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β-adrenergic receptors (βARs) are well established for conveying the signal from catecholamines to adipocytes. Acting through the second messenger cyclic adenosine monophosphate (cAMP) they stimulate lipolysis and also increase the activity of brown adipocytes and the 'browning' of adipocytes within white fat depots (so-called 'brite' or 'beige' adipocytes). Brown adipose tissue mitochondria are enriched with uncoupling protein 1 (UCP1), which is a regulated proton channel that allows the dissipation of chemical energy in the form of heat. The discovery of functional brown adipocytes in humans and inducible brown-like ('beige' or 'brite') adipocytes in rodents have suggested that recruitment and activation of these thermogenic adipocytes could be a promising strategy to increase energy expenditure for obesity therapy. More recently, the cardiac natriuretic peptides and their second messenger cyclic guanosine monophosphate (cGMP) have gained attention as a parallel signaling pathway in adipocytes, with some unique features. In this review, we begin with some important historical work that touches upon the regulation of brown adipocyte development and physiology. We then provide a synopsis of some recent advances in the signaling cascades from β-adrenergic agonists and natriuretic peptides to drive thermogenic gene expression in the adipocytes and how these two pathways converge at a number of unexpected points. Finally, moving from the physiologic hormonal signaling, we discuss yet another level of control downstream of these signals: the growing appreciation of the emerging roles of non-coding RNAs as important regulators of brown adipocyte formation and function. In this review, we discuss new developments in our understanding of the signaling mechanisms and factors including new secreted proteins and novel non-coding RNAs that control the function as well as the plasticity of the brown/beige adipose tissue as it responds to the energy needs and environmental conditions of the organism.

Alveolar macrophages (AMs) are multitasking cells that maintain lung homeostasis by clearing apoptotic cells (efferocytosis) and performing antimicrobial effector functions. Different PRRs have been described to be involved in the binding and capture of non-opsonized Streptococcus pneumoniae, such as TLR-2, mannose receptor (MR) and scavenger receptors (SRs). However, the mechanism by which the ingestion of apoptotic cells negatively influences the clearance of non-opsonized S. pneumoniae remains to be determined. In this study, we evaluated whether the prostaglandin E2 (PGE) produced during efferocytosis by AMs inhibits the ingestion and killing of non-opsonized S. pneumoniae. Resident AMs were pre-treated with an E prostanoid (EP) receptor antagonist, inhibitors of cyclooxygenase and protein kinase A (PKA), incubated with apoptotic Jurkat T cells, and then challenged with S. pneumoniae. Efferocytosis slightly decreased the phagocytosis of S. pneumoniae but greatly inhibited bacterial killing by AMs in a manner dependent on PGE production, activation of the EP2-EP4/cAMP/PKA pathway and inhibition of HO production. Our data suggest that the PGE produced by AMs during efferocytosis inhibits HO production and impairs the efficient clearance non-opsonized S. pneumoniae by EP2-EP4/cAMP/PKA pathway.

Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1β production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1β/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1β/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1β inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.

Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been accepted widely as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain-of-function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen, which induced an Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase 4 (PDE4), leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy, and identifies a unique probe of downstream-of-Smo Hh modulation.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE2-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.

The dysregulation of glucose-inhibited glucagon secretion from the pancreatic islet α-cell is a critical component of diabetes pathology and metabolic disease. We show a previously uncharacterized [Ca(2+)]i-independent mechanism of glucagon suppression in human and murine pancreatic islets whereby cAMP and PKA signaling are decreased. This decrease is driven by the combination of somatostatin, which inhibits adenylyl cyclase production of cAMP via the Gαi subunit of the SSTR2, and insulin, which acts via its receptor to activate phosphodiesterase 3B and degrade cytosolic cAMP. Our data indicate that both somatostatin and insulin signaling are required to suppress cAMP/PKA and glucagon secretion from both human and murine α-cells, and the combination of these two signaling mechanisms is sufficient to reduce glucagon secretion from isolated α-cells as well as islets. Thus, we conclude that somatostatin and insulin together are critical paracrine mediators of glucose-inhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose.
Copyright © 2015 the American Physiological Society.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca(2+) influx. Ca(2+)-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions.
Copyright © 2014 Elsevier Inc. All rights reserved.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that has an antioxidative protective effect on various tissues. Here, we determined whether GLP-1 has a role in the pathogenesis of diabetic nephropathy using nephropathy-resistant C57BL/6-Akita and nephropathy-prone KK/Ta-Akita mice. By in situ hybridization, we found the GLP-1 receptor (GLP-1R) expressed in glomerular capillary and vascular walls, but not in tubuli, in the mouse kidney. Next, we generated C57BL/6-Akita Glp1r knockout mice. These mice exhibited higher urinary albumin levels and more advanced mesangial expansion than wild-type C57BL/6-Akita mice, despite comparable levels of hyperglycemia. Increased glomerular superoxide, upregulated renal NAD(P)H oxidase, and reduced renal cAMP and protein kinase A (PKA) activity were noted in the Glp1r knockout C57BL/6-Akita mice. Treatment with the GLP-1R agonist liraglutide suppressed the progression of nephropathy in KK/Ta-Akita mice, as demonstrated by reduced albuminuria and mesangial expansion, decreased levels of glomerular superoxide and renal NAD(P)H oxidase, and elevated renal cAMP and PKA activity. These effects were abolished by an adenylate cyclase inhibitor SQ22536 and a selective PKA inhibitor H-89. Thus, GLP-1 has a crucial role in protection against increased renal oxidative stress under chronic hyperglycemia, by inhibition of NAD(P)H oxidase, a major source of superoxide, and by cAMP-PKA pathway activation.

AIMS - Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts.
RESULTS - Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available.
INNOVATION - HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a.
CONCLUSIONS - PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

PROBLEM - Clostridium sordellii causes endometrial infections, but little is known regarding host defenses against this pathogen.
METHOD OF STUDY - We tested the hypothesis that the immunoregulatory lipid prostaglandin (PG) E2 suppresses human macrophage clearance of C. sordellii through receptor-induced increases in intracellular cyclic adenosine monophosphate (cAMP). The THP-1 macrophage cell line was used to quantify C. sordellii phagocytosis.
RESULTS - PGE2 increased cAMP levels, activated protein kinase A (PKA), and inhibited the class A scavenger receptor-dependent phagocytosis of C. sordellii. Activation of the EP2 and EP4 receptors increased intracellular cAMP and inhibited phagocytosis, with evidence favoring a more important role for EP4 over EP2. This was supported by EP receptor expression data and the use of pharmacological receptor antagonists. In addition, the PKA isoform RI appeared to be more important than RII in mediating the suppression of ingestion of C. sordellii.
CONCLUSION - The endogenous lipid mediator PGE2 impairs human innate immune responses against C. sordellii.
© 2013 John Wiley & Sons Ltd.