CK2 as a novel regulator of M3 muscarinic receptor-mediated insulin release GPCRs regulate many aspects of beta-cell function including insulin release. We previously demonstrated that beta-cell M3 muscarinic receptors (M3Rs) play a key role in facilitating insulin secretion and maintaining euglycemia. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and CK2. At present, it remains unknown whether such phosphorylation events are physiologically relevant for the regulation of beta-cell activity. Recently, we showed that inhibition of CK2 in pancreatic beta-cells, knockdown of CK2alpha expression, or genetic deletion of CK2lphain beta-cells selectively facilitated M3R-stimulated insulin release in vitro and in vivo. This stimulatory effect on insulin secretion was associated with an M3R-mediated increase in intracellular calcium levels (in eta-cells). Treatment of mouse pancreatic islets with a selective CK2 inhibitor (CX4945) led to a strong reduction in muscarinic agonist-induced phosphorylation of beta-cell M3Rs, indicative of CK2-mediated phosphorylation of the M3R in beta-cells. Similarly, inhibition of CK2 also greatly enhanced M3R-stimulated insulin secretion in human islets. Interestingly, we found that CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance. This effect required the presence of M3Rs, since it was not observed with M3R-deficient mice. These findings support the novel concept that kinases acting on beta-cell GPCRs represent potential novel therapeutic targets to stimulate insulin secretion for therapeutic purposes. (Rossi M, Ruiz de Azua I, Barella LF, Sakamoto W, Zhu L, Cui Y, Lu H, Rebholz H, Matschinsky FM, Doliba NM, Butcher AJ, Tobin AB, Wess J. CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo. Proc Natl Acad Sci USA 112, E6818-24, 2015) Use of designer GPCRs to study GPCR regulation of key metabolic pathways Armbruster et al. (PNAS 104, 5163-8, 2007) first described a set of muscarinic receptor-based designer GPCRs which are now generally referred to as DREADDs ('designer receptors exclusively activated by designer drugs'). These designer receptors are unable to bind the endogenous muscarinic receptor agonist, acetylcholine, due to two single point mutations introduced into the transmembrane receptor core. Importantly, DREADDs can be efficiently activated by a compound called clozapine-N-oxide (CNO), an agent that is otherwise pharmacologically inert. The first DREADDs that were developed represent GPCRs that selectively activate G proteins of the Gq or Gi family, respectively. We subsequently generated additional DREADDs endowed with different coupling properties, including a Gs DREADD and a functionally promiscuous DREADD (Guettier et al., PNAS 106, 19197-202, 2009). More recently, we generated an M3R-based DREADD that is uncoupled from G proteins but retains arrestin-dependent signaling (Nakajima and Wess, Mol Pharmacol 82, 575-82, 2012). Analogously, we recently designed an M3R-based DREADD that shows the opposite coupling profile: lack of arrestin recruitment but efficient coupling to Gq-type G proteins (Hu et al., J Biol Chem 291, 7809-20, 2016; Wess, Trends Endocrin Metab, 2016 Jul 2, Epub ahead of print). We are currently in the process of expressing DREADDs with different coupling properties in various metabolically relevant cell types. These cell types include adipocytes, pancreatic beta-cells, skeletal muscle cells, hepatocytes, and certain neuronal subpopulations of the hypothalamus. Preliminary results indicate that CNO treatment of some of these mutant mouse strains has pronounced effect on glucose and energy homeostasis (unpublished results). A novel, central GPCR signaling pathway that triggers sustained orexigenic effects Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP, GABA, and neuropeptide Y. As is the case with essentially all other cell types, the activity of AgRP neurons is predicted to be regulated by cell surface receptors belonging to the superfamily of GPCRs which are linked to distinct functional classes of G proteins. So far, the functional consequences of activating the various GPCR/G protein signaling pathways in AgRP neurons have not been studied systematically. Recently, we examined the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons. We demonstrated that activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also identified a novel signaling pathway that links the stimulation of this class of receptors to the observed feeding phenotype. A key feature of this pathway is the PKA-dependent activation of the Klf4 transcription factor which promotes Agrp expression. We also showed that this Gs-mediated orexigenic pathway is clearly distinct from other GPCR signaling cascades that regulate the activity of AgRP neurons. These new findings suggest that agents that are able to inhibit this orexigenic pathway may become useful as novel appetite-suppressing drugs. (Nakajima K, Cui Z, Li C, Meister J, Cui Y, Fu O, Smith AS, Jain S, Lowell BB, Krashes MJ, Wess J. Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake. Nat Commun 7:10268, 2016) The data summarized below were obtained in a collaborative study. Role of Gq in brown and white fat formation and function Brown adipose tissue (BAT) dissipates energy in the form of heat. Interestingly, recent studies suggest that BAT activity correlates with leanness in human adults. Thus, agents that enhance BAT function may prove useful to reduce body fat mass in obese individuals. Since GPCRs represent excellent drug targets, we, in collaboration with Dr. Alexander Pfeifer's lab, examined which GPCRs are expressed in murine BAT. We found that 1/5 of the 300 non-sensory GPCRs expressed by murine BAT are linked to Gq-type G proteins. Administration of a Gq inhibitor or knockdown of Galpha-q expression promoted the differentiation of human and murine brown adipocytes. On the other hand, activation of Gq signaling by a Gq-coupled designer GPCR (Gq DREADD) or by a constitutively active form of Galpha-q abolished BAT differentiation. We found that the Gq-coupled endothelin A receptor, which is highly expressed in BAT, acts as an endogenous inhibitor of brown adipogenesis. Expression of a constitutively active form of Galpha-q in murine BAT reduced UCP1 expression and whole-body energy expenditure and inhibited the browning of subcutaneous white adipose tissue in mice. We also noted that Gq expression levels showed an inverse correlation with UCP1 expression in human subcutaneous white adipose tissue. These new findings indicate that Gq signaling is a critical regulator of the activity of brown and beige adipocytes. Our data also suggest that pharmacological inhibition of Gq signaling may represent a useful strategy to enhance BAT function for therapeutic purposes. (Klepac K, Kili A, Gnad T, Brown LM, Herrmann B, Wilderman A, Balkow A, Glde A, Simon K, Lidell ME, Betz MJ, Enerbck S, Wess J, Freichel M, Blher M, Knig G, Kostenis E, Insel PA, Pfeifer A. The Gq signalling pathway inhibits brown and beige adipose tissue. Nat Commun 7:10895, 2016.)

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Zhu, Lu; Rossi, Mario; Cui, Yinghong et al. (2017) Hepatic ?-arrestin 2 is essential for maintaining euglycemia. J Clin Invest 127:2941-2945
Zhu, Lu; Almaça, Joana; Dadi, Prasanna K et al. (2017) ?-arrestin-2 is an essential regulator of pancreatic ?-cell function under physiological and pathophysiological conditions. Nat Commun 8:14295
Klepac, Katarina; Kili?, Ana; Gnad, Thorsten et al. (2016) The Gq signalling pathway inhibits brown and beige adipose tissue. Nat Commun 7:10895
Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia et al. (2016) Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake. Nat Commun 7:10268
Wess, Jürgen (2016) Use of Designer G Protein-Coupled Receptors to Dissect Metabolic Pathways. Trends Endocrinol Metab 27:600-603
Rossi, Mario; Cui, Zhenzhong; Nakajima, Ken-Ichiro et al. (2015) Virus-Mediated Expression of DREADDs for In Vivo Metabolic Studies. Methods Mol Biol 1335:205-21
Rossi, Mario; Ruiz de Azua, Inigo; Barella, Luiz F et al. (2015) CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo. Proc Natl Acad Sci U S A 112:E6818-24
Nakajima, Kenichiro; Jain, Shalini; Ruiz de Azua, Inigo et al. (2013) Minireview: Novel aspects of M3 muscarinic receptor signaling in pancreatic ýý-cells. Mol Endocrinol 27:1208-16
Wess, Jürgen; Nakajima, Kenichiro; Jain, Shalini (2013) Novel designer receptors to probe GPCR signaling and physiology. Trends Pharmacol Sci 34:385-92
Jain, Shalini; Ruiz de Azua, Inigo; Lu, Huiyan et al. (2013) Chronic activation of a designer G(q)-coupled receptor improves ? cell function. J Clin Invest 123:1750-62

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