A.D. Strosberg

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Mapping of a functional autoimmune epitope on the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.

The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta 2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the beta 1-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the beta 1 receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the beta 1-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications.

Molecular characterization of the mouse beta 3-adrenergic receptor: relationship with the atypical receptor of adipocytes.

The gene encoding the murine beta 3‐adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2‐‐‐‐8A4 in mouse, and 8p11‐‐‐‐8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta‐adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1‐ and beta 2‐adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue.

Mediation of most atypical effects by species homologues of the β3‐adrenoceptor

1 A wide panel of compounds acting on β‐adrenoceptors active either in mammalian heart or in rodent digestive tract and adipose tissues, were investigated for their effects on Chinese hamster ovary cells transfected with the human or murine β3‐adrenoceptor gene. 2 The β3‐agonists, bucindolol, CGP 12177A and pindolol exhibited the highest binding affinities; BRL 37344, LY 79771, ICI 201651 and SR 58611A presented high potencies in stimulating adenylyl cyclase; bupranolol appeared as the most efficient β3‐antagonist. 3 This pharmacological analysis further established that the β3‐adrenoceptor is the prototype of the adipose tissue atypical β‐adrenoceptor, since these receptors share a number of pharmacological properties which differ strikingly from those of β1‐ and β2‐adrenoceptors: low affinities for conventional β‐adrenoceptor agonists and antagonists, high potencies for novel compounds active in adipose tissues, partial agonistic activites for several β1/β2‐antagonists. 4 Although the pharmacological profiles of the human and murine β3‐receptor were very similar, some quantitative or even qualitative differences were observed for particular compounds such as propranolol, which exhibited weak and partial agonistic effects at the human β3‐receptors and antagonistic effects at the murine β3‐receptors. These differences may result from key amino‐acid substitutions between the human and the murine β3‐receptor sequences, which may alter the binding site or signal processing. 5 Compounds active on atypical β‐sites of other tissues such as heart and digestive tract were also potent on the β3‐adrenoceptor expressed in Chinese hamster ovary cells, suggesting that this receptor mediates most of the atypical properties described in various tissues, and that differences in ligand effects may result from tissue‐related specificities.

Coupling of ETB endothelin receptor to mitogen-activated protein kinase stimulation and DNA synthesis in primary cultures of rat astrocytes

Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET‐1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ETA‐R), and IRL1620, an agonist selective for the ET receptor subtype B (ETB‐R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ETB‐R is the predominant subtype in these cells. Inhibition of forskolin‐stimulated cyclic AMP production was observed under ETB‐R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ETB‐R via Gi protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen‐activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ETB‐R, but through PTX‐insensitive G protein. IRL1620‐induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf‐1. This study reveals that the various effects of ET‐1 in astrocytes are mediated by the ETB‐R, which couples to multiple signaling pathways including the MAPK cascade.

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity‐purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty‐four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor‐bearing membranes. The M‐35b antibodies interacted only with native digitonin‐solubilized receptors, and not with denatured receptors. The M‐23c antibodies did not react with active digitonin‐solubilized receptors but recognized the denatured form. The M‐23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M‐35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.

β-adrenergic receptor-dependent and -independent stimulation of adenylate cyclase is impaired during severe sepsis in humans

Objectives: a) To investigate the functional consequences of sepsis on the β-adrenergic signal transduction in human circulating lymphocytes; b) to appreciate sepsis-associated catecholamine and cytokine release. Design: Experimental, comparative study. Setting: Research laboratory in a university hospital. Subjects: Healthy controls (n = 10); critically ill patients who were not septic (n = 7); septic patients with severe sepsis or septic shock (n = 11). Measurements and main results: Experiments were carried out using freshly isolated peripheral blood mononuclear cells (PBMC). We measured β-adrenergic receptor (βAR) number and affinity, and intracellular cAMP content at baseline and after the pharmacological stimulation of each component of the β -adrenergic complex: βAR with isoproterenol, Gs-protein with sodium fluoride (NaF), adenylate cyclase with forskolin. Catecholamine (adrenaline, noradrenaline) and cytokine (TNFα, IL-1α, IL-1β, IL-6) serum levels were measured. In both septic and non-septic patients we observed a similar 40 % down-regulation of βARs compared to controls, and a reduced basal and isoproterenol-stimulated cAMP accumulation (p < 0.05). The cAMP production elicited by NaF or forskolin was lower in septic patients than in the controls (p < 0.01). Forskolin-stimulated cAMP accumulation was significantly lower in septic patients than it was in non-septic ones (p < 0.001). Catecholamine serum concentrations were increased in the two patient groups without any significant difference. Elevated cytokine serum levels were detected in 45 % of the septic patients (versus 14 % of non-septic patients p < 0.05). Conclusions: Patients presenting with severe sepsis or septic shock have extended postreceptor defects of the β-adrenergic signal transduction. This finding suggests a heterologous desensitization of adenylate cyclase stimulation.

Are Several G Proteins Involved in the Different Effects of Endothelin-1 in Mouse Striatal Astrocytes?

Abstract: High‐affinity specific receptors of endothelin (ET‐1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I‐ET‐1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET‐1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide‐binding proteins.

Demonstration of muscarinic acetylcholine receptor-like immunoreactivity in the rat forebrain and upper brainstem

SummaryThe distribution of muscarinic acetylcholine receptor protein (mAChR) in the rat forebrain and upper brainstem was described by using a monoclonal antibody (M35) raised against mAChR purified from bovine forebrain homogenates. A method is investigated for light microscopic (LM) and electronmicroscopic (EM) immunocytochemical visualization of reactivity to mAChR-proteins. Putative cholinoceptive neurons including their dendrites were found immunoreactive in the cortical mantle, hippocampus, basal ganglia, amygdala, thalamus and several midbrain regions. In the neocortex, immunoprecipitate with M35 was mainly present in layer 5 pyramidal cells, some layer 3 pyramidal neurons and layer 2 stellate cells, all including their characteristic dendritic profiles of both basal and apical dendrites. In the hippocampus, a variety of pyramidal, granular and non-pyramidal celltypes were stained in various hippocampal cell layers, in the dentate hilus and in stratum oriens of cornu ammonis. Moreover, positively reacting cells occurred in central and lateral amygdala, all parts of the basal ganglia and ventral pallidum. The thalamus was very richly provided with labeled neurons in several nuclei but notably numerous in the ventrolateral, anteroventral and geniculate nuclei. In cortex and hippocampus also some staining of astrocytes occurred. Electron microscopic study of the intracellular distribution of M35 immunoreactivity in all cases showed dense precipitates in the soma cytoplasm in close association with the golgi apparatus, but conspicuous absence near the endoplasmic reticulum. Immunoprecipitate can be followed within the dendritic tree along the microtubular transport system, up to proximal and distal postsynaptic membrane positions, apposing non labeled presynaptic endings. Muscarinic receptor subtype recognition by M35 will be discussed by comparing M35 distribution with cholinergic innervation patterns, muscarinic receptor ligand binding studies and localization of muscarinic receptor subtype mRNAs.

Visualization of cholinoceptive neurons in the rat neocortex : colocalization of muscarinic and nicotinic acetylcholine receptors

The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in the rat neocortex became apparent. A striking similarity in distribution of the two cholinergic receptor types was found in the frontal/motor and parietal cortex. Accordingly, we observed a high degree of colocalization of muscarinic and nicotinic acetylcholine receptors within immunopositive cortical neurons. Approximately 90% of the cholinoceptive neurons expressed both types of receptors. The current data demonstrate that (i) the distribution of muscarinic and nicotinic cholinoceptive neurons in the neocortex is present in identical laminar patterns and represent the same type of cells, (ii) both classes of cholinergic receptors are highly colocalized within cholinoceptive neurons, which points at individual neurons as a likely site of interaction between muscarinic and nicotinic acetylcholine receptor-mediated processes.