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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 May 23;176(13):2292–2305. doi: 10.1111/bph.14678

Oral administration of a new HRI activator as a new strategy to improve high‐fat‐diet‐induced glucose intolerance, hepatic steatosis, and hypertriglyceridaemia through FGF21

Mohammad Zarei 1,2,3,[Link], Eugènia Pujol 1, Tania Quesada‐López 3,4,5, Francesc Villarroya 3,4,5, Emma Barroso 1,2,3, Santiago Vázquez 1, Javier Pizarro‐Delgado 1,2,3, Xavier Palomer 1,2,3, Manuel Vázquez‐Carrera 1,2,3,✉
PMCID: PMC6555855  PMID: 30927369

Abstract

Background and Purpose

FGF21 has emerged as a therapeutic strategy for treating type 2 diabetes mellitus due to its antidiabetic effects, and this has led to the development of long‐acting analogues of FGF21. However, these compounds have some limitations, including a need to be administered by s.c. injection and their prolonged pharmacodynamic effect compared with native FGF21, which might be responsible for their reported side effects.

Experimental Approach

We have previously demonstrated that i.p. administration of haem‐regulated eukaryotic translation initiation factor 2α kinase (HRI) activators increases hepatic and circulating levels of FGF21. In this study, we examined the effects of p.o. administration of a new HRI activator, EPB‐53, on high‐fat diet (HFD)‐induced glucose intolerance, hepatic steatosis, and hypertriglyceridaemia, and compared them with those of metformin.

Key Results

EPB‐53 administration for the last 2 weeks, to mice fed a HFD for 10 weeks, reduced body weight gain, improved glucose intolerance, and prevented hepatic steatosis and hypertriglyceridaemia, whereas metformin only ameliorated glucose intolerance. Moreover, EPB‐53, similar to the reported effects of FGF21, reduced lipogenesis in cultured human hepatocytes and in the liver of mice fed a HFD. Administration of EPB‐53 to Fgf21‐knockout mice had no effects, demonstrating that its efficacy is dependent on this hormone.

Conclusions and Implications

Overall, the findings of this study demonstrate that p.o. administration of HRI activators, by increasing FGF21, is a promising strategy for the treatment of type 2 diabetes mellitus and non‐alcoholic fatty liver disease.

Abbreviations

Acox

acyl‐CoA oxidase

ATF4

activating transcription factor 4

BTCtFPU

1‐(benzo[d][1,2,3]thiadiazol‐6‐yl)‐3‐(4‐chloro‐3‐(trifluoromethyl)phenyl)urea

BTdCPU

1‐(benzo[d][1,2,3]thiadiazol‐6‐yl)‐3‐(3,4‐dichlorophenyl)urea

Chop

C/EBP homologous protein

Cpt‐1α

carnitine palmitoyltransferase 1α

eIF2α

eukaryotic translation initiation factor 2α

HFD

high‐fat diet

Hmgcs2

3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2

HRI

haem‐regulated eukaryotic translation initiation factor 2α kinase

Hsd3b5

3‐β‐hydroxysteroid dehydrogenase type 5

Mcad

medium‐chain acyl‐CoA dehydrogenase

Mup1

major urinary protein 1

NAFLD

non‐alcoholic fatty liver disease

PGC‐1α

PPARγ co‐activator 1α

What is already known

  • Targeting FGF21 is an emerging therapeutic strategy for treating type 2 diabetes mellitus.

What this study adds

  • Oral administration of HRI activators improve glucose intolerance and prevented hepatic steatosis by increasing FGF21.

What is the clinical significance

  • The use of an oral drug to increase endogenous FGF21 levels might have advantages over FGF21 analogues.

1. INTRODUCTION

FGF21 is a secreted protein belonging to the FGF19 subfamily (Goetz et al., 2007). It elicits its actions through binding to a plasma membrane receptor complex consisting of the FGF receptor 1c isoform (FGFR1c) and β‐klotho co‐receptor (Goetz et al., 2007; Ogawa et al., 2007). Circulating FGF21 derives from the liver (Markan et al., 2014), and serum FGF21 levels correlate with its hepatic expression (Hale et al., 2012). FGF21 was originally identified as a fasting‐induced hormone that promotes increased glucose uptake in adipocytes (Kharitonenkov et al., 2005). Later studies demonstrated that pharmacological administration of FGF21 to animal models of obesity and/or diabetes improved glucose tolerance and insulin sensitivity, reduced hepatic and serum triglyceride levels, and caused weight loss (Coskun et al., 2008; Ding et al., 2012; Inagaki et al., 2007; Kharitonenkov et al., 2005; Xu et al., 2009). Despite these pharmacological effects, serum FGF21 levels are paradoxically increased in obesity, in both rodents (Fisher et al., 2010; Hale et al., 2012; Muise et al., 2008; Satapati et al., 2008; Zhang et al., 2008) and humans (Chavez et al., 2009; Chen et al., 2008; Mraz et al., 2009), and especially in type 2 diabetes mellitus (T2DM; Badman, Kennedy, Adams, Pissios, & Maratos‐Flier, 2009; Zhang et al., 2008). The presence of high endogenous FGF21 levels in obesity has led to this condition being considered as an FGF21‐resistant state (Fisher et al., 2010). However, this assumption is controversial, since, as mentioned above, exogenous pharmacological administration of FGF21 is effective in genetic and diet‐induced animal models of obesity (Hale et al., 2012). The increase in circulating FGF21 levels in obesity and other metabolic alterations probably reflects the depositing of fat in the liver (Hale et al., 2012; Maratos‐Flier, 2017), and consistent with this, serum FGF21 levels correlate with non‐alcoholic fatty liver disease (NAFLD) in humans (Dushay et al., 2010; Yilmaz et al., 2010). This assumption is also supported by the fact that administration of exogenous FGF21 reduces its expression in liver as plasma and hepatic triglyceride levels decrease or as adiposity and insulin resistance improve in animal models of obesity and insulin resistance (Hale et al., 2012).

The beneficial effects of pharmacological administration of FGF21 have led to the establishment of FGF21 as a therapeutic target for the treatment of metabolic diseases (Gimeno & Moller, 2014; Kharitonenkov & DiMarchi, 2015). This has encouraged the development of FGF21 analogues to treat human metabolic disorders such as obesity, dyslipidaemia, and T2DM (A. M. Kim, Somayaji, et al., 2017; Reitman, 2013; Talukdar et al., 2016). However, because of their peptidic origin, these analogues require parenteral administration, and therefore, there is a need for more convenient orally available drugs for targeting FGF21 to treat metabolic disorders. In addition, native FGF21 has a short half‐life, and to overcome this issue, long‐acting FGF21 analogues, with prolonged pharmacodynamics compared to native FGF21, have been designed (Huang et al., 2013; Weng et al., 2015). However, prolonged activation of the FGF21 receptor by these long‐acting FGF21 analogues might be responsible for their side effects, including bone loss (Talukdar et al., 2016), and increases in BP and heart rate (A. M. Kim, Somayaji, et al., 2017).

Recently, we reported that i.p. administration of a haem‐regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI) activator increases hepatic Fgf21 expression and reduces lipid‐induced hepatic steatosis and glucose intolerance in mice fed a high‐fat diet (HFD; Zarei et al., 2016). These effects were dependent on FGF21, since they were abolished in Fgf21‐null mice (Zarei et al., 2016). The activation of HRI resulted in the phosphorylation of eIF2α and the subsequent increase in the activity of activating transcription factor (ATF) 4, which is essential for inducing the expression of Fgf21 (De Sousa‐Coelho, Marrero, & Haro, 2012). This indicates that HRI activators, which are small molecules, are potential candidates for an p.o. treatment of T2DM. In this study, we compared the effects of the p.o. administration of a new HRI activator, EPB‐53 (Figure 1a), with those of metformin, on glucose tolerance, hepatic steatosis, and hypertriglyceridaemia in mice fed a HFD. Our findings show that EPB‐53 treatment reduces body weight gain, glucose intolerance, hepatic steatosis, and hypertriglyceridaemia and that these effects are dependent on FGF21.

Figure 1.

Figure 1

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EPB‐53 increases the expression of FGF21 in human Huh‐7 hepatocytes. (a) Molecular structure of EPB‐53. (b) FGF21 mRNA abundance in human Huh‐7 hepatocytes exposed to 10 μM of BTCtFPU, CTdCPU, and EPB‐53 for 24 hr. mRNA levels are presented as the mean ± SD (n = 6 per group). *P < .05 versus control (CT). # P < .05 versus BTCtFPU‐treated cells. † P < .05 versus BTdCPU‐treated cells

2. METHODS

2.1. Mice

Male C57BL/6 mice (10–12 weeks old; Harlan Ibérica S.A., Barcelona, Spain) were housed and maintained under a constant temperature (22 ± 2°C) and humidity (55%). The mice had free access to water and food and were subjected to 12‐hr light–dark cycles. After 1 week of acclimatization, mice were randomly distributed into two experimental groups (n = 6 each), and received either one daily p.o. gavage of vehicle (2% w/v, (2‐hydroxypropyl)‐β‐cyclodextrin) or one daily p.o. dose of 300 mg·kg−1·day−1 of the HRI activator EPB‐53 dissolved in the vehicle (volume administered 1 ml·kg−1) for 4 days. This high dose was selected because of the presence of two parameters that may lower efficacy, the poor solubility of the compound (ClogP = 5.5) and its high plasma protein binding (99.85%). In a second study, male C57BL/6 mice (10–12 weeks old) were randomly distributed into four experimental groups (n = 6 each) and fed either standard chow (one group) or a HFD (45% fat mainly form hydrogenated coconut oil, Product D08061110, Research Diets Inc.) for 10 weeks. Mice fed standard chow and one of the groups of mice fed the HFD received one daily p.o. gavage of vehicle, 2% w/v, (2‐hydroxypropyl)‐β‐cyclodextrin, meanwhile the remaining two groups fed the HFD received one daily p.o. dose of either the HRI activator EPB‐53 (300 mg·kg−1·day−1) or metformin (150 mg·kg−1·day−1; Y. S. Kim, Kim, et al., 2017), for the last 2 weeks. In a third study, male Fgf21 knockout (Fgf21 −/−) mice (8–10 weeks old; B6N;129S5‐Fgf21tm1Lex/Mmcd, obtained from the Mutant Mouse Regional Resource Centre) and their wild‐type littermates (Fgf21 +/+) were randomly distributed into three experimental groups (standard chow, HFD, and HFD + EPB‐53; n = 5 each) and fed the different diets for 3 weeks. The standard chow and HFD groups received one daily p.o. gavage of vehicle, 2% w/v, (2‐hydroxypropyl)‐β‐cyclodextrin, whereas the HFD + EPB‐53 group received one daily p.o. administration of the HRI activator EPB‐53 (300 mg·kg−1·day−1) for the last week. These last conditions were replicated for the dose–response study, where male C57BL/6 mice (10–12 weeks old) fed a HFD for 3 weeks were treated during the last week with EPB‐53 (100, 200, and 300 mg·kg−1·day−1).

For the glucose tolerance test, animals received 2 g·kg−1 body weight of glucose by i.p. injection, and blood was collected from the tail vein after 0, 15, 30, 60, and 120 min.

The experiments complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85‐23, revised 1996). All procedures were approved by the University of Barcelona Bioethics Committee, as stated in Law 5/July 21, 1995, passed by the Generalitat de Catalunya. The animals were treated humanely, and all efforts were made to minimize the animals' suffering and the animal numbers. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology.

2.2. Pharmacokinetics

CD‐1 male mice were treated with EPB‐53 p.o. at a single dose of 20 mg·kg−1. Plasma samples were obtained from the vena cava at 0, 0.5, 1, 3, 5, and 24 hr post‐administration (three mice per point). Analytical measurements were performed by LC/MS/MS. Pharmacokinetic parameters were calculated by means of non‐compartmental analysis, Phoenix 7.0 (WinNonlin 6.3).

2.3. Cell culture

Human Huh‐7 cells (RRID:CVCL_0336; a kind gift from Dr Mayka Sánchez, of the Josep Carreras Leukaemia Research Institute) were cultured in DMEM supplemented with 10% serum, at 37°C/5% CO2. Hepatocytes were exposed to a concentration of 10 μM of each diarylurea, as previously reported (Chen et al., 2011).

2.4. RNA preparation and quantitative RT‐PCR

The relative levels of specific mRNAs were assessed by real‐time RT‐PCR, as previously described (Zarei et al., 2016). The results for the expression of specific mRNAs were normalized to the expression of a control gene to avoid unwanted sources of variation. The primer sequences used are displayed in Table S1.

2.5. Immunoblotting

The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018). Total protein extracts were isolated as described previously (Zarei et al., 2016). Proteins (30 μg) were separated by SDS‐PAGE on 10% acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed using antibodies against activating transcription factor 4 (ATF4; sc‐390063), GAPDH (sc‐32233), HRI (sc‐365239; RRID:AB_10843794), very low‐density lipoprotein receptor (VLDLR; sc‐18824; Santa Cruz Inc., Heidelberg, Germany), VLDLR (AF2258; R&D Systems, Minneapolis, MN), AMPK (2532), phospho‐AMPK Thr172 (2535), eIF2α (9722), phospho‐eIF2α (Ser51; 9721; Cell Signaling Technology Inc., Danvers, MA), β‐actin (A5441), and tubulin (T9026; Sigma‐Aldrich). Detection was performed using the Western Lightning® Plus‐ECL chemiluminescence kit (PerkinElmer, Waltham, MA). The equal loading of proteins was assessed by Ponceau S staining. The size of the proteins detected was estimated using protein molecular mass standards (Bio‐Rad, Barcelona, Spain). The results for protein quantification were normalized to the levels of a control protein to avoid unwanted sources of variation.

2.6. Haematoxylin–eosin and Oil Red O staining

We performed haematoxylin–eosin and Oil Red O (ORO) staining as previously reported (Zarei et al., 2016).

2.7. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. For in vivo experiments, animals were randomly distributed between groups and experimenters blinded for liver analysis purposes. Results are expressed as means ± SD. Significant differences were established by ANOVA, using the GraphPad Prism programme (V6.01; RRID:SCR_002798; GraphPad Software Inc., San Diego, CA). When significant variations were found by one‐way ANOVA, the Tukey–Kramer multiple comparison posttest was performed only if F achieved P < .05 and there was no significant variance inhomogeneity. Differences were considered significant when P < .05.

2.8. Reagents

N,N′‐diarylureas, 1‐(benzo[d][1,2,3]thiadiazol‐6‐yl)‐3‐(3,4‐dichlorophenyl)urea (BTdCPU) and 1‐(benzo[d][1,2,3]thiadiazol‐6‐yl)‐3‐(4‐chloro‐3‐(trifluoromethyl)phenyl)urea (BTCtFPU) were synthesized as previously described (Zarei et al., 2016). The synthesis of EPB‐53 is included in Supplementary Data S1. Triglyceride (Sigma‐Aldrich, Madrid, Spain), aspartate aminotransferase (AST), alanine aminotransferase (ALT; Spinreact, Girona, Spain), and FGF21 (Millipore, Bedford, MA) levels were measured using a commercial kit.

2.9. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Cidlowski et al., 2017; Alexander, Fabbro et al., 2017).

3. RESULTS

3.1. EPB‐53 increases FGF21 expression in human Huh‐7 hepatocytes and in liver and serum of mice

It has previously been reported that N,N′‐diarylureas, including BTCtFPU and BTdCPU, are activators of HRI and induce eIF2α phosphorylation (Chen et al., 2013). We demonstrated that i.p. administration of both BTCtFPU and BTdCPU increased mouse hepatic and serum FGF21 levels (Zarei et al., 2016). Then, we synthesized a series of compounds featuring a diarylurea scaffold and screened these new compounds for their capacity to increase FGF21 expression in human Huh‐7 hepatoma cells. Of all these new compounds, we selected EPB‐53 as the best candidate for in vivo studies, since it showed a significant increase in FGF21 mRNA levels compared to the previous HRI activators BTCtFPU and BTdCPU (Figure 1b), and it displayed a good pharmacokinetic profile in mice (Table S2). Notably, the half‐life of the elimination phase of EPB‐53 by p.o. administration was 5.4 hr in mice, thus allowing for a substantial period of activity after its administration.

As a first step to check the effects of EPB‐53 on mice, we examined whether acute p.o. administration of EPB‐53 for 4 days activated the HRI–eIF2α–ATF4 pathway and increased hepatic expression and serum levels of FGF21. HRI is activated by autophosphorylation (Lu, Han, & Chen, 2001), and EPB‐53 increased hepatic phospho‐levels of HRI, compared to those of mice treated with the vehicle alone (Figure 2a). Consistent with the eIF2α kinase role of HRI, EPB‐53 significantly enhanced phospho‐eIF2α and ATF4 levels in liver (Figure 2a). Likewise, EPB‐53 increased the hepatic expression and serum levels of FGF21 (Figure 2b,c).

Figure 2.

Figure 2

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Oral administration of the HRI activator EPB‐53 for 4 days increases the hepatic expression and serum levels of FGF21 in mice. Mice received one daily p.o. gavage of vehicle (2% w/v cyclodextrin) or one daily p.o. dose of the HRI activator EPB‐53 for 4 days. (a) Liver cell lysate extracts were assayed via western blot analysis with antibodies against total and phospho‐HRI, total and phospho‐ eIF2α, and ATF4. (b) Fgf21 mRNA abundance in the liver. (c) Serum FGF21 levels. (d) Vldlr and Chop mRNA abundance in the liver. (e) Pgc‐1α, Pparα, Cpt‐1α, Mcad, and Fas mRNA abundance in the liver. Data are presented as the mean ± SD (n = 6 per group) relative to control (CT) mice. *P < .05 versus CT mice

Next, we analysed the expression of two additional ATF4‐target genes: VLDLR (Jo et al., 2013) and CCAAT/enhancer‐binding protein homologous protein (CHOP). VLDLR binds apolipoprotein E triglyceride‐rich lipoproteins such as chylomicrons, VLDL, and intermediate‐density lipoproteins, leading to lipid entry into the cell. Notably, hepatic VLDLR up‐regulation plays an essential role in the triglyceride‐lowering effect of fenofibrate (Gao et al., 2014). Meanwhile, CHOP induces cell‐cycle arrest and apoptosis (Zinszner et al., 1998). EPB‐53 treatment significantly increased the expression of Vldlr, but it did not affect Chop mRNA levels (Figure 2d).

It has been suggested that acute administration of FGF21 up‐regulates the expression of the transcriptional co‐activator PGC‐1α (Fisher et al., 2011), which in turn controls the activity of the master regulator of hepatic fatty acid oxidation, PPARα (Kersten & Stienstra, 2017). Consistent with the increase in FGF21 levels following EPB‐53 treatment, Pgc‐1α expression tended towards up‐regulation, but the differences did not reach statistical significance. Although the expression of Pparα was not affected, the increase in the expression of its target genes, Cpt‐1α and Mcad, suggested an increase in its activity (Figure 2e). Moreover, it has been reported that FGF21 controls hepatic triglyceride content in liver by reducing de novo lipogenesis (Xu et al., 2009; Zhang et al., 2011). Consistent with the increase in FGF21 levels, the expression of the lipogenic gene fatty acid synthase (Fas) was down‐regulated (Figure 2e).

3.2. EPB‐53 administration improves glucose intolerance, hepatic steatosis, and hypertriglyceridaemia in mice fed a HFD

Next, we examined the effects of EPB‐53 on mice fed a HFD, a model of diet‐induced obesity, and T2DM. First, mice were fed the HFD for 10 weeks and for the last 2 weeks they were treated with the vehicle alone, EPB‐53, or metformin. Mice treated with EPB‐53 did not show any sign of discomfort or toxicity. We compared the effects of EPB‐53 with those of metformin, the first‐line drug treatment for T2DM. Mice fed the HFD for 10 weeks showed an increase of 17.2 ± 1.6 g in body weight, compared to the 7.6 ± 0.8 g observed in mice fed the standard diet (Figure 3a). EPB‐53 treatment significantly reduced body weight gain (10.9 ± 1.3 g), whereas the reduction observed with metformin was of lower intensity (14.3 ± 1.8 g; Figure 3a). Drug treatment did not affect food intake (Figure S1a). The HFD also increased basal glucose levels, and this was prevented by EPB‐53 and metformin (Figure 3b). In addition, glucose intolerance caused by the HFD was prevented by EPB‐53 and by metformin (Figure 3c).

Figure 3.

Figure 3

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Oral administration of EPB‐53 reduces body weight gain and improves glucose intolerance in mice fed a high‐fat diet (HFD). Mice were fed standard chow, a HFD for 10 weeks, or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks. (a) Body weight and body weight gain. (b) Basal glucose levels. (c) Glucose tolerance test and AUC. Data are presented as the mean ± SD (n = 6 per group). *P < .05 versus control (CT) mice treated with the vehicle alone. # P < .05 versus mice fed a HFD and treated with the vehicle alone

Interestingly, EPB‐53 administration abolished the hepatic steatosis caused by the HFD, as demonstrated by ORO and haematoxylin–eosin staining and quantification of hepatic triglyceride levels (Figure 4a,b). In contrast, the trend towards a reduction in the accumulation of hepatic triglycerides caused by metformin did not reach statistical significance. Likewise, the 106% increase in serum triglyceride levels caused by the HFD was nearly completely abolished by treatment with EPB‐53, whereas metformin merely tended towards a slight reduction, which was not significant (P < .05 vs. mice fed the standard diet). Consistent with the reduction in hepatic triglyceride levels caused by EPB‐53, this compound also prevented the increase in serum ALT and AST caused by the HFD, whereas metformin only significantly reduced ALT (Figure 4d,e).

Figure 4.

Figure 4

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Oral administration of EPB‐53 prevents fatty liver in mice fed a high‐fat diet (HFD). Mice were fed standard chow, a HFD for 10 weeks, or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks (n = 6 per group). (a) Haematoxylin–eosin (H&E) and Oil Red O staining of livers. Scale bar: 100 μm. (b) Liver triglyceride levels. (c) Serum triglyceride levels. (d) Serum alanine aminotransferase (ALT) levels. (e) Serum aspartate aminotransferase (AST) levels. *P < .05 versus control (CT) mice treated with the vehicle alone. # P < .05 versus mice fed a HFD and treated with the vehicle alone. † P < .05 versus mice fed a HFD and treated with EPB‐53

When we examined the HRI–eIF2α pathway, we observed that EPB‐53 increased the levels of phospho‐HRI and phospho‐eIF2α, indicating that this compound activated this pathway, whereas metformin did not (Figure 5a). As expected, feeding mice a HFD increased the expression and serum levels of FGF21, and this was exacerbated in mice fed the HFD and treated with metformin (Figure 5b,c), which is consistent with the reported effects of metformin on FGF21 in liver and plasma (K. H. Kim et al., 2013). Surprisingly, mice fed the HFD and treated with EPB‐53 showed Fgf21 mRNA and serum levels similar to those present in the control group (Figure 5b,c). However, this is consistent with the lack of hepatic steatosis in both groups, since FGF21 levels reflect the depositing of fat in the liver (Hale et al., 2012; Maratos‐Flier, 2017). Moreover, when we assessed the expression of two FGF21 target genes negatively regulated by this hormone, 3‐β‐hydroxysteroid dehydrogenase type 5 (Hsd3b5), and major urinary protein 1 (Mup1; Inagaki et al., 2008), we observed that the expression of these genes was reduced in the liver of mice fed the HFD and treated with EPB‐53 (Figure 5d,e), suggesting a previous increase in the activity of FGF21. In contrast, the increase in FGF21 in mice fed the HFD or the higher increase in mice fed the HFD and treated with metformin did not reduce the expression of these genes, suggesting the presence of FGF21 resistance.

Figure 5.

Figure 5

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Oral administration of EPB‐53 increases the hepatic levels of phospho‐HRI and phospho‐eIF2α in mice fed a high‐fat diet (HFD). Mice were fed a standard chow, a HFD for 10 weeks, or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks. (a) Liver cell lysate extracts were assayed via western blot analysis with antibodies against total and phospho‐HRI, and total and phospho‐eIF2α. (b) Fgf21 mRNA abundance in the liver. (c) Serum FGF21 levels. (d) Mup1 mRNA abundance in the liver. (e) Hsd3b5 mRNA abundance in the liver. (f) Liver cell lysates extracts were assayed via western blot analysis with antibodies against total and phospho‐AMPK (p‐AMPK), and very‐LDL receptor (VLDLR). Data are presented as the mean ± SD (n = 6 per group) relative to control (CT) mice. *P < .05 versus CT mice treated with the vehicle alone. # P < .05 versus mice exposed to an HFD and treated with the vehicle alone. † P < .05 versus mice fed a HFD and treated with EPB‐53

Metformin acts via AMPK‐dependent mechanisms, although additional mechanisms have been reported (Rena, Hardie, & Pearson, 2017). Consistent with this, treatment with this drug increased hepatic phospho‐AMPK levels (Figure 5f). EPB‐53 did not significantly affect AMPK phosphorylation, but it increased the protein levels of the ATF4 target gene VLDLR (Figure 5f). Neither the HFD nor the drug treatment affected the expression of the ATF4 target gene Trb3 or β‐klotho (Figure S1b,c). In contrast, EBP‐53 significantly reduced the mRNA levels of Chop, and both HFD and EPB‐53 reduced the expression of Fgfr1c (Figure S1d,e, respectively). When we examined the expression of genes involved in fatty acid oxidation, the mRNA levels of Pparα and Cpt‐1α were up‐regulated by the HFD, whereas the expression of the latter gene was not further increased by either EPB‐53 or metformin treatment (Figure 6a). These findings suggest that an increase in fatty acid oxidation is not involved in the effects of EPB‐53 in hepatic steatosis. When we examined the expression of lipogenic genes, we observed that mice fed the HFD and those fed the HFD and also treated with metformin showed an increase in the mRNA levels of the lipogenic transcription factor Srebp1c (Figure 6b), whereas this was not observed in mice treated with EPB‐53. Interestingly, EPB‐53 treatment reduced the expression of the lipogenic genes stearoyl‐CoA desaturase 1 (Scd1), Fas, and glycerol phosphate acyltransferase (Gpat; Figure 6b). EPB‐53 also reduced the expression of the transcription factor carbohydrate‐responsive element‐binding protein (ChREBP): a major mediator of glucose action on lipogenic gene expression and a key determinant of lipid synthesis in vivo (Postic, Dentin, Denechaud, & Girard, 2007). Consistent with the increase in hepatic triglycerides, the levels of the protein FAS were increased in the liver of mice fed the HFD, but this increase was attenuated by EPB‐53 (Figure 6c). Moreover, the protein levels of PPARγ and its target gene CD36, both involved in lipid accumulation, were increased in the livers of mice fed the HFD, and this increase was prevented by EPB‐53 but not by metformin. The effects of EPB‐53 seemed not to be mediated by the reduction in body weight, since when we treated cultured human Huh‐7 hepatoma cells with EPB‐53, a strong reduction in FAS and CD36 expression was observed (Figure 6d), which is consistent with the strong increase in FGF21 observed in these cells following exposure to EPB‐53 (Figure 1b). Exposing hepatocytes to EPB‐53 also increased the expression of VLDLR (Figure 6d).

Figure 6.

Figure 6

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Oral administration of EPB‐53 decreases hepatic lipogenesis in mice fed a high‐fat diet (HFD). Mice were fed standard chow, a HFD for 10 weeks, or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks. (a) Pgc‐1α, Pparα, Cpt‐1α, Acox1, and Mcad mRNA abundance in the liver. (b) Srebp1c, Scd1, Fas, Gpat, and Chrebp mRNA abundance in the liver. (c) Liver cell lysate extracts were assayed via western blot analysis with antibodies against FAS, PPARγ, and CD36. Data are presented as the mean ± SD (n = 6 per group) relative to control (CT) mice. (d) FAS, CD36, and VLDLR mRNA abundance in human Huh‐7 hepatocytes exposed to 10 μM EPB‐53 for 24 hr. mRNA levels are presented as the mean ± SD (n = 5 per group). *P < .05 versus CT mice or CT cells treated with the vehicle alone. # P < .05 versus mice fed a HFD and treated with the vehicle alone. † P < .05 versus mice fed a HFD and treated with EPB‐53

3.3. Effects of EPB‐53 on glucose intolerance, hepatic steatosis, and hypertriglyceridaemia are dependent on FGF21

Next, to examine whether EPB‐53 displayed dose–response behaviour, we fed mice a HFD for 3 weeks and for the last week the mice were treated with three different doses of EPB‐53 (100, 200, or 300 mg·kg−1·day−1). EPB‐53 showed a dose–response trend in the parameters assessed (glucose intolerance, liver triglyceride content, and hepatic Fgf21 and Fas expression; Figure 7a–d).

Figure 7.

Figure 7

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EPB‐53 shows a dose–response relationship. Mice were fed a standard chow or a high‐fat diet (HFD) for 3 weeks and the last week they received one daily p.o. gavage of the vehicle or three different doses of EPB‐53. (a) Glucose tolerance test and AUC. (b) Liver triglyceride levels. (c) Fgf21 and (d) Fas mRNA abundance in the liver. Data are presented as the mean ± SD (n = 5 per group). *P < .05 versus mice fed a standard diet and treated with the vehicle alone. # P < .05 versus mice fed an HFD and treated with EPB‐53

Finally, we examined whether the effects of EPB‐53 were dependent on FGF21 by taking advantage of the use of Fgf21‐knockout mice. In this experiment, both wild‐type and Fgf21‐knockout mice were fed a HFD for 3 weeks, the last week of which they were treated with either vehicle alone or EPB‐53. This shorter period of treatment was selected to examine whether EPB‐53 increased the expression and serum levels of FGF21 in mice fed a HFD. In fact, the HFD increased the mRNA levels of FGF21 in the liver of wild‐type mice and also the serum levels of this hormone (Figure 8a), but these changes were even higher in mice fed the HFD and treated with EPB‐53. When we examined glucose intolerance (Figure 8b) and hepatic steatosis by quantification of hepatic triglyceride levels and ORO and haematoxylin–eosin staining (Figure 8c,d), we observed that EPB‐53 ameliorated these changes in wild‐type mice fed a HFD but not in Fgf21‐knockout mice. Similarly, the reduction in serum triglycerides observed in wild‐type mice fed the HFD and treated with EPB‐53 compared with mice fed the HFD and treated with the vehicle alone disappeared in Fgf21‐knockout mice (Figure 8e). Finally, EPB‐53 attenuated the increase in serum levels of ALT and AST (Figure 8f,g) caused by the HFD in wild‐type mice, but this effect was absent in mice lacking FGF21.

Figure 8.

Figure 8

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The effects of the HRI activator on high‐fat diet (HFD)‐induced glucose intolerance and hepatic steatosis are dependent on FGF21. Fgf21 −/− mice and their wild‐type (WT) littermates (Fgf21 +/+) were fed a standard chow or a HFD for 3 weeks and for the last week they received one daily p.o. gavage of the vehicle or EPB‐53. (a) Fgf21 mRNA abundance in the liver and serum FGF21 levels. (b) Glucose tolerance test and AUC. (c) Liver triglyceride levels. (d) Haematoxylin–eosin (H&E) and Oil Red O staining of livers. Scale bar: 100 μm. (e) Serum triglyceride levels. (f) Serum alanine aminotransferase (ALT) and (g) aspartate aminotransferase (AST) levels. Data are presented as the mean ± SD (n = 5 per group). *P < .05 versus WT mice fed a standard diet and treated with the vehicle alone. # P < .05 versus WT mice fed a HFD and treated with the vehicle alone. † P < .05 versus Fgf21‐null mice fed a standard diet and treated with the vehicle alone

4. DISCUSSION

FGF21 is a potential new target for obesity, T2DM, and associated co‐morbidities (Gimeno & Moller, 2014). This has aroused interest in the potential of FGF21 to treat metabolic diseases. However, the use of wild‐type native FGF21 is challenging as it has several limitations. One of these limitations is its short half‐life (Kharitonenkov et al., 2007), which has led to the development of long‐acting FGF21 analogues with prolonged pharmacodynamic effects compared to native FGF21 (Huang et al., 2013; Hecht et al., 2012). However, this increase in the potency of long‐acting FGF21 analogues may exacerbate some of the unwanted effects of FGF21, such as the reported increase in bone loss caused by this hormone (Wei et al., 2012). In fact, concerns raised following treatment with potent long‐acting FGF21 analogues in humans include changes in multiple markers of bone turnover (Talukdar et al., 2016) and an increase in both BP and heart rate (A. M. Kim, Somayaji, et al., 2017). Another limitation to the use of FGF21 in humans is the need for its administration to be s.c., because, due to its simplicity and convenience, p.o. administration improves patient compliance. Orally bioavailable drugs that increase the levels of native FGF21 might overcome all these limitations. We have previously reported that i.p. administration of HRI activators increases hepatic and plasma levels of FGF21 through activation of the eIF2α–ATF4 pathway (Zarei et al., 2016). Based on these findings, we have developed new p.o. bioavailable HRI activators with enhanced potency to increase FGF21 expression in human hepatocytes, and we selected EPB‐53 for an in vivo proof of concept. The administration of EPB‐53 for 4 days to normal mice increased the hepatic expression and circulating levels of FGF21 and slightly up‐regulated the expression of genes involved in fatty acid oxidation, whereas the expression of the lipogenic gene Fas was markedly down‐regulated. In mice fed a HFD for 10 weeks, administration of EPB‐53 for the last 2 weeks attenuated body weight gain caused by the HFD. This effect is consistent with the well‐known reported effect of FGF21 administration on body weight (Xu et al., 2009).

Treatment with EPB‐53 also reduced the serum levels of these lipids, whereas metformin did not. The reduction in serum triglycerides caused by EPB‐53 might be dependent, at least partially, on an increase in the levels of the ATF4‐target gene VLDLR (Jo et al., 2013). In fact, the up‐regulation of hepatic VLDLR via PPARα is required for the triglyceride‐lowering effect of fenofibrate (Gao et al., 2014). Thus, we can envisage that the up‐regulation of hepatic VLDLR by EPB‐53 increases the delivery of triglycerides transported by VLDL to the liver, reducing the availability of these lipids to be delivered to peripheral tissues, such as white adipose tissue. This action may contribute to the reduction in body weight. Notably, hepatic VLDLR up‐regulation following EPB‐53 treatment does not result in hepatic steatosis, suggesting that the uptake of triglycerides from plasma cannot compensate for the reduction in lipogenesis.

Both EPB‐53 and metformin also reduced glucose intolerance in mice fed a HFD. However, whereas metformin did not prevent either hepatic steatosis or hypertriglyceridaemia, EPB‐53 prevented both alterations completely. The reduction in hepatic steatosis in these mice seems to be the result of inhibition of hepatic lipogenesis, which is consistent with the effects observed following FGF21 administration (Wei et al., 2012). This effect of EPB‐53 was also observed in vitro in human hepatocytes. This finding rules out the possibility that the reduction in lipogenesis observed in vivo is secondary to body weight reduction. However, when we examined the levels of hepatic Fgf21 expression and serum levels in EPB‐53‐treated mice, no changes were observed compared to control mice, although the phosphorylated levels of HRI and eIF2α were increased. The lack of an increase in FGF21 in mice treated with EPB‐53 for 2 weeks might be explained by the following mechanism. As mentioned above, the administration of FGF21 reduces its expression in liver, as plasma and hepatic triglyceride levels decrease (Hale et al., 2012). This suggests that a similar mechanism might also operate with EPB‐53. Since EPB‐53 reduced hepatic triglyceride accumulation to values similar to those present in control mice, once the normal hepatic lipid content is achieved, the effect of EPB‐53 on FGF21 up‐regulation would be attenuated. In fact, a similar effect has been reported with inhibitors of fibroblast activation protein (FAP; Sánchez‐Garrido et al., 2016). This enzyme cleaves FGF21 and its inhibition increases FGF21 levels, thereby lowering body weight and improving glucose tolerance in mice fed a HFD, but these effects are much less intense in lean mice (Sánchez‐Garrido et al., 2016). Moreover, the reduction in the hepatic expression and serum levels of FGF21 might be explained by the presence of negative feedback by which enhanced levels of FGF21 inhibit the eIF2α–ATF4 pathway (Jiang et al., 2014). This negative feedback mechanism would control FGF21 levels, thereby avoiding excessive production of this hormone following EPB‐53 administration when plasma and hepatic triglyceride levels reach normal values. Notably, this feedback mechanism might avoid the development of the side effects (A. M. Kim, Somayaji, et al., 2017; Talukdar et al., 2016) reported with long‐acting FGF21 analogues due to overactivation of the FGF21 receptor. Surprisingly, and in contrast to EPB‐53, metformin treatment was accompanied by increased hepatic and circulating levels of FGF21, although this increase neither resulted in an amelioration of hepatic steatosis nor affected the expression of genes negatively regulated by FGF21, such as Hsd3b5 and Mup1, which were down‐regulated by EPB‐53. The increase in FGF21 levels following metformin treatment has been demonstrated to be dependent on the inhibition of mitochondrial complex I activity and the subsequent activation of the PKR‐like endoplasmic reticulum kinase (PERK)–eIF2α–ATF4 pathway (K. H. Kim et al., 2013). We currently have no explanation for the lack of effect of the increase in FGF21 levels caused by metformin compared to EPB‐53, although changes in the levels of total and intact serum FGF21 caused by differences in the activity of FAP might be implicated (Sánchez‐Garrido et al., 2016).

Confirmation of the dependence of EPB‐53 effects on FGF21 was obtained using Fgf21‐knockout mice. In mice deficient in FGF21, the effects of EPB‐53 were abolished. Oral administration of EPB‐53 for 1 week up‐regulated the hepatic expression and serum levels of FGF21, supporting the notion that for shorter treatments, EBP‐53 increases the levels of FGF21, whereas after longer treatments (2 weeks), this up‐regulation of FGF21 is attenuated.

Overall, the findings of this study demonstrate that p.o. administration of HRI activators is a potential strategy for the treatment of T2DM and NAFLD since it increases FGF21. Whether the use of HRI activators to increase FGF21 levels avoids the side effects reported with long‐acting FGF21 analogues remains to be studied by investigating the effects of long‐term treatment with these activators. In the clinical setting, the use of an oral drug to increase endogenous FGF21 levels might have advantages over FGF21 analogues for the treatment of insulin resistance, T2DM, and NAFLD.

CONFLICT OF INTEREST

M.Z., E.P., S.V., and M.V.‐C. are inventors of the Universitat de Barcelona patent application on HRI activators WO2018/010856. None of the other authors have competing interests.

AUTHOR CONTRIBUTIONS

M.Z., E.B., X.P., T.Q.‐L., J.P.‐D., and M.V.‐C. performed the experiments. E.P. and S.V. synthesized the compounds. M.V.‐C. and F.V. analysed the data and revised the results. M.Z. and M.V.‐C. designed the experiments and revised the results. M.V.‐C. was primarily responsible for writing the manuscript. All authors contributed to manuscript editing and approved the final version.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Data S1: Supporting information

Click here for additional data file. (115.2KB, pdf)

Figure S1: Effects of p.o. administration of EPB‐53 in mice fed a HFD. Mice were fed standard chow, a HFD for 10 weeks or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks (n = 6 per group). A, food intake. Trb3 (B), β‐Klotho (C), Chop (D), and Fgfr1c (E) mRNA abundance in the liver. Data are presented as the mean ± S.D. (n = 6 per group) relative to control mice. *p < 0.05 vs. control (CT) mice treated with the vehicle alone. #p < 0.05 vs. mice exposed to a HFD and treated with the vehicle alone. †p < 0.05 vs. mice fed a HFD and treated with EPB‐53.

Click here for additional data file. (844.2KB, jpg)

Data S2: Supporting information

Click here for additional data file. (74KB, pdf)

Table S1: Supporting information

Click here for additional data file. (108.8KB, pdf)

Table S2: Supporting information

Click here for additional data file. (100.2KB, pdf)

ACKNOWLEDGEMENT

We would like to thank the University of Barcelona's Language Advisory Service for revising the manuscript.

This study was partly supported by funds from the Spanish Ministry of the Economy and Competitiveness (SAF2015‐64146‐R and RTI2018‐093999‐B‐100) and the European Union European Regional Development Fund. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) and CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERObn) are Carlos III Health Institute projects. T.Q.‐L. is supported by a CONACyT (National Council for Science and Technology in Mexico) PhD scholarship. E.P. thanks the Institute of Biomedicine of the University of Barcelona (IBUB) for a PhD grant.

Zarei M, Pujol E, Quesada‐López T, et al. Oral administration of a new HRI activator as a new strategy to improve high‐fat‐diet‐induced glucose intolerance, hepatic steatosis, and hypertriglyceridaemia through FGF21. Br J Pharmacol. 2019;176:2292–2305. 10.1111/bph.14678

REFERENCES

  1. Alexander, S. P. H. , Cidlowski, J. A. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , … CGTP Collaborators . (2017). The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology, 174(S1), S208–S224. 10.1111/bph.13880 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander, S. P. H. , Fabbro, D. , Kelly, E. , Marrion, N. V. , Peters, J. A. , Faccenda, E. , … CGTP Collaborators . (2017). The Concise Guide to PHARMACOLOGY 2017/18: Enzymes. British Journal of Pharmacology, 174(S1), S272–S359. 10.1111/bph.13877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Badman, M. K. , Kennedy, A. R. , Adams, A. C. , Pissios, P. , & Maratos‐Flier, E. (2009). A very low carbohydrate ketogenic diet improves glucose tolerance in ob/ob mice independently of weight loss. American Journal of Physiology. Endocrinology and Metabolism, 297, E1197–E1204. 10.1152/ajpendo.00357.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chavez, A. O. , Molina‐Carrion, M. , Abdul‐Ghani, M. A. , Folli, F. , Defronzo, R. A. , & Tripathy, D. (2009). Circulating fibroblast growth factor‐21 is elevated in impaired glucose tolerance and type 2 diabetes and correlates with muscle and hepatic insulin resistance. Diabetes Care, 32, 1542–1546. 10.2337/dc09-0684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen, T. , Takrouri, K. , Hee‐Hwang, S. , Rana, S. , Yefidoff‐Freedman, R. , Halperin, J. , … Aktas, B. H. (2013). Explorations of substituted urea functionality for the discovery of new activators of the heme‐regulated inhibitor kinase. Journal of Medicinal Chemistry, 56, 9457–9470. 10.1021/jm400793v [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen, T. , Ozel, D. , Qiao, Y. , Harbinski, F. , Chen, L. , Denoyelle, S. , … Aktas, B. H. (2011). Chemical genetics identify eIF2α kinase heme‐regulated inhibitor as an anticancer target. Nature Chemical Biology, 7(9), 610–616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen, W. W. , Li, L. , Yang, G. Y. , Li, K. , Qi, X. Y. , Zhu, W. , … Boden, G. (2008). Circulating FGF‐21 levels in normal subjects and in newly diagnose patients with type 2 diabetes mellitus. Experimental and Clinical Endocrinology & Diabetes, 116, 65–68. [DOI] [PubMed] [Google Scholar]
  8. Coskun, T. , Bina, H. A. , Schneider, M. A. , Dunbar, J. D. , Hu, C. C. , Chen, Y. , … Kharitonenkov, A. (2008). Fibroblast growth factor 21 corrects obesity in mice. Endocrinology, 149, 6018–6027. 10.1210/en.2008-0816 [DOI] [PubMed] [Google Scholar]
  9. De Sousa‐Coelho, A. L. , Marrero, P. F. , & Haro, D. (2012). Activating transcription factor 4‐dependent induction of FGF21 during amino acid deprivation. The Biochemical Journal, 443, 165–171. 10.1042/BJ20111748 [DOI] [PubMed] [Google Scholar]
  10. Ding, X. , Boney‐Montoya, J. , Owen, B. M. , Bookout, A. L. , Coate, K. C. , Mangelsdorf, D. J. , & Kliewer, S. A. (2012). βKlotho is required for fibroblast growth factor 21 effects on growth and metabolism. Cell Metabolism, 16, 387–393. 10.1016/j.cmet.2012.08.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dushay, J. , Chui, P. C. , Gopalakrishnan, G. S. , Varela‐Rey, M. , Crawley, M. , Fisher, F. M. , … Maratos‐Flier, E. (2010). Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology, 139, 456–463. 10.1053/j.gastro.2010.04.054 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fisher, F. M. , Chui, P. C. , Antonellis, P. J. , Bina, H. A. , Kharitonenkov, A. , Flier, J. S. , & Maratos‐Flier, E. (2010). Obesity is a fibroblast growth factor 21 (FGF21)‐resistant state. Diabetes, 59, 2781–2789. 10.2337/db10-0193 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fisher, F. M. , Estall, J. L. , Adams, A. C. , Antonellis, P. J. , Bina, H. A. , Flier, J. S. , … Maratos‐Flier, E. (2011). Integrated regulation of hepatic metabolism by fibroblast growth factor 21 (FGF21) in vivo. Endocrinology, 152, 2996–3004. 10.1210/en.2011-0281 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gao, Y. , Shen, W. , Lu, B. , Zhang, Q. , Hu, Y. , & Chen, Y. (2014). Upregulation of hepatic VLDLR via PPARα is required for the triglyceride‐lowering effect of fenofibrate. Journal of Lipid Research, 55, 1622–1633. 10.1194/jlr.M041988 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gimeno, R. E. , & Moller, D. E. (2014). FGF21‐based pharmacotherapy—Potential utility for metabolic disorders. Trends in Endocrinology and Metabolism, 25, 303–311. 10.1016/j.tem.2014.03.001 [DOI] [PubMed] [Google Scholar]
  16. Goetz, R. , Beenken, A. , Ibrahimi, O. A. , Kalinina, J. , Olsen, S. K. , Eliseenkova, A. V. , … Mohammadi, M. (2007). Molecular insights into the klotho‐dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Molecular and Cellular Biology, 27, 3417–3428. 10.1128/MCB.02249-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hale, C. , Chen, M. M. , Stanislaus, S. , Chinookoswong, N. , Hager, T. , Wang, M. , … Xu, J. (2012). Lack of overt FGF21 resistance in two mouse models of obesity and insulin resistance. Endocrinology, 153, 69–80. 10.1210/en.2010-1262 [DOI] [PubMed] [Google Scholar]
  18. Harding, S. D. , Sharman, J. L. , Faccenda, E. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucl Acids Res, 46, D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hecht, R. , Li, Y. S. , Sun, J. , Belouski, E. , Hall, M. , Hager, T. , … Xu, J. (2012). Rationale‐based engineering of a potent long‐acting FGF21 analog for the treatment of type 2 diabetes. PLoS ONE, 7, e49345 10.1371/journal.pone.0049345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huang, J. , Ishino, T. , Chen, G. , Rolzin, P. , Osothprarop, T. F. , Retting, K. , … Levin, N. (2013). Development of a novel long‐acting antidiabetic FGF21 mimetic by targeted conjugation to a scaffold antibody. The Journal of Pharmacology and Experimental Therapeutics, 346, 270–280. 10.1124/jpet.113.204420 [DOI] [PubMed] [Google Scholar]
  21. Inagaki, T. , Dutchak, P. , Zhao, G. , Ding, X. , Gautron, L. , Parameswara, V. , … Kliewer, S. A. (2007). Endocrine regulation of the fasting response by PPARα‐mediated induction of fibroblast growth factor 21. Cell Metabolism, 5, 415–425. 10.1016/j.cmet.2007.05.003 [DOI] [PubMed] [Google Scholar]
  22. Inagaki, T. , Lin, V. Y. , Goetz, R. , Mohammadi, M. , Mangelsdorf, D. J. , & Kliewer, S. A. (2008). Inhibition of growth hormone signaling by the fasting‐induced hormone FGF21. Cell Metabolism, 8, 77–83. 10.1016/j.cmet.2008.05.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jiang, S. , Yan, C. , Fang, Q. C. , Shao, M. L. , Zhang, Y. L. , Liu, Y. , … Jia, W. P. (2014). Fibroblast growth factor 21 is regulated by the IRE1α–XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress‐induced hepatic steatosis. The Journal of Biological Chemistry, 289, 29751–29765. 10.1074/jbc.M114.565960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jo, H. , Choe, S. S. , Shin, K. C. , Jang, H. , Lee, J. H. , Seong, J. K. , … Kim, J. B. (2013). Endoplasmic reticulum stress induces hepatic steatosis via increased expression of the hepatic very low‐density lipoprotein receptor. Hepatology, 57, 1366–1377. 10.1002/hep.26126 [DOI] [PubMed] [Google Scholar]
  25. Kersten, S. , & Stienstra, R. (2017). The role and regulation of the peroxisome proliferator activated receptor alpha in human liver. Biochimie, 136, 75–84. 10.1016/j.biochi.2016.12.019 [DOI] [PubMed] [Google Scholar]
  26. Kharitonenkov, A. , & DiMarchi, R. (2015). FGF21 revolutions: Recent advances illuminating FGF21 biology and medicinal properties. Trends in Endocrinology and Metabolism, 26, 608–617. 10.1016/j.tem.2015.09.007 [DOI] [PubMed] [Google Scholar]
  27. Kharitonenkov, A. , Shiyanova, T. L. , Koester, A. , Ford, A. M. , Micanovic, R. , Galbreath, E. J. , … Shanafelt, A. B. (2005). FGF‐21 as a novel metabolic regulator. The Journal of Clinical Investigation, 115, 1627–1635. 10.1172/JCI23606 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kharitonenkov, A. , Wroblewski, V. J. , Koester, A. , Chen, Y. F. , Clutinger, C. K. , Tigno, X. T. , … Etgen, G. J. (2007). The metabolic state of diabetic monkeys is regulated by fibroblast growth factor‐21. Endocrinology, 148, 774–781.774. 10.1210/en.2006-1168 [DOI] [PubMed] [Google Scholar]
  29. Kilkenny, C. , Browne, W. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: The ARRIVE guidelines. British Journal of Pharmacology, 160, 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kim, A. M. , Somayaji, V. R. , Dong, J. Q. , Rolph, T. P. , Weng, Y. , Chabot, J. R. , … Calle, R. A. (2017). Once‐weekly administration of a long‐acting fibroblast growth factor 21 analogue modulates lipids, bone turnover markers, blood pressure and body weight differently in obese people with hypertriglyceridaemia and in non‐human primates. Diabetes, Obesity & Metabolism, 19, 1762–1772. 10.1111/dom.13023 [DOI] [PubMed] [Google Scholar]
  31. Kim, K. H. , Jeong, Y. T. , Kim, S. H. , Jung, H. S. , Park, K. S. , Lee, H. Y. , & Lee, M. S. (2013). Metformin‐induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation. Biochemical and Biophysical Research Communications, 440, 76–81. 10.1016/j.bbrc.2013.09.026 [DOI] [PubMed] [Google Scholar]
  32. Kim, Y. S. , Kim, M. , Choi, M. Y. , Lee, D. H. , Roh, G. S. , Kim, H. J. , … Choi, W. S. (2017). Metformin protects against retinal cell death in diabetic mice. Biochemical and Biophysical Research Communications, 492, 397–403. 10.1016/j.bbrc.2017.08.087 [DOI] [PubMed] [Google Scholar]
  33. Lu, L. , Han, A. P. , & Chen, J. J. (2001). Translation initiation control by heme‐regulated eukaryotic initiation factor 2α kinase in erythroid cells under cytoplasmic stresses. Molecular and Cellular Biology, 21, 7971–7980. 10.1128/MCB.21.23.7971-7980.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maratos‐Flier, E. (2017). Fatty liver and FGF21 physiology. Experimental Cell Research, 360, 2–5. 10.1016/j.yexcr.2017.05.006 [DOI] [PubMed] [Google Scholar]
  35. Markan, K. R. , Naber, M. C. , Ameka, M. K. , Anderegg, M. D. , Mangelsdorf, D. J. , Kliewer, S. A. , … Potthoff, M. J. (2014). Circulating FGF21 is liver derived and enhances glucose uptake during refeeding and overfeeding. Diabetes, 63, 4057–4063. 10.2337/db14-0595 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mraz, M. , Bartlova, M. , Lacinova, Z. , Michalsky, D. , Kasalicky, M. , Haluzikova, D. , … Haluzik, M. (2009). Serum concentrations and tissue expression of a novel endocrine regulator fibroblast growth factor‐21 in patients with type 2 diabetes and obesity. Clinical Endocrinology, 71, 369–375. 10.1111/j.1365-2265.2008.03502.x [DOI] [PubMed] [Google Scholar]
  37. Muise, E. S. , Azzolina, B. , Kuo, D. W. , El‐Sherbeini, M. , Tan, Y. , Yuan, X. , … Wong, K. K. (2008). Adipose fibroblast growth factor 21 is up‐regulated by peroxisome proliferator‐activated receptor gamma and altered metabolic states. Molecular Pharmacology, 74, 403–412. 10.1124/mol.108.044826 [DOI] [PubMed] [Google Scholar]
  38. Ogawa, Y. , Kurosu, H. , Yamamoto, M. , Nandi, A. , Rosenblatt, K. P. , Goetz, R. , … Kuro‐o, M. (2007). βKlotho is required for metabolic activity of fibroblast growth factor 21. Proceedings of the National Academy of Sciences of the United States of America, 104, 7432–7437. 10.1073/pnas.0701600104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Postic, C. , Dentin, R. , Denechaud, P. D. , & Girard, J. (2007). ChREBP, a transcriptional regulator of glucose and lipid metabolism. Annual Review of Nutrition, 27, 179–192. 10.1146/annurev.nutr.27.061406.093618 [DOI] [PubMed] [Google Scholar]
  40. Reitman, M. L. (2013). FGF21 mimetic shows therapeutic promise. Cell Metabolism, 18, 307–309. 10.1016/j.cmet.2013.08.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rena, G. , Hardie, D. G. , & Pearson, E. R. (2017). The mechanisms of action of metformin. Diabetologia, 60, 1577–1585. 10.1007/s00125-017-4342-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sánchez‐Garrido, M. A. , Habegger, K. M. , Clemmensen, C. , Holleman, C. , Müller, T. D. , Perez‐Tilve, D. , … Kharitonenkov, A. (2016). Fibroblast activation protein (FAP) as a novel metabolic target. Mol Metab, 5, 1015–1024. 10.1016/j.molmet.2016.07.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Satapati, S. , He, T. , Inagaki, T. , Potthoff, M. , Merritt, M. E. , Esser, V. , … Burgess, S. C. (2008). Partial resistance to peroxisome proliferator‐activated receptor‐α agonists in ZDF rats is associated with defective hepatic mitochondrial metabolism. Diabetes, 57, 2012–2021. 10.2337/db08-0226 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Talukdar, S. , Zhou, Y. , Li, D. , Rossulek, M. , Dong, J. , Somayaji, V. , … Calle, R. A. (2016). A long‐acting FGF21 molecule, PF‐05231023, decreases body weight and improves lipid profile in non‐human primates and type 2 diabetic subjects. Cell Metabolism, 23, 427–440. 10.1016/j.cmet.2016.02.001 [DOI] [PubMed] [Google Scholar]
  45. Wei, W. , Dutchak, P. A. , Wang, X. , Ding, X. , Wang, X. , Bookout, A. L. , … Wan, Y. (2012). Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator‐activated receptor γ. Proceedings of the National Academy of Sciences of the United States of America, 109, 3143–3148. 10.1073/pnas.1200797109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weng, Y. , Chabot, J. R. , Bernardo, B. , Yan, Q. , Zhu, Y. , Brenner, M. B. , … Talukdar, S. (2015). Pharmacokinetics (PK), pharmacodynamics (PD) and integrated PK/PD modeling of a novel long acting FGF21 clinical candidate PF‐05231023 in diet‐induced obese and leptin‐deficient obese mice. PLoS ONE, 10, e0119104 10.1371/journal.pone.0119104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xu, J. , Lloyd, D. J. , Hale, C. , Stanislaus, S. , Chen, M. , Sivits, G. , … Veniant, M. M. (2009). Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet‐induced obese mice. Diabetes, 58, 250–259. 10.2337/db08-0392 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yilmaz, Y. , Eren, F. , Yonal, O. , Kurt, R. , Aktas, B. , Celikel, C. A. , … Avsar, E. (2010). Increased serum FGF21 levels in patients with nonalcoholic fatty liver disease. European Journal of Clinical Investigation, 40, 887–892. 10.1111/j.1365-2362.2010.02338.x [DOI] [PubMed] [Google Scholar]
  49. Zarei, M. , Barroso, E. , Leiva, R. , Barniol‐Xicota, M. , Pujol, E. , Escolano, C. , … Vázquez‐Carrera, M. (2016). Heme‐regulated eIF2α kinase modulates hepatic FGF21 and is activated by PPARβ/δ deficiency. Diabetes, 65, 3185–3199. 10.2337/db16-0155 [DOI] [PubMed] [Google Scholar]
  50. Zhang, X. , Yeung, D. C. , Karpisek, M. , Stejskal, D. , Zhou, Z. G. , Liu, F. , … Xu, A. (2008). Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes, 57, 1246–1253. 10.2337/db07-1476 [DOI] [PubMed] [Google Scholar]
  51. Zhang, Y. , Lei, T. , Huang, J. F. , Wang, S. B. , Zhou, L. L. , Yang, Z. Q. , & Chen, X. D. (2011). The link between fibroblast growth factor 21 and sterol regulatory element binding protein 1c during lipogenesis in hepatocytes. Molecular and Cellular Endocrinology, 342, 41–47. 10.1016/j.mce.2011.05.003 [DOI] [PubMed] [Google Scholar]
  52. Zinszner, H. , Kuroda, M. , Wang, X. , Batchvarova, N. , Lightfoot, R. T. , Remotti, H. , … Ron, D. (1998). CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes & Development, 12, 982–995. 10.1101/gad.12.7.982 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: Supporting information

Click here for additional data file. (115.2KB, pdf)

Figure S1: Effects of p.o. administration of EPB‐53 in mice fed a HFD. Mice were fed standard chow, a HFD for 10 weeks or a HFD for 10 weeks plus EPB‐53 or metformin during the last 2 weeks (n = 6 per group). A, food intake. Trb3 (B), β‐Klotho (C), Chop (D), and Fgfr1c (E) mRNA abundance in the liver. Data are presented as the mean ± S.D. (n = 6 per group) relative to control mice. *p < 0.05 vs. control (CT) mice treated with the vehicle alone. #p < 0.05 vs. mice exposed to a HFD and treated with the vehicle alone. †p < 0.05 vs. mice fed a HFD and treated with EPB‐53.

Click here for additional data file. (844.2KB, jpg)

Data S2: Supporting information

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Table S1: Supporting information

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Table S2: Supporting information

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