Research Article Steviol Glycosides Modulate Glucose...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 348169 11 pageshttpdxdoiorg1011552013348169

Research ArticleSteviol Glycosides Modulate Glucose Transport inDifferent Cell Types

Benedetta Rizzo1 Laura Zambonin2 Cristina Angeloni1 Emanuela Leoncini1

Francesco Vieceli Dalla Sega2 Cecilia Prata2 Diana Fiorentini2 and Silvana Hrelia1

1 Department for Life Quality Studies Alma Mater Studiorum University of Bologna Corso Augusto 237 47921 Rimini Italy2 Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Via Irnerio 48 40126 Bologna Italy

Correspondence should be addressed to Laura Zambonin laurazamboninuniboit

Received 25 July 2013 Revised 27 September 2013 Accepted 30 September 2013

Academic Editor Tullia Maraldi

Copyright copy 2013 Benedetta Rizzo et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Extracts from Stevia rebaudiana Bertoni a plant native to Central and South America have been used as a sweetener since ancienttimes Currently Stevia extracts are largely used as a noncaloric high-potency biosweetener alternative to sugar due to the growingincidence of type 2 diabetes mellitus obesity andmetabolic disorders worldwide Despite the large number of studies on Stevia andsteviol glycosides in vivo little is reported concerning the cellular andmolecularmechanisms underpinning the beneficial effects onhuman healthThe effect of four commercial Stevia extracts on glucose transport activity was evaluated inHL-60 human leukaemiaand in SH-SY5Y human neuroblastoma cellsThe extracts were able to enhance glucose uptake in both cellular lines as efficiently asinsulin Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3KAkt pathway sincetreatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt Furthermore Stevia extracts wereable to revert the effect of the reduction of glucose uptake caused by methylglyoxal an inhibitor of the insulin receptorPI3KAktpathway These results corroborate the hypothesis that Stevia extracts could mimic insulin effects modulating PI3KAkt pathway

1 Introduction

Stevia rebaudiana Bertoni is a weak perennial shrub belong-ing to Asteraceae (Compositae) family native to subtropicalregions of Brazil and Paraguay Its leaves have been used as asweetener since ancient times and for many other medicinalpurposes in Latin America and the Orient for centuries[1 2] The ldquosweet herbrdquo has gained increasing interest fromnutritional researchers and commercial area in the last yearsdue to the growing need to find new natural calorie-freesweeteners alternative to sugar Indeed in both industrializedand developing countries the incidence of type 2 diabetesmellitus and obesity is sharply increasing as a result ofdietary behaviours reduced physical activities and ageingThese metabolic disorders have become major public healthproblems worldwide [3 4]

Glycemic control is fundamental to the management ofdiabetes since it is associated with significantly decreased

rates of retinopathy nephropathy neuropathy and cardiovas-cular disease the most common cause of death in diabeticpatients The effort to achieve near-normoglycemia throughthe key strategy of glycemic control includes recommenda-tions for prevention and control of diabetes for examplemonitoring carbohydrate intake and limiting the consump-tion of sugar-sweetened beverages [5]

Stevia leaves and extracts are natural noncaloric sweeten-ers that can substitute sucrose The main sweet componentsin leaves approximately 200ndash400 times sweeter than sucroseas shown by organoleptic tests [1 6] are stevioside andrebaudioside A steviol glycosides differing only by oneglucosemoiety Stevioside is formed by 3molecules of glucoseand one molecule of the aglycone steviol a diterpenic car-boxylic alcohol rebaudioside A holds one additional glucosemolecule [7] (Figure 1)

Steviol glycosides have been recently authorised as com-mercial sweeteners US Food and Drug Administration

2 Oxidative Medicine and Cellular Longevity

O

O O

O

O O

O

CH3 CH2

OH

OH

OH

OH

OH

OHH3C

HO

HO HO

HO

HO

H

H

H

H

H

(a)

HO

O

O O

OO

O O

O

CH3 CH2

OH

OH

OH

OH

OH

H3CHO

HO HO

HO

H

H

H

H

H

O

OH

OH

OH

HO

H

(b)

Figure 1 Chemical structure of stevioside (a) and rebaudioside A (b)

(FDA) has allowed the use of Stevia extracts containing notless than 95 total steviol glycosides Recently the EuropeanFood Safety Authority (EFSA) approved the use of steviolglycosides as food additive [8 9] Considering the availabletoxicity data (in vitro and in vivo animal studies and somehuman tolerance studies) steviol glycosides are considerednot carcinogenic genotoxic or associated with any repro-ductivedevelopmental toxicity Joint Expert Committee onFood Additives (JECFA) established an accepted daily intake(ADI) for steviol glycosides (expressed as steviol equivalents)of 4mgkg bwday [10 11]

Besides sweetness steviol glycosides in particular stevio-side have been shown to possess beneficial effects on humanhealth [7 12 13] Briefly pharmacological activities and ther-apeutical benefits include antitumour and anticancer anti-inflammatory antihyperglycemic antihypertensive antidiar-rheal immunomodulatory diuretic and enzyme inhibitoryactions Stevia has also been used to help control weight inobese subjects [14] moreover antioxidant properties havebeen described [15 16] Stevioside rebaudioside A and theirmetabolite steviol have been mostly investigated in in vivoanimal studies and at a lesser extent in humans Resultssuggest that stevioside and related compounds affect plasmaglucose modulating insulin secretion and sensitivity whichincrease glucose removal from the plasma [17 18] In addi-tion it seems likely that stevioside inhibits gluconeogenesisin the liver of diabetic rats [19 20] These antihyperglycemicinsulinotropic and glucagonostatic effects especially forrebaudioside A are largely plasma glucose level dependentrequiring high glucose levels [21 22] Despite the large num-ber of studies on Stevia and steviol glycosides very little is

reported concerning the cellular and molecular mechanismsunderpinning these effects

In the present study we examined the role of steviolglycosides on cellular glucose transport in cultured cellsGlucose is a polar molecule and requires specific carrierproteins located in the plasma membrane to cross thelipid bilayer and enter the cell Glucose is transported intothe cells through two different types of membrane associ-ated carrier proteins the Na+-coupled glucose transporters(SGLT) and the facilitative glucose transporters (GLUT)The human GLUT family is integral membrane proteinswidely distributed in probably all mammalian cells thatregulate the movement of glucose between extracellular andintracellular compartments maintaining a constant supply ofglucose available for metabolism [23] To date GLUT familyis constituted by 14 distinct isoforms differently distributedin human tissues [23 24] GLUT1 is considered responsiblefor the basal uptake in many cell types representing themost ubiquitously expressed isoform GLUT4 is responsiblefor insulin-stimulated glucose uptake in peripheral tissuesbut its expression has also been reported in the brain [2526] where glucose is an essential substrate for cerebraloxidative metabolism It has recently been reported that ina human neuronal cell line SH-SY5Y GLUT1 translocationin response to insulin-like growth factor (IGF-I) occurs[27] and for the first time in a neuronal cell system alsoGLUT4 is translocated to the plasma membrane in responseto insulin [28] We have been studying for a long timethe glucose transport activity in many leukaemia cell linesexpressing mainly GLUT1 demonstrating that also in thesecell types GLUT1 is recruited on the plasma membrane from

Oxidative Medicine and Cellular Longevity 3

intracellular compartments in response to different stimuligreatly enhancing the rate of glucose uptake [29 30] More-over it is well known that impaired GLUT4 translocationis causally linked to insulin resistance and consequently tononinsulin-dependent diabetes mellitus [31 32]

Starting from this knowledge and this background wechose the neuroblastoma SH-SY5Y and the promyelocyticleukaemia line HL-60 both expressing insulin and insulin-like growth factor-1 (IGF-I) receptors [28 33] to test somecommercial Stevia extracts in order to evaluate a possibleeffect of these compounds on glucose transport and to clarifythe molecular mechanism of action

2 Materials and Methods

21 Chemicals and Reagents Dulbeccorsquos modified Eaglersquosmedium (DMEM) foetal calf serum (FCS) penicillinstrep-tomycin 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetra-zolium bromide (MTT) staurosporine 2101584071015840-dichlorodihy-drofluorescein diacetate (H

2DCFDA dichlorofluorescin di-

acetate) 2-deoxy-glucose (DOG) phloretin CelLytic Mmammalian protease inhibitor mixture primary antibodyto 120573-actin methylglyoxal (MG) hydrogen peroxide bovineserum albumin (BSA) rebaudioside A standard (StReb)stevioside standard (StStev) and all other chemicals ofthe highest analytical grade were purchased from Sigma-Aldrich Roswell Park Memorial Institute (RPMI) 1640medium (with Hepes with L-glutamine) was purchasedfrom PAA 2-Deoxy-D-[2 3]-glucose and Ultima Gold MVscintillation cocktail were from PerkinElmer PhosSTOP aphosphatase inhibitor cocktail was obtained from RocheDiagnostic Nitrocellulose membranes and Amersham ECLAdvance Western Blotting Detection Reagents were fromGE-Healthcare Primary antibodies against phospho-Akt(Ser473) (no 4058) total Akt (no 9272) and horseradishperoxidase-conjugated secondary antibodies anti-rabbit (no7074) and anti-mouse (no 7076) were purchased from CellSignaling Technologies Anti-GLUT1 (sc-1603) anti-GLUT4(sc-1606) antibodies and anti-goat IgG conjugated tohorseradish peroxidase (sc-2020) were obtained from SantaCruz Biotechnology Primary antibody anti-phospho-PI3Kinase p85 pTyr458p55 pTyr199 (no PA5-17387) was fromThermo Scientific Anti-PI3 Kinase (no 06-195) antibodywas purchased from Millipore PageRuler Prestained proteinladder was from FermentasmdashThermo Fisher Scientific

Extracts from Stevia rebaudiana Bertoni were kindlysupplied by Eridania Sadam SpA

According to FDA and EFSA [8ndash11] total content ofsteviol glycosides in commercial Stevia extracts has to be atleast 95 (ww) and rebaudioside A plus stevioside must beat least 75 The four extracts tested differ by the relativecontent of rebaudioside A and stevioside In particularaccording to the certificates of analysis of each sweetener RebA (R97) contains 97-98 rebaudioside A Stevia RA60 (R60)contains about 60 rebaudiosideA and about 20 steviosideSteviol Glycosides SG95 (SG) contains 50 rebaudioside Aand at least 25 stevioside Truvia (TRU) contains a mixtureof steviol glycosides not analytically quantified

22 Cell Culture SH-SY5Y humanneuroblastoma cells weregrown at 37∘C in a humidified incubator with 5 CO

2

in Dulbeccorsquos modified Eaglersquos medium (DMEM) supple-mented with 10 (vv) foetal bovine serum (FBS) 2mMglutamine 50UmL penicillin and 50 120583gmL streptomycinas reported in [34] HL-60 acute myeloid leukaemia cellswere cultured inRPMI-1640medium supplementedwith 10FBS 2mM glutamine 100UmL penicillin and 100 120583gmLstreptomycin at 37∘C in a humidified atmospheremaintainedat 5 CO

2

23 Cell Viability Cells were treated with different con-centrations of steviol glycosides (05 to 5mgmL) or 1mM(corresponding to 1mgmL) StReb or 1mM (correspondingto 08mgmL) StStev for 24 h Cell viability was evaluatedby the MTT assay as reported in [35] SH-SY5Y and HL-60cells were incubated with 05mgmL MTT for 4 h at 37∘Cin multiwell plates At the end of the incubation blue-violetformazan salt crystals were formed and dissolved by addingthe solubilisation solution (10 SDS 001M HCl) then theplates were incubated overnight in humidified atmosphere(37∘C 5 CO

2) to ensure complete lysis The absorbance at

570 nm was measured using a multiwell plate reader (WallacVictor2 PerkinElmer)

24 Lactate Dehydrogenase Assay SH-SY5Y and HL-60 cellswere incubated with 1mgmL of each Stevia extract for24 h Lactate dehydrogenase (LDH) release from cells wasmonitored by collecting aliquots of medium LDH activitywas assayed by a spectrophotometric method based on thereduction of pyruvate to lactic acid coupled to NADH oxi-dationThe decrease in absorbance at 340 nm was monitoredat 37∘C 100 120583M H

2O2for 30 minutes was used as a positive

control

25 Assay for Caspase 3 Activity Caspase 3 activity inthe cell lysates was measured using a colorimetric assaykit by following the instructions from the manufacturer(Sigma) as described in [36] Cells were incubated with orwithout steviol glycosides (1mgmL) for 1 6 or 24 h After24 h cells were collected and lysed using the lysis bufferprovided in the kit (250mM HEPES pH 74 containing25mM CHAPS and 25mM DTT) The assay was based onthe hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) by caspase 3resulting in the release of free AMCmoiety The fluorescenceof AMC was read using a multiwell plate reader (WallacVictor2 PerkinElmer) excitation and emission wavelengthswere 360 nm and 460 nm respectively

The concentration of the AMC released was calculatedusing an AMC standard curve Caspase 3 activity wasexpressed in nmole of AMC released per min per mL of celllysate and normalised for total protein content in the lysateResults are reported as percentage with respect to the controlStaurosporine (1120583gmL) was used as an apoptosis inducer(positive control)


26 Measurement of Intracellular Reactive Oxygen Species(ROS) Levels ROS intracellular level was evaluated by usingthe fluorescent probe 2101584071015840-dichlorodihydrofluorescein diac-etate (H

2DCFDA) SH-SY5Y andHL-60 cells were incubated

with 5mgmL of each Stevia extract for 1 h and then subjectedor not to oxidative stress generated by 100 120583M H

2O2for

30 minutes Successively cells were washed twice in PBSand incubated with 5 120583M H

2DCFDA for 20min at 37∘C

H2DCFDA is a small nonpolar nonfluorescent molecule that

diffuses into the cells where it is enzymatically deacetylatedby intracellular esterases to a polar nonfluorescent com-pound that is oxidised to the highly green fluorescent 2101584071015840-dichlorofluorescein (DCF) The fluorescence of oxidizedprobe was measured using a multiwell plate reader (WallacVictor2 PerkinElmer) Excitation wavelength was 485 nmand emission wavelength was 535 nm Fluorescence valueswere reported as the percentage of intracellular ROS withrespect to control

27 Glucose Transport Assay Glucose transport assay wasperformed as described in [37 38] Cells were incubated ornot with different compounds (1mgmL) for 1 h then theywere washed twice in PBS and treated for 10min (SH-SY5Y)or 2min (HL-60) at 37∘C with a mixture of 2-deoxy-D-[2 3] glucose (08120583Ciassay) and 10mM unlabeled glucoseanalogue under conditions where the uptake was linearat least for 20min The transport was stopped by addingphloretin (final concentration 03mM) a potent inhibitorof glucose transport activity Radioactivity was measuredby liquid scintillation counting (Tri-Carb liquid scintillationanalyser PerkinElmer)

28 Immunoblotting Analysis After treatments cells werewashed with ice-cold PBS and lysed on ice using CelLytic Mcontaining mammalian protease and phosphatase inhibitormixtureThe resulting lysed cells were left on ice to solubilizefor 45min The lysates were centrifuged at 5000 g for 5minat 4∘C to remove unbroken cell debris and nuclei Celllysate protein concentration was determined by the Bio-RadBradford protein assay (Bio-Rad Laboratories) Samples werekept at 95∘C for 5min prior to separation on 10 SDS-PAGE Mini-Protean TGX precast gels using a Mini-Proteanapparatus (Bio-Rad Laboratories) Proteins (15 120583glane) wereelectrophoretically transferred to nitrocellulose membrane(Hybond-C GE Healthcare) in Tris-glycine buffer at 110Vfor 90min Membranes were then incubated in blockingbuffer containing 5 (wv) albumin in Tris-buffered saline(TBS)Tween to avoid nonspecific binding and incubatedovernight at 4∘C with primary antibodies (anti-GLUT1 anti-GLUT4 anti-phospho-Akt anti-total Akt anti-phospho-PI3K anti-total-PI3K or anti-120573-actin as internal normalizer)Nitrocellulose membranes were then washed 3 times withTBSTween and incubated with secondary antibodies inTBSTween containing 5 albumin for 60min at roomtemperature and successively washed with TBSTween Theresults were visualized by chemiluminescence using ECLAdvance reagent according to the manufacturerrsquos protocol(GE Healthcare) Images of the blots were obtained using a

CCD imager (ChemiDoc MP System Bio-Rad) Bands wereacquired and analysed by using Image Lab analysis software

29 Statistical Analysis Results are expressed as means plusmn SDDifferences among themeanswere determined byBonferronimultiple comparison test following one-way ANOVA andwere considered significant at 119875 lt 005

3 Results and Discussion

The effect of different commercial extracts from Steviarebaudiana Bertoni on glucose transport was investigated inboth SH-SY5Y neuroblastoma andHL-60myeloid leukaemiahuman cells

Glucose is the primary source of energy used by the brainand it is constantly delivered to individual cells (glial cellsand neurons) [39] In brain the relationship among glucosemetabolism GLUT isoforms modulation of glucose uptakerole of insulin and distribution of insulin receptor (IR) is verycomplex being dependent on specific regions of the brainand playing a key role also in cognitive functions Recentstudies report a close correlation between impaired glucoseuptakemetabolism and neurodegenerative diseases such asAlzheimerrsquos disease [40ndash42]

It is also recognised that cancer cells frequently over-express the GLUT family members due to the uncon-trolled proliferation requiring elevated energy and they oftenexpress GLUT isoforms not present in normal conditionsMoreover large hypoxic areas into the tumour cause anincrease in glucose utilization by cancer cells through glycol-ysis The requirement for energy is satisfied by an augmentedsugar intake realised by an increase in GLUT expression andan increment in the translocation of the transporters to theplasma membrane [23] For these reasons cancer cells are auseful model system to study the glucose transport activityand its signalling transduction pathway allowing to clarifythe molecular mechanism underlying steviosides biologicaleffects on glucose metabolism

The first aim of our paper was to evaluate the effect of fourdifferent Stevia extracts on cellular viability assessed byMTTassay SH-SY5Y and HL-60 cells were treated with differentconcentrations (05ndash5mgmL corresponding to 05ndash5mMfor R97 which can be assumed as a pure compound) of Steviaextracts for 24 h Data reported in Figure 2 show that theextracts did not affect cell viabilityproliferation confirmingthat they are not cytotoxic within the concentration rangetested Same results were obtained with similar concentra-tions of StReb or StStev (data not shown)

Cytotoxicity was also evaluated by lactate dehydroge-nase (LDH) assay which indicated that cell membraneintegritywas not compromised and excluded cellular necrosis(Figure 3)

In order to evaluate a possible effect on apoptosis theactivity of caspase 3 was measured Caspases play a cen-tral role in mediating various apoptotic responses and areactivated in a sequential cascade of cleavages To detect theenzymatic activity of caspase 3 the fluorogenic substrate Ac-DEVD-AMC was employed Treatments of cells with Stevia


C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 050

25

50

75

100

125

R97 R60 SG TRU

(mgmL)

Viab

ility

( o

f con

trol)

(a)

0

25

50

75

100

125

(mgmL)

Viab

ility

( o

f con

trol)

R97 R60 SG TRU

C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 05

(b)

Figure 2 Effect of steviol glycosides on cell viabilityproliferation SH-SY5Y (a) and HL-60 (b) cells were treated for 24 hours with differentconcentrations of the four compounds (01mgmL to 5mgmL) Viabilityproliferation was evaluated byMTT assay as described in Section 2and compared to control (C) Results are expressed asmeansplusmn SDof three independent experiments (119899 = 8) Statistical analysiswas performedby Bonferroni multiple comparison test following one-way ANOVA Significant differences were not revealed

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

lowast

H2O2

(a)

lowast

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

H2O2

(b)

Figure 3 Effect of steviol glycosides treatment on lactate dehydrogenase (LDH) activity LDH activity was measured by LDH assay asdescribed in Section 2 SH-SY5Y (a) and HL-60 (b) cells were treated with different compounds at 1mgmL final concentration for 24 hoursor cells were treated with 100 120583M H

2O2for 30min as control of LDH activity Results are expressed as means plusmn SD of three independent

experiments each performed in triplicate Statistical analysis was performed by Bonferroni multiple comparison test following one-wayANOVA lowast119875 lt 005 significantly different from control cells

extracts for 1 hour 6 hours (data not shown) or for 24 hours(Figure 4) did not influence caspase 3 activity indicating thatthe compounds did not induce programmed cell death

Since antioxidant properties of Stevia extracts have beendescribed [15 16] the antioxidant activity of the commercialextracts was investigated in SH-SY5Y and HL-60 cells Reac-tive oxygen species (ROS) levels were measured with the cell-permeant probe H

2DCFDA commonly used to detect free

radicalROS production in cells owing to the intracellularconversion to the highly green fluorescent DCF [43] Asshown in Figure 5 the compounds did not exhibit anyantioxidant activity since they were neither able to decreasebasal ROS level at the highest concentration used nor tocounteract intracellular ROS raise due to exogenous oxidativestress (100 120583M H

2O2for 30min) This lack of antioxidant

activity is in contrast with the data reported by other authors

[15 16] probably because the compounds used in the presentstudy are commercial sweeteners containing 95ndash98 steviolglycosides with no appraisable amounts of polyphenolsnaturally present in Stevia leaves likely responsible for Steviaantioxidant activity

Stevia extracts are largely used as a noncaloric high-potency biosweetener substitute for sugar The effect of fourStevia extracts on glucose transport activity was evaluated inHL-60 human leukaemia cells expressing principallyGLUT1the basal glucose transporter and in SH-SY5Y humanneuroblastoma cells expressing also GLUT4 the insulin-sensitive one Figure 6 shows that all the extracts and the twostandard compounds were able to enhance glucose uptake atsimilar extent after 1-hour incubation in both cellular lines

Since the increase in glucose uptake obtained with stan-dards is consistent with that shown by the whole extracts we


C R97 R60 SG TRU Stauro

Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)

Figure 4 Caspase 3 activity in SH-SY5Y (a) and HL-60 (b) cells was evaluated in the presence of 1mgmL steviol glycosides incubated for24 hours Staurosporine (Stauro 1 120583gmL for 4 hours) was used as positive control Caspase 3 activity was measured spectrofluorimetricallyafter 24 hours in cell lysates as reported in Section 2 Each column represents the mean plusmn SD of three independent experiments Statisticalanalysis was performed by Bonferroni multiple comparison test following one-way ANOVA lowast119875 lt 005 significantly different from controlcells

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)

Figure 5 Effect of steviol glycosides on ROS levels in SH-SY5Y and HL-60 cells SH-SY5Y (a) and HL-60 (b) cells were treated for 1 hourwith different compounds (5mgmL) then basal ROS levels were measured by means of H

2DCFDA assay as described in Section 2 Results

are expressed as means plusmn SD of four independent experiments (119899 = 8) Statistical analysis was performed by Bonferroni multiple comparisontest following one-way ANOVA Significant differences were not revealed SH-SY5Y (c) andHL-60 (d) cells were preincubated for 1 hour withdifferent compounds (5mgmL) and then exposed to oxidative stress generated by 100120583M H

2O2for 30min ROS levels were measured by

means of H2DCFDA assay as described in Section 2 Results are expressed as means plusmn SD of four independent experiments (119899 = 8) Statistical

analysis was performed by Bonferroni multiple comparison test following one-way ANOVA Significant differences were not revealed amongH2O2treated cells


0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)

Figure 6 Effects of Stevia extracts on glucose transport activity compared to the effect of insulin SH-SY5Y (a) and HL-60 (b) cells weretreated with steviol glycosides (1mgmL) with 100 nM insulin (I) with steviol glycosides and insulin simultaneously or 1mM standardcompounds (StReb StStev) Glucose uptake was assayed as described in Section 2 Results are expressed as means plusmn SD of three independentexperiments each performed in triplicate Statistical analysis was performed by Bonferroni multiple comparison test following one-wayANOVA lowast119875 lt 005 significantly different from control cells ∘119875 lt 005 significantly different from the corresponding cells not stimulatedwith insulin

can hypothesize that this effect is due to rebaudioside A andstevioside the two major components of Stevia extracts

Furthermore the influence of Stevia extracts on glucosetransport activity was compared to the effect of 100 nMinsulin (Figure 6) Results reveal that Stevia extracts andinsulin behave similarly being Stevia extracts as efficient asinsulin in increasing glucose uptake The cotreatment withinsulin and Stevia extracts causes a rise of glucose transportsignificantly higher than the increase due to insulin alone

It is well known that insulin induces the translocationof GLUT4 from cytosolic storage vesicles to the plasmamembrane enhancing glucose transport Recently this phe-nomenon was observed also for neural cells in vitro inparticular the human neuroblastoma SH-SY5Y cells througha phosphatidylinositol 3-kinase (PI3K)ndashdependent mecha-nism similar to the mechanism described in muscle or adi-pose tissues [28] Translocation of GLUTs to the membranehas been reported as a consequence of various stimuli inmany cellular types in addition changes in the expres-sion of GLUTs have been described in response to severalmetabolic and oxidative stresses and in various physiologicalor pathological conditions For example insulin and ischemiainduce GLUT1 movement to the membrane in rat heart[44] doxorubicin recruits GLUT1 to the plasma membraneby a ROS-mediated mechanism in cardiomyocytes [37] L-cysteine increases glucose uptake and GLUT3 levels in SH-SY5Y cells [45] growth factors cytokines hydrogen perox-ide and cholesterol depletion are able to increase glucoseuptake and GLUT1 translocation in leukaemia cell lines [46ndash49] The expression of GLUT1 and GLUT4 in neuroblastomaand leukaemia cells following treatments with Stevia extractsor insulinwas assessed byWestern blot analysis on cell lysatesFigure 7 reports representative immunoblots for GLUT iso-forms and the densitometric analysis in both cell lines Itcan be seen that the increase in GLUT1 and GLUT4 content

obtained following exposure to Stevia extracts is similar tothat obtained by insulin stimulation These results are inaccordance with those observed in the evaluation of glucosetransport activity

To clarify the molecular mechanism by which Steviaextracts enhance glucose transport the phosphorylationstatus of PI3K and Akt was evaluated following Steviaextract treatment and insulin stimulation Insulin activatesthe PI3KAkt pathway critical for neuronal survival andgrowth synaptic plasticity and development and learning[50 51] Indeed stimulation of insulin receptor localized inlipid rafts [52 53] produces the phosphorylation of tyrosinereceptor kinases and the activation of a signal transductionpathway involving PI3K and Akt The interaction of insulinwith its receptor is a regulator of growth and differentiation ofleukaemia cells [54 55]Highly specific insulin receptors havebeen identified on human promyelocytic leukaemia cells HL-60 [56] Immunoblotting results (Figure 8) show an increaseof phosphorylated forms of both PI3K and Akt following thetreatment with insulin or Stevia extracts indicating a possiblesimilar mechanism of action or at least a common signallingpathway

To better characterize the mechanisms of glucose uptakeinduction by steviol glycosides we used methylglyoxal (MG)as an inhibitor of the insulin receptorPI3KAkt pathwayMG is a reactive ketoaldehyde product of many metabolicpathways primarily glycolysis which is considered the mostrelevant and reactive glycation agent in vivo Advancedglycation end products (AGEs) have been implicated indevelopment and progression of several diseases includingdiabetes and its associated vascular complications renal fail-ure cirrhosis aging and recently also in diabetic neuropathyand in Alzheimerrsquos disease [57] Furthermore recent studiessuggest a correlation between MG and insulin resistance[58]


SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)

HL-60 cellsC R97I R60 SG TRU

42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

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TRU

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SH-SY5Y cells HL-60 cells

GLU

T1(fo

ld ch

ange

)

lowast

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(c)

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lin R97

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GLU

T4(fo

ld ch

ange

)

SH-SY5Y cells HL-60 cellslowast

lowast

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lowast lowast lowastlowast

(d)

Figure 7 Effects of Stevia extracts on GLUT content SH-SY5Y and HL-60 cells treated with insulin or with steviol glycosides were lysedwith CelLyticM as described in Section 2 Cell lysates were electrophoresed and immunoblotted with the indicated antibodies as described inSection 2 120573-Actin detection was used as a control Immunoblots representative of three independent experiments are reported for SH-SY5Y(a) and HL-60 (b) cell lines densitometric analysis (normalized for 120573-actin content and expressed as fold of control) is shown for GLUT1 (c)and GLUT4 (d) lowast119875 lt 005 significantly different from control cells

p-PI3K

PI3K

p-Akt

Akt

SH-SY5Y cellsC R97I R60 SG TRU

120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

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20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast

lowastlowastlowastlowastlowast

(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

Figure 8 Effects of Stevia extracts on PI3KAkt pathway SH-SY5Y andHL-60 cells treated with insulin or with steviol glycosides were lysedwith CelLyticM as described in Section 2 Cell lysates were electrophoresed and immunoblotted with the indicated antibodies as described inSection 2 120573-Actin detection was used as a control Immunoblots representative of three independent experiments are reported for SH-SY5Y(a) and HL-60 (b) cell lines densitometric analysis of PI3K phosphorylation status is expressed as phospho-PI3Ktotal PI3K ratio (c) anddensitometric analysis of Akt phosphorylation status is expressed as phospho-Akttotal Akt ratio (d) lowast119875 lt 005 significantly different fromcontrol cells


0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)

Figure 9 Effects of Stevia extracts on glucose transport activity compared to the effect of 01mM of methylglyoxal SH-SY5Y (a) and HL-60 (b) cells were treated with steviol glycosides with insulin (I) with methylglyoxal (MG 2 hours) and with steviol glycosides or insulinin the presence of MG Glucose uptake was assayed as described in Section 2 Results are expressed as means plusmn SD of three independentexperiments each performed in triplicate Statistical analysis was performed by Bonferroni multiple comparison test following one-wayANOVA lowast119875 lt 005 significantly different from control cells ∘119875 lt 005 significantly different from cells treated with MG alone

Figure 9 shows that MG incubation (2 hours) inducesa significant decrease in glucose transport and that thesubsequent treatment with Stevia extracts or insulin is ableto raise the glucose uptake corroborating the hypothesis thatStevia extracts could act via PI3KAkt pathway similar toinsulin As MG is a reactive aldehyde toxic to cells MTTassay was performed to verify that the MG concentration(01mM) used to inhibit glucose transport did not influencecell viability (data not shown)

4 Conclusions

In this study we evaluated the effects of steviol glyco-sides extracted from Stevia rebaudiana Bertoni leaves onglucose transport activity in two different cell lines Wedemonstrated for the first time to our knowledge thatrebaudioside A and stevioside the major glycosides in Steviaextracts are able to enhance glucose uptake in both SH-SY5Y neuroblastoma and HL-60 myeloid leukaemia humancells the raise being similar to that induced by insulinOur data suggest that steviol glycosides act by modulat-ing GLUT translocation through the PI3KAkt pathwayAlthough further experiments are needed these results sup-port the hypothesis that steviol glycosides and insulin couldshare a similar mechanism in regulating glucose entry intocells In conclusion Stevia extracts commercialised as zero-calorie natural sweeteners are involved in insulin regulatedglucose metabolism These findings suggest that the use ofStevia extracts goes beyond their sweetening power andmay also offer therapeutic benefits supporting the use ofbotanicals dietary supplements to improve the quality oflife

Authorsrsquo Contribution

Benedetta Rizzo and Laura Zambonin contributed equally tothis paper

Acknowledgments

Authors are grateful to Eridania Sadam SpA that kindlyprovided Stevia rebaudiana Bertoni extracts This work wassupported by grants from Italian Ministry for EducationUniversity and Research (MIUR) from the Italian Ministryof EconomicDevelopment (Project ldquoMade in Italy-over 50rdquo)and from Fondazione del Monte di Bologna e Ravenna ItalyThe funders had no role in study design data collectionand analysis decision to publish or paper preparation Theauthors declare that there is no conflict of interests regardingthe publication of this paper

References

[1] J RHanson andBHDeOliveira ldquoStevioside and related sweetditerpenoid glycosidesrdquo Natural Product Reports vol 10 no 3pp 301ndash309 1993

[2] A D Kinghorn andD D Soejarto ldquoDiscovery of terpenoid andphenolic sweeteners from plantsrdquo Pure and Applied Chemistryvol 74 no 7 pp 1169ndash1179 2002

[3] P Z Zimmet D J McCarty and M P De Courten ldquoThe globalepidemiology of non-insulin-dependent diabetes mellitus andthe metabolic syndromerdquo Journal of Diabetes and its Complica-tions vol 11 no 2 pp 60ndash68 1997

[4] R A DeFronzo ldquoThe triumvirate 120573-cell muscle liver Acollusion responsible for NIDDMrdquo Diabetes vol 37 no 6 pp667ndash687 1988


[5] ldquoAmerican Diabetes Association Standards of Medical Care inDiabetes-2013rdquo Diabetes Care vol 36 supplement 1 pp S11ndashS66 2013

[6] B Crammer and R Ikan ldquoSweet glycosides from the Steviaplantrdquo Chemistry in Britain vol 22 no 10 pp 915ndash917 1986

[7] V Chatsudthipong and C Muanprasat ldquoStevioside and relatedcompounds therapeutic benefits beyond sweetnessrdquo Pharma-cology andTherapeutics vol 121 no 1 pp 41ndash54 2009

[8] EFSA Panel on Food Additives and Nutrient Sources (ANS)ldquoScientific Opinion on safety of steviol glycosides for theproposeduses as a food additiverdquo EFSA Journal vol 8 no 4p 1537 2010

[9] European Food Safety Authority ldquoRevised exposure assessmentfor steviol glycosides for the proposed uses as a food additiverdquoEFSA Journal vol 9 no 1 p 1972 2011

[10] JECFA (Joint FAOWHO Expert Committee on Food Addi-tives)Compendium of Food Additive Specifications Monograph5 Steviol glycosides 2008 httpwwwfaoorgagagnjecfa-additivesdetailshtmlid=898

[11] JECFA (Joint FAOWHO Expert Committee on Food Addi-tives) ldquoSafety evaluation of certain food additives Prepared bythe 69th meeting of the Joint FAOWHO Expert Committee onFood Additivesrdquo WHO Food Additives Series vol 66 pp 183ndash220 2009

[12] S K Goyal S Samsher and R K Goyal ldquoStevia (Steviarebaudiana) a bio-sweetener a reviewrdquo International Journal ofFood Sciences and Nutrition vol 61 no 1 pp 1ndash10 2010

[13] G Brahmachari L C Mandal R Roy S Mondal and A KBrahmachari ldquoStevioside and related compoundsmdashmoleculesof pharmaceutical promise a critical overviewrdquo Archiv derPharmazie vol 344 no 1 pp 5ndash19 2011

[14] M Suttajit U Vinitketkaumnuen U Meevatee and D Bud-dhasukh ldquoMutagenicity and human chromosomal effect ofstevioside a sweetener from Stevia rebaudiana Bertonirdquo Envi-ronmental Health Perspectives vol 101 no 3 pp 53ndash56 1993

[15] S Tavarini and L G Angelini ldquoStevia rebaudiana Bertoni asa source of bioactive compounds the effect of harvest timeexperimental site and crop age on steviol glycoside contentand antioxidant propertiesrdquo Journal of the Science of Food andAgriculture vol 93 no 9 pp 2121ndash2212 2013

[16] N Shivanna M Naika F Khanum and V K Kaul ldquoAntiox-idant anti-diabetic and renal protective properties of Steviarebaudianardquo Journal of Diabetes and Its Complications vol 27no 2 pp 103ndash113 2013

[17] P B Jeppesen S Gregersen C R Poulsen and K Her-mansen ldquoStevioside acts directly on pancreatic 120573 cells to secreteinsulin actions independent of cyclic adenosine monophos-phate and adenosine triphosphate-sensitive K+-channel activ-ityrdquoMetabolism vol 49 no 2 pp 208ndash214 2000

[18] N Lailerd V Saengsirisuwan J A Sloniger C Toskulkaoand E J Henriksen ldquoEffects of stevioside on glucose transportactivity in insulin-sensitive and insulin-resistant rat skeletalmusclerdquoMetabolism vol 53 no 1 pp 101ndash107 2004

[19] P Chan K-L Wong I-M Liu T-F Tzeng T-L Yang and J-T Cheng ldquoAntihyperglycemic action of angiotensin II receptorantagonist valsartan in streptozotocin-induced diabetic ratsrdquoJournal of Hypertension vol 21 no 4 pp 761ndash769 2003

[20] T-H Chen S-C Chen P Chan Y-L Chu H-Y Yang and J-TCheng ldquoMechanism of the hypoglycemic effect of stevioside aglycoside of Stevia rebaudianardquo PlantaMedica vol 71 no 2 pp108ndash113 2005

[21] P B Jeppesen S Gregersen K K Alstrup and K HermansenldquoStevioside induces antihyperglycaemic insulinotropic andglucagonostatic effects in vivo studies in the diabetic Goto-Kakizaki (GK) ratsrdquo Phytomedicine vol 9 no 1 pp 9ndash14 2002

[22] R Abudula V V Matchkov P B Jeppesen H Nilsson CAalkjaeligr and K Hermansen ldquoRebaudioside A directly stim-ulates insulin secretion from pancreatic beta cells a glucose-dependent action via inhibition of ATP-sensitive K+-channelsrdquoDiabetes Obesity and Metabolism vol 10 no 11 pp 1074ndash10852008

[23] R A Medina and G I Owen ldquoGlucose transporters expres-sion regulation and cancerrdquo Biological Research vol 35 no 1pp 9ndash26 2002

[24] R Augustin ldquoThe protein family of glucose transport facilita-tors itrsquos not only about glucose after allrdquo IUBMB Life vol 62no 5 pp 315ndash333 2010

[25] C Leloup M Arluison N Kassis et al ldquoDiscrete brain areasexpress the insulin-responsive glucose transporter GLUT4rdquoMolecular Brain Research vol 38 no 1 pp 45ndash53 1996

[26] C Choeiri W Staines and C Messier ldquoImmunohistochemicallocalization and quantification of glucose transporters in themouse brainrdquo Neuroscience vol 111 no 1 pp 19ndash34 2002

[27] V C Russo K Kobayashi S Najdovska N L Baker and GA Werther ldquoNeuronal protection from glucose deprivation viamodulation of glucose transport and inhibition of apoptosis arole for the insulin-like growth factor systemrdquo Brain Researchvol 1009 no 1-2 pp 40ndash53 2004

[28] Y Benomar N Naour A Aubourg et al ldquoInsulin and leptininduce Glut4 plasma membrane translocation and glucoseuptake in a human neuronal cell line by a phosphatidylinositol3-kinase-dependent mechanismrdquo Endocrinology vol 147 no 5pp 2550ndash2556 2006

[29] T Maraldi D Fiorentini C Prata L Landi and G HakimldquoStem cell factor and H

2O2induce GLUT1 translocation in

M07e cellsrdquo BioFactors vol 20 no 2 pp 97ndash108 2004[30] T Maraldi D Fiorentini C Prata L Landi and G Hakim

ldquoGlucose-transport regulation in leukemic cells how can H2O2

mimic stem cell factor effectsrdquo Antioxidants and Redox Signal-ing vol 9 no 2 pp 271ndash279 2007

[31] F Ismail-Beigi ldquoMetabolic regulation of glucose transportrdquoJournal of Membrane Biology vol 135 no 1 pp 1ndash10 1993

[32] A R Saltiel and C R Kahn ldquoInsulin signalling and theregulation of glucose and lipid metabolismrdquo Nature vol 414no 6865 pp 799ndash806 2001

[33] A EWHendrickson PHaluska P A Schneider et al ldquoExpres-sion of insulin receptor isoform A and insulin-like growthfactor-1 receptor in human acutemyelogenous leukaemia effectof the dual-receptor inhibitor BMS-536924 in vitrordquo CancerResearch vol 69 no 19 pp 7635ndash7643 2009

[34] A Tarozzi F Morroni A Merlicco et al ldquoSulforaphane as aninducer of glutathione prevents oxidative stress-induced celldeath in a dopaminergic-like neuroblastoma cell linerdquo Journalof Neurochemistry vol 111 no 5 pp 1161ndash1171 2009

[35] A Minarini A Milelli V Tumiatti et al ldquoCystamine-tacrinedimer a new multi-target-directed ligand as potential thera-peutic agent for Alzheimerrsquos disease treatmentrdquo Neuropharma-cology vol 62 no 2 pp 997ndash1003 2012

[36] C Angeloni E Motori D Fabbri et al ldquoH2O2preconditioning

modulates phase II enzymes through p38MAPK and PI3KAktactivationrdquo American Journal of Physiology vol 300 no 6 ppH2196ndashH2205 2011


[37] S Hrelia D Fiorentini T Maraldi et al ldquoDoxorubicin inducesearly lipid peroxidation associated with changes in glucosetransport in cultured cardiomyocytesrdquo Biochimica et BiophysicaActa vol 1567 pp 150ndash156 2002

[38] D Fiorentini C Prata T Maraldi et al ldquoContribution ofreactive oxygen species to the regulation of Glut1 in twohemopoietic cell lines differing in cytokine sensitivityrdquo FreeRadical Biology andMedicine vol 37 no 9 pp 1402ndash1411 2004

[39] D D Clarke and L Sokoloff ldquoCirculation and energymetabolismof the brainrdquo inBasicNeurochemistry G J Siegel BW Agranoff RW Albers S K Molinoff P B Fisher andM DUhler Eds pp 637ndash669 Lippincott-Ratven Philadelphia PaUSA 1999

[40] F Pasquier A Boulogne D Leys and P Fontaine ldquoDiabetesmellitus and dementiardquoDiabetes and Metabolism vol 32 no 5pp 403ndash414 2006

[41] K F Neumann L Rojo L P Navarrete G Farıas P Reyesand R B Maccioni ldquoInsulin resistance and Alzheimerrsquos diseasemolecular links amp clinical implicationsrdquo Current AlzheimerResearch vol 5 no 5 pp 438ndash447 2008

[42] E Adeghate T Donath and A Adem ldquoAlzheimer disease anddiabetes mellitus do they have anything in commonrdquo CurrentAlzheimer Research vol 10 no 6 pp 609ndash617 2013

[43] E Eruslanov and S Kusmartsev ldquoIdentification of ROS usingoxidized DCFDA and flow-cytometryrdquo Methods in MolecularBiology vol 594 pp 57ndash72 2010

[44] S Egert N Nguyen and M Schwaiger ldquoMyocardial glu-cose transporter GLUT1 translocation induced by insulin andischemiardquo Journal of Molecular and Cellular Cardiology vol 31no 7 pp 1337ndash1344 1999

[45] V Gazit R Ben-Abraham O Vofsi and Y Katz ldquoL-cysteineincreases glucose uptake in mouse soleus muscle and SH-SY5YcellsrdquoMetabolic Brain Disease vol 18 no 3 pp 221ndash231 2003

[46] T Maraldi C Prata D Fiorentini L Zambonin L Landiand G Hakim ldquoSignal processes and ROS production in glu-cose transport regulation by thrombopoietin and granulocytemacrophage-colony stimulation factor in a human leukaemiccell linerdquo Free Radical Research vol 41 no 12 pp 1348ndash13572007

[47] C Prata TMaraldi L Zambonin D Fiorentini G Hakim andL Landi ldquoROS production and Glut1 activity in two humanmegakaryocytic cell linesrdquo BioFactors vol 20 no 4 pp 223ndash233 2004

[48] C Prata TMaraldi D Fiorentini L Zambonin G Hakim andL Landi ldquoNox-generated ROS modulate glucose uptake in aleukaemic cell linerdquo Free Radical Research vol 42 no 5 pp405ndash414 2008

[49] C Caliceti L Zambonin C Prata et al ldquoEffect of plasmamem-brane cholesterol depletion on glucose transport regulation inleukaemia cellsrdquo PLoSONE vol 7 no 7 Article ID e41246 2012

[50] L P van der Heide GM J Ramakers andM P Smidt ldquoInsulinsignaling in the central nervous system learning to surviverdquoProgress in Neurobiology vol 79 no 4 pp 205ndash221 2006

[51] R R Girgis J A Javitch and J A Lieberman ldquoAntipsychoticdrug mechanisms links between therapeutic effects metabolicside effects and the insulin signaling pathwayrdquo MolecularPsychiatry vol 13 no 10 pp 918ndash929 2008

[52] C Wu S Butz Y-S Ying and R G W Anderson ldquoTyrosinekinase receptors concentrated in caveolae-like domains fromneuronal plasma membranerdquo Journal of Biological Chemistryvol 272 no 6 pp 3554ndash3559 1997

[53] J Sanchez-Wandelmer A Davalos G de la Pena et alldquoHaloperidol disrupts lipid rafts and impairs insulin signalingin SH-SY5Y cellsrdquoNeuroscience vol 167 no 1 pp 143ndash153 2010

[54] T J Chaplinski T E Bennett and J F Caro ldquoAlteration ininsulin receptor expression accompanying differentiation ofHL-60 leukaemia cellsrdquo Cancer Research vol 46 no 3 pp1203ndash1207 1986

[55] J M Lord C M Bunce R J Duncan et al ldquoChanges ininsulin receptor expression in HL60 cells induced to differen-tiate towards neutrophils or monocytesrdquo Journal of MolecularEndocrinology vol 1 no 3 pp 197ndash201 1988

[56] J P Abita C Gauville and F Saal ldquoCharacterization of insulinreceptors in human promyelocytic leukaemia cell HL60rdquo Bio-chemical andBiophysical ResearchCommunications vol 106 no2 pp 574ndash581 1982

[57] M Sousa Silva R A Gomes A E Ferreira A Ponces Freireand C Cordeiro ldquoThe glyoxalase pathway the first hundredyears and beyondrdquo Biochemical Journal vol 453 no 1 pp1ndash15 2013

[58] PMatafome C Sena and R Seica ldquoMethylglyoxal obesity anddiabetesrdquo Endocrine vol 43 no 3 pp 472ndash484 2013

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


O

O O

O

O O

O

CH3 CH2

OH

OH

OH

OH

OH

OHH3C

HO

HO HO

HO

HO

H

H

H

H

H

(a)

HO

O

O O

OO

O O

O

CH3 CH2

OH

OH

OH

OH

OH

H3CHO

HO HO

HO

H

H

H

H

H

O

OH

OH

OH

HO

H

(b)

Figure 1 Chemical structure of stevioside (a) and rebaudioside A (b)

(FDA) has allowed the use of Stevia extracts containing notless than 95 total steviol glycosides Recently the EuropeanFood Safety Authority (EFSA) approved the use of steviolglycosides as food additive [8 9] Considering the availabletoxicity data (in vitro and in vivo animal studies and somehuman tolerance studies) steviol glycosides are considerednot carcinogenic genotoxic or associated with any repro-ductivedevelopmental toxicity Joint Expert Committee onFood Additives (JECFA) established an accepted daily intake(ADI) for steviol glycosides (expressed as steviol equivalents)of 4mgkg bwday [10 11]

Besides sweetness steviol glycosides in particular stevio-side have been shown to possess beneficial effects on humanhealth [7 12 13] Briefly pharmacological activities and ther-apeutical benefits include antitumour and anticancer anti-inflammatory antihyperglycemic antihypertensive antidiar-rheal immunomodulatory diuretic and enzyme inhibitoryactions Stevia has also been used to help control weight inobese subjects [14] moreover antioxidant properties havebeen described [15 16] Stevioside rebaudioside A and theirmetabolite steviol have been mostly investigated in in vivoanimal studies and at a lesser extent in humans Resultssuggest that stevioside and related compounds affect plasmaglucose modulating insulin secretion and sensitivity whichincrease glucose removal from the plasma [17 18] In addi-tion it seems likely that stevioside inhibits gluconeogenesisin the liver of diabetic rats [19 20] These antihyperglycemicinsulinotropic and glucagonostatic effects especially forrebaudioside A are largely plasma glucose level dependentrequiring high glucose levels [21 22] Despite the large num-ber of studies on Stevia and steviol glycosides very little is

reported concerning the cellular and molecular mechanismsunderpinning these effects

In the present study we examined the role of steviolglycosides on cellular glucose transport in cultured cellsGlucose is a polar molecule and requires specific carrierproteins located in the plasma membrane to cross thelipid bilayer and enter the cell Glucose is transported intothe cells through two different types of membrane associ-ated carrier proteins the Na+-coupled glucose transporters(SGLT) and the facilitative glucose transporters (GLUT)The human GLUT family is integral membrane proteinswidely distributed in probably all mammalian cells thatregulate the movement of glucose between extracellular andintracellular compartments maintaining a constant supply ofglucose available for metabolism [23] To date GLUT familyis constituted by 14 distinct isoforms differently distributedin human tissues [23 24] GLUT1 is considered responsiblefor the basal uptake in many cell types representing themost ubiquitously expressed isoform GLUT4 is responsiblefor insulin-stimulated glucose uptake in peripheral tissuesbut its expression has also been reported in the brain [2526] where glucose is an essential substrate for cerebraloxidative metabolism It has recently been reported that ina human neuronal cell line SH-SY5Y GLUT1 translocationin response to insulin-like growth factor (IGF-I) occurs[27] and for the first time in a neuronal cell system alsoGLUT4 is translocated to the plasma membrane in responseto insulin [28] We have been studying for a long timethe glucose transport activity in many leukaemia cell linesexpressing mainly GLUT1 demonstrating that also in thesecell types GLUT1 is recruited on the plasma membrane from











2


2






control







2O2for

















C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 050

25

50

75

100

125

R97 R60 SG TRU

(mgmL)

Viab

ility

( o

f con

trol)

(a)

0

25

50

75

100

125

(mgmL)

Viab

ility

( o

f con

trol)

R97 R60 SG TRU

C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 05

(b)


C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

lowast

H2O2

(a)

lowast

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

H2O2

(b)















Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)


C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)








0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2


2






control







2O2for

















C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 050

25

50

75

100

125

R97 R60 SG TRU

(mgmL)

Viab

ility

( o

f con

trol)

(a)

0

25

50

75

100

125

(mgmL)

Viab

ility

( o

f con

trol)

R97 R60 SG TRU

C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 05

(b)


C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

lowast

H2O2

(a)

lowast

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

H2O2

(b)















Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)


C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)








0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2O2for

















C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 050

25

50

75

100

125

R97 R60 SG TRU

(mgmL)

Viab

ility

( o

f con

trol)

(a)

0

25

50

75

100

125

(mgmL)

Viab

ility

( o

f con

trol)

R97 R60 SG TRU

C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 05

(b)


C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

lowast

H2O2

(a)

lowast

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

H2O2

(b)















Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)


C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)








0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 050

25

50

75

100

125

R97 R60 SG TRU

(mgmL)

Viab

ility

( o

f con

trol)

(a)

0

25

50

75

100

125

(mgmL)

Viab

ility

( o

f con

trol)

R97 R60 SG TRU

C 5 3 1 05 5 3 1 05 5 3 1 05 5 3 1 05

(b)


C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

lowast

H2O2

(a)

lowast

C R97 R60 SG TRU0

100

200

LDH

activ

ity (

of c

ontro

l)

H2O2

(b)















Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)


C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)








0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Casp

ase 3

activ

ity (

of c

ontro

l)lowast

0

100

200

(a)

lowast


Casp

ase 3

activ

ity (

of c

ontro

l)

0

100

200

(b)


C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(a)

C R97 R60 SG TRU0

25

50

75

100

125

ROS

( o

f con

trol)

(b)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(c)

C C R97 R60 SG TRU0

100

200

ROS

( o

f con

trol)

H2O2

(d)








0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

Glu

cose

upt

ake (

o

f con

trol)

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

∘ ∘

∘

∘

(a)

0

100

200

Glu

cose

upt

ake (

o

f con

trol)

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

∘ ∘

∘

∘

C

Insu

lin R97

R97+

I

R60

R60+

I

SG+

I

SG

TRU

TRU+

I

StRe

b

StSt

ev

(b)









SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SH-SY5Y cells

GLUT 1

GLUT 4

C R97I R60 SG TRU

120573-Actin

(a)


42 kDa

55 kDa

55 kDa

(b)

CIn

sulin R9

7R6

0SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25


GLU

T1(fo

ld ch

ange

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowast

(c)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SG

TRU

00

05

10

15

20

25

GLU

T4(fo

ld ch

ange

)


lowast

lowast

lowastlowast

lowast


(d)


p-PI3K

PI3K

p-Akt

Akt


120573-Actin

(a)


85 kDa

85 kDa

60 kDa

60 kDa

42 kDa

(b)

00

05

10

15

20

25


p-PI

3KP

I3K

ratio

(fol

d ch

ange

)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowast


(c)

00

05

10

15

20

25


p-A

ktA

kt ra

tio (f

old

chan

ge)

C

Insu

lin R97

R60

SG

TRU C

Insu

lin R97

R60

SGTR

U

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)



0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 Conclusions




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200G

luco

se u

ptak

e (

of c

ontro

l)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowast

lowastlowast lowast

lowast

∘∘∘∘∘

(a)

0

50

100

150

200

Glu

cose

upt

ake (

o

f con

trol)

C

MG

Insu

lin

MG+

I

R97

R97+

MG

R60

R60+

MG SG

SG+

MG

TRU

TRU+

MG

lowast

lowastlowast

lowast lowastlowast

∘

∘∘∘∘

(b)



4 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
















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