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Experimental Studies |
Obesity Unit, Fourth Department of Medicine (V.S., M.K., V.H.), Charles University, Prague 2, Czech Republic; Laboratoire des Adaptations de l’Organisme à l’Exercice Musculaire, Service d’Exploration de la Fonction Respiratoire et de Médecine du Sport, Hôpital Purpan, and INSERM Unité 317, Institut Louis Bugnard, Hôpital Rangueil, Faculté de Médecine (I.H., I.G., F.C., M.B., M.D., D.R., M.G., M.L., D.L.), Université Paul Sabatier, 31403 Toulouse Cédex 4, France; and Department of Cell and Molecular Biology (C.H.), Lund University, 22100 Lund, Sweden
Address all correspondence and requests for reprints to: Dr. Dominique Langin, Institut National de la Santé et de la Recherche Médicale U317, Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, F-31403 Toulouse Cédex 4, France. E-mail: langin{at}rangueil.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Obesity is a heterogeneous phenotype with the involvement of multiple genes and their interactions with nongenetic factors. Studies of monozygotic twins who have been reared apart revealed that the genetic contribution to the body-mass index (weight in kilograms divided by the square of the height in meters) was as high as 70% (9). Other genetic epidemiology approaches have shown that body fat mass and body mass index are characterized by a genetic contribution of 25–40% (10). Moreover, individuals, i.e. genotypes, respond differently to changes in environmental and lifestyle conditions. For example, genetic factors are likely to be involved in the tendency to gain weight during overfeeding periods (11). Similarly, an interaction between genotype and calorie restriction is possible.
The aim of the present study was to measure the variations in adipose tissue lipolysis during VLCD and to evaluate the genetic contribution to these changes. For that purpose, pairs of female obese identical twins were studied. The in vivo metabolic response to a ß-adrenergic stimulation was assessed by infusing isoproterenol. The lipolytic effect of catecholamines on isolated fat cells was studied with agents acting on adrenoceptors and at the postreceptor level. The level of hormone-sensitive lipase expression also was determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight pairs of female obese monozygotic twins were selected (40.1 ± 7.7 yr old). Zygoty of the subjects was established by history, physical appearance and identity of blood groups, red cell antigens, human leukocyte antigen system of A, B, and C loci, and apolipoprotein-B3 hypervariable region (data not shown). None of the patients was taking drugs before the study and during the diet period. The participants were sedentary. The patients received a 1600 kJ/day (383 kcal/day) liquid formula diet for 28 days (12). The formula included 36 g protein, 50 g carbohydrate, 4 g fat, and the recommended daily allowance of vitamins and minerals (National Research Council, 1989). The subjects stayed at the Obesity unit of Prague University Hospital during 1 week before the beginning of the diet (to perform entry examinations) and throughout the diet period. The examinations mentioned below were carried out during the first week of the hospital stay, i.e. before the diet, and at days 27 and 28 of VLCD. All subjects had given their informed consent before the study, and the investigation protocol was approved by the Ethical Committee of Prague University Hospital.
In vivo experimental protocol
Subjects were examined in the morning after an overnight fast. Height, body weight, and waist-to-hip ratio were measured. Percentage of fat was determined by hydrostatic weighing (13). Subsequent examinations were performed with subjects in a lying position. Heart rate and blood pressure were monitored continuously. Two iv catheters were inserted into antecubital veins, one in each arm. One catheter was used for isoproterenol infusion, the other one for blood sampling. After a 20-min resting period, a first blood sample was drawn for determination of basal values. Thereafter, the infusion of isoproterenol, diluted in saline solution, was started. Protection of the isoproterenol solution from light degradation was ensured by wrapping aluminum foil around the syringe, and the line was connected to the subject. Three sequential doses of isoproterenol were used: 0.02, 0.04, and 0.06 µg/kg lean body mass. Each dose was perfused for 10 min, and blood sampling was performed during the last minute of each 10-min interval. The infusion was interrupted if the subject’s heart rate exceeded 130 beats/min, which happened in two subjects at the highest rate of infusion.
Plasma glucose and free fatty acids were determined with a glucose oxidase technique (Biotrol, Paris, France) and an enzymatic procedure (Unipath, Dardilly, France), respectively. Plasma insulin concentrations were measured using a RIA kit (Pasteur Institute, Paris, France). Glycerol was analyzed using a sensitive radiometric method (14).
Fat cell isolation and measurement of lipolysis
An sc abdominal fat biopsy (300–400 mg) was performed under
local anesthesia (10–15 cm from the umbilic), and adipocytes were
isolated using collagenase (15). Digestion was performed for 40–50 min
at 37 C in a Krebs-Ringer bicarbonate buffer, pH7.4, containing 90 mg
glucose/100 mL and 4% BSA with 1 mg/mL collagenase. At the end of
digestion, the fat cell suspension was filtered and rinsed three times.
Fat cell volume and cell surface area were determined from 
200
adipocytes for each subject before and during VLCD (15, 16). The
quantity of glycerol released into the medium was measured using an
ultrasensitive bioluminescent technique to estimate adipocyte lipolysis
(17). All measurements were performed in triplicate. Drug potency was
estimated by calculating half-maximum effective concentration and pD2
value [-log(half-maximum effective concentration)].
Determination of hormone-sensitive lipase activity
The assay was performed essentially as previously described (18). Isolated adipocytes were rinsed twice with saline phosphate buffer. Cells were then homogenized at 4 C in 0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L dithioerythritol, and the protease inhibitors, leupeptin and antipain (both at 20 µg/mL). The samples were centrifuged at 100,000 x g for 45 min at 4 C. The fat-free infranatant was recovered for analysis of enzyme activity using 1(3)-mono[3H]oleoyl-2-oleylglycerol as substrate. All samples were incubated in triplicate on one occasion for 30 min at 37 C. A diacylglycerol analogue was used as substrate for enhancing assay activity, because hormone-sensitive lipase has 10-fold higher activity toward diacylglycerol than triacylglycerol. The diacylglycerol lipase activity is not dependent upon the phosphorylation state of the enzyme. Moreover, because this substrate has only one hydrolyzable ester bond at the 1(3)-position, neither the diacylglycerol analogue nor its hydrolysis products are substrates for monoacylglycerol lipase, which is abundant in adipose tissue. Furthermore, under conditions of the assay, i.e. pH 7.0 and no apoCII present, very low lipoprotein lipase activity was measured (19). One unit of enzyme activity is defined as 1 µmol fatty acid released per min at 37 C. Lipase activity was normalized to the protein concentration of the infranatant, which was measured according to Lowry, using BSA as standard (20).
Western blot analysis
Fat-depleted infranatants (50 µg protein) were subjected to SDS-PAGE (8%) and electroblotting to nitrocellulose membranes. Western blot analysis was performed using a chicken antibody directed against recombinant rat hormone-sensitive lipase (HSL) as primary antibody (21), a horseradish peroxidase conjugated antichicken IgG (Sigma-Aldrich, L’isle d’abeau, France) as secondary antibody, and Enhanced Chemiluminescence detection system (Amersham, Little Chalfont, Buckinghamshire, UK).
Statistical analysis
Results are expressed as mean ± SEM. Similarities within pairs of twins in response to VLCD were calculated according to Bouchard and co-workers (22). The effects of treatment (VLCD) and the interactions between genotypes and VLCD were assessed with a two-way ANOVA for repeated measures on one factor (time). Twins were considered nested within the pair, whereas the treatment effect was defined as fixed. The intraclass correlation coefficient for the changes of lipolysis rates in response to VLCD treatment was computed from the between-pairs and within-pairs means of square. This coefficient provides a quantitative estimate of the similarity within pairs in response to VLCD. An intraclass coefficient close to 1 indicates a perfect within-pair resemblance in response to VLCD, whereas a coefficient close to 0 would imply an absence of within-pair resemblance in response to the treatment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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presents the clinical data of eight pairs
of female monozygotic twins before and during VLCD. The mean body mass
index was reduced by more than 3 kg/m2 in response to VLCD.
Decreases of waist-to-hip ratio, fat mass, and fat cell volume also
were observed. Systolic and diastolic blood pressures were decreased
during VLCD. Regarding metabolic variables, only the level of blood
glucose was lower after 4 weeks of VLCD.
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). Isoproterenol induced a higher increase of plasma
free fatty acid and glycerol concentrations during vs.
before VLCD. VLCD had no effect on the increase of glucose (0.38
± 0.02 vs. 0.42 ± 0.02 mmol/L) and insulin (14.8
± 1.1 vs. 10.5 ± 0.7 mU/L) levels promoted by
isoproterenol at 0.06 µg/min·kg lean body mass.
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). The agents used were isoproterenol, adenosine
deaminase (which hydrolyzes endogenous adenosine and thereby relieves
its in vitro antilipolytic effect), isobutylmethylxanthine
(a phosphodiesterase inhibitor having adenosine A1-receptor antagonist
activity), and dibutyryl cAMP (a stable analogue of cAMP). These
compounds promoted higher rates of lipolysis during vs.
before VLCD. The effect was not caused by the agents per se
but to a 2-fold increase in basal lipolysis. If the agent-induced
lipolysis was expressed after subtraction of basal lipolysis, there was
no difference caused by VLCD. Before VLCD, a significant intrapair
resemblance was seen for basal and stimulated lipolyses when the
results were expressed on a per-cell basis and when they were corrected
for cell surface area. When considering VLCD-induced changes,
significant intrapair resemblance was found, per cell number and per
cell surface, for basal lipolysis and for lipolysis stimulated with
agents acting on cell surface receptors (Table 4).
Figure 1 illustrates the twin pair similarity for
VLCD-induced changes in basal and isoproterenol-stimulated
lipolyses.
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). Epinephrine, in the
presence of the 
2-adrenergic antagonist, RX821002, was
significantly more lipolytic between 1 and 100 nmol/L
(P < 0.05) during vs. before VLCD,
indicating an increased sensitivity of the ß-adrenergic component of
epinephrine effect. Accordingly, VLCD provoked a 3-fold increase in
isoproterenol potency (pD2 values from 8.5 ± 0.1 before VLCD to
8.9 ± 0.1 during VLCD, P < 0.01). There was no
significant intrapair resemblance for isoproterenol and epinephrine
potencies in response to VLCD. To investigate the
2-adrenergic antilipolytic pathway, increasing
concentrations of epinephrine were tested in the presence of adenosine
deaminase and the ß-adrenergic antagonist, propranolol. After
correction for the difference in basal lipolysis, no modification of
epinephrine sensitivity was observed during VLCD.
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). The
material available from the biopsies allowed the analysis of only ten
subjects. The intensity of the immunoreactive band corresponding to
human hormone-sensitive lipase was stronger during VLCD in nine of the
ten subjects. In one subject, there was no change in the intensity of
the band (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During calorie deprivation, triglycerides stored in adipose tissue are
hydrolyzed into glycerol and free fatty acids, which become, with
ketone bodies, the preferred fuels in the body. Under these conditions,
an increase of plasma concentrations of glycerol and free fatty acids
could be expected. In agreement with previous in vivo
studies (24, 25), no change in the plasma levels of the metabolites was
observed (Table 1
). The increase of glycerol and free fatty acid
concentrations induced by isoproterenol (Table 2) was higher during
vs. before VLCD. In agreement with these results, acute
exercise, a condition known to activate the sympathetic nervous system,
has been shown to induce a higher rise of plasma glycerol during
vs. before calorie restriction (24). However, the
VLCD-induced increase of plasma glycerol and free fatty acid
concentrations in response to isoproterenol may reflect more than the
changes in in vivo adipose tissue lipolytic rates because
the metabolite plasma levels are determined by both lipolysis and
utilization rates.
At the cellular level, a moderate increase of ß-adrenergic
sensitivity was observed (Fig. 2). The most prominent change was the
2-fold rise in basal lipolysis. During VLCD, an increase of basal
lipolysis rate expressed on a per-cell basis has been reported in early
studies (5, 6, 8). In some situations, basal and catecholamine-induced
lipolyses are positively correlated with fat cell size when lipolysis
rates are expressed per cell number (26, 27). To correct for fat cell
size variations, results were expressed per cell surface area (Table 3). The VLCD-induced increase of lipolysis was similar using either
mode of expression. These data show that the increase of lipolysis was
independent of change in fat cell size. In the rat, a fasting-induced
increase in basal lipolysis is associated with an increase of
hormone-sensitive lipase expression (28). A similar relationship is
found in man. Because hormone-sensitive lipase catalyzes the
rate-limiting step in adipose tissue lipolysis, the increase in basal
lipolysis during VLCD is likely to be caused by the increased
hormone-sensitive lipase expression (Fig. 3). We have shown recently
that long-term treatment of mouse adipocytes with cAMP produced a
decrease of hormone-sensitive lipase activity and mRNA levels (29).
Weight loss or energy restriction causes a decrease of basal
sympathetic activity, as measured by lower basal norepinephrine level
and appearance rate (30, 31). It remains to be seen whether a low
sympathetic tone promotes an increase of hormone-sensitive lipase
expression.
A major goal of the present study was to evaluate whether variations in
the lipolytic response of sc adipocytes of individuals subjected to
VLCD were related to the genotype of the subject. Before the treatment,
significant intrapair resemblance was seen for basal and stimulated
lipolysis rates expressed per cell number and per cell surface (Table 4). A similar observation has been reported by Bouchard and co-workers
(22) for basal lipolysis of male monozygotic twins. The extent of the
genetic contribution in basal lipolysis before VLCD is difficult to
appreciate because similarities within twin pairs may result from
shared genes as well as from shared family environment during childhood
and youth. In our study, most pairs of twins had been reared together
but were living apart at the time of the study. A significant
genotype-calorie restriction interaction effect was seen for basal
lipolysis and for lipolysis stimulated with agents acting on plasma
membrane receptors. No intrapair resemblance was found with dibutyryl
cAMP. These data suggest that the genetic factors controlling adipose
tissue lipolysis increase during VLCD interact with proximal components
of the lipolytic cascade. The precise level of interaction between
genetic factors and fat cell lipolysis, however, remains to be
determined.
In conclusion, energy restriction in moderately obese females resulted in an increased in vivo effect of the ß-adrenergic agonist isoproterenol on plasma free fatty acid and glycerol levels and a moderately increased in vitro sensitivity of the ß-adrenergic component of adipocyte lipolysis. The basal rates of lipolysis and hormone-sensitive lipase expression were increased during VLCD. The data also suggest that the genotype influences changes in sc adipose tissue lipolysis in response to VLCD.
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Acknowledgments |
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Footnotes |
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Received July 31, 1996.
Revised November 6, 1996.
Accepted November 22, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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lfors P, Nilsson NÖ,
Belfrage P. 1981 Hormone-sensitive lipase from adipose tissue of
rat. Methods Enzymol. 71:636–646.
2-adrenoceptors in obese women: effect of exercise and caloric
restriction. Clin Sci. 78:81–87.[Medline]
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) and during (
) VLCD. A, Lipolysis induced by epinephrine
in the presence of 10-5 mol/L 