Research Article Eleutheroside E, An Active …downloads.hindawi.com/journals/ecam/2013/934183.pdfEleutheroside E, An Active Component of Eleutherococcus senticosus , Ameliorates Insulin

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 934183 9 pageshttpdxdoiorg1011552013934183

Research ArticleEleutheroside E An Active Component ofEleutherococcus senticosus Ameliorates InsulinResistance in Type 2 Diabetic dbdb Mice

Jiyun Ahn1 Min Young Um1 Hyunjung Lee1 Chang Hwa Jung1

Seok Hyun Heo2 and Tae Youl Ha1

1 Metabolism and Nutrition Research Group Korea Food Research Institute 516 BaekhyunBundang Seongnam Gyeonggi 463-746 Republic of Korea

2 Research Division Korea Health Supplement Institute Seongnam 463-400 Republic of Korea

Correspondence should be addressed to Tae Youl Ha tyhapkfrirekr

Received 16 January 2013 Accepted 15 March 2013

Academic Editor Evan Paul Cherniack

Copyright copy 2013 Jiyun Ahn 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

Eleutheroside E (EE) a principal component of Eleutherococcus senticosus (ES) has anti-inflammatory and protective effects inischemia heart However it is unknown whether it ameliorates insulin resistance and reduces hyperglycemia in diabetes Thisstudy investigated the effect of EE-containing ES extracts as well as EE on hyperglycemia and insulin resistance in dbdb miceEE increased the insulin-provoked glucose uptake in C2C12 myotubes Moreover EE improved TNF-120572-induced suppression ofglucose uptake in 3T3-L1 adipocytes Five-week-old dbdb mice were fed a diet consisting of ES extract or EE for 5 weeks Bothwere effective in improving serum lipid profiles and significantly decreased blood glucose and serum insulin levels ES and EEsupplementation effectively attenuated HOMA-IR Glucose tolerance and insulin tolerance tests showed that EE increased insulinsensitivity Immunohistochemical staining indicated that ES and EE protected pancreatic alpha and beta cells fromdiabetic damageIn addition ES and EE improved hepatic glucose metabolism by upregulating glycolysis and downregulating gluconeogenesis inobese type 2 diabetic mice These data suggest that EE mediates the hyperglycemic effects of ES by regulating insulin signaling andglucose utilization The beneficial effects of EE may provide an effective and powerful strategy to alleviate diabetes

1 Introduction

Diabetes is a metabolic disorder characterized by hyper-glycemia and caused by increased hepatic glucose produc-tion abnormal glucose utilization in peripheral tissues andinadequate insulin secretion [1] Type 2 insulin-resistantdiabetes mellitus (T2DM) accounts for 90ndash95 of all casesof diabetes This heterogeneous disorder afflicts an estimated6 of the adult population in Western societies [2] Sev-eral approaches have been recommended to reduce hyper-glycemia including increasing pancreatic insulin releaseby sulfonylureas decreasing hepatic glucose production bymetformin enhancing insulin action by thiazolidinedionesand suppressing gut glucose absorption by 120572-glucosidaseHowever these treatments have limited efficacy a high

likelihood of tolerability issues and significant mechanism-based side effects [1]

Eleutherococcus senticosus (Rupr ampMaxim) is also calledHarms (ES) Acanthopanax senticosus Siberian ginseng orGasiogapi in Korea It is a well-known tonic and sedativeChinese herb that affects various diseases with its antibacte-rial antifatigue [3] antioxidant [4] and immunomodulating[5] activities The hypoglycemic activity of ES methanolextract has also been reported [6] However which functionalcomponent mediates the antidiabetic effect of ES remainsunknown

It has been reported that ES has several active con-stituents including lignans (sesamine eleutheroside E)glycans (eleutherans eleutheroside D) triterpene saponins

2 Evidence-Based Complementary and Alternative Medicine

(eleutheroside I K L and M) steroid glycosides (eleuthero-side A) hydroxycoumarins (isoflaxidin) phenylacrylic acidderivatives (syringin) and flavones [7]

Eleutheroside E (EE) is known to reduce physical fatigueand to enhance endurance performance [3] In addition it hasalso reported to have anti-inflammatory effects by inhibitingNF-120581B [8] and protecting against myocardial infarction [9]However to date the effects of EE on glucose uptake andinsulin resistance have not yet been studied

In the present study we measured the effect of EE on glu-cose uptake inmyotubes and insulin-resistant adipocytesWealso explored the hypoglycemic effect of ES and EE in T2DMdbdb mice The insulin signaling pathway in skeletal muscleand mRNA expression of hepatic metabolism-related geneswere evaluated to determine themolecularmechanisms of ESand EE-induced activity

2 Materials and Methods

21 Preparation of the Plant Extract and HPLC Analyses TheES stemwas supplied byHambackje Agricultural Association(Nonsan Republic of Korea) and identified by Professor YongMok Park College of Life Sciences Cheongju University(Cheongju Republic of Korea) It was ground in a milland passed through 50-mesh sieve ES powder (100 g) wasextracted with 150mL 50 ethanol for 5 hours at roomtemperature The extracted solutions were concentrated at50∘C using a rotary vacuum evaporator (BUCHI FlawilSwitzerland) and lyophilized The extraction yield was 413(ww) To determine the content of active constituents in theextract high performance liquid chromatography (HPLC)analyses were performed using a HPLC apparatus equippedwith a PU-2089 plus quaternary gradient pump auto samplerand an ultraviolet and refractive index detector (Jasco TokyoJapan) Syringin chlorogenic acid eleutheroside E andisoflaxidin were purchased from Sigma-Aldrich (St LouisMO USA) The standard solutions were prepared in 70methanol The ES extract was dissolved in HPLC-grademethanol filtered through a 045 120583m membrane filter anddegassed in an ultrasonic bath Separation was performedusing an XTerra RP C18 column (250 times 46mm 50120583mWaters Milford MA USA) at 35∘C The mobile phasesconsisted of 1 phosphoric acid (A) and 100 acetonitrile (B)at a flow rate of 10mLmin Elution was performed using thefollowing programmed gradient elution 0ndash40min elutionwith gradually increasing concentrations of solvent B (5ndash40) 40ndash50min isocratic elution with 40 solvent B and50ndash60min elution with gradually decreasing concentrationsof solvent B (40ndash5) Detection was performed at 216 nm

22 Glucose Uptake Assays C2C12 (ATCC Manassas VAUSA) cells were maintained in DMEM supplemented with10 FBS 30 120583gmL penicillin and 100 120583gmL streptomycinC2C12 myoblast differentiation was induced by switchingconfluent cells to DMEM supplemented with 2 horseserum and allowing formation of myotubes with dailymedia changes Cells were used in experiments 4 days afterdifferentiation C2C12 cells were exposed to 10120583M syringinEE or isoflaxidin for 24 h

3T3-L1 fibroblasts (ATCC) were maintained and dif-ferentiated as described previously [10] To induce insulinresistance differentiated 3T3-L1 adipocytes were treated with20 ngmL recombinant mouse TNF-120572 (Sigma Aldrich) for6 hThe insulin-resistant 3T3-L1 adipocytes were treated with10 120583M EE for 24 h

A fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-13-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG Invitro-gen Carlsbad CA USA) was used to measure glucoseuptake After exposure to the compounds described above500120583M 2-NBDG was added to the culture media for a10min incubation Cells were washed with Krebsrsquos bufferand incubated with 100 nM insulin for 10min To stop theresponse cells were washed with ice-cold Krebsrsquos buffer andthe fluorescence intensity of 2-NBDG was measured at anexcitation wavelength of 480 nm and an emission wavelengthof 540 nm

23 Animals Intraperitoneal Glucose Tolerance Tests(IPGTTs) and Insulin Tolerance Tests (IPITTs) Five-week-old male dbdb mice were obtained from SLC (HamamatsuJapan) The dbdb mouse has the mutation of leptin receptorand is a model of metabolic syndrome with T2DM [11] Afteracclimation for 1 week mice were maintained for 5 weeks onAIN-76 based diet (DM) or a diet containing 005 or 01ES extract (ESL ESH resp) or 0003 EE The body weightand 4 h fasting blood glucose of each mouse were monitoredweekly

Five weeks after feeding intraperitoneal glucose toler-ance tests (IPGTTs) and insulin tolerance tests (IPITTs)were performed IPGTT was determined in response tointraperitoneal administration of 2 g D-glucosekg bodyweight following a 4-hour fast Blood glucose was measuredfrom the tail vein 0 15 30 60 90 and 120 minutes afterglucose administration IPITT was determined in responseto intraperitoneal administration of 12 IU human insulinkgbody weight following a 4-hour fast Blood glucose was mea-sured 0 15 30 60 90 and 120 minutes after insulin adminis-trationThe area under the curve (AUC) was calculated usingthe trapezoidal method After 5 weeks on the diet mice weresacrificed following a 12-hour fast All animal studies wereconducted in accordance with a protocol approved by theInstitutional Animal Care and Use Committee of the KoreaFood Research Institute

24 Blood Parameters Blood glucose triglycerides (TG) freefatty acids (FFAs) total cholesterol (TC) and high-densitylipoprotein (HDL) levels were measured enzymatically usingcommercial kits (Shinyang Chemical Co Busan Republic ofKorea) Serum insulin levels were measured using an ELISAkit (ALPCO Diagnostics Salem NH USA) Homeostasismodel assessment for insulin resistance (HOMA-IR) wascalculated using the following formula

HOMA-IR (mmolL times 120583UmL)

= fasting glucose (mmolL)

times fasting insulin (120583UmL) 225

(1)

Evidence-Based Complementary and Alternative Medicine 3

0119864+00

1119864+05

15119864+05

2119864+05

25119864+05

3119864+05

(120583V

)

5 10 15 20 25 30(min)

05119864+05

(a)

0119864+001119864+052119864+053119864+054119864+055119864+056119864+05

(120583V

)

5 10 15 20 25 30(min)

Syrin

gin

Chlo

roge

nic a

cid

Eleu

ther

osid

e EIs

oflax

idin

(b)

Figure 1 Representative HPLC chromatogram of the E senticosus (ES) extract and its functional standard compounds (a) HPLCchromatogram of the ES extract (b) HPLC chromatogram of the major compounds including syringin chlorogenic acid eleutheroside Eand isoflaxidin

25 Histological Examination For histological analyses pan-creatic tissues were fixed in 10 buffered formalin embeddedin paraffin sectioned and stained with hematoxylin andeosin The stained areas were observed using a light micro-scope (Olympus Tokyo Japan) with a magnifying powerof times200 For immunohistochemistry pancreatic sectionswere deparaffinized and rehydrated and incubated for 25minutes in 70 methanol and hydrogen peroxide (H

2O2)

After washing with Tris-buffered-saline (TBS pH 73) thesections were incubated overnight at 4∘Cwith an anti-insulinantibody (BioGenex Fremont CA USA) diluted 1 1000 inTBS containing 10 bovine serum Sections were incubatedfor 90 minutes with an anti-guinea pig antibody (VectastainVector Servion Switzerland) in TBS containing 10 bovineserum (dilution 1 200) After washing sections were incu-bated with an avidin-peroxidase complex (Vectastain) for 15minutes and washed againThe sections were stained with 33diaminobenzidine (DAB) for 5 minutes and counter-stainedwith hematoxylin for 30 seconds

Relative beta cell volume in the pancreas was describedas the number of points corresponding to the anti-insulinantibody-stained areanumber of points corresponding toremaining pancreatic area

26 Insulin Signaling For insulin signaling experimentsmice were intraperitoneally injected with 5Ukg humaninsulin (Sigma Aldrich) following an overnight fast After 5minutes muscle tissues were removed and frozen in liquidnitrogen Tissues were lysed in RIPA buffer and westernblot analyses were performed as previously described [10]Primary antibodies used included phospho-AKT phospho-P70S6 kinase (P70S6K) phospho-insulin receptor beta sub-unit (IR120573) and 120573-actin (Cell Signaling Danvers MA USA)

27 Quantitative Real Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) Livers wereexcised immediately snap-frozen and stored at minus80∘Cuntil use Total RNA was extracted from 20ndash30mg of eachliver tissue using a NucleoSpin RNA II kit (Macherey-Nagel GmbH amp Co KG Duren Germany) according tothe manufacturerrsquos protocol RNA quantification in theextracted samples was performed using NanoDrop (ThermoScientific) RNA purity was assessed by measuring the

119860260

119860280

ratio Values of 19ndash21 were used in experimentsTotal RNA (1120583g) was used for cDNA synthesis with theMaxime RT-PCR PreMix Kit (Intron Seongnam Republicof Korea) per the manufacturerrsquos instructions The totalreaction volume was 20 120583L qPCR was conducted on anStepOnePlus Real-time PCR system (Applied Biosystems)using SYBR Green Real-time PCR Master Mix (TOYOBOOsaka Japan) with a 95∘C predenaturation for 5 minutesfollowed by 40 cycles of 95∘C for 15 seconds 60∘C for 15seconds and 75∘C for 45 seconds The PCR reaction wasperformed in a 20120583L reaction volume containing 2120583L cDNAtemplate and 10 pmol primers (forward and reverse resp)The primers used were as follows glucokinase forward 51015840-AGAAGGCTCAGAAGTTGGAGAC-31015840 reverse 51015840-GGA-TGGAATACATCTGGTGTTTCG-31015840 6-phosphofructo-kinase forward 51015840-TGTGGTCCGAGTTGGTATCTT-31015840reverse 51015840-GCACCTCCAATCACTGTGCC-31015840 glucose-6-phosphatase (G6Pase) forward 51015840-GAGTCTTGTCAG-GCATTGCT-31015840 reverse 51015840-GGTACATGCTGGAGTTGA-GG-31015840 phosphoenolpyruvate carboxykinase (PEPCK)forward 51015840-AAAAGCCTTTGGTCAACAAC-31015840 reverse51015840-AAACTTCATCCAGGCAATGT-31015840 and 18S forward 51015840-CTCAACACGGGAAACCTCAC-31015840 reverse 51015840-CGCTCC-ACCAACTAAGAACG-31015840

28 Statistical Analyses Results are expressed as the mean plusmnstandard deviation (SD) for cell studies and the mean plusmnstandard error (SEM) for animal studies Statistical analyseswere performed using GraphPad Prism 5 software (SanDiego CA USA) One-way analysis of variance (ANOVA)was used to compare quantitative data among groups TheBonferroni post hoc test was used if ANOVA indicatedsignificance (119875 lt 005)

3 Results

31 Analyses of the Functional Components of ES Thefunctional compounds of the 50 ethanol extract of ESwere analyzed using an established HPLC method Thechromatogram of the ES extract is shown in Figure 1 Thecontent of each active compound is presented in Table 1The ES extract contained 1678 648 and 1072mgg extractof syringin chlorogenic acid and EE respectively Previous


0

50

100

150

200

250

CTL SY EE ISO CTL SY EE ISO

Basal

lowast

2-N

BDG

( o

f CTL

)

+Insulin

(a)

0

2

4

6

8

TNF-120572 minus minus + + + +

lowast

EE minus minus minus minus + +

Insulin minus + minus + minus +

lowast

lowast

2-N

BDG

(fol

d ov

er co

ntro

l)

(b)

Figure 2The effect of functional ES components on glucose uptake inC2C12myotubes and insulin-resistant 3T3-L1 cells (a) C2C12myotubeswere treated for 24 h with 10120583M of each compound and glucose uptake was measured in basal and insulin exposure conditions lowast119875 lt 005versus basal control 119875 lt 005 versus insulin-treated control CTL control SY syringin EE eleutheroside E ISO isoflaxidin (b) For insulinresistance conditions 20 ngmLTNF-120572was added for 6 h in differentiated 3T3-L1 cells After treatment with 10120583MEE for 24 h glucose uptakewas measured lowast119875 lt 005 versus control Results are the mean plusmn SD of 6 wells in each group Each experiment was repeated at least threetimes

Table 1 The functional constituents in E senticosus extracts

(mgg extractww) Syringing Chlorogenic acid Eleutheroside E

1678 plusmn 018 6480 plusmn 079 1072 plusmn 019Data are expressed as the mean plusmn SD from at least three measurements

research indicates that syringin EE chlorogenic acid andisoflaxidin are the major components contributing to thepharmacological effects of ES [12] However isoflaxidin wasnot detected in our ES samples

32 Eleutheroside E Increases Insulin-Evoked Glucose UptakeFirst we examined the effects of the functional componentsof ES on glucose uptake in muscle myotubes As shown inFigure 2(a) syringin increased basal glucose uptake in C2C12myotubes EE noticeably amplified the insulin-stimulatedglucose uptake

To evaluate the effect of EE on impaired glucose uptakein an insulin-resistant state we induced insulin resistanceusing TNF-120572 treatment in 3T3-L1 adipocytes and measuredthe effect of EE on TNF-120572-mediated suppression of glucoseuptake Treatment of 10 120583M EE for 24 h increased basalglucose uptake aswell as improvedTNF-120572-mediated suppres-sion of glucose uptake (Figure 2(b)) These data suggest thatEE increases glucose uptake and could have a beneficial rolein insulin-resistant diabetes

33 Diet Containing Eleutheroside E Ameliorates Diabetes indbdb Mice To confirm the effect of EE on hyperglycemiaand glucose intolerance we supplemented dbdb mice withan experimental diet containing ESL ESH or EE for 5weeks Changes in the body weights of animals during the

experimental period are shown in Figure 3(a) There was aprogressive increase in body weight for 5 weeks and the finalbody weights of animals in the experimental groups (ESLESH and EE) were significantly higher than control animals

As shown in Table 2 serum TC and TG levels weresignificantly decreased after ESL and ESH treatment EEeffectively lowered TG levels Serum FFA levels were signifi-cantly reduced by ESL and EE treatment

The diabetic control group showed higher fasting bloodglucose and insulin levels and ESL ESH and EE treatmentseffectively decreased fasting blood glucose and insulin levelsMoreover ESL ESH and EE treatment significantly reducedHOMA-IR values compared to the diabetic control group

To evaluate the effect of ES and EE on the insulinresistance of dbdb mice we performed IPGTT and IPITTat 5 weeks of treatment After intraperitoneal glucose injec-tion blood glucose levels were monitored As shown inFigure 3(c) both ES and EE improved impaired glucosetolerance although ES treatment did not reach statisticalsignificance The area under curve (AUC) decreased 177226 and 439 in the ESL ESH and EE groups respec-tively compared with the diabetic control group

For IPITT (Figure 3(d)) ES and EE treatment modestlyameliorated the impaired insulin action compared with thediabetic control group The AUC of IPITT was significantlydecreased in EE-treated mice compared to the diabeticcontrol mice Collectively these results indicate that ES andEE have hypoglycemic effects and improve glucose tolerance

We hypothesized that the improvement of glucose tol-erance by ES and EE resulted from protection of 120573-cellfunction To test this hypothesis we examined the effectof ES and EE on the function of pancreatic 120572- and 120573-cells(Figure 4(a)) The insulin content of 120573-cells was determinedusing immunochemistry and we found that ESL and EE


0 1 2 3 4 525

30

35

40

45

50

DMESL

ESHEE

Weeks

Body

wei

ght (

g)

lowast

lowast

lowastlowast

lowastlowast

(a)

120

200

280

360

440

520

Bloo

d gl

ucos

e (m

gdL

)

0 1 2 3 4 5

DMESL

ESHEE

Weeks

lowastlowast

lowastlowast

lowast

lowast

lowast

(b)

IP-GTT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

0 15 30 60 90 1200

200

400

600

800 IP-GTT

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowast

lowastlowast lowast

DMESL

ESHEE

(c)

0 15 30 60 90 120100

200

300

400

500

600

DMESL

ESH

IP-ITT

EE

Time (min)

Bloo

d gl

ucos

e lev

el (m

gdL

)

IP-ITT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

lowast

(d)

Figure 3 The effects of ESL ESH and EE on body weight fasting blood glucose levels and impaired glucose tolerance in dbdb mice (a)Body weight measurements during the experimental period (b) Blood glucose levels after 4-hour fast After feeding the experimental dietfor 5 weeks IPGTT (c) and IPITT (d) were performed The area under curve (AUC) during IPGTT is also shown Each value represents themean plusmn SEM (119899 = 8) lowast119875 lt 005 versus diabetes mellitus (DM) group lowastlowast119875 lt 001 versus DM group


DM

ESL

EE

H and E Anti-insulin Antiglucaton

(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)

Figure 4The effect of ESL and EE on pancreatic insulin and glucagon production (a) Pancreatic tissue sections fromDM ESL and EE groupwere stained with hematoxylin and eosin anti-insulin and antiglucagon antibodies respectively The scale bar is 100120583m for each panel (b)Relative 120573-cell volume in the pancreas was calculated as described inMaterials andMethods lowast119875 lt 005 versus diabetes mellitus (DM) group

Table 2 The effect of ES and EE on the lipid profile of dbdb mice

DM ESL ESH EETotal cholesterol (mgdL) 37511 plusmn 7479 23819 plusmn 8642lowast 24729 plusmn 4591lowast 33059 plusmn 2117Triglyceride (mgdL) 18024 plusmn 5719 13789 plusmn 3577 11393 plusmn 2017lowast 11235 plusmn 2975lowast

FFA (uEqL) 141278 plusmn 7428 108016 plusmn 16021lowast 119008 plusmn 10230 107238 plusmn 26991lowast

Glucose (mgdL) 486 plusmn 2153 3978 plusmn 2930lowast 3892 plusmn 2124lowast 3150 plusmn 2467lowast

Insulin (ngmL) 8 plusmn 18 281 plusmn 071lowast 428 plusmn 129lowast 356 plusmn 041lowast

HOMA-IR 7589 plusmn 1364 3649 plusmn 1222lowast 4279 plusmn 1595lowast 335 plusmn 663lowast

All values are expressed as the mean plusmn SEM Six-week-old dbdb mice were fed experimental diet containing none 005ndash001 E senticosus (ES) extract and01 eleutheroside E (EE) for 5 weeksDM diabetes mellitus group ESL 005 ES extract supplemented group ESH 01 ES extract supplemented group EE 0003 eleutheroside E supplementedgrouplowastP lt 005 when compared to the DM group using one-way ANOVA followed by post hoc Bonferronirsquos tests

effectively prevented diabetic loss of 120573-cells (Figure 4(b))Furthermore ESL and EE ameliorated impairment of 120572-cells as confirmed by immunohistological glucagon staining(Figure 4(a)) These results suggest that supplementationof ES and EE effectively prevent diabetic impairment ofpancreatic 120572- and 120573-cells

To further explore the effects of ES and EE on insulin sen-sitivity insulin signaling was evaluated after insulin injectionin muscle of overnight-fasted mice from the ESL- and EE-treated groups As shown in Figure 5(a) IR120573phosphorylationwas decreased in diabetic mice Phosphorylation of AKT andits downstream target P70S6K was also reduced HoweverESL supplementation maintained insulin-induced phospho-rylation of IR120573 AKT and P70S6K Similar to ESL EE treat-ment increased insulin-dependent phosphorylation of IR120573AKT and P70S6K compared to diabetic mice (Figure 5(b))

Finally we examined the effect of ES and EE on hep-atic glucose metabolism by measuring the expression ofgenes involved in glycolysis and gluconeogenesis As shownin Figure 6(a) ESL and EE supplementation significantlyincreased the mRNA expression of glucokinase and 6-phosphofructokinase In contrast ESL and EE markedlydecreased the mRNA expression of G6Pase and PEPCK

(Figure 6(b)) These data indicate that ES and EE improvedglucose metabolism by upregulating glycolysis and downreg-ulating gluconeogenesis in diabetic mice

4 Discussion

T2DM is a common metabolic disorder characterized bychronic hyperglycemia and dyslipidemia resulting fromperipheral tissue insulin resistance and impaired insulinsecretion from the pancreas [13] It is estimated that T2DMpatients lose 15 years of an average life expectancy [14]Therefore intensive treatment to control hyperglycemia isneeded in diabetes However because clinical therapies havelimited efficacy and significantmechanism-based side effectsthere have been enormous increases in the use of medicinalplants for diabetes management [15]

In the present study we have shown that treating diabeticdbdb mice with ES and its main component EE improvesglycemic control and insulin resistance We also reportthat ES and EE have protective effects on pancreatic 120573-cellfunction This study provides the first evidence that the EE-induced positive effects are mediated by the enhancement of


Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL

minus minus minus minus ++++

(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

Figure 5 The effect of ESL (a) and EE (b) on insulin signaling inskeletal muscles Analyses of insulin-induced phosphorylation ofIR120573 AKT and P70S6K were performed by western blot Followingan overnight fast mice were either sacrificed for or injected with5Ukg of insulin Five minutes after injection muscle tissues werecollected and total protein was analyzed The phosphorylation sitein each protein is indicated

insulin signaling and the recovery of dysregulated glucosemetabolism

ES has been reported to contain syringin eleutheroside Eisoflaxidin and chlorogenic acid as themain compounds [12]In accordancewith previous studies our ES extract was foundto contain syringin chlorogenic acid and eleutheroside EHowever isoflaxidin was not detected in our sample

The hypoglycemic activity of the ES extracts was demon-strated and syringin [6] and chlorogenic acid [16] weresuggested to be the bioactive compounds However littleattention has been given to the role of EE on hyperglycemiaand glucose utilization Thus we focused on how EE affectsglucose uptake and insulin resistance

Impaired glucose uptake to peripheral tissues resultsin high circulating glucose levels [17] Therefore the mainstrategy is to stimulate glucose uptake to skeletal muscleliver and adipocytes that consume most plasma glucose EEincreased insulin-evoked glucose uptake in C2C12myotubesMoreover EE recovered impaired glucose uptake in TNF-120572 induced insulin-resistant adipocytes These results suggestthat EE has the potential to alleviate insulin resistance

To investigate this hypothesis further we examinedthe effect of dietary EE on hyperglycemia and glucosemetabolism using dbdb mice We found that ES as wellas EE inhibited hyperglycemia and glucose tolerance Inaddition hypertriglyceridemia and increased FFAs wereimproved upon ES and EE treatment Plasma FFAs are

derived almost exclusively from adipose tissue throughlipolysis of TG It has been demonstrated that increasedplasma FFA concentration causes insulin resistance and isassociated with T2DM [18]These findings led us to concludethat EE is one of the functional ES components for theattenuation of diabetes

A key component of the pathophysiology of T2DMinvolves defective insulin secretion from pancreatic islet 120573-cells This defect leads to the failure of the 120573-cell to compen-sate for insulin resistance and successive hyperglycemia [19]Our animal model dbdb mice is characterized by reducedproliferation and increased apoptosis in pancreatic 120573-cells[20]The present study reveals the pharmacological effects ofEE in pancreatic islet function and pathological developmentCompared with diabetic mice higher numbers of insulinsecreting 120573-cells and glucagon producing 120572-cells as well asfunctional integrity islet morphology were observed in ES-or EE-treated mice The protective effect of ES and EE on 120573-cell destruction and the subsequent enhancement of 120573-cellfunction improved glycemic control

Insulin resistance is an important risk factor in thedevelopment of T2DM It results from multiple defects insignal transduction pathways including reduced tyrosinephosphorylation of IR and subsequently decreased phospho-rylation of AKT and its direct target P70S6K [21] Increasingthe phosphorylation of these molecules enhances insulin-mediated signal transduction We performed western blotanalyses and confirmed that ES and EE treatment recoveredimpaired insulin signaling in skeletal muscle tissues byincreasing the insulin-induced phosphorylation of IR120573 AKTand P70S6K

The liver has a critical role in regulating endogenousglucose production from de novo synthesis (gluconeogenesis)or glycogen catabolism (glycogenolysis) Increased hepaticglucose production is predominantly responsible for thedevelopment of hyperglycemia in diabetes [22] Thus reduc-ing excessive hepatic glucose production is a good strategyto alleviate diabetes [23] Several enzymes that regulate rate-controlling steps in gluconeogenic or glycolytic pathwaysare obvious molecular targets for therapeutic treatment Inour study ES and EE were shown to upregulate glucokinaseand 6-phosphofructokinase and downregulate G6Pase andPEPCK thereby upregulating glycolysis and downregulatinggluconeogenesis respectively

In conclusion the present study provides evidence thatEE ameliorates type 2 diabetes by enhancing glucose uptakeimproving insulin resistance and pancreatic islet cell func-tion and regulating glucose metabolism These results sug-gest that EE may be partially responsible for the antidiabeticeffects of ES

Abbreviations

ES Eleutherococcus senticosusEE Eleutheroside ET2DM Type 2 insulin-resistant diabetes mellitusHPLC High performance liquid chromatography2-NBDG 2-[N-(7-nitrobenz-2-oxa-13-diazol-4-yl)

amino]-2-deoxyglucose


DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont

rol) lowastlowastlowast

lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)

Figure 6 The effect of ESL and EE on hepatic glucose metabolism (a) mRNA expression of the glycolysis-related genes glucokinase and 6-phosphofructokinase is shown (b) mRNA expression of gluconeogenesis-related genes G6Pase and PEPCK is shown After normalizationof each gene to the 18S gene mRNA levels are expressed as the percentage of diabetic mice Values indicate the mean plusmn SEM lowast119875 lt 005 versusDM group lowastlowast119875 lt 001 versus DM group

IPGTTs Intraperitoneal glucose tolerance testsIPITTs Intraperitoneal insulin tolerance testsAUC Area under the curveTG TriglyceridesFFAs Free fatty acidsTC Total cholesterolHDL High-density lipoproteinHOMA-IR Homeostasis model assessment for insulin

resistanceP70S6K P70S6 kinaseIR120573 Insulin receptor beta subunitG6Pase Glucose-6-phosphatasePEPCK Phosphoenolpyruvate carboxykinaseANOVA One-way analysis of variance

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgment

This study was supported by Korea Food Research Instituteand RampD program of MKEKEIT-10033818

References

[1] D E Moller ldquoNew drug targets for type 2 diabetes and themetabolic syndromerdquo Nature vol 414 no 6865 pp 821ndash8272001

[2] A F Amos D J McCarty and P Zimmet ldquoThe rising globalburden of diabetes and its complications estimates and projec-tions to the year 2010rdquoDiabetic Medicine vol 14 supplement 5pp S1ndashS85 1997

[3] S Nishibe H Kinoshita H Takeda and G Okano ldquoPhenoliccompounds from stem bark of Acanthopanax senticosus andtheir pharmacological effect in chronic swimming stressed ratsrdquoChemical and Pharmaceutical Bulletin vol 38 no 6 pp 1763ndash1765 1990

[4] C Y Yu S H Kim J D Lim M J Kim and I M ChungldquoIntraspecific relationship analysis byDNAmarkers and in vitrocytotoxic and antioxidant activity inEleutherococcus senticosusrdquoToxicology in Vitro vol 17 no 2 pp 229ndash236 2003

[5] M W Schmolz F Sacher and B Aicher ldquoThe synthesis ofRantes G-CSF IL-4 IL-5 IL-6 IL-12 and IL-13 in humanwhole-blood cultures is modulated by an extract from Eleuthe-rococcus senticosus L rootsrdquo Phytotherapy Research vol 15 no3 pp 268ndash270 2001


[6] H S Niu I M Liu J T Cheng C L Lin and F L Hsu ldquoHypo-glycemic effect of syringin from Eleutherococcus senticosus instreptozotocin-induced diabetic ratsrdquo Planta Medica vol 74no 2 pp 109ndash113 2008

[7] Y L Sun L D Liu and S KHong ldquoEleutherococcus senticosusas a crude medicine review of biological and pharmacologicaleffectsrdquo Journal of Medicinal Plants Research vol 5 no 25 p 72011

[8] T Yamazaki S Shimosaka H Sasaki T Matsumura TTukiyama and T Tokiwa ldquo(+)-Syringaresinol-di-O-120573-d-glucoside a phenolic compound from Acanthopanax senticosusHarms suppresses proinflammatory mediators in SW982human synovial sarcoma cells by inhibiting activating protein-1andor nuclear factor-120581B activitiesrdquo Toxicology in Vitro vol 21no 8 pp 1530ndash1537 2007

[9] Y Bu ZH Jin S Y Park et al ldquoSiberian ginseng reduces infarctvolume in transient focal cerebral ischaemia in Sprague-Dawleyratsrdquo Phytotherapy Research vol 19 no 2 pp 167ndash169 2005

[10] J Ahn H Lee S Kim and T Ha ldquoCurcumin-induced suppres-sion of adipogenic differentiation is accompanied by activationof Wnt120573-catenin signalingrdquo American Journal of Physiologyvol 298 no 6 pp C1510ndashC1516 2010

[11] S Koletsky ldquoPathologic findings and laboratory data in anew strain of obese hypertensive ratsrdquo American Journal ofPathology vol 80 no 1 pp 129ndash142 1975

[12] Y S Chan L N Cheng J H Wu et al ldquoA review of thepharmacological effects of Arctium lappa (burdock)rdquo Inflam-mopharmacology vol 19 no 5 pp 245ndash254 2011

[13] A R Saltiel ldquoNew perspectives into themolecular pathogenesisand treatment of type 2 diabetesrdquo Cell vol 104 no 4 pp 517ndash529 2001

[14] E T Rhodes L A Prosser T J Hoerger et al ldquoEstimatedmorbidity and mortality in adolescents and young adultsdiagnosedwithType 2 diabetesmellitusrdquoDiabeticMedicine vol29 no 4 pp 453ndash463 2012

[15] G Y Yeh D M Eisenberg T J Kaptchuk and R S PhillipsldquoSystematic review of herbs and dietary supplements forglycemic control in diabetesrdquo Diabetes Care vol 26 no 4 pp1277ndash1294 2003

[16] K W Ong A Hsu and B K Tan ldquoChlorogenic acid stimulatesglucose transport in skeletal muscle via AMPK activation acontributor to the beneficial effects of coffee on diabetesrdquo PLoSOne vol 7 no 3 Article ID e32718 2012

[17] D B Sacks and J M McDonald ldquoThe pathogenesis of typeII diabetes mellitus a polygenic diseaserdquo American Journal ofClinical Pathology vol 105 no 2 pp 149ndash156 1996

[18] G Boden ldquoRole of fatty acids in the pathogenesis of insulinresistance and NIDDMrdquo Diabetes vol 46 no 1 pp 3ndash10 1997

[19] M F Saad S E Kahn R G Nelson et al ldquoDisproportion-ately elevated proinsulin in Pima Indians with noninsulin-dependent diabetes mellitusrdquo Journal of Clinical Endocrinologyand Metabolism vol 70 no 5 pp 1247ndash1253 1990

[20] D A Gapp E H Leiter D L Coleman and R WSchwizer ldquoTemporal changes in pancreatic islet composition inC57BL6J-dbdb (diabetes) micerdquo Diabetologia vol 25 no 5pp 439ndash443 1983

[21] M Elchebly P Payette E Michaliszyn et al ldquoIncreased insulinsensitivity and obesity resistance in mice lacking the proteintyrosine phosphatase-1B generdquo Science vol 283 no 5407 pp1544ndash1548 1999

[22] R A DeFronzo R C Bonadonna and E Ferrannini ldquoPatho-genesis of NIDDM a balanced overviewrdquoDiabetes Care vol 15no 3 pp 318ndash368 1992

[23] I Magnusson D L Rothman L D Katz R G Shulman andG I Shulman ldquoIncreased rate of gluconeogenesis in type IIdiabetes mellitus A 13C nuclear magnetic resonance studyrdquoJournal of Clinical Investigation vol 90 no 4 pp 1323ndash13271992

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


(eleutheroside I K L and M) steroid glycosides (eleuthero-side A) hydroxycoumarins (isoflaxidin) phenylacrylic acidderivatives (syringin) and flavones [7]

Eleutheroside E (EE) is known to reduce physical fatigueand to enhance endurance performance [3] In addition it hasalso reported to have anti-inflammatory effects by inhibitingNF-120581B [8] and protecting against myocardial infarction [9]However to date the effects of EE on glucose uptake andinsulin resistance have not yet been studied

In the present study we measured the effect of EE on glu-cose uptake inmyotubes and insulin-resistant adipocytesWealso explored the hypoglycemic effect of ES and EE in T2DMdbdb mice The insulin signaling pathway in skeletal muscleand mRNA expression of hepatic metabolism-related geneswere evaluated to determine themolecularmechanisms of ESand EE-induced activity

2 Materials and Methods

21 Preparation of the Plant Extract and HPLC Analyses TheES stemwas supplied byHambackje Agricultural Association(Nonsan Republic of Korea) and identified by Professor YongMok Park College of Life Sciences Cheongju University(Cheongju Republic of Korea) It was ground in a milland passed through 50-mesh sieve ES powder (100 g) wasextracted with 150mL 50 ethanol for 5 hours at roomtemperature The extracted solutions were concentrated at50∘C using a rotary vacuum evaporator (BUCHI FlawilSwitzerland) and lyophilized The extraction yield was 413(ww) To determine the content of active constituents in theextract high performance liquid chromatography (HPLC)analyses were performed using a HPLC apparatus equippedwith a PU-2089 plus quaternary gradient pump auto samplerand an ultraviolet and refractive index detector (Jasco TokyoJapan) Syringin chlorogenic acid eleutheroside E andisoflaxidin were purchased from Sigma-Aldrich (St LouisMO USA) The standard solutions were prepared in 70methanol The ES extract was dissolved in HPLC-grademethanol filtered through a 045 120583m membrane filter anddegassed in an ultrasonic bath Separation was performedusing an XTerra RP C18 column (250 times 46mm 50120583mWaters Milford MA USA) at 35∘C The mobile phasesconsisted of 1 phosphoric acid (A) and 100 acetonitrile (B)at a flow rate of 10mLmin Elution was performed using thefollowing programmed gradient elution 0ndash40min elutionwith gradually increasing concentrations of solvent B (5ndash40) 40ndash50min isocratic elution with 40 solvent B and50ndash60min elution with gradually decreasing concentrationsof solvent B (40ndash5) Detection was performed at 216 nm

22 Glucose Uptake Assays C2C12 (ATCC Manassas VAUSA) cells were maintained in DMEM supplemented with10 FBS 30 120583gmL penicillin and 100 120583gmL streptomycinC2C12 myoblast differentiation was induced by switchingconfluent cells to DMEM supplemented with 2 horseserum and allowing formation of myotubes with dailymedia changes Cells were used in experiments 4 days afterdifferentiation C2C12 cells were exposed to 10120583M syringinEE or isoflaxidin for 24 h

3T3-L1 fibroblasts (ATCC) were maintained and dif-ferentiated as described previously [10] To induce insulinresistance differentiated 3T3-L1 adipocytes were treated with20 ngmL recombinant mouse TNF-120572 (Sigma Aldrich) for6 hThe insulin-resistant 3T3-L1 adipocytes were treated with10 120583M EE for 24 h

A fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-13-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG Invitro-gen Carlsbad CA USA) was used to measure glucoseuptake After exposure to the compounds described above500120583M 2-NBDG was added to the culture media for a10min incubation Cells were washed with Krebsrsquos bufferand incubated with 100 nM insulin for 10min To stop theresponse cells were washed with ice-cold Krebsrsquos buffer andthe fluorescence intensity of 2-NBDG was measured at anexcitation wavelength of 480 nm and an emission wavelengthof 540 nm

23 Animals Intraperitoneal Glucose Tolerance Tests(IPGTTs) and Insulin Tolerance Tests (IPITTs) Five-week-old male dbdb mice were obtained from SLC (HamamatsuJapan) The dbdb mouse has the mutation of leptin receptorand is a model of metabolic syndrome with T2DM [11] Afteracclimation for 1 week mice were maintained for 5 weeks onAIN-76 based diet (DM) or a diet containing 005 or 01ES extract (ESL ESH resp) or 0003 EE The body weightand 4 h fasting blood glucose of each mouse were monitoredweekly

Five weeks after feeding intraperitoneal glucose toler-ance tests (IPGTTs) and insulin tolerance tests (IPITTs)were performed IPGTT was determined in response tointraperitoneal administration of 2 g D-glucosekg bodyweight following a 4-hour fast Blood glucose was measuredfrom the tail vein 0 15 30 60 90 and 120 minutes afterglucose administration IPITT was determined in responseto intraperitoneal administration of 12 IU human insulinkgbody weight following a 4-hour fast Blood glucose was mea-sured 0 15 30 60 90 and 120 minutes after insulin adminis-trationThe area under the curve (AUC) was calculated usingthe trapezoidal method After 5 weeks on the diet mice weresacrificed following a 12-hour fast All animal studies wereconducted in accordance with a protocol approved by theInstitutional Animal Care and Use Committee of the KoreaFood Research Institute

24 Blood Parameters Blood glucose triglycerides (TG) freefatty acids (FFAs) total cholesterol (TC) and high-densitylipoprotein (HDL) levels were measured enzymatically usingcommercial kits (Shinyang Chemical Co Busan Republic ofKorea) Serum insulin levels were measured using an ELISAkit (ALPCO Diagnostics Salem NH USA) Homeostasismodel assessment for insulin resistance (HOMA-IR) wascalculated using the following formula

HOMA-IR (mmolL times 120583UmL)

= fasting glucose (mmolL)

times fasting insulin (120583UmL) 225

(1)


0119864+00

1119864+05

15119864+05

2119864+05

25119864+05

3119864+05

(120583V

)

5 10 15 20 25 30(min)

05119864+05

(a)

0119864+001119864+052119864+053119864+054119864+055119864+056119864+05

(120583V

)

5 10 15 20 25 30(min)

Syrin

gin

Chlo

roge

nic a

cid

Eleu

ther

osid

e EIs

oflax

idin

(b)



2O2)





119860260

119860280



3 Results



0

50

100

150

200

250


Basal

lowast

2-N

BDG

( o

f CTL

)

+Insulin

(a)

0

2

4

6

8


lowast



lowast

lowast

2-N

BDG

(fol

d ov

er co

ntro

l)

(b)
















0 1 2 3 4 525

30

35

40

45

50

DMESL

ESHEE

Weeks

Body

wei

ght (

g)

lowast

lowast

lowastlowast

lowastlowast

(a)

120

200

280

360

440

520

Bloo

d gl

ucos

e (m

gdL

)

0 1 2 3 4 5

DMESL

ESHEE

Weeks

lowastlowast

lowastlowast

lowast

lowast

lowast

(b)

IP-GTT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

0 15 30 60 90 1200

200

400

600

800 IP-GTT

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowast

lowastlowast lowast

DMESL

ESHEE

(c)

0 15 30 60 90 120100

200

300

400

500

600

DMESL

ESH

IP-ITT

EE

Time (min)

Bloo

d gl

ucos

e lev

el (m

gdL

)

IP-ITT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

lowast

(d)



DM

ESL

EE


(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)













4 Discussion




Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)






Acknowledgment


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0119864+00

1119864+05

15119864+05

2119864+05

25119864+05

3119864+05

(120583V

)

5 10 15 20 25 30(min)

05119864+05

(a)

0119864+001119864+052119864+053119864+054119864+055119864+056119864+05

(120583V

)

5 10 15 20 25 30(min)

Syrin

gin

Chlo

roge

nic a

cid

Eleu

ther

osid

e EIs

oflax

idin

(b)



2O2)





119860260

119860280



3 Results



0

50

100

150

200

250


Basal

lowast

2-N

BDG

( o

f CTL

)

+Insulin

(a)

0

2

4

6

8


lowast



lowast

lowast

2-N

BDG

(fol

d ov

er co

ntro

l)

(b)
















0 1 2 3 4 525

30

35

40

45

50

DMESL

ESHEE

Weeks

Body

wei

ght (

g)

lowast

lowast

lowastlowast

lowastlowast

(a)

120

200

280

360

440

520

Bloo

d gl

ucos

e (m

gdL

)

0 1 2 3 4 5

DMESL

ESHEE

Weeks

lowastlowast

lowastlowast

lowast

lowast

lowast

(b)

IP-GTT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

0 15 30 60 90 1200

200

400

600

800 IP-GTT

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowast

lowastlowast lowast

DMESL

ESHEE

(c)

0 15 30 60 90 120100

200

300

400

500

600

DMESL

ESH

IP-ITT

EE

Time (min)

Bloo

d gl

ucos

e lev

el (m

gdL

)

IP-ITT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

lowast

(d)



DM

ESL

EE


(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)













4 Discussion




Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)






Acknowledgment


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250


Basal

lowast

2-N

BDG

( o

f CTL

)

+Insulin

(a)

0

2

4

6

8


lowast



lowast

lowast

2-N

BDG

(fol

d ov

er co

ntro

l)

(b)
















0 1 2 3 4 525

30

35

40

45

50

DMESL

ESHEE

Weeks

Body

wei

ght (

g)

lowast

lowast

lowastlowast

lowastlowast

(a)

120

200

280

360

440

520

Bloo

d gl

ucos

e (m

gdL

)

0 1 2 3 4 5

DMESL

ESHEE

Weeks

lowastlowast

lowastlowast

lowast

lowast

lowast

(b)

IP-GTT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

0 15 30 60 90 1200

200

400

600

800 IP-GTT

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowast

lowastlowast lowast

DMESL

ESHEE

(c)

0 15 30 60 90 120100

200

300

400

500

600

DMESL

ESH

IP-ITT

EE

Time (min)

Bloo

d gl

ucos

e lev

el (m

gdL

)

IP-ITT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

lowast

(d)



DM

ESL

EE


(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)













4 Discussion




Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)






Acknowledgment


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 525

30

35

40

45

50

DMESL

ESHEE

Weeks

Body

wei

ght (

g)

lowast

lowast

lowastlowast

lowastlowast

(a)

120

200

280

360

440

520

Bloo

d gl

ucos

e (m

gdL

)

0 1 2 3 4 5

DMESL

ESHEE

Weeks

lowastlowast

lowastlowast

lowast

lowast

lowast

(b)

IP-GTT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

0 15 30 60 90 1200

200

400

600

800 IP-GTT

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowast

lowastlowast lowast

DMESL

ESHEE

(c)

0 15 30 60 90 120100

200

300

400

500

600

DMESL

ESH

IP-ITT

EE

Time (min)

Bloo

d gl

ucos

e lev

el (m

gdL

)

IP-ITT

DM ESL ESH EE0

25

50

75

100

125

AUC

( o

f con

trol)

lowast

(d)



DM

ESL

EE


(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)













4 Discussion




Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)






Acknowledgment


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DM

ESL

EE


(a)DM ESL EE

0

10

20

30

40

lowastlowast

Rela

tive120573

-cel

l vol

ume (

)

(b)













4 Discussion




Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

ove

r con

trol)

DM ESL EE0

03

06

09

12

PEPC

K (fo

ld o

ver c

ontro

l)

lowastlowast

lowast

lowastlowast

lowast

(b)






Acknowledgment


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Insulin 5 Ukg

p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)

120573-actin

DM DM + ESL


(a)

Insulin 5 Ukg

120573-actin

DM

(b)

DM + EE


p-IR120573 (Tyr-1146)

p-AKT (Ser-473)

p-P70S6K (Thr-389)












Abbreviations




DM ESL EE0

2

4

6

8

10

12

6-ph

osph

ofru

ctok

inas

e (fo

ld o

ver c

ontro

l)

DM ESL EE0

05

1

15

2

25

3G

luco

kina

se (f

old

over

cont


lowastlowast

lowast

(a)

DM ESL EE0

02

04

06

08

1

G6P

ase (

fold

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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















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OncologyJournal of





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Documents

Research Article Eleutheroside E, An Active …downloads.hindawi.com/journals/ecam/2013/934183.pdfEleutheroside E, An Active Component of Eleutherococcus senticosus , Ameliorates Insulin