Oxysterol Sensing Through GPR183 Triggers Endothelial Senescence in Hypertension.

BACKGROUND: Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown. METHODS: Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study. RESULTS: Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period circadian regulator 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice. CONCLUSIONS: This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

Targeting Neuronal GPR65 With Delayed BTB09089 Treatment Improves Neurorehabilitation Following Ischemic Stroke.

BACKGROUND: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown. METHODS: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65-/-) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65-/- and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting. RESULTS: BTB09089 significantly promoted motor outcomes in WT but not in GPR65-/- mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65-/- mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65-/- mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65-/- mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice. CONCLUSIONS: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.

G Protein-Coupled Receptors: A Century of Research and Discovery.

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and ß-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of ß-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

Genetic Deletion of ß-Arrestin 2 From the Subfornical Organ and Other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure.

BACKGROUND: ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the AT1R [angiotensin type 1 receptor])-mediated AT1R in the subfornical organ. Recently, we demonstrated that ARRB2 (ß-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT1R signaling. METHODS: Male and female Arrb2FLOX mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (Arrb2ICV-Cre). Arrb2FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2ICV-Control). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl. RESULTS: Arrb2ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2ICV-Cre mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, Arrb2ICV-Cre mice exhibited elevated saline intake. CONCLUSIONS: Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical AT1R responses to both exogenous and endogenous ANG. Stimulation of the AT1R/ARRB axis in the brain may represent a novel strategy to treat hypertension.

Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?

BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].

Faecalibacterium prausnitzii Attenuates CKD via Butyrate-Renal GPR43 Axis.

BACKGROUND: Despite available clinical management strategies, chronic kidney disease (CKD) is associated with severe morbidity and mortality worldwide, which beckons new solutions. Host-microbial interactions with a depletion of Faecalibacterium prausnitzii in CKD are reported. However, the mechanisms about if and how F prausnitzii can be used as a probiotic to treat CKD remains unknown. METHODS: We evaluated the microbial compositions in 2 independent CKD populations for any potential probiotic. Next, we investigated if supplementation of such probiotic in a mouse CKD model can restore gut-renal homeostasis as monitored by its effects on suppression on renal inflammation, improvement in gut permeability and renal function. Last, we investigated the molecular mechanisms underlying the probiotic-induced beneficial outcomes. RESULTS: We observed significant depletion of Faecalibacterium in the patients with CKD in both Western (n=283) and Eastern populations (n=75). Supplementation of F prausnitzii to CKD mice reduced renal dysfunction, renal inflammation, and lowered the serum levels of various uremic toxins. These are coupled with improved gut microbial ecology and intestinal integrity. Moreover, we demonstrated that the beneficial effects in kidney induced by F prausnitzii-derived butyrate were through the GPR (G protein-coupled receptor)-43. CONCLUSIONS: Using a mouse CKD model, we uncovered a novel beneficial role of F prausnitzii in the restoration of renal function in CKD, which is, at least in part, attributed to the butyrate-mediated GPR-43 signaling in the kidney. Our study provides the necessary foundation to harness the therapeutic potential of F prausnitzii for ameliorating CKD.

Short-Chain Fatty Acid Receptors and Blood Pressure Regulation: Council on Hypertension Mid-Career Award for Research Excellence 2021.

The gut microbiome influences host physiology and pathophysiology through several pathways, one of which is microbial production of chemical metabolites which interact with host signaling pathways. Short-chain fatty acids (SCFAs) are a class of gut microbial metabolites known to activate multiple signaling pathways in the host. Growing evidence indicates that the gut microbiome is linked to blood pressure, that SCFAs modulate blood pressure regulation, and that delivery of exogenous SCFAs lowers blood pressure. Given that hypertension is a key risk factor for cardiovascular disease, the examination of novel contributors to blood pressure regulation has the potential to lead to novel approaches or treatments. Thus, this review will discuss SCFAs with a focus on their host G protein-coupled receptors including GPR41 (G protein-coupled receptor 41), GPR43, and GPR109A, as well as OLFR78 (olfactory receptor 78) and OLFR558. This includes a discussion of the ligand profiles, G protein coupling, and tissue distribution of each receptor. We will also review phenotypes relevant to blood pressure regulation which have been reported to date for Gpr41, Gpr43, Gpr109a, and Olfr78 knockout mice. In addition, we will consider how SCFA signaling influences physiology at baseline, and, how SCFA signaling may contribute to blood pressure regulation in settings of hypertension. In sum, this review will integrate current knowledge regarding how SCFAs and their receptors regulate blood pressure.

GPR40 Agonism Modulates Inflammatory Reactions in Vascular Endothelial Cells.

Endothelial dysfunction is strongly linked with inflammatory responses, which can impact cardiovascular disease. Recently, G protein-coupled receptor 40 (GPR40) has been investigated as a modulator of metabolic stress; however, the function of GPR40 in vascular endothelial cells has not been reported. We analyzed whether treatment of GPR40-specific agonists modulated the inflammatory responses in human umbilical vein endothelial cells (HUVECs). Treatment with LY2922470, a GPR40 agonist, significantly reduced lipopolysaccharide (LPS)-mediated nuclear factor-kappa B (NF-κB) phosphorylation and movement into the nucleus from the cytosol. However, treatment with another GPR40 agonist, TAK875, did not inhibit LPS-induced NF-κB activation. LPS treatment induced expression of adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and attachment of THP-1 cells to HUVECs, which were all decreased by LY2922470 but not TAK875. Our results showed that ligand-dependent agonism of GPR40 is a promising therapeutic target for overcoming inflammatory reactions in the endothelium.

Extended Phenotyping and Functional Validation Facilitate Diagnosis of a Complex Patient Harboring Genetic Variants in MCCC1 and GNB5 Causing Overlapping Phenotypes.

Identifying multiple ultra-rare genetic syndromes with overlapping phenotypes is a diagnostic conundrum in clinical genetics. This study investigated the pathogenicity of a homozygous missense variant in GNB5 (GNB5L; NM_016194.4: c.920T > G (p. Leu307Arg); GNB5S; NM_006578.4: c.794T > G (p. Leu265Arg)) identified through exome sequencing in a female child who also had 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency (newborn screening positive) and hemoglobin E trait. The proband presented with early-onset intellectual disability, the severity of which was more in keeping with GNB5-related disorder than 3-MCC deficiency. She later developed bradycardia and cardiac arrest, and upon re-phenotyping showed cone photo-transduction recovery deficit, all known only to GNB5-related disorders. Patient-derived fibroblast assays showed preserved GNB5S expression, but bioluminescence resonance energy transfer assay showed abolished function of the variant reconstituted Gß5S containing RGS complexes for deactivation of D2 dopamine receptor activity, confirming variant pathogenicity. This study highlights the need for precise phenotyping and functional assays to facilitate variant classification and clinical diagnosis in patients with complex medical conditions.

Peptidyl and Non-Peptidyl Oral Glucagon-Like Peptide-1 Receptor Agonists.

Glucagon-like peptide-1 (GLP-1) receptor agonists are efficacious glucose-lowering medications with salient benefits for body weight and cardiovascular events. This class of medications is now recommended as the top priority for patients with established cardiovascular disease or indicators of high risk. Until the advent of oral semaglutide, however, GLP-1 receptor agonists were available only in the form of subcutaneous injections. Aversion to needles, discomfort with self-injection, or skin problems at the injection site are commonly voiced problems in people with diabetes, and thus, attempts for non-invasive delivery strategies have continued. Herein, we review the evolution of GLP-1 therapy from its discovery and the development of currently approved drugs to the unprecedented endeavor to administer GLP-1 receptor agonists via the oral route. We focus on the pharmacokinetic and pharmacodynamic properties of the recently approved oral GLP-1 receptor agonist, oral semaglutide. Small molecule oral GLP-1 receptor agonists are currently in development, and we introduce how these chemicals have addressed the challenge posed by interactions with the large extracellular ligand binding domain of the GLP-1 receptor. We specifically discuss the structure and pharmacological properties of TT-OAD2, LY3502970, and PF-06882961, and envision an era where more patients could benefit from oral GLP-1 receptor agonist therapy.

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

BACKGROUND: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear. METHODS: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM-Gprc5b-KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo. RESULTS: Mesenteric arteries from SMC-specific Gprc5b-KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM-Gprc5b-KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap. CONCLUSIONS: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.

Effects of ICI 182, 780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells / Efeitos do antagonista seletivo do REα e do REß ICI 182, 780 e do agonista de GPER G-1 no processo de autofagia em células de câncer de mama

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Understanding Bile Acid Signaling in Diabetes: From Pathophysiology to Therapeutic Targets.

Diabetes and obesity have reached an epidemic status worldwide. Diabetes increases the risk for cardiovascular disease and non-alcoholic fatty liver disease. Primary bile acids are synthesized in hepatocytes and are transformed to secondary bile acids in the intestine by gut bacteria. Bile acids are nutrient sensors and metabolic integrators that regulate lipid, glucose, and energy homeostasis by activating nuclear farnesoid X receptor and membrane Takeda G protein-coupled receptor 5. Bile acids control gut bacteria overgrowth, species population, and protect the integrity of the intestinal barrier. Gut bacteria, in turn, control circulating bile acid composition and pool size. Dysregulation of bile acid homeostasis and dysbiosis causes diabetes and obesity. Targeting bile acid signaling and the gut microbiome have therapeutic potential for treating diabetes, obesity, and non-alcoholic fatty liver disease.

Homology modeling of FFA2 identifies novel agonists that potentiate insulin secretion.

Critical aspects of maintaining glucose homeostasis in the face of chronic insulin resistance and type 2 diabetes (T2D) are increased insulin secretion and adaptive expansion of beta cell mass. Nutrient and hormone sensing G protein-coupled receptors are important mediators of these properties. A growing body of evidence now suggests that the G protein-coupled receptor, free fatty acid receptor 2 (FFA2), is capable of contributing to the maintenance of glucose homeostasis by acting at the pancreatic beta cell as well as at other metabolically active tissues. We have previously demonstrated that Gαq/11-biased agonism of FFA2 can potentiate glucose stimulated insulin secretion (GSIS) as well as promote beta cell proliferation. However, the currently available Gαq/11-biased agonists for FFA2 exhibit low potency, making them difficult to examine in vivo. This study sought to identify Gαq/11-biased FFA2-selective agonists with potent GSIS-stimulating effects. To do this, we generated an FFA2 homology model that was used to screen a library of 10 million drug-like compounds. Although FFA2 and the related short chain fatty acid receptor FFA3 share 52% sequence similarity, our virtual screen identified over 50 compounds with predicted selectivity and increased potency for FFA2 over FFA3. Subsequent in vitro calcium mobilization assays and GSIS assays resulted in the identification of a compound that can potentiate GSIS via activation of Gαq/11 with 100-fold increased potency compared with previously described Gαq/11-biased FFA2 agonists. These methods and findings provide a foundation for future discovery efforts to identify biased FFA2 agonists as potential T2D therapeutics.

[The research progress of LGR4 in the eye development and related diseases].

LGR4, also known as GPR48, is a member of the leucine-rich, G protein-coupled receptor family. It is widely expressed in tissues of the reproductive system, urinary system, sensory organs, digestive system, and central nervous system. LGR4 plays an important role in the development of various organs and cancer development and progression by modulating multiple signaling pathways. Recent studies have revealed that LGR4 is related with many kinds of human diseases such as gastrointestinal carcinomas. Eye development is a dynamic process regulated by a number of growth factors and cytokines. LGR4 is extensively expressed in the eyes in a finely tuned spatiotemporal pattern. Mice lacking LGR4 have been found to display anterior segment dysgenesis, including microphthalmia, iris hypoplasia, iridocorneal angle malformation and corneal dysgenesis, cataract and other defects. Here we review the role of LGR4 in the eye development and related molecular mechanisms. (Chin J Ophthalmol, 2017, 53: 236-240).

Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension.

BACKGROUND: Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. METHODS: In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr1]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. RESULTS: ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and ß-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in monocrotaline-exposed rats. CONCLUSIONS: These results show that ELA is an endogenous agonist of the human apelin receptor, exhibits a cardiovascular profile comparable to apelin, and is downregulated in human disease and rodent PAH models, and exogenous peptide can reduce the severity of cardiopulmonary remodeling and function in PAH in rats. This study provides additional proof of principle that an apelin receptor agonist may be of therapeutic use in PAH in humans.

The Expanding Complexity of Estrogen Receptor Signaling in the Cardiovascular System.

Estrogen has important effects on cardiovascular function including regulation of vascular function, blood pressure, endothelial relaxation, and the development of hypertrophy and cardioprotection. However, the mechanisms by which estrogen mediates these effects are still poorly understood. As detailed in this review, estrogen can regulate transcription by binding to 2 nuclear receptors, ERα and ERß, which differentially regulate gene transcription. ERα and ERß regulation of gene transcription is further modulated by tissue-specific coactivators and corepressors. Estrogen can bind to ERα and ERß localized at the plasma membrane as well as G-protein-coupled estrogen receptor to initiate membrane delimited signaling, which enhances kinase signaling pathways that can have acute and long-term effects. The kinase signaling pathways can also mediate transcriptional changes and can synergize with the ER to regulate cell function. This review will summarize the beneficial effects of estrogen in protecting the cardiovascular system through ER-dependent mechanisms with an emphasis on the role of the recently described ER membrane signaling mechanisms.

Critical Roles of STAT3 in ß-Adrenergic Functions in the Heart.

BACKGROUND: ß-Adrenergic receptors (ßARs) play paradoxical roles in the heart. On one hand, ßARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of ßARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac ßAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by ß-adrenergic agonists. To follow up this finding, we analyzed ßAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute ßAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic ß-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for ßAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of ßAR pathway, including ß1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in ßAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for ßAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.

The F-BAR Protein NOSTRIN Dictates the Localization of the Muscarinic M3 Receptor and Regulates Cardiovascular Function.

RATIONALE: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. OBJECTIVE: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. METHODS AND RESULTS: The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. CONCLUSIONS: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.