Fibroblast Growth Factor 21 mRNA Expression Research Articles

Treatment with dapagliflozin increases FGF-21 gene expression and reduces triglycerides content in myocardial tissue of genetically obese mice.

The association between obesity and some cardiovascular complications such as heart failure (HF) is well established, and drugs affecting adiposity are supposed to be promising treatments for these conditions. The sodium-glucose cotransporter-2 inhibitors (SGLT2i) are antidiabetic drugs showing benefits in patients with HF, despite the underlying mechanisms have not been completely understood yet. SGLT2i are supposed to promote systemic effects, such as triglycerides mobilization, through the enhancement of fibroblast growth factor-21 (FGF-21) activity. So, in this study, weevaluated the effects of dapagliflozin treatment on FGF-21 and related receptors (FGF-Rs) gene expression and on lipid content in myocardial tissue in an animal model of genetically induced obesity to unravel possible metabolic mechanisms accounting for the cardioprotection of SGLT2i. Six-week-old C57BL/6J wild-type mice and B6.V-LEP (ob/ob) mice were randomly assigned to the control or treatment group (14 animals/group). Treatment was based on the administration of dapagliflozin 0.15mg/kg/day for 4weeks. The gene expression of FGF-21 and related receptors (FGF-R1, FGF-R3, FGF-R4, and β-klotho co-receptor) was assessed at baseline and after treatment by real-time PCR. Similarly, cardiac triglycerides concentration was measured in the control group and treated animals. At baseline, FGF-21 mRNA expression in the heart did not differ between lean and obese ob/ob mice. Dapagliflozin administration significantly increased heart FGF-21 gene expression, but only in ob/ob mice (p < 0.005). Consistently, when measuring the amount of triglycerides in the cardiac tissue, SGLT2i treatment reduced the lipid content in obese ob/ob mice, while no significant effects were observed in treated lean animals (p < 0.001). The overall expression of the FGF-21 receptors was only minimally affected by dapagliflozin treatment both in obese ob/ob mice and in lean controls. Dapagliflozin administrationincreases FGF-21gene expression and reduces triglyceride content in myocardial tissue of ob/ob mice, while no significant effect was observed in lean controls. These results might help understand the cardiometabolic effects of SGLT2i inducing increased FGF-21 synthesis while reducing lipid content in cardiomyocytes as a possible expression of the switch to different energy substrates. This mechanism could represent a potential target of SGLT2i in obesity-related heart diseases.

Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet.

Mammals respond to amino acid (AA) deficiency by initiating an AA response pathway (AAR) that involves the activation of general control nonderepressible 2 (GCN2), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and activation of transcription factor 4 (ATF4). In this study, the effects of protein (N) and/or phosphorus (P) restriction on the GCN2/eIF2α/ATF4 pathway in the liver and the induction of fibroblast growth factor 21 (FGF21) in young goats were investigated. An N-reduced diet resulted in a decrease in circulating essential AA (EAA) and an increase in non-essential AA (NEAA), as well as an increase in hepatic mRNA expression of GCN2 and ATF4 and protein expression of GCN2. Dietary N restriction robustly increased both hepatic FGF21 mRNA expression and circulating FGF21 levels. Accordingly, numerous significant correlations demonstrated the effects of the AA profile on the AAR pathway and confirmed an association. Furthermore, activation of the AAR pathway depended on the sufficient availability of P. When dietary P was restricted, the GCN2/eIF2α/ATF4 pathway was not initiated, and no increase in FGF21 was observed. These results illustrate how the AAR pathway responds to N- and/or P-reduced diets in ruminants, thus demonstrating the complexity of dietary component changes.

Sodium Propionate or Sodium Butyrate Promotes Fatty Acid Oxidation in HepG2 Cells Under Oxidative Stress.

The beneficial effects of sodium butyrate (NaB) and sodium propionate (NaP) on fatty acid oxidation (FAO) genes and production of proinflammatory cytokines related to nonalcoholic fatty liver disease (NAFLD) were evaluated using HepG2 human liver hepatocellular carcinoma cells exposed to palmitate/oleate or lipopolysaccharides (LPSs) as a model. The results showed that NaP or NaB was able to promote FAO, regulate lipolysis, and reduce reactive oxygen species production by significantly increasing the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase 1 alpha (CPT1α), fibroblast growth factor 21 (FGF21), and uncoupling protein 2 (UCP2) in HepG2 cells. Together, NaP and NaB may produce greater effects by increasing CPT1α, PPARα, and UCP2 mRNA expression in LPS-treated HepG2 cells and by increasing CPT1α and ATGL mRNA expression in palmitate-/oleate-treated HepG2 cells. Only NaP treatment significantly increased FGF21 mRNA expression in palmitate-/oleate-treated HepG2 cells. The enzyme-linked immunosorbent assay results revealed that only pretreatment with LPSs and not palmitate/oleate significantly increased tumor necrosis factor alpha (TNF-α) expression in HepG2 cells. NaP alone or in combination with NaB significantly decreased TNF-α expression in LPS-induced HepG2 cells. The expression of interleukin-8 in both models showed no significant differences in all treatments. NaP and NaB show potential for in vivo studies on NAFLD.

PSUN311 JTV-519 Alleviates the Impairment of FGF21 Axis Signaling after Spinal Cord Contusion in Mice

Abstract Spinal cord injury (SCI) causes adverse changes in body composition and metabolic profile due to dramatic loss of skeletal muscle mass and an extremely sedentary lifestyle. These abrupt and permanent changes increase the risk for cardiovascular disease, liver disorders, and type 2 diabetes compared to the general population. We have reported that metabolic dysregulation following SCI due to transection is associated with impaired fibroblast growth factor 21 (FGF21) and adiponectin (ADPN) signaling [1], two important regulators of metabolism [2, 3]. JTV519, which stabilizes ryanodine receptors and blocks calcium uptake via SERCA [4], has been reported to have a strong cardioprotective effect via stabilization of ryanodine receptor 2. We examined the effects of SCI caused by contusion (a more common cause of SCI), with or without JTV519 administration, on FGF21 and ADPN signaling in tissues collected from mice at 56 days after SCI. SCI reduced serum and hepatic mRNA levels of FGF21 by 75% and 45%, respectively, compared to sham mice. SCI also significantly diminished FGF21 receptor (FGF-R1) and coreceptor (beta-klotho, KLB) mRNA expression in adipose tissue. In addition, SCI decreased serum ADPN levels, particularly its high molecular weight (HMW) isoform which is the most potent form of the protein. These inhibitory effects were associated with decreased hepatic mRNA expression for PPARalpha (55%), type-2 adiponectin receptor (AdipoR2; 70%), and Glut4 (75%). In addition, SCI reduced mRNA expression of leptin (by 85%) and UCP1 (by 95%) in adipose tissue. In skeletal muscle (gastrocnemius), SCI lowered mRNA expression of PGC1alpha (by 38%), ADPN (by 80%), FGF-R1 (by 35%) and AdipoR1 (by 20%) compared to sham mice. Treating sham-mice with JTV519 upregulated FGF21 and ADPN serum and mRNA levels as well as the expression of the associated metabolic genes of interest (mentioned above). Likewise, SCI-mice treated with JTV519 also demonstrated upregulation of serum levels of FGF21, the HMW isoform of ADPN, and hepatic FGF21 mRNA expression compared to vehicle-treated SCI-mice. Decreased hepatic mRNA expression of PPARalpha, AdipoR2, Glut4 and FGF-R1 and KLB mRNA in adipose tissue were also reversed after JTV519 administration, with levels approximately equivalent to those observed in sham-vehicle treated mice but lower than sham-JTV519 treated mice. Moreover, JTV519 treatment protected against SCI-induced decreases of PGC1alpha, ADPN and FGF-R1 mRNA expression in the gastrocnemius. These data imply that JTV-519 is able to alleviate SCI-induced impairments of FGF21/ADPN signaling. Therefore, JTV519 could be a potential pharmacological agent to treat metabolic dysfunction after SCI.

Activating transcription factor 4 aggravates angiotensin II-induced cell dysfunction in human vascular aortic smooth muscle cells via transcriptionally activating fibroblast growth factor 21.

Abdominal aortic aneurysm (AAA) is a life-threatening disorder worldwide. Fibroblast growth factor 21 (FGF21) was shown to display a high level in the plasma of patients with AAA; however, its detailed functions underlying AAA pathogenesis are unclear. An in vitro AAA model was established in human aortic vascular smooth muscle cells (HASMCs) by angiotensin II (Ang-II) stimulation. Cell counting kit-8, wound healing, and Transwell assays were utilized for measuring cell proliferation and migration. RT-qPCR was used for detecting mRNA expression of FGF21 and activating transcription factor 4 (ATF4). Western blotting was utilized for assessing protein levels of FGF21, ATF4, and markers for the contractile phenotype of HASMCs. ChIP and luciferase reporter assays were implemented for identifying the binding relation between AFT4 and FGF21 promoters. FGF21 and ATF4 were both upregulated in Ang-II-treated HASMCs. Knocking down FGF21 attenuated Ang-II-induced proliferation, migration, and phenotype switch of HASMCs. ATF4 activated FGF21 transcription by binding to its promoter. FGF21 overexpression reversed AFT4 silencing-mediated inhibition of cell proliferation, migration, and phenotype switch. ATF4 transcriptionally upregulates FGF21 to promote the proliferation, migration, and phenotype switch of Ang-II-treated HASMCs.

The Association Between FGF21 and Diabetic Erectile Dysfunction: Evidence from Clinical and Animal Studies.

Erectile dysfunction (ED), a complication of diabetes mellitus (DM), affects 50–75% of men with diabetes. Fibroblast growth factor 21 (FGF21) is a liver-derived metabolic regulator which plays a role in insulin-independent glucose uptake in adipocytes. We designed a clinical study and an animal experiment to investigate the relationship between FGF21 and DM-induced ED. The clinical study enrolled 93 participants aged > 18 years (61 patients with type 2 DM and 32 healthy controls) from Taian City Central Hospital (TCCH) in Shandong Province, China, amongst whom the association between serum FGF21 and diabetic ED was analyzed. To further validate this association, we developed animal model of diabetic ED using Sprague-Dawley (SD) rats. Serum FGF21 concentration and FGF21 mRNA expression in penile samples of the rats were determined with Western blotting and quantitative real-time PCR. Among the 93 participants, the level of serum FGF21 was negatively correlated with the IIEF-5 score (r = -0.74, P < 0.001). The analysis on the performance of FGF21 for ED diagnosis showed that the area under the receiver operating characteristic (ROC) curve was 0.875 (95% confidence interval [CI]: 0.803 to 0.946). In the animal experiment, the levels of serum FGF21, 2-Δ Δ Ct values of FGF21 mRNA expression, and relative levels of FGF21 in penile samples were higher in the ED group compared to the DM and control groups. Our findings demonstrated an association between the FGF21 level and diabetic ED, indicating the potential of this cytokine in predicting diabetic ED.

FGF21 promotes non-small cell lung cancer progression by SIRT1/PI3K/AKT signaling

AimsLung cancer is a key contributor to the cancer-related death throughout the world. FGF21 (fibroblast growth factor 21) has been found to regulate various pulmonary diseases, whereas, the role and mechanism of FGF21 in lung cancer remain unclear. The aim of this research was to explore the expression and function of FGF21 in lung cancer. Main methodsThe mRNA and protein expression of FGF21 were analyzed through qRT-PCR and western blot, respectively. Cell proliferation, apoptosis and migration were analyzed by CCK-8 assay, flow cytometry and wound-healing assay, respectively. ROS, SOD, LDH and CK were examined with respective commercially kit. Key findingsFGF21 level was increased in lung cancer tissue samples and cell lines at both mRNA and protein levels. Overexpressing FGF21 promoted cell growth and migration significantly. It also increased SOD and reduced ROS, LDH and CK contents. By contrast, down-regulated FGF21 presented the opposite effect on lung cancer cells. Furthermore, FGF21 may function as a tumor promotor by activating the SIRT1/PI3K/AKT signaling pathway in lung cancer. SignificanceThis study demonstrated that FGF21 was a tumor promoter in lung cancer development, serving as a feasible therapeutic target in the treatment of lung cancer.

1957-P: Intermittent Ketogenic Diet Ameliorates Diet-Induced Obesity and Fatty Liver with Improved FGF21 Signaling

Recently, ketogenic diet has been found to be effective on improving obesity. Fibroblast growth factor 21 (FGF-21) is an endocrine factor mainly synthesized in the liver and shown to impact the metabolism of multiple tissues. Here, we investigated the effect of intermittent ketogenic diet on obesity and the sensitivity of FGF-21. Eight-week-old C57BL/6 mice were subjected to a chow diet (CD group), a high fat diet (HFD group), an intermittent chow diet (iCD group) or an intermittent ketogenic diet (iKD group) for 16 weeks. The intermittent diet intervention began with an HFD and was alternated with a chow diet or a ketogenic diet biweekly. Although the average energy intake of iKD group was highest in the four groups, the body weight, fasting blood glucose, the body fat mass and the weight of liver, epididymal adipose tissues (eWAT), subcutaneous adipose tissue (sWAT) and brown adipose tissue (BAT) of iKD group were ameliorated remarkably when compared to HFD group. The insulin sensitivity of iKD group was greatly improved as well. Both the histological results and the quantification of liver triglyceride showed that hepatic steatosis was significantly alleviated in iKD group. Western blotting showed that lipid metabolism in liver was greatly improved by iKD. What’s more, the cold exposure test showed that the iKD group had the best cold tolerance in the four groups. The mRNA expression of FGF-21 was greatly upregulated and hepatic inflammation was ameliorated in livers of iKD group. HE staining showed that the adipocytes in eWAT and sWAT were smaller in iKD group. The whitening of BAT was also improved in iKD group. Both mRNA and protein levels of UCP1 and PGC1α were remarkably upregulated by iKD in eWAT, sWAT and BAT. The mRNA levels of FGFR1 and KLB, which are related to FGF-21 sensitivity, were observed to be improved in eWAT and sWAT. Our data demonstrated that iKD ameliorates diet-induced obesity and fatty liver, and improved insulin sensitivity and cold tolerance with improved FGF-21 signaling. Disclosure W. Guo: None. W. Wang: None. W. Li: None.

1896-P: Adipocyte-Derived Fibroblast Growth Factor 21 (FGF21) Contributes to Circulating FGF21 Pools in Diet-Induced Obese Mice

Fibroblast growth factor 21 (FGF-21) is a hormone derived from hepatocytes and adipocytes that plays a role in energy homeostasis, and promotes weight loss when given pharmacologically. Nutritional stress such as fasting increases liver-derived FGF-21 expression, whereas over-nutrition (i.e., obesity) promotes adipocyte-derived FGF-21 (Ad-FGF-21) production. Despite the current dogma that Ad-FGF-21 acts only in a paracrine manner, obesity is associated with elevated circulating FGF-21 levels which correlate with adiposity and adipose tissue FGF-21 mRNA expression. To date, a concise role for Ad-FGF-21 in obesity-associated energy balance has not been identified. To determine whether Ad-FGF-21 plays a pathological role in obesity, and whether it contributes to circulating FGF-21 levels, we generated mice with Ad-FGF-21 deficiency (Ad-FGF-21 KO) and placed them on an obesogenic high fat high sucrose diet for 18 weeks. Body composition, energy expenditure, glucose and insulin tolerance were assessed. Male and female Ad-FGF-21 KO mice were protected from obesity, gained less body fat, and exhibited improved glucose metabolism over wild type (WT) controls. Circulating FGF-21 levels were higher in obese WT mice than age-matched chow-fed WT or obese Ad-FGF-21 KO mice with no changes in hepatic FGF-21, suggesting that adipocytes are a source of FGF-21 in obesity. Moreover, adipose tissue explants from obese WT mice secreted higher levels of FGF-21 than explants from chow-fed mice, an effect that was blunted in Ad-FGF-21 KO mice. Finally, expression of fibroblast activation protein (FAP), an enzyme that cleaves FGF-21 to blunt its activity, was reduced from obese adipose tissue, providing a potential mechanism by which adipocyte-derived FGF-21 circulates in obesity. In summary, adipocyte-derived FGF-21 may contribute to obesity-associated increases in circulating FGF-21, with the potential to influence the pathology of obesity. Disclosure S. Wang: None. L. Goodspeed: None. K. Ogimoto: None. L. den Hartigh: None. Funding National Institutes of Health (HL092969)

Mechanisms of Action of Methionine Restriction and Fibroblast Growth Factor 21 (FGF21)

Abstract Objectives Many of the metabolic benefits of methionine restriction (MR) are mediated by FGF21. MR activates protein kinase R-like ER kinase (PERK) and nuclear respiratory factor 2 (NRF2) signaling pathways, which have also been tied to FGF21 production. Aim 1 is to elucidate the mechanism by which MR increases hepatic FGF21 production. FGF21 signaling in the brain is required for its metabolic effects. However, the neuronal circuits and mechanisms mediating FGF21’s metabolic effects remain unclear. Aim 2 is to determine the effects of FGF21 on neurons in the paraventricular nucleus (PVN). Methods Aim 1: HepG2 cells were treated with vehicle or NRF2 inhibitor (brusatol; 80 nM) for 4 h or PERK inhibitor (GSK2606414; 1 µM) for 2 h. Cells were then incubated in control or MR media for 16 h. FGF21 mRNA expression was measured. Aim 2: WT and Fgf21−/− mice were fed high-fat control or MR diets for 14 wk. 4 mice per group were perfused with formalin for immunohistochemistry. Patch-clamp study: Sympathetic, PVN neurons projecting to the rostral ventrolateral medulla were retrogradely-labeled and subsequently patched in acute hypothalamic slices and treated with vehicle or the FGF21 analog, LY2405319 (LY; 500 nM). Firing activity of PVN-RVLM neurons and the activity of GABAergic inhibitory inputs to these neurons were measured. Results Aim 1: MR increased FGF21 mRNA in HepG2 cells by 18-fold on average (P = 0.02). The ability of MR to increase FGF21 is abolished when cells are treated with the NRF2 inhibitor but is not affected by PERK inhibition. Aim 2: MR increased ERK phosphorylation in the PVN of WT, but not Fgf21−/− mice. LY either increased (33% of recorded neurons) or decreased (67% of recorded neurons) the firing activity of PVN-RVLM neurons suggesting that FGF21 effects may depend on their projecting targets. LY inhibited the frequency of GABA inhibitory postsynaptic current (IPSC) (42.02% decrease, P &amp;lt; 0.05). Conclusions In vitro analysis suggests that the ability of MR to increase FGF21 expression is dependent upon NRF2 activity, but independent of PERK. Our data support the hypothesis that FGF21 signals through the PVN modulating sympathetic-related PVN neuronal activity to produce its metabolic benefits. Funding Sources GSU Research Initiation Grant; Center for Neuroinflammation and Cardiometabolic Diseases (CNCD), GSU.

Increased Expression of FGF-21 Negatively Affects Bone Homeostasis in Dystrophin/Utrophin Double Knockout Mice.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy seen in children. In addition to skeletal muscle, DMD also has a significant impact on bone. The pathogenesis of bone abnormalities in DMD is still unknown. Recently, we have identified a novel bone-regulating cytokine, fibroblast growth factor-21 (FGF-21), which is dramatically upregulated in skeletal muscles from DMD animal models. We hypothesize that muscle-derived FGF-21 negatively affects bone homeostasis in DMD. Dystrophin/utrophin double-knockout (dKO) mice were used in this study. We found that the levels of circulating FGF-21 were significantly higher in dKO mice than in age-matched WT controls. Further tests on FGF-21 expressing tissues revealed that both FGF-21 mRNA and protein expression were dramatically upregulated in dystrophic skeletal muscles, whereas FGF-21 mRNA expression was downregulated in liver and white adipose tissue (WAT) compared to WT controls. Neutralization of circulating FGF-21 by i.p. injection of anti-FGF-21 antibody significantly alleviated progressive bone loss in weight-bearing (vertebra, femur, and tibia) and non-weight bearing bones (parietal bones) in dKO mice. We also found that FGF-21 directly promoted RANKL-induced osteoclastogenesis from bone marrow macrophages (BMMs), as well as promoted adipogenesis while concomitantly inhibiting osteogenesis of bone marrow mesenchymal stem cells (BMMSCs). Furthermore, fibroblast growth factor receptors (FGFRs) and co-receptor β-klotho (KLB) were expressed in bone cells (BMM-derived osteoclasts and BMMSCs) and bone tissues. KLB knockdown by small interfering RNAs (siRNAs) significantly inhibited the effects of FGF21 on osteoclast formation of BMMs and on adipogenic differentiation of BMMSCs, indicating that FGF-21 may directly affect dystrophic bone via the FGFRs-β-klotho complex. In conclusion, this study shows that dystrophic skeletal muscles express and secrete significant levels of FGF-21, which negatively regulates bone homeostasis and represents an important pathological factor for the development of bone abnormalities in DMD. The current study highlights the importance of muscle/bone cross-talk via muscle-derived factors (myokines) in the pathogenesis of bone abnormalities in DMD. © 2019 American Society for Bone and Mineral Research.

Translational Control of FGF21 mRNA Expression is Responsive to Nutritional Stress

Fibroblast growth factor 21 (FGF21) is a peptide hormone associated with the pathogenesis of several metabolic disorders such as Non‐alcoholic Fatty Liver Disease (NAFLD), Type‐2‐Diabetes, and obesity. Notably, FGF21 treatment is associated with enhanced insulin sensitivity, and in rodents, attenuation of the metabolic defects associated with consumption of a high‐fat diet. Because the majority of circulating FGF21 is secreted by the liver in mammals, understanding the mechanisms that regulate hepatic expression of the hormone is critical. Hepatic FGF21 expression has been shown to be regulated in part through changes in FGF21 gene transcription. However, recent studies have shown transcription‐independent changes in FGF21 secretion from isolated hepatocytes, implying that FGF21 protein expression is also regulated at the level of mRNA translation. Thus, the goal of the present study was to assess whether or not the FGF21 mRNA is translationally regulated using H4IIE hepatoma cells as a model system. In these studies, cells were treated with rapamycin as a mimic of starvation and translation of the FGF21 mRNA was assessed using sucrose density gradient centrifugation to separate mRNAs that are poorly translated, i.e. mRNAs associated with 3 or fewer ribosomes (“light” polysomes) from well‐translated mRNAs, i.e. mRNAs associated with four or more ribosomes (“heavy” polysomes). As a positive control, mRNAs bearing a terminal oligopyrimidine tract (TOP) at the 5′‐end of the message were also assessed, as previous studies have shown them to be translationally downregulated under the conditions used in this study. As expected rapamycin treatment resulted in repressed translation of the TOP mRNAs, as demonstrated by a shift of the mRNAs from heavy to light polysomes. In contrast, translation of the FGF21 mRNA was enhanced by rapamycin, suggesting that it is regulated through an mTORC1‐dependent mechanism distinct from, and opposite to, mRNAs with a TOP motif. Current efforts are directed toward identifying the regulatory element(s) in the FGF21 mRNA that engenders sensitivity to rapamycin‐induced translational upregulation.Support or Funding Information(Supported by NIH grant DK13499)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

HDAC3 inhibition in diabetic mice may activate Nrf2 preventing diabetes-induced liver damage and FGF21 synthesis and secretion leading to aortic protection.

Vascular complications are common pathologies associated with type 1 diabetes. In recent years, histone deacetylation enzyme (HDAC) inhibitors have been shown to be successful in preventing atherosclerosis. To investigate the mechanism for HDAC3 inhibition in preventing diabetic aortic pathologies, male OVE26 type 1 diabetic mice and age-matched wild-type (FVB) mice were given the HDAC3-specific inhibitor RGFP-966 or vehicle for 3 mo. These mice were then euthanized immediately or maintained for an additional 3 mo without treatment. Levels of aortic inflammation and fibrosis and plasma and fibroblast growth factor 21 (FGF21) levels were determined. Because the liver is the major organ for FGF21 synthesis in diabetic animals, the effects of HDAC3 inhibition on hepatic FGF21 synthesis were examined. Additionally, hepatic miR-200a and kelch-like ECH-associated protein 1 (Keap1) expression and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were measured. HDAC3 inhibition significantly reduced aortic fibrosis and inflammation in OVE26 mice at both 3 and 6 mo. Plasma FGF21 levels were significantly higher in RGFP-966-treated OVE26 mice compared with vehicle-treated mice at both time points. It also significantly reduced hepatic pathologies associated with diabetes, accompanied by increased FGF21 mRNA and protein expression. HDAC3 inhibition also increased miR-200a expression, reduced Keap1 protein levels, and increased Nrf2 nuclear translocation with an upregulation of antioxidant gene and FGF21 transcription. Our results support a model where HDAC3 inhibition may promote Nrf2 activity by increasing miR-200a expression with a concomitant decrease in Keap1 to preserve hepatic FGF21 synthesis. The preservation of hepatic FGF21 synthesis ultimately leads to a reduction in diabetes-induced aorta pathologies.

High non-esterified fatty acid concentrations promote expression and secretion of fibroblast growth factor 21 in calf hepatocytes cultured in vitro.

Negative energy balance is considered as the pathological basis of energy metabolic disorders in periparturient dairy cows. Serum non-esterified fatty acids (NEFA) are one of the most important indicators of energy balance status. Fibroblast growth factor 21 (FGF21) has been identified as a hepatokine involved in regulation of metabolic adaptations, such as promoting hepatic lipid oxidation and ketogenesis, during energy deprivation. However, the direct effects of NEFA on FGF21 expression and secretion in bovine hepatocytes are not entirely clear. The objective of this study was to investigate the effects of different NEFA concentrations on FGF21 expression and secretion in calf hepatocytes cultured in vitro. NEFA were added to the culture solution at final concentrations of 0.6, 1.2, 1.8 and 2.4mmol/L. After 24hr of continuous culture, FGF21 mRNA and protein expression levels in the hepatocytes were determined by real-time PCR and Western blot respectively. FGF21 secretion in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results showed that expression and secretion of FGF21 at 0.6mmol/L NEFA-treated hepatocytes was higher than that of the control group (p<.05). The FGF21 expression and secretion were similar at 1.2, 1.8 and 2.4mmol/L NEFA-treated hepatocytes and significantly higher than those observed for controls (p<.01). These data suggest that high concentrations of NEFA significantly promote FGF21 expression and secretion in bovine hepatocytes. In particular, this promotion occurs in a dose-dependent manner and may be involved in the pathological processes of energy metabolism disorders of dairy cows in the peripartum period.

Differentiated embryo chondrocyte 1 (DEC1) is a novel negative regulator of hepatic fibroblast growth factor 21 (FGF21) in aging mice

Human differentiated embryo chondrocyte expressed gene 1 (DEC1) is frequently used as a marker of senescence in vivo. Fibroblast growth factor 21 (FGF21), a novel endocrine-like member of the FGF superfamily, is highly expressed in the liver, and FGF21-transgenic mice have extended lifespans. Thus, we hypothesized that FGF21 may play a role in the DEC1-mediated aging process. In this study, DEC1 knockout (KO) mice were used to characterize the mechanism by which FGF21 protects mice from aging. Aging is strongly diminished in DEC1 KO mice, which is reflected by decreased lipid levels and oxidative stress, leading to an amelioration of liver function and structure. The expression of FGF21 decreased with aging in wild-type (WT) mice, whereas ATF4, Phospho-ERK and Phospho-p38 expression was maintained and was accompanied by a compensatory rise of FGF21 mRNA and protein expression in DEC1 KO mice. Over-expression of DEC1 markedly abolished the hepatic expression of FGF21, and siRNA-mediated inhibition of endogenous DEC1 increased the expression of FGF21. DEC1 further diminished the expression of ATF4 in HepG2 cells over-expressing DEC1. The induction of FGF21 and ATF4 at the mRNA and protein levels during the course of aging supports the view that DEC1 KO mice are able to restore the age-related imbalance of metabolism. Collectively, the data obtained in this study suggest that DEC1 is a novel negative regulator of hepatic FGF21 expression.

Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows.

In rodents, fibroblast growth factor 21 (FGF21) has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits further study, and in particular the mechanisms regulating transcription in response to changes in energy balance and feed intake.

Placental fibroblast growth factor 21 is not altered in late-onset preeclampsia.

BackgroundPreeclampsia (PE) is associated with alterations of placental function. The incidence of PE is higher in insulin resistant states. Women with PE have high circulating levels of the metabolic regulator fibroblast growth factor 21 (FGF21). FGF21 is synthesized in the placenta. The aim of this study was to compare the expression of FGF21, its receptors, downstream targets and transcriptional regulators in placental tissue from pregnancies with and without late-onset PE. Circulating FGF21 in maternal and cord blood was also studied.MethodsmRNA expression was determined by semi-quantitative real-time PCR and normalized for cellular composition in 17 women with and 20 without PE. Protein expression was quantified by Western Blot. FGF21 levels were measured by ELISA in maternal and cord serum of ten mother-baby dyads per condition.ResultsPlacental FGF21 mRNA and protein expression were similar in PE compared with control. Placental mRNA expression of the FGF receptors (1–4) and the co-receptor beta-Klotho was not different between the groups. There was no difference in the expression of the glucose transporters GLUT1, 3 or 4. PPAR-alpha but not PPAR-gamma expression was decreased in PE. Maternal FGF21 serum levels were not significantly different in PE. FGF21 was detected in cord blood of 6 infants (4 PE, 2 controls) but was undetectable in 14 infants.ConclusionsLate-onset PE is not associated with major changes to the expression of FGF21, its receptors or metabolic targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0006-3) contains supplementary material, which is available to authorized users.

Alpha lipoic acid induces hepatic fibroblast growth factor 21 expression via up-regulation of CREBH

Hepatic expression of fibroblast growth factor 21 (FGF21), one of the most promising therapeutic candidates for metabolic syndrome, is induced by multiple factors associated with fasting, including cyclic AMP response element-binding protein H (CREBH). Alpha lipoic acid (ALA), a naturally occurring thiol antioxidant, has been shown to induce metabolic changes that are similar to those induced by FGF21, including weight loss and increased energy expenditure. Here, we investigated the effect of ALA on hepatic FGF21 expression. ALA treatment enhanced CREBH and FGF21 mRNA expression and protein abundance in cultured hepatocytes. ALA increased FGF21 promoter activity by up-regulating CREBH expression and increasing CREBH binding to the FGF21 promoter, indicating that ALA up-regulates FGF21 at the transcriptional level. Moreover, inhibition of endogenous CREBH expression by siRNA attenuated ALA-induced FGF21 expression. Finally, treatment of mice with ALA enhanced fasting-induced up-regulation of CREBH and FGF21 in the liver and inhibited feeding-induced suppression of their expression. Consistently, ALA increased serum FGF21 levels in both fasted and fed mice. Collectively, these results indicate that ALA increases hepatic FGF21 expression via up-regulation of CREBH, identifying ALA as a novel positive regulator of FGF21.

Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis

Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF21 mRNA expression in H9C2 cardiomyotubes. Cardiac FGF21 expression was also induced upon fasting of healthy mice, implicating a role of FGF21 in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF21 (CM-Fgf21). FGF21 was efficiently secreted from cardiomyocytes of CM-Fgf21 mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF21 in cardiac energy metabolism. Together, our results show that FGF21 expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF21 levels interferes with cardiac and whole body energy homeostasis.