg body weight) in mice after an overnight fast induced plasma levels of FGF21 and β-hydroxybutyrate, indicating butyrate-enhanced Lipids (high-fat diet, fatty acids, retinoic acid) ketogenesis in mice [89]. Other exogenously supplied short-chain fatty acids, such as β-hydroxybutyrate and High-fat diet and polyunsaturated fatty acids hexanoate, did not induce FGF21 mRNA or FGF21 protein Hepatic FGF21 gene expression and plasma FGF21 levels levels, suggesting the specific role of butyrate. Functional were induced in mice fed a corn oil-based high-fat diet analysis of the FGF21 gene promoter (–1497/+5) revealed compared to control mice fed a high-carbohydrate low-fat the putative involvement of a bezafibrate-responsive Erickson and Moreau: Nutritional regulation of FGF21 gene expression 7 distal PPRE site and HDAC3 in the mechanism of butyrate. p38MAPK/SPZ1 axis may play a central role in the mecha- The use of small interfering RNA (siRNA) to block HDAC3 nism of α-LA. enhanced the activity of the promoter, suggesting that α-LA is known to induce Phase II detoxification butyrate induced FGF21 gene expression through the inhi- enzymes in a nuclear factor E2-related factor 2 (Nrf2)- bition of HDAC3, but not because of the short-chain fatty dependent manner by eliciting Nrf2 nuclear transloca- acid structure of butyrate. Li et al. [89] examined the regu- tion through the enhancement of NRF2 mRNA translation latory role of butyrate on FGF21 in mouse epididymal fat and the increase of Nrf2 protein half-life [102, 103]. Nrf2 and did not observe any effect. plays a central role in recruiting cellular defense systems α-Lipoic acid (α-LA) is a naturally occurring enzyme against oxidative and electrophilic stress and in maintain- cofactor synthesized from octanoic acid in most prokary- ing redox, lipid, and energy homeostasis. Cellular Nfr2 is otic and eukaryotic microorganisms as well as plant and sequestered in a cytosolic complex with the negative regu- animal mitochondria and plant plastids [90, 91]. α-LA can lator Kelch-like ECH-associated protein 1 (Keap1). In one also be absorbed from foods (leafy green vegetables and study, Nrf2 induction by the genetic knockdown of Keap1 red meats) and dietary supplements. Animal studies indi- increased hepatic FGF21 expression and plasma FGF21 cated that dietary supplementation of (R)-α-LA induced levels in diabetic db/db mice and diet-induced obese mice hepatic FGF21 expression and raised FGF21 blood levels [104]. A synthetic inducer of Nrf2 reproduced these effects above the levels induced by fasting alone [92–94]. Moreo- in these mice. As a transcription factor, Nrf2 regulates ver, feeding (R)-α-LA to rats reproduced the metabolic antioxidant/electrophile responsive element (ARE/EpRE)- effects of FGF21, including the lowering of liver and blood containing genes. Several ARE/EpREs were identified in triacylglycerol concentrations, and the decreased expres- the 5′-regulatory region of the mouse FGF21 gene [104]. sion of lipogenic genes including acetyl-CoA carboxylase 1 However, chromatin immunoprecipitation (ChIP) analysis (ACACA), fatty acid synthase (FASN), acyl-CoA desaturase did not reveal significant Nrf2 binding [104]. 1 (SCD1), elongation of very long chain fatty acids protein 6 (ELOV6), and glycerol-3-phosphate acyltransferase 1 (GPAM) [92–94]. (R)-α-LA (50 μM) stimulated the tran- Retinoic acid scription and secretion of FGF21 from human hepatocel- Li et al. [30] identified FGF21 as a novel target gene of lular carcinoma HepG2 cells [31]. A comparable outcome all-trans-retinoic acid (t-RA) through the action of the was observed in mouse hepatocyte AML12 cells treated retinoic acid receptor (RAR). They showed that adenovi- with 1 mM α-LA [95]. In these cells, Bae et al. [95] found ral overexpression of RARβ in mouse liver (i) stimulated that α-LA increased FGF21 promoter activity by upregu- hepatic mRNA and protein levels of FGF21, (ii) induced lating CREBH expression and increasing CREBH binding hepatic mRNA levels of carnitine palmitoyltransferase 1 to the FGF21 promoter. The knockdown of CREBH with α (CPT1α) (fatty acid catabolism), medium-chain acyl- siRNA attenuated the α-LA-driven induction of the FGF21 CoA dehydrogenase (MCAD, a β-oxidation enzyme), gene and protein expression. In the same study, the peri- 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2, a toneal injection of α-LA (100 mg/kg body weight per day ketogenic enzyme), and 3-hydroxy-3-methylglutaryl-CoA for 7 days) in mice induced hepatic CREBH and FGF21 lyase (HMGCL, also a ketogenic enzyme), (iii) increased transcript and protein levels, and raised serum FGF21 plasma levels of ketone body and β-hydroxybutyrate, levels in the fed and fasted states [95]. In HepG2 cells, and iv) raised whole-body energy expenditure. These α-LA altered the chromatin structure of a distal 5′-flanking effects were consistent with the physiological response 400-bp region (–1524/–1124 bp) of the FGF21 gene [31]. The to fasting observed upon FGF21 administration, except 400-bp fragment was enriched in the acetylated histone that in the study of Li et al. [30], the mice were sacrificed H3, which indicated its transcriptional competency to while having free access to food. Li et al. [30] further iden- bind transcriptional factors. In silico analysis of putative tified a putative RAR response element (RARE) separated transcription factors ranked the testis-specific bHLH-Zip by one base (DR1) in the human FGF21 gene located at transcription factor (SPZ1) as a lead candidate with three –645/–632, and two putative RARE sites spaced by five mixed E/G-boxes located at –1524/–1513, –1273/–1283, and nucleotides (DR5) located at –537/–521 and –602/–586 –1161/–1151 bp in the 5′-flanking region. Reports have indi- in the regulatory region of the mouse FGF21 gene. The cated that α-LA stimulates the cAMP/PKA signaling [96] functional relevance of this RARE-like site was estab- and other protein kinases downstream of cAMP, includ- lished when point mutations in the RARE abolished the ing ERK1/2 [97, 98] and p38MAPK [99, 100]. As SPZ1 has transactivity of t-RA/RAR for the FGF21 gene. Moreover, been shown to act downstream of MAPK [101], the cAMP/ the induction of FGF21 by the PPARα agonist, GW7647, 8 Erickson and Moreau: Nutritional regulation of FGF21 gene expression combined with t-RA appeared to be synergistic. The Through their inductive and repressive activities, the knockdown of PPARα abolished GW7647-mediated induc- first order clock-controlled genes RORs and REV-ERBs tion of FGF21 gene expression, whereas the knockdown of coordinate the circadian expression of FGF21 directly RARβ did not, indicating that FGF21 transcription is inde- [105, 106] as well as indirectly through PPARα/FGF21 [107] pendently upregulated by PPARα and RARβ [30]. (Figure 2). RORs and REV-ERBs are activated by the core The retinoic acid receptor-related receptors (RORs) clock genes aryl hydrocarbon receptor nuclear transloca- also play an important regulatory role in lipid metabo- tor-like protein 1 (BMAL1) and circadian locomotor output lism. RORs regulate gene expression by binding DNA as cycles kaput (CLOCK) and, in turn, provide feedback to monomers to ROR response elements (ROREs) consisting the core clock genes BMAL1/CLOCK and period circadian of an AGGTCA half-site preceded by a 5′ AT-rich sequence. protein homologue 1 (PER)/cryptochrome 1 (CRY) [108]. Wang et al. [105] identified a conserved classical RORE While the expression of FGF21 in the mouse liver exhib- site positioned at –1563/–1554 in the 5′ regulatory region ited a circadian rhythm, the oscillation pattern remains of the human FGF21 gene and –1223/–1213 upstream the ill defined. Wild-type male Jcl:ICR mice had to be fed mouse gene. This RORE site locates > 1000-bp upstream bezafibrate (a PPARα agonist) to exhibit temporal oscil- of two additional putative proximally located RORE sites lation in the transcription of hepatic FGF21, which peaked (–105/–96 and –88/–82 in the human FGF21 promoter, at the time of the dark-to-light transition (ZT0, ZT is Zeit- and –87/–78 and –69/–64 in the mouse FGF21 promoter). geber time within a 24-h cycle composed of 12 h of light These proximal sites mediated REV-ERBα repression of and 12 h of darkness). For comparison, the 24-h profile of the FGF21 gene in mouse primary hepatocytes [106]. FGF21 mRNA was flat in control Jcl:ICR mice not receiving

Figure 2: Schematic representation of the role of first order clock-controlled genes in the circadian regulation of FGF21 gene expression. The model depicts the activating and repressing transcriptional activity of core clock protein complexes BMAL1/CLOCK and PER/CRY onto their own genes (the core circadian feedback loop) and first order clock-controlled genes (ROR, E4BP4, REV-ERB, PPARα) mediated by E-boxes. The model also depicts the activating and repressing transcriptional activity of first order clock-controlled genes onto the core clock genes and onto FGF21, a second order clock-controlled gene, mediated by RORE, D-box, and PPRE. Erickson and Moreau: Nutritional regulation of FGF21 gene expression 9 bezafibrate [107]. In contrast, Tong et al. [109] found that, hepatocellular carcinoma HepG2 cells [126]. The underly- in C57BL6 mice, FGF21 mRNA peaked around circadian ing transcriptional mechanism is currently unknown and time CT6 in the first 24-h cycle and at CT12 in the second should be a matter of further investigation. 24-h cycle. In the latter study, mice were maintained on a 12 h/12 h light/dark cycle with free access to food and water for 2 weeks prior to being transferred to the dark for 48 h. Under these conditions, mice were sacrificed every Expert opinion 4 h for two 24-h cycles. Hence, CT6 represents the 6-h Recombinant FGF21 (rFGF21) administration is an experi- time point after lights were turned on during the 2-week mental polypeptide therapy against type 2 diabetes and feeding. In this study, the oscillation of FGF21 mRNA was lipid anomalies. However, the high costs of producing clearly anti-phase to that of E4-binding protein 4 gene rFGF21 and the mode of delivery by injection are impor- (E4BP4), which encodes a bHLH-Zip transcription factor tant limitations to the wide therapeutic use of engineered also called nuclear factor interleukin 3 regulated (NFIL3). FGF21. The stimulation of endogenous FGF21 production E4BP4 has a role in the regulation of circadian rhythm by through diet should be explored as a practical and cost- repressing the expression of a core clock gene periodic cir- effective alternative approach. Among dietary factors, cadian protein homologue 1 (PER). E4BP4 is regulated by high dextrose, low protein, methionine restriction, short- RORs and REV-ERBs, explaining why its circadian expres- chain fatty acids (butyric acid and lipoic acid), and all- sion profile is in phase with that of BMAL1 and CLOCK trans-retinoic acid have been shown to induce FGF21 [108, 110]. E4BP4 suppressed FGF21 gene expression by expression and circulating levels. These effects, though, direct binding to a D-box element in the distal promoter are usually more pronounced in liver tissue and isolated (–1033 to –1015) [109]. In mouse liver, temporal FGF21 hepatocytes than they are in adipose tissue and isolated expression was low during the light phase and steadily fat cells. This observation calls for further investigation of increased during the dark phase (ZT12–ZT24) when the the regulation of the FGF21 gene in non-hepatic tissues by animals actively fed [111]. This profile was exacerbated in dietary and non-dietary compounds. If new approaches mice (TSC1lox/lox Alb-CreTg/0) in which liver mamma- were to emerge from this research, they could be com- lian target of rapamycin complex 1 (mTORC1) activity was bined with liver-effective strategies to assess broader induced by the knockdown of its repressor hamartin, also efficacy. known as tuberous sclerosis 1 protein (TSC1) [111]. FGF21 expression was induced secondarily to PPARγ coactivator- 1α (PGC-1α) activation in response to mTORC1-mediated depletion of glutamine in the liver. In these mice, RORα Outlook and RORγ expression displayed a circadian rhythm in Given that all macronutrients and a growing list of struc- phase with FGF21, while that of REV-ERBα and REV-ERBβ turally diverse dietary molecules have been shown to reg- were anti-phase to FGF21. ulate FGF21 transcription in recent years, it is likely that other dietary compounds will be shown to alter FGF21 gene expression in the future. Regulation of FGF21 tran- Polyphenol (curcumin) scripts by non-coding RNAs is expected to develop from its current rudimentary beginnings. It is expected that Curcumin is the major polyphenol pigment isolated from further research will be devoted to elucidate the cause and the plant Curcuma longa, commonly known as turmeric. significance of interindividual variations in serum FGF21 Curcumin has a variety of pharmacologic properties. It has levels among human subjects. been demonstrated to have systemic anti-inflammatory activity and beneficial effects in several types of cancers both in animals and humans [112–117], and in individuals with dermatitis [118], Crohn’s disease [119], and ulcera- Highlights tive colitis [120]. In addition, curcumin has been shown to improve metabolic disorders associated with hyperlipi- –– Anti-miR-577-based approaches may boost the insuli- demia and hyperglycemia [121–125]. Curcumin gavage to notropic action of FGF21. mice stimulated FGF21 expression in liver [126]. A small –– CREBH and PPARα synergize to activate FGF21 gene dose of curcumin (2 μM) also increased FGF21 protein expression through the binding of the CREBH-PPARα- abundance in mouse primary hepatocytes and human RXR complex. 10 Erickson and Moreau: Nutritional regulation of FGF21 gene expression

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