Myricetin Ameliorates Defective Post-Receptor Insulin Signaling via

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1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 211, Article ID 15752, 9 pges doi:1.193/ecm/neq17 Originl Article Ameliortes Defective Post-Receptor Insulin Signling vi β-endorphin Signling in the Skeletl Muscles of Fructose-Fed Rts Thing-Fong Tzeng, 1 Shorong-Shii Liou, 2 nd I-Min Liu 2 1 Deprtment of Internl Medicine, Po Chien Hospitl, Ping Tung City, Chin 2 Deprtment of Phrmcy & Grdute Institute of Phrmceuticl Technology, Tjen University, Yen-Pou, Ping Tung Shien, Tiwn Correspondence should be ddressed to I-Min Liu, iml@mil.tjen.edu.tw Received 3 My 29; Accepted 25 Jnury 21 Copyright 211 Thing-Fong Tzeng et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. β-endorphin plys mjor role in the meliortion of insulin resistnce. The present study documents tht myricetin (3,5,7,3,4,5 -hexhydroxyflvone) meliortes insulin resistnce by enhncing β-endorphin production in insulin-resistnt rts. The rts were induced for insulin resistnce by feeding them diet contining 6% fructose for 6 weeks. The degree of insulin resistnce ws mesured by the homeostsis model ssessment of bsl insulin resistnce (HOMA-IR). The plsm levels of insulin nd β-endorphin were mesured by n enzyme-linked immunosorbent ssy. The insulin receptor-relted signling meditors in the soleus muscles of rts were evluted by immunoprecipittion or immunoblotting. ws injected dily (1 mg kg 1 per injection, thrice dily) for 14 dys. Consequently, the high-glucose plsm levels in fructose-fed rts decresed significntly concomitnt with n increse in plsm β-endorphin. The reduction of the elevted HOMA-IR index following tretment with myricetin ws subsequently inhibited by the dministrtionof β-funltrexmine hydrochloride () t doses sufficient to block μ-opioid receptors (MOR). The myricetin tretment ws lso observed to ffect the phosphoryltionof the insulinreceptor, insulin receptor substrte-1, Akt nd Akt substrte of 16 kd, with subsequent effects on glucose-trnsporter subtype 4 trnsloction, ll of which were blocked by pretretment. These results indicted tht enhncement of β-endorphin secretion, which in turn leds to peripherl MOR ctivtion, is involved in the ction of myricetin on the meliortion of impired signling intermedites downstrem of insulin receptors. 1. Introduction (3,5,7,3,4,5 -hexhydroxyflvone) is flvonoid commonly found in te, berries, fruits, vegetbles nd medicinl herbs. hs been shown to possess ntioxidtive nd cytoprotective properties [1, 2]. A therpeutic effect of myricetin in ptients with crdiovsculr diseses ssocited with dibetes mellitus hs lso been reported [1, 2]. An insulinomimetic effect of myricetin on lipogenesis nd glucose trnsport in the dipocytes of rts with noninsulin-dependent dibetes mellitus hs been observed [3]. hs been found to reduce hyperglycemi in dibetic rts, possibly through its bility to increse heptic glycogen synthesis nd to normlize hypertriglyceridemi [4]. hs lso demonstrted the bility to improve glucose utiliztion, lowering plsm-glucose levels in type 1 dibetes-like niml model [5]. Furthermore, myricetin displys the chrcteristics of rosiglitzone, demonstrting improved glucose utiliztion nd meliorting the impired insulin-signling pthwy in insulin-resistnt rts induced by the high intke of fructose. However, unlike rosiglitzone, it does not cuse n increse in body weight [6], suggesting tht myricetin cn serve s therpeutic djunct for the tretment of insulin-resistnt ptients nd/or for ptients susceptible to the thizolidinediones-induced side effects of weight gin nd edem [7]. Insulin resistnce in geneticlly obese Zucker rts ws shown to improve upon exercise, nd β-endorphin is implicted in this exercise-induced improvement of insulin resistnce [8]. The β-endorphin hs been shown to

2 2 Evidence-Bsed Complementry nd Alterntive Medicine reverse the impired insulin-stimulted glucose disposl in insulin-resistnt rts induced by fructose-rich diet [9]. Furthermore, it ws documented tht ctivtion of μ-opioid receptors (MOR) by lopermide reversed insulin-stimulted glucose uptke impired by cytokines in C 2 C 12 cells, with mrked recovery of insulin signling [1], nd cn meliorte defective postreceptor insulin signling, reversing the impirment of insulin-stimulted glucose utiliztion in obese Zucker rts [11]. A direct role hs been demonstrted for peripherl MOR ctivtion in the improvement of insulin resistnce induced by high intke of fructose, s this improvement is more rpid in MOR knockout mice thn in wild-type mice [12]. These results led to the ide tht ctivtion of MOR on insulin-trgeted orgns my hve beneficil effect in reducing insulin resistnce. We hve shown tht ctivtion of MORs on the soleus muscle, in response to incresed endogenous β-endorphin secretion, is essentil to the plsm glucose-lowering ction of myricetin in dibetic rts lcking insulin [13]. This study ws undertken to scertin whether elevtion of circulting β-endorphin nd/or ctivtion of peripherl MORs could medite the ction of myricetin nd meliorte insulin resistnce. 2. Methods 2.1. Mterils. Stndrd rt chow contining 6% vegetble strch, 5% ft nd 18% protein (Ct. #218) nd fructoserich rt chow contining 6% fructose, 5% ft nd 18% protein (Ct. #TD 89247) were obtined from Hrln Tekld (Mdison, WI, USA). (purity >96%; Ct. #M676) nd protein A-sephrose beds were purchsed from Sigm- Aldrich, Inc. (St. Louis, Missouri, USA). β-funltrexmine hydrochloride (; Ct. #926) ws purchsed from Tocris Bioscience (Bristol, UK). The dignostic kit used for determintion of plsm glucose levels (Ct. #COD1253) ws purchsed from BioSystem (Cost Brv, Brcelon, Spin). The rt insulin enzyme-linked immunosorbent ssy (ELISA) kit ws obtined from LINCO Reserch, Inc. (St. Chrles, MO, USA; Ct. #EZRMI-13K). The ELSA kit used for ssying β-endorphin-like immunorectivity (BER) ws obtined from Peninsul Lbortories Inc. (Belmont, CA, USA; Ct. #T-44). The kit used for the protein dye-binding ssy ws purchsed from Bio-Rd Lbortories (CA, USA). Anti-insulin receptor (IR) β-subunit (Ct. #MS-634 for immunoprecipittion; Ct. #MS-636 for western blotting), nti-insulin receptor substrte (IRS)-1 (Ct. #MS-63) nd nti-phosphotyrosine (Ct. #MS-445) ntibodies were obtined from NeoMrkers (Fremont, CA, USA). Anti-p85 subunit of phosphtidylinositol (PI) 3- kinse (p85) (Ct. #4292), nti-akt (Ct. #9272), ntiphospho-ser 473 Akt (Ct. #9271), nti-phospho-thr 38 Akt, nti-phospho-(ser/thr) Akt substrte (Ct. #9611) nd ntiglucose-trnsporter subtype 4 (GLUT 4) (Ct. #2299) ntibodies were cquired from Cell Signling Technology, Inc. (Beverly, MA, USA). The nti-as16 (Rb GAP) ntibody ws purchsed from Upstte (Chrlottesville, VA; Ct. #7-741). ECL Western Blotting Systems were obtined from Amershm Corp. (Brunschweig, Germny). Bovine insulin ws obtined from Novo Nordisk (Bgsverd, Denmrk). All other regents were obtined from stndrd sources Animl Models. Eight-week-old, mle Wistr rts were obtined from the Ntionl Lbortory Animl Center (Tipei, Tiwn). They were mintined in temperturecontrolled room (25 ± 1 C) nd kept on 12 : 12 lightdrk cycle (light on t 6: h) in our niml center. Rts received the fructose-rich rt chow for six dditionl weeks to induce insulin resistnce [6]. Food nd wter were vilble d libitum. All niml procedures were performed ccording to the Guide for the Cre nd Use of Lbortory Animls of the Ntionl Institutes of Helth s well s the guidelines of the Animl Welfre Act Tretment Procedures. The fructose-fed rts were divided into three experimentl groups. It hs been documented tht fructose-fed rts receiving intrvenous (i.v.) injections of myricetin in the lterl til vein every 8 h, thrice dily (t 6:., 14: nd 22: h) t 1 mg kg 1 per injection for 14 consecutive dys were found to demonstrte effectively meliorted insulin resistnce [6]. Thus, one group of fructose-fed rts ws given i.v. injections of 1mgkg 1 myricetin ccording to the tretment regimen described bove. The second group of fructose-fed rts received injection of myricetin plus [14]. ws i.v. injected 3 min prior to dministrtion of myricetin. The remining fructose-fed rts given the sme volume of vehicle (sline) used to dissolve the test medictions were considered s the vehicle-treted group. The injection volume ws controlled in tht 1 ml kg 1 ws routinely dministered. The injection ws given slowly to void s much pin nd shock s possible nd thereby helping to exclude non-predictive outcomes. The rts were mintined on fructose diet during the tretment period. Wter ws mde vilble d libitum throughout the experiment. After 2-week tretment, blood smples (.1 ml) were collected from the til vein of rts when deeply nesthetized by sodium pentobrbitl (3 mg kg 1, intrperitonel). Blood smples were used to evlute glucose, insulin nd β-endorphin plsm levels. The homeostsis model ssessment of bsl insulin resistnce (HOMA-IR) ws used to clculte n index from the product of the fsting concentrtions of plsm glucose (mmol L 1 )ndplsm insulin (μuml 1 ) divided by 22.5 [15]. Lower HOMA-IR vlues indicted greter insulin sensitivity, wheres higher HOMA-IR vlues indicted lower insulin sensitivity (insulin resistnce) Plsm Anlysis. Plsm glucose concentrtions were mesured using the glucose oxidse method by mens of commercilly vilble kit. The ELISA technique ws employed to quntify the plsm levels of insulin nd BER. All smples were nlyzed in triplicte In Vivo Insulin Receptor Activtion. To ssess the chnges in insulin-receptor ctivtion in vivo,rtsinthefedsttewere

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 nesthetized with sodium pentobrbitl t the end of the 2- week tretment period. A bolus of insulin (1 U kg 1 )ws then injected into the portl vein, s described previously [6]. Approximtely 12 s fter insulin injection, rts were scrificed nd the soleus muscle ws immeditely extirpted, wshed with cold phosphte buffer nd cut into 2 3 mg portions, which were then stored seprtely t 8 Cfor subsequent experiments Muscle Processing. Cytosol nd membrne frctions were prepred ccording to the previous method [6, 16]. Briefly, muscles used for mesuring insulin signling were weighed while still frozen nd homogenized (Polytron, Brinkmnn Instruments, Inc., Westbury, NY, USA) in.4 ml homogenizing buffer contining 25 mmol L 1 sucrose, 2 mmol L 1 Tris (ph: 7.5), 2 mmol L 1 ethylenediminetetrcetic cid (EDTA),.5 mmol L 1 ethylene glycol tetrcetic cid (EGTA), 2 μgml 1 leupeptin, 1 μgml 1 protinin, μg L 1 phenylmethylsulfonyl fluoride nd 2 mmol L 1 dithiothreitol. The homogente ws centrifuged t 1 g for 1 h t 4 C. The superntnt (cytosolic extrct) ws trnsferred to tube kept on ice, wheres the pellet ws resuspended in.45 ml homogenizing buffer contining 5% Triton X-1. The resuspended pellet frction ws then centrifuged t 14 g for 5 min t 4 C, nd the pellet ws discrded. The superntnt from this spin constitutes the membrne extrct. Protein concentrtions were determined by the BioRd protein dye-binding ssy. The superntnt ws stored t 8 C until used in immunoprecipittion nd Western immunoblotting Immunoprecipittion. Muscle lystes (5 μg) were subjected to immunoprecipittion with the nti-ir β-subunit, nti-irs-1 or nti-as16 (Rb GAP) ntibody t 4 C overnight, followed by shking with protein A-Sephrose beds for 1 h. The bed-protein A-ntibody-ntigen complexes were precipitted by brief centrifugtion. The pellets were wshed thrice in ice-cold buffer (.5% Triton X- 1, 1 mmol L 1 Tris, ph: 7.4, 1 mmol L 1 EDTA nd 2 mmol L 1 sodium vndte), resuspended in Lemmli smple buffer nd boiled for 5 min. The sephrose beds were precipitted by brief centrifugtion nd the superntnt prepred for sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE, 1% crylmide gel) using Bio-Rd Mini-Protein II system (55 nd 13 V during the stcking nd seprtion phses, respectively). Protein ws trnsferred to polyvinylidene difluoride (PVDF) membrne using Bio-Rd Trns-Blot system (2 h t 2 V in 25 mmol L 1 Tris, 192 mmol L 1 glyceine nd 2% methnol (MeOH)). Following trnsfer, the membrne ws probed with nti-ir β-subunit, nti-irs-1 nd ntiphosphotyrosine ntibodies Immunoblotting. Equl mounts of protein (5 μg) were prepred from muscle homogentes, subjected to SDS-PAGE, trnsferred to PVDF membrne s described bove nd probed with nti-pi3-kinse p85, nti-akt nd nti-glut 4 ntibodies ccording to the mnufcturer s instructions. Protein phosphoryltion of Akt ws mesured using the nti-phospho-ser 473 Akt nd nti-phospho- Thr 38 Akt ntibodies. Phosphoryltion of Akt substrte of 16 kd (PAS-AS16) ws detected using the nti-phospho- (Ser/Thr) Akt substrte ntibody. PAS-AS16 recognizes Akt phosphoryltion-motif peptide sequences (RXRXXpT/S). After three 5-min wshes in Tris Buffered Sline Tween- 2 (TBST; 2 mmol L 1 Tris-HCl, ph: 7.5, 15 mmol L 1 sodium chloride (NCl) nd.5% Tween 2), membrnes were incubted with the pproprite peroxidse-conjugted secondry ntibodies. The membrnes were then wshed thrice in TBST nd visulized on X-ry film using the ECL Western Blotting System. Densities of the obtined immunoblots were quntified using lser densitometer. The men vlue for smples from the vehicle-treted group on ech immunoblot, expressed in densitometry units, ws djusted to vlue of 1. All experimentl smple vlues were then expressed reltive to this djusted men vlue Sttisticl Anlysis. Dt re expressed s the men ± SEM for ech group of nimls t the number indicted in the tbles. Sttisticl differences mong groups were determined using two-wy repeted-mesures nlysis of vrince (ANOVA). Dunnett rnge post hoc comprisons were used to determine the source of significnt differences where pproprite. A P-vlue <.5 ws considered sttisticlly significnt. 3. Results 3.1. Generl Chrcteristics of Fructose-Fed Rts. Following the injection of myricetin, plsm glucose levels in fructosefed rts fell to vlue significntly lower thn in the vehicle-treted group (P <.5), showing plsm glucoselowering ctivity of 18.1 ± 3.2% (Tble 1). Menwhile, n elevtion of the plsm BER level in fructose-fed rts ws observed following myricetin tretment (Tble 1). In the presence of (1 μgkg 1 ), the plsm glucose-lowering ction of myricetin in fructose-fed rts ws eliminted to pproch the vlues of the vehicle-treted group (Tble 1). -induced secretion of plsm BER ws unffected in fructose-fed rts pretreted with t ny dosge (Tble 1). Additionlly, the HOMA-IR score in fructosefed rts receiving 2 weeks of myricetin tretment showed decreseof 7% of the score observed in the vehicletreted group (Tble 1). Pretreted fructose-fed rt with β- FNA (1 μgkg 1 ) ws observed to bolish this myricetininduced reduction of the HOMA-IR (Tble 1) Protein Levels nd Degrees of Insulin Receptor (IR) nd Insulin Receptor Substrte (IRS)-1 Tyrosine Phosphoryltion. There were no significnt differences in the expression of IR protein in the soleus muscle of fructose-fed rts between ny of the groups (Figure 1). Two-week myricetin tretment slightly elevted the levels of IRS-1 protein expression in the soleus muscles of fructose-fed rts, nd this ws ntgonized by (Figure 1). The quntifiction of the immunoblots is summrized in Tble 2.

4 4 Evidence-Bsed Complementry nd Alterntive Medicine Tble 1: Generl chrcteristics of fructose-fed rts fter 14-dy tretment with myricetin or myricetin plus MOR ntgonist. Vehicle + (1 μgkg 1 ) + (5 μgkg 1 ) + (1 μgkg 1 ) Plsm glucose (mg dl 1 ) ± ± 5.1 b ± ± ± 6.2 Plsm insulin (μuml 1 ) 53.8 ± ± ± ± ± 5.9 Plsm BER (pg ml 1 ) 49.3 ± ± 7.2 b 92.8 ± ± ± 5.6 HOMA-IR score 18.2 ± ± ± ± ± 2. Fructose-fed rts received i.v. injection of myricetin (1 mg kg 1 ), thrice dily for 14 dys. ws i.v. injected 3 min prior to dministrtion of myricetin. The vehicle used to dissolve the tested drugs ws given t the sme volume. Vlues (men ± SEM) were obtined from seven rts. P <.5 nd b P <.1 compred to the vlues of the vehicle-treted group, respectively. Tble 2: Quntifiction of the protein expression of specific insulin receptor-relted signling meditors in soleus muscles of fructose-fed rts receiving 14-dy tretment with myricetin or myricetin plus MOR ntgonist. Reltive units Fructose-fed rts Vehicle + IR 1. ± ±.6.99 ±.5 IRS ± ±.5 ±.8 p ±.5 1 ± ±.4 Akt 1. ± ±.6.96 ±.9 AS ± ± ±.6 Fructose-fed rts received i.v. injection of myricetin (1 mg kg 1 ), thrice dily for 14 dys. (1 μgkg 1 ) ws i.v. injected 3 min prior to dministrtion of myricetin. The vehicle used to dissolve the tested drugs ws given t the sme volume. Vlues (men ± SEM) were obtined from 5 rts. P <.5 represents the level of significnce compred to the vlues of the vehicle-treted group. IR IRS-1 p85 effects were not observed in rts pretreted with (Figure 2). The extent of IR nd IRS-1 tyrosine phosphoryltion in the soleus muscles of fructose-fed rts in response to insulin stimultion ws mrkedly elevted in the 2-week myricetin-treted group. filed to induce similr chnges in IR nd IRS-1 tyrosine phosphoryltion in the soleus muscles of fructose-fed rts pretreted with (Figure 2). Akt AS16 + Figure 1: Protein expression of insulin receptor-relted signling meditors in the soleus muscles of fructose-fed rts receiving 14-dy tretment with myricetin (1 mg kg 1 per i.v. injection, thrice dily) or myricetin plus. (1 μgkg 1 per injection) ws i.v. injected 3 min prior to dministrtion of myricetin. Rts tht did not receive ny tretment were given the sme volume of vehicle used to dissolve the test medictions. Findings were reproduced on four seprte occsions. The quntifiction of the dt is shown in Tble 2. Under non-insulin stimulting conditions, the degree of tyrosine phosphoryltion of IR nd IRS-1 in the soleus muscles of fructose-fed rts ws slightly elevted following the 2-week myricetin tretment, but these myricetin-induced 3.3. Chnges in Protein Levels nd Amount of the p85 Subunit of PI3-Kinse Associted with IRS-1. Two-week tretment of fructose-fed rts with myricetin improved the expression of the p85 subunit of PI3-kinse in the soleus muscles, nd this effect ws bolished by pretretment (Figure 2). The quntifiction of the immunoblots is summrized in Tble 2. Following 2-week myricetin tretment, the bsl degree of p85 ssocited with IRS-1 in the soleus muscles of fructose-fed rts ws slightly higher thn the levels of the vehicle-treted controls, nd myricetin filed to induce similr chnges in fructose-fed rts pretreted with (Figure 3). The higher degree of insulin-stimulted p85 ssocited with IRS-1 in myricetin-treted rts ws clerly prevented by pretretment (Figure 3) Protein Levels nd Degree of Akt nd Akt Substrte of 16 kd (AS16) Phosphoryltion. After 2 weeks of myricetin tretment, the bsl protein levels of Akt nd AS16 in the soleus muscles of fructose-fed rts were higher to those of their vehicle-treted group, nd this difference ws eliminted by pretretment (Figure 2). The quntifiction of the immunoblots is summrized in Tble 2.

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 Bsl Insulin stimultion Bsl Insulin stimultion IR-pY p85/irs-1 IR-pY (rbitrry units) p85/irs-1 (rbitrry units) b + IRS-1-pY (rbitrry units) IR-1-pY Bsl + () (b) Insulin stimultion + Figure 2: Tyrosine phosphoryltion (py) of insulin receptor (IR)() nd insulin receptor substrte (IRS)-1(b) in the soleus muscles of fructose-fed rts receiving 14-dy tretment with myricetin (1 mg kg 1 per i.v. injection, thrice dily) or myricetin plus. (1 μgkg 1 per injection) ws i.v. injected 3 min prior to dministrtion of myricetin. Rts tht did not receive ny tretment were given the sme volume of vehicle used to dissolve the test medictions. Findings were reproduced on four seprte occsions. Quntifiction of protein levels re expressed s men ± SEM (n = 5 per group) in ech column. P <.5 represents the level of significnce compred to the bsl vlues of the vehicletreted group. Figure 3: The mount of the p85 subunit of PI3-kinse ssocited with IRS-1(p85/IRS-1) in the soleus muscles of fructose-fed rts receiving 14-dy tretment with myricetin (1 mg kg 1 per i.v. injection, thrice dily) or myricetin plus. (1 μgkg 1 per injection) ws i.v. injected 3 min prior to dministrtion of myricetin. Rts tht did not receive ny tretment were given the sme volume of vehicle used to dissolve the test medictions. Findings were reproduced on four seprte occsions. Quntifiction of protein levels expressed s men ± SEM (n = 5 per group) in ech column. P <.5 nd b P <.1 represent the level of significnce compred to the bsl vlues of the vehicle-treted group. to 1.8-fold nd 2.2-fold reltive to tht of the vehicletreted controls, respectively (Figure 4()). Furthermore, the degree of insulin-stimulted AS16 phosphoryltion in the soleus muscles of myricetin-treted fructose-fed rts ws mrkedly higher thn the vlue of their vehicle-treted group (Figure 4(b)). These myricetin-induced effects were eliminted by pretretment (Figure 4) Insulin-Stimulted Glucose-Trnsporter Subtype 4 (GLUT 4) Trnsloction. At the termintion of the 2-week myricetin tretment, insulin-stimulted GLUT 4 protein expression in the membrne frction of the soleus muscles from fructosefed rts ws incresed 1.8-fold of tht observed in their vehicle-treted group; conversely, the protein levels in the cytosolic frction of the sme smples decresed to 6% of tht observed in the vehicle-treted controls (Figure 5). The myricetin-medited chnges in insulin-stimulted GLUT 4 trnsloction were reversed by pretretment (Figure 5). 4. Discussion It ws observed tht the degree of bsl phosphoryltion of Akt (Thr 38 /Ser 473 ) nd AS16 ws incresed in the soleus muscles of fructose-fed rts receiving 2-week myricetin tretment (Figure 4). The degree of insulinstimulted phosphoryltion of Akt tyrosine (Thr 38 )ndakt serine (Ser 473 ) ws lso significntly elevted by myricetin In the present study, 2-week tretment regimen with myricetin (1 mg kg 1 per i.v. injection, thrice dily) ws found to significntly increse plsm BER in mnner tht prlleled the lowering of plsm glucose in rts fed for 6 weeks with fructose chow, highlighting the possibility tht incresed plsm BER nd the reduction of plsm

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Bsl Insulin stimultion pakt (Thr 38 ) pakt (Ser 473 ) Akt phosphoryltion (rbitrry units) pakt (Thr 38 ) pakt (Ser 473 ) + b b + pas16 (rbitrry units) pas Bsl + Insulin stimultion b + () (b) Figure 4: Phosphoryltion of Akt () nd AS16 (b) in the soleus muscles of fructose-fed rts receiving 14-dy tretment with myricetin (1 mg kg 1 per i.v. injection, thrice dily) or myricetin plus. (1 μgkg 1 per injection) ws i.v. injected 3 min prior to dministrtion of myricetin. Rts tht did not receive ny tretment were given the sme volume of vehicle used to dissolve the test medictions. Findings were reproduced on four seprte occsions. Quntifiction of protein levels expressed s men ± SEM (n = 5per group) in ech column. P <.5 nd b P <.1 represent the level of significnce compred to the bsl vlues of the vehicle-treted group. Membrne frction Cytosolic frction GLUT 4 2. GLUT 4 Membrne GLUT 4 protein (rbitrry units) Cytosolic GLUT 4 protein (rbitrry units) () (b) Figure 5: Insulin-stimulted GLUT 4 protein expression in the membrne () nd cytosolic (b) frctions in the soleus muscles of fructosefed rts receiving 14-dy tretment with myricetin (1 mg kg 1 per i.v. injection, thrice dily) or myricetin plus. (1 μgkg 1 per injection) ws i.v. injected 3 min prior to dministrtion of myricetin. Rts tht did not receive ny tretment were given the sme volume of vehicle used to dissolve the test medictions. Findings were reproduced on four seprte occsions. Quntifiction of protein levels expressed s men ± SEM (n = 5 per group) in ech column. P <.5 represents the level of significnce compred to the vlues of the vehicle-treted group.

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 β-endorphin Insulin MOR IR GLUT 4 trnsloction PI3-kinse IRS-1 Akt/PKB GLUT 4 AS16 Skeletl muscle Figure 6: The possible mechnisms of myricetin ction on the meliortion of defective post-receptor insulin signling in the skeletl muscle of fructose-fed rts. glucose concentrtions re relted phenomen in this model. Although the effect of myricetin on the lowering of plsm glucose ws suppressed by blockde of MOR vi pretretment, this ntgonist hd no influence on the myricetin-induced secretion of plsm BER. As illustrted by the findings tht myricetin promoted n increse in plsm BER concurrent with lowering of plsm glucose concentrtions, direct effect of this flvonol on glucose homeostsis in insulin-resistnt rts seems unlikely. In humns nd other mmmls, skeletl muscle normlly ccounts for 75% of whole-body, insulin-stimulted glucose trnsport [17]. An impired bility of skeletl muscles to respond to insulin is, therefore, disruptive to systemic glucose homeostsis. Actully, the increse in insulin ction on skeletl muscle is likely to be relted to incresed protein expression nd/or functionl ctivity of severl key components of the insulin signl-trnsduction pthwy; defects in the insulin-signling cscde, which led to impired glucose utiliztion, re believed to ply key role in the pthogenesis of insulin resistnce [18]. It is conceivble tht IRS-1 tyrosine phosphoryltion (in response to insulin stimultion) generlly increses the ssocition of IRS-1 with the p85 subunit of PI3-kinse, resulting in incresed PI3- kinse ctivity. In turn, this would led to the ctivtion of serine/threonine kinse Akt (protein kinse B) nd, ultimtely, ccelerted rtes of GLUT 4 trnsloction, such tht GLUT 4 mnifests predominntly t the cell surfce nd enhnces insulin-stimulted glucose disposl [19]. It hs been documented tht the ction of insulin on glucose uptke nd metbolism is much greter in skeletl muscles composed primrily of oxidtive fibers (e.g., the soleus) s compred to glycolytic fibers (e.g., the epitrochleris nd extensor digitorum longus), even though the soleus muscle represents smll portion of the totl muscle mss [2]. Activtion of MOR locted on the soleus muscle, resulting in reversl of the impirment of insulin-stimulted glucose disposl in obese Zucker rts, hs been documented; this improvement in insulin resistnce ws ssocited with the meliortion of the post-receptor insulin-signling cscde, including downstrem effectors of the PI3-kinse signling pthwy involved in glucose-trnsporter trnsloction [11]. We found tht incresing the doses of negtively correlted with the effect of myricetin on the reduction of higher HOMA-IR in fructose-fed rts, lthough the higher plsm levels of BER were still pprent in the myricetintreted group. This rised the possibility tht n elevtion of circulting β-endorphin nd/or ctivtion of peripherl MOR might be the mechnisms by which myricetin meliortes the defective insulin ction. With this in mind, soleus muscle smples were prepred from ll nimls fter insulin stimultion. A substrte of Akt hs been identified tht my provide link between insulin signling nd GLUT 4 trfficking. This protein, AS16, hs GAP homology domin nd is phosphorylted by Akt in response to insulin [21]. In this study we showed tht myricetin tretment hd cler effects on the IRS-1/PI3-kinse/Akt/AS16 signling cscde (t both the protein expression nd phosphoryltion levels) nd subsequent GLUT 4 trfficking, which were ll suppressed by MOR ntgonism. These findings suggested tht the effect of myricetin on the improvement of post-receptor insulin signling might be dependent on the ctivtion of MOR locted on peripherl insulin-sensitive tissues, such s skeletl muscle. Additionlly, these results supported the essentil role of opioids during glucose homeostsis. Furthermore, opioids or opioid receptor ctivtion, especilly of the μ- subtype, my hve key functions in glycemic control. Although β-endorphin is relesed with drenocorticotropic hormone from the pituitry glnd [22], the pituitry glnd-independent relese of endogenous opioids occurs in other orgns, such s the drenl glnd [9, 23]. We provided new evidence tht ctivtion of α 1 -drenoceptors on the drenl glnd my increse β-endorphin secretion vi the phospholipse C-protein kinse C pthwy, which in turn ctivtes peripherl MOR to modify glucose metbolismssocited genes, thereby leding to improved peripherl

8 8 Evidence-Bsed Complementry nd Alterntive Medicine glucose utiliztion nd decresed heptic gluconeogenesis for meliortion of severe hyperglycemi in type 1-like dibetes [24]. Secretion of β-endorphin from the drenl glnd ws observed to be essentil to the plsm glucoselowering ction of myricetin in streptozotocin-induced (STZ)-dibetic rts [13]. It is not cler if myricetin-induced β-endorphin secretion in fructose-fed rts is medited by pituitry glnd-dependent or -independent pthwys; however, it ws strongly demonstrted tht the myricetinmedited ction converged t point between the downstrem trgets of the peripherl MOR nd the IRS-PI3-kinse signling pthwy, subsequently ffecting insulin-induced GLUT 4 trnsloction (Figure 6). A widely recommended pproch for the control of hyperglycemi is to dminister prenterl insulin preprtion tht provides constnt source of circulting insulin for h to ugment or replce deficient endogenous insulin secretion, lthough insulin resistnce is eventully developed in these insulin-treted ptients [25]. The mechnisms underlying the concentrtion- nd time-dependent induction of insulin resistnce re complex nd incompletely defined. Thus, it is impertive to identify new trgets tht cn chieve equl nd/or superior effects on the control of glycemi with reduction in insulin resistnce. The findings of our study provide new insights into the chemicl compounds or herbl products tht might enhnce β- endorphin secretion nd/or stimulte MOR in peripherl insulin-sensitive tissue, which might serve s potentil gents or djuvnts for trgeting glucose metbolism in insulinresistnt individuls. 5. Conclusion The survey showed tht the ction of myricetin on the meliortion of defective post-receptor insulin signling, especilly s it relted to IRS-1-ssocited PI3-kinse nd GLUT 4 trnsloction, required β-endorphin s meditor to ctivte peripherl MOR. Funding Ntionl Science Council Grnt (NSC B-127-5) of Tiwn, Republic of Chin. References [1] K. C. Ong nd H.-E. Khoo, Biologicl effects of myricetin, Generl Phrmcology, vol. 29, no. 2, pp , [2] C.-H.Ko,S.-C.Shen,T.J.F.Lee,ndY.-C.Chen, inhibits mtrix metlloproteinse 2 protein expression nd enzyme ctivity in colorectl crcinom cells, Moleculr Cncer Therpeutics, vol. 4, no. 2, pp , 25. [3] K. C. Ong nd H.-E. Khoo, Insulinomimetic effects of myricetin on lipogenesis nd glucose trnsport in rt dipocytes but not glucose trnsporter trnsloction, Biochemicl Phrmcology, vol. 51, no. 4, pp , [4] K.C.OngndH.-E.Khoo, Effects of myricetin on glycemi nd glycogen metbolism in dibetic rts, Life Sciences, vol. 67, no. 14, pp , 2. [5] I.-M. Liu, S.-S. Liou, T.-W. Ln, F.-L. Hsu, nd J.-T. Cheng, s the ctive principle of Abelmoschus moschtus to lower plsm glucose in streptozotocin-induced dibetic rts, Plnt Medic, vol. 71, no. 7, pp , 25. [6] I.-M.Liu,T.-F.Tzeng,S.-S.Liou,ndT.-W.Ln,, nturlly occurring flvonol, meliortes insulin resistnce induced by high-fructose diet in rts, Life Sciences, vol.81, no , pp , 27. [7]M.C.Grnberry,J.B.Hwkins,ndA.M.Frnks, Thizolidinediones in ptients with type 2 dibetes mellitus nd hert filure, Americn Journl of Helth-System Phrmcy, vol. 64, no. 9, pp , 27. [8] C.F. Su, Y. Y. Chng, H. H. Pi, I. M. Liu, C. Y. Lo, nd J. T. Cheng, Medition of bet-endorphin in exercise-induced improvement in insulin resistnce in obese Zucker rts, Dibetes/Metbolism Reserch nd Reviews, vol. 21, pp , 25. [9] C.-F. Su, Y.-Y. Chng, H.-H. Pi, I.-M. Liu, C.-Y. Lo, nd J.-T. Cheng, Infusion of β-endorphin improves insulin resistnce in fructose-fed rts, Hormone nd Metbolic Reserch, vol. 36, no. 8, pp , 24. [1]T.-F.Tzeng,I.-M.Liu,ndJ.-T.Cheng, Activtionofopioid μ-receptors by lopermide to improve interleukin-6-induced inhibition of insulin signls in myoblst C 2C12 cells, Dibetologi, vol. 48, no. 7, pp , 25. [11] T.-F. Tzeng, C.-Y. Lo, J.-T. Cheng, nd I.-M. Liu, Activtion of μ-opioid receptors improves insulin sensitivity in obese Zucker rts, Life Sciences,vol. 8, no.16, pp ,27. [12] J.-T. Cheng, I.-M. Liu, nd C. F. Hsu, Rpid induction of insulin resistnce in opioid μ-receptor knock-out mice, Neuroscience Letters, vol. 339, no. 2, pp , 23. [13] I.-M. Liu, S.-S. Liou, nd J.-T. Cheng, Medition of β-endorphin by myricetin to lower plsm glucose in streptozotocin-induced dibetic rts, Journl of Ethnophrmcology, vol. 14, no. 1-2, pp , 26. [14] J. U. Adms, C. A. Pronis, nd S. G. Holtzmn, Assessment of reltive intrinsic ctivity of mu-opioid nlgesics in vivo by using β-funltrexmine, Journl of Phrmcology nd Experimentl Therpeutics, vol. 255, no. 3, pp , 199. [15] D. R. Mtthews, J. P. Hosker, A. S. Rudenski, B. A. Nylor, D. F. Trecher, nd R. C. Turner, Homeostsis model ssessment: insulin resistnce nd bet-cell function from fsting plsm glucose nd insulin concentrtions in mn, Dibetologi, vol. 28, pp , [16] E. Rodríguez,N.Pulido,R.Romero,F.Arriet,A.Pndero, nd A. Rovir, Phosphtidylinositol 3-Kinse ctivtion is required for sulfonylure stimultion of glucose trnsport in rt skeletl muscle, Endocrinology, vol. 145, no. 2, pp , 24. [17] M. Björnholm nd J. R. Zierth, Insulin signl trnsduction in humn skeletl muscle: identifying the defects in Type II dibetes, Biochemicl Society Trnsctions, vol. 33, no. 2, pp , 25. [18] D. M. Muoio nd C. B. Newgrd, Mechnisms of disese: moleculr nd metbolic mechnisms of insulin resistnce nd bet-cell filure in type 2 dibetes, Nture Reviews Moleculr Cell Biology, vol. 9, pp , 28. [19] E. Crvlho, C. Rondinone, nd U. Smith, Insulin resistnce in ft cells from obese Zucker rts evidence for n impired ctivtion nd trnsloction of protein kinse B nd glucose trnsporter 4, Moleculr nd Cellulr Biochemistry, vol. 26, no. 1-2, pp. 7 16, 2.

9 Evidence-Bsed Complementry nd Alterntive Medicine 9 [2] X.M.Song,J.W.Ryder,Y.Kwno,A.V.Chiblin,A.Krook, nd J. R. Zierth, Muscle fiber type specificity in insulin signl trnsduction, Americn Journl of Physiology, vol. 277, no. 6, pp. R169 R1696, [21] H. F. Krmer, C. A. Witczk, E. B. Tylor, N. Fujii, M. F. Hirshmn, nd L. J. Goodyer, AS16 regultes insulinnd contrction-stimulted glucose uptke in mouse skeletl muscle, JournlofBiologiclChemistry, vol. 281, no. 42, pp , 26. [22] R. Guillemin, T. Vrgo, J. Rossier et l., β-endorphin nd drenl corticotropin re secreted concomitntly by the pituitry glnd, Science,vol. 197,pp , [23] J. G. Lin, S. L. Chng, nd J. T. Cheng, Relese of betendorphin from drenl glnd to lower plsm glucose by the electrocupuncture t Zhongwn cupoint in rts, Neuroscience Letters, vol. 326, pp. 17 2, 22. [24] I. M. Liu nd J. T. Cheng, Medition of endogenous β- endorphin in the plsm glucose-lowering ction of herbl products observed in type 1-like dibetic rts, Evidence-Bsed Complementry nd Alterntive Medicine. In press. [25]P.Reichrd,B.Y.Nilsson,ndU.Rosenqvist, Theeffect of intensive tretment of dibetes on the development nd progression of long-term complictions in insulin-dependent dibetes mellitus, The New Englnd Journl of Medicine, vol. 329, pp , 1993.

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