Beta 2-adrenoceptor mRNA Research Articles

Conjugated Linoleic Acid Induces Uncoupling Protein 1 in White Adipose Tissue of ob/ob Mice

Conjugated linoleic acid (CLA) reduces body weight and adipose mass in a variety of species. The mechanisms by which CLA depletes adipose mass are unclear, but two independent microarray analyses indicate that in white adipose tissue (WAT), uncoupling protein 1 (UCP1) was among genes most changed by CLA. The objective of this study was to determine whether CLA induces ectopic expression of UCP1 in WAT, which may contribute to increased energy expenditure and weight loss. Six-week old, male ob/ob mice were fed either a control diet (CON) or a diet supplemented with 1.5% mixed isomer CLA (CLA) for 4 weeks. A third group of mice (LEPTIN) was fed the control diet and received daily injections of recombinant leptin as a positive control for adipose depletion in ob/ob mice. CLA did not alter several mRNA markers of lipid oxidation in epididymal white adipose tissue (eWAT) , but significantly increased carnitine palmitoyltransferase-1b (CPT1b) and PPAR gamma coactivator-1alpha (PGC1alpha) expression. Notably, CLA increased both mRNA and protein expression of uncoupling protein-1 (UCP1). beta3-adrenoceptor mRNA and phosphorylated-p38 mitogen activated protein kinase (MAPK) protein levels were not affected by CLA, but were upregulated by LEPTIN. These data suggest the increased CPT1b, PGC1alpha, and UCP1, in WAT of CLA-fed mice may contribute to the depletion of adipose, and CLA does not appear to increase UCP1 through beta3-adrenergic signaling. Future studies will focus on understanding how CLA increases mitochondrial oxidation and energy dissipation in white adipose tissue.

Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2‐adrenoceptor mechanisms

Picomolar concentrations of the beta3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via beta2-adrenoceptors. Effects of BRL37344 and beta2-adrenoceptor agonists are compared. Mouse soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, [1-(14)C]-palmitate or [2-(14)C]-pyruvate, and BRL37344, beta2-adrenoceptor agonists and selective beta-adrenoceptor antagonists. Formation of 2-deoxy[1-(14)C]-glucose-6-phosphate or (14)CO2 was measured. 2-Deoxy[1-(14)C]-glucose uptake and beta-adrenoceptor mRNA were measured in C2C12 cells. 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33-54%. The effect of BRL37344 was prevented by 1 microM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 microM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 stimulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only beta2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via beta2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of β-adrenergic responses

Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.

Differential Effects of .BETA.2-Adrenoceptor Desensitization on the IgE-Dependent Release of Chemical Mediators from Cultured Human Mast Cells

In the present study, we examined the inhibitory effects of the beta2-adrenoceptor agonists isoproterenol, salbutamol, fenoterol, and clenbuterol, on the release of chemical mediators from cultured human mast cells after prolonged treatment with the agonists. Although preincubation of sensitized mast cells for 10 min with beta2-adrenoceptor agonists potently inhibited mediator release, prolongation of the preincubation period up to 240 min attenuated the inhibition. The attenuation of histamine release inhibition was potent when compared with that of prostaglandin D2 (PGD2) and cysteinyl leukotriene (LT) release inhibition. In contrast, forskolin inhibited mediator release and the inhibition increased gradually in proportion to the preincubation period. The reduced inhibition by the beta2-adrenoceptor agonists was compensated for by simultaneous treatment with cholera toxin. The beta2-adrenoceptor agonists elevated intracellular cAMP levels after 10-min incubation and the elevated levels were almost comparable to those after 240-min incubation. Forskolin elevated the intracellular cAMP levels more potently after incubation for 240 min than after 10 min. When mast cells were incubated for 3 d with the beta2-adrenoceptor agonists, similar attenuation of mediator release inhibition was observed. Elevation of intracellular cAMP levels was also attenuated, although beta2-adrenoceptor mRNA expression was potentiated. The present results collectively indicate that the attenuation of mediator release inhibition by beta2-adrenoceptor agonists under the present experimental conditions involves uncoupling between beta2-adrenoceptors and Gs proteins. Furthermore, the beta2-adrenoceptor desensitization causes differential attenuating effects on the inhibition of histamine, PGD2, and LT release, suggesting that downstream events involved in each inhibitory pathway have different sensitivity to receptor desensitization.

Characterization of lymphocyte beta 2-adrenoceptor signalling in patients with left ventricular volume overload disease.

1. Studies using animal experimental models have suggested that the beta2-adrenoceptor is uncoupled in association with alterations in the expression of G-protein-coupled receptor kinases (GRK) 2/3 in heart failure. However, the functional expression of the components of this pathway in human disease has not been fully elucidated yet. In the present study, we evaluated the possibility that the regulation of beta2-adrenoceptor signalling components in patients with left ventricular volume overload (VOL) depends on the severity of the overload. 2. We characterized the lymphocyte GRK 2-6, beta-arrestins 1 and 2, beta2-adrenoceptor expression at the mRNA and protein levels, as well as the activity of adenylyl cyclase, protein kinases (PK) A and PKC in patients with VOL using healthy blood donors as controls. 3. In the patient group, GRK2 mRNA was increased by 61% (P < 0.001), GRK3 was increased by 54% (P < 0.005), GRK5 was increased fivefold (P < 0.001) and the beta-arrestin 2 mRNA was increased by 40% (P < 0.05). These increases were paralleled with a sixfold increase in GRK2, a twofold increase in GRK3 and a 1.3-fold increase in GRK5 protein levels. These changes were associated with a significant decrease in beta2-adrenoceptor mRNA, the basal, catalytic and receptor-mediated activity of adenylyl cyclase and sensitization of the forskolin-stimulated activity towards augmented inhibition by guanylimidodiphosphate. In general, the increase in GRK2 and 5 mRNA exhibited a positive correlation with the gravity of the haemodynamic load, as determined by changes in left ventricular fractional shortening. 4. The results suggest that VOL induces an increase in the expression of lymphocyte beta2-adrenoceptor-specific GRK and beta-arrestin 2 in association with an attenuation in beta2-adrenoceptor levels. It can be speculated that the cardiac circulatory system adapts itself to altered haemodynamic functional demands partly by altering beta2-adrenoceptor signalling.

Lymphocyte glucocorticoid receptor mRNA correlates negatively to serum leptin in normal weight subjects.

The aim of the present study was to test the hypothesis that glucocorticoid receptor mRNA concentrations decreased with increasing fatness in normal subjects. Serum leptin concentrations, fat distribution parameters, lymphocyte glucocorticoid (GCR) mRNA, beta2-adrenoceptor mRNA and c-fos mRNA concentrations measured by RT-PCR-HPLC. Fifteen healthy non-obese young subjects with a mean body mass index (BMI) of 23. 5+/-0.3 (+/-s.e.m.) kg/m2. Lymphocyte GCR and beta2-adrenoceptor mRNA concentrations averaged 4.2+/-0.2 (+/-s.e.m. ) amol/microg total RNA and 1.4+/-0.1 amol/microg total RNA, respectively. There was a significant negative correlation between serum leptin and lymphocyte GCR mRNA (P<0.01). Serum leptin correlated positively with the waist-hip ratio (P<0.03), whereas lymphocyte GCR mRNA correlated negatively to the waist-hip ratio (P<0.04). Serum cortisol correlated with the weight of the subjects but not the waist-hip ratio or GCR mRNA. It is suggested that the decrease in lymphocyte GCR mRNA concentration with increasing serum leptin concentrations is a counterregulatory response to an increased body fat content. Further studies are warranted, especially to elucidate the relationship between GCR mRNA in lymphocytes and in fat cells and to clarify the mechanism of the decrease in GCR mRNA.

Interaction of amiodarone and triiodothyronine on the expression of beta-adrenoceptors in brown adipose tissue of rat.

1. This study was undertaken to evaluate in vivo the influence of amiodarone on the effects of triiodothyronine (T3) in brown adipose tissue (BAT) which are independent of thyroid hormone synthesis and of the conversion of thyroxine (T4) to T3. Thyroidectomized rats were given a replacement dose of T3 (0.5 mg kg(-1) p.o. daily for 3 days) with or without amiodarone (50 mg kg(-1) p.o. daily for 1 week). 2. As assessed by RT-PCR, treatment of thyroidectomized rats with T3 caused a 2 fold decrease in beta3-adrenoceptor (beta3-AR) mRNA levels and a 2 fold increase in beta1-AR mRNA levels. 3. Binding studies using [3H]-CGP 12177 as a ligand showed that treatment of thyoidectomized rats with T3 resulted in a 70% decrease in beta3-AR number and in an 80% increase in beta1-AR in BAT membranes. 4. T3-treatment abolished the increase in BAT adenylyl cyclase (AC) activity induced by CGP12177 in thyroidectomized rats. It also decreased the amount of Gi protein (ADP-ribosylation) by 30%. 5. At variance with the literature on the heart, amiodarone administration did not inhibit the positive effect of T3 on beta1-AR expression in BAT in thyroidectomized rats. However, it antagonized the effect of T3 on beta3-AR number, but not on AC activity or on Gi expression. 6. These results indicate that the effects of thyroid hormones on the responsiveness of BAT to catecholamines involves both receptor and post-receptor mechanisms, they also suggest that interaction between amiodarone and thyroid hormones is highly tissue-specific and depends on the beta-AR subtype.

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.

Functional and molecular evidence for beta 1-, beta 2- and beta 3- adrenoceptors in human colon.

1. Relaxation of carbachol pre-contracted human colonic muscle to (-)-isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the beta-adrenoceptor subtypes involved. beta 1-, beta 2- and beta 3-Adrenoceptor mRNAs were also measured in colonic muscle and mucosa. 2. (-)-isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50-7.39 +/- 0.12, and this response was inhibited by both propranolol (0.1 microM, pKB 8.55 +/- 0.12) and the selective beta 1-antagonist, CGP 20712A (0.1 microM, pKB 8.80 +/- 0.20), while the selective beta 2-antagonist, ICI 118551 (0.1 microM) failed to inhibit isoprenaline relaxation consistently. 3. (-)-Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70 +/- 0.17. Propranolol (0.1 microM). CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre-contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized. 4. beta 1- and beta 2-Adrenoceptor mRNAs were present in circular longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. beta 3-mRNA was also present in both muscle preparations but was not detected in human colonic mucosa. 5. In summary, beta 1-adrenoceptors are the predominant subtype mediating isoprenaline-induced relaxation of the thin longitudinal smooth muscle of human colon, while beta 1-receptors do not appear to be involved in these responses. However, beta 3-adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. beta 3-Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.

Effect of short- and long-acting beta 2-adrenoceptor agonists on pulmonary beta 2-adrenoceptor expression in human lung.

beta-Adrenoceptor agonists induce the down-regulation of beta 2-adrenoceptors and mRNA expression in animal lung. The down-regulation of beta 2-adrenoceptors may limit the therapeutic efficacy of beta 2-adrenoceptor-mediated bronchodilators in obstructive airways disease. We examined the effects of three selective beta 2-adrenoceptor agonists, salbutamol, salmeterol and formoterol on beta 2-adrenoceptor binding and mRNA levels in human lung in vitro. Human lung was obtained from cardiac transplantation donors. Peripheral lung was chopped and incubated with three selective beta 2-adrenoceptor agonist for 3 h or 24 h at three different concentrations (0.1, 1 and 10 microM). The affinity and density of beta 2-adrenoceptors was determined by [125I]iodocyanopindolol equilibrium binding in a lung membrane preparation in the presence of 0.1 microM CGP 20712 A (1-{2-[(3-carbamoyl-4-hydroxy)phenoxy] ethylamino}-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-propan-2-ol), a selective beta 1-adrenoceptor antagonist. Although treatment with salbutamol for 3 h did not change beta 2-adrenoceptor density, both salmeterol and formoterol reduced beta 2-adrenoceptor density, and exposure to each agonist for 24 h reduced beta 2-adrenoceptor density at all concentrations. Treatment with 10 microM salmeterol increased the equilibrium dissociation constant (Kd), and also shifted the competition curves of (-)-isoprenaline to the left, beta 2-Adrenoceptor mRNA, measured by Northern blot analysis using a human beta 2-adrenoceptor cDNA probe, was reduced after exposure to all beta 2-adrenoceptor agonists at 3 h. Our data provide evidence for down-regulation of beta 2-adrenoceptor protein and mRNA after selective beta 2-adrenoceptor agonist treatment in human lung.

Transgenic mice carrying the human beta 2-adrenergic receptor gene with its own promoter overexpress beta 2-adrenergic receptors in liver.

Up to now, transgenic mice models created to study the physiological impact of alterations in the human beta-adrenoceptor system have only focused on cardiac tissues and carried hybrid transgenes with strong cardiac promoters. We have developed a transgenic mouse strain (F28) carrying the human beta 2-adrenoceptor gene with its natural promoter region with the aim of producing a model that more closely reproduces the natural human beta 2-adrenoceptor tissue expression pattern. By means of northern blot analyses, using the appropriate probes, we have obtained evidence that (a) the human beta 2-adrenoceptor's structural gene is transcribed in several tissues of F28 mice; (b) the tissue distribution pattern of human beta 2-adrenoceptor mRNA in F28 mice completely differs from that of mouse beta 2-adrenoceptor mRNA; and (c) the tissue distribution pattern of mouse beta 2-adrenoceptor mRNA in F28 mice is very similar to that observed in their non-transgenic littermates. Like humans, F28 mice express human beta 2-adrenoceptor mRNA in liver, lung, brain, heart, and muscle. However, unlike humans, F28 mice do not accumulate human beta 2-adrenoceptor mRNA in kidney and spleen. By using [125I]iodocyanopindolol to label all beta-adrenoceptors and ICI 118,551 to discriminate between the binding to beta 2- and beta 1-adrenoceptors we have demonstrated that the beta 2-adrenoceptor binding activity increases over control values in F28 mouse tissues that accumulate transgenic mRNA. Accordingly, the number of beta 2-adrenoceptors increased slightly over the control values in muscle, heart, brain, and lung of F28 mice, while in liver these receptors were strongly overexpressed. We further showed that transgene beta 2-adrenoceptors couple to GTP-binding proteins, mediate beta-adrenoceptor agonist-stimulated adenylyl cyclase activation, and cause a strong enhancement of this response in liver membranes of F28 versus control mice. Finally, F28 mice show a phenotype of depressed ponderal development and perturbed hindquarter movements. This unique model should be useful to further investigate beta 2-adrenoceptor causal relationships with human pathologies.

Triiodothyronine regulates beta 3-adrenoceptor expression in 3T3-F442A differentiating adipocytes.

The effect of thyroid hormones on the beta 3-adrenoceptor expression was studied in the preadipose 3T3-F442A cell line. As assessed by molecular and pharmacological analyses, triiodothyronine addition to differentiating 3T3-F442A cells caused a 2.3-fold increase in beta 3-adrenoceptor mRNA levels, which was correlated with a parallel induction of beta 3-adrenoceptor number and of beta 3-adrenoceptor coupling to the adenylate cyclase system. Nuclear transcription experiments showed that triiodothyronine did not significantly alter the transcription rate of the beta 3-adrenoceptor gene. By contrast, the hormone increased by 36% the half-life of beta 3-adrenoceptor mRNA. Triiodothyronine exhibited a discrete effect on beta 3-adrenoceptor expression when added to mature 3T3-F442A adipocytes. This study indicates that thyroid hormones exert a differentiation-dependent and post-transcriptional regulation of beta 3-adrenoceptor expression in adipocytes.

Respective degree of expression of β1, β2‐ and β3‐adrenoceptors in human brown and white adipose tissues

1. The possible existence of a beta 3-adrenoceptor in human brown and white adipose tissues was investigated by mRNA expression and binding studies. 2. The relative amounts of beta 1-, beta 2- and beta 3-adrenoceptor mRNA, as determined by total RNA Northern blot analysis in newborn brown adipose tissue, were 28, 63 and 9% respectively of the total beta-adrenoceptor mRNA. 3. The beta 1/beta 2-adrenoceptors of human brown adipose tissue plasma membranes were characterized using [3H]-CGP 12177 as a ligand. Their Kd and Bmax values were 1.9 nM and 156 fmol mg-1 of membrane proteins, respectively. The beta 3-adrenoceptor was characterized by use of the new specific radioligand [3H]-SB 206606. The binding of this ligand was stereospecifically displaced by the active R,R- or the inactive S,S-enantiomer of BRL 37344 up to a concentration of about 10 microM. The Kd and Bmax values of the brown adipose tissue membrane beta 3-adrenoceptors were 87 nM and 167 fmol mg-1 of proteins, respectively. A low affinity [3H]-CGP 12177 binding site population was also detected in these membranes. 4. In human omental white adipose tissue, no beta 3-adrenoceptor mRNA could be detected in total RNA Northern blots and the beta 1-and beta 2-adrenoceptor mRNAs represented 9 and 91%, respectively of the total beta-adrenoceptor mRNA, and no specific binding of [3H]-SB 206606 could be measured.

Expression of beta3-adrenoceptor and stimulation of glucose transport by beta3-agonists in brown adipocyte primary culture.

Precursor cells of brown adipocytes were isolated from the interscapular brown fat of newborn rats and cultured on collagen-coated dishes. When confluent cells were treated with dexamethasone, mRNAs for muscle/adipocyte type of glucose transporter, hormone-sensitive lipase, and CCAAT/enhancer binding protein alpha were increased remarkably, confirming a predominant effect of dexamethasone on the terminal differentiation of the cultured cells. Effects of dexamethasone on the expression of three subtypes of beta-adrenoceptor were also examined. beta1- and beta2-adrenoceptor mRNAs remained constant regardless of dexamethasone-treatment, while beta3-adrenoceptor mRNA was present only in dexamethasone-treated differentiated cells. To assess the metabolic response mediated by beta3-adrenoceptor, glucose transport into the cells was estimated. Norepinephrine enhanced glucose transport in dexamethasone-treated differentiated cells, but not in undifferentiated cells. beta3-Adrenergic agonists mimicked completely the stimulatory effect of norepinephrine at concentrations lower by two orders of magnitude. These results suggest that the beta3-adrenoceptor is expressed during the course of differentiation in brown adipocytes and plays a significant role in the response of glucose transport to adrenergic stimulation.

Effect of glucocorticosteroid treatment on glucocorticoid receptor expression in human adipocytes.

The influence of glucocorticoid excess on expression of the glucocorticoid receptor (GR) and beta-adrenoceptor subtype was studied in isolated adipocytes obtained by sc fat biopsies from 17 healthy individuals. The biopsies were taken before and after 7 days of treatment with 25 mg prednisolone, given orally. GR and beta 1- and beta 2-adrenoceptor messenger ribonucleic acid (mRNA) levels were measured with a solution hybridization assay, and the number of beta 1- and beta 2-adrenoceptor binding sites was determined in radioligand binding experiments. GR protein levels were determined by Western blot analysis using an anti-GR antibody. Both GR protein and GR mRNA levels decreased significantly (P < 0.05) by about 50% after treatment, whereas no significant changes were demonstrated in either beta 1- or beta 2-adrenoceptor mRNA levels. The number of beta 2-adrenoceptor-binding sites, however, increased by 70% after treatment (P < 0.05), whereas the number of beta 1-adrenoceptor binding sites was not affected. The affinity of each receptor subtype was not significantly altered by steroid treatment. In conclusion, an in vivo glucocorticoid excess decreases GR mRNA as well as GR protein levels and selectively increases beta 2-adrenoceptor density in sc fat cells of healthy individuals. This indicates that glucocorticoids modulate human adipose metabolism by altering the expression of regulatory proteins at various mRNA and post-mRNA levels.

Expression of β3‐adrenoceptor mRNA in rat brain

The reverse transcription/polymerase chain reaction was used to demonstrate beta 3-adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum.

Molecular mechanisms underlying regional variations of catecholamine-induced lipolysis in rat adipocytes

The mechanisms underlying catecholamine control of lipolysis were studied in rat white adipocytes from epididymal, retroperitoneal, and subcutaneous fat depots. Sensitivity of subcutaneous adipocytes to selective beta 3-adrenoceptor agonists was lower than that of internal adipocytes. beta 3-Adrenoceptor mRNA levels were lower in subcutaneous adipocytes. A decreased beta 1/beta 2-adrenoceptor-mediated lipolysis was also observed in these adipocytes, and the number of beta 1/beta 2-adrenoceptors was lower than in the internal adipocytes. The number of alpha 2-adrenoceptors was higher in subcutaneous adipocytes without a marked difference in alpha 2-adrenoceptor-mediated antilipolysis between the depots. Subcutaneous adipocytes were also characterized by a lower maximal lipolytic response to drugs acting at different levels of the lipolytic cascade, suggesting differences at the postreceptor level. Lower hormone-sensitive lipase activity and mRNA levels in subcutaneous adipocytes were in agreement with the lipolysis data. These results suggest that the pattern of expression of the genes of the lipolytic pathway varies with the anatomic location of the fat depot.

Regulation of beta 3-adrenoceptor expression in white fat cells.

Catecholamines (adrenaline and noradrenaline) stimulate adipocyte lipolysis via three beta-adrenoceptor subtypes beta 1, beta 2 and beta 3. beta 3-adrenoceptor-mediated lipolysis varies according to the species. Rodent adipocytes exhibit the strongest response to beta 3 agonists while human fat cells are poorly responsive. The species-related differences can partly be explained by lower beta 3-adrenoceptor mRNA levels in human adipocytes compared to rat adipocytes. Poor coupling efficiency of human adipocyte beta 3-adrenoceptors cannot, however, be ruled out. The regulation of beta 3-adrenoceptor gene expression has been studied in the adipocytes of the murine cell line 3T3-F442A which express high levels of beta 3-adrenoceptors. Insulin and glucocorticoids down-regulate beta 3-adrenoceptor expression through a transcriptional effect. The impairment of beta 3-adrenoceptor gene expression in adipocytes of congenitally obese ob/ob mice could be related to the higher glucocorticoid plasma levels when compared to lean littermates although the direct involvement of glucocorticoids remains to be demonstrated. In the rat and the rabbit, the beta 3-adrenergic responsiveness varies according to the anatomical location of the fat pad. There is a marked decrease in beta 3-adrenergic response in rabbit retroperitoneal fat cells during ageing. cAMP modulates the beta 3-adrenergic response in white adipocytes at different levels. Human beta 3-adrenoceptor expression seems to be up-regulated by cAMP through an interaction with the promoter of the gene. It has been shown in cells transfected with cDNAs for the different beta-adrenoceptors that the beta 3-adrenoceptor is less prone to desensitization than the beta 1 and beta 2-subtypes. This observation is in agreement with the absence of desensitization of the beta 3-adrenoceptor response in isolated rat fat cells. Continuous infusion of noradrenaline for six days into hamsters does not lead to an alteration of the beta-adrenergic response. A similar treatment undertaken in the guinea pig, a species, unlike the hamster, devoid of beta 3-adrenoceptor responsiveness, promoted strong desensitization of the beta-adrenergic response through down-regulation of beta 1- and beta 2-adrenoceptors. From these observations, it could be hypothesized that the beta 3-adrenoceptor, that shows a low affinity for catecholamines, is the "emergency" beta-adrenoceptor which is essential under conditions of strong and sustained sympathetic nervous system activation.

Quantification of beta-adrenoceptors and beta-adrenoceptor kinase on protein and mRNA levels in heart failure.

The alterations of the beta-adrenoceptor adenylyl cyclase are reviewed. In failing myocardium, the down-regulation of beta 1-adrenoceptors is accompanied by a decrease in steady state mRNA levels, as studied with quantitative polymerase chain reactions in dilated and ischaemic cardiomyopathy. The density of beta 2-adrenoceptors and beta 2-adrenoceptor mRNA was unchanged in both pathological conditions compared to non-failing myocardium. In addition to down-regulation of beta 1-receptor protein and mRNA, an increased activity of the beta-adrenoceptor kinase (beta-ARK) was observed. Correspondingly, quantification of beta-ARK mRNA by a 5' and a middle portion PCR-product suggests that increased enzyme activity could be due to increased transcription. In summary, decreased steady state levels of beta 1-adrenoceptor mRNA could contribute to reduced beta-adrenoceptor density in failing myocardium. The known uncoupling of beta-adrenoceptors could be due to an increased mRNA expression and activity of beta-ARK.

Long-term exposure to norepinephrine results in down-regulation and reduced mRNA expression of pulmonary beta-adrenergic receptors in guinea pigs.

We studied the down-regulation of beta 2-adrenoceptors in guinea pig lung after chronic exposure to a beta-agonist. Guinea pigs were exposed to either norepinephrine (NE) or vehicle for 7 days. The density and affinity of beta-adrenoceptors were determined by Scatchard analysis of [125I]cyanopindolol binding in a lung membrane preparation and beta 1- and beta 2-adrenoceptor subtypes were studied in the presence of 0.1 microM ICI 118,551, a selective beta 2-adrenoceptor antagonist, or 0.1 microM CGP 20712 A, a selective beta 1-adrenoceptor antagonist, respectively. beta 2-Adrenoceptor mRNA was examined by Northern blot analysis. The tissue distribution of beta 2-adrenoceptors and of beta 2-adrenoceptor mRNA in lung after NE infusion were determined using receptor autoradiography and in situ hybridization, respectively. The functional significance of the decrease in beta 2-adrenoceptor was tested by measuring the relaxation response to a beta 2-agonist in isolated parenchymal strips. NE treatment resulted in decreases of 75 +/- 9%, 84 +/- 4%, and 66 +/- 9% of total beta-, beta 1-, and beta 2-adrenoceptors in the lung, compared with control animals. The administration of NE had only minimal effects on the dissociation constant (Kd) of the receptor for the radioligand. beta 2-Adrenoceptors mRNA was decreased by 46 +/- 13% after NE treatment. There was a correspondence between the distribution of beta 2-adrenoceptors and beta 2-adrenoceptor mRNA localization in both control and NE-treated guinea pigs. NE treatment reduced alveolar beta 2-adrenoceptors by 70 +/- 3% and its mRNA expression by 78 +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)