Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms

Human fat cell lipolysis is primarily regulated

by inhibitory modulators acting through distinct

mechanisms.

H Kather, … , K Aktories, K H Jakobs

J Clin Invest.

1985;

76(4)

:1559-1565.

https://doi.org/10.1172/JCI112137

.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of

lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6

micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated

with an increase in glycerol release averaging 150-200% above basal levels. Clonidine,

N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent

inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of

adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold

increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused

either no further increase or only a marginal additional increase of lipolysis after

pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in

controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate

cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were

impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes

pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect

on cyclic AMP after pertussis toxin treatment. The findings support the view that the

antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase

action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human

fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal […]

Research Article

Find the latest version:

Human Fat Cell Lipolysis

Is

Primarily Regulated by Inhibitory

Modulators Acting through

Distinct Mechanisms

H. Kather, W. Bieger, G. Michel,K.Aktories,and K. H.Jakobs

KlinischesInstitutfarHerzinfarktforschungander Medizinischen Universitdtsklinik, MedizinischePoliklinik derUniversitdt, and PharmakologischesInstitut der UniversitdtHeidelberg, D-6900Heidelberg,FederalRepublic ofGermany

Abstract

The effects of adenosine deaminase and ofpertussistoxin on hormonal regulation of lipolysis were investigated in isolated humanfat cells.Adenosine deaminase(1.6 5tg/ml)caused a two-tothreefold increaseincyclic AMP,which was associated with anincreaseinglycerolreleaseaveraging150-200% above basal levels. Clonidine,

N'-phenylisopropyladenosine,

prostaglandin

E2, andinsulin caused adose-dependent inhibition ofglycerol

releaseinthe presenceof adenosinedeaminase.

Pretreatment of adipocytes with pertussis toxin(5,ug/ml)

for 180 min resulted ina five-to sevenfold increase in cyclic AMP.Glycerolreleasewasalmostmaximaland

isoproterenol

causedeither no further increase or only a marginal additional increase oflipolysis after pretreatment with pertussis toxin,

whereascyclicAMPlevelswere500 times

higher

than in

con-trols. Theeffectsof

antilipolytic

agents knowntoaffect lipolysis

by inhibition ofadenylate cyclase activity, i.e., clonidine,

N6-phenylisopropyladenosine, and

prostaglandin

E2,were

impaired.

Incontrast, the

antilipolytic

actionofinsulinwas

preserved

in

adipocytespretreated with

pertussis

toxin. As incontrols, the

peptidehormonehadnodetectable effecton

cyclic

AMP after

pertussis

toxintreatment.

Thefindingssupportthe viewthattheantilipolyticeffect of

insulindoes notrequire adenylatecyclaseor

phosphodiesterase

action.Inaddition, theresults demonstratethat, upon relief of

endogenous

inhibition,

humanfat cell

lipolysis proceeds

at

con-siderable (adenosine

deaminase)

oralmost maximal(pertussis toxin)rates. A certain

degree

of

inhibition, therefore,

appears tobe necessaryforhumanfat celllipolysistobe

susceptible

for hormonalactivation.

Introduction

Human

adipose

tissue respondsto a

variety

of humoral, hor-monal, and neural factors (1). Mostof these regulatory

influ-encesappear to bemediated via activationorinhibition of

ad-enylate cyclase (1,

2).

Inhumanfat

cells,

stimulators of

cyclic

AMP (cAMP) formation include

_{f3-adrenergic}

agonists and a

single peptide hormone, namely

parathyroid

hormone

(2).

a2-Adrenergic amines, adenosine,and

prostaglandins

oftheE type, areinhibitorsofcAMP

production

(3-7). Insulin also inhibits lipolysis (1).However, themechanismbywhich theantilipolytic effect ofinsulin ismediated isnotunderstood. It iscontroversial, for instance, whether insulin regulates adenylate cyclase and

Receivedforpublication11June 1984 and in revisedform 31 May1985.

whether themodulationof intracellular cAMP concentrations

by the hormoneplays aprimaryrole (1).

Inadditionto theuncertainties regardingthemechanism(s) of action ofsome hormonal regulators, such asinsulin, ithas

beenfrequently difficultto relate theeffects ofhormonesinvitro

tosystemic metabolism in vivo. Itis still notclearwhich

hor-mones areresponsible for lipid mobilization during fasting. Li-polysis could be elevated by the drop in plasma insulin that occursduring fastingor by anincreasedreleaseof lipolytic

hor-mones such asepinephrine (1), twomechanisms ofregulation thatarefundamentally different. Ifa dropininsulin, i.e., relief of inhibition, were responsible for increased rates of lipolysis

during

starvation, this would imply that unrestrained basal

li-polysis proceedsatconsiderable ifnotmaximalrates. By contrast,

basal

lipolysis

could below or even negligible if starvation-in-duced activation oflipolysis were brought about by lipolytic

hormones.

Invitrobasal ratesof lipolysisare usually low andsubstantial

ratesof lipid mobilization are observed onlyinthe presenceof lipolytic hormones, suggesting thatlipolysis might, in fact, be

primarily regulated by activation. However, fatcells

sponta-neously produce and release inhibitory compounds such as

adenosine and prostaglandins (2-5, 8). In order to discern

whether basal lipolysis proceeds at maximal or near-maximal rates, it is necessary to eliminate the influence of inhibitory compoundsproducedduring incubation in vitro. Adenosinecan

be

easily

removedbyenzymic degradation using adenosine de-aminase.

Recently, atoxinhas beenidentifiedandcharacterizedfrom supernatants of cultures of Bordetella pertussis that elicits a va-riety of biologicalresponses including induction of

lymphocy-tosis, sensitization ofanimals to the lethal effects of histamine,

stimulation

of insulinsecretion, and lipid mobilization (9). Pre-treatment ofrats or hamsters with pertussis toxin impairs or

abolishes

theinvitro effectsonfatcelllipolysis of various

an-tilipolytic

agents,includinga-adrenergic agonists, prostaglandin

E2,adenosineanalogues, and nicotinic acid(10-13).Incontrast tothe effect ofthe latter compounds, treatment with pertussis

toxin (10,

11)

preserves the antilipolytic effect of insulin. The

toxin offeredthe opportunity to eliminate not only the effects

of adenosinebut also ofother endogenous inhibitors such as

prostaglandins.

Byusingboth adenosine deaminase and the bacterial toxin, we were able to demonstrate that basal human fat cell lipolysis proceeds atconsiderable rates upon relief of endogenous

inhi-bition, suggestingthat lipid mobilization from human fatcells

isprimarilyregulated by inhibition. As in the rat, the antilipolytic

action ofinsulin was preserved afterpretreatment of human

adipocytes withpertussis toxin, indicating that themechanisms

underlying

theantilipolytic effects of insulin are alsodifferent

from those of other inhibitors such as prostaglandin E2,

N'-phenylisopropyladenosine, and

a2-adrenergic

amines in human fat cells.

J.Clin. Invest.

©TheAmerican Society for ClinicalInvestigation, Inc.

0021-9738/85/10/1559/07 $1.00

Methods

Subjects. Subcutaneous adipose tissuewasobtained from 20 obese sub-jectsundergoing elective abdominalsurgery.The patientswerenot

se-lectedonthebasis ofage, sex,ordisease. Surgerywasperformed after anovernight fast. Anesthesiawasinitiated withashort-acting barbiturate andmaintained withoxygen,nitrous oxide, and halothane. Tissue

spec-imens ( 1-10gof tissue)wereobtainedatthestartof theoperation. Preparation offat cells and preincubation. Tissue specimenswere

cutinto small pieces and fat cellswereisolatedby collagenase digestion

accordingtoRodbell (14)using Krebs-Henseleit bicarbonate buffer, pH 7.4,containing 5 mM glucose, 40 g/liter of humanserumalbumin, and

0.5mg/ml of collagenase (Sigma Chemical Co., St. Louis, MO,typeII). After45 min of incubationat370C thefat cellswerewashed byflotation

(threetimes) and resuspended in thesamemedium (except that

colla-genasewasomitted)ataconcentration of50,000-200,000 cells/ml, and preincubated for 180minat370C underanatmosphereof 95%02and

5%CO2(vol/vol). Where indicated, the mediumwasfortifiedwith5

,g/

mlof pertussis toxin. Control cellswerehandled inanidenticalmanner exceptthat notoxin wasadded. Preincubations wereterminated by

washingwith fresh medium without pertussis toxin. Pertussis toxinwas alsoomitted during subsequent incubations.

Incubations. When glycerol release servedasthe only endpoint (see

Figs. 2 and 3), cell densitieswereadjustedto5,000-20,000 cells/mland

incubationswerecarriedoutin stoppered plastic vials inatotalvolume of0.05 ml underanatmosphere of 95%02and 5% CO2 (vol/vol) fora

further 180min. Hormones and adenosine deaminasewereaddedatthe

startof the final incubations in these latter experiments. Reactionswere

terminated by heating (950C, 5min).

Inexperiments where cAMP levels and glycerol releasewere

deter-mined concomitantly, higher cell densities (approximately10 cells/ml)

andlargerassayvolumes(6ml)wereusedduring thefinalincubations. Preliminary experiments indicated that theonsetof the antilipolytic effect

of insulinwaspreceded byalagof- 10min.Unless otherwisestated,

time-course experimentswere,therefore, started 15 minafterhormones

andadenosinedeaminase had beenadded. At the timesindicated, aliquots

of0.5and 0.05 mlwerewithdrawninthese latter experimentsandassayed

for cAMP andglycerol, respectively.

Pertussis toxin. Pertussis toxinwaspurifiedtoapparenthomogeneity accordingtoCowelletal.(15)fromsupernatantsof Bordetellapertussis

suspensions kindly donated byDrs. L. Robbel and F. Backkolb(Behring Werke, Marburg, Federal Republic of Germany). Preliminary

experi-mentsindicated that for human adipocytes relatively high concentrations (1-5,ug/ml) ofthe toxinwereneededinordertoachievecompletereversal ofthe antilipolytic effects of prostaglandin E2, clonidine, or N6-phe-nylisopropyladenosine within 180 minat37°C.Therefore,a

concentra-tion of5 ug/mlofpertussistoxinwasusedthroughout inthe present studies. As in other cell typesorspecies, pertussistoxinincreased basal

glycerol releasewithalagof 90-120minunder these conditions.

Assays. Theglycerolcontentofthedeproteinizedmediawasmeasured

byabioluminescent methodusinganautomaticluminescenceanalyzer (BertholdLB950T).Amanualversionofthis method has been described indetailelsewhere (16, 17).Theuseofanautomaticanalyzer required

someminor modifications.Briefly,0.05mloftheappropriatelydiluted

samples (2-10 times)wereaddedtoanequalvolumeofamedium

com-posedof 28nmol/litertriethanolamine-HCl, 1.1 mmol/liter KH2PO4,

20 nmol/liter Na3AsO4, 1.1 mmol/liter dithiotreitol, 2.9 mmol/liter MgCl2,1.54mmol/liter ATP,8.0mmol/liter NAD,7U/ml glycerokinase,

19U/ml glyceraldehyde 3-phosphate dehydrogenase,2.3U/ml glycerol 3-phosphate-dehydrogenase, and 33 U/mltriosephosphate-isomerase.

Afterincubationfor 120minat37°C,thesampleswerefurther diluted

(sixtimes)and0.01-mlaliquotsof the diluted mediawereassayedfor

NADH.Theassaycocktail for the luciferase reaction contained 0.5mmol/

litertetradecanal 1.1

Amol/liter

flavinmononucleotide, 15U/liter

bac-terialluciferase,and 8.5U/liter NAD(P)H:flavinmononucleotide oxi-doreductase. Theassaycocktailwasprepareddailyand heldat +2°C duringmeasurements. Light productionwasmeasured bya

microcom-puter-controlled automaticluminescence analyzer.The vialscontaining

0.01 mlof sample were automatically moved into the measuringposition.

Thereaction was started byautomatic injectionof the assay cocktail.

Measuringtime was 15 s. Theintegralofcounts between 10 and 15s wastakenas a measureofNADHconcentration.

cAMPwasdetermined afteracetylationusingacommercially avail-able radioimmunoassay (NEN Chemicals, Dreieich,FederalRepublic

ofGermany). Fatcell number was determined bycountingall cellsin appropriatelydiluted aliquots (10 Ml) of the cellsuspension.

Chemicals.Collagenase(typeII) wasfromSigma Chemical Co. All other enzymes and coenzymes and N6-phenylisopropyladenosine were fromBoehringer Mannheim,Federal Republic of Germany. Ammonium sulfatewasremovedfrom enzyme suspensions by centrifugation. Pros-taglandin E2 and porcine insulin were from Serva AG,

Heidelberg.

Highly purified human serum albumin was from Behring Werke, Marburg. Epi-nephrine andisoproterenol were purchased from Merck AG, Darmstadt, and RothAG, Karlsruhe, respectively. Clonidinewas agift ofBoehringer Ingelheim.

Statistics. Statistical analysiswas by the Wilcoxon test forpaired

observations.

Results

Effects

ofpertussis toxinandof adenosine deaminaseonbasal and isoproterenol-activated cAMPaccumulation and

lipolysis.

The time course of cAMP accumulation during exposure of

hu-manadipocytes tohormonal activators of lipolysis is less well defined than for rat adipocytes. Therefore, experiments were

done inwhich cAMP and glycerol release were measured

con-comitantly at different time intervals (Fig. 1). Basal levels of cAMP averaged 20 pmol/106 cells in this experiment. Removal ofendogenous adenosine resulted in approximately a threefold increase inbasal cAMP levels (Fig. I A), which was associated with a corresponding activation of glycerol release averaging 200%above basal levels (Fig. I C). The time course of cAMP accumulation in cells exposed to isoproterenol (I ,mol/liter) showedarapid increase tomaximum levels that remainedat

the same increased level for 60 min of incubation under all conditions employed. cAMP levels rose to more than 1,000

pmol/ 106 cells in the presence of isoproterenol, while the rate

ofglycerol release seen under these conditions exceeded that induced by adenosine deaminase by only 25%. When isopro-terenol andadenosine deaminase were added together, cAMP levels were further increased (from 1,000 pmol/ 106 cells to

_5,000pmol/106 cells). However,nofurther increase in glycerol

release was observed under these conditions.

Pretreatment of fat cells with pertussis toxin alsoresultedin an increase in cAMP levels which, though moderate in

com-parison to isoproterenol, exceeded that seen inthe presence of adenosine deaminasebyafactor of 2-3(Fig. 2B;TableI).In

the presence ofisoproterenolcAMPlevels rose to 10,000pmol/ 106 cells after pretreatment of fat cells with pertussis toxin. Adenosine deaminase had no effect on cAMP levels under these latterconditions, irrespective of whetherit wasaddedalone or

incombinationwithisoproterenol (Fig. 2 B).

After pretreatment with pertussistoxin,basal rates ofglycerol release werecomparable with those seenin thepresenceof

iso-proterenolincontrols(Fig.2D).Neitheradenosine deaminase

nor isoproterenol had any furtherstimulatory effect. The

ob-servations that both additionofadenosinedeaminase and

pre-treatment of human adipocytes with pertussis toxin induced

jic) 1- 1 01 20 30405060

t(min)

C. Control

3.0_

2.0-1.0

-01 20 304050 60

t(min)

I

B. Pertussis Toxin

C

._

0 A

~0

I

0

CD

0~*

Pertussis Toxin.

ao

2.

PO.

O 10 20304050 60 t(min)

D. Pertussis Toxin

a0

-

2.C0-O 10 203040 5060

t(niin)

Figure 1. Effects of adenosine deaminase and of pertussis toxinon

cAMP levels andonglycerol release.Fat cellswereisolatedfrom

adi-posetissue ofonesubject. The cell suspensionwasdivided intotwo setswhichwerepreincubated for 180 mininthepresenceorabsence ofpertussis toxin (5 ,g/ml)asdescribed in Methods. After washing,

the cell concentrationswereadjustedto

101

cells/ml, and both

suspen-sionsweredivided into aliquots (6 ml) containingnoadditions (.),

adenosine deaminase (1.6 jg/ml; o),orisoproterenol (1I mol/liter) ei-ther alone (-)orin combination with adenosine deaminase (A). Aden-osinedeaminasewasadded 15 min before thestartof the time-course

experiment,andtheglycerol accumulating during this intervalwas

subtracted from subsequent determinations. Isoproterenolwasadded

atthestartof the experiment. At the times indicated aliquots of 0.05 mlwerewithdrawn forassayof cAMP and glycerol, respectively. This

representative experimentwasrepeated five times.

onlymodestly increased,wereconfirmedineight separate

ex-periments(TableI,Fig. 2).

Fig. 2 shows dose-responsecurvesfor isoproterenolin

non-treated and pertussis toxin-non-treated adipocytes.Basallipolyticrates

wererelatively high in these experiments, averaging 1.5 ,umol/

106 cellsper 180 min. It hasbeen common, however, to find higher nonstimulated glycerol release in obese comparedtolean subjects, whereas maximalrates oflipolysisaresimilar in lean andobese subjects (18). In the absence of adenosine deaminase, the ,B-adrenergic amine caused aconcentration-dependent

in-creaseinlipolysis. Maximal activation occurredat1 ,umol/liter

and averaged 200% above basal levels. Removal of endogenous adenosine andpretreatmentof fat cells with pertussistoxin

in-creased basal lipolytic rates without substantially altering the maximalratesachieved in thepresenceof isoproterenol. There-fore, addition of isoproterenol caused eitherasmall (adenosine

deaminase) oronlyamarginal (pertussis toxin) additional in-creaseoflipolysis under these conditions.

Influenceofpertussis toxin and ofadenosinedeaminaseon

theeffects ofantilipolyticagents.Fig.3 shows the effectofvarious

antilipolytic agents in nontreated cells and in adipocytes

pre-treatedwith pertussis toxin.Adenosine deaminase (1.6

_jg/ml)

waspresentthroughouttheseexperiments. Incontrast to

con-d1 1 , , Iz

C.PertussisToxrin

-T T '-'i.

JLJ , 1

9 8 7 6 5 00 9 8 7 6 5 00 9 8 7 6 5

Ispoten (-log mol/liter)

Figure2.Influenceof adenosine deaminase and ofpertussistoxin treatment on thelipolyticaction ofisoproterenol.Fat cells were

prein-cubated for 180minin theabsence andpresenceof 5

;&g/ml

pertussis

toxin. Afterwashing,celldensities wereadjustedto5,000-20,000

cells/ml, andfatcells wereincubated with increasing concentrationsof

isoproterenoleither alone(A)orincombinationwithadenosine

deam-inase(B andC)inatotalvolumeof 0.05 ml for another 180 min.

When present, adenosinedeaminase(1.6 ug/ml) andisoproterenol

were added at the startofthefinal incubations.Values are means±standard error of four(A)tofivepaired experiments (Band C). For other details see Methods. Abbreviation: Adenos.Deam.,

adenosine deaminase.

trols, theantilipolytic effects of those agents known to affect lipolysis via inhibition of adenylate cyclase (N6-phenylisopro-pyladenosine, prostaglandin E2, clonidine) were abolished in fat cells pretreated with pertussis toxin.

Amongthe agents tested, only insulin was capable of

inhib-iting lipolysis in toxin-treated cells. The antilipolytic action of insulin was completely unaffected with respect to effective

con-centration range and degree of inhibition. Time course ofglycerol release andconcomitant determinationsof cAMP levels in the presence and absence of insulin and prostaglandin E2, respec-tively, are shown in Fig. 4. In this experiment the medium

con-tained 1.6

.g/ml

ofadenosine deaminase throughout. In control cells cAMP levels (in the presence of adenosine deaminase) rangedfrom 55 to 65pmol/106cells andglycerolrelease averaged

Table I. cAMP Levels inNontreated and Pertussis Toxin-pretreatedHumanAdipocytes

cAMP levels

Additions Control Pertussis toxin

pmol/10cells pmol/106cells

27±5 160±20§

Adenosine deaminase

(1.6

iig/ml)

70±15* 180±25§

Values aremeans±SE of eight separate experiments carried out with fat cells from different subjects. cAMP levels were determined at either 10 or 12minof incubation. For experimental details, see Methods and legend to Fig. 2.

* Significantly higherthan basal levels(P. 0.05). §Significantlyhigherthan controls (P.0.05).

A.

Control

1,00C

a.

a5

2

I .5

0 500

---I

7.0- A. Prostaiondin E2

6

.0-.j

3.0-

i

2.0-k

1.0

_

c 1 10 9 8 7 6

o(-lg moI/liter)

s 7.0 C. aonickne

;5j 0-

i

4.0 _+

j

r

B.

_Insulin

6.01

5.0

4.0 3.0

2.0

1.0 0

--41

c 13 12 11 10 9 8

(-log mo/liter)

300

_A.

Control 250

, 200(

$

150

v o L I I I

-5 0 10 2030 40 50 60

tlmin)

2.0

I

-I

9 8 7 6 5 "c 11 10 9 8 7 6 (-log mol/liter) (-log mol/liter)

Figure 3.Effects ofpertussis toxin on theantilipolytic action of

pros-taglandin E2, insulin, clonidine,andN6-phenylisopropyladenosine.

Untreated andpertussis toxin-treated fatcells wereincubated with

in-creasing concentrations ofvariousantilipolyticagentsfor180 min at

37°C.Antilipolyticagents were added at the startoftheincubations.

Themediumcontained 1.6jg/mlofadenosinedeaminase.Values are means±standard erroroffour (CandD)tosix(AandB)paired

ex-periments carriedout induplicatewithfat cellsfrom different sub-jects.Forfurther detailsseeMethods.Opensymbolsrefertopertussis toxin-treated cells;closedsymbolsshow therespectivecontrols. Ab-breviation:PIA,N6-phenylisopropyladenosine.

1.6Mmol/ 106 cellsper60 min.

Prostaglandin

E2 (50 nmol/liter) depressed cAMP levels to 30 usmol/ 106 cells.

Concomitantly,

glycerol releasewasdecreasedto0.4

Mmol/106

cells per 60min. Insulin reduced therateof glycerol release from 1.6

M4mol/

106 cellsper60minto 1.0

_Amol/

106cellsper60min.Incontrast

to

prostaglandin

E2, insulin hadnomeasurable effectoncAMP

levels under the

conditions employed,

indicating

thatin human

C. Control

I-O.

_l

-1 0 10 20 30 4050 60

t(minn

200k 150

B. Pertussis xin

*___ 4-- --a

1ooF

50

0L * * *

-5 0 10 20 3040 50 60 t(min)

Toxin

2.0

1.0

-5 0102030405060

t(min)

Figure4.Effects of insulin and of prostaglandin E2 on cAMP accumu-lation and glycerol release in nontreated and pertussis toxin-treated

adipocytes. Experimentalconditions were similar to thosedescribed

forFig. I except that not only adenosine deaminase (1.6 ug/ml) but alsohormones were added 15 min before the start of the experiment.

Glycerolaccumulating between - 15 min and 0 min wassubtracted

from subsequent determinations. This representative experiment was repeated sixtimes (Table II). For further details see legend to Fig. I and Methods.Symbols: *, control; *,insulin(10nmol/liter);*,

pros-taglandin E2(50nmol/liter).

fat cells insulin can exert antilipolyticeffects although cAMP levels remainunchanged.

In cells treated with pertussis toxin, glycerol release averaged 2.5 ,mMol/106 cells per 60 min. The inhibitory action of

pros-taglandin E2wascompletelyabolished,consistent with the data shown in Fig. 4, whereas the antilipolytic effect of insulin was

preserved in toxin-treated cells. Not only insulin but also pros-taglandin E2 failed todecrease cAMP after the cells had been treated with pertussis toxin.

The lack of effect of insulin on cAMP levels was confirmed in six separate experimentscarriedout with different cell

prep-TableII.Effects ofInsulin andProstaglandin E2oncAMP Accumulation and

LipolysisinNontreated and PertussisToxin-treatedHuman

_Adipocytes

Control* Pertussis toxin

cAMP cAMP

Additions at 12min at24 min Glycerolrelease at12 min at24mm Glycerolrelease

pmol/10' cells pmol/10' cells jsmol/10'cellsper 60 min pmol/10'cells pmol/10'cells jumol/lO'cells per 60 min

- 65±15 72±20 1.8±0.1 200±30 220±40 2.8±0.3

Prostaglandin E2

(50nmol/liter) 35±10* 40±10t 0.3±0.05* 193±30 200±40 2.9±0.2 Insulin

(10nmol/liter) 73±15 80±15

1.1±0.1t

210±30 215±30 1.8±0.1*

* Values are

means±standard

error of six paired

experiments

carried

outinduplicatewithfat cellsof differentsubjects.Themediumcontained 1.6

_;g/ml

of adenosine deaminase under both conditions. Forexperimentaldetails,seeMethods andlegendto

_Fig.

2. *

Significantly

lower than control values(P=0.05).

300

arations(Table II). For comparison, the effects of prostaglandin E2,which is knownto actvia

inhibition

of adenylate cyclase,

were tested simultaneously. Prostaglandin E2 consistently re-duced cAMP levels by -50% whereas insulin failed to induce a detectableinhibition of cAMP levels under the conditions em-ployed. Theeffects of insulinon cAMP accumulation are com-plex and havefrequently beenshown to depend on the insulin

concentrationused (1).Therefore, additional time-course studies (n= 3)werecarriedoutusing four different insulin

concentra-tions ranging from 10 pmol/liter to 10 nmol/liter. However, evenunder theseconditionsnodecreaseof cAMP levelscould

be detected(notshown).Inordertoexclude the possibility that

insulinonlytransiently decreased cAMP levels, threeother

ex-perimentswere carriedoutin which cAMP levelswere

deter-minedat 1, 5, and 10 minafterthe addition of thehormone

(10 nmol/liter). However, noshort-termchangeincAMP

con-centrationscould bedetected.

Discussion

Effects

ofpertussis toxin and

of

adenosine deaminase

onbasal and

isoproterenol-activated

rates

of

cAMP

accumulation

and

glycerol release. Fat cells produce

and extrude

inhibitory

regu-lators such asadenosineorprostaglandins of theE type(1-8).

As reportedpreviouslyand showninFig.1and2,evenextremely dilute suspensions ofhuman adipocytes produceadenosine in amountssufficienttodepressglycerol release

substantially

( 19).

In the presentstudies, bothremoval of endogenous adenosine andtreatmentof adipocytes with pertussis toxin resultedin an

increase ofcAMP. Lipolysiswasconsiderably activated when cAMP levels were increased two- to threefold (adenosine

de-aminase)and proceeded atmaximal ratesaftertreatmentwith pertussis toxin whichwas

associated

with aboutafive-to

sev-enfold increase in cAMP levels. These resultsareconsistent with

thewidely held belief that onlyasmallincrease in total

cyclic

AMPisnecessary tostimulate lipolysis fully. The fact that pre-treatmentof fat cells with

pertussis

toxin resultedinhighercAMP

levels thanremoval of endogenous

adenosine

couldsuggesteither

that sometightly-bound adenosine isnotaccessibletoadenosine deaminaseorthatadenosineisnottheonly inhibitorproduced during incubations in vitro. Support for the latter

possibility

comesfrom studies of Changet al.(8), who showed thathuman

adipocytes also produce prostaglandins ofthe Eand F types.

Similarexplanations mayapplyto theobservationthat cAMP levelselevated by isoproterenol (in the presence of adenosine

deaminase)

werefurther increasedby pertussistoxintreatment.

The lattereffect of pertussis toxincan also beexplained by

as-suming

that

binding

of

isoproterenol

toitsreceptor causes not

only activation ofcAMPformation via the stimulatory nucleo-tide

binding-protein

of adenylate cyclase

_(Nj),

but also

simul-taneous inhibition via the inhibitory coupling protein

(N1),

as proposed by Murayama and Ui (20) for rat adipocytes.

Reversal by pertussis toxin of the inhibitory

effects

of

N6-phenylisopropyladenosine,

prostaglandin E2, and clonidine. As

in the rat fat cell, pretreatment of human adipocytes with per-tussis toxin abolishedthe antilipolytic effects of

N6-phenyliso-propyladenosine and prostaglandin E2 (10, 11, 13).

Concomi-tantlythe decreaseofcAMPlevels produced by both agents was reversed. Rat adipocytes contain no functional a2-receptors. In human fat cells the

a2-adrenergic

effects of

_epinephrine

(not

shown)

and of clonidine were also blocked

(Fig. 3).

Pertussis

toxin catalyzes the ADP ribosylation in various cell types in-cluding fat cells ofa41,000-molwtproteinthat is apparentlya

subunit of

Ni,

the inhibitoryguanine nucleotidebinding-protein

ofadenylate cyclase(for reviewseeReferences21 and22). The covalent modificationinhibitsthefunctionalabilityofinhibitory

receptors to depress cAMP formation (21-23). cAMP levels were increased in adipocytes pretreated with pertussis toxin and

re-versalofthe antilipolytic effect of prostaglandin E2 was associated

withacorrespondingincrease ofcAMP,suggestingthatadenylate

cyclase is in fact involved. However, several effectsofpertussis

toxin have been describedwhich are independent of cyclic nu-cleotide changes (21, 22, 24). In addition, the concentration of

pertussis toxinused in this study was more than 10-fold higher

thanthat used by others with rat adipocytes (13). Therefore, we cannot exclude the possibility thatmechanisms other than

mod-ification ofadenylate cyclase are also important.

The roleof adenosineandofE-typeprostaglandinsas

phys-iologicregulatorsof adipose tissue is unknown at present. How-ever, thereismounting evidencefrom in vitro experiments

sug-gestingthatlocal regulatorsareimportant in long-term regulation

oflipolysis of different species includinghumans(1-3, 25-29).

Previous studies with adipocytes of starved subjects led us to conclude that the increase in basallipolytic rate seen in starvation

ismainly due to reliefof endogenous inhibition (19). The results

of thepresentstudiesareconsistent with this view. They dem-onstratethat relief of endogenousinhibitory influences results in almost maximal stimulation of glycerol release, suggesting thatthe primary functionalstatewhich is attained in the absence

ofany regulatorsis characterized by highratesof glycerolrelease.

Therefore,acertaindegreeof inhibitionappears tobe necessary

for fat cell lipolysistobesusceptibleto activation.

Effects

of insulin. In contrast to all otherinhibitorstested, theantilipolytic action of insulin waspreserved after pretreat-mentof adipocytes with pertussis toxin.Inaddition, although

the

antilipolytic

effectwasclearlymanifested, the peptide hor-monehadnoinhibitory effectsoncAMPaccumulation regard-lessofwhether the cells had or had not been treatedwithpertussis toxin.

The ,-oligomer of pertussis toxin has been shownto have

insulinlike effects onglucose oxidation in rat adipocytes (24).

The observations ofthe present studies, however, are almost

certainlynotrelatedtothissubunit, which mediates bindingof theholotoxintothe cellsurface, because basalglycerolrelease

should have beendecreased rather thanincreasedifthe

3-oligo-mer, inadditiontoitseffectson glucoseoxidationalso had

in-sulinlikeantilipolytic effects.

The antilipolytic effect of insulin hasoftenbeen correlated

witha decreasein cAMP concentrations in adipose tissue, and some

investigators

haveproposed that this decrease incAMP level fully explainsthe antilipolytic action of thishormone in

fat

cellsof different

species including

human adipocytes (30-33; for reviewseeReference 34).

ThecAMP response of adipocytes to lipolytic agentsoften

does not reach a steady state, but shows a peakfollowed bya

decline(1,35).Thesharpness ofthepeakhas been foundtobe very variable (1, 35). A possibleexplanation for this variability comesfrom studies of Engfeldt et al. (35), who showed that

phosphodiesterase activity isreduced by collagenase digestion ofhuman adipose tissue. In the present studies, cAMP accu-mulation in cells exposed to isoproterenol showed a rapid

reported from hamster adipocytes (36). Insulin is knownto

ac-tivate alow-Km phosphodiesterase (1). In view of thefindings

ofEngfeldt et al. (35), wecannot exclude the possibility that phosphodiesterase activity was sufficiently reducedtopreclude anyinsulin-induced activation of phosphodiesterase. In addition, incultured 3T3-Ll adipocytes insulin activation of

phospho-diesterase is prevented by pertussis toxintreatment(37). If

ap-plicable to human adipocytes, these latter findings wouldexplain

why cAMP levels were not changed by insulin aftertreatment

with pertussistoxin. In addition, the effects of insulin were

as-sessed only with glucose inthe medium.Hence,wedo notknow whether changes of cAMP would have occurred in the absence ofglucose. We, therefore, do not suggest that insulin has not

effects oncAMPlevels at all. However, ourobservations dem-onstrate that changes in cAMP levelscannotofferasatisfactory explanation for the mechanisms of the antilipolytic action of

insulin underall conditions.This conclusion is consistent with recent reports in which rat adipocytes were employed that had been made permeable by digitonin, orpharmacologic

manip-ulationstobypass endogenouscAMP production and utilization

(38-40). These latter studies consistently showedthatthe

anti-lipolytic effect of insulin doesnot require adenylate cyclaseor

phosphodiesterase action(38-40).

In conclusion,the results show that the antilipolyticeffect ofinsulin is not necessarily related to measurable decreases of cAMP. Additional evidence that the mechanisms underlying

theantilipolytic actionof insulin is different from the

mecha-nisms ofotherantilipolyticcompounds comes from the

obser-vationthat pertussis toxin blocks the inhibitory action of

pros-taglandin E2, N6-phenylisopropyladenosine, and clonidine

without affectingthe antilipolytic action of insulin. The peptide

hormoneis theonlyantilipolyticregulatorshown to be physi-ologically important in vivo. In vitro, lipolysis is severely

re-strained by endogenous inhibitors, of which adenosineis prob-ablythe mostimportant. Unrestrained lipolysis proceeds at al-mostmaximalrates,suggestingthat acertaindegreeof inhibition might benecessaryfor fatcelllipolysisto besusceptible to stim-ulation. Further information about the concentrations of

aden-osine and prostaglandins within adipose tissuein vitro and in

vivo, therefore,would be of great valuein gainingmoredetailed

insights

into the mechanisms that adjust lipid mobilizationto

the actual demands oftheorganism.

Acknowledgments

Theauthors aregrateful to Miss E. Messmer and Miss U. Biehl for skillful technical assistanceand are indebtedtoMrs. C. Brownfor careful preparation ofthemanuscript.Wewishto thank Dr. P. D.Wood, Stan-fordUniversity, Stanford, California,forproofreadingthemanuscript.

This work wassupported by grants of the Deutsche

Forschungsge-meinschaft, Bonn-Bad Godesberg; theThyssen-Stiftung,Cologne; and theBundesministerium fur Forschung und Technologie, Bonn, Federal Republic of Germany.

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