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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 867893 9 pageshttpdxdoiorg1011552013867893

Research ArticleBoehmeria nivea Stimulates Glucose Uptake byActivating Peroxisome Proliferator-Activated Receptor Gammain C2C12 Cells and Improves Glucose Intolerance in Mice Feda High-Fat Diet

Sung Hee Kim Mi Jeong Sung Jae Ho Park Hye Jeong Yang and Jin-Taek Hwang

Korea Food Research Institute 516 Baekhyun-dong Bundang-ku Seongnam Gyeonggi-do 463-746 Republic of Korea

Correspondence should be addressed to Jin-Taek Hwang jthwangkfrirekr

Received 9 January 2013 Revised 12 March 2013 Accepted 12 March 2013

Academic Editor Ravirajsinh N Jadeja

Copyright copy 2013 Sung Hee 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

We examined the antidiabetic property of Boehmeria nivea (L) Gaud Ethanolic extract of Boehmeria nivea (L) Gaud (EBN)increased the uptake of 2-[N-(nitrobenz-2-oxa-13-diazol-4-yl)amino]-2-deoxy-d-glucose in C2C12 myotubes To examine themechanisms underlying EBN-mediated increase in glucose uptake we examined the transcriptional activity and expression ofperoxisome proliferator-activated receptor gamma (PPAR-120574) a pivotal target for glucose metabolism in C2C12 myotubes Wefound that the EBN increased both the transcriptional activity and mRNA expression levels of PPAR-120574 In addition we measuredphosphorylation and expression levels of other targets of glucose metabolism such as AMP-activated protein kinase (AMPK) andprotein kinase B (AktPKB)We found that EBNdid not alter the phosphorylation or expression levels of these proteins in a time- ordose-dependent manner which suggested that EBN stimulates glucose uptake through a PPAR-120574-dependent mechanism Furtherwe investigated the antidiabetic property of EBN using mice fed a high-fat diet (HFD) Administration of 05 EBN reduced theHFD-induced increase in body weight total cholesterol level and fatty liver and improved the impaired fasting glucose level bloodinsulin content and glucose intoleranceThese results suggest that EBN had an antidiabetic effect in cell culture and animal systemsand may be useful for preventing diabetes

1 Introduction

Diabetes is a serious health issue that affects the life spanof humans Of those diagnosed with diabetes approximately5ndash10 have type 1 diabetes which is characterized by theloss of insulin production in pancreatic beta-cells whereas90ndash95 have type 2 diabetes which is characterized byinsulin resistance Although various drugs have been devel-oped for diabetes treatment their side effects remain obsta-cles to their use Natural ingredients are widely distributedin the plant kingdom They have been traditionally usedto treat a variety of human diseases including metabolicdisorders It is believed that natural ingredients are safer thansynthetic compounds because they have been used for a longtime [1 2] The results of a number of studies conductedby other researchers and our previous study also suggested

that naturally occurring compounds could exert beneficialhealth effects in the treatment of diabetes by modulating cel-lular signaling pathways [2ndash5] Among signaling moleculesperoxisome proliferator-activated receptor gamma (PPAR-120574) regulates fatty acid and glucose metabolism PPAR-120574 hasbeen implicated in the pathology of obesity and diabetes[3ndash5] PPAR-120574 agonists such as glitazone have been usedto treat hyperglycemia [6] In addition PPAR-120574 agonistsderived from natural herbs may prevent diabetes For exam-ple Cornus kousa F Buerger ex Miquel a medicinal plantincreases PPAR-120574 activity and stimulates glucose uptake Inaddition Aegle marmelos fruit aqueous extract Syzygiumaromaticum flower bud (clove) extract and Sambucus nigra(elderflower) extract exert antidiabetic effects by increasingPPAR-120574 activation or expression [7ndash9] AMP-activated pro-tein kinase (AMPK) and Akt protein are other important

2 Evidence-Based Complementary and Alternative Medicine

signaling molecules in glucose homeostasis AMPK is aninsulin-independent regulator of glucose uptake By contrastthe PI3 kinaseAkt pathway is an insulin-dependent regulatorof glucose uptake Thus AMPK and Akt are also therapeutictargets for metabolic disorders such as obesity and diabetes[10 11]

Boehmeria nivea (L) Gaud a flowering plant in thenettle family Urticaceae has been widely grown in east Asiancountries such as Korea India and China The edible partsof this plant the leaves and roots have been reported tohave anti-inflammatory antioxidant and antifungal effects[12 13] However the antidiabetic effect of B nivea has notbeen clearly elucidated Therefore in this study we evaluatedthe antidiabetic potential of ethanol extract of B nivea (EBN)and its signaling mechanisms by using in vitro and in vivoapproaches

2 Materials and Methods

21 Materials An authenticated B nivea sample was pro-vided by a public officer from the Seocheon County Office(Seocheon Republic of Korea) where a voucher specimenhas been deposited C2C12 and HEK293 cells were pur-chased from the American Type Culture Collection (Man-assas VA USA) Dulbeccorsquos modified Eaglersquos medium(DMEM) and fetal bovine serum (FBS) were purchasedfrom Welgene (Daegu Republic of Korea) Horse serumwas purchased from Life Technologies (Seoul Republicof Korea) 2-[N-(Nitrobenz-2-oxa-13-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) was purchased from Invitrogen(Carlsbad CA USA) PPAR-120574 and glyceraldehyde phosphatedehydrogenase (GAPDH) primers were designed based onsequence data from the NCBI database and were purchasedfrom Bioneer (Daejeon Republic of Korea) Phospho-AMP-activated protein kinase (pAMPK) phospho-Akt (pAKT)PPAR-120574 and AMPK were purchased from Cell SignalingTechnology (Beverly MA USA) 120573-actin and horseradishperoxidase-conjugated secondary antibody were purchasedfrom Santa Cruz Biotechnology (Santa Cruz CA USA)

22 Extraction and Lyophilization High-quality B niveaspecimens were selected and mixed by a specialist and theethanol extract was prepared Leaves of B nivea were pul-verized and extracted with 70 ethanol by shaking for 24 hat 25∘C and the precipitates were removed by centrifugationat 8000 timesg for 30min (Beckman USA) Supernatants werelyophilized using a freeze dryer (Il Shin DongducheonRepublic of Korea) The yield of ethanol extract from theleaves of B nivea was 100 (ww) Ethanol extract from theleaves of B nivea (EBN) was dissolved in distilled water andsterilized by passage through a 045 120583m Millipore filter unitfor use in the experiments

23 Muscle Cell Differentiation and Glucose Uptake AssayC2C12 cells were cultured inDMEMcontaining 10FBSThecells were maintained at 37∘C in a humidified 5 CO

2envi-

ronment After the cells reached confluency the mediumwaschanged toDMEM supplemented with 2 horse serum until

the cells were entirely differentiated For the experiments thecells were starved in low-glucose serum-free DMEM for 12 hand then treated with or without 50120583M 2-NBDG or with 2-NBDG with EBN at the indicated concentrations (200 400800 and 1200120583gmL) for 24 h Cellular uptake of 2-NBDGwas measured using a fluorometer at excitation and emissionwavelengths of 465 and 540 nm respectively

24 PPAR-120574 Transcriptional Activity Assay PPAR-120574 tran-scriptional activity was measured as described previously[14] HEK293 cells were cultured in DMEM containing10 FBS Cells were transiently transfected with 1 120583g oftotal DNA (expression plasmids for PPAR-120574 retinoid Xreceptor 120572 (RXR120572) PPAR response elements (PPREs) and120573-galactosidase) by using SuperFect Transfection Reagent(Qiagen Valencia CA USA) according to themanufacturerrsquosinstructions After transfection the cells were treated withEBN at the indicated concentrations (200 400 800 and1200120583gmL) in the absence or presence of rosiglitazone aPPAR-120574 agonist for 24 h PPAR-120574 transcriptional activity wasexamined using a luciferase reporter gene assay with theLuciferase Assay System (Promega Madison WI USA) andwas normalized to the 120573-galactosidase activity

25 RNA Isolation and Reverse Transcriptase-PolymeraseChain Reaction Differentiated C2C12 cells were treated withvarious concentrations of EBN for 6 h and total RNA wasisolated using TRIzol reagent (Invitrogen) according to themanufacturerrsquos instructions cDNA was synthesized fromisolatedRNAandwas amplified by polymerase chain reaction(PCR) in a PCR thermal cycle device by using specificprimers PPAR-120574 (sense) 51015840-ACC ACT CGC ATT CCT TTFAC-31015840 PPAR-120574 (antisense) 51015840-TCA GCG GGA AGG ACTTTA TG-31015840 120573-actin (sense) 51015840-TCA CCC ACA CTG TGCCCA TCT ACG A-31015840 and 120573-actin (antisense) 51015840-GGA TGCCAC AGG ATT CCA TAC CCA-31015840

26 Protein Extraction and Western Blot Analysis Total pro-tein was extracted from EBN-stimulated cells by using lysisbuffer (50mMTris-HCl pH 80 5mMEDTA 150mMNaCl1 NP-40 1mM PMSF a protease-inhibitor cocktail anda phosphatase-inhibitor cocktail) Equal amounts of protein(30 120583g) were separated using 10 SDS-PAGE and weretransferred onto a nitrocellulose membrane The membranewas blocked with 5 skim milk in Tris-buffered saline (TBS)for 1 h andwas incubated in primary antibody diluted in TBSAfter washing with TBST (TBS with 01 Tween 20) themembrane was incubated with HRP-conjugated secondaryantibody for 1 h at room temperature Blots were developedwith an enhanced chemiluminescence (ECL) kit (AmershamBuckinghamshire UK)

27 Animal Experiments Male C57BL6J mice (age 3 weeks)were obtained from Nara Biotech (Seoul Republic ofKorea) and housed under a 12 h light12 h dark cycle in atemperature- and humidity-controlled room (24∘C plusmn 1∘C at50 relative humidity) After adaptation for 1 week the micewere freely fed a 10 fat normal diet (ND D12450B Research

Evidence-Based Complementary and Alternative Medicine 3

Diets New Brunswick NJ USA) a 60 kcal high-fat diet(HFD D12492 Research Diets New Brunswick NJ USA) ora 60kcal high-fat diet plus 05EBN (HFD+05EBN) for9 weeks Food intake and body weight were measured everyweek After 9 weeks the mice were fasted overnight and thenkilledTheblood sampleswere collected from the orbital veinAll animal experiments were approved by the InstitutionalAnimal Care andUse Committee of the Korea Food ResearchInstitute

28 Glucose Tolerance Test The glucose tolerance test (GTT)was performed at 8 weeks After overnight fasting micewere intraperitoneally administered glucose (1 gkg of bodyweight) and blood was collected from the tail vein every30min from 0min to 150min after injection Blood glucoselevels were examined by an Accu-Chek glucometer (RocheBasel Switzerland)

29 Quantitation of Serum Total Cholesterol and Insulin Lev-els Fasting serum levels of total cholesterol (TC) and insulinwere determined by enzymatic methods using commercialkits (Asan Pharm Seoul Republic of Korea)

210 Histopathology Liver tissue was fixed in 4 bufferedformalin and cut into 4 120583m thick sections The sections werestained with hematoxylin and eosin (HampE) and examined bymicroscopy Fat content was scored semiquantitatively withthe following parameters as described previously [15] 0 = nofat 1+ = lt25 2+ = 25ndash50 3+ = 51ndash75 4+ = 76ndash95 and5+ = 100

211 Cytotoxicity Test Cells were starved with low-glucoseserum-free DMEM for 12 h and treated with EBN at theindicated concentrations (200 400 800 and 1200120583gmL)for 24 h The medium was removed and the cells wereincubated with 100120583L 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich St Louis MO USA) in PBS at 5mgmL for 4 h Theabsorbance was measured using an ELISA reader at 540 nm

212 Statistical Analysis The data have been expressed as themean plusmn standard deviation (SD) values of at least 3 indepen-dent experiments Statistical analyses were performed usingSPSS version 90 (SPSS Inc Chicago IL USA) Differencesbetween means were evaluated using two-way analysis ofvariance (ANOVA) followed by the Bonferroni test Valuesof P lt 005 were considered significant

3 Results

31 EBN Stimulates Glucose Uptake in C2C12 MyotubesWe first performed the 2-NBDG uptake assay to examinethe antidiabetic activity of EBN in C2C12 myotubes EBNsignificantly increased 2-NBDG uptake in a dose-dependentmanner (Figure 1(a)) Cytotoxic effects of EBN were notobserved below 1200 120583gmL (Figure 1(b))This result suggeststhat EBN increases glucose uptake in myotubes

Table 1 Effect of EBN on high-fat diet-induced body weightchanges serum cholesterol and insulin

ND HFD EBNInitial body weight (g) 203 plusmn 09 200 plusmn 11 201 plusmn 09Final body weight (g) 288 plusmn 30 406 plusmn 38a 357plusmn46b

SerumTotal cholesterol (mgdL) 172 plusmn 14 209 plusmn 9a 196 plusmn 9b

Insulin (ngmL) 0331 plusmn 0112 122 plusmn 021a 0812 plusmn 0201b

ND normal diet HFD high-fat diet EBN high-fat diet plus 05 EBNDataare expressed as mean plusmn SD (119899 = 10) Statistical significance aP lt 005 forthe ND versus HFD bP lt 005 for the HFD versus HFD + 05 EBN

32 Activation of PPAR-120574 rather than AMPK and Akt IsInvolved in EBN-Stimulated Glucose Uptake To determinethe molecular mechanisms underlying EBN-stimulated glu-cose uptake we measured the activities of PPAR-120574 AMPKand Akt PPAR-120574 is a critical target of a number of insulin-sensitizing drugs [3ndash5] Treatment with EBN significantlyincreased the transcriptional activity of PPAR-120574 in HEK293cells (Figure 2(a)) Under the same conditions 25120583M rosigli-tazone was used as a positive control for PPAR-120574 activationIn addition we measured the expression of PPAR-120574 inEBN-treated C2C12 myotubes EBN increased the expres-sion of PPAR-120574 in C2C12 myotubes (Figure 2(b)) Thenwe examined whether AMPK and Akt signaling pathwayswere involved in EBN-stimulated glucose uptake AMPK isanother target for the antidiabetic effect of metformin andAkt is a critical mediator of the insulin-sensitizing effect [1011] EBN did not increase the phosphorylation or expressionof AMPK and Akt in a dose- and time-dependent manner inC2C12myotubes (Figures 2(c) and 2(d)) 5-Aminoimidazole-4-carboxamide 1-120573-D-ribofuranoside (AICAR) was used asa positive control for AMPK activation These results indi-cate that EBN stimulates glucose uptake at least in partthrough activation of PPAR-120574 AMPK and Akt signalingpathways however were not involved in EBN-stimulatedglucose uptake in C2C12 myotubes

33 Administration of EBN Improves Body Weight Body FatMass Liver Fat Content and Serum TC Levels in Mice Fed aHFD We performed an in vivo experiment to confirm theantidiabetic effect of EBN The initial body weights of micein each group were not statistically different (Table 1) Thefinal bodyweights were lower in the EBN group (Table 1)Thebody weights in the HFD group were higher than those in theNDgroup (Figure 3(a))Thebodyweights ofmice in theHFD+ 05 EBN group were lower than those in the HFD groupThe total food intake was not statistically different in eachgroup (Figure 3(b)) In addition we assessed body fat massby using computed tomography (CT) imaging and foundthat the whole body fat mass was higher in the HFD groupthan in the ND group which is shown in red in Figure 3(c)Administration of 05 EBN significantly reduced the wholebody fat mass in mice fed a HFD (Figure 3(c)) Furtherserumcholesterol levels were significantly lower in theHFD+05 EBN group than in the HFD group (Table 1) Moreoverthe concentration of fat in the liver tissue was significantly


0

05

1

15

2

EBN

None 200 400 800 1200

lowast

lowastlowast

2-N

BDG

upt

ake (

fold

indu

ctio

n)

(120583gmL)

(a)

0

25

50

75

100

None 200 400 800 1200(120583gmL)

MTT

assa

y (

of c

ontro

l)EBN

(b)

Figure 1 Effect of EBN on glucose uptake in C2C12myotubes Differentiated C2C12 cells were treated with EBN and 2-[N-(nitrobenz-2-oxa-13-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) for 24 h Then the 2-NBDG assay was performed as described in Section 2 (a) Aftertreatment of EBN for 24 h in C2C12 myotubes the cytotoxicity was measured by MTT assay as described in Section 2 The result of MTTassay was expressed as percentage of control (None) (b) Data are expressed as mean plusmn standard deviation (SD) 119899 = 3 lowastP lt 005 versus None

lower in the HFD + 05 EBN group than in the HFDgroupThe histopathology scores also showed that 05 EBNsignificantly decreased fat accumulation stimulated by HFD(Figure 3(d))

34 Effect of EBN on Fasting Glucose Levels and GlucoseIntolerance Wedetermined fasting glucose and insulin levelsand performed a GTT to determine the antidiabetic effectof EBN in mice fed a HFD The fasting glucose level wassignificantly higher in the HFD group than in the ND groupThe fasting glucose level was lower in the HFD + 05 EBNgroup than in theHFDgroup (Figure 4(a) andTable 1) Com-pared to the ND group the HFD group showed an abnormalincrease in insulin levels but the insulin levels significantlydecreased in the HFD + 05 EBN group (Table 1) TheGTT test showed that the HFD led to glucose intoleranceAdministration of 05 EBNmarkedly decreased the glucoseintolerance induced by the HFD (Figures 4(b) and 4(c))These results show that EBN improves glucose tolerance andinsulin resistance

4 Discussion

In this study we showed that EBN exerts an antidiabeticeffect in C2C12 myotubes and in a mouse model of aHFD In addition we showed that PPAR-120574 activation ratherthan AMPK and Akt signaling was associated with theantidiabetic activity of EBN Our findings demonstrate that

EBN has antidiabetic and antiobesity effects in vitro andin vivo Compared to the findings of a recent study thatshowed that B nivea inhibits alpha-glucosidase or beta-glucosidase our findings better substantiate the antidiabeticeffect of EBN [16] Although the recent study showed theglucosidase inhibitory effect of B nivea in vitro it did notdescribe the mechanisms underlying this effect our studyis based on these data and we performed experimentsto confirm the antidiabetic effects of B nivea Throughcell and animal experiments we demonstrated the preciseantidiabetic efficacy of B nivea and the mechanisms under-lying the effect The total phenolic content of EBN was3640mg100 g Several phenolic compounds such as rutin(4648mg100 g) chlorogenic acid (196mg100 g) luteolin-7-glucoside (1129mg100 g) naringin (113mg100 g) hes-peridin (2369mg100 g) and tangeretin (159mg100 g) havealready been found in EBN [17]

We showed that EBN-stimulated glucose uptake isaccompanied by PPAR-120574 activation and expression but is notassociated with AMPK and Akt activation PPAR-120574 partic-ipates in glucose uptake and PPAR-120574 agonists increase thesensitivity of insulin receptors [3ndash5] In addition to thiazo-lidinedione a PPAR-120574 agonist that is used clinically naturallyoccurring ingredients or compounds have also been used asPPAR-120574 agonists to significantly improve insulin resistance[3 18 19] For example Cornus kousa F Buerger ex Miquela medicinal plant increases PPAR-120574 ligand binding activityand stimulates glucose uptake by an AMPK-independentmechanism [7] A number of studies have shown that


0

1

2

3

4

None EBN Rosi

PPA

R-120574

tran

scrip

tiona

l act

ivity

(fol

d in

duct

ion)

200120583gmL

400120583gmL

800120583gmL

1200 120583gmL

lowast

lowast

lowastlowast

lowast

(a)

None

EBN

200 400 800 1200(120583gmL)

120573-actin

PPAR-120574

(b)

pAMPK

pAKT

AMPK

AKT

None

EBN

200 400 800 1200(120583gmL)

120573-actin

(c)

AICAR

pAMPK

pAKT

AMPK

AKT

0 05 1 3

EBN (1200 120583gmL)

6h

120573-actin

(d)

Figure 2 The effect of EBN on transcriptional activity and expression of peroxisome proliferator-activated receptor gamma HEK293 cellswere transiently transfected with luciferase construct containing peroxisome proliferator-activated receptor gamma (PPAR-120574) retinoid Xreceptor (RXR120572) PPAR-response element (PPRE) and 120573-galactosidase Then the cells were treated with ethanol extract of Boehmeria nivea(EBN) (200 400 800 and 1200120583gmL) for 24 h Luciferase assay was performed and the activity was normalized with that of 120573-galactosidaseactivity (a)The differentiated C2C12 cells were exposed to EBN in a dose-dependentmanner for 6 hThemRNA level of PPAR-120574 andGAPDHwas measured by reverse transcriptase-polymerase chain reaction (RT-PCR) (b) Differentiated C2C12 cells were treated withEBN in a dose-and time-dependent manner AMP-activated protein kinase (AMPK) and Akt expressions were determined by western blot analysis ((c)(d)) 1mMAICARwas used as positive control for AMPK activation Data are expressed as mean plusmn standard deviation (SD) 119899 = 3 Statisticalsignificance lowastP lt 005 for the none versus EBN or rosiglitazone

aqueous extract from the fruits of Aegle marmelos Syzygiumaromaticum flower bud (clove) extract and Sambucus nigra(elderflower) extract improve insulin resistance by increasingPPAR-120574 activation or expression [8 9] Therefore consistentwith previous studies PPAR-120574 activation and expression

could explain the antidiabetic effect of EBN observed in ourstudy AMPK and Akt were not altered by EBN under ourexperimental conditions AMPK is a key regulator of glucosemetabolism and is a target ofmetformin an antidiabetic drug[10] Like PPAR-120574 AMPK is a target for naturally occurring


15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN

lowastlowastlowastlowast

lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)

Figure 3 The effect of EBN on body weight total food intake and accumulation of fat in the liver of mice fed a high-fat diet Mice were fednormal diet (ND) high-fat diet (HFD) and high-fat diet with 05 EBN (HFD + 05 EBN) Body weight and food intake were measuredevery week ((a) (b)) Body fat mass was assessed using computed tomography (CT) during the 8 weeks (c) In addition we measured theaccumulation of fat in the liver as described in Section 2 (d) Data are expressed as the mean plusmn standard deviation (SD) (119899 = 10) Statisticalsignificance lowastP lt 005 for the HFD versus HFD + 05 EBN

ingredients or compounds for the prevention of diabetes[20] Insulin-dependent PI3KAkt signaling can also improvediabetes by activating insulin receptor [21] Once insulinbinds to insulin receptor insulin receptor substrate 1 (IRS-1) is activated Activated IRS-1 triggers insulin signaling cas-cades involving PI3K phosphoinositide-dependent kinase-1

(PDK-1) and Akt and increases glucose transporter 4 (Glut-4) translocation and glucose uptake [22] EBN treatment didnot alter insulin-dependent PI3KAkt signaling and AMPKsignaling (Figures 2(c) and 2(d)) These results indicatethat EBN-stimulated glucose uptake is mediated by PPAR-120574which suggests that EBN does not mimic the effect of insulin


0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)

Figure 4 The effect of EBN on fasting glucose levels and glucose intolerance in mice fed a high-fat diet Mice were starved for 12 h andblood was drawn from the orbital vein and we measured the fasting glucose levels as described in Section 2 (a) Glucose tolerance test wasperformed at the end of 8 weeks Mice were injected with glucose (1 gkg of body weight) and blood glucose levels were examined every30min from 0min to 150min after injection (b) Area under curve (AUC) during glucose tolerance test (c) Data are expressed as mean plusmnstandard deviation (SD) (119899 = 10) Statistical significance lowastP lt 005 for the high-fat diet (HFD) versus HFD plus 05 EBN

through the insulin receptor or the effect of metformin onAMPK activation To our knowledge this is the first studyto show the antidiabetic effect and mechanisms of EBNHowever it is still unclear how EBN acts as a PPAR-120574 agonistOne possibility hinted at by early reports is that PPAR-120574 agonists stimulate glucose uptake by increasing Glut4expression andmRNA levels [23] In addition elevated levelsof PPAR-120574 transcript are accompanied by enhanced Glut-4 transcription and glucose uptake suggesting that glucoseuptake and Glut-4 transcription are induced through theactivation of PPAR-120574 by PPAR-120574 agonists [24]Therefore theantidiabetic effect of EBN may be due to increased PPAR-120574 and Glut-4 transcription However further studies arerequired to explain the cascades downstream from EBN-mediated activation of PPAR-120574 that stimulate glucose uptake

Administration of 05 EBN significantly reduced bodyweight body fat mass liver fat content and serum totalcholesterol levels In addition EBN significantly improvedHFD-induced fasting glucose levels and glucose toleranceA number of studies have described a link between HFD-induced obesity and metabolic disorders including diabeteshyperlipidemia and hypercholesterolemia [25 26] Obesitycauses insulin resistance and thus results in type 2 diabetes

We have shown that a HFD accelerated obesity and glucoseintolerance Administration of EBN dramatically improvedthe glucose intolerance caused by a HFD Sancheti et alshowed that the root extract of B nivea exerts an antidiabeticeffect against streptozotocin- (STZ-) induced diabetes in rats[16] In fact the STZ-induced diabetic model is generallyused to identify antidiabetic drugs or ingredients that offsettype 1 diabetes In the present study we investigated theantidiabetic effect of the leaf extract of B nivea We used theleaf extract because the leaves of B nivea have traditionallybeen ingested and used for the prevention of certain diseasesin Oriental countries The results reported by Sancheti et alon the antidiabetic effect of the root extract of B nivea in atype 1 diabetes model indicated that B niveamay be effectivefor the prevention of metabolic disorders such as diabetesobesity and fatty liver [27] As expected we found that EBNwas effective in obesity-induced type 2 diabetes mice Takentogether these findings indicate that the preventative effectsof EBN are applicable to obesity-induced insulin resistancesimilar to that in type 2 diabetes In addition the serum levelsof total cholesterol and the fat content of the liver decreased inthe EBN-treated group An elevated total cholesterol level is arisk factor for obesity and cardiovascular diseases Previous


papers have also reported that a number of ingredientsextracted from plants exert a cholesterol-lowering effect bylowering the absorption of cholesterol and increasing fecalsterol excretion [28 29] In addition several compoundsfound in plants inhibit cholesterol synthesis and reduce low-density lipoprotein- (LDL-) cholesterol levels in the plasma[30 31] Sancheti et al have also reported that B nivea exertsan inhibitory effect on cholinesterase in vitro suggesting thatB nivea has the potential to lower cholesterol levels [16]Cholinesterase plays an important role in the metabolismof lipids Elevation of the serum cholinesterase level isaccompanied by high serum cholesterol levels [16] Thus thecholesterol-lowering effect of EBN may be due to increasedcholesterol excretion or decreased cholesterol synthesis inthe liver The cholesterol lowering effect of EBN may also bedue to reduced serum levels of cholinesterase However themechanisms by which EBN lowers serum cholesterol levelsshould be elucidated further in future studies

5 Conclusions

Our study showed that EBN exerted an antidiabetic effect bytargeting PPAR-120574 signaling in myotubes In addition EBNimproved the abnormal increase in body weight liver fat andserum cholesterol level observed inHFD-fedmiceThereforeB nivea may be useful for preventing diabetes Howeveradditional studies are required to explain the precise signalingcascades underlying the antidiabetic effect of B nivea in bothcell and animal models

Acknowledgments

This work was supported by the Seocheon County Office andKorea Food Research Institute Republic of Korea

References

[1] C Zhang ldquoNatural compounds that modulate BACE1-processing of amyloid-beta precursor protein in Alzheimerrsquosdiseaserdquo Discovery Medicine vol 14 no 76 pp 189ndash197 2012

[2] J THwangD YKwon and SH Yoon ldquoAMP-activated proteinkinase a potential target for the diseases prevention by naturaloccurring polyphenolsrdquo New Biotechnology vol 26 no 1-2 pp17ndash22 2009

[3] A Shehzad T Ha F Subhan and Y S Lee ldquoNew mechanismsand the anti-inflammatory role of curcumin in obesity andobesity-related metabolic diseasesrdquo European Journal of Nutri-tion vol 50 no 3 pp 151ndash161 2011

[4] Q Liu L Chen L Hu Y Guo and X Shen ldquoSmall moleculesfrom natural sources targeting signaling pathways in diabetesrdquoBiochimica et Biophysica Acta vol 1799 no 10ndash12 pp 854ndash8652010

[5] T Deng S Shan Z B Li et al ldquoA new retinoid-like compoundthat activates peroxisome proliferator-activated receptors andlowers blood glucose in diabetic micerdquo Biological and Pharma-ceutical Bulletin vol 28 no 7 pp 1192ndash1196 2005

[6] B Cariou B Charbonnel and B Staels ldquoThiazolidinedionesand PPAR120574 agonists time for a reassessmentrdquo Trends inEndocrinology and Metabolism vol 23 no 5 pp 205ndash215 2012

[7] A K Sharma S Bharti S Goyal et al ldquoUpregulation of PPAR120574by Aegle marmelos ameliorates insulin resistance and 120573-celldysfunction in high fat diet fed-streptozotocin induced type 2diabetic ratsrdquo Phytotherapy Research vol 25 no 10 pp 1457ndash1465 2012

[8] M kuroda Y Mimaki T Ohtomo et al ldquoHypoglycemic effectsof clove (Syzygium aromaticumflower buds) on genetically dia-betic KK-Ay mice and identification of the active ingredientsrdquoJournal of Natural Medicines vol 66 no 2 pp 394ndash399 2012

[9] K B Christensen R K Petersen K Kristiansen and LP Christensen ldquoIdentification of bioactive compounds fromflowers of black elder (Sambucus nigra L) that activate thehuman peroxisome proliferator-activated receptor (PPAR) 120574rdquoPhytotherapy Research vol 24 supplement 2 pp S129ndashS1322010

[10] D G Hardie ldquoSensing of energy and nutrients by AMP-activated protein kinaserdquoAmerican Journal of ClinicalNutritionvol 93 supplement 4 pp 891Sndash896S 2011

[11] I Hers E E Vincent and J M Tavare ldquoAkt signalling in healthand diseaserdquo Cellular Signalling vol 23 no 10 pp 1515ndash15272011

[12] X Y Tian M Xu B Deng et al ldquoThe effects of Boehmerianivea (L) Gaud on embryonic development in vivo and in vitrostudiesrdquo Journal of Ethnopharmacology vol 134 no 2 pp 393ndash398 2011

[13] Q M Xu Y L Liu X R Li X Li and S L Yang ldquoThree newfatty acids from the roots of Boehmeria nivea (L) Gaudich andtheir antifungal activitiesrdquoNatural Product Research vol 25 no6 pp 640ndash647 2011

[14] M S Kim H J Hur D Y Kwon and J T Hwang ldquoTangeretinstimulates glucose uptake via regulation of AMPK signalingpathways in C2C12 myotubes and improves glucose tolerancein high-fat diet-induced obese micerdquo Molecular and CellularEndocrinology vol 358 no 1 pp 127ndash134 2012

[15] B J Song K H Moon N U Olsson and N Salem Jr ldquoPre-vention of alcoholic fatty liver and mitochondrial dysfunctionin the rat by long-chain polyunsaturated fatty acidsrdquo Journal ofHepatology vol 49 no 2 pp 262ndash273 2008

[16] S Sancheti S Sancheti and S Y Seo ldquoEvaluation of antigly-cosidase and anticholinesterase activities of Boehmeria niveardquoPakistan Journal of Pharmaceutical Sciences vol 23 no 2 pp236ndash240 2010

[17] M J Sung M Davaatseren S H Kim M J Kim and J THwang ldquoBoehmeria nivea attenuates LPS-induced inflamma-tory markers by inhibiting p38 and JNK phosphorylations inRAW2647 macrophages rdquo Pharmaceutical Biology In press

[18] M S Kim S Kung T Grewal and B D Roufogalis ldquoMethod-ologies for investigating natural medicines for the treatment ofnonalcoholic fatty liver disease (NAFLD)rdquo Current Pharmaceu-tical Biotechnology vol 13 no 2 pp 278ndash291 2012

[19] R K Petersen K B Christensen A N Assimopoulou et alldquoPharmacophore-driven identification of PPAR120574 agonists fromnatural sourcesrdquo Journal of Computer-Aided Molecular Designvol 25 no 2 pp 107ndash116 2011

[20] S L Huang R T Yu J Gong et al ldquoArctigenin a natural com-pound activates AMP-activated protein kinase via inhibition ofmitochondria complex I and amelioratesmetabolic disorders inobobmicerdquo Diabetologia vol 55 no 5 pp 1469ndash1481 2012

[21] B D Manning and L C Cantley ldquoAKTPKB signaling navigat-ing downstreamrdquo Cell vol 129 no 7 pp 1261ndash1274 2007

[22] M Ridderstrale ldquoSignaling mechanism for the insulin-likeeffects of growth hormonemdashanother example of a classical


hormonal negative feedback looprdquo Current Drug Targets vol 5no 1 pp 79ndash92 2005

[23] W T Garvey L Maianu J H Zhu J A Hancock and AM Golichowski ldquoMultiple defects in the adipocyte glucosetransport system cause cellular insulin resistance in gestationaldiabetes heterogeneity in the number and a novel abnormalityin subcellular localization of GLUT4 glucose transportersrdquoDiabetes vol 42 no 12 pp 1773ndash1785 1993

[24] A Shimaya E Kurosaki K Shioduka R NakanoM Shibasakiand H Shikama ldquoYM268 increases the glucose uptake celldifferentiation and mRNA expression of glucose transporter in3T3-L1 adipocytesrdquo Hormone and Metabolic Research vol 30no 9 pp 543ndash548 1998

[25] L E C van Meijl R Vrolix and R P Mensink ldquoDairy productconsumption and the metabolic syndromerdquo Nutrition ResearchReviews vol 21 no 2 pp 148ndash157 2008

[26] N Irwin I A Montgomery and P R Flatt ldquoEvaluation of thelong-term effects of gastric inhibitory polypeptide-ovalbuminconjugates on insulin resistance metabolic dysfunction energybalance and cognition in high-fat-fed micerdquo British Journal ofNutrition vol 108 no 1 pp 46ndash56 2012

[27] S Sancheti S SanchetiM Bafna H R Kim Y H You and S YSeo ldquoEvaluation of antidiabetic antihyperlipidemic and antiox-idant effects of Boehmeria nivea root extract in streptozotocin-induced diabetic ratsrdquo Brazilian Journal of Pharmacognosy vol21 no 1 pp 146ndash154 2011

[28] Z Y Chen R Jiao and Y M Ka ldquoCholesterol-loweringnutraceuticals and functional foodsrdquo Journal of Agricultural andFood Chemistry vol 56 no 19 pp 8761ndash8773 2008

[29] Y Lin M A Vermeer and E A Trautwein ldquoTriterpenic acidspresent in hawthorn lower plasma cholesterol by inhibitingintestinal ACAT activity in hamstersrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2011 Article ID 8012729 pages 2011

[30] S K Shin T Y Ha R A McGregor and M S Choi ldquoLong-term curcumin administration protects against atherosclerosisvia hepatic regulation of lipoprotein cholesterol metabolismrdquoMolecularNutrition FoodResearch vol 55 no 12 pp 1829ndash18402011

[31] T Sanclemente I Marques-Lopes J Puzo and A L Garcıa-Otın ldquoRole of naturally-occurring plant sterols on intestinalcholesterol absorption and plasmatic levelsrdquo Journal of Physi-ology and Biochemistry vol 65 no 1 pp 87ndash98 2009

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


signaling molecules in glucose homeostasis AMPK is aninsulin-independent regulator of glucose uptake By contrastthe PI3 kinaseAkt pathway is an insulin-dependent regulatorof glucose uptake Thus AMPK and Akt are also therapeutictargets for metabolic disorders such as obesity and diabetes[10 11]

Boehmeria nivea (L) Gaud a flowering plant in thenettle family Urticaceae has been widely grown in east Asiancountries such as Korea India and China The edible partsof this plant the leaves and roots have been reported tohave anti-inflammatory antioxidant and antifungal effects[12 13] However the antidiabetic effect of B nivea has notbeen clearly elucidated Therefore in this study we evaluatedthe antidiabetic potential of ethanol extract of B nivea (EBN)and its signaling mechanisms by using in vitro and in vivoapproaches

2 Materials and Methods

21 Materials An authenticated B nivea sample was pro-vided by a public officer from the Seocheon County Office(Seocheon Republic of Korea) where a voucher specimenhas been deposited C2C12 and HEK293 cells were pur-chased from the American Type Culture Collection (Man-assas VA USA) Dulbeccorsquos modified Eaglersquos medium(DMEM) and fetal bovine serum (FBS) were purchasedfrom Welgene (Daegu Republic of Korea) Horse serumwas purchased from Life Technologies (Seoul Republicof Korea) 2-[N-(Nitrobenz-2-oxa-13-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) was purchased from Invitrogen(Carlsbad CA USA) PPAR-120574 and glyceraldehyde phosphatedehydrogenase (GAPDH) primers were designed based onsequence data from the NCBI database and were purchasedfrom Bioneer (Daejeon Republic of Korea) Phospho-AMP-activated protein kinase (pAMPK) phospho-Akt (pAKT)PPAR-120574 and AMPK were purchased from Cell SignalingTechnology (Beverly MA USA) 120573-actin and horseradishperoxidase-conjugated secondary antibody were purchasedfrom Santa Cruz Biotechnology (Santa Cruz CA USA)

22 Extraction and Lyophilization High-quality B niveaspecimens were selected and mixed by a specialist and theethanol extract was prepared Leaves of B nivea were pul-verized and extracted with 70 ethanol by shaking for 24 hat 25∘C and the precipitates were removed by centrifugationat 8000 timesg for 30min (Beckman USA) Supernatants werelyophilized using a freeze dryer (Il Shin DongducheonRepublic of Korea) The yield of ethanol extract from theleaves of B nivea was 100 (ww) Ethanol extract from theleaves of B nivea (EBN) was dissolved in distilled water andsterilized by passage through a 045 120583m Millipore filter unitfor use in the experiments

23 Muscle Cell Differentiation and Glucose Uptake AssayC2C12 cells were cultured inDMEMcontaining 10FBSThecells were maintained at 37∘C in a humidified 5 CO

2envi-

ronment After the cells reached confluency the mediumwaschanged toDMEM supplemented with 2 horse serum until

the cells were entirely differentiated For the experiments thecells were starved in low-glucose serum-free DMEM for 12 hand then treated with or without 50120583M 2-NBDG or with 2-NBDG with EBN at the indicated concentrations (200 400800 and 1200120583gmL) for 24 h Cellular uptake of 2-NBDGwas measured using a fluorometer at excitation and emissionwavelengths of 465 and 540 nm respectively

24 PPAR-120574 Transcriptional Activity Assay PPAR-120574 tran-scriptional activity was measured as described previously[14] HEK293 cells were cultured in DMEM containing10 FBS Cells were transiently transfected with 1 120583g oftotal DNA (expression plasmids for PPAR-120574 retinoid Xreceptor 120572 (RXR120572) PPAR response elements (PPREs) and120573-galactosidase) by using SuperFect Transfection Reagent(Qiagen Valencia CA USA) according to themanufacturerrsquosinstructions After transfection the cells were treated withEBN at the indicated concentrations (200 400 800 and1200120583gmL) in the absence or presence of rosiglitazone aPPAR-120574 agonist for 24 h PPAR-120574 transcriptional activity wasexamined using a luciferase reporter gene assay with theLuciferase Assay System (Promega Madison WI USA) andwas normalized to the 120573-galactosidase activity

25 RNA Isolation and Reverse Transcriptase-PolymeraseChain Reaction Differentiated C2C12 cells were treated withvarious concentrations of EBN for 6 h and total RNA wasisolated using TRIzol reagent (Invitrogen) according to themanufacturerrsquos instructions cDNA was synthesized fromisolatedRNAandwas amplified by polymerase chain reaction(PCR) in a PCR thermal cycle device by using specificprimers PPAR-120574 (sense) 51015840-ACC ACT CGC ATT CCT TTFAC-31015840 PPAR-120574 (antisense) 51015840-TCA GCG GGA AGG ACTTTA TG-31015840 120573-actin (sense) 51015840-TCA CCC ACA CTG TGCCCA TCT ACG A-31015840 and 120573-actin (antisense) 51015840-GGA TGCCAC AGG ATT CCA TAC CCA-31015840

26 Protein Extraction and Western Blot Analysis Total pro-tein was extracted from EBN-stimulated cells by using lysisbuffer (50mMTris-HCl pH 80 5mMEDTA 150mMNaCl1 NP-40 1mM PMSF a protease-inhibitor cocktail anda phosphatase-inhibitor cocktail) Equal amounts of protein(30 120583g) were separated using 10 SDS-PAGE and weretransferred onto a nitrocellulose membrane The membranewas blocked with 5 skim milk in Tris-buffered saline (TBS)for 1 h andwas incubated in primary antibody diluted in TBSAfter washing with TBST (TBS with 01 Tween 20) themembrane was incubated with HRP-conjugated secondaryantibody for 1 h at room temperature Blots were developedwith an enhanced chemiluminescence (ECL) kit (AmershamBuckinghamshire UK)

27 Animal Experiments Male C57BL6J mice (age 3 weeks)were obtained from Nara Biotech (Seoul Republic ofKorea) and housed under a 12 h light12 h dark cycle in atemperature- and humidity-controlled room (24∘C plusmn 1∘C at50 relative humidity) After adaptation for 1 week the micewere freely fed a 10 fat normal diet (ND D12450B Research








3 Results










0

05

1

15

2

EBN

None 200 400 800 1200

lowast

lowastlowast

2-N

BDG

upt

ake (

fold

indu

ctio

n)

(120583gmL)

(a)

0

25

50

75

100

None 200 400 800 1200(120583gmL)

MTT

assa

y (

of c

ontro

l)EBN

(b)




4 Discussion





0

1

2

3

4

None EBN Rosi

PPA

R-120574

tran

scrip

tiona

l act

ivity

(fol

d in

duct

ion)

200120583gmL

400120583gmL

800120583gmL

1200 120583gmL

lowast

lowast

lowastlowast

lowast

(a)

None

EBN

200 400 800 1200(120583gmL)

120573-actin

PPAR-120574

(b)

pAMPK

pAKT

AMPK

AKT

None

EBN

200 400 800 1200(120583gmL)

120573-actin

(c)

AICAR

pAMPK

pAKT

AMPK

AKT

0 05 1 3

EBN (1200 120583gmL)

6h

120573-actin

(d)





15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN


lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)





0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results










0

05

1

15

2

EBN

None 200 400 800 1200

lowast

lowastlowast

2-N

BDG

upt

ake (

fold

indu

ctio

n)

(120583gmL)

(a)

0

25

50

75

100

None 200 400 800 1200(120583gmL)

MTT

assa

y (

of c

ontro

l)EBN

(b)




4 Discussion





0

1

2

3

4

None EBN Rosi

PPA

R-120574

tran

scrip

tiona

l act

ivity

(fol

d in

duct

ion)

200120583gmL

400120583gmL

800120583gmL

1200 120583gmL

lowast

lowast

lowastlowast

lowast

(a)

None

EBN

200 400 800 1200(120583gmL)

120573-actin

PPAR-120574

(b)

pAMPK

pAKT

AMPK

AKT

None

EBN

200 400 800 1200(120583gmL)

120573-actin

(c)

AICAR

pAMPK

pAKT

AMPK

AKT

0 05 1 3

EBN (1200 120583gmL)

6h

120573-actin

(d)





15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN


lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)





0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

EBN

None 200 400 800 1200

lowast

lowastlowast

2-N

BDG

upt

ake (

fold

indu

ctio

n)

(120583gmL)

(a)

0

25

50

75

100

None 200 400 800 1200(120583gmL)

MTT

assa

y (

of c

ontro

l)EBN

(b)




4 Discussion





0

1

2

3

4

None EBN Rosi

PPA

R-120574

tran

scrip

tiona

l act

ivity

(fol

d in

duct

ion)

200120583gmL

400120583gmL

800120583gmL

1200 120583gmL

lowast

lowast

lowastlowast

lowast

(a)

None

EBN

200 400 800 1200(120583gmL)

120573-actin

PPAR-120574

(b)

pAMPK

pAKT

AMPK

AKT

None

EBN

200 400 800 1200(120583gmL)

120573-actin

(c)

AICAR

pAMPK

pAKT

AMPK

AKT

0 05 1 3

EBN (1200 120583gmL)

6h

120573-actin

(d)





15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN


lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)





0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

None EBN Rosi

PPA

R-120574

tran

scrip

tiona

l act

ivity

(fol

d in

duct

ion)

200120583gmL

400120583gmL

800120583gmL

1200 120583gmL

lowast

lowast

lowastlowast

lowast

(a)

None

EBN

200 400 800 1200(120583gmL)

120573-actin

PPAR-120574

(b)

pAMPK

pAKT

AMPK

AKT

None

EBN

200 400 800 1200(120583gmL)

120573-actin

(c)

AICAR

pAMPK

pAKT

AMPK

AKT

0 05 1 3

EBN (1200 120583gmL)

6h

120573-actin

(d)





15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN


lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)





0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

NDHFD

Time (weeks)

Body

wei

ght (

g)

HFD + 05 EBN


lowastlowast

(a)

0

1

2

3

4

Tota

l foo

d in

take

(gd

ay)

ND HFD HFD + 05 EBN

(b)

0

5

10

15

20

25

30

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

times103

Who

le b

ody

fat m

ass (

mm

3)

lowast

(c)

0

1

2

3

4

5

Hist

opat

holo

gy sc

ore

ND

ND

HFD

HFD

HFD + 05 EBN

HFD + 05 EBN

lowast

HE

(d)





0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

ND HFD HFD + 05 EBN

Fasti

ng g

luco

se (m

gdL

)lowast

(a)

0

100

200

300

400

500

0 30 60 90 120 150

ND

HFD

Time (min)

Bloo

d gl

ucos

e (m

gdL

)

lowastlowast

lowast

(b)

0

400

800

1200

1600

2000

AUC

(au

)

ND HFD HFD + 05 EBN

lowast

(c)







5 Conclusions


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


Acknowledgments


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 Boehmeria nivea Stimulates Glucose …downloads.hindawi.com/journals/ecam/2013/867893.pdf · Research Article Boehmeria nivea Stimulates Glucose Uptake by ... distribution,