Transcription of the human uncoupling protein 3 gene is governed by a complex interplay between the promoter and intronic sequences.

AIMS/HYPOTHESIS: Uncoupling protein (UCP) 3 is an inner mitochondrial membrane transporter mainly produced in skeletal muscle in humans. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. In humans, UCP3 content is higher in fast-twitch glycolytic muscle than in slow-twitch oxidative muscle and is dysregulated in type 2 diabetes. Here, we studied the molecular mechanisms determining human UCP3 levels in skeletal muscle and their regulation by fasting in transgenic mice. METHODS: We produced a series of transgenic lines with constructs bearing different putative regulatory regions of the human UCP3 gene, including promoter and intron sequences. UCP3 mRNA and reporter gene expression and activity were measured in different skeletal muscles and tissues. RESULTS: The profile of expression and the response to fasting and thyroid hormone of human UCP3 mRNA in transgenic mice with 16 kb of the human UCP3 gene were similar to that of the endogenous human gene. Various parts of the UCP3 promoter did not confer expression in transgenic lines. Inclusion of intron 1 resulted in an expression profile in skeletal muscle that was identical to that of human UCP3 mRNA. Further dissection of intron 1 revealed that distinct regions were involved in skeletal muscle expression, distribution among fibre types and response to fasting. CONCLUSIONS/INTERPRETATION: The control of human UCP3 transcription in skeletal muscle is not solely conferred by the promoter, but depends on several cis-acting elements in intron 1, suggesting a complex interplay between the promoter and intronic sequences.

Functional beta3-adrenoceptor in the human heart.

Beta3-adrenoceptors are involved in metabolism, gut relaxation, and vascular vasodilation. However, their existence and role in the human heart have not been documented. We investigated the effects of several beta-adrenoceptor agonists and antagonists on the mechanical properties of ventricular endomyocardial biopsies. In the presence of nadolol, a beta1- and beta2-adrenoceptor antagonist, isoprenaline produced consistent negative inotropic effects. Similar negative inotropic effects also resulted from the action of beta3-adrenoceptor agonists with an order of potency: BRL 37344 > SR 58611 approximately CL 316243 > CGP 12177. The dose-response curve to BRL 37344-decreasing myocardial contractility was not modified by pretreatment with nadolol, but was shifted to the right by bupranolol, a nonselective beta-adrenoceptor antagonist. Beta3-adrenoceptor agonists also induced a reduction in the amplitude and an acceleration in the repolarization phase of the human action potential. Beta3-adrenoceptor transcripts were detected in human ventricle by a polymerase chain reaction assay. These results indicate that: (a) beta3-adrenoceptors are present and functional in the human heart; and (b) these receptors are responsible for the unexpected negative inotropic effects of catecholamines and may be involved in pathophysiological mechanisms leading to heart failure.

White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways.

OBJECTIVE: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes. METHODS: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a ß3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue. RESULTS: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response. CONCLUSIONS: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

Changes in plasma catecholamine and neuropeptide Y levels after sympathetic activation in dogs.

1. Plasma levels of noradrenaline (NA) and neuropeptide Y (NPY) were evaluated in two experimental models associated with an increase in sympathetic tone: conscious dogs which were subject to either sinoaortic denervation or acute administration of the alpha 2-adrenoceptor antagonist yohimbine. 2. Dogs that had undergone sinoaortic denervation exhibited a two fold increase in plasma NA without any change in NPY levels. 3. Yohimbine (0.05 mg kg-1 i.v. as a bolus) produced similar effects. A higher dose of yohimbine (0.5 mg kg-1 i.v.) increased both plasma NA (7 fold) and NPY (6.5 fold) levels. 4. The present results indicate that changes in plasma catecholamines and NPY are not always concomitant. They suggest that the simultaneous release of NA and NPY is only observed under in vivo conditions for a marked increase in sympathetic tone.

Release of neuropeptide Y and noradrenaline during afferent nerve stimulation.

The present study was carried out to investigate the possibility that noradrenaline (NA) and neuropeptide Y (NPY) are co-released after afferent vagal or saphenous stimulation (1, 5, 10 and 20 Hz) in chloralose-anaesthetized dogs. Electrical stimulation of the vagus elicited an increase in plasma NA levels for the 5, 10 and 20 (but not 1) Hz frequencies. Blood pressure only increased after a 20-Hz stimulation. In contrast, no change in plasma NPY levels was observed whatever the frequency of stimulation. Electrical stimulation of the saphenous nerve failed to change plasma NA and NPY levels. The present data suggest that (1) the release of NA varies according to the frequency of stimulation of nociceptive fibres, (2) NPY release does not seem to be involved in the pressor effect elicited by the stimulation of nociceptive-sensitive fibres, and (3) NPY and NA release are not necessarily linked.

Is neuropeptide Y co-released with catecholamines in experimental arterial hypertension following sinoaortic denervation?

The release of catecholamines and their coneurotransmitter neuropeptide Y (NPY) was investigated in conscious dogs with neurogenic arterial hypertension elicited by sinoaortic denervation. One month after denervation, an elevation of catecholamine levels (measured by HPLC) without elevation of NPY-like immunoreactivity (NPY-LI) levels in plasma (evaluated by RIA) has been found. This dissociation could be explained by 1) a transient release of NPY during the first weeks after surgery, 2) a depletion of neuronal NPY due to the permanent sympathetic stimulation, or 3) an insufficient increase in sympathetic tone. To test these hypotheses, we investigated the time courses of catecholamine and NPY-LI levels in arterial plasma during the first five weeks after sinoaortic denervation and responses to yohimbine (an alpha 2 antagonist which enhances transmitter release). Resting NPY-LI levels in plasma remained normal during the first five weeks after sinoaortic denervation. In normal dogs, a high dose of yohimbine (0.5 mg/kg i.v.) elevated both catecholamine (6-fold) and NPY-LI levels (1.5-fold), whereas a lower dose (0.05 mg/kg i.v.) induced a two fold elevation of catecholamine levels without changing NPY-LI concentrations. In sinoaortically denervated dogs, yohimbine elicited elevation of both catecholamines and NPY-LI whatever the dose used. Thus, neurogenic arterial hypertension in dogs seems to involve catecholamines but not NPY. Moreover, the present work suggests that a high level of sympathetic stimulation is required for a co-release of catecholamines and NPY.

Effects of clonidine, dihydralazine and splanchnic nerve stimulation on the release of neuropeptide Y, MET-enkephalin and catecholamines from dog adrenal medulla.

Various neuropeptides are costored together with catecholamines in the adrenal medulla. The concurrent release (evaluated by adrenal vein plasma levels) of these neuropeptides [neuropeptide Y (NPY), met-enkephaline (ME)] and catecholamines [adrenaline (A) and noradrenaline (NA)] from the adrenal gland was examined in chloralose-anesthetized dogs after intravenous administration of clonidine (10 micrograms/kg) and dihydralazine (1 mg/kg). These results were compared to those obtained after the stimulation of the right splanchnic nerve at 1, 5 and 10 Hz frequencies. The increment in the release of catecholamines and neuropeptides was evaluated for dihydralazine and splanchnic nerve stimulation. Dihydralazine (at its maximal effect) induced a significant preferential increase in catecholamines (expressed as mean (SEM): NA: 17.3 (5.4) fold, A: 13.1 (2.6) fold) and ME (16.0 (7.1) fold) versus basal values. However, the significant increase in NPY-LI was only 2.0 (0.4) times the baseline. Splanchnic nerve stimulation induced a frequency-dependent increase in catecholamines and neuropeptides. When the stimulation frequency was increased from 1 Hz to 5 Hz, NA and A levels increased 17.9 (4.3) and 14.0 (2.2) fold, respectively and ME levels 14.1 (3.0) fold. By contrast, NPY-LI was increased only 2.3 (0.3) fold under the same conditions. Increasing the stimulation frequency from 5 Hz to 10 Hz resulted in similar elevations of NA, ME, and NPY-LI adrenal plasma levels (about 4 times) whereas A only increased twice. Clonidine decreased catecholamine and ME adrenal plasma levels (the maximal percent decrease when compared with control values was about 75%) whereas NPY adrenal plasma levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A study of alpha 1- and alpha 2-adrenoceptor responsiveness in diabetic insipidus dogs.

The present study was performed to investigate the participation of circulating vasopressin in alpha-adrenoceptor responsiveness. Thus, we compared the pressor responses induced by selective alpha 1-or alpha 2-adrenoceptor stimulation in two groups of conscious dogs: a) normal animals and b) animals with surgically-induced diabetes insipidus. In addition, platelet alpha 2-adrenoceptors labelled with (3H)RX821002 were compared in the two groups. The pressor response to alpha 1-adrenoceptor stimulation [ie successive doses of noradrenaline (0.5, 1, 2, 4 micrograms/kg i.v.) after propranolol (1 mg/kg i.v.) plus yohimbine (0.5 mg/kg i.v.)] was significantly (P < 0.05) less pronounced in diabetic insipidus than in normal dogs. In contrast, the magnitude of the pressor effects of alpha 2-adrenoceptor stimulation [ie noradrenaline after propranolol plus prazosin (1 mg/kg i.v.)] was the same in the two groups of animals. Bmax and Kd values for (3H)RX821002 binding on platelets were similar in diabetic insipidus and normal dogs. This study shows that alpha 1- (but not alpha 2-) adrenoceptor responsiveness is decreased in diabetic insipidus suggesting the involvement of vasopressin in the mechanisms of the vascular alpha 1-adrenoceptor pressor response.

Effects of multiple reference points in spatial stimulus-response compatibility.

In two experiments, spatial stimulus-response compatibility effects in situations where the stimulus could appear in eight different locations were investigated. The locations were obtained as a result of orthogonal manipulation of hemispace, visual hemifield within hemispace, and relative position within hemifield. In the first experiment, only relative position within hemifield was relevant for selecting one of two responses (left or right). The results showed that both hemifield and relative position formed the basis of compatibility effects. In the second experiment, which was in most respects identical to the first, all spatial information was irrelevant. Only the geometrical shape of the stimulus determined the correct response. The results showed three S-R compatibility effects, based on hemispace, hemifield, and relative position. These results contradict earlier findings, and have implications for models of stimulus-response compatibility.

[Co-release of neuropeptides and catecholamines by adrenal medulla]. / Colibération des neuropeptides et des catécholamines par la médullosurrénale.

Different neuropeptides are costored together with catecholamines in the adrenal medulla. The concurrent release of these neuropeptides [neuropeptide Y (NPY), met-enkephalin (ME)] and catecholamines (adrenaline and noradrenaline) from the adrenal gland was examined in chloralose-anesthetized dogs after intravenous administration of dihydralazine (1 mg/kg) and insulin (0.3 U/kg). These results were compared to those obtained after the stimulation of the right splanchnic nerve at 1, 5 and 10 Hz frequencies. Baroreflex involvement or hypoglycemia induced a significant preferential increase in CA and ME versus basal values: around 16 fold for dihydralazine and 28 fold after insulin administration. In opposite, increase in NPY was only two times baseline. Splanchnic nerve stimulation induced a frequency-dependent increase in catecholamines and neuropeptides. At the lower frequencies (1 to 5 Hz), splanchnic nerve stimulation elicited a parallel increase in catecholamines and ME (13 to 17 fold basal values). By contrast, NPY increases 2 fold in the same conditions. At the higher frequencies (5 to 10 Hz), we observed a parallel (4 fold) increase in NA, ME and NPY adrenal plasma levels. In conclusion, the present data indicate that both adrenal ME and catecholamines (mainly NA) always exhibit a parallel fashion of corelease which is not the case for NPY and that different populations of chromaffin vesicles could be preferentially mobilized according to different physiological and pharmacological patterns.

[Differential regulation of the release of norepinephrine and neuropeptide Y]. / Régulation différentielle de la libération de noradrénaline et de neuropeptide Y.

The release of catecholamines and their co-neurotransmitter neuropeptide Y was investigated in conscious dogs with neurogenic arterial hypertension elicited by sinoaortic denervation. One month after denervation, an elevation of catecholamine levels (measured by HPLC) without elevation of neuropeptide Y levels in plasma (evaluated by RIA) has been found. This dissociation could be explained by a transient release of neuropeptide Y during the first weeks after surgery; a depletion of neuronal neuropeptide Y due to the permanent sympathetic stimulation; or an insufficient increase in sympathetic tone. To test these three hypotheses, we investigated the time courses of catecholamine and neuropeptide Y levels in arterial plasma during the first five weeks after sinoaortic denervation; and the responses to yohimbine (an alpha 2 antagonist which enhances transmitter release). Resting neuropeptide Y levels in plasma remained normal during the first five weeks after sinoaortic denervation. In normal dogs, a high dose of yohimbine (0.5 mg/kg i.v.) elevated both catecholamine (6-fold) and neuropeptide Y levels (1.5-fold), whereas a lower dose (0.05 mg/kg i.v.) induced a two fold elevation of catecholamine levels without changing neuropeptide Y concentrations. In sinoaortically denervated dogs, yohimbine elicited elevation of both catecholamines and neuropeptide Y whatever the dose used. Thus, neurogenic arterial hypertension in dogs seams to involve catecholamines but not neuropeptide Y. Moreover, the present work suggests that a high level of sympathetic stimulation is required for a co-release of catecholamines and neuropeptide Y.

[Neuropeptide Y, orthosympathetic nervous system, hypertension and alpha-2 adrenergic receptors]. / Neuropeptide Y, système nerveux orthosympathique, hypertension arterielle et récepteurs alpha 2-adrénergiques.

Neuropeptide Y (NPY) is coreleased with noradrenaline (NA) from sympathetic nerve endings. In vitro data suggest that NPY is coreleased during high stimulation frequencies. The present study investigates plasma levels of catecholamines and neuropeptide Y (NPY) during changes in sympathetic nervous activity in conscious dogs. Increase in sympathetic tone: arterial hypertension elicited by sinoaortic denervation induced an increase (X 2) in plasma noradrenaline (NA) but no change in NPY levels. High (0.5 mg/kg i.v.) but not low (0.05 mg/kg i.v.) doses of yohimbine rose plasma NPY concentrations. Decrease in sympathetic tone: clonidine (10 micrograms/kg i.v.) but not beta-blocking agents (propranolol or atenolol: 1 mg/kg i.v.) reduced plasma NPY levels. These results show that NPY is correleased in vivo from sympathetic nerve endings during marked and rapid increases in sympathetic tone. They suggest a lack of relationship between NA and NPY release. Alpha 2-adrenoceptors are involved in the presynaptic control of NPY release from sympathetic tone. Finally, some antihypertensive drugs (clonidine but not beta-blocking agents) are able to decrease plasma NPY levels.

[Is there any desensitization of presynaptic alpha 2-adrenergic receptors in hypertension? Experimental and clinical studies]. / Existe-t-il une désensibilisation des récepteurs alpha 2-adrénergiques présynaptiques dans l'hypertension artérielle? Etudes expérimentales et cliniques.

Several authors have discussed an alteration of adrenergic receptivity in arterial hypertension. De Champlain (Hypertension 1990; 8: S77-S85) suggested that postsynaptic alpha 1-adrenergic functions became dominant while beta-adrenergic functions are attenuated in arterial hypertension. However, the status of presynaptic alpha 2-adrenoceptors remains unknown. The present study investigates presynaptic alpha 2-adrenoceptors in hypertension through the measurement of plasma levels of noradrenaline after administration of yohimbine, an alpha 2-adrenoceptor antagonist, in essential hypertension. Yohimbine (0.2 mg/kg per os) induced a 73% increase of plasma levels of noradrenaline in hypertensive patients (n = 12) and a 178% one in normotensive subjects (n = 6, p < 0.05). A similar significant difference was found in experimental neurogenic hypertension observed in awake dogs 3 weeks after sinoaortic denervation: the increase in plasma concentrations of noradrenaline after yohimbine (0.5 mg/kg i.v.) was +279% in hypertensive versus +642% in normotensive dogs (p < 0.05). The results show that the magnitude of the yohimbine-induced sympathetic activation is lower in hypertensives than in normotensives. They suggest the existence of a presynaptic alpha 2-adrenoceptor desensitization in arterial hypertension. The abnormality of this presynaptic inhibitory mechanism can increase the sympathetic tone and help to develop and maintain arterial hypertension.

Resistance to high-fat-diet-induced obesity and sexual dimorphism in the metabolic responses of transgenic mice with moderate uncoupling protein 3 overexpression in glycolytic skeletal muscles.

AIMS/HYPOTHESIS: Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein expressed predominantly in glycolytic skeletal muscles. Its role in vivo remains poorly understood. The aim of the present work was to produce a mouse model with moderate overproduction and proper fibre-type distribution of UCP3. METHODS: Transgenic mice were created with a 16 kb region encompassing the human UCP3 gene. Mitochondrial uncoupling was investigated on permeabilised muscle fibres. Changes in body weight, adiposity and glucose or insulin tolerance were assessed in mice fed chow and high-fat diets. Indirect calorimetry was used to determine whole-body energy expenditure and substrate utilisation. RESULTS: Transgenic mice showed a twofold increase in UCP3 protein levels specifically in glycolytic muscles. Mitochondrial respiration revealed an increase of uncoupling in glycolytic but not in oxidative muscles. Transgenic mice gained less weight than wild-type littermates due to lower adipose tissue accretion when fed a high-fat diet. Animals showed a sexual dimorphism in metabolic responses. Female transgenic mice were more glucose-sensitive than wild-type animals, while male transgenic mice with high body weights had impaired glucose and insulin tolerance. Measurements of RQs in mice fed chow and high-fat diets suggested an impairment of metabolic flexibility in transgenic male mice. CONCLUSIONS/INTERPRETATION: Our data show that physiological overproduction of UCP3 in glycolytic muscles results in mitochondrial uncoupling, resistance to high-fat diet-induced obesity and sex specificity regarding insulin sensitivity and whole-body substrate utilisation.

Endotoxin exposure in asthmatic children and matched healthy controls: results of IPEADAM study.

UNLABELLED: Children spend increasing time indoors. Exposure to environmental factors may contribute to the development or exacerbation of the asthmatic phenotype. Inter-relationships between these factors might influence the manifestation of asthma. Endotoxin exposure has been shown to have pro-inflammatory and protective effects in different situations. We investigated the exposure to several indoor pollutants (endotoxin, Der p 1, damp, ETS, PM2.5) in asthmatic and healthy children. The children were recruited from two primary care centers according to their response to a validated questionnaire. Asthmatic children were matched for sex, age and sib-ship size with children living in asthma free households. Of 90 matched pairs, higher levels of endotoxin were found in the living room carpets, but not the bedroom carpet or mattresses of the asthma compared with the control homes (STATA analysis OR: 1.88 (1.11-3.18); P=0.018). Asthmatic children were also more likely to live as part of a single parent family, in a house where the parents self-reported the presence of damp, and where the living room had been redecorated in the 12 months prior to the sampling visits. This study suggests that endotoxin in urban homes is a risk factor for the development of asthma. Moreover, this study found that there were no statistically significant interactions between environmental factors. PRACTICAL IMPLICATIONS: This study has demonstrated that the home environments of English children (4-17) with asthma and without the disease do not differ greatly. With the exception of endotoxin, the parameters examined in this study, including house dust mite allergens, nitrogen dioxide, ETS and damp are unlikely to be related to the development of asthma. Avoidance of these pollutants may not be beneficial in preventing asthma in this age group.

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions.

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.

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.

The positive chronotropic effect induced by BRL 37344 and CGP 12177, two beta-3 adrenergic agonists, does not involve cardiac beta adrenoceptors but baroreflex mechanisms.

The presence of a beta-3 adrenoceptor (in addition to the classical beta-1 and beta-2 adrenoceptors) and its involvement in the control of heart rate was investigated in the dog. Experiments were carried out in conscious normal and sinoaortic denervated dogs (i.e. animals deprived of baroreceptor pathways). In normal dogs, infusion of isoproterenol, BRL 37344 (4-[-[(2-hydroxy-(3-chlorophenyl) ethyl)-amino]propyl]phenoxyacetate) (a beta-3 adrenergic agonist) or CGP 12177 (4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2- one) (a beta-1 beta-2 adrenergic antagonist reported to act as an agonist for the beta-3 adrenergic receptor) increased heart rate with an order of potency: BRL 37344 > isoproterenol >> CGP 12177. [125I]Cyanopindolol binding (2-2000 pM) was saturable and Scatchard analysis indicated the presence of an homogenous population of binding sites. KD was 12.8 +/- 18.5 pM and maximum binding was 94.2 +/- 12.5 fmol/mg of protein. Competition binding studies on dog heart membranes using 150 pM [125I] cyanopindolol indicated an order of potency (CGP 12177 > isoproterenol > BRL 37344) different from that observed in cardiovascular studies. Isoproterenol stimulated adenylate cyclase activity in heart membranes from normal dogs, whereas CGP 12177 and BRL 37344 were without any stimulating action. The positive chronotropic effects of isoproterenol, BRL 37344 and CGP 12177 were accompanied with a reduction in arterial blood pressure. In sinoaortic denervated animals, isoproterenol infusion provoked tachycardia and hypotension. BRL 37344 and CGP 12177 were without any significant effect on heart rate but induced a rapid and dramatic hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of beta3-adrenoceptors with low lipolytic action in human subcutaneous white adipocytes.

Beta3-Adrenoceptors are involved in the control of catecholamine-induced lipolysis in rodent adipose tissues. The expression and function of human beta3-adrenoceptors were investigated in subcutaneous white adipocytes of young healthy women. In these cells, beta3-adrenoceptor mRNAs represent 20% of total amount of beta-adrenoceptor transcripts and less than half of beta1-adrenoceptor transcripts. Among beta3-agonists known to stimulate beta3-adrenoceptor-mediated lipolysis in rodent fat cells, only CGP12177 was able to mediate such activity in human fat cells. In in vitro lipolysis experiments and in situ microdialysis studies, CGP12177 had a 4- to 5-times lower lipolytic efficacy than isoprenaline, a nonselective beta-adrenoceptor agonist. CGP12177-induced lipolysis was antagonized in vitro by bupranolol, a beta-adrenergic antagonist potent on rodent beta3-adrenoceptors but not by nadolol, a beta1- and beta2-adrenoceptor antagonist. The in vitro blockade of isoprenaline-stimulated lipolysis by nadolol showed that the agonist acted solely via beta1- and beta2-adrenoceptors. Isoprenaline and CGP12177 were able to increase the nutritive blood flow suggesting an interaction of these molecules with receptors present in adipose tissue vessels. In conclusion, beta3-adrenoceptors are expressed in human subcutaneous white adipocytes but do not significantly contribute to isoprenaline-induced lipolysis.