Hormonal Control of Substrate Cycling in Humans

Transcription

1 Hormonl Control of Substrte Cycling in Humns Hiroshi Miyoshi, Gerld 1. Shulmn,* Edwrd J. Peters, Mrt H. Wolfe, Driush Elhi,t nd Robert R. Wolfe The Shriners Burns Institute nd The University of Texs Medicl Brnch, Glveston, Texs 7755; *Deprtment ofmoleculr Biophysics & Biochemistry, Yle Medicl School, New Hven, Connecticut 652; tbeth Isrel Hospitl nd thrvrd Medicl School, Boston, Msschusetts 2138 Abstrct Recent studies hve estblished the existence of substrte cycles in humns, but fctors regulting the rte of cycling hve not been identified. We hve therefore investigted the cute response of glucose/glucose-6p-glucose (glucose) nd triglyceride/ftty cid (TG/FA) substrte cycling to the infusion of epinephrine (.3 gg/kg min) nd glucgon. The response to high dose glucgon infusion (2 gg/kg min) ws - tested, s well s the response to low dose infusion (5 ng/kg. min), with nd without the simultneous infusion of somtosttin (.1 gg/kg min) nd insulin (.1 mu/kg min). Additionlly, the response to chronic prednisone (5 mg/d) ws evluted, both lone nd during glucgon (low dose) nd epinephrine infusion. Finlly, the response to hyperglycemi, with insulin nd glucgon held constnt by somtosttin infusion nd constnt replcement of glucgon nd insulin t bsl rtes, ws investigted. Glucose cycling ws clculted s the difference between the rte of ppernce (R.) of glucose s determined using 24k- nd 6,642-glucose s trcers. TG/FA cycling ws clculted by first determining the R. glycerol with d5-glycerol nd the R. FFA with 11-'3Cqplmitte, then subtrcting R. FFA from three times R. glycerol. The results indicte tht glucgon stimultes glucose cycling, nd this stimultory effect is ugmented when the insulin response to glucgon infusion is blocked. Glucgon hd miniml effect on TG/FA cycling. In contrst, epinephrine stimulted TG/FA cycling, but ffected glucose cycling minimlly. Prednisone hd no direct effect on either glucose or TG/FA cycling, but blunted the stimultory effect of glucgon on glucose cycling. Hyperglycemi, per se, hd no direct effect on glucose or TG/FA cycling. Clcultions reveled tht stimultion of TG/FA cycling theoreticlly mplified the sensitivity of control of ftty cid flux, but no such mplifiction ws evident s result of the stimultion of glucose cycling by glucgon. Introduction Substrte cycles exist when opposing, nonequilibrium rections, ctlyzed by different enzymes, re ctive simultneously. The net result of substrte cycling is thermogenesis, nd mplifiction of enzymtic control cn lso occur (1). During the pst ten yers, number of studies hve investi- Address correspondence to Dr. Wolfe, Shriners Burns Institute, 61 Texs Ave., Glveston, TX Receivedfor publiction 16 July 1987 nd in revisedform 3 November J. Clifi. Invest. The Americn Society for Clinicl Investigtion, Inc /88/5/1545/11 $2. Volume 81, My 1988, gted the regultion of substrte cycles in in vitro systems or in niml studies (e.g., references 2 to 7). More recently, studies of glucose nd fructose cycling in humn subjects hve been undertken by ourselves (8, 9) nd others (1-14). We hve lso investigted triglyceride/ftty cid (TG/FA)' cycling in humns (15). This involves the processes of lipolysis nd reesterifiction occurring simultneously. In our study of glucose nd TG/FA cycling in severely burned ptients, we found tht both cycles were incresed three- to fivefold bove norml (9). Since glucgon ws mrkedly elevted in these burned ptients, nd the extent of the hypermetbolic response in burn ptients hs recently been found to be directly relted to the glucgon concentrtion (16), it ws logicl to suspect glucgon s the meditor of the incresed glucose cycling. However, the role of glucgon in controlling the rte of substrte cycling in humns is not cler. In vitro experiments hve generlly indicted tht glucgon inhibits cycling (3), but Issekutz concluded tht glucgon stimulted heptic glucose cycling in the conscious dog (6). It is lso possible tht glucgon contributes to incresed TG/FA cycling in vivo. In vitro experiments hve documented tht glucgon stimultes lipolysis (17, 18). Coupled with n incresed reesterifiction of FFA due to secondry hyperglycemi (15), it is possible tht n elevtion in glucgon could increse TG/FA cycling. However, in vivo experiments hve not determined the effect of glucgon on the rtes of relese of glycerol (s n index of lipolysis) or of FFA. The ction of other hormones on substrte cycling in humns is lso uncler. Wheres bet blockde with proprnolol in severely burned ptients decresed TG/FA cycling nd did not ffect glucose cycling (7), it is not known how either cycle is ffected by epinephrine infusion. Issekutz (6) reported tht cortisol hd direct stimultory effect on glucose cycling in the dog, nd tht cortisol mplified the stimultory effect of glucgon. Similr studies hve not been performed in humns, nor hs ny in vivo ssessment of the effect of cortisol on TG/FA cycling. To better understnd the roles of glucgon, epinephrine, nd cortisol in the regultion ofglucose nd TG/FA cycling in humns, we hve performed series of experiments using stble isotopic trcers to quntify the rtes of cycling. The response to glucgon nd epinephrine infusion hs been evluted before nd fter severl dys of tretment with prednisone. In ddition, the response to glucgon hs been evluted in the setting of constnt insulin concentrtion (by virtue of inhibition of secretion with somtosttin nd replcement t constnt bsl rte), nd the role of hyperglycemi, per se, hs been tested by infusing unlbeled glucose while holding both glucgon nd insulin constnt with somtosttin nd pproprite hormonl replcement. 1. Abbrevitions used in this pper: GEMS, gs chromtogrph mss spectrometer; m/e, mss/chrge rtio; TG/FA, triglyceride/ftty cid. Substrte Cycling 1545

2 Methods Studies were performed in norml volunteers rnging in ge from 2 to 32 (x = 24±1) yr. They were given complete physicl exmintion nd considered to be in good helth, nd they hd no history of ny metbolic disese or prolonged mediction. They were ll in the norml rnge of weight for height. The verge height nd weight ws 7±2 in. nd 71.8±2 kg. The protocol ws pproved by the Institutionl Review Bord of The University of Texs Medicl Brnch, nd informed consent ws obtined from ech volunteer. All volunteers were dmitted to the Clinicl Reserch Center ofthe bove institution the dy before the study. They were given stndrd hospitl mel in the evening, nd the study ws performed the following morning fter 14 to 16 h of fsting. Teflon ctheters were plced into the ntecubitl vein of one rm for infusions nd into contrlterl dorsl hnd vein for rterilized venous smpling, using the heted-hnd technique (19). After the bseline blood smple, constnt infusion of 2-d1-glucose nd 6,6-d2-glucose (Cmbridge Isotope Lbortories, Woburn, MA), d5-glycerol (Merck Isotopes, Montrel, Cnd) nd [I-'3C]plmitte (Trcer Technology, Newton, MA) were given. A stock solution of glucose isotope ws mde in sterile wter. For infusion, the pproprite mount of concentrted stock solution ws diluted in.9% sline. The plmitte ws bound to 5% humn lbumin (Cutter Biologicl, Almed, CA), s described previously (2), nd the glycerol ws dded directly to the lbumin-plmitte mixture. All isotope solutions were infused by mens of clibrted syringe pumps (Hrvrd Apprtus Co., Inc., Ntick, MA). The glucose isotopes were infused through ctheters with.22-,um filters in the line (Brd, North Reding, MA); this ws not possible with the plmitte mixture, due to the lbumin not esily pssing through the filter. Both the potssium slt of plmitte (dissolved in wter) nd d5-glycerol were pssed through.22-,um filter when injected into the bottle contining the sterile lbumin solution. The pproximte infusion rtes were s follows: 2-d1- nd 6,6-d2- glucose,.22 gmol/kg. min; d5-glycerol,.1 mol/kg- min; nd [1-'3C]plmitte,.4 gmol/kg-min. In ech experiment the exct infusion rte ws determined by mesuring the concentrtion of isotope in the infusion mixture nd multiplying tht vlue by the infusion rte of the solution (milliliters per minute). At the strt ofthe constnt infusions, priming doses of 2-d1- nd 6,6-d2-glucose were given (17.8 jsmol/kg). Glycerol nd plmitte primes were not needed. Experimentl protocols Glucgon experiments. In two protocols (n = 4 in ech), the first 9 min involved only the infusion of the glucose, glycerol, nd plmitte trcers. Then, without interruption of the isotope, glucgon ws infused for the next 6 min. Two rtes of glucgon were used; in one cse, the glucgon infusion rte (2 ug/kg. min) ws high enough to elicit the mximum response to glucgon. The other infusion rte (5 ng/kg min) ws designed to test the response to n elevted glucgon concentrtion in the physiologicl rnge. In the third group of studies the cute response to glucgon (n 4) = ws evluted while endogenous insulin relese ws blocked. Before the strt of the isotope infusion, somtosttin (Serono, Inc., Denens, Switzerlnd) ws infused t the rte of.1 g/kg- min in order to inhibit endogenous insulin nd glucgon secretion. Bsl replcement of insulin (.1 mu/kg. min) nd glucgon (1 ng/kg. min) ws then ccomplished by infusion. For h, the exct replcement rte of insulin ws djusted slightly so tht stble, norml blood glucose concentrtion could be chieved. After tht ws ccomplished no further chnge ws mde in the bsl replcement rtes. The isotope infusions described bove were then strted. After 9 min the glucgon infusion rte ws incresed from 1 to 6 ng/kg. min nd mintined t tht rte for the next 6 min. Thus, the increse in glucgon infusion rte (bove bsl) ws 5 ng/kg. min, which ws the sme infusion rte s in the protocol without somtosttin (described bove). Epinephrine experiments. In six subjects the response to low dose epinephrine infusion ws exmined. After the initil bsl period, in which only trcers (2-d1, 6,6-d2-glucose, d5-glycerol, nd [1-'3Clplmitte) were infused, epinephrine ws given s constnt infusion t the rte of.3 g/kg- min for 6 min. This rte ws selected becuse Clutter et l. (21) hd previously shown tht this rte of epinephrine infusion ws dequte to stimulte both glucose production nd lipolysis, yet the resulting plsm concentrtion of epinephrine (- 4 pg/ml) is in the sme rnge s tht observed in severely stressed ptients (such s burned ptients) (9). Prednisone experiments. The response to prednisone tretment ws ssessed in six subjects. All hd prticipted in the epinephrine study, s described bove, nd four lso prticipted in the low dose glucgon study. After the initil studies, they received 5 mg prednisone dy for 4 d (totl of2 mg). On the third dy of prednisone tretment, the glucgon (low dose) experiment ws repeted, nd on the fourth dy of prednisone tretment the epinephrine experiment ws repeted. To ssess the effect of prednisone lone, the vlues obtined in the two bsl periods fter prednisone ws verged nd compred with the verge bsl vlue for tht individul before prednisone tretment. Similrly, the interctive effects of prednisone nd epinephrine, nd prednisone nd glucgon, were ssessed by compring the responses with hormone infusion fter prednisone tretment to the responses observed before prednisone ws given. Hyperglycemi experiments. In ech of the bove protocols, the plsm glucose concentrtion incresed s result of the tretment. Therefore, we performed nother protocol in four subjects in which the response to hyperglycemi, independent of chnges in insulin or glucgon, ws ssessed. Somtosttin, bsl insulin, bsl glucgon, nd ll isotopes were infused s described bove. After bsl vlues were obtined, unlbeled glucose ws infused t the rte of2 mg/kg min for the next 9 min. The 9-min infusion of glucose ws used to ensure tht new physiologicl stedy stte would be chieved. Glucgon nd insulin were obtined from Eli Lilly & Co. (Indinpolis, IN) nd E. R. Squibb & Sons, Inc. (Princeton, NJ), respectively. Epinephrine ws supplied by Upjohn Co. (Klmzoo, MI). Ech hormone-infusion mixture ws mixed freshly ech dy. The epinephrine ws diluted in sline contining scorbic cid to prevent oxidtion. Prednisone (Elkins Sinn, Cherry Hill, NJ) ws given orlly in 25-mg tblets. In ech experiment blood smple ws drwn before the strt of the isotope infusion. After n initil 6 min for isotope equilibrium to be estblished, smples were drwn t 1-min intervls until the end of the experiment. In ll cses period I ended fter 9 min, mening smples drwn t 6, 7, 8, nd 9 min were used to obtin bsl vlues. The smples were then drwn t 1-min intervls throughout the experimentl perturbtion in period II. Smples were nlyzed for isotopic enrichment nd concentrtion of glucose, FFA, nd glycerol, s well s plsm concentrtions of insulin, glucgon, nd epinephrine. Anlysis ofsmples The smples for glucgon nd insulin nlysis were collected in iced tubes contining.5 ml of 15% EDTA nd.2 mg of protinin nd centrifuged immeditely. Plsm concentrtion of glucgon nd insulin were determined by rdioimmunossy using 6-15 ntibody obtined from Dr. Jonthn Jspn of the University of Chicgo (Chicgo, IL) nd kit from Micromedic Systems Inc. (Horshm, PA), respectively. Plsm epinephrine concentrtion ws mesured by rdioenzymtic ssy (22). Plsm glucose concentrtion ws mesured on Beckmn glucose utonlyzer. Blood for nlysis of glucose enrichment ws collected in heprinized prechilled tubes. The blood ws centrifuged immeditely nd the plsm ws frozen until the time ofnlysis. The plsm proteins were precipitted nd the superntnt pssed sequentilly through Dowex AG-1-X8 nd AG-5W-X8 nion nd ction exchnge columns. For the determintion of the enrichment of 6,6-d2-glucose, the pentcette derivtive ws formed; for the determintion of 2-d1-glucose enrichment, the trimethylsilyl derivtive ws formed. Mesurement of 1546 H. Miyoshi, G. I. Shulmn, E. J. Peters, M. H. Wolfe, D. Elhi, nd R. R. Wolfe

3 isotopic enrichment of the two derivtives ws performed using Hewlett-Pckrd 5985 gs chromtogrph mss spectrometer (GCMS). The ions t mss/chrge rtio (m/e) 171:169 were selectively monitored using the chemicl ioniztion mode to determine 6,6-d2- glucose enrichment, nd ions t m/e 25:24 were selectively monitored using electron impct ioniztion to determine 2-d1-glucose enrichment. By using these ions, the moleculr frgments included the crbon position of interest (e.g., the 6 position), while excluding the other lbeled position in the molecule (e.g., the 2 position). We hve previously described the detils of the mss spectrometry procedures (8). Smples to be nlyzed for plmitte enrichment were first processed for quntittive gs chromtogrphy, with heptdecnoic cid (.5 mg/ml plsm) being dded s n internl stndrd for subsequent mesurement of FFA concentrtion by gs chromtogrphy using Hewlett-Pckrd gs chromtogrph (22). After conversion to the methyl ester, [1-'3C]plmitic cid enrichment ws determined by GCMS s described previously (2). The processing of smples for glycerol involved the ddition of [2-'3C]glycerol s n internl stndrd for concentrtion mesurement, precipittion of proteins with B(OH)2 nd ZnSO4, nd pssing the resultnt superntnt sequentilly through nion nd ction exchnge columns. The trimethylsilyl derivtive ws formed nd isotopic enrichment determined by GCMS using electron-impct ioniztion (23). Ions were selectively monitored t m/e 25, 26, nd 28 in order to quntify the isotopic enrichment in the plsm resulting from the d5-glycerol infusion (m/e 28) nd the enrichment resulting from the in vitro ddition of [2-'3C]glycerol (m/e 26) for the purpose of determining concentrtion. Clcuitions The rte of ppernce of plmitte (R plmitte), glycerol (R glycerol), nd glucose (R glucose) were clculted using the sme generl pproch. In the bsl period (smples 6-9 min), physiologicl nd isotopic stedy stte existed, mening tht neither concentrtion of substrte nor isotopic enrichment ws chnging over time. Under these conditions, R ws clculted s: R (micromoles per kilogrm times minutes) = ([IEf]/[IEp] - l)f, where F is the isotope infusion rte (micromoles per kilogrm times minutes), IEf is the isotopic enrichment of the infuste, nd IEp is the isotopic enrichment of plsm (mole percent excess) t isotopic equilibrium. We hve discussed in detil elsewhere the use ofthis eqution with stble isotope trcers (24). Since plmitte is thought to be typicl of other long-chin ftty cids, the reltionship between flux nd concentrtion of the other ftty cids in plsm ws ssumed to be the sme s for plmitte. Therefore, R of totl FFA = R of plmitte/[plmitte]/[totl FFA], where [plmitte] nd [totl FFA] re the concentrtions of plmitte nd FFA, respectively. The concentrtion of plsm glycerol ws determined s follows: unlbeled glycerol (micromoles per milliliter) =.4,mol/ml/MPE -.4, when.4 Mmol of [2-'3C]glycerol ws dded to 1 ml ofech smple, nd MPE is the mole percent excess of ech smple s compred with smple from the sme subject without [2-'3C]glycerol dded. After the dministrtion of glucgon or epinephrine or unlbeled glucose, the stedy stte ws disturbed. In this cse, the nonstedy stte form of the Steele eqution ws used (fter curve fitting of rw dt) (25). R = F - V[(C2 + C,)/2] [(IE2 - IE,)/(t2 - t1)]/(ie2 + IE1/2), where C2 nd C, re the concentrtions t times t2 nd t1, respectively; IE2 nd 1E, re the enrichments t times t2 nd t,, respectively; nd V is the effective volume of distribution ofthe substrte. V ws ssumed to be 4 ml/kg for plmitte, 21 ml/kg for glycerol, nd 15 ml/kg for glucose. During the hyperglycemi experiments, the rte of endogenous glucose production ws clculted by subtrction of the glucose infusion rte from the clculted R. Rte of substrte cycling ws clculted s follows: rte of glucose substrte cycling (micromoles per kilogrm times minute) = R(2-dj) - R(6,6d2), where R(24di) is the R of glucose s clculted by the 2-di-glucose, nd R(6,64d2) is the R of glucose s clculted by the 6,6-drglucose: rte of TG/FA substrte cycling (micromoles per kilogrm times minutes) = 3 X R. glycerol - R FFA. The rtionle for this clcultion is tht for every glycerol relesed from triglyceride, three ftty cids must lso be relesed from triglyceride. Thus, if ftty cids pper in the plsm t rte less thn three times R glycerol, some reesterifiction of ftty cid must hve occurred. Sttisticl nlysis The vlue fter tretment ws compred sttisticlly with the bsl vlue for the sme group using the pired t test. Comprison between groups were mde using the nonpired t test. Results In ll cses, n isotopic nd physiologic stedy stte existed in period I. A representtive exmple of isotope enrichment is shown in Fig. 1. Averge vlues for isotopic enrichment do not reflect the stedy stte sitution s clerly, since the stndrd error of ech point does not distinguish between intrsubject vribility (smple to smple) t equilibrium nd intersubject vribility resulting from different isotopic infusion rtes nd different turnover rtes. Becuse stedy stte ws judged to hve been chieved in ech cse, the bsl vlues were consequently clculted by verging the four bsl enrichment vlues, nd dividing into the isotopic infusion rte. Glucgon experiments. The high dose infusion incresed the glucgon concentrtion to > 5, pg/ml, wheres the low dose infusion incresed the glucgon concentrtion to form bsl vlue of 153±35 to 453±56 pg/ml (men±sem). The plsm glucose concentrtion significntly incresed fter both glucgon infusions (Fig. 2). The infusion of somtosttin nd insulin did not ffect the extent to which low dose glucgon cused the glucose concentrtion to increse. The hyperglycemi during the high dose glucgon ws more exggerted thn during the low dose protocols. w. E mu * 2d1 Glucose o d2 Glucose Minutes Of Isotope Infusion I d5 Glycerol I I. I * 1-13C Plmitte Figure 1. Representtive exmple of glucose, plmitte, nd glycerol enrichment from experiment in which the first 9 min involved only the trcer infusion, nd from 9 to 15 min glucgon ws infused t the rte of 6 ng/kg. min. Isotope infusion rtes for this experiment were s follows: 2-d1-glucose,. 183,umol/kg. min; 6,6-d2-glucose,.178 gmol/kg * min; [ 1-'3C]plmitte,.36 /mol/kg * min; nd d5- glycerol,.97,umol/kg. min. Units for enrichment re mole percent excess (MPE). Substrte Cycling 1547

4 I Mumol/ml 1, O High Dose Glucgon...i Low Dose Glucgon + Somtosttin A*- Low Dose Glucgon Bsl Minutes Of Glucgon Infusion Figure 2. Glucose concentrtion before (bsl) nd during infusion of glucgon t 5 ng/kg * min (low dose glucgon), or 2 gg/kg * min (high dose glucgon). In the group given somtosttin (.1 pg/kg- min), insulin ws replced t the rte of.1 mu/kg - min throughout the entire experiment. Glucgon ws infused t I ng/kg. min in the bsl period, nd then the infusion ws incresed to 6 ng/kg- min from to 6 min of "glucgon infusion" period. In the bsl period, the insulin concentrtions were comprble in ll three groups (high dose glucgon, 1±1 yiu/ml; low dose glucgon, 8±1,U/ml; low dose glucgon plus somtosttin nd insulin replcement, 8±1 gu/ml; men±se). After the high dose glucgon, insulin rose to n verge vlue of 167±9,U/ml. The insulin concentrtion rose to 2±2,U/ml fter the low dose glucgon infusion. Insulin remined constnt throughout the somtosttin protocol (9±1 uu/ml fter glucgon infusion). These dt re presented in Fig. 3. c C 5 U C) C IC,umol/ml 17- -? 15- / $ 13- / 11- // 9~~~.1-~~~~~~~~~~~~~~ 2- / / I I I I I Bsl Minutes Of Glucgon Infusion Figure 3. Insulin concentrtion during the infusion of high dose glucgon (o), low dose glucgon (A), or low dose glucgon plus somtosttin nd replcement insulin (i). tsignificntly different from low glucgon groups, P <.1. *Significntly different from low glucgon nd somtosttin group. The bsl vlues for R(2-di)- nd R(6,6-d2)-glucose, nd glucose cycling, were similr in ll groups (Figs. 4-7) (Tble I). Both R(2-dl)- nd R(6,6-d2)-glucose were initilly stimulted by glucgon infusion in ll groups (Figs. 4-6). Without somtosttin infusion, these vlues peked t 2 min nd declined therefter in both low nd high dose glucgon groups. When somtosttin plus constnt bsl insulin ws infused, the R,(6,6-d2)-glucose response ws similr to the response during the low dose infusion without somtosttin, but the increse in R(2-dl)-glucose ws sustined throughout the entire infusion of glucgon (Fig. 6). The responses of glucose cycling to glucgon re shown in Fig. 7. All three groups hd significnt increses in cycling fter glucgon infusion. The pek vlue for glucose cycling ws significntly higher (P <.5) in ll cses thn the corresponding bsl vlue, s ws the verge vlue throughout the 6 min of glucgon infusion (Tble I). Comprison of the low glucgon group versus the low glucgon plus somtosttin nd bsl insulin replcement group indicted prolonged stimultion of cycling with somtosttin, since the vlues in the somtosttin group t 5 nd 6 min were significntly greter thn the corresponding vlues in the low dose glucgon group (P <.5). No significnt differences between the high nd low dose glucgon groups could be determined. Low dose glucgon did not significntly ffect R glycerol or R. FFA (Fig. 8). Similrly, R glycerol ws constnt during high dose glucgon but R FFA decresed from bsl vlue of 7.4±.8,umol/kg min to low vlue of 3.±.5,umol/ kg min. The fll in R FFA ws not immedite but corresponded to the inverse of the pttern of the chnge in glucose concentrtion. Similrly, low dose glucgon hd no effect on TG/FA cycling (Tble II). The high dose glucgon group hd n increse in TG/FA cycling, due to n incresed rte of reesterifiction of ftty cids, while lipolysis (R glycerol) remined constnt. Low dose glucgon with somtosttin hd no significnt effect on R glycerol, R FFA, nd TG/FA cycling (Tble II). Epinephrine experiments. The epinephrine concentrtion incresed from bsl vlue of 16±19 to 453±56 pg/ml (men±se) during the epinephrine infusion. Epinephrine infusion cused modest increse in both R(2-dl)- nd R(6,6-d2)-glucose (Fig. 9), but did not cuse significnt effect on glucose cycling (Tble I). Plsm glucose concentrtion nonetheless incresed significntly, from bsl vlue of 4.8±.2,mol/ml to pek vlue of 5.9±.3,umol/ml (men±se, P <.5). Insulin concentrtion did not increse significntly. On the other hnd, the stimultory effect of epinephrine on TG/FA cycling ws significnt (Tble II), owing to the fct tht three times R. glycerol incresed more thn did R. FFA (Fig. 1). However, the effect of epinephrine on lipolysis ws trnsient, nd therefore the overll verge rte of cycling during epinephrine infusion ws not significntly elevted (Tble II). Prednisone experiments. Prednisone tretment cused significnt increse in the plsm glucose concentrtion, from 4.8±.2,mol/ml before prednisone to 5.4±.2 Mmol/ml fter prednisone (P <.5). Insulin concentrtion lso significntly incresed from 9±1 uu/ml before prednisone to 17±2 gu/ml fter prednisone (P <.5). There were no significnt effects of prednisone lone on bsl glucose or TG/FA cycling (Tbles I nd II). The response to glucgon infusion of R(2-dl)- nd 1548 H. Miyoshi, G. I. Shulmn, E. J. Peters, M. H. Wolfe, D. Elhi, nd R. R. Wolfe

5 Tble I. Glucose Substrte Cycling Low dose glucgon Prednisone plus low Prednisone plus Glucose plus Low dose glucgon High dose glucgon plus somtosttin Epinephrine dose glucgon epinephrine somtosttin (n = 4) (n = 4) (n = 4) (n = 6) (n = 4) (n = 6) (n = 4) Bsl 5.3±.9 4.4±.8 6.8± ±.8 4.9±.7 2.9±.8 6.4±1.3 Pek vlue 18.±1.9* 25.8±6.* 23.5±2.4* 6.9± ± ± ±3.8 Averge vlue 12.4±1.9* 19.2±4.8* 15.8±2.7* 5.7± ± ±2.3 7.±3. Units re micromoles per kilogrm times minutes. Vlues re men±se. * Significntly different from bsl vlue (P <.5). R(6,6d2)-glucose, s well s the rte of glucose cycling, ws mrkedly blunted by prednisone (Fig. 4) (Tble I). R FFA nd R glycerol were not ffected by glucgon infusion fter prednisone tretment (Fig. 8). Prednisone tretment did not modify the response of either glucose cycling or TG/FA cycling to epinephrine infusion (Tbles I nd II) (Figs. 9 nd 1). Hyperglycemi experiment. The role of hyperglycemi in regulting glucose nd TG/FA cycling ws ssessed, since ll hormone infusions cused increses in glucose concentrtion. Plsm glucose concentrtion incresed from bsl vlue of 5.3±.4,umol/ml to vlue of 9.3±.6,mol/ml (men±se, P <.5) in this group. Bsl vlues for R. in this group were comprble with corresponding vlues in other groups (Figs. 11 nd 12). Hyperglycemi cused significnt suppression of endogenous glucose production when ssessed with 6,6-d2- glucose (P <.5), but the verge reduction in R(2-d1)-glucose ws not sttisticlly significnt (Fig. 1 1). Consequently, the clculted vlue for glucose cycling incresed slightly, but not significntly (Tble I). Hyperglycemi, in the bsence of chnges in glucgon nd insulin, cused slight but significnt reductions in both R glycerol nd R FFA (P <.5) (Fig. 12), but the clculted increse in TG/FA cycling ws not significnt (Tble II). Discussion Although substrte cycles were proposed over twenty yers go to be importnt in the regultion ofenergy metbolism (1), the ssessment of substrte cycling in humn subjects hs only recently been undertken (8-14). Wheres these recent studies hve estblished the existence of substrte cycling in humns, there is little evidence regrding the fctors regulting the rte of cycling. In this study we hve ssessed the cute response to elevtions in glucgon nd epinephrine, s well s the response to chronic elevtion in glucocorticoid ctivity. Our results indicte tht glucgon mrkedly stimultes glucose cycling, but hs little direct effect on TG/FA cycling. In contrst, epinephrine stimultes TG/FA cycling, but hs miniml effect on glucose cycling. The glucgon effect on glucose cycling is countercted by n inhibitory ction of insulin, but hyperglycemi does not ffect either glucose or TG/FA cycling. We did not observe direct effect of glucocorticoid tretment on either glucose or TG/FA cycling, but the response of glucose cycling to glucgon ws blunted fter glucocorticoid tretment. The rtionle for the use of glucose lbeled in different positions to quntify glucose cycling derives from the different Mmol/kg-min A jamol/kg-min 71 B,umonl/kg-mln 7, 5" * R 2dj, Glucose O R 66d2, Glucose S , Minutes of Glucgon Infusion Bsl Figure 4. R of glucose clculted with different trcers to low dose glucgon infusion before (A) nd fter (B) 3 d of tretment with prednisone. I -- BASAL io Minutes Of Glucgon Infusion Figure 5. Effect of high dose glucgon infusion on glucose R clculted with different trcers. Substrte Cycling 1549

6 jmol/kgmin 1 7u- * R 2d,,Glucose o R 66d2 Glucose,ugo/kg-mln s5-12- IV@9- S A Jmol/kg-min B 15, ir 4: * R FFA o R Glycerol 4- Is v Mk.,I BASAL Minutes of Glucgon Infusion Figure 6. Glucose R. clculted with different trcers during somtosttin, insulin, nd glucgon infusion. The rte of glucgon infusion ws incresed from I to 6 ng/kg. min fter bsl vlues were obtined. steps in the metbolism of glucose t which the hydrogen (or deuterium) is lost. The deuterium t position 2 is lost in the phosphoglucose isomerse step, so most lbel will be removed before glucose-6-phosphte leves to become either glycogen or dvnces through glycolysis, or recycles bck to glucose. Therefore, molecule tht cycles through the glucose/glucose- 6-phosphte cycle will lose the deuterium t position 2, but not,umoi/kgmln 4, cm 3 > 3- O o 1 bii co IC A -- High Dose Glucgon *.--- Low Dose Glucigon + Somtosttin -- Low Dose Glucgon BASAL Minutes of Glucgon Infusion Figure 7. Rte of gluconeogenic/glycolytic substrte cycling fter glucgon infusion. #Significnt difference (P <.5) from low dose glucgon infusion (without somtosttin). Bl 1 2 ;4 5O6 Minutes Of Glucgon Infusion Bsl Figure 8. Effect of low dose glucgon infusion before (A) nd fter (B) prednisone tretment on the R of glycerol nd FFA. the deuterium from position 6. The deuterium from position 6 of glucose is lost during crboxyltion of pyruvte into oxlocette, nd equilibrium with dicrboxylic cids. Thus, if molecule cycles through the glucose cycle (glucose/glucose- 6P-glucose) it will lose the lbel in the 2 position, but not in the 6 position. Consequently, the difference between the R of glucose s clculted using the different trcers will reflect the rte of glucose cycling. There is little possibility tht this method of estimting the rte of glucose cycling will overestimte the true vlues by virtue of glucose going to glycerol-6p, being incorported into triglyceride, then relesed s glycerol nd reincorported into glucose. Although this pthwy would theoreticlly dd to the clcultion of R2-R6 (since the 2D would be lost but the 6D would be retined), the quntittive spect of this cycle is virtully nil. Using high rte of infusion of [1-'3C]glucose, we found tht < 1% of the glycerol flux is derived from plsm glucose lbeled (15). Since the percentge contribution ofglycerol to glucose output in postbsorptive mn is < 5% (26), the mount of pprent glucose substrte cycling tht might ctully be occurring by this lterntive route is insignificnt. The interprettion tht the difference between R2 nd R6 equls the rte of glucose cycle ctivity vries from our previous clim tht R2 - R6 equled the sum ofthe glucose nd the fructose cycles (8). The rtionle for our previous interprettion ws tht substrte cycle in the metbolism of glucose could be quntified if lbel in one position of glucose is lost in tht cycle, while lbel from nother position is not lost. In tht cse, the rte of the glucose cycle would equl R2 - R3 (since the 3 deuterium is not lost in the glucose cycle), nd the rte of the fructose cycle would equl R3 - R6 (since the 6 deuterium is lost in the fructose cycle, nd the 6 deuterium re not lost). This generl pproch hs been used by others for clcultion ofboth the rte ofthe glucose cycle (14, 27, 28) nd the fructose cycle (29). If ech substrte cycle is clculted s described bove, then, the sum of the two cycles would be equl (R2 - R3) + (R3 - R6) = R2 - R6. However, wheres there is generl greement tht R3 - R6 provides reflection of fructose cycle ctivity, it is now evident to us tht 155 H. Miyoshi, G. L Shulmn, E. J. Peters, M. H. Wolfe, D. Elhi, nd R. R. Wolfe

7 Tble II. TG/FA Substrte Cycling Low dose glucgon Prednisone plus low Prednisone plus Glucose plus Low dose glucgon High dose glucgon plus somtosttin Epinephrine dose glucgon epinephrine somtosttin (n = 4) (n = 4) (n = 4) (n = 6) (n = 4) (n = 6) (n = 4) Bsl.7±1..9±.9 1.2±.3.4±.4.5±.3.4±.5.4±.3 Pek vlue.6±.8 4.8±.7* 1.4±.1 4.9±.9* 2.1±.9 6.4±2.7* 1.±.6 Averge vlue.1±.2 3.8±.7* 1.1±.2 1.±.5.7±.4 2.5±2.2.7±.3 Units re micromoles per kilogrm times minutes. Vlues re men±se. * Significntly different from bsl vlue (P <.5). the use of R52 - R53 s reflection of glucose cycle ctivity is vlid only when heptic fructose cycling is zero. Although this ppers to be the cse in postbsorptive humns (1 1), the lck of universlity of the definition of R.2 - R.3 invlidtes the extrpoltion described bove tht led us to the conclusion tht R52 - R6 equls the sum ofglucose plus fructose cycling. A more pproprite mnner in which to interpret the glucose dt is tht R2 mesures ll new glucose production, plus tht which hs cycled through the glucose cycle, while R.6 mesures only new glucose production. Therefore, R.2 - R6 equls the rte of glucose cycle ctivity. There is generl greement tht glucose cycling occurs in norml humn subjects in the postbsorptive stte, but there is some discrepncy in the precise quntittion oftht rte. Two other studies in which glucose cycling ws determined with rdioisotopes in norml volunteers hve recently been reported (1, 1 1), nd both ppers reported rtes of cycling tht were lower thn the vlue we hve reported using stble isotopes. We hve recently found tht systemtic difference between rdioisotopes nd stble isotopes cnnot explin this discrepncy. In seprte study we hve compred the R of glucose s determined simultneously with 2-D-glucose (R52D) nd [2-3H]glucose (R523H) in conscious rts. The verge vlues for R.2D nd R23H were 5.85±.29 nd 5.98±.34 jmol/kg-min i bl-- - L z.1 A Bsl S ymol/kg-min cr- Minutes of EpInephrine Infuslon B * R 2d1, Glucose O R 66d2, Glucose Men ± SE Bsl Figure 9. Effect of epinephrine infusion (.3 /Ag/kg * min) on R of glucose clculted with different trcers before (A) nd fter (B) prednisone tretment. mg/kg min, respectively (unpublished observtion). These vlues re obviously not different. It is possible tht the differences in cycling rtes between the vrious studies in humns simply reflects the difficulty of precise quntittion of ll necessry fctors, rther thn ny systemic differences. Thus, Bell et l. (1) reported vlue for R23H of 15,mol/kg-min, lmost precisely equl to our overll verge vlue of 14.6 Amol/kg * min. Although Krlnder et l. (1 1) reported lower vlue for R23H (12.1 Asmol/kg* min), their vlue for R63H of 1.3 timol/kg. min ws similr to our vlue for R6D of 9.8 Amol/kg* min. We re confident tht our method of nlysis induced no bis tht would hve resulted in n overestimtion ofr2 - R6. First, there ws no possibility of crosscontmintion of isotopic enrichment t the two positions. The frgment used to nlyze the enrichment of 2-D-glucose hs 2 3 structure of [(CH3)3SiOCHCHOSi(CH3)3]+, wheres the structure ofthe frgment used to nlyze 6,6-d2-glucose enrichment is (C2H32CH2CCCHC22H3)+. Thus, enrichment t the 2 position could not ffect the pprent enrichment t the 6 position, or vice vers. Since both frgments exclude the 1 position, ny deuterium tht ws not lost from the 6 position nd ws subsequently recycled into glucose would not be considered in the mesurement of the enrichment of 2-D-glucose. Furthermore, the 6-D enrichment would not be significntly ffected by such recycling either, since the mss increse due to recycling would be reflected by n increse in the rtio of m/e 17:169, nd would not hve noticeble effect on the rtio of Mmol/kgmmin 15I I S 3g- A //'S O >9Q-Q ~~3- Bsl 1 io 3' i 5 61 wnmol/kg-min 15, // B * R FFA O R Glycerol IB l - I I I I Minutes Of Epinephrine Infusion Bsl Figure 1. Effect of epinephrine infusion on the R of FFA nd glycerol before (A) nd fter (B) prednisone tretment. Substrte Cycling 1551

8 JAmsioll/Kg.mln IU possibility of recycled 3H from the 6 position contributing to 1-the pprent enrichment t the 2 position is not excluded. This would cuse n underestimtion of R23H. In contrst, the * R 2d, GLUCOSE detritition procedure used by Bell et l. (1) to determine the o R 6W2 GLUCOSE specific ctivity in position 2 should exclude ny enrichment in position 1. This could be the explntion of why Krlnder! - et l. (1 1) obtined slightly lower vlue for R2 thn either Fii~ gure 12. Effect of glucose infusion on the R of FFA nd glycerol. Bell et l. (1) or ourselves. Another possible problem in the rdioctive technique is tht, regrdless of the method of seprtion ofthe 2 nd 6 positions, the untestble ssumption must be mde tht with ech smple the ion exchnge chromtogr- 1 <>9 w I T phy successfully elimintes 1% of other lbeled compounds, such s lctte nd lnine. If this is not ccomplished, the difference between R2 nd R6 will be underestimted with either technique. Another potentil problem in the rdioctive 1A'_- T techniques is the uncertin extent to which complete sepr-, -'- _ tion of the ctivity of the 2 nd 6 positions is ccomplished. Bsl This ws reported to vry between 86 nd 97% by Krlnder et l. (1 1), but it is not cler how the recovery of ech individul Minutes of Glucose Infusion smple processed ws determined. Also, due to limittions in Figure 11. Effect of glucose infusion (2 mg/kg. min) on rte of en- the mount of rdioctivity tht cn be dministered to dog,enous glucose production clculted with different trcers. humn subjects, low counts re obtined, leding to less precision in determining specific ctivity. Finlly, it is erroneous to ssume tht the true vlue for glucose cycling in the volunteers e 6,6 m/l 171:169, which ws used to monitor the enrichment of studied by Krlnder et l. (I 1) ws the sme s for our sub- -d2-glucose. The technique we used to mesure isotopic en- jects, s our subjects were much younger (ge rnge, 2 to 32 ridhment cn relibly determine the known levels of nturl yr) thn their subjects (ge rnge, 33 to 6 yr). Given ll the buindnce of stble isotopes of glucose stndrd, nd the potentil pitflls of both the stble nd rdioisotope tech- of vrition on five repet nlyses of the sme niques, nd the differences in the subjects studied, it is interest- coefficient snnple (in the rnge of isotopic enrichment used in this study) ing tht the clculted vlues from the different studies re s is c :onsistently below 2%. Finlly, becuse of mss spectrum close s they re. In the context of the present study, we plce identifiction, there is little possibility of contmintion of the more emphsis on the reltive chnges in cycling rtes induced glulcose pek with nother compound tht would cuse spuri- by specific tretments thn with the exct rte of glucose cy- nlysis of glucose enrichment. cling in bsolute terms. ouss There re severl potentil sources of errors in the nlysis The interprettion of the 2-d1- nd 6,6-d2-glucose dt of specific ctivity of different positions of glucose. In the would be complicted if some 6,6-d2-glucose ws incorported method used by Krlnder et l. (11), the rdioctivity in the 1 into heptic glycogen during the bsl period, then relesed s possition is not distinguished from tht in position 2, thus the consequence of hormone infusion. In tht cse, the pprent stimultion of glucose cycling during hormone infusion would ii' mol/kg.min not reflect the simultneous ctivity of glucokinse nd glu- 9 cose-6-phosphtse, but rther only the ctivity of glucose-6- phosphtse, since the lbeled glucose coming from glycogen * R FFA brekdown would hve been incorported into the glycogen in o R Glycerol the preceding period. However, our results rgue ginst this being n importnt problem. In the fsting (bsl) stte, little 6- glycogen synthesis would be expected. Secondly, epinephrine is known to be potent stimultor ofglycogen brekdown, yet only minor increse in glucose cycling ws observed during Yv T epinephrine infusion. Thirdly, the mgnitude of the increse in glucose cycling fter glucgon ws lrge compred with ny rtifct due to relese of isotope from glycogen. In the bsl 3- period, glucose uptke verged 1 I gmol/kg. mm, or 1, gmol/kg over the 9-min period. Since the volunteers were 2-t_5! _2_ g g id fsting, the ssumption tht 1% of this glucose uptke (1 gtmol/kg) went directly to glycogen would lmost certinly be n overestimte. Nonetheless, even if tht lrge estimte of heptic glycogen synthesis during fsting is ssumed, nd in Bsl the further unlikely event tht glucgon infusion stimulted the complete relese of ll of tht newly synthesized glycogen, this would still ccount for < 1% of the increse in glucose cycling over the 6 min during the high dose glucgon infusion 1552 H. Miyoshi, G. L Shulmn, E. J. Peters, M. H. Wolfe, D. Elhi, nd R. R. Wolfe

9 (1,14 gmol/kg) (Tble I). Consequently, there is little doubt tht glucgon stimultes glucose cycling. The stimultion of glucose cycling by glucgon might hve been expected, on the one hnd, since the stimultion of glucose-6-phosphtse by glucgon is well estblished (3). On the other hnd, incresed glucose cycling lso requires simultneous increse in glucokinse ctivity. Although it hs been shown tht glucgon must be present for the mintennce of the regulr ctivity of glucokinse (3), direct stimultory effect of glucgon on glucokinse hs not been demonstrted. However, hyperglycemi does stimulte glucokinse (3). Consequently, the incresed glucose cycling during glucgon infusion my hve been the combined result ofdirect stimultion of glucose-6-phosphtse by glucgon, nd the stimultion ofglucokinse by the resulting hyperglycemi. Ifhyperglycemi ws responsible for the stimultion of glucokinse, n insulin response to the hyperglucgonemi/hyperglycemi ws not necessry for such n effect. In fct, comprison of the glucose cycling responses in the low dose glucgon groups with nd without somtosttin plus insulin replcement leds to the conclusion tht insulin inhibits glucose cycling. Glucose cycling decresed during the low dose glucgon infusion coincidentl with the rise in insulin concentrtion, nd when the insulin concentrtion ws held constnt throughout the glucgon infusion, glucose cycling continued to increse throughout the infusion period. The finding of n inhibitory effect of insulin on glucose cycling is consistent with the results reported by Efendic et l. (14), who found tht elevted glucose cycling ws chrcteristic of erly type two dibetes. These investigtors concluded tht normlly functioning insulin is necessry for the suppression of glucose cycling (14). However, the results from our experiment in which high dose glucgon ws infused might pper to be t odds with this conclusion, since the glucose cycling rte ws elevted despite lrge increse in insulin concentrtion. Alterntively, it is likely tht this insulin response dmpened wht would hve been n even lrger, nd more sustined, increse in cycling hd the insulin response been blocked with somtosttin. The pek in glucose cycling during high dose glucgon ws reched by 2 min of infusion, t time when the insulin response hd not yet reched mximum (Fig. 3). As the insulin reched the mximum levels lter in the infusion, the glucose cycling decresed, despite persistently high concentrtion of glucgon. A potentil compliction of the use of somtosttin plus insulin replcement is tht growth hormone secretion is lso suppressed by somtosttin. It is possible tht growth hormone might ffect glucose cycling, s it hs recently been reported tht cromeglic ptients with mrked increses in growth hormone concentrtion hd significnt increse in glucose cycling (12). However, the mgnitude of the increse in these ptients ws smll (1. gmol/kg min), rising the possibility tht less extreme vritions in growth hormone would not ffect glucose cycling t ll. This possibility is supported by the fct tht growth hormone infusion in norml volunteers hd no effect on bsl glucose production (31, 32). Furthermore, in our study when somtosttin ws infused growth hormone ws suppressed throughout both the bsl nd experimentl periods, so ny effect of incresing the glucgon infusion rte could not be scribed to chnge in growth hormone. Thus, it is extremely unlikely tht filure to replce growth hormone when somtosttin ws infused in ny wy influenced our conclusions regrding the effect of glucgon or glucose substrte cycling. Two potentil types oftg/fa cycle ctivity re possible in vivo: "intrcellulr," in which the FFA never leve the dipocyte before reesterifiction, nd "extrcellulr," in which the FFA re relesed into the plsm, whereupon they re clered from the plsm nd reesterified. We hve previously described how both of these pthwys cn be determined in humns (15). The clcultion of the extrcellulr recycling requires knowledge ofthe totl rte of ft oxidtion, which cn be determined by indirect clorimetry. In the present study, however, we were unble to quntify the rte of totl ft oxidtion during hormone infusion. The response to ech hormone ws trnsient, nd our bility to mesure oxygen consumption nd CO2 production ws not sufficiently ccurte to quntify substrte oxidtion in rpidly chnging nonstedy stte. Consequently, we hve clculted only intrcellulr TG/FA cycling. Since it is ssumed tht ll ftty cids tht do not enter the plsm re reesterified, the clcultion of intrcellulr recycling overestimtes the process by the mount of ftty cids tht do not enter the smpled plsm pool before oxidtion. Nonetheless, in our experience, chnges in intrcellulr cycling correspond closely to chnges in totl cycling (9, 15). The results from in vitro studies hve suggested stimultory effect of glucgon on lipolysis (17, 18). Our results indicte tht this effect my occur in vivo s well, since R glycerol ws mintined despite the elevtion in plsm glucose nd insulin concentrtion. Increses in either insulin or glucose concentrtion would hve been expected normlly to reduce R glycerol by themselves (15 ), s seen in Fig. 12. However, the physiologicl significnce of the glucgon effect on lipolysis is miniml, since the mount of FFA mobilized during glucose infusion ctully decresed modestly, presumbly reflecting the direct inhibitory effect of glucose on lipolysis (Fig. 12), s well s the stimultory effect of glucose nd insulin on reesterifiction (15). The stimultory effect of high dose glucgon on TG/FA cycling ws lmost certinly due to the resultnt hyperglycemi nd hyperinsulinemi stimulting reesterifiction, since R glycerol ws no higher thn during low dose glucgon infusion, when cycling ws not significntly incresed nd the plsm glucose nd insulin levels were much lower. Wheres the predominnt effect of glucgon is on glucose kinetics, epinephrine primrily exerts its effect on lipid kinetics. However, the direct effect of epinephrine on lipolysis (s reflected by R glycerol) cn only be observed in the erly phse of response (first 2 min), becuse the resulting increse in plsm glucose concentrtion countercts the lipolytic effect of epinephrine. It seems likely tht epinephrine exerts n effect not only on lipolysis but lso on reesterifiction. Although plsm glucose concentrtion rose during epinephrine infusion, the pek in TG/FA cycling occurred t 1-2 min - of epinephrine infusion. At this time the plsm glucose concentrtion hd risen only modestly. Thus, it my be tht stimultion of lipolysis is ccompnied by certin increse in direct reesterifiction due to n increse in intrcellulr ftty cid concentrtion. Wheres hyperglycemi my ugment the reesterifiction process, it does not pper to be necessry for n incresed rte of reesterifiction during epinephrine infusion. Substrte Cycling 1553

10 The direct effect of epinephrine on both lipolysis nd reesterifiction is supported by recent study we performed in severely burned ptients in whom plsm epinephrine concentrtion ws significntly elevted (9). In the bsl stte, these ptients hd elevted rtes of lipolysis nd of TG/FA cycling (fivefold elevtion). When they were given bet drenergic blockde, the rte of lipolysis ws reduced significntly, s ws the rte of TG/FA cycling, yet there ws miniml effect on plsm glucose concentrtion, glucose production, or glucose cycling (9). We hd nticipted both direct effect of glucocorticoid tretment on substrte cycling s well s n interctive effect of glucocorticoids with both glucgon nd epinephrine. The primry bsis for this expecttion ws study by Issekutz in conscious dogs (6). Issekutz observed n eightfold increse in glucose cycling with methylprednisolone tretment lone, nd significnt mplifiction ofthe stimultory effect ofglucgon on substrte cycling when it ws given fter methylprednisolone tretment. In contrst to the finding of Issekutz (6), we observed no effect of prednisone tretment lone on either glucose or TG/FA cycling. Our observtion is consistent with the results of other investigtors who found no effect ofcortisol infusion on totl glucose production in both dogs (33) nd mn (34). Furthermore, rther thn synergistic effect with glucgon, we found tht prednisone tretment blunted the response of glucose kinetics to glucgon infusion (Tble I). Owing to the inhibitory effect of insulin on glucose cycling, it seems likely tht the insulin response to the prednisone tretment countercted ny direct effect of prednisone on glucose cycling. Furthermore, this insulin response to prednisone ws pprently pronounced enough to lso block the norml stimultory effect of glucgon. This explntion could only be proven by repeting the studies involving prednisone tretment while simultneously inhibiting the insulin response. Wheres the prednisone tretment blunted the response of glucose cycling to glucgon, it did not ffect the stimultion of TG/FA cycling by epinephrine (Fig. 1 B nd Tble II). This is consistent with our conclusion cited bove tht epinephrine works directly to stimulte TG/FA cycling, rther thn being dependent on chnges in glucose nd/or insulin concentrtion to stimulte reesterifiction. The insulin concentrtion during epinephrine infusion ws much higher fter prednisone tretment thn before, yet the response of both R glycerol nd R FFA were lmost exctly the sme on ech study dy (Fig. 1, A nd B). We hve ttempted to elucidte the role of hyperglycemi, per se, in the control ofsubstrte cycling, becuse the hormone tretments tested cused the blood glucose concentrtion to increse significntly. We observed no significnt effect of hyperglycemi on glucose cycling, which is consistent with the recent report of Bell et l. (1). As reported in the dog (35), hyperglycemi hd n inhibitory effect on lipolysis nd corresponding effect on FFA mobiliztion. In contrst, we hve previously observed tht when glucose is infused nd insulin is llowed to increse, reesterifiction is stimulted more thn lipolysis is inhibited, so tht TG/FA cycling increses (15). By deduction, it ppers insulin is necessry for hyperglycemi to effectively increse reesterifiction. One of the mjor physiologicl roles of substrte cycles hs been proposed to be their contribution to overll energy expenditure. Since ATP is hydrolyzed with ech revolution of cycle, but there is no net conversion of substrte to product, there is n increse in thermogenesis nd energy expenditure (1). As explined bove, in this study we were not ble to relte the observed chnges in substrte cycling with directly mesured rtes of energy expenditure. Nonetheless, the chnges in substrte cycling tht we observed re consistent with other experiments ssessing the role of hormones on metbolic rte. It hs recently been shown tht glucgon stimultes metbolic rte in norml volunteers nd tht this effect is countercted by insulin (36). Our dt indicte tht incresed glucose cycling my be biochemicl bsis for glucgon-induced increse in energy expenditure. Similrly, it seems likely tht sympthetic nervous system stimultion increses metbolic rte in mn (37). Our dt suggest tht incresed TG/FA cycling my contribute to this response. Since, by definition, the existence of substrte cycles results in incresed energy expenditure without ny chnge in either the mount of substrte or product, the physiologicl significnce of these cycles hs been debted. One perspective is tht these "futile" cycles serve no purpose beyond thermogenesis (2). Alterntively, Newsholme nd Crbtree (1) hve demonstrted tht substrte cycling cn provide mechnism ofvrible sensitivity for the control of flux. They hve shown theoreticlly tht cycling will mplify the control of flux to given chnge in regultor (e.g., hormone) concentrtion by 1 + C/J, where C is the rte of cycling nd J is the rte of flux. Although this clcultion my not be strictly pplicble to the in vivo sitution, it nonetheless is useful in ddressing the issue of whether metbolic cycling is futile, or if it serves n importnt role in metbolic regultion. Inspection of the dt from the TG/FA dt in our study revels tht sensitivity ws mplified by stimultion of cycling by epinephrine. Epinephrine infusion incresed 1 + C/J from 1.6 to 1.37 when given lone, nd from 1.7 to 1.51 when infused fter prednisone tretment. The 3-5% mplifiction of sensitivity of control of FFA flux by TG/FA cycling induced by epinephrine is consistent with the fct tht severely burned ptients given proprnolol hd reduction in 1 + C/J (for TG/FA cycling nd FFA flux) from 1.84 to 1.39 (9). The finding tht epinephrine increses the sensitivity of control of the rte ofrelese offfa by mens of stimulting TG/FA cycling in humns is consistent with studies done in nimls (7), nd confirms the importnce of this substrte cycle in the regultion of metbolism. In contrst, the mrked stimultion of glucose cycling by glucgon ws not ccompnied by concomitnt increse in 1 + C/J. Although 1 + C/J incresed fter glucgon by 8-12% (verge over 1 h) in ech group, this is smll mount when compred with the mgnitude of increse in cycling rte (from 15 to 3%). Furthermore, it hs been pointed out tht incresed glucose cycling contributes to the lck ofglycemic control fter glucose infusions in type II dibetes (13), nd since glucose cycling is elevted in burned ptients (9), it is likely tht the hyperglycemi tht results during glucose infusions in those ptients is lso in prt the consequence of incresed glucose cycling. Thus, it my be mistke to generlize s to whether substrte cycling in humns is futile, or if it plys n importnt role in mplifying the enzymtic control of substrte flux. Clerly, chnges in TG/FA cycling cn modify control of FFA flux, but it is uncertin if there re conditions in which stimultion of the glucose cycle would result in n mplifiction of metbolic control of heptic glucose output H. Miyoshi, G. I. Shulmn, E. J. Peters, M. H. Wolfe, D. Elhi, nd R. R. Wolfe

11 Acknowledments The uthors wish to cknowledge the help of the nursing stff of the Clinicl Reserch Center in performing the infusions. Excellent technicl ssistnce ws provided by Susn Fons, Esther Surrig, nd Nitin Sehgl, nd we thnk Judy Chdwick for the typing nd preprtion of the mnuscript. This work ws supported by Ntionl Institutes of Helth grnt DK nd GCRC grnt 73, nd by grnt from the Shriners Hospitls of North Americ. References 1. Newsholme, E. A., nd B. Crbtree Substrte cycles in metbolic regultion nd in het genertion. Biochem. Soc. Symp. 41: Ktz, J., nd R. Rognstd Futile cycles in the metbolism of glucose. Curr. Top. Cell. Regul. 1: Hue, L The role of futile cycles in the regultion of crbohydrte metbolism in the liver. Adv. Enzymol. 52: Clrk, D. G., R. Rognstd, nd J. Ktz Isotopic evidence for futile cycles in liver cells. Biochem. Biophys. Res. Commun. 54: Dunn, A., J. Ktz, S. Golden, nd M. 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