research article melatonin-mediated intracellular insulin...

Research ArticleMelatonin-Mediated Intracellular Insulin during2-Deoxy-d-glucose Treatment Is Reduced through Autophagyand EDC3 Protein in Insulinoma INS-1E Cells

Han Sung Kim1 Tae-Young Han2 and Yeong-Min Yoo1

1Department of Biomedical Engineering Yonsei-Fraunhofer Medical Device Laboratory Yonsei University WonjuGangwon-do 26493 Republic of Korea2Fraunhofer Institute IKTS-MD Maria-Reiche-Straszlige 2 01109 Dresden Germany

Correspondence should be addressed to Yeong-Min Yoo yyeongmhanmailnet

Received 2 March 2016 Accepted 21 June 2016

Academic Editor Karolina Szewczyk-Golec

Copyright copy 2016 Han Sung Kim 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

2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways and then activates autophagy viaactivation of AMPK and endoplasmic reticulum (ER) stress We investigated whether 2-DG reduced intracellular insulin increasedby melatonin via autophagyEDC3 in insulinoma INS-1E cells p-AMPK and GRP78BiP level were significantly increased by 2-DG in the presenceabsence of melatonin but IRE1120572 level was reduced in 2-DG treatment Levels of p85120572 p110 p-Akt (Ser473Thr308) and p-mTOR (Ser2481) were also significantly reduced by 2-DG in the presenceabsence ofmelatoninMn-SOD increasedwith 2-DG plus melatonin compared to groups treated withwithout melatonin alone Bcl-2 was decreased and Bax increasedwith 2-DG plus melatonin LC3II level increased with 2-DG treatment in the presenceabsence of melatonin Intracellular insulinproduction increased in melatonin plus 2-DG but reduced in treatment with 2-DG withwithout melatonin EDC3 was increasedby 2-DG in the presenceabsence of melatonin Rapamycin an mTOR inhibitor increased GRP78BiP and EDC3 levels in adose-dependent manner and subsequently resulted in a decrease in intracellular production of insulin These results suggest thatmelatonin-mediated insulin synthesis during 2-DG treatment involves autophagy and EDC3 protein in rat insulinoma INS-1E cellsand subsequently results in a decrease in intracellular production of insulin

1 Introduction

Pancreatic 120573-cells are the only source of the peptide hormoneinsulin which is essential for stimulating glucose uptakein peripheral tissue and inhibiting glucose production inliver Pancreatic 120573-cell dysfunction and death result froma combination of genetic predisposition and exposure toenvironmental risk factors and contribute to type 2 diabetes(T2D) [1] The endoplasmic reticulum (ER) contains theprotein-folding machinery for secretory proteins and istherefore crucial for insulin biosynthesis Disruption of 120573-cell ER function by elevated free fatty acid levels and insulinresistance can result in an imbalance in protein homeostasisand ER stress which has been recognized as an importantmechanism for T2D [2 3] Recent reports have shown thatautophagy is activated in response to ER stress in pancreatic120573-cells [3ndash5]

AMP-activated protein kinase (AMPK) is a centralmetabolic regulator which balances energy productionthrough the AMP ATP ratio in eukaryotic cells and func-tions as a therapeutic target for anticancer and metabolicdiseases in diabetes mellitus and lipid metabolism [6ndash8]AMPK appears to be involved in insulin signaling pathwaysby positively regulating AktProtein Kinase B (PKB) inendothelial cells and cardiomyocytes [9 10]However AMPKactivation has also been reported to negatively affectAktPKBand insulin production [11] Activation or phosphorylationof AMPK is associated with activation or phosphorylation ofAktPKB and mammalian target of rapamycin (mTOR) forinsulin synthesis or secretion in pancreatic 120573-cells [12 13]

Melatonin can control insulin secretion both in vivo andin vitro via MT1 or MT2 receptors [14 15] Gi protein [16]cyclic AMP-response element-binding protein (CREB) [17]

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 2594703 11 pageshttpdxdoiorg10115520162594703

2 Oxidative Medicine and Cellular Longevity

and Gq proteinsphospholipase CIP3 [18] Circadianrhythms are influenced by melatonin and also induce a phaseshift in insulin secretion [19] Melatonin directly influencesdiabetes and associated metabolic diseases [20] The genesand proteins involved in direct regulation of insulin biosyn-thesis and secretion in pancreatic 120573-cells have been reportedIRE1120572 [21] Sirt1 [22] and Sirt4 [23] Recently melatonin wasshown to directly influence insulin biosynthesis and secretionunder ER stress via the RNA-binding protein human antigenD (HuD) in rat pancreatic INS-1E cells suggesting thatnuclear HuD may be an important negative regulator ininsulin production and secretion [13] However an interac-tion between the enhancer of mRNA decapping complex 3(EDC3) [24] and insulin production has not been reported

The glucose analogue 2-deoxy-d-glucose (2-DG) ismetabolized by hexokinase and acts as an inhibitor ofglycolysis [25] 2-DG triggers glucose deprivation withoutaltering other nutrients or metabolic pathways [26] and thenactivates autophagy via activation of AMPK [27] and endo-plasmic reticulum (ER) stress [28] Therefore 2-DG appearsto be an ideal tool to understand the interactions betweenautophagy and ER stress In this study we provide the firstdemonstration that 2-DG reduces intracellular insulin whichwas increased by melatonin via autophagy and EDC3 ininsulinoma INS-1E cells

2 Materials and Methods

21 Cell Culture INS-1E cells a clonal pancreatic 120573-cell linewere obtained from Professor Claes B Wollheim and werecultured in RPMI 1640 medium (Invitrogen Carlsbad CAUSA) containing 11mM glucose supplemented with 10mMHEPES (pH 73) 10 heat-inactivated fetal bovine serum(FBS Invitrogen) 50 120583M 120573-mercaptoethanol 1mM sodiumpyruvate 50 120583gmL penicillin and 100 120583gmL streptomycinat 37∘C with 5 CO

2in a humidified incubator

22 Treatment with 2-DG Melatonin and Rapamycin INS-1E cells were cultured in RPMI 1640 medium plus 2 heat-inactivated FBS in a 37∘C and 5 CO

2incubator with or

without melatonin (Sigma-Aldrich St Louis MO USA)andor 2-DG (5mM) (Sigma-Aldrich) for 24 hr or withrapamycin (20 to 80 nM) (Calbiochem SanDiegoMOUSA)for 24 hr

23 Western Blot Analysis Cells were harvested washedtwice with ice-cold phosphate buffered saline (PBS) and thenresuspended in 20mM Tris-HCl buffer (pH 74) containingprotease inhibitors (01mM phenylmethylsulfonyl fluoride5 120583gmL aprotinin 5120583gmLpepstatinA and 1120583gmL chymo-statin) and phosphatase inhibitors (5mMNa

3VO4and 5mM

NaF) Whole cell lysate was prepared using 20 strokes of aDounce homogenizer followed by centrifugation at 13000timesgfor 20min at 4∘CThe protein concentration was determinedusing the BCA assay (Sigma) Proteins (40120583g) were sep-arated by 12 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and the detection of insulinproteins was performed by 165 tricine SDS-PAGE Thesegels transferred onto a polyvinylidene difluoride (PVDF)

membrane The membrane was incubated with antibodies(diluted as indicated in brackets) directed against the fol-lowing proteins p-AMPK and AMPK (1 500) (Santa CruzBiotechnology) IRE1120572 (1 500) (Santa Cruz Biotechnology)p-PERK (1 1000) (Cell Signaling Technology Beverly MAUSA) GRP78BiP (1 1000) (Cell Signaling Technology)p85120572 and p85120573 (1 500) (Santa Cruz Biotechnology) p110(1 500) (Santa Cruz Biotechnology) p-Akt (T308 S473)and Akt (1 1000) (Cell Signaling Technology) p-mTOR(Ser2448 2481) andmTOR (1 1000) (Cell Signaling Technol-ogy) CuZn-SOD (1 500) (Santa Cruz Biotechnology) Mn-SOD (1 500) (Santa Cruz Biotechnology) catalase (1 500)(Santa Cruz Biotechnology) Bcl-2 (1 500) (Santa CruzBiotechnology) Bax (1 500) (Santa Cruz Biotechnology)insulin (1 500) (Santa Cruz Biotechnology) EDC3 (1 500)(Santa Cruz Biotechnology) and Actin (1 1000) (AssayDesigns Ann Arbor MI USA) Immunoreactive proteinswere visualized by exposure to X-ray film Protein bandswere analyzed by image-scanning and optical density wasmeasured using ImageJ analysis software (version 137WayneRasbandNIH BethesdaMDUSA)The data were correctedfor background subtraction and normalized by includingActin as an internal control

24 Statistical Analysis Significant differences were detectedby ANOVA followed by Tukeyrsquos test for multiple compar-isons Analysis was performed using the Prism Graph Padv40 (Graph Pad Software Inc San Diego CA USA) Valuesare expressed as means plusmn SD of at least three separatedexperiments in which case a representative experiment isdepicted in the figures 119901 values lt 005 were consideredstatistically significant

3 Results

31 Expressions of p-AMPK GRP78BiP IRE1120572 and p-PERK Proteins We investigated whether 2-DG reducedinsulin synthesis which had been increased by melatoninvia autophagy in rat insulinoma INS-1E cells 2-DG activatesautophagy via reactive oxygen species-mediated activation ofAMPK [27] andER stress [28]Therefore we first investigatedwhether 2-DG andor melatonin induced phosphorylationof AMPK and ER stress in rat insulinoma INS-1E cellsPhosphorylation of AMPK was significantly increased bytreatmentwith 2-DG in the presence or absence ofmelatonincompared to treatment with melatonin or FBS only (Figures1(a) and 1(b)) ER stress marker GRP78BiP protein wasalso significantly increased by treatment with 2-DG in thepresence or absence of melatonin (Figures 1(c) and 1(d)) butexpression of IRE1120572 protein was reduced (Figures 1(c) and1(d)) and p-PERK protein expression was not significantlyaffected by 2-DG treatment (Figures 1(c) and 1(e))

32 Expressions of p-PI3K AktPKB and mTOR ProteinsWe determined how 2-DG andor melatonin affected knownregulators of insulin signaling phosphoinositide 3-kinase(PI3K) AktPKB and mTOR in rat insulinoma INS-1Ecells Class IA PI3K is composed of a heterodimer of p110catalytic subunit and p85 regulatory subunit [29] 2-DG

Oxidative Medicine and Cellular Longevity 3

p-AMPK

AMPK

Melatonin (100120583M) ++

+

+

++

minusminus

minus

minus

minus minus

+

+

minus

minus2-Deoxy-d-glucose (5 120583M)

(a)

lowastlowast

lowastlowast


++

minusminus

minus minus

0

50

100

150

Relat

ive a

mou

nts o

f p-A

MPK

2-Deoxy-d-glucose (5 120583M)

(b)


+

+

++

minusminus

minus

minus

minus minus

+

+

minus

minus

Actin

GRP78BiP

p-PERK

IRE11205722-Deoxy-d-glucose (5 120583M)

(c)

Relat

ive a

mou

nts o

f IRE

1120572

lowastlowastlowastlowastlowastlowast

0

50

100


++

minusminus

minus minus

150


(d)

Rela

tive a

mou

nts o

f p-P

ERK

lowastlowastlowast

0

50

100

150

200

250


++

minusminus

minus minus2-Deoxy-d-glucose (5 120583M)

(e)

100

lowast

lowastlowastlowastlowastlowast

0

200

Relat

ive a

mou

nts o

f GRP

78B

IP


++

minusminus


(f)

Figure 1 Phosphorylation of AMPK IRE1120572 protein expression phosphorylation of PERK andGRP78BiP protein expression in the presenceor absence of melatonin andor 2-DG in rat insulinoma INS-1E cells INS-1E cells were incubated in RPMI 1640 medium supplemented with2 FBS withwithout melatonin (100120583M) andor 2-DG (5mM) for 24 hr at 37∘Cwith 5 CO

2

p-AMPK expression was analyzed byWesternblot (a c) The relative amount of p-AMPK (b) IRE1120572 protein (d) p-PERK (e) and GRP78BiP protein (f) was quantified as described inSection 2 Data represent mean plusmn SD of three experiments (119899 = 3) lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 versus 2 FBS 119901 lt 001119901 lt 0001 melatonin versus melatonin andor 2-DG


p110

Actin

p85120572

p85120573


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

lowastlowast

lowastlowastlowast

Relat

ive a

mou

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f p85120572

0

25

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75

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++

minusminus


(b)

lowastlowastlowast

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tive a

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(c)


0

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Relat

ive a

mou

nts o

f p11

0


++

minusminus


(d)

Figure 2 Expression of p85120572 p85120573 and p110 proteins in the presence or absence of melatonin andor 2-DG in rat insulinoma INS-1E cellsINS-1E cells were incubated in RPMI 1640 medium supplemented with 2 FBS withwithout melatonin (100120583M) andor 2-DG (5mM) for24 hr at 37∘C with 5 CO

2

p85120572 p85120573 and p110 proteins were analyzed byWestern blot (a) The relative amounts of p85120572 (b) p85120573 (c) andp110 proteins (d) were quantified in Section 2 Data represent mean plusmn SD of three experiments (119899 = 3) lowastlowast119901 lt 005 and lowastlowastlowast119901 lt 0001 versus2 FBS 119901 lt 0001 melatonin versus melatonin andor 2-DG

treatment with or without melatonin caused a significantreduction in expression of PI3K subunit p85120572 and p110 anda significant increase in p85120573 protein expression (Figures2(a)ndash2(d)) 2-DG also significantly decreased expression ofp-Akt (Ser473 Thr308) (Figures 3(a)ndash3(c)) and p-mTOR(Ser2481) (Figures 3(d)ndash3(f)) proteins in the presence orabsence of melatonin These results indicated that ER stressin the presence of 2-DG or 2-DG-plus melatonin negativelyregulates the PI3KAktmTOR pathway

33 Expressions of SOD Catalase Bcl-2 and Bax ProteinsNext to characterize intracellular and mitochondrial condi-tions according to antioxidant and prooxidant proteins weexamined how 2-DG andor melatonin affected superoxidedismutase (SOD) catalase Bcl-2 and Bax proteins in ratinsulinoma INS-1E cells CuZn-SOD was not significantlyaffected (Figures 4(a) and 4(b)) butMn-SODprotein expres-sion was increased by treatment with 2-DG plus melatonin

compared to groups treated withwithout melatonin alone orthe nontreated control group (Figures 4(a) and 4(c)) Catalaseprotein expression was decreased by 2-DG treatment inthe presence of melatonin (Figures 4(a) and 4(d)) Bcl-2 protein expression was decreased by treatment with 2-DG plus melatonin compared to treatment withwithoutmelatonin (Figures 4(e) and 4(f)) and Bax protein expressionwas increased in cells treated with 2-DG andor melatonincompared to nontreated control group (Figures 4(e) and4(g)) These results suggested that 2-DG or 2-DG-plus mela-tonin influenced mitochondria thus resulting in a change ofantioxidant and prooxidant protein levels

34 Expressions of LC3II Insulin and EDC3 Proteins Sub-sequently we investigated whether 2-DG reduced insulinsynthesiswhich had been increased bymelatonin via EDC3 inrat insulinoma INS-1E cells Expression of autophagymarkersLC3 II was increased by 2-DG treatment in the presence


p-Akt (S473)

Akt

p-Akt (T308)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (S

473)



++

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(b)

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100

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ive a

mou

nts o

f p-A

kt (T

308)

lowastlowastlowast lowastlowastlowast


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minusminus


(c)

mTOR

p-mTOR (S2448)

p-mTOR (S2481)


+

+

++

minus

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minusminus

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+


(d)

lowast

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Relat

ive a

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TOR

(S24

48)


++

minusminus


(e)

0

50

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Relat

ive a

mou

nts o

f p-m

TOR

(S24

81)

lowastlowastlowast

lowastlowastlowast


++

minusminus


(f)

Figure 3 Phosphorylation of Akt (Ser473) Akt (Thr308) mTOR (Ser2448) and mTOR (Ser2481) proteins in the presence or absence ofmelatonin andor 2-DG in rat insulinoma INS-1E cells INS-1E cells were incubated in RPMI 1640 medium supplemented with 2 FBSwithwithout melatonin (100120583M) andor 2-DG (5mM) for 24 hr at 37∘Cwith 5CO

2

p-Akt was analyzed byWestern blot (a d)The relativeamounts of p-Akt (Ser473) (b) p-Akt (Thr308) (c) mTOR (Ser2448) (e) andmTOR (Ser2481) (f) were quantified in Section 2 Data representmean plusmn SD of three experiments (119899 = 3) lowast119901 lt 005 lowastlowastlowast119901 lt 001 versus 2 FBS 119901 lt 005 119901 lt 0001 melatonin versus melatonin andor2-DG


Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

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100

CuZ

n-SO

D


(b)

lowast


++

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Rela

tive a

mou

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Mn-

SOD


(c)

lowast


++

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of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

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+


(e)

lowastlowastlowast


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Relat

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(f)

lowastlowastlowast

lowastlowastlowast

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++

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Relat

ive a

mou

nts o

f Bax


(g)

Figure 4 Expression of CuZn-SOD Mn-SOD catalase Bcl-2 and Bax proteins in the presence or absence of melatonin andor 2-DG in ratinsulinoma INS-1E cells INS-1E cells were incubated in RPMI 1640 medium supplemented with 2 FBS withwithout melatonin (100120583M)andor 2-DG (5mM) for 24 hr at 37∘C with 5 CO

2

CuZn-SOD Mn-SOD and catalase proteins were analyzed by Western blot (a e) Therelative amounts of CuZn-SOD (b) Mn-SOD (c) catalase proteins (d) Bcl-2 (f) and Bax proteins (g) were quantified in Section 2 Datarepresent mean plusmn SD of three experiments (119899 = 3) lowast119901 lt 005 lowastlowastlowast119901 lt 0001 versus 2 FBS 119901 lt 005 119901 lt 001 and

119901 lt 0001 melatoninversus melatonin andor 2-DG


Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

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Relat

ive a

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II

lowastlowastlowast

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(b)

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ive a

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ulin

lowastlowast



++

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(c)

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Rela

tive a

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C3

lowastlowast

lowastlowastlowast


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(d)

Figure 5 Expression of LC3 insulin and EDC3 proteins in the presence or absence of melatonin andor 2-DG in rat insulinoma INS-1Ecells INS-1E cells were incubated in RPMI 1640 medium supplemented with 2 FBS withwithout melatonin (100120583M) andor 2-DG (5mM)for 24 hr at 37∘C with 5 CO

2

LC3 Insulin and EDC3 proteins were analyzed by Western blot (a) The relative amounts of LC3 (b) insulin(c) and EDC3 proteins (d) were quantified in Section 2 Data represent mean plusmn SD of three experiments (119899 = 3) lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001versus 2 FBS 119901 lt 0001 melatonin versus melatonin andor 2-DG

or absence of melatonin (Figures 5(a) and 5(b)) Insulinsynthesis was increased by melatonin treatment compared tonontreated melatonin group but it was reduced by treatmentwith 2-DG (Figures 5(a) and 5(c)) Expression of EDC3 anegative regulator in insulin synthesis was increased by 2-DG in the presence or absence of melatonin (Figures 5(a)and 5(d)) suggesting that the decrease in intracellular insulinsynthesis was associated with an increase of autophagy andEDC3 protein expression

35 The Relationship of Insulin Production with Autophagyand EDC3 Protein Lastly to confirm the relationship ofinsulin production with autophagy and EDC3 proteinwe investigated whether the mTOR inhibitor rapamycininduced autophagy and expression of GRP78BiP and EDC3proteins in rat insulinoma INS-1E cells Rapamycin treat-ment resulted in an increase in GRP78BiP (Figures 6(a)and 6(c)) and EDC3 proteins in a dose-dependent man-ner (Figures 6(a) and 6(d)) and a subsequent decrease in

insulin production (Figures 6(a) and 6(b)) These resultssuggested that melatonin-mediated insulin synthesis during2-DG treatment involved autophagy-induced ER stress andEDC3 protein in rat insulinoma INS-1E cells subsequentlyresulting in a decrease in insulin protein biosynthesis

4 Discussion

ER dysfunction has been implicated in insulin resistanceand is an important cause of T2D [2ndash4] Autophagy in ERstress-induced pancreatic 120573 cells plays a role as an importantregulator of insulin production and secretion [2ndash5 13] Inaddition autophagy in pancreatic 120573 cells is essential to main-tain normal morphology cell mass and function of 120573-cellsand is regarded as a crucial stress response factor to protect120573-cells under insulin-resistant states [4 5] The glucose analog2-DG acts as a glycolytic inhibitor leading to ER stress and anunfolded protein response [26ndash28] Although 2-DG-inducedER stress has been shown to activate autophagy the precise


Cellular insulinRapamycin (nM) 0 0 20 20 40 40 60 60 8080

EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rapamycin (nM)0 20 40 60 80

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Relat

ive a

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f ins

ulin

(b)



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Relat

ive a

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f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

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Relat

ive a

mou

nts o

f ED

C3

(d)

Figure 6 Expression of insulin GRP78BiP and EDC3 proteins with or without rapamycin treatment in rat insulinoma INS-1E cells INS-1Ecells were incubated in RPMI 1640medium supplemented with 2 FBS withwithout rapamycin (20 to 80 nM) for 24 hr at 37∘Cwith 5CO

2

Insulin GRP78BiP and EDC3 proteins were analyzed by Western blot (a) The relative amounts of insulin (b) GRP78BiP (c) and EDC3proteins (d) were quantified in Section 2 Data represent mean plusmn SD of three experiments (119899 = 3) lowast119901 lt 005 lowastlowastlowast119901 lt 0001 versus 1 FBS

mechanism is not fully understood Here we demonstratedthe relevance of ER stress-mediated autophagy caused by 2-DG for insulin synthesis in pancreatic 120573 cells Our previousreport showed that ER stress caused by melatonin espe-cially in the presence of thapsigargin decreased intracellularinsulin biosynthesis and that extracellular secretion of insulinmay be regulated by melatonin in rat insulinoma INS-1E cells[13] Here we found that melatonin reduced insulin produc-tion in the presence of 2-DG via autophagy-induced ER stressin rat insulinoma INS-1E cells (Figures 1(c)ndash1(f) and 6(a)ndash6(d)) mTOR inhibitor rapamycin induced autophagy andthen subsequently decrease in insulin production (Figure 6)Therefore autophagy can regulate insulin production at thecellular level through physiological and biochemical changesof ER homeostasis involving the unfolded protein response[30 31]

GRP78BiP is localized in the ER and its expression isincreased by environmental stresses in many types of cellsOther studies have showed that conditions including glucosedeprivation 2-DG treatment and hypoxia or increase ofintracellular Ca2+ concentration induce GRP78BiP expres-sion [32ndash35] Our previous research demonstrated thatexpression of the ER-stress proteinGRP78BiP is significantly

decreased by tunicamycinmelatonin treatment and remainsalmost unperturbed with thapsigarginmelatonin treatmentwhen compared to controlmelatonin in rat pancreatic INS-1E cells [13] This suggests that GRP78BiP protein can beregulated by several cellular stresses which perturb ER func-tion and homeostasis including tunicamycin thapsigarginand calcium ionophore A23187 [36] 2-DG is an especiallypotent inducer of GRP78BiP protein expression [37] In thisstudy we found that melatonin significantly regulated insulinproduction through the expression of GRP78BiP proteinwhich induced ER stress under conditions of 2-DG or 2-DG plus melatonin treatment in rat insulinoma INS-1E cells(Figures 1(c) and 1(f)) Therefore GRP78BiP accompaniedwith ER stress is a crucial element in insulin biosynthesis andsecretion in rat pancreatic 120573-cells [38]

The interaction between 2-DG and the AMPKmTORsignal pathway has not been reported in pancreatic 120573 cellsActivation of AMPK and inactivation of mTOR signal path-way have only been studied under conditions of nutrientdeprivation including glucose andor leucine starvation inpancreatic beta-cells rat skeletal muscle cells and hepa-tocytes [12 13 39 40] Gleason et al [12] reported thatbecause glucose and amino acids stimulate insulin release


from pancreatic 120573 cells AMPK represents an importantfactor for control of 120573-cell function In addition the acti-vationphosphorylation of AMPK in 120573-cells by low glucosecorrelateswith inactivation of themTORpathway as a cellularsensor for nutritional conditions in 120573 cells [12 41] The othercondition that is lack of amino acid leucine causes anincrease in the activationphosphorylation of AMPK and adecrease in the activation of mTOR pathway [12 39 40]It is therefore unlikely that the ability of AMPK and themTOR signal pathway to sense both glucose and amino acidsplays a role in regulation of insulin synthesis or secretion[12 41] 2-DG induces glucose deprivation and results in theactivationphosphorylation of AMPK [26 27] In our studyphosphorylation of AMPK was significantly increased (Fig-ures 1(a) and 1(b)) and phosphorylation of mTOR (Ser2481)was significantly decreased (Figures 3(d) and 3(f)) by 2-DG inthe presence or absence of melatonin showing that 120573-cells doindeed possess a mechanism by which glucose derivation cansuppress the mTOR signal pathway via activation of AMPKThis result suggests that a connection between AMPK andmTOR in the 120573-cell is of particular relevance to insulinsynthesissecretion during 2-DG treatment The finding thatAMPK and the mTOR signal pathway can sense both glucoseand amino acids may apply to 2-DG treatment conditions inthe regulation of insulin synthesis or its secretion from 120573-cells

The genesproteins involved in insulin biosynthesis andsecretion in pancreatic 120573-cells have been studied IRE1120572 [21]Sirt1 [22] Sirt4 [23] and HuD [13] The positive regulatorsof insulin biosynthesis and secretion are IRE1120572 Sirt1 andWSF1 whereas Eny2 Sirt4 and HuD are negative regulatorsOur recent study suggests that nuclear HuD under ER stressconditions with thapsigargin and melatonin functions as animportant negative regulator of insulin production from ratpancreatic INS-1E cells [13] However our current studydemonstrated that HuD under autophagy and ER stress with2-DG and melatonin could not negatively regulate insulinproduction (data not shown) Instead EDC3 was involvedin insulin synthesis during 2-DGmelatonin or rapamycintreatment and subsequently induced a decrease in insulinprotein (Figures 5 and 6) An interaction between EDC3 andinsulin production has not yet reported [24] Our study is thefirst to suggest that EDC3 functions as a negative regulator ofinsulin production in rat pancreatic INS-1E cells

Glucose deprivation by mechanisms including 2-DGinduces the production of reactive oxygen species (ROS)to activate AMPK in pancreatic 120573 cells [42] Hypoxia andhyperoxia trigger AMPK activation via mitochondrial ROSproduction [43] These studies support a role for AMPK asan important redox sensor through a mitochondrial ROSmechanismWe showed that 2-DG andormelatonin affectedSOD catalase Bcl-2 and Bax proteins in rat insulinomaINS-1E cells (Figure 4) suggesting that 2-DG or 2-DG plusmelatonin influences antioxidant and prooxidant proteinsthrough a mechanism involving mitochondrial ROS

Class IA PI3K is composed of a p110 catalytic subunit anda p85 regulatory subunit and plays a pivotal role in insulin sig-naling [29 44] We found that 2-DG treatment significantlyreduced expression of both PI3K subunits in the presence or

absence of melatonin (Figure 2)This result indicated that thep85 proteins may bind and stabilize the p110 subunit [44]

We report that melatonin may directly regulate the intra-cellular biosynthesis of insulin in the presence of 2-DG inrat insulinoma INS-1E cells Intracellular insulin productionmay be partially regulated by the expression of ER stressGRP78BiP-induced autophagy and EDC3 protein underconditions of 2-DG treatment

5 ConclusionsThese results suggest that melatonin-mediated insulin syn-thesis during 2-DG treatment involves autophagy and EDC3protein in rat insulinoma INS-1E cells and subsequentlyresults in a decrease in intracellular production of insulin

Competing InterestsThe authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by the Leading ForeignResearch Institute Recruitment Program through theNational Research Foundation of Korea (NRF) funded by theMinistry of Science ICT amp Future Planning (2010-00757)

References

[1] S Orahilly ldquoHuman genetics illuminates the paths to metabolicdiseaserdquo Nature vol 462 no 7271 pp 307ndash314 2009

[2] D L Eizirik A K Cardozo and M Cnop ldquoThe role forendoplasmic reticulum stress in diabetes mellitusrdquo EndocrineReviews vol 29 no 1 pp 42ndash61 2008

[3] S H Back and R J Kaufman ldquoEndoplasmic reticulum stressand type 2 diabetesrdquo Annual Review of Biochemistry vol 81 pp767ndash793 2012

[4] J-J Yin Y-B Li Y Wang et al ldquoThe role of autophagy inendoplasmic reticulum stress-induced pancreatic 120573 cell deathrdquoAutophagy vol 8 no 2 pp 158ndash164 2012

[5] A Bartolome C Guillen and M Benito ldquoAutophagy playsa protective role in endoplasmic reticulum stress-mediatedpancreatic 120573 cell deathrdquoAutophagy vol 8 no 12 pp 1757ndash17682012

[6] G A Rutter G Da Silva Xavier and I Leclerc ldquoRoles of 51015840-AMP-activated protein kinase (AMPK) in mammalian glucosehomoeostasisrdquo Biochemical Journal vol 375 part 1 pp 1ndash162003

[7] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005

[8] R J Shaw ldquoGlucose metabolism and cancerrdquo Current Opinionin Cell Biology vol 18 no 6 pp 598ndash608 2006

[9] S L Longnus C Segalen J Giudicelli M P Sajan R VFarese and E Van Obberghen ldquoInsulin signalling downstreamof protein kinase B is potentiated by 51015840AMP-activated proteinkinase in rat hearts in vivordquo Diabetologia vol 48 no 12 pp2591ndash2601 2005

[10] L Bertrand A Ginion C Beauloye et al ldquoAMPK activationrestores the stimulation of glucose uptake in an in vitro


model of insulin-resistant cardiomyocytes via the activation ofprotein kinase Brdquo American Journal of PhysiologymdashHeart andCirculatory Physiology vol 291 no 1 pp H239ndashH250 2006

[11] S Kovacic C-L M Soltys A J Barr I Shiojima KWalsh andJ R B Dyck ldquoAkt activity negatively regulates phosphorylationof AMP-activated protein kinase in the heartrdquo The Journal ofBiological Chemistry vol 278 no 41 pp 39422ndash39427 2003

[12] C E Gleason D Lu L A Witters C B Newgard and M JBirnbaum ldquoThe role of AMPK and mTOR in nutrient sensingin pancreatic 120573-cellsrdquo The Journal of Biological Chemistry vol282 no 14 pp 10341ndash10351 2007

[13] Y-M Yoo ldquoMelatonin-mediated insulin synthesis during endo-plasmic reticulum stress involves HuD expression in rat insuli-noma INS-1E cellsrdquo Journal of Pineal Research vol 55 no 2 pp207ndash220 2013

[14] E Muhlbauer E Albrecht I Bazwinsky-Wutschke and EPeschke ldquoMelatonin influences insulin secretion primarily viaMT(1) receptors in rat insulinoma cells (INS-1) and mousepancreatic isletsrdquo Journal of Pineal Research vol 52 no 4 pp446ndash459 2012

[15] E Muhlbauer E Albrecht K Hofmann I Bazwinsky-Wutschke and E Peschke ldquoMelatonin inhibits insulin secretionin rat insulinoma 120573-cells (INS-1) heterologously expressing thehuman melatonin receptor isoform MT2rdquo Journal of PinealResearch vol 51 no 3 pp 361ndash372 2011

[16] E Peschke E Muhlbauer U Muszlighoff V J Csernus EChankiewitz and D Peschke ldquoReceptor (MT1) mediated influ-ence ofmelatonin on cAMPconcentration and insulin secretionof rat insulinoma cells INS-1rdquo Journal of Pineal Research vol 33no 2 pp 63ndash71 2002

[17] I Bazwinsky-Wutschke S Wolgast E Muhlbauer E Albrechtand E Peschke ldquoPhosphorylation of cyclic AMP-responseelementndashbinding protein (CREB) is influenced by melatonintreatment in pancreatic rat insulinoma 120573-cells (INS-1)rdquo Journalof Pineal Research vol 53 no 4 pp 344ndash357 2012

[18] C B Wollheim and T J Biden ldquoSecond messenger func-tion of inositol 145-trisphosphate Early changes in inos-itol phosphates cytosolic Ca2+ and in insulin release incarbamylcholine-stimulated RINm5F cellsrdquoThe Journal of Bio-logical Chemistry vol 261 no 18 pp 8314ndash8319 1986

[19] G Boden J Ruiz J-L Urbain and X Chen ldquoEvidence fora circadian rhythm of insulin secretionrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 271 no 2 part1 pp E246ndashE252 1996

[20] E Peschke ldquoMelatonin endocrine pancreas and diabetesrdquoJournal of Pineal Research vol 44 no 1 pp 26ndash40 2008

[21] K L Lipson S G Fonseca S Ishigaki et al ldquoRegulation ofinsulin biosynthesis in pancreatic beta cells by an endoplasmicreticulum-resident protein kinase IRE1rdquoCellMetabolism vol 4no 3 pp 245ndash254 2006

[22] L Bordone M C Motta F Picard et al ldquoSirt1 regulates insulinsecretion by repressing UCP2 in pancreatic beta cellsrdquo PLoSBiology vol 4 no 2 article e31 2006

[23] C Argmann and J Auwerx ldquoInsulin Secretion SIRT4 Gets inon the Actrdquo Cell vol 126 no 5 pp 837ndash839 2006

[24] M Larance A F Rowland K L Hoehn et al ldquoGlobalphosphoproteomics identifies a major role for AKT and 14-3-3 in regulating EDC3rdquo Molecular and Cellular Proteomics vol9 no 4 pp 682ndash694 2010

[25] A Sole and R K Crane ldquoThe inhibition of brain hexokinaseby adenosinediphosphate and sulfhydryl reagentsrdquo Journal ofBiological Chemistry vol 206 no 2 pp 925ndash936 1954

[26] F Aghaee J P Islamian andB Baradaran ldquoEnhanced radiosen-sitivity and chemosensitivity of breast cancer cells by 2-deoxy-D-glucose in combination therapyrdquo Journal of Breast Cancervol 15 no 2 pp 141ndash147 2012

[27] W Jiang Z Zhu andH JThompson ldquoModulation of the activ-ities of AMP-activated protein kinase protein kinase B andmammalian target of rapamycin by limiting energy availabilitywith 2-deoxyglucoserdquo Molecular Carcinogenesis vol 47 no 8pp 616ndash628 2008

[28] H Xi M Kurtoglu H Liu et al ldquo2-Deoxy-D-glucose activatesautophagy via endoplasmic reticulum stress rather than ATPdepletionrdquoCancer Chemotherapy and Pharmacology vol 67 no4 pp 899ndash910 2011

[29] P K Vogt J R Hart M Gymnopoulos et al ldquoPhosphatidyli-nositol 3-kinase the oncoproteinrdquo Current Topics in Microbiol-ogy and Immunology vol 347 no 1 pp 79ndash104 2010

[30] D T Rutkowski and R S Hegde ldquoRegulation of basal cellularphysiology by the homeostatic unfolded protein responserdquoTheJournal of Cell Biology vol 189 no 5 pp 783ndash794 2010

[31] S S Cao and R J Kaufman ldquoUnfolded protein responserdquoCurrent Biology vol 22 no 16 pp R622ndashR626 2012

[32] G Kuznetsov K T Bush P L Zhang and S K Nigam ldquoPertur-bations inmaturation of secretory proteins and their associationwith endoplasmic reticulum chaperones in a cell culture modelfor epithelial ischemiardquo Proceedings of the National Academy ofSciences of the United States of America vol 93 no 16 pp 8584ndash8589 1996

[33] A S Lee ldquoMammalian stress response induction of theglucose-regulated protein familyrdquo Current Opinion in CellBiology vol 4 no 2 pp 267ndash273 1992

[34] W W Li Y Hsiung Y Zhou B Roy and A S Lee ldquoInductionof the mammalian GRP78BiP gene by Ca2+ depletion andformation of aberrant proteins activation of the conservedstress-inducible grp core promoter element by the humannuclear factor YY1rdquo Molecular and Cellular Biology vol 17 no1 pp 54ndash60 1997

[35] Z Yu H Luo W Fu and M P Mattson ldquoThe endoplasmicreticulum stress-responsive protein GRP78 protects neuronsagainst excitotoxicity and apoptosis suppression of oxidativestress and stabilization of calcium homeostasisrdquo ExperimentalNeurology vol 155 no 2 pp 302ndash314 1999

[36] H Sasaya T Utsumi K Shimoke et al ldquoNicotine suppressestunicamycin-induced but not thapsigargin-induced expres-sion of GRP78 during ER stress-mediated apoptosis in PC12cellsrdquo Journal of Biochemistry vol 144 no 2 pp 251ndash257 2008

[37] S Kishi K Shimoke Y Nakatani et al ldquoNerve growth factorattenuates 2-deoxy-d-glucose-triggered endoplasmic reticulumstress-mediated apoptosis via enhanced expression of GRP78rdquoNeuroscience Research vol 66 no 1 pp 14ndash21 2010

[38] L Zhang E Lai T Teodoro and A Volchuk ldquoGRP78 but notprotein-disulfide isomerase partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancre-atic 120573-cellsrdquoThe Journal of Biological Chemistry vol 284 no 8pp 5289ndash5298 2009

[39] A K Saha X J Xu E Lawson et al ldquoDownregulation of AMPKaccompanies leucine- and glucose-induced increases in proteinsynthesis and insulin resistance in rat skeletal musclerdquoDiabetesvol 59 no 10 pp 2426ndash2434 2010

[40] F Xiao Z Huang H Li et al ldquoLeucine deprivation increaseshepatic insulin sensitivity via GCN2mTORS6K1 and AMPKpathwaysrdquo Diabetes vol 60 no 3 pp 746ndash756 2011


[41] C Tokunaga K-I Yoshino and K Yonezawa ldquomTOR inte-grates amino acid- and energy-sensing pathwaysrdquo Biochemicaland Biophysical Research Communications vol 313 no 2 pp443ndash446 2004

[42] Y Cai G A Martens S A Hinke H Heimberg D Pipeleersand M Van de Casteele ldquoIncreased oxygen radical formationand mitochondrial dysfunction mediate beta cell apoptosisunder conditions of AMP-activated protein kinase stimulationrdquoFree Radical Biology andMedicine vol 42 no 1 pp 64ndash78 2007

[43] N MMazure and J Pouyssegur ldquoHypoxia-induced autophagycell death or cell survivalrdquo Current Opinion in Cell Biology vol22 no 2 pp 177ndash180 2010

[44] KUeki D A Fruman CM Yballe et al ldquoPositive and negativeroles of p85120572 and p85120573 regulatory subunits of phosphoinositide3-kinase in insulin signalingrdquo The Journal of Biological Chem-istry vol 278 no 48 pp 48453ndash48466 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and Gq proteinsphospholipase CIP3 [18] Circadianrhythms are influenced by melatonin and also induce a phaseshift in insulin secretion [19] Melatonin directly influencesdiabetes and associated metabolic diseases [20] The genesand proteins involved in direct regulation of insulin biosyn-thesis and secretion in pancreatic 120573-cells have been reportedIRE1120572 [21] Sirt1 [22] and Sirt4 [23] Recently melatonin wasshown to directly influence insulin biosynthesis and secretionunder ER stress via the RNA-binding protein human antigenD (HuD) in rat pancreatic INS-1E cells suggesting thatnuclear HuD may be an important negative regulator ininsulin production and secretion [13] However an interac-tion between the enhancer of mRNA decapping complex 3(EDC3) [24] and insulin production has not been reported

The glucose analogue 2-deoxy-d-glucose (2-DG) ismetabolized by hexokinase and acts as an inhibitor ofglycolysis [25] 2-DG triggers glucose deprivation withoutaltering other nutrients or metabolic pathways [26] and thenactivates autophagy via activation of AMPK [27] and endo-plasmic reticulum (ER) stress [28] Therefore 2-DG appearsto be an ideal tool to understand the interactions betweenautophagy and ER stress In this study we provide the firstdemonstration that 2-DG reduces intracellular insulin whichwas increased by melatonin via autophagy and EDC3 ininsulinoma INS-1E cells

2 Materials and Methods

21 Cell Culture INS-1E cells a clonal pancreatic 120573-cell linewere obtained from Professor Claes B Wollheim and werecultured in RPMI 1640 medium (Invitrogen Carlsbad CAUSA) containing 11mM glucose supplemented with 10mMHEPES (pH 73) 10 heat-inactivated fetal bovine serum(FBS Invitrogen) 50 120583M 120573-mercaptoethanol 1mM sodiumpyruvate 50 120583gmL penicillin and 100 120583gmL streptomycinat 37∘C with 5 CO

2in a humidified incubator

22 Treatment with 2-DG Melatonin and Rapamycin INS-1E cells were cultured in RPMI 1640 medium plus 2 heat-inactivated FBS in a 37∘C and 5 CO

2incubator with or

without melatonin (Sigma-Aldrich St Louis MO USA)andor 2-DG (5mM) (Sigma-Aldrich) for 24 hr or withrapamycin (20 to 80 nM) (Calbiochem SanDiegoMOUSA)for 24 hr

23 Western Blot Analysis Cells were harvested washedtwice with ice-cold phosphate buffered saline (PBS) and thenresuspended in 20mM Tris-HCl buffer (pH 74) containingprotease inhibitors (01mM phenylmethylsulfonyl fluoride5 120583gmL aprotinin 5120583gmLpepstatinA and 1120583gmL chymo-statin) and phosphatase inhibitors (5mMNa

3VO4and 5mM

NaF) Whole cell lysate was prepared using 20 strokes of aDounce homogenizer followed by centrifugation at 13000timesgfor 20min at 4∘CThe protein concentration was determinedusing the BCA assay (Sigma) Proteins (40120583g) were sep-arated by 12 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and the detection of insulinproteins was performed by 165 tricine SDS-PAGE Thesegels transferred onto a polyvinylidene difluoride (PVDF)

membrane The membrane was incubated with antibodies(diluted as indicated in brackets) directed against the fol-lowing proteins p-AMPK and AMPK (1 500) (Santa CruzBiotechnology) IRE1120572 (1 500) (Santa Cruz Biotechnology)p-PERK (1 1000) (Cell Signaling Technology Beverly MAUSA) GRP78BiP (1 1000) (Cell Signaling Technology)p85120572 and p85120573 (1 500) (Santa Cruz Biotechnology) p110(1 500) (Santa Cruz Biotechnology) p-Akt (T308 S473)and Akt (1 1000) (Cell Signaling Technology) p-mTOR(Ser2448 2481) andmTOR (1 1000) (Cell Signaling Technol-ogy) CuZn-SOD (1 500) (Santa Cruz Biotechnology) Mn-SOD (1 500) (Santa Cruz Biotechnology) catalase (1 500)(Santa Cruz Biotechnology) Bcl-2 (1 500) (Santa CruzBiotechnology) Bax (1 500) (Santa Cruz Biotechnology)insulin (1 500) (Santa Cruz Biotechnology) EDC3 (1 500)(Santa Cruz Biotechnology) and Actin (1 1000) (AssayDesigns Ann Arbor MI USA) Immunoreactive proteinswere visualized by exposure to X-ray film Protein bandswere analyzed by image-scanning and optical density wasmeasured using ImageJ analysis software (version 137WayneRasbandNIH BethesdaMDUSA)The data were correctedfor background subtraction and normalized by includingActin as an internal control

24 Statistical Analysis Significant differences were detectedby ANOVA followed by Tukeyrsquos test for multiple compar-isons Analysis was performed using the Prism Graph Padv40 (Graph Pad Software Inc San Diego CA USA) Valuesare expressed as means plusmn SD of at least three separatedexperiments in which case a representative experiment isdepicted in the figures 119901 values lt 005 were consideredstatistically significant

3 Results

31 Expressions of p-AMPK GRP78BiP IRE1120572 and p-PERK Proteins We investigated whether 2-DG reducedinsulin synthesis which had been increased by melatoninvia autophagy in rat insulinoma INS-1E cells 2-DG activatesautophagy via reactive oxygen species-mediated activation ofAMPK [27] andER stress [28]Therefore we first investigatedwhether 2-DG andor melatonin induced phosphorylationof AMPK and ER stress in rat insulinoma INS-1E cellsPhosphorylation of AMPK was significantly increased bytreatmentwith 2-DG in the presence or absence ofmelatonincompared to treatment with melatonin or FBS only (Figures1(a) and 1(b)) ER stress marker GRP78BiP protein wasalso significantly increased by treatment with 2-DG in thepresence or absence of melatonin (Figures 1(c) and 1(d)) butexpression of IRE1120572 protein was reduced (Figures 1(c) and1(d)) and p-PERK protein expression was not significantlyaffected by 2-DG treatment (Figures 1(c) and 1(e))

32 Expressions of p-PI3K AktPKB and mTOR ProteinsWe determined how 2-DG andor melatonin affected knownregulators of insulin signaling phosphoinositide 3-kinase(PI3K) AktPKB and mTOR in rat insulinoma INS-1Ecells Class IA PI3K is composed of a heterodimer of p110catalytic subunit and p85 regulatory subunit [29] 2-DG


p-AMPK

AMPK


+

+

++

minusminus

minus

minus

minus minus

+

+

minus


(a)

lowastlowast

lowastlowast


++

minusminus

minus minus

0

50

100

150

Relat

ive a

mou

nts o

f p-A

MPK


(b)


+

+

++

minusminus

minus

minus

minus minus

+

+

minus

minus

Actin

GRP78BiP

p-PERK


(c)

Relat

ive a

mou

nts o

f IRE

1120572


0

50

100


++

minusminus

minus minus

150


(d)

Rela

tive a

mou

nts o

f p-P

ERK

lowastlowastlowast

0

50

100

150

200

250


++

minusminus


(e)

100

lowast


0

200

Relat

ive a

mou

nts o

f GRP

78B

IP


++

minusminus


(f)


2



p110

Actin

p85120572

p85120573


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

lowastlowast

lowastlowastlowast

Relat

ive a

mou

nts o

f p85120572

0

25

50

75

100


++

minusminus


(b)

lowastlowastlowast

lowastlowastlowast

0

100

200

Rela

tive a

mou

nts o

f p85120573


++

minusminus


(c)


0

25

50

75

100

125

Relat

ive a

mou

nts o

f p11

0


++

minusminus


(d)


2







p-Akt (S473)

Akt

p-Akt (T308)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (S

473)



++

minusminus


(b)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (T

308)



++

minusminus


(c)

mTOR

p-mTOR (S2448)

p-mTOR (S2481)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(d)

lowast

0

25

50

75

100

125

Relat

ive a

mou

nts o

f p-m

TOR

(S24

48)


++

minusminus


(e)

0

50

100

150

Relat

ive a

mou

nts o

f p-m

TOR

(S24

81)

lowastlowastlowast

lowastlowastlowast


++

minusminus


(f)


2



Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

50

100

CuZ

n-SO

D


(b)

lowast


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

25

50

75

100

Mn-

SOD


(c)

lowast


++

minusminus

minus minus

0

25

50

75

100

of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(e)

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

Relat

ive a

mou

nts o

f Bcl-

2


(f)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

100

125

Relat

ive a

mou

nts o

f Bax


(g)


2




Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-AMPK

AMPK


+

+

++

minusminus

minus

minus

minus minus

+

+

minus


(a)

lowastlowast

lowastlowast


++

minusminus

minus minus

0

50

100

150

Relat

ive a

mou

nts o

f p-A

MPK


(b)


+

+

++

minusminus

minus

minus

minus minus

+

+

minus

minus

Actin

GRP78BiP

p-PERK


(c)

Relat

ive a

mou

nts o

f IRE

1120572


0

50

100


++

minusminus

minus minus

150


(d)

Rela

tive a

mou

nts o

f p-P

ERK

lowastlowastlowast

0

50

100

150

200

250


++

minusminus


(e)

100

lowast


0

200

Relat

ive a

mou

nts o

f GRP

78B

IP


++

minusminus


(f)


2



p110

Actin

p85120572

p85120573


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

lowastlowast

lowastlowastlowast

Relat

ive a

mou

nts o

f p85120572

0

25

50

75

100


++

minusminus


(b)

lowastlowastlowast

lowastlowastlowast

0

100

200

Rela

tive a

mou

nts o

f p85120573


++

minusminus


(c)


0

25

50

75

100

125

Relat

ive a

mou

nts o

f p11

0


++

minusminus


(d)


2







p-Akt (S473)

Akt

p-Akt (T308)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (S

473)



++

minusminus


(b)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (T

308)



++

minusminus


(c)

mTOR

p-mTOR (S2448)

p-mTOR (S2481)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(d)

lowast

0

25

50

75

100

125

Relat

ive a

mou

nts o

f p-m

TOR

(S24

48)


++

minusminus


(e)

0

50

100

150

Relat

ive a

mou

nts o

f p-m

TOR

(S24

81)

lowastlowastlowast

lowastlowastlowast


++

minusminus


(f)


2



Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

50

100

CuZ

n-SO

D


(b)

lowast


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

25

50

75

100

Mn-

SOD


(c)

lowast


++

minusminus

minus minus

0

25

50

75

100

of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(e)

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

Relat

ive a

mou

nts o

f Bcl-

2


(f)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

100

125

Relat

ive a

mou

nts o

f Bax


(g)


2




Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p110

Actin

p85120572

p85120573


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

lowastlowast

lowastlowastlowast

Relat

ive a

mou

nts o

f p85120572

0

25

50

75

100


++

minusminus


(b)

lowastlowastlowast

lowastlowastlowast

0

100

200

Rela

tive a

mou

nts o

f p85120573


++

minusminus


(c)


0

25

50

75

100

125

Relat

ive a

mou

nts o

f p11

0


++

minusminus


(d)


2







p-Akt (S473)

Akt

p-Akt (T308)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (S

473)



++

minusminus


(b)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (T

308)



++

minusminus


(c)

mTOR

p-mTOR (S2448)

p-mTOR (S2481)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(d)

lowast

0

25

50

75

100

125

Relat

ive a

mou

nts o

f p-m

TOR

(S24

48)


++

minusminus


(e)

0

50

100

150

Relat

ive a

mou

nts o

f p-m

TOR

(S24

81)

lowastlowastlowast

lowastlowastlowast


++

minusminus


(f)


2



Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

50

100

CuZ

n-SO

D


(b)

lowast


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

25

50

75

100

Mn-

SOD


(c)

lowast


++

minusminus

minus minus

0

25

50

75

100

of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(e)

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

Relat

ive a

mou

nts o

f Bcl-

2


(f)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

100

125

Relat

ive a

mou

nts o

f Bax


(g)


2




Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-Akt (S473)

Akt

p-Akt (T308)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (S

473)



++

minusminus


(b)

0

100

200

Relat

ive a

mou

nts o

f p-A

kt (T

308)



++

minusminus


(c)

mTOR

p-mTOR (S2448)

p-mTOR (S2481)


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(d)

lowast

0

25

50

75

100

125

Relat

ive a

mou

nts o

f p-m

TOR

(S24

48)


++

minusminus


(e)

0

50

100

150

Relat

ive a

mou

nts o

f p-m

TOR

(S24

81)

lowastlowastlowast

lowastlowastlowast


++

minusminus


(f)


2



Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

50

100

CuZ

n-SO

D


(b)

lowast


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

25

50

75

100

Mn-

SOD


(c)

lowast


++

minusminus

minus minus

0

25

50

75

100

of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(e)

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

Relat

ive a

mou

nts o

f Bcl-

2


(f)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

100

125

Relat

ive a

mou

nts o

f Bax


(g)


2




Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Actin

CuZn-SOD

Catalase

Mn-SOD


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

50

100

CuZ

n-SO

D


(b)

lowast


++

minusminus

minus minus

Rela

tive a

mou

nts o

f

0

25

50

75

100

Mn-

SOD


(c)

lowast


++

minusminus

minus minus

0

25

50

75

100

of ca

tala

seRe

lativ

e am

ount

s


(d)

Actin

Bcl-2

Bax


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(e)

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

Relat

ive a

mou

nts o

f Bcl-

2


(f)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


++

minusminus

minus minus

0

25

50

75

100

125

Relat

ive a

mou

nts o

f Bax


(g)


2




Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Actin

Insulin

EDC3

LC3 III


+

+

++

minus

+

minusminus

minus

minus

minus minus

+


(a)

0

25

50

75

100

Relat

ive a

mou

nts o

f LC3

II

lowastlowastlowast

lowastlowastlowast


++

minusminus


(b)

0

25

50

75

100

125

Relat

ive a

mou

nts o

f ins

ulin

lowastlowast



++

minusminus


(c)

0

10

20

30

40

50

60

70

80

90

Rela

tive a

mou

nts o

f ED

C3

lowastlowast

lowastlowastlowast


++

minusminus


(d)


2





4 Discussion




EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















EDC3

GRP78BiP

Actin

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

50

100

150

Relat

ive a

mou

nts o

f ins

ulin

(b)



0

50

100

150

Relat

ive a

mou

nts o

f GRP

78B

iP

(c)

lowastlowastlowast

lowast

lowastlowastlowast


0

100

200

Relat

ive a

mou

nts o

f ED

C3

(d)


2















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article melatonin-mediated intracellular insulin...

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