J Food Biochem. 2019;00:e13049. wileyonlinelibrary.com/journal/jfbc  | 1 of 11https://doi.org/10.1111/jfbc.13049

© 2019 Wiley Periodicals, Inc.

Received:24June2019  |  Revised:23August2019  |  Accepted:4September2019DOI: 10.1111/jfbc.13049

F U L L A R T I C L E

Berberine chloride ameliorated PI3K/Akt‐p/SIRT‐1/PTEN signaling pathway in insulin resistance syndrome induced in rats

Marwa El‐Zeftawy1,2  | Doaa Ghareeb2,3  | Eman R. ElBealy4 | Rasha Saad2 | Salma Mahmoud2 | Nihal Elguindy5  | Attalla F. El‐kott6,7  | Mohamed El‐Sayed5

1Biochemistry Department, Faculty of Veterinary Medicine, The New Valley University, New Valley, Egypt2BiologicalScreeningandPreclinicalTrialLab, Biochemistry Department, Faculty of Science,AlexandriaUniversity,Alexandria,Egypt3PharmaceuticalandFermentationIndustries Development Center, General AuthorityofCityofScientificResearchandTechnologyApplications,Alexandria,Egypt4BiologyDepartment,CollegeofScienceforgirls,KingKhalidUniversity,Abha,SaudiArabia5Biochemistry Department, Faculty of Science,AlexandriaUniversity,Alexandria,Egypt6Biology Department, College of Science,KingKhalidUniversity,Abha,SaudiArabia7Zoology Department, College of Science,DamanhourUniversity,Damanhour,Egypt

CorrespondenceMarwa El‐Zeftawy, Biochemistry Department, Faculty of Veterinary Medicine, The New Valley University, New Valley, Egypt.Email: marwa@vetnv.au.edu.eg, marwa_3_1983@yahoo.com

Funding informationdeanshipofScientificResearchatKingKhalidUniversity,Abha,KSAforsupported this work under grant number (R.G.P.1/117/40),Grant/AwardNumber:R.G.P.1/117/40

AbstractThe liver is the main organ involved in lipid metabolism process and it helps in drug detoxification.Insulinresistanceisconsideredoneofriskreasonswhichleadtosev‐eralmetabolicdiseases.Currently,berberine(BER)occupiesahugechallengeagainstmultiplediseaseswithnotoxiceffect.Thepresentworkwasaimedtoidentify,doesBER‐chloride has a poisonous influence on the liver? and investigating the outcome ofBER‐chlorideonPI3K/Akt‐p/SIRT‐1/PTENpathwayduringinsulinresistancesyn‐drome.The insulin resistancemodelwasachieved inexperimental female ratsviahigh‐fatdiet (HFD).Glucose, insulin, lipidprofiles,andhepaticoxidativestresspa‐rameterswereassessed.PI3K,AKt‐p,SIRT‐1,andPTENlevelsinhepatictissueweredetermined at genome and protein levels. Further adiponectin concentration was performed in serum, hepatic, and white adipose tissues. Molecular study of fold al‐terationininsulin,insulinreceptor,andretinolbindingprotein‐4(RBP4)inliverwasdone.

Practical applicationsObesity syndrome causes multiple obstacles in modern years. The current results revealed elevation the body weight of rats, plasma glucose, homeostatic model as‐sessment, glycated hemoglobin, insulin, and lipid profiles concentrations in a group of rats, which nourished HFD for 8 weeks and this rise, was diminished after 2 weeks from BER‐chloride administration. Further, BER‐chloride improved transaminases enzymes, pro‐oxidant, and antioxidant defense system, PI3K, AKt‐p, SIRT‐1, andPTENintheliver,withdownregulationofhepaticRBP4.Hence,thesedataprovideacrucial message that BER‐chloride enhanced both hepatic function and insulin signal‐ing pathways that might be of therapeutic importance to insulin resistance with no harmful effect on the liver. BER‐chloride is predicted to be a drug of choice for obe‐sity complications cure.

K E Y W O R D S

berberinechloride,hepatotoxicity,insulinreceptor,liver

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1  | INTRODUC TION

Obesity represents one of the severe health trouble worldwide and normally linked with hepatic insulin resistance and hyperglycemia (Kahn, Hull, & Utzschneider, 2006; Ren, Zhou, Huang, Wang, & Li, 2019).Multiplemetabolicsyndromesashyperlipidemia,typeIIdia‐betes mellitus, nonalcoholic fatty liver, and cardiovascular disorders areaccompaniedtoinsulinresistancesyndrome.Allthesemetabolicsyndromes may lead to bad prognosis and early mortality in some diseasedindividuals(David,Rifkin,Rabbani,&Ceradini,2017).PI3K/Aktisoneofthepathwayswhichinvolvedinmetabolicconsequenceofinsulin(Gaoetal.,2016).Thoughnumerouspharmaceuticaldrugsare available for chronic diseases cure, but these drugs show harmful side effects with long term of use due to their poisonous effect on bodyorgans(Kocetal.,2019;Peter,Basha,Giridharan,Lavinya,&Sabina,2017).

Berberine (BER) is a natural alkaloid isoquinoline sequesteredfrom different plants like Berberis vulgaris(Jiang,Li,&Li,2015).BERis a strong base which is usually unstable when present in free form so it usually accompanied with chloride ion in the form of BER‐chlo‐ride(Bodiwala,Sabde,Mitra,Bhutani,&Singh,2011;Li,Yuan,Shang,&Guo,2017).BERactsasananticancer (Liuetal.,2015),anti‐in‐flammatory(Liuetal.,2015;Vuddanda,Chakraborty,&Singh,2010),anti‐leishmanial (Vuddanda et al., 2010; Zhang et al., 2016), andanti‐immunodeficiencyvirusinhumans(Dongetal.,2011).BERalsoimproved some cardiac syndromes and intestinal infections (Zhang etal.,2016).RecentlyBERusedasaneuroprotectiveagentagainstsome neurodegenerative disorders as it has the capability of passing theblood–brainbarrier (Jiangetal.,2015).To investigatewhetherBER‐chloride has a protective influence on insulin resistance syn‐drome, the current study sets up in vivo animal model using high‐fat diet (HFD) feeding. The effects ofBER‐chloride on the numerousinsulin signaling pathway were investigated.

2  | MATERIAL S AND METHODS

2.1 | Materials

BER‐chloridewasgotfromSigma‐AldrichChemicalCo.(USA).Kitsofbloodglucoselevel(BGL),protein,lipidprofiles(totalcholesterol“TC,” triacylglycerol “TG,” and high density lipoprotein cholesterol “HDL‐c”),glycatedhemoglobin(HbA1C),aswellascreatinine,urea,alanine aminotransfersase (ALT), and aspartate aminotransferase(AST)kitswerepurchasedfromSpinreact(Spain),Human(Germany),Biosystem (Egypt), and Biolabo (France), respectively. Ribonucleicacid(RNA)extractionkit,Maximereversetranscription(RT)premixkit,2xTaqmastermix,deoxyribonucleicacid(DNA)Ladder,RNase‐freewater,andtheprimersequencesofβ‐actin, insulin, insulin recep‐tor (IR),andretinolbindingprotein‐4 (RBP4)werepurchasedfromQiagen (Germany), Intron Biotechnology (Korea) and Fermentas,Thermofisherscientific(Germany),respectively.Additionally,prim‐ersofphosphatidylinositol‐3‐kinase(PI3K),phosphorylatedproteinkinaseB(Akt‐p),sirtuintype1(SIRT‐1),andphosphataseandtension

homolog(PTEN)werepurchasedfromBiovision(USA).ELISAkitofinsulin,PI3K,AKt‐p,SIRT‐1,PTEN,andadiponectinwerepurchasedfromDRG(USA),WuhanFineBiologicalTechnologyCo.(China),RayBiotech (Georgia), MyBiosource (USA), Abcam (USA), Bioscience(USA), respectively. Foline reagent, thiobarbituric acid (TBA), re‐ducedglutathione(GSH),5′5′dithio‐bis‐2‐nitrobenzoicacid(DTNB),cumenehydroxide,methylgreen,polyethyleneglycol (PEG),nitro‐cellulosemembrane,sodiumdodecylsulfate,acrylamide,andBCIP/NBTsubstratesolution,premixedwereobtainedfromSigma‐AldrichChemicalCo. (St.Louis,Mo,USA).Organicsolvents,namelyetha‐nol95%,methanol,andformalinsolutionswereofHPLC‐gradeandbroughtfromMerck(USA).Otherreagentswereobtainedwithhighgrade.Primaryantibodiesofβ‐actin,Akt‐p,andPTENweregotfromBiotechne(USA),whilePI3KandSIRT‐1wereobtainedfromAbcam(USA).AlsomouseandrabbitsecondaryantibodieswerepurchasedfromBiotechne (USA) andAbcam (USA). Proteinmarkerwas pur‐chasedfromBiovision(USA).

2.2 | Experimental animal protocol and sample preparation

FemalealbinoSprague‐Dawleyrats(Rattus norvegicus),withweightrangedfrom130to150gandaged(10–12)weeksold,weretakenfromtheexperimentalanimalhouseofMedicalResearchInstitute,AlexandriaUniversity,Egypt.Ratswerekeptinpolycarbonatecages(sixratspercage).Theywerekeptunderconventionalenvironmentsof temperature and humidity with a 12‐hr photoperiod. Diet and waterwere provided ad libitum. The experimental ratswere han‐dled in agreement with the National Organizations of Health Guide fortheCareandUseofLaboratoryAnimals(NIH1996).Thisworkwas done in firm accordance with the approvals in the Guide for the CareandUseofLaboratoryAnimals.Theprotocolwasapprovedac‐cordingtotheEthicsofAnimalHouseinMedicalTechnologyCenter,AlexandriaUniversity,Egypt.Alltheanimalswereadapted1weekbeforetheexperimentbegins.

Afterthat,30ratsweredividedinto5groups(sixratspergroup),Group1(Shamcontrolgroup)thatwerehealthyandfreefromanydisease and they fed the standard food for 10 weeks, Group 2 (Controlvehicle)fedonlow‐fatdiet(LFD)for8weeksthenreceived20%PEGby intragastric tube (10ml/kgBwt) for 2weeks.Group3 (Control BER‐chloride) fed LFD for 8 weeks, then orally givenBER‐chloridedissolvedin20%ofPEG(100mg/kgBwt)for2weeks.Group4(Inductiongroup)fedonHFDfor8weeks,thenorallygiven20%ofPEG(10ml/kgBwt)for2weeks.Group5(Inductiontreatedgroup) fed HFD for 8 weeks, then orally given BER‐chloride dis‐solvedin20%ofPEG(100mg/kgBwt)for2weeks.DosesofBER‐chlorideandPEGwhichwereusedinthisprotocolreferredtootherpreviousstudies(El‐Sayed,Ghareeb,Talat,&Sarhan,2013;Ghareebetal.,2015;Saleh,Attia,&Ghareeb,2018).

Rat's body weights were noticed in the first and last week of ther‐apy. Blood sampling and animal scarification were performed under anesthesia,andalleffortsweremadetodecreasesuffering.Aftertheendoftheexperimentalperiod,ratswerefastedovernightafter8‐hr,

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blood samples were collected in sodium fluoride tubes for measur‐ingoffastingBGL.Afterafullfastingperiod(12‐hr),bloodsampleswere collected, and then centrifuged at 3,000 rpm for 10 min. The obtainedserumwaskeptat−20°Cuntilanalyzed.Hepatictissuesandwhiteadiposetissuefromallgroupswereexcised immediatelyandwashed with ice‐cold saline. Homogenization was performed in 0.1M ofsodiumphosphate‐buffer,pH7.4(forhepatictissue)andin0.15Mofpotassiumchloride(forwhiteadiposetissue)(Steinberg,Vaughan,&Margolis, 1961). The homogenatewas centrifuged at 4,000 rpmfor15minat4°Candsupernatantwaskeptat−80°Cuntilanalysis(Pandian, Anuradha, &Viswanathan, 2002). In each group, part ofhepatictissuewaspreservedinliquidnitrogen,andstoredat−80°Cfor totalRNA isolationandpolymerasechain reaction (PCR)analy‐sis.Anotherpartofliverandwhiteadiposetissuewerefixedat10%neutralbufferedformalinsolutionforhistopathologicalexamination.

2.3 | Biochemical, molecular, and histopathological studies

2.3.1 | Biochemical and molecular studies

BGL was determined by a glucose assay kit that is dependent on glucose oxidase‐peroxidase method. Serum insulin was analyzedthroughtheDRGinsulinELISAkit.Theinsulinresistancewasevalu‐atedbycalculatingtheHOMA‐IRaspreviouslydescribed(Hanetal.,2018).HbA1C%wasassessedbyBiosystemkit.Totalproteincon‐centration was measured spectrophotometrically using Beirut assay basedkit.Serumlipidprofile(TC,TG,andHDL‐c),urea,creatinine,ALT,andASTwereperformedaccordingtotheinstructionsoftheircommercial kits. LDL‐c and vLDL‐c levels were calculated according to previous studied formula by Friedewald, Levy, and Fredrickson (1972).Standardizedmethodswereusedtodeterminetheconcen‐trationofthiobarbituricacidreactivesubstances(TBARS)(Tappel&Zalkin,1959)andGSHinliver(Ellman,1959).

Inaddition,hepaticactivitiesofxanthineoxidase(XO)(Litwack,Bothwell,Williams,&Elvehjem,1953),glutathioneperoxidase(GPx)(Chiu,Stults,&Tappel,1976;Paglia&Valentine,1967),andadeninetriphosphatase (ATPase) (Candeias,Abreu,Pereira,&Cruz‐Morais,2009)weredone.PI3K,Akt‐p,SIRT‐1,andPTENconcentrationsinhepatic tissue homogenate and adiponectin level in serum, liver ho‐mogenate,andwhiteadiposetissuewereestimatedusingELISAkits.Thesetestsemploythequantitativesandwichenzymeimmunoassaytechnique.

TotalRNAwasisolatedfromliverusingtotalRNAextractionKitandprocessedaccordingtokitmanufacturer'sdirections.AfterthattheleveloftotalRNAwasmeasuredbyspectrophotometerat260and280nm.OnemicrogramoftheisolatedRNAwasreversetran‐scribed into complementaryDNA (cDNA) using reverse transcrip‐tase(MaximeRTPre‐Mixkit,Fermentas,EU).

Primers used for PCR technique were constructed using theknown sequences for the respective genes. The gene‐specific primer sequences were as follows: β‐actin forward primer 5′‐CAT CACTATCGGCAATGAGC‐3′ and reverse primer 5′‐GACAG

CACTGTGTTGGCATA‐3′, RBP4 forward primer 5′‐TTTTCTGTG GACGAGAAGGGT‐3′ and reverse primer 5′‐TGGTCATCGTTTCCT CGCTTG‐3′,IRforwardprimer5′‐TGACAATGAGGAATGTGGGGAC‐3′andreverseprimer5′‐GGGCAAACTTTCTGACAATGACTG‐3′, insulin forward primer 5′‐TTCTACACACCCAAGTCCCGTC‐3′ and reverse primer 5′‐ATCCACAATGCCACGCTTCTGC‐3′, PI3K forward primer 5′‐AACACAGAAGACCAATACTC‐3′, and reverse primer 5′‐TTCGCC ATCTACCACTAC‐3′,Akt‐pforwardprimer5′‐GTGGCAAGATGTGTAT GAG‐3′,andreverseprimer5′‐CTGGCTGAGTAGGAGAAC‐3′,SIRT‐1forwardprimer5′‐TGAAGCTGTTCGTGGAGATATTTTT‐3′,andreverse primer5′‐CATGATGGCAAGTGGCTCAT‐3′,andPTENforwardprimer 5′‐CCCAGTTTGTGGTCTGCCAGC‐3′andreverseprimer5′‐ATGAGCTTGTCCTCCCGCCG‐3′.

PCRprogramwasoptimizedfortheprimerpairandthePCRpro‐tocolwas40cycles,eachconsistedof30sdenaturationat95°C,30sannealingat52.5°C,51.5°C,50°C,52°C,58°C,58.8°C,63.9°C,and68.9°Cforβ‐actin,RBP4,IR,insulin,PI3K,Akt‐p,SIRT‐1,andPTEN,respectively,and60sextensionat72°C.TheprimerswererunontheMiniCycler(Eppendorf,Labcaire,Germany).ThePCRproductswere run on 1.5% agarose gel. Gels were stained with ethidium bro‐mide,visualizedby30nmUltravioletRadiator(Alpha‐Chem.Imager,USA),andphotographicrecordweremade.TheopticaldensityandthemicrogramcontentofbandswerecalculatedbytheUVIBANDMAXsoftwareprogram.

Westernblotwasperformedtodeterminetheproteinlevelex‐pressionofPI3K,Akt‐p, SIRT‐1, andPTEN inhepatic tissue.Afterseparation of protein (100 μgofeachsamplewasused)bysodiumdodecyl sulfate poly acrylamide gel electrophoresis (SDS‐PAGE)technique, the gel was transferred to nitrocellulose membrane.The membrane was incubated with labeled antibodies specific for the protein of interest and β‐actin was used as an internal control. Following adding the secondary antibody and incubation time, the unbound antibody was washed off, leaving only antibody which bounded to the protein of interest so only one band was visible and its thickness was corresponded to the amount of protein which pres‐entinasampleanditwasdetectedusingBCIP/NBTsubstratesolu‐tion. The intensities of obtaining bands were estimated using image analyzingsystem(UVIBANDMAXsoftware).

2.3.2 | Histopathological study

Hepatic and white adipose tissues of each rat from each group was excised and immediately fixed at 10% neutral buffered formalinsolution after washing with ice‐cold normal saline. The resultant fixed tissue samples were used for histopathological examinationin the Histopathology Laboratory of Medical Technology Center, Alexandria University, using the routine procedures developed inthe respective laboratories. The tissue was cut at 3 mm thick, and the blocks were embedded in paraffin. Using a rotary microtome, sections of 8 µm thickness were cut. The sections were stained with hematoxylin and eosin and examined under Olympusmicroscope(Olympus,Tokyo,Japan)at(100X)magnificationforanyhistopatho‐logical changes.

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2.4 | Statistical analysis

DatawereanalyzedusingPrimerofBiostatisticssoftwareprogram(Version5.0)byone‐wayANOVA.Significanceofmeans±SD was detected via using the multiple comparisons Student–Newman–keuls test at p<.05.AdiponectincorrelationwasanalyzedbySPSS(Version20.0)softwareprogram,usingpersoncoefficient(r).

3  | RESULTS

3.1 | Body weight, BGL, and insulin resistance parameters

Feeding of HFD for 8‐weeks leads to body weight elevation more than control levels. Fasting BGL, homeostatic model assessment in‐sulinreceptor(HOMA‐IR),andHbA1Cwerealsoelevated1.4,3.7,and45.2‐folds, in comparison to sham control rats. Moreover, elevation of insulin concentration was noticed in HFD group 1.0‐fold in serum and 72.3‐foldinliverhomogenate.AdministrationofBER‐chlorideleadstoreductionoffastingBGL,seruminsulin,HOMA‐IR,andHbA1Cto0.5,0.2,0.6,and0.2‐folds,respectively,comparedwithHFDrats(Table1).Moreover,HFDup regulatedexpressionof insulingene in liverandBER‐chloridetherapyfor2weekswasn'tsignificant(Figure1).

3.2 | Lipid profiles parameters

Lipid profile showed a significant rise in the HFD group than that of sham control where TC increased 1.2‐fold and TG, LDL‐c, and vLDL‐c were 0.9‐fold increase, while HDL‐c was significantly decreased by 43%. Lipid profiles were partially repaired after using BER‐chloride astherapyfor2weeks(Table2).

3.3 | Oxidative stress markers, serum transaminases activity, and kidney function tests

ExperimentalHFDratsexhibitedanelevationofTBARSandXO(1.5and0.8‐folds)incomparisontoshamcontrol.BothTBARSandXOwere decreased nearly 0.4‐ and 0.2‐folds after 2 weeks of BER‐chlo‐ridetreatment.However,GSH,GPx,andATPaseweredecreasedby

50.9%, 41.9%, and 35.2% in the HFD group compared to sham con‐trol and after administration of BER‐chloride therapy; those previ‐ous parameters were increased by percentage 42.9, 33.7, and 26.1, respectively (Table 3). Also, HFD intake increased liver transami‐nases (ALT and AST) and kidney function tests (urea and creati‐nine)comparingtoshamcontrolone.Administrationof100mg/kg

TA B L E 1  Effectofberberine(BER)chlorideonbodyweight,bloodglucoselevel(BGL),seruminsulin,homeostaticmodelassessmentinsulinresistance(HOMA‐IR),andglycatedhemoglobin(HbA1C)afterthetreatmentofdiabetic‐inducedexperimentalanimals

Groups Body weight (g) BGL (mg/dl)Serum insulin (µIU/ml) HOMA‐IR (mg/dl) HbA1C (%)

Shamcontrol 141.0±7.4a 76.0±1.4a 10.0±0.2a 1.91±0.061a 4.2±0.4a

Controlvehicle(LFD+20%PEG) 142.2±8.04a 73.33±2.6a 10.1±0.6a 1.9±0.13a 4.4±0.42a

Control BER‐chloride 144.0±6.07a 75.2±3.31a 10.45±0.33a 1.92±0.12a 4.0±0.24a

Induction(HFD+20%PEG(10ml/kgBwt)

202.3±5.39b 180.1±4.38c 20.17±2.93c 8.97±1.37c 6.1±0.39c

Induction treated with BER‐chloride

200.5±8.02b 97.7±5.61b 15.67±2.42b 3.76±0.48b 5.1±0.18b

Note: Valuesrepresentthemean±SDofsixrats.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

F I G U R E 1  Effectofberberine(BER)chlorideonthefoldchangeofinsulin,insulinreceptor(IR)andretinolbindingprotein‐4(RBP4)genes.(A)Agrosegelelectrophoresisofgeneexpressionofinsulin(293bp),IR(129bp),andRBP4(392bp)comparedtoβ‐actin (300bp).(B)Foldchangeofgeneexpressioninliverhomogenateafterthetreatmentofdiabetic‐inducedexperimentalanimalsrepresentedas6rats±SE.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),(c),and(d)werestatistically represented compared to sham control group as a at p < .001, b at p < .01, c at p < .05, and d at p > .05

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Bwt BER‐chloride in HFD rats for 2 weeks leads to decrease liver transaminases activities and kidney function tests nearly 0.3‐ and 0.2‐folds(Table4).

3.4 | Insulin signaling pathway parameters

As shown inTable5 andFigures1‒3 themarked significantde‐clineofPI3K,AKt‐pandSIRT‐1by57.1%,42.9%,and61.9%,wasnoticedinHFDratscomparedtoshamcontrolatELISAlevel,alsothey decreased by 76.2%, 77.5%, and 93.1% at genome level and by 92.9%, 42.1%, and 76.0% at protein level. Administration of

BER‐chloridetothatgroupofrats leadsto increasePI3K,AKt‐p,andSIRT‐1to84.4%,49%,and120%,respectively,atELISAlevel,also they 4.4‐, 0.78‐, 10.5‐ and 0.5‐folds increased at genome level and also they 8.5‐, 1.7‐ and 0.5‐folds raised at protein level. However,PTENwasincreasedinHFDratsby66.6%inELISAleveland 1.25‐ and 21‐folds elevated at genome and protein levels, respectively. After BER‐chloride treatment PTEN diminished to27.5%atELISAlevel,50%atgenomelevel,and74.2%atproteinlevel. Additionally, RBP4 was reduced from 1.5‐fold to 0.4‐foldafter BER‐chloride treatment. However, BER‐chloride failed to af‐fecttheHFDsideeffectonIRexpression.

TA B L E 2  Effectofberberine(BER)chlorideontotalcholesterol(TC),triacylglycerol(TG),high‐densitylipoproteincholesterol(HDL‐c),low‐densitylipoproteincholesterol(LDL‐c),andverylow‐densitylipoproteincholesterol(vLDL‐c)afterthetreatmentofdiabetic‐inducedexperimentalanimals

Groups TC (mg/dl) TG (mg/dl) HDL‐c (mg/dl) LDL‐c (mg/dl) vLDL‐c (mg/dl)

Shamcontrol 130.5±2.79a 110.3±3.6a 59.33±3.14a 110.33±3.62a 22.1±0.7a

Controlvehicle(LFD+20%PEG) 127.3±4.41a 110.0±4.47a 64.3±3.01a 41.0±3.74a 22.3±1.2a

Control BER‐chloride 129.3±2.25a 111.2±2.40a 63.2±2.56a 43.9±4.24a 21.77±0.5a

Induction(HFD+20%PEG (10ml/kgBwt)

282.2±3.31c 210.5±3.73c 33.3±2.88c 206.7±2.78c 42.9±0.4c

Induction treated with BER‐chloride

164.5±4.14b 141.5±2.26b 42.8±3.37b 93.4±6.20b 28.133±0.7b

Note: Valuesrepresentthemean±SDofsixrats.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

TA B L E 3  Effectofberberine(BER)chlorideonhepatocytethiobarbituricacidreactivesubstances(TBARS),reducedglutathione(GSH),xanthineoxidase(XO),glutathioneperoxidase(GPx),andadeninetriphosphatase(ATPase)statusafterthetreatmentofdiabetic‐inducedexperimentalanimals

GroupsTBARS (mmol/mg protein)

GSH (mg/mg protein)

XO (IU/mg protein)

GPx (U/mg protein)

ATPase (µmol/pi/min/mg protein)

Shamcontrol 2.1±0.02a 0.285±0.05a 128.0±1.6a 7.06±0.3a 0.71±0.02a

Controlvehicle(LFD+20%PEG) 2.03±0.04a 0.29±0.06a 130.17±4.26a 7.02±0.55a 0.73±0.01a

Control BER‐chloride 2.12±0.28a 0.29±0.05a 129.67±4.46a 7.17±0.22a 0.71±0.01a

Induction(HFD+20%PEG (10ml/kgBwt)

5.25±0.35c 0.14±0.02c 231.5±7.56c 4.1±0.33c 0.46±0.05c

Induction treated with BER‐chloride

3.15±0.24b 0.20±0.03b 171.3±3.83b 5.48±0.33b 0.58±0.03b

Note: Valuesrepresentthemean±SDofsixrats.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

TA B L E 4  Effectofberberine(BER)chlorideonserumalanineaminotransferase(ALT)andaspartateaminotransferase(AST)activitiesandkidneyfunctiontestsafterthetreatmentofdiabetic‐inducedexperimentalanimals

Groups ALT (U/ml) AST (U/ml) Urea (mg/dl) Creatinine (mg/dl)

Shamcontrol 33.5±2.1a 46.0±3.2a 33.4±2.3a 0.71±0.1a

Controlvehicle(LFD+20%PEG) 34.2±4.6a 49.3±1.8a 31.83±4.6a 0.72±0.04a

Control BER‐chloride 32.2±1.94a 49.5±3.78a 29.67±2.16a 0.71±0.04a

Induction(HFD+20%PEG(10ml/kgBwt) 58.5±5.09c 85.7±5.68c 56.17±2.14c 0.90±0.05b

Induction treated with BER‐chloride 40.8±3.71b 62.5±6.80b 39.83±3.49b 0.75±0.06a

Note: Valuesrepresentthemean±SDofsixrats.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

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3.5 | Adiponectin concentration

Adiponectinpercentageinserum,liver,andwhiteadiposetissueofHFDratswasreduced(52%,80%,and45%),andthispercentagewas

elevated after BER‐chloride administration for 2 weeks (48%, 385%, and65.3%),respectively(Table6).

3.6 | Histopathological results

The biochemical and molecular alterations were confirmed by the histopathological study in both liver and white adipose tissue (FiguresS1a–eandS2a–e).

TA B L E 5  Effectofberberine(BER)chlorideonphosphatidylinositol‐3‐kinase(PI3K),phosphorylatedproteinkinaseB(Akt‐p),Sirtuintype1(SIRT‐1),andphosphataseandtensionhomolog(PTEN)levelsinhepatocyteafterthetreatmentofdiabetic‐inducedexperimentalanimals

Groups PI3K (pg/g) Akt‐p (pg/g) SIRT‐1 (ng/g) PTEN (pg/g)

Shamcontrol 493.0±12.3c 350.0±23.5c 105.0±10.2c 1,200±28.9a

Controlvehicle(LFD+20%PEG) 486.0±12.0c 335.0±19.3c 110.0±6.9c 1,250.0±25.3a

Control BER‐chloride 491.0±23.5c 342.0±18.9c 109.0±9.7c 1,150.0±65.3a

Induction(HFD+20%PEG(10ml/kgBwt)

211.5±21.5a 200.0±12.1a 40.0±1.2a 2,000.0±28.9c

Induction treated with BER‐chloride 390.0±31.2b 298.0±14.2b 86.0±8.5b 1,450.0±23.5b

Note: Valuesrepresentthemean±SDofsixrats.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

F I G U R E 2  Effectofberberine(BER)chlorideonthefoldchangeofphosphatidylinositol‐3‐kinase(PI3K),phosphorylatedproteinkinaseB(Akt‐p),Sirtuintype1(SIRT‐1),andphosphataseandtensionhomolog(PTEN)genes.(A)AgrosegelelectrophoresisofgeneexpressionofPI3K(195bp),Akt‐p(195bp),SIRT‐1(82bp),andPTEN(79bp)comparedtoβ‐actin(300bp).(B)Foldchangeofgeneexpressioninliverhomogenateafterthetreatmentofdiabetic‐inducedexperimentalanimalsrepresentedas6rats±SE.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),(c)and(d)werestatisticallyrepresentedcomparedtosham control group as a at p < .001, b at p < .01, c at p < .05, and d at p > .05

F I G U R E 3  Effectofberberine(BER)chlorideontheproteinlevelsofphosphatidylinositol‐3‐kinase(PI3K),phosphorylatedproteinkinaseB(Akt‐p),Sirtuintype1(SIRT‐1),andphosphataseandtensionhomolog(PTEN).(A)WesternblotofproteinexpressionofPI3K(−85kDa),Akt‐p(−60kDa),SIRT‐1(−120kDa),andPTEN(−54kDa)comparedtoβ‐actin(−45kDa).(B)Proteinlevelsinliverhomogenateafterthetreatmentofdiabetic‐inducedexperimentalanimalsrepresentedas6rats±SE.ANOVA(one‐way)followedbyStudent–Newman–keulstest.Meanswithletters(a),(b),(c),and(d)were statistically represented compared to sham control group as a at p < .001, b at p < .01, c at p < .05, and d at p > .05

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FigureS1a–ediscoveredliver'shistopathologicaldataofallstud‐ied groups. The liver architecture in control vehicle and control BER‐chloride was normal in comparison with a sham control group (Figure S1a–c).Onthecontrary,pathologicalstudiesrevealedthattheliverof the HFD group contained obvious fat droplets, necrosis, and in‐flammation(FigureS1d).ForHFDtreatedgroupwithBER‐chloride,the necrosis and inflammation sites disappeared, and the steatosis decreasedfrommoderatetomildgrade(FigureS1e).

Further histopathological results of white adipose tissue were demonstratedin(FigureS2,a‐e).Controlrat'sexposednormaltissue(FigureS2a).BothPEGandBER‐chlorideadministratedgroupsweresimilartocontrolrats(FigureS2bandS2c).However,adiposetissueof HFD rats showed multiple fibrosis and degeneration for the archi‐tectureoftheadipocytes(FigureS2d).TreatmentofHFDratswithBER‐chloride for 2 weeks lead to the regeneration of the cells and reductionofthelipiddropletsinit(FigureS2e).

4  | DISCUSSION

In recent years, insulin resistance has a huge challenge, it repre‐sented one of the dangerous metabolic conditions, and in many casesitoccursduetobaddietaryhabits(Haslam&James,2005)orduetosomediseasesliketypeIIdiabetesmellitus(Sekoetal.,2018).Inthecurrentstudy,HFDfeedingfor8weeksinducedhy‐perinsulinaemia which is attributed to inability of liver to utilize the secreted insulin, although the normal role of pancreatic β‐cell (Arnoldetal.,2018).Duringthistimetheratsbecomemoreobesecompared to LFD controls due to elevation of insulin concentra‐tionwhichinhibitfattyacidoxidationsofatsaccumulatedmainlyintheliverbecauseitisthemainorganofoxidationprocess(Liuetal.,2017).Higherlevelsoffastinginsulin,glucose,andHOMA‐IRindexconfirmingthestateof insulinresistance.Furthermore, in‐creasingofHbA1cisanotherindicatorofinsulinresistanceandcor‐related with renal function parameters elevation (Fiorentino et al., 2017).Additionally,theresultsofthisstudyshowedupregulationofRBP4expressioninHFDrats.FromprevioushypothesisRBP4

elevates gluconeogenesis in the liver, so hyperglycemia is occur‐ring(Hutchison,Harrison,Stepto,Meyer,&Teede,2008),hindersthe insulin signaling in muscle and decreases the uptake of glucose by reduction the actionofPI3K (Yousefi&TaheriChadorneshin,2018).DefectsinPI3Kreportedinotherfindings(Jiang&Zhang,2002). Preceding studies showed the highest concentration ofRBP4 are accompanied by increase in body mass index, insulinresistance, and hypertriglyceridemia (Domingos, Queiroz, Lotufo, Bensenor,&Titan,2017).StudyofPTENhadasignificantimpor‐tance, PTEN is a lipid phosphatase inhibits insulin signaling bydephosphorylatingphosphatidylinositol(3,4,5)triphosphate(PIP3)tophosphatidylinositol(4,5)diphosphate(PIP2)(Cully,You,Levine,&Mak,2006).Hence,PTENisantagonizingtheactionofPI3KandinhibitsAktasappearincurrentresults,whereHFDratsexhibitedanelevationinPTENconcentrationandthedeclineofPI3K(Yao&Nyomba,2008).

It is known that, SIRT1 is a key regulator of lipidmobilizationthrough its action together with adenosine monophosphate‐acti‐vatedproteinkinase (AMPK) through increasing fattyacidmetab‐olism(Merksameretal.,2013).So,decreaseinSIRT1concentrationin HFD rats is linked by hyperlipidemia and insulin resistance due toreductionofphosphorylatedand/oractivatedAMPKisresultingin lipid synthesis increase (Boulet et al., 2015). The results of thepresent study are in agreement with previous works (Deng, Chen, & Li,2007).Further,SIRT1hasnumerousrolesininsulinsignalingpath‐way it regulates insulin secretion from β‐cell of pancreas via reduc‐tionuncouplingprotein‐2(UCP2)geneexpressionandimprovementthe depolarization in β‐cellofpancreas(Liang,Kume,&Koya,2009)and regulates the insulin signaling pathway through deacetylation of insulin receptorsubstrate‐2 (IRS2)andactivationofAkt incells(Yoshizakietal.,2009).FromthosementionedmechanismsofSIRT1andPTEN,thecurrentstudyexhibiteddecreasedofAkt‐pconcen‐tration in hepatic cells of HFD rats. Moreover adiponectin has a vital role in the insulin resistance pathway; it has a regulating influence on insulinsensitivity (Wu,Ni,&Lu,2016). Itwasnoticedthatdis‐turbance in lipidmetabolismandexcessive fatdeposition leads toabnormalsynthesisofadiponectin(Burnstock&Gentile,2018).HFD

Groups

Adiponectin (ng/ml)

Serum Hepatic tissueWhite adi‐pose tissue

Shamcontrol 1.42±0.49a 11.03±0.97a 21.42±1.65a

Control vehicle (LFD + 20% PEG)

1.39±0.49a 10.67±0.97a 22.97±1.12a

Control BER‐chloride 1.59±0.26a 10.77±1.15a 23.43±1.51a

Induction(HFD+20%PEG(10ml/kgBwt)

0.682±0.11b 2.0±0.7b 11.78±1.89b

Induction treated with BER‐chloride

1.01±0.18a 9.7±0.9a 19.47±1.67a

Note: Valuesrepresentthemean±SDofsixrats.SPSS(Version20.0).Meanswithletters(a),(b),and(c)werestatisticalrepresentedcomparedtoshamcontrolgroupasfollow:a=p<.001,b=p<.01,c=p < .05.

TA B L E 6   Effect of berberine (BER)chlorideonadiponectinlevelinserum, liver, and white adipose tissue homogenates after the treatment of diabetic‐inducedexperimentalanimals

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rats associated with decline of adiponectin concentration in serum, hepatic, and adipose tissues which may be attributed to disruption of both adiponectin receptors‐1 and 2 leading to elevation of glucose concentrationandreducetheactionofperoxisomeproliferatoracti‐vated receptor α‐signaling pathways, respectively, and finally insulin resistanceoccurs(S.Li,Shin,Ding,&vanDam,2009).Further,therearesometheoriessuggestingtheroleofSIRT1intheregulationofadiponectin secretion from the adipocytes by deacetylating of fork head transcription factor O1 (FOXO‐1) protein and enhance thetranscription of gene that encodes adiponectin in adipocytes (Qiao & Shao,2006).Hence,SIRT1declineeffectonadiponectinsecretion.

TheroleofSIRT1isextendedtocontroltheproductionofreac‐tiveoxygenspecies(ROS)(Colaketal.,2011)asSIRT1isconsideredone of the important proteins that protect cells from stress damage (Dengetal.,2007).UndernormalconditionthehepatocytebalancetheoxidativestressthroughtheactionofantioxidantenzymessuchasGPxwhichconvertshydrogenperoxide(H2O2)towater(Flores,Adhami, &Martins‐Green, 2016). Rats have insulin resistance areshowing lowGPx activity soH2O2 accumulated and hepatic cells damaged. These results were confirmed by elevation of liver trans‐aminases(ALTandAST).H2O2 accumulation also affected on renal tissue, which confirmed by an increase both urea and creatinine lev‐els.AnothercauseoftheelevationofROSincaseof insulinresis‐tance is attributed to the dysregulated production of adipocytokines where plasma adiponectin concentration is inversely correlated with systemicoxidativestress(Furukawaetal.,2004).

In recent years with regard to the side effects of artificial med‐icines, increasing attention has been paid by researchers to herbal medicines. Our in vivo study revealed that treatment with BER‐chloride has negative influence on insulin resistance through ac‐tivating two proteins involved in several physiological processes, SIRT‐1 and AMPK (Lin et al., 2018). Those two proteins able toactivateeachother,AMPKactivateSIRT‐1byelevationthecon‐centrationofnicotinamidephosphoribosyltransferaseandSIRT‐1stimulates AMPK through deacetylation of serine‐threoninekinase LKB1 (Hardie, 2015; Sun et al., 2014). Also, BER has thecapacity to recover insulin resistance syndrome through other mechanisms, where it protects β‐cell of islets of Langerhans from damage, allows glucose uptake by skeletal muscle, improves he‐patic gluconeogenesis and decreases the concentration of lipids in the blood (Pirillo & Catapano, 2015; Yang et al., 2014). As aresultofSIRT‐1andAMPKpathwayactivation,adiponectincon‐centration was restored after BER‐chloride therapy in our study which similar to previous results (Wu et al., 2015). Elevation ofadiponectin concentration is linked by regulation of β‐oxidationoffattyacidsandglucosemetabolism(Lin&Li,2012;Yamauchietal.,2002).Hence, treatedratswithBER‐chlorideshowedsignificantdecline of lipid profiles and BGL.

Also, BER‐chloride ameliorates hyperlipidemia results from in‐sulin resistance via different mechanisms, BER lowers blood cho‐lesterol levels through inhibiting cholesterol uptake and absorption in the intestine (Wang et al., 2014), reduces TC secretion from

F I G U R E 4  Schematicdiagramfortheeffectofberberine(BER)chlorideoninsulinsignalinginhigh‐fatdiet(HFD)insulinresistanceinduced rats

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enterocytestothecirculationbydownregulationacetylCoAtrans‐feraseIIenzyme(Wangetal.,2017)andincreasestheregulationofLDL‐receptor and hence, BER decreases the concentration of LDL‐c (Abidi,Zhou,Jiang,&Liu,2005).Moreover,BERchlorideisabletoimprove glucose control by stimulation the glycolysis in peripheral tissue, inhibition of FOXO‐1 and hepatic nuclear factor 4, lead tosuppression of glucose‐6‐phosphatase and phosphoenolpyruvate carboxykinaseenzymeswhichresponsibleforlivergluconeogenesis(Kimetal.,2009;Xiaetal.,2011)andactivationofglucosetrans‐port‐1(GLUT1)(Coketal.,2011).

The current study revealed BER‐chloride has the ability to raise the concentration of antioxidant enzymes (GSH, GPx, andXO) by reducing the high level of lipid peroxidation (Lao‐ong,Chatuphonprasert, Nemoto, & Jarukamjorn, 2012). Also BER hasthe ability to prevent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is the main source of ROS production(Chengetal.,2013).Theseresultsare inagreementwithprevioustheorieswhichprovedthatBERisastrongantioxidantmoleculeduetoitsabilitytoscavengefreeradicals(Kumaretal.,2015).Moreover,BER‐chloride exerts protective effect againstROS throughSIRT‐1activationwhereSIRT‐1iscapabletomodulateNOX4/NADPHox‐idativesubunit(Karbasforooshan&Karimi,2018).FallofROSpro‐duction lead to decrease the level of liver transaminases activity in rats, group administrated BER‐chloride. In the present study, BER‐chloridedownregulatesRBP4whichactsasaneffectiveinsulinsen‐sitizingfunction(Zhang,Xu,Guo,Meng,&Li,2008).Frompreviousstudies, itwasnoticedthatreductionofRBP4concentrationisre‐lated to elevation of HDL‐c and decrease TG levels in some patients. Also,asa resultof thedeclineofSIRT‐1 inHFDrats,elevationofTBARSandXO,anddecreaseinGPxandGSHwasreportedandthiswas improved after BER‐chloride administration. From this study, we conclude that BER‐chloride can be considered one of therapeutics used to help the treatment of insulin resistance syndrome through itsinfluenceonmultipleinsulinsignalingpathways.Aschematicrep‐resentationwasdesigned(Figure4)tosummarizethemodificationof insulin resistance by BER‐chloride.

ACKNOWLEDG MENTS

TheauthorsextendtheirappreciationtothedeanshipofScientificResearch at KingKhalidUniversity, Abha, KSA for supported thisworkundergrantnumber(R.G.P.1/117/40)

CONFLIC T OF INTERE S T

The authors declared that they have no conflict of interest.

ORCID

Marwa El‐Zeftawy https://orcid.org/0000‐0002‐2884‐8148

Doaa Ghareeb https://orcid.org/0000‐0003‐3327‐4377

Nihal Elguindy https://orcid.org/0000‐0002‐5705‐1349

Attalla F. El‐kott https://orcid.org/0000‐0001‐5060‐0790

R E FE R E N C E S

Abidi,P.,Zhou,Y.,Jiang,J.D.,&Liu,J.(2005).Extracellularsignal‐regu‐lated kinase‐dependent stabilization of hepatic low‐density lipopro‐tein receptormRNAbyherbalmedicineberberine.Arteriosclerosis, Thrombosis, and Vascular Biology, 25(10), 2170–2176. https://doi.org/10.1161/01.ATV.0000181761.16341.2b

Arnold, S. E.,Arvanitakis, Z.,Macauley‐Rambach, S. L., Koenig,A.M.,Wang,H.‐Y.,Ahima,R.S.,…Nathan,D.M.(2018).Brain insulinre‐sistance in type 2 diabetes and Alzheimer disease: Concepts andconundrums. Nature Reviews Neurology, 14(3),168–181.https://doi.org/10.1038/nrneu rol.2017.185

Bodiwala,H.S.,Sabde,S.,Mitra,D.,Bhutani,K.K.,&Singh,I.P.(2011).Synthesis of 9‐substituted derivatives of berberine as anti‐HIVagents. European Journal of Medicinal Chemistry, 46(4), 1045–1049.https ://doi.org/10.1016/j.ejmech.2011.01.016

Boulet,M.M.,Chevrier,G.,Grenier‐Larouche,T.,Pelletier,M.,Nadeau,M., Scarpa, J., … Tchernof, A. (2015). Alterations of plasmame‐tabolite profiles related to adipose tissue distribution and car‐diometabolic risk. American Journal of Physiology‐Endocrinology and Metabolism, 309(8), E736–E746. https://doi.org/10.1152/ajpendo.00231.2015

Burnstock, G., & Gentile, D. (2018). The involvement of purinergicsignalling in obesity. Purinergic Signal, 14(2), 97–108. https://doi.org/10.1007/s11302‐018‐9605‐8

Candeias,M.F.,Abreu,P.,Pereira,A.,&Cruz‐Morais,J.(2009).EffectsofstrictosamideonmousebrainandkidneyNa+,K+‐ATPaseandMg2+‐ATPase activities. Journal of Ethnopharmacology, 121(1), 117–122.https ://doi.org/10.1016/j.jep.2008.08.032

Cheng, F.,Wang, Y., Li, J., Su, C.,Wu, F., Xia,W.‐H.,… Tao, J. (2013).Berberine improves endothelial function by reducing endothelial microparticles‐mediated oxidative stress in humans. International Journal of Cardiology, 167(3), 936–942. https://doi.org/10.1016/j.ijcard.2012.03.090

Chiu,D.T.,Stults,F.H.,&Tappel,A.L.(1976).Purificationandpropertiesofratlungsolubleglutathioneperoxidase.Biochimica et Biophysica Acta, 445(3),558–566.https://doi.org/10.1016/0005‐2744(76)90110‐8

Cok,A., Plaisier,C., Salie,M. J.,Oram,D. S.,Chenge, J.,& Louters, L.L.(2011).Berberineacutelyactivatestheglucosetransportactivityof GLUT1. Biochimie, 93(7), 1187–1192. https://doi.org/10.1016/j.biochi.2011.04.013

Colak,Y.,Ozturk,O.,Senates,E.,Tuncer,I.,Yorulmaz,E.,Adali,G.,…Enc,F. Y. (2011). SIRT1 as a potential therapeutic target for treatmentof nonalcoholic fatty liver disease. Medical Science Monitor, 17(5),HY5‐9.https://doi.org/10.12659/MSM.881749

Cully,M.,You,H.,Levine,A.J.,&Mak,T.W.(2006).BeyondPTENmu‐tations:ThePI3Kpathwayasanintegratorofmultipleinputsduringtumorigenesis. Nature Reviews Cancer, 6(3), 184–192. https://doi.org/10.1038/nrc1819

David,J.A.,Rifkin,W.J.,Rabbani,P.S.,&Ceradini,D.J.(2017).TheNrf2/Keap1/AREPathwayandOxidativeStressasaTherapeuticTargetinType II Diabetes Mellitus. Journal of Diabetes Research, 2017, 1–15. https ://doi.org/10.1155/2017/4826724

Deng, X. Q., Chen, L. L., & Li, N. X. (2007). The expression ofSIRT1 in nonalcoholic fatty liver disease induced by high‐fatdiet in rats. Liver International, 27(5), 708–715. https://doi.org/10.1111/j.1478‐3231.2007.01497.x

Domingos,M.A.M.,Queiroz,M.,Lotufo,P.A.,Bensenor,I.J.,&Titan,S.M.O.(2017).SerumRBP4andCKD:Associationwithinsulinre‐sistance and lipids. Journal of Diabetes and Its Complications, 31(7),1132–1138. https ://doi.org/10.1016/j.jdiac omp.2017.04.013

Dong,S.F.,Hong,Y.,Liu,M.,Hao,Y.Z.,Yu,H.S.,Liu,Y.,&Sun, J.N.(2011).Berberine attenuates cardiacdysfunction inhyperglycemicand hypercholesterolemic rats. European Journal of Pharmacology, 660(2–3),368–374.https://doi.org/10.1016/j.ejphar.2011.03.024

10 of 11  |     EL‐ZEFTAWY ET AL.

Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82(1), 70–77. https://doi.org/10.1016/0003‐9861(59)90090‐6

El‐Sayed,M.M.,Ghareeb,D.A.,Talat,H.A.,&Sarhan,E.M.(2013).Highfat diet induced insulin resistance and elevated retinol binding pro‐tein 4 in female rats; treatment and protection with Berberis vulgaris extract and vitamin A.Pakistan Journal of Pharmaceutical Sciences, 26(6),1189–1195.

Fiorentino,T.V.,Marini,M.A.,Succurro,E.,Sciacqua,A.,Andreozzi,F.,Perticone,F.,&Sesti,G.(2017).Elevatedhemoglobinglycationindexidentify non‐diabetic individuals at increased risk of kidney dysfunc‐tion. Oncotarget., 8(45), 79576–79586. https://doi.org/10.18632/oncot arget.18572

Flores, C., Adhami, N., & Martins‐Green, M. (2016). THS tox‐ins induce hepatic steatosis by altering oxidative stress andSIRT1 levels. Journal of Clinical Toxicology, 6, 318. https ://doi.org/10.10.4172/2161‐0495.1000318

Friedewald,W.T.,Levy,R.I.,&Fredrickson,D.S.(1972).Estimationofthe concentration of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clinical Chemistry, 18(6),499–502.

Furukawa,S.,Fujita,T.,Shimabukuro,M.,Iwaki,M.,Yamada,Y.,Nakajima,Y.,…Shimomura,I.(2004).Increasedoxidativestressinobesityandits impact on metabolic syndrome. Journal of Clinical Investigation, 114(12),1752–1761.https://doi.org/10.1172/jci21625

Gao,Y.F.,Zhang,M.N.,Wang,T.X.,Wu,T.C.,Ai,R.D.,&Zhang,Z.S. (2016).HypoglycemiceffectofD‐chiro‐inositol in type2diabe‐tesmellitusratsthroughthePI3K/Aktsignalingpathway.Molecular and Cellular Endocrinology, 433, 26–34. https ://doi.org/10.1016/j.mce.2016.05.013

Ghareeb, D. A., Khalil, S., Hafez, H. S., Bajorath, J., Ahmed, H. E. A.,Sarhan, E.,… El‐Demellawy,M.A. (2015). Berberine reduces neu‐rotoxicity related to nonalcoholic steatohepatitis in rats.Evidence‐Based Complementary and Alternative Medicine, 2015, 1–13. https ://doi.org/10.1155/2015/361847

Han, S. J., Boyko, E. J., Kim, S. K., Fujimoto, W. Y., Kahn, S. E., &Leonetti,D.L.(2018).AssociationofThighMuscleMasswithInsulinResistance and Incident Type 2 Diabetes Mellitus in Japanese Americans.Diabetes & Metabolism Journal, 42(6),488–495.https://doi.org/10.4093/dmj.2018.0022

Hardie,D.G.(2015).AMPK:Positiveandnegativeregulation,anditsrolein whole‐body energy homeostasis. Current Opinion in Cell Biology, 33, 1–7. https ://doi.org/10.1016/j.ceb.2014.09.004

Haslam,D.W.,&James,W.P.(2005).Obesity.Lancet, 366(9492),1197–1209.https://doi.org/10.1016/S0140‐6736(05)67483‐1

Hutchison,S.K.,Harrison,C.,Stepto,N.,Meyer,C.,&Teede,H.J.(2008).Retinol‐binding protein 4 and insulin resistance in polycystic ovary syndrome. Diabetes Care, 31(7),1427–1432.https://doi.org/10.2337/dc07‐2265

Jiang,G.,&Zhang,B.B.(2002).Pi3‐kinaseanditsup‐anddown‐streammodulators as potential targets for the treatment of type II diabetes. Frontiers in Bioscience, 7, d903–907. https ://doi.org/10.2741/jiang

Jiang,W.,Li,S.,&Li,X.(2015).Therapeuticpotentialofberberineagainstneurodegenerative diseases. Science China Life Sciences, 58(6),564–569. https ://doi.org/10.1007/s11427‐015‐4829‐0

Kahn,S.E.,Hull,R.L.,&Utzschneider,K.M.(2006).Mechanismslinkingobesity to insulin resistance and type 2 diabetes. Nature, 444(7121),840–846. https ://doi.org/10.1038/natur e05482

Karbasforooshan,H.,&Karimi,G.(2018).TheroleofSIRT1indiabeticretinopathy. Biomedicine & Pharmacotherapy, 97, 190–194. https ://doi.org/10.1016/j.biopha.2017.10.075

Kim,W.S.,Lee,Y.S.,Cha,S.H.,Jeong,H.W.,Choe,S.S.,Lee,M.‐R.,…Kim,J.B.(2009).BerberineimproveslipiddysregulationinobesitybycontrollingcentralandperipheralAMPKactivity.American Journal of

Physiology‐Endocrinology and Metabolism, 296(4),E812–819.https://doi.org/10.1152/ajpen do.90710.2008

Koc, K., Cerig, S., Ozek, N. S., Aysin, F., Yildirim, S., Cakmak, O., …Geyikoglu,F. (2019).Theefficacyofoleuropeinagainstnon‐ste‐roidal anti‐inflammatory drug induced toxicity in rat kidney.Environmental Toxicology, 34(1), 67–72. https://doi.org/10.1002/tox.22658

Kumar,A.,Ekavali,Chopra,K.,Mukherjee,M.,Pottabathini,R.,&Dhull,D.K.(2015).Currentknowledgeandpharmacologicalprofileofber‐berine:Anupdate.European Journal of Pharmacology, 761, 288–297. https ://doi.org/10.1016/j.ejphar.2015.05.068

Lao‐ong, T., Chatuphonprasert, W., Nemoto, N., & Jarukamjorn, K. (2012). Alteration of hepatic glutathione peroxidase and superox‐ide dismutase expression in streptozotocin‐induced diabetic miceby berberine. Pharmaceutical Biology, 50(8),1007–1012.https://doi.org/10.3109/13880 209.2012.655377

Li, J.‐W., Yuan, K., Shang, S.‐C., & Guo, Y. (2017). A safer hypoglyce‐mic agent for type 2 diabetes—Berberine organic acid salt. Journal of Functional Foods, 38, 399–408. https ://doi.org/10.1016/j.jff.2017.09.031

Li,S.,Shin,H.J.,Ding,E.L.,&vanDam,R.M.(2009).Adiponectinlevelsandriskoftype2diabetes:Asystematicreviewandmeta‐analysis.JAMA, 302(2),179–188.https://doi.org/10.1001/jama.2009.976

Liang,F.,Kume,S.,&Koya,D.(2009).SIRT1andinsulinresistance.Nature Reviews Endocrinology, 5(7), 367–373. https://doi.org/10.1038/nrendo.2009.101

Lin, H., & Li, Z. (2012). Adiponectin self‐regulates its expression andmultimerization in adipose tissue: An autocrine/paracrine mecha‐nism? Medical Hypotheses, 78(1), 75–78. https://doi.org/10.1016/j.mehy.2011.07.063

Lin,Y.,Sheng,M.,Ding,Y.,Zhang,N.,Song,Y.,Du,H.,…Yu,W.(2018).Berberineprotectsrenaltubularcellsagainsthypoxia/reoxygenationinjuryviatheSirt1/p53pathway.Journal of Natural Medicines, 72(3),715–723. https ://doi.org/10.1007/s11418‐018‐1210‐1

Litwack,G.,Bothwell,J.W.,Williams,J.N.J.,&Elvehjem,C.A.(1953).Acolorimetricassayforxanthineoxidase inrat liverhomogenates.Journal of Biological Chemistry, 200(1),303–310.

Liu,Q., Xu, X., Zhao,M.,Wei, Z., Li, X., Zhang, X.,… Shao, C. (2015).Berberine induces senescence of human glioblastoma cells by down‐regulating the EGFR‐MEK‐ERK signaling pathway. Molecular Cancer Therapeutics, 14(2), 355–363. https://doi.org/10.1158/1535‐7163.MCT‐14‐0634

Liu,R.,Li,H.,Fan,W.,Jin,Q.,Chao,T.,Wu,Y.,&Yang,X.(2017).Leucinesupplementation differently modulates branched‐chain amino acid catabolism, mitochondrial function and metabolic profiles at the dif‐ferent stage of insulin resistance in rats on high‐fat diet. Nutrients, 9(6),https://doi.org/10.3390/nu9060565

Merksamer,P.I.,Liu,Y.,He,W.,Hirschey,M.D.,Chen,D.,&Verdin,E.(2013). The sirtuins, oxidative stress and aging: An emerging link.Aging, 5(3),144–150.https://doi.org/10.18632/aging.100544

Paglia,D.E.,&Valentine,W.N.(1967).Studiesonthequantitativeandqualitative characterizationof erythrocyte glutathioneperoxidase.Journal of Laboratory and Clinical Medicine, 70(1),158–169.

Pandian, R. S., Anuradha, C. V., & Viswanathan, P. (2002).Gastroprotective effect of fenugreek seeds (Trigonella foe‐num graecum) on experimental gastric ulcer in rats. Journal of Ethnopharmacology, 81, 393–397. https ://doi.org/10.1016/S0378‐8741(02)00117‐4

Peter, S. J., Basha, S. K.,Giridharan, R., Lavinya, B.U.,& Sabina, E. P.(2017). Suppressive effect of Spirulina fusiformis ondiclofenac‐in‐duced hepato‐renal injury and gastrointestinal ulcer in Wistar al‐bino rats: A biochemical and histological approach. Biomedicine & Pharmacotherapy, 88, 11–18. https ://doi.org/10.1016/j.biopha.2017.01.032

     |  11 of 11EL‐ZEFTAWY ET AL.

Pirillo,A.,&Catapano,A.L.(2015).Berberine,aplantalkaloidwithlipid‐and glucose‐lowering properties: From in vitro evidence to clinical studies. Atherosclerosis, 243(2),449–461.https://doi.org/10.1016/j.ather oscle rosis.2015.09.032

Qiao,L.,&Shao,J.(2006).SIRT1regulatesadiponectingeneexpressionthrough Foxo1‐C/enhancer‐binding protein alpha transcriptionalcomplex. Journal of Biological Chemistry, 281(52), 39915–39924.https ://doi.org/10.1074/jbc.M6072 15200

Ren, L., Zhou, X.,Huang, X.,Wang,C.,& Li, Y. (2019). The IRS/PI3K/Akt signaling pathway mediates olanzapine‐induced hepatic insu‐lin resistance in male rats. Life Sciences, 217, 229–236. https ://doi.org/10.1016/j.lfs.2018.12.015

Saleh,S.R.,Attia,R.,&Ghareeb,D.A.(2018).Theamelioratingeffectofberberine‐rich fraction against gossypol‐induced testicular inflam‐mation andoxidative stress.Oxid Med Cell Longev, 2018, 1056173. https ://doi.org/10.1155/2018/1056173

Seko,Y.,Sumida,Y.,Tanaka,S.,Mori,K.,Taketani,H.,Ishiba,H.,…Itoh,Y.(2018).Insulinresistanceincreasestheriskofincidenttype2diabetesmellitus in patients with non‐alcoholic fatty liver disease. Hepatology Research, 48(3),E42–E51.https://doi.org/10.1111/hepr.12925

Steinberg,D.,Vaughan,M.,&Margolis,S.(1961).Studiesoftriglyceridebiosynthesis in homogenates of adipose tissue. Journal of Biological Chemistry, 236(6),1631–1637.

Sun, Y., Li, J., Xiao, N. A.,Wang,M., Kou, J., Qi, L., … Liu, K. (2014).Pharmacological activation of AMPK ameliorates perivascular ad‐ipose/endothelial dysfunction in a manner interdependent on AMPKandSIRT1.Pharmacological Research, 89, 19–28. https ://doi.org/10.1016/j.phrs.2014.07.006

Tappel,A.L.,&Zalkin,H. (1959). Inhibitionof lipidperoxidation inmi‐tochondria by vitamin E. Archives of Biochemistry and Biophysics, 80, 333–336.https://doi.org/10.1016/0003‐9861(59)90259‐0

Vuddanda, P. R., Chakraborty, S.,& Singh, S. (2010). Berberine:A po‐tential phytochemical with multispectrum therapeutic activities. Expert Opinion on Investigational Drugs, 19(10), 1297–1307.https://doi.org/10.1517/13543 784.2010.517745

Wang,H.,Zhu,C.,Ying,Y.,Luo,L.,Huang,D.,&Luo,Z.(2017).Metforminand berberine, two versatile drugs in treatment of common metabolic diseases. Oncotarget, 9(11),10135–10146.https://doi.org/10.18632/oncot arget.20807

Wang,Y.,Yi,X.,Ghanam,K.,Zhang,S.,Zhao,T.,&Zhu,X.(2014).Berberinedecreases cholesterol levels in rats through multiple mechanisms, including inhibition of cholesterol absorption. Metabolism ‐ Clinical and Experimental, 63(9),1167–1177.https://doi.org/10.1016/j.metabol.2014.05.013

Wu,Q.M.,Ni,H.X.,&Lu,X.(2016).Changesofadipocytokineexpres‐sion after diabetic rats received sitagliptin and the molecular mecha‐nism. Asian Pacific Journal of Tropical Medicine, 9(9),893–897.https://doi.org/10.1016/j.apjtm.2016.07.014

Wu,U.,Cha,Y.,Huang,X.,Liu,J.,Chen,Z.,Wang,F.,&Ding,H.(2015).Protectiveeffectsofberberineonhighfat‐inducedkidneydamageby increasing serum adiponectin and promoting insulin sensitivity. International Journal of Clinical and Experimental Pathology, 8(11),4486–4492.

Xia,X.,Yan,J.,Shen,Y.,Tang,K.,Yin, J.,Zhang,Y.,…Weng,J. (2011).Berberine improves glucose metabolism in diabetic rats by inhibi‐tion of hepatic gluconeogenesis. PLoS ONE, 6(2),e16556.https://doi.org/10.1371/journ al.pone.0016556

Yamauchi, T., Kamon, J.,Minokoshi, Y., Ito, Y.,Waki,H., Uchida, S., …Kadowaki,T.(2002).Adiponectinstimulatesglucoseutilizationandfatty‐acid oxidation by activating AMP‐activated protein kinase.Nature Medicine, 8(11),1288–1295.https://doi.org/10.1038/nm788

Yang,T.C.,Chao,H.F.,Shi,L.S.,Chang,T.C.,Lin,H.C.,&Chang,W.L. (2014). Alkaloids from Coptis chinensis root promote glucoseuptake in C2C12 myotubes. Fitoterapia, 93, 239–244. https ://doi.org/10.1016/j.fitote.2014.01.008

Yao, X. H., & Nyomba, B. (2008). Hepatic insulin resistance inducedby prenatal alcohol exposure is associated with reduced PTENand TRB3 acetylation in adult rat offspring. American Journal of Physiology‐Regulatory, Integrative and Comparative Physiology, 294(6),R1797–R1806. https ://doi.org/10.1152/ajpre gu.00804.2007

Yoshizaki,T.,Milne,J.C.,Imamura,T.,Schenk,S.,Sonoda,N.,Babendure,J.L.,…Olefsky,J.M.(2009).SIRT1exertsanti‐inflammatoryeffectsand improves insulin sensitivity in adipocytes. Molecular and Cellular Biology, 29(5),1363–1374.https://doi.org/10.1128/MCB.00705‐08

Yousefi,M.R.,&TaheriChadorneshin,H. (2018).Theeffectofmoder‐ate endurance training on gastrocnemius retinol‐binding protein 4 and insulin resistance in streptozotocin‐induced diabetic rats. Interventional Medicine and Applied Science, 10(1),59–63.https://doi.org/10.1556/1646.9.2017.34

Zhang,S.,Wang,X.,Yin,W.,Liu,Z.,Zhou,M.I.,Xiao,D.,…Peng,D.(2016).Synthesisandhypoglycemicactivityof9‐O‐(lipophilicgroupsubsti‐tuted)berberinederivatives.Bioorganic & Medicinal Chemistry Letters, 26(19),4799–4803.https://doi.org/10.1016/j.bmcl.2016.08.027

Zhang,W.,Xu,Y.C.,Guo, F. J.,Meng,Y.,&Li,M. L. (2008).Anti‐dia‐betic effects of cinnamaldehyde and berberine and their impacts onretinol‐bindingprotein4expressioninratswithtype2diabetesmellitus. Chinese Medical Journal, 121(21), 2124–2128. https://doi.org/10.1097/00029 330‐20081 1010‐00003

SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article: El‐Zeftawy M, Ghareeb D, ElBealy ER,etal.BerberinechlorideamelioratedPI3K/Akt‐p/SIRT‐1/PTENsignalingpathwayininsulinresistancesyndromeinduced in rats. J Food Biochem. 2019;00:e13049. https ://doi.org/10.1111/jfbc.13049