Type 2 diabetes is characterized by hyperglycemia,

Transcription

1 ORIGINL RTICLE Ftty cid Incubtion of Myotubes From Humns With Type Dietes Leds to Enhnced Relese of -Oxidtion Products ecuse of Impired Ftty cid Oxidtion Effects of Tetrdecylthiocetic cid nd Eicospentenoic cid ndres J. Wenss, 1 rild C. Rustn, Mrlene Just, 3 Rolf K. erge, Christin. Drevon, 1 nd Michel Gster 3 OJECTIVE Incresed vilility of ftty cids is importnt for ccumultion of intrcellulr lipids nd development of insulin resistnce in humn myotubes. It is unknown whether different types of ftty cids like eicospentenoic cid () or tetrdecylthiocetic cid () influence these processes. RESERCH DESIGN ND METHODS We exmined ftty cid nd glucose metolism nd gene expression in cultured humn skeletl muscle cells from control nd type dietic individuls fter dys of preincubtion with or. RESULTS Type dietes myotubes exhibited reduced formtion of CO from plmitic cid (P), wheres relese of -oxidtion products ws unchnged t bseline but significntly incresed with respect to control myotubes fter preincubtion with nd. Preincubtion with enhnced both complete (CO ) nd -oxidtion of plmitic cid, wheres incresed only -oxidtion significntly. mrkedly enhnced tricylglycerol (TG) ccumultion in myotubes, more pronounced in type dietes cells. TG ccumultion nd ftty cid oxidtion were inversely correlted only fter preincubtion, nd totl level of cyl-co ws reduced. Glucose oxidtion (CO formtion) ws enhnced nd lctte production decresed fter chronic exposure to nd, wheres glucose uptke nd storge were unchnged. nd especilly incresed the expression of genes involved in ftty cid uptke, ctivtion, ccumultion, nd oxidtion. CONCLUSIONS Our results suggest tht 1) mitochondril dysfunction in dietic myotubes is cused by disturbnces downstrem of ftty cid -oxidtion; ) promoted ccumultion of TG, enhnced -oxidtion, nd incresed glucose From the 1 Deprtment of Nutrition, Institute of sic Medicl Sciences, Fculty of Medicine, University of Oslo, Oslo, Norwy; the Deprtment of Phrmceuticl iosciences, School of Phrmcy, University of Oslo, Oslo, Norwy; the 3 Deprtment of Endocrinology, Odense University Hospitl, Odense, Denmrk; nd the Lipid Reserch Group of Medicine, Hukelnd University Hospitl, University of ergen, ergen, Norwy. Corresponding uthor: ndres J. Wenss,.j.wenss@medisin.uio.no. Received 31 July nd ccepted 19 November. Published hed of print t on 9 December. DOI: 1.337/db by the mericn Dietes ssocition. Reders my use this rticle s long s the work is properly cited, the use is eductionl nd not for profit, nd the work is not ltered. See -nc-nd/3./ for detils. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 1 U.S.C. Section 173 solely to indicte this fct. oxidtion; nd 3) improved complete plmitic cid oxidtion in dietic myotubes, opposed incresed lipid ccumultion, nd incresed glucose oxidtion. Dietes :7 3, 9 Type dietes is chrcterized by hyperglycemi, reduced ility to oxidize ft, nd ccumultion of tricylglycerol (TG) in skeletl muscle fibers. The incresed deposition of intrmyocellulr TG (imtg) hs received specil interest, becuse severl studies hve demonstrted positive ssocition between insulin resistnce nd imtg storge (1,). ccumultion of imtg depends on the vilility nd uptke of ftty cids, the rte of ftty cid oxidtion, nd the rte of synthesis nd hydrolysis of TG. Incresed vilility of plsm free ftty cid (FF) during lipid infusion or high-ft feeding is ssocited with development of insulin resistnce nd ccumultion of imtg in vivo (3). Moreover, studies hve shown impired cpcity for ftty cid oxidtion in skeletl muscle from insulin-resistnt/ type dietic individuls (,), nd reduced mitochondril ftty cid oxidtion in skeletl muscle nd myotubes is ssocited with incresed deposition of imtg ( ). Ftty cids my promote insulin resistnce vi intrcellulr intermedites such s cyl-co, dicylglycerol (DG), nd cermides, interfering with insulin signling nd glucose metolism (9). Previous studies hve demonstrted positive effects on skeletl muscle insulin sensitivity of mono- nd polyunsturted ftty cids (PUFs) compred with sturted ftty cids (1 1). Very long chin -3 ftty cids, including eicospentenoic cid (), my protect ginst skeletl muscle insulin resistnce cused by high-ft feeding in vivo (1,13). PUFs my lso promote incresed TG ccumultion without impiring insulin-stimulted glucose uptke in myotubes (1,11). The sulfur-substituted ftty cid nlog tetrdecylthiocetic cid () is pn peroxisome prolifertor ctivted receptor (pn- PPR) ctivtor tht reduces plsm lipids nd enhnces heptic ftty cid oxidtion in rodents (1). Dul nd pn-ppr gonists re currently being developed for tretment of type dietes (1), nd hs been shown to improve glucose metolism in insulin-resistnt rts (1) nd to stimulte mitochondril prolifertion in rt DIETES, VOL., MRCH 9 7

2 MYOTUES ND FTTY CID OXIDTION skeletl muscle (17). We hve recently demonstrted tht my increse ftty cid oxidtion in humn myotubes similr to the PPR -specific gonist GW11 (1). Skeletl muscle metolism is influenced by physicl ctivity, hormonl sttus, nd muscle fiber type, rendering it difficult to determine the impct of nd on bsl nd insulin-stimulted intermediry metolism. Cultured humn myotubes disply the morphologicl, metolic, nd biochemicl properties of dult skeletl muscle (19) nd offer unique model to distinguish between genetic nd environmentl fctors in the etiology of insulin resistnce (). We nd others hve reported severl potentil intrinsic deficiencies in myotubes from individuls with type dietes, including lower bsl plmitte oxidtion (1) nd impired insulin-stimulted glucose metolism (,). It is unknown whether or my improve insulin resistnce or other chrcteristics of type dietes, such s decresed lipid oxidtion in myotubes. To identify the potentil effects of nd on the intermediry energy metolism nd insulin resistnce, we compred the effect of,, nd oleic cid in myotubes estlished from obese individuls with type dietes nd obese helthy subjects. RESERCH DESIGN ND METHODS Dulbecco s modified Egle s medium (DMEM)-Glutmx, FCS, Ultroser G, penicillin-streptomycin-mphotericin, nd trypsin-edt were obtined from Gibco, Invitrogen (Pisley, U.K.). -[ 3 H(G)]deoxy-D-glucose (. Ci/ mmol) nd D-[ 1 C(U)]glucose ( mci/mmol) were purchsed from PerkinElmer, NEN (oston, M). [1-1 C]cette ( mci/mmol), [1-1 C]P ( mci/mmol), nd D-[1-1 C]glucose ( mci/mmol) were obtined from mericn Rdioleled Chemicls (St. Louis, MO). Plmitic cid, oleic cid, S (essentilly ftty cid free), cytochlsin, nd extrcellulr mtrix gel were purchsed from Sigm-ldrich (St. Louis, MO). Insulin ctrpid ws from Novo-Nordisk (gsverd, Denmrk). gilent Totl RN Isoltion Mini kit ws purchsed from Mtriks (Oslo). The primers, SYR Green nd TqMn reverse-trnscription regents kit were obtined from pplied iosystems (Wrrington, U.K.), nd the protein ssy kit ws purchsed from iord (Copenhgen). ws purchsed from NU-Chek-Prep, Id kjemi (Ås, Norwy). nd [1-1 C] were provided by R.K.. iopsies. Skeletl muscle biopsies were obtined from eight obese type dietic ptients (9. 1. yers) nd eight obese control subjects (.3 1. yers) tken from musculus vstus lterlis in the fsted stte by needle biopsy under locl nesthesi. Only sedentry subjects were recruited. Dietic individuls were treted either with diet lone or in combintion with sulfonylure, metformin, or insulin, which ws withdrwn 1 week before tking the biopsies. The ptients hd no dietic complictions prt from simple retinopthy. The control subjects hd norml glucose tolernce nd no fmily history of type dietes. The groups were mtched with respect to ge nd MI but differed by fsting plsm glucose concentrtions (.. vs mmol/l, P.1, t test), fsting serum insulin levels (.. vs pmol/l, P.), glucose disppernce rte during euglycemichyperinsulinemic clmp (. 3. vs mg min 1 m, P.1), nd 1C (..1 vs. 7..%, P.). ll subjects gve written informed consent, nd the locl ethics committee of Funen nd Vejle County pproved the study. Cell culture nd ftty cid incubtion. Myotubes were estlished from stellite cells (19). Humn myoblsts from control nd type dietic subjects were differentited to myotubes t physiologicl concentrtion of insulin ( pmol/l) nd glucose (. mmol/l) for dys. Myotubes were then exposed to S ( mol/l), oleic cid (1 mol/l), (1 mol/l), or (1 mol/l) for nother dys. Stock solutions of ftty cid sodium slts ( mmol/l) nd S (. mmol/l) were heted to C nd rpidly mixed (molr rtio of.:1). Only opticlly cler solutions were used. There were no differences between the myotubes fter preincubtion with ftty cids s evluted by microscopic inspection serching for floting cells or cells with lipid droplets. The myotubes hd similr protein content independent of incubtion medium. Plmitic cid uptke nd distribution of rdiolel in lipids Plmitic cid uptke. Myotubes were incubted in DMEM supplemented with. mmol/l S,. mmol/l L-crnitine, mmol/l HEPES, [1-1 C]P (. Ci/ml,. mmol/l), nd pmol/l or 1. mol/l insulin for h, to study bsl nd insulin-medited uptke nd lipid distribution of plmitte. De novo lipid synthesis. Myotubes preincubted with ftty cids were further incubted hinmedium contining [1-1 C]cette (3 Ci/ml, 1 mol/l). fter incubtion, myotubes were plced on ice, wshed three times with 1 ml PS, scrped twice with l distilled wter, nd homogenized. The cell homogentes were ssyed for protein nd extrcted for lipids, nd the rdioleled lipids were seprted by thin-lyer chromtogrphy (1) nd quntified by liquid scintilltion. Totl lipid uptke is the sum of oxidtion nd storge products. Mesures of ftty cid oxidtion cid-soluble metolites. Myotubes were incubted in DMEM supplemented with. mmol/l S,. mmol/l L-crnitine, [1-1 C]P (. Ci/ml,. mmol/l), nd pmol/l or 1. mol/l insulin for htostudy cellulr relese of excess 1 C plmitic cid derived cid-soluble metolites (SMs) to the medi. The incubtion medi were trnsferred to new tubes nd ssyed for leled SMs, which minly re byproducts of -oxidtion remining in solution fter precipittion of the rdioleled ftty cid with perchloric cid (PC) (3). CO nd totl oxidtion. Myotubes were incubted in DMEM supplemented with. mmol/l S,. mmol/l L-crnitine, mmol/l HEPES, [1-1 C]P (. Ci/ml,. mmol/l), nd pmol/l insulin for htostudy CO formtion (1). PC ws dded to the cells to mesure both intr- nd extrcellulr (totl) SMs from plmitic cid. Thus, totl plmitic cid oxidtion is the sum of CO nd totl SMs. Glucose metolism. Glucose uptke, glycogen synthesis, nd glucose oxidtion were determined s previously described (7). RN isoltion nd rel-time RT-PCR. Myotubes were wshed nd centrifuged to pellet before totl RN ws isolted by Totl RN Isoltion Mini kit (gilent), ccording to the supplier protocol. Totl RN (. g/ l) ws reversely trnscribed with TqMn reverse-trnscription regents kit (7). Quntittive RT-PCR ws performed using n I PRISM 7 Detection System. DN expression ws determined by SYR Green (7), nd primers for uncoupling protein (UCP), ftty cid trnslocse (CD3/FT), crnitine plmitoyl trnsferse (CPT)-1, CPT, ftty cid binding protein 3 (FP3), long-chin cyl-coenzyme synthetse 1 (CSL1), CSL3, mitochondril trifunctionl protein -subunit (HDH), cyl-coenzyme dehydrogense medium-chin (CDM), DGT1, DGT, hexokinse II (HKII), GLUT1, GLUT, ctin- (CT ), nd ribosoml protein, lrge, P (RPLP) (supplementl Tle 1, ville in n online ppendix t db-13) were designed using Primer Express (pplied iosystems). In ddition, expression of CPT1b, citrte synthse (CS), mlte dehydrogense (MDH), CC, UCP3, steroyl-coenzyme desturse 1 (SCD1), PGC1, CT, nd RPLP (supplementl Tle 1) were nlyzed with the inventoried TqMn gene expression ssys of pplied iosystems on 79HT Fst Rel-Time PCR system with 9-well block module following stndrd protocols. The trnscription levels were normlized to CT nd RPLP. nlysis of cyl-co frctions. Cells were hrvested by centrifugtion nd wshed twice with 1 mmol/l PC nd stored t C until long-chin ftty cyl-co (LCF-Co) extrction. Heptdecnoyl-Co (33 pmol) nd H O were dded to finl volume of l before ddition of 3 ml chloroform: methnol (:1). Cells were then homogenized with n Ultrturrx homogenizer. Chloroform (1 ml) nd H O (1 ml) were dded nd vortexed vigorously. fter centrifugtion (3, rpm/1 min/ C), the interphse ws recovered nd dried under strem of N. The LCF-Cos were further extrcted nd derivtized to fluorescent cyl etheno-co esters using. mol/l chlorocetldehyde,.% SDS, nd.1 mol/l citrte, ph ( l), nd then seprted nd nlyzed by reverse-phse chromtogrphy (). Pek res were integrted using Chromeleonc version. (Dionex) nd quntified reltive to the internl heptdecnoyl-co stndrd. Identifiction of individul LCF-Co ws performed using stndrd LCF-Co mixtures. The detection level of our methods ws. pmol/l. Scintilltion proximity ssy. We pplied this method becuse it provides ccurte nd reproducible cell dt with limited use of resources for quite extensive nlyses (). In short, rdioleled substrtes tken up nd ccumulted by dherent cells re concentrted close to the scintilltor embedded in the plstic bottom of ech well nd thus provide stronger signl thn the rdiolel dissolved in the medium lone (). Myotubes were preincubted with 1 mol/l oleic cid for dys with ddition of 1 mol/l the lst h. Then myotubes were wshed with PS, medium contining [1-1 C]P (1 Ci/ml, 1 mol/l) ws dded, nd myotubes were incubted up to h. Ftty cid ws bound to S t rtio of.:1. Sttisticl nlysis. The effect of group nd incubtions ws evluted by liner mixed models nlysis in SPSS (v1..1), monitoring group nd tretment effects bsed on ll ville dt from ech experiment nd tking into ccount the correltion between mesurements/myotubes from the sme subjects. Some cyl-co vlues were below detection limit nd were ssigned DIETES, VOL., MRCH 9

3 .J. WENSS ND SSOCITES Uptke of [1-1 C]P (nmol/mg cell protein) 1 1 ObControl c ObTD 1 CO from [1-1 C]P (nmol/mg cell protein) 1 CO s % of totl oxidtion (CO +SM) C b FIG. 1. Plmitte uptke nd oxidtion. Differentited myotubes from type dietic or control subjects were preincubted with mol/l S, 1 mol/l oleic cid, 1 mol/l, or 1 mol/l for dys nd incubted with. mmol/l [1-1 C]P for h. The pnels show plmitte uptke, which is the sum of totl recovered CO, SMs, nd totl lipids (); oxidtion to CO (); CO from plmitic cid s percentge of totl oxidtion (C). Results present mens SE (n )., P <. vs. bseline; b, P <. vs. oleic cid; c, P <. vs. ; P <. vs. ObControl, nlyzed by liner mixed models sttistics (SPSS), which lso clculted the llover results bsed on ll ville dt. TD, type dietes. the vlue. pmol/l in ccordnce to the detection level. Nonprmetric sttisticl nlyses (Mnn-Whitney nd Wilcoxon s signed-rnk test) were performed on the cyl-co dt for evlution of group nd tretment differences, respectively. P vlue. ws considered significnt. RESULTS Effect of ftty cids on uptke nd oxidtion of ftty cids. We observed enhnced uptke of 1 C plmitic cid in cultured myotubes from controls nd type dietic subjects fter dys of preincubtion with oleic cid,, or (Fig. 1), showing tht ftty cid vilility is importnt for cellulr lipid uptke. y dding 1 mol/l insulin cutely, 1 C plmitic cid ccumultion ws further enhnced by 1 3% (dt not shown; P.). There ws n llover significnt % reduction in 1 C plmitic cid derived CO relesed from type dietes vs. control myotubes (Fig. 1), indicting tht type dietes cells exhibit reduced complete plmitic cid oxidtion. However, fter incubtion with, control nd type dietes myotubes showed enhnced 1 CO production from 1 C plmitic cid compred with bseline or preincubtion with oleic cid (Fig. 1), suggesting tht my improve mitochondril ftty cid oxidtion. The percentge of plmitic cid tht ws oxidized completely to CO compred with totl oxidtion [sum of complete (CO ) plus incomplete ftty cid oxidtion (SMs)] ws lower in type dietes myotubes, especilly fter preincubtion with (Fig. 1C). Preincubtion with nd enhnced 1 C plmitic cid derived SMs recovered in the medi, nd the response ws significntly stronger in type dietes myotubes ( 1% nd 1%) compred with control myotubes ( % nd 7%; Fig. ). In the presence of insulin, preincubtion with,, nd oleic cid enhnced SM relese, with stronger llover response in type dietes compred with control myotubes (Fig. ). We lso observed similr effects of nd on SMs derived from 1 C oleic cid, with significntly stronger relese from type dietes thn control myotubes in the presence of insulin (dt not shown). ccumultion of TG. Preincubtion of myotubes with oleic cid nd enhnced 1 C plmitic cid ccumultion into lipids compred with bseline by 3 3% nd 9 3%, respectively (results not shown; P.). The [1-1 C]P-derived SM relese (nmol/mg cell protein ObControl ObTD [1-1 C]P-derived SM relese (nmol/mg cell protein FIG.. Plmitte-derived SMs relese to the medium. Differentited myotubes from type dietes or control subjects were preincubted with ftty cids s described in Fig. 1 nd then cutely incubted with. mmol/l [1-1 C]P 1 mol/l insulin for h. The pnels show SMs relesed from the cells, derived from 1 C plmitic cid without () nd with () 1 mol/l insulin. Results represent mens SE (n 7)., P <. vs. bseline; b, P <. vs. oleic cid; P <. vs. ObControl, nlyzed by liner mixed models sttistics (SPSS), which lso clculted the llover results bsed on ll ville dt. DIETES, VOL., MRCH 9 9

4 MYOTUES ND FTTY CID OXIDTION [1-1 C]P-derived TG s % of bseline 3 1 c Chnges in P-derived TG reltive to bseline 3 1 ObControl ObTD Chnges in P oxidtion reltive to bseline FIG. 3. Chnges in TG ccumultion from 1 C plmitic cid reltive to bseline () nd negtive correltion between chnges in 1 C plmitic cid oxidtion nd TG ccumultion fter preincubtion with (). Differentited myotubes from type dietic or control subjects were preincubted with ftty cids s described in Fig. 1 nd then cutely incubted with [1-1 C]P (. mmol/l) for h. The pnels show chnges in 1 C plmitic cid derived TGs s percentge of bseline (S) () nd negtive correltion between chnges in totl 1 C plmitic cid oxidtion nd TG ccumultion in myotubes preincubted with (r.9, P <.1). Results represent mens SE (n )., P <. vs. bseline; b, P <. vs. oleic cid; c, P <. vs. ; P <. vs. ObControl, nlyzed by liner mixed models sttistics (SPSS), which lso clculted the llover results bsed on ll ville dt. enhnced lipid ccumultion might be explined by strong increment in TG ccumultion fter preincubtion with oleic cid (7. nd 7.9 nmol/mg cell protein, control, nd type dietes, respectively) nd (1.9 nd 17. nmol/mg cell protein, control, nd type dietes, respectively) compred with bseline (results not shown; P.). The reltive increse in TG ccumultion ws significntly higher in type dietes thn control myotubes fter preincubtion with (Fig. 3). Preincubtion with did not increse totl 1 C plmitic cid lipid ccumultion, lthough rdioleled TG ws slightly incresed compred with bseline but reduced compred with oleic cid nd (Fig. 3). Similr effects of oleic cid,, nd on lipid synthesis were observed using 1 C oleic cid (dt not shown). Moreover, preincubtion with cused negtive correltion between reltive chnge in totl oxidtion nd TG synthesis from 1 C plmitic cid (r.9, P.) (Fig. 3), but not with oleic cid or (results not shown). Gene expression relted to mitochondril function nd lipid metolism. There were no differences between the two groups in expression of severl genes importnt for mitochondril function nd ftty cid metolism (Tle 1). Preincubtion with ftty cids incresed the expression of CPT1- / (1.- to -fold) nd HDH (3%). However, UCP nd -3 were modertely incresed with respect to bseline by 3 nd %, respectively, only fter preincubtion with. Expression of CD3/FT, CSL, nd FP3 ws incresed fter preincubtion with more so thn oleic cid nd (Tle 1). Preincubtion with lso promoted.-fold induction in SCD1 compred with the other ftty cids (Tle 1). DG cyltrnsferse (DGT), responsible for the finl step in TG synthesis, ws incresed similrly by ll ftty cids in control myotubes, wheres type dietes myotubes displyed reduced response, especilly during incubtion with (Fig. ). The nucler trnscription fctor PPR coctivtor (PGC1 ) hd similr expression pttern to DGT, with enhnced expression fter ftty cid preincubtion in control cells nd reduced response in type dietes myotubes (Fig. ). cyl-co frctions. Totl cyl-co ws incresed reltive to bseline fter preincubtion with, wheres preincubtion with reduced cyl-co by % in type dietes myotubes but significntly less in control cells (Fig. ). Preincubtion with reduced C1:1- Co by n verge of 1% (not shown; P.) nd C1:-Co by % (Fig. ) reltive to bseline, wheres C1:1-Co ws mrkedly incresed (Fig. C). lso substntilly reduced the level of C1:-Co reltive to bseline in type dietes myotubes, to level significntly lower thn in control myotubes (Fig. D). Preincubtion with oleic cid reduced C1:-Co by 71 % (Fig. ) nd incresed C1:1-Co by n verge of 7% (not shown; P.) reltive to bseline. -Co nd C1:-Co hd similr retention times nd therefore TLE 1 Gene expression reltive to bseline (Ct no.) Oleic cid CPT1 1. (7) CPT1 1. (33) c CPT 1. (3) CDM 1. () HDH 1. () CS 1. () b 1.1. c MDH 1. () b 1.. UCP 1. () c UCP3 1. (3) b 1..7 c CD3/FT 1. (9) c CSL1 1. () CSL3 1. () c FP3 1. () c SCD1 1. () b.. c DGT1 1. (3) Dt re mens SE (n 1). Individul results were normlized to bseline (set to 1; verge bseline Ct numbers for the investigted genes re shown in brckets, giving n impression of reltive expression levels). P. vs. bseline; b P. vs. oleic cid; c P. vs. nlyzed by liner mixed models sttistics (SPSS). 3 DIETES, VOL., MRCH 9

5 .J. WENSS ND SSOCITES DGT mrn expression reltive to bseline ppered in the sme pek on the chromtogrm. The reltive increse in cyl-co fter preincubtion with (Fig. ) could be scribed to mrked increse in the combined /C1:-Co pek compred with C1:-Co t bseline (Fig. ). Interestingly, C:-Co ws decresed in type dietes compred with control myotubes fter ftty cid preincubtions nd significntly fter (Fig. E). Glucose metolism. Dietic myotubes displyed n llover (bsl nd insulin-stimulted) 1% decresed glucose oxidtion compred with control cells (results not shown; P.). sl nd insulin-medited glucose oxidtion ws enhnced fter preincubtion with (%) nd (%) in myotubes from both groups (results not shown, P.). Glucose uptke nd glycogen synthesis, however, were mostly unffected by preincubtions with ftty cids (dt not shown). Thus, PGC1α mrn expression reltive to bseline ObControl ObTD FIG.. Gene expression of DGT nd PGC1. Pnels show chnges in DGT () nd PGC1 () gene expression reltive to bseline (set to 1) for control nd type dietes myotubes. Cells were preincubted s described in RESERCH DESIGN ND METHODS. Results represent mens SE (n )., P <. vs. bseline; b, P <. vs. oleic cid; P <. vs. ObControl, nlyzed by liner mixed models sttistics (SPSS), which lso clculted the llover results bsed on ll ville dt. preincubtion with nd my hve reduced nonoxidized glucose metolites like lctte nd pyruvte, ccounting for the incresed glucose oxidtion. Moreover, the mrn expression of GLUT, GLUT1, nd HKII were unchnged fter ftty cid preincubtions (results not shown). inhibits the oleic cid medited increse of cell-ssocited 1 C plmitic cid. We monitored incorportion of 1 C- into complex cellulr lipids nd found tht only miniml mount ws recovered in the TG frction, wheres most ws found in phospholipids (not shown). De novo synthesis of ftty cids from 1 C-cette ws similr fter dys of preincubtion of myotubes with compred with bseline (Fig. ), but the rtio between rdioctivity recovered in DG nd TG ws sevenfold higher fter (Fig. ). To further elucidte the impct of on cellulr ftty cid ccumultion, we monitored the time course for cell-ssocited 1 C plmitic cid by scintilltion proximity ssy (SP). s expected, dys of preincubtion with oleic cid cused enhnced cell-ssocited 1 C plmitic cid (Fig. ). itself cused no chnge compred with bseline, nd ddition of for the lst h of the oleic cid preincubtion period ws sufficient to inhibit the oleic cid medited increse in cell-ssocited 1 C plmitic cid (Fig. ). Together, these dt suggest tht or -Co my reduce ftty cid stimulted TG synthesis, possibly by inhibiting DGT ctivity s lso suggested previously in rt livers (). DISCUSSION Cultured humn myotubes is the most similr cell system to intct skeletl muscle tht cn be modulted ex vivo. Compred with rodent models, they express the right genetic bckground nd the specific skeletl muscle phenotype. The extrcellulr environment cn be controlled precisely nd kept reltively constnt over time, without interference by systemic homeosttic compenstory mechnisms. We nd others hve reported severl potentil intrinsic deficiencies in myotubes from individuls with type dietes ( ). These differences pper most obvious when compring myotubes estlished from len with obese type dietic subjects but re not lwys cyl-co s % of bseline ObControl b ObTD c c cyl-co (pmol/mg cell protein) c c cyl-co (pmol/mg cell protein) C c c cyl-co (pmol/mg cell protein) D c cyl-co (pmol/mg cell protein) E FIG.. Cellulr cyl-co reltive to bseline. Differentited myotubes were preincubted s described in Fig. 1 before the cells were hrvested for mesurement of cyl-co. : shows totl cyl-co reltive to bseline; nd E: disply C1:-Co (nd -Co where preincubted with ), C1:1-Co, C1:-Co, nd C:-Co, respectively. Results represent mens SE (n )., P <. vs. bseline; b, P <. vs. oleic cid; c, P <. vs.. P <. vs. ObControl. Evluted by nonprmetric Mnn-Whitney nd Wilcoxon s signed-rnk test. DIETES, VOL., MRCH 9 31

6 MYOTUES ND FTTY CID OXIDTION Chnges reltive to bseline sum lipid synthesis DG TG rtio DG/TG b b c bseline bseline c c bseline bseline bseline (1 µm) (1 µm) (1 µm) + (1 µm) lst h 1 3 Incubtion time (h) FIG.. Prtitioning of de novo synthesized ftty cids in DG nd TG () nd effect of on oleic cid medited increse in cell-ssocited 1 C plmitic cid (). : Distribution of de novo synthesized 1 C-cette derived ftty cids fter hincomplex lipids (DG nd TG) fter -dy preincubtion of humn myotubes from helthy donors with mol/l ftty cid free S (bseline), 1 mol/l oleic cid, 1 mol/l, or 1 mol/l (n ). : Cell-ssocited 1 C plmitic cid mesured by SP in humn myotubes from helthy donors preincubted dys with mol/l S (bseline), 1 mol/l oleic cid, 1 mol/l, or 1 mol/l oleic cid 1 mol/l the lst dy (n ). Results represent mens SE., P <. vs. bseline; b, P <. vs. oleic cid; c, P <. vs., nlyzed by liner mixed models sttistics (SPSS). Cell-ssocited [1-1 C]P (nmol/mg cell protein) significnt when compring obese with obese type dietes (7). We studied the effects of nd on the intermediry energy metolism nd insulin resistnce in myotubes estlished from obese subjects with type dietes nd obese helthy subjects. We hve previously demonstrted reduced cpcity for complete oxidtion of leled plmitic cid to CO in myotubes from obese type dietic subjects compred with obese control myotubes (1), nd this dysfunction ws reproduced in our present study. Here, we observed tht CO production from leled plmitic cid ws reduced for dietic myotubes (Fig. 7 vs. ). Furthermore, the relese of ftty cid -oxidtion products (SMs) ws incresed by type dietes compred with control myotubes fter preincubtion with ftty cids (Fig. 7C vs. D). Our dt suggest tht mitochondril dysfunction in type dietes myotubes is probly cused by disturbnces downstrem of ftty cid -oxidtion leding to excess production nd relese of SMs during incresed influx nd/or -oxidtion of ftty cids. In support for this, severl studies hve suggested mitochondril dysfunction in the etiology of insulin resistnce nd type dietes (,,). Moreover, incresed levels of -oxidtion products hve been found in both murine skeletl muscle nd plsm, probly reflecting mitochondril overlod due to excess vilility of ftty cids (9,3). Excess FF tken up by myotubes my either be oxidized or stored s TG. PGC1 is n importnt regultor of muscle oxidtive cpcity, mitochondril content, nd fiber type (31,3), nd insulin resistnce my be negtively ssocited with PGC1 expression in skeletl muscle (33,3). In the present study, gene expression of PGC1 ws incresed in response to ftty cids, but the reltive response ws blunted in dietic myotubes compred with control cells. In CC1 myotubes, PGC1 expression ws enhnced fter h but reduced fter 1 h of incubtion with plmitic cid (3), wheres unsturted ftty cids like oleic cid nd linoleic cid enhnced PGC1 expression in humn myotubes (3). Our dt suggest tht the reduced cpcity for complete mitochondril ftty cid oxidtion observed for type dietes myotubes might be relted to suboptiml responses in expression of PGC1, e.g., when the myotubes re exposed to ftty cids. DGT, which is involved in TG formtion (37), showed similr expression pttern to PGC1, nd the expression of these genes correlted well (r.1, P.1). Reduced DGT gene expression fter preincubtion with ftty cids in dietic cells, together with impired complete ftty cid oxidtion, my contribute to enhnced levels of potentilly lipotoxic intrcellulr ftty cid intermedites nd might ply role in lipid-induced insulin resistnce. Preincubtion of myotubes with led to incresed production of SMs, wheres incresed both SMs nd CO production (Fig. 7C nd D). Incresed formtion of -oxidtion intermedites my cuse mitochondril stress nd induce skeletl muscle insulin resistnce (3,3). However, we did not observe ny detrimentl effects of chronic preincubtion with either or on insulin-stimulted glucose metolism in humn myotubes. This does not exclude ny such link, becuse we did not coincubte with ftty cids during the glucose experiments tht might be prerequisite for negtive effects to occur. Nevertheless, Power et l. (39) showed tht supplementtion with L-crnitine could meliorte rther thn worsen insulin resistnce in mice, despite incresed levels of -oxidtion products in plsm nd skeletl muscle. Furthermore, preincubtion with nd incresed glucose oxidtion, suggesting improved insted of reduced cpcity for glucose hndling (Fig. 7C nd D). 3 DIETES, VOL., MRCH 9

7 .J. WENSS ND SSOCITES Obese TD Glucose FF Extrcellulr Glucose FF Extrcellulr Intrcellulr Intrcellulr Glucose--P cyl-co Glucose--P cyl-co Pyruvte Pyruvte β-oxidtion DG β-oxidtion DG NOG CO SM TG NOG CO SM TG Oxidtion Storge Oxidtion Storge C Obese fter nd vs. D TD fter nd vs. Glucose FF Extrcellulr Glucose FF Extrcellulr Intrcellulr Intrcellulr Glucose--P cyl-co Decresed by Glucose--P cyl-co Decresed by Pyruvte Pyruvte NOG & CO β-oxidtion & SM DG TG NOG & CO β-oxidtion & SM DG TG Oxidtion Storge Oxidtion Storge FIG. 7. Chnges in energy metolism in humn myotubes fter preincubtion with ftty cids. We observed reduced oxidtive metolism (CO formtion) of ftty cids nd glucose in obese type dietes myotubes compred with obese control cells ( vs. ). Incubtion with or promoted incresed -oxidtion in dietic cells (C vs. D). enhnced lipid uptke, ccumultion of TG (especilly in dietic cells), nd ftty cid -oxidtion in both groups of myotubes (C nd D). normlized complete lipid oxidtion in dietic myotubes to bseline level in control cells ( nd vs. D) nd opposed incresed lipid ccumultion (C nd D). Glucose oxidtion ws enhnced fter chronic exposure to nd, reducing nonoxidized glucose (NOG; C nd D). NOG consists mostly of lctte nd pyruvte. dotted rrows, reduced; fortified rrows, incresed; boxes leled with nd, effects of preincubtion. Preincubtion of myotubes with the nturlly occurring ftty cids enhnced 1 C plmitic cid uptke in line with previous observtions (,1), nd ws more effective thn oleic cid (Fig. 7C nd D). Preincubtion with oleic cid, nd especilly, lso promoted incresed incorportion of 1 C plmitic cid into TG, nd the response to ws more pronounced in dietic myotubes (Fig. 7C vs. D). We observed negtive correltion between totl ftty cid oxidtion nd TG synthesis in the presence of, suggesting tht my redirect excess ftty cids into TG. Storge of excess ftty cids such s TG my protect ginst cell dmge due to neutrliztion of lipotoxic intermedites (,1). Furthermore, reduced the totl mount of cyl-co, in prticulr C1:- Co, nd the reduction ws gretest for dietic myotubes. Incresed levels of cyl-co nd especilly C1:-Co hve been ssocited with insulin resistnce (,3). Moreover, mrkedly incresed the level of C1:1-Co, which hs recently been shown to function s lipokine improving peripherl insulin ction () nd glucose metolism in L rt myotubes (). In ddition, incubtion of myotubes from norml len subjects with improved glucose uptke, despite n incresed incorportion of 1 C oleic cid into TG (1). In rts, lso improved insulin resistnce while incresing imtg levels (). Preincubtion with incresed complete oxidtion of 1 C plmitic cid to CO to similr degree in control nd dietic myotubes. Thus, restored complete plmitic cid oxidtion in dietic cells to bseline level of control myotubes (Fig. 7 nd vs. D). The uptke of plmitic cid ws incresed fter preincubtion with but did not led to similr ccumultion of 1 C plmitic cid into TG compred with preincubtion with oleic cid or. The reduced TG ccumultion my reflect enhnced ftty cid oxidtion, nd we hve dt showing incresed TG ccumultion from 1 C plmitic cid fter preincubtion when myotubes were cutely coincubted with the CPT1 inhibitor etomoxir (.J.W.,.C.R., unpublished observtions). lterntively, -Co my inhibit DGT ctivity (), s lso suggested by the mrkedly incresed cellulr DG-to-TG recovery rtio of DIETES, VOL., MRCH 9 33

8 MYOTUES ND FTTY CID OXIDTION 1 C-cette in the presence of, without chnges in lipid synthesis. Furthermore, we observed tht 1 C- itself could not be efficiently incorported into TG in humn myotubes nd tht coincubtion with prevented the oleic cid induced increse in cell-ssocited 1 C plmitic cid. Moreover, myotubes preincubted with hd enhnced levels of cyl-co nd -Co probly ccounted for most of the difference becuse 3-thi ftty cids cnnot undergo -oxidtion (1). However, preincubtion of myotubes with neither impired insulin-stimulted glucose uptke nor glycogen synthesis, wheres glucose oxidtion ws incresed in cells from both donor groups (Fig. 7C nd D). my enhnce mitochondril prolifertion in skeletl muscle (17), nd ctivtion of PPR nucler receptors probly medites much of the effects of on gene expression (1,1,). ecuse there were no differences in induction of CPT1 expression fter preincubtion with reltive to oleic cid nd, the enhnced -oxidtion fter my be explined by the twofold higher induction of CD3/FT expression. CD3 my fcilitte ftty cid trnsport cross the plsm membrne in response to contrction nd insulin (7), but CD3 my lso reside on the outer mitochondril membrne () in colocliztion with CPT1 to fcilitte mitochondril ftty cid influx (,9). In rts, the PPR gonist rosiglitzone incresed skeletl muscle ftty cid oxidtion nd mitochondril CD3 mount, but not CPT1 protein expression or ctivity (9), which is in line with our observtions. Prt of the incresed oxidtion of 1 C plmitic cid to CO fter preincubtion with my be relted to the incresed UCP expression nd mitochondril uncoupling (). Thus, preincubtion with the synthetic ftty cid nlog enhnced complete ftty cid nd glucose oxidtion in dietic myotubes nd opposed the incresed lipid ccumultion observed fter preincubtion with oleic cid nd especilly. In summry, type dietes myotubes seem to hve n impired mitochondril cpcity for ftty cid oxidtion nd glucose (Fig. 7 vs. ), linked to defects downstrem of ftty cid -oxidtion, leding to incresed relese of SMs during high ftty cid vilility (Fig. 7C vs. D). Reduced response of PGC to ftty cids in dietic myotubes my be ssocited with reduced mitochondril function nd reduced DGT ctivity, creting lipid intermedites nd consequently insulin resistnce. nd might execute beneficil effects by incresing complete ftty cid oxidtion nd TG formtion, respectively (Fig. 7C nd D), thereby improving overll energy metolism nd ftty cid hndling in type dietes skeletl muscle. CKNOWLEDGMENTS Lipgene (Integrted Project th Frmework Progrmme, Food Qulity, nd Sfety; FOOD-CT-3-9), Frei Chocolde Friks Medicl Foundtion, The John Throne Holst Foundtion for Nutrition Reserch, The Dnish Medicl Reserch Council, the Dnish Dietes ssocition, nd the Novo-Nordisk Foundtion provided finncil support. R.K.. nd the University of ergen, Hukelnd University Hospitl, re shreholders of THI MEDIC, which holds ptents regrding effects of. No other potentil conflicts of interest relevnt to this rticle were reported. We thnk Irene Lynfort for excellent technicl ssistnce. We thnk Kurt Højlund nd Klus Levin for the muscle biopsies nd Jens Knudsen for ssisting in mesurements of cyl-co. REFERENCES 1. Storlien LH, Jenkins, Chisholm DJ, Pscoe WS, Khouri S, Kregen EW: Influence of dietry ft composition on development of insulin resistnce in rts: reltionship to muscle triglyceride nd -3 ftty cids in muscle phospholipid. Dietes : 9, Pn D, Lillioj S, Kriketos D, Milner MR, ur L, ogrdus C, Jenkins, Storlien LH: Skeletl muscle triglyceride levels re inversely relted to insulin ction. Dietes :93 9, oden G: Interction between free ftty cids nd glucose metolism. Curr Opin Clin Nutr Met Cre : 9,. Simoneu J, Kelley DE: ltered glycolytic nd oxidtive cpcities of skeletl muscle contribute to insulin resistnce in NIDDM. J ppl Physiol 3:1 171, He J, Wtkins S, Kelley DE: Skeletl muscle lipid content nd oxidtive enzyme ctivity in reltion to muscle fiber type in type dietes nd obesity. Dietes :17 3, 1. Petersen KF, efroy D, Dufour S, Dziur J, riyn C, Rothmn DL, DiPietro L, Cline GW, Shulmn GI: Mitochondril dysfunction in the elderly: possible role in insulin resistnce. Science 3:11 11, 3 7. Kse ET, Wenss J, s V, Hojlund K, Levin K, Thoresen GH, eck- Nielsen H, Rustn C, Gster M: Skeletl muscle lipid ccumultion in type dietes my involve the liver X receptor pthwy. Dietes :11 111,. Gster M, eck-nielsen H: Tricylglycerol ccumultion is not primrily ffected in myotubes estlished from type dietic subjects. iochim iophys ct 171:1 11, 9. Kregen EW, Cooney GJ: Free ftty cids nd skeletl muscle insulin resistnce. Curr Opin Lipidol 19:3 1, 1. s V, Rokling-ndersen MH, Kse ET, Thoresen GH, Rustn C: Eicospentenoic cid (: n-3) increses ftty cid nd glucose uptke in cultured humn skeletl muscle cells. J Lipid Res 7:3 37, 11. Lee JS, Pinnmneni SK, Eo SJ, Cho IH, Pyo JH, Kim CK, Sinclir J, Febbrio M, Wtt MJ: Sturted, but not n- polyunsturted, ftty cids induce insulin resistnce: role of intrmusculr ccumultion of lipid metolites. J ppl Physiol 1:17 17, 1. Coll T, Eyre E, Rodriguez-Clvo R, Plomer X, Snchez RM, Merlos M, Lgun JC, Vzquez-Crrer M: Olete reverses plmitte-induced insulin resistnce nd inflmmtion in skeletl muscle cells. J iol Chem 3: , 13. Storlien LH, Kregen EW, Chisholm DJ, Ford GL, ruce DG, Pscoe WS: Fish oil prevents insulin resistnce induced by high-ft feeding in rts. Science 37:, erge RK, Tronstd KJ, erge K, Rost TH, Wergedhl H, Gudbrndsen, Skorve J: The metolic syndrome nd the heptic ftty cid dringe hypothesis. iochimie 7:1, 1. lkumr P, Rose M, Gnti SS, Krishn P, Singh M: PPR dul gonists: re they opening Pndor s ox? Phrmcol Res :91 9, 7 1. Mdsen L, Guerre-Millo M, Flindt EN, erge K, Tronstd KJ, ergene E, Sebokov E, Rustn C, Jensen J, Mndrup S, Kristinsen K, Klimes I, Stels, erge RK: Tetrdecylthiocetic cid prevents high ft diet induced diposity nd insulin resistnce. J Lipid Res 3:7 7, 17. Totlnd GK, Mdsen L, Klementsen, Vgenes H, Kryvi H, Froylnd L, Hexeberg S, erge RK: Prolifertion of mitochondri nd gene expression of crnitine plmitoyltrnsferse nd ftty cyl-co oxidse in rt skeletl muscle, hert nd liver by hypolipidemic ftty cids. iol Cell 9:317 39, 1. Løvås K, Røst TH, Skorve J, Ulvik RJ, Gudbrndsen, ohov P, Wenss J, Rustn C, erge RK, Husebye ES: Tetrdecylthiocetic cid ttenutes dyslipidemi in mle ptients with type dietes mellitus, possibly by dul PPR / ctivtion nd incresed mitochondril oxidtion. Dietes Obes Met. In press. ville from Gster M, Kristensen SR, eck-nielsen H, Schroder HD: cellulr model system of differentited humn myotubes. PMIS 19:73 7, 1. Henry RR, brms L, Nikoulin S, Cirldi TP: Insulin ction nd glucose metolism in nondietic control nd NIDDM subjects: comprison using humn skeletl muscle cell cultures. Dietes :93 9, Gster M, Rustn C, s V, eck-nielsen H: Reduced lipid oxidtion in skeletl muscle from type dietic subjects my be of genetic origin: evidence from cultured myotubes. Dietes 3:, 3 DIETES, VOL., MRCH 9

9 .J. WENSS ND SSOCITES. Gster M, Petersen I, Hojlund K, Poulsen P, eck-nielsen H: The dietic phenotype is conserved in myotubes estlished from dietic subjects: evidence for primry defects in glucose trnsport nd glycogen synthse ctivity. Dietes 1:91 97, 3. Skrede S, remer J, erge RK, Rustn C: Stimultion of ftty cid oxidtion by 3-thi ftty cid reduces tricylglycerol secretion in cultured rt heptocytes. J Lipid Res 3:139 1, 199. Just M, Fergemn NJ, Knudsen J, eck-nielsen H, Gster M: Long-chin cyl-co is not primrily incresed in myotubes estlished from type dietic subjects. iochim iophys ct 17: 7,. Wenss J, Rustn C, Lovstedt K, Kull, Wikstrom S, Drevon C, Hllen S: Cell-bsed multiwell ssys for the detection of substrte ccumultion nd oxidtion. J Lipid Res :91 97, 7. erge RK, Mdsen L, Vgenes H, Tronstd KJ, Gottlicher M, Rustn C: In contrst with docoshexenoic cid, eicospentenoic cid nd hypolipidemic derivtives decrese heptic synthesis nd secretion of tricylglycerol by decresed dicylglycerol cyltrnsferse ctivity nd stimultion of ftty cid oxidtion. iochem J 33: , Gster M: Insulin resistnce nd the mitochondril link: lessons from cultured humn myotubes. iochim iophys ct 177:7 7, 7. Mogensen M, Shlin K, Fernstrom M, Glintborg D, Vind F, eck-nielsen H, Hojlund K: Mitochondril respirtion is decresed in skeletl muscle of ptients with type dietes. Dietes :19 199, 7 9. Koves TR, Li P, n J, kimoto T, Slentz D, Ilkyev O, Dohm GL, Yn Z, Newgrd C, Muoio DM: Peroxisome prolifertor-ctivted receptorgmm co-ctivtor 1lph-medited metolic remodeling of skeletl myocytes mimics exercise trining nd reverses lipid-induced mitochondril inefficiency. J iol Chem :33 339, 3. Koves TR, Ussher JR, Nolnd RC, Slentz D, Mosedle M, Ilkyev O, in J, Stevens R, Dyck JR, Newgrd C, Lopschuk GD, Muoio DM: Mitochondril overlod nd incomplete ftty cid oxidtion contribute to skeletl muscle insulin resistnce. Cell Met 7:, 31. Wu Z, Puigserver P, ndersson U, Zhng C, delmnt G, Mooth V, Troy, Cinti S, Lowell, Scrpull RC, Spiegelmn M: Mechnisms controlling mitochondril biogenesis nd respirtion through the thermogenic coctivtor PGC-1. Cell 9:11 1, Lin J, Wu H, Trr PT, Zhng CY, Wu Z, oss O, Michel LF, Puigserver P, Isotni E, Olson EN, Lowell, ssel-duby R, Spiegelmn M: Trnscriptionl co-ctivtor PGC-1 lph drives the formtion of slow-twitch muscle fibres. Nture 1:797 1, 33. Mooth VK, Lindgren CM, Eriksson KF, Subrmnin, Sihg S, Lehr J, Puigserver P, Crlsson E, Ridderstrle M, Luril E, Houstis N, Dly MJ, Ptterson N, Mesirov JP, Golub TR, Tmyo P, Spiegelmn, Lnder ES, Hirschhorn JN, ltshuler D, Groop LC: PGC-1lph-responsive genes involved in oxidtive phosphoryltion re coordintely downregulted in humn dietes. Nt Genet 3:7 73, 3 3. Ptti ME, utte J, Crunkhorn S, Cusi K, erri R, Kshyp S, Miyzki Y, Kohne I, Costello M, Sccone R, Lndker EJ, Goldfine, Mun E, DeFronzo R, Finlyson J, Khn CR, Mndrino LJ: Coordinted reduction of genes of oxidtive metolism in humns with insulin resistnce nd dietes: potentil role of PGC1 nd NRF1. Proc Ntl cd Sci US 1: 71, 3 3. Coll T, Jove M, Rodriguez-Clvo R, Eyre E, Plomer X, Snchez RM, Merlos M, Lgun JC, Vzquez-Crrer M: Plmitte-medited downregultion of peroxisome prolifertor-ctivted receptor- coctivtor 1 in skeletl muscle cells involves MEK1/ nd nucler fctor- ctivtion. Dietes :779 77, 3. Stiger H, Stiger K, Hs C, Weisser M, Mchico F, Hring HU: Ftty cid-induced differentil regultion of the genes encoding peroxisome prolifertor-ctivted receptor-gmm coctivtor-1lph nd -1bet in humn skeletl muscle cells tht hve been differentited in vitro. Dietologi :11 11, 37. Mn WC, Miyzki M, Chu K, Ntmbi J: Colocliztion of SCD1 nd DGT: implying preference for endogenous monounsturted ftty cids in triglyceride synthesis. J Lipid Res 7: , 3. n J, Muoio DM, Shiot M, Fujimoto Y, Cline GW, Shulmn GI, Koves TR, Stevens R, Millington D, Newgrd C: Heptic expression of mlonyl-co decrboxylse reverses muscle, liver nd whole-niml insulin resistnce. Nt Med 1: 7, 39. Power R, Hulver MW, Zhng JY, Dubois J, Mrchnd RM, Ilkyev O, Muoio DM, Myntt RL: Crnitine revisited: potentil use s djunctive tretment in dietes. Dietologi : 3, 7. Listenberger LL, Hn X, Lewis SE, Cses S, Frese RV Jr, Ory DS, Schffer JE: Triglyceride ccumultion protects ginst ftty cid-induced lipotoxicity. Proc Ntl cd Sci U S 1:377 3, 3 1. Liu L, Zhng Y, Chen N, Shi X, Tsng, Yu YH: Upregultion of myocellulr DGT1 ugments triglyceride synthesis in skeletl muscle nd protects ginst ft-induced insulin resistnce. J Clin Invest 117:179 19, 7. Ellis, Poynten, Lowy J, Furler SM, Chisholm DJ, Kregen EW, Cooney GJ: Long-chin cyl-co esters s indictors of lipid metolism nd insulin sensitivity in rt nd humn muscle. m J Physiol Endocrinol Met 79:E E, 3. Yu C, Chen Y, Cline GW, Zhng D, Zong H, Wng Y, ergeron R, Kim JK, Cushmn SW, Cooney GJ, tcheson, White MF, Kregen EW, Shulmn GI: Mechnism by which ftty cids inhibit insulin ctivtion of insulin receptor substrte-1 (IRS-1)-ssocited phosphtidylinositol 3-kinse ctivity in muscle. J iol Chem 77:3 3,. Co H, Gerhold K, Myers JR, Wiest MM, Wtkins SM, Hotmisligil GS: Identifiction of lipokine, lipid hormone linking dipose tissue to systemic metolism. Cell 13:933 9,. Dimopoulos N, Wtson M, Skmoto K, Hundl HS: Differentil effects of plmitte nd plmitolete on insulin ction nd glucose utiliztion in rt L skeletl muscle cells. iochem J 399:73 1,. Kusunoki M, Tsutsumi K, Hr T, Ogw H, Nkmur T, Miyt T, Skkibr F, Fukuzw Y, Sug T, Kto K, Hirook Y, Nky Y: Ethyl icospentte (omeg-3 ftty cid) cuses ccumultion of lipids in skeletl muscle but suppresses insulin resistnce in OLETF rts: Otsuk Long- Evns Tokushim Ftty. Metolism :3 3, 3 7. Luiken JJ, Dyck DJ, Hn XX, Tndon NN, rumugm Y, Gltz JF, onen : Insulin induces the trnsloction of the ftty cid trnsporter FT/CD3 to the plsm membrne. m J Physiol Endocrinol Met :E91 E9,. Cmpbell SE, Tndon NN, Woldegiorgis G, Luiken JJ, Gltz JF, onen : novel function for ftty cid trnslocse (FT)/CD3: involvement in long chin ftty cid trnsfer into the mitochondri. J iol Chem 79:33 31, 9. enton CR, Hollowy GP, Cmpbell SE, Yoshid Y, Tndon NN, Gltz JF, Luiken JJ, Spriet LL, onen : Rosiglitzone increses ftty cid oxidtion nd ftty cid trnslocse (FT/CD3) but not crnitine plmitoyltrnsferse I in rt muscle mitochondri. J Physiol :17 17,. Echty KS, Winkler E, Frischmuth K, Klingenberg M: Uncoupling proteins nd 3 re highly ctive H( ) trnsporters nd highly nucleotide sensitive when ctivted by coenzyme Q (ubiquinone). Proc Ntl cd Sci U S 9:11 11, 1 DIETES, VOL., MRCH 9 3