On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans

Transcription

1 Am J Physiol Endocrinol Metb 289: E849 E856, First published June 21, 2005; doi: /jpendo On the suppression of plsm nonesterified ftty cids by insulin during enhnced intrvsculr lipolysis in humns André C. Crpentier, 1 Frédérique Frisch, 1 Denis Cyr, 2 Philippe Généreux, 1 Bruce W. Ptterson, 3 Robert Giguère, 2 nd Jen-Ptrice Billrgeon 1 1 Deprtment of Medicine, Division of Endocrinology nd 2 Deprtment of Clinicl Biochemistry, Centre hospitlier universitire de Sherbrooke, Université de Sherbrooke, Quebec, Cnd; nd 3 Center for Humn Nutrition, Deprtment of Internl Medicine, Wshington University School of Medicine, St. Louis, Missouri Submitted 22 Februry 2005; ccepted in finl form 15 June 2005 Crpentier, André C., Frédérique Frisch, Denis Cyr, Philippe Généreux, Bruce W. Ptterson, Robert Giguère, nd Jen-Ptrice Billrgeon. On the suppression of plsm nonesterified ftty cids by insulin during enhnced intrvsculr lipolysis in humns. Am J Physiol Endocrinol Metb 289: E849 E856, First published June 21, 2005; doi: /jpendo During the fsting stte, insulin reduces nonesterified ftty cid (NEFA) ppernce in the systemic circultion mostly by suppressing intrcellulr lipolysis in the dipose tissue. In the postprndil stte, insulin my lso control NEFA ppernce through enhnced trpping into the dipose tissue of NEFA derived from intrvsculr triglyceride lipolysis. To determine the contribution of suppression of intrcellulr lipolysis in the modultion of plsm NEFA metbolism by insulin during enhnced intrvsculr triglyceride lipolysis, 10 helthy nonobese subjects underwent pncretic clmps t fsting vs. high physiologicl insulin level with intrvenous infusion of heprin plus Intrlipid. Nicotinic cid ws dministered orlly during the lst 2 h of ech 4-h clmp to inhibit intrcellulr lipolysis nd ssess insulin s effect on plsm NEFA metbolism independently of its effect on intrcellulr lipolysis. Stble isotope trcers of plmitte, cette, nd glycerol were used to ssess plsm NEFA metbolism nd totl triglyceride lipolysis in ech prticipnt. The glycerol ppernce rte ws similr during fsting vs. high insulin level, but plsm NEFA levels were significntly lowered by insulin. Nicotinic cid significntly blunted the insulin-medited suppression of plsm plmitte ppernce nd oxidtion rtes by 60 nd 70%, respectively. In contrst, nicotinic cid did not ffect the mrked stimultion of plmitte clernce by insulin. Thus most of the insulin-medited reduction of plsm NEFA ppernce nd oxidtion cn be explined by suppression of intrcellulr lipolysis during enhnced intrvsculr triglyceride lipolysis in helthy humns. Our results lso suggest tht insulin my ffect plsm NEFA clernce independently of the suppression of intrcellulr lipolysis. intrcellulr lipolysis; lipid oxidtion; postprndil stte; lipid metbolism ABNORMAL PLASMA NONESTERIFIED FATTY ACID (NEFA) metbolism is felt to ply n importnt role in the development of type 2 dibetes (23). Prolonged experimentl elevtion of plsm NEFA reduces insulin-medited glucose utiliztion in muscle, impirs glucose-medited insulin secretion, nd increses endogenous glucose production in vivo in humns (8, 31, 38). One of the erliest identifible metbolic defects in ptients t high risk of developing type 2 dibetes is elevtion of postprndil levels of plsm triglycerides nd blunting of erly postprndil lowering of plsm NEFA levels (2). Impired postprndil suppression of plsm NEFA could be implicted in the development of insulin resistnce nd type 2 dibetes through incresed exposure of extr-dipose tissues to NEFA in individuls t high risk of developing type 2 dibetes (23). In contrst to the fsting stte, where insulin reduces extrdipose tissue exposure to NEFA minly by suppressing intrcellulr lipolysis in dipose tissues, the effect of insulin on circulting NEFA metbolism in the postprndil stte my be more complex. First, insulin stimultes lipoprotein lipse (LPL)-medited lipolysis of chylomicrons in the microcircultion of the dipose tissue, mjor mechnism by which this hormone cn stimulte preferentil prtitioning of ftty cids in the dipose tissue during the postprndil stte (13). Second, insulin suppresses intrcellulr lipolysis in dipose tissue, thereby reducing plsm NEFA ppernce rte (10, 27). Third, insulin my stimulte the esterifiction of NEFA in the dipose tissue, potentilly contributing to the dipose tissue uptke nd trpping of plsm NEFA tht re generted from intrvsculr triglyceride lipolysis (10, 12, 15, 27). Insulin my lso reduce postprndil lipid oxidtion independently of chnge in plsm NEFA vilbility possibly by incresing intrcellulr glucose flux (35). Becuse of these potentil effects of insulin, the reltive role of suppression of intrcellulr lipolysis on in vivo plsm NEFA metbolism in the postprndil stte cnnot redily be predicted. The im of the present study ws to determine the reltive role of the suppression of intrcellulr lipolysis in the modultion of NEFA metbolism by insulin in the presence of enhnced intrvsculr triglyceride lipolysis in vivo in humns. To tht im, we propose n experimentl prdigm bsed on the following ssumptions: 1) n intrvenous heprin Intrlipid infusion mximlly stimultes the production of plsm NEFA from intrvsculr triglyceride lipolysis by ctivting LPL sitting t the endothelium into the microcircultion of tissues nd by supplying chylomicron-like prticles in the circultion (9); 2) nicotinic cid given orlly in humns is very effective t suppressing intrcellulr lipolysis in dipose tissues (7), llowing us to determine ny effect of insulin on systemic NEFA flux independently of its suppressing effect on intrcellulr lipolysis. Under these conditions, it would be expected tht ny non-lpl-medited effect of insulin in stimulting NEFA esterifiction (i.e., intrcellulr trpping) nd in limiting the entry of NEFA into the systemic circultion generted from intrvsculr triglyceride lipolysis in the di- Address for reprint requests nd other correspondence: A. Crpentier, Division of Endocrinology, Centre hospitlier universitire de Sherbrooke, Sherbrooke, Quebec, Cnd J1H 5N4 (e-mil: ndre.crpentier@usherbrooke.c). The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. The rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct /05 $8.00 Copyright 2005 the Americn Physiologicl Society E849

2 E850 pose tissue circultion would become more pprent. This experimentl prdigm llowed us to ssess the reltive importnce of suppression of intrcellulr lipolysis vs. enhnced trpping of plsm NEFA derived from intrvsculr triglyceride lipolysis in the dipose tissue in the suppression of plsm NEFA ppernce nd oxidtion by insulin. In cse of predominnt effect of the suppression of intrcellulr lipolysis in cusing insulin-medited reduction of plsm NEFA ppernce nd oxidtion, one would expect tht ny difference observed in these prmeters between fsting nd high plsm insulin levels would be bolished to lrge extent during nicotinic cid intke. MATERIALS AND METHODS Subjects. Ten helthy nonobese Cucsin subjects (men BMI kg/m 2, 3 femles, 7 mles) ged 21 to 56 yr (men yr) prticipted in the studies. None hd dibetes, bsed on repeted ssessment of fsting glucose concentrtion (1). None were tking ny mediction, hd ny current medicl condition known to ffect lipid levels or insulin sensitivity, or hd known crdiovsculr disese. The three women were premenopusl, nd the studies were conducted during the folliculr phse of their menstrul cycle. Informed written consent ws obtined from ll prticipnts in ccordnce with the Declrtion of Helsinki nd the protocol ws pproved by the Humn Ethics Committee of the Centre hospitlier universitire de Sherbrooke. Experimentl protocols. All subjects prticipted in four studies 3 4 wk prt, nd they voided chnge in lifestyle nd weight throughout the study. They were provided with dietry instructions to mintin eucloric diet nd follow 3-dy food diry prescribed by registered dieticin, nd complince ws scertined on the morning of ech study. The prticipnts were told to void strenuous exercise for 48 h before ech study, s described previously (9). The subjects were dmitted t our metbolic investigtion center on ech occsion between 0730 nd 0830 fter 12-h overnight fst nd remined fsting for the durtion of the study. On rrivl, body weight nd height were mesured, nd len body mss ws determined by electricl bioimpednce (Hydr ECF/ICF; Xitron Technologies, Sn Diego, CA). An intrvenous ctheter ws plced in one forerm for infusions, nd nother ws plced in retrogrde fshion in the contrlterl rm mintined in heting box ( 55 C) for blood smpling. Protocols A nd C nd B nd D (Fig. 1) were designed to produce similr nd sustined elevtion of intrvsculr lipolysis of triglycerides during 4hbymens of n intrvenous infusion of heprin (250 U/h) nd 20% Intrlipid (40 ml/h) (9) nd triglyceride emulsion composed of 50% linolete, 26.5% olete, 10.5% plmitte, 8.5% linolente, nd 3.5% sterte. In protocols A/C, fsting insulin ws mintined by continuous low (0.05 mu kg 1 min 1 ) insulin infusion (Novolin R, Novo Nordisk). In protocols B/D, high insulin ws obtined using primed (0.8 mu/kg) continuous high (1.2 mu kg 1 min 1 ) insulin infusion (with 10 meq/h KCl to void insulin-induced hypoklemi). We (9) hve previously shown tht intrvenous Intrlipid plus heprin infusion results in smll but significnt increse in plsm glucose t fsting insulin level. Therefore, protocol A or C ws lwys performed first to mtch s precisely s possible the expected smll increse in plsm glucose levels between the A/C nd B/D studies by using vrible infusion of 20% dextrose djusted ccording to plsm glucose level. Octreotide (30 g/h Sndosttin, Sndoz) nd humn growth hormone (GH; 3 ng kg 1 min 1 Nutropin, Roche) were dministered in ll four protocols (20). Glucgon ws not replced, becuse it cn result in insulin secretion brekthrough t low insulin infusion rte (20) nd becuse it hs miniml effect on NEFA metbolism in humns (3, 16, 29). Fig. 1. Experimentl protocols. All of the prticipnts underwent 4 experimentl protocols (A, B, C, nd D) with iv infusion of heprin Intrlipid during 4 h from time 0 to stimulte intrvsculr triglyceride lipolysis. After 30-min bseline period during which blood smples, indirect clorimetry, nd breth smples were performed, n iv bolus of [ 13 C]NH 13 CO 3 ws injected t time 0 in ll 4 protocols. A 4-h euglycemic pncretic clmp with iv octreotide growth hormone (GH) nd low (protocols A nd C) vs. high (protocols B nd D) -dose insulin infusions to mintin fsting vs. high plsm insulin levels, respectively, ws performed from time 0. Intrvenous infusion of 20% dextrose (D20%) nd potssium chloride (KCl) were dministered during protocols B nd D to mintin plsm glucose t fsting level nd to void decrese in serum potssium level induced by insulin. In protocols A nd B, primed continuous iv infusion of [1,1,2,3,3-2 H 5]glycerol nd continuous iv infusion of [U- 13 C]plmitte were dministered during 4 h from time 0. Inprotocols C nd D, continuous iv infusion of [1,2-13 C]cette ws dministered during 4 h from time 0. During the lst 2hofthe4-hclmp in ll 4 protocols, prticipnts received nicotinic cid orlly every 30 min to suppress dipose tissue intrcellulr lipolysis. Blood nd breth smples nd indirect clorimetry were performed between 90 nd 120 min nd between 210 nd 240 min of the clmp. At time 0 min in protocols A nd B, constnt infusion (0.01 mol kg 1 min 1 )of[u- 13 C]K plmitte (Cmbridge Isotope Lbortories, Andover, MA; in 100 ml of 5% humn serum lbumin) ws dministered during the 4 h of the study (5), preceded by bolus infusion of sterile [1-13 C]NH 13 CO 3 (1.2 mol/kg, Cmbridge Isotope Lbortories) to prime the bicrbonte pool (41). The choice of plmitte trcer in our experimentl protocol ws bsed on the following: 1) plmitte, olete, nd linolete, the most prevlent NEFAs in humn plsm nd in Intrlipid, hve similr clernce rtes in humns (28); nd 2) plmitte trcer hd been previously used with success to mesure totl plsm NEFA turnover in humns fter orl ft intke (27). A primed (1.6 mol/kg) continuous (0.11 mol kg 1 min 1 ) infusion of [1,1,2,3,3-2 H 5]glycerol (Cmbridge Isotope Lbortories) ws lso dministered through m Millipore filter for the durtion of the study to quntify plsm glycerol flux s reflection of whole body lipolysis (22). During protocols C nd D, [1,2-13 C]sodium cette (Cmbridge Isotope Lbortories) ws infused (0.08 mol kg 1 min 1 ) insted of the plmitte nd glycerol trcers to determine the cette retention fctor (5, 33) in conditions identicl to those of protocols A nd B (see below). All trcers were tested for sterility nd pyrogenicity prior to use. During the lst 2hoftheprotocols, nicotinic cid ws given orlly (100 mg t 120 nd 200 min, nd 150 mg t 150 nd 180 min), protocol previously shown to result in stedy suppression of intrcellulr lipolysis for up to 4 h (7). The effect of insulin on NEFA metbolism ttributble to its inhibitory effect on intrcellulr lipolysis vs. tht ttributble to other possible effects, such s enhnced trpping of NEFA derived from intrvsculr triglyceride lipolysis,

3 E851 could then be ssessed by compring nicotinic cid-medited vs. insulin-medited chnge in NEFA metbolism. After 30-min bed rest, blood smples were tken t 10-min intervls t bseline nd during the lst 30 min of the first 2 h (without nicotinic cid), nd the lst 30 min of the 4-h clmp period (with nicotinic cid). Blood ws collected in tubes contining N 2EDTA nd Orlistt (30 g/ml; Roche, Mississug, ON, Cnd) to prevent in vitro triglyceride lipolysis. Urine nitrogen excretion ws mesured throughout the studies (19). After 10-min equilibrtion, oxygen uptke (V O2) nd crbon dioxide production (V CO2) were mesured during 30-min bseline period nd during the lst 30 min of the period with nd without nicotinic cid to determine totl body crbohydrte nd lipid oxidtion by indirect clorimetry (Vmx29n, Sensormedics) (14). Expirtory gses were collected t bseline nd t 10-min intervls into 10-ml exetiners (Lbco) throughout these periods to determine 13 CO 2 to 12 CO 2 rtio by isotope rtio mss spectrometry (IRMS) (34). Lbortory ssys. Glucose ws ssyed t bedside (Beckmn Glucose Anlyzer II; Beckmn Instruments, Fullerton, CA). Insulin, glucgon, nd growth hormone (GH) were mesured by specific rdioimmunossys (Linco, St. Chrles, MO, nd Nichols Institute Dignostics, Sn Jun Cpistrno, CA). Totl plsm NEFA nd triglycerides were mesured using colorimetric ssys (Wko Industrils nd Thermo DMA, respectively). Plsm glycerol ws extrcted nd derivtized with bis(trimethylsilyl)trifluorocetmide 10% trimethylchlorosilne (Regis Technologies, Morton Grve, IL), nd plsm [1,1,2,3,3-2 H 5]glycerol enrichment ws mesured by GC-MS using n Agilent GC model 5890A (Agilent Technologies, Avondle, PA) coupled to n MS detector (model 5971 qudrupole MSD, Agilent) equipped with Supelco SPB-5 fused silic column (25 m 0.20 mm, 0.33 m) nd splitless injector. Electron impct ioniztion with n electron bem energy of 70 ev ws used in selected ion monitoring mode to monitor mss-to-chrge rtio (m/z) 117, 205 for glycerol, m/z 120, 208 for [1,1,2,3,3-2 H 5]glycerol, nd m/z 118, 206 for [1-13 C]glycerol (internl stndrd). To mesure plsm plmitte, linolete, olete, nd [U- 13 C]plmitte enrichment, heptdecnoic cid ws dded s n internl stndrd to 100 l of plsm nd mixed with 500 l of methnol. After centrifugtion, the superntnt ws filtered nd injected on Hypersil ODS column (5 m, mm) on n LC/MSD series 1100 (Agilent) with monitoring of ions 279 (C18:2), 281 (C18:1), 255 (C16:0), 271 (C16:0 M 16), nd 269 (C17, internl stndrd). Stndrd curves were generted for C16:0, C18:1, C18:2, nd C16:0 M 16 enrichment by use of purified stndrds of known concentrtion. The intr- nd interssy coefficients of vrition were 6.1% for ll of these ssys. Breth 13 CO 2/ 12 CO 2 ws determined using gs isotope rtio mss spectrometer (Delt XL; Finnign, Bremen, Germny). Clcultions. The plsm plmitte ppernce rte (R plmitte) ws clculted from the C16:0 M 16 enrichment of plsm plmitte from bckground nd the trcer infusion rte (6): R plmitte F/TTR plmitte, where F is the C16:0 M 16 infusion rte determined during ech experiment, nd TTR plmitte is the plsm plmitte C16:0 M 16- to-c16:0 M 0 rtio during trcer infusion, corrected for bckground (which is zero in this cse). The totl plsm NEFA R ws determined by multiplying the plmitte R by the rtio of concentrtion of plsm NEFA to plsm plmitte level (21). This pproch ssumes tht the clernce of plsm plmitte is good estimte of the clernce of the other plsm long-chin free NEFA (except for sterte) (18, 28, 37). To estimte totl body triglyceride lipolysis, the plsm glycerol R ws lso determined from plsm glycerol M 5 enrichment from bseline nd the trcer infusion rte, s described in (22), corrected for the infusion rte of free glycerol contined in the Intrlipid infuste (4). Clernce rtes of plsm plmitte nd glycerol were determined by dividing their respective R by their plsm concentrtions. The frctionl plsm plmitte oxidtion ws determined (6): F ox plmitte (V CO2 TTR CO2 ) / (INF [U- 13 C]plmitte 16 k), where V CO2 is expressed in micromoles per kilogrm of len body mss (LBM) per minute, TTR CO2 is the breth 13 CO 2 trcer-to-trcee rtio, INF [U- 13 C]plmitte is the plmitte trcer infusion rte in micromoles per kilogrm LBM per minute, nd k is the cette recovery fctor s clculted by the following (5): k V CO2 TTR CO2 ) / (INF [1,2-13 C]cette 2), where INF [1,2-13 C]cette is the cette trcer infusion rte in micromoles per kilogrm of LBM per minute nd V CO2 nd TTR CO2 were mesured during protocol C or D. Sttisticl nlyses. The dt t bseline nd with nd without nicotinic cid intke were verged nd re expressed s mens SE. All metbolic prmeters mesured during the high- vs. fstinginsulin experiments without nd with nicotinic cid were exmined using n ANOVA for repeted mesures, nd Scheffé s test for multiple comprisons ws performed whenever the P vlue ws significnt. To ssess the effect of high- vs. fsting-insulin level nd whether nd to wht extent inhibition of intrcellulr lipolysis contributes to insulin s effect, the vlues of ll of the prmeters mesured during the high-insulin study were subtrcted from the corresponding vlue during the fsting insulin study. These differences with nd without nicotinic cid intke were compred by pired t-test. For ll nlyses, two-tiled P vlue of 0.05 ws considered significnt. All nlyses were performed with the SAS softwre for Windows, version 8.02 (SAS Institute, Cry, NC). RESULTS Plsm glucose, NEFA, triglycerides, nd hormone levels t fsting nd high insulin levels. By design, plsm glucose levels were mtched between the fsting nd high-insulin conditions. As expected, there ws smll but not significnt (NS) increse in plsm glucose level from bseline to the end of the 4-h infusion of heprin-intrlipid t fsting insulin (Tble 1). Plsm glucose levels were similr between fsting nd high-insulin studies with or without nicotinic cid. Also by design, plsm insulin levels were significntly incresed from bseline in the high-insulin study but did not chnge in the fsting-insulin study. Nicotinic cid did not ffect plsm insulin levels during the clmps. As expected, totl plsm NEFA levels were significntly incresed more thn twofold t fsting insulin, lthough high insulin resulted in stbiliztion of NEFA t their bseline level. Totl plsm NEFA levels did not chnge significntly with nicotinic cid intke t fsting nd t high insulin levels. Plsm triglyceride levels were lso significntly incresed during Intrlipid-heprin infusion vs. bseline but were not ffected by insulin or nicotinic cid. Plsm glucgon levels were reduced from bseline to similr levels during fsting nd high-insulin clmps nd were not chnged by nicotinic cid intke. Plsm GH levels were not ffected by insulin level or nicotinic cid intke. Plsm concentrtion of individul ftty cids, glycerol, nd trcer enrichment t fsting nd high insulin levels. Men plsm plmitte levels were significntly incresed by 70% from bseline t fsting insulin (P 0.05) nd tended to be reduced by 25% from bseline (NS) t high insulin (Tble 2). Nicotinic cid intke reduced the plsm plmitte concentrtion by 20% vs. without nicotinic cid t fsting (P 0.05) but not t high insulin. Men plsm M 16 plmitte TTR ws not significntly ffected by insulin or nicotinic cid intke. Plsm olete concentrtions were elevted by 50% from

4 E852 Tble 1. Plsm glucose, NEFA, triglycerides, nd hormone levels Protocol Phse Bseline No NA NA P, ANOVA* Glucose, mmol/l Fsting insulin High insulin Insulin, pmol/l Fsting insulin High insulin NEFA, mol/l Fsting insulin High insulin TG, mmol/l Fsting insulin High insulin Glucgon, ng/l Fsting insulin High insulin GH, g/l Fsting insulin High insulin Dt re mens SE. GH, growth hormone; NA, nicotinic cid; NEFA, nonesterified ftty cids; TG, triglycerides. *P vlues re from ANOVA for repeted mesures with post hoc Scheffé s test performed whenever the P vlue from the model ws significnt. Scheffé s test P 0.05 vs. bseline. Scheffé s test P 0.05 vs. fsting insulin. bseline t fsting insulin (P 0.05) nd tended to be reduced by 30% from bseline t high insulin, but this difference ws not significnt. Nicotinic cid resulted in significnt reduction of plsm olete towrd bseline levels t fsting insulin (P 0.05 vs. without nicotinic cid) but did not ffect plsm olete levels t high insulin. Plsm linolete levels were incresed more thn fourfold from bseline t fsting (P 0.05) but significntly less so t high insulin level ( 2.2-fold elevtion, P 0.05 vs. fsting insulin). Plsm linolete levels were not significntly ffected by nicotinic cid intke. Plsm glycerol levels were similrly nd significntly incresed from bseline t fsting nd t high insulin (P 0.05) but were unffected by nicotinic cid. Plsm glycerol M 5 TTR ws not significntly ffected by insulin level or nicotinic cid. Plsm glycerol nd NEFA metbolism t fsting nd high insulin levels. Plsm glycerol ppernce nd clernce rtes were unffected by chnge in plsm insulin level or by nicotinic cid intke (Tble 3). Plsm plmitte R ws significntly reduced t high vs. fsting insulin without nicotinic cid intke (P 0.05). However, during nicotinic cid intke this difference ws reduced nd not significnt nymore. Totl plsm NEFA ppernce ws not significntly ffected by insulin or nicotinic cid intke. High insulin level ws ssocited with significnt increse of plsm plmitte clernce rte by 40% (P 0.05) compred with fsting insulin. Nicotinic cid intke did not ffect plsm plmitte clernce rte. Frctionl plmitte oxidtion ws not significntly ffected by plsm insulin level or nicotinic cid intke [ vs t high vs. fsting insulin, respectively, without nicotinic cid intke; nd vs % t high vs. fsting insulin, respectively, with nicotinic cid intke (P NS)]. In contrst, plsm plmitte oxidtion rte ws significntly reduced t high vs. fsting insulin without nicotinic cid intke (P 0.05), but this difference ws not significnt nymore during nicotinic cid intke. The cette recovery fctor ws not significntly different t high vs. fsting insulin ( vs %, respectively) nd ws significntly higher during nicotinic cid intke ( nd % t high nd fsting insulin, respectively, P 0.05 vs. no nicotinic cid intke). Indirect clorimetry nd totl body crbohydrte nd lipid oxidtion t fsting nd high insulin levels. There ws no significnt chnge from bseline of V O2,V CO2, nd respirtory quotient (RQ) during intrvenous heprin-intrlipid infusion t fsting insulin (Tble 4). Therefore, there ws no significnt chnge in totl body crbohydrte nd lipid oxidtion rtes ssocited with heprin-intrlipid infusion t fsting insulin. V CO2, RQ, nd, consequently, totl body crbohydrte oxid- Tble 2. Plsm concentrtion of glycerol, individul ftty cids, nd plmitte M 16 nd glycerol M 5 TTR Phse Protocol Bseline No NA NA P, ANOVA* Plmitte, mol/l Fsting insulin High insulin Plmitte TTR Fsting insulin High insulin Olete, mol/l Fsting insulin High insulin Linolete, mol/l Fsting insulin High insulin Glycerol, mol/l Fsting insulin High insulin Glycerol TTR Fsting insulin High insulin Dt re mens SE. TTR, trcer-to-trcee rtio. *P vlues re from ANOVA for repeted mesures with post hoc Scheffé s test performed whenever the P vlue from the model ws significnt. Scheffé s test P 0.05 vs. bseline. Scheffé s test P 0.05 vs. fsting insulin. Scheffé s test P 0.05 vs. no NA.

5 Tble 3. Plsm glycerol nd NEFA metbolism E853 Phse Protocol No NA NA P, ANOVA* R glycerol Fsting insulin High insulin b Cl glycerol Fsting insulin High insulin R plmitte Fsting insulin High insulin R NEFA Fsting insulin High insulin b Cl plmitte Fsting insulin High insulin Ox plmitte Fsting insulin High insulin Dt re mens SE. Cl, clernce; Ox, oxidtion; R, ppernce rte. Expressed in mol kg len body mss (LBM) 1 min 1. b Expressed in ml kg LBM 1 min 1.*P vlues re from ANOVA for repeted mesures with post hoc Scheffé s test performed whenever the P vlue from the model ws significnt. Scheffé s test P 0.05 vs. fsting insulin. tion were significntly higher t high vs. fsting insulin, wheres totl body ft oxidtion ws significntly reduced (P 0.05). Nicotinic cid intke hd no effect on V O2,V CO2, RQ, nd, consequently, on totl body crbohydrte nd ft oxidtion. Effect of insulin on NEFA metbolism ttributble to suppression of intrcellulr lipolysis. Suppression of intrcellulr lipolysis with nicotinic cid resulted in significnt blunting of the insulin-medited suppression of plsm plmitte ( 68 8 vs mol/l with vs. without nicotinic cid, respectively, P 0.05; Fig. 2A), olete ( vs mol/l with vs. without nicotinic cid respectively, P 0.05), nd totl NEFA levels ( vs mol/l with vs. without nicotinic cid respectively, P 0.05; Fig. 2B), but not linolete level ( vs mol/l with vs. without nicotinic cid respectively, P NS). Nicotinic cid intke lso resulted in significnt 60% blunting of the reduction of plmitte ppernce induced by insulin ( vs mol kg LBM 1 min 1 with vs. without nicotinic cid, respectively, P 0.05; Fig. 2C), but only tended to blunt insulin-medited reduction of totl plsm NEFA ppernce ( vs mol kg LBM 1 min 1 with vs. without nicotinic cid, respectively, P NS; Fig. 2D). In contrst, nicotinic cid intke hd no effect on insulin-medited stimultion of plmitte clernce ( vs ml kg LBM 1 min 1 with vs. without nicotinic cid respectively, P NS; Fig. 2E). Nicotinic cid lso significntly blunted the insulin-medited reduction of plsm plmitte oxidtion by 70% ( vs mol kg LBM 1 min 1 with vs. without nicotinic cid, respectively, P 0.05; Fig. 2F). DISCUSSION Our experimentl protocol using intrvenous infusion of heprin plus Intrlipid resulted in stedy nd similr increse in intrvsculr triglyceride lipolysis t fsting nd high insulin levels, supported by no significnt chnge in glycerol level nd ppernce rte t fsting vs. high insulin levels. This llowed us to determine the effect of insulin on plsm NEFA ppernce independently of insulin-medited chnge in LPL ctivity in the dipose tissue. In this condition, it is expected tht ny non-lpl-medited stimulting effect of insulin on the esterifiction of NEFA originting from intrvsculr triglyceride lipolysis in the dipose tissue would become more pprent. In the present study, the reduction of plsm plmitte ppernce nd oxidtion by insulin during enhnced intrvsculr triglyceride lipolysis ws only 40%, in contrst with the profound ( 80%) suppression of plsm NEFA ppernce by insulin observed in the postbsorptive stte in humns (28). Thus, s Tble 4. Indirect clorimetry nd totl crbohydrte nd lipid oxidtion rte Phse Protocol Bseline No NA NA P, ANOVA* V O2 Fsting insulin V CO2 High insulin Fsting insulin High insulin RQ Fsting insulin High insulin b Ox CHO Fsting insulin High insulin b Ox totl FA Fsting insulin High insulin Dt re mens SE. CHO, crbohydrte; FA, ftty cid. Expressed in ml kg LBM 1 min 1. b Expressed in mol kg LBM 1 min 1.*P vlues re from ANOVA for repeted mesures with post hoc Scheffé s test performed whenever the P vlue from the model ws significnt. Scheffé s test P 0.05 vs. bseline. Scheffé s test P 0.05 vs. fsting insulin.

6 E854 Fig. 2. Insulin-medited chnge of plsm plmitte level (A), totl plsm nonesterified ftty cid (NEFA or FFA) level (B), plsm plmitte ppernce rte (R ) (C), totl plsm NEFA R (D), plsm plmitte clernce (E), nd plsm plmitte oxidtion rte (F) without (filled brs) nd with nicotinic cid intke (open brs). In other words, open brs illustrte the effect of insulin independent of its suppressive effect on intrcellulr lipolysis. *P 0.05 vs. without nicotinic cid intke. P vlues re from pired t-tests. LBM, len body mss; Dt re mens SE. expected during Intrlipid-heprin intrvenous infusion, lrge frction of plsm NEFA vilble to the systemic circultion ws derived from intrvsculr triglyceride lipolysis. This ws lso suggested by the bsence of significnt modultion of plsm linolete levels by nicotinic cid intke, since lrge frction of plsm linolete is expected to derive from intrvsculr lipolysis of Intrlipid. Nevertheless, we found tht hyperinsulinemi reduced plsm plmitte ppernce nd oxidtion rtes mostly through inhibition of intrcellulr lipolysis, s nicotinic cid reduced by t lest 60 nd 70%, respectively, the suppression of plsm plmitte ppernce nd oxidtion by insulin. It hs been previously demonstrted tht some frction of NEFA derived from intrvsculr triglyceride lipolysis in vivo in humns is vilble in the systemic circultion during the fsting (26) nd the postprndil stte (32). According to our results, insulin does not predominntly regulte the systemic NEFA flux originting from intrvsculr triglyceride lipolysis through non-lpl-medited stimultion of trpping of NEFA into the dipose tissue in helthy individuls, lthough defect in this mechnism in pthophysiologicl sttes cnnot be excluded. Thus, s during fsting, suppression of intrcellulr lipolysis ppers to be the most importnt mechnism by which insulin suppresses systemic NEFA flux during enhnced intrvsculr triglyceride lipolysis. Our dt lso suggest tht insulin increses plsm plmitte clernce independently of inhibition of intrcellulr lipolysis. Interestingly, hyperinsulinemi ws lso ssocited with enhnced plsm NEFA clernce in previous humn studies during the postbsorptive (28) nd the postprndil stte (27). However, the reltive contribution of NEFA ppernce vs. clernce nd of the reduction of intrcellulr lipolysis in the modultion of postprndil plsm NEFA levels by insulin ws not ddressed in previous studies. A direct cute effect of insulin on trnsport of NEFA into tissues hs been demonstrted in some (11, 24, 30) but not ll (42) ex vivo nd in vivo studies in nimls. No in vivo study in humns, including the present study, reported the effect of insulin on NEFA clernce independent of insulin-medited reduction in NEFA levels. To our knowledge, whether plsm NEFA clernce cn be sturted t high vs. low physiologicl NEFA levels hs not been formlly estblished in vivo in humns. Thus cution should be exerted before concluding tht insulin per se nd not reduction of NEFA levels by insulin through inhibition of intrcellulr lipolysis ws the cuse of the enhnced NEFA clernce during the hyperinsulinemic clmp in the present study. In

7 vivo humn studies re underwy in our lbortory to ddress this issue. Plsm NEFA ppernce rte is elevted in the postprndil stte in individuls with viscerl obesity (17) nd in obese ptients with type 2 dibetes compred with younger nd lener helthy individuls (27). The precise mechnism for enhnced postprndil plsm NEFA flux in these individuls remins to be estblished, but defective control of NEFA flux by insulin is n obvious trget. Whether impired insulinmedited suppression of intrcellulr lipolysis or impired insulin-medited trpping of ftty cids derived from intrvsculr lipolysis during the postprndil stte could ply role in the incresed vilbility of NEFA to extr-dipose tissues in obesity nd type 2 dibetes remins to be estblished. Our results lso indicte tht impired insulin-stimulted clernce of NEFA is nother potentil mechnism for incresed postprndil plsm NEFA levels in insulin-resistnt sttes. Interestingly, defect in postprndil plsm NEFA clernce hs been demonstrted in ptients with type 2 dibetes (25, 39). Whether defect in insulin-stimulted clernce of NEFA occurs in vivo in humns with type 2 dibetes remins to be determined. In the present study, we used intrvenous heprin to mximlly stimulte LPL ctivity t both fsting nd high plsm insulin levels. However, we cnnot totlly exclude tht hyperinsulinemi did not result in smll increse in dipose tissue LPL ctivity vs. fsting insulin conditions despite the use of heprin. If this hd occurred, it would hve resulted in enhnced LPL-medited NEFA ppernce into the dipose tissue during hyperinsulinemi, mking more pprent ny insulin-medited increse in post-lpl dipose tissue NEFA trpping in limiting systemic NEFA flux. In other words, this would hve reduced the pprent role of inhibition of intrcellulr lipolysis in the reduction of systemic NEFA flux by insulin. Therefore, this possible limittion would nevertheless strengthen our conclusion tht insulin reduces NEFA ppernce during enhnced intrvsculr lipolysis mostly by inhibition of dipose tissue intrcellulr lipolysis. Nicotinic cid intke significntly blunted the insulin-medited reduction of plsm plmitte oxidtion rte but did not ffect insulin-medited reduction of totl lipid oxidtion. An increse in intrcellulr lipolysis in muscle could offer t lest prt of the explntion for this observtion, since this phenomenon hs been shown to mintin lipid oxidtion during reduction of plsm NEFA by nicotinic cid intke in vivo in humns (40). The lck of effect of nicotinic cid on totl lipid oxidtion ws in contrst to the significnt reduction induced by insulin. Thus, during enhnced intrvsculr triglyceride lipolysis, the inhibitory effect of insulin on totl lipid oxidtion ws not solely dependent on the reduction of plsm NEFA ppernce, in ccord with previous results from Sidossis nd Wolfe. (36). The protocol of dministrtion of nicotinic cid tht we used hs been shown to reduce fsting plsm NEFA level by 80% (7). However, it is possible tht nicotinic cid did not suppress intrcellulr lipolysis s profoundly s insulin did. This would hve resulted in underestimtion of the insulinmedited suppression of plsm plmitte ppernce ttributed to inhibition of intrcellulr lipolysis. Therefore, this limittion would not ffect our conclusion tht most of the insulin-medited suppression of plsm NEFA ppernce nd E855 oxidtion during enhnced intrvsculr triglyceride lipolysis in helthy humns is due to inhibition of intrcellulr lipolysis. We conclude tht, in helthy humns, suppression of intrcellulr lipolysis is the mjor mechnism by which insulin reduces the plsm NEFA ppernce nd oxidtion rtes during enhnced intrvsculr triglyceride lipolysis. 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