¹INSERM Unité 317, Laboratory of Medical and Clinical Pharmacology, Faculty of Medicine, Toulouse, France

²Department of Sport Medicine and Obesity Unit, Charles University, Prague, Czech Republic

Correspondence to: M Berlan, INSERM U 317, Laboratoire de Pharmacologie Médicale et Clinique, Faculté de Médecine, 37 Allées Jules Guesde, 31073 Toulouse cedex, France. E-mail: berlan@cict.fr

OBJECTIVE: We recently demonstrated that natriuretic peptides (NP) are involved in a pathway inducing lipolysis in human adipose tissue. Atrial NP (ANP) and brain NP (BNP) operate via a cGMP-dependent pathway which does not involve phosphodiesterase-3B inhibition or cAMP. The study was performed to evaluate the effect of ANP on lipid mobilization in obese women and secondly to examine the possible effect of a low-calorie diet (LCD) on the lipolytic response of subcutaneous abdominal fat cells to NP and on the lipid mobilization induced by ANP infusion (1 µg/m² min for 60 min).

SUBJECTS: Ten obese women from 40.5±3.4 y old were selected for this study. Their body weight was 96.4±5.7 kg and their BMI was 35.3±1.7 kg/m². They received a 2.5-2.9 MJ/day formula diet for 28 days.

DESIGN: Before and during the LCD, an adipose tissue biospy was performed for in vitro studies and, moreover, ANP was perfused i.v. to evaluate its lipid mobilizing action in toto and in situ in subcutaneous abdominal adipose tissue (SCAAT) using microdialysis.

RESULTS: The lipolytic effects of isoproterenol, ANP, BNP and bromo-cGMP (an analogue of cGMP) on fat cells increased by about 80-100% during LCD. The lipid mobilization during i.v. ANP infusion, assessed by plasma non-esterified fatty acids (NEFA) increase was enhanced during the LCD. However, during LCD, ANP infusion induced a biphasic effect on glycerol concentration in plasma and interstitial fluid of SCAAT; a significant increase was observed in glycerol levels during the first 30 min infusion period, followed by a steady decrease. The concentration of glycerol was lower during the post-infusion period than during the baseline period. This effect was stronger in obese subjects submitted to the LCD with a low-carbohydrate composition. Other plasma parameters were weakly increased (noradrenaline) or not modified (insulin, glucose) by ANP infusion and no difference was found before and during LCD treatment.

CONCLUSION: The present study shows that NP are powerful lipolytic agents in subcutaneous fat cells and that both isoproterenol- and NP-induced lipolysis increase during LCD, in obese women. These changes seem to be associated with an improvement of the lipolytic pathway at a post-receptor level. Moreover, i.v. administration of ANP induced a lipid mobilizing effect which was enhanced by a LCD in these objects.

International Journal of Obesity (2002) 26, 24-32. DOI: 10.1038/sj/ijo/0801845

ANP; BNP; adipocytes; fat cells; lipolysis; microdialysis

Introduction

The treatment of obesity often includes a hypocaloric diet which promotes lipid mobilization/oxidation as the major energy source. The hydrolysis of triglyceride stores, which releases non-esterified fatty acids (NEFA) and glycerol from adipose tissue, is a key step in the metabolic process leading to the decrease of fat mass. Catecholamines are powerful regulators of lipid mobilization in humans and are considered to stimulate lipolysis mainly through the 1- and 2- adrenoceptors (AR)¹ and previous data have shown that hypocaloric diets enhance catecholamine-induced lipid mobilization.^2,3,4,5,6

We recently demonstrated that natriuretic peptides (NP) are involved in a new pathway controlling human adipose tissue lipolysis.⁷ They operate via a cGMP-dependent pathway which does not involve phosphodiesterase-3B inhibition or cAMP production and atrial natriuretic peptide (ANP) was the most potent compound among the three NPs-ANP, BNP (brain-NP) and CNP (C-type NP). Moreover we have also reported in a previous clinical investigation, that ANP infusion in men promotes various metabolic effects, ie local increase of interstitial glycerol concentration in subcutaneous abdominal adipose tissue (SCAAT), increase in plasma glycerol, and NEFA, noradrenaline and insulin levels which were similar in lean and obese young healthy subjects.⁸ A decreased lipolytic response to catecholamines has been reported in obese subjects.^5,9,10 Nevertheless, we have demonstrated that a very low calorie diet (VLCD) increases the in situ lipolytic response to isoproterenol and to dobutamine in the subcutaneous adipose tissue of obese subjects.¹¹

The aim of the present work was to compare in vitro -adrenergic and NP lipolytic effects on isolated adipocytes and to investigate in situ (microdialysis of SCAAT) and in toto the lipomobolizing effect of an i.v. infusion of ANP in obese subjects before and during a low-calorie diet (LCD).

Methods

Patients

Ten obese women from 22 to 57 y old (mean 40.5±3.4 y) were selected for this study. Their body weight was 96.4±5.7 kg (range 75.6-116.3 kg) and had been stable for at least 3 months before the beginning of the study. Their body mass index (BMI) was 35.2±1.7 kg/m² (range 30.2-46.3 kg/m²). All the patients received a 2.5-2.9 MJ/day formula diet (Dietifine^Ò Lab. Bio2, Lyon, France) for 28 days. The formula included 92 g protein, 22 g carbohydrate and 16 g fat and the recommended daily allowance of vitamins and minerals. The subjects were in-patients throughout the duration of the diet and their usual medication was maintained. All subjects had given their informed consent before the study and the investigation protocol was approved by the Ethical Committee of Prague University Hospital.

Experimental protocol

The subjects were investigated at 8 am after an overnight fast and were maintained in the supine position during the experimental period. An indwelling polyethylene catheter was inserted into the antecubital vein of each arm. At 8.30 am, a needle micro-biopsy (200-400 mg) of adipose tissue was performed under local anesthesia 10-15 cm from the umbilicus. The ANP was infused through the i.v. catheter placed in the right arm using an Auto-Syringe infusion pump. Blood samples were withdrawn from a catheter placed in the left arm. Resting baseline measurements were performed during the first 60 min. Immediately after the baseline period, ANP was administered with isotonic saline as vehicle by an infusion at a constant rate of 1 µg/m²/min for 60 min. The total volume infused was <40 ml. Appropriate doses of ANP were selected on the basis of changes in metabolic and cardiovascular parameters previously determined by us⁸ and other groups.^11,13,14 During the baseline period and ANP infusion, the heart rate was continuously recorded using a standard three-lead ECG. Systolic and diastolic blood pressures were evaluated every 15 min using a Dynamap device.

Two microdialysis probes (Carnegie Medecin, Stockholm, Sweden) of 20´0.5 mm and 20 000 MW cut-off were inserted percutaneously after epidermal anesthesia (200 µl of 1% lidocaine, Roger-Bellon, Neuilly-s-Seine, France) into the abdominal SCAAT at a distance of 10 cm to the right of the umbillicus and separated by at least 10 cm. The probes were connected to a microinjection pump (Harvard Apparatus, Les Ulis, France) and perfused with Ringer's solution (139 mmol/l sodium, 2.7 mmol/l potassium, 0.9 mmol/l calcium, 140.5 mmol/l chloride, 2.4 mmol/l bicarbonate, 5.6 mmol/l glucose). One probe was perfused with Ringer supplemented with 0.1 mmol/l propranolol (-adrenergic receptor antagonist). The perfusate solutions were supplemented with ethanol (1.7 g/l). Ethanol was added to the perfusate in order to estimate changes in the blood flow, as previously described.¹⁵ After a 30 min equilibration period, a 30 min fraction of dialysate was collected at a flow rate of 0.5 µl/min. Then, the infusion was set at 2.5 µl/min for the remaining experimental period. The calibration procedure using various infusion rates was applied for interstitial glycerol concentration determination in SCAAT as previously described by our group.^11,15 A simplified, but relevant and less time-consuming method was selected in this study. The estimated interstitial glycerol concentrations were calculated by plotting (after log-transformation) the concentration of glycerol in the dialysate measured at 0.5 and 2.5 µl/min against the infusion rates. After the calibration of the probes, two 15 min fractions of the outgoing dialysate were collected and then ANP was infused i.v. for 60 min. Dialysate samples were collected for each 15 min period during infusion and during the 60 min post-infusion period. Water intake was allowed ad libitum during the experimental periods.

Adipocyte preparation and lipolysis measurements

Isolated adipocytes were obtained by collagenase digestion of adipose tissue fragments in Krebs Ringer bicarbonate-Hepes buffer containing albumin (3.5 g/100 ml; KRBA) and glucose (5.6 mmol/l) at pH 7.4 and under gentle shaking at around 60 cycles/min at 37°C.⁷ Then, the fat cells were filtered through a silk screen and washed three times with KRBA buffer to eliminate collagenase, isolated adipocytes were brought to a suitable dilution (1000-2000 cells/assay) in KRBA buffer for lipolysis assays and incubated with pharmacological agents in a final volume of 100µ l for 90 min at 37°C as previously described.⁷ At the end of the incubation 20-50 µl aliquots of the infranatant were taken for glycerol assay to determine the lipolytic index. Concentration-response assays were performed using isoproterenol (non-selective -AR agonist), ANP, BNP and bromo-cGMP (an analogue of cGMP) was used at the concentration of 10 mmol/l.

Drugs and biochemical determinations

The following reagents were used for fat cell isolation and lipolysis measurement: bovine serum albumin; fraction V, fatty-acid free; collagenase from Clostridium histolyticum; bromo-cGMP (Boehringer-Mannheim, Meylan, France); isoprenaline hydrochloride (Sigma, Saint Quentin Fallavier, France); human synthetic ANP (Neosystem, Strasbourg, France). ANP Clinalfa for biomedical and clinical research perfused in subjects came from France Biochem, Meudon, France. Plasma ANP concentrations were determined using a RIA kit from Phoenix Pharmaceuticals, Inc (Belmont, USA). Plasma noradrenaline and adrenaline were assayed by high performance liquid chromatography using electrochemical (amperometric) detection, as previously described.⁸ Ethanol in dialysate and perfusate (5 µl) was determined using an enzymatic method.¹⁶ Glycerol was determined in plasma and in dialysate using an ultrasensitive radiometric method.¹⁷ Plasma glucose was assayed with a glucose oxidase technique (Biotrol, Paris, France). NEFA were assayed with an enzymatic method (Unipath, Dardilly, France). Plasma insulin was measured using a Bi-insulin IRMA kit from Sanofi Diagnostics Pasteur (Marnes-La-Coquette, France).

Statistical analysis

All the values are means±s.e.m. Flow-rate calibration data were analysed by linear regression, tested for goodness of fit. Statistical comparison of in vitro data on isolated fat cells before and during LCD was performed using two-way Anova analysis for repeated measures. During ANP infusion, plasma and interstitial response curves were calculated as the total integrated changes over baseline values (area under the curves, AUC) using the trapezoidal method. Statistical comparison of the effect of LCD was done on AUC using Student's paired t-test. The effects of ANP were analysed by one-way ANOVA with time as the factor of the analysis followed by a Bonferroni-Dunett post-hoc test, taking basal values as control. P<0.05 was considered statistically significant.

Results

Changes induced by LCD

The changes in body weight and in various biological parameters observed on the 28th day of LCD for the whole population are depicted in Table 1. LCD induced a weight loss and a significant reduction of fasting plasma insulin levels. Plasma NEFA, glucose and noradrenaline concentrations were unchanged. Plasma and interstitial glycerol levels tended to be lower during LCD, but did not reach a significant difference.

In vitro lipolysis of isolated fat cells

Spontaneous glycerol release (basal lipolysis) was found to be significantly increased by the LCD; values expressed in µmol glycerol released by 100 mg lipid for 90 min were 0.052±0.08 and 0.073±0.02 before and during LCD, respectively. The lipolytic effects of ANP, BNP and bromo-cGMP were compared to those of isoproterenol (-AR agonist). The maximal lipolytic effect of isoproterenol measured before the LCD was taken as a reference for the lipolytic effect induced by all drugs before and during the LCD. The results depicted in Figure 1 show that before LCD, maximal ANP- or BNP-induced lipolysis was 60% of the isoproterenol effect and that the lipolytic effect of bromo-cGMP was 40%. The LCD induced an 80-90% increase of the maximal lipolytic effect of isoproterenol. It was also found that the LCD induced a significant increase of about 100-110% of the maximal lipolytic response initiated by ANP and BNP. Before LCD, in the presence of bromo-cGMP, glycerol production was 0.152±0.026 µmol/100 mg lipid/90 min and 0.224±0.028 during LCD (P=0.04). The LCD did not modify the potency of isoproterenol or NP: calculated pD2 (-log EC₅₀) for isoproterenol, ANP and BNP effect were similar before and during LCD (6.93±0.15 vs 7.17±0.15, for isoproterenol, 8.73±0.37 vs 9.02±0.30, for ANP and 7.41±0.11 vs 7.33±0.14 for BNP).

Plasma metabolite concentrations

During the baseline period, plasma concentrations of ANP were similar before and during LCD (68.5±9.1 vs 87.5±14.3 pg/ml, respectively). During the first 30 min of ANP infusion plasma ANP concentrations were similar before and during LCD (641.4±52.3 vs 646.1±31.6 pg/ml) and were also identical at the end of the 60 min infusion (658.8±42.6 vs 659.6±27.9 pg/ml).

Both before and during LCD, plasma NEFA and glycerol concentrations increased after 15 min of ANP infusion and reached a maximum value at t=30 min (Figure 2). Thirty minutes after stopping the infusion, plasma NEFA concentrations returned to basal values. The calculated AUC for the plasma NEFA values during the ANP infusion was different before and during LCD (P=0.004). The time-course of changes in plasma glycerol levels differed strikingly from that of NEFA levels. Before LCD, it was observed that the ANP-induced effect was weak and did not reach a significant level whatever the infusion period. During LCD, a significant rise in plasma glycerol levels was observed, the maximal level being reached 30 min after the beginning of the infusion, then a steady decrease in plasma glycerol concentrations was noticed until the end of infusion (Figure 2). Finally, at cessation of infusion the plasma concentration of glycerol was significantly lower than during the baseline period. However, the calculated AUC for the plasma glycerol values during the ANP infusion were different before and during LCD (P=0.02).

Other plasma metabolite concentrations are depicted in Table 2. No significant variations of plasma glucose level were observed in either period during the ANP infusion. A weak but non-significant increase in plasma insulin was observed during ANP infusion. The calculated AUC for insulin variations in the plasma during the ANP infusion did not show significant differences before or during LCD. ANP infusion also induced a slight increase in plasma noradrenaline levels; this effect was not significant whatever the infusion period and no difference was observed when compared to the values measured before or during LCD. Plasma lactate concentrations were decreased during the first 30 min of ANP and during the post-infusion period. The decrease was significantly higher during LCD than before.

Glycerol concentrations in SCAAT and ethanol changes in the dialysate

In basal conditions, the interstitial glycerol concentration in subcutanous abdominal adipose tissue was similar in the control probe and in the probe perfused with propranolol, before LCD (180±7 vs 169±16 µmol/l) or during the LCD (197±20 vs 182±26 µmol/l). In both dialysis probes and before LCD, infusion of ANP induced a weak increase in interstitial glycerol concentrations, the maximal effect being reached after 30 min (Figure 3). Thirty minutes after stopping the infusion, interstitial glycerol concentration returned to basal values. As found for plasma glycerol, it was observed that, during LCD, the glycerol increase was significantly higher than before LCD in the control probe as well as in the probe with propranolol during the first 30 min of ANP infusion. Then, a progressive decrease in glycerol concentrations was observed during the end of the infusion period. Finally during the post-infusion period, the interstitial levels of glycerol decreased to values significantly lower than those determined during the baseline period. The AUC calculated for the interstitial glycerol concentrations during the ANP infusion period was not different before or during LCD in either probe (P=0.15 for the control probe P=0.06 for the probe perfused with propranolol).

Adipose tissue blood flow was assessed using ethanol outflow/inflow ratios (ethanol concentration measured in the dialysate divided by the ethanol concentration measured in the perfusate´100) from the probes and the results are depicted in Figure 4. Before and during LCD, the ethanol outflow/inflow ratio was similar in the control probe (79±4 vs 81±2, respectively) and in the probe perfused with propranolol (84±3 vs 83±3, respectively). Before LCD, a significant decrease of the ethanol outflow/inflow ratio was observed starting from t=30 min of ANP infusion in the two probes. During LCD, the decrease in ethanol ration started from t=15 min in both probes. The AUC calculated for ANP-induced vasodilation were different when comparing the corresponding AUC before and during LCD in the control probe (P=0.03) and in the probe perfused with propranolol (P=0.05).

Cardiovascular responses to the ANP infusion

The LCD induced a significant decrease in heart rate, systolic and diastolic blood pressure (Table 1). ANP infusion induced a discrete increase in heart rate, this effect being unchanged before and during LCD. In both situations, systolic and diastolic blood pressure were unchanged by ANP infusion (not shown).

Discussion

The present investigation shows that NP induces a lipolytic effect in subcutaneous fat cells and ANP increases lipid mobilization in obese women. However, hypocaloric diet promoted both an increase of NP-induced lipolysis in fat cells and of ANP-mediated lipid mobilization.

In vitro, isolated adipocytes spontaneously release more glycerol during hypocaloric diets, as shown previously.^3,6 However, our data showed that fasting plasma glycerol and NEFA concentrations did not change during the hypocaloric diet. The calculated interstitial levels of glycerol in the subcutaneous adipose tissue were also found to be quite similar before and after 28 days of LCD. In agreement with values previously reported by Jansson et al¹⁸ in obese patients (with a different calibration method, ie no-net flux method), by us¹⁹ and others^18,20 using the present method, the interstitial levels of glycerol were about 1.7-fold higher than in the plasma. The values were also similar to those found in the venous blood draining the SCAAT.²¹

Animal studies have shown that fasting or energy restriction increase the lipolytic response promoted by catecholamines.^2,22 In humans, conflicting results have been reported. Spontaneous lipolysis is currently found to be enhanced by energy restriction but lipolytic responses to catecholamines have been shown to be increased,^2,3,6 unmodified or diminished in isolated subcutaneous fat cells from obese subjects during fasting²³ or after a 15-day LCD (3.3 MJ/day).²⁴ This is also in contrast with in vivo studies where an increased responsiveness of adipose tissue to catecholamine infusion has been demonstrated after short-term fasting.^4,5 A VLCD (2 MJ/day, for 4 weeks) led to an increased lipolytic response to exercise in obese subjects.²⁵ We have found that a VLCD in obese subjects improved the in situ lipolytic response to isoproterenol.¹¹ These discrepancies between in vivo, in vitro and in situ studies could reflect regional differences in the adaptative mechanisms in adipose tissue depending how few calories were in the diet and the sex of the patients. However, our results clearly show that, in women, the -adrenergic lipolytic pathway was largely increased and that, similarly, ANP- and BNP-induced lipolysis were also significantly increased during the LCD. NP represent a new pathway controlling human adipose tissue lipolysis; they operate via a cGMP-dependent mechanism which does not involve phosphodiesterase-3B inhibition or cAMP production.⁷ It was shown in the present study that bromo-cGMP induced a higher lipolytic effect during LCD than before. A previous study carried out in women submitted to a VLCD showed that the lipolytic effect of dibutyryl-cAMP (an analog of cAMP) was also increased.⁶ Several mechanisms have been proposed to explain the increased -adrenergic sensitivity of adipose tissue after VLCD including up-regulation of -AR and/or an increase in the efficiency of the coupling between -AR and adenylyl cyclase and an increased expression of hormone-sensitive lipase (HSL). Previous data have shown that the increase in HSL activity during VLCD is of the same order as the increase of basal lipolysis and that an increase of protein content paralleled the increase of total activity.⁶ In addition, unpublished data from our group have shown that the HSL is phosphorylated and activated by ANP stimulation in human fat cells. Taken together, these results suggest that the increase of both isoproterenol and NP lipolytic effects could be related to an increased expression and/or activity of the HSL induced by the LCD. The parallel improvement of both pathways suggests that the same pool of HSL is probably affected by cGMP and cAMP-dependent stimulation's.

A plasma NEFA concentration increase initiated by ANP infusion was observed before LCD and markedly raised during LCD (Figure 1). These results fit with the in vitro effects. Since the potency (pD2 values) of ANP is not changed during LCD, the enhancement of the lipid mobilizing action of ANP, based on the plasma NEFA levels, is probably a reflection of the increased HSL activity initiated by the LCD. It is not related to an increased sensitivity of the ANP lipolytic pathway per se. Concerning glycerol, before LCD, ANP infusion had a weak effect on plasma or interstitial glycerol concentration and LCD promoted a significant increase in plasma and interstitial glycerol levels. The in vivo responsiveness to ANP infusion, assessed through the rates of appearance in the circulation of NEFA and glycerol, exhibited different profiles, particularly during the LDC. The first period of glycerol increase (lasting 30 min) was followed by a rapid and sustained decrease of its level in the plasma and in the interstitial compartment too. The post-infusion plasma levels of glycerol were found to be lower than those measured before infusion. The change in local blood flow, known to influence the concentration of the interstitial metabolites, is an important variable in microdialysis studies.^26,27 The ANP-mediated lipolytic response was associated with vasodilatation, which seems to be enhanced during the diet (Figure 4). Thus, the effect on local blood flow could partly mask the action of ANP on lipolysis in SCAAT since an increment of glycerol drainage in the circulation was promoted simultaneously with the lipolytic action.

A theoretical explanation of the decrease of plasma glycerol levels can be proposed. During fasting or LCD, there may be a rapid removal of glycerol from the circulation and its re-utilization mainly by the liver and to a lesser extent by the kidney.^28,29 In these situations it has been shown that plasma glycerol is almost completely converted into glucose.²⁹ A higher proportion of glycerol and protein converted into glucose has been reported in obese subjects during fasting periods ²⁹ and the ability of obese individuals to spare protein breakdown during acute starvation has previously been reported.³⁰ To interpret the evolution of the plasma glycerol concentration, during and after ANP infusion, it can be proposed that when lipolysis is increased under ANP infusion, a rapid uptake of glycerol for neoglucogenesis occurring in the liver contributes to the decrease in its plasma level. This effect is stronger in obese subjects submitted to LCD containing a high proportion of protein. Likewise, this hypothesis is supported by the decrease in plasma lactate concentration also observed after ANP infusion and particularly during LCD. Lactate also converted into glucose mainly by the liver.

It cannot be excluded that a part of the lipid mobilizing effect promoted by ANP is related to sympathetic nervous system activation. In fact, at the dose used, the increase in plasma noradrenaline is very weak. The present study suggests that this effect is mainly related to the direct action of ANP on fat cells because propranolol (added at high concentrations to the microdialysis perfusate) did not modify the increase in interstitial glycerol promoted by ANP infusion (Figure 3). It has been shown that at these concentrations, propranolol had no effect on basal glycerol release^8,31 but could blunt the lipolytic effect of noradrenaline or adrenaline infused in the microdialysis probe.³²

The decrease of plasma insulin levels, described during LCD treatment,^33,34 could also be an important factor for the regulation of lipolytic responsiveness. The improvement of the lipolytic response to -AR stimulation and to ANP could be related to the modification of insulin status particularly in subjects with an insulin resistance syndrome. In fact, we have demonstrated that the lipolytic effect of ANP on human fat cells is independent of the level of insulin.⁷ The subjects included in the present study did not have abnormal fasting plasma insulin levels. ANP infusion moderately increased the plasma insulin level but the effect was only significant during the LCD. A significant increase of plasma insulin level has previously been noted when a five-fold higher dose of ANP than that used in the present study was infused.¹³ Although ANP binding sites are present in rat pancreatic islets, no direct insulinotropic effect of ANP has been clearly established in pancreatic islets. In fact, ANP was able to augment cGMP levels in isolated pancreatic islets without affecting insulin secretion at various glucose concentrations.³⁵ A weak effect, dependent on the secretagogues used has been reported when working on isolated perfused rat pancreas.³⁶ Therefore, although studies do not exist in humans, it is probable that in vivo elevation of plasma insulin levels in response to ANP are an indirect rather than a direct effect and represent a minor contribution to the lipid mobilizing responses reported in the present study.

Several epidemiological and clinical studies have shown an association of obesity and hypertension. It has recently been shown that short-term caloric restriction enhances ANP-induced diuresis and decreases arterial blood pressure.¹⁴ Previous studies in animals help to interpret these findings and our results. Rat and human adipose tissue contain high levels of NPr mesenger RNA^37,38 and large numbers of clearance receptor binding sites (NPr-C). A dramatic reduction of the expression of NPr-C has been reported during fasting.³⁹ This specific reduction of NPr-C gene expression in adipose tissue appeared to be accompanied by an increased biological activity of ANP. Our results are also consistent with the hypothesis that the reduction of NPr-C leads to reduced uptake of ANP allowing improved binding to biologically active NPr-A or NPr-B receptors in fat cells and higher efficiency on lipolysis during LCD.

Conclusion

In conclusion, hypocaloric diet concomitantly promotes increased -AR- and NP-induced lipolysis in fat cells and an increased lipid mobilizing effect of ANP in obese women. These changes seem to be associated to an improvement of the lipolytic pathway at the post-receptor level. Secondly, the results suggest that ANP induces an increase of glycerol metabolism in tissues other than adipose tissue during during LCD.

Acknowledgements

The authors wish to express their gratitude to M-T Canal for its contribution to the study. The study was financially supported by the Commission of the European Communities RDT Programme (FATLINK: dietary fat, body weight control, and links between obesity and cardiovascular disease), the Fondation pour la Recherche Médicale, the Direction Générale de la Coopération Internationale et du Développement (Programme d'Action Intégré Franco-Tchéque), and a grant from the Czech Republic (grant IGA 4674).

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Figure 1 Lipolytic effects of isoproterenol, ANP and BNP in abdominal subcutaneous isolated fat cells before and during LCD. Lipolysis expressed in percentage of maximal glycerol production induced by isoproterenol, before LCD (100%). Data are expressed as means±s.e.m. of experiments carried out on adipose tissue.

Figure 2 Changes in plasma non-esterified fatty acids (NEFA) and glycerol concentrations during the i.v. infusion of ANP in obese women before and during LCD. Values are expressed as percentage of pre-infusion values. Baseline values (µmol/l) were 633±52 and 668±89 for NEFA and 132±16 and 109±14 for glycerol before and during LCD, respectively. Data are expressed as means ±s.e.m. *P<0.05 when compared to per-infusion values.

Figure 3 Changes in interstitial glycerol in subcutaneous adipose tissue during the i.v. infusion of ANP in obese women before and during LCD. One probe (A) was perfused with Ringer alone and the other (B) with Ringer plus 100 µmol/l propranolol. Before LCD, baseline values (µmol/l) were 181±21 and 169±16 µmol/l in the control probe and in the probe with propranolol, respectively. During LCD, corresponding baseline values were 198±20 and 182±26 µmol/l respectively. Data are expressed as means±s.e.m. *P<0.05 when compared to preinfusion values.

Figure 4 Changes in ethanol ratio (ethanol dialysate concentration/ethanol perfusate level´100) in subcutaneous adipose tissue during the i.v. infusion of ANP in obese women before and during LCD. One probe (A) was perfused with Ringer alone and the other (B) with Ringer plus 100 µmol/l propranolol. After a calibration period, 2.5 µl min flow rate was maintained and dialysate fractions were collected at 15 min intervals. Data are expressed as means±s.e.m. *P<0.05 when compared to preinfusion values. +P<0.05 when compared to corresponding values measured before LCD.

Table 1 Morphological, cardiovascular and biological characteristics of obese women before and after 28 days of LCD

Table 2 Effect of ANP infusion on plasma insulin, glucose, lactate and noradrenaline concentrations before and during LCD