Differential adrenergic regulation of the gene expression of the beta-adrenoceptor subtypes beta1, beta2 and beta3 in brown adipocytes.
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ABSTRACT:
SUBMITTER: Bengtsson T
PROVIDER: S-EPMC1220999 | biostudies-other | 2000 May
REPOSITORIES: biostudies-other
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Project description:Background and purposeThere are two important properties of receptor-ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor-ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 beta-adrenoceptor agonists at the three human beta-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.Experimental approachStable clonal CHO-K1 cell lines, transfected with either the human beta(1), beta(2) or beta(3)-adrenoceptor, were used, and whole-cell [(3)H]-CGP 12177 radioligand binding and [(3)H]-cAMP accumulation were measured.Key resultsSeveral agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the beta(2)-adrenoceptor over the beta(1) or beta(3)), while others (e.g. isoprenaline) had little affinity-selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for beta(1); clenbuterol, AZ 40140d, salbutamol for beta(2)) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the beta(1)- and beta(3)-adrenoceptors.Conclusions and implicationsThere are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.
| S-EPMC2936015 | biostudies-literature
Project description:Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.
| S-EPMC2196435 | biostudies-literature
Project description:Objective3',5'-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. β-adrenergic receptors (β-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes.MethodsWe measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual β-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with β-AR stimulated lipolysis and analysed the expression of β-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots.ResultsUsing pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control β1-AR-induced cAMP. Unexpectedly, β3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by β1-AR associated with PDE3 and PDE4, while another pool is centred around β3-AR and is much less controlled by these PDEs. Functionally, lower control of β3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by β1-AR and is under tight control of PDE3 and PDE4.ConclusionsWe have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of β1-and β3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.
| S-EPMC7191645 | biostudies-literature
Project description:Boosting energy expenditure by harnessing the activity of brown adipocytes is a promising strategy for combatting the global epidemic of obesity. Many studies have revealed that the β3-adrenergic receptor agonist is a potent activator of brown adipocytes, even in humans, and PKA and p38 MAPK have been demonstrated for regulating the transcription of a wide range of critical genes such as Ucp1. We previously revealed that the PKA-ASK1-p38 axis is activated in immature brown adipocytes and contributes to functional maturation. However, the downstream mechanisms of PKA that initiate the p38 MAPK cascade are still mostly unknown in mature brown adipocytes. Here, we identified the ASK family as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. Additionally, PKA also activates ASK2, which contributes to MAPK regulation. These lines of evidence provide new details for tailoring a βAR-dependent brown adipocyte activation strategy.
| S-EPMC7591029 | biostudies-literature
Project description:Lipid species patterns are conserved within cells to maintain physicochemical properties of membranes and cellular functions. We present the lipidome, including sterols, glycerolipids (GLs), glycerophospholipids (GPLs), and sphingolipids (SLs), of primary ex vivo differentiated (I) white, (II) brite, and (III) brown adipocytes derived from primary preadipocytes isolated from (I) epididymal white, (II) inguinal white, and (III) intrascapular brown adipose tissue. Quantitative lipidomics revealed significantly decreased fractions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), with longer (C > 36) and more polyunsaturated species, as well as lower levels of cardiolipin (CL) in white than in brite and brown adipocytes. Together, the brite and brown lipidome was comparable and indicates differences in membrane lipid packing density compared with white adipocytes. Changes in ceramide species profile could be related to the degree of browning. Beta-adrenergic stimulation of brown adipocytes led to generation of saturated lyso-PC (LPC) increasing uncoupling protein (UCP) 1-mediated leak respiration. Application of stable isotope labeling showed that LPC formation was balanced by an increased de novo synthesis of PC.
| S-EPMC6692052 | biostudies-literature
Project description:In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control.
| S-EPMC1218133 | biostudies-other
Project description:Adrenergic stimulation of electrogenic K+ secretion in isolated mucosa from guinea pig distal colon required activation of two beta-adrenergic receptor subtypes (beta-AdrR). Addition of epinephrine (epi) or norepinephrine (norepi) to the bathing solution of mucosae in Ussing chambers increased short-circuit current (Isc) and transepithelial conductance (Gt), consistent with this cation secretion. A beta-adrenergic classification was supported by propranolol antagonism of this secretory response and the lack of effect by the alpha-AdrR antagonists BE2254 (alpha1-AdrR) and yohimbine (alpha2-AdrR). Subtype-selective antagonists CGP20712A (beta1-AdrR), ICI-118551 (beta2-AdrR), and SR59320A (beta3-AdrR) were relatively ineffective at inhibiting the epi-stimulated Isc response. In combination, CGP20712A and ICI-118551 inhibited the response, which supported a synergistic action by beta1-AdrR and beta2-AdrR. Expression of mRNA for both beta1-AdrR and beta2-AdrR was indicated by RT-PCR of RNA from colonic epithelial cells. Protein expression was indicated by immunoblot showing bands at molecular weights consistent with monomers and oligomers. Immunoreactivity (ir) for beta1-AdrR and beta2-AdrR was prominent in basolateral membranes of columnar epithelial cells in the crypts of Lieberkühn as well as intercrypt surface epithelium. Cells in the pericryptal sheath also had beta1-AdrR(ir) but did not have discernable beta2-AdrR(ir). The adrenergic sensitivity of K+ secretion measured by Isc and Gt was relatively low as indicated by EC(50)s of 41 +/- 7 nM for epi and 50 +/- 14 nM for norepi. Adrenergic activation of electrogenic K+ secretion required the involvement of both beta1-AdrR and beta2-AdrR, occurring with an agonist sensitivity reduced compared with reported values for either receptor subtype.
| S-EPMC2724081 | biostudies-literature
Project description:The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.
| S-EPMC2386676 | biostudies-literature
Project description:cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon beta-adrenergic stimulation under stress. Both beta(1)- and beta(2)-adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that beta(2)ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the beta(2)AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the beta(2)AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the beta(2)AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of beta(2)AR. Our data underscore the complex regulation of intracellular cAMP by beta(2)AR-associated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.
| S-EPMC2797152 | biostudies-literature
Project description:Mice and other small mammals heavily rely on UCP1-mediated non-shivering thermogenesis in the cold, and thus the unexpected cold resistance of UCP1-knockout (UCP1KO) mice has always been and still is an unresolved mystery inextricably intertwined with alternative means of thermogenesis and futile substrate cycles. Several potentially thermogenic futile substrate cycles in adipose tissue of WT and UCP1KO mice have been reported in the literature. We hypothesized that isoproterenol treatment of brown adipocytes from UCP1KO mice would cause an immediate acceleration of futile substrate cycling, and entail a consecutive upregulation of the involved enzymes to recruit additional capacity. In this study, we analyzed changes in the proteome of wild type (WT) and UCP1KO adipocytes during acute adrenergic stimulation to detect characteristic molecular signatures, which allowed us to narrow down our search to potential candidates related to Ca2+ and lipid metabolism.
2022-04-25 | PXD025854 | Pride