Changes in catecholamine induced lipolysis in isolated human fat cells during the first year of life

Changes in catecholamine-induced lipolysis in

isolated human fat cells during the first year of

life.

C Marcus, … , T Sonnenfeld, P Arner

J Clin Invest.

1987;

79(6)

:1812-1818.

https://doi.org/10.1172/JCI113022

.

Catecholamine-induced lipolysis in isolated human adipocytes during the first year of life

was investigated. During this period fat cell size increased markedly. Basal and

catecholamine-induced glycerol release were positively correlated with age when lipolysis

was expressed per cell. However, when lipolysis was expressed per unit of cell surface

area (micrometer squared), this correlation was observed only for noradrenaline. Basal

lipolysis and the effect of the pure beta-agonist, isoprenaline, were identical in infants and

adults. From 0 to 2 mo of age noradrenaline had very little lipolytic effect. The addition of the

alpha-2-adrenoceptor antagonist, yohimbine, to noradrenaline equalized lipolysis per

micrometer squared in infants and adults and the alpha-2-adrenoceptor sensitivity was

significantly enhanced in infants. In both groups the lipolytic adrenoceptor was of the beta-1

type. In conclusion, adipocytes from infants have a poor lipolytic response to noradrenaline

partly because of the small fat cells but mainly because of an enhanced

alpha-2-adrenoceptor activity.

Research Article

Find the latest version:

Changes

in

Catecholamine-induced Lipolysis

in

Isolated Human

Fat

Cells

during the First Year of Life

ClaudeMarcus, Bengt Karpe, Per Bolme, Tomas Sonnenfeld, and Peter Amer

Karolinska Institute, Departments ofPediatrics, Surgery, and Medicine, and the Research Center, Huddinge University Hospital; and DepartmentofPediatric Surgery, St. Gdrans Hospital, Stockholm, Sweden

Abstract

Catecholamine-induced

lipolysis

in isolated humanadipocytes

during thefirst year of life was investigated. During this period fat cell size increased markedly. Basal and catecholamine-induced glycerolrelease were

positively

correlated with age when lipolysis wasexpressed per cell. However, whenlipolysiswas expressed per unit of cell surface area(micrometer squared), this correlation was observedonlyfornoradrenaline. Basal lipolysis and the effect of the purebeta-agonist, isoprenaline, were identical in infants andadults. From0to2moof age noradrenaline had very little lipolyticeffect. The addition of the alpha-2-adrenoceptor

antag-onist, yohimbine, to noradrenaline equalized lipolysis per mi-crometer squared in infants and adults and the alpha-2-adre-noceptorsensitivity wassignificantly enhancedininfants. In both groups thelipolytic adrenoceptor was of the beta-l type. In con-clusion, adipocytes frominfants have a poor lipolytic response tonoradrenaline partly because of the small fat cells but mainly because ofanenhancedalpha-2-adrenoceptor activity.

Introduction

Adipose tissue plays a central role as a source of energy-rich substrates inman.Afterthe neonatalperiodandduringtherest of thefirst year oflife, the subcutaneous adipose tissue mass

increases considerably. The average bodyweightincreases ap-proximately three times from birth to 1 yrof age. During that

timetheadipose tissue weight increases sixfold, orfrom 11 to 16%of the body weight, to 24-28% (1). During the 1styearof life the adipose tissue mass expands mainly through an increase infat cellsize, although an increase inadipocyte number also takesplace(1,2).Thefactors regulating fatcellvolume are un-known. Since triacylglycerols constitute >95% of the fat cell volume,it is possible thatfactors which regulate the breakdown oftriacylglycerols (lipolysis)are ofimportancefor the control

of fatcellsize.Ourknowledge oftheregulation mechanism of lipolysis during infancyis poor; this is due to the difficulty in

obtainingmorethan verysmall amountsoffat for in vitro studies. However,it is wellestablished that the sympathetic nervous sys-tem, via noradrenaline andcirculating catecholamines, is of sig-nificance in theregulation of lipolysis in human adults. Cate-cholamines and insulin are the only hormones with a pro-nounced acute effectonlipolysis in adult human adipocytes (3).

Addresscorrespondence to Dr. Marcus, Department of Pediatrics,

Hud-dinge Hospital, S-14186 Huddinge, Sweden.

Receivedforpublication 13 August 1986 and in revisedform 23 De-cember1986.

Furthermore,catecholamines,whicharesolely lipolyticin most species, have different effects on lipolysisin man: stimulation via beta-adrenoceptors andinhibition viaalpha-2-adrenoceptors (4). The physiological meaning of dual regulation ofa single metabolic pathway byasingleclass of hormonesisnot under-stood.

The purpose of the presentinvestigationwas to study the catecholamineregulation oflipolysisin fat cellsduringthe 1st yearof life. The effects of differentadrenergic agentsonlipolysis duringthis phase of life were evaluatedby means ofrecently developed sensitivemethods that enabled thelipolysistobe ex-aminedindetail in small humanadipose samples (5).

Methods

Subjects.Thestudycomprised36childrenaged3 wkto 12moand 12 adults between17and 45yrofage. ItwasapprovedbytheEthics Com-mittee oftheKarolinskaInstitute. Both male and femalesubjectswere

investigated. All subjects underwent electiveinguinalherniaoperations.

Noneof thecaseshad inflamedorincarcerated hernia. Theywere oth-erwise healthy and of normalweight.The infantswerearbitrarilydivided into three age groups, 0-2 mo, 2-6 mo, and 6-12iro, without any

correctionforgestationalage.Inguinalherniasare morefrequentin

pre-maturely born infants(6), but those whoweredifficulttoplace in the abovementionedagegroupswereexcluded.

Afteranovernightfast,anesthesiawasinducedwiththiopentalsodium andmaintained withfentanylandamixture ofoxygenandnitrous oxide. Pancuronewasgiven as amuscle-relaxingagent. Insomeinfantsa1o% wt/vol glucosesolutionwasgiven intravenously during the 3hbefore the removalof fat.

Isolationofadipocytesand determinationoflipolysis.Subcutaneous adipose tissue (100-300 mg)wasremovedfrom thesurgicalincisionat

thestartof theoperation.Therewas novisualevidence of fat cell necrosis

orothermorphologicalabnormalities in theareaof the fatbiopsy. Fat cellswereisolated by collagenasedigestion usingRodbell'smethod(7). Theadipocyteswereincubatedinduplicatefor 2hat370Cin Kreb's-Ringer phosphate buffer (pH 7.4)containingalbumin (40 g/liter), glucose (1 g/liter), and ascorbic acid (0.1g/liter),with airasthegasphase.The final fat cell concentration was 1% vol/vol, which corresponded to

- 5,000-10,000 cells/ml.Atthe endof theincubation, analiquotof

themediumwasremoved for theanalysisofglycerolrelease,whichwas

usedas anindexoflipolysisandwasdeterminedbyakinetic

biolumi-nescencemethod (5).Fatcell diameterwasmeasured(8).Meanfat cell volume andfat cell surfacearea werecalculated.Theformulausedto

calculatethe fat cell surfacearea was(mean diameter of the fat cell2

+thestandarddeviation ofthediameter) X r.Thetheoretical back-groundto the formulas used hasbeen discussed in detail previously

(9,

10).

Lipolysisinsegmentsofadiposetissue. In somemethodological

ex-periments lipolysiswasinvestigatedinfatfragments. Adiposetissuewas cutintopieceseachweighing 10-20 mg. About20mgof tissue was

incubatedin 1mlof mediumasdescribed above.

Expressions ofthe results. Lipolysis was expressed per cell or per unit ofcellsurfacearea. Themaximalbeta-agonist-induced lipolysis (responsiveness)wascalculatedfrom each individual dose-responsecurve asthedifferencebetween basalglycerol release andglycerolreleaseat

1812 C.Marcus,B.Karpe,P.Bolme, T. Sonnenfeld, and P. Amer J.Clin. Invest.

© The AmericanSocietyfor Clinical Investigation,Inc.

the maximumeffectiveagonist concentration. The concentration of

ag-onist that produced 50% ofthemaximum effect

_(ED50)

wascalculated graphically from the individual dose-response curves.

Chemicals.Noradrenaline, adrenaline, isoprenaline, clonidine, pro-pranolol, andyohimbinewere obtained from Sigma Chemical Co., St.

Louis,MO. The collagenase was prepared from clostridium histolyticum

andwasofSigmatype 1.Dialyzedbovineserumalbumin (fraction V) was purchased from the Armour Pharmaceutical Co., Eastbourne,

England. The same batch was used in all experiments. All other chemicals wereofthehighest purity gradecommerciallyavailable.

Statistical methods. The values presented are the means and the standarderrorofthe mean(SE).Thestatisticalmethods used were

Stu-dent's unpairedt test andlinearregression analysis.

Results

Age, sex, and fat cell size. Age and fat cell size in the groups studied are shown in Table I. There was a positive correlation between the ageof the subjects and fat cellsizeduring the first yearof life (r =0.685, P<0.001), but no such correlation in the groupof adult subjects (r=0.014). Theincrease in fat cell volume was most pronounced during the first 6 mo (2.5-fold

increase in cell volume). The further increase in cell volume from the 6-12-mo-old infants to the adults was less marked (1.5-fold), but the difference was significant (P < 0.05). Ap-proximately 10% of the subjects investigated were female; how-ever, wefound no difference in lipolysis between males and fe-males.

Basallipolysis. The basal (unstimulated) lipolysis in fat cells

from thedifferentagegroupsstudied isshownin Table II. Ex-pressed per cell there was anincrease inlipolysiswithincreasing age(r= 0.58, P <0.001). However, when basal lipolysis was expressed perunitofcellsurfaceareathere was no such corre-lation with age (r = 0.067), and the basal lipolytic rates were similar in all the groups. Furthermore,fatcell volume correlated

significantlywithlipolysisper cell(r=0.75, P<0.001), but not with lipolysis per cell surface area.

Lipolytic

effect

of

catecholamines.

The effects of the pure beta-agonist isoprenaline and the combined alpha- and

beta-agonist noradrenaline on lipolysis in the fourage groups are shown inFig. 1.Inall groupsthelipolyticresponseto

isopren-aline expressedper cellwassignificantand dosedependent (Fig.

1A).Apositiveinfluence ofage wasobserved at allisoprenaline

concentrations,and themaximumlipolyticeffectshoweda

pos-itivecorrelation withage(r=_{0.60). However,}when isoprenaline-inducedlipolysiswasexpressed perunit ofcellsurfacearea, the

dose-response curves werealmostidentical in the different age groups.Thus, therewas nosignificant differencein the response atanyconcentration of the drug (Fig. 1 C).Isoprenaline

sensi-Table I. Characteristics ofthe Age Groups

0-2mo 2-6mo 6-12mo Adults

(M/F) 9/1 10/1 8/1 7/3

Age 6±2 wk 13±4wk 39±10wk 31±7 yr

Adipocyte

volume(pl) 134±50 200±93 329±100 497±168

Thesubjectsweredivided into fouragegroups:infants<2 mo, in-fants>2moand . 6mo,infants>6moand .12 mo,and adults. Values aremeans+SD. M,male;F.female.

Table

II.

BasalLipolysisRate inInfantsand Adults

Glycerol release Correlation between age 0-2mo 2-6mo 6-12mo Adults andlipolysis

Mumol

per

Am2per

2hx 10" 1.3±0.2 1.0±0.3 1.5±0.3 1.2±0.2 r=0.067

Mmol

per

I07 cells

per2h 1.6±0.2 1.8±0.5 3.2±0.6 3.6±0.6 r=0.58

Theage groupsare thesame asinTable I.Lipolysis was expressed per cellsurfacearea orpercell. Thecorrelation between basal glycerol

re-lease and age wasexamined in the whole material, using linear regres-sion analysis.

tivity

was not age dependent. The ED50 was obtained at 1 nmol/liter of isoprenaline in allgroups.

The lipolytic effect of noradrenalinewas poorintheyoungest groupofinfants, witha mean maximalresponseof80% over

the basal values. Theresponseincreased gradually in the older infants and reached adult levels (- 2.5-fold stimulation ofbasal lipolysis) in the 6-12-moagegroup.However, contrarytowhat wasfound concerning unstimulated and isoprenaline-induced lipolysis, the noradrenaline-induced lipolysis showedapersistent agedependency whether itwasexpressedperunit ofcell surface area orperfat cell(Fig. 1, B and D). Therewasalsoapositive correlation between age and the individual maximal lipolytic response tonoradrenaline in the entire infantgroup(r=0.605), but not in theadult group (r = 0.167),when it wasexpressed percellsurfacearea.

The sensitivity of the cells to noradrenaline, expressed as

ED50,

did not vary significantly between the age groups. The

ED50 value indicated for the youngest group is, nevertheless, uncertain, sincethepoorlipolytic responseinthisgroupcauses considerableerrors intheEDs0valuesfornoradrenaline.

Effectofyohimbineoncatecholamine-induced lipolysis. The

lipolyticresponse of noradrenalinewaslower than that of iso-prenaline inhuman adults, since the catecholamines activate both the lipolytic beta-adrenoceptors and the antilipolytic alpha-2-adrenoceptors (4). The difference in lipolysis between age groupsshown in Fig. 1 seems tobeduepartlytothewell-known factthat there is apositivecorrelation between fat cell sizeand lipolysis when the metabolicrateisexpressed percell(10). Con-versely, factors unrelated tofat cell size mayalso be involved. Toinvestigate whether the poorlipolyticeffect ofnoradrenaline inearlyinfancy,whichpersistedwhencompensationwasmade forthe differencesin fatcell size(Fig. 1D),wasdue to enhanced alpha-2-adrenoceptor-mediated activity,the effect of the addi-tion ofyohimbine (selective alpha-2-antagonist)was studiedin twosubgroupsof the material: six infants 1-4 mo ofageandsix adults(Fig. 2). Theyohimbine concentration used(l0-4 mol/ liter) was chosen togivea complete blockade of the alpha-2-adrenoceptors (1 1). Without yohimbinethe noradrenaline-in-ducedlipolysisin the infantgroupwasin the samerangeasin the 2-6-mo-oldgroupshown in Fig. 1-i.e., - _{50% of the}

re-sponse in the adult group.Withyohimbineaddedtothemedium, the differences between theagegroupsdisappearedand the

A

25

Ue

3 d 20

0

-

'-I-0-,

C C

10

04 cv

.u

4--~0

-cnH

z:

X

-11

B

I

I....**,*III **IIII III

-10 -9 -8 -7 -6

LOG DRUG CONCENTRATION, MOL/L

asit had onnoradrenaline-induced lipolysisin thetwogroups.

Yohimbine alone had noeffecton lipolysis (data notshown). Sincethedifferencebetweenmaximallipolyticresponsesto

nor-adrenaline andisoprenalinewasdue to alpha-2-adrenoceptor-mediated inhibitionboth ininfantsand adults, the percent

alpha-2reductionin

noradrenaline-induced

lipolysiscan becalculated asisoprenalineminusnoradrenalinedividedbyisoprenalinein each subject. This value for alpha-2 inhibition, which is not

dependentontheexpressionoflipolysis (percellorsurfacearea),

was84±12% in the youngestinfantgroup(0-2 mo) compared with 50±16% in the adult group.

The lipolytic potency of isoprenaline, noradrenaline, and adrenaline togetherwithyohimbine (expressed as a percentage of the' maximal effectto avoid the influence of cell size on

li-polysis) is shown in Fig. 3. With yohimbrine in the medium

actingas analpha-2-adrenoceptor blocker, the catecholamines

interactalmostexclusively with beta-adrenoceptors. The relative orderof potency between isoprenaline and the catecholamines was:isoprenaline>noradrenaline=adrenaline, bothin infants and adults. These results are in agreement with the classical

pharmacological

definition of the

beta-l-adrenoceptor

both in infantsandadults.

Antilipolytic

effect ofcatecholamines.

Theantilipolyticeffect of catecholamineswas

investigated

intwoways:bystudyingthe

u)

0_En

Pr.,

U

H

z

--4

Ia

x

C-'

c0 N.,

Figure1.Isoprenaline- and nor-adrenaline-induced lipolysis dur-ing the first year of life and at adult age. Adipocytes were incu-bated withincreasing concentra-tions of noradrenaline and iso-prenaline. The glycerol release to themedium was determined. Li-polysis is expressed per cell in A andB, and per unit of cell sur-face area in C and D.

ED5o

of noradrenalineareindicatedwith

arrowsin D. The

ED~v

of

iso-prenaline was 1.3±0.7 nmol/liter. Thebasalglycerol releasewas

subtractedfrom the catechol-amine-inducedvalues sothat net glycerol release is given. The

val-ues aremean±SE. (x) 0-2 mo; (!) 2-6mo;(A)6-12 mo; and(0)

adults.

NORADRENALINE CONCENTRATION

log mol/l

Figure2.Effect ofyohimbineonnoradrenaline-inducedlipolysis.(A andC)Isoprenaline;(B andD) noradrenaline. Isolated fat cellsfrom sixinfants 0-4mo(triangles)andsix adults (squares) were incubated withindicatedconcentrations of noradrenaline alone (opensymbols)

orwith theaddition of10-4mol/literyohimbine(filled symbols).The characteristics of the age groups were(a) infants,five males and one female,age 9±3wk, fat cell volume 180±86 pl; and(b)adults, six males, age 31±7 yr, fat cell volume 497±168 pl.

100 10l- Figure3.Sensitivity of

catecholamine-A

/ B r induced lipolysis. (A) 0-4 mo; (B) adults.

5cl/4

i.

8g

g / Therelative order ofpotencyof

(o)

isopren-/ / / aline, (A, A) adrenaline, and (x) noradrena-7sf /x - / line inducedlipolysis in fat cells obtained

>

s0- /60 / from infants and adults. Fat cellswere ob-tained from the same subjects as in Fig.2

_/./ Sf andincubated with or without increasing

40-

_40/

_{concentrationsof catecholamines.}

Yohim-bine(10- mol/liter) was added to the me-0 20- 20- S / dium to block the

alpha-2-adrenoceptor-mediated antilipolytic effect of the catechol-amines.Lipolysis is expressed here as a

per-II _7-1 - X_1111111. centage of the maximallipolyticeffect of

-10 -9 -8 -7 -8 -5 -10 -9 -8 -7 -6 -5 each catecholamine. The individual

ED50

values, calculated as described in Methods, LOG DRUG CONCENTRATION, MOL/L werealso determined: isoprenaline, infants

1.7±0.7 nmol/liter and adults 1.2±0.4 nmol/liter; adrenaline, infants 215±85 nmol/liter and adults 85±38 nmol/liter; and noradrenaline, infants 412±255 nmol/liter and adults 88±32 nmol/liter.

antilipolytic effect of the combined alpha- and beta-agonists, noradrenaline and adrenaline, during beta-adrenoceptor block-ade(12),andby meansofthe synthetic selective alpha-2-adre-noceptor agonist, clonidine. Theophyllamine was added to the incubation medium as a lipolytic agent, as suggested previously (12). Propranolol was added to noradrenaline and adrenaline at aconcentration(lo-4 mol/liter), which gives a complete

beta-adrenoceptorblockade (13). When propranolol was added alone to the fat cellsit hadno effect on lipolysis (data not shown). Infants aged 0-4 mo and adults were investigated.

Fig.4shows the maximal inhibitory effect of noradrenaline,

adrenaline, andclonidine on lipolysis induced with theophyl-lamine

(l0-'

mol/liter). There were no significant differences in maximum inhibition of lipolysis either between the drugs or betweenthe age groups when glycerol releasewasexpressed per cellsurfacearea. Furthermore, the theophyllamine-induced

li-

6-En

0

To

w;

10

W-\

d

N

0

4-2-~

THEO NA A CLO

Figure4.Antilipolyticeffect ofnoradrenaline, adrenaline,and cloni-dine.Adipocytesfrom six infants and sixadults(openbars)were

incu-batedwithincreasing concentrations of noradrenaline(NA),

adrena-line (A),andclonidine (CLO) withtheophyllamine (THEO, l0-3mol/ liter)aslipolytic agent.Propranolol

(10'

mol/liter)wasusedtoblock thebeta-adrenoceptoractivityofNAand A.Glycerolrelease in the absence orinthepresence of maximum effective concentrations ofthe

variousantilipolytic agents isgiven.Thesameinfantsas inFig.2were

investigated.

polysisin the absenceof inhibitingagent was not significantly

different statisticallyininfantsandadults.

For the studyof the sensitivity of the drugs, the dose-response curves wereplotted aspercent ofmaximal effect toavoid the influence of the expression of the rate of lipolysis (per cell or

surface area). These results are given in Fig. 5. All the dose-response curves wereshifted tothe leftin the infant group as compared with the adults. To evaluatestatisticallythe differences insensitivity between the groups, ED50 for eachindividual dose-responsecurve wasdetermined. The means of the ED50 values weresignificantly lower for noradrenaline, adrenaline, and

clon-idineininfants as compared with adults (Table III).

Intheory, theinitialratesof lipolysismay influence the shape

ofthedose-response curvesofantilipolyticdrugs. Thereforeit wasof importance to exclude the possibility that small variations in the lipolytic effect of theophyllamine between infants and adults had amajor influenceonthe ED50 values.Usingthedata in Table IV, we compared the three infants who had the highest

lipolytic effects of theophyllaminewith the three adults whohad the lowestlipolytic effectsoftheophyllamine. Thedifference in ED50 valuesforallcatecholaminesbetweeninfantsand adults

persistedin these extreme subgroups (valuesnotshown). Thus,

small differencesintheophyllamine-induced lipolysis between

infants and adults have no bearing on the results with

cate-cholaminesensitivity.

Effects

ofadenosine deaminase on

lipolysis.

Theeffects of1

U/ml ofadenosine deaminase

(ADA),'

which inactivates

en-dogenousadenosine,wasexaminedinfat cellsofthreechildren

of0-4 mo of age. The cells were incubated with or without

different concentrations of noradrenaline.

Lipolysis

was

ex-pressedasmicromoles ofglycerolper 2 h per1m2X

10-11.

ADA increased the basallipolyticratefrom 1.3±0.4to2.2±0.6. How-ever, it didnotalter the maximallipolyticresponse of noradren-aline(1.7±0.7withoutADAand 1.6±0.7 with

ADA). Likewise,

ADA had no effect on noradrenaline-induced

lipolysis

in fat cellsofadultsubjects (datanotshown).

Effect of collagenase

incubation

of

adipose

tissue. It is

re-portedthatthe

adipocyte

isolation process with

collagenase

has effectson

lipolysis

inhumanadults

(14).

Catecholamine-induced

1. Abbreviation usedinthis paper:ADA,adenosine deaminase.

Catecholamine-inducedLipolysisduringInfancy

r-

100-

60-

40-

20--S -8 -7 -6

--5 -10

100-

80-60-~

40-

20-<1-T 1 T r T r TT l 1 1 1-1 -9 -8 -7 -8 -5

IT

-10 -9 -8 -7 -6 -5

LOG

DRUG

CONCENTRATION,

MOL/1

Figure 5. Sensitivity of the catecholamine- and clonidine-induced anti-lipolysis. The dose-response curves for (left) noradrenaline, (middle) adrenaline, and (right) clonidine from the investigationinFig.4 were

plotted as a percentage of the maximal inhibitory effect. The latter was

lipolysisis increased in isolated fat cells ascomparedwith fat segments, andthisis duetoinhibitionofphosphodiesterase by

collagenase. Intheory, infant adipose tissue may respond

dif-ferently fromadult tissuetocollagenase. Therefore, the lipolytic effects of catecholamines in isolated adipocyteswerecompared with those infragments of adipose tissue fromtwo2-3-mo-old

infants.Thefragmentshad thesamebasallipolysisastheisolated cells.Noradrenalinestimulated basallipolysis 14% infragments

and 91% inisolated cells.Isoprenalinestimulated basallipolysis 5.6-fold infragmentsand 7.8-fold in cells. Thus, thelipolytic

effectof catecholamines is increasedinisolatedinfantadipocytes asit isinisolated adult cells.

Effect ofglucose

infusion

onlipolysis. Infantshave ingeneral

ahigher metabolicturnoverthanadults. Todeterminewhether the fasting period had any influenceonthe lipolyticrate, we gave infants 2-4 mo ofage glucose infusions during the 3 h

beforethestartof the operation. However, there wereno

dif-ferencesinbasal,maximalnoradrenaline,ormaximal isopren-aline-induced lipolysis in fat cells obtainedfrom theseinfants

compared with age- and weight-matched infantswhodid not receive glucoseinfusions beforetheoperation (Table IV).

Discussion

In the present study the effect of catecholamines on lipolysis duringthe 1st yearof lifehas beeninvestigated forthefirst time

Table III.ConcentrationofDrugs Giving

Half-MaximumInhibitionofLipolysis

(ED50)

ED50(nmol/liter)

Study

group Noradrenaline Adrenaline Clonidine

Infants 64±18 30±12 3±1

Adults 440±185 156±42 23±4

P <0.01 <0.05 <0.01

TheexperimentsinFig. 5 were used to calculateED50from each dose-responsecurve.Thestatisticaldifference was tested using Student's unpairedt test.

calculatedasglycerol release with only theophyllamine and proprano-lol present in the medium, minus glycerol release at the maximum ef-fectiveinhibitory concentration of the hormone. Thesameinfants aged 0-4mo(x) and adults (o)asinFig.2 wereinvestigated.

insomedetail.Itwasobserved that theadipocytesizeincreased markedlyduring this period of life. This isin accordance with what haspreviouslybeenshownby others (1, 2), althoughsome authors(15)havefound

divergent

results withonlyaslight in-crease in fat cell sizeduring this period. The reason for this

discrepancyis unclear. In the latter paper, all the ageswere

ad-justed for

gestational

age. This could, perhaps together with

methodological variationsin theestimates offat cell size

(16),

explainthedifferencesin results.

Ofgreaterimportance, however,is the marked age

depen-dencyoflipolysis. Thisagedependencyhadtwodifferent

char-acteristics. First,basal and catecholamine-inducedlipolysis in-creased gradually duringthe 1st year, when

lipolysis

was

ex-pressed per cell number. This correlated significantly with the

increaseinfat cell size duringthe1st yearof

life,

whichconfirms

the well-known observation thatlipolysis per cell is relatedto the sizeoftheadipocyte (10). This isnot asurprising finding,

since itseemsphysiologicalthatalargecell hasamoremarked

metabolic activity than a small one. The dualeffects of

cate-cholamineson

lipolysis

inhumanfat cells, stimulationvia beta-adrenoceptors andinhibition via alpha-adrenoceptors,have been knownforalongtime (17, 18).However, the increase inlipolysis

per cell

during

the 1styearoflife is probablynotmainlydueto

TableIV.Effect of Glucose InfusiononLipolysisinInfants

Glycerol release(jumol/nmn per 2 h X1(")

Group Basal Noradrenaline Isoprenaline

Glucose 0.9;0.8 1.2; 1.8 7.4; 5.2 (mean0.9) (mean1.5) (mean6.3) Fasted 1.0±0.3 1.5±0.6 6.5±1.1

Twoinfants (2 mo,4mo) received 10% wt/vol glucose solution (3ml/ kg bodywtper h) duringthe3 hbefore removal of fat. Fatcellswere incubated withorwithoutvariousconcentrations of noradrenalineor

isoprenaline. Glycerol releaseatmaximum effective catecholamine concentration isgiven.The results(mean±SE)in 10children aged 2-4

mo who werefasted overnight are given for comparison.

1816 C.Marcus, B. Karpe, P.Bolme, T.Sonnenfeld, andP. Amer

100-E-4 1W

80-Pz4 0

60-

40-O

20-_

I.

altered adrenoceptor activity. The effect of isoprenaline (pure beta-agonist), as well as that of noradrenaline (combined alpha-and beta-agonist), increased markedly during this period. In the first place, the properties of the lipolytic beta-adrenoceptor seemed to be unchanged during this period.

Catecholamine-in-duced lipolysis in adults is mainly mediated via beta-l-adre-noceptors (19), and the relative order of potency in the lipolytic

effectsof isoprenaline, noradrenaline, and adrenaline during al-pha-2-blockade in the present study was similar in infants and adults. This suggests that the beta-l-adrenoceptor activity is also predominant during infancy. Secondly, isoprenaline sensitivity was similar in infants and adults. Finally, both basal and

cate-cholamine-inducedlipolysis increased markedly with age. The increase in the rate of catecholamine-induced lipolysis during infancy seems to be at least partly due to the increase in fat cell

sizeand thus not only to altered physiological properties in the cells.

Conversely, marked differences between the various lipolytic

activitieswere observed during the 1st year of life when lipolysis was related to fat cell surface area. With this expression of

li-polysisboth the basal and theisoprenaline-stimulatedrates were almostidentical ininfantsand adults. However, a marked age dependency was observed with the endogenous catecholamine

noradrenaline.The lipolytic effect of the hormone was very poor inisolated fat cells from infants below 2 mo of age, but it

grad-ually increasedininfantsup to 6-12 mo of age, when it reached almost the same levels as in adults. This difference in

noradren-alineeffect betweeninfantsand adults could hardly be explained

by an alteration in the beta-adrenoceptor activity. The results

of methodological experimentsindicate that it is not due to

dif-ferent sensitivitiesto collagease treatment between infant and adultfatcells. Instead, our data clearly indicate that the decreased noradrenaline effect in infants, which thus persisted whether

lipolysiswasexpressed per unit of cell surface area or per cell, was mainly due to alterations in the effect mediated via the alpha-2-adrenoceptors. There is much evidence for this assumption.

First,thedifferencesin the lipolytic effects of noradrenaline be-tweenthe age groups disappeared completely when the alpha-2

blocking agent, yohimbine, waspresent in the medium. Sec-ondly, in theinfantgroup there was an increased sensitivity to both naturalcatecholamines and the synthetic selective alpha-2-adrenoceptor against,clonidine,with regard to theantilipolytic activity.Thirdly, thenoradrenaline/isoprenalinequotient (which represents alpha-adrenoceptor activity) was enhanced in infancy. Theenhancedalpha-2-adrenoceptor-mediated antilipolytic effectin the youngest age group may have severalexplanations.

It maybe due to amodulation of thebindingof the

catechol-aminestothealpha-2-adrenoceptor-i.e., alterationsin receptor number oraffinity. It may also be due to changes beyond the receptorlevel, such as in theinteraction between the receptor

and the GTP-sensitive inhibitory coupling protein, or in the interaction between the couplingproteinand thecatalytic com-ponent ofadenylate cyclase. Unfortunately, it isnot atpresent

possibletoclarifythesequestions.The study ofcatecholamine binding, of coupling proteins, and ofadenylate cyclase in fat cellsrequiresmuchlargeramountsofadipose tissuethan, for ethical reasons, it ispossibleto removefrom infants. However, the observedalteration in thealpha-2-adrenergic sensitivity

in-dicates thatchanges in ornear thealpha-2-adrenoceptor may beofimportance.

It hasrecentlybeensuggestedthatadenosine,whichactsas aparacrine modulatorofthecyclicAMP-system, is one of the

factorsresponsible for the poor lipolytic effect ofcatecholamines

in the adipose tissue of obese adult subjects during caloric

re-striction(20). The poor effect of noradrenaline during infancy is probably not due to variations in the endogenous adenosine levels. The addition of ADA, which effectively breaks down adenosine to inosine, did not alter the catecholamine-induced lipolysis either in the infants or in the adult group.

Inthis study adipose tissue was obtained after an overnight

fast,andit iswell known thatcatecholamine-induced lipolysis

can be modulated by prolonged fastingin man (3). Intheory, infant fat cells may be more sensitive in this respect than adult

adipocytes. However,forseveral reasonsthisseems not to be of importance for the present results. First, the increase in the basal rate of lipolysis is the principal finding during fasting (3), and this was not observed in infants. Secondly, the results with glucose infusion did not differ from those observed with overnight fasting. Little is known about the regulation of lipolysis during

in-fancy.In thefew studiespublishedsofar the methods used to determine lipolysis have proved insensitive and only a small number of subjects have been studied (21, 22). Novak et al. (23) reported that the adipose tissue of newborn infants is less sensitive tocatecholaminesthan is adipose tissue from old persons. It is alsoof interest that both free fatty acids and glycerol are reported tobe very low at the timeofbirth (24, 25), although the cate-cholamineconcentrationhas been reported to be high in blood samplesfromthefetal scalpduring deliveryand from the

um-bilicalcord (26, 27).Apoorcorrelationbetweenumbilicalcord

concentrations of catecholaminesandglycerol is also reported (26, 28). It has to be emphasized that the results in this study wereobtained during in vitro conditions that cannot be directly

extrapolatedtoin vivosituations.Furthermore, the present study

did not directly concern the neonatal period. Nevertheless, it seems reasonable to believe that the increase in the alpha-2-adrenoceptorinhibitionof lipolysis isonefactorbehind the

im-paired effect of catecholamines during deliveryand after birth. It may also be viewed as aphysiologicallyprotectivemechanism against excessive lipid mobilization in situations with a high catecholamine secretion.

The enhancement of alpha-2-adrenoceptoractivityin fat cells

during infancymay also beofphysiological importancefor the growthof fatcells duringthis period of life. Inhibition ofthe

lipolytic effect of catecholaminesprotects the fat cell from ex-haustionofits lipid stores and thereby favors anexpansion of the fat cell volume. Thus, the enhanced alpha-2-adrenoceptor activity may bepartly responsiblefor the rapidincrease in fat cellsizeobservedduringthefirst6moof life.

Insummary,we havefoundthatnoradrenaline duringthe first months oflife has a very limitedlipolytic effect in vitro

owingto the enhanced alpha-2-adrenoceptor

activity.

The

li-polytic effect of noradrenalineincreases

gradually

with age and

attains the same effectasin adultswithin the 1st yearof life.

This phenomenon may beofimportancefor the

regulation

of lipolysis during early infancy. There is alsoa generalincrease in thelipolytic activity during the first year of life that seems

relatedtofat cellgrowth.

Acknowledgments

Theauthors wishtothank Gunilla Micha-Johansson forherexcellent technical assistance.Theinvaluablecooperationof the staffatthe

De-partmentof PediatricAnaesthesiologyandPediatricSurgeryatSt.

Gor-an'sHospitalisgreatly

appreciated.

This study was supported by grantsfrom the Swedish Medical Re-searchCouncil (19x 01034), Karolinska Institute, theSamariten Foun-dation, the Osterman FounFoun-dation,theSwedish Society of Medicine, and theFoundation for Old Female Servants.

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