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Calorigenic Effect of Norepinephrine Correlated with Plasma Free Fatty Acid Turnover and Oxidation Daniel Steinberg, … , Elsworth R. Buskirk, Ronald H. Thompson J Clin Invest. 1964;43(2):167-176. https://doi.org/10.1172/JCI104901. Research Article Find the latest version: https://jci.me/104901/pdf
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Page 1: Calorigenic Effect of Norepinephrine Correlated with ...

Calorigenic Effect of Norepinephrine Correlated with PlasmaFree Fatty Acid Turnover and Oxidation

Daniel Steinberg, … , Elsworth R. Buskirk, Ronald H. Thompson

J Clin Invest. 1964;43(2):167-176. https://doi.org/10.1172/JCI104901.

Research Article

Find the latest version:

https://jci.me/104901/pdf

Page 2: Calorigenic Effect of Norepinephrine Correlated with ...

Journal of Clinical InvestigationVol. 43, No. 2, 1964

Calorigenic Effect of Norepinephrine Correlated with PlasmaFree Fatty Acid Turnover and Oxidation *

DANIEL STEINBERG, PAUL J. NESTEL,t ELSWORTHR. BUSKIRK,4 ANDRONALDH. THOMPSON

(From the Laboratory of Metabolism, National Heart Institute, and the Metabolic DiseaseBranch, National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.)

The increase in oxygen consumption caused byadministration of epinephrine has been extensivelystudied in animals and in man. Many physiologi-cal and biochemical correlations have been madeand many different hypotheses have been ad-vanced to explain this so-called calorigenic effect,but there is still no clearly established consensusregarding the mechanism or mechanisms involved(1, 2).

In recent years it has become clear that one ofthe most striking metabolic effects of the cate-cholamines is their ability to stimulate mobiliza-tion of depot fat in the form of free fatty acids(FFA) (3). Studies by Fritz, Davis, Holtrop,and Dundee (4) and by Eaton and Steinberg (5)have shown, moreover, that the rate of oxidationof labeled FFA by isolated skeletal muscle prepa-rations in vitro is a function of the concentrationof FFA in the medium. Similar effects of FFAconcentration on FFA oxidation have been dem-onstrated in rat liver slices (6) and in perfusedrat liver (7). We have previously suggested,therefore, that the calorigenic action of catechol-amines might be due, at least in part, to their ef-fect in mobilizing FFA (5).

The present clinical studies were undertaken toexplore this possibility further. Because the meta-bolic effects of epinephrine are somewhat morecomplex than those of norepinephrine, in thatthe former actively mobilizes glucose as well asFFA, the present studies were done primarilywith norepinephrine. It is shown that infusionof norepinephrine increases the rate of oxygen

* Submitted for publication August 12, 1963; acceptedOctober 3, 1963.

tPresent address: Department of Medicine, RoyalMelbourne Hospital, Melbourne, Australia.

t Present address: Laboratory of Applied Physiology,Pennsylvania State University, State College, UniversityPark, Pa.

consumption in man and that this is accompaniedby an increase in turnover of plasma FFA and anincrease in rate of conversion of labeled plasmaFFA to respiratory C1402. All of these effects ofnorepinephrine are abolished or radically re-duced when the patient has first been given an in-travenous dose of pronethalol [2-isopropylamino-1-(2-naphthyl)ethanol hydrochloride], a drug thatinhibits catecholamine stimulation of adrenergic,8-receptors (8, 9) and that prevents the FFAmobilization induced by catecholamines (10).1 Apreliminary report of these findings has been made(11).

MethodsThe subjects studied were young, adult, normal, con-

trol volunteers on regular ward diets, with their totalcaloric intake adjusted to maintain constant body weight.Metabolic studies were all done after a 12- to 16-hourfast, and subjects remained at rest in bed on the morn-ings of studies. Indwelling plastic catheters were placedin an antecubital vein of each arm after the skin had beenanesthetized with Xylocaine (2-diethylamino-2',6'-aceto-xylidide). A slow drip of 0.85% NaCl was maintainedto keep the catheters open. The subj ects were thentaken to the metabolic chamber in a wheel chair. Theconstruction and operation of the metabolic chamberhave been fully described by Buskirk, Thompson, Moore,and Whedon (12). At least 30 minutes was allowed toestablish stable control values for rate of oxygen con-sumption. Blood samples were drawn from one armthrough a 3-way stopcock and delivered into a chilledtube containing heparin. Infusions of drugs, or labeledpalmitate, or both, were given into the opposite armwith Bowman finger pumps.

Plasma FFA were determined by Dole's method (13)using isobctane (2,2,4-trimethylpentane) in place of hep-

1 Since these studies were completed, the Research De-partment of Imperial Chemical Industries, Ltd., Maccles-field, England, manufacturers of pronethalol (Alderlin),has found that mice on chronic high dosage show a sig-nificant incidence of locally malignant lymphosarcomataof the thymus gland. Further use of the drug in clini-cal investigation should probably await thorough eva'u-ation of these preliminary findings.

167

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168 STEINBERG, NESTEL, BUSKIRK, AND THOMPSON

TABLE I

Effects of infused norepinephrine on FFA levels, FFA turnover, andoxygen consumption in normal control subjects

Norepinephrine Plasma Plasma Percentage 02 PercentageSubject, administration FFA FFA change in con- change insex, age, Dosage Blood Pulse concen- turn- FFA sump- 02 con-

wt rate Time* pressure rate tration over turnover tion sumption

,ug/min min mmHg pEq/ml pEq/min % ml/min %Control values 100/70 60 0.69 326 225

C.H. (I), 6 12 118/85 47 0.85 459 + 41 245 + 9M, 21, 6 22 1.14 533 + 64 255 +1366.6 kg 12 15 130/90 45 1.29 544 + 66 245 + 9

12 26 1.38 540 + 66 265 +18

Control values 95/65 70 0.36 215C.H. (II), Placebo

M, 21, infusionj 10 90/65 55 0.33 210 - 266.6 kg Control values 0.46 211

12 10 130/90 47 0.93 235 +11

Control values 100/60 55 0.46 205B.L., 10.5 15 110/80 48 1.16 221 + 8

M, 23, Control valuest 105/60 47 0.54 20566.1 kg 10.5 16 120/80 47 1.42 229 +12

Control values 110/65 58 0.48 226 192B.L., 6 10 115/80 48 0.69 280 + 24 202 + 5

M, 23, 6 27 1.65 677 +200 210 + 966.1 kg 12 22 124/84 46 1.61 573 +154 231 +20

20 18 150/90 50 1.81 250 +30

L.C.,M, 25, Control values 100/76 60 0.90 333 23572.6 kg 18 16 140/110 60 1.57 506 + 52 286 +22

S.P., Control values 90/60 50 0.39 155F, 17, 7.2 13 100/70 48 1.21 165 + 660.3 kg 14.4 14 1.69 181 +17

14.4 25 120/80 60 1.82 180 +16

* Times indicate the cumulative number of minutes from the start of administration at each dosage rate. Except where indicated by inter-vening control values, dosage rates were changed without interrupting the infusion.

t Saline infused by Bowman pump with identical routine of blood pressure readings and blood samplings that accompanied norepinephrineinfusions. Infusion identified to patient as "norepinephrine."

t Obtained 53 minutes after discontinuing first norepinephrine infusion.

tane. Blood glucose was measured enzymatically by theglucose oxidase method described by Keston (14).Reagents (Glucostat) were obtained commercially.2Plasma lactate levels were determined enzymatically (15).

Plasma FFA turnover was determined by using a con-stant intravenous infusion of palmitate-1-C'4. The basisfor this method has been fully discussed by Armstrongand associates (16). The infusion solution was preparedby adding a tracer amount of sodium palmitate-1-C'4 toa solution of human serum albumin (5 to 7.5%o). Thesolutions were prepared for clinical use and were testedfor sterility and absence of pyrogens.3 The labeled pal-mitate was diluted in 100 to 150 ml of 0.85%o NaCl andinfused at a rate of 1 to 1.5 ml per minute. Within 15to 20 minutes the total FFA radioactivity per milliliterof plasma had reached a plateau value that did not thenvary more than + 10% during the control period.Plasma FFA radioactivity was measured by first ex-

2 Worthington Biochemical Corp., Freehold, N. J.3 Performed by Mr. William H. Briner, Radiopharma-

ceutical Service, National Institutes of Health, Bethesda,Md.

tracting by Dole's method, re-extracting the F-FA fromthe isooctane layer into alcoholic KOH, acidifying thelatter, and finally re-extracting the FFA into isooctane.The isooctane was dried in a counting vial under astream of N2, the residue was redissolved in toluenecontaining 0.5% 2,5-diphenyloxazole, and radioactivitywas assayed in a Packard Tri-Carb liquid scintillationspectrometer. In several experiments the lipids in thefinal isodctane extract were fractionated on silicic acidcolumns (17). No significant amounts of radioactivitywere recovered with the phospholipid fraction.

For measurement of C1402 production, a measuredfraction of the gas stream from the metabolic chamberwas diverted and collected in 4- to 5-minute periods inDouglas bags. The gas from these bags was slowlypulled through a gas-flow meter and delivered througha sintered glass diffuser into a column of NaOH. ABa(OH)2 trap connected in series showed that collectionof CO, was complete. Three-ml samples of the NaOHwere added to 1 g of fluorescence-grade anthracene,5 and

4 Packard Instrument Co., La Grange, Ill.5 Distillation Products, Inc., Rochester, N. Y.

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CALORIGENIC EFFECT OF NOREPINEPHRINEAND FFA TURNOVER

radioactivity was assayed in the liquid scintillation spec-trometer ( 18).

Pronethalol (Alderlin) was generously donated.0Norepinephrine (Levophed) was diluted in 0.85%o NaClto a final concentration of 6.6 ,ug per ml (expressed asthe base).

Results

The changes in blood pressure, pulse rate, FFAconcentration, and oxygen consumption produced

6 Ayerst Laboratories, New York, N. Y.

by intravenous infusion of norepinephrine aresummarized in Tables I and II. In Table lI theresponses to a second norepinephrine infusion,given immediately after administration of pro-nethalol, are shown for ready comparison with theresponses in the control state. The effects ofnorepinephrine in the control state will be dis-cussed first.

Oxygen consumption. In every case infusionof norepinephrine at dosage rates of 10 to 21 ug

TABLE II

Blocking effect of pronethalol on the increased FFA turnover andhypermetabolism induced by norepinephrine

Norepinephrine Plasma Plasma Percentage 02 PercentageSubject, administration FFA FFA change in con- change insex, age, Dosage Blood Pulse concen- turn- FFA sump- 02 con-

wt rate Time* pressure rate tration over turnover tion sumption

pg/misn minBefore pronethalol

Control values18 12

W.S., 18 16M, 23, +2t65.1 kg After pronethalol

Control values18 818 16

Before pronethalolControl values

D.C., 10 15M, 27, After pronethalol79.1 kg Control values

10 710 910 15

Before pronethalolControl values

16 12B.L. (I), After pronethalol

M, 23, Control values66.1 kg 16 11

Control valuesi16 9

Before pronethalolControl values

R.B., 13 18M. 35, 21 1486.4 kg After pronethalol

Control values21 14

Before pronethalolControl values

B.L. (II). 18 13M, 23, After pronethalol66.1 kg Control values

18 718 14

mmHg MEq/ml pEqlmin % mi/min %

110/65 74 0.69140/85 52 0.86

1.36

110/75 70 0.860.73

142/90 50 0.69

96/64150/100

95/58115/80

130/85

110/68140/95

112/60140/100

420500

790

430430400

75 0.37 25885 0.90 444

72 0.39 29843 0.41 172

48 0.64 302

55 0.7042 1.05

59 0.6837 0.64

118/58 65 0.68140/105 38 0.59

100/68125/90140/105

110/68140/90

77 0.8655 1.1345 1.34

83 0.8644 0.69

270300

270190

270

450588572

460298

220+ 19.0 246

+135.5 262

2350.0 197

- 7.0 230

247+ 72.0 344

274- 42.3 260

220+ 1.3 295

205+ 11.1 256

184- 29.6 156

215140

215+ 30.7 235+ 27.4 245

229- 35.2 221

110/60 78 0.52155/83 57 1.16

0.85160/90 45 0.56

0.68

240306

236208229

169

+11.8

+19.1

-16.2- 2.1

+39.2

- 5.1-19.7+ 7.7

+24.8

-15.2

-23.9

+ 9.3+13.9

- 3.5

+27.5

-12.6- 3.8

* Times indicate cumulative number of minutes from start of infusion at each dosage rate. Dosage rates were changed without interruptingthe infusion.

t Values determined 2 minutes after discontinuing the norepinephrine infusion.Complete record for this study shown in Figure 1.I Control values obtained 29 minutes after discontinuing first infusion of norepinephrine.

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STEINBERG, NESTEL, BUSKIRK, AND THOMPSON

per minute (0.13 to 0.31 ptg per kg per minute)significantly increased the rate of oxygen con-sumption. The value at the peak of the response,reached toward the end of the 15- to 25-minuteinfusion period, was 21 ± 8% above control val-ues (11 cases shown in Tables I and II). Theresponses ranged from + 11 to + 397o.

FFA levels and turnover. Basal FFA levels inthe plasma, determined after the subjects were inposition in the metabolic chamber and just beforethe norepinephrine infusion was started, were0.58 ± 0.19 uEq per ml. At the time when themaximal response in oxygen consumption wasreached, FFA levels had risen to 1.28 ± 0.27 uEqper ml, an increase of 127 ± 57%o over basalvalues.

In seven subjects FFA turnover was deter-mined before and during norepinephrine treat-ment by using a continuous intravenous infu-sion of palmitate-1-C14. In every case norepi-nephrine caused an increase in turnover, but theresponse was variable, ranging from 11 to 154%oabove control values with a mean rise of 74 ±53%o (Tables I and II). The percentage increasein FFA turnover was greater than the percentageincrease in oxygen consumption in six of theseven cases. The mean percentage increase inFFA turnover for the seven cases studied (74%)was more than three times the mean percentageincrease in oxygen consumption (24%o).

Pulse and blood pressure. Within a minute ortwo after the infusion was started, blood pres-sure rose and pulse rate decreased. The brady-cardia, presumably a reflex response to the risein blood pressure, was generally most marked dur-ing the first 5 minutes, the heart rate returningtoward control values when the infusion wascontinued at a constant rate. At the time whenthe maximal changes in oxygen consumption wereobserved, the mean decrease in pulse rate was 9beats per minute. At that time systolic bloodpressure had risen 36 mmand diastolic 26 nmm ofHg above control levels.

A few patients reported headache or poundingin the head, and a few noted difficulty in "gettinga deep breath" during the first few minutes of theinfusion. These symptoms were transient, andmost patients had no discomfort during the latterpart of the infusion period. There were no otheradverse reactions.

Because fear and anxiety can cause elevationsof FFA and might lead to increases in oxygenconsumption, we restudied patient C. H., a some-what apprehensive individual, following the stand-ard protocol but infusing only 0.85%o sodium chlo-ride. The usual control blood samples were taken,the Bowmanpump was started, and the recordingtechnician was told to mark "norepinephrinestarted" at that time. There were no changes ofany significance in plasma FFA level or oxygenconsumption (Table I, C. H., II). After a restinginterval of 20 minutes, the same protocol was fol-lowed, this time with norepinephrine in the infu-sion. Both plasma FFA and oxygen consumptionrose significantly.

Conversion of palmiitate-l-C14 to C1402. Intwo studies measurements of respiratory C1402were made before and during norepinephrine ad-ministration as shown in Table III. In both casesthe net rate of production of C1402 increased.The plasma FFA levels rose and the plasma FFAspecific radioactivity fell at the same time. Thusthe calculated minimal rate at which plasma FFAwas being converted to C1402 rose to a greaterextent than would be indicated by considering theincrease in absolute rate of C1402 productionalone. The changes in oxidation of plasma FFAcalculated from concurrent measurements of FFAspecific radioactivity in plasma and total C1402 inrespiratory gas represented increases of approxi-mately 200% (Table III).

These calculations do not take account of thelarge size and relatively slow turnover of the bodybicarbonate pool. This will introduce a lag be-tween the time that stimulation of the rate of FFAoxidation in the tissues begins and the time whena new steady state rate of appearance of C14 inrespiratory CO2 is attained. The measurements inthe control period were made at least 30 minutesafter starting the infusion of labeled palmitate.The C1402 output in successive collections duringthe control period was very nearly the same, indi-cating that a steady state had been achieved, i.e.,the specific radioactivity of the bicarbonate poolwas relatively constant. The norepinephrine infu-sions, however, extended over a limited time pe-riod (14 and 17 minutes), and the specific radio-activity of the bicarbonate pool probably had notyet reached a new steady state level. Thus theestimated norepinephrine effects on the rate of

170

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CALORIGENIC EFFECT OF NOREPINEPHRINEAND FFA TURNOVER

TABLE III

Effects of norepinephrine on turnover and oxidation of serum FFA;modification by pronethalol treatment

Norepinephrine, Plasma Plasma Rate of MinimalSubject, administration FFA Plasma FFA respiratory oxidationsex, age, Dosage concen- FFA turn- C1402 of plasma 02 con-

wt rate Time* tration SA over production FFAt sumption

pg/min min pg/ml cpm/pEq pEqi cpm/min pEqi mil/minmin min

Before pronethalolControl values 0.36 308 201 3,500 11.3 231

0.41 273 264 3,800 13.9 25010 7 0.66 254 288 4,100 16.1 318

D.C., 10 14 0.84 146 416 4,900 33.6 332M, 27,79.1 kg After pronethalol

Control values 0.40 228 316 4,000 17.5 26810 9 0.58 258 279 4,000 15.5 26010 13 0.61 248 290 4,800 19.3 278

Control values 0.82 138 312 2,750 20.0 228L.C., 0.90 129 333 2,750 21.3 235

M, 25, 18 12 1.31 114 403 5,750 50.3 29372.6 kg 18 17 1.57 85 506 6,000 70.6 286

* Times indicate cumulative number of minutes from start of infusion at given dose rate.t Rate of production of respiratory C1402 divided by mean specific radioactivity of serum FFA determined at start

and at end of 4-minute collection of respiratory CO2 (counts per minute/minute)/(counts per minute/microequivalezit).

palmitate oxidation calculated in Table III mayunderestimate the magnitude of the true stimula-tion.

Effects of pronethalol. In six experiments pro-nethalol was given intravenously (75 to 100 mgover a 10-minute period) after the rate of oxygenconsumption had returned to control levels fol-lowing discontinuing of the control norepinephrineinfusion. A second infusion of norepinephrinewas then given at the same dosage rate used inthe first infusion, and the same parameters of re-sponse were measured (Tables II and III).

Pronethalol treatment did not materially alterthe changes in pulse rate and blood pressure in-duced by norepinephrine. On the other hand,the rises in plasma FFA levels and the rises inrate of oxygen consumption were radically re-duced or entirely abolished. The data from arepresentative experiment (Case B.L., I, in TableII) are presented graphically in Figure 1. Therise in blood pressure and the slowing of the pulserate were essentially the same with and withoutprior pronethalol treatment. In this case thesharp rise in plasma FFA level caused by nor-epinephrine was completely blocked by pronethaloltreatment. The first norepinephrine infusion ele-vated the rate of oxygen consumption by almost25%. As in most of the other studies, the eleva-

llJ ELIX,

80_7060 _50 _40_30_

170

I 130 -

E 90-50L_1.1_

<N., 0.9 -

LA UJi 0.7

z2 290

C Z 240C-H8rb 90 -

140_

B. L.M a23

I0-0.~~~N

NEAR NEO START STOP START STOP-NE_ NEN NE NE NE

- :

PRONETHALOL

0 15 30 45 60 75 90 105 120 135MINUTES

FIG. 1. EFFECTS OF NOREPINEPHRINEON OXYGENCON-SUMPTION, PLASMA FFA LEVELS, BLOOD PRESSURE, ANDPULSE RATE BEFORE AND AFTER ADMINISTRATION OF PRO-NETHALOL. Data from study of subject B.L. (I) (seeTable II), a 23-year-old male weighing 66.1 kg. Arrowsindicate points at which norepinephrine infusion was be-gun (START NE) and ended (STOP NE). Pro-nethalol, 100 mg, was given intravenously over the 10-minute period indicated. Norepinephrine was infusedat a rate of 16 ,ug per minute during each of the threeexperimental periods, one before and the other two afterpronethalol administration.

171

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STEINBERG, NESTEL, BUSKIRK, AND THOMPSON

tion persisted after the end of the infusion, andthe return to basal levels was rather slow. Inthis study two infusions of norepinephrine weregiven after the administration of pronethalol.The rate of oxygen consumption, instead of ris-ing, actually fell during these infusions and thenshowed some "rebound" above basal levels whenthe infusion was stopped. If the oxygen con-sumption during and after the norepinephrine in-fusion is integrated, the net change is close tozero.

In Tables II and III the responses are shownat the time of the peak in oxygen consumptionduring the control infusion of norepinphrine andthen at the corresponding time during the post-pronethalol infusion of norepinephrine. The "re-bound" is not shown, but was noted in all casesand was in no case greater in relative magnitudethan that shown in Figure 1. Plasma FFA levelsfell during the postpronethalol infusion of nor-epinephrine in four of the six experiments. Thelevels then rose to values equal to or greater thancontrol values when the norepinephrine infusionwas stopped.

In five studies the changes in FFA turnoverinduced by norepinephrine were measured be-fore and after administration of the blocker.Whereas the turnover was increased by norepi-nephrine given before pronethalol treatment, itwas either not changed or actually fell when nor-epinephrine was given after pronethalol treatment.

These effects of pronethalol on the responses tonorepinephrine are summarized in Table IV.

Changes in blood glucose and serum lactate.In four studies the blood glucose level was de-termined at 5- to 10-minute intervals. Controlvalues were 83, 86, 92, and 92 mg per 100 ml;peak values, reached just at the end, or 5 to 10

TABLE IV

Effects of pronethalol on responses to norepinephrine

Mean percent-age change in-

duced bynorepinephrine

Afterpron-

Control ethalol

Percentage change in FFA level [6]* +100 +19Percentage change in FFA turnover [5] + 71 - 8Percentage change in 02 consumption [6] + 28 - 4

* Figures in brackets indicate number of studies summarized.

minutes after the end of the norepinephrine infu-sion, were 94, 100, 104, and 109 mg per 100 ml,respectively. To test whether hyperglycemia perse might elevate metabolic rate, two of these foursubjects were restudied in the metabolic chamberon another day. Glucose was given intravenouslyat a rapid rate, and blood glucose levels rose towell over 200 mg per 100 ml. There was nomeasurable increase in oxygen consumption.

Plasma lactate levels were determined in threestudies. Control values were 0.92, 0.92, and 1.00/%Eq per ml; the peak values reached duringnorepinephrine infusion were 1.06, 1.12, and 1.01,uEq per ml, respectively.

Discussion

In the present studies norepinephrine was shownto increase the rate of oxygen consumption inevery subject tested. This calorigenic effect ofnorepinephrine has been demonstrated in severalspecies of experimental animals (19-23). Weareaware of only one previous report of positive re-sults in man (24), however, although several in-vestigators have reported no effect of norepineph-rine on oxygen consumption in man (25-28). Thecalorigenic action of norepinephrine has been foundto be smaller than that of epinephrine in rats(19), guinea pigs (20), and rabbits (21), andthis is our experience in recent clinical studies(29). The results presented above, however, es-tablish that under the conditions used norepineph-rine has a significant calorigenic action in man.

With the rise in oxygen consumption there wasa marked increase in the rate of fat mobilizationmanifested by an increase in concentration andrate of turnover of plasma FFA. Plasma FFAconcentrations rose on the average by 127% andcalculated turnover by 74%. Conversion of pal-mitate-1-C'4 to C1402 was simultaneously in-creased.

The question arises as to whether the rise inoxygen consumption, directly or indirectly, wasa consequence of the FFA mobilization inducedby norepinephrine. Pilkington, Lowe, Robinson,and Titterington (10) showed that pronethalol, arather specific inhibitor of adrenergic receptors ofthe /8 class (8, 9), blocked the FFA-mobilizingaction of epinephrine. As shown here, the FFA-mobilizing action of norepinephrine was similarlyblocked. The associated rise in oxygen consump-

172

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CALORIGENIC EFFECT OF NOREPINEPHRINEAND FFA TURNOVER

tion, the calorigenic action, was also blocked.Preliminary studies have shown that the calori-genic action of epinephrine in man is likewiseblocked by pronethalol (29). The concomitance ofthe two effects and the fact that both are blockedby pronethalol is compatible with, but does notestablish, a cause and effect relationship betweenthe FFA-mobilizing action and the calorigenic ac-

tion of catecholamines. The catecholamines havemany physiological activities in addition to theirfat-mobilizing activity. The possibility that othercatecholamine effects are more relevant to thecalorigenic action and that these are simultaneouslyinhibited by pronethalol cannot be ruled out. Atleast, the fat-mobilizing action of the hormonesand their activities relevant to the calorigenic ef-fect are both blocked by pronethalol, and the re-

ceptors involved thus fall operationally into thecategory of f8 receptors.

The degree to which FFA are diluted in poolsof nonradioactive fatty acids after leaving theplasma and before being oxidized is not known.It is therefore not possible to draw firm con-

clusions regarding the rate at which fatty acidsare being oxidized from data of the kind presentedhere. A minimal figure can be calculated by as-

suming no dilution, and as shown in Table III,such a calculation gives values only 5 to 10%that of the total turnover in the plasma com-

partment. Oxidation of this small amount ofFFA would account for only a very small frac-tion of the observed oxygen consumption, eitherin the basal state or during administration ofnorepinephrine. It seems highly probable thatthere is considerable dilution by extravascularpools of fatty acids (nonesterified and esterified)and that the actual amounts being oxidized are

much higher.If the extra amount of FFA mobilized under the

influence of norepinephrine does indeed representthe extra substrate being oxidized, it should bepossible to correlate in some measure the observedincrement in oxygen consumption with the amount

of oxygen needed to oxidize the newly mobilizedFFA. From the tabulated data, it can be calcu-lated that in eight of the nine studies in whichturnover was measured, the amount of oxygen

needed to oxidize the extra FFA mobilized duringnorepinephrine administration equals or exceedsthe observed increment in oxygen consumption.

(In the exceptional case [B.L., I, Table II], theresponse in FFA turnover was less than 25% ofthe next lowest response and only 10% of theresponse observed in the same patient given thesame dose of norepinephrine on another occasion.)Two points should be considered in evaluating thesignificance of this relationship. First; as dis-cussed above, we cannot be certain about the trueextent to which the FFA turned over in theplasma compartment are being oxidized. Second,the absolute values for turnover by the constantinfusion method may be too low. If there wereany important re-entry of radioactive FFA fromtissues into plasma, this would constitute an ad-ditional "input" of labeled material over andabove that being infused and should be added tothe value for "counts per minute infused per min-ute." The fact that a stable value for plasmaFFA specific radioactivity is reached within 10 to20 minutes indicates that any such tissue sourcesmust quickly reach a plateau specific radioactivity.Still there might be considerable cycling into andout of a radioactive extravascular pool, and thisturnover would go undetected. The fact that theabsolute values for turnover obtained in the pres-ent study are in the same range as those obtainedby Fredrickson and Gordon (30) using the singleinjection technique suggests that the potential er-ror discussed cannot be large, but further studiesare in progress to evaluate this. In any case, thetrue turnover would, if anything, be higher thanthat estimated here, and the extra substrate madeavailable could, even if only partially oxidized,account for the observed increments in oxygenconsumption.

Previous studies have shown that the rate ofoxidation of labeled long-chain fatty acids bymuscle and by liver in vitro is increased whentheir concentration in the incubation medium isincreased (4-6). Recently, we have been able todemonstrate an increase in rate of oxygen utiliza-tion by perfused rat liver exposed to perfusingfluid containing a high concentration of FFA(7). The possibility that the calorigenic effectof the catecholamines and, by analogy, the hyper-metabolism of the hyperthyroid state, are directlyattributable to an increase in utilization of FFAmade available to the peripheral tissues at highconcentrations has been suggested (5). Earlierstudies by Cori and Cori (31) led to the conclu-

17

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sion that the larger fraction of the extra caloricconsumption after epinephrine administration wasattributable to combustion of fat. Carlson andHsieh have proposed that norepinephrine may beof great importance in the elevated heat produc-tion of cold-adapted animals (32, 33). Recentstudies show that adipose tissue from cold-adaptedrats releases FFA more rapidly than tissue fromcontrol rats (34, 35). Cold-adapted rats show anexaggerated calorigenic response to norepineph-rine, and their adipose tissue appears to be moresensitive to the fat-mobilizing effects of norepi-nephrine (34-37). Thus there is considerable cir-cumstantial evidence relating the elevated meta-bolic rate of rats exposed to cold to catecholamine-stimulated fat-mobilization.

It is possible that the association between theFFA-mobilizing action and the calorigenic actionof catecholamines is indirect, even though causallyrelated. For example, the extra FFA madeavailable may be converted to another substrateform before oxidation. The work of Havel andco-workers has shown that FFA are very rapidlyincorporated into triglycerides in the liver andthen resecretedi in low-density lipoproteins (38).Studies by Gidez, Roheim, and Eder (39) and byNestel and Steinberg (7) show that the outputof triglycerides by the isolated perfused rat liveris increased when the FFA concentration of theperfusing fluid is elevated. It would be useful toknow if the turnover of the triglycerides in low-density lipoproteins is increased by administrationof catecholamines. A second possibility is thatthe FFA are first converted to ketone bodies inthe liver and then further oxidized by peripheraltissues. The ketone body levels are indeed some-what elevated during catecholamine administrationto normal subjects (40). The increment in oxy-gen consumption may partly reflect the energyused for the breakdown and resynthesis of tri-glycerides within the adipose tissue, as proposedby Ball and Jungas (41), and the energy re-quired for re-esterification of FFA mobilized andthen taken up again in the liver and other tissues.

Lundholm showed that the calorigenic effect ofepinephrine is well-correlated with the rise in se-rum lactate levels accompanying its administra-tion and that infusion of lactate into rabbits in-duced a rise in oxygen consumption (21). In thepresent studies, with norepinephrine, there waslittle or no change in serum lactate levels. Brew-

ster, Issacs, Osgood, and King found that epi-nephrine and norepinephrine had approximatelyequal calorigenic potency in dogs but, as in thepresent studies, norepinephrine did not raise se-rum lactate levels (23). The hyperglycemia ac-companying epinephrine administration has alsobeen proposed as a factor in the calorigenic re-sponse (1). Norepinephrine is much less po-tent than epinephrine in stimulating glycogenoly-sis, and at the dosages used in the present stud-ies, there was only a minimal increase in bloodglucose levels (maximal levels of 94 to 109 mgper 100 ml). Intravenous administration of glu-cose at rates that raised blood glucose levels tovalues much higher than those reached duringnorepinephrine infusion (over 250 mg per 100ml) did not cause any increase in oxygen con-sumption. Although hyperglycemia and hyper-lacticacidemia may play some role in the hyper-metabolic action of epinephrine, it seems unlikelythat these play an important role in the calori-genic action of norepinephrine as used in thepresent studies.

Another factor that might contribute to thecalorigenic action of norepinephrine is an increasein cardiac work. Since cardiac metabolism onlyaccounts for about 5 % of total body oxygen con-sumption in man (42), it seems unlikely that thiscould be increased enough to cause the increasesin rate of oxygen consumption induced by nor-epinephrine in these studies. Moreover, pre-treatment with pronethalol did not importantlymodify the changes in blood pressure and pulserate induced by norepinephrine, whereas it didabolish the rise in oxygen consumption.

SummaryIntravenous infusion of norepinephrine (0.13

to 0.31 ug per kg per minute) significantly in-creased oxygen consumption in fasting youngadults, the mean increase being 21 + 8% abovecontrol values in 11 studies. Concomitantly,plasma FFA levels rose 127 ± 57%o above con-trol values, and turnover of plasma FFA rose74 + 53%o. Blood glucose levels increased onlyvery slightly (mean increase of 14 mg per 100 mlin four cases), and changes in plasma lactatelevels were of marginal significance. In twocases conversion of palmitate-1-C14 to C140, wasmeasured and was shown to be increased 2 to 3times by norepinephrine administration.

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CALORIGENIC EFFECT OF NOREPINEPHRINEAND FFA TURNOVER

When the subjects were given an intravenousdose of 75 to 100 mg of pronethalol, a beta-adrenergic blocking agent, before the norepineph-rine infusion, the rise in oxygen consumption andthe increase in FFA levels and turnover wereradically reduced or entirely abolished. Thechanges in blood pressure and pulse rate wereessentially the same as those observed in the ab-sence of the blocking agent.

The present findings are compatible with thehypothesis that the calorigenic action of norepi-nephrine is causally related to its ability to in-crease the rate of FFA mobilization. This maybe direct, reflecting an increased rate of utilizationof the FFA themselves, or indirect, reflecting uti-lization of triglycerides or ketone bodies formedin the liver at an increased rate as a result ofmore rapid hepatic uptake of FFA. In addition,the increase in oxygen consumption associatedwith an increased turnover of fatty acids withinthe adipose tissue, as previously described, maycontribute to the increase in over-all metabolicrate.

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ANNOUNCEMENTOF MEETINGS

The American Federation for Clinical Research will hold itsTwenty-first Annual Meeting in Atlantic City, N. J., at the CasinoTheater on the Steel Pier on Sunday, May 3, 1964, at 9:00 a.m. Jointsectional meetings with The American Society for Clinical Investigationwill be held on Sunday afternoon at Chalfonte-Haddon Hall, and additionalmeetings sponsored by The American Federation for Clinical Researchwill be held on Sunday evening.

The American Society for Clinical Investigation, Inc., will hold itsFifty-sixth Annual Meeting in Atlantic City, N. J., on Monday, May 4,at 9:00 a.m., at the Casino Theater on the Steel Pier, and in simultaneousprograms with The American Federation for Clinical Research on Sundayafternoon, May 3, in Chalfonte-Haddon Hall.

The Association of American Physicians will hold its Seventy-seventh Annual Meeting in Atlantic City, N. J., at the Casino Theater onthe Steel Pier on Tuesday, May 5. at 9:30 a.m., and in the Vernon Room,Chalfonte-Haddon Hall, on Wednesday, May 6, at 9:30 a.m.

The American Society for Clinical Nutrition will hold its FourthAnnual Meeting in Atlantic City, N. J., at the Colton Manor Hotel onSaturday, May 2, from 1 :00 to 5:00 p.m.

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