Antidiabetic Effects of Pterosin A, a Small-Molecular-Weight Natural Product, on Diabetic Mouse Models.

1.6 MB·English

by  Hsu, Feng-Lin

ORIGINALARTICLE
Antidiabetic Effects of Pterosin A, a Small-Molecular-
Weight Natural Product, on Diabetic Mouse Models
Feng-Lin Hsu,1 Chun-Fa Huang,2 Ya-Wen Chen,3,4 Yuan-Peng Yen,5 Cheng-Tien Wu,5 Biing-Jiun Uang,6
Rong-Sen Yang,7 and Shing-Hwa Liu5,8
progressionofhyperglycemiaintype2diabeticpatients(4).
The therapeutic effect of pterosin A, a small-molecular-weight
natural product, on diabetes was investigated. Pterosin A, Hypoglycemia and weight gain are the main side effects of
administered orally for 4 weeks, effectively improved hypergly- conventionalinsulinsecretagoguessulfonylureas(5).Among
cemia and glucose intolerance in streptozotocin, high-fat diet– the risks of the thiazolidinediones, including rosiglitazone
fed, and db/db diabetic mice. There were no adverse effects in andpioglitazone,areweightgain,edema,anemia,pulmonary
normal or diabetic mice treated with pterosin A for 4 weeks. edema, and congestive heart failure (5). Therefore, develop-
Pterosin A significantly reversed the increased serum insulin ment of new antihyperglycemic agents with safety and effec-
and insulin resistance (IR) in dexamethasone-IR mice and in
tiveness is an urgentneed.
db/dbmice.PterosinAsignificantlyreversedthereducedmuscle
Traditional medicines derived from medicinal plants are
GLUT-4 translocation and the increased liver phosphoenol-
usedby;70%oftheworld’spopulation(6).Asalternatives
pyruvate carboxyl kinase (PEPCK) expression in diabetic mice.
Pterosin A also significantly reversed the decreased phosphory- to synthetic chemical compounds, plants are an important
lations of AMP-activated protein kinase (AMPK) and Akt in source for hypoglycemic drugs, which are widely used in
musclesofdiabeticmice.ThedecreasedAMPKphosphorylation several systems of traditional medicine to prevent diabetes
and increased p38 phosphorylation in livers of db/db mice were (7,8). Several fern plants have been shown to possess hypo-
effectively reversed by pterosin A. Pterosin A enhanced glucose lipidemic,hypoglycemic,vascularprotective,anti-inflammatory,
uptake and AMPK phosphorylation in cultured human muscle and antinociceptive activities (9–12). Several pterosin
cells. In cultured liver cells, pterosin A inhibited inducer- compounds isolated from fern plants have been shown to
enhanced PEPCK expression, triggered the phosphorylations
have smooth muscle relaxant, leishmanicidal, and anti-
of AMPK, acetyl CoA carboxylase, and glycogen synthase cancer activities in vitro (13–15). The antidiabetic activity
kinase-3, decreased glycogen synthase phosphorylation, and
increasedtheintracellularglycogenlevel.Thesefindingsindi- ofpterosincompoundsstillremainsunclear.PterosinA,a
catethatpterosinAmaybeapotentialtherapeuticoptionfor natural product with a molecular weight of 248.3, can be
diabetes. Diabetes 62:628–638, 2013 isolated from several fern plants (chemical structure is
shown in Fig. 1A).
Here,weinvestigatedtheeffectandpossiblemechanism
D of pterosin A, which was isolated from a fern plant Hypo-
iabetes is a worldwide and national threat. The lepis punctata (Thunb.) Mett., on glucose metabolism in
total number of people with diabetes is pro- diabeticanimals.TheantidiabeticactivityofpterosinAwas
jected to rise from 366 million in 2011 to an investigated in several diabetic mouse models. Blood glu-
estimated 552 million by 2030 (1). Diabetes is coselevelsaremaintainedbythebalancebetweenglucose
a known major risk factor for cardiovascular disease, uptake by peripheral tissues and glucose secretion by the
metabolic syndrome, dyslipidemia, and end-stage renal liver.Wealsotestedtheinvolvementofglucoseuptakeand
disease(2).Patientswithdiabetesrequireoptimalglycemic gluconeogenesis-related signaling molecules in the anti-
control and prevention of diabetes complications to im- hyperglycemiceffectofpterosinA.Theresultsshowedthat
prove their quality of life (3). Pharmacological therapy, as the antihyperglycemic effect of pterosin A is associated
monotherapy or combination therapy, is often necessary with inhibited gluconeogenesis in the liver and enhanced
to achieve glycemic control in the management of di- glucose consumption in peripheral tissues.
abetes. However, it has been reported that neither sulfo-
nylureas nor biguanides can significantly alter the rate of
RESEARCHDESIGNANDMETHODS
PreparationofpterosinA.ThefreshwholeplantsofHypolepispunctata
From the 1Graduate Institute of Pharmacognosy, Taipei Medical University, (Thunb.)Mett. were extractedwithmethanoltoaffordthecrudeextracts,
Taipei,Taiwan;the2GraduateInstituteofChineseMedicalScience,School whichwerethenpartitionedbetweenn-hexane,ethylacetate,andHO.The
ofChineseMedicine,ChinaMedicalUniversity,Taichung,Taiwan;the3De- ethylacetatefractionwassubjecttochromatographiestoisolateand2purify
partment of Physiology, China Medical University, Taichung, Taiwan; the
the tested compound pterosin A. The purity of pterosin A was 99.7% as
4Graduate Institute of Basic Medical Science, China Medical University,
Taichung, Taiwan; the 5Institute of Toxicology, College of Medicine, Na- determined by high-performance liquid chromatography. The structure of
tional Taiwan University, Taipei, Taiwan; the 6Department of Chemistry, pterosinA(Fig.1A)waselucidatedusingone-andtwo-dimensionalnuclear
College of Science, National Tsing Hua University, Hsinchu, Taiwan; the magneticresonancespectroscopyandmassspectrometry;thedataarepre-
7DepartmentofOrthopaedics,CollegeofMedicine,NationalTaiwanUniver- sentedbelow:
sity,Taipei,Taiwan;andthe8DepartmentofUrology,CollegeofMedicine, PterosinA:C H O,m.p.122–123°C,[a] 245.0°(c=0.1,MeOH),atan
15 20 3
NationalTaiwanUniversityHospital,Taipei,Taiwan. amorphouspowder.
Correspondingauthor:Shing-HwaLiu,[email protected]. 1H-NMR(Methanol-d,500MHz)d,ppm:1.08(s,3H,H-10),2.43(s,3H,H-15),
Received5May2012andaccepted15August2012. 4
2.65(s,3H,H-14),2.71(d,J=17.1Hz,1H,H-3),2.98(t,2H,J=7.6Hz,H-12),3.22
DOI:10.2337/db12-0585 (d,J=17.1Hz,1H,H-3),d3.45(d,J=10.7Hz,1H,H-11),3.60(t,2H,J=7.6Hz,
F.-L.H.andC.-F.H.contributedequallytothiswork.
(cid:1)2013bytheAmericanDiabetesAssociation.Readersmayusethisarticleas H-13),d3.70(d,J=10.7Hz,1H,H-11),7.15(s,1H,H-5).
longastheworkisproperlycited,theuseiseducationalandnotforprofit, 13C-NMR (Methanol-d4, 125 MHz) d, ppm: 13.8 (C-14), 21.0 (C-15), 21.4
and the work is not altered. See http://creativecommons.org/licenses/by (C-10), 31.8 (C-12), 36.9 (C-3), 50.6 (C-2), 61.6 (13C), 68.2(11C),125.9 (C-5),
-nc-nd/3.0/fordetails. 131.6(C-9),135.1(C-7),138.3(C-8),145.0(C-6),152.3(C-4),211.9(C-1).
628 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
F.-L.HSUANDASSOCIATES
FIG.1.A:ChemicalstructureofpterosinA.EffectsofpterosinAonhyperglycemia,glucoseintolerance,andbodyweightinaSTZ-induceddiabetic
mouse(DM)model.TheSTZ-induceddiabeticmicewereorallytreatedwithpterosinA(10–100mg/kg)for4weeks,andtheoralglucosetolerance
testwasperformed(B)andthebodyweightsweremeasured(C).D:MicewerepretreatedwithpterosinAfor3days,STZwasinjected,andthe
oralglucosetolerancetestwasperformedafter1week.Dataarepresentedasmean6SEM(n=8).*P<0.05vs.diabeticmicewithoutpterosin
A;#P<0.05vs.diabeticmicealone.
Experimental animals. The protocols in this study were approved by the Dexamethasone-induced insulin-resistance (IR) mouse model. Mice
institutionalanimalcareandusecommittee,andthecareanduseoflabo- weredailytreatedwithdexamethasone(1mg/kg,i.p.)inthepresenceorab-
ratory animals were conducted in accordance with the guidelines of the senceofpterosinA(100mg/kg,oral)ormetformin(300mg/kg,oral)for1week.
animal research committee of the College of Medicine, National Taiwan Age-matchedmiceweregivenanequalvolumeofnormalsaline.
University.Themiceusedinthisstudyweretreatedhumanelyandwithregard db/db diabetic mice. Male C57BL6 db/db mice and nondiabetic littermate
foralleviationofsuffering. controldb/mmice(6weeksold)wereobtainedfromTheJacksonLaboratory
Crj:CD-1(ICR)mice.MaleICRmice(4weeksold)werepurchasedfromthe (BarHarbor,ME).Micewerehousedinaroomataconstanttemperatureof
AnimalCenteroftheCollegeofMedicine,NationalTaiwanUniversity,Taipei, 2262°Cwith12-hlight/darkcycles.MicewereorallytreatedwithpterosinA
Taiwan.Themicewerehousedfivepercageunderstandardlaboratorycon- (100mg/kg)orvehiclefor4weeksandthenwerekilledbyexsanguinationunder
ditionsataconstanttemperature2262°Cwith12-hlight/darkcycles(1). anesthesiaafterbloodsampleswerecollected.Fastingbloodglucoseandinsulin
Experimentaldiabeticmice.Micewerefastedovernightandthenwerein- weremeasured.ThehomeostasismodelassessmentIR(HOMA-IR)indexwas
traperitoneallyinjectedwith100mg/kgstreptozotocin(STZ;Sigma).STZwas calculatedasfollows:fastingglucose(mmol/L)3fastinginsulin(mU/L)/22.5.
dissolved in sodium citrate buffer (pH 4.5) and injected within 15 min of Oralglucosetoleranceandinsulintolerancetests.Theoralglucosetol-
preparation.OneweekafterSTZinjection,thebloodglucoselevelsreachedmore erancetestwasperformedasdescribedpreviously(16).Micewithorwithout
than400mg/dL.Age-matchedmiceweretreatedwithvehicle.TheSTZ-induced drug treatment received an oral glucose challenge (1 g/kg). Blood samples
diabeticmicewerethenorallytreatedwithpterosinA(10–100mg/kg)orvehicle were collected before and at 15, 45, 75, and 105 min after delivery of the
for4weeks(2). glucoseload.Theinsulintolerancetestwasperformedafteran8-hfast,and
High-fatdiet(HFD)–induceddiabeticmice.Themicehadfreeaccessto insulin(1unit/kg)wasadministeredbyintraperitonealinjection.Bloodsam-
standardrodentchow(fatcontent,2%kcal;age-matchedcontrolmice)oran pleswerecollectedfromtheorbitalsinusofeachmouseat0,30,and60min
HFD(fatcontent,60%kcalbasedonlard;TestDiet,Richmond,IN)for8weeks aftertheinsulininjection.
and then were orally treated with pterosin A (100 mg/kg) or vehicle for 4 Measurements of blood glucose and insulin. Blood glucose levels were
weeks(3). determinedusingtheSURESTEPbloodglucosemeter(LifeScan).Tomeasure
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 629
ANTIDIABETICEFFECTSOFPTEROSINA
theamountofinsulin,aliquotsofsampleswerecollectedfromtheserumat anti–phospho-GSK3-a/b,anti-glycogensynthase(GS),anti–phospho-GS(Cell
indicated intervals and analyzed by an insulin antiserum immunoassay Signaling Technology), anti-phosphoenolpyruvate carboxyl kinase (PEPCK),
accordingtotheinstructionsofthemanufacturer(MercodiaAB). anti-p38, anti–phospho-p38, and anti–a-tubulin (Santa Cruz Biotechnology,
Cellculture Inc.)antibodies.AfterthemembraneswerewashedinPBSand0.01%Tween-
RathepaticH4-IIEcellsandhumanhepaticHepG2cells.Thecellswere 20,therespectivesecondaryantibodiesconjugatedtohorseradishperoxidase
culturedinahumidifiedchamberwitha5%CO and95%airmixtureat37°C wereappliedfor1h.Theantibody-reactivebandswereidentifiedbyenhanced
2
and maintained in Dulbecco’s modified Eagle’s medium containing glucose chemiluminescencereagents(AmershamPharmaciaBiotech)andexposedon
(4.5 g/L) and L-glutamine (2 mmol/L), supplemented with 10% FBS and 1% Kodakradiographicfilm.
penicillin-streptomycin. Real-time quantitative PCR analysis. H4-IIE cells were cultured with or
Human primary skeletal muscle cells. Skeletal muscle biopsy samples withoutpterosinAinthepresenceorabsenceofPEPCKinducers8-bromo-
(;0.2g)wereobtainedfrompatientsinthecourseoforthopedicsurgery,with cAMP (62.5 mmol/L) and dexamethasone (62.5 nmol/L) (19) for 24 h. Total
institutionalethicalcommitteeapprovalandinformedconsent,attheNational RNAwasisolatedfromthecellswithTrizolreagent.TherelativemRNAex-
TaiwanUniversityHospital,Taipei,Taiwan.Humanmyoblastswereisolated pression for PEPCK was determined by real-time quantitative PCR as pre-
anddifferentiatedtomyotubes,aspreviouslydescribed(17).Thecellswere viouslydescribed(20).Briefly,0.5–1mgtotalRNAwasusedforthereverse
cultured in Ham’s F-10 supplemented with 20% FBS, 2.5 ng/mL basic fibro- transcriptionofRNAtocDNAusingavianmyeloblastosisvirusreversetran-
blasticgrowthfactor,and1%penicillin-streptomycinin5%CO at37°C. scriptase (Promega). Each sample (2 mL cDNA) was tested with real-time
2
Rat b-cell line RINm5F cells. The cells were cultured in a humidified SYBR Green PCR reagent (Invitrogen) with specific primers (forward and
chamberwitha5%CO and95%airmixtureat37°CandmaintainedinRPMI reverse[59to39]):PEPCK:GAGTGCCCATCGAAGGCATandCCAGTGCGC-
2
1640mediumsupplementedwith10%FBSand1%penicillin-streptomycin. CAGGTACTTG;b-actin: GCCCTAGACTTCGAGC andCTTTACGGATGTCA-
Westernblotting.Theexperimentswereperformedasdescribedpreviously ACGT. Amplification was performed using an ABI StepOnePlus sequence
(18).Theequivalentof30–50mgtotalproteinwassubjectedtoelectrophoresis detection system (PE, Applied Biosystems), and data were analyzed using
on10%SDS-polyacrylamidegels.Themembranewasblockedfor1hinPBS StepOne2.1software(AppliedBiosystems).
and0.01%Tween-20containing5%nonfatdrymilkandincubatedwithanti– Measurementofintracellularglycogen.H4-IIEcellswereseededat13106
GLUT-4,anti–phospho-AMP–activatedproteinkinase(AMPK),anti–phospho- cells/wellina6-wellplateandallowedtoadhereandrecoverovernight.Cells
acetylCoAcarboxylase(ACC),anti-glycogensynthasekinase3(GSK3)-a/b, werechangedtofreshmediaandincubatedwithorwithoutpterosinA.After
FIG.2.EffectsofpterosinAonhyperglycemia,glucoseintolerance,andbodyweightinHFD-fed–inducedanddb/dbdiabeticmousemodels.The
HFD-fed–induced(AandB)ordb/db(CandD)diabeticmicewereorallytreatedwithpterosinA(100mg/kg)for4weeks,oralglucosetolerance
testswereperformed,andbodyweightsweremeasured.Dataarepresentedasmean6SEM(n=8).*P<0.05vs.diabeticmicewithoutpterosinA.
630 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
F.-L.HSUANDASSOCIATES
24-htreatment,intracellularglycogenwasextractedaspreviouslydescribed RESULTS
(21), and the amount of glycogen was determined by a glycogen assay kit Effects of pterosin A on hyperglycemia and glucose
(BioVision). intoleranceinseveraldiabeticanimalmodels.Wefirst
Cell viability assay. Cell proliferation was determined by a colorimetric
investigatedtheantihyperglycemiceffectsofpterosinAon
assay using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT; Sigma). This assay measures the activity of living cells via mito- several well-known mouse models of diabetes. Pterosin A
chondrialdehydrogenaseactivitythatreducesMTTtopurpleformazan.The (10–100mg/kg)administeredorallyfor4weekseffectively
formazan was solubilized by DMSO, and its absorbance at 570 nm was improved hyperglycemia and glucose intolerance in sev-
measured. eral diabetic mouse models, including STZ injection (Fig.
Measurement of glucose uptake. A fluorescent glucose analog 2-(N-[7-
1B), HFD-fed (Fig. 2A), and db/db diabetic mice (Fig. 2C).
nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; Invitrogen)
Pretreatment with pterosin A (100 mg/kg) before STZ in-
wasusedtomeasureglucoseuptakeinprimaryhumanskeletalmusclecells.A
totalof104cells/wellwereseededinablack96-welltissuecultureplate.Cells jection also provided protection against STZ-induced hy-
were washed with Krebs-Ringer buffer. Cells were preincubated for 30 min perglycemia (Fig. 1D). Pterosin A reversed the decreased
withpterosinA(10–150mg/mL)or/andinsulin(100nmol/L)at37°C.2-NBDG body weight in STZ diabetic mice (Fig. 1C) and the in-
wasaddedatafinalconcentrationof100mmol/L,theuptakeproceededfor30 creased body weight in HFD-fed mice (Fig. 2B) and db/db
minat37°C,andthencellswerewashedwithPBS.Fluorescenceinthecells
diabetic mice (Fig. 2D).
wasmeasuredatanexcitationwavelengthof485nmandanemissionwave-
lengthof535nmwithafluorescencemicroplatereader. Pterosin A showed no influence on food intake in di-
Measurement of nitric oxide (NO) production. Nitrite (NO2) concen- abetic mice (data not shown). The increased serum levels
2
trations in the culture supernatants were measured by the Griess reagent of total cholesterol and LDL-cholesterol in HFD- and STZ-
(Activemotif),andabsorbancewasdeterminedat540nm. induced diabetic mice were also reversed by the pterosin
Statisticalanalysis.Thedataaregivenasmeans6SEM.Whenmorethan
A treatment (Fig. 3A and B). Pterosin A was capable of
one group was compared with one control, significance was evaluated
accordingtoone-wayANOVA.Duncan’sposthoctestwasappliedtoidentify decreasing the increased serum blood urea nitrogen, cre-
groupdifferences.Probabilityvaluesof,0.05wereconsideredsignificant. atinine (Fig. 3C), aspartate aminotransferase, and alanine
FIG. 3. Effects of pterosin A on serum levels of lipid, blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), and alanine
aminotransferase (ALT) in HFD-fed or STZ-induced diabetic mouse (DM) models. The HFD-feeding or STZ-induced diabetic mice were orally
treatedwithpterosinA(100mg/kg)for4weeks.Serumlipids(totalcholesterol,triglyceride,LDL-cholesterol,HDL-cholesterol)(AandB),BUN
andcreatinine(C),andASTandALT(D)levelsweremeasuredinfastingmice.Dataarepresentedasmean6SEM(n=8).*P<0.05vs.control.
#P<0.05vs.STZorHFDdiabeticmicewithoutpterosinA.
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 631
ANTIDIABETICEFFECTSOFPTEROSINA
aminotransferase levels (Fig. 3D) in STZ-induced diabetic Effects of pterosin A on glucose uptake and
mice. No adverse effects were observed in normal control gluconeogenesis-related signaling molecules in diabetic
mice treated with pterosin A (100 mg/kg) for 4 weeks, in- miceandculturedmuscleandlivercells.Next,wetested
cludingbehavior,bloodpressure,heartrate,bloodglucose, whetherglucoseuptakeandgluconeogenesis-relatedsignal
organweights,bloodbiochemicalmarkers,andpathological moleculesareinvolvedintheeffectofpterosinA.Asshown
examination(datanotshown).Wealsocompletedanacute in Fig. 6, pterosin A (100 mg/kg) administered orally for
orallimittestforpterosinA,atadoseof5,000mg/kg,andno 4 weeks was capable of reversing the reduced GLUT-4
deathsorothertoxicsymptomsappeared(datanotshown). translocation from the cytosol to the membrane fraction
Effect of pterosin A on IR in the mouse models. We and the decreased phosphorylated AMPK and phosphory-
next test the effect of pterosin A on IR in dexamethasone- lated Akt protein expressions in the skeletal muscles of
treated mice and db/db diabetic mice. As shown in Fig. 4, STZ-induced diabetic mice (Fig. 6A) and db/db diabetic
serum insulin levels, HOMA-IR index, and insulin intoler- mice (Fig. 6B). Moreover, pterosin A (50 mg/mL) signifi-
ancewereincreasedinadexamethasone-inducedIRmouse cantly increased 2-NBDG uptake (Fig. 6Ca) and phosphor-
model. Pterosin A (100 mg/kg) and metformin (300 mg/kg, ylated AMPK expression (Fig. 6Cb) in cultured primary
as a positive control) administered orally for 1 week signi- human skeletal muscle cells.
ficantlyreversedIRinthedexamethasone-treatedmice.The PterosinA(100mg/kg)wasalsocapableofreversingthe
levelsofseruminsulin,HbA ,andtheHOMA-IRindexwere increased PEPCKexpressioninthe livers(Fig. 7A) of STZ-
1c
markedlyenhancedindb/dbdiabeticmice,whichwerealso induced diabetic mice. Moreover, pterosin A (100 mg/kg)
significantlyreversed by the oraladministration of pterosin reversed the reduced phosphorylations of AMPK and Akt,
A (100 mg/kg) for 4 weeks (Fig. 5A–C). Moreover, immuno- increased p38 phosphorylation, and increased PEPCK and
histochemical insulin staining for islet hypertrophy showed GLUT-2 protein expressions in the livers of db/db diabetic
that pterosin A treatment effectively reversed the islet hy- mice (Fig. 7B). However, the results of in vitro studies
pertrophy in pancreas of db/db mice (Fig. 5D). showedthatpterosinA(50–150mg/mL)effectivelytriggered
FIG.4.EffectsofpterosinAonseruminsulin,HOMA-IR,andinsulinintoleranceinadexamethasone-inducedIRmousemodel.Micewerein-
traperitoneallytreatedwithdexamethasone(Dex,1mg/kg)for1weekandthenwereorallytreatedwithpterosinA(Pte,100mg/kg)ormetformin
(Met,300mg/kg)for1week,andseruminsulin(A),HOMA-IR(B),andinsulintolerancetest(C)weredetected.Dataarepresentedasmean6
SEM(n=5).*P<0.05vs.control.#P<0.05vs.dexamethasonegroupwithoutpterosinA.
632 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
F.-L.HSUANDASSOCIATES
the phosphorylations of AMPK and its downstream signal DISCUSSION
ACC (Fig. 7Ca) and increased the GSK3-a/b phosphoryla- Inthisstudy,wedemonstrateforthefirsttimethatpterosin
tion and decreased the GS phosphorylation (Fig. 7Cb) in A,asmall-molecular-weightnaturalproduct,caneffectively
culturedrathepaticcelllineH4-IIEcells.Similarly,pterosin alleviate the hyperglycemia, glucose intolerance, insulin
A (150 mg/mL) from a plant extract source and pterosin A resistance, dyslipidemia, and islet hypertrophy in diabetic
(150 mg/mL) from a chemically synthesized source both mouse models. Pterosin A improves diabetes through me-
effectivelytriggeredthephosphorylationsofAMPKandAkt chanismsin the inhibition of liver gluconeogenesisand the
proteins in cultured human hepatic cell line HepG2 cells enhancementofglucosedisposal.Moreover,pterosinAcan
(Fig. 7D). Moreover, pterosin A (50–150 mg/mL) effectively improve the decreased body weight in STZ-diabetic mice
inhibited 8-bromo-cAMP/dexamethasone-enhanced PEPCK and the increased body weight in HFD-fed mice and in
mRNA expression in a dose-dependent manner (Fig. 8A) db/dbdiabeticmice,whichmaybeattributedtotheability
and significantly enhanced the intracellular glycogen con- ofpterosinAtosignificantlyimproveglucosehomeostasis
tents (Fig. 8B) in H4-IIE cells. and insulin resistance (22,23).
Effects of pterosin A on cell viability and NO pro- Glucosehomeostasisincirculationismaintained bythe
ductioninb-cells. Pterosin A (10–150mg/mL) effectively balance between the rate of glucose entering the circula-
reversed the STZ-reduced cell viability and STZ-increased tion(glucoseappearance)andtherateofglucoseremoval
NO production in b-cell line RINm5F cells in a dose- from the circulation (glucose disappearance). In a normal
dependent manner (Fig. 8Ca and b). Moreover, pterosin A individual under the fed state, insulin suppresses gluco-
(10–150mg/mL)alsosignificantlyreversedtheinterleukin- neogenesis and glycogenolysis in the liver and enhances
1b–increasednitriteproductioninRINm5Fcellsinadose- glucose disposal in the peripheral tissues; however, in a
dependent manner (Fig. 8Cc). diabeticindividual,hepatic glucose production iselevated
FIG.5.EffectsofpterosinAonseruminsulin,HbA ,HOMA-IRindex,andislethypertrophyindb/dbdiabeticmice.Diabeticmicewereorally
1c
treatedwithpterosinA(100mg/kg)for4weeks,andtheseruminsulin(A),HbA (B),HOMA-IR(C),andimmunohistochemicalinsulinstaining
1c
forpancreaticislethypertrophy(D)weredetected.Dataarepresentedasmean6SEM(n=8).*P<0.05vs.control.#P<0.05vs.diabeticmice
withoutpterosinA.(Ahigh-qualitycolorrepresentationofthisfigureisavailableintheonlineissue.)
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 633
ANTIDIABETICEFFECTSOFPTEROSINA
andglucoseappearanceexceedsglucosedisappearancein a rate-limiting enzyme in gluconeogenesis, is negatively re-
thecirculation,causingpostprandialhyperglycemia(24).To gulatedbyinsulinandishighlyresistantinhyperinsulinemic
control blood glucose levels, insulin promotes glucose up- diabetic models (31,32). It has been mentioned that
take into adipocytes and skeletal muscle cells through an acquired or genetic defects in insulin or other signal trans-
activation of a cascade of signal transduction, which stim- duction pathways may lead to increased PEPCK gene ex-
ulates GLUT-4, an insulin-responsive glucose transporter pression and gluconeogenesis (31). Gómez-Valadés et al.
protein, from intracellular sites (cytosol) to the cell mem- (33)demonstratedthathepaticPEPCKpartialsilencingwith
brane (25,26). In type 2 diabetic patients, impaired glucose RNA interference significantly lowered hyperglycemia and
transport in skeletal muscles has been demonstrated to be improved glucose tolerance in diabetic mice. Moreover,
a major factor responsible for reduced glucose uptake in GLUT-2 is known to transport glucose across the plasma
whole body (27,28). Over-expression of GLUT-4 in skeletal membrane of liver (34). GLUT-2 in the liver of a type 2 di-
muscleshasbeenshowntoimproveglucosehomeostasisin abetic animal model was found to be upregulated (35).
several diabetic animal models and protect against the de- In the current study, we found that pterosin A signifi-
velopment of diabetes (28). GLUT-4 has been suggested as cantly reversed the reduced GLUT-4 translocation to mem-
a therapeutic target for pharmacological intervention strat- brane in the skeletal muscles and the increased protein
egies to control diabetic hyperglycemia (26,28). expressions of PEPCK and GLUT-2 in the livers of diabetic
In addition, phosphoenolpyruvate gluconeogenesis and mice. The in vitro studies also showed that pterosin A sig-
hepatic glucose production have been found to be in- nificantly increased glucose uptake in cultured human
creased in type 2 diabetic patients (29). The excess glu- skeletalmusclecellsandeffectivelyinhibited8-bromo-cAMP/
coneogenesisofthefastingstatehasalsobeendemonstrated dexamethasone–enhanced PEPCK mRNA expression
tobecarriedovertotheinsulinizedstate,causingtoglucose in cultured liver cells. These results indicate that the in-
overproduction in type 2 diabetic patients (30). PEPCK, hibition in liver gluconeogenesis and the enhancement
FIG.6.EffectsofpterosinAontheglucoseuptake–relatedsignalproteinsinskeletalmusclesofdiabeticmice(DM)andculturedprimaryhuman
skeletalmusclecells.TheSTZ-induceddiabeticmice(A)anddb/dbdiabeticmice(B)wereorallytreatedwithpterosinA(100mg/kg)for4weeks.
A:ProteinexpressionsofmuscleGLUT-4(a),phosphorylatedAMPK(b),andphosphorylatedAkt(c)weredeterminedbyWesternblotting.C:
Uptakeof2-NBDG(a)andphosphorylatedAMPKexpression(b)inculturedprimaryhumanskeletalmuscleweredetected.DatainAandCaare
presentedasmean6SEM(n=4).*P<0.05vs.control.#P<0.05vs.diabeticmicewithoutpterosinAorwithpterosinAaloneinculturedcells.B
andCb:Representativeimagesofthreeindependentexperimentsareshown.
634 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
F.-L.HSUANDASSOCIATES
in muscle glucose disposal are involved in the pterosin phosphorylation(Ser-9),andfurtherreducedcAMP-response
A-induced antidiabetic action. element-binding protein (CREB) phosphorylation (Ser-129)
The intracellular AMPK signaling cascade is a cellular and PEPCK gene expression in the liver (41).
energy status sensor. AMPK activity is switched on when GSK3 inhibition has also been found to improve oral
the intracellular AMP-to-ATP ratio is elevated. Activation glucose disposal by increasing liver glycogen synthesis in
of AMPK is known to downregulate several biosynthetic atype2diabeticratmodel(42).AselectiveGSK3inhibitor,
pathways,suchasfattyacidsynthesisandgluconeogenesis L803-mts,hasalsobeenshowntosuppressCREB-regulated
intheliver,andtoswitchonseveralcatabolicpathwaysfor PEPCKgeneexpression,increasetheintracellularglycogen
ATP generation such as glucose uptake (upregulation of level in the liver, and upregulate GLUT-4 expression in the
GLUT-4)andglycolysis(36,37).ActivationofAMPKtriggers skeletal muscle of ob/ob diabetic mice (43).
intracellular metabolic changes that would be beneficial However,AMPK-regulatedACCinhibitionreducesmalonyl
in diabetic patients, such as increased glucose uptake in CoA content and subsequently leads to reduced fatty acids
musclesandothertissues,decreasedglucoseproductionin synthesis and increased mitochondrial fatty acid oxidation
the liver, and decreased synthesis and increased fatty acid in the liver (35,36). In the current study, we found that
oxidation (36,37). It has been shown that p38 MAPK medi- pterosin A significantly reversed the decreased phosphory-
ates pH-responsive induction of PEPCK mRNA in a gluco- latedAMPKandphosphorylatedAktinthemusclesofSTZ-
neogenic and pH-responsive renal proximal tubule-like cell inducedanddb/dbdiabeticmice.PterosinAalsoeffectively
line (38). Inhibition of p38 signaling has also been demon- reversedthedecreasedAMPK,increasedphospho-p38,and
strated to suppress gluconeogenesis along with the ex- increasedPEPCKexpressionsintheliversofdb/dbdiabetic
pressionsofgenesofPEPCKandglucose-6-phosphatasein mice. In cultured human skeletal muscle cells, pterosin
the liver (39). Berasi et al. (40) have demonstrated that AmarkedlyincreasedthephosphorylationofAMPK.Invitro
AMPKactivationinhibits gluconeogenesisthrough anearly study in liver cells also showed that pterosin A inhibits
growth response 1–activated dual-specificity protein phos- 8-bromo-cAMP/dexamethasone-enhanced PEPCK mRNA
phatase 4–inhibited p38 MAPK pathway in the hepatocyte expression, triggers the phosphorylations of AMPK and
cell lines. In addition, Horike et al. (41) showed that acti- GSK3, decreases the phosphorylation of glycogen syn-
vationofAMPKcouldtriggertheinactivationofGSK3-bby thase, and increases intracellular glycogen levels. Pterosin
FIG.7.EffectsofpterosinAontheliver gluconeogenesis–relatedsignalproteinsindiabeticmice(DM)andculturedlivercells.STZ-induced
diabetic mice(A) anddb/dbdiabetic mice(B)wereorally treatedwith pterosin A(100 mg/kg)for 4weeks.The proteinexpressionsofliver
gluconeogenesis–related signal proteins (PEPCK, phosphorylated AMPK, phosphorylated Akt, and phosphorylated p38) and GLUT-2 were de-
terminedbyWesternblotting.PhosphorylationsofAMPK,Akt,ACC,GSK3-a/b,andGSinculturedH4-IIE(C)andHepG2(D)livercellstreated
withpterosinA(50–150mg/mL)for0.5–4hweredetected.Db:PterosinAfromachemicallysynthesizedsourcewasused.A:Dataarepresentedas
mean6SEM(n=4).*P<0.05vs.control(C).#P<0.05vs.diabeticmicewithoutpterosinA.B–D:Representativeimagesofthreeindependent
experimentsareshown.
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 635
ANTIDIABETICEFFECTSOFPTEROSINA
A suppressed lipogenic enzyme-ACC activity in the liver pancreatic b-cell apoptosis through the pathways of Jun
cells that may lead to decreased lipogenesis. These results NH -terminal kinase activation and Akt suppression (48).
2
suggestthatanAMPK-relatedsignalingpathwayisinvolved However, Liu et al. (49) have suggested that apoptosis and
in the antidiabetic activity by pterosin A. necrosis are both involved in the b-cell death induced by
Diabetes is known a metabolic disorder and is compli- cytokines in which apoptosis is mostly NO-independent,
cated by multiorgan deterioration. The increasing evidence whereas necrosis is NO-dependent. It has been indicated
has suggested that oxidative stress and inflammation may that antioxidants could decrease diabetes complications by
playmajorrolesindiabetescomplications(44).Obesityhas protecting from oxidative stress (50). In the current study,
been shown to be associated with a state of chronic low- we found that pterosin A is capable of decreasing the in-
level inflammation (45). It has been found that the pro- creased serum levels of blood urea nitrogen and creatinine
duction of reactive oxygen species (ROS) is elevated in (markersof renal function) and aspartate aminotransferase
obesity, which may further activate the inflammatory path- and alanine aminotransferase (markers of liver function)
ways(46).Anassociationofearlydiabeticnephropathywith in STZ-induced diabetic mice, indicating that pterosin A
increasedintrarenalNOgenerationhasbeensuggested(47). possesses hepatorenal protective action during diabetes.
NO has also been found to contribute to cytokine-induced Moreover, in vitro study in cultured b-cells showed that
FIG.8.EffectsofpterosinAonPEPCKmRNAexpressionandintracellularglycogeninculturedlivercellsandoncellviabilityandnitritepro-
ductioninculturedb-cells.A:HepaticH4-IIEcellsweretreatedwithpterosinA(50–150mg/mL)inthepresenceorabsenceofPEPCKinducerfor
24h,andtheexpressionsofPEPCKmRNAweredeterminedbyreal-timePCR.B:H4-IIEcellsweretreatedwithpterosinA(50–150mg/mL)for
24h,andintracellularglycogenlevelsweremeasured.C:b-CelllineRINm5FcellsweretreatedwithpterosinA(10–150mg/mL)for24hinthe
presenceorabsenceofSTZ(5mmol/L;a,b)orinterleukin(IL)-1b(5ng/mL;c),andthenthecellviability(a)andnitrite(b,c)productionwere
measured.Alldataarepresentedasmean6SEMofthreetofiveindependentexperiments.*P<0.05vs.control.#P<0.05vs.PEPCKinducer
aloneorSTZaloneorIL-1balone.
636 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
F.-L.HSUANDASSOCIATES
pterosin A effectively reversed STZ-reduced cell viability, 12. LeiYF,ChenJL,WeiH,XiongCM,ZhangYH,RuanJL.Hypolipidemicand
STZ-increasedNOproduction,andinterleukin-1b–increased anti-inflammatorypropertiesofAbacopterinAfromAbacopterispenangiana
NO production. These results imply that pterosin A may in high-fat diet-induced hyperlipidemia mice. Food Chem Toxicol 2011;49:
protect pancreatic b-cells from NO-induced cell damage. 3206–3210
13. Sheridan H, Frankish N, Farrell R. Smooth muscle relaxant activity of
However, whether pterosin A can decrease diabetes com- pterosinZandrelatedcompounds.PlantaMed1999;65:271–272
plications by protecting from oxidative stress needs to be 14. TakahashiM,FuchinoH,SekitaS,SatakeM.Invitroleishmanicidalac-
clarified in the future. tivityofsomescarcenaturalproducts.PhytotherRes2004;18:573–578
In conclusion, pterosin A reverses the reduced GLUT-4 15. Ouyang DW, Ni X, Xu HY, Chen J, Yang PM, Kong DY. Pterosins from
translocation from cytosol to membrane in skeletalmuscle Pterismultifida.PlantaMed2010;76:1896–1900
16. HuangCF,ChenYW,YangCY,etal.Extractoflotusleaf(Nelumbonu-
andtheincreasedPEPCKexpressionintheliverofdiabetic
cifera) and its active constituent catechin with insulin secretagogue ac-
mice. An AMPK-regulated signaling pathway is involved in tivity.JAgricFoodChem2011;59:1087–1094
the activation of muscle GLUT-4 and inhibition of liver 17. YenYP,TsaiKS,ChenYW,HuangCF,YangRS,LiuSH.Arsenicinhibits
PEPCKexpressionbypterosinA.Moreover,pterosinAalso myogenic differentiation and muscle regeneration. Environ Health Per-
increases GSK3 phosphorylation and decreases the GS spect2010;118:949–956
phosphorylation that further enhances intracellular glyco- 18. ChenYW,HuangCF,TsaiKS,etal.Theroleofphosphoinositide3-kinase/
Aktsignalinginlow-dose mercury-inducedmousepancreatic b-celldys-
gen synthesis in liver cells. We also found that pterosin A
functioninvitroandinvivo.Diabetes2006;55:1614–1624
effectively reversed islet hypertrophy in db/db mice; how-
19. Sasaki K, Cripe TP, Koch SR, et al. Multihormonal regulation of phos-
ever, the mechanism needs to be clarified in the future. phoenolpyruvatecarboxykinasegenetranscription.Thedominantroleof
Takentogether,thepterosinA-inducedantidiabeticactivity insulin.JBiolChem1984;259:15242–15251
isassociatedwithinhibitedgluconeogenesisintheliverand 20. LuTH,HsiehSY,YenCC,etal.Involvementofoxidativestress-mediated
enhancedglucoseconsumptioninperipheraltissues.These ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic
findings also indicate that pterosin A may be a potential signalsinmethylmercury-inducedneuronalcellinjury.ToxicolLett2011;
204:71–80
therapeutic option for diabetes.
21. RyuHS,ParkSY,MaD,ZhangJ,LeeW.TheinductionofmicroRNAtargeting
IRS-1isinvolvedinthedevelopmentofinsulinresistanceunderconditionsof
mitochondrialdysfunctioninhepatocytes.PLoSONE2011;6:e17343
ACKNOWLEDGMENTS
22. Swanston-FlattSK,DayC,BaileyCJ,FlattPR.Traditionalplanttreatments
This study was supported by a grant from the National for diabetes. Studies in normal and streptozotocin diabetic mice. Dia-
Science Council of Taiwan (NSC 99-2323-B-002-007). betologia1990;33:462–464
No potential conflicts of interest relevant to this article 23. LazarMA.Howobesitycausesdiabetes:notatalltale.Science2005;307:
were reported. 373–375
24. Aronoff SL, Berkowitz K, Shreiner B, Want L. Glucose metabolism and
F.-L.H. prepared and provided the testing samples,
regulation:beyondinsulinandglucagon.DiabetesSpectr2004;17:183–190
analyzedtheresearchdata,andcontributedtodiscussion.
25. KlipA.Themanywaystoregulateglucosetransporter4.ApplPhysiolNutr
C.-F.H. and Y.-W.C. collected and analyzed the research Metab2009;34:481–487
dataandcontributedtodiscussion.Y.-P.Y.,C.-T.W.,B.-J.U., 26. Morgan BJ, Chai SY, Albiston AL. GLUT4 associated proteins as thera-
and R.-S.Y. prepared and analyzed the testing samples. peutictargetsfordiabetes.RecentPatEndocrMetabImmuneDrugDiscov
S.-H.L.designedtheexperimentsandwrote,reviewed,and 2011;5:25–32
27. Zierath JR, He L, Gumà A, Odegoard Wahlström E, Klip A, Wallberg-
edited the manuscript. F.-L.H. and S.-H.L. are the guaran-
HenrikssonH.Insulinactiononglucosetransportandplasmamembrane
tors of this work, and, as such, had full access to all the
GLUT4contentinskeletalmusclefrompatientswithNIDDM.Diabetologia
datainthestudyandtakeresponsibilityfortheintegrityof 1996;39:1180–1189
data and the accuracy of data analysis. 28. Wallberg-Henriksson H, Zierath JR. GLUT4: a key player regulating glu-
cosehomeostasis?Insightsfromtransgenicandknockoutmice(review).
MolMembrBiol2001;18:205–211
REFERENCES 29. ConsoliA,NurjhanN,CapaniF,GerichJ.Predominantroleofgluconeo-
1. UnwinN,WhitingD,GuariguataL,GhyootG,GanD,Eds.IDFDiabetes genesisinincreasedhepaticglucoseproductioninNIDDM.Diabetes1989;
Atlas.5thEd.Brussels,Belgium,InternationalDiabetesFederation.2011. 38:550–557
Availablefromhttp://www.idf.org/diabetesatlas.Accessed11April2012. 30. Gastaldelli A, Toschi E, Pettiti M, et al. Effect of physiological hyper-
2. Martinez-HervasS,RomeroP,HevillaEB,etal.Classicalcardiovascularrisk insulinemiaongluconeogenesisinnondiabeticsubjectsandintype2di-
factorsaccordingtofastingplasmaglucoselevels.EurJInternMed2008;19: abeticpatients.Diabetes2001;50:1807–1812
209–213 31. FriedmanJE,SunY,IshizukaT,etal.Phosphoenolpyruvatecarboxykinase
3. LaskerRD.Thediabetescontrolandcomplicationstrial.Implicationsfor (GTP)genetranscriptionandhyperglycemiaareregulatedbyglucocorti-
policyandpractice.NEnglJMed1993;329:1035–1036 coidsingeneticallyobese db/db transgenicmice.JBiol Chem 1997;272:
4. KrentzAJ, Bailey CJ.Oral antidiabetic agents: currentrole intype 2 di- 31475–31481
abetesmellitus.Drugs2005;65:385–411 32. Quinn PG, Yeagley D. Insulin regulation of PEPCK gene expression:
5. ChengAY,FantusIG.Oralantihyperglycemictherapyfortype2diabetes amodelforrapidandreversiblemodulation.CurrDrugTargetsImmune
mellitus.CMAJ2005;172:213–226 EndocrMetabolDisord2005;5:423–437
6. MishraR,ShuaibM.Shravan,MishraPS.Areviewonherbalantidiabetic 33. Gómez-ValadésAG,Vidal-AlabróA,MolasM,etal.Overcomingdiabetes-
drugs.JAppPharmSci2011;1:235–237 inducedhyperglycemiathroughinhibitionofhepaticphosphoenolpyruvate
7. JaraldE,JoshiSB,JainDC.Diabetesandherbalmedicines.IranJPharm carboxykinase(GTP)withRNAi.MolTher2006;13:401–410
Ther2008;7:97–106 34. Burcelin R, Dolci W, Thorens B. Glucose sensing by the hepatoportal
8. WaisM,NazishI,SamadA,etal.Herbaldrugsfordiabetictreatment:an sensorisGLUT2-dependent:invivoanalysisinGLUT2-nullmice.Diabetes
updatedreviewofpatents.RecentPatAntiinfectDrugDiscov2012;7:53–59 2000;49:1643–1648
9. Ajikumaran Nair S, Shylesh BS, Gopakumar B, Subramoniam A. Anti- 35. SliekerLJ,SundellKL,HeathWF,etal.Glucosetransporterlevelsintis-
diabetes and hypoglycaemic properties of Hemionitis arifolia (Burm.) suesofspontaneouslydiabeticZuckerfa/farat(ZDF/drt)andviableyellow
Mooreinrats.JEthnopharmacol2006;106:192–197 mouse(Avy/a).Diabetes1992;41:187–193
10. Chen J, Chen X, Lei Y, et al. Vascular protective potential of the total 36. HardieDG.Minireview:theAMP-activatedproteinkinasecascade:thekey
flavanolglycosidesfromAbacopterispenangianaviamodulatingnuclear sensorofcellularenergystatus.Endocrinology2003;144:5179–5183
transcription factor-kB signaling pathway and oxidative stress. J Ethno- 37. HardieDG.TheAMP-activatedproteinkinasepathway—newplayersup-
pharmacol2011;136:217–223 streamanddownstream.JCellSci2004;117:5479–5487
11. HaiderS,NazreenS,AlamMM,GuptaA,HamidH,AlamMS.Anti-inflammatory 38. FeifelE,ObexerP,AndratschM,etal.p38MAPKmediatesacid-induced
andanti-nociceptiveactivitiesofethanolicextractanditsvariousfractions transcriptionofPEPCKinLLC-PK(1)-FBPase(+)cells.AmJPhysiolRenal
fromAdiantumcapillusvenerisLinn.JEthnopharmacol2011;138:741–747 Physiol2002;283:F678–F688
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 637