brady_effects of fasting on body composition.pdf

7/30/2019 brady_effects of fasting on body composition.pdf

1/10

The Effects of Fasting on Body Composition,G lucose Turnover, Enzym es and M etabolitesin the Chicken1LINDA J. BRADY, DALE R. ROMSOS,PAUL S. BRADY, WERNER G. BERGENANDGIL BER T A . LE VEILLED epartm ents o f F ood Science and H um an N utritionand A nim al H usband ry, M ichig an State U niversity,E ast Lan sing, M ich igan 4 8824

ABSTRACT Chickens (1,200 g) were fasted 1, 4, or 8 days. S ignificantdecreases occurred in total body protein and fat with fasting, with thegreatest energy loss from fat. G lucose production determ ined with either[2-3H ] or [6-3H ]g lucose injected sim ultaneously w ith [U -14C ]glucose rem ained constant with fasting at 10 to 13 m g/m inute/kg body weight whichis m uch higher than reported for m ammals. B lood lactate and glycerol w ereunchanged with fasting, while pyruvate increased and plateaued. Plasm aalanine, serine and glycine levels w ere extrem ely high com pared to valuesin fasted m ammals. B lood /3-hydro xybutyrate increased dram atically w ithfasting (350 to 3,500 nm /m l), while acetoacetate rem ained constant. Thehepatic lactate : pyruvate ratio was unchanged with fasting, while the/3-hydroxybutyrate : acetoacetate ratio increased. These ratios have beenreported to influence ph osphoeno lpyruvate (P EP ) and glucose pro ductionin mammals. Hepatic and renal phosphoenolpyruvate carboxykinase(P EPCK) levels rem ained constant, w hile hepatic lactate dehydrogenasein cre ase d w ith fa stin g. -H yd ro xy bu ty ra te d eh yd ro ge na se le ve ls w ere v erylow at all tim es. The results indicate little glucose sparing adaptation perkg in the fasting chicken. J. Nutr. 108: 648-657, 1978.INDEXING KEY WORDS chicken enzymes glucose turnover m eta bo lite s p ro lo ng ed fa st

The fasting state in the human and dog species: rat, rabbit, guinea pig and pigeonis characterized by decreased plasma glu- (7, 8, 10-14).cose levels (1-3), decreased glucose turn- The chicken, however, has been re-over (3, 4), decreased plasma gluconeo- ported to maintain plasma glucose levelsgenie am ino acid levels (3, 5), and in- and glucose turnover rates during fastingcreased blood ketone levels (2, 3, 6). The (15, 16). An oxidized mitochondrial redoxsubcellular location of a key regulatory en- state is associated w ith increased phos-zyme in hepatic glucose production, phosphoenolpyruvate (PEP) and glucose pro-phoenolpyruvate carboxykinase (PEPCK), duction in guinea pig liver, in which 50%is also sim ilar in these two species, di- of PEPCK is m itochondrial, and it hasvided approximately equally between the been proposed that in other species w ithc yto so lic a nd m ito ch on dria l c ompa rtm en ts(8, 9). The subcellular location of PEPCK .. , .. ,,...,. , ,1077. n , n r i i-i- Received for publication August 22, 1977.influences the flux of carbon and reducing i s upported in part by NIH AM 13957 and GMequivalents between mitochondria ana om.DRR ^^^^^^L^CytOSOl in the gluconeogenic State in many priment Station Journal Article No. 8224.

64 8


2/10

EFFECT OF FASTING IN THE CHICKEN 649m itochondrial PEPCK, an oxidized m ito-chondrial redox state would be favorableto PEP and glucose production (8). Thisis in contrast to the rat, where a reducedm itochondrial redox state has been shownm ost favorable to PEP and glucose production (8). The pigeon and chicken possessa lm ost e ntire ly in tramito ch on dria l PEPCKin liver, which in the pigeon, has been reported to remain constant with fasting(14). Thus, it was of interest to examinethe effect of fasting on body com position,g lucose turnover, m eta bolites used in redoxe stim atio n, g lu co ne og en ic m eta bo lite s, a ndenzym es involved in glucose productionand mediating redox reactions in thechicken.

METHODSOne day old male broiler chicks wereobtained from a commercial source2 andfe d a n on -p urifie d h ig h-c arb oh yd ra te d ie t.3Water was available ad libitum . Roomlights were on 24 hours a day. When thechickens reached approxim ately 1,200 g,the experiments were started. Measurements were made in the fed state, and at1, 4, and 8 days of fasting.Experiment 1. Chickens (seven pergroup) were killed on the appropriatedays and whole body composition was obtained. The carcasses were dried 48 hoursat 80and ground twice through a meatgrinder (feathers included) to a homogenous consistency. Total nitrogen (17) wasdeterm ined; protein was assum ed to equalnitrogen X 6.25. Total fat w as determ inedg ra vime tric all y a fte r c hlo ro fo rm :meth an ol(2 :1 ) e xtra ctio n.E xperim ent 2. C hickens (five per g roup)w ere divided into 3 groups upon reaching1,200 g. G lucose turnover m easurem entswere performed on days 1, 4, and 8 offasting. Indwelling catheters4 were implanted 1 hour before the start of the experiment. Forty fiCi of [2-3H] or [6-3H]-glucose (specific activity500 mCi/mmole) w as injected sim ultaneously w ith5 jtiC i [U -1 4C ]g lu co se (sp ec ific a ctiv ity 180 mCi/mmole) into chickens on appropriate days. Blood samples were drawn attimed intervals and samples processed aspreviously desc ribed (15).Experiment 3. Chickens (10 per group)

w ere divid ed in to five grou ps. T he chick ensin the first group were bled from a wingvein in the fed state and at 1, 4, and 8 daysof fasting. Five m l of blood were drawnfrom each bird in G roup 1 on appropriatedays and placed in iced tubes containingheparin. After shaking, 2 ml of wholeblood were precipitated in 8 ml of 6% tri-chloroacetic acid (TCA). The tubes werecentrifuged at 3,000 X g for 15 minutesand the resulting supernatant neutralizedw ith 20% KOH. The tubes were centrifuged again at 3,000 X g for 15 minutesand assays done on the resulting supernatant. Three ml heparinized blood werealso centrifuged to obtain plasma foram ino acid analyses. Plasm a was precipitated w ith 1 0 volum es of 20% sulfosalicylicacid. N orleucine w as added as an internalstandard. Analyses were performed in alithium citrate buffer system using anautoanalyzer.5Groups 2-5 were used for hepatic andrenal m etabolite determ inations in the fedstate and at 1, 4, and 8 days of fasting. Inthese groups cervical dislocation w as perform ed sim ultaneously w ith a m id ventralincision below the sternum . The sternumand overlying m uscle were rapidly raisedand the liver was exposed and freezeclamped with tongs precooled in liquidnitrogen. The w hole process took no m orethan 5 seconds. Im mediately after freeze-clamping the liver, a kidney was rapidlyremoved, rinsed in buffer, blotted, andfrozen in liquid nitrogen. A pproxim ately15 to 20 seconds elapsed before kidneysw ere frozen. Frozen tissues w ere w eighedin previously tared tubes containing 6%TCA and were homogenized.6 The ho-mogenate was centrifuged at 10,000 X gfor 15 m inutes and the supernatant was decanted and neutralized as with blood samples. Before analyses, supernates weretreated to rem ove flavins.7L actate (18), p yruvate (19), -hydroxy -butyrate (20) and acetoacetate (21) levels2 F airview Farm s, R em ington, Indiana.Master Mix Chick Starter, Central Soya, FortW ayn e, I nd ia na .4 B ecton-D icklnson, R utherford, N ew Je rsey.5 T echnicon Instrum ents, Tarrytown, New Y ork." P olytron, Brinkm an Instruments, W estbury, NewYork.7 F ioristi, Fisher Laboratory C hem icals, Falrlawn,N ew Je rse y.


3/10

65 0 BRA DY ET A L .TA BLE l

B od y w eig ht a nd c om po sitio n o f c hic ke ns fa sted fo r 0 , 1 , 4 , o r 8 d ays1 (e xp erim en t 1 )Day s f as te d

B ody w eight (g)L iv er w eight (g)K idney w eight (g)T otal protein (g)T otal f at (g )1 ,2 11

15 12)30 .5 1 .510)2 .3 0 .110)NDND1,083

14b(10)25. 0 0 .88 )2.4 0.3 (8)216 5 7)1 48 11 *( 7) 92 2

15 11)18 .2 0 .8C (11)1 .40 .111)197 4' (7)109 1P (6)778

18' ' (11)11. 9 0 .8 " ( 10 )1 .0 0 .1< (10 )187 4" (7)61 12' (7)1M eariS EM .T he num be r o f c hic ken s is in dic ated in p aren th eses . N um bers in th e sam e lin e w ith d if fe ren tsuperscript letters are signif icantly dif ferent (P < 0.05). N D = not determ ined.

w ere determ ined by standard enzym aticm ethods. In addition, glutam ine and glutam ate (22) and a-k etoglutarate lev els (23)w ere determ ined in k idney and liv er.Experim ent 4. Chickens ( five per group )w ere either fed or fasted 1, 4, or 8 day s. Onthe appropriate day , chick ens w ere k illedand liv er and k idney rem oved for enzym eanaly sis. L iv er w as hom ogeniz ed8 in 10v olum es 0.25 M sucrose containing 1 HIMreduced glutathione and 1 HIM EDTA .K idney s w ere hom ogeniz ed in 5 volum esof the sam e buf fer. Hom ogenates w erecentrifuged at 600 X g for 15 m inutes, andthe resulting supernatant w as recentri-f uged at 18,000 X g for 15 m inutes. T hesupernatant f rom this centrifugation w asused to assay lactic dehydrogenase. T hepellet obtained from this centrifugationw as resuspended in 10 v olum es of ice w aterand sonicated.8 T his suspension w as usedf or assay o f p ho sp ho en olp yru vate carb ox y -k in ase, g lu tam ate d eh yd ro gen ase an d /8 -h y-drox ybuty rate dehy drogenase. Enz ym esw ere determ ined by the f ollow ing m ethods :lactic dehy drogenase (EC 1.1.1.27) (25),glutam ate dehy drogenase (EC 1.4.1.2)(2 6), and -h y dro x y bu ty rate d eh y droge nase (EC 1.1.1.30) (27) by ox idation ofN A DH. PEPCK (EC 4.1.1.32) w as determ ined by the m ethod of Helm rath andB ieber (24) w hich inv olv ed f ix ation of 14C -bicarbonate into acid stable m alate. Enz ym e activ ities w ere ex pressed per m g protein. Protein w as determ ined by them ethod of L ow ry et al. (28).Data w ere analy zed by analy sis of v ariance (com pletely random ized design).T uk ey 's


4/10

EFFECT OF FASTING IN THE CHICKEN 65 1approxim ation of futile cycling a t th is pointi n me tabo li sm .G lucose replacem ent rates w ere significantly greater at every time period for[2-3H ]g lucose than [6-3 H]glucose. T herew ere no differences at any tim e in [U -14C ]-glucose replacem ent rates (m g/m inute/kg). Neither tritiated tracer suggested adecreased glucose production per kg bodyw eight w ith fasting. H ow ev er, glucose production per to tal anim al decre ased betw eendays 1 and 4 of fasting as the chickenslos t we ight .The percent "C -carbon recycling, w hichhas been used to estim ate the percentageof tricarbon units originally derived fromglucose that return to glucose, was of thesame order as found for dogs (3), andslightly higher than ponies (32), rats (33),and rabbits (34). It is suggested fromthese data that the chicken may not increase the am ount of tricarbon units fromglucose returning to glucose with a longfast.The constancy of glucose m etabolism isfurther reflected in the body glucose m ass.Most values agree very closely, w ith theexception of the [2-3H] glucose 8 dayvalue. H ow ever, this m ean w as influencedby one very high value in com parison withth e o th ers .Levels of circulating metabolites arepresented in table 3. G lycerol levels re

mained relatively constant with fasting.Lactate levels were higher than reportedfor rats (3, 35), w ith no significant changeduring fasting. Pyruvate levels increasedsignificantly betw een fed and 1 day of fastin g, b ut d id n ot in cre ase sig nific an tly th ereafter. Thus, the plasm a lactate :pyruvateratio declined from fed to 1 day of fasting,but did not decline further w ith fasting.B lood 3-hydr oxybu ty ra te l evel s i nc reas edsubstantially from fed to 1 day of fasting;blood acetoacetate levels rem ained fairlyconstant during the fast. Consequently,th e b lo od / J-h yd ro xy bu ty ra te : ac eto ac eta teratio increased markedly from the fed tothe fasted sta te.Plasm a am ino acid levels are presentedin table 4. Alanine and serine levels decreased slightly, but not significantly during the fast. G lycine levels rem ained relatively constant. T he c onstant levels of th esepossible gluco neogenic am ino acids agreeswell w ith constant levels of glycerol, pyruvate, and lactate and a constant rate ofglucose utilization during the fast. Threo-nine and methionine increased betweendays 1 and 4 while % cystine increased between days 4 and 8. Aspartate, glutamateand glutam ine, tyrosine, phenylalanine,and lysine exhibited no significant differences with fasting. As noted by others(36, 37), plasma lysine levels were highcom pared to m ammalian levels. B ranched

TABLE 2Glucos e me ta bo li sm in f as te d c hi ck en s1 * ( ex pe riment 2 )Day s f as te d

B ody w eight (g)P la sm a g lu co se (mg /1 00l)Glucoser ep la ceme nt r at e~2-3H]glucose(mg/min/kg)J6-3Hlglucose(mg/min/kg)|U-14Cjglucose(mg/min/kg)6-3H]glucose(mg/min/chick)Percent14Cecycling3Bodyg lu co se ma ss[2-3H]glucose(mg/kg)RPHJglucose(mg/kg)[U-^CJglucose(mg/kg)1,083

20813.110.78.311.522588551648922"2211.49.27.68.5'14572538645778'2413.311.07.88.528816'6977161.01.00.50.73686856

1Mea n fo r five c hic ken s. [2 -3 H]g lu cos e re pla ceme nt ra te w as g re ater th an [6 -3 H]glu co se re pla ceme nt ra teat all tim es. [U -"C Jvalues w ere com bined for both groups as there w as no significant difference betw eenthem . 2 N um bers in the sam e line w ith different superscript letters are significantly different (P < 0.05).3 Percent recycling = [6-3H ]glucose replacem ent rate m inut C U-14C ]glucose replacem ent rate divided byt he [ 6- 3H3g lu co se r ep la ceme nt r ate .


5/10

6 52 BRA DY ET A L .TA BLE 3

Ci rc ul at in g l ev el s o f me ta bo li te s i n f ed and fas te d chi ck en s1 -* ( ex pe rimen t 3 )Day s f as te d

Me tab oli te (nmol es /m llood)GlycerolLactatePyruvateLac tat e : py ru vat e/3-HydroxybutyrateAcetoacetate/3-Hydroxybutyrate :acetoacetate0ND

4,75018526362"18721180

3,040309"10 "2,4006103'23l4130

3,42021316 "3,690C556678110

4,03029814 "2,610"12221SEM40

4602033002091 M ean for 10 chick ens. N D = not determ ined,le tters are sig nif ican tly dif feren t (P < 0 .05 ).

chain am ino acids did not all respond inthe sam e m anner. V aline and leucine increased from 1 to 4 day s, then changedn o f urth er. Iso leu cin e f ell sig nif ican tly w ithf asting. T hree-M eth ylhistidin e increasedm ark edly w ith f asting, probably as a resultof increased m uscle breakdow n (38), although d ecreased ex cretion could also contrib ute to th e ris in g le ve ls . -N -meth y lly sin ealso in cre ased w ith f as tin g. U ric acid lev elstended to rem ain constant in agreem entw ith a prev io us stud y w ith f asting chick ens(16).

TA BLE 4Am in o a cid a nd u ri ca cid le ve ls i n t he f as te d c hic ke n1( ex pe riment 3 )

DaysastedAlanineSerineGlycinei

CystineThreonineAspartaeGlu+ GluH>ProlineMethionineTyrosinePhenylalanineV alineIsoleucineLeucineLysinee-N-MethyllysineArginineHistidine3-MethylhistidineUric

Ac id1plasr1,4492,3311,211462,981931,034535892261794953564062,8581313902347.22.948

(nmoles/ml)1 ,3182,0941,276444,258*1121,075539131216203823266 5482,436 418

1 M ean f or 10 ch ick ens. N um bers in the sam e line w ith diff e re nt s up ers cri pt l it te rs are s ig n if ic an tl y d if f ere nt ( P < O .OS ).

2 N um bers in the sam e line w ith dif ferent superscriptS ince the hepatic redox state has beenim plicated in control of PEP and glucoseform ation in som e m am m als (7, 8, 10, 11),m etabolites com monly used to estim ate

compartm e ntal red ox states w e re m e asu red(table 5). L iv er lactate rem ained quiteconstant, as did liv er py ruvate. T he liv erlac tate : py ru v ate ratio was not s ig nif ic an tlydif ferent durin g the f ast. L iv er /J-hy drox y-buty rate increased dram atically f rom f edto fasted, w hile acetoacetate lev els rem ained constant, thus the increased / -hy -drox ybuty rate :acetoacetate ratio. L iv erglutam ate and a-k etoglutarate increasedw ith fasting, as did glutam ine. T he glutam ate :a-k etoglutarate ratio decreasedw i th f as ti ng .T able 5 also contains data for k idneym etabo lite lev els. L actate v alues tended tobe higher than in liv er during fasting,possibly ref lecting increased anaerobicgly coly sis as this organ w as not f reeze-stopped as quick ly as liv er. Py ruv ate lev elsw ere of the sam e order as in liv er, as w asth e lac tate :p y ru v at e ratio . -Hydro x ybuty -rate and acetoacetate lev els and the /8-hy -d ro xy bu ty rate : aceto acetate ratio w e re similar to liv er. a-K etoglutarate did notincrease as m ark edly as in liv er, but glutam ate and glutam ine w ere higher, possiblydue to the critical role of k idney s in m aintaining acid-base balance, as w ell as theirpossible role in glucose production (39).T able 6 contains enzym e data for fedand fasted chick ens. L iv er and k idneyPEPCK ac tiv it y remain ed c on stan t th ro ughout the f ast. L iv er lactate dehy drogenaseactiv ity w as high and increased signif icantly w ith fasting, w hile k idney activ ity


6/10

EFFECT OF FASTING IN THE CHICKEN 653TABLE 5

L iv er a nd k id ne y m eta bo li te s in fa st ed c hi ck en s1 ( ex pe rim en t 3 )Days f as ted

Metabol it e ( nmole s/ g t is sue ) 1 SE MLiverLactatePyruvateLactate:yruvate/3-hydroxybutyrateAcetoacetate0-hydroxybutyrate

:cetoacetateGlutamatea-ketoglutarateGlutamate:-ketoglutarateGlutamineKidneyLactatePyruvateLactate

:yruvate/3-hydroxybutyrateAcetoacetatejS-hydroxybutyrate:cetoacetateGlutamatea-ketoglutarateGlutamate

:-ketoglutarateGlutamine1,570111143272921.13,62080451,0901,570166926126918,600

4,500"402^112,7201,20015081,120307

1Mean f or 10 chic kens . Numbers i n th e s ame li ne w ith d iff er en t s up er sc rip t le tte rs a re s ig ni fi ca ntly d ifferent (P < 0.05).remained constant. Liver and kidney glutam ate dehydro genase tended to be highestat 4 days, with no differences otherwise.L iver /3-hydrox ybutyrate dehyd rogenaseactivity was low at all tim es. Neither liverprotein concentration, nor kidney protein

concentrationfasting. varied significantly withDISCUSSIONThe response to fasting elicited in theyoung chicken was the maintenance of a

TABLE 6Enz yme a cti vi tie s in f as te d c hi ck en s1 ( ex pe rim en t 4 )

Days f as tedEnzyme (nmoles/m in/mg protein) SEM

LiverPEPCKLactatedehydrogenaseGlutamatedehydrogenase/3-hydroxybutyratedehydrogenasePro te in :m i tochondr ia (mg/gissue)Protein:cytosol(mg/gissue)KidneyPEPCKLactate

dehydrogenaseGlutamateehydrogenaseProtein:mi tochondr ia (mg/gissue)Protein:cytosol (mg/g tissue)6501,3409219"54547722,25094134425102,240"14935

51*499302,620

117103505005,300e22310

44508752,140

1348874k4705,44011216

46558252,36078102'4510

131281Mean f or 10 chic kens . Number s in th e s ame lin e w ith d iff er en t s up er sc rip t l ette rs a re s ig nif ic an tl y d ifferent (P < 0.05).


7/10

65 4 BRADY ET AL.constant rate of glucose utilization perkilogram body weight from 1 to 8 days. Inthe dog there is an initial drop in glucoseutilization from 1 day fast to 1 week, anda constant level from then on (3). Thisadaptation apparently did not occur in thechicken during the tim e period m onitored.In the chicken, the rate of glucose utilization per kg body w eight rem ained constantas w ell as plasm a levels of possible glucosep recursors. H ow eve r, total glucose u tilization per animal decreased with fasting inboth chickens (present study) and dogs(3), as both groups of animals lost bodyweight. Increased plasma levels of N-m ethyllysine and 3-m ethylh istidine c ouldbe indicative of increased muscle breakdown (38, 41) to provide amino acids forglucose production, energy, and essentialp ro te in sy nth esis. In cre asin g le ve ls o f th esemetabolites occurred concurrently withdecreasing carcass nitrogen from 1 to 4days. Plasma uric acid production did notin cre ase sig nific an tly , a n o bs erv atio n n ote dby others (16).Providing that the assum ptions of tracerm ethodology are correct ( 30, 31 ) and thatthe difference between the replacem entra te w ith [2 -3H]g lu co se a nd [6 -3H]g lu co seis indicative of "futile cycling" (40), itappears that the chicken possesses anactive "futile cycle" or "substrate cycle" atth e g lu co se -g lu co se -6 -p ho sp ha te sta ge . A lthough statistics cannot be applied to themean differences at each time period obtained in the present experiment, valuesobtained are quite sim ilar: 2 .3, 2.2, and 2.3mg/minute/kg for 1, 4, and 8 days, re-specively. A lthough this "substrate cycle"has been thought of as ATP wasting, a different interpretation h as recently bee n p roposed (40). This interpretation suggeststhat the m ulti-functional character of glu-cose-6-phosphatase is at least partly responsible for glucose cycling at the glu-cose-glucose-6-phosphate step, and thatthis is an ex change reaction w hich requiresn o ene rg y.The percent 14C-carbon recycling suggests that a co nstant level of tricarbon u nitsoriginally derived from glucose w as againreincorporated into glucose. The calculation is the difference betw een replacem entrate w ith [6-3H ]glucose and replacem ent

rate with [U-14C]glucose, but its significance is not entirely clear at this point dueto several problems. One problem in theuse of 14C to estimate carbon recycling ispossible dilution of the 14C label by un-labeled CO2 and acetyl CoA in the Kreb'scycle. This dilution leads to underestimates of carbon flow back into glucose(33). It is also possible that 14 CO 2derivedfrom labeled glucose may be reincorporated back into glucose, thus increasingthe specific activity of glucose in the blood(42).L evels of possible glucose precursors invivo in the plasm a also rem ained constantthroughout the fast: lactate, pyruvate,glycerol and alanine. In one study plasm aglycerol levels were reported to increasein chickens fasted 24 hours w ith no furtherchange between 24 to 96 hours of fasting( 1 6 ). H ow ever, le vels o f plasm a precu rsorsare often not a reliable index of turnover.Hall et al. (43) found that fasting increased glycerol production without significant changes in blood glycerol. A lthough fed plasm a alanine levels were notobtained in this study, others have shownthat these are also quite high when compared to m ammals (36, 37, 44).Total blood ketone levels increaseddram atically in this study betw een fed andfasted chickens. The significance of thisin regard to fuel utilization is unclear atthis point. Perhaps levels need to build upto a certain threshold for transfer andutilization in tissues to occ ur.T he re do x s ta te o f c ellu la r c ompa rtm en tshas been estim ated by the cellular lactate:pyruvate ratio (cytosol) and the -hy-droxybutyrate :acetoacetate ratio (m itochondria) (45). The assum ptions inherentin these measurements are that; 1) them etab olites resid e in one com partm ent onlyor have free access across m em branes suchthat equal distribution can occur; 2) theactivity of the mediating enzyme be highenough to keep the reaction close toequilibrium ; 3) the enzym e m ediating thereaction m ust be confined to one com partm ent. A lterations in these ratios have beenshown to be correlated with changes inPEP and glucose production in rat, rabbit,guinea pig and pigeon (7, 10-14).In species which possess a cytosolic


8/10

EFFECT OF FASTING IN THE CHICKEN 655PEPCK, a reduced mitochondrial redoxstate is associated with increased PEP andglucose production. However, in speciesw ith p re domin an tly m ito ch on dria l PEPCK ,lik e th e c hic ke n, a n o xid iz ed m ito ch on dria lstate is associated with increased PEP orglucose production (7, 10-14). The datapresented here show m aintenance of glucose production, but an increased /3-hy-droxybutyrate : acetoacetate ratio in liverand kidney in a species w ith m itochondrialPEPCK. This appears anomalous but ac lo se lo ok a t -h yd ro xy bu ty ra te d eh yd ro -genase activity could explain these data atleast partially. The activity reported inchicken liver in this study is very low.This has also been shown to be true inpigeon liver (46). Thus, this pair of metabolites does no t satisfy criteria for a redoxm etabolite indicator pair because the lowe nz ym e a ctiv ity d oe s n ot fu lfill a ssump tio n2) above. On the other hand, glutamatedeh ydrogenase a nd lactate dehydro genaseshowed higher activities which allowedtheir use in redox estimation. The glutam ate : a-keto glutarate ratio decreased w ithfasting, as expected for a species withm itochondrial PEPCK . The lactate :pyru-vate ratio was constant with fasting. It hasbeen found that dietary alterations affecthepatic lactate : pyruvate ratios. In the rat,fa stin g in cre ase s th e la cta te :p yru va te ra tio ;while in the chick, 2 hours fasting decreased the ratio (47). In this study, nofurther decrease occurred after 1 day offasting.Liver and kidney PEPCK activities w erequite high in the chickens and suggested ah igh capacity for gluco se production w hichw as reflected in the glucose turnover ratesobtained. If enzym e activities obtained forvarious species are compared, it appearsthat avian capacity is very high. Soling( 14 ) com pared rat, guinea pig, and pigeontotal PEPCK activity and found pigeonliver enzym e activity 2 to 30 tim es greaterdepending on dietary state. Furtherm ore,both rat and guinea pig cytosolic enzymeactivity increased with fasting, whilepigeon liver m itochondrial enzyme remained constant. In this study, chickenliver and kidney enzyme activities alsorem ained constant at a high level. Guineapig m itochondrial enzym e has been shown

to increase with fasting in one study (48).In summary, the results show that thefasting chicken did not decrease glucoseutilization per kg body weight, but diddecrease glucose utilization per anim al asw eigh t loss occurred. P lasm a lactate, pyruvate, glycero l, and a lanine levels rem ainedconstant with fasting. Blood /3-hydroxy-butyrate values increased dram atically reflectin g increased production or decreasedutilization and/or excretion. The differences in avian versus m ammalian m etabolism in fasting provide a basis for com parison under other types of nutritional alteration.L ITERATURE C ITED1. Owen, O., Feiig, P., Morgan, A ., Wahren,G . & Canili, G . (1969) Liver and kidneym etabolism during prolonged starvation. J.C lin. Invest. 48, 574-582.2. C ahill, G ., H errera, M ., M organ A ., Soeldner,J., S teinke, J., L evy, P ., R eichard, G . & K ipnis,D . (1966) H orm one-fuel interrelationshipsduring fasting. J. C lin. Invest. 45, 1751-1769.3. Brady, L., Armstrong, M ., Muiruri, K .(Bergen, W . G., Romsos, D . R. & Leveille,G . A . ( 1977 ) Influence of prolonged fasting on glucose turnover and blood metabolites in the dog. J. Nutr. 107, 1053-1060.4. Kreisberg, R., Pennington, L. & Boshell, B.(1970) Lactate turnover and gluconeogene-sis in normal and obese humans: effect ofs ta rva tio n. D iab ete s 1 9, 5 3-6 3.5. Felig, P ., Owen, O., Wahren, J. & Cahill, G .(1969) Amino acid metabolism during prolonged starvation. J. Clin. Invest. 48, 584-594.6. W iener, R ., Hirsch, H . & Spitzer, J. (1971)

C erebral extraction of ketones and their penetration into the CSF in the dog. Am. J.P hy si ol. 2 20 , 1 54 2- 15 46 .7. Hanson, R. (1974) The choice of animalspecies for studies of m etabolic regulation.Nutr. Rev. 32, 1-8.8. Soling, H . & Kleineke, J. (1976) Speciesdependent regulation of hepatic gluconeo-genesis in higher anim als. In: G luconeogene-sisIts Regulation in M amm alian Species(Hanson, R. W . & Mehlman, M . A., eds.),W iley & Sons, New York.9. Belo, P. S., Romsos, D . R. & Leveille, G . A.( 1 976 ) Influence of diet on glucose tolerance, on the rate of glucose utilization, ando n glu co neo ge nic e ri2 ym e a ctiv ities in th e d og .J. N utr. 106, 1465-1474.10. Garber, A . & Hanson, R. (197I) The control of phosphoenolpyruvate formation byrabbit liver m itochondria. J. Biol. C hem . 246,5555-5562.11. Garber, A . & Hanson, R. (1971) The interrelationships of the various pathways forming gluconeogenic precursors in guinea piglever m itochondria. J. B iol. Chem . 246, 589-598.


9/10

65 6 BRADY ET AL.12. Arinze, L , Garber, A . & Hanson, R. (1973)The regulation of gluconeogenesis in mamm alian liver. J. B iol. C hem . 248, 2266-2274.13. Soling, H ., W illms, B ., Kleineke, J. & Gehl-hoff, M . (1970) Regulation of gluconeogenesis in guinea pig liver. Eur. J. B iochem .

16, 2 89 -3 02 .14. Soling, H ., Kleineke, J., W illm s, B., Janson,G. & Khn,A. (1973) Relationship betw een intracellular distribution of phospho-enolpyruvate carboxykinase, regulation ofgluconeogenesis, and energy cost of glucoseform ation. Eur. J. B iochem . 37, 233-243.15. Belo, P. S., Romsos, D . R. & Leveille, G . A.( 1976 ) Blood metabolites and glucose metabolism in the fed and fasted chicken. J.N utr. 1 06 , 11 35 -1 14 3.16. Evans, R . & Scholz, R . (1971) M etabolicresponses of chicks during adaptation to ahigh protein "carbohydrate-free" diet. J.N utr. 1 01 , 11 27 -1 13 6.17. Horwitz, W . (ed.) (1960) Official M ethodsof Analysis of the Association of OfficialA gricu ltu ra l C hem is ts, pp . 6 43 -64 4.18. Gutman, I. & W ohlfield, A . (1974) Lclate.In: M ethods of Enzymatic Analysis (Berg-meyer, H ., de.), pp. 1464-1468, AcademicPress, N ew Y ork.19. Czok, R. & Lamprecht, W . (1974) Pyru-v ate , p hos pho eno lp yru va te , an d D -g ly ce ra te-3-phosphate. In: Methods of EnzymaticA nalysis (Bergm eyer, H ., d.), p p. 1446-1451, A cadem ic Press, N ew Y ork.20. W illiamson, D. & Mellanby, J. (1965)/3 -h yd ro xy bu ty ra te. In : M eth od s o f E nz ym aticA na ly sis (B ergm ey er, H ., d.),p p. 4 59 -4 61 ,A cadem ic Press, N ew Y ork.21. Mellanby, J. & Williamson, D. (1965)Acetoacetate. In: M ethods of EnzymaticA nalysis (B ergm eyer, H ., d.),pp. 454-457,A cadem ic P ress, N ew Y ork.22. Lund, P. (1974) Determ ination with gluta-

m inase and glutamate dehydrogenase. In:M ethods of E nzym atic A nalysis (B ergm eyer,H ., d.), pp. 1719-1722, Academic Press,N ew Y ork.23. Bergmeyer, H . & Bemt, E. (1974) 2-Oxo-glutarate. In: M ethods of Enzym atic A nalysis[Bergmeyer, H ., d.), pp. 1577-1580, Academ ic Press, N ew Y ork.24. Helmrath, T. & Bieber, L. (1974) Developm ent of gluconeogenesis in neonatal pig liver.A m. J. P hysiol. 227, 1306-1313.25. Bergmeyer, H . & Bemt, E . (1974) Lactatedehydrogenase. In: M ethods of EnzymaticA naly sis (B ergme yer, H ., d .),p p. 5 74-5 79 ,A cadem ic Press, N ew Y ork.26. Schmidt, E. (1974) Glutamate dehydrogenase. In: M ethods of Enzymatic Analysis(Bergmeyer, H ., d.), pp. 650-656, Academ ic Press, N ew Y ork.27. W ilkinson, J. (1974) 2-Hydroxybutyratedehydrogenase. In: M ethods of EnzymaticA na ly sis (B ergme ye r, H ., d.),p p. 60 3-6 07,A cadem ic Press, N ew Y ork.28. Lowry, O., Rosebrough, N., Fair, A . & Randall, R . (1951) Protein measurement with

the Folin phenol reagent. J. B iol. Chem .193 , 2 65 -2 75 .29. Steele, R . & Torrie, J. (1960) Principlesand Procedures of Statistics, M cGraw-HillCo., New York, pp. 99-109.30. Katz, J., Rostami, H . & Dunn, A. (1974)Evaluation of glucose turnover, body massand recycling w ith reversible and irreversibletracers. B iochem . J. 142, 161-170.31. Katz, J., Dunn, A., Chenoweth, M . & Golden,S. ( 1974 ) Determ ination of synthesis, recycling, and body mass of glucose in ratsand rabbits in vivo with 3H and "C labeledg lu cos e. B ioc hem . J . 1 42 , 1 71 -1 83 .32. Anwer, M ., Chapman, T. & Gronwall, R .(1976) Glucose utilization and recycling inponies. A m. J. P hysiol. 230, 138-142.33. Katz, J., Golden, S. & Chenoweth, M . ( 1976)E stim ation of glucose turnover in rats in vivow ith tritium labeled glucose. H oppe-S eyler'sP hy siol. C hem. 3 57, 1 38 7-1 39 4.34. Dunn, A., Katz, J., G olden, S. & Chenoweth,M . (1976) Estimation of glucose turnoverand recycling in rabbits using various ("H,"C ) glucose labels. A m. J. P hysiol. 230, 534-539.35. Romsos, D . R., Belo, P. S . & Leveille, G . A.(1974) Effect of 1,3-butanediol on hepaticfatty acid synthesis and metabolite levels inthe rat. J. N utr. 104, 1438-1445.36. Hill, D . & Olsen, E. (1963) Effect of starvation and nonprotein diet on blood plasmaamino acids, and observations on the detection of amino acids lim iting growth in chicksfed purified diets. J. N utr. 79, 303-310.37. Tasahi, I. & Takuo, O. (1971) Effect ofdietary protein level on plasma free am inoacids in the chicken. J. N utr. 101, 1225-1232.38. Young, V. (1970) The role of skeletal andcardiac muscle in the regulation of proteinm etabolism . In: M am malian Protein M etabolism (M unro, H ., d.), pp. 622-625, Academ ic Press, N ew Y ork.

39. Sykes, A . ( 1971 ) Formation and composition of urine. In: Physiology and Biochemistry of the Domestic Fowl (Bell, D . J. & Freeman, B. M ., eds.), pp. 266-271, AcademicP ress, N ew Y ork.40. Issekutz, B . (1977) Studies on hepaticg lu co se cy cles in n orm al a nd m ethy lp red niso -lone treated dogs. M etabolism 26, 157-170.41. Hardy, M . & Perry, S. (1969) In vitromethylation of m uscle proteins. Nature 223,300-301.42. Shipley, R . & Gibbon, A. (1975) Rate ofincorporation of C O2 carbon into glucose andother body constituents in vivo. Can. J.P hy sio l. P ha rm . 53 , 8 95 -9 02 .43. Hall, S., Hall, A ., Layberry, R., Berman, M .& Hetenyi, G . (1976) Effects of age andfasting on gluconeogenesis from glycerol indogs. A m. J. Physiol. 230, 362-367.44. Felig, P., Owen, O ., Wahren, J. & Cahill, G .(1969) Amino acid metabolism during prolonged starvation. J. Clin. Invest. 48, 584-594.45. Gumaa, K ., McLean, P. & Greenbaum , A.(1971) Compartmentation in relation to


10/10

EFFECT OF FASTING IN THE CHICKEN 657metabolic control in liver. In: Essays in Bio- 47. Yeh, Y. & Leveille, G . A. (1971) Studieschem istry, Vol. 7 (Campbell, P. & Dickens, on the relationship between lipogenesis andF., eds.), pp. 39-86, Academ ic Press, New the level of coenzyme A derivatives, lactateYork. and pyruvate in chick liver. J. Nutr. 101,46. Bailey, E. & Hrne, J. (1972) Formation 911-918.and utilization of acetoacetate and o-3-hy- 48. Elliott, K . & Pogson, C. ( 1 977 ) The effectsdroxybutyrate by various tissues of the adult of starvation and experimental diabetes onpigeon. Comp. Biochem. Physiol. 42B, 659- phosphoenolpyruvate carboxykinase in the667. guinea p ig. Biochem . J. 164, 357-361.

brady_effects of fasting on body composition.pdf

Documents