Dataset Information

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β₁ and β₃-adrenoceptor signalling.

ABSTRACT:

Objective

3',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.

Methods

We 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.

Results

Using 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.

Conclusions

We 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.

SUBMITTER: Kannabiran SA

PROVIDER: S-EPMC7191645 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In brown adipocytes, fundamental cellular processes (cell proliferation, differentiation and apoptosis) are regulated by adrenergic stimulation, notably through beta-adrenergic receptors. The presence of all three beta-receptor subtypes has been demonstrated in brown adipose tissue. Due to the significance of the action of these receptors and indications that the subtypes govern different processes, the adrenergic regulation of the expression of the beta(1)-(,) beta(2)- and beta(3)-adrenoceptor genes was examined in murine brown-fat primary cell cultures. Moderate levels of beta(1)-receptor mRNA, absence of beta(2)-receptor mRNA and high levels of beta(3)-receptor mRNA were observed in mature brown adipocytes (day 6 in culture). Noradrenaline (norepinephrine) addition led to diametrically opposite effects on beta(1)- (markedly enhanced expression) and beta(3)-gene expression (full cessation of expression, as previously shown). beta(2)-Gene expression was induced by noradrenaline, but only transiently (<1 h). The apparent affinities (EC(50)) of noradrenaline were clearly different (7 nM for the beta(1)-gene and</=1 nM for the beta(3)-gene), as were the mediation pathways (solely via beta(3)-receptors and cAMP for the beta(1)-gene and via beta(3)-receptors and cAMP, as well as via alpha(1)-receptors and protein kinase C, for the beta(3)-gene). The half-lives of the corresponding mRNA species were very short but different (17 min for beta(1)-mRNA and 27 min for beta(3)-mRNA), and these degradation rates were not affected by noradrenaline, implying that the mRNA levels were controlled by transcription. Inhibition of protein synthesis also led to diametrically opposite effects on beta(1)- and beta(3)-gene expression, but - notably - these effects were congruent with the noradrenaline effects, implying that a common factor regulating beta(1)-gene expression negatively and beta(3)-gene expression positively could be envisaged. In conclusion, very divergent effects of adrenergic stimulation on the expression of the different beta-receptor genes were found within one cell type, and no unifying concept of adrenergic control of beta-receptor gene expression can be formulated, either concerning different cell types, or concerning the different beta-receptor subtype genes.

Project description:Background and purposeStress-related catecholamines have a role in cancer and β-adrenoceptors; specifically, β2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, β3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which β3 -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β3 -adrenoceptors in immune-tolerance regulation.Experimental approachA mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of β-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β-blockers (propranolol and SR59230A) and specific β-adrenoceptor siRNAs targeting β2 - or β3 -adrenoceptors were used.Key resultsOnly β3 -, but not β2 -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and β3 -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and β3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes.Conclusions and implicationsOur data suggest that β3 -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth.Linked articlesThis article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

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Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β₁ and β₃-adrenoceptor signalling.

Objective

Methods

Results

Conclusions

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Dataset Information

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β1 and β3-adrenoceptor signalling.

Objective

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β₁ and β₃-adrenoceptor signalling.