Research Article Combination Therapy with Oleanolic Acid...

Research ArticleCombination Therapy with Oleanolic Acid and Metforminas a Synergistic Treatment for Diabetes

Xue Wang1 Yupeng Chen1 Daoud Abdelkader1 Waseem Hassan1

Hongbin Sun2 and Jun Liu1

1National Drug Screening Center and Jiangsu Key Laboratory of TCM Evaluation and Translational ResearchChina Pharmaceutical University 24 Tongjiaxiang Nanjing 210009 China2Center for Drug Discovery State Key Laboratory of Natural Medicines China Pharmaceutical University24 Tongjiaxiang Nanjing 210009 China

Correspondence should be addressed to Jun Liu junliucpueducn

Received 13 August 2014 Revised 13 December 2014 Accepted 15 December 2014

Academic Editor Giuseppe Paolisso

Copyright copy 2015 Xue Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aims and Background Type 2 diabetes is a chronic disease that cannot be treated adequately using the known monotherapiesespecially when the disease progresses to an advanced stage In this study we explore the possibility of treating the disease with anovel combination approach of oleanolic acid (OA) a glycogen phosphorylase (GP) inhibitor andmetforminMethods Dbdbmicewere randomly divided into four groups a dbdb control group dbdb mice treated with OA (250mgkg) dbdb mice treated withmetformin (100mgkg) and dbdbmice treatedwith a combination ofOA andmetformin Allmicewere treated for fourweeksTheeffects of the treatments on glucose homeostasis were measured using an OGTT an assessment of insulin sensitivity and signalingin the liver and the hepatic glucose production Results Combination therapy with OA and metformin significantly reduced theblood glucose and insulin levels and improved the liver pathology compared with that for the monotherapy in the dbdb diabeticmouse model We also found that the combination therapy significantly increased the mRNA expression of glycogen synthesis anddecreased theGP PGC-1120572 PEPCK1 andG-6-Pase levels In addition the combination therapywithOAandmetformin significantlyincreased the phosphorylation of AKT PI3K AMPK and ACC and decreased the protein expression levels of G-6-Pase PEPCK1and TORC compared with those for either monotherapy The combination therapy also reduced the phosphorylation of mTORand CREB Conclusions Our results suggest that the combination therapy with OA and metformin has synergistic effects on thesymptoms of dbdb diabetic mice by improving glucose and insulin homeostasis

1 Introduction

Diabetes mellitus and the associated microvascular andmacrovascular complications are the major threat to diabeticpatients Numerous studies have clearly shown that effectiveblood glucose control is the key to preventing and treatingdiabetic complications [1ndash3] In addition to insulin resistanceone of the major reasons for hyperglycemia in diabeticpatients is increased hepatic glucose production Suppressinghepatic glucose production is a viable approach for treatingdiabetes [4] Because the balance between gluconeogenesisand glycogenolysis determines the hepatic glucose produc-tion either of the pathways could be targeted to treat diabetes

Metformin has been the first-line drug for treatingdiabetes for the past five decades The major mechanismby which metformin lowers blood glucose levels is throughthe inhibition of hepatic gluconeogenesis [5] We previouslyreported that pentacyclic triterpene inhibits glycogen phos-phorylase (GP) and can be used as a new strategy to lowerblood glucose [6] using natural substances with low toxicity[7ndash11]

Oleanolic acid (OA) is a natural component ofmany plantfoods and medicinal herbs and also a pentacyclic triterpenethat exerts beneficial effects against diabetes and metabolicsyndrome [12ndash15] OA is therapeutically effective withoutinducing significant side effects [16ndash18] Our previous studies

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2015 Article ID 973287 12 pageshttpdxdoiorg1011552015973287

2 Journal of Diabetes Research

HO

H

COOH

Figure 1 The structure of OA

showed that OA can inhibit GP and its hypoglycemic effect ispartially mediated by the inhibition of glycogenolysis in vivo[17 18]

The complicated pathogenesis of diabetes suggests that acombination therapy may be more effective in treating thisdisease In this study we test whether a combination therapyusing OA and metformin would have synergistic effects onimproving glucose metabolism in diabetic conditions

2 Materials and Methods

21 Drugs and Chemicals Oleanolic acid (structure shown inFigure 1) was obtained from the Center for Drug Discoveryat China Pharmaceutical University Metformin (purity gt98) was obtained from Sigma (St Louis MO USA)The mouse insulin enzyme immunoassay ELISA kit waspurchased from Invitrogen Serum triglyceride (TG) totalcholesterol (TC) high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) kits werepurchased from Whitman Biotech (Nanjing Ltd China)Antibody (Ab) for G-6-Pase was purchased from Santa CruzBiotechnology (Santa Cruz CA USA) TORC2 was fromMillipore (Billerica MA USA) and phospho-CREB and120573-actin were from Abcam (Cambridge MA USA) TheAbs against protein kinase B (PKBAkt) phospho-Ser473-Akt (pAkt) PI3K phospho-PI3K AMPK phosphor-AMPKACC phosphor-ACC PEPCK1 mTOR and phospho-mTORwere purchased from Cell Signaling Technology (BeverlyMA USA) The HRP-conjugated secondary Abs affinity-purified mouse anti-rabbit IgG and rabbit anti-mouse IgGwere purchased from Sigma (St Louis MO USA)

22 Animals and Treatments Male C57BLKsJ-Lepdb (dbdb) mice were obtained from the Model Animal ResearchCenter of Nanjing University China The animals weremaintained in a 12 h light-dark cycle at a temperature of 25∘Cwith free access to water and regular chow To investigatethe effect of OA and metformin the seven-week-old dbdbmale mice were divided into four groups (119899 = 6group)

The mice were given vehicle (025 wv CMC) OA(250mgkg) metformin (100mgkg) or a combination of thetwo drugs once daily by intragastric administration for 28days The body weight and food intake were recorded reg-ularly After 8 hours of fasting blood glucose concentrationsweremonitored using tail vein blood and a glucometer (One-Touch Ultra Lifescan) every week The mice were fasted for12 hours for an oral glucose tolerance test (OGTT day 21)with the slight modifications described in the figure legendBlood samples were obtained from the tail vein and theserum insulin concentration was measured using an ELISAkit At the end of the experiment (day 28) the mice werefasted for 8 hours and blood samples were collected from theorbital venous plexus and centrifuged for serum separationThe animals were euthanized by CO

2inhalation and the

liver tissue was excised and stored in 4 paraformaldehydefor further analysis All of the animal experiments wereconducted according to the Guidelines of ExperimentalAnimal Care issued by the Committee for the Purpose ofControl and Supervision of Experiments on Animals

23 Biochemical Parameter Analysis At the end of the exper-iment the blood glucose levels and the serum levels of triglyc-eride (TG) total cholesterol (TC) high-density lipoproteincholesterol (HDL-C) and low-density lipoprotein cholesterol(LDL-C) were measured using commercially available kitsfollowing the manufacturerrsquos protocol The serum concen-tration of insulin was measured using ELISA followingthe protocol provided in the kit The homeostatic modelassessment (HOMA) value was calculated as described byMatthews et al [19] Insulin resistance was determined usingthe HOMA method and the following equation HOMAvalue for insulin resistance (HOMA-IR) = fasting insulin(mUmL) lowast fasting glucose (mmolL)225 The liver indexwas calculated as follows and used to assess the organhealth and hepatic histological damage liver index = liverweightbody weight To evaluate the glycogen content of thetissue the method of Lillie and Greco was used [20] Liversamples were homogenized in 50mM TrisHCl buffer atpH 74 containing 150mM NaCl 1mM EDTA and 1mMPMSF The liver triglyceride and total cholesterol contentswere analyzed using the previously described protocol

24 Hepatic Histology The tissue was subsequently embed-ded in paraffin and sectioned to a thickness of 5mm usinga microtome Tissue sections prepared on aminosilane-treated slides were deparaffinized and dehydrated using agraded alcohol series and distilled water The tissue sectionswere stained with hematoxylin and eosin The glycogencontent was determined by comparative PAS staining withor without prior amylase treatment (Histoserv GermantownMD) For the detection of immunofluorescence the slideswere incubated with a mouse monoclonal 120572-actin antibody(1 1000 Santa Cruz Biotechnology Inc) together with a rab-bit antibody for G-6-Pase (1 500 Santa Cruz BiotechnologyInc) and a rabbit antibody for PEPCK1 (Cell Signaling)followed by a TRITC-conjugated goat anti-mouse antibodyand a FITC-conjugated goat anti-rabbit antibody (1 100Huawei ImmunoResearch Laboratories Inc) After thorough

Journal of Diabetes Research 3

Table 1 Sequences of primers for the genes

Gene Sense primer (51015840 to 31015840) Antisense primer (51015840 to 31015840)PGC-1120572 CAACAGCAAAAGCCACAAAG GGGGTCAGAGGAAGAGATAAPEPCK1 GAACAAGGAGTGGAGACC TTCATAGACAAGGGGGACG-6-Pase GTGGGCATCAATCTCCTC GACTTCCTGGTCCGGTCTGP TAAAAAAGAACTTCAACCGC TGTAGAACTCCAAAGACAGGGS CTCTGTGTCCTCGCTTCCA GTCACCTTCGCCTTCGTCTGLUT2 ATGAAGAAAGAAAAGGAAGA TGCTGGTTGAATAGTAAAATGAPDH CATGTTCGTCATGGGTGTGAAC CAGTCTTCTGGGTGGCAGTGAT

washing the slides were mounted and visualized using afluorescent microscope

25 Western Blotting Lysis buffer (25mM Tris-HCl pH 75100mM NaCl 25mM EDTA and EGTA 20mM NaF1mM Na

3VO4 20mM Na 120573-glycerophosphate 10mM Na

pyrophosphate 05 Triton X-100 01 120573-mercaptoethanoland protease inhibitor cocktail (Roche)) was used to lysethe tissue SDS-PAGE-resolved proteins were transferred tonitrocellulose membranes (GE Healthcare) and incubatedwith Abs to detect the G-6-Pase TORC2 phospho-CREBAkt phospho-Ser473-Akt PI3K phospho-PI3K AMPKphosphor-AMPK ACC phosphor-ACC PEPCK1 mTORand phospho-mTOR protein levels After the incubation withthe HRP-conjugated secondary Ab the signals were detectedusing an ECL kit All Western blot results are representativeof at least two independent experiments

26 RNA Extraction and Quantitative RT-PCR Total RNAfrom the liver was extracted using TRIZOL Reagent (Invit-rogen) The RNA concentration was determined using UVspectrophotometry RT-PCR was performed using Thunder-bird SYBRMaster Mix (TAKARA Japan) The PCR was per-formed on a Real-Time PCRDetection System (StepOnePlusABI) with the following cycles 95∘C for 1min followed by40 cycles of 95∘C for 15 s 58∘C for 15 s and 72∘C for 45 s todetect the GP GS PGC-1120572 G-6-Pase PEPCK1 and GLUT2gene levels (sequences shown in Table 1) GAPDH expressionwas used as an internal controlThe 2minusΔΔCT was calculated forevery sample and normalized to GAPDH All RT-PCR resultsare representative of three independent experiments

27 Statistical Analysis Data are expressed as the mean plusmnSEM unless otherwise indicated A two-tailed Studentrsquos 119905-testand one-way ANOVA were performed to test for statisticallysignificant differences A value of 119875 lt 005 was consideredsignificant at a 95 confidence level

3 Results

31 Effects of the Combined Treatment on Food Consump-tion Body Weight Liver Weight and Serum BiochemicalParameters in dbdb Mice Vehicle (05 CMC-Na) OA(250mgkg) metformin (100mgkg) or a combination of thetwo drugs was intragastrically administered to 7-week-old

obese dbdb male mice once daily for 4 weeks No significantalterations in the overall body weight gain liver index andliver biochemical parameters were found However signifi-cant differences in food intake water intake and the levels ofserum biochemical parameters were observed (Table 2)

32 Effects of the Combined Treatment on the Fasting SerumInsulin (FSI) Level Fasting Serum Glucose (FSG) Leveland Other Pharmacodynamics Biomarkers in dbdb MiceAt baseline the vehicle OA metformin and combinationgroups were matched with respect to fasting blood glucoselevel After four weeks of treatment the fasting blood glucoselevel was significantly lower in the OA metformin andcombination groups than in the vehicle group (Figure 2(a))Importantly the mice treated with the combination of OAand metformin achieved a more significant decrease inthe fasting blood glucose level compared with that for themice that received the monotherapies Similarly the fastingserum insulin levels were significantly decreased in themetformin group and combination group compared withthat in the vehicle group (Figure 2(b)) In addition theinsulin sensitivity (as indicated by the insulin resistance indexHOMA-IR) was also improved in the mice treated witheither monotherapy or the combination therapy and thegreatest improvement occurred in the animals treated withthe combination of the two agents (Figure 2(c))

33 Effect of the OA Metformin and OA-Metformin Com-bination Therapy on the Oral Glucose Tolerance Test Resultsin dbdb Mice To further evaluate the changes in glucosemetabolism and insulin secretion in dbdb mice an OGTTwas performed 3 weeks after the start of the treatmentsAlthough the glucose levels during the OGTT in the OA-treated mice were similar to those of the metformin-treatedmice (Figures 3(a) and 3(b)) the combination therapy-treatedmice showed a marked decrease in blood glucose levelscompared with those in the vehicle-treated group

34 Liver Inflammation The HampE-stained liver samplesobtained from the four groups showed signs of liver inflam-mation Although the vehicle group showed lobular inflam-mation and hepatocyte ballooning the OA monotherapygroup showed slightly reduced levels of inflammatory cellinfiltrates and hepatocyte degeneration The metforminmonotherapy group showed an even greater reduction in


Table 2 Comparison of characteristics of dbdb mice with or without OA andor metformin treatment

Parameters Vehicle group Oleanolic acid(100mgkg)

Metformin(250mgkg)

Combination(350mgkg)

Weight gain (g) 413 plusmn 232 592 plusmn 198 560 plusmn 184 666 plusmn 159Food intake (gmouse) 749 plusmn 059 642 plusmn 045 603 plusmn 135 570 plusmn 133lowast

Water intake (mLmouseday) 285 plusmn 031 186 plusmn 052lowast 208 plusmn 047 166 plusmn 016lowast

Serum lipid (mmolL)Triglyceride 196 plusmn 045 134 plusmn 031 134 plusmn 039lowast 112 plusmn 015lowastlowastlowast

Total cholesterol 559 plusmn 043 545 plusmn 025 451 plusmn 038lowast 364 plusmn 049lowastlowastlowastΔ

HDL-cholesterol 902 plusmn 097 754 plusmn 082lowastlowast 587 plusmn 074lowastlowast 501 plusmn 072lowastlowastlowast

LDL-cholesterol 193 plusmn 034 161 plusmn 054 103 plusmn 028lowast 095 plusmn 014lowastlowast

Liver changesLiver weight (g) 172 plusmn 023 158 plusmn 025 174 plusmn 037 157 plusmn 014Liver index 00417 plusmn 00033 00389 plusmn 00039 00407 plusmn 00060 00408 plusmn 00030Liver triglyceride (mgg) 251 plusmn 046 235 plusmn 034 205 plusmn 036 191 plusmn 019Liver total cholesterol (mgg) 01012 plusmn 00507 00904 plusmn 00282 00727 plusmn 00229 00730 plusmn 00264

Data are expressed as the mean plusmn SEM lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 indicate significant differences for comparisons with the vehicle group119875 lt 001 and

119875 lt 0001 indicate significant differences for comparisons with the OAmonotherapy group and Δ119875 lt 005 indicates significant differencesfor comparisons with the metformin monotherapy group

Bloo

d gl

ucos

e con

cent

ratio

n 40

30

20

10

0Vehicle Oleanolic acid Metformin Combination

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(mm

olL

)

(a)

Vehicle Oleanolic acid Metformin Combination

lowastlowastlowast

8

6

4

2

0

Seru

m in

sulin

conc

entr

atio

n (120583

gL) lowast

(b)


lowastlowastlowast

lowastlowastlowastHO

MA-

IR

400

300

200

100

0

lowast

(c)

Figure 2 The effect of oleanolic acid metformin and their combination on the FSI level FPG level and HOMA-IR in dbdb mice (a) TheFPG level was measured after 8 h of fasting in dbdb mice treated with vehicle oleanolic acid metformin or the combination for 4 weeks(b) The FSI level was measured after 8 h of fasting in dbdb mice treated with vehicle oleanolic acid metformin or the combination for 4weeks (c) The HOMA-IR index was calculated for each group Data are shown as the mean plusmn SEM lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001indicate significant differences for comparisons with the vehicle group 119875 lt 005 indicates significant differences for comparisons with themonotherapy group (119899 = 6)


VehicleOleanolic acid

MetforminCombination

80

60

40

20

0Bloo

d gl

ucos

e con

cent

ratio

n (m

mol

L)

0 30 60 90 120 150

Time (min)

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

8000

6000

4000

2000

0

Are

a und

er cu

rve

(mm

ollowast0

ndash120

min

)

(b)

Figure 3 Chronic treatment results in normalized hyperglycemia and improved glucose tolerance in dbdb mice (a) Glucose tolerance testin oleanolic acid- metformin- and combination-treated and untreated dbdb mice After three weeks of treatment with OA metforminand the combination mice were fasted for 12 hours and then given glucose (15 gkg) The blood glucose levels in the tail tip blood weremeasured at 0 15 30 60 and 120 minutes after glucose administration (b) One-way ANOVA of the area under curve showed that the drugscan significantly improve glucose tolerance in dbdb mice compared with that in untreated dbdb mice Data are shown as the mean plusmn SEMlowast

119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 indicate significant differences for comparisons with the vehicle group 119875 lt 0001 indicatessignificant differences for comparisons with the monotherapy group (119899 = 6)

these parameters Furthermore the combination therapygroup showedminimal inflammatory cell infiltrates and onlya small amount of hepatocyte degeneration (Figure 4(a))The PAS stain showed that the liver glycogen level in thecombination therapy group was significantly higher thanthose in the vehicle group or the OA and metformin groups(Figure 4(b)) The liver glycogen content in the combinationtherapy group was significantly higher than that in thevehicle group and either monotherapy group (Figure 4(c))As shown in Figures 5(a) and 5(b) the G-6-Pase and PEPCK1immunoreactivity in liver was significantly decreased in thecombination group compared with that in the vehicle group

35 Effect of the OA Metformin and OA-Metformin Combi-nation Therapy on Hepatic mRNA Expression Levels of KeyRegulators Associated with Gluconeogenesis and Glycogenol-ysis In the present study we also examined the effect ofeach treatment on the expression of several important genesfor controlling hepatic gluconeogenesis and glycogenolysisincluding PGC-1120572 GP G-6-Pase and PEPCK1 As shown inFigure 6 the combination therapy with OA and metforminsignificantly inhibited the mRNA expression of PGC-1120572and decreased the mRNA expression levels of GP G-6-Pase and PEPCK1 However the treatment increased theGS mRNA expression level in dbdb mice (Figure 6) Theseresults demonstrate that compared with the monothera-pies the combination therapy inhibits gluconeogenesis andglycogenolysis more extensively in dbdb mice

36 Liver Protein Expression Levels of Markers Associated withGluconeogenesis and Glycogenolysis We have also examinedthe effects of the OA metformin and combination therapy

on the insulin signaling pathway induced phosphorylationof PI3K Akt and mTOR and on the AMPK pathway(Figure 7(a)) These treatment groups also showed activationof AMPK phosphorylation compared with that in the dbdbcontrol diabetic group This phosphorylation can also stimu-late the downstream phosphorylation of ACC (Figure 7(b))We investigated whether OA metformin or the combi-nation therapy inhibited gluconeogenesis by reducing G-6-Pase PEPCK1 TORC and p-CREB The protein levelsof TORC and p-CREB were increased in the dbdb miceThe administration of OA metformin or a combination ofthe two drugs lowered the protein levels of TORC and p-CREB Furthermore the protein expression of G-6-Pase andPEPCK1 decreased (Figure 7(c))

4 Discussion

The current study investigated a therapy that combined OAandmetformin as a synergistic treatment for diabetesmellitusand attempted to evaluate the underlying mechanisms Thecombination therapy (OA and metformin) was found topossess different but complementary mechanisms of actionon the plasma glucose and insulin levels that prevented thedevelopment of diabetes In addition to the improved liverpathology the combination therapy potently decreased themRNA expression levels of GP PGC-1120572 PEPCK1 and G-6-Pase and increased the synthesis of glycogen Interestinglyseveral of these genes showed statistically significant additiveor synergistic changes We also found that the combinationtherapy could partly activate the AMPK and AKT signalingpathway to a greater extent compared with the activation byeither of the monotherapies

Metformin is a widely used hypoglycemic agent for thetreatment of diabetes with a well-known ability to reduce



(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)

Figure 4 Histological analysis using HampE and PAS staining after 4 weeks of treatment with oleanolic acid metformin and the combination(a) Representative photomicrographs depicting the HampE staining of liver sections in untreated and treated dbdb mice (b) Representativephotomicrographs depicting the PAS staining of liver sections in untreated and treated dbdb mice (c) Liver glycogen lowast119875 lt 001 versusuntreated dbdb mice lowastlowastlowast119875 lt 0001 versus untreated dbdb mice 119875 lt 0001 versus oleanolic acid-treated dbdb mice Each bar representsthe mean plusmn SEM of the group (119899 = 6)

hepatic gluconeogenesis At the hepatocellular level met-formin is known to improve insulin-mediated glycogensynthesis and inhibit gluconeogenesis [21] As a natural triter-penoid OA has been used in traditional Chinese medicineto treat hepatitis since the 1970s In recent years OA hasbeen reported to effectively lower blood glucose levels bypromoting insulin signal transduction [22 23] In additionNgubane et al [24] reported that OA restored depletedglycogen levels and thus provided another approach fortreating diabetesDue to their differentmechanisms of actionthe administration of metformin and OA together appearsto be an effective strategy for lowering blood glucose in asynergistic manner

To gain the deeper insight into diabetes mellitus severalanimal models have been developed in the last severaldecades these models include the streptozotocin-induceddiabetic rat model and conventional or genetically modifiedmodels [25] The dbdb mouse has been the most commonlyemployed model that has been used to investigate the patho-genesis of diabetes mellitus in many studies since 1966 [26]The dbdb mouse model which is characterized by deficientleptin receptor activity is known to cause deleterious changesin various tissues triggered by hyperglycemia dyslipidemia

obesity insulin resistance and advanced glycation [27]Among these pathogenic factors in diabetes hyperinsuline-mia and insulin resistance are associated with diabetes riskand increases in these particular factors have been shown insubjects that have a metabolic abnormality [28 29]

The mice under this model have a clear obese phenotypewith enhanced food intake and increased fasting blood glu-cose and lipid levels after 7 weeks afterwards the symptomsgradually worsen to become severe diabetic complicationsThe results clearly showed amarked reduction inwater intakein the mice that only received OA and the combinationtherapy group had a particularly significant change in foodand water intake compared with that in the monotherapygroups This finding indicated that OA could partly improveenergymetabolismdisorders andmaintain body stabilityThechanges in the food and water intake might be interpreted asa reflex biological effect that is induced by the combinationtherapy or the changes might be a compensatory responseto the level of some other parameter We also found thateither the metformin or OA monotherapy tended to atten-uate diabetic symptoms however the combination therapyusing both compounds exerted synergistic effects on lipidand glucose metabolism Compared with the monotherapy


Combination

DAPI Skelemin G-6-Pase Merge

Vehicle

Oleanolic

Metformin

acid

(a)

Combination

DAPI Skelemin PEPCK1 Merge

Vehicle

Metformin

Oleanolicacid

(b)

Figure 5 Histological analysis using immunofluorescence staining after 4 weeks of treatment with oleanolic acid metformin or thecombination (a) Double immunofluorescence of 120572-actin (green fluorescence) and G-6-Pase (red fluorescence) from the pancreas ofdifferent groups merged figures are shown on the right (b) Double immunofluorescence of 120572-actin (green fluorescence) and PEPCK1 (redfluorescence) from the pancreas of different groups merged figures are shown on the right



15

10

05

00

Gly

coge

n ph

osph

oryl

ase

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

mRN

A ex

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sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

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sion

(b)


15

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00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast

(c)Vehicle Oleanolic acid Metformin Combination

15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

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K1m

RNA

expr

essio

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(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n

lowastlowastlowast lowastlowastlowast lowastlowastlowast

(f)

Figure 6 Analysis of the liver mRNA expression levels of markers associated with glucose metabolism

the combination therapy clearly inhibited the elevated lipidlevels induced by the dbdb mouse model Metformin hadonly a slight advantage in reducing triglycerides and totaland HDL-cholesterol levels compared with the reductionby the OA treatment but the combination therapy showedan extremely potent synergistic action Significant improve-ments were also observed in the plasma glucose and insulinlevels and in the homeostasis model assessment-insulinresistance (HOMA-IR) index for the combination therapygroupThe attenuation of diabetic pathology characterized bya reduction in the serum glucose levels and an improvementin insulin resistance could possibly result from the synergisticeffects by which metformin and OA inhibit both gluconeo-genesis and glycogenolysis We demonstrated the therapycombining OA and metformin exerted favorable effectsthe improvement in hyperglycemia and lipid metabolisminduced by the combination therapy was greater than that for

either of the monotherapies or the combination treatmentwas successful in this spontaneous animal model of diabetesmellitus

Liver glycogen is synthesized in response to elevatedconcentrations of glucose and insulin [30] Many genes playa crucial role in the process of hepatic glycogen synthesisGlycogen phosphorylase (GP) is the enzyme responsible forthe synthesis of glucose-1-phosphate the source of energyfor muscles and the rest of the body [31] G-6-Pase catalyzesthe hydrolysis of glucose 6-phosphate (G6P) to glucoseand inorganic phosphate [32] Our results suggest that thetreatment groups especially the combination therapy grouphad fewer droplets than the number of droplets in the controlgroup The combination therapy significantly improved theglycogen content in the liver compared with that in either ofthe monotherapy groups Signaling pathways were studiedand the combination therapy effectively reduced hepatic


Vehicle Metformin Combination

p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin

Vehicle Metformin CombinationOleanolic acid

p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)

Figure 7 Analysis of the liver protein expression levels of markers associated with glucose metabolism (a) Effect of OA metformin andthe combination on the insulin-stimulated phosphorylation of Akt in mouse livers After the experiment the livers were harvested andthe phosphorylation of Akt PI3K and mTOR was measured using Western blotting (b) Effect of OA metformin and the combinationon the phosphorylation of AMPK and PCC in dbdb mouse livers was evaluated using Western blotting After the experiment the liverswere harvested and the Akt phosphorylation was measured using Western blotting (c) Effect of OA metformin and the combination ongluconeogenesis in mouse livers The protein expression of PEPCK1 G-6-Pase TORC and phospho-CREB was evaluated using Westernblotting

gluconeogenesis by decreasing the mRNA expression level ofPGC-1120572 G-6-Pase and PEPCK In addition the combinationtherapy significantly inhibited the mRNA levels of GP andGLUT2 and increased themRNA level of GS thus decreasinghepatic glucose production

AMPK is a heterotrimer composed of alpha-catalytic andbeta- and gamma-regulatory subunits some genes in thissignaling pathway are subject to alternative splicing whichincreases the range of possible heterotrimer combinations[33 34] Cellular stresses that inhibit ATP production orincrease its consumption change the AMP ATP ratio andactivate the pathway [35] A well-known role of AMP kinase

(AMPK) is its participation in the regulation of manymetabolic processes including glucose uptake and fatty acidoxidation in muscle and fatty acid synthesis and gluconeo-genesis in the liver Due to its roles in energy regulation theAMPK pathway is regarded as a potential therapeutic targetfor obesity and type 2 diabetes In this study we found thatOA and metformin induced AMPK and ACC phosphoryla-tionMetformin andOA inducedAMPKandACCactivationreduced the downregulation of lipogenic enzymes such asFAS and increased pyruvate kinase activity thus improvinginsulin resistance The activation of AMPK inhibited boththe transcription and translocation of PEPCK1 andG-6-Pase


Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR

Figure 8 Proposed mechanism by which oleanolic acid and metformin inhibit diabetes

resulting in an increase in insulin-stimulated glucose uptakeIn addition each of the monotherapies negatively regulatedseveral proteins that are central to ATP-consuming processessuch as processes that involve p-CREB and TORC2 resultingin the downregulation or inhibition of gluconeogenesis Asexpected the combination therapy group significantly acti-vated the phosphorylation of AMPK and ACC thus improv-ing the metabolic outcomes Meanwhile the group thatreceived the combination treatment had lower G-6-PasePEPCK1 TORC and p-CREB levels compared with thosein the diabetic control group or either monotherapy groupOverall these effects reduce gluconeogenesis

Akt is essential for insulin stimulating events such asglucose uptake and glycogen synthesis [36] Our resultssuggest that the combination therapy stimulates PI3K whichphosphorylates AKT and downregulates p-mTOR to improveinsulin resistance mTOR is the target of both components ofthe combination therapy and connects the AMPK and AKTpathways [37 38] and mTOR might be the key that inducedthe difference in action

In conclusion the present study shows the efficacy of acombination therapy using OA and metformin this combi-nation controls hypoglycemia by reducing both gluconeo-genesis and glycogenolysis In addition to reducing bloodglucose this therapeutic approach has a key advantage in itsmodulation of glycogen synthesis which may also have pre-ventive and therapeutic benefits against diabetes The combi-nation therapy-treated group showed a markedly enhancedphosphorylation of AMPK and AKT and the treatmentinduced decreased expression of proteins and genes thatare relevant to glucose metabolism Thus our combinationtherapy results in a net inhibition of gluconeogenesis and

glycogenolysis and reduces hepatic glucose production(Figure 8) these effects show its interesting potential as atreatment for diabetes

Abbreviations

GS Glycogen synthaseGP Glycogen phosphorylasePEPCK1 Phosphoenolpyruvate carboxykinase 1G-6-Pase Glucose-6-phosphataseGLUT2 Glucose transporter type 2PGC-1120572 PPAR120574 coactivator 1120572AMPK AMP-activated protein kinaseACC Acetyl-CoA carboxylaseTORC Transducer of regulated CREBmTOR Mammalian target of rapamycinCREB cAMP-response element-binding protein

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the National Natural ScienceFoundation of China (Grant 81373303)

References

[1] B Paulweber P Valensi J Lindstr NM Lalic andC J GreavesldquoEurope evidence-based guidline for the prevention of type 2


diabetesrdquoHormone andMetabolic Research vol 42 supplement1 pp S3ndashS36 2010

[2] R A DeFronzo D Simonson and E Ferrannini ldquoHepatic andperipheral insulin resistance a common feature of Type 2 (non-insulin-dependent) and Type 1 (insulin-dependent) diabetesmellitusrdquo Diabetologia vol 23 no 4 pp 313ndash319 1982

[3] C Postic R Dentin and J Girard ldquoRole of the liver in the con-trol of carbohydrate and lipid homeostasisrdquo Diabetes andMetabolism vol 30 no 5 pp 398ndash408 2004

[4] D M Nathan J B Buse M B Davidson and et al ldquoMedicalmanagement of hyperglycemia in type 2 diabetes a consensusalgorithm for the initiation and adjustment of therapy aconsensus statement of the American Diabetes Association andthe European Association for the Study of Diabetesrdquo DiabetesCare vol 32 no 3 pp 193ndash203 2009

[5] M Foretz S Hebrard J Leclerc et al ldquoMetformin inhibits hep-atic gluconeogenesis inmice independently of the LKB1AMPKpathway via a decrease in hepatic energy staterdquo The Journal ofClinical Investigation vol 120 no 7 pp 2355ndash2369 2010

[6] X A Wen P Zhang J Liu et al ldquoPentacyclic triterpenesPart 2 synthesis and biological evaluation of maslinic acidderivatives as glycogen phosphorylase inhibitorsrdquo Bioorganicand Medicinal Chemistry Letters vol 16 no 3 pp 722ndash7262006

[7] J Liu H Sun W Duan D Mu and L Zhang ldquoMaslinic acidreduces blood glucose in KK-Ay micerdquo Biological and Pharma-ceutical Bulletin vol 30 no 11 pp 2075ndash2078 2007

[8] J Liu H B Sun X F Wang D Mu H Liao and L ZhangldquoEffects of oleanolic acid and maslinic acid on hyperlipidemiardquoDrug Development Research vol 68 no 5 pp 261ndash266 2007

[9] J Chen J Liu L Zhang et al ldquoPentacyclic triterpenes Part 3synthesis and biological evaluation of oleanolic acid derivativesas novel inhibitors of glycogen phosphorylaserdquo Bioorganic andMedicinal Chemistry Letters vol 16 no 11 pp 2915ndash2919 2006

[10] J Liu T He Q Lu J Shang H B Sun and L Y Zhang ldquoAsiaticacid preserves beta cell mass and mitigates hyperglycemiain streptozocin-induced diabetic ratsrdquo DiabetesMetabolismResearch and Reviews vol 26 no 6 pp 448ndash454 2010

[11] L Y Zhang J Chen Y C Gong J Liu W Hua and H SunldquoSynthesis and biological evaluation of asiatic acid derivativesas inhibitors of glycogen phosphorylasesrdquo Chemistry amp Biodi-versity vol 6 no 6 pp 864ndash874 2009

[12] X Wen H Sun J Liu et al ldquoNaturally occurring pentacyclictriterpenes as inhibitors of glycogen phosphorylase synthe-sis structure-activity relationships and X-ray crystallographicstudiesrdquo Journal ofMedicinal Chemistry vol 51 no 12 pp 3540ndash3554 2008

[13] J Liu ldquoPharmacology of oleanolic acid and ursolic acidrdquo Journalof Ethnopharmacology vol 49 no 2 pp 57ndash68 1995

[14] J Liu ldquoOleanolic acid and ursolic acid research perspectivesrdquoJournal of Ethnopharmacology vol 100 no 1-2 pp 92ndash94 2005

[15] P DzubakM Hajduch D Vydra et al ldquoPharmacological activ-ities of natural triterpenoids and their therapeutic implicationsrdquoNatural Product Reports vol 23 no 3 pp 394ndash411 2006

[16] L Z Xu and Z X Wan ldquoThe effect of oleanolic acid on acutehepatitis (70 cases)rdquoHumane Medicine vol 7 pp 50ndash52 1980

[17] S L Xu ldquoEffects of oleanolic acid on chronic hepatitis 188 casereportsrdquo in Symposium on Oleanolic Acid pp 23ndash25 1985

[18] D M Minich J S Bland J Katke et al ldquoClinical safety andefficacy of NG440 a novel combination of rho iso-alpha acidsfrom hops rosemary and oleanolic acid for inflammatory

conditionsrdquo Canadian Journal of Physiology and Pharmacologyvol 85 no 9 pp 872ndash883 2007

[19] D R Matthews J P Hosker A S Rudenski B A Naylor D FTreacher and R C Turner ldquoHomeostasis model assessmentinsulin resistance and 120573-cell function from fasting plasmaglucose and insulin concentrations in manrdquo Diabetologia vol28 no 7 pp 412ndash419 1985

[20] R D Lillie and J Greco ldquoMalt diastase and ptyalin in place ofsaliva in the identification of glycogenrdquoBiotechnicampHistochem-istry vol 22 no 2 pp 67ndash70 1947

[21] N Wollen and C J Bailey ldquoInhibition of hepatic gluconeo-genesis by metformin Synergism with insulinrdquo BiochemicalPharmacology vol 37 no 22 pp 4353ndash4358 1988

[22] X Wang X-L Ye R Liu et al ldquoAntioxidant activities ofoleanolic acid in vitro possible role of Nrf2 and MAP kinasesrdquoChemico-Biological Interactions vol 184 no 3 pp 328ndash3372010

[23] X Wang Y-L Li H Wu et al ldquoAntidiabetic effect of oleanolicacid a promising use of a traditional pharmacological agentrdquoPhytotherapy Research vol 25 no 7 pp 1031ndash1040 2011

[24] P S Ngubane B Masola and C T Musabayane ldquoThe effectsof syzygium aromaticum-derived oleanolic acid on glycogenicenzymes in streptozotocin-induced diabetic ratsrdquoRenal Failurevol 33 no 4 pp 434ndash439 2011

[25] K Sharma P McCue and S R Dunn ldquoDiabetic kidney diseasein the dbdbmouserdquoTheAmerican Journal of PhysiologymdashRenalPhysiology vol 284 no 6 pp 1138ndash1144 2003

[26] K S Oh E Y Kim M Yoon and C M Lee ldquoSwim trainingimproves leptin receptor deficiency-induced obesity and lipiddisorder by activating uncoupling proteinsrdquo Experimental andMolecular Medicine vol 39 no 3 pp 385ndash394 2007

[27] N Ghilardi S Ziegler A Wiestner R Stoffel M H Heim andR C Skoda ldquoDefective STAT signaling by the leptin receptor indiabetic micerdquo Proceedings of the National Academy of Sciencesof the United States of America vol 93 no 13 pp 6231ndash62351996

[28] S Chandran F Yap and K Hussain ldquoMolecular mechanismsof protein induced hyperinsulinaemichypoglycaemiardquo WorldJournal of Diabetes vol 5 no 5 pp 666ndash677 2014

[29] T Pelikanova ldquoInsulin resistancemdashits causes and therapy pos-sibilitiesrdquo Vnitrnı Lekarstvı vol 60 no 9 pp 746ndash755 2014

[30] L Agius ldquoPhysiological control of liver glycogen metabolismlessons from novel glycogen phosphorylase inhibitorsrdquo Mini-Reviews in Medicinal Chemistry vol 10 no 12 pp 1175ndash11872010

[31] NGaboriaud-Kolar andA-L Skaltsounis ldquoGlycogenphospho-rylase inhibitors a patent review (2008ndash2012)rdquo Expert OpiniononTherapeutic Patents vol 23 no 8 pp 1017ndash1032 2013

[32] J C Hutton and R M OrsquoBrien ldquoGlucose-6-phosphatase cat-alytic subunit gene familyrdquoThe Journal of Biological Chemistryvol 284 no 43 pp 29241ndash29245 2009

[33] D Stapleton K I Mitchelhill G Gao et al ldquoMammalian AMP-activated protein kinase subfamilyrdquo The Journal of BiologicalChemistry vol 271 no 2 pp 611ndash614 1996

[34] A Woods P C F Cheung F C Smith et al ldquoCharacterizationof AMP-activated protein kinase 120573 and 120574 subunits assembly ofthe heterotrimeric complex in vitrordquo The Journal of BiologicalChemistry vol 271 no 17 pp 10282ndash10290 1996

[35] W W Winder and D G Hardie ldquoInactivation of acetyl-CoAcarboxylase and activation of AMP-activated protein kinase in


muscle during exerciserdquo The American Journal of PhysiologymdashEndocrinology and Metabolism vol 270 no 2 pp E299ndashE3041996

[36] E Hajduch G J Litherland and H S Hundal ldquoProtein kinaseB (PKBAkt)mdasha key regulator of glucose transportrdquo FEBSLetters vol 492 no 3 pp 199ndash203 2001

[37] E Sarnowska A Balcerak M Olszyna-Serementa D KotlarekT J Sarnowski and J A Siedlecki ldquoAmp-activated proteinkinase (AMPK) as therapeutic targetrdquo Postępy Higieny i Medy-cyny Doswiadczalnej vol 67 pp 750ndash760 2013

[38] A R Aroor SMcKarns V G Demarco G Jia and J R SowersldquoMaladaptive immune and inflammatory pathways lead to car-diovascular insulin resistancerdquoMetabolism Clinical and Exper-imental vol 62 no 11 pp 1543ndash1552 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


HO

H

COOH

Figure 1 The structure of OA

showed that OA can inhibit GP and its hypoglycemic effect ispartially mediated by the inhibition of glycogenolysis in vivo[17 18]

The complicated pathogenesis of diabetes suggests that acombination therapy may be more effective in treating thisdisease In this study we test whether a combination therapyusing OA and metformin would have synergistic effects onimproving glucose metabolism in diabetic conditions

2 Materials and Methods

21 Drugs and Chemicals Oleanolic acid (structure shown inFigure 1) was obtained from the Center for Drug Discoveryat China Pharmaceutical University Metformin (purity gt98) was obtained from Sigma (St Louis MO USA)The mouse insulin enzyme immunoassay ELISA kit waspurchased from Invitrogen Serum triglyceride (TG) totalcholesterol (TC) high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) kits werepurchased from Whitman Biotech (Nanjing Ltd China)Antibody (Ab) for G-6-Pase was purchased from Santa CruzBiotechnology (Santa Cruz CA USA) TORC2 was fromMillipore (Billerica MA USA) and phospho-CREB and120573-actin were from Abcam (Cambridge MA USA) TheAbs against protein kinase B (PKBAkt) phospho-Ser473-Akt (pAkt) PI3K phospho-PI3K AMPK phosphor-AMPKACC phosphor-ACC PEPCK1 mTOR and phospho-mTORwere purchased from Cell Signaling Technology (BeverlyMA USA) The HRP-conjugated secondary Abs affinity-purified mouse anti-rabbit IgG and rabbit anti-mouse IgGwere purchased from Sigma (St Louis MO USA)

22 Animals and Treatments Male C57BLKsJ-Lepdb (dbdb) mice were obtained from the Model Animal ResearchCenter of Nanjing University China The animals weremaintained in a 12 h light-dark cycle at a temperature of 25∘Cwith free access to water and regular chow To investigatethe effect of OA and metformin the seven-week-old dbdbmale mice were divided into four groups (119899 = 6group)

The mice were given vehicle (025 wv CMC) OA(250mgkg) metformin (100mgkg) or a combination of thetwo drugs once daily by intragastric administration for 28days The body weight and food intake were recorded reg-ularly After 8 hours of fasting blood glucose concentrationsweremonitored using tail vein blood and a glucometer (One-Touch Ultra Lifescan) every week The mice were fasted for12 hours for an oral glucose tolerance test (OGTT day 21)with the slight modifications described in the figure legendBlood samples were obtained from the tail vein and theserum insulin concentration was measured using an ELISAkit At the end of the experiment (day 28) the mice werefasted for 8 hours and blood samples were collected from theorbital venous plexus and centrifuged for serum separationThe animals were euthanized by CO

2inhalation and the

liver tissue was excised and stored in 4 paraformaldehydefor further analysis All of the animal experiments wereconducted according to the Guidelines of ExperimentalAnimal Care issued by the Committee for the Purpose ofControl and Supervision of Experiments on Animals

23 Biochemical Parameter Analysis At the end of the exper-iment the blood glucose levels and the serum levels of triglyc-eride (TG) total cholesterol (TC) high-density lipoproteincholesterol (HDL-C) and low-density lipoprotein cholesterol(LDL-C) were measured using commercially available kitsfollowing the manufacturerrsquos protocol The serum concen-tration of insulin was measured using ELISA followingthe protocol provided in the kit The homeostatic modelassessment (HOMA) value was calculated as described byMatthews et al [19] Insulin resistance was determined usingthe HOMA method and the following equation HOMAvalue for insulin resistance (HOMA-IR) = fasting insulin(mUmL) lowast fasting glucose (mmolL)225 The liver indexwas calculated as follows and used to assess the organhealth and hepatic histological damage liver index = liverweightbody weight To evaluate the glycogen content of thetissue the method of Lillie and Greco was used [20] Liversamples were homogenized in 50mM TrisHCl buffer atpH 74 containing 150mM NaCl 1mM EDTA and 1mMPMSF The liver triglyceride and total cholesterol contentswere analyzed using the previously described protocol

24 Hepatic Histology The tissue was subsequently embed-ded in paraffin and sectioned to a thickness of 5mm usinga microtome Tissue sections prepared on aminosilane-treated slides were deparaffinized and dehydrated using agraded alcohol series and distilled water The tissue sectionswere stained with hematoxylin and eosin The glycogencontent was determined by comparative PAS staining withor without prior amylase treatment (Histoserv GermantownMD) For the detection of immunofluorescence the slideswere incubated with a mouse monoclonal 120572-actin antibody(1 1000 Santa Cruz Biotechnology Inc) together with a rab-bit antibody for G-6-Pase (1 500 Santa Cruz BiotechnologyInc) and a rabbit antibody for PEPCK1 (Cell Signaling)followed by a TRITC-conjugated goat anti-mouse antibodyand a FITC-conjugated goat anti-rabbit antibody (1 100Huawei ImmunoResearch Laboratories Inc) After thorough










3 Results









Metformin(250mgkg)











Bloo

d gl

ucos

e con

cent

ratio

n 40

30

20

10



lowastlowastlowast

(mm

olL

)

(a)


lowastlowastlowast

8

6

4

2

0

Seru

m in

sulin

conc

entr

atio

n (120583

gL) lowast

(b)


lowastlowastlowast


MA-

IR

400

300

200

100

0

lowast

(c)





80

60

40

20

0Bloo

d gl

ucos

e con

cent

ratio

n (m

mol

L)

0 30 60 90 120 150

Time (min)

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

8000

6000

4000

2000

0

Are

a und

er cu

rve

(mm

ollowast0

ndash120

min

)

(b)







4 Discussion





(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)







Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results









Metformin(250mgkg)











Bloo

d gl

ucos

e con

cent

ratio

n 40

30

20

10



lowastlowastlowast

(mm

olL

)

(a)


lowastlowastlowast

8

6

4

2

0

Seru

m in

sulin

conc

entr

atio

n (120583

gL) lowast

(b)


lowastlowastlowast


MA-

IR

400

300

200

100

0

lowast

(c)





80

60

40

20

0Bloo

d gl

ucos

e con

cent

ratio

n (m

mol

L)

0 30 60 90 120 150

Time (min)

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

8000

6000

4000

2000

0

Are

a und

er cu

rve

(mm

ollowast0

ndash120

min

)

(b)







4 Discussion





(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)







Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Metformin(250mgkg)











Bloo

d gl

ucos

e con

cent

ratio

n 40

30

20

10



lowastlowastlowast

(mm

olL

)

(a)


lowastlowastlowast

8

6

4

2

0

Seru

m in

sulin

conc

entr

atio

n (120583

gL) lowast

(b)


lowastlowastlowast


MA-

IR

400

300

200

100

0

lowast

(c)





80

60

40

20

0Bloo

d gl

ucos

e con

cent

ratio

n (m

mol

L)

0 30 60 90 120 150

Time (min)

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

8000

6000

4000

2000

0

Are

a und

er cu

rve

(mm

ollowast0

ndash120

min

)

(b)







4 Discussion





(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)







Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















80

60

40

20

0Bloo

d gl

ucos

e con

cent

ratio

n (m

mol

L)

0 30 60 90 120 150

Time (min)

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

8000

6000

4000

2000

0

Are

a und

er cu

rve

(mm

ollowast0

ndash120

min

)

(b)







4 Discussion





(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)







Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

(b)

Live

r gly

coge

n co

ncen

trat

ion

(mg

g)

lowastlowastlowast

25

20

15

10

5


(c)







Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Combination


Vehicle

Oleanolic

Metformin

acid

(a)

Combination


Vehicle

Metformin

Oleanolicacid

(b)




15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















15

10

05

00

Gly

coge

n ph

osph

oryl

ase


lowastlowastlowast

mRN

A ex

pres

sion

(a)


15

10

05

00

20

Gly

coge

n sy

ntha

se

lowastlowast

mRN

A ex

pres

sion

(b)


15

10

05

00

PGC-1120572

mRN

A ex

pres

sion

lowastlowastlowast

lowast


15

10

05

00

G-6

-Pas

em

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

lowast

(d)


15

10

05

00

PEPC

K1m

RNA

expr

essio

n

lowastlowastlowast

lowastlowastlowast

(e)


15

10

05

00

GLU

T2m

RNA

expr

essio

n


(f)







p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















p-PI3K

p-AKT

t-PI3K

p-mTOR

t-mTOR

t-AKT

Oleanolic acid

120573-actin

(a)

120573-actin


p-AMPK

p-ACC

t-AMPK

t-ACC

(b)


120573-actin

PEPCK

TORC

G-6-Pase

p-CREB

(c)






Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Insulin

Insulinreceptor

Glucose

GLUT2

Metformin

OCT1

[AMP][ATP]

Oleanolic acid

AMPK

P ACC

P TORC2

Nucleus CREBP

PGC-1120572

PEPCK

Gluconeogenesis

SREBP-1

GS

Lipogenesis

Steatosis

Insulin resistance

Glucose

G-6-Pase

Glycogen

PI3K

AKT

mTOR






Abbreviations




Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Combination Therapy with Oleanolic Acid...

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