antiobesity and antidiabetes effects of a cudrania tricuspidata … · 2016-05-17 ·...

Research ArticleAntiobesity and Antidiabetes Effects of a Cudrania tricuspidataHydrophilic Extract Presenting PTP1B Inhibitory Potential

Dae Hoon Kim1 Sooung Lee2 Youn Wook Chung3 Byeong Mo Kim4 Hanseul Kim56

Kunhong Kim6 and Kyung Mi Yang6

1New Drug Discovery Laboratory Hyundai Pharmaceutical Co Ltd Gyeong-Gi Bio-Center Lui-dongYeongtong-gu Suwon Gyeonggi-do Republic of Korea2Chuncheon Bioindustry Foundation Natural Resources Commercialization Center Chuncheon Gangwon-do Republic of Korea3Yonsei Cardiovascular Research Institute Yonsei University College of Medicine Seoul Republic of Korea4Severance Integrative Research Institute for Cerebral amp Cardiovascular Diseases (SIRIC)Yonsei University College of Medicine Seoul Republic of Korea5Brain Korea 21 Project for Medical Science Yonsei University Seoul Republic of Korea6Department of Biochemistry and Molecular Biology Yonsei University College of Medicine Seoul Republic of Korea

Correspondence should be addressed to Kunhong Kim kimkh34yuhsac and Kyung Mi Yang kyungmi yangyuhsac

Received 26 October 2015 Revised 19 January 2016 Accepted 20 January 2016

Academic Editor Maciej Banach

Copyright copy 2016 Dae Hoon Kim et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Diabetes and obesity represent the major health problems and the most age-related metabolic diseases Protein-tyrosine phos-phatase 1B (PTP1B) has emerged as an important regulator of insulin signal transduction and is regarded as a pharmaceutical targetfor metabolic disorders To find novel natural materials presenting therapeutic activities against diabetes and obesity we screenedvarious herb extracts using a chip screening allowing the determination of PTP1B inhibitory effects of the tested compounds usinginsulin receptor (IR) as the substrate Cudrania tricuspidata leaves (CTe) had a strong inhibitory effect on PTP1B activity andsubstantially inhibited fat accumulation in 3T3-L1 cells CTe was orally administrated to diet-induced obesity (DIO) mice oncedaily for 3 weeks after which changes in glucose insulinmetabolism and fat accumulationwere examinedHepatic enzymemarkers(aspartate aminotransferase AST and alanine aminotransferase ALT) and total fat mass and triglyceride levels decreased in CTe-treated mice whereas body weight and total cholesterol concentration slightly decreased CTe increased the phosphorylation ofIRS-1 and Akt in liver tissue Furthermore CTe treatment significantly lowered blood glucose levels and improved insulin secretionin DIO mice Our results strongly suggest that CTe may represent a promising therapeutic substance against diabetes and obesity

1 Introduction

Many metabolic diseases such as diabetes hypertensioncirculatory diseases Alzheimerrsquos disease and malignantcancer are associated with obesity [1ndash5] Protein-tyrosinephosphatases (PTPs) are important risk factors for metabolicdiseases PTPs play an important role in maintaining theproper tyrosine phosphorylation state of proteins [6] Abnor-mal tyrosine phosphorylation has been implicated in obesityand diabetes Thus PTP inhibitors have been investigated aspotential drugs against these diseases Protein-tyrosine phos-phatase 1B (PTP1B) is a negative regulator of insulin signalingIncreasedPTP1B activity results in the development of insulin

resistance subsequently leading to type 2 diabetes and obesity[7] The insulin receptor (IR) is the most important substrateof PTP1B On binding to its receptor insulin induces activa-tion of the insulin-receptor kinase (IRK) through autophos-phorylation Recruitment of insulin-receptor substrate (IRS)proteins induces activation of Akt leading to glucose uptakein muscle [8] PTP1B dephosphorylates IR thereby deactivat-ing it and contributing to insulin resistance PTP1B is alsofound in the brain where it plays an important role in energyregulation by interfering with the leptin receptor therebyaffecting the body weight status [6] PTP1B knockout animalsshow increased autophosphorylation of the insulin receptorand their myocytes and hepatocytes are more sensitive to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 8432759 11 pageshttpdxdoiorg10115520168432759

2 BioMed Research International

insulin than those of wild-type animals [9 10] Additionallythese animals present a low body fat content and highresistance to obesity from excessive nutritional intake PTP1Boverexpression is associated with an increase in insulin levels[11] PTP1B is considered as an attractive target for the devel-opment of new treatments for obesity and related metabolicsyndromes Although synthetic PTP1B inhibitors such asthiazolidinediones phosphorus-containing phosphotyrosylmimetics dephostatin and bidentate have been developed[12] little is known about the PTP1B inhibitory activity ofdrugs derived from natural resources

The increasing incidence of obesity and diabetes is aserious socioeconomic problem in many countries To over-come this problem multinational pharmaceutical companiesare aggressively performing studies to develop competitivedrugs Natural product-based therapeutic approaches pro-vide a medicinal herb or fruitful source for safe effectiveand less expensive therapeutic drugs In addition naturalproducts have been attracting attention because they presenta multipronged mechanism of action and contain manyhydrophilic active ingredients [13] Many herbal extractshave been used for the treatment of obesity and dia-betes Extracts derived from Peucedanum japonicum ThunbPhalaris canariensis and Panax ginseng have been used totreat obesity [14ndash16] Animal models and clinical studiesshowed that plant extracts from Apocynaceae Capparisspinosa Indigofera spicata Forssk andAntidesma bunius pos-sess antidiabetic effects [17ndash20] Therefore natural productsare considered as promising sources for PTP1B inhibitorsSince the discovery of flavonoids as natural PTP1B inhibitorsvarious natural substances with an inhibitory effect on PTPactivity have been reported [21ndash23] Until recently approx-imately 300 PTP1B inhibitors have been identified fromnatural resources [24] However further studies are needed toevaluate the effects of natural products with PTP1B inhibitoryactivity against diabetes and obesity

Cudrania (C) tricuspidata a small tree of the Moraceaefamily is widely used as an ingredient in oriental medicineSeveral reports demonstrated that C tricuspidata extract(CTe) has various effects including inhibition of pancre-atic lipase lipopolysaccharide-induced nitric oxide produc-tion prostaglandin E2 production in macrophages IL-1120573-induced rheumatoid synovial fibroblast proliferation andmatrix metalloproteinase production [25ndash27] In this studywe selected a CTe from among many plant extracts usinga chip screening platform that allowed us to determinePTP1B inhibitory effects of various extracts using IR as thesubstrate Furthermore we investigated the effects of CTe onmetabolic disturbances associated with obesity and diabetesusing a high-fat diet-induced obesity (DIO) mouse modelOur results provide a basis for developing novel antiobesityand antidiabetic medications and functional foods

2 Materials and Methods

21 Extract Preparation C tricuspidata leaves were obtainedfrom traditional Koreanmedical center (GoseongGangwon-do Korea) which were pulverized using a blender Thepulverized leaves were subjected to reflux extraction twice

using 20 times its volume of distilled water at 100∘C for8 h The extracts were filtered using Whatman filter papernumber 2 (pore size 8 120583m) concentrated by using a rotaryvacuum evaporator (EYELA Tokyo Japan) and lyophilizedusing a freezer dryer (PVTF20R IlshinbiobaseDongduchunKorea) CTe extraction and processing were performed ina good manufacturing practice (GMP) facility according toindustry standards The CTe yield was 287

22 PTP1B Inhibitory Assay Using a Protein Chip ScreeningMethod The PTP1B inhibitory activity of the tested extractswas evaluated using recombinant IR protein as a substrateThe IR was incubated at 30∘C for 1 h for autophosphoryla-tion For the screening chip the IR was spotted to a slide(Proteogen Co Chuncheon Korea) and coated overnight at4∘C A mixture containing 005 120583g PTP1B (RampD system) andCTe was used to assess CTe inhibitory activity on PTP1BWe used orthovanadate (Na

3VO4) as the positive control and

10 PEG as the negative control to evaluate the quality ofour screening The final volume of the mixture was 1 120583L Themixturewas added to the IR-coated slide and incubated for 1 hin a 30∘C incubator Afterwashing the anti-phospho-insulin-receptor antibody (used at a 1 100 dilution in dilution buffer10 bovine serum albumin and 30 glycerol in phosphatebuffered saline (PBS) pH 74 Invitrogen Carlsbad CAUSA)was added and incubated in a 30∘C incubator for 1 h Thesecondary antibody used for visualization was an anti-rabbitIgG-Cy5 (used at a 1 100 dilution in dilution buffer 10bovine serum albumin and 30 glycerol in PBS buffer pH7 Invitrogen) and incubated in a 30∘C incubator for 1 h ThePTP1B inhibitory activity of the extracts was visualized usinga microarray scanner system (GenePix 4300A MolecularDevices Sunnyvale CA USA)The data were analyzed usingGraphPad Prism4 software (GraphPad Software San DiegoCA USA)

23 3T3-L1 Cell Proliferation andDifferentiation 3T3-L1 pread-ipocytes (CL-173trade ATCCManassas VAUSA)were culturedin Dulbeccorsquos modified eagle medium (DMEM) medium(Sigma-Aldrich Saint Louis MO USA) supplemented with10 fetal bovine serum (FBS Gibco Co Carlsbad CA USA)and maintained at 37∘C with 5 CO

2 To induce differen-

tiation 3T3-L1 cells were treated with methylisobutylxan-thine dexamethasone and insulin (MDI) inductionmedium(1 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine(IBMX) and 10 120583gmL insulin in 10 DMEM medium) atapproximately 100 confluenceThe adipocytes were allowedto differentiate for 8 days in cultureThe accumulation of lipiddroplets was examined

24 Staining with Oil Red O and Quantification After 8 daysof culture to allow cell differentiation the culture mediumwas removed and the cells were fixed in 10 formalin atambient temperature for 2 h and then washed once with60 isopropanol The washed cells were stained for 10minat ambient temperature by using the Oil Red O stain (Sigma-Aldrich St Louis MO USA) which specifically reacted withlipid droplets The cells were washed with distilled waterand observed under a microscope (IX71 Olympus Tokyo

BioMed Research International 3

Japan) For quantitative measurement of lipid droplets theOil Red O stain was extracted with 100 isopropanol and theabsorbancewasmeasured at 492 nmThebackground controlwas 100 isopropanol

25 Diet-Induced Obesity (DIO) Mouse Model and Treat-ment The experimental animals were 4-week-old C57BL6mice They were divided into the normal group and obe-sitydiabetes group The animals were allowed to acclimatizefor 1 week The mice were fed a 60 high-fat diet (HFD) for12 weeks which increased the body weight These animalswere then used as the DIO mice The lean group comprisedC57BL6 mice fed a normal chow PBS was used as theplacebo treatment for 3 weeks prior to treatment with CTeAt 16 weeks the CTe treatment was conducted for 3 weeksThe CTe concentrations administered to the mice were 20and 100mgkg The effects of CTe were compared to those ofsitagliptin an antidiabetic medication and sibutramine anantiobesity medication which were administered at 10mgkgand 5mgkg respectively to the control animalsTheCTe andthe drugs were administered orally once daily

26 Measurement of Body Weight Body Fat Content andPlasma Lipids After 12 weeks on the high-fat diet micewere grouped The mice in the vehicle group were treatedwith 05 carboxymethylcellulose solution (vehicle) Themice in the experimental groups were administered 20 or100mgkg of CTe dissolved in the vehicle solution Changesin body weight were measured for 3 weeks every 2 daysby using an animal scale The animals were fasted for 24 hand were then anesthetized using ether Blood was collectedfrom the abdominal vena cava and the plasma was obtainedby centrifugation at 3000 rpm at 4∘C for 10min Afterblood sampling the inguinal fat and epididymal fat wereremoved washed with saline solution dried and weighedPlasma fatty acid levels such as triglyceride (TRIG) high-density lipoprotein- (HDL-) cholesterol and low-densitylipoprotein- (LDL-) cholesterol were measured using therespective kits purchased from Roche and by using anautomatic analyzer (Cobas C111 Roche Basel Switzerland)

27 Measurement of Serum ALT and AST Levels Plasma ala-nine aminotransferase (ALT) and aspartate aminotransferase(AST) were analyzed using commercially available test kits(Thermo Scientific Middletown VA)

28 Measurement of Glucose and Insulin Levels DIO micewere fasted for 1 day and orally administered with vehicleor CTe After 30min a glucose bolus (2 gkg dissolved insaline Sigma-Aldrich) was administered to each mouse andblood was drawn from the tail vein at 0 30 60 90 and120min after glucose administrationTheblood glucose levelswere measured using an Accu-Chek glucometer (Roche)and the insulin level was measured using an enzyme-linkedimmunosorbent assay (ELISA) kit (Abcam Cambridge UK)

29 Oral Glucose Tolerance Test DIO mice were fasted for6 h and then orally administered 20 and 100mgkg of CTedissolved in 05 CMC After 30min a glucose bolus (2 gkg

dissolved in saline Sigma-Aldrich) was administered to eachmouse Blood glucose levels were measured at indicated timepoints from tail vein bleeding by Accu-Chek glucometer(Roche) The area under the curve (AUC) was calculated foreach animal using the Trapezoid method

210 Pathscan Sandwich ELISA Assay Tissues were homog-enized in RIPA buffer (50mM Tris-HCl (pH 75) 150mMNaCl 1 NP-40 1 sodium deoxycholate 01 SDS2mM EDTA 1mM sodium orthovanadate 2mM sodiumpyrophosphate and 10 120583gmL leupeptin) and centrifuged tomake the supernatant available for sandwich ELISA assayThe assay was carried out according to the manufacturerrsquosinstructions (Cell signaling Danvers MA USA) Briefly onehundred 120583L of supernatant was added to eachwell of the anti-p-IRS-1 (panTyr) or anti-p-Akt (Ser473) coated microwellplate and then incubated for 2 h at 37∘C After being washedthe bound protein was incubated with specific antibodyfollowed by exposure to the HRP- (horseradish peroxidase-)linked secondary antibody and the TMB (tetramethylbenzi-dine) substrate Finally stop solution was added to each welland the absorbance at 450 nm was read by microplate reader(Molecular Device Sunnyvale CA USA)

211 Statistical Analysis Data are presented as themean plusmn SDfor each group Studentrsquos t-test was used to evaluate statisticalsignificance

3 Results

31 Inhibitory Effects of CTe on PTP1B Activity in the ProteinChip Screening The inhibitory potency of CTe on the PTP1Bwas evaluated with an IC

50value above 65 120583gmL (data not

shown) To identifymedicinal herbs that inhibit PTP1B activ-ity phosphorylation inhibitory reactions were performedas described in Section 2 As shown in Figure 1(a) PTP1Bpotently inhibited IR phosphorylation The CTe presenteda high inhibitory activity against PTP1B similar to that ofthe positive control Na

3VO4 The CTe contained potent

inhibitors of PTP1B and displayed a high selectivity towardsPTPs

32 CTe Prevented Lipid Accumulation in 3T3-L1 Cells Toexamine the effect of CTe on lipid accumulation differenti-ated 3T3-L1 cells were treated with 50 100 and 200120583gmL ofCTe and the lipid droplets formed in the cells were stainedwith Oil Red O stain (Figures 1(b) and 1(c)) Compared tountreated differentiated cells in which many lipid dropletswere observed CTe-treated differentiated cells presented fewlipid droplets in a concentration-dependent manner (119901 lt005 and 119901 lt 0001 resp) Taken together these resultsshowed that CTe treatment inhibited lipid accumulation in3T3-L1 cells in a dose-dependent manner

33 Effects of CTe on the BodyWeight and Body Fat Content ofDIOMice To explore the effects of CTe on glucose toleranceand further characterize the kinetics of the effects of CTe onglucose metabolism we generated DIO mice using C57BL6mice (Figure 2(a)) First we determined the effects of CTe


PEGPTP1B

CTe

++

200minus

++

100minus

++

50minus

+++minus

++

minus

minus

+

minus

minus

minus

Na3VO4

(a)

0 0 50 100 200

MDI

CTe (120583gmL)

(b)

0

20

40

60

80

100

120

Lipi

d ac

cum

ulat

ion

( o

f MD

I con

trol)

0 0 50 100 200

MDI

CTe (120583gmL)

lowastlowastlowastlowast

(c)

Figure 1 Effect of CTe on PTP1B activity and lipid droplets (a) The substrate (IR insulin receptor) was subjected to autophosphorylationfor 1 h followed by spotting on a chip slide Reaction mixture was incubated for 24 h in the presence of PTP1B (100mgmL) withsodium orthovanadate (Na

3

VO4

800 120583M) CTe (50 100 or 200 120583gmL) or 10 PEG alone The fluorescent signal corresponding to thephosphorylated IR incubated with an anti-phospho-IR followed by incubation with an anti-Cy5 antibody was measured using a microarrayscanner system (b) CTe inhibition of fat droplet formation in 3T3-L1 cells 3T3-L1 cells were stained with Oil Red O dye and examined usinga light microscope (c) Quantification of lipid accumulation in differentiated 3T3-L1 cells by measuring the absorbance of the cell extract at520 nm Statistical significance was determined by Studentrsquos 119905-test lowast119901 lt 005 and lowastlowastlowast119901 lt 0001 versus MDI treated cells

on weight gain in the DIO mouse model As shown inFigure 2(b) no significant difference was observed in bodyweight between the vehicle- and CTe-treated groups Con-sistent with previous studies mice treated with sibutramineshowed a marked decrease in body weight [28] However thebody weight of mice treated with CTe at both concentrations(20 and 100mgkg) was unchanged for up to 25 dayscompared to that of the vehicle group (119899 = 7) AdditionallyCTe treatment had no effect on the weights of the liverspleen or kidney when compared to vehicle treated-DIOmice (data not shown) Weights of the total fat (Figure 3(a))inguinal fat (Figure 3(b)) and epididymal fat (Figure 3(c))were significantly affected by the 100mgkg CTe treatment(119901 lt 005 119901 lt 001 and 119901 lt 0001 resp)

34 CTe Enhanced Phosphorylation of Signal TransductionMolecules in DIO Mice Liver In order to further confirm the

action mode of the CTe in vitro Pathscan sandwich ELISAassay was conducted PTP1B is involved in regulatory step ofsignal transduction by dephosphorylation of cellular proteintherefore CTe probably increases phosphorylation by theinhibition of PTP1B Pathscan sandwich ELISA assay analysisshowed that CTe enhanced phosphorylation of IRS-1 and Aktsignal transduction proteins in DIO mice liver (Figures 3(d)and 3(e)) (119901 lt 005 119901 lt 001 and 119901 lt 0001 resp) SimilarlyCTe treatment increased the phosphorylation of IRS-1 andAkt in 3T3-L1 cells (data not shown) These results revealedthat CTe has may be effectively contributed to the inhibitionof PTP1B

35 Effects of CTe on Plasma Lipids and Liver Enzymes Threeweeks after CTe administration we evaluated the effect ofCTe on the concentration of plasma lipids Total CHOL(Figure 4(a)) and LDL (Figure 4(b)) and HDL (Figure 4(c))


Vehicle group

Acclimatization

1 week 13 weeks

High-fat diet

16 weeks

Placebo treatment

19 weeks

CTe or PC treatment

0 weeks

Acclimatization

1 week 13 weeks

High-fat diet Placebo treatment

19 weeks0 weeks

CTe-treated group

(a)

Vehicle

lowast

lowast

lowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

3 6 9 12 15 18 21 241Days

42

44

46

48

50Bo

dy w

eigh

t (g)

CTe 100mgkgCTe 20mgkg

Sibu 5mgkg

(b)

Figure 2 Time line of the experimental procedures using the diet-induced obesity (DIO) mouse model and effects of CTe on the bodyweight of DIO mice (a) C57BL6 mice were treated daily with vehicle or 60 high fat diet (HFD) by oral gavage for 12 weeks Week 0 is thestarting point beginning on the 1st week with normal chow The mice were treated with a placebo for 3 weeks prior to CTe treatment Thevehicle group received the placebo treatment for 6 weeks without oral administration of CTe (upper panel) The HFD-fed mice were orallyadministered CTe or a positive control (PC sibutramine or sitagliptin) for 1 month (lower panel) (b) Body weights of DIOmice treated withvehicle or CTe (20 or 100mgkg) for 3 weeks Sibutramine was used as a positive control Data are presented as the mean plusmn SD for each group(119899 = 7) lowast119901 lt 005 and lowastlowastlowast119901 lt 0001 versus vehicle administered DIO mice

concentrations were not affected by CTe treatment HoweverTRIG (Figure 4(d)) concentration was lower in the 20 or100mgkg CTe-fed mice (119901 lt 001 and 119901 lt 0001 resp) Thecellular lipid accumulation in skeletal muscle has been asso-ciated with dyslipidemia and insulin resistance Our resultsshowed that TRIG content was significantly decreased inDIOmice by CTe treatment In line with these findings plasmalevels of AST were lower in CTe-fed DIOmice in comparisonwith DIO mice fed with vehicle (Figure 4(e)) (119901 lt 005)However plasma ALT levels showed no differences betweenthe two groups (Figure 4(f))

36 Effects of CTe on Glucose and Glucose Tolerance AfterHFD consumption mice presented significantly elevatedfasting blood glucose concentrations when compared withanimals that consumed the normal diet We next performedan oral glucose tolerance test (OGTT) to evaluate whetherCTe supplementation improved the glycemic control in DIOmice OGTT was performed on DIO mice exposed to asingle oral administration of CTe at 20 and 100mgkg for2 h CTe improved glucose tolerance In fact significantly

lower blood glucose levels at 30 and 60min (Figure 5(a)) aswell as a reduced AUC (Figure 5(b)) were observed after CTeadministration following glucose bolus when compared tovehicle-treated mice The sitagliptin-treated positive controlmice showed a greater decrease in blood glucose at all time-points and at 120min the blood glucose level was very lowTherefore we concluded that CTe was effective in loweringblood glucose levels These results revealed that CTe func-tions as an efficient blood glucose lowering natural productagent

37 CTe Improved Insulin Levels We further assessedwhether CTe can prevent diabetes in DIO mice C57BL6mice were fed the vehicle or a HFD containing 20 or100mgkg of CTe As shown in Figure 5(c) the level ofinsulin secreted 20min after the administration of CTe wasincreased in the 20 and 100mgkg CTe groups which were16- and 20-fold higher respectively than that of the controlgroup The level of insulin in the sitagliptin-treated positivecontrol groupwas higher than that of the CTe-treated groupsBecause CTe treatment resulted in an increase in insulin in


20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)

lowastlowastlowast daggerdaggerdagger

(a)

20 100CTe (mgkg)

Lean Vehicle

lowastlowast daggerdagger

0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle

lowastlowastlowast daggerdagger

0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)

Figure 3 Effects of CTe on fat mass and insulin signaling in DIO mice The HFD-fed mice were orally administered vehicle or CTe (20 or100mgkg) for 3 weeks At 28 h after the final CTe treatment fat was collected from DIO mice (a) Total fat weight (b) Inguinal fat weight(c) Epididymal fat weight (d) p-IRS-1 and (e) p-Akt detection was estimated by Pathscan sandwich ELISA assay Data are presented as themean plusmn SD for each group (119899 = 7) Statistical significance was determined by Studentrsquos t-test lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 versus lean micedagger119901 lt 005 daggerdagger119901 lt 001 and daggerdaggerdagger119901 lt 0001 versus vehicle administered DIO mice


Lean VehicleCTe (mgkg)

100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)

lowastlowastlowastdagger

(e)


100200

20

40

ALT

(L)

(f)

Figure 4 The HFD-fed mice were orally administered CTe (20 or 100mgkg) for 3 weeks Blood was collected from the abdominal vein (a)Levels of total cholesterol (CHOL) (b) low-density lipoprotein (LDL) (c) high-density lipoprotein (HDL) and (d) triglycerides (TRIG) wereanalyzed in triplicate using a serum biochemical analyzer Liver tissue was collected for toxicity test (e) Aspartate aminotransferase (AST)and (f) alanine aminotransferase (ALT) levels were monitored by clinical chemistry analyzer Data are presented as the mean plusmn SD for eachgroup (119899 = 7) Statistical significance was determined by Studentrsquos t-test lowastlowastlowast119901 lt 0001 versus lean mice dagger119901 lt 005 and daggerdaggerdagger119901 lt 0001 versusvehicle administered DIO mice


LeanVehicle CTe 100mgkg

CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)

0 30 60 90 120minus30Time after glucose loading (min)


lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)

20 40 60 80 100 1200Time after glucose loading (min)

(c)

Figure 5 Effect of CTe on blood glucose and insulin levels in DIO mice The HFD-fed mice were orally administered CTe (20 or 100mgkg)for 3 weeks (a) The effect of CTe on plasma glucose levels in DIO mice (b) The area under the plasma glucose concentration-time curve for2 h (AUC

0ndash2 h) in an OGTT (c)The effect of CTe on plasma insulin in DIOmice Sitagliptin was used as a positive control Data are presentedas the mean plusmn SD for each group (119899 = 7) Statistical significance was determined by Studentrsquos t-test lowastlowastlowast119901 lt 0001 versus vehicle administeredDIO mice

the DIO mouse model we believe that CTe treatment couldprevent diabetes

4 Discussion

Obesity a metabolic disorder concomitant with diabetes isa serious challenge that needs to be overcome in the agingsociety The recent trend of drug development is to identifyplant-derived drugs effective in controlling both diabetesand obesity [7] PTP1B is a well-established antidiabetes

and antiobesity therapeutic target [24] PTP1B plays a majorrole in regulating insulin signaling Normally insulin causesphosphorylation events through IR autophosphorylationwhich enhances IR kinase activity followed by activation ofphosphatidylinositide-3-kinase (PI3K) and subsequent Aktphosphorylation cascade which plays a central role in thecontrol of cell growth survival and metabolism [29] Micelacking the Ptp1b gene presented improved insulin sensitivitywith increased tyrosine phosphorylation of IR and did notdevelop type II diabetes or obesity PTP1B deletion prevents


myocardial anomalies in HFD-induced obesity [30] Micewith whole body PTP1B deletion were protected againstthe development of obesity and diabetes PTP1B inhibitionin peripheral tissues has been useful for the treatment ofmetabolic syndrome and reduction of cardiovascular risks inaddition to diabetes [31] PTP1B-directed antisense oligonu-cleotides are already tested in phase II clinical trials [32]These studies indicate that PTP1B inhibitors may be promis-ing candidates for the development of novel antidiabetic andantiobesity drugs Natural products garnered the attentionfor the long-term treatment of metabolic diseases becausethey have a multipronged mode of action and contain mul-tifunctional ingredients [33] Therefore there is a significantinterest in natural products that inhibit PTP1B activity fortheir potential use in the treatment of obesity and diabetes

In this study we demonstrated that the oral adminis-tration of CTe induces protective effects against obesity anddiabetic induction in the DIO mouse model To obtain anatural substance containing active ingredients we selectedcrude extracts from natural sources that have a potentialinhibitory effect on PTP1B activity by monitoring their effecton protein phosphorylation levels through chip screeningAmong the examined hydrophilic herb extracts CTe hada strong inhibitory capacity towards PTP1B activity in thescreening platform (Figure 1(a)) We also elucidated theantiobesity effects of CTe on fat accumulation in 3T3-L1adipocytes by staining with Oil Red O (Figures 1(b) and1(c)) Measurement of the blood lipid profile after oraladministration of CTe showed that fat mass and TRIG weresignificantly decreased (Figures 3 and 4(d)) in DIO miceAlthough known natural products with PTP1B inhibitoryactivity have been isolated and identified fromvarious naturalresources many reagents are not effective and present poorbioavailability [24] Our current results indicate that CTe is apotential natural product PTP1B inhibitor for the treatmentof obesity and diabetes risk factors

Some reagents showed good properties but compared tothe existing antidiabetic agents such as rosiglitazone met-formin and pioglitazone oral PTP1B inhibitors are difficultto design [34] Long-term administration of nonspecific T-cell protein-tyrosine phosphatase (TC-PTP) inhibitors canbe a problem New antibody-based treatment strategies arebeing developed for diabetes and obesity [35] The recentlydeveloped shRNA PTP1B inhibitor has yielded satisfactoryresults [7 36]However studies on the development of PTP1Binhibitors are still in their early stages and the development ofa safe oral substance is required

Synthetic PTP1B inhibitors that are currently in use forthe treatment of obesity have limitations because of theirside effects Moreover their clinical application is still limitedbecause the stability and the dosage efficacy of syntheticinhibitors have not been clearly examined Therefore therehas been a growing interest in natural substances CTe isan appropriate oral medication because of its high solubilityAdditionally it exhibits good activity towards PTPs in vitrowithout hepatic toxicity C tricuspidata is a well-knownnatural product with a variety of therapeutic effects Thechemical constitution and the pharmacological effects such asanti-inflammatory and antiproliferative action antioxidant

activities and apoptosis induction of C tricuspidata havebeen demonstrated [37ndash40] Similarly Kim et al reportedthat the ethanol extract of C tricuspidata exhibited aninhibitory effect on lipolysis activity [25] These results showthat C tricuspidata is a promising antiobesity agent Severalstudies reported the antiobesity antidiabetic and antihyper-glycemic effects of various natural materials [41ndash43] Meta-analysis studies have shown that supplement of naturalproduct in human also helps improvement on glucose leveland plasma lipid profile [44 45] The focus of many recentstudies on obesity and diabetes is to determine the efficacyof natural compounds with the dual function of increasingserum insulin levels and decreasing glucose levels Addi-tionally we found that CTe treatment effectively decreasedblood glucose levels and increased insulin (Figure 5) Theadvantages of drugs derived from natural substance such asC tricuspidata include fewer side effects cost-effectivenessand the possibility of oral administration CTe can be used asan alternative to synthetic drugs for the treatment of diabetesThese studies showed that hydrophilic extracts containingpotent PTP1B inhibitors may be promising reagents for thedevelopment of novel antidiabetic and antiobesity healthenhancing drugs Further studies should be performed toisolate the single active compound from CTe that is effectivefor diabetes treatment

Conflict of Interests

None of the authors has any conflict of interests in thegeneration or publication of the data in this paper

Acknowledgments

This work was supported by the Chuncheon BioindustryFoundation Korea and the National Research Foundationof Korea (NRF) Grant funded by the Korean Government(MSIP) (no NRF-2011-0030086)

References

[1] O H Requejo and M C R Rodriguez ldquoNutrition and cancerrdquoNutricion Hospitalaria vol 32 supplement 1 pp 67ndash72 2015

[2] V Kotsis S Stabouli S Papakatsika Z Rizos and G ParatildquoMechanisms of obesity-induced hypertensionrdquo HypertensionResearch vol 33 no 5 pp 386ndash393 2010

[3] C J Lavie R V Milani and H O Ventura ldquoObesity andcardiovascular disease risk factor paradox and impact ofweight lossrdquo Journal of the American College of Cardiology vol53 no 21 pp 1925ndash1932 2009

[4] N Toda K Ayajiki and TOkamura ldquoObesity-induced cerebralhypoperfusion derived from endothelial dysfunction one ofthe risk factors for Alzheimerrsquos diseaserdquo Current AlzheimerResearch vol 11 no 8 pp 733ndash744 2014

[5] J Kaur ldquoA comprehensive review on metabolic syndromerdquoCardiology Research and Practice vol 2014 Article ID 94316221 pages 2014

[6] J M Zabolotny K K Bence-Hanulec A Stricker-Krongradet al ldquoPTP1B regulates leptin signal transduction in vivordquoDevelopmental Cell vol 2 no 4 pp 489ndash495 2002


[7] E Panzhinskiy J Ren and S Nair ldquoPharmacological inhibitionof protein tyrosine phosphatase 1B a promising strategy forthe treatment of obesity and type 2 diabetes mellitusrdquo CurrentMedicinal Chemistry vol 20 no 21 pp 2609ndash2625 2013

[8] T O Johnson J Ermolieff andM R Jirousek ldquoProtein tyrosinephosphatase 1B inhibitors for diabetesrdquo Nature Reviews DrugDiscovery vol 1 no 9 pp 696ndash709 2002

[9] I Nieto-Vazquez S Fernandez-Veledo C de Alvaro C MRondinone A M Valverde and M Lorenzo ldquoProtein-tyrosinephosphatase 1B-deficient myocytes show increased insulin sen-sitivity and protection against tumor necrosis factor-120572-inducedinsulin resistancerdquo Diabetes vol 56 no 2 pp 404ndash413 2007

[10] A Gonzalez-Rodriguez J E Clampit O Escribano M BenitoC M Rondinone and A M Valverde ldquoDevelopmental switchfrom prolonged insulin action to increased insulin sensitiv-ity in protein tyrosine phosphatase 1B-deficient hepatocytesrdquoEndocrinology vol 148 no 2 pp 594ndash608 2007

[11] RDi Paola L Frittitta GMiscio et al ldquoA variation in 31015840 UTRofhPTP1B increases specific gene expression and associates withinsulin resistancerdquo The American Journal of Human Geneticsvol 70 no 3 pp 806ndash812 2002

[12] A K Tamrakar C K Maurya and A K Rai ldquoPTP1B inhibitorsfor type 2 diabetes treatment a patent review (2011ndash2014)rdquoExpert Opinion on Therapeutic Patents vol 24 no 10 pp 1101ndash1115 2014

[13] C D Egan ldquoAddressing use of herbal medicine in the primarycare settingrdquo Journal of the American Academy of Nurse Practi-tioners vol 14 no 4 pp 166ndash171 2002

[14] R N Nugara M Inafuku K Takara H Iwasaki and H OkuldquoPteryxin a coumarin in Peucedanum japonicumThunb leavesexerts antiobesity activity through modulation of adipogenicgene networkrdquo Nutrition vol 30 no 10 pp 1177ndash1184 2014

[15] R M Perez Gutierrez D Madrigales Ahuatzi C HorcacitasMdel E Garcia Baez T Cruz Victoria and J M Mota-FloresldquoAmeliorative effect of hexane extract of Phalaris canariensison high fat diet-induced obese and streptozotocin-induceddiabetic micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2014 Article ID 145901 13 pages 2014

[16] S O YangH R Park E S Sohn et al ldquoClassification of ginsengberry (Panax ginseng CA MEYER) extract using 1H NMRspectroscopy and its inhibition of lipid accumulation in 3 T3-L1 cellsrdquo BMCComplementary and AlternativeMedicine vol 14article 455 2014

[17] E M Birru M Abdelwuhab and Z Shewamene ldquoEffect ofhydroalcoholic leaves extract of Indigofera spicata Forssk onblood glucose level of normal glucose loaded and diabeticrodentsrdquo BMC Complementary and Alternative Medicine vol15 article 321 2015

[18] W H El-Tantawy N D Soliman D El-naggar and AShafei ldquoInvestigation of antidiabetic action ofAntidesma buniusextract in type 1 diabetesrdquo Archives of Physiology and Biochem-istry vol 121 no 3 pp 116ndash122 2015

[19] M Kazemian M Abad M R Haeri M Ebrahimi and RHeidari ldquoAnti-diabetic effect of Capparis spinosa L root extractin diabetic ratsrdquo Avicenna Journal of Phytomedicine vol 5 no4 pp 325ndash332 2015

[20] A C Pereira A B D Pereira C C LMoreira et al ldquoHancorniaspeciosa Gomes (Apocynaceae) as a potential anti-diabeticdrugrdquo Journal of Ethnopharmacology vol 161 pp 30ndash35 2015

[21] J Li L Gao F Meng et al ldquoPTP1B inhibitors from stems ofAngelica keiskei (Ashitaba)rdquo Bioorganic amp Medicinal ChemistryLetters vol 25 no 10 pp 2028ndash2032 2015

[22] Z Q Liu T Liu C Chen et al ldquoFumosorinone a novelPTP1B inhibitor activates insulin signaling in insulin-resistanceHepG2 cells and shows anti-diabetic effect in diabetic KKAymicerdquo Toxicology and Applied Pharmacology vol 285 no 1 pp61ndash70 2015

[23] A Pitschmann M Zehl A G Atanasov V M Dirsch E Heissand S Glasl ldquoWalnut leaf extract inhibits PTP1B and enhancesglucose-uptake in vitrordquo Journal of Ethnopharmacology vol 152no 3 pp 599ndash602 2014

[24] C-S Jiang L-F Liang and Y-W Guo ldquoNatural productspossessing protein tyrosine phosphatase 1B (PTP1B) inhibitoryactivity found in the last decadesrdquo Acta Pharmacologica Sinicavol 33 no 10 pp 1217ndash1245 2012

[25] Y S Kim Y Lee J Kim et al ldquoInhibitory activities of Cudraniatricuspidata leaves on pancreatic lipase in vitro and lipolysis invivordquo Evidence-Based Complementary andAlternativeMedicinevol 2012 Article ID 878365 8 pages 2012

[26] E-G Lee S-L Lee H-J Chae S J Park Y C Lee and W-HYoo ldquoEthyl acetate fraction from cudrania tricuspidata inhibtsIL-1120573-induced rheumatoid synovial fbroblast proliferation andMMPs COX-2 and PGE2 productionrdquo Biological Research vol43 no 2 pp 225ndash231 2010

[27] G Yang K Lee M Lee I Ham and H-Y Choi ldquoInhibition oflipopolysaccharide-induced nitric oxide and prostaglandin E2production by chloroform fraction of Cudrania tricuspidata inRAW 2647 macrophagesrdquo BMC Complementary and Alterna-tive Medicine vol 12 article 250 2012

[28] G Hansen J Jelsing and N Vrang ldquoEffects of liraglutideand sibutramine on food intake palatability body weight andglucose tolerance in the gubra DIO-ratsrdquo Acta PharmacologicaSinica vol 33 no 2 pp 194ndash200 2012

[29] S Guo ldquoInsulin signaling resistance and metabolic syndromeinsights from mouse models into disease mechanismsrdquo Journalof Endocrinology vol 220 no 2 pp T1ndashT23 2014

[30] M R Kandadi E Panzhinskiy N D Roe S Nair D Hu andA Sun ldquoDeletion of protein tyrosine phosphatase 1B rescuesagainst myocardial anomalies in high fat diet-induced obesityrole of AMPK-dependent autophagyrdquo Biochimica et BiophysicaActa (BBA)mdashMolecular Basis of Disease vol 1852 no 2 pp299ndash309 2015

[31] M Delibegovic D Zimmer C Kauffman et al ldquoLiver-specificdeletion of protein-tyrosine phosphatase 1B (PTP1B) improvesmetabolic syndrome and attenuates diet-induced endoplasmicreticulum stressrdquo Diabetes vol 58 no 3 pp 590ndash599 2009

[32] H Cho ldquoProtein tyrosine phosphatase 1B (PTP1B) and obesityrdquoVitamins amp Hormones vol 91 pp 405ndash424 2013

[33] I Ikeda ldquoMultifunctional effects of green tea catechins onprevention of the metabolic syndromerdquo Asia Pacific Journal ofClinical Nutrition vol 17 supplement 1 pp 273ndash274 2008

[34] S-X Sun X-B LiW-B Liu et al ldquoDesign synthesis biologicalactivity and molecular dynamics studies of specific proteintyrosine phosphatase 1B inhibitors over SHP-2rdquo InternationalJournal of Molecular Sciences vol 14 no 6 pp 12661ndash126742013

[35] I N Foltz S Hu C King et al ldquoTreating diabetes andobesity with an FGF21-mimetic antibody activating the120573KlothoFGFR1c receptor complexrdquo Science TranslationalMedicine vol 4 no 162 Article ID 162ra153 2012

[36] S Vakili S S S Ebrahimi A Sadeghi et al ldquoHydrodynamic-based delivery of PTP1B shRNA reduces plasma glucose levelsin diabetic micerdquo Molecular Medicine Reports vol 7 no 1 pp211ndash216 2013


[37] K H Park Y-D Park J-M Han et al ldquoAnti-atheroscleroticand anti-inflammatory activities of catecholic xanthones andflavonoids isolated from Cudrania tricuspidatardquo Bioorganic andMedicinal Chemistry Letters vol 16 no 21 pp 5580ndash5583 2006

[38] T-J Kim H-J Han Y Lim et al ldquoAntiproliferative action ofcudraflavone B isolated from Cudrania tricuspidata throughthe downregulation of pRb phosphorylation in aortic smoothmuscle cell proliferation signalingrdquo Journal of CardiovascularPharmacology vol 53 no 4 pp 341ndash348 2009

[39] Y J Lee S Kim S J Lee I Ham and W K Whang ldquoAntiox-idant activities of new flavonoids from Cudrania tricuspidataroot barkrdquo Archives of Pharmacal Research vol 32 no 2 pp195ndash200 2009

[40] Y-H Rho B-W Lee K-H Park and Y-S Bae ldquoCudrafla-vanone A purified from Cudrania tricuspidata induces apop-totic cell death of human leukemia U937 cells at least in partthrough the inhibition of DNA topoisomerase I and proteinkinase C activityrdquo Anti-Cancer Drugs vol 18 no 9 pp 1023ndash1028 2007

[41] H-S Wu D-F Zhu C-X Zhou et al ldquoInsulin sensitizingactivity of ethyl acetate fraction of Acorus calamus L in vitroand in vivordquo Journal of Ethnopharmacology vol 123 no 2 pp288ndash292 2009

[42] W-K Lee S-T Kao I-M Liu and J-T Cheng ldquoIncrease ofinsulin secretion by ginsenoside Rh2 to lower plasma glucosein Wistar ratsrdquo Clinical and Experimental Pharmacology andPhysiology vol 33 no 1-2 pp 27ndash32 2006

[43] T Anwer M Sharma K K Pillai andM Iqbal ldquoEffect ofWith-ania somnifera on insulin sensitivity in non-insulin-dependentdiabetes mellitus ratsrdquo Basic and Clinical Pharmacology andToxicology vol 102 no 6 pp 498ndash503 2008

[44] C Serban A Sahebkar D Antal S Ursoniu and M BanachldquoEffects of supplementation with green tea catechins on plasmaC-reactive protein concentrations a systematic review andmeta-analysis of randomized controlled trialsrdquo Nutrition vol31 no 9 pp 1061ndash1071 2015

[45] S Ursoniu A Sahebkar M C Serban and M Banach ldquoLipidprofile and glucose changes after supplementation with astax-anthin a systematic review and meta-analysis of randomizedcontrolled trialsrdquo Archives of Medical Science vol 11 no 2 pp253ndash266 2015


insulin than those of wild-type animals [9 10] Additionallythese animals present a low body fat content and highresistance to obesity from excessive nutritional intake PTP1Boverexpression is associated with an increase in insulin levels[11] PTP1B is considered as an attractive target for the devel-opment of new treatments for obesity and related metabolicsyndromes Although synthetic PTP1B inhibitors such asthiazolidinediones phosphorus-containing phosphotyrosylmimetics dephostatin and bidentate have been developed[12] little is known about the PTP1B inhibitory activity ofdrugs derived from natural resources

The increasing incidence of obesity and diabetes is aserious socioeconomic problem in many countries To over-come this problem multinational pharmaceutical companiesare aggressively performing studies to develop competitivedrugs Natural product-based therapeutic approaches pro-vide a medicinal herb or fruitful source for safe effectiveand less expensive therapeutic drugs In addition naturalproducts have been attracting attention because they presenta multipronged mechanism of action and contain manyhydrophilic active ingredients [13] Many herbal extractshave been used for the treatment of obesity and dia-betes Extracts derived from Peucedanum japonicum ThunbPhalaris canariensis and Panax ginseng have been used totreat obesity [14ndash16] Animal models and clinical studiesshowed that plant extracts from Apocynaceae Capparisspinosa Indigofera spicata Forssk andAntidesma bunius pos-sess antidiabetic effects [17ndash20] Therefore natural productsare considered as promising sources for PTP1B inhibitorsSince the discovery of flavonoids as natural PTP1B inhibitorsvarious natural substances with an inhibitory effect on PTPactivity have been reported [21ndash23] Until recently approx-imately 300 PTP1B inhibitors have been identified fromnatural resources [24] However further studies are needed toevaluate the effects of natural products with PTP1B inhibitoryactivity against diabetes and obesity

Cudrania (C) tricuspidata a small tree of the Moraceaefamily is widely used as an ingredient in oriental medicineSeveral reports demonstrated that C tricuspidata extract(CTe) has various effects including inhibition of pancre-atic lipase lipopolysaccharide-induced nitric oxide produc-tion prostaglandin E2 production in macrophages IL-1120573-induced rheumatoid synovial fibroblast proliferation andmatrix metalloproteinase production [25ndash27] In this studywe selected a CTe from among many plant extracts usinga chip screening platform that allowed us to determinePTP1B inhibitory effects of various extracts using IR as thesubstrate Furthermore we investigated the effects of CTe onmetabolic disturbances associated with obesity and diabetesusing a high-fat diet-induced obesity (DIO) mouse modelOur results provide a basis for developing novel antiobesityand antidiabetic medications and functional foods

2 Materials and Methods

21 Extract Preparation C tricuspidata leaves were obtainedfrom traditional Koreanmedical center (GoseongGangwon-do Korea) which were pulverized using a blender Thepulverized leaves were subjected to reflux extraction twice

using 20 times its volume of distilled water at 100∘C for8 h The extracts were filtered using Whatman filter papernumber 2 (pore size 8 120583m) concentrated by using a rotaryvacuum evaporator (EYELA Tokyo Japan) and lyophilizedusing a freezer dryer (PVTF20R IlshinbiobaseDongduchunKorea) CTe extraction and processing were performed ina good manufacturing practice (GMP) facility according toindustry standards The CTe yield was 287

22 PTP1B Inhibitory Assay Using a Protein Chip ScreeningMethod The PTP1B inhibitory activity of the tested extractswas evaluated using recombinant IR protein as a substrateThe IR was incubated at 30∘C for 1 h for autophosphoryla-tion For the screening chip the IR was spotted to a slide(Proteogen Co Chuncheon Korea) and coated overnight at4∘C A mixture containing 005 120583g PTP1B (RampD system) andCTe was used to assess CTe inhibitory activity on PTP1BWe used orthovanadate (Na

3VO4) as the positive control and

10 PEG as the negative control to evaluate the quality ofour screening The final volume of the mixture was 1 120583L Themixturewas added to the IR-coated slide and incubated for 1 hin a 30∘C incubator Afterwashing the anti-phospho-insulin-receptor antibody (used at a 1 100 dilution in dilution buffer10 bovine serum albumin and 30 glycerol in phosphatebuffered saline (PBS) pH 74 Invitrogen Carlsbad CAUSA)was added and incubated in a 30∘C incubator for 1 h Thesecondary antibody used for visualization was an anti-rabbitIgG-Cy5 (used at a 1 100 dilution in dilution buffer 10bovine serum albumin and 30 glycerol in PBS buffer pH7 Invitrogen) and incubated in a 30∘C incubator for 1 h ThePTP1B inhibitory activity of the extracts was visualized usinga microarray scanner system (GenePix 4300A MolecularDevices Sunnyvale CA USA)The data were analyzed usingGraphPad Prism4 software (GraphPad Software San DiegoCA USA)

23 3T3-L1 Cell Proliferation andDifferentiation 3T3-L1 pread-ipocytes (CL-173trade ATCCManassas VAUSA)were culturedin Dulbeccorsquos modified eagle medium (DMEM) medium(Sigma-Aldrich Saint Louis MO USA) supplemented with10 fetal bovine serum (FBS Gibco Co Carlsbad CA USA)and maintained at 37∘C with 5 CO

2 To induce differen-

tiation 3T3-L1 cells were treated with methylisobutylxan-thine dexamethasone and insulin (MDI) inductionmedium(1 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine(IBMX) and 10 120583gmL insulin in 10 DMEM medium) atapproximately 100 confluenceThe adipocytes were allowedto differentiate for 8 days in cultureThe accumulation of lipiddroplets was examined

24 Staining with Oil Red O and Quantification After 8 daysof culture to allow cell differentiation the culture mediumwas removed and the cells were fixed in 10 formalin atambient temperature for 2 h and then washed once with60 isopropanol The washed cells were stained for 10minat ambient temperature by using the Oil Red O stain (Sigma-Aldrich St Louis MO USA) which specifically reacted withlipid droplets The cells were washed with distilled waterand observed under a microscope (IX71 Olympus Tokyo











3 Results









PEGPTP1B

CTe

++

200minus

++

100minus

++

50minus

+++minus

++

minus

minus

+

minus

minus

minus

Na3VO4

(a)

0 0 50 100 200

MDI

CTe (120583gmL)

(b)

0

20

40

60

80

100

120

Lipi

d ac

cum

ulat

ion

( o

f MD

I con

trol)

0 0 50 100 200

MDI

CTe (120583gmL)


(c)


3

VO4







Vehicle group

Acclimatization

1 week 13 weeks

High-fat diet

16 weeks

Placebo treatment

19 weeks

CTe or PC treatment

0 weeks

Acclimatization

1 week 13 weeks


19 weeks0 weeks

CTe-treated group

(a)

Vehicle

lowast

lowast

lowast




lowastlowastlowast

lowastlowastlowast

3 6 9 12 15 18 21 241Days

42

44

46

48

50Bo

dy w

eigh

t (g)


Sibu 5mgkg

(b)







20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)


(a)

20 100CTe (mgkg)

Lean Vehicle


0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)




100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References


























































3 Results









PEGPTP1B

CTe

++

200minus

++

100minus

++

50minus

+++minus

++

minus

minus

+

minus

minus

minus

Na3VO4

(a)

0 0 50 100 200

MDI

CTe (120583gmL)

(b)

0

20

40

60

80

100

120

Lipi

d ac

cum

ulat

ion

( o

f MD

I con

trol)

0 0 50 100 200

MDI

CTe (120583gmL)


(c)


3

VO4







Vehicle group

Acclimatization

1 week 13 weeks

High-fat diet

16 weeks

Placebo treatment

19 weeks

CTe or PC treatment

0 weeks

Acclimatization

1 week 13 weeks


19 weeks0 weeks

CTe-treated group

(a)

Vehicle

lowast

lowast

lowast




lowastlowastlowast

lowastlowastlowast

3 6 9 12 15 18 21 241Days

42

44

46

48

50Bo

dy w

eigh

t (g)


Sibu 5mgkg

(b)







20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)


(a)

20 100CTe (mgkg)

Lean Vehicle


0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)




100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References

















































PEGPTP1B

CTe

++

200minus

++

100minus

++

50minus

+++minus

++

minus

minus

+

minus

minus

minus

Na3VO4

(a)

0 0 50 100 200

MDI

CTe (120583gmL)

(b)

0

20

40

60

80

100

120

Lipi

d ac

cum

ulat

ion

( o

f MD

I con

trol)

0 0 50 100 200

MDI

CTe (120583gmL)


(c)


3

VO4







Vehicle group

Acclimatization

1 week 13 weeks

High-fat diet

16 weeks

Placebo treatment

19 weeks

CTe or PC treatment

0 weeks

Acclimatization

1 week 13 weeks


19 weeks0 weeks

CTe-treated group

(a)

Vehicle

lowast

lowast

lowast




lowastlowastlowast

lowastlowastlowast

3 6 9 12 15 18 21 241Days

42

44

46

48

50Bo

dy w

eigh

t (g)


Sibu 5mgkg

(b)







20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)


(a)

20 100CTe (mgkg)

Lean Vehicle


0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)




100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References

















































Vehicle group

Acclimatization

1 week 13 weeks

High-fat diet

16 weeks

Placebo treatment

19 weeks

CTe or PC treatment

0 weeks

Acclimatization

1 week 13 weeks


19 weeks0 weeks

CTe-treated group

(a)

Vehicle

lowast

lowast

lowast




lowastlowastlowast

lowastlowastlowast

3 6 9 12 15 18 21 241Days

42

44

46

48

50Bo

dy w

eigh

t (g)


Sibu 5mgkg

(b)







20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)


(a)

20 100CTe (mgkg)

Lean Vehicle


0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)




100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References

















































20 100CTe (mgkg)

Lean Vehicle0

1

2

3

4

5To

tal f

at w

eigh

t (g)


(a)

20 100CTe (mgkg)

Lean Vehicle


0

05

1

15

2

Ingu

inal

fat w

eigh

t (g)

dagger

(b)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

Epid

idym

al fa

t wei

ght (

g)

(c)

20 100CTe (mgkg)

Lean Vehicle


0

1

2

3

4

5

6

p-IR

S-1

(fold

chan

ge)

(d)

20 100CTe (mgkg)

Lean Vehicle


dagger

0

1

2

3

4

p-A

kt (f

old

chan

ge)

(e)




100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References


















































100200

100

200

300

Tota

l CH

OL

(mg

dL)

lowastlowastlowast

(a)


100200

20

40

60

LDL

(mg

dL)

lowastlowastlowast

(b)


100200

50

100

150

200

HD

L (m

gdL

)

lowastlowastlowast

(c)


100200

50

100

150

TRIG

(mg

dL)

lowastlowastlowast

dagger

daggerdaggerdagger

(d)


100200

20

40

60

80

100

120

140

AST

(UL

)


(e)


100200

20

40

ALT

(L)

(f)




CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References


















































CTe 20mgkg

Sita 10mgkg

0

100

200

300

400

500

600Bl

ood

gluc

ose (

mg

dL)



lowastlowastlowast

lowastlowastlowast

(a)



lowastlowastlowast

100200

200

400

600

Plas

ma g

luco

seAU

C0

2hndash

800

1000

Sita

(b)


CTe 20mgkg

Sita 10mgkg

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

Plas

ma i

nsul

in (n

gm

L)


(c)




4 Discussion











Acknowledgments


References
























































Acknowledgments


References
















































antiobesity and antidiabetes effects of a cudrania tricuspidata … · 2016-05-17 ·...

Documents