Louisin Stte University LSU Digitl Commons LSU Mster's Theses Grdute School 2013 Insulin Secretgogue Activity of Ellgic Acid-Rich Muscdine (Vitis Rotundifoli) nd Indin Gooseerry (Emlic Officinlis) Extrcts on Pncretic Bet Cells Sriknth Erpin Louisin Stte University nd Agriculturl nd Mechnicl College, serpi1@tigers.lsu.edu Follow this nd dditionl works t: http://digitlcommons.lsu.edu/grdschool_theses Prt of the Life Sciences Commons Recommended Cittion Erpin, Sriknth, "Insulin Secretgogue Activity of Ellgic Acid-Rich Muscdine (Vitis Rotundifoli) nd Indin Gooseerry (Emlic Officinlis) Extrcts on Pncretic Bet Cells" (2013). LSU Mster's Theses. 1770. http://digitlcommons.lsu.edu/grdschool_theses/1770 This Thesis is rought to you for free nd open ccess y the Grdute School t LSU Digitl Commons. It hs een ccepted for inclusion in LSU Mster's Theses y n uthorized grdute school editor of LSU Digitl Commons. For more informtion, plese contct gcoste1@lsu.edu.
INSULIN SECRETAGOGUE ACTIVITY OF ELLAGIC ACID-RICH MUSCADINE (VITIS ROTUNDIFOLIA) AND INDIAN GOOSEBERRY (EMBLICA OFFICINALIS) EXTRACTS ON PANCREATIC BETA CELLS A Thesis Sumitted to the Grdute Fculty of the Louisin Stte University nd Agriculturl nd Mechnicl College in prtil fulfillment of the requirements for the degree of Mster of Science in The Deprtment of Food Science y Sriknth Erpin B.Tech., Achry N G Rng Agriculturl University, 2009 Decemer 2013
I dedicte this work to my eloved Prents, Mr. Stynryn Erpin nd Mrs. Venkt Rmn Erpin, my Guru Sri Brhmsri Chgnti Koteswrro gru, nd the Almighty ii
ACKNOWLEDGEMENTS I sincerely thnk the Almighty for estowing his lessings on me throughout my life. I owe the position where I stnd tody to his holy feet. I cknowledge tht without HIS utmost love on me, I would hve chieved nothing. I would like to express my deepest sense of grtitude for my mentor Dr. Jck N. Losso for his invlule guidnce during my study towrds chievement of Mster s degree. His dvices hve een insightful nd not only limited to my thesis work ut lso towrds severl spects of my creer. I cn never forget the encourgement he hs given me to lwys strive for the etterment. I would lso tke this opportunity to thnk my dvisory committee memers, Dr. John W. Finley nd Dr. Sit Aggrwl for their gret support nd vlule suggestions ll through my reserch. My specil thnks to Dr. Sit Aggrwl for lierlly providing me the NIT- 1 pncretic β-cells for my work. I m quite grteful to Ms. Kren Mcdonough, Nmrt Krki, Alfredo Prudente, Adrin Soto, Din Crvjl Aldz, Dmir Torrico, Behnm Keshvrz, Mrjn Mohtshmin nd Luke Mumphrey for their help towrds my reserch. Finlly, my hertfelt thnks to my reltives, Krishn Prsd Mtukumlli, Sireesh Mtukumlli, Venkt Suresh Ged, Sunith Ged nd friends, Yswnth Chintn, Si Sriplli, Srvn kumr Dunn, Murli Krishn Gopu, Prdeep Kumr Mnth, Rjeswri Atl, Venkt Reddy Atl, Chndrmouli Vddeplli, Siv Krthik Ntesn, Steesh Kumr Kodvli, Hri Chintlpudi nd Prveen Chinni, without whose unconditionl support I wouldn t hve reched this stge of my life. iii
TABLE OF CONTENTS ACKNOWLEDGEMENTS...iii ABSTRACT...vi CHAPTER 1. INTRODUCTION...1 CHAPTER 2. LITERATURE REVIEW 4 2.1. Dietes 4 2.1.1. Types of Dietes..4 2.2. Risk fctors for dietes...5 2.2.1. Hyperglycemi, free ftty cids nd type 2 dietes...5 2.3. Oxidtive stress nd β-cells..7 2.4. Inflmmtion nd β-cells.....8 2.4.1. Interleukin-1β (IL-1β) nd β-cells.8 2.5. Dietes Mngement.....12 2.5.1. Therpeutic pproch...12 2.5.2. Dietry pproch.....13 2.5.3. Muscdine or ml ioctives nd dietes 13 2.5.3.1. Muscdine.13 2.5.3.2. Aml (Indin gooseerry).15 2.5.3.3. Ellgic cid nd urolithins 16 2.5.3.4. Isorhmnetin.18 CHAPTER 3. MATERIALS AND METHODS...20 3.1. Regents. 20 3.2. Fruit extrcts preprtion 20 3.3. Determintion of ellgic cid content in muscdine or ml extrcts... 21 3.4. NIT-1 cell line 21 3.5. Conjugtion of plmitic cid with ovine serum lumin (PA-BSA).. 22 3.6. Cell viility ssy 22 3.7. Interction etween inducers nd inhiitors of inflmmtion in NIT-1 cells 23 3.8. Anlysis of IL-1β.. 23 3.9. Anlysis of insulin concentrtion.. 23 3.10. Sttisticl nlysis 24 CHAPTER 4. RESULTS & DISCUSSION 25 4.1. Ellgic cid content in muscdine nd ml extrcts 25 4.2. Effect of EA, UR, IS, MS or AM on the viility of NIT-1 cells treted with Glu, PA or Glu+PA 26 4.3. Effect of EA, UR, IS, MS or AM on Glu-induced IL-1β.... 31 4.4. Effect of EA, UR, IS, MS or AM on PA-induced IL-1β... 35 4.5. Effect of EA, UR, IS, MS or AM on Glu+PA-induced IL-1β.. 39 4.6. Effect of EA, UR, IS, MS or AM on insulin secretion in Glu-treted NIT-1 cells... 42 4.7. Effect of EA, UR, IS, MS or AM on insulin secretion in PA-treted NIT-1 cells 46 iv
4.8. Effect of EA, UR, IS, MS or AM on insulin secretion in Glu+PA-treted NIT-1 cells 50 CHAPTER 5. CONCLUSIONS... 54 REFERENCES. 55 VITA. 67 v
ABSTRACT Dietes is n inflmmtory disese ssocited with hyperglycemi. Chronic exposure of pncretic β-cells to glucolipotoxicity stimultes low-grde inflmmtion ssocited with the relese of pro-inflmmtory cytokines such s interleukin-1β (IL-1β). Incresed levels of IL-1β cn initilly led to decresed insulin secretion nd finlly β-cell deth. Ellgic cid nd quercetin hve een reported to e nti-inflmmtory in severl studies. Vitis rotundifoli (muscdine) or Emlic officinlis (ml) re good sources of ellgic cid nd quercetin. Ellgic cid or quercetin is ioville s is or re metolized into ioville urolithins or isorhmnetin, respectively. The ojective of this study ws to evlute the effect of ellgic cid, urolithin A, isorhmnetin, muscdine or ml extrcts stndrdized to their ellgic cid content on glucose-, plmitic cid-, or glucose + plmitic cid-induced IL-1β nd insulin secretion. Acid-hydrolyzed nd neutrlized extrcts of muscdine or ml were prepred nd the ellgic cid content in the extrcts ws mesured y HPLC. The ellgic cid content in muscdine nd ml extrcts ws 9.4 ± 2.3 mg/g nd 19.4 ± 1.5 mg/g, respectively. NIT-1 pncretic β-cells were incuted with 33.3 mm glucose, 250 µm plmitic cid or 33.3 mm glucose + 250 µm plmitic cid for 24 h followed y ddition of 0.01 10 µm ech of ellgic cid, urolithin A, isorhmnetin, extrcts of muscdine or ml stndrdized to its ellgic cid content followed y dditionl incution for 72 h. All incutions were performed t 37 o C in 5% CO 2 humidified incutor. IL-1β nd insulin were nlyzed in the superntnts y ELISA. Glucose, plmitic cid or glucose + plmitic cid up-regulted IL-1β nd reduced insulin secretion in NIT-1 cells. Glucose induced more IL-1β secretion thn plmitte nd reduced insulin secretion thn plmitic cid. Ellgic cid, muscdine or ml extrcts contining ellgic vi
cid equivlent dose-dependently inhiited IL-1β secretion. Urolithin A or isorhmnetin did not significntly inhiit IL-1β. Ellgic cid, urolithin A, isorhmnetin, or extrcts from muscdine or ml dose-dependently incresed insulin secretion. Muscdine or ml extrcts stndrdized to their ellgic cid content showed higher stimultion of insulin secretion nd IL-1β downregultion compred to pure ellgic cid. A tenttive explntion for this inhiition is the presence of other ioctive compounds in the muscdine or ml extrcts. The results of this study show tht ellgic cid, muscdine or ml re effective modultors of glucose-, plmitic cid- or glucose+plmitic cid-induced IL-1β secretion nd promoters of insulin secretion in β- cells. Keywords: NIT-1 cells; inflmmtion; glucose; plmitic cid; muscdine; ml; ellgic cid; urolithin A; isorhmnetin; IL-1β; insulin vii
CHAPTER 1 INTRODUCTION Dietes is chronic degenertive disese tht develops due to defects in insulin secretion function of pncretic β-cells resulting from excess intke of nutrients such s crohydrtes or fts (1-3). The presence of high levels of glucose or free ftty cids increses free rdicl production nd therey induce oxidtive stress (4, 5) tht cn e trnslted into chronic inflmmtory process y the relese of cytokines (6, 7). The comintion of glucose toxicity nd lipid toxicity is referred to s glucolipotoxicity. Interleukin-1β (IL-1β) is the mjor proinflmmtory cytokine relesed under glucose, lipid or glucolipotoxicity (8). Elevted IL-1β levels led to n initil fulty insulin secretion (9, 10) nd ultimtely β-cell filure (11). Synthetic nti-dietic drugs or plnt foods tht cn modulte inflmmtion cn help in dietes mngement. The demnd for functionl foods hs een incresing due to the growing pulic wreness on the potentil helth enefits of dietry io-ctives (12). Phenolic compounds in plnt foods hve een scried with nti-oxidnt (13), nti-inflmmtory (14) nd nti-dietic (15) potentils. Ellgic cid is phenolic ioctive compound with high nti-oxidnt ctivity present in plnt foods s ellgitnnins (16). Ellgitnnins re hydrolyzed y the intestinl microflor into ellgic cid nd further metolized to urolithins (17, 18). Ellgic cid showed hypo-glycemic nd insulin stimulting effects upon dministrtion to dietic rts (19, 20). On the other hnd, isorhmnetin is metolite of quercetin with nti-inflmmtory (21), hypoglycemic, nd nti-oxidnt ctivities (22). The inhiition of IL-1β s the possile mechnism ehind the potentil of ellgic cid, urolithins or isorhmnetin on insulin stimultion needs to e explored. 1
Muscdine (Vitis rotundifoli) is ntive fruit of the southern prt of the United Sttes. It is good source of ellgic cid nd quercetin (23-25). The nti-dietic ctivity of muscdine hs een demonstrted in n in vivo study (26). Tretment with muscdine extrcts inhiited dietes ssocited complictions such s dvnced glyction end products (27, 28) nd enzymes relted to crohydrte nd lipid metolism (29). Aml (Emlic officinlis) is fruit ntive to Indi nd hs nti-inflmmtory nd nti-oxidnt properties (30, 31). Aml is good source of ellgic cid nd quercetin (32, 33). In vivo studies in dietic rts (34) or humn sujects (35-38) showed tht ml exhiited strong nti-dietic ctivity y lowering lood glucose levels. The studies, however, did not exmine the effect of ml on inflmmtory cytokines s potentil mechnism involved in lowering the lood glucose. The ojective of the present study ws to investigte the effect of ellgic cid, urolithin A, isorhmnetin, or muscdine or ml stndrdized to their ellgic cid content on glucose-, plmitic cid- or glucose + plmitic cid-induced IL-1β nd insulin secretion. To chieve this ojective, the following steps were followed: 1. Determine the ellgic cid content in muscdine nd ml extrcts 2. Determine the effect of ellgic cid, urolithin A, isorhmnetin, or muscdine or ml extrcts on cell viility of glucose-, plmitte- or glucose+plmitic cid-treted NIT- 1 cells 3. Investigte the effect of ellgic cid, urolithin A, isorhmnetin, or muscdine or ml extrcts on the glucose-, plmitte- or glucose+plmitic cid-induced IL-1β in NIT-1 cells 2
4. Study the effect of ellgic cid, urolithin A, isorhmnetin, or muscdine or ml extrcts on the insulin secretion in NIT-1 cells exposed to glucose, plmitte or glucose + plmitic cid. 3
CHAPTER 2 LITERATURE REVIEW 2.1. Dietes Dietes is group of diseses mrked y high levels of lood glucose resulting from defects in insulin secretion, insulin ction or oth (39). It is nticipted tht the numer of dietics in the world will increse 1.5-fold from 371 millions in 2012 to 552 millions y 2030 nd the USA hs one of the highest deth rtes (183,633/yer) from dietes in the world (40). According to the Ntionl Dietes Fct Sheet from CDC, 8.3% (25.8 millions) of Americns re dietics. Among them, 10.9 millions re those ged ove 65 yers, 14.7 millions re those ged etween 20 nd 65 nd 215,000 people ged less thn 20 yers hd dietes in 2010 (39). The estimted costs ssocited with dietes in the US hve risen to $245 illion in 2012 from $174 illion in 2007 (41). The reports stress the necessity to develop strtegic therpeutic pln ssocited with dietry mngement to ttenute the disese nd restore insulin ctivity. 2.1.1. Types of Dietes There re siclly two types of dietes, Type 1 dietes (T1D) nd Type 2 dietes (T2D). T1D is n utoimmune medited process, which results in the destruction of pncretic β- cells therey impiring insulin secretion nd leding to hyperglycemi. Exogenous insulin injection is required to control the metolic disorder (42). T2D is lifestyle-relted, developing s result of excess pressure on β-cells to mintin glucose homeostsis generted y incresed energy consumption. The excess clories from crohydrtes induce hyperinsulinemic condition which leds to chronic insulin resistnce. Dietry lnce in comintion with physicl ctivity nd insulin therpy or insulin secretgogue dministrtion cn improve the condition (43, 44). Norml pncretic tissues contin nerly one million islets (1-2% of totl pncres) nd pproximtely one illion β-cells (50% of islets), of which t lest 40% of the β-cell mss is 4
required for the mintennce of glucose homeostsis (45, 46). Under norml helthy conditions nd predietic stte, the β-cell mss is mintined through the self-repliction of β-cells nd regenertion from other pncretic cells. The incresed mss helps the β-cells to mintin stle glucose levels nd compenste for insulin resistnce (47). The stress of continuous hyperglycemic lod eventully overwhelms the pncres, leds to the filure of β-cell functionlity nd regenertion. The initil hyperinsulinemic conditions ultimtely cuses other tissues to ecome insulin resistnt which leds to clinicl dietes (45, 48). Dietes is ssocited with 40% - 60% decrese in islet volume (49-51), reduced β-cell re y 24% (52), nd 30% lower concentrtion of pncretic insulin (50) due to loss in β-cells compred to non-dietic condition. The defective mitochondri in the β-cells in dietic condition led to decrese in the cellulr ATP/ADP rtio to 40%. (53). 2.2. Risk fctors for dietes Genetics is non-modifile risk fctor for dietes. Identifiction of cndidte genes hs een postulted s relile method to identify genes susceptile to development of the disese. However, it is chllenging to segregte group of common genes s they vry depending on ethnic groups, exposure to environment nd gene-environment interctions (54). Life style fctors such s excessive consumption of high levels of sturted fts, sugr-sweetened everges nd strchy foods, low fier diets nd sedentry lifestyle contriute to the development of dietes (55). 2.2.1. Hyperglycemi, free ftty cids nd type 2 dietes The durtion of exposure of β-cells to high concentrtions of nutrients such s glucose or ftty cids will determine the effect of the lter on the β-cells. Acute exposure to high concentrtion of nutrients stimultes insulin secretion wheres chronic exposure impirs β-cell function nd ffects β-cell survivl s oserved in type 2 dietic ptients (56). Type 2 dietes 5
is chrcterized y filure to increse β-cell mss tht cn compenste for the incresed demnd for insulin (51, 57). The comintion of glucose toxicity nd lipid toxicity is referred to s glucolipotoxicity. Pncretic β-cells re highly responsive to stimuli from glucose in serum. Increse in glucose levels cn led to β-cell hyperctivity to mintin glucose homeostsis. (58). Chronic hyperctivity pressurizes β-cells to dpt to provide sufficient insulin. This process increses the demnd for production of more mount of denosine tri phosphte (ATP) through glycolytic nd mitochondril ctivity. Hyperctivity of mitochondri increses ROS production. Additionlly, the inherent low levels of key nti-oxidnt proteins in pncretic islets mke β-cells highly susceptile to metolic stress (59). Oesity nd hyperlipidemi re the primry environmentl risk fctors leding to dietes. Dietry ft nd dipose tissue re the source of free ftty cids in plsm. Incresed dipose tissue mss under oese conditions increses the concentrtion of free ftty cids tht serves s the mjor risk fctor ssocited with hyperlipidemi in dietes (1-3, 60). Chronic exposure of β-cells to free ftty cids hs shown impired glucose-stimulted insulin secretion nd insulin synthesis (61). Moreover, free ftty cids contriute to insulin resistnce nd lso prevent the β-cell response to overcome insulin resistnce y secreting more insulin (62). The comintion of insulin resistnce nd β-cell dysfunction leds to inility to control lood glucose levels nd s result dietes develops (63). High levels of free ftty cids (FFA) resulting from hyperlipidemi lso cuse n influx of immune cells such s mcrophges nd T- cells in the pncres. Due to this, low-grde inflmmtory process develops through the production of inflmmtory cytokines such s IL-1β which otherwise is usully trnsient or milder in individuls tht re not t risk of developing type 2 dietes (64). 6
Long-term exposure to sturted free ftty cids hs deleterious effects on β-cells. Cultured humn islets exposed to 500 µm plmitic cid for 4 dys resulted in incresed levels of β-cell poptosis mrkers, decresed β-cell prolifertion nd suppression of insulin secretion (65). Rt pncretic islets exposed to 250 µm plmitic cid for 48 h displyed type 2 dietic chrcteristics such s decresed insulin synthesis nd impired glucose stimulted insulin secretion in β-cells (66). 2.3. Oxidtive stress nd β-cells Imlnce etween cellulr nti-oxidnt levels nd superfluous genertion of rective oxygen species (ROS) is defined s oxidtive stress. The genertion of ROS tkes plce either due to incresed demnd for ATP leding to incomplete oxygen reduction or y controlled reduction yielding the highly rective superoxides, hydroxyl rdicls nd hydrogen peroxide, most of which re degrded y nti-oxidnt enzymes such s superoxide dismutse, ctlse, glutthione peroxidse nd peroxiredoxin (67, 68). Free rdicls induce chronic degenertive diseses y dmging components such s lipids, proteins nd nucleic cids (69). Glucose serves s the source of energy for β-cell, signls the production of insulin within the cell, nd provides the signl for insulin secretion (70). β-cells mintin glucose homeostsis y the conversion of stimuli from glucose to insulin secretion (71). During hyperglycemic conditions, incresed glucose influx provides the sustrte for the increse of mitochondril respirtory ctivity nd susequent reduction of moleculr oxygen to free rdicls yielding superoxide nions (72, 73). Insulin secretion process is highly susceptile to the ROS. Diets rich in crohydrtes or ftty cids cn led to hyperctivity of respirtory metolic process nd generte ROS (4). Even fleeting exposure to oxidtive stress due to glucolipotoxicity cn cuse inctivtion of mitochondri nd interception of signls involved in glucose stimulted insulin secretion (74). 7
Concurrently, the β-cells re quite fril in their expression of nti-oxidnts posing themselves susceptile to fil even to smller degree of exposure to oxidtive stress unlike other orgns of the ody (75, 76). 2.4. Inflmmtion nd β-cells Inflmmtion is response to n injury of tissue. It is identified y the invsion of immune cells, relese of cytokines nd dmge to tissue structurlly nd even functionlly in few cses. The positive effects of inflmmtion include prevention of infection nd regenertion of ffected tissue. Persistence of inflmmtion over long period cn induce excess production of cytokines nd ggrvte the disese (77). Exposure of pncretic β-cells to hyperglycemi or hyperlipidemi nd susequent oxidtive stress cn initilly stimulte n inflmmtory process tht helps for the β-cell repir nd regenertion. Uncontrolled levels of high glucose or free ftty cids cn cuse chronic inflmmtion which involves infiltrtion of immune cells, relese of cytokines nd eventully result in poptosis followed y firosis (6). The slow development of the inflmmtory process nd the rpid clernce of the ded cell deris y the immune cells offers resistnce to rpid detection of inflmmtion in vivo (78). Among the group of inflmmtory cytokines tht ffect β-cell functionlity, IL-1β is the most prominent one (8). 2.4.1. Interleukin-1β (IL-1β) nd β-cells Cytokines re products nd effectors of inflmmtory process tht cuse oxidtive stress nd cell dysfunction (79, 80). IL-1β is the mjor proinflmmtory cytokine produced under stress conditions of high concentrtions of glucose or free ftty cids. The cytokine cn enhnce its production y uto stimultion (8). Endogenous IL-1β relesed y β-cells cn ttrct mcrophges tht cn contriute to the production of dditionl mount of IL-1β. Increse in IL- 1β concentrtion ultimtely results in fulty insulin secretory process nd β-cell filure. 8
Furthermore, it cn lso induce the expression of other cytotoxic fctors tht led to β-cell destruction (9, 81). IL-1β medited inflmmtory process cn e exmined y n initil oxidtive stress followed y down regultion of insulin signl trnsduction. This effect cn e worse in the presence of other cytokines such s tumor necrosis fctor-α (TNF-α) nd interleukin-6 (IL-6) (10). Exposure to the cytokines, TNF-α nd IL-6 relesed y dipocytes due to inflmmtion cn impir β-cell functionlity (82, 83). IL-1β nd TNF-α relesed from infiltrting immune cells in dietic conditions cn cuse significnt destruction of β-cells (84). IL-1β toxicity is mplified in the presence of TNF-α nd oth IL-1β nd TNF-α cn induce the ctivtion of poptotic pthwys (85). Pncretic β-cells cultured with recominnt TNF-α exhiited impired insulin ction. (86). However the negtive effect of TNF-α ws neutrlized when the cells were treted with slicyltes. High glucose or free ftty cids cleve pro IL-1β to generte IL-1β (73, 87-93). Cspse- 1 ctivtion y sturted free ftty cids nd resulting cleving of pro IL-1β occurs through mitochondril ROS production in NLRP3 dependent mnner (94). The genertion of IL-1β y high glucose or free ftty cids is illustrted in Figure 1. The first step of IL-1β ctivtion involves the ccumultion of electrons resulting from ctolism of excess glucose or ftty cids on the electron crriers such s nictotinmide denine dinucleotide (NADH), flvin denine dinucleotide (FADH 2 ). The electrons re then trnsported to the oxygen molecules involved in mitochondril respirtory chin. Free rdicls re produced through the single electron reduction of oxygen molecule leding to ROS tht cuse oxidtive stress (73, 87). 9
Thioredoxin plys n importnt role in cell prolifertion nd prevention of poptosis. In norml cells, thioredoxin inds the thioredoxin intercting protein (TXNIP) therey msking its vilility for interction with nucleotide inding domin nd leucine rich repet contining protein 3 (NLRP3), n ggregtion of proteins tht medites the utoctlytic ctivtion of cysteine protese, cspse-1 (88). However, ROS produced y oxidtive stress sets the TXNIP free enling its inding to NLRP3 (89). Moreover, 10-fold increse in TXNIP protein levels hs een relted to n equivlent rise in the clevge of the poptotic protein, cspse-3. This effect of TXNIP lso signifies its involvement in β-cell dysfunction (90). The ctivtion of the NLRP3 inflmmsome protein complex requires the efflux of K + ions from the cell. During hyperctivity of β-cell, extrcellulr ATP levels increse nd led to ctivtion of the P2X7 receptor. Activted P2X7 receptor pves the wy for efflux of K + ions from the cell which cn e oserved y the drop in K + ions in β-cell to 70 mm during stress conditions compred to the concentrtion round 140 mm under norml conditions (91). Another protein involved in this pthwy is the poptosis-ssocited speck-like protein (ASC). The shift in cytoplsmic K + ions result in the interction of the NLRP3 nd ASC followed y the interction of ASC nd procspse 1 through their cspse recruiting domins (CARDs). It results in the dimeriztion of procspse 1 to ctive cspse 1 nd the clevge of inctive pro IL-1β is fcilitted y ctive cspse 1. The IL-1β is then relesed y the β-cell which is medited y n influx of C +2 ions (92, 93). The IL-1β relesed into extrcellulr mtrix cn re-enter the cell nd induce the ctivtion of pro IL-1β y n uto stimultion process. This uncontrolled production of IL-1β leds to greter degree of inflmmtion nd vicious cycle of inflmmtory process (95). 10
High glucose / FFA Extrcellulr ATP Rective oxygen species Activtion of P2X7 receptor Thioredoxin Thioredoxin intercting protein (TXNIP) Nucleotide inding domin nd leucine rich repet contining protein 3 (NLRP3) Efflux of K + ions Dimeriztion of ASC nd cspse 1 Active cspse 1 Clevge of pro IL-1β to ctive IL-1β Auto stimultion Influx of C +2 ions Relese of IL-1β from β cell Figure 1. Activtion nd relese of IL-1β y glucose or free ftty cids (73, 87-93). Therefter, the relesed IL-1β destilizes the insulin production ctivity of β-cell nd prompts the ctivtion of NF-κB nd its nucler trnsloction resulting in DNA frgmenttion. Such phenomenon ws oserved in the pncretic sections of type 2 dietic ptients due to the 11
IL-1β relesed y β-cells. Moreover, induction of nucler trnsloction of NF-κB y IL-1β on its own or y comining with IFN-γ hs lso een reported (11, 96). Keeping the ove informtion in view, the deleterious effects of inflmmtory cytokines, oxidtive stress nd IL-1β in prticulr, therpies imed t suppression of these cytokines my e eneficil in conserving β-cell functionlity. 2.5. Dietes Mngement 2.5.1. Therpeutic pproch Dietes hs no cure. Severl short nd long term therpies imed t preserving β-cell function nd improvement of insulin secretion re currently in use. These include insulin injections nd orl nti-dietic gents such s sulfonylure, igunides nd lph-glucosidse inhiitors. Dietic conditions hve een reported to e restored upon usge of tretments over short term. Besides, such tretments hve een oserved to e detrimentl to the β-cell mss (97). Elevted cytoplsmic C +2 levels cn cuse excitotoxicity of β-cells nd depolrize them y direct closure of K ATP chnnels upon long term tretment with drugs such s sulphonylure. This cn led to norml ctivity of β-cells (98). On the other hnd, long term tretments proved to e effective y exerting stimulting effects on β-cell prolifertion. However, the ssocited side effects such s hypoglycemi nd weight gin due to sulfonylures, gut prolems, liver toxicity due to thizolidinediones nd skin rshes due to insulin injection in some cses nd cost re the limiting fctors (99-103). Immunosuppression therpy y the usge of pncretic islet cell implnttion increses the metolic stress on β-cells nd susequently drins their ility to produce insulin. These effects re the limiting fctors in using islet implnttion s n solute cure for dietes (104). Considering the ove fctors, dietry pproch cn e promising lterntive to mitigte the deleterious effects of inflmmtion in dietes. 12
2.5.2. Dietry pproch The role of diet in dietes mngement is quite significnt. For decdes, trditionl medicines from plnts formed the min sources of dietry mngement of dietes efore the introduction of therpeutic tretment with insulin (105). Consumption of fruits nd vegetles hs een ssocited with decresed occurrence of chronic oxidtive stress-relted diseses such s dietes, cncer nd crdiovsculr diseses, esides n enhnced cellulr nti-oxidnt defense (106-108). Plnt foods re good lterntives to synthetic nti-dietic drugs ecuse the side effects ssocited with plnt food use re negligile (30, 109). Phenolics nd flvonoids re the focus of nti-dietic dietry compounds. This reserch is focused on muscdine nd ml s sources of dietry ioctives for type 2 dietes mngement. 2.5.3. Muscdine or ml ioctives nd dietes 2.5.3.1. Muscdine Vitis rotundifoli is grpe tht is ntive to southestern region of the United Sttes. Muscdine hs een cultivted since the 16 th century nd is considered s southern specilty product. It grows over wide rnge extending from Delwre to the Gulf of Mexico nd towrds west from Missouri to Texs (110). The highly resistnt nture of muscdine to Pierce s disese nd thriving ility in soil nd climtic conditions unfvorle for other grpe vrieties plces it in unique position in contriuting to the economy of southestern sttes (23). Muscdine skins re rich in phenolics such s ellgic cid, myricetin, quercetin nd kempferol wheres gllic cid, ctechin nd epictechin re the mjor phenolics in seeds (23, 111). Yi et l. identified ellgic cid, quercetin, myricetin nd kempferol in muscdine grpes (24) nd the presence of those compounds were lso reported y Wng et l. in muscdine pomce extrcts (69). The concentrtion of ellgic cid in vrious products of muscdine hs een studied to rnge etween 36-91 mg/100 g fresh weight in fruits (25), 8-84 mg/l in juices 13
nd 2-65 mg/l in wines (112). Muscdine grpes re consumed s juices, jms, jellies, or wines (69, 113). They re good source of dietry fier (114). The rich rom nd unique tste re the positive fctors, wheres the tough skins re the limiting fctor for muscdine to e ccepted y the consumer the wy other grpes hve een ccepted. Greenspn et l. reported tht muscdine skin extrcts dose-dependently inhiited the relese of TNF-α, IL-1β nd IL-6, nd superoxides stimulted y LPS (lipopolyscchride) in lood mononucler cells (110). Muscdine pomce extrcts showed dose-dependent DPPH free rdicl scvenging ctivity (69). Investigtion on the nti-dietic efficcy of muscdine juice, wine or delcoholized wine in type 2 dietic sujects showed tht consumption of delcoholized muscdine grpe wine for 28 dys ltered lood insulin levels in type 2 dietic sujects nd the fsting lood glucose: insulin rtio incresed from 8.5 to 13.1 (26). A low glucose: insulin rtio of less thn 7 is predictive of insulin resistnce nd delcoholized wine consumption hs improved the rtio indicting etter response to insulin. Consumption of wine or juice did not show ny effect on fsting lood glucose, insulin, or glycted hemogloin levels, wheres the tricylglycerols nd low density lipoprotein levels hve een reduced. Hyperglycemi increses protein glyction nd formtion of dvnced glyction end products (AGEs). Dicronyls such s glyoxl nd methylglyoxl rect with proteins to form AGEs tht hve een reported to e custive fctor for dietic complictions (115). Incresed free rdicls production, ltered gene expression nd relese of proinflmmtory cytokines re the dietic complictions promoted y AGEs. Moreover, the concentrtion of methyglyoxl in dietic ptients ws found to e five times higher thn in helthy ones (116). Wng et l. investigted AGEs inhiition nd methylglyoxl scvenging ctivities of muscdine extrcts (27). AGEs genertion ws inhiited y pproximtely 53% nd methylglyoxl y 46% therey 14
suggesting the eneficil effects of muscdine in preventing AGEs ssocited chronic diseses such s dietes. The suppressive effect of ethnolic extrcts of muscdine skins on AGEs formtion hs een reported (28). Muscdine skin extrcts inhiited the AGEs formtion y 40%, ut did not ffect methyglyoxl-medited glyction of lumin. You et l. reported the potentil inhiitory effect of muscdine extrcts on crohydrte hydrolyzing nd lipid metolizing enzymes tht re linked to ply role in dietes. (29). The study exmined the inhiition of α-glucosidse nd pncretic lipse inhiition y phenolics in muscdine extrcts. Among the phenolics of muscdine extrcts tested, ellgic cid nd quercetin were reported to e the most potent enzyme inhiitors. 2.5.3.2. Aml (Indin gooseerry) Emlic officinlis, commonly clled ml or Indin gooseerry, elongs to the Euphorecee fmily. The edile fruit is generlly consumed in rw, cooked or pickled forms. In the trditionl Indin medicine or Ayurved, sthm, dirrhe, rheumtic pins cn e treted with ml (117, 118). The nti-inflmmtory, nti-oxidnt nd rdicl scvenging ctivity of ml hs een oserved in studies on niml models (30, 31). Aml is good source of gllic cid (32), ellgic cid (33), quercetin (33) nd quercetin- 3-O-β-D-glucoside (119). Orl dministrtion of commercil enzymtic or polyphenol rich extrcts of ml to dietic rts dose-dependently reduced the expression of cretinine nd 5- hydroxy-methyfurfurl tht re indictors of oxidtive stress (120). Dietic rts fed with ml juice for 8 weeks hd higher plsm levels of nti-oxidnt enzymes, superoxide dismutse nd ctlse thn rts tht were not fed with ml juice. Aml juice dministrtion lso ttenuted hyperglycemi nd hypercholesterolemi in the dietic rts (34). Aml hs een used to tret type 2 dietes ptients. Consumption of ml juice y dietic ptients for 3 months long with other plnt extrcts showed reduced fsting lood 15
glucose levels (35). Dietic sujects given with 500 mg tlets consisting of 25% ml for 3 months showed reduced fsting lood sugr levels (36). Consumption of medium sized ml fruit (35 g) for 2 months induced hypoglycemi (37). Orl consumption of ml powder for 21 dys dose-dependently decresed lood glucose nd cholesterol levels nd improved the levels of high-density lipoprotein cholesterol in type 2 dietes ptients (38). The ove reported studies support ml s promising dietry source for ttenuting dietic condition. The mechnism y which ml improves dietic condition nd the ioctive compounds responsile for such n ctivity, need to e investigted t the moleculr level. 2.5.3.3. Ellgic cid nd urolithins Ellgic cid exists in food s hydrolysle tnnins of the non-flvonoid polyphenol group known s ellgitnnins, which re comintion of hexhydroxydiphenic cid nd glucose. Hydrolysis of ellgitnnins sets the cid group free, which therefter undergoes spontneous cycltion nd through the formtion of internl lctone ridges, yields ellgic cid. The in vitro high nti-oxidnt ctivity exhiited y ellgitnnins is ttriuted to the phenolic hydroxyls groups in the ortho position of their structure (16). Figure 2. Ellgic cid structure. Figure 3. Urolithin A. Studies on the physiologicl chnges of ellgic cid from different portions of intestine hd confirmed its relese from ellgitnnins in the smll intestine nd the microil trnsformtion to urolithins in jejunum, where they re sored. Ellgic cid metolize into 16
tetr, tri, di nd monohydroxy-dienzopyrzone derivtives through the loss of lctone rings of ellgic cid nd successive removl of hydroxyls, forming the di nd mono forms usully referred to s urolithin A nd B, respectively (16, 17, 121). The rte of conversion of ellgic cid to its metolites depends on the composition of gut microorgnisms nd cn differ sed on n individul s microil profile (18). Cerd et l. reported tht concentrtions of ellgic cid cn rech up to 100 µm in the intestine. Despite such higher concentrtions of ellgic cid in the gstrointestinl trct, most of the ellgic cid is converted to metolites. The plsm concentrtion of ellgic cid metolites including urolithin A nd B in plsm cn rech up to 5 µm (121). Ellgic cid is ioville t micromolr concentrtions. Consumption of 180 ml pomegrnte juice contining 25mg ellgic cid y humn suject showed tht ellgic cid concentrtion in plsm ws 31.9 ng/ml (0.1 µm) fter 1h of consumption nd eliminted fter 4 h. Low iovililtiy of ellgic cid ws ttriuted to its poor wter soluility nd trnsformtion y intestinl cteri to metolites (122-124). Twenty helthy volunteers were fed with 40 mg ellgic cid cpsules. Plsm concentrtion of ellgic cid ws 30-50 ng/ml (0.1-0.2 µm) fter 15 min nd 200 ng/ml (0.7 µm) fter 1h. Men serum elimintion hlf-life ws round 8.4h (125). The mximum plsm level of urolithin A ws reported to rnge etween 14-25 µm fter 6 8h nd vried mong volunteers confirming the metolism of ellgic cid into urolithin A nd B (126). The nti-dietic ctivity of ellgic cid in vivo hs een reported in dietic rts. Ellgic cid dministrtion for 35 dys restored the plsm insulin tht ws suppressed y streptozotocin nd decresed the streptozotocin-induced increse of plsm glucose, lood 17
glycosylted hemogloin nd hexokinse ctivity (19). Orl dministrtion of ellgic cid to streptozotocin-nicotinmide-induced dietic rts dose-dependently inhiited plsm glucose levels. Mximum reduction of glucose y ellgic cid ws t 2 h. Ellgic cid stimultion of β- cell for incresed insulin secretion ws proposed to e the possile mechnism ehind the hypoglycemic effect of ellgic cid (127). The iovilility nd in vivo nti-dietic ctivity of ellgic cid hs een reported in erlier studies. However, investigtion on the ttenution of dietes development y ellgic cid in vitro cn help to elucidte the mechnism involved in its nti-dietic ctivity. 2.5.3.4. Isorhmnetin Isorhmnetin (3, 4, 5, 7-tetrhydroxy-3 -methoxyflvone) is n ctive flvonol glycone nd metolite of quercetin. Onions, pples, te or red wine re mong the est sources of quercetin (128). Orl dministrtion of the isorhmnetin diglucoside from mustrd lef to dietic rts lowered lood glucose nd 5-hydroxymethylfurfurl expressing ntidietic nd nti-oxidnt ctivities. (22). However, intrperitonl dministrtion did not show ny such inhiition. It hs een hypothesized tht the oserved inhiitory ctivity of isorhmnetin diglucoside might hve een due to the glycone form, isorhmnetin formed during metolic conversion in the gut. Intrperitonel dministrtion of isorhmnetin exhiited similr effects s oserved y orl dministrtion of isorhmnetin diglucoside. Figure 4. Isorhmnetin structure. 18
The concentrtion of quercetin or its metolites t 10µM tested ginst IL-1β-induced nitrite production nd insulin secretion suppression in rt pncretic cells showed tht quercetin ws effective ut quercetin metolites were not (129). Interestingly, ll the metolites considered were glucosides including isorhmnetin 3-glucuronide, suggesting tht reduced efficcy ws ssocited with glucoronidtion (22). Aglycone forms of quercetin metolites hve nti-inflmmtory nd nti-oxidnt properties. Lipopolyscchride-stimulted IL-1β, IL-6 nd TNF-α mrna levels long with nitric oxide synthse in mcrophges were suppressed y quercetin nd isorhmnetin t 10µM level, ut quercetin-3-glucuronide did not (21). Under physiologicl conditions, quercetin glycosides in foods re metolized to sulftes nd glucuronides nd glycones of quercetin nd isorhmnetin (130). Quercetin is ioville in its originl form or s metolite. The mximum ttinle plsm level of pure quercetin hs een estimted to e 0.1 to 0.4 µm (131). Keeping this in view, studies should exmine the efficcy of quercetin or its metolites such s isorhmnetin s dietic ttenutors t concentrtions lower thn 10 µm. 19
CHAPTER 3 MATERIALS AND METHODS 3.1. Regents HPLC grde methnol nd cetic cid were purchsed from Fisher Scientific (Fir Lwn, NJ). Acetonitrile ws purchsed from J.T Bker (Phillipsurg, NJ) nd hydrochloric cid ws otined from VWR (Bridgeport, NJ). Dimethyl sulfoxide (DMSO) ws purchsed from RESEARCH ORGANICS (Clevelnd, OH). Urolithin A (UR) ws gift from Dr. Toms Brern, F.A., (CEBAS, Spin). Glucose (Glu), plmitic cid (PA), ellgic cid (EA), isorhmnetin (IS), nd ovine serum lumin (BSA) were otined from SIGMA-ALDRICH (St. Louis, MO). F12k medium for NIT-1 cells ws otined from ATCC (Mnsss, VA) nd fetl ovine serum (FBS) ws purchsed from ATLANTA Biologicls (Lwrencevillie, GA). Sodium icronte nd glutmte used for medium preprtion were purchsed from Life Technologies (Grnd Islnd, NY). Ison vriety of muscdine (MS) pomce ws otined from Felicin Cellrs (Jckson, LA) nd frozen fruits of ml (AM) were purchsed from Deep Foods Inc. (Union, NJ). 3.2. Fruit extrcts preprtion MS pomces or AM fruits were lended, freeze dried nd stored t - 20 o C until use. Five grms of freeze dried MS or AM ws tken nd dissolved in 100 ml of 80% methnol + 20% 6N HCl. Acid hydrolysis ws crried out in wter th (Lline orit microprocessor shker th) t 60 o C nd 200 rpm for 2 h for the conversion of flvonoid glycosides to glycones. Smples were then sonicted (Brnson 2510, Dnury, CT) for 10 min to mximize the extrction (111). The extrcts were vcuum filtered through Whtmn No.1 filter pper using Buchner funnel. The solvents were removed in rotry evportor (Buchi Rotvpor, New Cstle, DE) nd neutrlized with 1N NOH. The resultnt extrcts were freeze dried (Genesis 35 XL lyophilizer, VirTis Co., NY) nd stored t -20 C until nlysis. 20
3.3. Determintion of ellgic cid content in muscdine or ml extrcts Agilent 1100 series HPLC (Agilent, Snt Crl, CA) equipped with n Agilent Zorx SB-C18 column (5 µm, 4.6 x 250 mm) connected to G1315B Diode Arry UV Detector ws used to estimte the ellgic cid content. Phenolic cids profiles nd contents in MS or AM extrcts were nlyzed using the method of Pstrn et l. with modifictions (111). One mg of freeze dried extrct MS or AM ws dissolved in 1ml of 80% methnol + 20% 6N HCl nd filtered through 0.2 µm nylon syringe filter (Fisher, GA) efore injecting into HPLC. Three moile phses were used nmely, solvent A, methnol/cetic cid/wter (10:2:88, v/v/v); solvent B, cetonitrile; nd, solvent C, wter. All the solvents were filtered through 0.2 µm filter pper nd sonicted (Brnson 2510, Dnury, CT) for 10 min prior to use. Grdient elution ws performed s follows: t 0 min, 100% solvent A; t 5 min, 90% solvent A nd 10% solvent B; t 25 min, 30% solvent A nd 70% solvent B; t 30 min, 100% solvent C; t 35 min, 100% solvent C; t 36 min, 100% solvent A nd 4 min postrun with 100% solvent A. Twenty µl of smple ws injected, the column temperture ws mintined t 40 o C nd the flow rte ws 1mL/min. The detection ws done t 280 nm using diode rry detector (DAD) (113). The smples were run in triplictes nd peks nd concentrtion of ellgic cid in fruit extrcts were confirmed y mtching with the retention times otined y running the clirtion curve of ellgic cid stndrd. 3.4. NIT-1 cell line This study ws conducted using NIT-1, pncretic β-cell line developed from nonoese dietic (NOD) mice, Mus musculus tht is trnsgenic for the SV40 T ntigen ( protooncogen cple of trnsforming lot of cell types) under control of the insulin promoter nd spontneously develops et cell denoms. These cells showed low percent (5%) of glucgon, no somtosttin nd positive for insulin when stined t pssge 18 indicting tht they re 21
good strin to e employed for in vitro studies (132). The cell line ws otined from Dr. Sit Aggrwl (Willim Hnsel Cncer Prevention Lortory, Pennington Biomedicl Reserch Center, Bton Rouge, LA). The cells were cultured nd mintined in F12k medium (ATCC, Mnsss, VA) contining 10% FBS, 2% sodium icronte nd 1% glutmte. 3.5. Conjugtion of plmitic cid with ovine serum lumin (PA-BSA) The conjugte of PA with BSA ws prepred s descried previously (133, 134). Thirty percent BSA ws prepred in DPBS (Dulecco s Phosphte Buffered Sline) tht contined 2.5% HEPES. The ph of the solution ws djusted to 7.2 with 1M KOH nd then sterile-filtered through 0.2 µm syringe filter. A stock solution of 12.5 mm PA ws prepred efore conjugting with BSA. Ten mg of cell culture grde PA ws septiclly weighed nd dissolved in hexne t 9% of finl volume of stock. The mixture ws vortexed for few seconds nd then dried under nitrogen to otin white, chlky powder. The slt thus otined ws immeditely re-dissolved in wrm sterile wter t 9% of finl volume of stock nd comined with 30% BSA t 91% of finl stock volume. The resultnt conjugte of PA ws flushed with rgon, liquoted into sterile vils nd stored t -20 o C. 3.6. Cell viility ssy NIT-1 cells were seeded t 10,000 cells per well in 96-well plte nd incuted for 24 h efore tretment. Stocks solutions of EA nd IS were prepred in methnol nd stock solutions of UR, MS, nd AM were prepred in DMSO. Stocks were diluted with medium to otin concentrtions of 100 µm nd 1 µm efore cell tretment. Glu stock ws prepred in the medium nd sterile-filtered with 0.2 µm nylon syringe filter (Fisher, GA). Cells were treted with 33.3 mm Glu, 250 µm PA, or comintion of 33.3 mm Glu nd 250 µm PA nd incuted for 24 h t 37 o C with 5% CO 2. Therefter, 0.01, 0.05, 1 nd 10 µm of EA, UR, IS, or MS or AM extrcts contining 0.01, 0.05, 1 nd 10 µm equivlent of EA were dded to the cells 22
followed y incution for 72 h. Cell viility ws then determined fter totl incution of 96 h using the MTS ssy with CellTiter 96 A queous One solution (Promeg, Mdison, WI) ccording to the mnufcturer s protocol. Asornce vlue of the MTS ssys were red in Bio-Rd Model 680 micro plte reder (Hercules, CA). All the tretments were performed in triplictes nd the results re presented s percentge of control. 3.7. Interction etween inducers nd inhiitors of inflmmtion in NIT-1 cells To study the effect of the inducer (Glu, PA or Glu+PA) nd inhiitor (EA, UR, IS, MS or AM) on inflmmtory mrkers, 3 x 10 5 NIT-1 cells were seeded in 6-well pltes nd incuted for 24 h in humidified tmosphere t 37 o C with 5% CO 2. The cells were treted with 33.3 mm Glu or 250 µm PA or comintion of 33.3 mm Glu nd 250 µm PA for 24 h. Therefter, 0.01, 0.05, 1 or 10 µm of EA, UR, IS, or MS or AM contining 0.01, 0.05, 1 or 10 µm equivlent of EA were dded to the wells followed y incution for 72 h. The superntnts were removed nd stored t -80 o C until further nlysis. All the tretments were performed in triplictes. 3.8. Anlysis of IL-1β IL-1β levels in the superntnts of control nd treted cells were nlyzed y sndwich ELISA using commercilly ville kits from Peprotech (Rock Hill, NJ) ccording to the mnufcturer s instructions. All of the smples were tested in duplictes. 3.9. Anlysis of insulin concentrtion The superntnts from control or treted cells were nlyzed for insulin secretion using the commercilly ville ultr-sensitive mouse insulin ELISA kit from Crystlchem (Downers Grove, IL). The ssy ws performed ccording to mnufcturer s protocol nd ll the smples were nlyzed in duplictes. 23
3.10. Sttisticl nlysis Results re expressed s mens ± stndrd devition. Sttisticl nlysis ws performed using Anlysis of vrince (ANOVA). Seprtion of mens nd difference etween control nd tretments ws determined y Tukey s nlysis (SAS, version 9.3). Sttisticl significnce ws set t P-vlue < 0.05. 24
CHAPTER 4 RESULTS AND DISCUSSION 4.1. Ellgic cid content in muscdine nd ml extrcts To quntify the EA content in MS nd AM, HPLC nlysis ws performed. The results re presented in Fig. 4.1. EA eluted t 12.6 min. HPLC nlysis showed tht the EA content in MS nd AM ws 9.4 ± 2.3 mg/g nd 19.4 ± 1.5 mg/g, respectively. On fresh weight (F.W.) sis, the estimted EA ws 88.23 mg/100g in MS nd 219 mg/100g in AM. Pstrn-Bonill identified ellgic cid, myricetin, quercetin nd kempferol s mjor phenolics in MS skins (111). Gllic cid nd ellgic cid re the mjor phenolics in AM (32, 33). Quercetin is minor phenolic in AM (33). The rnge of EA concentrtion in MS found in this study ws 36-91 mg/100 g on F.W. sis nd fit within the rnge reported y Torronen et l (25). The verge EA content in different vrieties of MS ws 16.5 mg/100g of F.W. EA content in Ison vriety of MS ws 8.7 mg/100g F.W in whole fruit nd 22 mg/100g of F.W in skins lone (111). Pltnov et l. reported tht HPLC nlysis of commercilly ville extrct of AM showed EA content etween 2.42 nd 1.29 mg/g (135). Thin lyer chromtogrphic (TLC) nlysis of wter extrct of AM showed tht the ellgic cid content in AM ws 6.45 g/100 g F.W (32). Figure 4.1.A. HPLC profile of muscdine fruit extrct. EA eluted t 12.6 min. 25
Figure 4.1.B. HPLC profile of ml fruit extrct. EA eluted t 12.6 min. The vrition in the mount of ellgic cid mong different studies my e ssocited to the difference in cultivrs, growing conditions nd hrvest time (136). 4.2. Effect of EA, UR, IS, MS or AM on the viility of NIT-1 cells treted with Glu, PA or Glu+PA To evlute the effects of Glu or PA on cell viility, NIT-1 cells were treted with Glu, PA or Glu+PA for 96 h. Cell viility ws nlyzed y MTS ssy nd expressed s percentge of vile cells in the control. The results re presented in Fig 4.2.1. The soluility of free ftty cids is high in queous solutions when conjugted to BSA. Moreover, BSA contins two high ffinity inding sites for long-chin free ftty cids (137). Therefore, PA conjugted to BSA ws used in the experiments. To confirm if BSA y itself did not ffect cell viility, cells were treted with BSA lone nd compred with control. The results showed tht BSA did not ffect NIT-1 cell viility nd Glu, PA or Glu+PA tretment exhiited prolifertive effect, which however ws not significntly different from control. Nevertheless, neither Glu nor PA ws toxic to NIT-1 cells. The results in this study with PA were in contrst to the reported decrese in cell viility nd incresed poptosis in the 26
pncretic β-cells treted with PA for 24 h (138). The present study ws conducted for 96 h nd inhiition of cell viility in PA treted cells ws not oserved. The effect of EA, UR, IS or extrcts of MS or AM long with Glu, PA or Glu+PA on cell viility ws lso tested. NIT-1 cells were exposed to Glu, PA or Glu+PA for 24 h nd treted with EA, UR, IS or extrcts of MS or AM followed y 72 h incution. The concentrtions of the EA, UR or IS tested were 0.01, 0.05, 1 nd 10 µm, respectively. For the tretments with extrcts of MS or AM, equivlent concentrtions of ellgic cid quntified y HPLC were used. Cell viility ws nlyzed using the MTS ssy nd expressed s percentge of control vile cells. The results re presented in Fig 4.2.2. to 4.2.6. The iovilility of phenolics dicttes their efficcy s ginst chronic diseses (16). Therefore, the concentrtions tested in the study were in the rnge of ioville levels of EA (0.1 µm - 0.7 µm) s reported in two studies (122, 125). The iovilility of UR differed mong studies. One study reported the mximum ttinle level s 5 µm (121), wheres nother study reported rnge etween 14-25 µm (126). The concentrtions tested in the present study were in the rnge of those reports. The viility of NIT-1 cells incuted with Glu nd treted with EA, UR, IS, MS or AM ws not significntly different from the viility of control cells. A dose-dependent decrese in viility ws oserved with increse in concentrtion of EA, UR, IS, MS or AM. However, the viility of treted cells ws not significntly different from control cells except for cells treted with 10 µm IS (Fig.4.2.4), suggesting tht the compounds were not cytotoxic to NIT-1 cells. The oserved dose-dependent decrese in cell viility in the present study is similr to the reported increse in cell deth from 3.8% t higher dilution of muscdine skin extrct of 1:400 to 8.7% t lower dilution of 1:100 (110). The difference in response of the ioctive compounds t 27
Cell viility (% of control) different concentrtions my e ssocited to the hormetic effect of these ntioxidnts tht show stimultory effect t low concentrtion nd toxic effect t high concentrtion (139, 140). The viility of cells exposed to PA or Glu+PA nd treted with EA, UR, IS, MS or AM showed tht there ws no significnt difference in cell viility compred to control cells. The concentrtions (0.1-10µM) tested either showed prolifertive effect or did not significntly inhiit NIT-1 cell viility. The dose-dependent decrese in cell viility ws similr to the response oserved with Glu nd EA, UR, IS, MS or AM-treted NIT-1 cells. 120 100 80 60 40 20 0 Control Glu PA Glu+PA BSA Figure 4.2.1. Effects of 33.3mM Glu, 250 µm PA, 33.3mM Glu + 250 µm PA, or BSA on the viility of NIT-1 cells. Cells were incuted with Glu, PA, Glu+PA or BSA for 96 h nd the viility ws determined y the MTS ssy. The vlues re expressed s percentge of untreted control. Results re presented s men ± S.D, (n=3). 28