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Insulin. Its role in the thermic effect of glucose.

L Christin, … , E Jéquier, K J Acheson

J Clin Invest.

1986;

77(6)

:1747-1755.

https://doi.org/10.1172/JCI112497

.

To investigate the possible role of insulin per se in the thermic response to glucose/insulin

infusions, respiratory exchange measurements were performed on eight healthy young men

for 45 min before and 210 min after somatostatin infusion. Two tests were performed on

separate days and each had two consecutive phases of 90 min each. Test 1. Two different

rates of glucose uptake were imposed, one at euglycemia (phase 1) and the other at

hyperglycemia (phase 2) while insulinemia was maintained constant throughout. Test 2.

Glucose uptake was maintained constant throughout while insulin was infused at two

different rates: 1 mU/kg per min with hyperglycemia (phase 1) and 6.45 mU/kg per min with

"euglycemia" (phase 2). The thermic effect of glucose and insulin, obtained from phase 1 in

both tests, was 5.9 +/- 1.2 and 5.8 +/- 0.5% (NS) of the energy infused, respectively. A step

increase in glucose uptake alone, test 1, phase 2, (0.469 +/- 0.039 to 1.069 +/- 0.094 g/min)

caused an increase in energy expenditure of 0.14 0.03 kcal/min (thermic effect 5.9

+/-1.1%). When insulin was increased by 752 +/- 115 microU/ml, with no change in glucose

uptake, energy expenditure rose by 0.05 +/- 0.02 kcal/min, which correlated with the

increase in plasma catecholamines. It is concluded that a large proportion of the thermic

response to […]

Research Article

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(2)

Insulin

Its Role inthe Thermic Effect of Glucose

L.Christin, C.-A. Nacht,0.Vemet,E.Ravussin,E.Jequier, andK.J. Acheson

Institute ofPhysiology, UniversityofLausanne, 1005LausanneandNestleResearchDepartment, 1800 Vevey,Switzerland

Abstract

Toinvestigate the possibleroleofinsulin persein thethermic

response to glucose/insulin infusions, respiratory exchange measurements wereperformed on eight healthy young menfor

45 min before and 210 min after somatostatin infusion. Two

testswere performed on separate days and each hadtwo

con-secutive phases of90min each. Test 1. Twodifferentrates of glucose uptake wereimposed, oneat euglycemia(phase 1)and the other at hyperglycemia (phase 2) while insulinemia was

maintained constant throughout.

Test 2.Glucoseuptake was maintainedconstantthroughout

while insulin was infused at twodifferent rates: 1 mU/kg per minwith hyperglycemia (phase 1) and6.45mU/kgper min with

"euglycemia" (phase

2).

The thermiceffectof glucose andinsulin, obtainedfrom phase

1 in both tests, was5.9±1.2 and 5.8±0.5% (NS) of the energy infused, respectively. A step increase in glucose uptake alone,

test1, phase2,

(0.469±0.039

to1.069±0.094g/min)causedan

increase in energy expenditure of0.14±0.03kcal/min (thermic effect 5.9±1.1%).When insulinwasincreasedby752±115

AUI

ml, withnochange in glucose uptake,energyexpenditure rose by 0.05±0.02

kcal/min,

which correlated with the increase in plasma catecholamines.

Itis concluded that a large proportion of the thermic response

toglucose/insulininfusions isduetoglucosemetabolism alone. Thethermic effect of insulin is small

aind

appearstobemediated by the sympatheticnervoussystem; thusatphysiologicalinsulin

concentrations,thethermic effect of insulin perseisnegligible.

Introduction

The

thermic

effect of glucose inleanindividuals either afteroral

ingestionorduring intravenous

infusions

isgreater(1, 2)than

canbeattributedto the energycostofstoring glucoseasglycogen (3). Onthe otherhand,obesesubjectsoften(4-8)butnotalways (9-11) present with a reducedthermic effect when compared

with lean controls, which is aggravated by increasing insulin

resistance(5).Althoughadiminished thermic effect offoodhas been proposedtocontributetothepathogenesisofobesity( 12-14), itis uncertain whether thisisacause or aconsequence of theobese state,particularlysinceithas been demonstrated that when the glucose uptakeis the same in lean and obeseindividuals

Addressreprint requests toDr. K.J.Acheson,Instituteof Physiology, University of Lausanne,Rue duBugnon 7, CH-1005, Lausanne, Swit-zerland.

Receivedfor publication 7November 1985 and in revised form 14

February1986.

thethermic effect ofglucose/insulin is also the same (15). Roth-well and Stock( 16)have reported that the increase in metabolic

rateinresponse to amealwasreduced instreptozotocin-induced

diabetic rats butwas reestablished in those receiving insulin. This has led to thesuggestion that insulin action is involved in thethermicresponse tofood ingestion (16, 17).

Since the effects of glucose and insulin metabolismare so

intimately related,it is difficult to conclude if it is a directeffect

of insulinindependentofglucosemetabolism thatisresponsible for the observed increase in thermogenesis. This study was

therefore designed toseparately measurethe thermic effect of

glucose alone and the influence of insulin itselfonenergy

ex-penditure in man. For this purposeeuglycemic and hypergly-cemicinsulin clampingwasperformed inhealthy youngmale

subjects with or without somatostatin infusionin combination

with continuousrespiratory exchangemeasurements. This

ap-proach allowed us todetermine whether insulin per se plays a rolein the thermic response toglucose/insulin infusions.

Methods

Subjects

Eight healthy young male subjects, whose physical characteristics are presented in TableI, were studied. No subject had a family history of diabetes mellitus andnone wastaking any medication during the

ex-perimentalprotocol. The protocolwassubmitted,reviewed, and accepted by the ethical committee of the Faculty ofMedicine, University of Lau-sanne.After receivingadetailed explanationof the experimental protocol, written consent was obtained from each volunteer before acceptance into the study.

Experimentalprotocol

For3 d before each test the subject was instructed to eat a diet containing atleast250 gof carbohydrate, supplemented with sugared fruit juice providing an additional 60 g of hexose sugars per day. Each subject was requested to keep a logofhis diet and physical activity during this period sothat he couldreplicatesimilar dietary and activitypatterns in the days preceding subsequent tests.

Eachsubject spent thenightbefore thetest attheInstitute. Inthe

morningthesubject was awakened at 6:30 a.m. and after voiding was transferredto the roominwhichthetest was tobe performed. Two venouslines were inserted. One was a venous catheter(Venflon, Viggo, AB,Helsingborg, Sweden) placed inanantecubital veinforinfusionof glucose and hormones and the other,a19-gauge Butterfly (Abbott Ireland Ltd,Sligo, Republic of Ireland)wasinsertedretrogradely into a hand vein forblood samplingand was kept patent with physiologicalsaline.

The handwas then placedinaheated box (-60°C) toachieve arterial-izationof thevenousblood (18).

Thesubject performedfourexperimentalprotocols within a period of2 mo. Thefirst and second (Fig. 1) and the third and fourth experiments wereperformed within 2 wk.

Testlawith constant insulin infusion at two successive steady state

plasmaglucose concentrations. This test was designed to study the effect ofanincrease in glucose uptake on energy expenditure, without a con-comitant changeininsulinemia.

After 45 min ofbaseline measurements, somatostatin (Stilamin,

Ser-ThermicEffect ofInsulin 1747

J.Clin. Invest.

© The American Society for Clinical Investigation, Inc. 0021-9738/86/06/1747/09 $ 1.00

(3)

Table LPhysicalCharacteristicsoftheSubjects

Name Age Weight Height Bodyfat FFM

yr kg cm %kg

C.M. 20 68.24 176.5 12.9 59.6

M.L. 20 78.45 185.5 16.2 65.74

N.F-Y. 21 65.05 175.5 0.6 58.2

C.C. 20 85.60 181.0 14.7 73.0

LU. 19 62.64 181.5 11.6 55.37

G.T. 22 67.75 186.0 12.0 59.62

B.P-Y. 22 81.72 194.5 15.5 69.05

E.K. 22 67.98 180.0 14.4 58.2

Mean±SD 20.8±1.2 72.2±8.5 182.7±6.1 13.5±2.0 62.3±6.2

onoS.A.,CH-1 170Aubonne,Switzerland)wasinfusedatacontinuous rate (7

iAg/min)

for

the.

next 2 hof theexperiment. Thisinfuision rate

waschosentoeffectivelyinhibitendogenousinsulinsecretion(19, 20).

After 30 min of somatostatin infusion aloneaprimingdose of

bio-synthetichumaninsulin(Huminsulin Normal,EliLilly, Indianapolis,

Euglycemic Hyperglycemic 1

clamp clamp

Insulin 1mrnWkg.min

G lucose 20%

M-0.47±0.4' M.1.07k009~

I 7pg/min

9

9

gg/min---I

Roespieatory Exchange Measurements

-60 Blood f

samples

Test2a

0 60 120 180minutes

Hyperglycemic 'Euglycemic'

clamp clamp

Insulin

G lucose 20%

M-0.88±t.10 F-- M-0-93±0O40

Somatosta,tin ,mn..1

~~~~~p/i 1~ 9p mn~

-60 Blood t

saimples

0 60 120 180 minutes

Figure1.Experimentaldesign.Thestudywasdivided intotwo tests:

(Test la)Plasmainsulin concentrationsweremaintainedat '--80UU/

mlthroughoutthetest.Gluco'seuptake (M)wasraised from 0.47±0.04to 1.07±0.09g/min (Mean±SEM)at120minin orderto

raiseglycemiafrom 91 to171 mg/dlandmaintain itatthatlevel for another90mmn.(Test2a)Mwasmaintainedconstant at ~--0.9g/min throughoutthetest. At 120minplasmainsulin concentrationswere

raised from 95to848usU/ml.Since Mwasconstant,glycemia de-creasedfrom 170to 1mg/dl.

IN)wasgiveninadecreasingmanner over aperiodof 10 minas

pre-viouslydescribed (18)andwastheninfusedatacontinuousrateof 1

mg/kgpermin foir the remainder of thetest.Euglycemiawasmaintained inarterializedvenousbloodsamplesobtainedat5-minintervals,during

the somatostatin and insulininfusions, byvaryingthe infusionrateofa

20%,glucosesolution(18).After90min ofhyperinsufinemic euglycemia,

aprimingdose ofthe20%glucosesolutionwasgiveninalogarithmically

decreasingmanner(18)andwasthenvaried in ordertoraise and maintain the blood glucoselevel at 170-180 mg/dlfor another 90 min. When hyperglycemiawasimposed,acondition that stimulatesendogenous in-sulinsecretion,therateof somatostatin infusionwasincreasedto9 utg/

mintoensurethat endogenous insulindid not escape somatostatin's inhibitoryeffects.

Test Ib: SameprotocolasIlabut without somatostatininfusion.This

test wasusedas acontrol fortest Ia, inordertocheck whether

somna-tostatin,whichwasgivenintest1a,hadaneffectonenergyexpenditure.

Test 2a with constantglucose infusion at two successive levels of insulinemia. After 45 min of baseline measurements, somatostatinwas

infusedat7

gg/min

for2 h. Whensomatostatin had been infused for 30 minaprimecontinuous infusion of Huminsulinwasbegunatarate

of 1 mU/kg perminasdescribed above (test la).Atthesametimea

primingdose ofa20%glucosesolutionwasinfused and then varied in ordertoraise and maintainglycemiaat170-180mg/dlfor 90 min. After 90 min ofhyperinsulinemichyperglycemia, the somatostatin infusion

ratewasincreasedto9

gg/min

and insulinemiawasincreasedbyaprime

continuousinfusion(6.45 mU/kgpermin)forafurther 90min, while

maintainingtheglucoseinfusionconstant.

Test2b: Sameprotocol'as2abut without somatostatininfuion.(This

test wasusedas acontrol fortest2a). Threesubjectswhoparticiae in the above

experiments

also volunteered fortwoadditionaltestswithout

somatostatin,inwhich either the 1-mU/kgperminhyperinsulinemic, euglycemic clampor thehyperinsulinemic, hyperglycemic clampwas

continued for the wholedurationof thetestin ordertoobserve 'whether the thermiceffect ofglucose/insulin infusionswerechanged withtime.

Duringeache'xperiment,continuousrespiratoryexchange measu're-mentswere performedfor the duration of thetest(baseline and test) usingaventilatedhood,open-circuit,indirectcalorimeter,thedetails of which have beendescribed elsewhere(21).

Heartratewasrecordedthroughoutthetestbymeansofaportable, lightweight, electronic device(heart rate memory, Difa BeneluxBY,

Breda,TheNetherlands).

Bloodsamplesweretaken every 5min afterthebaseline forglucose analysis. Samples werealsotakenatregularintervals (Fig. 1)for free fattyacid,bloodureanitrogen,and hormoneanalyses.

Urinewascollectedatthebeginningand end of thetestandanalyzed

forglucose, nitrogen, and catecholamines.

Analyses

BloodglucosewasanalyzedinduplicateonaBeckmanIIglucose analyzer (BeckmanInstrumnents,Inc.,Fullerton, CA).Plasmasampleswerealso

analyzedfor insulin(22), C-peptide (kit,Byk-Mallinckrodt, Diezenback,

FederalRepublico'fGermany)andglucagon (23) byradioimmufioassay,

freefattyacids(24)onthe Doleextract(25),andcatecholaminesbyhigh performance liquidchromatography(26, 27).

Urinarynitrogenwasanalyzed,afterdigestion,on aTechnicon

au-toanalyzer (Technicon Instruments, Corp., Tarrytown, NY) (28),and

urinary catecholamines were measured by fluorometry (29). Urinary glucosewasa'nalyzedwithglucoseteststicks(Gluketur-test, Boehringer, Mannheim, Federal Republic ofGermany). Glucosureawasonly ob-tainedinonetestandwasfurtheranalyzedinduplicateontheBeckman IIglucose analyzer.

Data

analysis

Fordatapresentation,themeanvaluesobtainedduringthe last 30 min ofeach relevantphaseineachprotocolwereconsidered.IntestsIlaand

lb,phase 1 representsthehyperinsulinemic (-.80

jtU/ml)

euglycemic

clam- an phAs21- th hyernulnei hyegye ic lamp (-' 171

mg/dl). In tests 2a and 2b, phase 1 represents thehyperinsulinemic, Teat la

I

(4)

hyperglycemic (- 170 mg/dl) clamp, and phase 2 the "euglycemic" ( 101 mg/dl)clampwithastep increaseininsulinemia (insulinemia

850,uU/ml).

Anindexofproteinoxidationwascalculatedfromthemeanurinary

nitrogenexcretionduringthe test, after correctionfor changesinnitrogen

in theureapool(30).

"Glucosestorage"ornonoxidative glucosedisposalwascalculated

bysubtractingtherateofglucose oxidationfrom therateofthe corrected

glucose uptake(M),i.e.,bytakingintoaccountthechangesin theglucose poolassumingadistribution volume of 0.21 liter/kgperbodywtand

subracing

anyurinaryglucoselosses.Urinary glucosewasonlyobserved

oncein Isubject,duringtest2b and amountedtoanexcretionrateof 0.01 mg/kgper min.Hepatic glucoseproductionwasnotmeasured in the presentstudyandwasassumedtobetotallysuppressedduringthe glucose/insulininfusions (31, 32).

Thethermiceffectofglucose/insulin

(TEe)

wascalculatedbydividing theincrease in energyexpenditure (EE)

(EEp.

I-EE.)bythe

energy content of the increase in glucose uptake (GU)

(GUp1,,

I

-

GQU,.tmd).

Testslaand 2a

TEG,=

EEp

-EE,

tw(GUpI

-GUw)X3.74X 100. Thethermic effect ofglucosealone(TEG)wascalculatedasfollows:

Test Ja

TEG

=

EEp

2- EE

1/(GUphm

2-

GUp,,

1)

X 3.74X

100,

where EE is thesteadystateenergyexpenditure kcal/min,GUis the glucose

uptakeg/min,and3.74 is the energy value Igglucosekcal/g.

Statistical analyses were performed using Student's paired t test. Stepwise regression analysiswasused to determine which parameter change (glucose uptake, plasma concentration of insulin, norepinephrine, andepinephrine) correlated with each change in energy expenditure. Results are expressed as mean±SEM unless stated otherwise.

Results

Theaim of the presentprotocols wereintest la, to maintain insulinemiaat a constantrateduringtwodifferent steadystate

conditions ofglucose uptake and intest2atomaintainaconstant

glucose uptakeduringtwodifferent steadystate conditions of insulinemia. The results of the blood parameters and heartrates

measuredduring the baseline and the different phasesofeach experimentaresummarized in TableII.

Testla. Itcanbeseenthat with somatostatin infusion there

was a slight butsignificant decrease in both glycemia and in-sulinemia between the baseline and somatostatin phases after which blood glucosewasmaintainedat91 ±1 mg/dl when plasma insulinwas80±4

AU/ml

(phase 1). As expected, C-peptide

con-centrations fell dramatically with somatostatin and decreased furtherto 0.18±0.01 ug/ml in phase 2atwhich time plasma insulin concentrations were 71±3 uU/ml.

Test lb. Glycemic values were similartothose in test la. Withoutsomatostatininfusion, C-peptideconcentrationswere moreelevated than in test Ia, especially during the hyperglycemic

TableI. ChangesinthePrincipal Blood Parameters andHeart Rate intheTestswith and withoutSomatostatin(n= 8) (Mean±SEM)

Baseline Somatostatin Phase I Phase 2 Baseline Phase 1 Phase 2 Parameter (-304 min) (0-30min) (90-120min) (180-210min) (-30-0min) (60-90min) (150-180 min)

Test ia(withsomatostatin) Testlb(without somatostatin)

Euglycemia followed by hyperglycemia

Glucose(mg/dl) 99±1* 95±2 NS

91±1t

171±2 101±2* 91±1* 170±3

Insulin

(,uU/ml)

11.2±0.9§ 6.7±1.0t 80±4t 71±3

12.0±0.9t

81±7t 108±9

C-peptide(ng/ml) 1.54±0.07t 0.59±0.02t 0.23±0.01t 0.18±0.01

1.74±0.2t

0.72±0.13t

3.70±0.30

Glucagon(pg/ml)

100±12t

46±6 NS 44±8 NS 43±5 81±9 NS

69±12t

53±10

Freefattyacids 249±25t 344±47t 128±9t 112±11 268±16t

135±8t

118±6

(Amollliter)

Norepinephrine 209±16NS 204±18 NS 229±19 NS 234±19 205±10§ 232±18 NS 242±13

(pg/ml)

Epinephrine(pg/ml) 74±5 NS 72±3NS 79±5 NS 82±6 71±3 NS 76±4 NS 77±3

Heartrate(beats/min) 67±3 NS 65±2NS 69±3* 73±3 68±2t 74±2* 76±2

Test 2a(withsomatostatin) Test2b(withoutsomatostatin)

Hyperglycemia followed byhyperinsulinemia

and euglycemia

Glucose(mg/dl) 101±2* 97±2* 170±2* 101±2 100±2t 171±1t 122±3

Insulin(AU/mlm 12.8±1.0t 6.0±1t 95±7t 848±121 10.9±1.6t 128±8* 843±43

C-peptide

(ng/mO

1.62±0.10t

0.55±0.04t 0.25±0.03§ 0.02±0.01 1.56±0.13t 4.40±0.21t 2.82±0.24

Glucagon(pg/ml

82±11t

41±6§ 31±4NS 36±7 86±9t 46±7 NS 44±5

Freefatty acids

(;tmol/

280±18* 439±46t 119±14 NS 111±12 259±30t

129±9*

114±7

liter)

Norepinephrine 208±15 NS 204±10 NS 207±15* 244±12 213±21 NS 225±17t 274±25

(pg/ml)

Epinephrine(pg/ml) 76±4 NS 75±4 NS 75±5* 84±4 76±5 NS 81±6t 91±6

Heart rate(beats/min) 63±2NS

62±1t

67±2t

73±2

65±2t

74±2t 80±3

*<0.01. t<0.005. § <0.05.

(5)

phase(3.7±0.3 vs. 0.18±0.01 ng/ml, P<0.001). Plasmainsulin concentrations were similar during theeuglycemic phase (phase 1) in tests la and Ib, but was much higher during the hypergly-cemicphase (phase 2) in test lb than in test la (108±9 vs. 71±9 ,uU/ml,respectively, P <0.001). Thesomatostatin infusion in test lathuseffectively inhibited endogenous insulin secretion

during hyperglycemia.

Test2a. During phase 1 (hyperinsulinemic hyperglycemic clamp)the plasmaglucoseconcentrationreached asimilarvalue tothatinphase 2 oftest la (170±2 vs. 171±2 mg/dl,

respec-tively).Attheendofphase2, thehighinsulin infusionrate(6.45 mU/kg per min) induced a very high insulinemia

(848±121,uU/

ml)withaconcomitant decrease in plasma glucose concentration towardseuglycemia (101±2 mg/dl). Plasma C-peptide

concen-trationwasmarkedly reduced by thesomatostatin infusionfrom 1.62±0.1 to 0.25±0.03 and 0.02±0.01 ng/ml during baseline, phase 1 and phase 2, respectively.

Test2b. Similar glycemicconcentrationswere obtainedin test2b asintest2a, whileinsulinemiawashigher duringphase 1 oftest2b (128±8 vs. 95±7

,uU/ml,

P<0.001 in tests 2b and 2a,respectively). Plasma C-peptideconcentrationsalsoincreased

during hyperglycemiabut weresignificantly inhibitedatthehigh insulinconcentrations (843±43 ,AU/ml) obtained in phase 2.

Plasma

concentration of norepinephrine and epinephrine.

Plasma norepinephrine concentrations in tests 2 were signifi-cantly increased during thehyperinsulinemic phase (phase 2) when comparedwith values in phase 1 (244±12 vs. 207±15 pg/ ml, P<0.01; 274±25 vs. 225±17 pg/ml, P<0.005,in tests 2a and2b, respectively). Similarly,plasmaepinephrine

concentra-tionswere slightly increasedin tests 2aand 2bduringthe

hy-perinsulinemic phase.

Plasma concentration of glucagon. The plasma glucagon

concentrationwaslowered bysomatostatininfusionsin tests la

and 2a. In test 2b, plasma glucagon concentrations were

de-creasedfrom 86±9 (baseline)to46±7pg/ml (phase 1; P<0.005)

following

the stepincreaseininsulinemia.

Glucose uptake, glucose oxidation, andglucose storage. The

changes inglycemia,glucose uptake (M), glucose oxidation, and

glucosestorage areillustratedin Fig. 2 for tests laand 2a. During thebaseline

period

glucoseoxidation was0.136±0.020 g/min

(test la) and0.112±0.021 g/min (test 2a). Lipid oxidation at

this timewas0.061±0.007 g/min in both tests. With

somato-statin,

glucose oxidation fellto0.110±0.017 g/min, P<0.02,

(test la)and0.098±0.015g/minNS (test2a)eventhoughglucose

uptake was 0.093±0.018 g/min and 0.094±0.017 g/min,

re-spectively.

Duringthe last 30 minof thehyperinsulinemic (80±4

,U/

ml),euglycemic clamp (test la)glucoseuptakewas0.469±0.039

E 12 .0

n

2 0.8

0 co

0

05

0.4,

O-L

-60 0 60 120 180

Time(min)

E

.L

0

D

V

0

-60 0

g/min, of which 0.255±0.021 g/min could be accounted for by oxidation and the remainder, 0.214±0.036 g/min, by glucose storage. At this time lipid oxidation had decreased to almost half the postabsorptive value 0.034±0.006g/min.

Astepchange in the rate ofglucose uptake from 0.469±0.039 g/min to 1.069±0.086 g/min resulted in an increase in both glucose oxidationto0.385±0.029 g/min (P < 0.005) and storage to 0.684±0.077 g/min (P < 0.001), while lipid oxidation de-creased to negligible values.

During the last 30 min of test 2a phase 1, where hypergly-cemia wasmaintainedat170±2mg/dl ataninsulinemia of 95±7

AU/ml

glucose uptake was 0.884±0.101 g/min, one-third of which was due to oxidation (0.284±0.029 g/min) and 0.600±0.086 g/min to glucose storage. The step change in plasma

insulin concentration from 95±7 to 848±121

AU/ml

(test 2a, phase 2) caused a slight but significant increase in glucose uptake from 0.884±0.101 to 0.926±0.102 g/min, P < 0.001, which was due toafallin glucoseconcentrationin the glucose space. The

disposal of thissmallincreasein glucose uptake could be entirely accounted for byanincrease inglucose oxidation since glucose storage remainedunchanged (0.570±0.080 g/min, NS).

During both clamp phases lipid oxidation decreased signif-icantly (0.020±0.004 g/min, P<0.001 and 0.006±0.006 g/min, P < 0.001, phases 1 and 2, respectively) when compared with the baseline (0.061±0.007 g/min) and somatostatin

(0.067±0.004 g/min)phases.

Table III presents the resultsforglucoseuptake,oxidation, and storageobtainedin the twotestswithout somatostatin. In theseconditions, although glucose uptakewasslightly greater, thecontribution of oxidation and storagetoglucose uptake was the same(within 4%) in each of the different phasesas in the

testswith somatostatin. Wheninsulinemiawasincreased from 128±8 to 843±43 ,uU/mlglucose uptakeincreased slightly and wasaccompaniedby asignificantincrease in

glucose

oxidation.

The changes in energyexpenditure, glucose uptake and in-sulinemia during the twosomatostatin tests are illustrated in

Fig. 3. Glucose uptake has been divided into itsconstituents, oxidation,and storage. Itcanbeseen(test la) thatanincrease in both glucose uptake andinsulinemia of0.377±0.044g/min

and 73±4

ALU/ml

(phase 1 minus somatostatin), respectively,

causedanincreasein energyexpenditure of0.08±0.02 kcal/min or athermiceffect of5.9±1.2%. When therateofglucose uptake

wasincreased by a further 0.599±0.07g/min, energy expenditure also increased by 0.14±0.03 kcal/min, which resulted in a thermic effectofglucosealone of 5.9± 1.1%.

Intest2a, theincreasein bothglucoseuptakeandinsulinemia

of0.791±0.110 g/minand 90±7 ,uU/ml, respectively, caused

anincrease in energyexpenditure of0.17±0.03kcal/min.Inthis

TEST 2a

plasma glucose (mg/d)|

1180

1140

100

60 Figure2.Changeinplasma glucose

.*,glucose

uptake

and its compo-C 9]

20nents,

oxidationu,andstorageoduring

20 thetwo testswithsomatostatin (n=8:

60 120 180 Mean+SEM).

(6)

Table III. Glucose Metabolism and Metabolic Variables during the Two Testswithout Somatostatin (n 8,Mean±SEM)

Baseline Phase I Phase 2

-300 min 6090 min 15S-180 min

Test Ib: Euglycemia followed by hyperglycemia

Glucose uptake(g/min) 0.524±0.040* 1.168±0.094

Glucoseoxidation(glmin) 0.116±0.017t 0.281±0.088t 0.378±0.024

Glucose storage(g/min) 0.243±0.026t 0.790±0.088

AEnergy expenditure(kcal/min) 0.14±0.03* 0.27±0.03

Thermic effect (%) 7.1±1.4 NS 6.0±0.5

Theoretically derived thermiceffect"l(%) 2.5±0.2 3.6±0.1

Lipid oxidation

(glmin)

0.066±0.006§ 0.032±0.008§ 0.006±0.004

Test 2b: Hyperglycemia followed by hyperinsulinemia

Glucose uptake(glmin) 1.076±0.088 NS 1.097±0.090

Glucose oxidation(glmin) 0.137±0.013t 0.360±0.023t 0.391±0.020

Glucose storage(glmin) 0.716±0.075 NS 0.705±0.075

A Energy expenditure(kcal/min) 0.25±0.02* 0.31±0.03

Thermic effect (%) 6.5±0.2§ 7.6±0.3

Theoreticallyderived thermic effect (%) - 3.5±0.1 3.4±0.1

Lipid oxidation (g/min) 0.058±0.004t 0.009±0.006 NS 0.003±0.005

* <0.005. t<0.01. § <0.05. 11The theoretically derived thermic effect=glucose storageX5.3 + glucose uptake, where 5.3% is the thermic

effect of glucose storage as glycogen (3).

condition glucose storage was 0.605±0.099 g/min, and the

thermic effectwas5.8±0.5%,whichwasthe same as that observed in phase 2 of test la. When glucose uptake and storage was

maintainedconstant andinsulinemia was increased by 752±115 ,uU/ml, energy expenditure increased by 0.05±0.02 kcal/min (P

<0.025).

Stepwise regression analysiswasperformedonthechanges

in glucose uptake, plasmainsulin, and catecholamine concen-trations,in the somatostatintests todeterminewhichparameters most influenced the observed increase inenergy expenditure

(Table IV). It can beseenthatintestla,astepchangeinglucose uptake correlated

significantly

with the increase in energy ex-penditure (r=0.823, P<0.01)and accounted for 68%

(r2)

of this response (P<0.025).The correlationimproved slightlyand

remained significantwhentheincreaseinplasma epinephrine concentrationswereaddedtotheequation(r=0.837,P<0.05). In test 2a whereglucose uptakewasmaintainedessentially constantand plasmainsulin concentrationswereincreased by

752±115 uU/ml, the change ininsulinemia didnotcorrelate

withthechange inenergyexpenditure. Althoughthe increases in both plasmanorepinephrineandepinephrine concentrations

TEST la

E

i-aco

a5

a

w

aGIucoseUptake

Q61.2 (g/min)

I

OA4 1JD

L020.4

iI

0

AIRII*ooO

(pu/mi)1

800

1600

1400

1200

10

correlated with energy expenditure (r = 0.745 and0.689, re-spectively, Fig. 4) only norepinephrine correlated significantly when analyzed bystepwiseregression analysis (P<0.05).

Table V presentsboththemetabolicand blood parameters ofthe tests where eithereuglycemiaorhyperglycemiawas main-tained continuously for3 h.These resultsdemonstrate thatonce

steady state conditions were attained, no further changes

oc-curredinanyofthemeasuredparameters, except in the

eugly-cemicclamp,where bothplasmainsulin and freefattyacid con-centrations fellslightlybutsignificantly.

Heartrate.Fig.5 presents thechangesinheartrateduring

theexperimentswith and without somatostatin. In the

postab-sorptivestate, theresting supineheartratewas66±1beats/min

in all tests. Whensomatostatinwasinfused,therewas a

transitory

fallthatwas

significant

(68±3 to65±2

beats/min,

P<0.02in test 1;63±2to62±1

beats/min,

P<0.05 intest2)and lasted

- 10-15min.Duringthedifferentclamp phases,the heartrate

increased significantly intratest. Although the heartratetended tobe lowerwithsomatostatinintest 1,thedifferenceswere not

significant.

However,intest2,the differencesweremoreevident andtheywere

significantly

different.

TEST 2a

Figure 3. Changes in energy

expen-diturem, glucose oxidation o, glu-cosestorage o, and plasma insulin concentrations a, over the somato-statin baseline values during phaseI ontheleft and phase2onthe right of each of the tests in which somato-statinwasinfused.Notethat glucose uptake is the sum of glucose oxida-tion +glucose storage (n=8;

mean±SEM).

(7)

TableIV.Simpleand Partial CorrelationCoefficients by Stepwise Regression Analysis Between

theChangesin EnergyExpenditure (Dependent Variable) and the ChangesinGlucose Uptake,

Plasma Insulin, Norepinephrine and Epinephrine Concentrations (Independent Variables) (n = 8)

Dependentvariable A energyexpenditure kcal/min

Independentvariables Simplecorrelation Partial correlation P

Test la:Steady state insulin concentrations with a step increase in

glucose uptake (phase 2-phasel) \

A Glucose uptake(glmin) 0.823 <0.025

A Plasma epinephrine(pg/ml) 0.450 0.837 <0.05

APlasma norepinephrine

(pg/lm)

-0.583 0.856 NS

Test 2a:Steady state glucose uptakewith astepincrease inplasma

insulin concentration (phase2 -phasel)\1

APlasma norepinephrine(pg/mI) 0.745 <0.05

APlasma epinephrine(pg/mI) 0.689 ) 0.754 NS

A Plasma insulin(uU/ml) 0.333 J 0.722 NS

Urinarycatecholamines. During test1aurinary norepineph-rine and epinephrine excretions were 1.67±0.45

gg/h

and 0.35±0.18

,ug/h,

respectively, and were notsignificantly different from the excretion rates observed in test 2a (1.49±0.41

ug/h

norepinephrineand 0.40±0.28ag/hepinephrine). In the control testswithoutsomatostatin the rate of norepinephrine excretion was1.60±0.45 and 1.92±0.37

,g/h

(testlb and 2b, respectively) andthatof epinephrine was 0.39±0.23 and 0.41±0.15

,ag/h

(test

lb and2b, respectively).

Discussion

This

study

investigated

theinfluence of

glucose

metabolismper seandinsulinper se uponthe thermiceffectof glucose/insulin

infusions. Theresults demonstrate that a step change in glucose

0-12

0.10

c008

E

"I

006

a004

002

0 l

- * 0

0 * 0

0 *

0

I

,,o'

-10 0 10 20 30 40 50 60 70

Aplasma Norepinephrine pg/ml

-5 0 5 10

A plasma Epinephrine pg/mi

15 20

Figure 4. Correlations between the increase inenergyexpenditure (mean kilocaloriesperminute)and thechangesinplasma catechol-amine concentrations withastepincrease in insulinemia of 752±115 ,uU/ml(test2a,phase2minus phase 1).Simplelinearcorrelation

analysisshowed that bothnorepinephrine (* *, y=0.00 lx + 0.008;r=0.745,P<0.05)andepinephrine

(-

-- -o,y=0.004x +0.015;r=0.689,P<0.05)weresignificantlycorrelated withmean

kilocaloriesperminute. Withstepwiselinearcorrelationanalysis only the increase inplasmanorepinephrineconcentrationswas

significantly

correlated withmeankilocaloriesperminute and couldaccountfor 57%(r2)of this increase (n=8).

uptakeishighlycorrelated with acorrespondingchangein energy

expenditure. Althoughastepchangeininsulinwasaccompanied byaslightincrease inenergyexpenditure,the latter was

signif-icantlycorrelated with aconcomitant rise inplasma catechol-amineconcentration rather than with the change in insulinemia. Theinfluence of insulinemia upon plasma norepinephrine concentrations supports the observations of Rowe et al. (33) that

hyperinsulinemia, rather than hyperglycemia, stimulates

sym-patheticnervousactivity.The fact that stepwise linear regression analysis showsasignificant correlation between the increasein

plasmanorepinephrine and that of energy expenditure suggests that, when present inlarge concentrations, insulin can cause a

small increase in energyexpenditure that is mediated via the sympatheticnervoussystem.Such a role for insulin has already beensuggested(34), possibly acting via receptors that have been found in theventromedialhypothalamus(35). Insulin

concen-trations of 840 ,U/ml are rarely or never observed, even in

pathological conditions. It is thus likely that physiological changes in insulin concentrations can account for onlyavery smallproportionof the observed change in energyexpenditure, i.e.,0.05kcal/minper 752

,U

per mlinsulinor0.007kcal/min

for every 100 gU/ml increase in insulin concentration ifone assumes alinearrelationship between the riseinplasmainsulin concentrationand theincreasein energyexpendituremediated via thesympathetic nervous system.

Flatt has calculated the energy cost of storing glucoseas gly-cogen basedontheratio of the numberofmolesof ATP used

to those produced during glucoseoxidation andglucose

con-version to glycogen, i.e., 5.3% of the energy content of glucose

or0.2 kcal/g (3). Bycomparingthethermic effects of

glucose/

insulin,glucosealone, and insulin aloneobtainedexperimentally

with those calculatedasabove, it ispossibletoobtainanindex of thecontributionof theobligatoryand facultativethermogenic

components(36) duringthedifferentphasesof the

study.

The

energy cost of glucose storage (obligatory

thermogenesis)

ac-counted for -50-60% of the measured thermic effect(Tables III and V) when no somatostatin was infused and 60% with

somatostatin infusionduring both hyperglycemic phases (test la,phase 2 andtest2a, phase 1).With astep increase in

insu-linemia, thecontribution of the facultative

thermogenesis

in-creasedfrom 42to51%with somatostatinand from 46to55%

withoutsomatostatin,demonstratingthatinsulinand/or

(8)

Table V.Blood Parameters andMetabolic VariablesduringEitherConstantEuglycemiaorHyperglycemia (n= 3, Mean±SEM)

Euglycemia Hyperglycemia

Baseline Phase I Phase 2 Baseline Phase 1 Phase 2 (-30-0 min) (60-90min) (150-180min) (-30-0min) (60-90min) (150-180 min)

Glucose uptake(g/min) - 0.505±0.026 NS 0.536±0.035 - 1.195±0.177 NS 1.178±0.152

Glucose oxidation

(glmin) 0.146±0.051* 0.286±0.037 NS 0.289±0.026 0.137±0.033* 0.407±0.059 NS 0.403±0.042

Glucosestorage

(glmin)

0.220±0.053 NS 0.247±0.061 - 0.788±0.135 NS 0.775±0.121

AEnergy expenditure

(kcal/min) 0.17±0.05NS 0.15±0.06 0.29±0.05 NS 0.28±0.07

Thermic effect (%) 8.9±2.2 NS 6.9±2.4 6.7±0.9 NS 6.3±1.3

Theoretically derived

thermiceffectt(%) 2.3±0.5 2.7±0.2 - 3.4±0.2 3.4±0.1

Lipidoxidation(g/min) 0.048±0.014 NS 0.028±0.009 NS 0.024±0.016 0.061±0.014 NS -0.002±0.014 NS 0.007±0.001

Glucose(mg/di) 95±1 NS 89±2 NS 93±3 98±0.4§ 170±1 NS 171±2

Freefatty acids

(Amol/liter)

273±76NS 142±14§ 130±13 267±37 NS 135±16 NS 133±19

Insulin(,U/mi) 12.6±1.8§ 84±6§ 78±7 12±2§ 128±22 NS 137±18

C-peptide (ng/ml) 1.58±0.17§ 0.72±0.16 NS 0.58±0.12 1.81±0.21§ 5.30±0.68 NS 5.88±0.74

Glucagon(pg/mI) 89±47 NS 63±28 NS 61±25 72±21§ 35±15 NS 33±13

Norepinephrine(pg/mi) 205±7§ 252±16 NS 239±7 233±30 NS 253±23 NS 242±30

Epinephrine (pg/ml) 64±10§ 75±11 NS 72±10 76±8 NS 84±7NS 77±7

Heart rate(beats/min) 60±4§ 69±4NS 69±5 61±3§ 70±5 NS 72±5

*<0.05. t Thetheoreticallyderivedthermic effect="glucose storage"X5.3 . glucoseuptake;where5.3 is thethermiceffectof

glucose

storage

asglycogen (3); § <0.005.

mediated factorscausedasmall increase in energyexpenditure

thatwasnotrelatedtoglucoseuptake.

Itisinterestingtonotethat the increase in energyexpenditure overthesomatostatin baseline duringthehyperglycemic, high

plasmainsulinclamp (test 2a, phase 2) was 0.23±0.03 kcal/min

at aglucoseuptake of 0.926±0.102 g/min of which 0.05±0.02 kcal/minor 22% can be accounted forbyinsulinand/or stim-ulation of the sympathetic nervous system. If one assumes,as

observed elsewhere(37), thatacertainsympatheticcomponent

totheincrease inenergyexpenditurewasalreadypresentbefore

the step increase in plasma insulin concentration, then these resultsconfirm ourpreviousobservations (36, 37) that stimu-lation ofthesympatheticnervous system canaccountfor - 30%

of theincreasein energyexpenditure during glucose/insulin

in-fusions.

Although it would appear thatinsulinviastimulation ofthe

sympatheticnervoussystemcanaccountfor20-25%ofthe

fac-TESTS la and lb

ultativethermogenesis (36)it is possiblethat otherfactorsmay beinvolvedbutwhichcannotbedetermined from ourdata.

In this study somatostatin wasused as a tool to suppress

endogenous insulin secretion. Itis unlikely that it influenced ourresultssubstantially sincetests lband2bcarriedoutwithout somatostatingavesimilar resultsof glucose metabolism.

Certainaspectsofsomatostatin actionswereobserved,

how-ever. Somatostatin infusion causedadecrease in bloodglucose concentration, which necessitatedtheinfusion of glucoseat a

rateof1.3 mg/kg per minin bothtests, tomaintain euglycemia.

Itiswelldocumented thatthis decreaseinglycemia is principally

duetoinhibition ofglucagon-stimulated glycogenolysisby so-matostatin, whichresults ina50-80%reduction inhepatic

glu-coseproduction (38, 39). Although hepatic glucose production

wasnotmeasured in the present study, one can assume a basal

hepatic glucose production of -2.2 mg/kg per min (40, 41);

somatostatin thus caused a 60% decrease in hepatic glucose

TESTS 2a and 2b

co0

$

-60 0 60 120 180

Time(min)

-60 0 60 120 180

FigureS.Change inrestingheartrate

with(- *) and without somatostatin (o o)infusionduringthe twoclamp protocols (n=8; mean±SEM).

Thermic EffectofInsulin 1753 5)

(a

cc

0

0)

(9)

production in our study consistent with the results of others (38, 39).

During the last 30 min of the hyperinsulinemic euglycemic clamp with somatostatin, glucose uptake was somewhat lower (6.5±0.4 mg/kg permin) than during thesamephase without somatostatin (7.2±0.5 mg/kg per min), but the differencewas

notsignificant. While otherstudieshavereported decreased pe-ripheral glucose utilization after long periods ofsomatostatin infusion (38, 42), they have beenquestionedby Bergman et al. (43) who observed an increased glucose clearancewith

soma-tostatin infusion andinsulin in dogs. They proposed that

so-matostatinincreases insulin receptor sensitivity to insulin but

theirrateof somatostatininfusionwasapproximately eight times

the rate used in the present study. Such large doses could cause nauseaandabdominal pain in humansubjects(44).

Theinitiation of somatostatin infusionnotonlyinfluenced

hepatic glucose production but other metabolicparameters as

well. Transient falls were also observed in metabolic rate and glucose oxidation, conversely fat oxidation rose concomitant withanincrease in plasmafreefatty acids. The latter was

prob-ably duetoincreasedlipolysisby the removaloftheinhibitory effect of insulin. Whether the fall in glucose oxidation was a resultof reduced hepatic glucose production (38, 39), inhibition

ofglucoseoxidationby the glucosefatty-acid cycle(45)orboth

cannotbedetermined fromourdata.

Theinfluenceof increasing insulin concentrations on plasma

catecholamine concentrationshas beenmentionedabove.Like

Rowe etal.(33), wefound that these hormonal changes corre-latedwellwithourheartrate measurements. An 11% increase

incirculating plasmacatecholamine concentrationwas

accom-panied byan 11%increasein heartrate.Itis of interestto note

that when thesomatostatin infusionwasinitiated, therewas a significantbuttransient fall inheartrate(seeFig. 5)thatlasted

10-15 min. This effect of somatostatin on the heart rate has beenobservedbefore (46, 47),butnosuggestionsweremadeto explainthisphenomenon. Since somatostatin inhibitspancreatic insulinandglucagonsecretion, thisdecrease in heartratecould be duetoinhibition ofthe positive inotropic action of insulin (48) and/orthepositive inotropic and chronotropic action of glucagon (49). Butthiswouldnotexplainthetransientnature ofthe response. Itisalsopossiblethatsomatostatinmight influ-ence the sympathetic nervous system. Somatostatin or soma-tostatin-likepeptideshave beenfoundin thebrain(50),andit has been suggested that somatostatin and endorphin neurons interacttomodulatesympathetic outflowtothe adrenal medulla

(51). Thesameauthorshave not,however, observed similar ef-fects of somatostatin onperipheral

sympathetic

nerve

endings,

norhasit beenfound to decrease plasmaepinephrine or

nor-epinephrineconcentrations (52); this is in agreement with the resultsof this study.Assuming that the slight decrease in heart

rateobservedwith somatostatininfusion represents a change in

sympatheticnervousactivity, this is ofatransitorynaturesince the

resting

heartratewasreestablished before the start of the

insulin infusion.

Inconclusionourresultsdemonstrate thatalargeproportion

of the thermic responseto glucose/insulin infusions is dueto

glucose metabolism alone. The contribution of insulin persein the stimulation of energyexpenditure is small and appearsto bemediated via thesympatheticnervoussystem, thusat phys-iologicalinsulinconcentrations,thethermogeniceffect of insulin

per seis negligible.

Acknowledgments

The authors wouldlike to thank Mss. D.Kock,E.Rossi,E.Temler,D.

Penseyresfortheir technical assistance andMs. J. Braissant fortyping

themanuscript.We would alsolike to thank Serono S.A CH- 1170

Au-bonne, Switzerland,for thegenerous giftof somatostatin(Stilamin)and

theNESTLECo.,Vevey,Switzerland,for its financialsupport.

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