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Original Studies |
Institut National de la Santé et de la Recherche Médicale U-317, Laboratoire de Pharmacologie Médicale et Clinique, Centre d’Investigation Clinique, and Laboratoire des Adaptations de l’Organisme à l’Exercice Musculaire, Université Paul Sabatier, 31073 Toulouse, France
Address all correspondence and requests for reprints to: Dr. Michel Berlan, INSERM U-317, Laboratoire de Pharmacologie Médicale et Clinique, Faculté de Médecine, 37 Allées Jules Guesde, 31073 Toulouse Cedex, France. E-mail: berlan{at}cict.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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2-AR-mediated antilipolytic effects of adrenaline were
not modified. Lymphocyte ß-AR number was unchanged during HDBR. Our
results demonstrate that a sustained sympathoinhibition induces an
increase in the lipolytic ß-adrenergic response in adipose tissue and
suggest that this hypersensitization is linked to an increase in the
postreceptor steps of the lipolytic cascade in the adipocyte rather
than to changes in ß-adrenoceptors. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In healthy humans, the consequence of low SNS activity on AR-mediated functions is poorly documented. A low SNS activity state can be achieved during maintained -6° head-down bed rest (HDBR) (9, 10, 11, 12, 13). HDBR suppress the SNS stimulation linked to the baroreflex stimulation induced by orthostatism. In these experimental conditions, the hemodynamic consequences of the resting state on the SNS during HDBR have been established (14, 15). Adipose tissue is an interesting model to investigate the low SNS activity state because human fat cells possess various ß-AR (ß1-, ß2-, and ß3-AR), the stimulation of which enhances adenylyl cyclase activity, controlling lipolysis (16). The ß-adrenergic function of adipose tissue can be explored in vitro on isolated adipocytes obtained by needle biopsy and in situ using the microdialysis technique (17, 18). To our knowledge, the effect of HDBR on the adaptation of ß-AR-mediated lipolysis in adipose tissue, has never been reported.
The present study was conducted to investigate the consequences of a sustained low SNS activity on the ß-adrenergic lipolytic responses of adipose tissue. For this purpose, lipolytic responsiveness was studied 1) in vitro on isolated fat cells using various adrenergic agents, and 2) in vivo using the changes in the interstitial glycerol concentration (as the lipolysis index) induced by in situ infusion of isoprenaline, as assessed by microdialysis. All investigations were conducted before and during the last 5 days of HDBR.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eight healthy young male subjects, 21–34 yr old (mean ± SEM, 27.2 ± 1.5 yr), who had not been subjected to any nutritional or pharmacological protocol before the study, gave their informed consent to participate in this clinical protocol. All were drug free. Their mean body weight and height were 70.6 ± 2.4 kg (range, 59–79 kg) and 175.5 ± 2.6 cm (range, 168–189 cm), respectively. All had stable weight during the previous 3 months, and their mean body mass index was 22.9 ± 0.6 kg/m2 (range, 21.7–25.5 kg/m2). All experimental procedures were approved by the ethical committee, and the investigations were carried out at the Center of Clinical Investigation of Purpan University Hospital.
Experimental protocol
The subjects underwent similar sets of investigations on days 1 and 6, i.e. before and during a 5-day period of -6° HDBR. For that, the volunteers were placed in beds propped up with blocks at the foot to achieve a -6° head down tilt. During this period, the subjects were supervised using a video camera to ensure that they remained in this position throughout the experiment. The mean daily caloric intake was 2341 ± 102 Cal. Dietary sodium and potassium intake were held constant at 90 and 80 mmol/day, respectively. Water intake was ad libitum (mean, 1236 ± 66 mL/day). The photoperiod was 16 h of light, 8 h of darkness, with lights off at 2300 h.
The day before the beginning of HDBR (day 1), the subjects entered the room at 2000 h and were maintained in a supine position. A venous blood sample of 20 mL for the preparation of leukocytes was drawn from an indwelling polyethylene catheter inserted in the cubital vein. A biopsy of abdominal sc adipose tissue was performed (after intradermal anesthesia with 1% lidocaine, Roger-Bellon, France) with a 2.3-mm diameter needle. By successive suctions, approximately 200–300 mg adipose tissue were drawn into a syringe.
At 0830 h, a microdialysis probe (20 x 0.5 mm; 20,000 dalton molecular mass cut-off; Carnegie Medicine, Stockholm, Sweden) was inserted percutaneously after light intradermal anesthesia (100 µL 1% lidocaine) into the abdominal sc adipose tissue 100 mm from the navel, on the opposite side of the biopsy. The probes was connected to a multichannel microinjection pump (Harvard Apparatus, South Natick, MA) and continuously perfused with sterile Ringer’s solution (154 mmol/L sodium, 4 mmol/L potassium, 2.5 mmol/L calcium, and 160 mmol/L chloride) supplemented with ethanol (40 mmol/L) at 0.8 µL/min.
No collection of the dialysate was performed during the first 30 min immediately after implantation of the probes. For each probe and during each experimental period, the in vivo recovery rate at 2.5 µL/min was assessed using the measure of dialysate glycerol concentrations at various perfusion rates as previously described (18, 19). Briefly, the probes were perfused at four successive rates (0.8, 1.5, 3.5, and 2.5 µL/min, respectively), and the dialysate glycerol concentrations were determined at the steady state for each perfusion rate. These concentrations were plotted (after log transformation) against the perfusion rates. Regression analysis was used to calculate the glycerol concentration at zero flow, corresponding to the extracellular glycerol concentration. The ratio (dialysate glycerol concentration at 2.5 µL/min/extracellular glycerol concentration) x 100 expressed the in vivo recovery rate of the probe at 2.5 µL/min. Previous studies have shown a good reproducibility of extracellular glycerol determinations over time when the same group of subjects is studied with 8- to 15-day intervals between experiments (18). After this calibration period, the perfusion flow rate was maintained at 2.5 µL/min, and 10-min fractions were collected. After three fractions were collected at baseline, the probe was perfused with increasing concentrations of isoprenaline, i.e. 0.01, 0.1, and 1 µmol/L. Three 10-min fractions were collected for each concentration. In each collected fraction, glycerol was assayed as the lipolysis index. The ethanol levels were measured in the perfusate and in the outgoing dialysate to assess the ethanol outflow/inflow ratio, indicating the changes in nutritive blood flow around the probes (20, 21). On day 6, the subjects performed an identical session of investigation, but in the head-down position.
Lipolysis measurements
After needle biopsy, adipocytes were isolated using the method
described by Rodbell (22) in a Krebs-Ringer bicarbonate HEPES solution
(pH 7.4) containing 2% BSA, 6 mmol/L glucose (KRBHA), and 0.5 mg/mL
collagenase. Isolated adipocytes were washed three times, and the cells
were used for lipolysis measurements in KRBHA buffer.
Concentration-response curves were performed using isoprenaline (a
nonselective ß-AR agonist), adrenaline alone or in the presence of 10
µmol/L RX 821002 (a selective 2-AR antagonist), and
dobutamine or terbutaline (selective ß1- and
ß2-AR-agonists, respectively). The lipolytic effect of
CGP 12177 (a selective ß3-AR agonist) was studied at 100
µmol/L. All pharmacological compounds were added to a 5-µL volume
at the start of the incubation performed with 2000–3000 isolated fat
cells in a final volume of 100 µL KRBHA. The incubation was run for
90 min, and 30 µL infranatant were removed for the determination of
glycerol (lipolytic index). Lipolytic activity was expressed as
micromoles of glycerol released per 106 adipocytes for 90
min. The average number of adipocytes added in 100 µL KRBHA was
measured by dividing the total lipid in 100 µL (determined
gravimetrically after lipid extraction) by the average cell volume
(determined by counting under the microscope). The concentration of the
agonist inducing a half-maximal lipolytic effect (EC50) was
determined using computer-fitting analysis of concentration-response
curves obtained with the various ß-AR agonists. The negative
logarithm of the EC50 value (pD2) was defined
as the AR sensitivity.
Preparation of leukocytes membranes and binding studies
Leukocytes were isolated from citrated blood samples. After
dilution (1:1, vol/vol) with a saline buffer (140 mmol/L NaCl, 14.5
mmol/L Tris, 0.54 mmol/L KCl, 0.098 mmol/L MgCl2, 0.005
mmol/L CaCl2, and 0.55 mmol/L glucose), the samples were
layered on 10 mL Ficoll-Paque (density = 1.077) and centrifuged
for 30 min at 150 x g at 20 C. The upper layer
containing the platelets was removed. The leukocytes remaining in a
thin lower band were diluted in buffer and centrifuged at 80 x
g for 10 min at 20 C, and the pellet was washed twice in
buffer and stored at -60 C until use. Binding experiments were carried
out using [125I]cyanopindolol. Lymphocyte membranes were
homogenized and washed in 30 mL 5 mmol/L Tris-HCl, 5 mmol/L
ethylenediamine tetraacetate, and 120 mmol/L NaCl buffer (pH 7.5)
before centrifugation (40,000 x g, 15 min, 4 C).
Binding studies were performed in a final volume of 200 µL of the
corresponding buffer. Incubations were carried out at 37 C for 40 min.
Nonspecific binding was determined in the presence of 200 µmol/L
adrenaline. The binding assay was stopped by the addition of 4 mL
ice-cold incubation buffer followed by rapid filtration through Whatman
GF/C glass fiber filters (Clifton, NJ). The filters were then washed
twice with 10-mL portions of ice-cold buffer. The radioactivity
retained on the filters was measured in a Packard 
-counter (Downers
Grove, IL) at an efficiency of 80%. Specific binding was defined as
total binding minus nonspecific binding.
Biochemical determinations
Plasma adrenaline and noradrenaline were assayed by high pressure liquid chromatography using electrochemical (amperometric) detection, as previously described (23). The detection limit was 20 pg/sample. Day to day variability was 4%, and within-run variability was 3%. Glycerol was determined in plasma, dialysate, or KRBHA samples using an ultrasensitive radiometric method (18); the intra- and interassay variabilities were 5.0% and 9.2%, respectively. The ethanol concentration in 5-µL fractions of dialysate and perfusate was determined with an enzymatic method (18); the intra- and interassay variabilities were 3.0% and 4.5%, respectively. Plasma glucose and nonesterified fatty acids were assayed with a glucose oxidase technique (Biotrol, Paris, France) and an enzymatic method (Unipath, Dardilly, France), respectively.
Drugs and analytical methods
Isoprenaline hydrochloride was obtained from Winthrop (Clichy,
France). Dobutamine hydrochloride was obtained from Lilly (Saint-Cloud,
France), and terbutaline sulfate was obtained from Astra (Nanterre,
France). CGP 12,177
(4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-one)
came from Ciba-Geigy (Basel, Switzerland). The 2-AR
antagonist RX 821002
[2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline] was a gift from
Reckitt and Colman Laboratories (Kingston-upon-Hull, UK).
[125I]Cyanopindolol and [-32P]ATP were
obtained from Amersham (Les Ullis, France) and ICN Biochemical (Orsay,
France), respectively. Crude collagenase and other products were
purchased from Boehringer Mannheim (Mannheim, Germany).
Statistical analysis
All values are given as the mean ± SEM. ANOVA for repeated measures with Scheffe’s post-hoc test and Wilcoxon’s paired test were used for comparisons when appropriate. P < 0.05 was considered statistically significant. All statistical comparisons were performed using a statistical software package (StatView 4.5, Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Fasting plasma noradrenaline and
adrenaline levels and other biological parameters were not modified
during the HDBR period. No change in resting heart rate or systolic
blood pressure and a weak significant increase in diastolic blood
pressure were observed. Body weight, fluid intake, and urine output
were unchanged during HDBR period (not shown). The saturation curves of
[125I]cyanopindolol on leukocyte membranes before and
during HDBR are depicted in Fig. 1. No
significant difference was found in either total number of binding
sites or affinity for [125I]cyanopindolol, evaluated
after Scatchard analysis (Table 1).
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The calculated interstitial glycerol concentrations were not
different before and during HDBR (mean of the first three fractions,
171 ± 18 and 177 ± 25 µmol/L, respectively). The in
vivo recovery rates of the probes at 2.5 µL/min were not
different either before or during HDBR (33 ± 2% and 31 ±
4%, respectively). Before HDBR, the addition of 0.01 µmol/L
isoprenaline (nonselective ß-AR agonist) into the perfusate did not
modify extracellular glycerol levels. Higher concentrations (0.1 and 1
µmol/L) significantly increased the extracellular glycerol
concentrations to 153 ± 12% and 237 ± 24% over the
baseline (100%), respectively (Fig. 2). During HDBR, isoprenaline
increased the extracellular glycerol levels to 144 ± 13% at 0.01
µmol/L, to 261 ± 27% at 0.01 µmol/L, and to 374 ± 44%
at 1 µmol/L (Fig. 2). The lipolytic
response to isoprenaline was significantly higher during than before
HDBR. In both periods, isoprenaline decreased the ethanol
outflow/inflow ratio, indicating an isoprenaline-dependent increase in
nutritive blood flow in adipose tissue (Fig. 2). The vasodilating
effects of isoprenaline were more pronounced during than before
HDBR.
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The distribution of fat cell sizes (expressed as the number of
cells for a given diameter) was not changed before or during HDBR (not
shown). Thus, the results of the lipolysis experiments are expressed as
micromoles of glycerol released per 106 cells. The
spontaneous release of glycerol (basal lipolysis) was significantly
increased during HDBR (Table 2). Concentration-response curves obtained
with isoprenaline, dobutamine (ß1-AR agonist), and
terbutaline (ß2-AR agonist), before and during HDBR, are
depicted in Figs. 3 and 4.
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. Before and during HDBR, adrenaline was
devoid of lipolytic effect until it reached a level of 10 µmol/L
(Fig. 5). In both situations a clear increase in the lipolytic effect
of adrenaline was observed when RX 821002 (selective 2
antagonist) was added to the incubation medium. The additive effect of
RX 821002 was not different before and during HDBR (86.3 ± 33.2%
and 74.6 ± 20.2% increase in lipolysis induced with 10 µmol/L
adrenaline, respectively). The lipolytic effect of adrenaline, measured
in the presence of RX 821002, was higher during than before HDBR
(F = 7.76; P = 0.01).
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. The results
were analyzed using two-way ANOVA, with drug effect (drug
vs. basal) and HDBR (during vs. before) as
factors of the analysis. In each case, there was an increase in
drug-induced glycerol release before as well as during HDBR (Table 2),
but no significant modification of the response was assessed by the
interaction term of the two factors (F = 2.01–4.11;
P = NS). The lipolytic sensitivities (pD2)
of isoprenaline, adrenaline, and the selective ß-AR agonists used
were unmodified during HDBR (Table 3).
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). dbcAMP increased the glycerol release before and after HDBR. The
lipolytic efficacy of dbcAMP was not changed during HDBR (interaction
term: F = 4.11; P = 0.06).
To rule out the postreceptor events on ß-AR-mediated lipolysis, the
lipolytic activities of fat cells were expressed as a percentage of the
dbcAMP effect at the maximal post-receptor stimulation. Using this
expression, the concentration curves of isoprenaline before and after
HDBR (Fig. 3B) were superimposable. Identical results were found with
the selective ß1- and ß2-selective agonists
or with adrenaline alone or in presence of RX 821002 (not shown). Table 2 reports the comparative maximal lipolytic effects of all of the
compounds, using a similar expression of the results; the lipolytic
effects of the selective ß-AR agonists were comparable before and
during HDBR.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previous reports have shown that HDBR promotes a sustained reduction of sympathoneural release and lower noradrenaline synthesis and turnover (10, 24, 25). The inhibition of SNS activity was not associated with a reduction of plasma catecholamine levels, as concurrent hypovolemia occurs (24). This was sustained by a decrease in urinary catecholamine excretion in the first 24 h of HDBR (10, 26). The accuracy of plasma catecholamine levels has been questioned, as a decreased plasma volume may maintain (10, 26) plasma catecholamine concentrations when spillover is reduced. However, the total circulating norepinephrine level was dramatically lowered when corrected by the plasma volume (13). The lack of changes in plasma catecholamine levels in our study is in agreement with these previous reports. Leukocytes are currently used as cell models for estimating the changes occurring in ß-AR expression in humans during HDBR. In our study, the number of lymphocyte ß-AR was not modified. This is in agreement with the findings of other researchers who measured either the number of ß-AR or the production of cAMP after ß-AR stimulation in leukocytes during 9 days of HDBR (26). Conversely, a selective increase in ß1-/ß2-AR responsiveness has recently been reported in the cardiovascular system after 14 days of HDBR (13).
In microdialysis experiments, infusion of increasing concentrations of
isoprenaline (nonselective ß-AR agonist) into the probes promoted a
dose-dependent increase in extracellular glycerol concentrations: this
lipolytic response was enhanced during HDBR (Fig. 2A). As
previously shown by our group (18), the in situ isoprenaline
infusions through microdialysis probes reduced the ethanol
outflow/inflow ratio, suggesting an increase in nutritive blood flow in
adipose tissue (Fig. 2B). The vasodilating effect of
isoprenaline was also increased during HDBR. It is noticeable that this
vascular effect could partly blunt the positive effect on lipolysis,
since an increment in glycerol drainage in the circulation was
simultaneously promoted (19).
In agreement with in situ results, the in vitro
studies of isolated fat cells showed that HDBR promoted an increase in
isoprenaline-induced glycerol release (Fig. 3). This effect during HDBR
was also found with selective agonists of the three ß-AR subtypes
(Fig. 4 and Table 2). The in vitro study revealed an
increase in basal lipolysis (i.e. spontaneous glycerol
release in the absence of any agonist). However, the basal lipolysis
can be largely artifacted, as adipocytes are not submitted in
vitro to their physiological regulation factors. It is noticeable
that no change in basal interstitial glycerol concentrations was
observed in microdialysis experiments. The results were expressed as a
percentage of maximal dbcAMP to target the modification of lipolysis
measurements (i.e. modification of the transduction system).
This expression seems to be more accurate than the use of basal
lipolysis. After this transformation, the stimulating effects of the
various ß-AR agonists were similar before and during HDBR (Fig. 3 and
Table 2). This strongly suggests that a low sympathetic activity state
led to a modification the lipolytic cascade, downstream from the
ß-AR/adenylyl cyclase system rather than a modification of ß-AR
number, affinity, or the coupling system with adenylyl cyclase. The
unchanged pD2 of ß-AR agonists on the lipolytic process
(Table 3) argues for this hypothesis, as does the absence of
modification of leukocyte ß-AR (Table 1), insofar as they reflect
those in adipocytes.
Thus, an increment in the activity of enzymes such as cAMP-dependent protein kinase and hormone-sensitive lipase or a reduced activity of the cAMP-dependent phosphodiesterase could explain the improved lipolytic effect of ß-AR agonists described in situ and in vitro. Larger amounts of adipose tissue, not available with the needle biopsy used in the present experiment, would be required to measure these enzymatic activities. The observed increase in basal or dbcAMP lipolysis during HDBR could be caused by the increased hormone-sensitive lipase activity (or expression) because this enzyme catalyzes the rate-limiting step in fat cell lipolysis. When considering the present results obtained in HDBR with other situations known to reduce SNS activity (fasting or calorie restriction), they fit with the appearance of an increased ß-AR sensitivity of adipose tissue: energy restriction increased the hormone-sensitive lipase expression, the in vitro sensitivity of the ß-adrenergic component of fat cell lipolysis (27), and the sensitivity of adipose tissue to exogenously infused (19, 28, 29, 30) or exercise-released catecholamines (31).
Adipocyte 2-AR are involved in the control of lipolysis
of human adipocytes. Their stimulation by physiological catecholamines
induces antilipolysis (32). They were responsible for the weak
lipolytic effect of adrenaline observed (Fig. 5). The blockade by an
2-AR antagonist unmasked the ß-adrenergic component of
the adrenaline effect. It was observed that the potentiating effect of
RX 821002 on adrenaline-induced lipolysis was of similar amplitude
before and during HDBR, suggesting that the 2-AR pathway
was not modifies by sympathoadrenal inhibition.
In conclusion, the present results demonstrate that a low sympathetic activity state induced by 5-day HDBR increases the responsiveness of lipolytic processes in adipose tissue assessed by in situ microdialysis assay. In vitro studies on adipocytes suggest an increased efficiency of the posttransductional steps of the lipolytic cascade rather than an alteration of adrenergic receptors. It should be noticed that the whole body sensitivity of lipid mobilization to physiological and pharmacological ß-AR agonists was not assessed in this study and needs further investigations.
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Acknowledgments |
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Footnotes |
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Received June 5, 1997.
Revised October 23, 1997.
Accepted October 31, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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2-adrenoceptors in regulating lipid
mobilization from human adipose tissue. J Clin Invest. 91:1997–2003.
2-adrenoceptors: the regulation of fat cell function and lipolysis. Endocr Rev. 16:716–738.[CrossRef][Medline]
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) or during (•) 5-day
HDBR. The inset shows Scatchard plots of specific
binding derived from the saturation curves. Values are the mean ±
SE.
and 
), on
glycerol release in sc isolated adipocytes before (open symbols) and
during (filled symbols) HDBR. Values are the mean ±
SEM. The effects of adrenaline, alone and with
10-5 mol/L RX 821002, were first analyzed in each period
using one-way ANOVA, with adrenaline concentration as the factor of the
analysis and Scheffe’s post-hoc test. Adrenaline alone
increased glycerol release (F = 11.09; P <
0.001); the increment was only significant with the 10-5
mol/L adrenaline concentration. In the presence of RX 821002,
adrenaline increased glycerol release (F = 26.89;
P < 0.001); the increment was significant from
10-7-10-5 mol/L adrenaline. Then a
statistical comparison of the curves was performed using ANOVA for
repeated measures, with HDBR period (before vs. during),
drugs (adrenaline vs. adrenaline and RX 821002), and
adrenaline concentrations as factors of the analysis and
Bonferroni-Dunn post-hoc test. HDBR increased the
lipolytic effect of adrenaline alone or in the presence of RX 821002
(F = 12.29; P < 0.001). The addition of RX
821002 increased the lipolytic of adrenaline (F = 10.26;
P < 0.001); this effect was not different during
HDBR (interaction term; F = 0.59; P = NS).