Prospect, New South Wales, Australia

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1 J. Phy8il. (1968), 198, pp With 5 textftgure8 Printed in Great Britain CHANGES IN PLASMA GLUCOSE, LACTATE AND FREE FATTY ACIDS IN LAMBS DURING SUMMIT METABOLISM AND TREATMENT WITH CATECHOLAMINES By G. ALEXANDER, S. C. MILLS AND T. W. SCOTT Frm the C.S.I.R.O., Divisin f Animal Physilgy, The Ian Clunies Rss Animal Research Labratry, Prspect, New Suth Wales, Australia (Received 21 Nvember 1967) SUMMARY 1. Expsure t cld increased the plasma levels f glucse and lactate in yung lambs (< 163 days ld). Free fatty acid cncentratins als increased, the increase being much greater in lambs lder than 2 days. 2. Intravenus infusins f adrenaline and nradrenaline int lambs under a neutral thermal envirnment caused large increases in plasma glucse, lactate and free fatty acids. 3. Adrenaline and nradrenaline infusins increased the cncentratin f palmitic, stearic and leic acids in the plasma f yung lambs. 4. Treatment f lambs in summit metablism with either r, adrenergic blcking agents (phentlamine r prpranll, respectively) caused the plasma levels f free fatty acids t fall. Phentlamine hydrchlride decreased the cncentratin f plasma glucse, but did nt change the lactate level. In cntrast, prpranll decreased the cncentratin f bth f these metablites in a significant prprtin f the experiments. 5. Inductin f muscular paralysis by suxamethnium chlride, in lambs expsed t cld, resulted in a rise in plasma free fatty acids, and a fall in glucse and lactate levels. 6. These bservatins suggest that the circulating level f plasma metablites in yung lambs is nt the limiting factr in the metablic respnse t cld expsure. INTRODUCTION In the preceding paper (Alexander & Williams, 1968), an attempt was made t assess the shivering and nnshivering thermgenic cmpnents f heat prductin in cld expsed lambs.

2 278 G. ALEXANDER, S. C. MILLS AND T. W. SCOTT In the same study, data were btained n the levels f varius plasma metablites (glucse, lactate and free fatty acids) in lambs under 'basal' and 'summit' metablic cnditins. Analyses were als undertaken n plasma metablites during the infusin f catechlamines, a and, adrenergic receptr inhibitrs and suxamethnium chlride. A preliminary accunt f this wrk has already been published (Mills, Sctt & Alexander, 1967). METHODS Details f lambs, the surgical preparatins, infusins f substances, bld sampling and ther techniques were described in the preceding paper (Alexander & Williams, 1968). Chemical analy8i8. Plasma glucse was measured by the glucse xidase prcedure f Huggett & Nixn (1957). Plasma lactate was determined using the lactic dehydrgenase technique (Lundhlm, MhmeLundhlm & Vams, 1963). Plasma free fatty acids (FFA) were estimated by the methd f Dle (1956) as mdified by Annisn (196). After titrating the alchlic extract (cntaining FFA) with 1 NNaOH, water (2. ml.) and excess alkali were added and the cntents shaken twice with 3 ml. f light petrleum (b.p. 4 6 C) t remve ther plasma lipids. The alchl:water mixture cntaining the sdium salts f the fatty acids was acidified with NHCl and the FFA was extracted int light petrleum (b.p. 46' C). The methyl esters f the fatty acids were prepared by the technique f Schlenk & Gellerman (196) and separated int individual cmpnents by gasliquid chrmatgraphy using prcedures described by Sctt, Bassett & Setchell (1967) and Sctt, Vglmayr & Setchell (1967). Stati8tical analy8i8. The binmial distributin with P = was used t assess whether the varius treatments had an effect n the plasma metablites in a significant prprtin f experiments n lambs f all ages. Prbability values were btained frm the tables f the Binmial Prbability Distributin, Natinal Bureau f Standards Applied Mathematic Series 6 (195). RESULTS Changes in plasma levels f glucse, lactate and FFA in lambs during basal and summit metablism. N changes were bserved in the cncentratins f plasma glucse, lactate and FFA while the lambs (1day r 4dayld) were under basal cnditins (Fig. I a). These lambs had indwelling catheters (see preceding paper, Alexander & Williams, 1968) and were receiving infusins f saline ver the entire perid. In cntrast, in a significant prprtin f lambs f all ages expsed t severe cld (i.e. during summit metablism), there was an increase in the cncentratin f plasma glucse (mean increase 123 mg/1 ml., P < 1) and lactate (mean increase 43 mg/1 ml., P < 1) (Fig. lb; Tables 13). This increase ccurred despite the inevitable variatins in the cncentratin f plasma metablites with age. In 1dayld lambs expsed t cld, seven ut f ten shwed small increases in plasma FFAs (Tables 13) and in the lder lambs (> 2 days) larger increases were bserved (Fig. 2; Tables 13). Cld expsure increased the plasma cncentratin in a significant number f experiments (mean increase 367.,uequivJl.. P < 5). It is apparent

3 METABOLIC CHANGES IN LAMBS 279 that the age f the lamb and variatins between animals cntributed t the difference in FFA levels (Tables 13), but it is mst unlikely that the change bserved, as a result f either cld expsure r ther treatments mentined belw, can be attributed t ther factrs. The animals were nt fasted fr any significant perid (see Alexander & Williams, 1968) and the values recrded were immediately befre and generally within 3 min after the treatment cmmenced. 2 (a) (b) E S 1t E S S.12 Pl : 12 I 4 _ 4 4 L7 ~.A Ta.. T p, 4% _': 2 _ / cz 4 15 F_ 1 & ^ A Time (min) I %% " 3 a Fig. 1. Plasma levels f glucse, lactate and free fatty acids in lambs during basal (cntrl) and summit (S) (cld expsure) metablism. (a) Basal:, lamb 175 (1dayld); A 1A, lamb 72 (4dayld). (b) Summit:, lamb 221 (< 1 dayld); El O, lamb 78 (1dayld). E = time lamb is placed in cld chamber. Effect f adrenaline and nradrenaline n the level f plasma metablites in lambs under basal cnditins. Infusins f adrenaline int ne lamb under a neutral thermal envirnment prduced dramatic increases in the cncentratin f plasma glucse, lactate and FFA (Fig. 3); similar results were bserved in tw ther lambs. Thus in a significant prprtin f the experiments n three lambs f varius ages, adrenaline increased plasma glucse (mean increase 9 mg/1 ml.; P < O1), lactate (mean increase 41 mg/1 ml.; P < 1) and FFA (mean increase 832,uequiv/l.; P < 1). Similarly, nradrenaline infusins (Fig. 4) als increased the cncentratins f glucse (mean increase 9 mg/1 ml.; P < 1),

4 28 G. ALEXANDER, S. C. MILLS AND T. W. SCOTT lactate (mean increase 19 mg/1 ml.; P <.1) and FFA (mean increase 1193,tequiv/1.; P < 1) in a significant prprtin f experiments. Adrenaline (Fig. 5a) and nradrenaline (Fig. 5b) increased the cntent f palmitate (16: ), stearate (18: ) and leate (18: 1) in the plasma f 7 r 6 I 4 I 3 F t x 1 I a 8 A x t & ~ A. x A~~~~~~ s 6 Lamb age (days) Fig. 2. Effect f cld expsure n the percentage change in plasma free fatty acids in lambs (163 days ld). *, Lamb 197; A, 218;, 46, x, 8; A, 194; L, 227; *, 224, +, 15; C), 29; V, 24. lamb 196 at 11 and 18 days ld, respectively. Smaller increases were bserved in linleate (18:2) and linlenate (18:3). The five fatty acids represented mre than 8 % f the plasma FFA fractin. Effect f a and f8adrenergic receptr inhibitrs n plasma glucse, lactate and FFA in lambs during summit metablism. Phentlamine hydrchlride was used as an areceptr blcking agent and in a significant pr

5 METABOLIC CHANGES IN LAMBS prtin f experiments it decreased the cncentratin f glucse (mean decrease 4 mg/1 ml., P <.3) and FFA (mean decrease 291,tequiv/l., P <.6), but had n effect n the level f lactate (P >.39) (Table 1). This trend was bserved in five lambs (Table 1). N age pattern was nted. Prpranll (flreceptr inhibitr) decreased the cncentratin f glucse (mean decrease 39 mg/1 ml., P < 6), lactate (mean decrease 4 (a) L (b) L (c) L L 8 44 I? ~ I 8~~ 7,ApA~tf 1 1 i3 ~> 15 3/16 13 ~~~~15 ~~~~ Time (min) Fig. 3. Effect f adrenaline n plasma levels f glucse, lactate and free fatty acids in lamb 196 under basal cnditins. (a) ldayld: 4, 3,tg/kg single injectin;s 1,ug/kg/min (27 min), fllwed by 5,utg/kg/min (25 min). (b) 11dayld: 4, 15,ug/ kg single injectin;, 5,ug/kg/min (2 min). (c) 39dayld, 4, 3 1g/kg single injectin: 1, 1,g/kg/min (3 min). mg/1 ml., P < 1)and FFA (mean decrease 652uequiv/1., P <.6) in a significant prprtin f experiments n lambs examined at different ages (Table 2). Effect f suxamethnium chlride n plasma levels f glucse, lactate and EPA in lambs during summit metablism. In a significant prprtin f experiments, guxamethnium chlride increased the cncentratin f plasma FFA (mean increase 56462equiv/l., P < 19), but decreased the levels f glucse (mean decrease 16 mg/1 ml., P < g19) and lactate (mean decrease 23 mg/1 ml., P < 19) (Table 3).

6 282 G. ALEXANDER, S. C. MILLS AND T. W. SCOTT (a) L (b). (c) e F s A F~~~~~3 t 3 ta L.~ 1 1 AA~ Fig Time (min) Effect f nradrenaline n plasma levels f glucse, lactate and free fatty acids in lamb 195 under basal cnditins. (a) 1dayld: 4, 17 pg/kg single injectin;, 33 ug/kg/min (3 min). (b) 8dayld: 4, 15 jug/kg single injectin; +, 5 ptg/kg/min (2 min), fllwed by 1 jig/kg/min (15 min). (c) 33dayld: 4,, 6 jig/kg single injectin, +, 1 jig/kg/min (45 min). 3 2 DISCUSSION The mst striking bservatin in the present studies is the dramatic increase in the cncentratin f plasma glucse and lactate in lambs during expsure t cld. Alexander (1962a) nted an increase in ttal reducing sugar in plasma frm 1 t 3dayld lambs during summit metablism. Dawkins & Hull (1964) als bserved increases in the cncentratin f glucse and lactate in the plasma f newbrn rabbits expsed t cld cnditins. On the ther hand, in nnmammalian species, Freeman (1966) fund n elevatin in bld sugar levels f 1 t 21dayld chicks during expsure t a temperature f 4 C and, in fact, reprted a fall in the 7dayld fwl. It is appaient that there are differences between species in their metablic respnses during expsure t cld. Mrever, there als appear t be cnsiderable differences in the metablic respnse f newbrn animals t catechlamines, which are thught t be the prime mediatrs f the nnshivering respnse during cld (Mre & Underwd, 1963; Carlsn, 1966). In newbrn kittens, rabbits and rats, nradrenaline prduced a much greater thermgenic respnse than adrenaline (Mre & Underwd, 1963)

7 METABOLIC CHANGES IN LAMBS 283 but n measurements were made n plasma metablites. Similar results in thermgenic respnse were btained by Scpes & Tizard (1963) fr newbrn kittens and these authrs als reprted that bth adrenaline and nradrenaline prduced very little increase in bld lactate (frm 1 t 1 mg/1 ml.), but elevated bld glucse by abut 4 mg/1 ml. 4 r 36 F 32 F 28 F (a) v F I F F K 16: 18: 18:1 FFA 18:2 18: : 18: 18:1 18:2 18:3 FFA Fig. 5a. Effect f adrenaline (3 jtg/kg single injectin fllwed by infusin at the rate f 1 jtg/kg/min fr 2 min) n the cncentratin f individual plasma free fatty acids in lamb 196 (11dayld). E, befre adrenaline; El, after adrenaline. Fatty acids (16: palmitic, 18: stearic, 18:1 leic, 18:2 linleic, 18:3 linlenic) were separated by gas liquid chrmatgraphy. b. Effect f nradrenaline (15,ug/kg single injectin fllwed by infusin at the rate f 5 Atg/kg/min fr 2 min, then 1 Atg/kg/min fr 2 min n the cncentratin f individual plasma free fatty acid in lamb 196 (18dayld)., befre nradrenaline; EO, after nradrenaline. Fatty acid nmenclature as described in a. In newbrn guineapigs (1 t 8dayld), expsed t a neutral thermal envirnment, adrenaline increased 2 cnsumptin t the same extent as nradrenaline (Dawes & Mestyan, 1963) but n bld analyses were reprted. In the present experiments, infusins f adrenaline and nradrenaline int lambs (1 t 4dayld) under basal cnditins caused I9 I n Phy. i98

8 284 G. ALEXANDER, S. C. MILLS AND T. W. SCOTT s P4 ) ~b * 1: 4 C :O1 t 1 cq c * 1 C 1 CO 1 C C C _ 1 = c1 1 1 CO C O CO C 1 4 1' CO 11 1 ~~~~~~~~~t _ w ) 1$ 34 _ CO 11=q CO CO C CO1' 1C 11 1 CO COm Uit 1 CO c CZ cq H CO CO CO CO 1 CO 1 11 CX = 1 CO CO 1 1_I P ~ 1 I' 1 r COC4 1 t'4 C 1 1 1q CB e t>4 t.1 1 CO H 1~~~ amc CO 1O CO 11 t' 1 1 CO 1 1.S OC) (M P4 C C 34 4 b * c CO 1 1 CO u: 1e4 CO 1 C i CO 11 aq N 1 CO r c r CO 1 1 m 1 C Q8 w,z c 4 _ C) n 34 3' ~~~~~~~~~~' v r _ v 11 q V c CO) COl b e b * 1

9 METABOLIC CHANGES IN LAMBS 285. ^Y =~~~~~~~~a. ~st N 1 m e~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 Q t m t:> di CD C Cli L. q _ nx P~~~~~~~~~~~~~~~~~n CO CO 1C 4S._~~~~~~~C rlp1 ci rc._ 8~~~~~~ t1l r E _ ~~~~P4 Q O4 1 q;l4 t r cl ri tx C P zlcs*4 l SCOH c* C 1 1*wC C4 X ~ ~~~~~~ CD 4 Cq COC Cl Ul' Q (M.d4 p _ N N al csc c qcs es cq c'l s A: aq cs cs cs cq m q c* q cq q aq e O O e _ e uz c~~q r tp 1 P IV c 1. * a a c = qcv m alc4 r Cq P4~> P D 1 P _4 4 _ P4 P4 4 P X. * > t ~~~~~~ 4 "I (M14 C) = P4 1 Cs~ e 192

10 286 C. ALEXANDER, S. C. MILLS AND P. W. SCOPT fri C X> >1 =N1 ~~~~~~~~~~~~~~ b.>.qccq et c a 4 " X 1 1 C 1 4 m O ~ C) Pr _ t b b 1 > e "X ;> za aq F c t A N.1 a _ C4 OC4 L (m b W 1 C 11 W P4 ~ ~ 1 c C C t ;_ r ~ ^ c] cacq cq c Pcq 5 2l t '44. r~~~~( cq( go 1 C),,Q r. _ * O b X X s b b X > > b2 C 1 in c 1 c 3.q l a. _ ': CD ) O ; H 4 r tj s l_ x1cc b_ X :;, > e 44~~~~~~~~~~~~~~~~~P ~~~~~~~~~~ >~ 4 OD l m a qd4 C) (m 4 ~~~~~~* ": cc 1 Cq bli~~~~

11 METABOLIC CHANGES IN LAMBS 287 similar increases in the plasma levels f glucse, lactate and free fatty acids, and, mrever, the thermgenic respnses were similar (Alexander & Williams, 1968). Thus, in sme species, adrenaline cannt be discunted as the mediatr f nnshivering thermgenesis. In newbrn lambs, it wuld appear that smewhat less than half f the heat prduced during summit metablism results frm nnshivering thermgenesis (Alexander & Williams, 1968), and this cntributin declines as the animal grws lder. In the newbrn rabbit, the site f nnshivering thermgenesis is in the brwn adipse tissue (Hull, 1966) and a similar situatin prbably exists in the newbrn lamb which cntains apprximately 24% fat (Alexander, 1962b; Bdy & Shrland, 1964), the majrity being f the brwn adipse type. Dawkins & Hull (1964) detected nly a small increase in the plasma levels f free fatty acids in cld expsed newbrn rabbits and, similarly, the increases bserved in plasma free fatty acids in the present studies were als small in the majrity f lambs (13 days ld) during expsure t cld; these results cnfirm thse reprted by Alexander (1962a). The result is cnsistent with the cnclusin f Ball & Jungas (1961) that the majr prtin f the fatty acids frmed frm triglyceride liplysis (via the activatin f a catechlaminesensitive lipase; Rizack, 1964) are either xidized r reesterified within the brwn adipse tissue cell, the net result being increased heat prductin. Hwever, as the animal grws lder, the prprtin f brwn adipse tissue decreases and that f white adipse tissue increases; thus, during cld expsure f lder lambs, there tends t be a larger increase in the cncentratin f plasma free fatty acids (Fig. 2) because the rate f liplysis exceeds the rate f reutilizatin within the adipse cell. The increase in the majr individual plasma free fatty acids (namely, palmitic, stearic and leic) in lambs fllwing infusins f adrenaline and nradrenaline suggests direct mbilizatin frm adipse tissue, since previus analysis by Sctt, Bassett & Setchell (1967) has shwn palmitic, stearic and leic acids accunt fr 18, 12 and 49 %, respectively, f the neutral lipid fatty acids in perirenal fat. The present results with specific adrenergic blcking agents exemplifies the cnclusin f Hagen & Hagen (1964) that it is nt pssible t classify the receptrs n which adrenaline and nradrenaline act t prduce their effects n carbhydrate metablism. Thus, infusins f phentlamine hydrchlride (xadrenergic inhibitr) int lambs, whse bld glucse and lactate cntent were already elevated as a result f cld expsure, decreased the cncentratin f glucse but had n effect n lactate levels in a significant prprtin f the experiments. On the ther hand, prpranll (/?adrenergic inhibitr) reduced bth lactate and glucse cncentratins in a significant number f bservatins n lambs which had

12 288 G. ALEXANDER, S. C. MILLS AND T. W. SCOTT elevated plasma levels as a result f summit cnditins. Mrever, bth phentlamine hydrchlride and prpranll decreased plasma levels f free fatty acids in lambs expsed t cld, suggesting that bth and, sympathetic receptrs are invlved r that these substances have a direct effect n fatty acid metablism (see Wenke, 1966). The increase in plasma free fatty acid cncentratin in lambs paralysed by infusin f suxamethnium chlride implies that the rate f fatty acid utilizatin by the muscle is decreased r, alternatively, that the drug itself has a direct r indirect effect n liplysis. In mst experiments, suxamethnium chlride prduced a fall in plasma lactate and glucse and this prbably reflects a decrease in the rate f muscle glycgenlysis. The high cncentratins f glucse and lactate bserved in the plasma f newbrn and yung lambs during cld expsure suggest that the circulating level f substrate is nt the limiting cmpnent in the capacity f the animal t respnd and it wuld appear that ther factrs are vital in the prcess f thermgenesis. REFERENCES ALEXANDER, G. (1962a). Temperaturn regulatin in the newbrn lamb. Aust. J. agric. Ree. 13, ALEXANDER, G. (1962b). Energy metablism in the starved newbrn lamb. V. Summit metablism. Aust. J. agric. Res. 13, ALEXANDER, G. & WILLIAMS, D. (1968). Shivering and nnshivering thermgenesis during summit metablism in yung lambs. J. Physil. 198, ANNISON, E. F. (196). Plasma nnesterified fatty acids in sheep. Aust. J. agric. Res. 11, BALL, E. G. & JUNGAS, R. L. (1961). On the actin f hrmnes which accelerate the rate f xygen cnsumptin and fatty acid release in rat adipse tissue in vitr. Prc. natn. Acad. Sci. U.S.A. 47, BODY, D. R. & SHORLAND, F. B. (1964). Maternal and fetal lipids f sheep. Nature, Lnd. 22, 769. CARLSON, L. D. (1966). The rle f catechlamines in cld adaptatin. Pharmac. Rev. 18, DAWES, G. S. & MESTYAN, G. (1963). Changes in xygen cnsumptin f newbrn guinea pigs and rabbits n expsure t cld. J. Physil. 168, DAWKINS, M. J. R. & HULL, D. (1964). Brwn adipse tissue and the respnw f newbrn rabbits t cld. J. Physil. 172, DOLE, V. P. (1956). A relatin between nnesterified fatty acids in plasma and the metablism f glucse. J. clin. Invest. 35, FREEMAN, B. M. (1966). The effects f cld, nradrenaline and adrenaline upn the xygen cnsumptin and carbhydrate metablism f the yung fwl (Gallus Dmesticus). Cmp. Bichem. Physil. 18, HAGEN, J. H. & HAGEN, P. B. (1964). Actins f adrenaline and nradrenaline n metablic systems. In Actins f Hrmnes n Metablic Prcesses, ed. LITWACK, G. & KRIT CHEVSKY, D., p Lndn: Jhn Wiley and Sns, Inc. HUGGET, A. ST G. & NIXON, D. A. (1957). The use f glucse xidase, perxidase, and dianisidine in determinatin f bld and urinary glucse. Lancet 273, HULL, D. (1966). The structure and functin f brwn adipse tissue. Br. med. Bull. 22, LUNDHOLM, L., MOHMELUNDHOLM, E. & VAMOS, N. (1963). Lactic acid assay with L( +) lactic acid dehydrgenase frm rabbit muscle. Acta physil. scand. 58,

13 METABOLIC CHANGES IN LAMBS 289 MILLS, S. C., SCOTT, T. W. & ALEXANDER, G. (1967). Influence f catechlamines n plasma levels f free fatty acids, glucse and lactate in newbrn lambs. Prc. Au8t. Bichem. Sc. p. 23. MOORE, R. E. & UNDERWOOD, M. C. (1963). The thermgenic effects f nradrenaline in newbrn and infant kittens and ther small mammals. A pssible hrmnal mechanism in the cntrl f heat prductin. J. Phy8il. 168, RIZACK, M. A. (1964). Activatin f an epinephrinesensitive liplytic activity frm adipse tissue by adensine 3',5'phsphate. J. bil. Chem. 239, SCHLENK, H. & GELLERMAN, J. L. (196). Esterificatin f fatty acids with diazmethane n a small scale. Analyt. Chem. 32, SCOPES, J. W. & TIZARD, J. P. M. (1963). The effect f intravenus nradrenaline n the xygen cnsumptin f newbm mammals. J. Phy8il. 165, SCOTT, T. W., BASSETT, J. M. & SETCHELL, B. P. (1967). Characterizatin and metablism f vine fetal lipids. Bichem. J. 14, SCOTT, T. W., VOGLMAYR, J. K. & SETCHELL, B. P. (1967). Lipid cmpsitin and metablism in testicular and ejaculated ram spermatza. Bichem. J. 12, WENKE, M. (1966). Effects f catechlamines n lipid mbilizatin. Adv. Lipid Re8. 4, 6915.