INSERM U 465, Centre Biomédical des Cordeliers, Paris, France

Correspondence to: R Bazin, INSERM U 465, Centre Biomédical des Cordeliers, 15 rue de I^'Ecole de Médecine, 75270 Paris, Cedex 06, France. E-mail: rbazin@bhdc.jussieu.fr

BACKGROUND: In fa/fa Zucker rats, leptin receptor deficiency is responsible for both a deficit of energy expenditure and hyperphagia which lead to massive obesity and insulin resistance in adulthood. This obesity is also characterised by alterations of the -adrenergic signaling pathway.

OBJECTIVE: To determine whether alterations in -adrenergic pathway could occur at the onset of obesity when fa/fa rats are not yet hyperinsulinemic.

ANIMALS: Fourteen-day-old suckling fa/fa and Fa/fa littermates (from heterozygous lean (Fa/fa) female and homozygous obese (fa/fa) male mating).

MEASUREMENTS: Membranes were prepared from isolated adipocytes after collagenase treatment of inguinal adipose tissue. The response of adenylyl-cyclase activity to stimulation by isoprenaline, GTP-S or forskolin was studied. B_max and K_d of (₁+₂) and of ₃ adrenoceptors were measured using ³H-CGP saturation binding experiments. mRNA concentration of ₁- and ₃-AR was determined by semi-quantitative RT-PCR. G_s protein was quantified by Western blotting and Gi protein by ADP-ribosylation.

RESULTS: Despite an almost normal body weight, inguinal fat pad weight was increased two-fold by the expression of fa mutation. This increase was entirely accounted for by fat cell hypertrophy (´2.5 in volume). In fa/fa compared to Fa/fa pups, response of adenylyl cyclase to isoprenaline was decreased two-fold but responses to GTPS or forskolin were unchanged. Density of (₁+₂) and ₃-AR was not affected by the fa/fa genotype, as well as G_s and Gi concentration.

CONCLUSION: Response of inguinal fat cells to catecholamines was decreased without any quantitative modifications of the different elements of the adenylyl cyclase cascade. This suggests an alteration in the coupling between -AR and G proteins. Due to the important increase in fat cell volume we hypothesize that changes in the physical properties of plasma membranes and/or changes in cytoskeleton-extracellular-matrix interactions could disturb the -adrenergic pathway responsiveness. In addition to the excess of lipid storage, which occurs very early at the onset of obesity, the impairment of the responsiveness to catecholamines reported in this study might worsen the obesity syndrome.

International Journal of Obesity (2001) 25, 1592-1598

₁-adrenoceptors; ₃-adrenoceptors; Gs protein; Gi protein; adenylyl cyclase; lipolysis

Introduction

Zucker strain of rats bears a recessive mutation in the gene encoding for the leptin receptor, and thus, does not respond to this hormone mainly secreted by adipose tissues.¹ Consequently, these rats exhibit a large leptinemia due to the lack of hormonal feedback on hypothalamus.² This mutation is called 'fa' for fatty; fa/fa rats develop obese phenotype while Fa/fa and Fa/Fa rats remain lean.

In fa/fa rats leptin receptor deficiency is responsible for both a deficit of energy expenditure and hyperphagia,³ which lead to a massive obesity in adulthood. Nevertheless, early after birth, while hyperphagia is not yet established,⁴ fa/fa rats already have a deficit of energy expenditure attributable to a deficiency of brown adipose tissue (BAT) functioning.⁵ This is easily explained by the inability of hypothalamic centers controlling BAT activity to respond to leptin.⁶ Moreover, -adrenergic signaling pathway is also altered in BAT of suckling fa/fa rats.⁷.

Since BAT is an important effector of body weight regulation in rats, these changes would be sufficient to account for the further development of obesity. However in fa/fa rats, some functions of white adipose tissue (WAT) are also modified early in life.³ Enzymatic activities involved in the storage of lipid, such as fatty acid synthase⁸ and lipoprotein lipase,⁹ are increased and therefore the over-development of WAT is, at least partly, attributable to an increased lipid accretion. Fat content of the adipocyte also depends on lipid mobilization which is mainly triggered by the sympathetic activation of the cell. Therefore, in adipocytes of fa/fa pups, an alteration in the response to sympathetic activation could also contribute to the increase of fat stores.

To date, the transduction of noradrenergic message has never been investigated in adipocyte of preobese suckling Zucker rats. Thus the aim of the present study was to examine the -adrenergic signaling pathway in isolated adipocytes from inguinal adipose tissue of lean and preobese 14-day-old Zucker rats. We studied expression and density of (₁+₂)- and ₃-adrenoceptors (AR) which are quantitatively and qualitatively the main adrenoceptors in rat adipocytes¹⁰ and the activity and level of Gs protein. We measured the activity of adenylyl-cyclase (AC) which is mainly controlled by sympathetic nervous system through -ARs and Gs protein. Since ₃-AR also interact with Gi, at least in adipose cell line,¹¹ we also studied the level of Gi.

Material and methods

Animals

Animals were 14-day-old Fa/fa and fa/fa rats obtained in our laboratory by crossing between Fa/fa females and fa/fa males. Litter sizes were standardized at 10 pups. Mothers were fed ad libitum with standard commercial diet (UAR, France). Rats were kept at 22°C and submitted to a 12:12 h light/dark cycle. Animals were maintained in accordance with the guidelines established by the European Union (86/609). Pups were weighed and killed by decapitation at 14 days post-partum. Inguinal WAT pads were dissected and immersed in a plastic bottle containing 0.9% NaCl (room temperature) and weighed. Phenotype was determined by plotting inguinal fat pad weight vs body weight.¹² Tissues were immediately used for cell isolation or stored at -80°C for further RNA analysis.

White adipocyte isolation

Cells were isolated by collagenase from 600 to 800 mg tissue (ie inguinal pads from 10-12 lean animals or five to six preobese animals) according to Rodbell.¹³ One aliquot (50 µl) was taken for measurement of cell size by a microphotographic method followed by a computer analysis of the frequency distribution curves of cell diameters (Perfect Image, Numeris, France).

Crude membrane preparation

Cell suspension was diluted four times in ice-cold hypotonic buffer (0.1 mM KHCO₃, 0.25 mM MgCl₂, 0.2 mM EGTA, 0.2 mM AEBSF(4-[2-Aminoethyl]-benzenesulfonylfluoride) and 0.15 mM Tris-HCl, pH 7.5), vigorously shaken and then centrifuged at 700 g for 10 min at 4°C. Supernatant below the fat cake was collected and centrifuged at 160 000 g for 45 min at 4°C. The final pellet was suspended in a 1 mM EDTA, 0.2 mM AEBSF, 25 mM Tris-HCl pH 7.5 buffer to obtain a final protein concentration of 2-5 µg/µl. Membranes were either immediately used for determination of AC activity and binding studies or frozen at -80°C for further analysis. Eight additional litters were used for measurement of both the DNA concentration¹⁴ and the membrane protein content from the same adipocytes suspension.¹⁵

Determination of adenylyl-cyclase (AC) activity in crude membranes

AC activity (E.C.4.6.1.1) was assayed in 20-30 µg protein by measurement of [³²P] cAMP production from [³²P] ATP (30 Ci mmol^-1, Amersham France) in a medium containing 0.2 mM ATP, 1 mM cyclic AMP, 10 mM phosphocreatine, 0.5 units of creatine phosphokinase, 5 µM GTP, 5 mM MgCl₂, 0.2 mM EDTA and 50 mM Tris-HCl (pH 7.5). [³²P] cAMP was separated from [³²P] ATP by column chromatography on alumina as previously described.⁷ Isoprenaline (a non-specific -adrenergic agonist) was used to activate AC. Activity of the AC catalytic subunit was estimated in the presence of 2 µM Mn²⁺, 100 µM guanosine 5^'-[-thio]diphosphate (GDP[S]) and without Mg²⁺ (to inhibit Gs activity) in basal conditions, or after stimulation by 100 µM diterpene forskolin. GTPS (a non-hydrolyzable GTP analog which stimulates -adrenergic pathway directly at the level of Gs) was used to estimate Gs activity. In several experiments, stimulation of AC activity with isoprenaline was also measured after incubation (15 min) of the membranes with pertussis toxin (34 µg/mg protein) preactivated in the presence of 2 mM dithiotreitol and 1.1 mM ATP, 60 min at room temperature.

₁- and ₃-AR mRNA analysis

Semi-quantitative RT PCR was performed as previously described¹⁶ on total RNA extracted from inguinal pads with the CsCl method. Amplification products had expected sizes of 308, 265 and 236 base pairs for ₃-AR, ₁-AR and -actin respectively. They were separated on a 2% agarose gel and visualized by ethidium bromide staining. cDNA amplification of ₃-AR and -actin with 25 cycles was linear up to 150 and 100 ng RNA, respectively, and that of ₁-AR was linear up to 300 ng RNA with 33 cycles.

Determination of ₁+₂ and ₃-AR densities

For binding assays to membrane fractions, 50 µg of protein were incubated for 30 min at 37°C with (-)-³H-[CGP] 12177 (NEN, NET 1061), at concentrations ranging from 0.25 to 100 nM, in the presence of 10 mM MgCl₂, 1 mM ascorbic acid, and 50 mM Tris-HCI, pH 7.4. 100 µM GTP was added to shift all receptors into the low affinity conformation for agonists.¹⁷ Non-specific binding was determined in the presence of 100 µM (-)-isoprenaline. Separation of bound from free ligand was achieved by filtration over glass fiber disks (Whatman GF/C). All assays were run in duplicate. Data generated from saturation binding experiments were analyzed by GraphPad Prism program (San Diego, CA, USA).

Quantitative determination of G_s protein

Western blot analysis was performed on 20 µg of membrane protein with an anti-G_s protein antibody (NET 805, NEN France).⁷

Quantitative determination of Gi protein

ADP-ribosylation of Gi with pertussis toxin and [³²P] NAD was performed on 50 µg membrane protein as previously described.¹⁶ Briefly, membranes were incubated at 37°C for 30 min in a final volume of 200 µl of reaction mix containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.2 mM ATP, 1 mM GTP, 3 µM [³²P] NAD, 10 mM phosphoenolpyruvate, 0.5 units/ml pyruvate kinase, 0.5 units/ml myokinase, 10 mM thymidine, 0.1% Lubrol, and activated PTX. The reaction was stopped by centrifugation and the resulting pellet was washed with ice-cold buffer (20 mM Tris-HCl) and than solubilized in Laemmli buffer. The labeled protein were resolved on SDS-polyacrylamide gels (12% wt/vol) and electroblotted onto nitro-cellulose. Blots were exposed to X-ray film, and the resulting autoradiograms were quantified by densitometry.

Lipolysis

Cells (0.5-1´10⁶) were suspended in 2.5 ml of Krebs Ringer bicarbonate buffer (pH 7.4) containing 5 mM glucose and 3% bovine serum albumin, and incubated at 37°C in an atmosphere of O₂/CO₂ (95:5) in stoppered Nalgene vials for 60 min. Then an aliquot (1 ml) of infranatant incubation medium was removed, acidified (100 µl of 30% TCA); neutralized (80 µl of 10% KOH) and essayed in duplicate for glycerol content using an enzymatic method (Glycerol kit from Roche). Before each incubation, one aliquot of isolated adipocytes was taken for further determination of adipocyte triglycerides content (Sigma Diagnosis kit).

Statistical analysis

Results are expressed as means±s.e.m. Statistical significances of differences were determined by using Student-Fisher t-test or Tukey test after ANOVA (Graph Pad Prism).

Results

Body weight of 14-day-old rats was only slightly increased by fa gene expression (23.33±0.16 vs 21.94±0.15 g for 171 fa/fa rats vs 168 Fa/fa, P<0.001). Nevertheless, in fa/fa rats, inguinal WAT was 2.3-fold heavier than in Fa/fa rats (57.7±1.95 vs 25.5±1.95 g pad^-1, 171 fa/fa vs 168 Fa/fa, P<0.001). This increase in fat pad weight was entirely accounted for by adipocyte hypertrophy since the cell volume was multiplied by 2.5 (25.4±1.02 vs 18.8±0.46 µm for diameter, ie 8.6±0.5 vs 3.5±0.05 pl for volume, in fa/fa vs Fa/fa, P<0.01, four experiments). Thus, 2-week-old fa/fa pups are already clearly engaged on the way to obesity and can be qualified as preobese animals.

When biological parameters are measured in isolated membranes, the most common reference used for result expression is membrane protein mass. However, expression of fa mutation induced fat cell hypertrophy, thus the quantity of membranes could differ in adipocytes from fa/fa and Fa/fa rats. To solve this problem, the correspondence between membrane protein mass and cell number was determined in the two genotypes. Identical aliquots of cell suspension sampled from the same isolated cell preparations where used either for the determination of their DNA content or for the determination of their membrane protein content. The ratio µg membrane protein-µg DNA was not different between obese fa/fa rats and their lean Fa/fa littermates (1.7±0.17 and 1.9±0.25 for 7 and 10 adipocyte preparations in lean and preobese respectively). This indicates that the hypertrophy of cells is mainly due to an enlarged triglyceride droplet without any change in total membrane protein levels. Thus, in the present study, the variations of parameters between Fa/fa and fa/fa rats are similar either when expressed per mg protein or per cell.

Figure 1 shows that basal activity of AC was not different between lean (Fa/fa) and preobese (fa/fa) pups. After maximal stimulation by isoprenaline, AC activity in fa/fa pups was half of that was observed in their lean counterparts. This suggests that, in preobese rats, alterations of AC cascade could occur at receptor and/or post-receptor levels. To investigate whether the variations in agonist-stimulated AC activity arose from modulation of -AR number, we measured -AR subtype densities in membrane fractions from isolated adipocytes (Table 1). The ₃-AR displays a low affinity for (-)-³H-[CGP] 12177¹⁸ and can thus be distinguished from the classical ₁- and ₂-AR subtypes which have a high affinity for this compound. Saturation experiments were performed on crude membrane fractions using a wide range of concentrations (0.25-100 nM) of hydrophilic radioligand (-)-³H-[CGP] 12177. In both Fa/fa and fa/fa rats, adipocytes possessed an heterogeneous -AR population resolved in a Scatchard plot into a major ₃-AR component (90%) of low affinity for (-)-³H-[CGP] 12177 and a minor ₁ plus ₂-AR component (10%) of high affinity for the ligand. Results presented in Table 1 also show that pharmacological characteristics of (-)-³H-[CGP] 12177 binding sites in white adipocyte membranes from 14-day-old Zucker pups resemble those reported in adult Wistar rats¹⁰ or in 3T3F442A adipose cells.¹⁹ No difference could be detected between fa/fa and Fa/fa pups either in ₃-AR density or in ₁ plus ₂-AR densities. In good agreement with the binding studies, the mRNA concentrations of ₁- and ₃-AR, measured by a semiquantitative RT PCR, were not changed by the fa gene expression (Figure 2). The next elements of the transduction system linking catecholamines to AC are the G proteins. Determination of G_s concentration in membranes was performed by Western blot analysis. The two isoforms of 42 and 45 kDa usually present in adipocytes were detected (Figure 3). No differences between lean and preobese rats were observed either for the 42 kDa or for the 45 kDa isoforms (Figure 3). The response of AC activity to the stimulation of membranes by GTPS was not affected by expression of the fa mutation (Figure 1). In 3T3F442A adipocytes Gi protein is also activated after ₃-AR stimulation,¹¹ so the concentration of Gi protein in membranes was measured. As for G_s, Gi level was unaffected by the expression of the fa mutation (Figure 3).

Although the amount of Gs and Gi was not different between preobese and lean rats, they could have altered functions. In order to remove an hypothetical Gi-dependent inhibition of AC activity, we investigate the effect of isoprenaline on AC activity after pretreatment of membranes with pertussis toxin (which blockades the interaction between receptors and Gi) Results presented in Figure 4 show that treatment with pertussis toxin did not affect the activation of AC by isoprenaline either in fa/fa or in Fa/fa rats.

AC basal activity was not affected by expression of the fa mutation; however, alteration in the AC catalytic subunit could not be excluded. Direct activation of AC catalytic subunit was performed following forskolin activation, a compound which directly stimulates this enzyme and allows the measurement of its maximal activity. Results presented in Figure 1 show that no difference in maximal AC activity could be detected between fa/fa and Fa/fa pups.

To determine whether anomalies in the -adrenergic signaling pathway observed in membrane had functional consequences, we investigated lipolytic activity in intact cells. Results presented in Table 2 clearly show that, in basal conditions, there was no significant difference between lean and preobese rats, whereas activation with isoprenaline resulted in a six-fold increase of lipolytic activity in Fa/fa and only a two-fold increase in fa/fa.

Discussion

In fa/fa rats, the leptin receptor deficiency is responsible for the development of obesity. In addition to its central effect on food intake,² leptin regulates UCP1 gene expression in BAT²⁰ and is also able to activate sympathetic nerve activity in WAT.²¹ Thus, in fa/fa rats, brown fat dissipates less energy as heat and the white fat synthesizes and takes up more lipids.³ The present study shows that, in addition, white fat of suckling fa/fa rats is characterized by an impaired -adrenergic signaling pathway, leading to a reduced lipolytic activity which could worsen the obesity syndrome.

Our data show that the membrane protein-DNA ratio is identical in preparations of isolated adipocytes from lean and preobese rats. This indicates that the comparison of a parameter between lean and obese rats, when results are expressed per mg protein, is equivalent to the comparison of the same parameter expressed per cell. Thus, per adipose cell, the amounts of -ARs and G_s protein, as well as the activities of Gs and AC catalytic subunit, are not modified in fa/fa compared to Fa/fa rats. Surprisingly, despite the apparent absence of effect of the fa gene expression on the different elements of the -adrenergic signaling pathway, we observed a large decrease of AC activity after stimulation by the non-specific -agonist isoprenaline. Such a defect has been reported in WAT of adult fa/fa rats²² and this was explained by a fall in both the number of -AR(s) and the concentration of G_s protein. The present study clearly show that these two abnormalities were not detectable in adipocytes of suckling fa/fa rats. One of the major differences between adult and suckling fa/fa rats is the level of their plasma insulin concentration. During the suckling to weaning transition, fa/fa rats develop hyperinsulinemia.²³ Thus insulin, which has been shown to control negatively the expression of both G_s²⁴ and ₃-AR,^25,26 could be responsible for the alterations of AC cascade reported in the adipocytes of adult hyperinsulinemic fa/fa rats. By contrast, in normoinsulinemic fa/fa pups,⁸ the absence of any change in both the number of -ARs and the amount of G_s together with the normal response of AC activity to GTPs and forskolin strongly suggest that the decreased AC responsiveness to -AR stimulation was not attributable to insulin.

When AC activity was stimulated by isoprenaline, the blockade of a putative coupling between -ARs and Gi with pertussis toxin had no effect. Thus, in mature adipocytes of Zucker rats, and at variance with what was observed in 3T3F442A cell line,¹¹ ₃-AR could not functionally couple with Gi. These data, together with the absence of any difference in Gi concentration between the two genotypes, demonstrate that alteration of AC cascade could not be ascribe to coupling of -ARs to Gi. Taken together, our data suggest that a defective coupling activity between -ARs and Gs was present in the adipocytes of the fa/fa pups.

Several changes in morphological structure of adipocytes from fa/fa rats could be responsible for this alteration. Expression of the fa mutation induces modifications in the fatty acid composition of phospholipids and in the physical properties of the plasma membrane of adipocytes²⁷ as well as to a large extent in the cell surface. These changes could modify protein anchorage in membrane and/or regulation of coupling activity between G protein and the -ARs.^28,29 The adipocyte is the only cell type the size of which is strongly modified even in physiological conditions. For example, a two-fold increase in the diameter (which is very common for adipose cells) leads to a eight-fold increase in the cell volume. Increasing cell size modifies both the adhesion of cells to extracellular matrix and the relationships between extracellular and intracellular spaces. Variation of cell size is associated with changes in cytoskeleton-extracellular-matrix interactions (for review see Boudreau and Jones³⁰) and several studies have shown that some of the G proteins subunits are associated with other cellular structures, including cytoskeleton, as well as with plasma membranes.³¹ Taken together, these data suggest that adipocytes hypertrophy could disturb the distribution of G proteins within the cell and thus could play a role in the impairment of the -adrenergic signaling pathway in preobese rats.

In conclusion, as early as 14 days of age, at a moment when only a few endocrine disorders are present in fa/fa rats, an alteration of the -adrenergic signaling pathway is detectable in adipocytes of preobese rats. This study provides evidence for a decreased capacity of AC to respond to catecholamines, probably due to a decrease in the coupling between -ARs and Gs protein. Thus, during the development of obesity, adipocyte hypertrophy would be first attributable to an increased fat deposition; this hypertrophy in turn would disturb the control of lipolysis, leading to a progressive aggravation of the obesity syndrome. However, further studies are needed to elucidate the mechanisms involved in the adipocyte adaptation to hypertrophy.

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Figure 1 Adenylyl-cyclase activity (AC) after stimulation by isoprenaline (ISO), GTPS or forskolin (FSK) in adipocytes of 14-day-old Zucker rats. AC activity in membranes of isolated adipocytes from Fa/fa (filled bars) or fa/fa (open bars) pups was determined in the absence (25 and 29 experiments for Fa/fa and fa/fa respectively) or in the presence of 10^-4 M ISO (six and seven experiments for Fa/fa and fa/fa respectively), 10^-4 M GTPS ISO (nine and 14 experiments for Fa/fa and fa/fa respectively) or 10^-4 M FSK (12 and 10 experiments for Fa/fa and fa/fa respectively). Values for fa/fa rats are presented as percentage of values for Fa/fa rats (normalized to 100%). Experimental values before these transformations were: 13.5±1.8 and 14±1.7, 87±11 and 36±4, 25±3 and 31±4, 89±21 and 112±20 pmol of cAMP produced per min per mg protein for Fa/fa and fa/fa rats, in basal conditions, with ISO, GTPS and FSK, respectively. All the results are presented as means±s.e.m.; **P<0.01 fa/fa vs Fa/fa.

Figure 2 ₁-Adrenoceptor (₁-AR) and ₃-adrenoceptor (₃-AR) expression in adipocytes of 14-day-old Zucker rats. ₁ and ₃-AR mRNA concentrations in adipocytes of Fa/fa (filled bars) or fa/fa (open bars) rats pups were determined by RT-PCR (see materials and methods). -AR mRNA levels were normalized to -actin mRNA content and are expressed in percentage of -actin signal. Results are means of 11, 15, 11 and 16 experiments for, respectively, Fa/fa ₁-AR, fa/fa ₁-AR, Fa/fa ₃-AR and fa/fa ₃-AR. Differences between fa/fa and Fa/fa for the same -AR, as well as differences between -AR subtypes for the same genotype were not statistically significant. One typical RT-PCR experiment is presented above for each -AR (L, Fa/fa and Ob, fa/fa; bp, sizes of mRNAs in base pairs).

Figure 3 Quantitative determination of Gi and of G_s isoforms in adipocyte membranes of 14-day-old Zucker rats. Gi quantification was performed by ADP ribosylation with pertussis toxin and ³²P-[NAD], and GS quantification by Western blotting. Results are presented as mean±s.e.m. of five experiments, they are expressed in arbitrary units resulting from densitometric scanning of the X-ray films. Differences between fa/fa (open bars) and Fa/fa (filled bars) were not statistically significant. Typical experiments are presented in inserts (L, Fa/fa and Ob, fa/fa; arrows indicate molecular weight in kDa).

Figure 4 Effect of pertussis toxin (PTX) on isoprenaline (Iso)-stimulated adenylyl-cyclase (AC) activity. AC activity was determined in adipocyte membranes from Fa/fa and fa/fa pups, in basal conditions and in the presence of 10^-4 M Iso, with or without pretreatment with activated PTX (34 µg/mg membrane proteins). Results are presented as mean±s.e.m. of three and four membrane preparations for Fa/fa and fa/fa, respectively; NS, difference between Iso and Iso+PTX not statistically significant.

Table 1 Characteristics of (-)-³H-[CGP] 12177 binding sites in white adipocyte membranes from 14-day-old Zucker pups

Table 2 Lipolysis in isolated white adipocytes from 14-day-old Zucker pups