adrenalina y lipolisis

J Physiol 587.13 (2009) pp 3393–3404 3393

Adrenaline but not noradrenaline is a determinantof exercise-induced lipid mobilization in humansubcutaneous adipose tissue

I. de Glisezinski1, D. Larrouy1, M. Bajzova3, K. Koppo3,4, J. Polak3, M. Berlan1,2, J. Bulow5, D. Langin1,3,M. A. Marques1, F. Crampes1,3, M. Lafontan1 and V. Stich3

1Institut National de la Sante et de la Recherche Medicale (INSERM), Unite 858, Laboratoire de recherches sur les Obesites, Institut de MedecineMoleculaire de Rangueil Toulouse, France; Universite Paul Sabatier, Toulouse, France2Laboratoire de pharmacologie medicale et clinique, Toulouse, France3INSERM, laboratoire Franco-Tcheque, Prague, Czech Republic4Faculty of Medicine and Health Sciences, Department of Pharmacology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium5Department of Clinical Physiology, Bispebjerg Hospital, Copenhagen University, DK-2400, Denmark

The relative contribution of noradrenaline (norepinephrine) and adrenaline (epinephrine) inthe control of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise wasevaluated in men treated with a somatostatin analogue, octreotide. Eight lean and eight obeseyoung men matched for age and physical fitness performed 60 min exercise bouts at 50% of theirmaximal oxygen consumption on two occasions: (1) during i.v. infusion of octreotide, and (2)during placebo infusion. Lipolysis and local blood flow changes in SCAT were evaluated usingin situ microdialysis. Infusion of octreotide suppressed plasma insulin and growth hormonelevels at rest and during exercise. It blocked the exercise-induced increase in plasma adrenalinewhile that of noradrenaline was unchanged. Plasma natriuretic peptides (NPs) level was higherat rest and during exercise under octreotide infusion in lean men. Under placebo, no differencewas found in the exercise-induced increase in glycerol between the probe perfused with Ringersolution alone and that with phentolamine (an α-adrenergic receptor antagonist) in lean subjectswhile a greater increase in glycerol was observed in the obese subjects. Under placebo, propranololinfusion in the probe containing phentolamine reduced by about 45% exercise-induced glycerolrelease; this effect was fully suppressed under octreotide infusion while noradrenaline was stillelevated and exercise-induced lipid mobilization maintained in both lean and obese individuals.In conclusion, blockade of β-adrenergic receptors during exercise performed during infusionof octreotide (blocking the exercise-induced rise in adrenaline but not that of noradrenaline)does not alter the exercise-induced lipolysis. This suggests that adrenaline is the main adrenergicagent contributing to exercise-induced lipolysis in SCAT. Moreover, it is the combined action ofinsulin suppression and NPs release which explains the lipolytic response which remains underoctreotide after full local blockade of fat cell adrenergic receptors. For the moment, it is unknownif results apply specifically to SCAT and exercise only or if conclusions could be extended to allforms of lipolysis in humans.

(Received 9 January 2009; accepted after revision 28 April 2009; first published online 5 May 2009)Corresponding authors M. Lafontan: Unite Inserm 858, Institut de Medecine Moleculaire de Rangueil, BP84225, 31432Toulouse cedex 4, France. Email: [email protected]; V. Stich: Franco-Czech Laboratory, Charles University,Praha, Czech Republic. Email: [email protected]

In humans, catecholamines (adrenaline andnoradrenaline) and insulin have been consideredto be the major regulators of lipolysis (Horowitz,2003; Bartness & Song, 2007). Neither glucagon norglucagon-like peptide-1 affects lipolysis in isolated fatcells or the rate of lipolysis in human subcutaneous

adipose tissue (SCAT) (Bertin et al. 2001). A number ofresults, although showing the unquestionable impact ofcatecholamines on lipid mobilization in humans, havenot convincingly established the relative contributionof both amines in the physiological control of lipidmobilization during exercise. It is clear that fatty

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3394 I. de Glisezinski and others J Physiol 587.13

acid mobilization increases during I.V. catecholamineadministration. Catecholamines act on three fat celladrenergic receptors that have been shown to play arole in catecholamine-stimulated lipolysis (Lafontan& Berlan, 1993; Lafontan, 1994; Arner, 1999). Localadministration of catecholamines or selective β- andα2-adrenergic receptor agonists and antagonists intoadipose tissue via a microdialysis probe have revealedthe contribution of the various adrenergic receptors tothe regulation of in vivo lipolysis (Arner et al. 1990b;Galitzky et al. 1993; Barbe et al. 1996; Stich et al. 2000b).Moreover, selective β-adrenergic receptor subtypeblockade has revealed differences in the contribution ofthe β-receptors to lipolysis (Lundborg et al. 1981; VanBaak, 1988; Arner et al. 1990a; Stich et al. 2000b). A directneuronal influence on lipolysis has been demonstrated byintraneural stimulation of the lateral femoral cutaneousnerve that innervates human subcutaneous adiposetissue. An increase in interstitial glycerol concentration,measured by microdialysis, was observed under nervestimulation (Dodt et al. 1999, 2000, 2003). Noradrenalinehas also been proposed to initiate cold-induced lipidmobilization, since cold exposure promotes a specificrise in plasma noradrenaline while exerting weak effectson the other glucose- and lipid-regulatory hormones(Koska et al. 2002). The role of sympathetic nerveactivity was elucidated in studies on paraplegic patientswith injured spinal cord (i.e. injury level at T3–T5).That study gave evidence for the hypothesis that it iscirculating catecholamines that are important for theexercise-induced increase in SCAT lipolysis (Stallknechtet al. 2001). Elevated levels of circulating adrenalinemay also explain the increased lipolysis observed duringmental stress (Lonnqvist et al. 1992).

Exercise is an excellent physiological challenge topromote sympathetic nervous system (SNS) activation;there is no doubt that it contributes to the control of lipidmobilization during exercise. Increased catecholaminelevels, promoted by exercise, stimulate both fat cell β1−2-and α2-adrenergic receptors (ARs) which stimulate andinhibit lipolysis, respectively (Berlan & Lafontan, 1982;Lafontan & Berlan, 1982). The simultaneous activationof both receptors modulates the intracellular cAMPconcentration, which activates cAMP-dependent proteinkinase, leading to the phosphorylation and activation ofthe hormone-sensitive lipase (HSL) (Langin & Lafontan,2004). Thus the lipolytic response depends on adrenergicreceptor expression in fat cells (Lafontan & Berlan, 1995).The relative importance of sympathetic nerve activity andcirculating catecholamines released by adrenal medullaremains poorly established during exercise since plasmalevels of both catecholamines are increased concomitantly.The plasma level of noradrenaline is an index of bothevents (nerve leakage and release by adrenal medullachromaffin cells). Adrenaline is predominantly expressed

by chromaffin cells while in vitro studies have revealedthat noradrenaline release is quite similar to that ofadrenaline. In rodents, there are apparently distinctpopulations of chromaffin cells, separately innervated, andsecreting each catecholamine; it is less known in humans(Bartness & Song, 2007). In normal-weight healthysubjects noradrenaline released from the adrenal medullais considered to be lower than noradrenaline leakingfrom vesicles in sympathetic nerve endings (Eisenhoferet al. 2004). Moreover, contrary to former beliefs, variousstudies have shown that oral β-adrenergic receptorblockade did not inhibit exercise-induced lipolysis at lowand moderate intensities of exercise (Jesek et al. 1990;Wijnen et al. 1993; Moro et al. 2004; Ormsbee et al. 2009).In addition to the effect of an increase in SNS activity andadrenal medulla secretion, the mobilization of lipids fromadipose tissue during exercise also results from the inter-play between several other secreted factors such as insulin,natriuretic peptides (NPs) and growth hormone (GH).It is well accepted that the reduction of plasma insulinlevels occurring during exercise enhances the lipolyticresponsiveness of fat cells. Moreover, NPs (i.e. atrialnatriuretic peptide (ANP) and B-type natriuretic peptide(BNP) released from the exercising heart) are potentactivators of lipolysis in human fat cells (Sengenes et al.2000). NPs stimulate fat cell plasma membrane receptors(NPR-A subtype) bearing an intrinsic guanylylcyclaseactivity and increase intracellular levels of cyclic GMP(cGMP) that activates a cGMP-dependent protein kinase(PKG). PKG-dependent phosphorylation of perilipin andHSL stimulates lipolysis (Sengenes et al. 2003). ANPincreases lipid mobilization from adipose tissue duringshort-exercise bouts and the effect is enhanced by oralβ-blockade (Moro et al. 2004). The GH response toacute exercise may increase lipolysis only during thepost-exercise recovery period (Mulla et al. 2000; Wee et al.2005; Enevoldsen et al. 2007).

The aim of the present study was to assess therelative contribution of adrenaline and noradrenalinein the control of lipolysis during exercise in men usingoctreotide, a somatostatin analogue, to block adrenalinerelease (Enevoldsen et al. 2007). Utilization of octreotidealso promotes inhibition of insulin and GH release (DiLuigi et al. 1997, 2001). Microdialysis was used to monitorlocal lipid mobilization (i.e. glycerol release) in SCATduring exercise. It is a recognized method for mechanisticexplorations of adipose tissue function in vivo.Intravenous infusion of octreotide was applied before,during and after exercise. Healthy lean young males, fastedovernight, performed exercise bouts over 60 min at 50% oftheir VO2max during placebo or octreotide administration.A pharmacological strategy was also used to support thephysiological approach. At the adipose tissue level, localblockade of fat cell β- and α2-adrenergic receptors bydirect infusion of the β- (i.e. propranolol) and α2- (i.e.

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society



J Physiol 587.13 Adrenaline is a major determinant of exercise-induced lipolysis 3395

Table 1. Effect of placebo or octreotide infusion on plasma insulin (μU ml−1), glucose(mmol l−1), glycerol (μmol l−1) and non-esterified fatty acids (NEFA; μmol l−1) during rest,exercise and recovery in lean subjects

Placebo or octreotide infusion

Rest Exercise Recovery

Time (min) 60 120 135 150 165 180 210

PlaceboInsulin 3.7 3.2 3.3 2.9 2.7∗ 2.1 2.5S.E.M. ±0.4 ±0.6 ±0.6 ±0.6 ±0,6 ±0.6 0.6Glucose 4.6 4.5 4.7 4.7 4.6 4.5 4.4S.E.M. 0.1 ±0.1 ±0.1 ±0.1 ±0.1 ±0.1 0.1Glycerol 71 75 142∗ 171∗ 210∗ 193∗ 88S.E.M. ±9 ±8 ±23 ±36 ±39 ±32 ±8NEFA 127 130 107∗ 121 132 148 199∗

S.E.M. ±13 ±15 ±10 ±20 ±17 ±16 ±23

OctreotideInsulin 3.3 0.3∗# 0.7∗# 0.8# 0.8∗# 0.8∗# 0.9∗#S.E.M. ±0.4 ±0.1 ±0.3 ±0.3 ±0.3 ±0.3 ±0.3Glucose 4.4 4.1∗ 5.1 5.4∗# 5.7∗# 5.5∗# 5.6∗

S.E.M. ±0.1 ±0.1 ±0.2 ±0.2 ±0.2 ±0.3 ±0.3Glycerol 87 123∗# 227∗# 273∗# 307∗# 317∗# 111S.E.M. ±12 ±14 ±36 ±50 ±41 ±27 ±9NEFA 128 274∗# 215∗# 264∗# 276∗# 287∗# 359∗#S.E.M. ±12 ±40 ±27 ±35 ±27 ±24 ±45

Probes were perfused for 60 min for an equilibration period and stabilization of the probesbefore the experimental period was begun. Octreotide or placebo was infused from time60 min until 210 min. Exercise was performed from time 120 to 180 min. ∗P < 0.05 whencompared to basal values before exercise. #P < 0.05 when compared to placebo infusion.

phentolamine) antagonists in the microdialysis probes wasused to delineate the part played by both catecholaminesduring exercise. We have previously shown that a strongα2-adrenergic responsiveness could be demonstratedin SCAT during exercise in obese subjects. Thephysiological stimulation of SCAT adipocyteα2-adrenergic receptors during exercise-inducedsympathetic nervous system activation contributes tothe blunted lipolysis noted in obese men (Stich et al.2000b). A group of obese subjects, performing similarexercises, was included to explore this aspect under ourexperimental conditions (i.e. under octreotide infusion).

Methods

Subjects

Eight healthy lean young men (age: 23.8 ± 0.4 years;body mass index (BMI): 23.5 ± 0.7; percentage fat mass:16.2 ± 0.5; fat-free mass: 67.9 ± 2.4 kg) who had notbeen enrolled into any pharmacological or nutritionalprotocol prior to the study were recruited. All had a stableweight during the previous 3 months. Eight obese youngmen (age 28.1 ± 1.8 years; BMI 33.5 ± 1.4; percentage fat

mass 27.2 ± 2.6; fat-free mass: 77.7 ± 4.7 kg) were alsoincluded in the study. Selection of the men was based on ascreening evaluation of detailed medical history, a physicalexamination, and several blood chemistry analyses.

One week before the investigation period, the maximaloxygen uptake (VO2max) was determined on an electricallybraked bicycle ergometer (Ergometrics 800s Ergoline) byuse of an incremental procedure (work rate increasing by30 W (3 min)−1). VO2max was measured using an OxyconPro (Jaeger) and the highest VO2 value was considered asVO2max. The mean VO2max was 3.28 ± 0.08 l min−1 in lean,and 3.05 ± 0.12 l min−1 in obese, respectively. The EthicalCommittee of the third Faculty of Medicine of Pragueapproved the study and all procedures conformed to thestandards set by the Declaration of Helsinki. All the subjectsgave their written informed consent for the experimentalconditions after detailed explanation.

Design of the study

Subjects were investigated at 8 a.m. after an overnightfast for 2 days separated by 1 week according to a doubleblind randomized crossover procedure. The subjectsentered the laboratory at 8 a.m. and were maintained





in a semi-recumbent position. Two indwelling poly-ethylene catheters were inserted into antecubital veins,one for blood sampling and the other for placebo oroctreotide (30 ng kg−1 min−1) infusion. At 8.30 a.m., threemicrodialysis probes (Carnegie Medicine, Stockholm,Sweden) of 20 mm × 0.5 mm and 20 000 MW cut-offwere inserted percutaneously after epidermal anaesthesia(200 μl of 1% lignocaine (lidocaine), Roger-Bellon,Neuilly-sur-Seine, France) into the SCAT at a distance of10 cm from the umbilicus. The probes were connected to amicroperfusion pump (Harvard apparatus, S.A.R.L.,Les Ulis, France) and perfused with Ringer solution(139 mmol l−1 sodium, 2.7 mmol l−1 potassium,0.9 mmol l−1 calcium, 140.5 mmol l−1 chloride,2.4 mmol l−1 bicarbonate). Ethanol (1.7 g l−1) wasadded to the perfusate in order to estimate changes inthe blood flow, as previously described (Galitzky et al.1993; Fellander et al. 1996). All the probes were perfusedat the rate of 2.5 μl min−1. One probe was perfusedwith Ringer solution. The second probe was perfusedwith 100 μmol l−1 phentolamine (an α1,2-adrenergicreceptor antagonist). The third probe was perfusedwith 100 μmol l−1 phentolamine plus 100 μmol l−1

propranolol (a non-selective β−adrenergic receptorantagonist). Probe calibration was checked and the meanrelative recovery found (28–32% range) was the same asthat reported in our previous studies (Stich et al. 2000a).

After insertion, probes were perfused for 60 min for anequilibration period and stabilization of the probes beforethe experimental period was begun. Four 15 min fractionsof the outgoing dialysate were collected from all probes.Immediately after, the subjects were infused with placeboor with 30 ng kg−1 min−1 octreotide over 150 min. Then,after 1 h of octreotide perfusion, the subjects exercisedfor 60 min at 50% of their VO2max. The exercise load wassimilar under placebo or octreotide infusion (96.9 ± 4.9and 80.0 ± 9.4 W in the obese subjects, respectively).During the exercise bouts and the 30 min recovery period,dialysate was collected every 15 min.

Heart rate was continuously monitored with a PolarAccurex Plus cardiometer. At rest and during exercise,blood pressure was measured with an exercise-adaptedmonitor (Tango Stress Test BP Monitor, Suntech MedicalInstruments, Raleigh, NC, USA). Water intake was allowedad libitum at rest, and during exercise and recoveryperiods.

Before and during exercise, blood was collectedfor the determination of endocrine and metabolicparameters. Plasma for ANP and GH determinationswas collected on EDTA (1 mg ml−1) plus aprotinin(Trasylol) (5 μmol ml−1). Two millilitres of blood was alsocollected in 50 μl (1 mmol l−1) of an antioxidant product(sodium metabisulfite, Sigma Aldrich, Saint-QuentinFallavier, France), in order to prevent the oxidationof catecholamines, and immediately centrifuged in a

refrigerated centrifuge. The plasma was stored at −80◦Cuntil analysis.

Analytical methods and drugs

Octreotide (Sandostatin) was obtained from ZenecaPharma (Cergy, France), phentolamine methanesulfonate(Regitine) from Novartis Pharma (Rueil-Malmaison,France) and propranolol (Avlocardyl) from ThervaMedical (Neuilly-sur-Seine, France). Glycerol in thedialysate and plasma was analysed by an enzymaticmethod (Sigma, Saint Louis, MO, USA). Ethanol inthe dialysate and perfusate (5 μl) was determinedwith an enzymatic method (Bernst & Gutman, 1974).Plasma glucose and non-esterified fatty acids (NEFA)were determined with a glucose oxidase technique(Biotrol, Paris, France) and an enzymatic procedure(Wako, Unipath, Dardilly, France), respectively. Plasmainsulin concentrations were measured using enzymeimmunoassay (EIA) kits from Mercodia (Uppsala,Sweden). Plasma adrenaline and noradrenaline weremeasured using EIA kits from Bio-Source Europe(Nivelles, Belgium). Plasma lactate was evaluated using anenzymatic method (BioMerieux, Marcy l’Etoile, France).Plasma ANP was determined using a radioimmunoassaykit and BNP using an EIA kit (extraction free) fromPeninsula Laboratories (San Carlos, CA, USA). Growthhormone was determined using EIA kits from R&Dsystems (Lille, France).

Statistical analysis

All values are means ± S.E.M. The responses to exercisewere analysed by two-way repeated measures ANOVA.When the ANOVA was significant, post hoc tests werefitted according to the Bonferroni procedure. Dialysateconcentration–response curves were calculated as the totalmean changes over baseline values. Significance values arequoted in the figures and tables. P < 0.05 was consideredstatistically significant.

Results

Effect of octreotide infusion on plasma values at restand during exercise

At rest, octreotide infusion decreased plasma insulin andplasma glucose within 60 min and increased plasma NEFAlevels (Table 1) whereas these parameters were unchangedwith the placebo (Table 1). During exercise, octreotideinfusion induced a large increase in plasma glycerol andNEFA concentrations when compared with the placebo(Table 1). During exercise, plasma insulin remained lowerduring octreotide infusion when compared with the





placebo condition. The exercise-induced rise in plasmaglycerol and NEFA was higher during octreotide infusionthan with the placebo. Plasma glucose increased duringexercise under octreotide infusion while it remainedunchanged with the placebo (Table 1).

During exercise, plasma noradrenaline concentrationsincreased similarly under placebo and octreotide infusion.Plasma adrenaline and GH concentrations increasedunder the placebo but remained unchanged withoctreotide (Fig. 1 and Table 2). One hour after thebeginning of the octreotide infusion, plasma ANP andBNP increased moderately at rest when compared withthe placebo infusion. During exercise, the increment ofANP and BNP was higher under octreotide than underplacebo administration (Table 2). Octreotide infusion didnot modify plasma lactate concentration at rest or duringexercise (Table 2).

Effect of octreotide on glycerol concentration in thedialysate from SCAT during exercise

The exercise-induced increase in glycerol release inSCAT was higher under octreotide infusion than underplacebo in lean subjects (Fig. 2). A similar effect ofoctreotide infusion was observed in the obese subjects(Fig. 3). In lean subjects, whether under placebo (Fig. 4A)or octreotide infusion (Fig. 4B), the exercise-inducedincrease in glycerol level in the probes infused withphentolamine was not different from that in the controlprobe (i.e. that perfused with Ringer solution alone); thepotentiating effect of octreotide observed in the Ringerprobe (Fig. 2) was maintained (Fig. 4B). Conversely andas previously reported by our group in the obese subjects,when the placebo was administered, the exercise-inducedincrease in glycerol output was considerably enhanced inthe probe with the phentolamine infusion (Fig. 3A).

The addition of propranolol into the probe containingphentolamine reduced the exercise-induced rise of glycerolin the dialysate by 48% in placebo conditions while nosuch reduction was observed during infusion of octreotide(Fig. 5A). On the contrary, when octreotide was infused,the concentration of dialysate glycerol in the probewith phentolamine plus propranolol was not modified(Fig. 5B). As previously mentioned, under octreotide,there was no exercise-induced increase in plasmaadrenaline levels (Fig. 1) while the exercise-inducedincrease in plasma noradrenaline was equivalent tothat observed with the placebo infusion. In the obesegroup, when propranolol was added to the probe withphentolamine, the exercise-induced increase in dialysateglycerol concentration was reduced by about 55%under placebo infusion (Fig. 3A). When octreotide wasinfused, propranolol did not modify the concentrationof dialysate glycerol in the probe with phentolamine plus

propranolol (Fig. 3B); the β-adrenergic component of theresponse (i.e. revealed by propranolol blockade) observedin placebo-treated obese subjects has disappeared(Fig. 3A).

Effect of octreotide infusion on adipose tissue bloodflow in SCAT at rest and during exercise

The local changes in adipose tissue blood flow (ATBF)were evaluated with the previously used ethanol washoutmethod. The ethanol ratio (%) was calculated asthe ethanol concentration measured in the dialysatedivided by the ethanol concentration measured in theperfusate × 100 and taken as an index of ethanol washout.A high ethanol ratio corresponds to a lower ethanolwashout and a lower regional ATBF. Whatever the probes,octreotide had no significant impact on ATBF. Duringtwo resting periods, the ethanol outflow/inflow ratio wasnot different between the probes with Ringer solutionalone or with added phentolamine and phentolamineplus propranolol. During the exercise bouts, as previouslyobserved, no significant change in the ethanol ratio was

Figure 1. Plasma noradrenaline and adrenaline concentrationsat rest, during exercise and the recovery period underintravenous infusion of placebo or octreotide(30 ng min−1 kg−1) in lean subjectsData are expressed as mean ± S.E.M. ∗P < 0.05 significant whencompared to values obtained with octreotide infusion.





Table 2. Effect of placebo or octreotide infusion on plasma atrial natriuretic peptide (ANP;pg ml−1), B-type natriuretic peptide (BNP; pg ml−1), growth hormone (GH; ng ml−1) andlactate (mmol l−1) during rest, exercise and recovery in lean subjects

Placebo or octreotide infusion

Rest Exercise Recovery

Time (min) 60 120 135 150 165 180 210

PlaceboANP 45.4 43.5 ND 54.4∗ ND 56.3∗ 45.9S.E.M. ±8.3 ±7.1 - ±8.1 - ±8.2 ±6.8BNP 672.3 616.1 ND 815.3 ND 1137.2 789.8∗

S.E.M. ±175.2 ±169.9 - ±221.4 - ±534.1 ±221.4GH 0.1 0.1 0.6∗ 2.5∗ 4.2∗ 4.2∗ 0.2∗

S.E.M. ±0.04 ±0.04 ±0.3 ±0.7 ±1.1 ±1.1 ±0.04Lactate 0.97 0.98 2.01∗ 1.86∗ 1.66∗ 1.53∗ 1.15S.E.M. ±0.1 ±0.1 ±0.3 ±0.4 ±0.3 ±0.2 ±0.1

OctreotideANP 46.1 49.7# ND 63.3∗# ND 66.5∗# 47.1S.E.M. ±6.3 ±5.2 - ±8.0 - ±8.8 ±8.1BNP 804.7 832 ND 1394∗# ND 2129.1∗# 1707.6S.E.M. ±105.2 ±120.5 ±199.1 - ±419.1 ±325.3GH 0.08 0.06 0.06# 0.09# 0.2# 0.4# 0.1#S.E.M. ±0.02 ±0.02 ±0.01 ±0.02 ±0.08 ±0.1 ±0.03Lactate 1.14 0.97 2.24∗ 1.89∗ 1.76∗ 1.57∗ 1.12S.E.M. ±0.2 ±0.1 ±0.5 ±0.3 ±0.3 ±0.2 ±0.1

Probes were perfused for 60 min for an equilibration period and stabilization of the probesbefore the experimental period was begun. Octreotide or placebo was infused from time60 min until 210 min. Exercise was performed from time 120 to 180 min. ∗P < 0.05 whencompared to resting values before exercise. #P < 0.05 when compared to placebo infusion.ND, not determined.

found in any of the probes in the various experimentalconditions (Fig. 6). Similar results were observed in theobese subjects (not shown).

Effect of octreotide infusion on heart rate at rest andduring exercise in normal weight subjects

The exercise load was similar under placebo or octreotideinfusion (96.9 ± 4.9 W). Before infusions, the basal heart

Figure 2. Dialysate glycerolconcentration from subcutaneousadipose tissue (SCAT) measured at rest,during exercise and the recovery periodunder intravenous infusion of placeboor octreotide (30 ng min−1 kg−1) in leansubjectsProbes were perfused with Ringer solutionalone. Data are expressed as mean ± S.E.M.∗P < 0.05 significant when compared tovalues obtained with octreotide infusion.

rate was 62.7 ± 0.7 and 61 ± 3.2 beats min−1, respectively.At the end of the exercise bouts, the heart rate reachedsimilar values under placebo or octreotide infusion(134.7 ± 5.6 and 135.0 ± 5.7 beats min−1, respectively at∼50% of maximal oxygen uptake).

Discussion

As previously observed in a number of studies, plasmalevels of adrenaline and noradrenaline increased during





Figure 3. Mean changes in dialysate glycerolconcentrations (DGC) in subcutaneous adiposetissue (SCAT) promoted by exercise in obesesubjects under intravenous infusion of (A) placeboand (B) octreotide (30 ng min−1 kg−1)One probe was perfused with Ringer solution alone(Ringer), the second with phentolamine (100 μmol l−1)(phentolamine) and the third one with Ringer solutionplus phentolamine (100 μmol l−1) plus propranolol(100 μmol l−1) (phentolamine + propranolol). Data areexpressed as mean ± S.E.M. P = 0.03 significant whencompared to control (Ringer) values and P = 0.04significant when compared to phentolamine values.

Figure 4. Dialysate glycerolconcentration from SCAT measured atrest, during exercise and the recoveryperiod under intravenous infusion ofplacebo (A) or octreotide(30 ng min−1 kg−1; B) in lean subjectsProbes were perfused with Ringer solutionalone or with Ringer solution plusphentolamine (100 μmol l−1). Data areexpressed as mean ± S.E.M.





the exercise bouts performed during the placebo infusion.Octreotide infused before and during the exercise hasprovided an experimental protocol to reassess therole of catecholamines in exercise-induced lipolysis.Octreotide blocks adrenaline, insulin and GH actionand enhances exercise-induced lipid mobilization inSCAT. When adrenaline release is blocked, it is thuspossible to delineate the part played by noradrenalinealone although plasma noradrenaline measurementsare not easily interpretable. Noradrenaline probablyoriginates from sympathetic nerve endings and/or fromchromaffin cells from the adrenal medulla which alsorelease noradrenaline (Eisenhofer et al. 2004). Sinceexercise-induced increment of noradrenaline is similarin placebo and octreotide-treated subjects (Fig. 1), itis impossible to evaluate the octreotide impact onnoradrenaline release by the adrenal medulla. Theimportant point is that in the absence of adrenaline, theβ-adrenergic effect disappears (i.e. the blocking effect ofpropranolol is lost) (Figs 3B and 5B). Thus, the plasmanoradrenaline increment is apparently without any effecton β-adrenergic receptor-dependent responsiveness in fatcells. Therefore, the exercise-induced lipid mobilizationobserved in SCAT under octreotide infusion cannot beattributed to noradrenaline in the SCAT of lean or obesesubjects. As discussed later, insulin suppression and NPs

Figure 5. Dialysate glycerolconcentration from subcutaneousadipose tissue (SCAT) measured at rest,during exercise and the recovery periodunder intravenous infusion of placebo(A) or octreotide (30 ng min−1 kg−1; B)in lean subjectsOne probe was perfused with Ringersolution plus phentolamine (100 μmol l−1)and the second one with Ringer solutionplus phentolamine (100 μmol l−1) pluspropranolol (100 μmol l−1). Data areexpressed as mean ± S.E.M. ∗P < 0.05significant when compared to valuesobtained in the probe with phentolamineplus propranolol and the Ringer probe withphentolamine alone.

are the most relevant hormonal factors responsible for thiseffect.

Octreotide binds to somatostatin receptor 5 (SSTR5on the pancreatic β-cell), inhibits calcium influx andattenuates the early phase of insulin secretion (Gordonet al. 1999; Mitra et al. 1999). It is probably a similarmechanism that explains the effect of octreotide inchromaffin cells of the adrenal medulla (Zink & Raue,1992). Octreotide infusion promotes a prompt declinein plasma insulin and glucagon concentrations (Albertiet al. 1973; Koerker et al. 1974; Sherwin et al. 1977).Glucagon is without effect on lipolysis and the lipolyticrate of human SCAT (Bertin et al. 2001). Infusion ofoctreotide with its potent effect on insulin secretion clearlyshows that, as expected, the large decrease in plasmainsulin is an important factor in the increase in plasmaglycerol and NEFA observed mainly during exercise(Fig. 2). Insulin is an anti-lipolytic hormone that activatesfat cell phosphodiesterase-3B (PDE-3B) which in turnreduces cAMP concentration and thence reduces lipolysis(Hagstrom-Toft et al. 1992; Arner et al. 1993).

In the case of GH, as previously demonstrated, underoctreotide infusion, plasma GH did not increase duringexercise (Enevoldsen et al. 2007). In contrast, underplacebo infusion, plasma GH increased considerably(Table 2). However, GH cannot be involved in the





enhancement of lipid mobilization during the exercisebout in the present study, since previous studies haveshown that the lipolytic effect of a GH injection appearedafter at least 2 h (Hansen et al. 2002; Djurhuus et al.2004). It has also been demonstrated that GH-inducedlipid mobilization only occurs after 60 min of exercise(Gibney et al. 2007). The GH response to acute exercisemay increase lipolysis during the post-exercise recoveryperiod only (Mulla et al. 2000; Wee et al. 2005; Enevoldsenet al. 2007); it cannot explain our results.

Exploration of catecholamine action requires theuse of suitable antagonists of adrenergic receptors; theagents used in the study have previously been validatedin microdialysis studies (Arner et al. 1990a; Moro et al.2004). It is well established that propranolol blocksβ1- and β2-adrenergic receptor-mediated effects at theconcentration used (100 μmol l−1) in the microdialysisprobes (Moro et al. 2004). Phentolamine was the onlyα-adrenergic receptor antagonist that was allowed forthe study. The α1/2-adrenergic receptor antagonist wasused in probes in the placebo and octreotide experimentsto remove any putative impact of catecholamines on fatcell α2-adrenergic receptors on exercise-induced lipidmobilization. At the concentration used, phentolaminehas been shown to fully exert its antagonist effects in vivo(Stich et al. 2000b; Polak et al. 2007). When the probeswere perfused with the α-antagonist, octreotide had noadditional impact on the exercise-induced lipolysis to thatobserved under control conditions (Fig. 2). In the leansubjects, phentolamine did not enhance lipidmobilization. It is a result confirming that the α2

effects are very weak, as previously demonstrated (Stichet al. 2000b; Polak et al. 2007). Nevertheless, as previouslyshown, a significant enhancing effect of phentolaminewas observed in the obese subjects (Fig. 3A). In the case ofthe β-adrenergic component of lipolysis, under placeboadministration, the exercise-induced lipid mobilizationwas partly suppressed by propranolol in both the lean(Fig. 5A) and obese subjects (Fig. 3B) as previouslyreported by us and others (Arner et al. 1990b; Moroet al. 2004). The part of lipid mobilization blocked bypropranolol corresponds to the catecholamine-dependenteffect on lipid mobilization that could be due to theaction of adrenaline and/or noradrenaline. The strikingpoint of our results is provided under octreotide infusionwhereby adrenaline secretion is inhibited during exercise(Fig. 1). When adrenaline release was suppressedby octreotide, the enhancing effect of phentolamineobserved in placebo-treated obese subjects is suppressed(Fig. 3B). Thus, the α2-adrenergic inhibiting componentin exercise-induced lipid mobilization usually observed inobese SCAT disappears. Moreover, the antagonistic effectof propranolol on exercise-induced lipid mobilizationdisappeared in both lean (Fig. 5B) and obese (Fig. 3B) sub-jects. Although the exercise-induced increment in plasma

noradrenaline persisted under octreotide, it is withouteffect on the α2- (i.e. in the obese) and β-adrenergic (i.e. inlean and obese) responsiveness of the adipose tissue. Thus,in the absence of adrenaline, the adrenergic componentof lipolysis disappears completely. This result revealsthe contribution of adrenaline and not noradrenaline inexercise-induced lipid mobilization in lean and obese sub-jects. Our results fit with a previous proposal originatingfrom studies on paraplegic patients with spinal cordinjuries (Stallknecht et al. 2001). In these studies, theauthors concluded that circulating catecholamines areimportant for the exercise-induced increase in sub-cutaneous adipose tissue lipolysis. Our study reveals thatit is adrenaline that is the determinant factor. It mustbe remembered that the β2-adrenergic receptor, which isa β-adrenergic receptor subtype of the human fat cell,is known to have a greater affinity for adrenaline thanfor noradrenaline (Lafontan, 1994); it is also true for theα2-adrenergic receptor (Lafontan & Berlan, 1982).

However, under such conditions exercise still promoteslipid mobilization that could be attributed to a decrease in

Figure 6. Ethanol ratio in SCAT measured at rest, duringexercise and the recovery period under intravenous infusion ofplacebo or octreotide (30 ng min−1 kg−1) in lean subjectsEach column represents the mean values before infusion and duringinfusion.





insulin and to another putative lipolytic factor. Since wehave shown in in vitro lipolysis assays in human fat cellsthat octreotide has no effect onβ-adrenergic responses (i.e.isoproterenol (isoprenaline)- and noradrenaline-inducedlipolysis is unaffected by octreotide) (not shown), theremaining possible lipolytic factor(s) able to explain theincrease in glycerol release are NPs. The plasma levels ofANP and BNP are higher under octreotide than underplacebo (Table 2). It has been demonstrated that insulininfusion decreased ANP release in lean healthy subjects(Nannipieri et al. 2002). Therefore, it could be post-ulated that the decrease in plasma insulin under octreotideinfusion could contribute to the increase in plasma NPsin addition to exercise. NPs are potent lipolytic factorsin human fat cells (Sengenes et al. 2000, 2003) and in invivo conditions (Galitzky et al. 2001; Birkenfeld et al. 2005;Lafontan et al. 2008).

To conclude, to our knowledge, the present studyexamines for the first time the impact on lipid mobilizationin SCAT of an exercise bout performed under conditionspreserving noradrenaline release and suppressing bothadrenaline and insulin release. It reveals that it is plasmaadrenaline rather than noradrenaline that is the mainadrenergic factor that contributes to the control ofexercise-induced lipid mobilization in SCAT. It is thecombined action of insulin suppression and NPs release(although the lack of a NPR-A receptor antagonist preventsdetermination of the precise part played by NPs) whichexplains the remaining lipolytic efficacy under octreotide.It cannot be excluded that the effects observed underour working conditions could depend on the anatomicallocation of the fat deposits and the intensity of exercise. Itmust be remembered that these results apply to SCAT andexercise, but we do not know how general this may be andif conclusions could be extended to all forms of lipolysisin humans.

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Acknowledgements

This study was supported by a grant from the DirectionGenerale de la Cooperation Internationale et du Developpement(Programme d’Action Integre Franco-Tcheque), from NovartisPharma S.A.S, by the FP6 project Hepadip, by a grant from theMinistry of Education of the Czech Republic (MSM 0021620814)and by grant 303/07/0840 of the Grant Agency of Czech Republic.




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