¹Institute of Normal Human Morphology-Anatomy, Faculty of Medicine, University of Ancona, Ancona, Italy

²Knoll Farmaceutici Spa, Muggiò Milano, Italy

Correspondence to: S Cinti, Institute of Normal Human Morphology, Faculty of Medicine, Via Tronto 10/A, 60020 Ancona, Italy. E-mail: cinti@popcsi.unian.it

OBJECTIVES: To evaluate morphological aspects and immunohistochemical markers of brown adipose tissue (BAT) activation following chronic treatment with sibutramine, a novel anti-obesity drug which increases thermogenesis and energy expenditure in mammals, and to establish whether chronic sibutramine treatment induces recruitment of BAT in white adipose tissue (WAT) depots.

DESIGN: Adult rats were administered 7 mg/kg/day oral sibutramine for 4 weeks. Body weight was monitored daily. At the end of the 4 weeks rats were perfused with buffered paraformaldehyde solution; interscapular BAT and retroperitoneal and epididymal WAT were carefully dissected for weight and volume measurements and processed for light microscopic studies and immunohistochemistry on paraffin-embedded sections. Where possible, semiquantitative morphometric analyses were performed.

RESULTS: Chronic sibutramine treatment determined a significant (about 8%) reduction in body weight. Compared with controls, sibutramine-treated rats showed: (1) interscapular brown adipocytes staining more intensely for uncoupling protein 1 (UCP1), the thermogenic mitochondrial protein; (2) a significantly larger number (about 45%) of brown adipocyte nuclei positive for peroxisome proliferator-activated receptor , the transcription factor driving UCP1 expression; (3) surprisingly, a significant reduction (about 30%) in BAT parenchymal noradrenergic nerve staining; and (4) a significant weight and volume reduction of WAT depots, but no significant signs of transdifferentiation of white into brown adipocytes.

CONCLUSION: This study confirms the ability of sibutramine to induce weight loss by selective and sustained activation of BAT in rodents without recruitment of brown fat in WAT depots. The parallel findings of a high level of brown adipocyte activation and low parenchymal noradrenergic innervation are discussed and a possible direct effect of sibutramine and/or its active metabolites on peripheral BAT sympathetic nerve terminals is hypothesized.

International Journal of Obesity (2002) 26, 354-360. DOI: 10.1038/sj/ijo/0801926

sibutramine; brown adipose tissue; thermogenesis; energy expenditure; sympathetic nerves; noradrenaline

Introduction

Sibutramine represents a new class of centrally acting pharmaceutical compounds referred to as serotonin-noradrenaline reuptake inhibitors, or SNRIs.^1,2 It is a tertiary amine and when administered to animals and humans, it is rapidly deaminated to form the secondary amine, metabolite 1 and the primary amine, metabolite 2, which are believed to exert the major pharmacological action of the drug.^2,3 Sibutramine has been shown to cause weight loss in humans⁴ and rats^5,6 by increasing satiety, thus reducing food intake,⁷ but its ability to induce a reduction in body weight is also linked to an increase in mammalian energy expenditure processes. Indeed, acute sibutramine treatment produced an increase in oxygen consumption, basal metabolic rate, and body temperature in rats.⁸ Using the labeled 2-deoxy-d-glucose method in anesthetized rats, sibutramine has been shown to cause a selective increase in glucose utilization in brown adipose tissue (BAT),⁹ the mammalian heat-producing tissue.¹⁰ Thus, sibutramine, mimicking the non-shivering thermogenic response to cold, selectively activates BAT; this metabolic effect is believed to result mainly from an increased sympathetic drive to brown adipocytes, likely to be due to raised central 5-HT levels.¹¹

In mammals, adipose depots are made up of two tissue types, white adipose tissue (WAT) and BAT, which are structurally interconnected and functionally inter-related in order to partition the energy contained in lipids between thermogenesis and other metabolic functions.¹² Brown adipocyte heat production is strongly regulated by noradrenaline released by sympathetic nerves via selective ₃-adrenoceptors stimulation.¹³ In small rodents, exposure to low temperatures^14,15,16 or treatment with ₃-adrenoceptor agonists^17,18 leads not only to the functional activation of BAT, but also to an increase in the number of brown adipocytes positive for the thermogenic mitochondrial uncoupling protein 1 (UCP1)^19,20,21 in WAT depots. Recent data suggest the possibility that fully differentiated white adipocytes can directly convert into heat-producing brown adipocytes following sympathetic stimulus.²² Based on the sustained effect of sibutramine on basal metabolic rate and thermogenesis,⁸ the aim of the present work was to evaluate morphological aspects and immunohistochemical indices of BAT functional activation in rats receiving chronic oral sibutramine. In addition, WAT depots were examined in order to detect possible aspects of conversion of white adipocytes into brown fat cells.

Methods

Animals and treatment

Male Sprague-Dawley rats aged 20 weeks and weighing 350-400 g were obtained from Harlan Italy (Milan, Italy). They were individually caged and kept at a constant temperature of 22°C, with free access to food and water. Lights were on 12 h daily (07:00-19:00). Animal care was in accordance with institutional guidelines and the experimental protocol was approved by the local Animal Care Ethical Committee.

Seven rats received 7 mg/kg/day oral (intragastric) sibutramine hydrochloride (kindly supplied by Knoll Pharmaceuticals, UK) for 4 weeks. Five rats were administered 0.9% (w/v) saline, ie the vehicle in which sibutramine was dissolved. Weight was monitored daily in each rat. At the end of treatment, animals were anesthetized and perfused intra-aortically with a 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.4). Interscapular BAT (IBAT) and retroperitoneal and periepididymal WAT were carefully dissected under a surgical microscope (Zeiss OPM 19, Carl Zeiss, Oberkochen, Germany) for weight and volume evaluations. They were then postfixed overnight in the same fixative by immersion, washed, dehydrated in ethanol and embedded in paraffin.

Immunohistochemistry

Immunoreactivity was assessed in 3 µm-thick serial sections according to the avidin-biotin-peroxidase (avidin-biotin complex, ABC) method: 0.3% H₂O₂ in methanol, 30 min at room temperature to block endogenous peroxidase; two washes in 0.015 M phosphate buffered saline (PBS; pH 7.4) of 15 min each; 1:75 normal rabbit serum (Pel-Freez, AR; UCP1 schedule), normal horse serum (Pel-Freez; PPAR schedule) or normal goat serum (Pel-Freez; neuropeptide schedule) in PBS, 20 min at room temperature to block aspecific sites. The primary antibodies were: polyclonal sheep anti-UCP1 (generously provided by Dr D Ricquier, Meudon, France); monoclonal mouse anti-peroxisome proliferator-activated receptor (PPAR; Santa Cruz, CA, USA); polyclonal rabbit anti-tyrosine hydroxylase (TH; Chemicon, CA, USA); and polyclonal rabbit anti-calcitonin gene-related peptide (CGRP; Amersham, UK). The sections with the primary antibodies were incubated overnight at 4°C; then two PBS washes of 15 min each; 1:200 IgG biotinylated serum rabbit anti-sheep (Vector Laboratories, CA, USA; UCP1 schedule), 1:200 IgG biotinylated serum horse anti-mouse (Vector; PPAR schedule); 1:200 IgG biotinylated serum goat anti-rabbit (Vector; neuropeptide schedule) in PBS, 30 min at room temperature; two PBS washes, 15 min each; ABC reagent (Vector), 60 min at room temperature; two PBS washes, 15 min each; 0.02% H₂O₂ and 0.075% diaminobenzidine (Sigma, Italy) in 0.05 M Tris buffer (pH 7.6), 5 min in a dark room; rinsing in distilled water; counterstaining with hematoxylin; mounting in Entellan. Negative controls were obtained in each instance by omitting the primary antibody and using preimmune serum instead of the primary antiserum. To test the specificity of the neuropeptide antisera, immunohistochemical reactions were performed in parallel with the positive controls: ovary for TH and CGRP and BAT of developing rats at embryonic day 18 for PPAR. As regards UCP1, no cross-reaction was observed in samples of liver, kidney, skeletal muscle and WAT, which are known to contain UCP2 and/or UCP3.²³ Thus, our antibody can be considered specific for UCP1.

Morphometry

For semiquantitative evaluations, PPAR and TH immunohistochemical reactions were performed in standardized conditions for all BAT samples. The number of PPAR-positive nuclei was evaluated on one representative section for each animal; for each section, 10 high-power fields were randomly selected and the percentage of positive nuclei vs total brown adipocyte nuclei was evaluated. The area occupied by noradrenergic nerves was evaluated on one representative section for each animal; for each section, 20 high-power fields were randomly selected and the area (µm²) occupied by the specific brownish precipitate, indicating both presence and amount of TH (area/field), was measured using a morphological imaging system (LUCIA, Version 4.5, Nikon Instruments, Italy).

Signs of BAT recruitment in visceral WAT were sought in 3 µm-thick serial sections obtained from the retroperitoneal and periepididymal depots. Five consecutive slices were collected every 200 µm at a total of 40-45 different levels per depot and per animal. Half of them were stained with hematoxylin and eosin to evidence the presence of multilocular cells among white adipocytes, whereas adjacent sections underwent immunohistochemistry for UCP1 (see above), the specific marker of brown adipocytes.

Statistical analysis

Results are presented as mean±s.e. Group means were compared by two-way ANOVA. Significance was defined at the 95% confidence level.

Results

Body weight

The mean body weight of control and sibutramine-treated rats over the experimental period is shown in Figure 1. In control animals body weight increased by about 4% over the 4 week treatment; this was less than their expected growth curve, and may have been due to the stressful situation of daily handling and drug administration. The sibutramine-treated rats immediately lost weight. Body weight plummeted during the first and, to a lesser extent, second week of treatment, in line with the strong initial effect on food intake previously observed in rats,⁸ and subsequently showed a variable tendency to recovery. At the end of the 4 weeks, treated rats exhibited a small but significant weight reduction of about 8% (419±5 vs 388±8 g, P<0.05).

Interscapular brown adipose tissue

There were no macroscopic (color, weight and volume) differences in the IBAT of treated compared with control rats. Serial sections stained with hematoxylin and eosin showed that in treated animals brown adipocytes were generally more eosinophilic and filled with small lipid droplets, suggesting a greater degree of functional activation compared with controls. Accordingly, UCP1 was more expressed in the brown adipocytes of treated (Figure 2B) than control rats (Figure 2A), and formed a Harlequin-like pattern (ie intensely stained brown adipocytes among less stained or completely negative ones), which in our experience is a valuable morphological index of sympathetic-dependent brown fat activation.¹² In control rats, UCP1 staining was less intense and more homogeneous.

PPAR is a transcription factor that drives the massive acquisition of the thermogenic phenotype in brown adipocytes and directly regulates UCP1 gene transcription in brown adipocytes.²⁴ PPAR immunohistochemistry showed numerous and intensely stained brown adipocyte nuclei in treated rats. Accordingly, PPAR-positive nuclei were significantly more numerous (by about 45%) in treated than in control rats (Figure 3A), also in line with the major expression of UCP1 we observed after chronic sibutramine administration.

The immunohistochemistry for TH, the marker of sympathetic noradrenergic nerves, showed in controls a large number of intensely stained noradrenergic nerves at the level of blood vessels and parenchyma (Figure 2C), as previously reported.²⁵ Surprisingly, TH-immunoreactive parenchymal nerves appeared to be less numerous in treated rats (Figure 2D) than in controls. Accordingly, the morphometric analysis of parenchymal innervation showed that the mean area/field occupied by noradrenergic nerves was significantly (about 30%) reduced in treated rats (Figure 3B).

By contrast, there were no differences between the two groups in the distribution of CGRP-containing, ie sensory, nerves²⁶ supplying IBAT blood vessels and parenchyma.²⁵

Visceral white adipose tissues

Compared with control animals, sibutramine-treated rats showed a significant (about 38%) reduction in the weight (Figure 4A) of the retroperitoneal adipose depot as well as in its volume (about 35%; Figure 4B). Similarly, they showed a reduction of about 16% in the weight (Figure 4B) and of about 33% in the volume (Figure 4D) of the epididymal adipose depot that were also significant. The different reactivity of the retroperitoneal and epididymal adipose depots after sibutramine administration may be ascribed to interdepot differences in vascularity, innervation and cell composition.

In periepididymal depots, hematoxylin-eosin-stained serial sections showed no sign of transdifferentiation of white into brown adipocytes, nor were multilocular cells detected in either treated or control specimens. White adipocytes exhibited the same unilocular appearance in both groups.

Only two treated rats showed some multilocular cells, grouped or scattered among white adipocytes, in retroperitoneal WAT. Of these multilocular cells, only 1 or 2% were positive for UCP1. Multilocular cells were not observed in the retroperitoneal WAT of the other five treated rats or in controls. No UCP1-positive unilocular cells were observed in these WAT depots in either treated or control rats.

No evident differences occurred between the two groups in vascular and parenchymal noradrenergic (TH) and sensory (CGRP) innervation.

Discussion

In mammals, BAT thermogenesis is induced not only by cold but also by diet.²⁷ Food intake activates BAT, which produces a certain amount of non-shivering thermogenesis that putatively counterbalances the energy surplus associated with feeding. Recent data in experimental animal models of obesity show a role for brown fat in the energy balance and the development of obesity.²⁸ Sibutramine, a novel anti-obesity drug, increases the feeling of satiety, thus reducing food intake,^5,6,7 and at the same time energy expenditure via selective activation of BAT thermogenesis in rodents.^8,9 Notably, sibutramine causes a significant increase in basal metabolic rate in lean subjects²⁹ and it limits the decline in energy expenditure associated with weight loss in obese subjects,³⁰ suggesting that an additional mechanism of increased energy expenditure may play a role in sibutramine-treated humans.

The present results show that sibutramine administration is able to induce significant and sustained activation of brown fat in vivo. After treatment for 4 weeks, we observed strong UCP1 staining of interscapular brown adipocytes matched by a high expression of PPAR, the transcription factor whose expression is regulated by noradrenaline and which directly drives UCP1 synthesis in brown fat cells.²⁴ Sibutramine is believed to activate BAT by increasing the central-dependent sympathetic drive to brown fat.¹¹ If this were the case, we should observe a denser noradrenergic innervation similar to that observed after cold exposure.¹⁰ By contrast, parenchymal noradrenergic nerves were not only less abundant in sibutramine-treated animals but also less densely stained considering the high amount of UCP1 expressed by adipocytes. In this context, it should be noted that sibutramine and its active metabolites are selective serotoninergic and noradrenergic reuptake inhibitors. A direct presynaptic action of sibutramine and/or its metabolites at peripheral sympathetic nerve terminals in BAT is an intriguing hypothesis. It could be speculated that the increased availability of noradrenaline in the synaptic cleft due to the inhibition of its presynaptic reuptake could result in adipocyte overstimulation (and thus in high expression of PPAR and UCP1 in brown adipocytes) and that this, in turn, would induce the overstimulation of presynaptic autoreceptors which downregulate noradrenaline synthesis in nerve terminals (resulting in low staining of BAT parenchymal noradrenergic nerves). Similar mechanisms widely occur in the synapses of the central nervous system following administration of other antidepressant drugs,³¹ and they have recently been demonstrated for sibutramine at hypothalamic level.³² Connoley et al³³ found that the ganglionic blocking agent, chlorisondamine, inhibited completely the thermogenic response to sibutramine and/or its active metabolites occurring within a few hours of their administration, but that it had no effect on the response to a thermogenic agonist that acts directly on ₃-adrenoceptors in BAT. Nevertheless, this was a contrived situation, and it cannot be ruled out that under normal conditions an additional peripheral mechanism of BAT sympathetic enhancement may strengthen the thermogenic properties of sibutramine. If this were true, the therapeutic and adverse effects of sibutramine should be reconsidered in the light of an additional peripheral action of the drug.

This study also aimed at establishing whether sibutramine is able to recruit brown adipocytes in WAT depots. We observed that administration of 7 mg/kg/day sibutramine over 4 weeks did not induce significant brown fat recruitment in WAT depots. In fact, only two of the seven treated animals showed weak signs of brown adipocyte recruitment in retroperitoneal WAT. Considering the high reactivity of BAT to numerous stimuli, this finding could be an independent phenomenon. Nevertheless, it cannot be excluded that an adequate protocol (higher dose and longer treatment) might induce significant BAT recruitment in WAT depots as a consequence of a central and/or peripheral action of sibutramine. This field clearly deserves further investigation, given its potential importance in the treatment of obesity in humans, where BAT is poorly represented but white fat has been shown to be able to convert into BAT.³⁴ In our experimental setting, however, sibutramine probably acts in the central nervous system on central pathways devoted to the activation of food intake-dependent brown fat thermogenesis, and its action indirectly confirms that the neural pathways and peripheral cues regulating BAT and WAT metabolism are different, and that their concerted activation is variably regulated. Cold provokes the sympathetic activation of BAT, but also an increased sympathetic drive to WAT³⁵ that is responsible for the recruitment of additional brown fat^14,15,36 and mobilization of fatty acids³⁷ in order to supply brown adipocytes with the substrate for heat production. By contrast, food intake activates BAT, but is associated with an increased WAT mass. Finally, there are conditions where one adipose tissue, but not the other, is activated by the sympathetic nervous system. For example, in response to starvation, the sympathetic drive to BAT is decreased, while the sympathetic drive on WAT is increased.³⁸ In this context, the significant reduction in weight and volume of retroperitoneal and epididymal WAT depots observed in our sibutramine-treated rats is probably related not only to reduced food intake, but also to an increased lipolysis, required for lipid substrates to be burned by the sustained BAT activation. Of note, activation of BAT results in compensatory hyperphagia.^39,40 Thus, it cannot be excluded that, especially in small mammals, where BAT is widely represented and importantly affects the energy balance, prolonged sibutramine-dependent BAT activation tends to counterbalance the behavioral action of sibutramine in enhancing satiety, and that the pharmacological effect of chronic sibutramine administration on the energy balance is significantly linked to its thermogenic properties.

Acknowledgements

The authors wish to thank Daniele Giannini and Rosanna Cangiano for their excellent technical assistance. This work was financed by Knoll Farmaceutici Spa Italia.

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Figure 1 Effect of 7 mg/kg/day oral sibutramine on rat body weight over 28 days of treatment. Bars represent the mean values±s.e. The difference in body weight between vehicle- and sibutramine-treated rats is significant (P<0.05) on day 4 and remains significant until the end of treatment.

Figure 2 Rat interscapular brown adipose tissue. Immunohistochemistry. In vehicle-treated rats (A) brown adipocyte staining for UCP1 was weak and homogeneous. After administration of 7 mg/kg/day sibutramine over 4 weeks (B), brown adipocytes stained intensely for UCP1, and exhibited a motley pattern (intensely stained cells among weakly stained or negative ones). In vehicle-treated rats (C), a rich parenchymal noradrenergic innervation was evident among brown adipocytes (arrows). After treatment with sibutramine (D), the density of parenchymal noradrenergic nerves appeared to be reduced, and nerve staining weaker. V, blood vessel. A, B, C, D 200´.

Figure 3 Rat interscapular brown adipose tissue. Morphometry. (A) The percentage of brown adipocytes showing PPAR-positive nuclei significantly increased after administration of 7 mg/kg/day sibutramine over 4 weeks. (B) The area/field occupied by parenchymal noradrenergic nerves significantly decreased after treatment with sibutramine. Bars represent the mean values±s.e. for each group. Significance was established at P<0.05.

Figure 4 Rat visceral white adipose tissues. The weight and volume of the retroperitoneal (A, B) and of the epididymal (C, D) adipose depots decreased significantly after administration of 7 mg/kg/day sibutramine over 4 weeks. Bars represent the mean values±s.e. for each group. Significance was established at P<0.05.