research article gelam honey attenuates the oxidative ... honey attenuates the oxidative...

Research ArticleGelam Honey Attenuates the Oxidative Stress-InducedInflammatory Pathways in Pancreatic Hamster Cells

Sher Zaman Safi1 Kalaivani Batumalaie1 Rajes Qvist1

Kamaruddin Mohd Yusof2 and Ikram Shah Ismail1

1Faculty of Medicine Department of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2Department of Molecular Biology and Genetics Faculty of Arts and Science Canik Basari University Samsun Turkey

Correspondence should be addressed to Sher Zaman Safi safinustyahoocomand Kalaivani Batumalaie kalaivanibatumalaiegmailcom

Received 10 September 2014 Revised 23 August 2015 Accepted 1 October 2015

Academic Editor Abir El-Alfy

Copyright copy 2016 Sher Zaman Safi et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose Type 2 diabetes consists of progressive hyperglycemia and insulin resistance which could result from glucose toxicityinflammatory cytokines and oxidative stress In the present study we investigated the effect of Gelam honey and quercetin on theoxidative stress-induced inflammatory pathways and the proinflammatory cytokines Methods HIT-T15 cells were cultured andpreincubated with the extract of Gelam honey (20 40 60 and 80120583gmL) as well as quercetin (20 40 60 and 80 120583M) prior tostimulation by 20 and 50 mM glucose ResultsHIT-T15 cells cultured under hyperglycemic condition showed a significant increasein the inflammatory pathways by phosphorylating JNK IKK-120573 and IRS-1 at Ser307 (119901 lt 005) There was a significant decreasein the phosphorylation of Akt at Ser473 (119901 lt 005) Pretreatment with Gelam honey and quercetin reduced the expression ofphosphorylated JNK IKK-120573 and IRS-1 thereby significantly reducing the expression of proinflammatory cytokines like TNF-120572IL-6 and IL-1120573 (119901 lt 005) At the same time there was a significant increase in the phosphorylated Akt showing the protectiveeffects against inflammation and insulin resistance (119901 lt 005) In conclusion our data suggest the potential use of the extract fromGelam honey and quercetin in modulating the inflammation induced insulin signaling pathways

1 Research Background

Diabetes is one of the most common noncommunicablediseases affecting millions of people globally [1 2] It is oneof the most challenging health problems in many developingand industrialized countries and the exact cause is unknownOne of the foremost challenges we now face is to accountmechanistically not only for the definition of hyperglycemiabut also for other physiological and biochemical abnormali-ties which are the characteristics of the disease [3 4]

Type 2 diabetes consists of progressive hyperglycemiainsulin resistance and pancreatic 120573 cell failure The patho-genesis of type 2 diabetes is complex and in most instancesclearly requires defects in both 120573 cell function and insulinsensitivity and together both of these abnormalities bringabout hyperglycemia Type 2 diabetes has a metabolic milieuwhich is characterized by insulin resistance and chronic acuteinflammation In the last decade a great deal of attention

has been focused on the understanding of insulin resistancewhich is an important contributor to the development andmaintenance of hyperglycemia in type 2 diabetes [5 6]Β cell dysfunction can result from glucose toxicity

inflammatory cytokines oxidative stress or lipotoxicity inthe presence of excess glucose Oxidative stress throughthe production of reactive oxygen species (ROS) has beenproposed as the root cause underlying the developmentof insulin resistance 120573 cell dysfunction impaired glucosetolerance and type 2 diabetes mellitus [3]

Oxidative stress is induced by reactive oxygen andnitrogen species produced by several biochemical pathwaysassociatedwith hyperglycemia (glucose autooxidation polyolpathway prostanoid synthesis and protein glycation) andis critically involved in the impairment of 120573 cell functionduring the development of type 2 diabetes [7 8] Reactiveoxygen species can function as signalingmolecules to activatea number of stress sensitive pathways and inflammatory

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 5843615 13 pageshttpdxdoiorg10115520165843615

2 Evidence-Based Complementary and Alternative Medicine

pathways leading to the production of inflammatory markerssuch as IL-6 and TNF-120572 that impair insulin signaling throughserine phosphorylation of IRS-1 [9]

Insulin is a pleiotropic hormone which has many func-tions that are exerted across a variety of insulin target tissuesthrough several intracellular signaling cascades Insulin sub-strate (IRS-1) mediated insulin signaling regulates peripheralinsulin action as well as pancreatic 120573 cell function by regulat-ing proliferation survival and insulin secretion The defectsin insulin signaling pathwaymainly involve IRS-1 Activationof the insulin receptor leads to tyrosine phosphorylation ofIRS-1 thereby initiating signal transduction Insulin receptortyrosine kinases can signal through the phosphatidylinositol3-kinase (PI3K) pathway which is mainly responsible for themetabolic actions of insulin The major target of PI3K is theserine threonine kinase Akt It has been shown that the Aktdependent mechanism in the distal events of exocytosis isresponsible for the defect in insulin secretion [7 10]

This transient exposure of 120573 cells to oxidative stressinterrupts the normal coupling of glucose metabolism toinsulin secretion by activating stress signaling pathways andthe inflammatory pathways [11] Despite the multiplicity ofinflammatory pathways the development of insulin resis-tance is often due to the activation of jun-N-terminal kinase(JNK) and the IkappaB kinase (IKK-120573) The JNK and IKK-120573 pathways are activated by ROS and various other factorsincluding the inflammatory cytokines such as TNF-120572 IL-6and IL-1betawhich are involved in the development of insulinresistance found in type 2 diabetes [12]

Reactive oxygen species and mitochondrial stress-induced [13] activation of JNK and IKK-120573 promotes thephosphorylation of IRS-1 at serine sites that negativelyregulate normal signaling through the insulin receptorIRS-1axis as seen in the phosphorylation of IRS-1 at serine 307(Ser307) It has been reported that serine phosphorylationof insulin receptor-1 (IRS-1) inhibits insulin stimulatedtyrosine phosphorylation of IRS-1 leading to an increase ininsulin resistance [10] Therefore one of the causes leadingto a defect in insulin signaling can be attributed to serinephosphorylation of IRS-1 at serine 307 residues whichactivate the jun-N-terminal kinase (JNK) and IKK-120573 thusproviding a plausiblemechanistic link between inflammationand insulin resistance [14]

Recently protein kinase B (PKB andAkt) has been shownto function in the insulin signaling cascade by phospho-rylating transcription factors which are responsible for thetranscription and expression of genes related to insulinsynthesis and secretion Previous studies have shown thatinactivation of Akt can lead to insulin resistance decreased120573 cell mass and impaired insulin secretion [15]

Numerous studies have shown that honey has antioxidantand scavenging properties which prevents the oxidativedamage caused by free radicals The antioxidant propertyis due to the phenolic compounds which are present inthe honey and it has been demonstrated that the biolog-ical activities correlate with the phenolic content of thehoney [16] The phenolic content of the Malaysian Gelamhoney has been shown to have both anti-inflammatoryand antioxidant properties [17] Quercetin is one of the

important components of Gelam honey that may be furtherelaborated in terms of its function and use as antidiabeticagent

Therefore the aim of our present study is to determinethe effect of Gelam honey extract and quercetin on theJNK and IKK-120573 inflammatory pathways and IRS-1 serinephosphorylation which causes insulin resistance and the Aktactivated insulin signaling pathway which improves insulinresistance

2 Research Design and Methods

21 Extraction of Phenolic Compounds from Honey by SolidPhase Extraction (SPE) Gelam honey samples (Departmentof Agriculture Parit Botak Johor Malaysia) were sub-jected to base hydrolysis and extracted with ethyl acetateas described by Wahdan [18] and Seo and Morr [19] Therecovered fractions were combined and dried under nitrogengas

22 Determination of the Phenolic Content Phenolic com-pounds from the extract were assayed using Folin-Ciocalteuassay [20] Briefly the extract (1mL) was added to 10 Folin-Ciocalteu reagent (Sigma F9252) and 05 sodium carbonateThe contents were thoroughly mixed and allowed to standfor 2 hours The absorbance of the blue color that developedafter 2 hours was read at 765 nm Results were expressed inmicrograms of Gallic acid per gram of the extract using astandard curve generated with Gallic acid (Sigma G7384)

23 Determination of the Flavonoid Content The totalflavonoid (TF) content was determined spectrophotometri-cally Briefly 1mL of honey extract or a standard solutionof quercetin (Sigma Q4951) (10 50 100 150 200 and250 120583gmL) in distilled water was added to a 10mL volumet-ric flask containing 4mL of double distilled water 300120583Lof NaNO

2(5 vv) and 300 120583L of 10 AlCl

3 The solution

was allowed to stand at room temperature in the dark for30 minutes and the absorbance was read at 430 nm The TFcontentwas determinedusing the standard curve of quercetin(120583gmL) and was expressed as 120583g of quercetin equivalents in1 g of extract

24 Cell Culture HIT-T15 cells were cultured according tothe instructions provided by ATCC (CRL-1777) On arrivalthe cells were cultured immediately in T-25 cm flask in theF12K medium (ATCC 30-2004) supplemented with 10 FBSand 1 penicillin and streptomycin at 37∘C (5 CO

2in

air) Cells (3rd passage) were trypsinized and subculturedfollowing which they were incubated for 5 days until theyreached 80 confluency

25 Treatment of HIT-T15 Cells with Quercetin and GelamHoney Extract HIT-T15 cells (5 times 105) were pretreatedwith Gelam honey extract (20 40 60 and 80 120583gmL) andquercetin (20 40 60 and 80 120583M) for 24 hours after whichthe medium was replaced with fresh medium Following thisglucose (SigmaG8769) (20 or 50mM)was added and the cellswere incubated for another 24 hours

Evidence-Based Complementary and Alternative Medicine 3

26 Cell Lysate Preparation After treatment the cells werewashed twice in PBS and lysed in mammalian cell lysis buffer(Sigma MCL-1 which was supplemented with protease andphosphatase inhibitors Insoluble materials were eliminatedby centrifugation (12000timesg 10min 4∘C) and the proteinconcentration in the supernatant was determined by Brad-ford assay (Bio-Rad Laboratories)

27 Western Blot Analysis Thirty micrograms of proteinextracts was loaded on 10 SDS-polyacrylamide gel andtransferred to activated nitrocellulose membrane The mem-branes were blocked with tris-buffered saline (TBS) contain-ing 5 nonfat milk and incubated with pAkt (Ser473) pIRS-1 (Ser307) p-JNK p-IKK-120572120573 IL-6 IL-1120573 and TNF alphaprimary antibodies (obtained from Santa Cruz Laboratories)overnight at 4∘C with a concentration of 1 1000 120573-actin wasused as a loading control After 3 times extensive washesin TBS (for 20ndash30m each) membranes were incubated for1 h at room temperature with the appropriate horseradishperoxidase-conjugated secondary antibodies We use theconcentration of 1 5000 for secondary antibodies Thenwe visualized the film using chemiluminescence substrateaccording to the manufacturerrsquos instructions (AmershamLife Sciences Little Chalfont UK) Quantitative analysis ofthe protein content was performed by Gel DocumentationSystem (Biospectrum 410 UVP)

28 Statistical Analyses Data were analyzed with one-wayANOVA using SPSS version 160 software The results wereexpressed as the mean plusmn standard deviation value lowast119901 lt 005was considered to be statistically significant

3 Results

31 Total Phenolic and Flavonoid Content 10 g of liquefiedfresh Malaysian Gelam honey (Apis mellifera) was extractedusing ethyl acetate and the extract was found to contain 52 120583gof Gallic acid per gram of extract of total phenolic contentand 692 120583g of quercetin per gram of extract of total flavonoidcontent

In our previous paper [21] we demonstrated the presenceof ROS by using 2101584071015840-dichlorodihydrofluorescein diacetate(DCFH-DA) reagent under hyperglycemic conditions andby the measurement of intracellular ROS in individualcells by image analysis Our data showed the antioxidanteffect of Gelam honey extract and the flavonoid compoundsquercetin luteolin and chrysin in the cultured cells bysignificantly inhibiting the production of ROS in a dosedependent manner Again our data showed the reduction ofROS in the single cells in a dose dependent manner afterpretreatment with the honey extract and the flavonoids

32 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on Phospho-JNKExpression under Normal andHyper-glycemic Conditions HIT-T15 cells were pretreated with thequercetin at concentrations of 20 40 60 and 80120583M andGelam honey extract at concentrations of 20 40 60 and80 120583gmL for 24 hours following which they were cul-tured with 20mM (Figures 1(a) 1(c) and 1(e)) or 50mM

(Figures 1(b) 1(d) and 1(f)) glucose to determine the phos-phorylation of JNK The data revealed that exposure of HIT-T15 cells to 20 and 50mM glucose significantly increasedthe level of phospho-JNK expression compared to controlPretreatment with quercetin and Gelam honey extract signif-icantly (119901 lt 005) reduced the ROS induced expression ofphospho-JNK under 20mM glucose (Figures 1(a) 1(c) and1(e)) by 60 and 42 in a dose dependent manner Pre-treatment with quercetin and Gelam honey extract reducedthe expression of phospho-JNK significantly (119901 lt 005) by64 and 50 respectively compared to the cells that werecultured with 50mM glucose (Figures 1(b) 1(d) and 1(f))alone The phospho-JNK protein levels from each samplewere normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treated group)(Figure 1)

33 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on Phospho-IKK-120573 Expression under Normal andHyperglycemic Conditions HIT-T15 cells were pretreatedwith the quercetin at concentrations of 20 40 60 and80 120583M and Gelam honey extract at concentrations of 2040 60 and 80 120583gmL for 24 hours following which theywere cultured with 20mM (Figures 2(a) 2(c) and 2(e)) or50mM (Figures 2(b) 2(d) and 2(f)) glucose to determinethe phosphorylation of IKK-120573 The phosphorylation of IKK-120573 was increased in HIT-T15 cells treated with 20mM and50mM glucose alone as compared with control Pretreat-ment with the quercetin and Gelam honey extract showed55 and 44 decrease (119901 lt 005) in the expression ofphosphorylated IKK-120573 in a dose dependent manner in thecells that were cultured in 20mM (Figures 2(a) 2(c) and2(e)) glucose Pretreatment with quercetin and Gelam honeyextract significantly (119901 lt 005) decreased the expression ofphosphorylated IKK-120573 under 50mM glucose (Figures 2(b)2(d) and 2(f)) by 60 and 47 in a dose dependent mannerThe phosphorylated IKK-120573 protein levels from each samplewere normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treated group)(Figure 2)

34 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on IL-6 Expression under Normal and HyperglycemicConditions HIT-T15 cells were pretreated with the quercetinat concentrations of 20 40 60 and 80 120583M and Gelam honeyextract at concentrations of 20 40 60 and 80120583gmL for 24hours following which they were cultured in 20mM (Figures3(a) 3(c) and 3(e)) or 50mM (Figures 3(b) 3(d) and 3(f))glucose to determine the expression of IL-6 The expressionwas increased in HIT-T15 cells treated with 20mM and50mMglucose alone as comparedwith control Pretreatmentwith the quercetin and Gelam honey extract showed 52and 40 decrease (119901 lt 005) in the expression of IL-6 ina dose dependent manner in the cells that were cultured in20mM glucose (Figures 3(a) 3(c) and 3(e)) Pretreatmentwith quercetin and Gelam honey extract significantly (119901 lt005) decreased the expression of IL-6 under 50mM glucose(Figures 3(b) 3(d) and 3(f)) by 60 and 47 in a dosedependent mannerThe IL-6 protein levels from each sample


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Figure 1 Effect of quercetin and Gelam honey extract on phospho-JNK expression Quantitative analysis and representative western blotanalysis of phospho-JNK in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and50mM ((b) (d) and (f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSSversion 160 software and are presented as themean plusmn standard deviation and represent themean of three different experiments (e) lowast119901 lt 005and 119901 lt 0005 quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin andhoney extract treated compared to the 50mM glucose alone

were normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treatedgroup)

35 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on IL-1120573 Expression under Normal and HyperglycemicConditions HIT-T15 cells were pretreated with the quercetinat concentrations of 20 40 60 and 80 120583M and Gelamhoney extract at concentrations of 20 40 60 and 80 120583gmLfor 24 hours following which they were then culturedwith 20 or 50mM glucose to determine the expressionof IL-1120573 IL-1120573 in HIT-T15 cells was markedly increasedfollowing 20mM (Figures 4(a) 4(c) and 4(e)) and 50mM(Figures 4(b) 4(d) and 4(f)) glucose treatment but the

trend was reversed after pretreatment with quercetin andGelam honey extract Pretreatment of the cells with differentconcentration of quercetin and honey extract for 24 hourssignificantly reduced the expression of IL-1120573 up to 55 and42 respectively compared to the cells that were culturedalone with 20mM glucose (Figures 4(a) 4(c) and 4(e))On the other hand pretreatment with quercetin and honeyextract reduced the expression of IL-1120573 significantly (119901 lt005) up to 63 and 54 respectively compared to the cellsthat were cultured alone with 50mM glucose (Figures 4(b)4(d) and 4(f)) The IL-1120573 protein levels from each samplewere normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treated group)Figure 3




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Figure 2 Effect of quercetin and Gelam honey extract on phospho-IKK-120573 expression Quantitative analysis and representative western blotanalysis of phospho-IKK-120573 in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and50mM ((b) (d) and (f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSSversion 160 software and are presented as themean plusmn standard deviation and represent themean of three different experiments (e) lowast119901 lt 005and 119901 lt 0005 quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin andhoney extract treated compared to the 50mM glucose alone

36 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on TNF Alpha Expression under Normal and Hyper-glycemic Conditions HIT-T15 cells were pretreated with thequercetin at concentrations of 20 40 60 and 80120583M andGelam honey extract at concentrations of 20 40 60 and80 120583gmL for 24 hours following which they were thencultured with 20mM (Figures 5(a) 5(c) and 5(e)) or 50mM(Figures 5(b) 5(d) and 5(f)) glucose to determine the TNFalpha The TNF alpha was increased in HIT-T15 cells treatedwith 20mM and 50mM glucose alone as compared withcontrol Pretreatment with the quercetin and Gelam honeyextract showed 43 and 55 decrease (119901 lt 005) in theexpression of TNF alpha in a dose dependent manner inthe cells that were cultured in 20mM glucose (Figures 5(a)

5(c) and 5(e)) Pretreatmentwith quercetin andGelamhoneyextract significantly (119901 lt 005) decreased the expression ofTNF alpha in a dose dependent manner in the cells culturedwith 50mM glucose (Figures 5(b) 5(d) and 5(f)) by 52and 66 The TNF alpha protein levels from each samplewere normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treated group)(Figure 5)

37 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on pIRS-1 (Ser307) Expression under Normal andHyperglycemic Conditions HIT-T15 cells were pretreatedwith the quercetin at concentrations of 20 40 60 and80 120583M and Gelam honey extract at concentrations of 20 40



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Figure 3 Effect of quercetin and Gelam honey extract on IL-6 expression Quantitative analysis and representative western blot analysis ofIL-6 in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and 50mM ((b) (d) and(f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSS version 160 softwareand are presented as the mean plusmn standard deviation and represent the mean of three different experiments (e) lowast119901 lt 005 and 119901 lt 0005quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin and honey extract treatedcompared to the 50mM glucose alone

60 and 80 120583gmL for 24 hours following which they werethen cultured with 20mM (Figures 6(a) 6(c) and 6(e)) or50mM (Figures 6(b) 6(d) and 6(f)) glucose to determinethe phosphorylation of IRS-1 (Ser307) The phosphorylationof IRS-1 (Ser307) was increased in HIT-T15 cells treated with20mM and 50mM glucose alone as compared with controlPretreatment with the quercetin and Gelam honey extractshowed 49 and 55 decrease (119901 lt 005) in the expressionof pIRS-1 (Ser307) in a dose dependent manner in the cellsthat were cultured in 20mM glucose (Figures 6(a) 6(c)and 6(e)) Pretreatment with quercetin and Gelam honeyextract significantly (119901 lt 005) decreased the expressionof pIRS-1 (Ser307) in cells that were cultured in 50mM

glucose (Figures 6(b) 6(d) and 6(f)) by 43 and 60 in adose dependent manner The pIRS-1 protein levels from eachsample were normalized to their respective 120573-actin proteinamounts (lowast119901 lt 005 and 119901 lt 0005 versus glucose treatedgroup) (Figure 6)

38 Effect of Pretreatment with Quercetin and Gelam HoneyExtract on pAkt (Ser473) Expression under Normal andHyperglycemic Conditions HIT-T15 cells were pretreatedwith the quercetin at concentrations of 20 40 60 and80 120583M and Gelam honey extract at concentrations of 20 4060 and 80 120583gmL for 24 hours following which they werethen cultured with 20 or 50mM glucose to determine the



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Figure 4 Effect of quercetin and Gelam honey extract on IL-1120573 expression Quantitative analysis and representative western blot analysisof IL-1120573 in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and 50mM ((b) (d) and(f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSS version 160 softwareand are presented as the mean plusmn standard deviation and represent the mean of three different experiments (e) lowast119901 lt 005 and 119901 lt 0005quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin and honey extract treatedcompared to the 50mM glucose alone

phosphorylation of Akt (Ser473) There was no change in theAkt (Ser473) phosphorylation in HIT-T15 cells when treatedwith 20 mM glucose (Figures 7(a) 7(c) and 7(e)) but it wasmarkedly reduced when the cells were treated with 50mM(Figures 7(b) 7(d) and 7(f)) glucose Pretreatment of thecells with different concentration of quercetin and honeyextract for 24 hours significantly increased the expression ofpAkt (Ser473) up to 38 and 70 respectively comparedto the cells that were cultured alone with 20mM glucose(Figures 7(a) 7(c) and 7(e))On the other hand pretreatmentwith quercetin and honey extract increased the expressionof pAkt (Ser473) significantly (119901 lt 005) up to 30 and54 respectively compared to the cells that were culturedalone with 50mM glucose (Figures 7(b) 7(d) and 7(f)) Thecells that were exposed to Akt inhibitor VIII prevented the

quercetin and honey extract induced Akt Ser473 phosphory-lation (Figure 7) The pAkt protein levels from each samplewere normalized to their respective 120573-actin protein amounts(lowast119901 lt 005 and 119901 lt 0005 versus glucose treated group)(Figure 7)

4 Discussion

In our previous study we investigated the antioxidant effectof the Malaysian Gelam honey and some of its flavonoidcomponents (chrysin luteolin and quercetin) individu-ally on pancreatic hamster cells (HIT-T15) cultured underhyperglycemic conditions Our data demonstrated that thecultured cells pretreated with the extract of the Gelamhoney and the different flavonoid components (quercetin


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Figure 5 Effect of quercetin andGelamhoney extract onTNF alpha expressionQuantitative analysis and representativewestern blot analysisof TNF alpha in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and 50mM ((b) (d)and (f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSS version 160software and are presented as the mean plusmn standard deviation and represent the mean of three different experiments (e) lowast119901 lt 005 and119901 lt 0005 quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin and honeyextract treated compared to the 50mM glucose alone

luteolin and chrysin) at varying concentrations for 24 hoursprotected the 120573 cell from oxidative damage caused by ROSinduced by hyperglycemia [21] Furthermore we investigatedthe effect of Gelam honey extract and quercetin on the stressactivated NF-120581B andMAPK pathways and IRS-1 serine phos-phorylation causing insulin resistance and the Akt activatedinsulin signaling pathway causing increase in insulin contentOur data demonstrated that the Gelam honey extract andquercetin had the best protective effect against hyperglycemiainduced oxidative stress by improving the insulin content andinsulin resistance [22]

The floral source of Gelam honey is Melaleuca cajuputiThis was the only source of Gelam honey that was used inKamaruddin et al laboratory and different students workedon the different aspects of this honey They were the first toestablish the detection of the phenolic compounds in Gelamhoney found in Malaysia using HPLC [23] Chromatogramof phenolic acid and flavonoids detected in Malaysian Gelamhoney using HPLC-UV absorption (1) 290 nm and (2)340 nm is shown in the paper of the same group [24] In ourprevious study we used three different flavonoids chrysinluteolin and quercetin [21 22] This shows that the quercetin


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(Ser307

)

Figure 6 Effect of quercetin on pIRS-1 (Ser307) expression Quantitative analysis and representative western blot analysis of pIRS-1 (Ser307)in HIT-T15 cells pretreated with quercetin and Gelam honey extract in cells cultured in 20mM ((a) (c) and (e)) and 50mM ((b) (d) and(f)) glucose The results were normalized with 120573-actin antibody Data were analyzed with one-way ANOVA using SPSS version 160 softwareand are presented as the mean plusmn standard deviation and represent the mean of three different experiments (e) lowast119901 lt 005 and 119901 lt 0005quercetin and honey extract treated compared to the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin and honey extract treatedcompared to the 50mM glucose alone

was themost effective amongst the three flavonoidsTheworkon caffeic acid on the same Gelam honey has already beenpublished by the above-mentioned group [25 26]

Therefore in our present study we determined the effectof Gelam honey extract and quercetin on the oxidative stressactivated JNK and IKK-120573 inflammatory pathways and IRS-1 serine phosphorylation causing insulin resistance and theAkt activated insulin signaling pathway which attenuates theinflammation thereby increasing insulin sensitivity

Several studies have demonstrated that flavonoids mayreduce hyperglycemia and exert protective effects againstnonenzymatic glycation of proteins in animals [27 28]The flavonoid quercetin in particular has been reported tohave anti-inflammatory properties in diabetic animalmodels

by protecting the retinal cell apoptosis in diabetes [29]Under diabetic conditions hyperglycemia induced ROS andinflammatory cytokines presumably lead to the activation ofthe JNK and IKK-120573 pathways It has been shown that IKK andJNK control the major inflammatory response pathways inhyperglycemia and that they are activated by oxidative stress[30 31]

Yuan et al [32] demonstrated that reduced signalingthrough the IKK-120573 pathway inhibition by sodium salicy-late in obese mice is accompanied by improved insulinsensitivity Again the study showed that phosphorylationof IKK-120573 was increased in diabetes but the blockadeof NF-120581B oxidative stress and the genetic deletion ofTNF-120572 attenuated phosphorylation of IKK-120573 suggesting


(a)

(b)

(c)

(d)

(e) (f)

20mM Q20 Q20Q40 Q60 Q80 Q80ControlAkt inhibitor VIII

50mM Q20 Q40 Q60 Q80Control Q20 Q80

Akt inhibitor VIII

20mMControl HE20 HE20HE40 HE60 HE80 HE80Akt inhibitor VIII

120573-actin

HE20 HE8050mMControl HE20 HE40 HE60 HE80Akt inhibitor VIII

lowastlowastlowastlowast

lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Figure 7 Effect of quercetin and Gelam honey extract on pAkt (Ser473) expression Quantitative analysis and representative western blotanalysis of pAkt (Ser473) inHIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20mM ((a) (c) and (e)) and 50mM((b) (d) and (f)) glucose An increase in the level of pAkt (Ser473) was observed after pretreatment with quercetin and honey extract Aktinhibitor VIII prevented the expression of Akt Ser473 phosphorylation induced by quercetin and honey extract The results were normalizedwith 120573-actin antibody Data were analyzed with one-way ANOVA using SPSS version 160 software and are presented as the mean plusmn standarddeviation and represent the mean of three different experiments (e) lowast119901 lt 005 and 119901 lt 0005 quercetin and honey extract treated comparedto the 20mM glucose alone (f) lowast119901 lt 005 and 119901 lt 0005 quercetin and honey extract treated compared to the 50mM glucose alone

that the phosphorylation of IKK-120573 is increased in dia-betic mice by the activation of oxidative stress and TNF-120572[33]

The increase in free fatty acids inflammatory cytokinesand oxidative stress under diabetic conditions activates theJNK pathway which causes insulin resistance and pancreatic120573 cell dysfunction [34] It has been reported that the JNK

pathway was abnormally activated in the liver muscle andadipose tissue in obese type 2 diabetic mice and that theinsulin resistance was substantially reduced when the micewere homozygous for a targeted mutation in the JNK1 gene

Our data showed that exposure of HIT-T15 cells to 20and 50mM glucose caused a significant increase in the levelof phosphorylated JNK (Figure 1) and phosphorylated IKK-120573


(Figure 2) expressions compared to the control Pretreatmentwith quercetin and Gelam honey extract significantly (119901 lt005) reduced the ROS induced inflammatory pathway ofphosphorylated JNK (Figure 1) and phosphorylated IKK-120573(Figure 2) when treated with 20 and 50mM glucose Thisgives further support to the fact that oxidative stress inducedby high glucose activates the major inflammatory pathways

It has been shown that hyperglycemia is proinflammatoryand is normally restrained by the anti-inflammatory effectof insulin secreted in response to that stimulus [35] Hyper-glycemia induced by glucose clamping in normal subjectsshowed a significant increase in the circulating levels ofinterleukin- (IL-) 6 IL-18 Il-1120573 and tumor necrosis factor-120572 (TNF-120572) These effects were more sustained in patientswith impaired glucose tolerance as well as in those after con-secutive pulses of intravenous glucose which were annulledby glutathione implicating an oxidative mechanism [31]Interestingly enough these cytokines have been implicatedin insulin resistance (TNF-120572 IL-6 and IL-1120573) atheroscle-rotic plaque destabilization (IL-18) and future cardiovascularevents (IL-6 IL-18 IL-1120573 and TNF-120572) So an increasedoxidative stress may be a likely mechanism linking stresshyperglycemia to increased inflammatory cytokine produc-tion [31 34] Several cross-sectional studies showed thatinsulin resistance and type 2 diabetes are associated withhigher levels of C-reactive protein (CRP) interleukin-6 (IL-6) interleukin-1120573 and tumor necrosis factor-120572 (TNF-120572)which are markers of subclinical systemic inflammation [35]Furthermore various longitudinal studies have shown thatelevated levels of CRP IL-1120573 IL-6 and TNF-120572 predict thedevelopment of type 2 diabetes [35]

Our data supported the above statement by showing thatTNF-120572 IL-6 and IL-1120573 expressions (Figures 3 4 and 5)in HIT-T15 cells were markedly increased following 20 and50mM glucose treatment but were reversed after pretreat-ment with quercetin and Gelam honey extract In additionour data showed that the expression of TNF-120572 IL-6 and IL-1120573 (Figures 3 4 and 5) was decreased

Our previous data demonstrated that Akt (Ser473) phos-phorylation was increased after pretreatment with quercetinand honey extract (REF) Effect of Gelam honey on theoxidative stress induced signaling pathways in pancreatichamster cells [22]

In the year 2001 it was reported thatmice lacking the pAkt(Ser473) protein were insulin resistant with impaired insulinsecretion [36] It was shown that Akt (Ser473) is necessaryfor normal pancreatic 120573 cell function including a novelregulatory role for Akt signaling in insulin secretion [37]Several studies reported that a decrease in insulin secretionand insulin resistance induced by hyperglycemia has beenassociated with decreased Akt activity [38ndash41]

In our previous papers [21 22] we showed that the cellscultured under 20mM glucose did not show any changes atthe level of the Akt phosphorylation while under 50mM theAkt (serine 473) phosphorylation was reduced significantlyThe phosphorylation was increased significantly after pre-treatment with quercetin and honey extract Akt inhibitorVIII prevented the expression of Akt Ser473 phosphorylationinduced by quercetin and honey extract When we studied

the insulin content our data demonstrated that there wasa significant increase in the insulin content (119901 lt 005)when the cells cultured under 20mMglucose were pretreatedwith quercetin and honey extract but there was a significantdecrease in the insulin content (119901 lt 005) when the cellswere treated with Akt inhibitor VIII before pretreating withquercetin and Gelam honey extract when the cells werecultured in 50mM glucose and there was a significantincrease in the insulin content (119901 lt 005 and 119901 lt 0005)when the cells were pretreated with quercetin and Gelamhoney extract A significant decrease was seen (119901 lt 005 and119901 lt 0005) when the cells were treatedwithAkt inhibitor VIIIbefore pretreating with quercetin and Gelam honey extract

Thus our data demonstrated the protective effects of Aktagainst 120573 cell dysfunction and insulin resistance which wasfurther supported by the fact that Akt inhibitor increasedthe insulin resistance by decreasing the quantity of insulinsecreted

It has been shown that activation of the IKK-120573 and JNKpathways increases IRS-1 serine phosphorylation which leadsto suppression of insulin signaling and that suppression ofthe IKK pathway decreases insulin resistance and amelioratesglucose intolerance in diabetic mice [29] Our data whichsupports the above statement showed that phosphorylationof IRS-1 (Ser307) was increased in HIT-T15 cells treatedwith 20 and 50mM glucose alone as compared with controlPretreatment with the quercetin and Gelam honey extractsignificantly decreased (119901 lt 005) the expression of pIRS-1(Ser307) (Figure 6) in a dose dependent manner in the cellsthat were cultured in 20 and 50mM glucose (Figure 6) Ourdata suggest that there is decrease in phosphorylation of IRS-1(Ser307) (Figure 6)

5 Conclusion

In conclusion our data suggest the potential use of the extractfrom Gelam honey in treating diabetes by modulating theinflammatory pathways The data provide further supportto the role of inflammation in 120573 cell dysfunction andinsulin resistance In addition since stress-induced JNK andIKK pathways are involved in the development of insulinresistance inflammation and 120573 cell dysfunction it is possiblethat such pathways could be a therapeutic target for type 2diabetes

Disclosure

Sher Zaman Safi and Kalaivani Batumalaie are both firstauthors of this paper in which the western blotting wasperformed by Kalaivani Batumalaie

Conflict of Interests

The authors declare no conflict of interests in this work

Authorsrsquo Contribution

Sher Zaman Safi contributed to conception and design ofthe study Rajes Qvist performed revision of paper Kalaivani


Batumalaie is the student who did the experiments of WBIkram Shah Ismail is Head of the Diabetic Group and revisedthe paper Sher Zaman Safi and Kalaivani Batumalaie havecontributed equally to this work

Acknowledgments

The authors would like to thank the University of Malayafor their financial support Grants nos RG516-13HTM andRG528-13HTMThe authors are also thankful to the IPPP forsupporting the postdoctoral fellowship

References

[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[2] S Z Safi R Qvist K Chinna M A Ashraf D Paramasivamand I S Ismail ldquoGene expression profiling of the peripheralblood mononuclear cells of offspring of one type 2 diabetic par-entrdquo International Journal of Diabetes in Developing Countriespp 1ndash13 2015

[3] A F Amos D J McCarty and P Zimmet ldquoThe rising globalburden of diabetes and its complications estimates and projec-tions to the year 2010rdquo Diabetic Medicine vol 14 no 5 pp S7ndashS85 1997

[4] S Z Safi H Shah G O S Yan and R Qvist ldquoInsulinresistance provides the connection between hepatitis C virusand diabetesrdquoHepatitisMonthly vol 15 no 1 Article ID e239412015

[5] S E Shoelson J Lee and A B Goldfine ldquoInflammation andinsulin resistancerdquoThe Journal of Clinical Investigation vol 116no 7 pp 1793ndash1801 2006

[6] S Z Safi R Qvist S Kumar K Batumalaie and I S BIsmail ldquoMolecular mechanisms of diabetic retinopathy generalpreventive strategies and novel therapeutic targetsrdquo BioMedResearch International vol 2014 Article ID 801269 18 pages2014

[7] D Giugliano A Ceriello and G Paolisso ldquoOxidative stress anddiabetic vascular complicationsrdquo Diabetes Care vol 19 no 3pp 257ndash267 1996

[8] S Z Safi R Qvist G O S Yan and I S B Ismail ldquoDifferentialexpression and role of hyperglycemia induced oxidative stress inepigenetic regulation of 1205731 1205732 and 1205733-adrenergic receptors inretinal endothelial cellsrdquo BMC Medical Genomics vol 7 article29 2014

[9] N Sattar C G Perry and J R Petrie ldquoType 2 diabetes asan inflammatory disorderrdquo The British Journal of Diabetes andVascular Disease vol 3 no 1 pp 36ndash41 2003

[10] V Aguirre E D Werner J Giraud Y H Lee S E Shoelsonand M F White ldquoPhosphorylation of Ser307 in insulin receptorsubstrate-1 blocks interactions with the insulin receptor andinhibits insulin actionrdquo Journal of Biological Chemistry vol 277no 2 pp 1531ndash1537 2002

[11] N Li T Brun M Cnop D A Cunha D L Eizirik and PMaechler ldquoTransient oxidative stress damages mitochondrialmachinery inducing persistent 120573-cell dysfunctionrdquoThe Journalof Biological Chemistry vol 284 no 35 pp 23602ndash23612 2009

[12] V T Samuel and G I Shulman ldquoMechanisms for insulinresistance common threads and missing linksrdquo Cell vol 148no 5 pp 852ndash871 2012

[13] S Z Safi K BatumalaieMMansor et al ldquoGlutamine treatmentattenuates hyperglycemia-induced mitochondrial stress andapoptosis in umbilical vein endothelial cellsrdquoClinics vol 70 no8 pp 569ndash576 2015

[14] B Draznin ldquoMolecular mechanisms of insulin resistanceSerine phosphorylation of insulin receptor substrate-1 andincreased expression of p85120572 the two sides of a coinrdquo Diabetesvol 55 no 8 pp 2392ndash2397 2006

[15] E Bernal-Mizrachi S Fatrai J D Johnson et al ldquoDefectiveinsulin secretion and increased susceptibility to experimentaldiabetes are induced by reduced Akt activity in pancreatic islet120573 cellsrdquo The Journal of Clinical Investigation vol 114 no 7 pp928ndash936 2004

[16] M A Mamary A A Meeri and M A Habori ldquoAntioxidantactivities and total phenolics of different types of honeyrdquoNutrition Research vol 22 no 9 pp 1041ndash1047 2002

[17] A Aljadi andM Kamaruddin ldquoEvaluation of phenolic contentsand antioxidant capacities of twoMalaysian floral honeysrdquo FoodChemistry vol 85 no 4 pp 513ndash518 2004

[18] HA LWahdan ldquoCauses of the antimicrobial activity of honeyrdquoInfection vol 26 no 1 pp 26ndash31 1998

[19] A Seo and C V Morr ldquoImproved high-performance liquidchromatographic analysis of phenolic acids and iso-flavonoidsfrom soybean protein productsrdquo Journal of Agricultural andFood Chemistry vol 32 no 3 pp 530ndash533 1984

[20] S Z Hussein K M Yusoff S Makpol and Y A YusofldquoAntioxidant capacities and total phenolic contents increasewith gamma irradiation in two types of Malaysian honeyrdquoMolecules vol 16 no 8 pp 6378ndash6395 2011

[21] B Kalaivani Q Rajes M Y Kamaruddin S I Ikram and D SShamala ldquoThe antioxidant effect of theMalaysian Gelam honeyon pancreatic hamster cells cultured under hyperglycemicconditionsrdquo Clinical and Experimental Medicine vol 14 no 2pp 185ndash195 2014

[22] B Kalaivani Z S Sher M Y Kamaruddin S I Ikram D SShamala and Q Rajes ldquoEffect of Gelam honey on the oxidativestress-induced signaling pathways in pancreatic hamster cellsrdquoInternational Journal of Endocrinology vol 2013 Article ID367312 10 pages 2013

[23] A M Aljadi and K M Yusoff ldquoIsolation and identification ofphenolic acids in Malaysian honey with anti-bacterial proper-tiesrdquo Turkish Journal of Medical Sciences vol 33 pp 229ndash2362003

[24] S Z Hussein K M Yusoff S Makpol and Y A M YusofldquoAntioxidant capacities and total phenolic contents increasewith gamma irradiation in two types of Malaysian honeyrdquoMolecules vol 16 no 8 pp 6378ndash6395 2011

[25] M Kassim M Mansor T A Kamalden et al ldquoCaffeic acidPhenethyl ester (CAPE) scavenger of peroxynitrite in vitro andin sepsis modelsrdquo Shock vol 42 no 2 pp 154ndash160 2014

[26] M Kassim M Mansor A Suhaimi G Ong and K M YusoffldquoGelam honey scavenges peroxynitrite during the immuneresponserdquo International Journal of Molecular Sciences vol 13no 9 pp 12113ndash12129 2012

[27] O Coskun M Kanter A Korkmaz and S Oter ldquoQuercetina flavonoid antioxidant prevents and protects streptozotocin-induced oxidative stress and 120573-cell damage in rat pancreasrdquoPharmacological Research vol 51 no 2 pp 117ndash123 2005

[28] N Y Nuraliev and G A Avezov ldquoThe efficacy of quercetin inalloxan diabetesrdquo Experimental and Clinical Pharmacology vol55 no 1 pp 42ndash44 1992


[29] I Jessica B Maia G Muraya and G Carlos ldquoQuercetin ame-liorates hyperglycemia-induced inflammation and apoptosis inthe retina and lateral geniculate nucleus in a rat model of type2 diabetes mellitusrdquo The FASEB Journal vol 28 no 1 article6888 2014

[30] E Katherine M Raffaele and G Dario ldquoStress hyperglycemiainflammation and cardiovascular eventsrdquo Diabetes Care vol26 no 5 pp 1650ndash1651 2003

[31] S Kumar S Z Safi R Qvist and I S Ismail ldquoEffect of agonistsof adenosine receptors on inflammatory markers in humanMuller cellsrdquo Current Science vol 106 no 4 pp 582ndash586 2014

[32] M Yuan N Konstantopoulos J Lee et al ldquoReversal of obesity-and diet-induced insulin resistance with salicylates or targeteddisruption of Ikk120573rdquo Science vol 293 no 5535 pp 1673ndash16772001

[33] H Kaneto N Katakami M Matsuhisa and T MatsuokaldquoRole of reactive oxygen species in the progression of Type 2diabetes and atherosclerosisrdquo Mediators of Inflammation vol2010 Article ID 453892 11 pages 2010

[34] H Kaneto Y Nakatani D Kawamori T Miyatsuka and T-A Matsuoka ldquoInvolvement of oxidative stress and the JNKpathway in glucose toxicityrdquoThe Review of Diabetic Studies vol1 no 4 pp 165ndash174 2004

[35] D R Nathalie P Rita D Jingzhong et al ldquoDiabetes hyper-glycemia and inflammation in older individuals the healthaging and body composition studyrdquo Diabetes Care vol 29 no8 pp 1902ndash1908 2006

[36] H Cho J Mu J K Kim et al ldquoInsulin resistance and a diabetesmellitus-like syndrome in mice lacking the protein kinase Akt2(PKB120573)rdquo Science vol 292 no 5522 pp 1728ndash1731 2001


[38] P Gual Y Le Marchand-Brustel and J-F Tanti ldquoPositiveand negative regulation of insulin signaling through IRS-1phosphorylationrdquo Biochimie vol 87 no 1 pp 99ndash109 2005

[39] D A Glauser and W Schlegel ldquoThe emerging role ofFOXO transcription factors in pancreatic 120573 cellsrdquo Journal ofEndocrinology vol 193 no 2 pp 195ndash207 2007

[40] A D Kohn S A Summers M J Birnbaum and R A RothldquoExpression of a constitutively active Akt SerThr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter4 translocationrdquoThe Journal of Biological Chemistry vol 271 no49 pp 31372ndash31378 1996

[41] K K Y Cheng K S L Lam D Wu et al ldquoAPPL1 potentiatesinsulin secretion in pancreatic 120573 cells by enhancing proteinkinase Akt-dependent expression of SNARE proteins in micerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 23 pp 8919ndash8924 2012

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


pathways leading to the production of inflammatory markerssuch as IL-6 and TNF-120572 that impair insulin signaling throughserine phosphorylation of IRS-1 [9]

Insulin is a pleiotropic hormone which has many func-tions that are exerted across a variety of insulin target tissuesthrough several intracellular signaling cascades Insulin sub-strate (IRS-1) mediated insulin signaling regulates peripheralinsulin action as well as pancreatic 120573 cell function by regulat-ing proliferation survival and insulin secretion The defectsin insulin signaling pathwaymainly involve IRS-1 Activationof the insulin receptor leads to tyrosine phosphorylation ofIRS-1 thereby initiating signal transduction Insulin receptortyrosine kinases can signal through the phosphatidylinositol3-kinase (PI3K) pathway which is mainly responsible for themetabolic actions of insulin The major target of PI3K is theserine threonine kinase Akt It has been shown that the Aktdependent mechanism in the distal events of exocytosis isresponsible for the defect in insulin secretion [7 10]

This transient exposure of 120573 cells to oxidative stressinterrupts the normal coupling of glucose metabolism toinsulin secretion by activating stress signaling pathways andthe inflammatory pathways [11] Despite the multiplicity ofinflammatory pathways the development of insulin resis-tance is often due to the activation of jun-N-terminal kinase(JNK) and the IkappaB kinase (IKK-120573) The JNK and IKK-120573 pathways are activated by ROS and various other factorsincluding the inflammatory cytokines such as TNF-120572 IL-6and IL-1betawhich are involved in the development of insulinresistance found in type 2 diabetes [12]

Reactive oxygen species and mitochondrial stress-induced [13] activation of JNK and IKK-120573 promotes thephosphorylation of IRS-1 at serine sites that negativelyregulate normal signaling through the insulin receptorIRS-1axis as seen in the phosphorylation of IRS-1 at serine 307(Ser307) It has been reported that serine phosphorylationof insulin receptor-1 (IRS-1) inhibits insulin stimulatedtyrosine phosphorylation of IRS-1 leading to an increase ininsulin resistance [10] Therefore one of the causes leadingto a defect in insulin signaling can be attributed to serinephosphorylation of IRS-1 at serine 307 residues whichactivate the jun-N-terminal kinase (JNK) and IKK-120573 thusproviding a plausiblemechanistic link between inflammationand insulin resistance [14]

Recently protein kinase B (PKB andAkt) has been shownto function in the insulin signaling cascade by phospho-rylating transcription factors which are responsible for thetranscription and expression of genes related to insulinsynthesis and secretion Previous studies have shown thatinactivation of Akt can lead to insulin resistance decreased120573 cell mass and impaired insulin secretion [15]

Numerous studies have shown that honey has antioxidantand scavenging properties which prevents the oxidativedamage caused by free radicals The antioxidant propertyis due to the phenolic compounds which are present inthe honey and it has been demonstrated that the biolog-ical activities correlate with the phenolic content of thehoney [16] The phenolic content of the Malaysian Gelamhoney has been shown to have both anti-inflammatoryand antioxidant properties [17] Quercetin is one of the

important components of Gelam honey that may be furtherelaborated in terms of its function and use as antidiabeticagent

Therefore the aim of our present study is to determinethe effect of Gelam honey extract and quercetin on theJNK and IKK-120573 inflammatory pathways and IRS-1 serinephosphorylation which causes insulin resistance and the Aktactivated insulin signaling pathway which improves insulinresistance

2 Research Design and Methods

21 Extraction of Phenolic Compounds from Honey by SolidPhase Extraction (SPE) Gelam honey samples (Departmentof Agriculture Parit Botak Johor Malaysia) were sub-jected to base hydrolysis and extracted with ethyl acetateas described by Wahdan [18] and Seo and Morr [19] Therecovered fractions were combined and dried under nitrogengas

22 Determination of the Phenolic Content Phenolic com-pounds from the extract were assayed using Folin-Ciocalteuassay [20] Briefly the extract (1mL) was added to 10 Folin-Ciocalteu reagent (Sigma F9252) and 05 sodium carbonateThe contents were thoroughly mixed and allowed to standfor 2 hours The absorbance of the blue color that developedafter 2 hours was read at 765 nm Results were expressed inmicrograms of Gallic acid per gram of the extract using astandard curve generated with Gallic acid (Sigma G7384)

23 Determination of the Flavonoid Content The totalflavonoid (TF) content was determined spectrophotometri-cally Briefly 1mL of honey extract or a standard solutionof quercetin (Sigma Q4951) (10 50 100 150 200 and250 120583gmL) in distilled water was added to a 10mL volumet-ric flask containing 4mL of double distilled water 300120583Lof NaNO

2(5 vv) and 300 120583L of 10 AlCl

3 The solution

was allowed to stand at room temperature in the dark for30 minutes and the absorbance was read at 430 nm The TFcontentwas determinedusing the standard curve of quercetin(120583gmL) and was expressed as 120583g of quercetin equivalents in1 g of extract

24 Cell Culture HIT-T15 cells were cultured according tothe instructions provided by ATCC (CRL-1777) On arrivalthe cells were cultured immediately in T-25 cm flask in theF12K medium (ATCC 30-2004) supplemented with 10 FBSand 1 penicillin and streptomycin at 37∘C (5 CO

2in

air) Cells (3rd passage) were trypsinized and subculturedfollowing which they were incubated for 5 days until theyreached 80 confluency

25 Treatment of HIT-T15 Cells with Quercetin and GelamHoney Extract HIT-T15 cells (5 times 105) were pretreatedwith Gelam honey extract (20 40 60 and 80 120583gmL) andquercetin (20 40 60 and 80 120583M) for 24 hours after whichthe medium was replaced with fresh medium Following thisglucose (SigmaG8769) (20 or 50mM)was added and the cellswere incubated for another 24 hours





3 Results








20mM Q20 Q40 Q60 Q80

Phospho-JNKControl

Phospho-JNK



Phospho-JNK

120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast lowast

lowast

lowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



Control

Control

50mM 20 40 60 80

35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



lowast

lowastlowast

lowastlowast

lowast lowast









120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast

lowast

lowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573







(a)

(b)

(c)

(d)

(e) (f)

IL-6

IL-6


IL-6



120573-actin

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-6 lowast

lowast

lowastlowast

lowast

Control 50mM 20 40 60 80



35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

IL-6







(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results








20mM Q20 Q40 Q60 Q80

Phospho-JNKControl

Phospho-JNK



Phospho-JNK

120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast lowast

lowast

lowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



Control

Control

50mM 20 40 60 80

35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



lowast

lowastlowast

lowastlowast

lowast lowast









120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast

lowast

lowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573







(a)

(b)

(c)

(d)

(e) (f)

IL-6

IL-6


IL-6



120573-actin

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-6 lowast

lowast

lowastlowast

lowast

Control 50mM 20 40 60 80



35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

IL-6







(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20mM Q20 Q40 Q60 Q80

Phospho-JNKControl

Phospho-JNK



Phospho-JNK

120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast lowast

lowast

lowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



Control

Control

50mM 20 40 60 80

35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

pho

spho

-JN

K



lowast

lowastlowast

lowastlowast

lowast lowast









120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast

lowast

lowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573







(a)

(b)

(c)

(d)

(e) (f)

IL-6

IL-6


IL-6



120573-actin

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-6 lowast

lowast

lowastlowast

lowast

Control 50mM 20 40 60 80



35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

IL-6







(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















120573-actin

50mMControl

(a)

(b)

(c)

(d)

(e) (f)

HE20 HE40 HE60 HE80

lowast

lowast

lowast

lowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Phospho-IKK-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573

Relat

ive e

xpre

ssio

n of

pho

spho

-IKK

-120573







(a)

(b)

(c)

(d)

(e) (f)

IL-6

IL-6


IL-6



120573-actin

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-6 lowast

lowast

lowastlowast

lowast

Control 50mM 20 40 60 80



35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

IL-6







(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

(b)

(c)

(d)

(e) (f)

IL-6

IL-6


IL-6



120573-actin

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-6 lowast

lowast

lowastlowast

lowast

Control 50mM 20 40 60 80



35

3

4

25

2

15

1

05

0

Relat

ive e

xpre

ssio

n of

IL-6







(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

(b)

(c)

(d)

(e) (f)

IL-1120573




120573-actin

lowast

lowastlowast

lowastlowast

lowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573

lowastlowast

lowastlowast

lowastlowast

lowast

3

25

4

35

2

15

1

05

0Control 50mM 20 40 60 80



Relat

ive e

xpre

ssio

n of

IL-1120573




4 Discussion



120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120573-actin


(a)


(b)


(c)


(d)

(e) (f)

lowastlowast

lowastlowast

lowastlowastlowast

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowast

lowast

lowast

lowast lowast

lowast

3

4

25

35

2

15

1

05

0Control 50mM 20 40 60 80



TNF-120572

TNF-120572

TNF-120572

TNF-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572

Relat

ive e

xpre

ssio

n of

TN

F-120572





(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(b)



(a)

(c)


(d)

(e) (f)


120573-actin

lowastlowast

lowast

lowastlowast

lowast

35

3

25

2

15

1

05

0Control 20mM 20 40 60 80



lowastlowast

lowastlowast

lowastlowast

3

4

2

1

0Control 50mM 20 40 60 80



pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

pIRS-1 (Ser307)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)

Relat

ive e

xpre

ssio

n of

pIR

S-1

(Ser307

)








(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)

(d)

(e) (f)



Akt inhibitor VIII


120573-actin



lowastlowast

lowast

25

2

15

1

05

0Control 20mM 20 40 60 80

Control20mM glucose


lowastlowast

lowast

lowastlowast

lowast

02

04

06

08

1

12

14

16

18

0Control 50mM 20 40 60 80

Control20mM glucose


pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

pAkt (Ser473)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)

Relat

ive e

xpre

ssio

n of

pA

kt (S

er473

)
















5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























5 Conclusion


Disclosure








Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Acknowledgments


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 gelam honey attenuates the oxidative ... honey attenuates the oxidative...

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