Ketone-Body Utilization by Adult and Suckling Rat Brain in vivo

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Bichem. J. (1971) 122, 13-18 13 Printed in Great Britain Ketne-Bdy Utilizatin by Adult and Suckling Rat Brain in viv By R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS Metablic Research Labratry, Nuffield Department f linical Medicine, Radcliffe Infirmary, xfrd X2 6HE, U.

1 Bichem. J. (1971) 122, Printed in Great Britain Ketne-Bdy Utilizatin by Adult and Suckling Rat Brain in viv By R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS Metablic Research Labratry, Nuffield Department f linical Medicine, Radcliffe Infirmary, xfrd X2 6HE, U.K. (Received 27 Nvember 197) 1. Ketne-bdy utilizatin in fed and starved adult and suckling rats has been investigated by measuring arteri-venus differences acrss the brain. Venus bld was cllected frm the cnfluence f sinuses and arterial bld frm the femral artery in adult rats and by cardiac puncture in suckling rats. 2. During starvatin the arteri-venus difference f ketne bdies increased in prprtin t their cncentratins in the bld and reached a value f.16mm at 48h. At a given cncentratin f the respective ketne bdies the arteri-venus differences f acetacetate were abut twice thse f 3-hydrxybutyrate. 3. Fed rats in which the cncentratins fketne bdies were raised by intravenus infusin fsdium acetacetate had the same arteri-venus differences as starved rats at crrespnding ketne-bdy cncentratins. Thus the ability f the rat brain t utilize ketne bdies is independent f the nutritinal state. 4. The cncentratins f glucse, acetacetate and 3-hydrxybutyrate were much lwer in the brain than in the arterial bld. The measured (bld cncentratin)/(brain cncentratin) rati was 4.4 fr glucse, 4.5 fr acetacetate and 8.1 fr 3-hydrxybutyrate in 48h-starved rats. 5. The mean arteri-venus difference f glucse acrss the brain was.51 mm in fed rats and.43mm in 96h-starved rats. 6. nversin f glucse int lactate rse frm negligible values in the fed state t.2mm after 48h starvatin and decreased t zer after 96h starvatin. 7. In day-ld suckling rats the arteri-venus differences f ketne bdies acrss the brain were als prprtinal t the ketne-bdy cncentratin, but they were abut 3-4 times greater than in adult rats at the same bld ketne-bdy cncentratin. 8. Arteri-venus differences f glucse were abut the same in adult and suckling rats. 9. The brain f fed suckling rats frmed mre lactate frm glucse than fed adult rats. 1. The results indicate that ketne bdies are majr metablic fuels f the brain f the suckling rat under nrmal cnditins. Ketne bdies are knwn t be xidized by rat and guinea-pig brain slices (Drahta, Hahn, Murek & Trjanva, 1965; Rllestn & Newshlme 1967; Ide, Steinke & ahill, 1969; Ith & Quastel, 197). Measurements f the arteri-venus differences in bese human patients underging prlnged starvatin (wen et al. 1967) indicate that the human brain can als utilize ketne bdies in viv. Hwever, the wrk f wen et al. (1967) leaves it pen whether the utilizatin f ketne bdies by brain is an adaptatin f this tissue t prlnged fasting r whether it is a cnsequence f the high cncentratins f ketne bdies in the bld. In rats the necessary enzymes fr ketnebdy utilizatin have been shwn t be present in the brain irrespective f the nutritinal state (Williamsn, Bates, Page & Krebs, 1971). T determine the imprtance f bld cncentratin as a factr in the regulatin f ketne-bdy utilizatin, acetacetate was infused int the femral vein f fed rats and arteri-venus differences acrss the brain were measured. MATERIALS AND METHDS Rat&. Female rats f the Wistar strain, weighing abut 2g and suckling rats, days ld were used. All rats were allwed free access t fd unless therwise indicated. Reagent8 and analytical methd8. The reagents and analytical methds were the same as thse described by Page, Krebs & Williamsn (1971) except where indicated. Sdium acetacetate fr infusin was prepared frm ethyl acetacetate as described by Krebs & Egglestn (1941). The final slutin was cncentrated t 4.5M under vacuum. It cntained n ethanl when analysed by the methd f Dickinsn & Dalziel (1967). xygen in bld was determined by the methd f Van Slyke & Neill (1924).

14 R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS 1971 llectin f bld 8amples. Adult rats were anaesthetized by intraperitneal injectin f a freshly prepared aqueus slutin f Nembutal (5mg/kg bdy wt.).

2 14 R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS 1971 llectin f bld 8amples. Adult rats were anaesthetized by intraperitneal injectin f a freshly prepared aqueus slutin f Nembutal (5mg/kg bdy wt.). A catheter (Prtex Nyln Intravenus annula, size 12FG 3, external diameter.75mm with a bevelled end; Prtex Ltd., Hythe, Kent, U.K.) was placed in the femral artery after surgical expsure and thrugh it a heparin slutin (1 units per kg bdy wt.) was injected. Then a small hle was drilled thrugh the skull ver the cnfluence f the sagittal and transverse sinuses. The dura was penetrated with a needle and.2 ml f bld was cllected. The sinuses drain primarily the cerebral hemispheres and measurements f sinus bld therefre reflect metablism f this area. Arterial bld (.2 ml) was sampled frm the femral artery immediately befre and after the sinus sample. Arterial bld (.1 ml) frm suckling rats was taken frm the left ventricle f the heart. Sinus bld (.1 ml) was btained within 3s by insertin f a 25 gauge needle int the cnfluence f the sinuses. Bld samples were treated with 3ml f 2% (w/v) H14. A neutral deprteinized slutin was btained by the standard methd. Infusin f acetacetate. Adult rats were infused thrugh a catheter intrduced int the femral vein and psitined int the inferir vena cava. Acetacetate (4.5 M) was infused with a mtr-driven syringe at a rate f 15,tml/ min (.2 ml/h) fr 35 min t attain cncentratins fketne bdies apprximating t thse f starvatin. Higher cncentratins were attained by infusing acetacetate at a rate f 65juml/min (.86ml/h) fr 15min and the cncentratin was maintained by infusin at a rate f 3 tlml/ min (.4 ml/h) fr an additinal 2min. The rate f infusins maintained a steady cncentratin f ketne bdies and bld samples were taken at 35 min. Brain metablites. Anaesthetized rats were frzen in liquid N2 and the brains remved as described by Mark, Gdin & Mander (1968). Further treatment was as described by Williamsn, Lund & Krebs (1967) fr liver. Glycgen was measured by the methd f Lwry, Passnneau, Hasselberger & Schulz (1964). RESULTS Arteri-venus differences f ketne bdies and glucse in the brain f adult rats. The mean arterivenus difference f glucse acrss the brain f fed animals was fund t be.51 mm whereas the differences fr lactate and pyruvate were negligible (Table 2). Althugh the cncentratin f acetacetate was lw in the fed state (Table 1) small but significant arteri-venus differences (mean.22mm) existed (Table 2). The xygen required fr the cmplete xidatin f glucse and ketne bdies remved (taking int accunt the lactate and pyruvate prduced) during passage thrugh the brain was 3.8mM, a value in apprximate agreement with the measured arteri-venus difference f 2.84mM (Table 2). In rats starved fr 48h the arteri-venus difference f ketne bdies rse abut sevenfld t.16mm. The arteri-venus difference fketne bdies did nt increase when the starvatin perid was extended t 96h. After 96h e 3 *-._ ) 6 PQ cc.~.3 c3 c c c s > X c,-4,- r-4 m t- N t +i -- -n _ c t c5 1e ).+ 1^ 4a nm c- qnt L c cici c1 l c = t- " N ) ') ; ; i ; c) n3t- s I- t c m q c q c.-!! V cc H -fl H H H H n " c. "I ~~ c) -a -- -f 4 r- M 6 c 6 HH H H ^ c c H H N: Ns >t aq cl m c cq c >e s n scq c s V - q ed A a ) c -cd c n c c- 6 nc; ; nc ci PA -4 cq ncc6 m..- i. p-' l -fl cn - t- cn n L -,,d t1 c La D (m 1-14 ) r-4 r- m c H,;~c im t e) a= e r- t 1- _f Ul t- t L L Ṙ 36 W_ D ncc ct~ ~ ~~t~ = e 3 c c-: P4 4 P4, c

3 , eq D 3 -ee3 ' t;.4g e. a-52 S. -.4 ~.Q.< 5 u a- + t as * p8 c c D Fs c It t SI D q a3 H+ + -f ck t -,,D l E- c 4 1 e c )H H- tl N c + + * * * * * * * * * * >- 21 v a2 e g ^.... ) * * *2 cz X ; ce -c;; cq q R Vl. 122 KETNE-BDY UTILIZATIN BY RAT BRAIN IN VIV 15 starvatin the arteri-venus difference f glucse a decreased abut 16%. In cntrast with the brains f fed rats, thse f animals starved fr 48h, cnverted abut 2% f the glucse taken up int lactate. n prlnged starvatin (96h) the lactate prductin returned t almst zer. The calculated G arteri-venus difference f xygen remained cll+ cnstant thrughut starvatin indicating that ketne bdies partially replaced glucse as a fuel. When ketne-bdy cncentratins were raised t starvatin values by infusin f acetacetate int fed rats the arteri-venus differences were cmparable with thse f starved rats (Tables 1 cllx and 2). Rats infused with acetacetate t attain abnrmally high arterial cncentratins, shwed larger arteri-venus differences f ketne bdies. Acetacetate and 3-hydrxybutyrate were remved frm bld apprximately in prprtin t their cncentratins in the physilgical range by bth starved and acetacetate-infused fed rats (Fig. 1 and 2). Hwever, at a given cncentratin the acetacetate arteri-venus cncentratin difference was twice that f 3-hydrxybutyrate. This caused the 3-hydrxybutyrate/acetacetate cncentratin rati t increase after passage f the bld thrugh the brain (Table 1). Fed rats, infused with acetacetate at a high rate did nt prduce lactate and the calculated arteri-venus cncentratin difference f xygen (3.96mm) was nt significantly different (P<.49) frm the measured value (3.13mM) btained n + different rats.. ncentratin gradients f metablites between I brain and arterial bld. The cncentratins f * ; glucse, acetacetate and 3-hydrxybutyrate were strikingly lwer in the brain than in bld. The measured (cncentratin in bld)/(cncentratin - ) Efw 5.2 Fed-infused - 48h starvednj.' Fed-infused _~~ ~~~~~~~ I E c c L I H H H,_ q I *_ v ' Fed, 72h starved 96h starved S 6 nen. f arterial acetacetate (mm) I c G t e3 L c Y ~~~c ~~ri)rr~~~~~~ ~~4 Fig. 1. ncentratin dependence f acetacetate uptake by brain f adult rats. The arteri-venus differences and arterial cncentratins are mean values (+ S.E.M.) expressed as,uml/ml. Fr ther experimental details see the text.

4 16 R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS 1971 in brain) rati was 4.4 fr glucse, 4.5 fr acetacetate and 8.1 fr 3-hydrxybutyrate in 48h- there was cnsiderable variatin between litters a culated xygen cnsumptin f the brain. Althugh starved rats (Table 3). Since brain-tissue cncentratins were nt crrected fr bld cntent (abut f acetacetate and arteri-venus cncentratin linear relatinship between arterial cncentratins 3%; Mark et al. 1968; Hindfelt & Siesj6, 197) r differences and an apprximately linear relatinship fr 3-hydrxybutyrate existed (Figs. 3 and 4). fr the cntent f cerebrspinal fluid (abut 12%; Hindfelt & Siesj6, 197), the actual cncentratin As in adult rats the rate f acetacetate uptake was gradients must be much greater. Similar cncentratins f glucse have been fund in brain (Gey, given cncentratin. Hwever, 3-hydrxybutyrate twice as great as that f 3-hydrxybutyrate at a 1956; Lwry et al. 1964; Mark et al. 1968). Large cntributed mre t ttal ketne-bdy uptake due cncentratin gradients fr ketne bdies have t its higher circulating cncentratins (Tables 4 als been bserved in muscle (Harrisn & Lng, 194). Thus penetratin f ketne bdies int brain may be a factr limiting the rate f their metablism. Arteri-venus differences f ketne bdies and glucse in the brain f suckling rats. The results with suckling rats (Tables 4 and 5) shw that ketne bdies accunt fr a high prprtin f the cal- and 5). At a given cncentratin the arteri-venus differences f bth acetacetate and 3-hydrxybutyrate were abut 3-4 times thse bserved in adult rats. Als as was bserved in adult rats, starvatin did nt enhance ketne-bdy uptake (Figs. 3 and 4). Glucse uptake by brain f suckling rats was cmparable t, r less than, adult values but in cntrast with adult rats a cnsiderable prtin was cnverted int lactate (Table 5). 5 ) ;.. t.2 ncn. f arterial 3-hydrxybutyrate (mm) Fig. 2. ncentratin dependence f 3-hydrxybutyrate uptake by brain f adult rats. The arteri-venus differences and arterial cncentratins are mean values (±.E.M.) expressed as,uml/ml. Fr ther experimental details see the text. DISUSSIN The present experiments indicate that ketnebdy utilizatin by brain becmes imprtant whenever these substances are present at a sufficiently high circulating cncentratin. A critical experiment is the demnstratin that the arteri-venuis difference f ketne bdies acrss the brain is independent f the nutritinal state. Thus the arteri-venus differences are the same whether circulating ketne-bdy cncentratins are raised by starvatin r by infusin f acetacetate int fed rats. This is in agreement with the finding that the enzymes f ketne-bdy utilizatin are nt altered by the nutritinal state (Williamsn et al. 1971). Whether this is als true fr the human brain (see wen et al. 1967) remains t be tested, but it seems unnecessary t pstulate an adaptive increase in the enzymes necessary fr ketne-bdy Table 3. Metablite cncentratins in brain and arterial bld The values are means (± S.E.M.) f fur bservatins and are expressed in,uml/g f brain r,uml/ml f arterial bld. Fed-infused rats were infused with acetacetate at the high rate as described in the Materials and Methds sectin. The values fr brain were nt crrected fr cntaminatin by bld r cerebrspinal fluid. Fed rats Fed-infused rats 48h-starved rats Metablite Glucse Glycgen Lactate Pyruvate 3ydrxybutyrate Acetacetate Brain 1.21 ± ± ± ±.8.94 ± ±.5 Brain 1. ± ± ±.15.47±.2.117± ±.62 Bld 4.43 ±.27.34± ± ± 1.26 [Bld] [Brain] Brain ±.23.46± ± ±.28 Bld 4.14 ± ± ± ±.68 [Bld] [Brain]

5 Vl. 122 KETNE-BDY UTILIZATIN BY RAT BRAIN IN VIV 17 c 2t N l, t- c _1-1 r1 _I _. 1 ; ; 1 4i i _1 t-. 'Ir. t-*.. * * *. 2) 2) p). as la. * 1c -4 -i: 1 ț -- ar 2 a N,-41 e a.2.2, 2 -~ i i c i Z 2b t- 2 Ni 4. ) R..,) 2 f 2 ) 46).2 c1 2 2) Nu' D ' 2 D :c; c; (6 c; c c : H c. 2 ". c 4 t-4 2 t c 12 t c 1 2c - t a I 24 ~ 1' e )(z -4 H H H H 2 - ~ g 1 t Le i _i ci cq ri r-4 i- r- i r4 MI a3 a R -4 E- da '2 E p..r : E 2)4 d t f _ - p.- e a g Id.)._! e! q - S + 2 4H _ H ) ; _I M~~~ _ R^ IyN H * 4F 4F * * * H N s 2 N = 4 Y). -4. c. It 2-4F I 1 L N.42. G. P. d p 2:uX5 22 t.2 t 22-2 t i 6! v 6-4 t- R ; < * * ;J ; 2 c 2. m N _I-1 -I li ( 1 '?ax *9 i -H H H P " li Ji ; li -! " H c; ; 5 ; a t c -r H 1 4 _ 4 N H _- t--i 4 a N 2..-* ḻ 4 (1) I;i -4 ;; I$ -.- c ad A w D. 4)I r4 z -M) 4) S -4.'a 4.'.') pq I '9 I -S' k A..5 G m-. -M -., -a-d -, ' m. _)2 1,f :3 ~ g ~. Q (1) H c a t- 2 d *_Y N t- Xi inln _ N QR LS a._~ c t R a" '., Id: 2L) k, - t, es > a2r2 Id P c 2 N e V _ p $ a - al) 41-4 t' ( Pa 1 t 2)-s c e. _ - as -1 t- am $ e c W

18 R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS 1971.2.1I Fed c: ; t ~~~~~~~24h starved 6/l 16h starved 4h starved.2.4.6.8 nen. f arterial acetacetate (mm) Fig. 3.

6 18 R. A. HAWKINS, D. H. WILLIAMSN AND H. A. KREBS I Fed c: ; t ~~~~~~~24h starved 6/l 16h starved 4h starved nen. f arterial acetacetate (mm) Fig. 3. ncentratin dependence f acetacetate uptake by brain f suckling rats. The arteri-venus differences and arterial cncentratins are mean values (±S.E.M.) expressed as u.ml/ml. Fr ther experimental details see the text. utilizatin t explain the bservatins n human brain in prlnged starvatin. In the brain f suckling rats ketne bdies appear t be at least as imprtant as glucse as a surce f metablic fuel. The larger arteri-venus differences bserved acrss the brain f suckling rat is paralleled by the greater enzymic capacity fr ketne-bdy utilizatin bserved at this age (Page et al. 1971) and are in agreement with a higher rate f ketne-bdy xidatin shwn fr brain slices by Ith & Quastel (197). The authrs thank Mr. R. S. Hughtn fr his help in the develpment f the bld-sample techniques. This wrk was supprted by grants frm the Medical Research uncil and frm the U.S. Public Health Service (Grant n. AM 11748). D. H. W. is a member f the external staff f the Medical Research uncil. R. A. H. hlds a United States Public Health Service Pstdctral Research Fellwship (F2-AM-3,273-1) Fed 4h starved.3 Fed Fed h starved 1h starved.i nen. f arterial 3-hydrxybutyrate (mm) Fig. 4. ncentratin dependence f 3-hydrxybutyrate uptake by brain f suckling rats. The arteri-venus differences and arterial cncentratins are mean values (±s.e.m.) expressed as,uml/ml. Fr ther experimental details see the text. REFERENES Dickinsn, F. M. & Dalziel, K. (1967). Bichem. J. 14, 165. Drahta, Z., Hahn, P., Murek, J. & Trjanv&, M. (1965). Phy8ilgia bhem8lv. 14, 134. Gey, K. F. (1956). Bichem. J. 64, 145. Harrisn, H.. & Lng,. N. H. (194). J. bil. hem. 133, 29. Hindfelt, B. & Siesj, B. K. (197). Life Sci. 9, 121. Ide, T., Steinke, J. & ahill, G. E., jun. (1969). J. bil. hem. 217, 784. Ith, T. & Quastel, J. H. (197). Bichem. J. 116, 641. Krebs, H. A. & Egglestn, L. V. (1941). Bichem. J. 39, 438. Lwry,. H., Passnneau, J. V., Hasselberger, F. X. & Schulz, D. W. (1964). J. bil. hem. 239, 18. Mark, J., Gdin, Y. & Mander, P. (1968). J. Neurchem. 15, 141. wen,. E., Mrgan, A. P., Kemp, H. G., Sullivan, J. M., Herrera, M. G. & ahill, G. F., jun. (1967). J. clin. Inve8t. 46, Page, A., Krebs, H. A. & Williamsn, D. H. (1971). Bichem. J. 121, 49. Rllestn, F. S. & Newshlme, E. A. (1967). Bichem. J. 14, 519. Van Slyke, D. D. & Neill J. M. (1924). J. bil. hem. 56, 523. Williamsn, D. H., Bates, M. W., Page, A. & Krebs, H. A. (1971). Bichem. J. 121, 41. Williamsn, D. H., Lund, P. & Krebs, H. A. (1967). Bichem J. 13, 514.

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