Research Article Protein Fractions from Korean Mistletoe ...downloads.hindawi.com/journals/ecam/2014/703624.pdf · Research Article Protein Fractions from Korean Mistletoe (Viscum

Research ArticleProtein Fractions from Korean Mistletoe(Viscum Album coloratum) Extract Induce InsulinSecretion from Pancreatic Beta Cells

Ki-Wook Kim12 Seung-Hoon Yang13 and Jong-Bae Kim1

1 School of Life Sciences Handong Global University Pohang Gyungbuk 791-708 Republic of Korea2Department of Microbial Pathogenesis Boyer Center for Molecular Medicine Yale University School of MedicineNew Haven CT 06510 USA

3Department of Biological Chemistry The Weizmann Institute of Science 76100 Rehovot Israel

Correspondence should be addressed to Jong-Bae Kim jbkimhandongedu

Received 23 February 2014 Accepted 22 April 2014 Published 14 May 2014

Academic Editor Cicero L T Chang

Copyright copy 2014 Ki-Wook Kim et al This 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

Mistletoe (Viscum Album coloratum) has been known as a medicinal plant in European and Asian countries Recent data show thatbiological activity of mistletoe alleviates hypertension heart disease renal failure and cancer development In this study we reportthe antidiabetic effect ofKoreanmistletoe extract (KME) KME treatments enhanced the insulin secretion from the pancreatic120573-cellwithout any effects of cytotoxicity PDX-1 and beta2neuroD known as transcription factors that regulate the expression of insulingenewere upregulated by treatment of theKMEprotein fractions isolated by ion-exchange chromatography after ammonium sulfateprecipitation Furthermore these KME protein fractions significantly lowered the blood glucose level and the volume of drinkingwater in alloxan induced hyperglycemic mice Taken together with the findings it provides new insight that KMEmight be servedas a useful source for the development of medicinal reagent to reduce blood glucose level of type I diabetic patients

1 Introduction

Insulin is an essential hormone that maintains the blood andurinary glucose level At transcriptional level insulin geneexpression is controlled by the specific transcription factorspancreatic and duodenal homeobox factor-1 (PDX-1) andbeta2neuroD [1ndash4] However abnormal immune responsedestroys pancreatic 120573 cell and impairs insulin secretionleading to insulin dependent type I diabetes [5 6] It leads tosevere complications such as vascular disease renal failureand heart disease [7ndash9] Although exogenous insulin therapythrough the insulin pump or subcutaneous insulin injectiontemporally delays the onset of complications it finally resultsin type I diabetes mellitus [10ndash13]

Mistletoe (Viscum album) is a semiparasitic plant whichgrows on deciduous trees in Asian and European countries

It has been used as a traditional therapeutic reagent to lowerblood pressure and modulate immune responses [14ndash18]Previously we reported that Korean mistletoe has a varietyof immunological activities such as cytokine induction NKcell activation and adjuvant activity [19ndash21] Specificallylectins isolated from KME had antitumor activity to suppresstumor growth and to inhibit tumor-induced angiogenesisor metastasis [22ndash24] Although it has been reported thatKME and its lectins played critical role for immunologicalactivity and antitumor activity [25] little is known about theantidiabetic effect of Korean mistletoe

In this study we revealed KME protein fractions withoutcellular damage induced to release insulin from the pancre-atic 120573 cell by activating transcription factors such as PDX-1and beta2neuroD required for insulin gene expression Pos-sibly due to the elevation of insulin release by KME protein

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 703624 8 pageshttpdxdoiorg1011552014703624

2 Evidence-Based Complementary and Alternative Medicine

fraction the blood glucose level and drinking water volumewere significantly reduced in alloxan induced hyperglycemicmice [26 27]

2 Materials and Methods

21 Reagents RPMI 1640 without glucose alloxan andKrebs-Ringer bicarbonate (KRB) buffer were purchased fromSigma Anti-PDX-1 and antineuroD all from Santa CruzBiotechnology were used for immunoblotting Rat insulinELISA Kit (SHIBAYAGI) was used for insulin secretionSYBR greenmastermix (Applied Biosystems) andRNAEasy-spin (iNtRON) were used for real-time PCR

22 Cell Culture and Treatment Rat pancreatic 120573 cellsRINm5F cells were provided by PohangUniversity of Scienceand Technology (POSTECH) RINm5F cells were culturedin a monolayer in RPMI 1640 medium (Gibco) supple-mented with 10 fetal bovine serum (FBS CAMBREX) 1penicillinstreptomycin (CAMBREX) and 55mMglucose at37∘C under a humidified 95ndash5 (vv) mixture of air andCO2 RINm5F cells were cultured in complete medium with

10 FBS to sim90 cell confluence and were then incubatedwith 05 FBS medium for 12sim16 hours After that RINm5Fcells were treated with KM extractThe supernatant was usedfor detection of insulin secretion and the attached cells wereused for immunoblotting

23 Animals Specific pathogen-free 7-week-old imprintingcontrol region (ICR) mouse strains were purchased from theDae-Han LaboratoryAnimal Center Republic of KoreaMicewere maintained in the Laboratory of Animal ExperimentInstitute of Bioscience Han-Dong Global University underlaminar air-flow conditions Water and diets were suppliedad libitum Each group was composed of 5 mice for checkingblood glucose level and drinking water volumes

24 Extraction and Purification of Korean Mistletoe Mistle-toe growing on oak (Quercus variabilis Blume) betweenDecember and February was harvested from Kangwon-doKorea and stored at minus80∘C until use The nonlectin fractions(NLF) were isolated from an extract of Korean mistletoeplants using hydrolyzed120572-lactose-Sepharose (Sigma) columnchromatography after ammonium sulfate precipitation asdescribed previously [24 25] Briefly the extracts were dis-solved in distilled water and filtered through a 045 120583mmem-brane (Whatmann) Then they adjusted to 70 saturationwith (NH

4)SO4 The precipitate was resuspended in a small

volume of phosphate-buffered saline (PBS) dialyzed againstthe same buffer and applied to an 120572-lactose-Sepharosecolumn which had been hydrolyzed for 25 hours To removethe Korean mistletoe lectin (KML-C) the adsorbed materialwas eluted with a lactose-containing buffer (01M lactose pH73) Unbounded proteins which were free of KML-C werepooled dialyzed against distilled water and freeze-driedEach process was performed at 4∘CNLFdissolved in distilledwater was partitioned by chloroform Chloroformic extractsdissolved in phosphate buffer (PBS 10mM phosphate buffer

containing 085 NaCl pH 74) were applied to the DEAE-Sepharose Fast flow column (AmershamPharmacia Biotech)which had previously equilibrated with the same buffer Aftercolumn was washed with phosphate buffer again remainingbound proteins were eluted with 05M 1M and 2M NaCl(DEAE 05M 1M and 2M fractions) DEAE fractionsdissolved in PBS were applied to the Sep-Pak column (WatersCorporation) which had been previously equilibrated withthe same buffer The column was washed with PBS andbound proteins were eluted with 20 ethanol (Sep-Pak20 SP20 fractions) and 60 ethanol (Sep-Pak 60 SP60fractions) Each eluted protein fraction was dialyzed againstdistilled water every 4 hours for 3 days and stored in minus20∘Cuntil use

25 FACS Analysis for Cytotoxicity Cytotoxicity was ana-lyzed by the annexin V and propidium iodide (PI Merck)using flow-cytometry (FACS) machine (Beckman CoulterEpic XL) RINm5F cells (2 times 106 cells) were seeded into 6-wellplates and incubated for 12 hours After washing KM extractwas treated with the cells and incubated in the 37∘C with 5CO2for 30 minutes Cells were carefully detached from the

plates and thendetached cellswere stainedwith the apoptosisdetection kit containing annexin V-FITC and PI at ice in thedark condition Stained cells werewashedwith FACS solutionand living or dead cells were measured by FACS machine

26 Measurement of Insulin 1mL of RINm5F cells (4 times105 cellswell) was seeded into the 24-well plates (Falcon) andcultured in RPMI-1640 containing 111mM glucose (Sigma)After incubation for 12 hr to allow attachment cells werewashed with Krebs-Ringer bicarbonate buffer (KRB) in 3timesThen cells were preincubated with 1mL of KRB bufferfor 30 minutes at 37∘C Cells were then incubated for 30minutes with KM extracts The supernatants were collectedfrom each well and stored at minus20∘C until use for insulinsecretion assay An assay for the measurement of insulinsecretion from RINm5F cells was carried out by rat insulinELISA Kit (SHIBAYAGI) and detected by ELISA reader at450 nm

27 Real-Time PCR Total RNA were extracted using easy-spin RNA extraction kit (Intron Korea) cDNA were pro-duced with 5 120583g of total RNA using Superscript (Invitrogen)In a florescent temperature cycler (LightCycler Roche Diag-nostics Ltd Lewes UK) 10 percent of each RT reaction wasamplified in a 20120583L PCR containing 4mMMgCl

2 4 pmol of

each primer and 1x SYBR green master mix Samples wereincubated in the LightCycler for an initial denaturation at94∘C for 30 seconds followed by 30 PCR cycles Each cycleconsisted of 95∘C for 15 seconds 55∘C for 32 seconds and72∘C for 32 seconds

119879ℎ119890 119874119897119894119892119900119899119906119888119897119890119900119905119894119889119890 119875119903119894119898119890119903119904

For insulin are

forward 51015840-ATA GAC CAT CAG CAA GCAGG-31015840

Evidence-Based Complementary and Alternative Medicine 3

01 1000

PI

Annexin VNon-treated

987

KM extract (1mgmL)

973

KM extract (2mgmL)

886

Single parameter

01 1000

PI

Annexin V

01 1 10 100 1000

01 1000

PI

Annexin V

0

51

102

153

204

DNA content

01

1000

01

1000

01

1000

(128times128)

(128times128)

(128times128)

(a)

Cel

l sur

viva

l (

)

20

40

60

80

100

120

01mgmL 2mgmL 50mM 100mM

KME Alloxan

(b)

Figure 1 Cytotoxicity of pancreatic 120573 cells by KME treatment (a) Representative FACS analysis for PI and annexin V staining of RINm5Fcell following KME treatment Nontreated RINm5F cells (left) KME (1mgmL) treated RINm5F cell (middle) and KME (2mgmL) treatedRINm5F cells (right) DNA contents of RINm5F cells under the KME treatment Blue nontreated red KME (1mgmL) and green KME(2mgmL) (b) The cytotoxic activity of KME- or alloxan-treated cells was measured by XTT assay

reverse 51015840-CTC CAG TTG TGG CAC TTGCG-31015840

For PDX-1 are

forward 51015840-TAC GCG GCC ACA CAG CTCTAC AAG GAC-31015840reverse 51015840-CCA CTT CAT GCG ACG GTTTTG GAA CCA GA-31015840

And for 120573-actin are

forward 51015840-AGG ATG CAG AAG GAG ATCACT G-31015840

reverse 51015840-GGG TGT AAC GCA ACT AAGTCA TAG-31015840

120573-actin was used as an endogenous internal control Toconfirm amplification of specific transcripts melting curveprofiles (cooling the sample to 65∘C for 15 seconds andheating slowly to 95∘C with continuous measurement of flu-orescence) were produced at the end of each PCR procedure

28 Western Blot Analysis For Western blotting analysiscells were lysed by resuspension in buffer A (25mM Tris-HCl pH 75 2mMNa orthovanadate 05mM EDTA 10mM


Insu

lin se

cret

ion

(pg

mL)

2

4

6

8

10

12times10

3

Media 0612255 101184167

KME (mgmL)Glucose (mM)42

lowast

lowastlowastlowast

lowastlowast

(a)

Insu

lin se

cret

ion

(pg

mL)

1

2

3

4

5

6

7

8

1256253131577942105025

Lectin (120583gmL)KMEMedia

times103

(b)

6

1

2

3

4

5

LFFNon treated KME

Insu

lin se

cret

ion

(pg

mL)

NStimes103

(c)

1

2

3

4

5

6In

sulin

secr

etio

n (p

gm

L)

Non treated 05M 1M 2MDEAE column fraction

LFF

times103

(d)

Figure 2 Insulin secretion of RINm5F cells by a variety of KME protein fractions (a) Insulin secretion of RINm5F cell by KME dosedependent manner (b) Insulin secretion of RINm5F cells by lectin (KML-C) isolated fromKoreanmistletoe (c) Insulin secretion of RINm5Fcells by the lectin-free KME and total KME (d) Insulin secretion of RINm5F cells by protein fractions from KME isolated by ion-exchangechromatography

NaF 10mM Na pyrophosphate 80mM 120573-glycerophosphate25mMNaCl 1 (vv) Nonidet P-40) containing 1x completeprotease inhibitor mixture (Roche Diagnostics) and thenboiled for 10min After sonication they were centrifugedfor 15min in 13000 timesg Protein concentration in the extractwas determined using the BCA kit (Pierce) Protein sampleswere separated on SDS-PAGE and blotted onto nitrocellulosemembrane (Amersham Biosciences) Blots were developedwith the ECL kit (Pierce)

29 Generation and Analysis of Hyperglycemic Mice ICRmice were grouped into several groups of five mice Alloxan(70mgkg) was injected into mice via tail vein Next day ofalloxan injection 200120583L of DEAE eluted protein fractionsfromKM extracts (50 100 200 and 400 120583gmL) was injectedintraperitoneally After 10 days the same volumes of DEAEeluted protein fraction were injected intraperitoneally againBlood was obtained from the tail vein of injected micethrough heparinized capillary tube Blood glucose levels weremeasured by the glucose measurement kit (Assan Pharma-cia) Drinking water volumes were measured by a decimeter

3 Results

31 KME Has No Cytotoxicity to Rat Pancreatic 120573 Cell Dueto the cytotoxicity of lectin A subunit contained in KMEwe examined whether KME has the cytotoxic activity on ratpancreatic 120573 cells (RINm5F) Apoptotic cells and necrotic cellwere measured by annexin V and PI staining As shown inFigure 1(a) most cells were alive after aqueous KM extract(1mgmL 2mgmL) treatment It indicates that RINm5 cellviability was not affected by KME treatment To evaluatethe cell proliferation as well as cytotoxicity we incubatedRINm5 cells with KME (1mgmL 2mgmL) for 48 hoursThe survival rate of RINm5 cell was measured by XTT assayWell known as a chemical compound to specifically destroypancreatic beta cells alloxan severely damaged the RINm5cells However RINm5 cells were not affected by KME at all(Figure 1(b)) These findings imply that KME is not toxic tothe rat pancreatic 120573 cells

32 Lectin-Free KME Protein Fractions Induce Insulin Secre-tion on Rat Pancreatic 120573 Cell To examine the potential tosecrete insulin by KME the insulin released from RINm5F


1

2

3

4

5

6

Relat

ive e

xpre

ssio

n 7

8

85310

9

Time (h)

Insulin genePDX-1

DEAE 05M

(a)

1

2

3

4

5

83510

Relat

ive e

xpre

ssio

n

Time (h)

Insulin genePDX-1

DEAE 1M

(b)

0 15 30 60 3hr 5hr 8hr12hrRINm5F cells

Neuro D (49KD)

Non specificPDX-1 (42KD)

Time (min)DEAE 05M

(c)


Neuro D (49KD)


Time (min)DEAE 1M

(d)

Figure 3 Insulin gene and transcription factor expression byDEAE-Sepharose fractionatedKMEprotein fractions ((a)-(b)) Time dependentmRNA expressions of insulin gene and PDX-1 gene in RINm5F cells under the treatment of (a) 05M DEAE protein fractions and (b) 1MDEAE ((c)-(d)) Time dependent PDX-1 and beta2neuroD protein expressions of RINm5F cells under the treatment of (d) 05M DEAEprotein fraction and (d) 1M DEAE protein fraction

cells was measured by ELISA after treatment of a severaldoses of KME While glucose concentration had no effectto secrete insulin KME had stimulatory effect on insulinsecretion in a dose dependent manner (Figure 2(a)) Nextwe investigated whether the lectin (KML-C) isolated fromKME has potential to secrete insulin on RINm5F cellsAlthough the lectin (KML-C) isolated by KME was treatedwith the RINm5 cells in a dose dependent manner (025sim125 120583gmL) for 12 hours insulin secretion was not detectedin RINm5F cells (Figure 2(b)) However lectin-free KMEprotein fraction (LFF) induced insulin secretion as similaras the KME treatments (Figure 2(c)) Thereafter LFF wasisolated from DEAE-Sepharose Column and finally dividedinto three fractions by concentration of 05M 1M and 2MNaCl elution buffer respectively Both 05M and 1M NaCleluted DEAE-Sepharose column fractions induced insulinsecretion from RINm5F cells whereas 2M NaCl elutedDEAE-Sepharose column fractions did not have insulinsecretion ability (Figure 2(d)) Therefore it suggested thatpurified protein factions from KME have an ability of insulininduction in rat pancreatic 120573 cells

33 Protein Fractions from KME Induce Insulin andPDX-1 Gene Expression The molecular mechanism of

insulin secretion by protein fraction of KME remainsunknown To determine whether protein fractions ofKME activate insulin gene in transcriptional levels weanalyzed the expression level of insulin gene and its relatedtranscription factor PDX-1 through the real-time PCRAfter the treatment of 05M DEAE protein fractionsinsulin genes were peaked at three hours and PDX-1genes were upregulated in an hour (Figure 3(a)) Althoughinsulin genes and PDX-1 genes in 1M-DEAE proteinfraction treated RINm5F cells were relatively shown inlow expression they were also upregulated in an hour(Figure 3(b))

Although the expression level of PDX-1 genes was notaltered within 60min the expression level of insulin geneswas significantly increased in real-time PCR analysis Toaddress this discrepancy we determined to analyze the PDX-1 and beta2neuroD expression in time dependent mannerWestern analysis of PDX-1 expression revealed that thistranscription factor was highly increased after 15min of 05Mor 1M DEAE protein fraction of KME treatment and itsexpression was gradually decreased Similar to the PDX-1beta2neuroD is highly expressed within 60min by the 05Mor 1MDEAEprotein fraction of KME (Figures 3(c) and 3(d))These results strongly suggested that protein fractions ofKME activate PDX-1 and beta2neuroD transcription factors


Insu

lin se

cret

ion

(pg

mL)

PBS Alloxan LFF DEAE 05M

300

600

900

1200

1500

1800

0

(a)

Bloo

d gl

ucos

e (m

gdL

)

Non treated Alloxan LFF DEAE 05M

100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600

0Non treated Alloxan

(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

0

Drin

king

wat

er v

ol

(mL

mic

eda

y)

Non treated Alloxan(100mM)

400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)

Figure 4 Antidiabetic effect of KME protein fractions on alloxan induced hyperglycemic mice ((a)-(b)) Restoration of (a) insulin leveland (b) blood glucose level of alloxan induced hyperglycemic mice by the lectin-free KME and 05M DEAE protein fraction of KME (c)Restoration of blood glucose level of alloxan induced mice by dose dependent 1M DEAE protein fractions (d) Reduction of drinking-waterconsumption of alloxan induced mice by 1M DEAE protein fractions

in early time point eventually resulting in the activation ofinsulin gene

34 Protein Fractions from KME Rescued the Insulin Secretionand Blood Glucose Level in Alloxan Induced HyperglycemicMice Next we examined whether insulin secretion andblood glucose level could be rescued by introduction of theprotein fractions of KME in vivo model To this end lectin-free fraction of KME or its 05M DEAE protein fractionwas injected into alloxan induced diabetic mice respectivelyInsulin secretion and serum glucose level were measuredafter 48 hours of KME fractionated protein injection Wefound that insulin secretion was severely impaired by alloxaninjection but it was partially restored by injection of the KMEprotein fractions (Figure 4(a)) Furthermore blood glucoselevel was restored by 05M DEAE protein fraction treatedmice compared to alloxan treated mice (Figure 4(b)) Alsoit was confirmed that blood glucose level of alloxan induceddiabeticmice was gradually reduced by dose dependentman-ner of 1M DEAE protein fraction treatment (Figure 4(c))

Similar to diabetic patients alloxan induced diabeticmice consumed much more water than normal mice Toinvestigate whether the consumption of drinking watercould be reduced by treatment of DEAE protein fractionsof KME we measured the volume of drinking water of

diabetic mice in the presence or absence of DEAE proteinfractions of KME As expected drinking water volume wasslightly decreased regardless of the dose of DEAE proteinfractions (Figure 4(d)) Collectively these findings suggestedthat DEAE-Sepharose fractionated proteins fromKM extracthave an effective antidiabetic activity in alloxan inducedhyperglycemia mice

4 Discussion

It has been well known that Korean mistletoe has strongimmunomodulatory effects such as immune cells activationcytokines induction and antitumor effects [19 20 22ndash24]Despite the use of this plant as an effective traditionaltreatment for diabetes little is known about the molecularmechanism for the antidiabetic effect of this plant [28]

In this study we demonstrated the antidiabetic effect ofthe various KME protein fractions in vitro and in vivomolec-ular mechanisms of insulin secretion First we examined theinduction ability of insulin secretion from pancreatic 120573 cellsby various protein fractions of KME through the activationof transcription factors such as PDX-1 and beta2neuroDwhich regulate the insulin genes expression Second wefound that pancreatic120573 cells were not impaired byKME treat-ment whereas these cells were severely destroyed by alloxantreatment Our data support that KME directly activates the


insulin genes and transcription factors associated with theinsulin gene expression for the first time

Initially it has been expected that lectin of Koreanmistletoe (KML-C) could have an effect on diabetes becauseof its diverse biological activities However the data showedin this study that KML-C is not related to the induction ofinsulin and lectin-free KMEprotein fractions (LFF) stimulatethe secretion of insulin Furthermore we investigated thefact that protein fractions from KME separated by severalpurification methods induce insulin secretion from rat pan-creatic 120573 cells Molecular signaling of insulin induction wascontrolled by activation of several transcription factors Herewe demonstrated that PDX-1 and beta2neuroD which playa critical role in induction of insulin genes expression wereupregulated in KME treated pancreatic 120573-cells Consistentwith in vitro data KME treatment restored the insulinsecretion in alloxan induced hyperglycemic mice Moreoverblood glucose level and drinking water volume were restoredby KME treatment in alloxan induced hyperglycemic miceThese in vivo data support that KME is able to rescuedestroyed pancreatic 120573 cells or delay the pancreatic 120573 cellsdestruction As reported in Lyu et al the lectin component ofKME is able to promote cell proliferation [29] Accordinglywe speculate that some specific components of KME regulatethe proliferation or regeneration of pancreatic 120573 cells but itsmolecular mechanism will be investigated in the future

In conclusion the present study provides evidence for thefirst time for insulin releasing protein fractions in Koreanmistletoe It is required for separating a large amount ofspecific single peptide from KME which has an insulin-secreting activity Together with the current findings it isthought that this single peptide from KME can representa source of potential new therapy for plant treatment ofdiabetes

Conflict of Interests

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

Authorsrsquo Contribution

Ki-Wook Kim and Seung-Hoon Yang contributed equally tothis work

References

[1] M Welsh D A Nielsen A J MacKrell and D F SteinerldquoControl of insulin gene expression in pancreatic 120573-cells andin an insulin-producing cell line RIN-5F cells II Regulationof insulin mRNA stabilityrdquoThe Journal of Biological Chemistryvol 260 no 25 pp 13590ndash13594 1985

[2] P O Berggren and I B Leibiger ldquoNovel aspects on signal-transduction in the pancreatic beta-cellrdquo Nutrition Metabolismamp Cardiovascular Diseases vol 16 supplement 1 pp S7ndashS102006

[3] PM Jones and S J Persaud ldquoProtein kinases protein phospho-rylation and the regulation of insulin secretion from pancreatic120573-cellsrdquo Endocrine Reviews vol 19 no 4 pp 429ndash461 1998

[4] S K Chakrabarti and R G Mirmira ldquoTranscription factorsdirect the development and function of pancreatic 120573 cellsrdquoTrends in Endocrinology and Metabolism vol 14 no 2 pp 78ndash84 2003

[5] ldquoDiagnosis and classification of diabetes mellitusrdquo DiabetesCare vol 27 supplement 1 pp S5ndashS10 2004

[6] E J Weringer and A A Like ldquoImmune attack on pancre-atic islet transplants in the spontaneously diabetic BioBreed-ingWorcester (BBW) rat is not MHC restrictedrdquo Journal ofImmunology vol 134 no 4 pp 2383ndash2386 1985

[7] W T Cade ldquoDiabetes-relatedmicrovascular andmacrovasculardiseases in the physical therapy settingrdquo Physical Therapy vol88 no 11 pp 1322ndash1335 2008

[8] M Brownlee ldquoBiochemistry and molecular cell biology ofdiabetic complicationsrdquo Nature vol 414 no 6865 pp 813ndash8202001

[9] A R Andersen J S Christiansen J K Andersen S Kreinerand T Deckert ldquoDiabetic nephropathy in type 1 (insulin-dependent) diabetes an epidemiological studyrdquo Diabetologiavol 25 no 6 pp 496ndash501 1983

[10] D E deWitt and I BHirsch ldquoOutpatient insulin therapy in type1 and type 2 diabetes mellitus scientific reviewrdquo Journal of theAmerican Medical Association vol 289 no 17 pp 2254ndash22642003

[11] Y Ohkubo H Kishikawa E Araki et al ldquoIntensive insulintherapy prevents the progression of diabetic microvascularcomplications in Japanese patients with non-insulin-dependentdiabetes mellitus a randomized prospective 6-year studyrdquoDiabetes Research and Clinical Practice vol 28 no 2 pp 103ndash117 1995

[12] M A Atkinson andG S Eisenbarth ldquoType 1 diabetes new per-spectives on disease pathogenesis and treatmentrdquo The Lancetvol 358 no 9277 pp 221ndash229 2001

[13] S C Shah J I Malone and N E Simpson ldquoA randomizedtrial of intensive insulin therapy in newly diagnosed insulin-dependent diabetes mellitusrdquo The New England Journal ofMedicine vol 320 no 9 pp 550ndash554 1989

[14] G Stein W Henn H von Laue and P Berg ldquoModulation ofthe cellular and humoral immune responses of tumor patientsbymistletoe therapyrdquo European Journal ofMedical Research vol3 no 4 pp 194ndash202 1998

[15] J Melzer F Iten K Hostanska and R Saller ldquoEfficacy andsafety of mistletoe preparations (viscum album) for patientswith cancer diseasesrdquo Forschende Komplementarmedizin vol16 no 4 pp 217ndash226 2009

[16] A M Gray and P R Flatt ldquoInsulin-secreting activity ofthe traditional antidiabetic plant Viscum album (mistletoe)rdquoJournal of Endocrinology vol 160 no 3 pp 409ndash414 1999

[17] B Gerber C Scholz T Reimer V Briese and W JannildquoComplementary and alternative therapeutic approaches inpatients with early breast cancer a systematic reviewrdquo BreastCancer Research andTreatment vol 95 no 3 pp 199ndash209 2006

[18] E C Lavelle G Grant A Pusztai et al ldquoMistletoe lectinsenhance immune responses to intranasally co-administeredherpes simplex virus glycoproteinD2rdquo Immunology vol 107 no2 pp 268ndash274 2002

[19] T J Yoon Y C Yoo T B Kang et al ldquoCellular and humoraladjuvant activity of lectins isolated from Korean mistletoe (Vis-cum album colaratum)rdquo International Immunopharmacologyvol 1 no 5 pp 881ndash889 2001


[20] T J Yoon Y C Yoo T B Kang et al ldquoProphylactic effect ofKorean mistletoe (Viscum album coloratum) extract on tumormetastasis is mediated by enhancement of NK cell activityrdquoInternational Journal of Immunopharmacology vol 20 no 4-5pp 163ndash172 1998

[21] T J Yoon Y C Yoo T B Kang et al ldquoLectins isolated fromKorean mistletoe (Viscum album coloratum) induce apoptosisin tumor cellsrdquo Cancer Letters vol 136 no 1 pp 33ndash40 1999

[22] T J Yoon Y C Yoo T B Kang et al ldquoAntitumor activityof the Korean mistletoe lectin is attributed to activation ofmacrophages andNKcellsrdquoArchives of Pharmacal Research vol26 no 10 pp 861ndash867 2003

[23] T J Yoon Y C Yoo O B Choi et al ldquoInhibitory effectof Korean mistletoe (Viscum album coloratum) extract ontumour angiogenesis and metastasis of haematogenous andnon-haematogenous tumour cells in micerdquo Cancer Letters vol97 no 1 pp 83ndash91 1995

[24] T B Kang Y C Yoo K H Lee et al ldquoKorean mistletoelectin (KML-IIU) and its subchains induce nitric oxide (NO)production in murine macrophage cellsrdquo Journal of BiomedicalScience vol 15 no 2 pp 197ndash204 2008

[25] T B Kang S K Song T J Yoon et al ldquoIsolation andcharacterization of two Korean mistletoe lectinsrdquo Journal ofBiochemistry and Molecular Biology vol 40 no 6 pp 959ndash9652007

[26] H-W Rho J-N Lee H-R Kim B-H Park and J-W ParkldquoProtective mechanism of glucose against alloxan-induced 120573-cell damage pivotal role of ATPrdquo Experimental and MolecularMedicine vol 32 no 1 pp 12ndash17 2000

[27] B-H Park and J-W Park ldquoThe protective effect of Amomumxanthoides extract against alloxan-induced diabetes throughthe suppression of NF120581B activationrdquo Experimental and Molecu-lar Medicine vol 33 no 2 pp 64ndash68 2001

[28] T Mandrup-Poulsen ldquoApoptotic signal transduction pathwaysin diabetesrdquo Biochemical Pharmacology vol 66 no 8 pp 1433ndash1440 2003

[29] S Y Lyu J H Choi H J Lee W B Park and G J KimldquoKoreanmistletoe lectin promotes proliferation and invasion oftrophoblast cells through regulation of Akt signalingrdquo Repro-ductive Toxicology vol 39 pp 33ndash39 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Journal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


fraction the blood glucose level and drinking water volumewere significantly reduced in alloxan induced hyperglycemicmice [26 27]

2 Materials and Methods

21 Reagents RPMI 1640 without glucose alloxan andKrebs-Ringer bicarbonate (KRB) buffer were purchased fromSigma Anti-PDX-1 and antineuroD all from Santa CruzBiotechnology were used for immunoblotting Rat insulinELISA Kit (SHIBAYAGI) was used for insulin secretionSYBR greenmastermix (Applied Biosystems) andRNAEasy-spin (iNtRON) were used for real-time PCR

22 Cell Culture and Treatment Rat pancreatic 120573 cellsRINm5F cells were provided by PohangUniversity of Scienceand Technology (POSTECH) RINm5F cells were culturedin a monolayer in RPMI 1640 medium (Gibco) supple-mented with 10 fetal bovine serum (FBS CAMBREX) 1penicillinstreptomycin (CAMBREX) and 55mMglucose at37∘C under a humidified 95ndash5 (vv) mixture of air andCO2 RINm5F cells were cultured in complete medium with

10 FBS to sim90 cell confluence and were then incubatedwith 05 FBS medium for 12sim16 hours After that RINm5Fcells were treated with KM extractThe supernatant was usedfor detection of insulin secretion and the attached cells wereused for immunoblotting

23 Animals Specific pathogen-free 7-week-old imprintingcontrol region (ICR) mouse strains were purchased from theDae-Han LaboratoryAnimal Center Republic of KoreaMicewere maintained in the Laboratory of Animal ExperimentInstitute of Bioscience Han-Dong Global University underlaminar air-flow conditions Water and diets were suppliedad libitum Each group was composed of 5 mice for checkingblood glucose level and drinking water volumes

24 Extraction and Purification of Korean Mistletoe Mistle-toe growing on oak (Quercus variabilis Blume) betweenDecember and February was harvested from Kangwon-doKorea and stored at minus80∘C until use The nonlectin fractions(NLF) were isolated from an extract of Korean mistletoeplants using hydrolyzed120572-lactose-Sepharose (Sigma) columnchromatography after ammonium sulfate precipitation asdescribed previously [24 25] Briefly the extracts were dis-solved in distilled water and filtered through a 045 120583mmem-brane (Whatmann) Then they adjusted to 70 saturationwith (NH

4)SO4 The precipitate was resuspended in a small

volume of phosphate-buffered saline (PBS) dialyzed againstthe same buffer and applied to an 120572-lactose-Sepharosecolumn which had been hydrolyzed for 25 hours To removethe Korean mistletoe lectin (KML-C) the adsorbed materialwas eluted with a lactose-containing buffer (01M lactose pH73) Unbounded proteins which were free of KML-C werepooled dialyzed against distilled water and freeze-driedEach process was performed at 4∘CNLFdissolved in distilledwater was partitioned by chloroform Chloroformic extractsdissolved in phosphate buffer (PBS 10mM phosphate buffer

containing 085 NaCl pH 74) were applied to the DEAE-Sepharose Fast flow column (AmershamPharmacia Biotech)which had previously equilibrated with the same buffer Aftercolumn was washed with phosphate buffer again remainingbound proteins were eluted with 05M 1M and 2M NaCl(DEAE 05M 1M and 2M fractions) DEAE fractionsdissolved in PBS were applied to the Sep-Pak column (WatersCorporation) which had been previously equilibrated withthe same buffer The column was washed with PBS andbound proteins were eluted with 20 ethanol (Sep-Pak20 SP20 fractions) and 60 ethanol (Sep-Pak 60 SP60fractions) Each eluted protein fraction was dialyzed againstdistilled water every 4 hours for 3 days and stored in minus20∘Cuntil use

25 FACS Analysis for Cytotoxicity Cytotoxicity was ana-lyzed by the annexin V and propidium iodide (PI Merck)using flow-cytometry (FACS) machine (Beckman CoulterEpic XL) RINm5F cells (2 times 106 cells) were seeded into 6-wellplates and incubated for 12 hours After washing KM extractwas treated with the cells and incubated in the 37∘C with 5CO2for 30 minutes Cells were carefully detached from the

plates and thendetached cellswere stainedwith the apoptosisdetection kit containing annexin V-FITC and PI at ice in thedark condition Stained cells werewashedwith FACS solutionand living or dead cells were measured by FACS machine

26 Measurement of Insulin 1mL of RINm5F cells (4 times105 cellswell) was seeded into the 24-well plates (Falcon) andcultured in RPMI-1640 containing 111mM glucose (Sigma)After incubation for 12 hr to allow attachment cells werewashed with Krebs-Ringer bicarbonate buffer (KRB) in 3timesThen cells were preincubated with 1mL of KRB bufferfor 30 minutes at 37∘C Cells were then incubated for 30minutes with KM extracts The supernatants were collectedfrom each well and stored at minus20∘C until use for insulinsecretion assay An assay for the measurement of insulinsecretion from RINm5F cells was carried out by rat insulinELISA Kit (SHIBAYAGI) and detected by ELISA reader at450 nm

27 Real-Time PCR Total RNA were extracted using easy-spin RNA extraction kit (Intron Korea) cDNA were pro-duced with 5 120583g of total RNA using Superscript (Invitrogen)In a florescent temperature cycler (LightCycler Roche Diag-nostics Ltd Lewes UK) 10 percent of each RT reaction wasamplified in a 20120583L PCR containing 4mMMgCl

2 4 pmol of

each primer and 1x SYBR green master mix Samples wereincubated in the LightCycler for an initial denaturation at94∘C for 30 seconds followed by 30 PCR cycles Each cycleconsisted of 95∘C for 15 seconds 55∘C for 32 seconds and72∘C for 32 seconds

119879ℎ119890 119874119897119894119892119900119899119906119888119897119890119900119905119894119889119890 119875119903119894119898119890119903119904

For insulin are

forward 51015840-ATA GAC CAT CAG CAA GCAGG-31015840


01 1000

PI


987

KM extract (1mgmL)

973

KM extract (2mgmL)

886

Single parameter

01 1000

PI

Annexin V

01 1 10 100 1000

01 1000

PI

Annexin V

0

51

102

153

204

DNA content

01

1000

01

1000

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(128times128)

(128times128)

(128times128)

(a)

Cel

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20

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60

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KME Alloxan

(b)



For PDX-1 are








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(c)

1

2

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4

5

6In

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times103

(d)




3 Results




1

2

3

4

5

6

Relat

ive e

xpre

ssio

n 7

8

85310

9

Time (h)

Insulin genePDX-1

DEAE 05M

(a)

1

2

3

4

5

83510

Relat

ive e

xpre

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n

Time (h)

Insulin genePDX-1

DEAE 1M

(b)


Neuro D (49KD)


Time (min)DEAE 05M

(c)


Neuro D (49KD)


Time (min)DEAE 1M

(d)







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(pg

mL)


300

600

900

1200

1500

1800

0

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d gl

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gdL

)


100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600


(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

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Drin

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(mL

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400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)






4 Discussion











References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















01 1000

PI


987

KM extract (1mgmL)

973

KM extract (2mgmL)

886

Single parameter

01 1000

PI

Annexin V

01 1 10 100 1000

01 1000

PI

Annexin V

0

51

102

153

204

DNA content

01

1000

01

1000

01

1000

(128times128)

(128times128)

(128times128)

(a)

Cel

l sur

viva

l (

)

20

40

60

80

100

120


KME Alloxan

(b)



For PDX-1 are








Insu

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(pg

mL)

2

4

6

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2

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(c)

1

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times103

(d)




3 Results




1

2

3

4

5

6

Relat

ive e

xpre

ssio

n 7

8

85310

9

Time (h)

Insulin genePDX-1

DEAE 05M

(a)

1

2

3

4

5

83510

Relat

ive e

xpre

ssio

n

Time (h)

Insulin genePDX-1

DEAE 1M

(b)


Neuro D (49KD)


Time (min)DEAE 05M

(c)


Neuro D (49KD)


Time (min)DEAE 1M

(d)







Insu

lin se

cret

ion

(pg

mL)


300

600

900

1200

1500

1800

0

(a)

Bloo

d gl

ucos

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gdL

)


100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600


(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

0

Drin

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wat

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(mL

mic

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400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)






4 Discussion











References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Insu

lin se

cret

ion

(pg

mL)

2

4

6

8

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12times10

3

Media 0612255 101184167


lowast

lowastlowastlowast

lowastlowast

(a)

Insu

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(pg

mL)

1

2

3

4

5

6

7

8

1256253131577942105025


times103

(b)

6

1

2

3

4

5

LFFNon treated KME

Insu

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(pg

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NStimes103

(c)

1

2

3

4

5

6In

sulin

secr

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n (p

gm

L)


LFF

times103

(d)




3 Results




1

2

3

4

5

6

Relat

ive e

xpre

ssio

n 7

8

85310

9

Time (h)

Insulin genePDX-1

DEAE 05M

(a)

1

2

3

4

5

83510

Relat

ive e

xpre

ssio

n

Time (h)

Insulin genePDX-1

DEAE 1M

(b)


Neuro D (49KD)


Time (min)DEAE 05M

(c)


Neuro D (49KD)


Time (min)DEAE 1M

(d)







Insu

lin se

cret

ion

(pg

mL)


300

600

900

1200

1500

1800

0

(a)

Bloo

d gl

ucos

e (m

gdL

)


100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600


(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

0

Drin

king

wat

er v

ol

(mL

mic

eda

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400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)






4 Discussion











References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

2

3

4

5

6

Relat

ive e

xpre

ssio

n 7

8

85310

9

Time (h)

Insulin genePDX-1

DEAE 05M

(a)

1

2

3

4

5

83510

Relat

ive e

xpre

ssio

n

Time (h)

Insulin genePDX-1

DEAE 1M

(b)


Neuro D (49KD)


Time (min)DEAE 05M

(c)


Neuro D (49KD)


Time (min)DEAE 1M

(d)







Insu

lin se

cret

ion

(pg

mL)


300

600

900

1200

1500

1800

0

(a)

Bloo

d gl

ucos

e (m

gdL

)


100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600


(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

0

Drin

king

wat

er v

ol

(mL

mic

eda

y)


400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)






4 Discussion











References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Insu

lin se

cret

ion

(pg

mL)


300

600

900

1200

1500

1800

0

(a)

Bloo

d gl

ucos

e (m

gdL

)


100

200

300

400

500

600

0

lowastlowastlowast

(b)

Bloo

d gl

ucos

e (m

gdL

)

100

200

300

400

500

600


(100mM)400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(c)

10

20

30

40

50

0

Drin

king

wat

er v

ol

(mL

mic

eda

y)


400120583gmL 200120583gmL 100120583gmL

DEAE 1M

(d)






4 Discussion











References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Protein Fractions from Korean Mistletoe ...downloads.hindawi.com/journals/ecam/2014/703624.pdf · Research Article Protein Fractions from Korean Mistletoe (Viscum