Hundreds of hormones and ligands stimulate cyclic AMP (cAMP) signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs). Although the functions and individual ...effectors of cAMP signaling are well characterized in many tissues, pleiotropic effects of GPCR agonists limit investigations of physiological functions of cAMP signaling in individual cell types at different developmental stages in vivo. To facilitate studies of cAMP signaling in specific cell populations in vivo, we harnessed the power of DREADD (designer receptors exclusively activated by designer drugs) technology by creating ROSA26-based knock-in mice for the conditional expression of a Gs-coupled DREADD (rM3Ds-green fluorescent protein GFP, or "GsD"). After Cre recombinase expression, GsD is activated temporally by the administration of the ligand clozapine N-oxide (CNO). In the same allele, we engineered a CREB-luciferase reporter transgene for noninvasive bioluminescence monitoring of CREB activity. After viral delivery of Cre recombinase to hepatocytes in vivo, GsD is expressed and allows CNO-dependent cAMP signaling and glycogen breakdown. The long-term expression of GsD in the liver results in constitutive CREB activity and hyperglycemia. ROSA26-Gs-DREADD mice can be used to study the physiological effects of cAMP signaling, acute or chronic, in liver or any tissue or cell type for which transgenic or viral Cre drivers are available.
G protein-coupled receptors (GPCRs) are the target of ~30% to 35% of all US Food and Drug Administration-approved drugs. The individual members of the GPCR superfamily couple to 1 or more functional ...classes of heterotrimeric G proteins. The physiological outcome of activating a particular GPCR in vivo depends on the pattern of receptor distribution and the type of G proteins activated by the receptor. Based on the structural and functional properties of their α-subunits, heterotrimeric G proteins are subclassified into 4 major families: Gs, Gi/o, Gq/11, and G12/13. Recent studies with genetically engineered mice have yielded important novel insights into the metabolic roles of Gi/o-type G proteins. For example, recent data indicate that Gi signaling in pancreatic α-cells plays a key role in regulating glucagon release and whole body glucose homeostasis. Receptor-mediated activation of hepatic Gi signaling stimulates hepatic glucose production, suggesting that inhibition of hepatic Gi signaling could prove clinically useful to reduce pathologically elevated blood glucose levels. Activation of adipocyte Gi signaling reduces plasma free fatty acid levels, thus leading to improved insulin sensitivity in obese, glucose-intolerant mice. These new data suggest that Gi-coupled receptors that are enriched in metabolically important cell types represent potential targets for the development of novel drugs useful for the treatment of type 2 diabetes and related metabolic disorders.
Impaired lung function caused by decreased airway diameter (bronchoconstriction) is frequently observed whether body weight is abnormally high or low. That these opposite conditions affect the ...airways similarly suggests that the regulation of airway diameter and body weight are intertwined. We show here that, independently of its regulation of appetite, melanocortin pathway, or sympathetic tone, leptin is necessary and sufficient to increase airway diameter by signaling through its cognate receptor in cholinergic neurons. The latter decreases parasympathetic signaling through the M3 muscarinic receptor in airway smooth muscle cells, thereby increasing airway diameter without affecting local inflammation. Accordingly, decreasing parasympathetic tone genetically or pharmacologically corrects bronchoconstriction and normalizes lung function in obese mice regardless of bronchial inflammation. This study reveals an adipocyte-dependent regulation of bronchial diameter whose disruption contributes to the impaired lung function caused by abnormal body weight. These findings may be of use in the management of obesity-associated asthma.
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► Leptin regulates airway diameter, independently of body weight and inflammation ► Leptin uses the parasympathetic tone to regulate airway diameter ► Modulating the parasympathetic tone can correct bronchoconstriction in obese mice
ABSTRACT
We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a ...reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1–5 mAChR) expression by using RT‐PCR in UBCs, recorded Ca2+i responses to a bitter stimulus in isolated UBCs of wild‐type and mAChR‐deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by M1, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR‐deficient mice. In cystometry, M5 mAChR‐deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.—Deckmann, K., Rafiq, A., Erdmann, C., Illig, C., Durschnabel, M., Wess, J., Weidner, W., Bschleipfer, T., Kummer, W. Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback. FASEB J. 32, 2903–2910 (2018). www.fasebj.org
Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the ...importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline 10 wild-type (WT) mice or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.
G-protein-coupled receptor (GPCR) kinases (GRKs) abrogate GPCR signaling by promoting receptor desensitization and internalization. Accumulating evidence suggests that GRK2 represents an important ...regulator of GPCR-mediated effects on systemic glucose metabolism, obesity, and insulin resistance. Despite the key role of the liver in maintaining euglycemia, the potential metabolic relevance of hepatic GRK2 has yet to be examined. Thus, the goal of this study was to explore the potential role of hepatic GRK2 in maintaining glucose homeostasis and other key metabolic functions.
To address this question, we generated mice that showed a ∼90% reduction in GRK2 protein expression selectively in hepatocytes (Hep-GRK2-KO mice) and subjected these mice, together with their control littermates, to systematic metabolic phenotyping studies.
We found that Hep-GRK2-KO mice maintained on regular chow did not differ significantly from their control littermates in glycemia, glucose tolerance, insulin sensitivity, in vivo gluconeogenesis, and glucagon-induced hyperglycemia. We obtained similar findings when we analyzed Hep-GRK2-KO mice and control littermates consuming an obesogenic high-fat diet. Likewise, plasma levels of insulin, glucagon, free fatty acids, and ketone bodies remained unaffected by the lack of hepatocyte GRK2. The same was true when we examined the expression levels of key genes regulating hepatic glucose and fatty acid metabolism.
In summary, our data suggest that hepatocyte GRK2 is dispensable for systemic glucose homeostasis and other key metabolic functions in both lean and obese mice. This finding suggests that drug development efforts aimed at inhibiting GRK2 to improve impaired glucose homeostasis and insulin sensitivity need to focus on other metabolically important tissues.
Abstract Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new ...classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell G s signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell G s signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α−cell G s -coupled A 2A adenosine receptors. Studies with α-cell-specific Gα s knockout mice showed that α-cell G s also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched G s -coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.
G protein-coupled receptors (GPCRs) regulate virtually all metabolic processes, including glucose and energy homeostasis. Recently, the use of designer GPCRs referred to as designer receptors ...exclusively activated by designer drug (DREADDs) has made it possible to dissect metabolically relevant GPCR signaling pathways in a temporally and spatially controlled fashion in vivo.
Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an ...important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.
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