Frédéric Preitner

β1/β2/β3-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet‐ and cold‐induced thermogenesis and of fasting‐induced lipolysis, through the β‐adrenoceptors (β1/β2/β3). To test this hypothesis, we generated β1/β2/β3‐adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold‐intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein‐1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β‐adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

Targeted gene disruption reveals a leptin-independent role for the mouse beta3-adrenoceptor in the regulation of body composition.

Targeted disruption of mouse beta3-adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the beta3-adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null (-/-) mice. In brown adipose tissue, beta3-adrenoceptor disruption induced a 66% decrease (P < 0.005) in beta1-adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in -/- mice, body fat accumulation was favored (+41%, P < 0.01), with a slight increase in food intake (+6%, NS). These effects were accentuated by high fat feeding: -/- mice showed increased total body fat (+56%, P < 0.025) and food intake (+12%, P < 0.01), and a decrease in the fat-free dry mass (-10%, P < 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in -/- and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed -/- mice suggests that the threshold of body fat content inducing leptin secretion is higher in -/- than in control mice. Taken together, these studies demonstrate that beta3-adrenoceptor disruption creates conditions which predispose to the development of obesity.

Effects of β-Adrenoceptor Subtype Stimulation on obese Gene Messenger Ribonucleic Acid and on Leptin Secretion in Mouse Brown Adipocytes Differentiated in Culture

The ob gene product is known to control food intake and energy expenditure. To determine whether thermogenic agents directly control ob gene expression, the effects ofβ -adrenoceptor agonists on the level of the ob gene messenger RNA (mRNA) and on leptin secretion have been studied in mouse brown adipocytes differentiated in culture. These cells highly expressed the β3-adrenoceptor, the uncoupling protein, and the ob gene mRNAs. The ob gene was expressed in mouse brown adipocytes earlier than in mouse white adipocytes under the same culture conditions and to a similar level. The β3-, β1-, andβ 2-adrenoceptor agonists BRL 37344, dobutamine, and terbutaline inhibited ob gene expression in mouse brown adipocytes differentiated in culture with EC50 values of 0.3, 1.0, and 85 nm, respectively. Leptin secretion by the cells under basal conditions was 78 ± 10 pg/μg DNA·4 h and was decreased by exposure to the β-adrenoceptor agonists. The ob gene mRNA half-life was 9.4 h and was decreased to 2.4 h by 1 nm BRL 373...

Metabolic response to various β-adrenoceptor agonists in β3-adrenoceptor knockout mice: Evidence for a new β-adrenergic receptor in brown adipose tissue

The β3‐adrenoceptor plays an important role in the adrenergic response of brown and white adipose tissues (BAT and WAT). In this study, in vitro metabolic responses to β‐adrenoceptor stimulation were compared in adipose tissues of β3‐adrenoceptor knockout and wild type mice. The measured parameters were BAT fragment oxygen uptake (MO2) and isolated white adipocyte lipolysis. In BAT of wild type mice (−)‐norepinephrine maximally stimulated MO2 4.1±0.8 fold. Similar maximal stimulations were obtained with β1‐,β2‐ or β3‐adrenoceptor selective agonists (dobutamine 5.1±0.3, terbutaline 5.3±0.3 and CL 316,243 4.8±0.9 fold, respectively); in BAT of β3‐adrenoceptor knockout mice, the β1‐ and β2‐responses were fully conserved. In BAT of wild type mice, the β1/β2‐antagonist and β3‐partial agonist CGP 12177 elicited a maximal MO2 response (4.7±0.4 fold). In β3‐adrenoceptor knockout BAT, this response was fully conserved despite an absence of response to CL 316,243. This unexpected result suggests that an atypical β‐adrenoceptor, distinct from the β1‐, β2‐ and β3‐subtypes and referred to as a putative β4‐adrenoceptor is present in BAT and that it can mediate in vitro a maximal MO2 stimulation. In isolated white adipocytes of wild type mice, (−)‐epinephrine maximally stimulated lipolysis 12.1±2.6 fold. Similar maximal stimulations were obtained with β1‐, β2‐ or β3‐adrenoceptor selective agonists (TO509 12±2, procaterol 11±3, CL 316,243 11±3 fold, respectively) or with CGP 12177 (7.1±1.5 fold). In isolated white adipocytes of β3‐adrenoceptor knockout mice, the lipolytic responses to (−)epinephrine, to the β1‐, β2‐, β3‐adrenoceptor selective agonists and to CGP 12177 were almost or totally depressed, whereas those to ACTH, forskolin and dibutyryl cyclic AMP were conserved.

Contribution of β‐adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wildtype and β3‐adrenoceptor knockout mice

The smooth muscle relaxant responses to the mixed β3‐, putative β4‐adrenoceptor agonist, (−)‐CGP 12177 in rat colon are partially resistant to blockade by the β3‐adrenoceptor antagonist SR59230A suggesting involvement of β3‐ and putative β4‐adrenoceptors. We now investigated the function of the putative β4‐adrenoceptor and other β‐adrenoceptor subtypes in the colon, oesophagus and ureter of wildtype (WT) and β3‐adrenoceptor knockout (β3KO) mice. (−)‐Noradrenaline and (−)‐adrenaline relaxed KCl (30 mM)‐precontracted colon mostly through β1‐and β3‐adrenoceptors to a similar extent and to a minor extent through β2‐adrenoceptors. In colon from β3KO mice, (−)‐noradrenaline was as potent as in WT mice but the effects were mediated entirely through β1‐adrenoceptors. (−)‐CGP 12177 relaxed colon from β3KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (−)‐noradrenaline and increase for (−)‐CGP 12177 indicate compensatory increases in β1‐ and putative β4‐adrenoceptor function in β3KO mice. In oesophagi precontracted with 1 μM carbachol, (−)‐noradrenaline caused relaxation mainly through β1‐and β3‐adrenoceptors. (−)‐CGP 12177 (2 μM) relaxed oesophagi from WT by 61.4±5.1% and β3KO by 67.3±10.1% of the (−)‐isoprenaline‐evoked relaxation, consistent with mediation through putative β4‐adrenoceptors. In ureter, (−)‐CGP 12177 (2 μM) reduced pacemaker activity by 31.1±2.3% in WT and 31.3±7.5% in β3KO, consistent with mediation through putative β4‐adrenoceptors. Relaxation of mouse colon and oesophagus by catecholamines are mediated through β1‐ and β3‐adrenoceptors in WT. The putative β4‐adrenoceptor, which presumably is an atypical state of the β1‐adrenoceptor, mediates the effects of (−)‐CGP 12177 in colon, oesophagus and ureter.