Influence of Sodium ~-Hydroxybutyrate on Glucose and Free Fatty Acid Metabolism in Normal Dogs*

Transcription

1 Dabetologa 3, (1967) nfluence of Sodum ~-Hydroxybutyrate on Glucose and Free Fatty Acd Metabolsm n Normal Dogs* E. BALASSE, E. COUTURER and J. R.M. F~ANCKSON Departments of Expermental Medcne and General Pathology, Unversty of Brussels Receved January 30, 1967 Summary. The netabolc effects of a 120 nn sodum fl.hydroxybutyrate nfuson (5 nlnoles/kg/h) were studed n healthy anaesthetzed dogs. -- Arteral blood sugar decreased progressvely, the fall averagng 25 ng/100 nl from the 80th nn onwards. A slght, transent ncrease n plasma nsuln concentraton was observed wth a mean peak of ~- 8 #U/ml at the 10 th nn of nfuson. The dsappearance rates of a 12C-glucose load or of a tracer of l~c-glucose were not modfed by the sodum fl-hydroxybutyrate nfuson. sotope dluton calculaton showed that hypoglycaelna was entrely the consequence of a reducton n hepatc glucose output. -- Plasma lqefa concentraton decreased durng the frst hour, the fal] averagng 180 # noles/1. Subsequently, despte the mantenance of the nfuson of ketone bodes, plasma NEFA returned to basal levels or above n some dogs, whle remanng at low levels n others. Turnover studes usng a constant nfuson of ~dc-pallntate, revealed that the decrease n NEFA concentraton was the consequence of both an nhbton of ~EFA outflow from adpose tssue and an ncrease n the rate of NEFA uptake by the tssues. -- Contrary to data of the lterature, our results do not suggest that the prolonged nhbtory effect of sodum ~-hydroxybutyrate on hepatc glucose output could be medated through the mnor pancreatc stmulaton. Ths could, however, explan the ~EFA changes observed. L, nfluence du fl-hydroxybutyrate de sodum sur le mdtabolsme du glucose ct des acdes gras lbres chez le chen normal. Rdsumd. Les Facton d'une perfuson de f-hydroxybutyrate de sodum (5 n noles/kg/h pendant 120 nn) sur les n@tabolslnes glucdque et lpdque du chen normal anesth@sd. -- La glycdlne art6relle d6crolt progressvelnent, la chute attegaant 25 rag/100 nl partr de la 80e nn. L'nsulndme s'accrot ldg~rement (d+s 8/,U/nl) ~ la 10 ~me nn et retourne ~ sa valeur basale la 30 eme nn de la perfuson. La vtesse de dsparton d'uue surcharge. v. en glucose-l~c ou en glucose-ltc n'est pas nodf6e par l'adlnnstraton des corps c~tonques. Le calcul de dluton sotopque nontre que l'hypoglycdme est ent~relnent secondare ~ une r6ducton du d4bt glucosd du foe. -- La concentraton plaslnatque moyenne des NEFA dmnue de 180 #M/1 pendant la ere heure de la perfuson. Ult@reurement, nalgr6 le nanten de celle-c, la lpacd6me s'@lsve chez certans anmaux jusqu'~ des taux ddpassant les valeurs basales, tands que l'abassement persste chez d'autres. La vtesse du ((turnover)) des NEFA, mesurde par ]a technque de perfuson contnue de pallntate-l-l~c nontre que la chute des NEFA rdsulte des effets conjuguds d'une basse de leur producton par le tssu adpeux et d'une auglnentaton de leur vtesse de captaton par ]es tssus consomlnateurs. -- Ces rdsultats sont en opposton avcc eertanes doun@es de la lttdratm'e parce qu'ls excluent que ]a chute de la glycdlne sot secondare ~ la fable stmulaton pancrdatque observde. Celle-c peut ccpendant rendre compte des modfcatons transtores de la lpacddme. Der Enflufl von Natrum fl-hydroxybutyrat auf den Stoffwechsel der Glucose und der freen.fettsauren bem normalen Hund. Zusammenfassung. An an~stheserten unden untersuchten wr de Stoffwechselwrkungen ener nfuson von fl-hydroxybutyrat (5 m/moles/kg/std) fber 2 Stunden. -- Wr beebachteten ene zunehmende Blutzuckersenkung, de nach der achtzgsten Mnute.D. 25 ng~o betrug. Ferner trat en lechter, vorfbergehender Ansteg der Plasmansuln-Konzentraton yon.d. 8 Mkroenheten/ml 10 nn nach Begnn der nfuson en. De Schwundraten von 12C-Glucose eder xac-glucosegaben Knderten sch unter der nfuson ncht. Berechnungen der sotopen-verdfnnung zegten, dab de Blutzuekersenkung ledglch auf ener verlnnderten Glucosefresetzung aus der Leber beruht. -- De Plasmakonzentraton der free1 FettsKuren sank w~hrend der ersten Stunde urn durehschnttlch 180 Mkrolnol/1. Danach kehrte sc be engen Hunden trotz Fortsetzung ter KetokSrper-nfuson anf den Ausgangswert oder sogar hshere l~onzentratonen zurfek, wkhrend se be anderen Teren ernedrgt bleb. Ulnsatzstuden mt Hflfe ener Dauernfuson yon x~cl~almtat ergaben, dal~ der Abfall der FFS-Konzentraton durch ene Verrngerung der FFS-Abgabe des Fettgewebes und ene verlnehrte FFS-Aufnahme n anderen Geweben bedngt war. ln Gegensatz zu den Angaben der Lteratur wesen unsere Ergebnsse ncht darauf bn, da$ de anhaltende Helnlnung der Glucose-Abgabe aus der Leber durch Natrnln fl-hydroxybutyrat fber de gerngfgge Stlnulerung des Pankreas zu erklkren st. Doch ksnnte dese de aufgetretenen ~nderungen der FFS-Konzentratonen bewrken..key.words: Sodum /5-hydroxybutyrate nfuson n dogs, effects on ~2C- and 14C-glucose, nsuln, NEFA, x~c-palmtate ntroducton Although the mechansms nvolved n hepatc ketogeness are ncompletely understood, t s well * Work performed under the Assocaton contract Euratoln-Unverstes of Psa and Brussels (n o BAC). Ths work has been presented at the Second Annual Meetng of the European Assocaton for the Study of Dabetes (Aarhus, July 6--8, 1966). establshed that the producton of ketone bodes s ncreased when the moblzaton of lpds s enhanced, as t s durng carbohydrate deprvaton. Conversely, recent work suggests that ketone bodes mght nterfere wth carbohydrate and lpd metabolsm. n vtro, ketone bodes added to the ncubaton medum nhbt glucose uptake and oxdaton by muscle [28, 14, 24] and decrease the lpolyss of adpose tssue [5]. MADSON and coworkers [21, 19] have shown that ketone bodes, when admnstered n

2 - 1-14C Vot. 3, No. 6, 1967 E. BASASSE eg al. : nfluence of Sodum fl-hydroxybutyrate 489 vvo, provoke metabolc dsturbances chefly characterzed by a stmulaton of nsuln release and a fall n the plasma concentratons of glucose and NEFA. The purpose of the present work s to study the knetcs of glucose and NEFA modfcaton durng sodum fl-hydroxybutyrate (Na fl-0h-b) nfusons n dogs by means of sotopc methods usng glucose and palmtate -1-14C. Materals and Methods The studes were performed on adult mongrel dogs weghng kg, fasted h. Anaesthesa was nduced wth Pento-barbtal (15 mg/kg) and mantaned by further smaller doses accordng to requrements. The dfferent expermental protocols used are defned n the fgures. The measured parameters were the followng: blood sugar concentraton, plasma concentraton of NEFA, nsuln and ketone bodes, "K value" of the ntravenous glucose tolerance test and turnover rates of glucose and NEFA estmated by sotopc dluton methods. Blood samples for all chemcal and sotopc determnatons were drawn from the femoral artery va an ndwellng Cournand needle. All the substances admnstered to the dogs were njected or nfused through ndwellng polyethylene catheters nserted n the saphenous vens. After a control perod, Na DL-fl-Ot-B was nfused as an aqueous soluton, the anmals recevng 5 mmole/kg/h under a constant flow of 73.2 ml/h. The pt of the Na f-0h-b soluton was MkMne (ph 8) and was not neutralzed before njecton, snce t has been proven that alkalnty has no nfluence on the metabolc effect of the substance [21]. Analytcal methods Blood sugar concentraton was determned on heparnzed blood samples contanng fluorde, by a modfcaton of to~'f~a~'s method [17], adapted to the Auto-AnMyser. The other chemcal determnatons were performed on plasma samples, whch were obtaned from heparnzed blood mmedately centrfuged under refrgeraton, and kept frozen at ~ untl the day of the analyss. NEt%'A concentraton was estmated followng DoL]~'s procedure [10], total ]cetone bodes followng a modfcaton [18] of MC~AnL's method [22] and plasma nsuln accordng to the method of MORGAN et M. [23]. The ntravenous glucose tolerance test was performed followng the technque descrbed by CONA~D et al. [8, 7]: rapd.v. glucose loadng (0.5 g/kg body weght n a 50~o soluton); calculaton of the glucose dsappearance rate by graphcal procedure from the 7 m tll the 31 st ran on a sem-logarthmc scale (log. of blood sugar concentraton as ordnate, tme as abscssa). Glucose turnover rate: sxty #C of g]ucose-l-14c (2 mc/mm) was rapdly njected by the venous route and allowed to equlbrate n the extracellular flud for 40 mn. From that tme onwards the lac-glucose content of the whole blood was followed by frequent samplng. The blood 14C-glucose was solated as 14C02 by a fermentaton method usng leueonostoc mesenterodes [4, 9]. The 14C0~ produced was trapped by tyamne and drectly counted by lqud scntllaton n a P.P.O. (4 g/l) -- P.O.P.O.P. (100 rag/l) -- toluene medum. The recovery of 14C02 from g]ucose--lac provded by ths method vared from 85 to 92 ~o. The countng effcency averaged 72~o. Results were corrected for countng effcency but not for ncomplete recovery of radoactvty. The l~c-glncose fractonal dsappearance rate was determned graphcally on a sem-logarthmc graph (tme as abscssa, lac-glucose content of the blood as logarthmc ordnate). The nflux-el flux rate of glucose was only calculated durng the steady state perods usng the formula: F=K*C where F s the nflux-efflux rate expressed n rag/100 ml/ ran; K* the fractonal dsappearance rate n % per ran; C, the blood glucose concentraton (mg/100 ml) averaged for each steady state perod. NE.FA turnover rate Preparaton of labelled palmtate for njecton: 20 mg of unlabelled palmtc acd was added as carrer to 500 #C of palmtc acd-l-l~c (36.6 mc/m/mole) n benzene soluton. The soluton was evaporated to dryness under a flow of ntrogen and redssolved n 1 ml ethanol. After addton of 5 ml of 0.02 N NaOH, the alcohol was evaporated at 100 ~ and the aqueous pa]mtate-14c soap soluton was dluted to a fnal volume of 100 ml wth a 10% soluton of crystallne bovne albumn n salne. The preparaton was dvded nto 10 alquots contanng 50 #C each and stored at -- 20~ untl use. On the day of the experment 50/~C of the 14C-pMmtate albumn complex was dluted to 100 ml wth salne, and nfused ntravenously throughout the experment at a constant flow of 37 ml/h regardless the weght of the dog. Estmaton of plasma NEFA and 14C-pamtate concentratons : from each plasma sample, duplcate 1 ml alquots were extracted followng DoLl's technque. One of them was used for measurements of unlabelled NEFA; the other one for estmaton of radoactve palmtate, accordng to the followng procedure adapted from WNC~L~ et al. [29] : the heptane phase was removed and pooled wth another 3 ml heptane extract of the underlyng phase ; 2 ml of 0.05 N NaOH n 30~o ethanol soluton was added; after mxng and centrfugng, the upper heptane phase contanng the neutral plasma lpds was removed and dscarded, and the lower alcoholc phase washed wth an addtonal 3 ml of heptane ; the alcoholc phase was then acdfed wth 0.5 ml of 0.5 N tt2s0 a and extracted twce wth 3 ml portons of heptane; these heptane extracts were pooled n a scntllaton-countng val together wth 6 ml of toluene contanng P. O. P. O. P (200 rag/l) and P. P. 0. (8 g/l) and counted wth an effcency of about 85%. When pmmtc acd-l-ltc was eomplexed wth albumn and then carred through the analytcal

3 490 E. BALASSE et al. : nfluence of Sodum ~-Hydroxybutyrate Dabetooga procedure, % of the radoactvty was recovered, l%esults were corrected for countng effcency but not for ncomplete recovery of radoactvty. Calculatons: when palmtate-tj4c was nfused at a constant rate, a plateau of 14C-palmtate concentraton was reached approxmately 10 mn after startng the nfuson. Fractonal turnover rates and total NEFA nflux or efflux have been calculated by the followng formula [16] durng the steady state perods (stable levels of both 14C-palmtate and unlabelled NEFA) : K-- q where K s the fractonal turnover rate (ran-l); the radoactvty nfused (d.p. m/ran); Q the total radoactvty n plasma NEFA (d.p.m) S.A. where F s the total nflux or effux of nonlabelled NEFA (#moles/ran); S.A., the plasma NEFA specfc actvty (d. p. m./# mole). l" Na/3-OH-B " 5m.moles/l<g/k ] m,g/loem,1, Blood glucose conc. ', 80 7o [Plasma nefa conc.] A moles/try1 ~ J 05 o.~f ~ ~.#//~_/z 1 6.0[ [ ~moles/l Ptasma ketches c~ 2,0 ~JJJJ::';~;;~",';~- 0,zLU/m~], P[asma lnsuln cone. t o,0 nn n n 0 ' r 3~0,, 60 ' 90' ' 120 " [50mn ]80 Fg. l. nfluence of 8, constant ~Ta ~-Ot{-B nfuson on blood glucose concentraton and plasma concentraton of NEFA, total ketone bodes and nsuln. Mean results for 9 dogs S.E.M. Results The mean values of blood glucose concentraton and of plasma concentraton of NEFA, total ketones and nsuln for the control perod and the Na fl-oh-b nfuson perod are llustrated n Fg. 1. The modfcatons nduced by the ketone bodes were the followng. 1. A decrease n blood sugar whch plateaued for the last 40 ran of the nfuson, representng a mean fal of 25 rag/100 ml. 2. A plasma NEFA decrease averagng 180 #moles/1 at the end of the frst hour of nfuson. Mter ths ntal declne, the behavor of the plasma NEFA was varable from dog to dog. n some of them, there occured a secondary rse brngng the NEFA levels up to basal values or above, though n others plasma NEFA remaned at low levels untl the end of the nfuson perod. 3. A progressve rse n ketonema reachng the mean value of 6 mmoles/l at the end of the nfuson. 4. A small ncrease n plasma nsuln concentraton, observed n each experment and statstcally hghly sgnfcant. Ths effect was transent reachng ts peak at the 1O th ran of nfuson (+ 8 FU/ml), and was not detectable after the 30 TM mn. '-- Nafl-OH-B 5 m, moles/kg'/h, a Blood glucose ~1 ~g/100w~ t.v. glucose /t.v. glucose 300 F f 0.Sg/kg fo.sg/kg" 20ol- / /~.! / "~;~K-2.9 _, [0 L. " [~.K o t [ f r o ~ ' - /,, 50F ~ ' ~! O0 120ran Fg. 2. Effect of & constant l~a /3-OH-B nfuson on the ntravenous glucose tolerance test n 6 dogs. Mean results 8.E.M. Fg. 2 shows the mean values of blood sugar concentraton observed durng two successve ntravenous glucose tolerance tests. The "K value" averaged 2,9% per ran n the basal perod and was unaffected by the Na fl-oh-b nfuson, despte ts major hypoglycaeme acton. The nfluence of Na fl-oh-b on glucose turnover s llustrated n Fg. 3. The amounts of radoglucose njected not beng proportonal to the body weght of the anmals, results are expressed n percentage of the blood actvtes (d. p. m/ml) extrapolated to tme zero. Durng the control perod and the 2h nfuson of Na fl-oh-b, the decrease n glucose-l-14c concentraton was lnear and constant, correspondng to a fractonal turnover rate of 1.1% per mn. Lver glucose output, estmated for the two steady state perods (control perod and the last 40 ran of the Na f-o-b nfuson), showed a sgnfcant decrease (p < 0.001) from mg/100 ml/mn to rag/100 ml/mn under the nfluence of the admnstraton of ketone bodes. These data reveal that the hypoglycaeme acton of Na f-ott-b s due only to a reducton n lver glucose output and not to any modfcaton of the overall glucose utlzaton rate.

4 Vol. 3, No. 6, 1967 E. BALASSE et al. : nfluence of Sodum f-hydroxybutyrate 491 Data pertanng to the turnover of NEFA are presented n fg. 4. Ten ran after startng the constant nfuson of radoactvty, the plasma concentraton of palmtate 1-1~C reached a plateau. From ths moment, lttle varaton n the concentraton and specfc r~g-/oo~t 5() 40 ',= t LBlood glucose conc.] Nafl-OH-B S~m8les/'kg/l~ ]Blood glucose-t-.'%one. Ol: [ % r "~ =, - F ~Glucose_[_14 c / 60,,~ C [Hepafc lucose outpuf] m,g/lo0 m,l/mn "~ 0.5 ~ ] rnn 210 Fg. 3. nfluence of a Na fl-oh-b constant nfuson on blood glucose concentraton, ltc-glucose fractonal dsappearance rate and estmated lver glucose output. The mean blood tc-glucose concentratons are expressed n percentage of the blood actvtes (d. p. m/ml) extrapolated to tme zero. Mean results for 8 dogs :~ S.E.M. NEFA uptake by the tssues; 2. an nhbton of NEFA nflux nto the blood from adpose tssue. The ndvdual data of these experments are summarzed n Table 1. The fractonal turnover rates and the nflux or efflux rate of nonlabelled NEFA are also gven; they are estmated for the last 20 ran of both the control perod and the ketone nfuson perod, whch corresponded to steady states. The fractonal!~ Pslmtate - J4C - nfuson ~, p_ Nap-OH-~ _..j [ nfuson f Plasma nefa cone. /~ moles/m,1, [ O.6r!... J,,,, \ ~o-*-~, o o...~ 03L r-. n --, 9! tpbsme palmffafs-t-r cone. 4.p.~/ng, 1700[ ~, l~.,~ 1500'-.~ 0"'~-'~,, ' "\ L d,.p.tn,/m,], / ~e~-.,...~ 4.4oo[40oo [Plasma nefa spec,ac~.] &200[ ~.~~ l"r/~'\,~.,--~ / t '0 ' ~ ran 120 Fg. 4. nfluence of a Na fl-oll-b nfuson on p]asma ]qefa concentraton, palmtate-l-14c concentraton and NEFA specfc actvty durng a constant nfuson of palmtate-l-14c. Mean results for 9 dogs Body nflux rate of Experment Weght 14C-palmtate Plasma Palmtate 14C No (kg) (d.p.m./mn) 1 cone (d.p.m./ml) Table t. nfluence of Na fl-oh-b on NEFA turnover Control S Plasma NEFA Fractonal turnover Nonlabelled NEFA Spee. Act (d.p.m./~mote) rate of NEFA (%/ran) nflux (;~mole/mn) Na fl-oe-b ~ Control 2 l~a fl-o-b ~ Control Nafl-0H-B 3 ControP Nafl-OH-B t Mean Calculated from the radoactvty recovered by the descrbed procedure (see text). Mean of 3 values obtaned durng the last 20 ran of the control perod (steady state). a Mean of 3 values obtaned durng the last 20 ran of the Na f-o--b nfuson perod (steady state). actvty of NEFA occured durng the control perod. Na f-oh-b nfuson nduced a decrease n both labelled and unlabelled plasma NEFA content and a rse n plasma specfc actvty. Thus, two dstnct mechansms are nvolved n the lowerng of plasma NEFA by ketone bodes: 1. an ncrease n the rate of turnover rates were calculated by assumng that the plasma volume represented 5 % of the body weght and remaned constant throughout the experment. For the control perod, the fractonal turnover rate averaged 35 % per mn, and was systematcally ncreased to gve a mean of 42.8% per ran, the rse beng statstcally Dabeto]oga, VoL 3 33

5 492 E. BALASSE e~ al. : nfluence of Sodum fl-hydroxybutyrate Dabetologa hghly sgnfcant (0.001 <p < 0.01). Smultaneously, the mean rate of nflux or efflux of unlabelled NEFA decreased from 243 #moles/mn to 185 #moles/ran. The fall occured n 8 of the 9 dogs studed and was also hghly sgnfcant (0.001 < p < 0.01). Sx control experments were performed usng nfuson of sodum chlorde (5 mmoles/kg/h) nstead of Na f-o--b (5 mmoles/kg/h). n these experments no sgnfcant modfcatons n the concentratons of ether blood glucose Or plasma labelled and unlabelled NEFA were observed. Dscusson The present studes afford quanttatve data regardng glucose and NEFA metabolsm durng nfusons of Na #-Ot-B. A. Glucose metabolsm n our expermental condtons, the hypoglycaemc acton of the nfused ketone bodes does not seem related to the observed fl-cell stmulaton. ndeed the ncrease n nsuln concentraton of perpheral blood was small and transent, whereas hypoglycaema was present throughout the nfuson. Moreover, f the hypoglycaema was medated through an nsulnc effect, an ncrease n the rate constant of glucose utlzaton should be observed; such an ncrease was not found whether the rate constant was measured by the.v. glucose tolerance test and or by the sotopc method. Lastly our data show that the hypoglycaema was brought about by a reducton n the glucose output of the lver. t has been proved that durng nsulne hypoglycaema n the normal anaesthetzed dog, the nhbtory nfluence of nsuln on the lver glucose output s always overwhelmed by the stmulatng effect of perpheral hypoglycaema [13, 27]. 0nly when hypoglycaema s reduced by glucose nfuson does the hepatc acton of nsuln become evdent n the normal dog [20, 26]. Snce hypoglycaema cannot be explaned by an nsulnc effect, the hypothess of a drect nhbtory acton of the ketone bodes upon lver glucose output must be consdered. The fndng that Na fl-0-b does not nhbt the rate constant of perpheral glucose uptake by the whole tssues s n contrast wth the effects observed n vtro on muscle,.e. wth solated daphragm or perfused heart. Our results are n many ways opposed to those obtaned by MADSON et al. [21, 19], who notced that the Na fl-o-b nfuson produces an mportant and prolonged pancreatc stmulaton, whch they hold responsble for the observed decrease n lver glucose output. They also suggested a ketone-nduced nhbton of nsuln acton on glucose uptake, snce n ther experments perpheral glucose utlzaton was altered despte hgher blood levels of nsuln. The dscrepances exstng between MADson's group conclusons and ours are probably due to dfferences n the expermental condtons: 1. these authors measured the nsuln concentraton n the pancreatc venous plasma and gave a heaver load of ketone bodes; 2: they performed ther experments on dogs wth porto-caval shunts, and ther assumpton regardng the acton of nsuln on lver glucose output obvously cannot be extended to the normal anaesthetzed dog, as mentonned above; and 3. they measured the perpheral glucose utlzaton wth an ndrect, non-sotopc method, based upon the comparson between the lver balance and the sze of the glucose pool, whereas our measurements were drect estmatons of glucose utlzaton rate by the whole tssues. Stll more elaborated are the conclusons of F~LTs et al. [12]. Usng sodum acetoacetate nstead of Na fl-ohh-b, they showed that the perpheral glucose utlzaton was enhanced when ketone bodes were nfused alone, whereas acetoacetate lowered glucose assmlaton rate when admnstered to dogs rendered hyperglycaemc by a constant glucose nfuson. nfusng acetoacetate at the rate of 1.52 mmoles/ kg/30 mn, F~;rAzcs et al. [11] observed no modfcatons n perpheral nsuln concentraton. These data are n far accordance wth our results, although a precse comparson s not possble, due to dfferences n the chemcal compound used, the doses njected and the speces used. B. 3TEFA Metabolsm n the dscusson of the acton of Na f-oh-b on NEFA metabolsm, t s necessary to consder successvely the behavour of plasma NEFA durng the frst and the second hour of n fuson of ketone bodes. Durng the frst hour of nfuson, a systematc drop of NEFA plasma concentraton was observed. The use of an sotopc method made t possble to show that ths drop was the consequence of both an ncrease n NEFA uptake by the tssues and a decrease of NEFA producton by adpose tssue. The small recorded rse n plasma nsuln concentraton can provde an explanaton for the observed NEFA modfcatons for the followng reasons : 1. small doses of nsuln havng a neglgble nfluence n vvo upon carbohydrate metabolsm may exhbt a clear-cut acton on NEFA metabolsm [30, 1]; and 2. nsuln may promote a reducton n NEFA producton by adpose tssue and an ncrease of ther uptake, n partcular at the hepatc level [25, 6]. Our results pertanng to the acton of ketone bodes on plasma NEFA are consstent wth the fndngs of MEBANE et al. [21]. Nevertheless, a drect acton of Na fl-0h-b on adpose tssue cannot be excluded. ndeed, BJo~xTo~P has recently provded evdence for an nhbtory effect of Na fl-o-b on rate of lpolyss of adpose tssue n vtro [5]. However the acton of Na fl-ot-b on adpose tssue seems to be a complex one, snce.az~son observed an ncrease n NEFA release n the presence of Na fl-oh-b [15]. Durng the second hour of nfuson, no unform response of the plasma NEFA was recorded: they remaned at low levels n some anmals and strkngly rose n others. Ths effect could be nterpreted ether as

6 Vol. 3, No. 6, 1967 E. BALASSE et al. : nfluence of Sodum f-hydroxybutyrate 493 the consequence of the return of the blood nsuln to basal level, or as resultng from the adaptatve reactons to hypoglycaema. When one consders the metabolc effects of Na f-0tt-b as a whole, one could be surprsed by ther smlarty wth those produced by sulfonylurea n dogs. Lke ketone bodes, sulfonylurea provokes a transent ncrease n nsuln secreton but ther prolonged hypoglycaeme acton s caused by an nhbton of the lver glucose output [3]. Ther acton on NEFA concentraton s also bphasc, wth an ntal fm1 nduced by the nsuln secreton, and a secondary rse that s probably the consequence of the prolonged non-hlsulnc hypoglycaema and hence to the glucose defcency at the perpheral level [2]. Acknowledgments. We are most grateful to Dr. H.A. 0oMs who performed the mmune-assays of nsuln. One of us, E.C., was supported by "Fends Lekme-1%opsy". References [1] BALASSE, E., 0. THYS et V. CONARD: Acton comparge de dff@rentes doses d'nsulne sur la glycdme et le taux des acdes gras lbres du plasma (F.F.A.) chez l'homme normal. Arch. nt. Physol. 71, (1963). [2] -- W. MALASSE, J.1%.M. FRANCKSON et V. CONARD : Acton du chlorpropamde sur les blans hdpatque et splanchnque des acdes gras du ehen normal. Arch. nt. Pharmacodyn. 155, (1965). [3] BELLENS, 1%. : Contrbuton ~ l'gtude des mdcansmes d'acton des drogues hypoglycdmantes. Acta Endocrn., Kbh. 38, (suppl.) (1961). [4] BERNSTEN,.A., K. LEUTZ, M. MALM, P. HAMBYE and H.G. WooD: Degradaton of glucose 14C wth leuconostoc mesenterodes; alternate pathways and tracer patterns. J. bol. Chem. 215, (1955). [5] BJORNTORP, P. : The effect of f-hydroxybutyrc acd on glycerol outflow h'om adpose tssue n vtro. Metabolsm 15, (1966). [6] COLWELL, J.A., and A. LEN: Effect of ste of admnstraton of nsuln on blood glucose and fatty acd concentratons. Dabetes 12, (1963). [7] CONA~D, V.: Mesure de l'assmlaton du glucose. Bases th@orques et applcatons clnques. Bruxelles, Acta reed. belg. 6d., [8] -- J.1%.M. FRANCKSON, P.A. BASTENE, J. KESTENS et L. KOVAOS: Etude crtque du trangle d'hyperglyc6me ntraveneuse chez l'homme normal et d6- termnaton d'un coeffcent d'assmlaton glucdque. Arch. nt. Pharmacodyn, 93, (1953). [9] DEMOSS, 1%.S., R.C. BARD and J.C. GUNSALUS: The mechansm of heterolactc fermentaton. J. Bact. 62, (1951). [10] DOLE, V.P.: A relaton between the non-esterfled fatty acds n plasma and the metabolsm of glucose. J. cln. nvest. 35, (1956). [11] FAJANS, S. S., J. C. FLOYD, Jr., 1%. F. KNOPF and J. W. CONY: A comparson of Leucne-and Acetoacetatenduced hypoglycema n Man. J. cln. nvest. 43, (1964). [12] FELTS, P.W., O.B. CROFFORD and C.1%. PARK: Effect of nfused ketone bodes on glucose utlzaton n the dog. J. cln. nvest. 43, (1964). [13] FRANCKSON, J. 1%.M., H.A. Oo~s, R. BELLENS, Y. ARNOULD and V. CONAlCD: Acton d'une perfuson ntraportale d'nsulne sur la glycgme art6rehe et le d6bt glucos6 h6patque du chen. Arch. nt. Pharmacodyn. 142, (1963). [14] HALL, L.M.: Preferental oxdaton of acetoacetate by the perfused heart. Bochem. bophys, res. cornman. 6, (1961). [ 15] HAYSON, 1%. W., and Z.Z. ZPORN 9 Factors"nfluencng the utlzaton of ketone bodes by mouse adpose tssue. J. Lpd. 1%es. 7, ~(1966). [ 16] HAVEL, 1%. J., A. NAMARK and C. F. B ORCHGREVNCK. Turnover rate and oxdaton of free fatty acds of blood plasma n man durng exercse: studes durng contnuous nfuson of palmtate -1-14C. J. cln. nvest. 42, (1963). [17] HOFFMANN, W. S. : A rapd photoelectrc method for the determnaton of glucose n blood and urne. J. bol. Chem. 120, (1937). [18] JOaNSON, 1%.E., F. SARGENT and 1%. PASSMORE: Normal varatons n total ketone bodes n serum and urne of healthy young men. Quart. J. exp. Physol. 43, (1958). [19] MADSON, L.L., D. MEBANE, 1%.H. UNGER and A. LOeH~VER: The hypoglycemc acton of ketones.. Evdence for a stmulatory feedback of ketones on the pancreatc beta cells. J. cln. nvest. 43, (1964). [20] MALASSE, W., Y. z~lgould, E. RASO, V. CONARD and J.R.M. FRANCKSON: Acton of a supramaxmal nsuln load on the glucose hepatc balance of anaesthetzed dogs under strct normoglycemc condtons. Dabetologa 1, (1965). [21] MEBANE, D., and L.L. MADSON: Hypoglycemc acton of ketones.. Effects of ketones on hepatc glucose output an perpheral glucose utlzaton. J. Lab. cln. Med. 63, (1964). [22] MCtAELS, G.D., S. MARGEN, G. LEBERT and L.W. KNSELL: Studes n fat metabolsm.. The colormetrc determnaton of ketone bodes n bologcal fluds. J. cln. nvest. 30, (1951). [23] MORGAN, C.R. and A. LAZA~OW: mmunoassay of nsuln; two antbody system. Dabetes 12, (1963). [24] RANDLE, P.J., C.N. MALES, P.B. GARLAND and E. A. NEWSHOLME: The glucose fatty-acd cycle. ts role n nsuln senstvty and the metabolc dsturbances of dabetes melltus. Lancet 1963 H, [25] SHOEMAKER, W.C., P.J. CARRUTHE~S, D.H. ELWYN and J. AS~ORE: Effect of nsuln on fatty acd transport and regonal metabolsm. Amer. J. Physol. 203, (1962). [26] STEELE, R., J.S. BSHOP, A. DUNN, N. ALTSZULER,. RATHGEB and R.C. de BoDe : nhbton by nsuln of hepatc glucose producton n the normal dog. Amer. J. Physol. 208, (1965). [27] TA~DNG, F. and P. SCHAMBYE: The acton of sulfonylureas and nsuln on the glucose output from the lver of normal dogs. Endocrnology 36, (1958). [28] WLLA~SON, J.R., and H.A. KREBS : Acetoacetate as fuel of respraton n the perfused rat heart. Bochem. J. 80, (1961). [29] WNCKLER, B., R. STEELE, N. ALTSZULER and R.C. DE BODe: Effect of growth hormone on free fatty acd metabolsm. Amer. J. Physol. 21}6, (1964): [30] ZERLE~, K.L., and D. 1%ABNOWTZ: Effect of very small concentratons of nsuln on forearm metabolsm. Persstence of ts acton on potassum and free fatty acds wthout ts effect on glucose. J. cln. nvest. 43, (1964). E. BALASSE Departments of Expermental Medcne and General Pathology Unversty of Brussels 115, Boulevard de Waterloo Bruxelles 1 - Belgum 33*