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Atrial natriuretic peptide contributes to physiological control of lipid mobilization in humans¹

CEDRIC MORO^*,, FRANCOIS CRAMPES^*, CORALIE SENGENES^*, ISABELLE DE GLISEZINSKI^*, JEAN GALITZKY^*, CLAIRE THALAMAS, MAX LAFONTAN^*,2 and MICHEL BERLAN^*,,2

^* Unité de recherches sur les Obésités, Institut National de la Santé et de la Recherche Médicale (INSERM U586); Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, Université Paul Sabatier;
Center of Clinical Investigation of Toulouse Hospital, France; and
Department of Clinical and Medical Pharmacology, Toulouse, France

²Correspondence: M. B., INSERM U 586, Laboratoire de Pharmacologie Médicale et Clinique, Faculté de Médecine, 37 Allées Jules Guesde, 31073 Toulouse, France. E-mail: berlan{at}cict.fr; and M. L., Unité de recherches sur les Obésités, Institut National de la Santé et de la Recherche Médicale (INSERM U586), Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, Université Paul Sabatier, Toulouse, France. E-mail: lafontan{at}toulouse.inserm.fr

SPECIFIC AIMS

In humans, lipid mobilization is considered to depend mainly on catecholamine action and interplay between fat cell ß- and ₂-adrenergic receptors (AR). Putative contribution of atrial natriuretic peptide (ANP) was hypothesized since we have previously shown that ANP is a potent lipolytic agent on human fat cells. During exercise, sympathetic nervous system (SNS) is activated and, concomitantly, ANP is released from heart. Control of lipid-mobilizing mechanisms in abdominal subcutaneous adipose tissue (SCAT) was investigated using microdialysis in men during two successive exercise bouts performed at 35% (E1) and 60% (E2) of their VO₂max. Experiments were performed after oral administration of placebo or tertatolol, a ß-AR antagonist that potentiates plasma ANP increment during exercise. In both situations, we studied the importance of local lipolysis through measurement of glycerol release in a control microdialysis probe and in a probe perfused with propranolol (ß-antagonist). In all cases, perfusate of the probe was complemented with an ₂-AR antagonist (phentolamine, 100 µmol/L) in order to block antilipolytic ₂-ARs. We also determined extracellular cGMP levels (reflecting ANP activity on fat cells) in dialysate and measured variations in plasma ANP and cGMP levels.

PRINCIPAL FINDINGS

1. Effect of placebo administration on extracellular glycerol concentration during exercise
Exercise-induced increase in extracellular glycerol concentration (EGC) was stronger during E2 than during E1 in SCAT (Fig. 1 a). A residual increase in EGC was observed in a probe supplemented with 100 µmol/L propranolol; it was higher during E2 than during E1. Mean change in EGC during E2 and the recovery period is given in the insert of Fig. 1a . Propranolol led to a significant limitation of exercise-induced EGC increase (–41% and –37% reduction during E1 and E2, respectively) but a lipid mobilizing action persisted.

View larger version (28K): [in this window] [in a new window]   Figure 1. a) Effect of local propranolol administration on extracellular glycerol concentration during two successive exercise bouts performed at 35% (E1) and 60% (E2) of VO2max and during recovery in SCAT after placebo treatment (insert, mean changes in extracellular glycerol concentration). *Significantly different when compared with control probe (P<0.05). b) Suppression of local lipid mobilization induced by increasing doses of isoproterenol after a local propranolol coadministration in the microdialysis probe in SCAT. *Significant when compared with isoproterenol alone (P<0.05).

To verify whether local infusion of propranolol was truly able to counteract ß AR-dependent lipolysis, we performed a perfusion through dialysis probes of two concentrations of isoproterenol in SCAT and at rest. Isoproterenol promoted a concentration-dependent increase in EGC. Addition of propranolol into the perfusate totally suppressed the lipolytic effect of isoproterenol without altering basal values (Fig. 1b ).

2. Effect of tertatolol administration on EGC during exercise
Ninety min after oral intake of tertatolol (5 mg), no difference in EGC increase was found between the two probes during exercise; EGC was higher during E2 than during E1 (Fig. 2 a). Mean changes of EGC are depicted in the insert of Fig. 2a . Propranolol was without effect on exercise-induced EGC increase during either exercise bout. We verified whether tertatolol administered orally was able to counteract lipolysis induced by a local activation of ß-ARs. Two concentrations of isoproterenol were perfused in SCAT through dialysis probes 90 min after subjects had taken tertatolol. Tertatolol totally suppressed the lipolytic effect of 0.1 or 1 µmol/L isoproterenol (Fig. 2b ).

View larger version (25K): [in this window] [in a new window]   Figure 2. a) Effect of local propranolol administration on extracellular glycerol concentration during two successive exercise bouts performed at 35% (E1) and 60% (E2) of VO2max and during recovery in SCAT after tertatolol treatment (insert, mean changes in extracellular glycerol concentration values). b) Suppression of isoproterenol-induced lipid mobilization in SCAT after oral administration of tertatolol.

3. Modifications of blood flow in SCAT during exercise bouts
Changes in blood flow occurring in SCAT microcirculation were evaluated by measuring ethanol escape from microdialysis probes. At rest, average ethanol ratios were not different in probes with phentolamine or with phentolamine plus propranolol. During exercise bouts, no significant changes in ethanol ratio were observed in any probes either after placebo or tertatolol administration.

4. Exercise-induced changes in extracellular and plasma levels of cGMP
To verify involvement of ANP in stimulation of lipolysis in SCAT, we first performed a perfusion of 10 µmol/L ANP during 60 min through a microdialysis probe inserted in SCAT in resting subjects. An increase in both cGMP concentrations (from 11.2±3 to 37±5 pmol/mL) and EGC (163±19 to 302±31 µmol/L) was observed in dialysate. A linear correlation was found between EGC values and cGMP concentrations when performing regression analysis from each data point (r=0.85; P<0.01). Taking into account these results, extracellular cGMP levels were determined from pooled dialysates, from perfusates supplemented with propranolol, and obtained during 30 min of E1 and E2 performed after placebo or tertatolol administration. Results show that exercise increased extracellular cGMP concentration; the highest increment was observed during E2 (24.8±2.8 vs. basal 16.2±0.9 pmol/mL) and after tertatolol administration (52.6±13.1 vs. basal 14.6±0.8 pmol/mL). Plasma cGMP concentrations were lower than in extracellular fluid and a parallel increase was observed. Correlation was found between values of plasma ANP and extracellular cGMP concentrations measured at rest and during each exercise bout after placebo or tertatolol administration (r=0.41; P<0.01). Regression analysis from each data point (36 determinations) shows a linear correlation between EGC values and extracellular cGMP concentrations in two experimental conditions, placebo (r=0.33; P<0.05) and tertatolol (r=0.49; P<0.02).

5. Exercise-induced changes in plasma parameters and cardiovascular responses
Increase in plasma norepinephrine and epinephrine concentrations was higher during E2 than during E1. Tertatolol administration enhanced plasma catecholamines increment. After placebo administration, E1 moderately increased plasma ANP concentrations (highest concentrations were reached during E2). Tertatolol administration caused a stronger increase in plasma ANP concentrations during E1 and E2. Tertatolol administration enhanced the increase in cortisol. Tertatolol decreased plasma insulin levels at rest and E1 and E2 successively induced a decrease in plasma insulin concentrations in both conditions. Plasma glucose values remained unchanged during exercise bouts whatever the treatment. Tertatolol administration did not modify plasma lactate changes during E1 and E2. Plasma NEFA and glycerol concentrations at rest were reduced 90 min after tertatolol administration when compared with placebo treatment (P<0.05). During exercise, plasma NEFA and glycerol concentrations remained significantly lower under tertatolol treatment (P<0.01) when compared to placebo values.

In the resting state, tertatolol administration lowered heart rate but did not modify systolic and diastolic blood pressure. During E1 and E2, heart rate increased after placebo or tertatolol administration but remained lower under tertatolol treatment. A similar pattern of changes was observed with systolic blood pressure,: the increase measured during E1 and E2 was weaker, as expected, after tertatolol administration. Finally, whatever the treatment, evolution of diastolic blood pressure was similar (results not shown).

CONCLUSIONS AND SIGNIFICANCE

For a long time, catecholamines released under SNS activation were considered major agents in control of lipid mobilization from adipose tissue during exercise in humans. However, discovery of peptides with lipolytic properties requires various aspects of the regulation of lipid mobilization to be reconsidered. NPs are involved in a cGMP-dependent pathway for control of human fat cell lipolysis in vitro. Present results reveal a contribution of NPs to the enhancement of lipid mobilization during exercise in human SCAT. Previous investigations have shown that circulating ANP concentrations increased 2- to 3-fold during short-exercise bouts of increasing intensities. Plasma ANP increment was enhanced (at a similar developed power of exercise) when subjects had been pretreated with tertatolol. The most striking argument supporting physiological contribution of ANP in control of lipid mobilization concerns exercise-induced lipid mobilization remaining under oral ß-AR-blockade, when highest levels of plasma ANP are attained. In absence of any other relevant factor, NPs represent reasonable candidates to explain exercise-induced lipid mobilization resistant to local propranolol (Fig. 1) and noticeable lipid-mobilizing action observed under oral ß-blockade in subjects performing physical exercise (Fig. 2) . Extracellular levels of cGMP are higher in extracellular fluid of SCAT than in plasma; a result suggesting that the second messenger originates from fat cell leakage. cGMP increment in extracellular space could be related to action of ANP on fat cells; correlation between both parameters supports this view. Finally, the relation found between the increment in extracellular cGMP concentration and EGC changes focus upon a possible link between cGMP and glycerol release (i.e., lipolysis). E2 performed under tertatolol promoted a large increase in extracellular cGMP. During exercise, putative release of biological lipolytic compounds should also be considered. Parathyroid hormone stimulates lipolysis in vitro in human fat cells. Its lipolytic effect is weak and only obtained with high concentrations. Moreover, moderate exercise had no action or slightly increased parathyroid hormone concentration in blood. Growth hormone (GH) secreted during exercise and i.v. administration of h-GH have shown a lipid mobilizing action. The time course of appearance of the lipolytic effect of GH is long in fat cell incubations in vitro and appears at least two hours after i.v. injection of GH in vivo. Thus, involvement of GH-related lipolytic effects cannot be proposed to interpret lipid mobilization in this study. Other putative agents, release of which could be affected by exercise, such as prostaglandins, adenosine, and neuropeptide Y are known to exert antilipolytic effects on human fat cells.

In conclusion, outside ANP, contribution of another lipid-mobilizing partner cannot be proposed. Direct infusion of ANP in a microdialysis probe promotes concomitant increment in cGMP and glycerol concentrations in extracellular fluid in adipose tissue. Determining relationships between concomitant changes in plasma ANP, and extracellular cGMP and glycerol, strongly sustain the initial hypothesis proposed that the NP lipolytic pathway contributes physiologically to mobilize lipids from adipose tissue in humans.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1086fje;

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