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Nonalcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge with activated GPCRs to modulate cell-signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of G protein α12 (Gα12) on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 levels in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12-KO liver revealed that the Gα12-signaling pathway regulated sirtuin 1 (SIRT1) and PPARα, which are responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12-KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of ubiquitin-specific peptidase 22 (USP22) via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12-KO mice fed a high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.

Current drugs for treating heart failure (HF), for example, angiotensin II receptor blockers and β-blockers, possess specific target molecules involved in the regulation of the cardiac circulatory system. However, most clinically approved drugs are effective in the treatment of HF with reduced ejection fraction (HFrEF). Novel drug classes, including angiotensin receptor blocker/neprilysin inhibitor (ARNI), sodium-glucose co-transporter-2 (SGLT2) inhibitor, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, soluble guanylyl cyclase (sGC) stimulator/activator, and cardiac myosin activator, have recently been introduced for HF intervention based on their proposed novel mechanisms. SGLT2 inhibitors have been shown to be effective not only for HFrEF but also for HF with preserved ejection fraction (HFpEF). In the myocardium, excess cyclic adenosine monophosphate (cAMP) stimulation has detrimental effects on HFrEF, whereas cyclic guanosine monophosphate (cGMP) signaling inhibits cAMP-mediated responses. Thus, molecules participating in cGMP signaling are promising targets of novel drugs for HF. In this review, we summarize molecular pathways of cGMP signaling and clinical trials of emerging drug classes targeting cGMP signaling in the treatment of HF. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8-mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8-producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI., Introduction Myocardial infarction (MI) is a major disease worldwide (1, 2). Following MI, cardiomyocytes receiving blood supply from occluded blood vessels die rapidly due to the shortage of oxygen and [...]

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver. [ABSTRACT FROM AUTHOR]

Abstract.: Overexpression of constitutively active (CA)-Gα13 significantly increased the expression of interleukin (IL)-1β and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1β mRNA induction by CA-Gα13 was suppressed by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, but not by BAPTA-AM, an intracellular Ca2+ chelator. In contrast, IL-6 mRNA induction by CA-Gα13 was suppressed by BAPTA-AM but not by DPI. However, both IL-1β and IL-6 mRNA induction was suppressed by nuclear factor κB (NF-κB) inhibitors. The CA-Gα13-induced NF-κB activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-Gα13 was suppressed by SK&F96365 (1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-Gα13 increased basal Ca2+ influx. These results suggest that Gα13 regulates IL-1β mRNA induction through the reactive oxygen species-NF-κB pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-κB pathway. Keywords:: cardiac fibroblast, Gα13 protein, nuclear factor of κB, Ca2+, interleukin

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR 1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/ MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKR1 predominates over PKR2. PKR1 was colocalized with G[[alpha].sub.11] and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Gall expression in H5V cells effectively inhibited prokineticin-2-induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/G[[alpha].sub.11] in this process. However, in conditions in which PKR2 predominated over PKR1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with G[[alpha].sub.12], and activated G[[alpha].sub.12], which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR 1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors. angiogenesis; signaling; G proteins doi:10.1152/ajpheart.00898.2009

Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels. [ABSTRACT FROM AUTHOR]

Author(s): Motohiro Nishida [1]; Yoshiko Maruyama [1]; Rie Tanaka [1]; Kenji Kontani [2]; Taku Nagao [1]; Hitoshi Kurose (corresponding author) [1] Reactive oxygen species (ROS) have been identified as central [...]

Internalization of G protein-coupled receptors (GPCRs) is a fundamental mechanism to maintain cellular homeostasis by switching off receptor-mediated signaling and controlling protein quality of these receptors ([1], [2]). In addition, two ITC groups were necessary to exert an inhibitory effect on P2Y SB 6 sb R. MRS2578 oligomerized P2Y SB 6 sb R, but other compounds with one ITC failed to oligomerize P2Y SB 6 sb R (fig. Dimethyl itaconate (DI), a cell-permeable itaconate analog, also caused P2Y SB 6 sb R degradation in HEK293 cells and decreased UDP-induced CCL2 release in RAW264.7 cells (fig. Because a MRS2578 derivative that lacks ITC residues loses the inhibitory action on P2Y SB 6 sb R ([9]), we investigated whether other ITC-containing compounds also inhibited P2Y SB 6 sb R-stimulated C-C motif chemokine 2 (CCL2) release in RAW264.7 cells. [Extracted from the article]

Myocardial infarction is one of the major causes of death worldwide. Many heart cells die during myocardial infarction through various processes such as necrosis, apoptosis, necroptosis, autophagy-related cell death, pyroptosis and ferroptosis. These dead cells in infarcted hearts expose the so-called 'eat-me' signals, such as phosphatidylserine, on their surfaces, enhancing their removal by professional and non-professional phagocytes. Clearance of dead cells by phagocytes in the diseased hearts plays a crucial role in the pathology of myocardial infarction by inhibiting the inflammatory responses caused by the leakage of contents from dead cells. This review focuses on the rapidly growing understanding of the molecular mechanisms of dead cell phagocytosis, termed efferocytosis, during myocardial infarction, which contributes to the pathophysiology of myocardial infarction. [ABSTRACT FROM AUTHOR]

Several studies are conducted to understand the molecular mechanisms associated with the constitutive expression of the glutathione S-transferase (GST) isozymes during differentiation of the Caco-2 cells in humans. The expression of the GST isozymes is shown to be highly regulated by the Kelch-like ECH-associated protein 1 (Keap1).

Heart failure (HF) is a heart disease that is classified into two main types: HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF). Both types of HF lead to significant risk of mortality and morbidity. Pharmacological treatment with β-adrenergic receptor (βAR) antagonists (also called β-blockers) has been shown to reduce the overall hospitalization and mortality rates and improve the clinical outcomes in HF patients with HFrEF but not HFpEF. Although, the survival rate of patients suffering from HF continues to drop, the management of HF still faces several limitations and discrepancies highlighting the need to develop new treatment strategies. Overstimulation of the sympathetic nervous system is an adaptive neurohormonal response to acute myocardial injury and heart damage, whereas prolonged exposure to catecholamines causes defects in βAR regulation, including a reduction in the amount of βARs and an increase in βAR desensitization due to the upregulation of G protein-coupled receptor kinases (GRKs) in the heart, contributing in turn to the progression of HF. Several studies show that myocardial GRK2 activity and expression are raised in the failing heart. Furthermore, β-arrestins play a pivotal role in βAR desensitization and, interestingly, can mediate their own signal transduction without any G protein-dependent pathway involved. In this review, we provide new insight into the role of GRKs and β-arrestins on how they affect βAR signaling regarding the molecular and cellular pathophysiology of HF. Additionally, we discuss the therapeutic potential of targeting GRKs and β-arrestins for the treatment of HF. [ABSTRACT FROM AUTHOR]

We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugatesin situin the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81μg/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten. [ABSTRACT FROM PUBLISHER]

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions. [ABSTRACT FROM AUTHOR]

Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI. [ABSTRACT FROM AUTHOR]

Hyperactivation of Gq signaling causes cardiac hypertrophy, and β-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of Gαs in neonatal rat cardiomyocytes, reduced Gαs expression, and suppressed cAMP response to β-adrenergic receptor stimulation. Ectopic expression of Gαq induced a similar suppression, which is due to the degradation of Gαs by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of Gαs, effectively canceled the Gαq-induced ubiquitination of Gαs and recovered the cAMP accumulation. In vitro, Gαq competes for the binding of Gαs to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure. [ABSTRACT FROM AUTHOR]

Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation. [ABSTRACT FROM AUTHOR]