Chromium Alleviates Glucose Intolerance, Insulin Resistance, and Hepatic ER Stress in Obese Mice

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1 nture pulishing group rticles intervention AND prevention Chromium Allevites Glucose Intolernce, Insulin Resistnce, nd Heptic ER Stress in Oese Mice Nir Sreejyn, Feng Dong, Mchender R. Knddi,2, Xioping Yng 3 nd Jun Ren Ojective: Chromium hs gined populrity s nutritionl supplement for dietic ptients. This study evluted the effect of chronic dministrtion of chromium complex of d-phenyllnine ( ) on glucose nd insulin tolernce in oese mice. The study tested the hypothesis tht suppresses endoplsmic reticulum (ER) stress nd insulin resistnce in these nimls. Methods nd Procedures: C57BL len nd oese mice were rndomly divided to orlly receive vehicle or (3.8 µg of elementl chromium/kg/dy) for 6 months. Insulin sensitivity ws evluted y glucose nd insulin tolernce tests. Protein levels of phosphorylted pncretic ER kinse (PERK), α suunit of trnsltion initition fctor 2 (eif2α) nd inositol-requiring enzyme- (IRE-), p-c-jun, nd insulin receptor sustrte- (IRS-) phosphoserine-37 were ssessed y western lotting. In vitro ER stress ws induced y treting cultured muscle cells with thpsigrgin in the presence or sence of. Results: mice showed poor glucose nd insulin tolernce compred to the len controls, which ws ttenuted y. Mrkers of insulin resistnce (phospho-c-jun nd IRS- phosphoserine) nd ER stress (p-perk, p-ire-, p-eif2α), which were elevted in mice, were ttenuted following tretment. Chromium tretment ws lso ssocited with reduction in liver triglyceride levels nd lipid ccumultion. In cultured myotues, ttenuted ER stress induced y thpsigrgin. Discussion: Orl tretment reduces glucose intolernce, insulin resistnce, nd heptic ER stress in oese, insulin-resistnt mice. Oesity (28) 6, doi:.38/oy Introduction Insulin resistnce, concomitnt with dietes, metolic syndrome, oesity, nd hypertension, is mjor risk fctor for the development nd progression of crdiovsculr diseses, the leding cuses of mortlity nd moridity (). The currently ccepted therpeutic regimens include phrmcologicl tretment (igunides, thizolidinediones), cloric restriction, nd physicl exercise, which re effective, lthough none is deemed the ultimte cure. In consequence, there hs een sustntil demnd for development of new gents trgeting the insulinsignling cscde to improve overll insulin sensitivity nd long-term enefit in the mngement of dietes, oesity, nd insulin resistnce. Minerl chromium, which is thought to ply key role in crohydrte metolism y potentiting the ction of insulin, hs drwn recent interest for its role in dietes nd oesity (2). Dietry deficiency of chromium is elieved to e positively ssocited with the risk of dietes nd its complictions (3). This is supported y findings from clinicl trils tht dietry chromium supplementtion cn lower lood-glucose levels nd improve lipid profile in dietic ptients (reviewed in ref. 4). Better iovilility of low-moleculr-weight orgnic chromium complexes, nd the identifiction of the iologiclly ctive form of chromium s complex with n oligopeptide, prompted the design nd evlution of low-moleculr-weight orgnic chromium complexes s potentil therpeutic gents in treting insulin in type 2 dietes (5). In severl niml nd humn studies, chromium complex of picolinic cid, the most populrly used dietry supplement, hs een shown to modulte intrcellulr pthwys of glucose metolism nd improve comoridities ssocited with insulin resistnce (6). In recent clinicl tril, Mrtin nd ssocites demonstrted n improvement in insulin sensitivity nd glycted hemogloin in sujects with type 2 dietes who received chromium Division of Phrmceuticl Sciences nd Center for Crdiovsculr Reserch nd Alterntive Medicine, School of Phrmcy, University of Wyoming, Lrmie, Wyoming, USA; 2 Deprtment of Phrmcology, Mnipl College of Phrmceuticl Sciences, Mnipl University, Mnipl, Krntk, Indi; 3 Division of Crdiothorcic Surgery, Deprtment of Surgery, School of Medicine, University of Colordo t Denver nd Helth Sciences Center, Denver, Colordo, USA. Correspondence: Nir Sreejyn (sreejy@uwyo.edu) Received 8 July 27; ccepted 3 Decemer 27; pulished online 3 April 28. doi:.38/oy oesity VOLUME 6 NUMBER 6 JUNE 28 33

2 picolinte (7). Despite these documented evidence for the eneficil effects of chromium, two recent studies suggested tht chromium tretment my not hve ny effect on insulin sensitivity or glycted hemogloin levels in oese ptients with poorly controlled dietes (8,9). Bsed on its evlutions of the existing scientific evidence, the US Food nd Drug Administrtion (FDA) hs llowed the following qulified helth clim: Chromium picolinte my reduce the risk of insulin resistnce, nd therefore possily my reduce the risk of type 2 dietes. FDA concludes, however, tht the existence of such reltionship etween chromium picolinte nd either insulin resistnce or type 2 dietes is highly uncertin. To understnd the role of chromium in tretment of dietes, these discrepncies wrrnt further extensive studies. Although the moleculr mechnisms leding to insulin resistnce remin elusive, emerging evidence suggests tht endoplsmic reticulum (ER) stress my ply pivotl role in this phenomenon (,). Ozcn nd collegues showed elevted ER stress mrkers in dipocytes nd liver cells of geneticlly oese nd high-ft fed mice (2). ER stress cuses ctivtion of c-jun N-terminl kinse (JNK) tht phosphoryltes, serine residue (37) on insulin receptor sustrte- (IRS-), leding to the suppression of insulin signling (2). Consequently, molecules tht suppress ER stress cn improve dietes nd ssocited comoridities (3,4). Bsed on this ckground, it is pertinent to ssess the potentil effect of chromium on ER stress. The present study evluted the impct of chronic tretment with chromium complex of the mino cid d-phenyllnine ( ) (5) on glucose tolernce nd insulin sensitivity in oese mice with type 2 dietes. The study lso ssessed the effect of chronic chromium therpy on plsm nd liver lipid levels. Further, in n ttempt to understnd the potentil mechnisms involved, the effect of chromium tretment on heptic ER stress nd cellulr mrkers of insulin resistnce ws investigted. Methods And Procedures Mterils All chemicls, unless stted otherwise, were otined from Sigm Chemicl (St. Louis, MO). The Micro BCA protein ssy kit ws purchsed from Pierce Chemicl (Rockford, IL). Synthesis of ws synthesized nd chrcterized s descried previously (5). Briefly, queous solutions of CrCl 3 6H 2 O (2.6 g, mmol in 5 ml wter) nd d-phenyllnine (4.8 g, 3 mmol in 5 ml wter) were mixed t 8 C nd refluxed for 4 h. The homogeneous green rection mixture ws freeze-dried. The greenish-violet solid otined ws wshed with cetone nd dried in n ir oven. Animl tretment protocol The experimentl procedures descried in this study were pproved y the University of Wyoming (Lrmie, WY) Animl Use nd Cre Committee. In rief, homozygous B6.V-lep<o>/J mle mice were otined from the Jckson Lortory (Br Hror, ME) t 5 weeks of ge nd were housed within the School of Phrmcy Animl Fcility t the University of Wyoming with free ccess to food nd wter. The o/ o oese (leptin deficient) mice nd ge-mtched wild-type C57BL/6J mle mice were rndomly divided to receive either vehicle (H 2 O) or, provided in drinking wter for 6 months strting t 2 months of ge. On the sis of clculted wter intke, ws dministered to provide dose of ~45 µg/kg/dy (corresponding to ~3.8 µg of elementl chromium/kg/dy). Plsm leptin, insulin, nd lipids mesurement Plsm leptin nd insulin levels were mesured y rdioimmunossy nd mouse insulin enzyme-linked immunossy using commercilly ville kits per mnufcturer s specifictions (Linco Reserch, St. Chrles, MO). Serum triglyceride nd cholesterol levels were mesured using n ssy kit from Dignostic Chemicls, Oxford, CT. Glucose nd insulin tolernce tests At the end of the tretment schedule, mice were sujected to the intrperitonel glucose tolernce test (GTT) s descried previously (6). Briefly, the mice were fsted overnight (~2 h), nd glucose chllenge ws initited with intrperitonel injection of glucose ( g/kg). Glucose levels were determined in lood drops otined y clipping the til of the mice immeditely efore glucose chllenge, s well s t 5-, 6-, nd 2- min intervls. Serum glucose levels were determined using ACCU- CHEK Advntge Glucose Anlyzer (Roche Dignostics, IN). An insulin tolernce test ws performed t the end of 5 months of tretment. Mice (without overnight strving) were given intrperitonel injections of insulin (5 U/kg). Blood-glucose levels were determined y the tilclip method t different time points s descried previously (7). Liver triglyceride ssy Liver triglyceride levels were determined s descried previously (8). Briefly, g liver tissue from ech niml ws homogenized with 2 ml of chloroform/methnol (/2 y volume). After vortexing for 5 min, ml of chloroform nd ml of wter were dded sequentilly, mixed, nd centrifuged riefly t 3, rpm to seprte the phses. The lower phse ws trnsferred to nother tue, nd the residue ws mixed with.3 ml chloroform for the second-step vortex nd centrifugtion. The lower phse otined y the centrifugtion ws mixed with the first chloroform phse in the sme tue. After evportion with nitrogen gs t 55 C, the lipid extrct ws dissolved in.3 ml 2- propnol. Triglyceride levels were mesured spectrophotometriclly (Spectr Mx 9 Microplte Spectrophotometer; Moleculr Devices, Sunnyvle, CA). The liver sections were stined with hemtoxylin nd eosin. Western lot nlysis for mrkers of ER stress nd insulin resistnce The liver of ech mouse ws rpidly removed nd homogenized in lysis uffer contining 2 mmol/l Tris (ph 7.4), 5 mmol/l NCl, mmol/l EDTA, mmol/l EGTA, % Triton,.% sodium dodecyl sulfte, nd protese inhiitor cocktil. Smples were then sonicted for 5 s nd centrifuged t 2,g for 2 min t 4 C. The protein concentrtion of the superntnt ws ssessed using the Protein Assy Regent (Bio-Rd Lortories, Hercules, CA). Equl mounts (5 µg protein/lne) of protein nd prestined moleculr weight mrker (Gico-BRL, Githersurg, MD) were loded onto 7% sodium dodecyl sulfte polycrylmide gels in minigel pprtus (Mini-PROTEAN II, Bio-Rd) efore eing seprted nd trnsferred to nitrocellulose memrnes ( µm pore size, Bio-Rd). Memrnes were incuted for h in locking solution contining 5% nonft milk in Tris-uffered sline, wshed in Tris-uffered sline nd incuted overnight t 4 C with ntiphospho-perk (:25), ntiphospho-ire, nti-ire (:,), nti-eif2α (:5) nd ntiphospho- eif2α (:5), nti phospho-irs- (:,), nti-irs- (:,), ntiphospho-c-jun (:,), nd ntitotl JNK (:,) ntiody. Rit polyclonl nti-p-ire nd nti-ire ntiodies were kindly provided y Fumihiko Urno, University of Msschusetts. All other ntiodies were otined from Cell Signling (Beverly, MA). After incution with the primry ntiody, lots were incuted with n ntirit IgG HRP-linked ntiody t dilution 332 VOLUME 6 NUMBER 6 JUNE 28

3 of :5, for h t room temperture. Immunorective nds were detected using the Super Signl West Dur Extended Durtion Sustrte (Pierce, Milwukee, WI). The intensity of nds ws mesured with scnning densitometer (Model GS-8; Bio-Rd), coupled with Bio- Rd PC nlysis softwre. IRS- immunoprecipittion Immunoprecipittion ws performed s descried previously (9). Briefly, cell lystes from the liver ( mg of totl protein) were prepred in ml rdioimmunoprecipittion ssy uffer in.5-ml microtues. Anti-IRS- ntiody ws dded ( µg) to ech smple nd incuted overnight t 4 C. The immunoprecipittes were cptured on Protein A/G Sephrose eds (Pierce, Rockford, IL) y incuting the eds for 2 h t 4 C. The eds were wshed three times with cold rdioimmunoprecipittion ssy uffer, followed y finl wsh with phosphte-uffered sline. The eds were then suspended in 2 Lemmli uffer nd heted t 95 C for 5 min, nd the superntnt ws used for western lotting. Cell culture nd tretment The skeletl muscle cell line C2C2 from dult mouse legs (Americn Type Culture Collection) ws grown in Dulecco s modified Egle s medium supplemented with % fetl ovine serum nd % penicillin-streptomycin under humidified tmosphere of 5% CO 2 in ir nd mintined t low confluence. To induce differentition, the culture medium ws chnged to % fetl ovine serum insted of % fetl ovine serum when cells reched 7 8% confluence. Once differentited into myotues, the cells were rendered quiescent y incuting them with serum-free medium for 24 h. During the lst 6 h of quiescence, the myotues were treted with thpsigrgin ( µmol/l) in the presence or sence of. At the end of the tretment period, the cells were lysed with rdioimmunoprecipittion ssy uffer nd western lotted with ntiphospho α suunit of trnsltion initition fctor 2 (eif2α) s descried ove. The complete experiment, including differentition of myotues, ws repeted three times. Dt nlysis Dt re expressed s men ± s.e.m. nd sttisticlly evluted using 2-wy ANOVA using the Scheffe F-test for post hoc nlysis (Jndel Scientific, Sn Rfel, CA). A P vlue of <5 ws considered to e sttisticlly significnt. Results Generl fetures of len nd mice treted with As expected, mice hd significntly higher ody, hert, liver, nd kidney mss compred to ge-mtched len controls. Chronic tretment with ws not ssocited with chnges in ody mss (Tle ). tretment on plsm leptin, insulin, nd lipids levels Plsm leptin levels were undetectle in mice, nd tretment with did not lter leptin levels in these nimls (Figure ), lthough smll increse in plsm leptin levels ws oserved in len mice treted with (Figure ). Blood levels of insulin, triglyceride, nd cholesterol were significntly elevted in oese mice compred to the len control, suggesting the presence of insulin resistnce nd hyperlipidemi. Chronic tretment with did not lter the sl serum levels of insulin, triglyceride, or cholesterol (Figure d). Tle Generl fetures of len nd mice with or without 6-month tretment of t 45 µg/kg/dy (3.8 µg of elementl chromium/kg/dy) for 6 months Body mss (g) Hert mss (g) Liver mss (g) Kidney mss (g) c control control 29.2 ±.5 3 ± ± ± ± 2 4 ±.3 ± 2 9 ± 6.5 ± 8.48 ± ± ± 7 ± 3 3 ± 2 7 ± 2.5 ± 4 Men + s.e.m., n = 4 mice per group, P < 5 vs. len control. Serum leptin level (ng/ml) Serum triglyceride level (mmol/l) Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 Serum insulin level (ng/ml) d tretment on glucose nd insulin tolernce tests Whole-ody insulin sensitivity ws ssessed y performing GTTs nd insulin tolernce tests in oth len nd oese mice chroniclly treted with (Figure 2,). The sl fsting lood-glucose level in the len mice ws 93.2 ± 3. mg/dl nd tht for the oese mice ws 4 ± 8.9 mg/dl, indicting overt hyperglycemi in the oese mice. Following glucose chllenge in the len nimls, lood-glucose levels peked t 5 min nd returned to ner-norml levels within 2 min. In contrst, mice exhiited severe hyperglycemi upon dministrtion of glucose nd exhiited impired glucose tolernce s evidenced y the high postchllenge lood glucose levels even t 2 min. Chronic ingestion of significntly improved glucose tolernce in mice, with glucose disposl curves showing significntly lower lood levels of glucose t 5, 6, nd 2 min. 2. Serum cholesterol level (mmol/l) Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 Figure Effect of 6-month tretment of on serum levels of () leptin, () insulin, (c) triglyceride, nd (d) cholesterol in len nd mice. Men ± s.e.m., n = 4 mice per group, P < 5 vs. len group, P < 5 vs. group. oesity VOLUME 6 NUMBER 6 JUNE

4 Plsm glucose (mg/dl) Plsm glucose (mg/dl) Time (min) control + Cr(D-phe) 3 control + Cr(D-phe) 3 Tretment with did not lter the glucose disposl rte in len mice. In oth oese nd len groups, chronic tretment with hd no pprent effect on the sl levels of fsting lood glucose. Oese nimls sujected to insulin chllenge showed mrked reduction in postchllenge lood-glucose levels (Figure 2). The lood-glucose levels in oese mice treted with chromium were significntly lower t 5 nd 6 min compred to the glucose levels t the corresponding time points in the oese mice receiving the vehicle. Surprisingly, in contrst to estlished reports, len mice sujected to insulin chllenge did not exhiit precipitous fll in lood-glucose levels. Not hving strved the nimls prior to the test my hve contriuted to this discrepncy. Tretment with did not lter the glucose disposl rtes in len nimls following insulin chllenge. tretment on indictors of heptic ER stress Insulin resistnce nd oesity re ssocited with n increse in ER stress (2). To test whether chromium complexes improve glucose tolernce nd insulin sensitivity vi reducing ER stress, ER stress indictors such s pncretic ER kinse (PERK), inositol-requiring enzyme- (IRE-), nd eif2α were ssessed in heptic tissues of oese nd len mice treted with or without. Consistent with previous reports (2,4), levels control + Cr(D-phe) 3 control + Cr(D-phe) Time (min) 9 2 Figure 2 Effect of chronic dministrtion of on glucose metolism nd insulin sensitivity in mice. (3.8 µg or elementl chromium/kg/dy) ws orlly dministered for 6 months to 2 3 months old mice ge- nd sex-mtched len control. () Blood-glucose levels following glucose chllenge ( g/kg) performed t the end of 6 months of tretment with chromium. () Blood-glucose levels following insulin chllenge (5 IU/kg) performed t the end of 5 months of tretment with chromium. Dt re represented s mens ± s.e.m., nd sterisks indicte sttisticl significnce compred to control (P < 5, P < 5). 2 p-ire/ire rtio..8 p-eif2α eif2α p-ire- IRE- P-PERK + Cr(D-phe) 3 Control Cr(D-phe) 3 Control Cr(D-phe) 3 d + Cr(D-phe) 3 p-perk expression (ritrry density) 4 kd 4 kd kd kd 7 kd of phosphorylted PERK (Thr98), eif2α, nd IRE- were significntly higher in oese mice compred to len controls (Figure 3). This oesity-ssocited increse in p-perk, p-ire, nd eif2α ws reduced in -treted oese mice. In the len group, chronic therpy with did not lter the protein levels of ER stress indictors s seen y unchnged levels of IRE- protein (Figure 3). tretment on mrkers of insulin resistnce Insulin resistnce nd oesity re ssocited with ctivtion of c-jun-n-terminl kinse (JNK) nd susequent JNK-medited serine phosphoryltion of IRS- t Ser-37 tht negtively regultes insulin signling (2). Accordingly, the levels of phospho-c-jun nd IRS- phospho-serine-37 were elevted in the livers of oese mice compred to len control. Tretment with Cr (d-phe) 3 suppressed tyrosine phosphoryltion of c-jun nd serine phosphoryltion of IRS- in livers of mice (Figure 4). c p-eif2fα/eif2fα rtio + Cr(D-phe) Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 Figure 3 Effect of 6-month tretment with chromium on mrkers of endoplsmic reticulum (ER) stress in liver tissues of len nd mice. () Phosphoryltion of pncretic ER kinse (PERK) (thr98), inositol-requiring enzyme- (IRE-) nd α suunit of trnsltion initition fctor 2 (eif2α) in liver tissues of treted mice nd len controls were ssessed y western lotting using specific ntiodies. ( d) Represent the reltive densities of phospho-perk (thr98), phospho-ire-, phospho-eif2α nd totl IRE- respectively. Vlues re men ± s.e.m., n = 4 per group, P < 5 vs. len group, P < 5 vs. group. 334 VOLUME 6 NUMBER 6 JUNE 28

5 Control Cr(D-phe) 3 Control Cr(D-phe) 3.2 p-c-jun JNK IP:IRS-;IB:IRS--Ser 37 IP:IRS-;IB:IRS- p-c-jun/jnk rtio p-irs-/irs rtio kd 54 kd 46 kd 8 kd 8 kd Liver triglyceride (µg/g)..8 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 + Cr(D-phe) 3 Figure 4 Effect of 6-month tretment with on mrkers of insulin resistnce in oese nd len mice. () Representtive figure showing protein levels of phosphorylted c-jun, totl Jun N-terminl kinse (JNK), serine phosphorylted insulin receptor sustrte- (IRS-) (Ser-37), nd totl IRS- protein from two nimls of ech group. () The grphs show the quntittion of phosphoryltion of ech protein. Dt re presented s men ± s.e.m., from four nimls per group, P < 5 vs. len group, P < 5 vs. group. tretment on liver triglyceride content nd morphology in mice Oesity nd insulin resistnce hs een ssocited with ltertions in liver lipid metolism (2). Anlysis of heptic triglyceride content showed significnt increse in liver triglycerides levels in the mice compred to len controls (Figure 5). -treted oese nimls hd significnt reduction in heptic triglyceride levels compred to vehicle control. Histologicl exmintion of the mouse livers reveled n increse in the numer nd size of intrcellulr vcuoles chrcteristic of lipid droplets (Figure 5). In contrst, the numer of vcuoles ws significntly lower in the chromium-treted nimls, indicting prtil reversl of oesity-induced ft ccumultion in liver y chromium tretment. on thpsigrgin-induced ER stress in cultured myotues Lystes from myotues treted with thpsigrgin (n gent tht promotes ER stress y depletion of clcium stores) cused significnt increse in phosphorylted e-if2α, n erly mrker of ER stress, which ws inhiited y treting the cells with (Figure 6). The inhiition ws evident even t nmol/l concentrtions of. Chromium tretment did not lter the totl protein levels of eif2α. Discussion The mjor findings from the present study re tht chronic tretment with improves glucose tolernce nd insulin resistnce in n oese mouse model of type 2 dietes. Control Cr(D-phe) 3 Figure 5 Liver triglyceride content nd morphology in nd len mice following tretment with. () Liver triglyceride content ws mesured in len nd mice treted with vehicle or the chromium complexes. Dt re presented s men ± s.e.m., n = 4 per group, P < 5 vs. len group, P < 5 vs. group. () Representtive hemtoxylin (from 3 5 sections per mouse liver, n = 4 per group) nd eosin stining of liver sections from len nd mice from the control nd chromium-treted groups. Cr(D-(phe)) 3 (µmol/l) p-eif2α eif2α Thpsigrgnin (µmol/l). 4 kd 4 kd Figure 6 Effect on the thpsigrgin-stimulted endoplsmic reticulum (ER)-stress in cultured myotuules. ER stress ws induced y treting quiescent myotues with thpsigrgin ( µmol/l) for 6 h. Levels of ER stress indictor, phospho α suunit of trnsltion initition fctor 2 (eif2α) ws ssessed y western lotting (upper pnel). The lot ws reproed for totl eif2α (lower pnel). The lot shown ove is representtive figure from three seprte experiments. Furthermore, the dt presented here suggest tht chromium suppresses mrkers of insulin resistnce such s serine phosphoryltion of IRS-, tyrosine phosphoryltion of JNK, nd heptic ER stress in oese nimls. Chromium tretment lso reduced triglyceride levels nd lipid ccumultion in the liver of oese nimls without ltering the totl ody mss of these nimls. In cultured myocytes, chromium inhiited thpsigrgin-induced ER stress. Tken together, these results demonstrte tht chronic chromium therpy my hve oesity VOLUME 6 NUMBER 6 JUNE

6 eneficil effects in the tretment of dietes nd insulin resistnce ssocited with oesity. The eneficil effects of chromium complexes in niml models of dietes nd insulin resistnce hve een previously reported. Using n insulin-resistnt, type 2 dietic rt model (JCR:LA-corpulent rt), Ceflu nd co-workers hve demonstrted tht supplementtion of chromium picolinte t dose of ~ µg chromium/kg/dy for 2 weeks improves glucose disposl rtes nd enhnces insulin-stimulted phosphoryltion of IRS- nd PI-3K ctivity in skeletl muscle. (The pper erroneously reported the chromium dose s 8 µg/kg/dy) (22). Their dt suggest tht chromium picolinte improves insulin ction y ugmenting insulin signl trnsduction, which is in ccordnce with the results of the current study. Clodfelder nd co-workers hve shown tht orl tretment with much higher doses of chromium (, µg Cr/kg) given s trinucler chromium propionte complex cuses reduction in plsm insulin, glycted hemogloin, nd plsm lipids in Zucker oese rts (model for erly stges of type 2 dietes) nd Zucker dietic ftty rts ( model for type 2 dietes). Despite the high dose of chromium used in these experiments, the nimls filed to show ny chnges in glucose levels following GTT, lthough there ws reduction in post-gtt plsm insulin levels (23). In rt model of dexmethsoneinduced dietes, Kim nd co-workers hve shown tht higher doses of chromium picolinte (3 mg/kg/dy corresponding to 3.7 mg/kg/dy of elementl chromium) cuse significnt decrese in plsm glucose levels following n insulin tolernce test (24). In ccordnce with the dt from Clodfelder nd co-workers (23), tretment with chromium did not lower lood-glucose levels following GTT lthough insulin levels were significntly reduced (24). It difficult to compre these studies with the present study owing to the different sources nd doses of chromium used nd differences in the niml species nd model employed. However, tken together, these results help to further sustntite the clim tht chromium nd its complexes my hve eneficil effects in the tretment or control of dietes nd insulin resistnce. Previous studies (7,22,23) hve demonstrted tht tretment with chromium my reduce serum lipid levels in dietic conditions. However, the present study fils to confirm ny lipid-lowering effect from chromium tretment in oese mice. This difference my e ttriuted to the low dose of chromium (~3.8 µg/kg/dy of elementl chromium) used in the current study. Although chromium tretment filed to reduce serum lipid levels in this study, it significntly reduced liver triglyceride levels nd liver lipid ccumultion. The disprity etween the serum nd liver lipid levels suggests tht chromium my e cting t the level of lipid trnsporters either enhncing the trnsport of lipids from the liver or preventing ctive reuptke of lipids y the liver. Recent studies show tht chromium ttenutes ABCA, protein tht regultes cholesterol efflux, suggesting role for chromium in lipid trnsport (25). Agents tht reduce ER stress hve een shown to enhnce insulin signling nd meliorte glucose intolernce (4). ER stress is thought to suppress insulin receptor signling through the hyper-ctivtion of c-jun N-terminl kinse (JNK) (26). Insulin signling is negtively regulted y JNK through serine phosphoryltion of residue 37 within IRS- in liver nd dipose tissues (27). The phosphoryltion of IRS- Ser 37 prevents ssocition of its protein tyrosine inding with insulin receptor β-suunit, therey preventing IRS- inding to the receptor nd insulin-dependent ctivtion of PI3-kinse (2). Additionlly, Ser 37 phosphoryltion of IRS- ccelertes its uiquitin-proteosome-medited degrdtion (28). Attenution of ER stress, JNK ctivtion, nd Ser 37 phosphoryltion of IRS- my represent novel mechnism y which chromium imprts its eneficil effects in improving insulin sensitivity. In summry, the present study shows tht chronic dministrtion of low-moleculr chromium complexes improves glucose tolernce nd ER stress in oese type 2 dietic mice. Chromium tretment lso ttenutes heptic triglyceride levels nd lipid ccumultion. These effects of chromium my e medited y ttenution of oesity nd insulin resistnce stimulted ER stress nd consequent reduction in JNK ctivtion. Thus, the ility of chromium complexes to llevite ER stress nd improve glucose tolernce provides further support for the use of chromium in tretment or prophylxis of dietes nd relted diseses. Acknowledgments This work ws supported y the Americn Dietes Assocition Junior Fculty Awrd to N.S. We thnk Ms Virgini L. Cole for editing the mnuscript. Disclosure The uthors declred no conflict of interest. 28 The Oesity Society References. Sowers JR, Frohlich ED. Insulin nd insulin resistnce: impct on lood pressure nd crdiovsculr disese. Med Clin North Am 24;88: Anderson RA. Chromium in the prevention nd control of dietes. Dietes Met 2;26: Jeejeehoy KN, Chu RC, Mrliss EB, Greenerg GR, Bruce-Roertson A. Chromium deficiency, glucose intolernce, nd neuropthy reversed y chromium supplementtion, in ptient receiving long-term totl prenterl nutrition. Am J Clin Nutr 977;3: Ceflu WT, Hu FB. Role of chromium in humn helth nd in dietes. Dietes Cre 24;27: Vincent JB. Recent dvnces in the nutritionl iochemistry of trivlent chromium. Proc Nutr Soc 24;63: Anderson RA, Cheng N, Bryden NA et l. Elevted intkes of supplementl chromium improve glucose nd insulin vriles in individuls with type 2 dietes. Dietes 997;46: Mrtin J, Wng ZQ, Zhng XH et l. 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