Search Results (84)

Over the past decades, bile acids have been recognized as important signaling molecules with significant roles in metabolic health and disease. Many of their beneficial effects are mediated through the activation of the Takeda G protein-coupled receptor 5 (TGR5), a G protein-coupled receptor ubiquitously expressed in both humans and animals. Upon activation, TGR5 stimulates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels and subsequent activation of protein kinase A (PKA). PKA then phosphorylates and activates several downstream signaling pathways, including exchange protein directly activated by cAMP (EPAC), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (AKT). Through these pathways, TGR5 acts as a key molecular link between bile acid signaling and the regulation of energy metabolism. TGR5 activation has been associated with body weight loss in obese models, primarily by reducing food intake, enhancing thermogenesis in adipose tissue and muscle to increase energy expenditure, and improving insulin secretion. This review highlights recent advances in our understanding of TGR5 biology and critically examines its therapeutic potential, limitations, and controversies in the context of energy metabolism, offering new perspectives and opportunities for treating metabolic disorders. Full article

Adenylyl cyclases (ACs) are key regulators of cyclic adenosine monophosphate (cAMP) signaling—a pathway critical for neuroregeneration, synaptic plasticity, and neuronal survival. In both the central and peripheral nervous systems, injury-induced activation of ACs promotes axonal outgrowth and functional recovery through the stimulation of protein kinase A (PKA), exchange proteins directly activated by cAMP (Epac), and cAMP-response element-binding protein (CREB). Among the various AC isoforms, calcium-sensitive AC1, AC8, and AC5, as well as bicarbonate-responsive soluble AC (sAC), have emerged as crucial mediators of neuroplasticity and axon regeneration. These isoforms coordinate diverse cellular responses—including gene transcription, cytoskeletal remodeling, and neurotransmitter release—to metabolic, synaptic, and injury-related signals. Dysregulation of AC activity has been implicated in the pathophysiology of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis, as well as in chronic pain syndromes. Pharmacological modulation of cAMP levels through AC activation, phosphodiesterase (PDE) inhibition, or pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor signaling has shown therapeutic promise in preclinical models by enhancing neurogenesis, remyelination, and synaptic repair. Conversely, targeted inhibition of specific AC isoforms, particularly AC1, has demonstrated efficacy in reducing maladaptive plasticity and neuropathic pain. This review highlights the diverse roles of ACs in neuronal function and injury response and discusses emerging strategies for their therapeutic targeting. Full article

Aim: Myricetin, a natural bioflavonoid, is reported as an anti-diabetic agent since it possesses the ability to inhibit α-glucosidase activity, stimulate insulin action and secretion, manage ROS, and prevent diabetes complications. Myricetin was identified as a new insulin secretagogue that enhances glucose-stimulated insulin secretion and seems like a better antidiabetic drug candidate. Here, we explored the insulinotropic mechanism(s) of myricetin in vitro in mice islets and in silico. Methods: Size-matched pancreatic islets were divided into groups and incubated in the presence or absence of myricetin and agonists/antagonists of major insulin signaling pathways. The secreted insulin was measured by ELISA. Molecular docking studies were performed with the key player of insulin secretory pathways. Results: Myricetin dose-dependently enhanced insulin secretion in isolated mice islets, and its insulinotropic effect was exerted at high glucose concentrations distinctly different from glibenclamide. Myricetin-induced insulin secretion was significantly inhibited using the diazoxide. Furthermore, myricetin amplified glucose-induced insulin secretion in depolarized and glibenclamide-treated islets. Myricetin showed an additive effect with forskolin- and IBMX-induced insulin secretion. Interestingly, H89, a PKA inhibitor, and MAY0132, an Epac-2 inhibitor, significantly inhibited myricetin-induced insulin secretion. The in silico molecular docking studies further validated these in vitro findings in isolated pancreatic islets. Conclusions: Myricetin, a potential natural insulin secretagogue, amplifies glucose-induced insulin secretion via the cAMP-PKA-Epac-2 signaling pathway. Full article

Respiratory viruses can cause life-threatening conditions such as sepsis and acute respiratory distress syndrome. However, vaccines and effective antivirals are available for only a limited number of infections. The majority of approved antivirals are direct-acting agents, which target viral proteins essential for infection. Unfortunately, mutations have already emerged that confer resistance to these antivirals. In addition, there is an urgent need for broad-spectrum antivirals to address the unpredictable emergence of new viruses with pandemic potential. One promising strategy involves modulating the innate immune response and cellular signaling. Imiquimod, a Toll-like receptor 7 (TLR7) agonist, has shown efficacy in murine models of influenza and respiratory syncytial virus (RSV). Additionally, it demonstrates antiviral activity against herpes simplex virus type 1 (HSV-1) and RSV independent of the TLR7/nuclear factor kappa B (NF-κB) pathway, with protein kinase A (PKA) as a crucial downstream effector. In this study, we demonstrate that imiquimod exhibits concentration-dependent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and canine coronavirus (CCoV) in epithelial cells, underscoring its broad-spectrum action against coronaviruses. Moreover, its anti-coronavirus effect appears to be independent of the TLR/NF-κB and PKA/exchange protein directly activated by cyclic adenosine monophosphate (EPAC) pathways and may instead be linked to the activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. The ability of imiquimod to inhibit coronavirus replication via the MEK/ERK pathway, coupled with its immunomodulatory properties, highlights its potential as a broad-spectrum antiviral. Full article

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants, young children, and immunocompromised individuals. Currently, FDA-approved monoclonal antibody therapies are limited to infants and young children with severe RSV disease. As a result, there is an urgent need for comprehensive studies of RSV pathogenesis to support the development of new therapeutic strategies. Exchange proteins directly activated by cAMP (EPAC) have recently emerged as key regulators in various viral infections. Our previous work identified EPAC isoform 2 (EPAC2) as a critical factor in RSV replication and host innate immune responses. However, the molecular mechanisms underlying EPAC2’s role in RSV infection remain unclear. In this study, we investigated EPAC2-mediated RSV infection by identifying EPAC2-interacting proteins. Proteomics and immunoprecipitation analyses revealed that junction plakoglobin (JUP) interacts with EPAC2 in both mock- and RSV-infected cells, with this interaction notably enhanced during RSV infection. To determine JUP’s role in RSV infection, we compared viral replication in JUP-deficient and control cells. JUP downregulation significantly reduced the production of infectious RSV particles, likely by impairing viral budding and viral gene transcription. Moreover, our findings indicate that JUP is essential for an effective cellular immune response to RSV infection. Together, these results suggest that EPAC2 and JUP may cooperatively regulate RSV replication and dissemination. Full article

Infection with Porphyromonas gingivalis (Pg), which is a major periodontal pathogen, causes a large number of systemic diseases based on chronic inflammation such as diabetes and Alzheimer’s disease (AD). However, it is not yet fully understood how Pg can augment local systemic immune and inflammatory responses during progression of AD. There is a strong association between depression and elevated levels of inflammation. Noradrenaline (NA) is a key neurotransmitter that modulates microglial activation during stress conditions. In this study, we have thus investigated the regulatory mechanisms of NA on the production of interleukin-1β (IL-1β) by microglia following stimulation with Pg virulence factors, lipopolysaccharide (LPS), and outer membrane vesicles (OMVs). NA (30–1000 nM) significantly enhanced the mRNA level, promoter activity, and protein level of IL-1β up to 20-fold in BV-2 microglia following treatment with Pg LPS (10 μg/mL) and OMVs (150 μg of protein/mL) in a dose-dependent manner. Pharmacological studies have suggested that NA synergistically augments the responses induced by Pg LPS and OMVs through different mechanisms. AP-1 is activated by the β₂ adrenergic receptor (Aβ₂R)-mediated pathway. NF-κB, which is activated by the Pg LPS/toll-like receptor 2-mediated pathway, is required for the synergistic effect of NA on the Pg LPS-induced IL-1β production by BV-2 microglia. Co-immunoprecipitation combined with Western blotting and the structural models generated by AlphaFold2 suggested that cross-coupling of NF-κB p65 and AP-1 c-Fos transcription factors enhances the binding of NF-κB p65 to the IκB site, resulting in the synergistic augmentation of the IL-1β promoter activity. In contrast, OMVs were phagocytosed by BV-2 microglia and then activated the TLR9/p52/RelB-mediated pathway. The Aβ₂R/Epac-mediated pathway, which promotes phagosome maturation, may be responsible for the synergistic effect of NA on the OMV-induced production of IL-1β in BV-2 microglia. Our study provides the first evidence that NA synergistically enhances the production of IL-1β in response to Pg LPS and OMVs through distinct mechanisms. Full article

Animal feeding has a great impact on the management of beef farms, also affecting the nutritional properties of the meat. Therefore, in this study, the following two forage-to-concentrate ratios were tested on twenty farmed Podolian young bulls: high forage-to-concentrate (HF:C) ratio of 65:35 vs. low forage-to-concentrate (LF:C) ratio of 45:55. The fatty acid profile, bioactive compounds, α-tocopherol content, and oxidative stability were evaluated on Longissimus thoracis muscle vacuum-packaged and aged at 2 °C for 11 and 18 days, respectively. Feeding the highest forage-to-concentrate ratio improved the fatty acid profile by decreasing the saturated fatty acids (p < 0.01) and increasing the monounsaturated (p < 0.05) and n-3 polyunsaturated fatty acids (p < 0.001). In particular, the percentages of linolenic acid (C18:3n-3), eicosapentaenoic acid (EPA-C20:5n-3), DPA (C22:5n-3), and docosahexaenoic acid (DHA-C22:6n3) were significantly higher (p < 0.01) in the HF:C group than the LF:C group. The highest forage-to-concentrate ratio also increased the contents of bioactive compounds, such as creatine (p < 0.001), carnosine (p < 0.01), and anserine (p < 0.05). This study suggests that a diet composed of 65% forage may be a feasible strategy to enrich meat with healthy bioactive compounds. Full article

Estrogen is a steroid hormone that plays a key role in regulating many physiological processes, such as follicle activation and development and oocyte maturation in mammals. Ca²⁺ is crucial in oogenesis, oocyte maturation, ovulation, and fertilization. However, the mechanism by which estrogen regulates Ca²⁺ during oocyte maturation in mice has not been reported. This study revealed that Ca²⁺ levels in oocytes significantly increase during the 4–12 h period in vitro. Oocytes treated with 0.1 µM estrogen and 1 µM G1, a G-protein-coupled estrogen receptor (GPER) agonist, showed significantly increased Ca²⁺ levels, while treatment with 1 µM G15, an antagonist of GPER, significantly decreased Ca²⁺ levels. Notably, estrogen regulates Ca²⁺ in oocytes through the GPER pathway and promotes the expression of the Ca²⁺-producing protein EPAC1. In addition, estrogen alleviates the inhibitory effect of the Ca²⁺ chelator BAPTA-AM during oocyte maturation by promoting Ca²⁺ production. Furthermore, estrogen can promote the expression of the mitochondrial generation-associated protein SIRT1 through the GPER pathway, alleviate mitochondrial oxidative damage caused by BAPTA-AM, and restore the mitochondrial membrane potential level. Collectively, this study demonstrates that estrogen can regulate Ca²⁺ through the GPER-EPAC1 pathway and promote the expression of SIRT1, which promotes oocyte mitochondrial function during oocyte maturation. Full article

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic β-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs. Full article

Ras-related Rap1A GTPase is implicated in pancreas β-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein β (Ero1lβ), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lβ and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15–30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function. Full article

Glucagon-like peptide-1 (GLP-1) is a multifunctional incretin hormone with various physiological effects beyond its well-characterized effect of stimulating glucose-dependent insulin secretion in the pancreas. An emerging role for GLP-1 and its receptor, GLP-1R, in brain neuroprotection and in the suppression of inflammation, has been documented in recent years. GLP-1R is a G protein-coupled receptor (GPCR) that couples to Gs proteins that stimulate the production of the second messenger cyclic 3’,5’-adenosine monophosphate (cAMP). cAMP, acting through its two main effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), exerts several anti-inflammatory (and some pro-inflammatory) effects in cells, depending on the cell type. The present review discusses the cAMP-dependent molecular signaling pathways elicited by the GLP-1R in cardiomyocytes, cardiac fibroblasts, central neurons, and even in adrenal chromaffin cells, with a particular focus on those that lead to anti-inflammatory effects by the GLP-1R. Fully elucidating the role cAMP plays in GLP-1R’s anti-inflammatory properties can lead to new and more precise targets for drug development and/or provide the foundation for novel therapeutic combinations of the GLP-1R agonist medications currently on the market with other classes of drugs for additive anti-inflammatory effect. Full article

The high mortality rate among patients with acute myocardial infarction (AMI) is one of the main problems of modern cardiology. It is quite obvious that there is an urgent need to create more effective drugs for the treatment of AMI than those currently used in the clinic. Such drugs could be enzyme-resistant peptide analogs of glucagon-like peptide-1 (GLP-1). GLP-1 receptor (GLP1R) agonists can prevent ischemia/reperfusion (I/R) cardiac injury. In addition, chronic administration of GLP1R agonists can alleviate the development of adverse cardiac remodeling in myocardial infarction, hypertension, and diabetes mellitus. GLP1R agonists can protect the heart against oxidative stress and reduce proinflammatory cytokine (IL-1β, TNF-α, IL-6, and MCP-1) expression in the myocardium. GLP1R stimulation inhibits apoptosis, necroptosis, pyroptosis, and ferroptosis of cardiomyocytes. The activation of the GLP1R augments autophagy and mitophagy in the myocardium. GLP1R agonists downregulate reactive species generation through the activation of Epac and the GLP1R/PI3K/Akt/survivin pathway. The GLP1R, kinases (PKCε, PKA, Akt, AMPK, PI3K, ERK1/2, mTOR, GSK-3β, PKG, MEK1/2, and MKK3), enzymes (HO-1 and eNOS), transcription factors (STAT3, CREB, Nrf2, and FoxO3), K_ATP channel opening, and MPT pore closing are involved in the cardioprotective effect of GLP1R agonists. Full article

Portugal’s EPAC (Empresa Pública do Abastecimiento dos Cereais) silo network, initially planned in the 1930s but constructed and utilised primarily in the 1970s, consisted of 31 silos with a total capacity of 841,100 t. The network’s usage declined, however, due to market liberalisation and Portugal’s accession to the European Economic Community in 1985. This study focuses on adapting a methodology to inventory and analyse the 31 silos in the EPAC network, considering their general features, construction, technological facilities, and socioeconomic aspects. The silos are situated in 30 cities and towns, predominantly in the country’s key grain-growing regions, particularly the Alentejo region. While there are variations in design and construction, most EPAC silos contain two or three rows of circular reinforced concrete cells and use the spaces between cells for storage. Their capacities range from 6000 to 35,000 t. Some are inland grain reception and storage silos, while others are larger-capacity port silos designed to unload grain rapidly onto ships using mechanical or pneumatic unloaders. These structures are a significant part of Portugal’s agro-industrial heritage and have, in some cases, been repurposed as museums or event venues. Compared to other agro-industrial buildings, silos pose unique conversion challenges due to their height and design complexities. Examples of successful reuse in countries like Spain and Italy may provide insights for potential silo projects in Portugal. However, analysis suggests that such proposals and similar initiatives may be viable only in the more highly populated towns. Full article

The G protein-coupled α₂-adrenoceptor subtype C (abbreviated α_2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet–hands, Raynaud’s phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α_2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein–protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α_2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α_2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α_2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy. Full article

The Ras/cAMP/PKA pathway regulates responses to nutrients’ availability and stress in budding yeast. The cAMP levels are subjected to negative feedback, and we have previously simulated a dynamic model of this pathway suggesting the existence of stable oscillatory states depending on the symmetrical and opposed activity of the RasGEF (Cdc25) and RasGAPs (Ira proteins). Noisy oscillations related to the activity of this pathway were reported by looking at the nuclear localization of the transcription factor Msn2, and sustained oscillations of the nuclear accumulation of Msn2 under the condition of limiting glucose were observed. We were able to reproduce the periodic accumulation of Msn2-GFP protein in a yeast cell under the condition of limiting glucose, and we also detected oscillations of cAMP. We used a sensor based on a fusion protein between YFP-Epac2-CFP expressed in yeast cells. The FRET between CFP and YFP is controlled by the cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in a single yeast cell over a long time. Using this method, we were able to detect noisy oscillations of cAMP levels in single yeast cells under conditions of nutritional stress caused by limiting glucose availability. Full article