Activation Of Gq Signaling Research Articles

AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

The choroid plexus (CP) is a source of trophic factors for the developing and mature brain. Insulin is produced in epithelial cells of the CP (EChPs), and its secretion is stimulated by Htr2c-mediated signaling. We modulated insulin expression in EChPs with intracerebroventricular injections of AAV5. Insulin overexpression in CP decelerates food intake, whereas its knockdown has the opposite effect. Insulin overexpression also results in reduced anxious behavior. Transcriptomic changes in the hypothalamus, especially in synapse-related processes, are also seen in mice overexpressing insulin in CP. Last, activation of Gq signaling in CP leads to acute Akt phosphorylation in neurons of the arcuate nucleus, indicating a direct action of CP-derived insulin on the hypothalamus. Taken together, our findings signify that CP is a relevant source of insulin in the central nervous system and that CP-derived insulin should be taken into consideration in future work pertaining to insulin actions in the brain.

Abstract 102: Mitoquinone Reduces Preeclamptic Phenotypes Associated With Syncytiotrophoblast-Specific Gq Signaling

Syncytiotrophoblast (STB) stress is theorized to drive the pathogenesis of preeclampsia (PE), and Gq-activating hormones implicated in PE have been associated with placenta oxidative damage, mitochondrial dysfunction, and maternal symptomology. As the causes and consequences of STB stress remain largely unknown, we used an in vivo model to determine whether excess STB-specific Gq signaling is sufficient to cause phenotypes of PE and if a mitochondrial-targeted antioxidant (mitoquinone) mitigates these effects. Activation of Gq signaling in STB cells was achieved by crossing dams harboring a cre-dependent Gq-coupled DREADD (hM3Dq) with Gcm1 -Cre +/- sires and injecting clozapine N-oxide (CNO) or saline mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gq activation resulted in maternal proteinuria (n=9-11 CNO 43±4 vs saline 28±3mg/day; p=0.01), mild segmental congestion of glomerular capillaries, and elevated circulating IP-10 (p=0.03), MCP-5 (p=0.04), and IL-12p40 (p=0.01). hM3Dq stimulation reduced labyrinth vascularization (n=10 Cre + 19±1%, Cre - 24±1%, saline 23±1%; p<0.01 Cre + vs each), spiral artery diameter (n=7-11 Cre + 110±11μm, Cre - 135±9, saline 149±12 μm; p=0.03), placenta mass (n=10-13 Cre + 0.12±0.01, Cre - 0.14±0.01, saline 0.13±0.01g; p<0.01), and fetal mass (n=10-13 Cre + 0.23±0.03, Cre - 0.30±0.02, saline 0.29±0.02g; p<0.01). Preliminary data suggest a placental oxidative defense response to Gq induction based on increased protein levels of SOD2 and catalase, and decreased malondyaldehyde. Mitoquinone (5mg/kg) corrected maternal proteinuria (n=8 22±4mg/day, p<0.01), decreased circulating IL-12p40 (p=0.03), and protected both labyrinth vascularization (n=8 23±1%, p=0.05) and spiral artery diameter (n=8 164±18μm, p=0.02). These data indicate that elevated Gq signaling in the STB layer is sufficient to cause pathological features of PE, via a mechanism involving mitochondrial stress.

32-OR: Receptor-Mediated Gq Signaling in Adipocytes as a Critical Modulator of Systemic Glucose and Lipid Homeostasis

Objective: In obesity, decreased insulin action leads to the accelerated breakdown of triglycerides stored in adipocytes, causing peripheral insulin resistance, a hallmark of type 2 diabetes. In this study, we investigated whether selective activation of Gq signaling in adipocytes might cause similar metabolic effects as insulin. Methods: We generated a novel mutant mouse strain that expressed a CNO-sensitive, Gq-coupled designer G protein-coupled receptor (Gq DREADD = QGD) selectively in adipocytes (adipo-GqD mice). DREADDs are stimulated by an exogenous agent (e.g., CNO) that is otherwise pharmacologically inactive. We also made another mouse strain that selectively lacked Galpha-q and -11 in adipocytes (adipo-Gq/11 KO mice). Both mutant mouse strains underwent a series of metabolic tests. Results: CNO-mediated activation of adipocyte Gq signaling in the adipo-GqD mice significantly improved glucose homeostasis and suppressed lipolysis. In contrast, adipo-Gq/11 KO mice showed the opposite metabolic phenotypes, impaired glucose metabolism and enhanced lipolysis. Consistent with these findings, in vitro studies with cultured adipocytes demonstrated that activation of Gq signaling enhanced glucose uptake and inhibited lipolysis in an AMPK-dependent manner. To test the hypothesis that activation of adipocyte Gq signaling might counteract impaired insulin signaling in adipocytes, we generated adipo-GqD mice that carried only one copy of the insulin receptor (IR) gene in adipocytes (adipo-GqD-IR+/- mice). Interestingly, activation of adipocyte Gq signaling by CNO ameliorated the metabolic deficits displayed by adipo-GqD-IR+/- mice maintained on a high-fat diet. Conclusion: Our data indicate that adipocyte Gq signaling represents an essential regulator of whole-body glucose and lipid homeostasis. This finding is likely to stimulate the development of novel drugs that can mimic the actions of insulin in the insulin-resistant state. Disclosure T. Kimura: None. S. Pydi: None. L. Wang: None. Y. Cui: None. O. Gavrilova: None. J. Wess: None.

1971-P: Activation of Adipocyte Gq Signaling Causes Improved Whole-Body Glucose Homeostasis

Objective: Obesity and type 2 diabetes have emerged as severe threats to human health worldwide. Adipocytes play a key role in the regulation of whole-body glucose and energy homeostasis. The aim of this study was to examine the metabolic effects of selective activation of Gq signaling in adipocytes in vivo. Methods: We generated a novel mutant mouse strain that expressed a CNO-sensitive designer G protein-coupled receptor (Gq DREADD = GqD) which couples to Gq selectively in adipocytes (adipo-GqD mice). Notably, DREADDs can only be activated by an exogenous agent (CNO) that is otherwise pharmacologically inert. We also created a mouse strain that selectively lacked Galpha-q and -11 in adipocytes (adipo-Gq/11 KO mice). Mice consuming either regular chow or a high-fat diet were subjected to a series of metabolic tests. Results: CNO-mediated activation of GqD in adipo-GqD mice caused significant improvements in glucose homeostasis and suppressed lipolysis, while adipo-Gq/11 KO mice showed opposite metabolic phenotypes (impaired glucose metabolism and enhanced lipolysis). In agreement with these findings, CNO treatment of GqD-expressing 3T3-F442A adipocytes resulted in enhanced glucose uptake and inhibited CL316,243-induced lipolysis. Conclusion: Studies with newly generated mouse models allowed us to evaluate the metabolic relevance of adipocyte Gq signaling in vivo. Most strikingly, Gq activation in adipocytes led to improved glucose homeostasis and reduced lipolysis in mice. These findings are of considerable relevance for the development of novel antidiabetic drugs. Disclosure T. Kimura: None. S. Pydi: None. L. Wang: None. Y. Cui: None. O. Gavrilova: None. J. Wess: None. Funding Japan Society for the Promotion of Science

Activation of Gq signaling by Pasteurella multocida toxin inhibits the osteoblastogenic-like actions of Activin A in C2C12 myoblasts, a cell model of fibrodysplasia ossificans progressiva.

The human disease fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling disorder of extensive heterotopic bone growth that is caused by a point mutation (R206H) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. The mutation leads to extensive BMP-signaling induced by Activin A, which is normally an antagonist for wildtype receptors, resulting in excessive and uncontrolled bone formation. Here, we studied the effects of Pasteurella multocida toxin (PMT), which activates osteoclasts and inhibits osteoblast activity, in C2C12 myoblasts expressing the mutant Alk2(R206H) receptor as model of FOP. In our study, we mainly used alkaline phosphatase (ALP) activity as marker to determine osteoblast differentiation. BMP-4 stimulated an increase in ALP activity in C2C12-Alk2wt and C2C12-Alk2(R206H) cells. By contrast, Activin A only induced ALP activity in C2C12-Alk2(R206H) cells. In both cases, PMT acted as a potent inhibitor of ALP activity. PMT-induced inhibition of ALP activity was paralleled by a constitutive activation of the heterotrimeric Gq protein. Expression of a permanently active Gαq blocked Activin A/Alk2(R206H)-dependent increase in ALP activity. Inactivation of Gq by specific inhibitor FR900359 blocked the PMT effect. Similarly, canonical second messengers and effectors of Gαq (e.g. ionophore A23187-induced increase in intracellular Ca2+ and activation of PKC by PMA (phorbol 12-myristate 13-acetate)) inhibited Alk2(R206H)-mediated induction of ALP activity. Notably, Activin A-induced increase in ALP activity in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine. PMT did not alter tail phosphorylation of the major downstream effectors of the Alk2 receptor, Smad1/5/9; neither did the toxin affect nuclear translocation of the Smad-complex. However, PMT diminished BMP responsive element-induced gene expression. The data indicate that PMT potently inhibits the induction of osteoblast markers in a FOP model via activation of G proteins. Moreover, our findings indicate that activation of G protein-coupled receptors and of G protein signaling might be a rationale for pharmacological therapy of FOP.

44-OR: Human Pseudo Islets Reveal Differential Effects of Gq Signaling in ß Cells in Low and High Glucose

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p<0.05 β and α cells). In perifusion, pseudoislets were similar to native islets in biphasic glucose-stimulated and cAMP-potentiated insulin secretion as well as in epinephrine and low glucose stimulation of and high glucose inhibition of glucagon release. Adenoviral-transduced dispersed human islet cells (>90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p<0.0001). In contrast, hM3Dq and control pseudoislets had similar insulin secretion in high glucose (11mM G + 10μM CNO; 24.8 vs. 23.2 ng/100IEQ/hr; p>0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.

Increased ubiquitination and the crosstalk of G protein signaling in cardiac myocytes: involvement of Ric-8B in Gs suppression by Gq signal

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.