Fibroblast Growth Factor 21 Secretion Research Articles

The good, the bad, and the unknown: Fructose and FGF21.

In this issue of Molecular Metabolism, Dushay et al. report new results demonstrating fructose ingestion in humans acutely and robustly raises circulating levels of fibroblast growth factor 21 (FGF21) [1], a recently discovered hormone that has been proposed to have beneficial effects on metabolic health, including reduction of body weight and improvements of glucose and lipid metabolism [2]. People suffering from the metabolic syndrome have higher baseline circulating FGF21 levels and exhibit a larger increase of plasma FGF21 concentrations following an oral fructose load. In contrast, the increase of FGF21 after ingestion of the same amount of glucose was delayed and much more modest. These results are paradoxical in that the metabolic effects of FGF-21 appear to be mainly beneficial while those of dietary fructose are generally deleterious [3]. The authors suggest the possibility that obese subjects with metabolic syndrome are resistant to the actions of FGF21 (akin to augmentation of circulating insulin and leptin concentrations and responses in insulin and leptin resistance) and that the exaggerated response to fructose ingestion may be a compensatory response [1]. The increases of FGF-21 in response to fructose ingestion across normal weight and overweight/obese subjects was significantly correlated with circulating insulin and glucose levels in response to oral glucose loads suggesting a relationship between FGF21 and insulin senstivity/glucose tolerance. Together, these novel results shed light on a potential mechanism related to the adverse metabolic effects of dietary fructose and raise new questions about the nutritional regulation of metabolic responses to dietary macronutrients, specifically sugars.

Abstract 1439: Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21)

Abstract Caloric Restriction (CR) is known to be the only model to consistently increase the lifespan and postpone age-related diseases in all species. CR increases glycogenolysis, lipolysis with fatty acid and ketone body utilization and accelerates protein catabolism. We previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing the growth of 4T1 mammary tumors and their metastases. 4T1 is a highly metastatic mouse breast cancer model which resembles breast cancer in patients. Obesity causes subclinical inflammation in adipose tissue that contributes to insulin resistance and cancer progression. Chronic inflammation predisposes to different forms of cancer and diabetes, and is correlated with increased risk of breast cancer. CR reduces insulin resistance, adiposity and inflammation and the anti-diabetic drug metformin (MET) is a CR mimetic agent. In this study, we evaluated whether the combination of MET with the anti-obesity drug orlistat (OR) enhances the CR mimetic and anti-cancer properties of MET on 4T1 mammary tumor cells. Female 8 week-old BALB/c mice received: vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/day) or MET + OR. After 3 weeks on drugs, 4T1 cells (105) were injected into mice and drugs continued till the end of the experiment. Treated-mice had a reduced adiposity (p<0.05) without any signs of toxicity. At 30 days, MET + OR co-treatment reduced tumor volume (p<0.01) and displayed a range of cellular alterations such as decreased proliferative index (p<0.05); increased apoptotic rate (p<0.01) and altered intra-tumor collagen deposit. MET + OR treatment reduced total vessel length (p<0.01) and the number of spontaneous lung metastases (p<0.01). In addition, combinations of MET (1-2.5mM) + OR (2.5-5µM) decreased in vitro 4T1 mammary cell proliferation (p<0.01), adhesion (p<0.05) and migration (p<0.01) compared to vehicle and drugs alone. Interestingly, we found that levels of fibroblast growth factor 21 (FGF21), an endocrine factor that regulates glucose and lipid metabolism, were significantly increased with MET + OR treatment (p<0.05). It has been shown that increased circulating levels of FGF21 correlated with decreased obesity and increased sensitivity to insulin; but up to date the role of FGF21 in tumor progression was not elucidated. We found that treatment of 4T1 cells with MET + OR increased the secretion of FGF21 (p<0.05) measured by ELISA assays. To elucidate the role of FGF21; we initially demonstrated that addition of recombinant protein FGF21 (0-10nM) did not change 4T1 cell proliferation. In contrast, the reduced cell proliferation and adhesion induced by MET + OR, were abolished by the pre- and co-treatment with anti-FGF21 neutralizing antibody; indicating that FGF21 may play an important role. Our results provide a new rationale basis for the use of MET plus OR as cancer therapy. Citation Format: Shobika Sivaram, Erdene Baljinnyam, Kousaku Iwatsubo, Lydia I. Puricelli, Mariana S. De Lorenzo. Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2014-1439

FGF21 expression and release in muscle cells: involvement of MyoD and regulation by mitochondria-driven signalling.

Although the liver is generally considered the main site of production of FGF21 (fibroblast growth factor-21), high FGF21 levels have been found to be associated with neuromuscular mitochondrial genetic diseases, and there are indications that the muscle may be a relevant site of FGF21 production under conditions of muscular mitochondrial stress. In the present study, we found that expression and release of FGF21 was associated with myogenic differentiation, and we identified MyoD as a major controller of FGF21 gene transcription. Mimicking mitochondrial dysfunction using respiratory chain/oxidative phosphorylation inhibitors resulted in enhanced expression and release of FGF21 by muscle cells. The increased production of reactive oxygen species, subsequent induction of p38 MAPK (mitogen-activated protein kinase) and activation of an ATF2 (activating transcription factor 2)-binding site at the proximal promoter region of the FGF21 gene was found to be a major mechanism linking mitochondrial dysfunction with enhanced FGF21 gene transcription in myogenic cells. The myogenic factor MyoD was required for the induction of FGF21 gene transcription by mitochondrial dysfunction, thus explaining the preferential response of muscle cells to mitochondrial dysfunction-induced FGF21 expression and secretion. FGF21 release by muscle cells in response to mitochondrial alterations may represent a physiological mechanism by which the sensing of internal energetic status by muscles results in the release of FGF21 to favour systemic metabolic adaptations.

Basal and postprandial change in serum fibroblast growth factor-21 concentration in type 1 diabetic mellitus and in healthy controls.

Fibroblast growth factor-21 (FGF-21) appears to have an important role in glucose and lipid metabolism. FGF-21 secretion is mainly determined by nutritional status. The aim of this study was to measure basal and postprandial FGF-21 and postprandial change of FGF-21 concentration in type 1 diabetes mellitus (T1DM) patients and in healthy controls, and to investigate the differences between the groups. The cross-sectional study included 30 C-peptide negative T1DM patients, median age 37 years (20-59), disease duration 22 years (3-45), and nine healthy controls, median age 30 years (27-47). Basal and postprandial FGF-21 concentrations were measured by ELISA. The associations of FGF-21 with glucose, lipids, and insulin were analyzed. Individuals with T1DM showed significantly lower basal FGF-21 concentration (P=0.046) when compared with healthy controls (median value 28.2 vs 104 pg/mL) and had significantly different postprandial change (∆ 30'-0') of FGF-21 (P=0.006) in comparison with healthy controls (median value -1.1 vs -20.5 pg/mL). The glucose and lipid status did not correlate with FGF-21. In healthy controls, postprandial insulin level correlated with basal FGF-21 (ρ=0.7, P=0.036). Multiple regression analysis showed that they are independently associated after adjustment for confounding factors (β=1.824, P=0.04). We describe the pathological pattern of basal and postprandial change of FGF-21 secretion not associated with glucose, lipid levels, or insulin therapy in patients with T1DM. Since FGF-21 has numerous protective metabolic effects in the experimental model, the lower basal FGF-21 concentration in T1DM patients opens the question about the potential role of recombinant FGF-21 therapy.

Glucagon stimulates hepatic FGF21 secretion through a PKA- and EPAC-dependent posttranscriptional mechanism.

Previous studies have shown that whole body deletion of the glucagon receptor suppresses the ability of starvation to increase hepatic fibroblast growth factor 21 (FGF21) expression and plasma FGF21 concentration. Here, we investigate the mechanism by which glucagon receptor activation increases hepatic FGF21 production. Incubating primary rat hepatocyte cultures with glucagon, dibutyryl cAMP or forskolin stimulated a 3-4-fold increase in FGF21 secretion. The effect of these agents on FGF21 secretion was not associated with an increase in FGF21 mRNA abundance. Glucagon induction of FGF21 secretion was additive with the stimulatory effect of a PPARα activator (GW7647) on FGF21 secretion. Inhibition of protein kinase A (PKA) and downstream components of the PKA pathway [i.e. AMP-activated protein kinase and p38 MAPK] suppressed glucagon activation of FGF21 secretion. Incubating hepatocytes with an exchange protein directly activated by cAMP (EPAC)-selective cAMP analog [i.e. 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME)], stimulated a 3.9-fold increase FGF21 secretion, whereas inhibition of the EPAC effector, Rap1, suppressed glucagon activation of FGF21 secretion. Treatment of hepatocytes with insulin also increased FGF21 secretion. In contrast to glucagon, insulin activation of FGF21 secretion was associated with an increase in FGF21 mRNA abundance. Glucagon synergistically interacted with insulin to stimulate a further increase in FGF21 secretion and FGF21 mRNA abundance. These results demonstrate that glucagon increases hepatic FGF21 secretion via a posttranscriptional mechanism and provide evidence that both the PKA branch and EPAC branch of the cAMP pathway play a role in mediating this effect. These results also identify a novel synergistic interaction between glucagon and insulin in the regulation of FGF21 secretion and FGF21 mRNA abundance. We propose that this insulin/glucagon synergism plays a role in mediating the elevation in FGF21 production during starvation and conditions related to metabolic syndrome.

ATF4- and CHOP-dependent induction of FGF21 through endoplasmic reticulum stress.

Fibroblast growth factor 21 (FGF21) is an important endogenous regulator involved in the regulation of glucose and lipid metabolism. FGF21 expression is strongly induced in animal and human subjects with metabolic diseases, but little is known about the molecular mechanism. Endoplasmic reticulum (ER) stress plays an essential role in metabolic homeostasis and is observed in numerous pathological processes, including type 2 diabetes, overweight, nonalcoholic fatty liver disease (NAFLD). In this study, we investigate the correlation between the expression of FGF21 and ER stress. We demonstrated that TG-induced ER stress directly regulated the expression and secretion of FGF21 in a dose- and time-dependent manner. FGF21 is the target gene for activating transcription factor 4 (ATF4) and CCAAT enhancer binding protein homologous protein (CHOP). Suppression of CHOP impaired the transcriptional activation of FGF21 by TG-induced ER stress in CHOP−/− mouse primary hepatocytes (MPH), and overexpression of ATF4 and CHOP resulted in FGF21 promoter activation to initiate the transcriptional programme. In mRNA stability assay, we indicated that ER stress increased the half-life of mRNA of FGF21 significantly. In conclusion, FGF21 expression is regulated by ER stress via ATF- and CHOP-dependent transcriptional mechanism and posttranscriptional mechanism, respectively.

Elevated FGF21 secretion, PGC-1α and ketogenic enzyme expression are hallmarks of iron–sulfur cluster depletion in human skeletal muscle

Iron-sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.

Functional thermogenic beige adipogenesis is inducible in human neck fat

Recent studies suggest human neck brown adipose tissue (BAT) to consist of 'brown adipocyte (BA)-like' or beige adipocytes. However, little is known about their thermogenic function. Within the beige adipocyte transcriptome, fibroblast growth factor-21 (FGF21) is a gene whose protein product acts as an adipokine, regulating cold-induced thermogenesis in animals. Here, we explored (i) the adipogenic potential, thermogenic function and FGF21 secretory capacity of beige adipocytes derived from human neck fat and (ii) the role of FGF21 in modulating adipose bioenergetics. Progenitors isolated from human cervical fat were differentiated into adipocytes with either a BA-like or white adipocyte (WA) phenotype. FGF21 secretion was measured by enzyme-linked immuosorbent assay. Real-time PCR/western blotting was used to determine cellular mRNA/protein levels. Extracellular flux bioanalyzer was used to quantify adipocyte oxygen consumption and fatty acid oxidation. Adipocyte heat production was measured by infrared thermography. Under hormonal manipulation, primary human neck pre-adipocytes differentiated into adipocytes with either BA-like or WA phenotypes, on gene/protein and functional levels. BA-like cells expressed beige but not classic BA markers. During BA differentiation, FGF21 gene expression and secretion were increased, and were augmented following norepinephrine exposure (a cold mimic in vitro). Differentiated WA expressed β-klotho, a critical co-factor mediating FGF21 action. Treatment of WA with FGF21-induced UCP1 expression and increased oxygen consumption, respiratory uncoupling, norepinephrine-mediated thermogenesis, fatty acid oxidation and heat production, thus recapitulating the association between cold-induced FGF21 secretion and cold-induced thermogenesis in vivo. Beige adipocytes are thermogenic in humans. FGF21 is a beige adipokine capable of promoting a brown fat-like thermogenic program in WAs. This study provides first evidence of inducible functional thermogenic beige adipogenesis in human neck fat. FGF21 holds promise as a cold-induced beige adipokine with metabolic benefits of therapeutic relevance through browning of white adipose tissue.

Retinoic Acid Receptor β Stimulates Hepatic Induction of Fibroblast Growth Factor 21 to Promote Fatty Acid Oxidation and Control Whole-body Energy Homeostasis in Mice

Activation of retinoic acid receptor (RAR) with all-trans-retinoic acid (RA) ameliorates glucose intolerance and insulin resistance in obese mice. The recently discovered fibroblast growth factor 21 (FGF21) is a hepatocyte-derived hormone that restores glucose and lipid homeostasis in obesity-induced diabetes. However, whether hepatic RAR is linked to FGF21 in the control of lipid metabolism and energy homeostasis remains elusive. Here we identify FGF21 as a direct target gene of RARβ. The gene transcription of Fgf21 is increased by the RAR agonist RA and by overexpression of RARα and RARβ, but it is unaffected by RARγ in HepG2 cells. Promoter deletion analysis characterizes a putative RA-responsive element (RARE) primarily located in the 5'-flanking region of the Fgf21 gene. Disruption of the RARE sequence abolishes RA responsiveness. In vivo adenoviral overexpression of RARβ in the liver enhances production and secretion of FGF21, which in turn promotes hepatic fatty acid oxidation and ketogenesis and ultimately leads to increased energy expenditure in mice. The metabolic effects of RA or RARβ are mimicked by FGF21 overexpression and largely abolished by FGF21 knockdown. Moreover, hepatic RARβ is bound to the putative RAREs of the Fgf21 promoter in a fasting-inducible manner in vivo, which contributes to FGF21 induction and the metabolic adaptation to prolonged fasting. In addition to other nuclear receptors, such as peroxisome proliferator-activated receptor α and retinoic acid receptor-related receptor α, RAR may act as a novel component to induce hepatic FGF21 in the regulation of lipid metabolism. The hepatic RAR-FGF21 pathway may represent a potential drug target for treating metabolic disorders.

Glucagon increases circulating fibroblast growth factor 21 independently of endogenous insulin levels: a novel mechanism of glucagon-stimulated lipolysis?

Glucagon reduces body weight by modifying food intake, glucose/lipid metabolism and energy expenditure. All these physiological processes are also controlled by fibroblast growth factor 21 (FGF-21), a circulating hepatokine that improves the metabolic profile in obesity and type 2 diabetes. Animal experiments have suggested a possible interaction between glucagon and FGF-21 however, the metabolic consequences of this crosstalk are not understood. The effects of exogenous glucagon on plasma FGF-21 levels and lipolysis were evaluated in healthy volunteers and humans with type 1 diabetes, as well as in rodents with streptozotocin (STZ)-induced insulinopenic diabetes. In vitro, the role of glucagon on FGF-21 secretion and lipolysis was studied using isolated primary rat hepatocytes and adipocytes. Fgf-21 expression in differentiated rat pre-adipocytes was suppressed by small interfering RNA and released FGF-21 was immunoneutralised by polyclonal antibodies. Glucagon induced lipolysis in healthy human volunteers, patients with type 1 diabetes, mice and rats with STZ-induced insulinopenic diabetes, and in adipocytes isolated from diabetic and non-diabetic animals. In addition, glucagon increased circulating FGF-21 in healthy humans and rodents, as well as in patients with type 1 diabetes, and insulinopenic rodents. Glucagon stimulated FGF-21 secretion from isolated primary hepatocytes and adipocytes derived from animals with insulinopenic diabetes. Furthermore, FGF-21 stimulated lipolysis in primary adipocytes isolated from non-diabetic and diabetic rats. Reduction of Fgf-21 expression (by approximately 66%) or immunoneutralisation of released FGF-21 markedly attenuated glucagon-stimulated lipolysis in adipocytes. These results indicate that glucagon increases circulating FGF-21 independently of endogenous insulin levels. FGF-21 participates in glucagon-induced stimulation of lipolysis.

Abstract 74: Heme Oxygenase-PPARα Induction of FGF21 in Hepatocytes Recruits pAMPK/pAKT and Attenuates Insulin Resistance in Obese Mice

The peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that is known to regulate glucose and lipid homeostasis and increases expression of the hormone fibroblast growth factor 21 (FGF21), which is secreted to blood, increases phosphorylation of AMPK and AKT in adipocytes and has been purported to stimulate adiponectin synthesis. The heme oxygenase (HO) system is functionally important for reducing ROS and inflammation whose induction has been shown to increase serum adiponectin levels and affect lowering of blood glucose and fatty acids. This study was designed to examine the hypothesis that a HO-1-PPARα crosstalk could elevate hepatic FGF21 thus leading to enhanced adiponectin secretion and abatement of metabolic imbalance. Preliminary experiments were conducted in human hepatocellular carcinoma cells (Hep G2) cultured in the absence or presence of the HO-1 inducer, cobalt protoporphyrin (CoPP, 2μmoles/l). A 2.0 fold induction of HO-1 in Hep G2 cells by CoPP (p<0.05) increased expression of FGF21 1.5 fold (n=3, p=0.0119 vs. vehicle control) without affecting PPARα expression. Additional experiments in obese (ob/ob) mice treated with CoPP (5mg/kg/day) resulted in 2 fold (p<0.05) increase of FGF21 mRNA expression in liver as compared to mice treated with the vehicle. These observations were complemented by blood glucose measurements, which showed significant attenuation in obese mice treated with CoPP (vehicle: 285.0±24 vs. CoPP: 160.0±27.3, p<0.05, n= 5), along with enhancement of adiponectin levels in CoPP treated obese mice (p<0.05). Taken together these results demonstrate that HO-1 could increase serum adiponectin levels and insulin sensitivity by elevating hepatic FGF21 levels. Our overall hypothesis is that the HO-1 can stimulate PPARα activity with resultant activation of hepatic FGF21 secretion and that this HO-PPARα-FGF21 axis could work in concert to modulate the development of insulin resistance and diabetes.

Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

Previous studies have shown that starvation or consumption of a high fat, low carbohydrate (HF-LC) ketogenic diet induces hepatic fibroblast growth factor 21 (FGF21) gene expression in part by activating the peroxisome proliferator-activated receptor-α (PPARα). Using primary hepatocyte cultures to screen for endogenous signals that mediate the nutritional regulation of FGF21 expression, we identified two sources of PPARα activators (i.e. nonesterified unsaturated fatty acids and chylomicron remnants) that induced FGF21 gene expression. In addition, we discovered that natural (i.e. bile acids) and synthetic (i.e. GW4064) activators of the farnesoid X receptor (FXR) increased FGF21 gene expression and secretion. The effects of bile acids were additive with the effects of nonesterified unsaturated fatty acids in regulating FGF21 expression. FXR activation of FGF21 gene transcription was mediated by an FXR/retinoid X receptor binding site in the 5'-flanking region of the FGF21 gene. FGF19, a gut hormone whose expression and secretion is induced by intestinal bile acids, also increased hepatic FGF21 secretion. Deletion of FXR in mice suppressed the ability of an HF-LC ketogenic diet to induce hepatic FGF21 gene expression. The results of this study identify FXR as a new signaling pathway activating FGF21 expression and provide evidence that FXR activators work in combination with PPARα activators to mediate the stimulatory effect of an HF-LC ketogenic diet on FGF21 expression. We propose that the enhanced enterohepatic flux of bile acids during HF-LC consumption leads to activation of hepatic FXR and FGF19 signaling activity and an increase in FGF21 gene expression and secretion.

Long-term high-fat diet links the regulation of the insulin-sensitizing fibroblast growth factor-21 and visfatin

High-fat diet (HFD) is associated with insulin resistance, hyperinsulinemia, elevated plasma free fatty acid (FFA), and increased risk for atherosclerotic vascular disease. However, the mechanisms underlying the HFD-induced insulin resistance have not been fully clarified. The aim of present study is to evaluate the effects of long-term HFD on the regulation of the insulin-sensitizing fibroblast growth factor-21 (FGF-21) and visfatin in ApoE−/− mice. A total of twenty male ApoE−/− mice were randomly divided into normal chow diet (NC) or HFD (HF) group for 16weeks. Euglycemic–hyperinsulinemic clamp was performed to evaluate insulin sensitivity in this animal model. Both mRNA and protein contents of FGF-21 and visfatin were assayed by Quantitative real-time PCR and Western blot. Long-term HFD resulted in the marked abnormality of glucose and lipid metabolism as well as a large decrease in whole-body insulin sensitivity. Accompanied by abnormal glucose–lipid metabolism and aggravated insulin resistance, FGF-21, β-klotho, FGFR1, FGFR3 and FGFR4 mRNA expressions were markedly up-regulated, whereas visfatin mRNA expression was markedly down-regulated in liver and/or adipose tissue of HFD-fed mice. In addition, Western blotting also revealed both up-regulation of the FGF-21 protein and down-regulation of visfatin protein in liver, adipose tissue and plasma of HFD-fed mice. Both FGF-21 and visfatin expression and secretion are regulated by a potent regulator, long-term HFD. And these adipokines are associated with glucose–lipid metabolism and insulin resistance.

Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

Fibroblast growth factor 21 (FGF21) has recently emerged as a metabolic hormone involved in regulating glucose and lipid metabolism in mouse, but the regulatory mechanisms and actions of FGF21 in humans remain unclear. Here we have investigated the regulatory mechanisms of the human FGF21 gene at the transcriptional level. A deletion study of the human FGF21 promoter (−1672 to +230 bp) revealed two fasting signals, including peroxisome proliferator-activated receptor α (PPARα) and glucagon signals, that independently induced human FGF21 gene transcription in mouse primary hepatocytes. In addition, two feeding signals, glucose and xylitol, also dose-dependently induced human FGF21 gene transcription and mRNA expression in both human HepG2 cells and mouse primary hepatocytes. FGF21 protein expression and secretion were also induced by high glucose stimulation. The human FGF21 promoter (−1672 to +230 bp) was found to have a carbohydrate-responsive element at −380 to −366 bp, which is distinct from the PPAR response element (PPRE). Knock-down of the carbohydrate response element binding protein by RNAi diminished glucose-induced human FGF21 transcription. Moreover, we found that a region from −555 to −443 bp of the human FGF21 promoter region exerts an important role in the activation of basic transcription. In conclusion, human FGF21 gene expression is paradoxically and independently regulated by both fasting and feeding signals. These regulatory mechanisms suggest that human FGF21 is increased with nutritional crisis, including starvation and overfeeding.

Transcriptional Repressor E4-binding Protein 4 (E4BP4) Regulates Metabolic Hormone Fibroblast Growth Factor 21 (FGF21) during Circadian Cycles and Feeding

Fibroblast growth factor 21 (FGF21) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF21 induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF21 and a key circadian output protein, E4BP4. Expression of Fgf21 displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf21 transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf21 expression, and overexpression of E4BP4 represses FGF21 secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf21 promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF21 expression during circadian cycles and feeding.

Regulation of FGF21 Expression and Secretion by Retinoic Acid Receptor-related Orphan Receptor α

Fibroblast growth factor 21 (FGF21) is a hormone produced by fat and the liver that plays an important role in lipid metabolism. FGF21 expression is induced by peroxisome proliferator-actived receptor alpha in response to physiological conditions requiring increased fatty acid oxidation. Retinoic acid receptor-related receptor alpha (RORalpha) is another nuclear receptor that plays a critical role in lipid metabolism as well as in regulation of the circadian rhythm. In this study we demonstrate that RORalpha directly regulates the expression and secretion of FGF21. A canonical ROR response element was identified in the proximal promoter of the FGF21 gene and shown to exhibit functional activity. Overexpression of RORalpha in HepG2 cells resulted in increased expression and secretion of FGF21. Suppression of RORalpha expression caused a decrease in FGF21 expression and secretion, suggesting that RORalpha contributes to the basal expression of FGF21. These data suggest that one mechanism by which RORalpha regulates lipid metabolism may be by modulation of FGF21 secretion. Furthermore, this study identifies a clear link between RORalpha, a key regulator of the mammalian clock, and FGF21, an important hormone regulating glucose and lipid homeostasis.