Research Article ATF4- and CHOP-Dependent Induction of...

Research ArticleATF4- and CHOP-Dependent Induction ofFGF21 through Endoplasmic Reticulum Stress

Xiao-shan Wan1 Xiang-hong Lu2 Ye-cheng Xiao3 Yuan Lin4

Hong Zhu5 Ting Ding2 Ying Yang3 Yan Huang3 Yi Zhang5 Yan-Long Liu3

Zhu-mei Xu3 Jian Xiao23 and Xiao-kun Li13

1 College of Basic Medical Sciences Jilin University Changchun 130021 China2 Translation Medicine Research Center Lishui Peoplersquos Hospital Wenzhou Medical University Lishui Zhejiang 323000 China3Molecular Pharmacology Research Center Key Laboratory of Biotechnology and Pharmaceutical EngineeringSchool of Pharmaceutical Science Wenzhou Medical University Wenzhou Zhejiang 325035 China

4Department of Ophthalmology Xiamen Hospital of Traditional Chinese Medicine Xiamen 361000 China5 Department of Endocrinology The First Affiliated Hospital Wenzhou Medical University Wenzhou 323000 China

Correspondence should be addressed to Jian Xiao xfxj2000126com and Xiao-kun Li xiaokunli163net

Received 16 January 2014 Accepted 22 April 2014 Published 11 May 2014

Academic Editor Yadong Wang

Copyright copy 2014 Xiao-shan Wan et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fibroblast growth factor 21 (FGF21) is an important endogenous regulator involved in the regulation of glucose and lipidmetabolism FGF21 expression is strongly induced in animal and human subjects with metabolic diseases but little is known aboutthe molecular mechanism Endoplasmic reticulum (ER) stress plays an essential role in metabolic homeostasis and is observedin numerous pathological processes including type 2 diabetes overweight nonalcoholic fatty liver disease (NAFLD) In thisstudy we investigate the correlation between the expression of FGF21 and ER stress We demonstrated that TG-induced ER stressdirectly regulated the expression and secretion of FGF21 in a dose- and time-dependent manner FGF21 is the target gene foractivating transcription factor 4 (ATF4) andCCAATenhancer binding protein homologous protein (CHOP) Suppression ofCHOPimpaired the transcriptional activation of FGF21 by TG-induced ER stress in CHOPminusminusmouse primary hepatocytes (MPH) andoverexpression of ATF4 and CHOP resulted in FGF21 promoter activation to initiate the transcriptional programme In mRNAstability assay we indicated that ER stress increased the half-life of mRNAof FGF21 significantly In conclusion FGF21 expression isregulated by ER stress via ATF- andCHOP-dependent transcriptionalmechanism and posttranscriptionalmechanism respectively

1 Introduction

Thefibroblast growth factor family contains 22members witha wide range of biological functions relevant to regulatingcell growth differentiation wound healing developmentand angiogenesis [1ndash3] Fibroblast growth factor 21 (FGF21)is a unique member of the FGF family and has broadmetabolic functions including stimulating glucose uptakeinsulin-independently and improving hyperglycemia anddyslipidemia [4ndash7] FGF21 has a protective effect on thepreservation of pancreatic 120573-cell function and promoteshepatic and peripheral insulin sensitivity via the preventionof lipolysis which improves insulin resistance [8ndash10] In

addition FGF21 can resist the diet-induced obesity andinduce fatty acid oxidation [8 11 12] At present FGF21 isconsidered as a novel metabolism regulator and has becomea focus of metabolic disease research

FGF21 is expressed predominantly in liver and to alower extent in white adipose tissue thymus skeletal muscleand pancreatic 120573-cells [4 9 13] Substantial clinical researchhas focused on detecting FGF21 expression levels in variouspathological states It has been reported that serum FGF21and hepatic mRNA expression levels in patients with NAFLDare significantly higher than levels in control subjects whichcorrelates with a substantial increase in liver triglyceridelevels [14ndash16] Plasma FGF21 was also found to elevate

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 807874 9 pageshttpdxdoiorg1011552014807874

2 BioMed Research International

in type 2 diabetic or impaired glucose tolerance patients[17ndash19] Circulating FGF21 levels were significantly higherin overweight subjects than those in lean individuals [2021] Animal studies have reported similar results showingincreased FGF21 mRNA levels and serum FGF21 concentra-tions in the hepatic and adipose tissue of high fat diet-inducedand genetically obese mice compared with wild-type mice[6 8 22] An increase in FGF21 mRNA levels is similarlyinduced by fasting [23ndash25] It seems likely that FGF21 levelsare unchanged in different physiological states but increasedwith stress in individuals who are either overweight or havetype 2 diabetes or NAFLD Based on these findings wepropose that the mechanism of increased FGF21 levels inmetabolism disease may be due to feedback regulation butthe mechanism responsible for the effect is still unclear

Numerous studies indicated that ER stress was closelyrelated to metabolic diseases and it contributed to triggeringinsulin resistance obesity and type 2 diabetes [26ndash29] ERis the site of synthesis folding and routing of proteins andit plays a prominent role in maintaining Ca2+ homeostasisin the cytosol ER stress is a compensatory process thataims to preserve cellular functions and survival and induceby hypoxia toxicity infection unfold protein accumula-tion and perturbation of Ca2+ homeostasis [30] ER stresstransducers including PKR-like ER kinase (PERK) acti-vating transcription factor 6 (ATF6) and inositol-requiringenzyme 1 (IRE1) can be activated [31] Phosphorylationof eukaryotic initiation factor 120572 (eIF2120572) via activationby PERK leads to translational induction of ATF4 BiP-free pATF6(p) is transported to the Golgi apparatus whereit is processed to a transcriptionally active nuclear formpATF6(N) Activated IRE1 site-specifically cleaves x-box-binding protein 1 (XBP1) mRNA precursor to create themature XBP1mRNA (XBP1-sp) ATF4 pATF6(N) andXBP1-sp then activate transcription of CCAAT enhancer bindingprotein homologous protein (CHOP) by binding to theappropriate promoter region and CHOP plays a crucialrole in ER stress-mediated apoptosis and in diseases includ-ing diabetes brain ischemia and neurodegenerative disease[32]

Several studies have shown that upregulation of FGF21is mediated by ATF4 under conditions causing cellularstress such as amino acid deprivation autophagy andmitochondrial dysfunction [33ndash36] ATF4 directly increasesFGF21 expression in cells with ER stress by binding toboth amino acid-responsive element 1 (AARE1) and aminoacid-responsive element 2 (AARE2) sequence on FGF21[35 37] ATF4 activates the CHOP gene downstreambut not much is known on the relationship betweenCHOP and FGF21 To investigate whether FGF21 is reg-ulated by ER stress via effects on AFT4 and CHOP weestablish an ER stress cell model using TG (thapsigar-gin) in which we detect FGF21 and ER stress-specificgene expression levels We then demonstrated that TG-induced ER stress upregulates the expression and secre-tion of FGF21 by influencing ATF4 and CHOP providinginsights on the mechanisms that link FGF21 and metabolicdiseases

2 Materials

Dulbeccorsquos modified Eaglersquos medium (DMEM) penicillin-streptomycin (p-s) newborn calf serum (NCS) and fetalbovine serum (FBS) were obtained from Gibco BRL (GrandIsland NY USA) TRIzol reagent was obtained from Invit-rogen (Carlsbad CA USA) High-Capacity cDNA ReverseTranscription Kits were obtained from Applied Biosys-tems (Foster City CA USA) QIAprep spin miniprepkits were obtained from Qiagen Restriction endonucleasesHind III and Xho I were purchased from NEB (IpswichMA USA) Vector pGL417-Luc Fugene HD reagents andLuciferase Assay System were obtained from Promega (Sun-nyvale CA USA) Mouse FGF21 ELISA Kits were obtainedfrom RampD Systems (Minneapolis MN USA) Isobutyl-1-methylxanthine (IBMX) Dexamethasone (DEX) InsulinThapsigargin (TG) Actinomycin D and all other chemicalreagents were obtained from Sigma-Aldrich (St Louis MO)

3 Methods

31 Cell Culture and Differentiation 3T3-L1 murinepreadipocytes were obtained from the American TypeCulture Collection (Manassas VA USA) Cells werecultured in DMEM containing 10 NCS and 1 p-s cellswere induced to differentiate with DMEM plus 10 FBS1 p-s 05mM IBMX 1 120583M of DEX and 17 120583M Insulinfor two days Then the induction medium was replaced byDMEMwith 10 FBS 1 p-s and 17 120583M Insulin for anothertwo days followed by 10 FBSDMEM medium which waschanged every two days After 5-6 additional days more than85 cells differentiated to mature adipocytes which can beused for the experiments

32 Isolation and Culture of Mouse Primary HepatocytesPrimary hepatocytes were isolated from C57BL6J wild type(WT) andCHOPknockout (CHOPminusminus)mice (male 8weeks)and cultured as described previously [38] Cells were main-tained in serum-free WilliamrsquoE medium containing 01120583MDex 1 penicillin and 1 120583M thyroxine Before treatmentcells were incubated at 37∘C in 5 CO

2for approximately

16 h or until they had attached

33 RNA Isolation and Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA wasextracted from 3T3-L1 adipocytes using the TRIzol reagentaccording to the manufacturerrsquos instructions Total RNA(2 120583g) was used as a template for first-strand cDNA synthesisusing the High-Capacity cDNA Reverse Transcription KitThe mRNA levels of ATF4 splicing of XBP1 (XBP1-sp)CHOP and FGF21 were quantified using the followingprimers ATF4 forward primer 51015840-CCT AGG TCT CTTAGA TGA CTA TCT GGA GG-31015840 ATF4 reverse primer51015840-CCA GGT CAT CCA TTC GAA ACA GAG CAT CG-31015840XBP1-sp forward primer 51015840-TGA GTC CGC AGC AGGTG-31015840 XBP1-sp reverse primer 51015840-GAC AGG GTC CAACTT GT-31015840 CHOP forward primer 51015840-GCT CCT GCC TTTCAC CTT GG-31015840 CHOP reverse primer 51015840-GGT TTT TGA

BioMed Research International 3

TTC TTC CTC TTC-31015840 FGF21 forward primer 51015840-GCAGTCCAGAAAGTC TCC-31015840 FGF21 reverse primer 51015840-TGTAAC CGT CCT CCA GCA G-31015840 iQ SYBR Green Supermixwas used as a fluorescent dye to detect the presence ofdouble-stranded DNA The mRNA levels of each target genewere normalized to an endogenous control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) GAPDH forwardprimer 51015840-GTC GTG GAT CTG ACG TGC C-31015840 GAPDHreverse primer 51015840-GAT GCC TGC TTC ACC ACC TT-31015840The ratio of normalized mean value for each treatment groupto vehicle control group (DMSO) was calculated

34 Enzyme-Linked Immunosorbent Assay (ELISA) of FGF213T3-L1 adipocytes were treated with TG (0 125 25 50 and100 nM) for 24 h or TG (100 nM) for 0 2 4 8 16 and24 h The accumulated FGF21 in the culture medium wasdetermined using ELISA Kit according to the manufacturerrsquosinstructions The total protein concentrations of viable cellswere determined using the Bio-Rad Protein Assay reagentThe total amounts of the FGF21 in medium were normalizedto the total protein amounts and reported as pgmg protein

35 Plasmids Construction and Luciferase Assay The mouseFGF21 promoter constructs minus1497+5 were generously pro-vided by Dr Wenke Feng (The University of LouisvilleLouisville USA) and subcloned into pGL417-Luc luciferasereport vector using Hind III and Xho I sites The expressionvector containing the coding sequence of ATF4 or CHOPwaspreserved in our laboratory All plasmids were propagatedin Escherichia coli DH5120572 and isolated using QIAprep spinminiprep kit (Qiagen) 293T cells were plated in 6-well plates24 h before transfection Cells were transfected with 2120583gof pGL417 promoter FGF21 (minus1497+5) 2120583g of ATF4 orCHOP expression vector using Fugene HD (Promega) 48 hafter transfection the cells were harvested and lysed andthe luciferase activity was measured using the LuciferaseAssay System (Promega) The transfection efficiency wasnormalized to cotransfection of 1 120583g of GFP vector

36 Assessment of FGF21 mRNA Stability 3T3-L1 matureadipocytes were treated with TG (100 nM)DTT or vehiclecontrol for 4 h then Actinomycin D (50 120583gmL) was addedto themedium (time 0 h)ThemRNA of the cells was isolatedafter added Actinomycin D for 05 1 2 4 and 6 h FGF21ATF4 XBP1-sp and CHOPmRNA levels were detected usingreal-time RT-PCR as described in the previous section theresults are expressed as the fold of themRNAvalue at the timeof Actinomycin D addition

37 Statistical Analysis All of the experiments were repeatedat least three times results were stated as the mean plusmnstandard error One-way ANOVA was employed to analyzethe differences between sets of data Statistics were performedusing GraphPad Pro A value of 119875 lt 005 was consideredsignificant

4 Results

41 ER Stress Increases FGF21 Expression To investigate theeffect of ER stress on FGF21 mRNA levels we treated 3T3-L1 adipocytes with TG a potent ER stress activator bydisturbing ER calcium homeostasis The mRNA levels of ERstress-specific genes (ATF4 XBP1-sp and CHOP) and FGF21were detected using real-time RT-PCR We observed thatTG increased FGF21mRNA expression in a time-dependentmanner (Figure 1(a)) However the expression levels at 24 hwere lower than those at 16 h perhaps due to cell toxic-ity As shown in Figure 1(b) after the 3T3-L1 adipocyteswere incubated with 125 25 and 100 nM TG for 16 h thelevels of FGF21 mRNA were significantly increased in aconcentration-dependent manner compared with the vehiclecontrol group

42 ER Stress Induces FGF21 Secretion Based on the abovefindings a model of TG-induced stress in 3T3-L1 adipocyteswas established and we used this model to examine whetherER stress increases FGF21 secretion Differentiated 3T3-L1cells were treated with TG the FGF21 protein levels in themedium were measured using ELISA As shown in Figures2(a) and 2(b) TG-induced ER stress led to increase insecreted FGF21 in a time- and dose-dependent manner TGinduced FGF21 protein level to a 40-fold rise at concentrationof 100 nM for 24 h

43 Knockout of CHOP Decreases FGF21 Expression CHOPis a major transcription factor involved in ER stress Todetermine whether CHOP expression contributes to ERstress-induced upregulation of FGF21 we isolatedMPH fromWT and CHOPminusminus mice and treated the cells with TG for24 h In WTMPH TG promoted the mRNA levels of CHOPand FGF21 However in CHOPminusminusMPH TG failed to induceFGF21 expression because ATF4 is upstream gene of CHOPand there is no effect of CHOP knockout on the activationof ATF4 Moreover much research indicated that ATF4 caninduce FGF21 expression under stress [33ndash37] As depictedin Figure 3 an absence of CHOP expression significantlyincreased FGF21 gene expression by 30 These resultsindicate that CHOP may be a key player in the mechanismby which TG-induces increased FGF21 expression in MPH

44 ATF4 and CHOP Increase FGF21 Promoter-Driven Tran-scription To address the mechanism of TG-induced stressregulating FGF21 expression we subcloned the FGF21 pro-moter (minus1497+5) into the pGL417-Luc luciferase report vec-tor andmeasured the ability of ATF4 or CHOP to regulate theactivation of the FGF21 promoter using a cotransfection assayA previous study has reported that FGF21 expression couldbe mimicked by overexpression of ATF4 [37] Unlike 3T3-L1cell line 293T cells can be transfected with high efficiency293T cells were cotransfected with pGL417-Luc luciferasereport vector which was inserted the mouse FGF21 pro-moter and expression vector for ATF4 or CHOP Luciferaseactivity was determined at 48 h after transfection Figure 4(a)demonstrates that compared with the control group ATF4


0

2

4

6

8

Relat

ive m

RNA

leve

ls(A

TF4)

Time (h)0 2 4 8 16 24

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowastlowastlowastlowast

Relat

ive m

RNA

leve

ls(X

BP1-

sp)

Time (h)0 2 4 8 16 24

0

20

40

60

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

2

4

6

8

Relat

ive m

RNA

leve

ls(F

GF2

1)

Time (h)0 2 4 8 16 24

lowastlowastlowast

Relat

ive m

RNA

leve

ls(C

HO

P)

Time (h)0 2 4 8 16 24

0

10

20

30

lowastlowast

lowastlowast

(a)

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(A

TF4)

0 25 50 100

Concentration (nM)

lowast

lowastlowast

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 25 50 100

Concentration (nM)

lowastlowastlowastlowast

Relat

ive m

RNA

leve

ls(X

BP1-

sp)

0

20

40

60

80

100

0 25 50 100

Concentration (nM)

lowastlowast

lowastlowastlowast

0 50 100 2000

5

10

15

20

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

lowast

lowastlowastlowast

(b)

Figure 1 ER stress increases FGF21 mRNA levels (a) 3T3-L1 adipocytes were treated with TG (100 nM) for 0 2 4 8 16 and 24 h (b) 3T3-L1adipocytes were treated with TG (25 50 and 100 nM) for 16 h Total cellular RNA was isolated The mRNA levels of ATF4 XBP1-sp CHOPand FGF21 were measured by real-time RT-PCR Values are mean plusmn SE of three independent experiments Statistical significance relative tovehicle control lowast

119875

lt 005 lowastlowast119875

lt 001 lowastlowastlowast119875

lt 0001


0 2 4 8 16 240

10

20

30

40

50

60

Time (h)

lowastlowastlowast

lowast

FGF2

1 le

vel

(pg

mg

prot

ein)

(a)

0 125 25 50 1000

10

20

30

40

50

lowastlowastlowast


TG (nM)

FGF2

1 le

vel

(pg

mg

prot

ein)

(b)

Figure 2 ER stress induces FGF21 secretion Differentiated 3T3-L1 cells were treated with 100 nMTG for 0 2 4 8 16 and 24 h (a) or differentconcentrations of TG for 24 h (b) at the end of treatment cell culture medium was collected The protein level of FGF21 was determined byELISA Values are mean plusmn SE of three independent experiments Statistical significance relative to vehicle control lowast

119875


lt 0001

0 50 100 2000

5

10

15

20

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

WTCHOPminusminus

(a)

0 50 100 2000

5

10

15

20

WT

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(F

GF2

1)

CHOPminusminus

(b)

Figure 3 Knockout of CHOP decreases FGF21 expression WT and CHOPminusminus mouse primary hepatocytes were treated for 24 h withincreasing concentration of TG Total cellular RNA was isolated and the mRNA levels of CHOP and FGF21 were measured by real-timeRT-PCR Values are mean plusmn SE of three independent experiments Statistical significance relative to WT vehicle control lowast

119875

lt 005lowastlowastlowast

119875

lt 0001 statistical significance relative of the same TG concentration between WT group and CHOPminusminus group 119875

lt 001

overexpression enhanced FGF21 promoter activitymore than3-fold This is consistent with the result of a previous study[37] that reported that there are two conservedATF4-bindingsites in the promoter region of the FGF21 gene and thatFGF21 expression can be mimicked by overexpression ofATF4 CHOP is one of the genes that is downstream of ATF4We hypothesized that CHOPwould induce FGF21 expressionsimilar to ATF4We transfectedHEK293 cells with the FGF21reporter construct and CHOP As shown in Figure 4(b) simi-lar toATF4 CHOPoverexpression significantly increased thetranscription of an FGF21 promoter-driven reporter Thesefindings indicate that ATF4 and CHOP upregulate FGF21

expression by activating the promoter in an environment ofTG-induced ER stress

45 ER Stress Increases FGF21 mRNA Stability Posttran-scriptional regulation is a major mechanism for the expres-sion of cytokines To determine whether TG- or DTT-(dithiothreitol-) induced ER stress increases FGF21 expres-sion by regulating mRNA stability we examined the effectsof TGDTT on the mRNA stability of FGF21 in 3T3-L1adipocytesThe results indicated that TG and DTT increasedthe half-life of mRNA of FGF21 significantly but had no effect


mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)

Figure 4 ATF4 and CHOP increase FGF21 promoter-driven transcription 293T cells were transfected with FGF21 promoter reporterconstruct along with the expression plasmid ATF4 or CHOP Values aremean plusmn SE of three independent experiments Statistical significancerelative to control vector lowast

119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)

Figure 5 ER stress increases FGF21 mRNA stability 3T3-L1 adipocytes were pretreated with 100 nMTGDTT or vehicle control (DMSO)for 4 h and then treated with 50120583gmL actinomycin D (time 0) Total cellular RNA was extracted at 0 05 1 2 4 and 6 h after actinomycinD addition FGF21 mRNA levels were determined by real-time RT-PCR Values are mean plusmn SE of three independent experiments

on ER stress-specific genes (Figure 5) This result suggestedTG- and DTT-induced ER stress activate FGF21 expressionby increasing mRNA stability specifically

5 Discussion

FGF21 acts as a hormone-like cytokine on multiple tissues tocoordinate carbohydrate and lipid metabolism [4] Clinicalresearch has shown that serum FGF21 levels are higher in

subjects who are overweight have NAFLD or are type 2diabetic [14ndash18 20] Similarly circulating FGF21 concentra-tions in dbdb mice were much higher than normal as werethe FGF21 mRNA levels in both the liver and white adiposetissue [6 8 22] Previous studies have reported that FGF21expression is mediated by several transcriptional activatorsand their DNA response elements Gene expression of FGF21is induced directly by PPAR120572 in response to starvation andketotic states and PPAR120572 agonists in liver [23 25] as well asin cultured adipocytes and adipose tissue by PPAR120574 [39ndash41]


Activation of the farnesoidX receptor (FXR) increased FGF21gene expression and secretion was mediated by FXRretinoidX receptor binding site in 51015840-flanking region of the FGF21gene [42] A study demonstrated that glucose activation ofcarbohydrate response element binding protein (ChREBP) isinvolved in the upregulation of FGF21 mRNA expression inliver [43] Retinoic acid receptor-related receptor 120572 (ROR120572)also induces expression and secretion of FGF21 and there isa canonical ROR response element in the proximal promoterof FGF21 gene that exhibits functional activity [44] PGC-1120572-mediated reduction of FGF21 expression is dependent on theexpression of its ligand ALAS-1 and Rev-Erb120572 [45]

In addition studies by Schaap et al suggest that FGF21expression is regulated by ER stress [37]The authors reportedthat FGF21 mRNA is increased by TG-induced ER stressin rat H4IIE cells and rat primary hepatocytes Moreoverintraperitoneal injection of the ER stressor tunicamycininduced hepatic FGF21 expression in mice and resulted inmarked elevation of serum FGF21 levels [37] Consistent withthese new findings we observed that TG-induced ER stresselevated FGF21 expression and secretion in murine 3T3-L1adipocytes along with increasing ATF4 expression

PERK (PKR-like ER kinase) is one of the major ER stresspathways PERK can induce CHOP via activating ATF4However there was no information regarding the regulationof FGF21 by CHOP We show for the first time that CHOPcan increase FGF21 expression by activating transcriptionvia promoter elements and enhancing mRNA stability in ERstress We analyzed mouse FGF21 (minus1497+5) promoter andconfirmed the absence of the conserved CHOP binding site51015840-(AG) (AG) TGCAAT (AC) CCC-31015840 Thus FGF21 wasnot directly responsive to CHOPdirectly To the contrary ourdata demonstrates that CHOP can induce the transcription ofa FGF21 promoter-driven reporter (Figure 4(b)) CHOPmayalso regulate the expression of FGF21 indirectly by activatingother cytokines and intracellular stress signaling pathwaysthough this remains to be determined conclusively

Gene expression can be regulated by posttranscriptionalcontrol of mRNA stability [46] The presence of AU-richelements (AREs) in the 31015840-untranslated region (31015840-UTR) isessential for stabilization or degradation of mRNA of inflam-matory factor [47] The RNA-binding proteins (RBPs) suchas HuR AUF1 and CUG-BP1 positively regulate stabilityof many target mRNA via binding AREs present in the 31015840-UTR [48 49] In this study we identified for the first timethat increased FGF21 mRNA stability through the binding ofRBPs to its target mRNAs is responsible for elevated FGF21levels by TG- orDTT-induced ER stress in differentiated 3T3-L1 cells

In conclusion these findings suggest that FGF21 is the tar-get gene for ATF4 and CHOP and transcription and mRNAstabilization are responsible for ATF4 and CHOP mediatedinduction of FGF21 expression in ER stressThus we indicateER stress is the key mechanism for regulating FGF21 inseveral metabolic diseases Moreover our studies provideimportant information about the FGF21 signaling pathwayand the clinical significance of FGF21 in the developmentof metabolic diseases Compared with WT MPH FGF21mRNA levels are reduced in CHOPminusminus MPH treated with

TG however the effects of CHOP overexpression on FGF21levels are not understood And it remains to be detectedthat the synergistic effect of ATF4 and CHOP on FGF21expression Moreover further prospective studies are neededto determine the specific RBPs and their binding sites inFGF21 31015840-UTR as well as the signaling pathway of CHOP-dependent activation of FGF21 in ER stress

Conflict of Interests

The authors confirm that the content of this paper has noconflict of interests

Acknowledgments

This work was supported by National Natural Science Fund-ing ofChina (81170813) ZhejiangNatural Science Foundation(Y2100048 Y2110328 and Y12H030018) Zhejiang ProvincialProject of KeyGroup (2010R50042) and Fujian Provincial forMedical Innovative Health Project (2011-CXB-44)

References

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[2] S K Olsen M Garbi N Zampieri et al ldquoFibroblast growthfactor (FGF) homologous factors share structural but notfunctional homology with FGFsrdquo The Journal of BiologicalChemistry vol 278 no 36 pp 34226ndash34236 2003

[3] P M Smallwood I Munoz-Sanjuan P Tong et al ldquoFibroblastgrowth factor (FGF) homologous factors new members ofthe FGF family implicated in nervous system developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 18 pp 9850ndash9857 1996

[4] A Kharitonenkov T L Shiyanova A Koester et al ldquoFGF-21as a novel metabolic regulatorrdquo Journal of Clinical Investigationvol 115 no 6 pp 1627ndash1635 2005

[5] A Kharitonenkov V J Wroblewski A Koester et al ldquoThemetabolic state of diabetic monkeys is regulated by fibroblastgrowth factor-21rdquo Endocrinology vol 148 no 2 pp 774ndash7812007

[6] T CoskunHA BinaMA Schneider et al ldquoFibroblast growthfactor 21 corrects obesity in micerdquo Endocrinology vol 149 no12 pp 6018ndash6027 2008

[7] J A Seo and N H Kim ldquoFibroblast growth factor 21 a novelmetabolic regulatorrdquoDiabetesampMetabolism Journal vol 36 no1 pp 26ndash28 2012

[8] J Xu D J Lloyd C Hale et al ldquoFibroblast growth factor21 reverses hepatic steatosis increases energy expenditureand improves insulin sensitivity in diet-induced obese micerdquoDiabetes vol 58 no 1 pp 250ndash259 2009

[9] W Wente A M Efanov M Brenner et al ldquoFibroblast growthfactor-21 improves pancreatic 120573-cell function and survival byactivation of extracellular signal-regulated kinase 12 and Aktsignaling pathwaysrdquo Diabetes vol 55 no 9 pp 2470ndash24782006

[10] P Arner A Pettersson P J Mitchell J D Dunbar A Kharito-nenkov and M Ryden ldquoFGF21 attenuates lipolysis in human


adipocytesmdasha possible link to improved insulin sensitivityrdquoFEBS Letters vol 582 no 12 pp 1725ndash1730 2008

[11] K Mai J Andres K Biedasek et al ldquoFree fatty acids linkmetabolism and regulation of the insulin-sensitizing fibroblastgrowth factor-21rdquo Diabetes vol 58 no 7 pp 1532ndash1538 2009

[12] Y Hotta H Nakamura M Konishi et al ldquoFibroblast growthfactor 21 regulates lipolysis in white adipose tissue but is notrequired for ketogenesis and triglyceride clearance in liverrdquoEndocrinology vol 150 no 10 pp 4625ndash4633 2009

[13] F L Mashili R L Austin A S Deshmukh et al ldquoDirect effectsof FGF21 on glucose uptake in human skeletal muscle impli-cations for type 2 diabetes and obesityrdquo DiabetesMetabolismResearch and Reviews vol 27 no 3 pp 286ndash297 2011

[14] H Li Q Fang F Gao et al ldquoFibroblast growth factor 21 levelsare increased in nonalcoholic fatty liver disease patients and arecorrelated with hepatic triglyceriderdquo Journal of Hepatology vol53 no 5 pp 934ndash940 2010

[15] J Dushay P C Chui G S Gopalakrishnan et al ldquoIncreasedfibroblast growth factor 21 in obesity andnonalcoholic fatty liverdiseaserdquo Gastroenterology vol 139 no 2 pp 456ndash463 2010

[16] Y Yilmaz F Eren O Yonal et al ldquoIncreased serum FGF21levels in patientswith nonalcoholic fatty liver diseaserdquoEuropeanJournal of Clinical Investigation vol 40 no 10 pp 887ndash8922010

[17] W-W Chen L Li G-Y Yang et al ldquoCirculating FGF-21 levelsin normal subjects and in newly diagnose patients with type 2diabetes mellitusrdquo Experimental and Clinical Endocrinology andDiabetes vol 116 no 1 pp 65ndash68 2008

[18] L Li G Yang H Ning M Yang H Liu andW Chen ldquoPlasmaFGF-21 levels in type 2 diabetic patients with ketosisrdquo DiabetesResearch and Clinical Practice vol 82 no 2 pp 209ndash213 2008

[19] A O Chavez M Molina-Carrion M A Abdul-Ghani FFolli R A DeFronzo and D Tripathy ldquoCirculating fibroblastgrowth factor-21 is elevated in impaired glucose tolerance andtype 2 diabetes and correlates with muscle and hepatic insulinresistancerdquo Diabetes Care vol 32 no 8 pp 1542ndash1546 2009

[20] X Zhang D C Y Yeung M Karpisek et al ldquoSerum FGF21levels are increased in obesity and are independently associatedwith the metabolic syndrome in humansrdquo Diabetes vol 57 no5 pp 1246ndash1253 2008

[21] MMrazM Bartlova Z Lacinova et al ldquoSerum concentrationsand tissue expression of a novel endocrine regulator fibroblastgrowth factor-21 in patients with type 2 diabetes and obesityrdquoClinical Endocrinology vol 71 no 3 pp 369ndash375 2009

[22] FM Fisher J L Estall ACAdams et al ldquoIntegrated regulationof hepatic metabolism by fibroblast growth factor 21 (FGF21) invivordquo Endocrinology vol 152 no 8 pp 2996ndash3004 2011

[23] M K Badman P Pissios A R Kennedy G Koukos J S Flierand E Maratos-Flier ldquoHepatic fibroblast growth factor 21 isregulated by PPAR120572 and is a key mediator of hepatic lipidmetabolism in ketotic statesrdquo Cell Metabolism vol 5 no 6 pp426ndash437 2007

[24] T Inagaki P Dutchak G Zhao et al ldquoEndocrine regulation ofthe fasting response by PPAR120572-mediated induction of fibroblastgrowth factor 21rdquo Cell Metabolism vol 5 no 6 pp 415ndash4252007

[25] T Lundasen M C Hunt L-M Nilsson et al ldquoPPAR120572 is akey regulator of hepatic FGF21rdquo Biochemical and BiophysicalResearch Communications vol 360 no 2 pp 437ndash440 2007

[26] U Ozcan Q Cao E Yilmaz et al ldquoEndoplasmic reticulumstress links obesity insulin action and type 2 diabetesrdquo Sciencevol 306 no 5695 pp 457ndash461 2004

[27] G S Hotamisligil ldquoEndoplasmic reticulum stress and theinflammatory basis of metabolic diseaserdquo Cell vol 140 no 6pp 900ndash917 2010

[28] L Ozcan and I Tabas ldquoRole of endoplasmic reticulum stressin metabolic disease and other disordersrdquo Annual Review ofMedicine vol 63 pp 317ndash328 2012

[29] S S Cao and R J Kaufman ldquoTargeting endoplasmic reticulumstress in metabolic diseaserdquo Expert Opinion on TherapeuticTargets vol 17 no 4 pp 437ndash448 2013

[30] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquo Genes and Development vol 13 no 10pp 1211ndash1233 1999

[31] S Oyadomari and M Mori ldquoRoles of CHOPGADD153 inendoplasmic reticulum stressrdquo Cell Death and Differentiationvol 11 no 4 pp 381ndash389 2004

[32] H Zinszner M Kuroda XWang et al ldquoCHOP is implicated inprogrammed cell death in response to impaired function of theendoplasmic reticulumrdquo Genes and Development vol 12 no 7pp 982ndash995 1998

[33] A L De Sousa-Coelho P F Marrero and D Haro ldquoActivatingtranscription factor 4-dependent induction of FGF21 duringamino acid deprivationrdquo Biochemical Journal vol 443 no 1 pp165ndash171 2012

[34] K H Kim Y T Jeong H Oh et al ldquoAutophagy deficiency leadsto protection from obesity and insulin resistance by inducingFgf21 as a mitokinerdquo Nature Medicine vol 19 no 1 pp 83ndash922013

[35] X Jiang C Zhang Y Xin et al ldquoProtective effect of FGF21 ontype 1 diabetes-induced testicular apoptotic cell death probablyvia both mitochondrial- and endoplasmic reticulum stress-dependent pathways in the mouse modelrdquo Toxicology Lettersvol 219 no 1 pp 65ndash76 2013

[36] Y Luo and W L McKeehan ldquoStressed liver and muscle call onadipocytes with FGF21rdquo Frontiers in Endocrinology vol 4 p194 2013

[37] F G Schaap A E Kremer W H Lamers et al ldquoFibroblastgrowth factor 21 is induced by endoplasmic reticulum stressrdquoBiochimie vol 95 no 4 pp 692ndash699 2013

[38] H Zhou E C Gurley S Jarujaron et al ldquoHIV proteaseinhibitors activate the unfolded protein response and disruptlipid metabolism in primary hepatocytesrdquo American Journal ofPhysiology vol 291 no 6 pp G1071ndashG1080 2006

[39] E S Muise B Azzolina D W Kuo et al ldquoAdipose fibroblastgrowth factor 21 is up-regulated by peroxisome proliferator-activated receptor 120574 and altered metabolic statesrdquo MolecularPharmacology vol 74 no 2 pp 403ndash412 2008

[40] J S Moyers T L Shiyanova F Mehrbod et al ldquoMoleculardeterminants of FGF-21 activitymdashsynergy and cross-talk withPPAR120574 signalingrdquo Journal of Cellular Physiology vol 210 no 1pp 1ndash6 2007

[41] HWang LQiang and S R Farmer ldquoIdentification of a domainwithin peroxisome proliferator-activated receptor 120574 regulatingexpression of a group of genes containing fibroblast growthfactor 21 that are selectively repressed by SIRT1 in adipocytesrdquoMolecular and Cellular Biology vol 28 no 1 pp 188ndash200 2008

[42] H A Cyphert X Ge A B Kohan et al ldquoActivation of the far-nesoid X receptor induces hepatic expression and secretion offibroblast growth factor 21rdquoThe Journal of Biological Chemistryvol 287 no 30 pp 25123ndash25138 2012


[43] K Iizuka J Takeda and Y Horikawa ldquoGlucose induces FGF21mRNA expression through ChREBP activation in rat hepato-cytesrdquo FEBS Letters vol 583 no 17 pp 2882ndash2886 2009

[44] Y Wang L A Solt and T P Burris ldquoRegulation of FGF21expression and secretion by retinoic acid receptor-relatedorphan receptor 120572rdquoThe Journal of Biological Chemistry vol 285no 21 pp 15668ndash15673 2010

[45] J L Estall J L Ruas C S Choi et al ldquoPGC-1120572 negatively reg-ulates hepatic FGF21 expression by modulating the hemeRev-Erb120572 axisrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 106 no 52 pp 22510ndash225152009

[46] B Schwanhuusser D Busse N Li et al ldquoGlobal quantificationof mammalian gene expression controlrdquo Nature vol 473 no7347 pp 337ndash342 2011

[47] C M Brennan and J A Steitz ldquoHuR and mRNA stabilityrdquoCellular and Molecular Life Sciences vol 58 no 2 pp 266ndash2772001

[48] I Raineri D Wegmueller B Gross U Certa and C MoronildquoRoles of AUF1 isoforms HuR and BRF1 in ARE-dependentmRNA turnover studied by RNA interferencerdquo Nucleic AcidsResearch vol 32 no 4 pp 1279ndash1288 2004

[49] J-Y Lu and R J Schneider ldquoTissue dstribution of AU-richmRNA-binding proteins involved in regulation of mRNAdecayrdquo The Journal of Biological Chemistry vol 279 no 13 pp12974ndash12979 2004

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


in type 2 diabetic or impaired glucose tolerance patients[17ndash19] Circulating FGF21 levels were significantly higherin overweight subjects than those in lean individuals [2021] Animal studies have reported similar results showingincreased FGF21 mRNA levels and serum FGF21 concentra-tions in the hepatic and adipose tissue of high fat diet-inducedand genetically obese mice compared with wild-type mice[6 8 22] An increase in FGF21 mRNA levels is similarlyinduced by fasting [23ndash25] It seems likely that FGF21 levelsare unchanged in different physiological states but increasedwith stress in individuals who are either overweight or havetype 2 diabetes or NAFLD Based on these findings wepropose that the mechanism of increased FGF21 levels inmetabolism disease may be due to feedback regulation butthe mechanism responsible for the effect is still unclear

Numerous studies indicated that ER stress was closelyrelated to metabolic diseases and it contributed to triggeringinsulin resistance obesity and type 2 diabetes [26ndash29] ERis the site of synthesis folding and routing of proteins andit plays a prominent role in maintaining Ca2+ homeostasisin the cytosol ER stress is a compensatory process thataims to preserve cellular functions and survival and induceby hypoxia toxicity infection unfold protein accumula-tion and perturbation of Ca2+ homeostasis [30] ER stresstransducers including PKR-like ER kinase (PERK) acti-vating transcription factor 6 (ATF6) and inositol-requiringenzyme 1 (IRE1) can be activated [31] Phosphorylationof eukaryotic initiation factor 120572 (eIF2120572) via activationby PERK leads to translational induction of ATF4 BiP-free pATF6(p) is transported to the Golgi apparatus whereit is processed to a transcriptionally active nuclear formpATF6(N) Activated IRE1 site-specifically cleaves x-box-binding protein 1 (XBP1) mRNA precursor to create themature XBP1mRNA (XBP1-sp) ATF4 pATF6(N) andXBP1-sp then activate transcription of CCAAT enhancer bindingprotein homologous protein (CHOP) by binding to theappropriate promoter region and CHOP plays a crucialrole in ER stress-mediated apoptosis and in diseases includ-ing diabetes brain ischemia and neurodegenerative disease[32]

Several studies have shown that upregulation of FGF21is mediated by ATF4 under conditions causing cellularstress such as amino acid deprivation autophagy andmitochondrial dysfunction [33ndash36] ATF4 directly increasesFGF21 expression in cells with ER stress by binding toboth amino acid-responsive element 1 (AARE1) and aminoacid-responsive element 2 (AARE2) sequence on FGF21[35 37] ATF4 activates the CHOP gene downstreambut not much is known on the relationship betweenCHOP and FGF21 To investigate whether FGF21 is reg-ulated by ER stress via effects on AFT4 and CHOP weestablish an ER stress cell model using TG (thapsigar-gin) in which we detect FGF21 and ER stress-specificgene expression levels We then demonstrated that TG-induced ER stress upregulates the expression and secre-tion of FGF21 by influencing ATF4 and CHOP providinginsights on the mechanisms that link FGF21 and metabolicdiseases

2 Materials

Dulbeccorsquos modified Eaglersquos medium (DMEM) penicillin-streptomycin (p-s) newborn calf serum (NCS) and fetalbovine serum (FBS) were obtained from Gibco BRL (GrandIsland NY USA) TRIzol reagent was obtained from Invit-rogen (Carlsbad CA USA) High-Capacity cDNA ReverseTranscription Kits were obtained from Applied Biosys-tems (Foster City CA USA) QIAprep spin miniprepkits were obtained from Qiagen Restriction endonucleasesHind III and Xho I were purchased from NEB (IpswichMA USA) Vector pGL417-Luc Fugene HD reagents andLuciferase Assay System were obtained from Promega (Sun-nyvale CA USA) Mouse FGF21 ELISA Kits were obtainedfrom RampD Systems (Minneapolis MN USA) Isobutyl-1-methylxanthine (IBMX) Dexamethasone (DEX) InsulinThapsigargin (TG) Actinomycin D and all other chemicalreagents were obtained from Sigma-Aldrich (St Louis MO)

3 Methods

31 Cell Culture and Differentiation 3T3-L1 murinepreadipocytes were obtained from the American TypeCulture Collection (Manassas VA USA) Cells werecultured in DMEM containing 10 NCS and 1 p-s cellswere induced to differentiate with DMEM plus 10 FBS1 p-s 05mM IBMX 1 120583M of DEX and 17 120583M Insulinfor two days Then the induction medium was replaced byDMEMwith 10 FBS 1 p-s and 17 120583M Insulin for anothertwo days followed by 10 FBSDMEM medium which waschanged every two days After 5-6 additional days more than85 cells differentiated to mature adipocytes which can beused for the experiments

32 Isolation and Culture of Mouse Primary HepatocytesPrimary hepatocytes were isolated from C57BL6J wild type(WT) andCHOPknockout (CHOPminusminus)mice (male 8weeks)and cultured as described previously [38] Cells were main-tained in serum-free WilliamrsquoE medium containing 01120583MDex 1 penicillin and 1 120583M thyroxine Before treatmentcells were incubated at 37∘C in 5 CO

2for approximately

16 h or until they had attached

33 RNA Isolation and Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA wasextracted from 3T3-L1 adipocytes using the TRIzol reagentaccording to the manufacturerrsquos instructions Total RNA(2 120583g) was used as a template for first-strand cDNA synthesisusing the High-Capacity cDNA Reverse Transcription KitThe mRNA levels of ATF4 splicing of XBP1 (XBP1-sp)CHOP and FGF21 were quantified using the followingprimers ATF4 forward primer 51015840-CCT AGG TCT CTTAGA TGA CTA TCT GGA GG-31015840 ATF4 reverse primer51015840-CCA GGT CAT CCA TTC GAA ACA GAG CAT CG-31015840XBP1-sp forward primer 51015840-TGA GTC CGC AGC AGGTG-31015840 XBP1-sp reverse primer 51015840-GAC AGG GTC CAACTT GT-31015840 CHOP forward primer 51015840-GCT CCT GCC TTTCAC CTT GG-31015840 CHOP reverse primer 51015840-GGT TTT TGA







4 Results






0

2

4

6

8

Relat

ive m

RNA

leve

ls(A

TF4)

Time (h)0 2 4 8 16 24

lowastlowastlowast



Relat

ive m

RNA

leve

ls(X

BP1-

sp)

Time (h)0 2 4 8 16 24

0

20

40

60

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

2

4

6

8

Relat

ive m

RNA

leve

ls(F

GF2

1)

Time (h)0 2 4 8 16 24

lowastlowastlowast

Relat

ive m

RNA

leve

ls(C

HO

P)

Time (h)0 2 4 8 16 24

0

10

20

30

lowastlowast

lowastlowast

(a)

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(A

TF4)

0 25 50 100

Concentration (nM)

lowast

lowastlowast

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 25 50 100

Concentration (nM)


Relat

ive m

RNA

leve

ls(X

BP1-

sp)

0

20

40

60

80

100

0 25 50 100

Concentration (nM)

lowastlowast

lowastlowastlowast

0 50 100 2000

5

10

15

20

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

lowast

lowastlowastlowast

(b)


119875



lt 0001


0 2 4 8 16 240

10

20

30

40

50

60

Time (h)

lowastlowastlowast

lowast

FGF2

1 le

vel

(pg

mg

prot

ein)

(a)

0 125 25 50 1000

10

20

30

40

50

lowastlowastlowast


TG (nM)

FGF2

1 le

vel

(pg

mg

prot

ein)

(b)


119875


lt 0001

0 50 100 2000

5

10

15

20

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

WTCHOPminusminus

(a)

0 50 100 2000

5

10

15

20

WT

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(F

GF2

1)

CHOPminusminus

(b)


119875


119875


lt 001





mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)


119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)



5 Discussion












Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







4 Results






0

2

4

6

8

Relat

ive m

RNA

leve

ls(A

TF4)

Time (h)0 2 4 8 16 24

lowastlowastlowast



Relat

ive m

RNA

leve

ls(X

BP1-

sp)

Time (h)0 2 4 8 16 24

0

20

40

60

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

2

4

6

8

Relat

ive m

RNA

leve

ls(F

GF2

1)

Time (h)0 2 4 8 16 24

lowastlowastlowast

Relat

ive m

RNA

leve

ls(C

HO

P)

Time (h)0 2 4 8 16 24

0

10

20

30

lowastlowast

lowastlowast

(a)

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(A

TF4)

0 25 50 100

Concentration (nM)

lowast

lowastlowast

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 25 50 100

Concentration (nM)


Relat

ive m

RNA

leve

ls(X

BP1-

sp)

0

20

40

60

80

100

0 25 50 100

Concentration (nM)

lowastlowast

lowastlowastlowast

0 50 100 2000

5

10

15

20

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

lowast

lowastlowastlowast

(b)


119875



lt 0001


0 2 4 8 16 240

10

20

30

40

50

60

Time (h)

lowastlowastlowast

lowast

FGF2

1 le

vel

(pg

mg

prot

ein)

(a)

0 125 25 50 1000

10

20

30

40

50

lowastlowastlowast


TG (nM)

FGF2

1 le

vel

(pg

mg

prot

ein)

(b)


119875


lt 0001

0 50 100 2000

5

10

15

20

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

WTCHOPminusminus

(a)

0 50 100 2000

5

10

15

20

WT

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(F

GF2

1)

CHOPminusminus

(b)


119875


119875


lt 001





mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)


119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)



5 Discussion












Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

2

4

6

8

Relat

ive m

RNA

leve

ls(A

TF4)

Time (h)0 2 4 8 16 24

lowastlowastlowast



Relat

ive m

RNA

leve

ls(X

BP1-

sp)

Time (h)0 2 4 8 16 24

0

20

40

60

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

2

4

6

8

Relat

ive m

RNA

leve

ls(F

GF2

1)

Time (h)0 2 4 8 16 24

lowastlowastlowast

Relat

ive m

RNA

leve

ls(C

HO

P)

Time (h)0 2 4 8 16 24

0

10

20

30

lowastlowast

lowastlowast

(a)

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(A

TF4)

0 25 50 100

Concentration (nM)

lowast

lowastlowast

0

2

4

6

8

10

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 25 50 100

Concentration (nM)


Relat

ive m

RNA

leve

ls(X

BP1-

sp)

0

20

40

60

80

100

0 25 50 100

Concentration (nM)

lowastlowast

lowastlowastlowast

0 50 100 2000

5

10

15

20

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

lowast

lowastlowastlowast

(b)


119875



lt 0001


0 2 4 8 16 240

10

20

30

40

50

60

Time (h)

lowastlowastlowast

lowast

FGF2

1 le

vel

(pg

mg

prot

ein)

(a)

0 125 25 50 1000

10

20

30

40

50

lowastlowastlowast


TG (nM)

FGF2

1 le

vel

(pg

mg

prot

ein)

(b)


119875


lt 0001

0 50 100 2000

5

10

15

20

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

WTCHOPminusminus

(a)

0 50 100 2000

5

10

15

20

WT

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(F

GF2

1)

CHOPminusminus

(b)


119875


119875


lt 001





mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)


119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)



5 Discussion












Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 2 4 8 16 240

10

20

30

40

50

60

Time (h)

lowastlowastlowast

lowast

FGF2

1 le

vel

(pg

mg

prot

ein)

(a)

0 125 25 50 1000

10

20

30

40

50

lowastlowastlowast


TG (nM)

FGF2

1 le

vel

(pg

mg

prot

ein)

(b)


119875


lt 0001

0 50 100 2000

5

10

15

20

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(C

HO

P)

WTCHOPminusminus

(a)

0 50 100 2000

5

10

15

20

WT

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

Concentration (nM)

Relat

ive m

RNA

leve

ls(F

GF2

1)

CHOPminusminus

(b)


119875


119875


lt 001





mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)


119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)



5 Discussion












Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


mFGF21-promoter

Control ATF-40

100

200

300

400

500

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)(minus1497+5)

(a)

Control CHOP0

200

400

600

800mFGF21-promoter

lowast

Nor

mal

ized

luci

fera

se ac

tivity

( co

ntro

l)

(minus1497+5)

(b)


119875

lt 005

ControlTGDTT

Relat

ive m

RNA

leve

ls(F

GF2

1)

0 1 2 3 4 5 6Time (h)

00

02

04

06

08

10

12

(a)

0 1 2 3 4 5 600

02

04

06

08

10

12

Time (h)

Relat

ive m

RNA

leve

ls(A

TF4)

ControlTGDTT

(b)



5 Discussion












Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










Acknowledgments


References

























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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