G protein-coupled receptor kinase 2 (GRK2) as a potential therapeutic target in cardiovascular and metabolic diseases

G protein-coupled receptor kinase 2 (GRK2) is a central signaling node involved in the modulation of many G protein-coupled receptors (GPCRs) and also displaying regulatory functions in other cell signaling routes. GRK2 levels and activity have been reported to be enhanced in patients or in preclinical models of several relevant pathological situations, such as heart failure, cardiac hypertrophy, hypertension, obesity and insulin resistance conditions, or non-alcoholic fatty liver disease (NAFLD), and to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Therefore, targeting GRK2 by different strategies emerges as a potentially relevant approach to treat cardiovascular disease, obesity, type 2 diabetes, or NAFLD, pathological conditions which are frequently interconnected and present as co-morbidities.Our laboratories are supported by Ministerio de Economía; Industria y Competitividad (MINECO) of Spain (grant SAF2017- 84125-R to FM and CM and SAF2016-80305-P to MS and AMB), CIBERCV-Instituto de Salud Carlos III, Spain (grants CB16/11/00278 and CB16/11/00286 to FM and MS, respectively, co-funded with European FEDER contribution), and Programa de Actividades en Biomedicina de la Comunidad de Madrid (grants B2017/BMD-3671-INFLAMUNE to FM and B2017/ BMD-3676-AORTASANA to MS)

Molecular physiopathology of obesity-related diseases: multi-organ integration by GRK2

Obesity is a worldwide problem that has reached epidemic proportions both in developed and developing countries. The excessive accumulation of fat poses a risk to health since it favours the development of metabolic alterations including insulin resistance and tissue inflammation, which further contribute to the progress of the complex pathological scenario observed in the obese. In this review we put together the different outcomes of fat accumulation and insulin resistance in the main insulin-responsive tissues, and discuss the role of some of the key molecular routes that control disease progression both in an organ-specific and also in a more systemic manner. Particularly, we focus on the importance of studying the integrated regulation of different organs and pathways that contribute to the global pathophysiology of this condition with a specific emphasis on the role of emerging key molecular nodes such as the G protein-coupled receptor kinase 2 (GRK2) signalling hubMinisterio Sanidad y Consumo-Instituto de Salud Carlos III, Spain; Grants SAF2014-55511-R and SAF2012-36400 from Ministerio de Economia y Competitividad (MINECO), Spain (to FM-CM and MS); S2010/BMD-2332 (INDISNET) from Comunidad de Madrid, Spain (to FM); an EFSDNovo Nordisk Grant (to FM) and Fundacion Ramon Areces (to CM and AMB)Peer Reviewe

G Protein-Coupled Receptor Kinase 2 (GRK2) as a Potential Therapeutic Target in Cardiovascular and Metabolic Diseases

G protein-coupled receptor kinase 2 (GRK2) is a central signaling node involved in the modulation of many G protein-coupled receptors (GPCRs) and also displaying regulatory functions in other cell signaling routes. GRK2 levels and activity have been reported to be enhanced in patients or in preclinical models of several relevant pathological situations, such as heart failure, cardiac hypertrophy, hypertension, obesity and insulin resistance conditions, or non-alcoholic fatty liver disease (NAFLD), and to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Therefore, targeting GRK2 by different strategies emerges as a potentially relevant approach to treat cardiovascular disease, obesity, type 2 diabetes, or NAFLD, pathological conditions which are frequently interconnected and present as co-morbidities

GRK2 regulates GLP-1R-mediated early phase insulin secretion in vivo

© The Author(s).[Background]: Insulin secretion from the pancreatic β-cell is finely modulated by different signals to allow an adequate control of glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) act as key physiological potentiators of insulin release through binding to the G protein-coupled receptor GLP-1R. Another key regulator of insulin signaling is the Ser/Thr kinase G protein-coupled receptor kinase 2 (GRK2). However, whether GRK2 affects insulin secretion or if GRK2 can control incretin actions in vivo remains to be analyzed. [Results]: Using GRK2 hemizygous mice, isolated pancreatic islets, and model β-cell lines, we have uncovered a relevant physiological role for GRK2 as a regulator of incretin-mediated insulin secretion in vivo. Feeding, oral glucose gavage, or administration of GLP-1R agonists in animals with reduced GRK2 levels (GRK2+/− mice) resulted in enhanced early phase insulin release without affecting late phase secretion. In contrast, intraperitoneal glucose-induced insulin release was not affected. This effect was recapitulated in isolated islets and correlated with the increased size or priming efficacy of the readily releasable pool (RRP) of insulin granules that was observed in GRK2+/− mice. Using nanoBRET in β-cell lines, we found that stimulation of GLP-1R promoted GRK2 association to this receptor and that GRK2 protein and kinase activity were required for subsequent β-arrestin recruitment. [Conclusions]: Overall, our data suggest that GRK2 is an important negative modulator of GLP-1R-mediated insulin secretion and that GRK2-interfering strategies may favor β-cell insulin secretion specifically during the early phase, an effect that may carry interesting therapeutic applications.We acknowledge support by Ministerio de Economía y Competitividad (MINECO/FEDER), Spain (grant SAF2017-84125-R to FM and CM and BFU2017-89336-R to MV); CIBER de Enfermedades Cardiovasculares (CIBERCV). Instituto de Salud Carlos III, Spain (grant CB16/11/00278 to F.M., co-funded with European FEDER contribution); CIBER de Diabetes y Enfermedades Metabólicas Asociadas (Ciberdem), Instituto de Salud Carlos III (CB07/08/0029 to MV); and Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to FM; Medical Research Council to AT and BJ

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

This is a pre-copyedited, author-produced pdf of an article accepted for publication in Journal of Molecular Cell Biology following peer review. The version of record Journal of Molecular Cell Biology 6.4 (2014) is available online at: http://dx.doi.org/10.1093/jmcb/mju025G protein-coupled receptor kinase 2 (GRK2) is an important serine/threonine-kinase regulating different membrane receptors and intracellular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts overexpression of a GRK2 kinase-deficient mutant (K220R) caused precocious differentiation of cells into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2- overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.This work was supported by Grants BFU2008-04043 (to M.L. and S.F.-V.), SAF2012-3618 (to S.F.-V.), and SAF2011-23800 (to F.M.) from Ministerio de Economía y Competitividad, Spain; S2010/BMD-2332 (INDISNET) from Comunidad de Madrid, Spain (to F.M.); CIBER de Diabetes y Enfermedades Metabólicas Asociadas and The Cardiovascular Network (RD06- 0014/0037 and RD12/0042/0012) from Ministerio Sanidad y Consumo-Instituto Carlos III, Spain (to F.M.); UAM-Banco de Santander (to C.M.); BFU2010-14884 (to M.R.-G.). S.F.-V. is recipient of a ‘Miguel Servet’ tenure track program (CP10/00438) co-financed by the European Regional Development Fund (ERDF). We also acknowledge the support of COST Action BM0602 from the European Commission (to M.L.) and institutional support from Fundación Ramón Arece

Cell-Type Specific GRK2 Interactomes: Pathophysiological Implications

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key hub in cell signaling cascades. In addition to modulating activated G protein-coupled receptors, GRK2 can phosphorylate and/or functionally interact with a complex network of cellular proteins in a cell-type and physiological context-dependent way. A combination of such canonical and noncanonical interactions underlies the participation of this kinase in the control of cell migration, proliferation or metabolism and in integrated processes at the tissue or whole organism levels, such as angiogenesis, cardiovascular function, or insulin resistance, among others. Its role as a signaling node and the fact that altered levels of GRK2 are detected in a variety of pathological conditions put forward this protein as a potentially relevant diagnostic and therapeutic targe

Influence of GRK2 levels in hepatic pathophysiology

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-04-2018Esta tesis tiene embargado el acceso al texto completo hasta el 27-04-2021G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes G protein coupled-receptors (GPCRs), and plays an important role in the development of both insulin resistance (IR) and obesity in vivo. In this work we further explore the role of GRK2 in these disorders, focusing on the establishment and progression of non-alcoholic fatty liver disease (NAFLD), a disease spectrum related to obesity and IR. We find that hepatic GRK2 protein levels are upregulated in different mouse models of steatosis and, conversely, we demonstrate that the development of NAFLD is prevented in mice with low levels of GRK2 (GRK2+/- mice) upon a high fat diet (HFD). Moreover, using an inducible GRK2 knock-out mouse model we demonstrate that decreasing GRK2 levels during the course of the high-fat feeding reverts the HFD-induced obese and insulin-resistant phenotype. GRK2 downmodulation prevents further body weight gain and fat mass accretion, causes beneficial effects in glucose tolerance and insulin sensitivity and increases the expression of markers of thermogenesis in brown adipose tissue after the HFD. In the liver, tamoxifen-induced GRK2-/- mice are protected from NAFLD, showing less signs of lipid accumulation and inflammation compared to control littermates after the HFD. We also used a methionine and choline deficient diet (MCD) as a model of non-alcoholic steatohepatitis (NASH) that is independent of fat mass accretion and IR. A blinded anatomopathological analysis revealed that GRK2+/- mice are protected from the development of NASH detected in their WT littermates. GRK2+/- mice show decreased hepatic triglyceride accumulation, inflammation and ER stress responses, while they preserve protective mechanisms against MCD-induced NASH that are lost in WT mice, as indicated by markers of autophagy or mitochondrial biogenesis and fusion. Interestingly, GRK2 protein and mRNA levels are upregulated in human liver biopsies from simple steatosis and NASH patients when compared to normal liver samples. Moreover, GRK2 overexpression is per se able to increase palmitic acid-induced steatosis in human hepatoma cells, thus establishing not only a parallelism but rather a cause-effect relationship between the expression levels of this kinase and lipid accumulation. Conversely, fasting, which induces a state of physiological hepatic steatosis, provokes a similar degree of lipid deposition in livers of WT and GRK2+/- mice, together with a marked decrease in hepatic GRK2 levels in both genotypes. However, GRK2+/- mice display higher levels of fasting serum fatty acids and glucose. Remarkably, glucagon-induced glucose production and mRNA expression of the gluconeogenic enzyme PEPCK are enhanced in mouse primary hepatocytes with silenced GRK2 demonstrating that this kinase can regulate glucagon actions in these cells. In sum, our results suggest that GRK2 acts as a central integrative node able to regulate different intracellular routes that control metabolic responses in vivo due to its unique ability to both directly modulate the insulin receptor cascade as well as key GPCRs related to metabolism (such as the glucagon receptor). These results consolidate GRK2 as a prospective therapeutic target for a potential clinical intervention in diseases with a metabolic component such as IR, obesity and NAFLD.La proteína GRK2 es una quinasa clásicamente conocida por su función en la desensibilización de receptores acoplados a proteínas G (GPCR). Sin embargo, se ha descrito que GRK2 desempeña un papel importante en el desarrollo de resistencia a la insulina (IR) y obesidad in vivo. En este trabajo, se analiza el papel de GRK2 en estas situaciones patológicas centrándonos específicamente en la enfermedad de hígado graso no alcohólica (NAFLD), una condición estrechamente relacionada con obesidad e IR. Así, encontramos que los niveles proteicos de GRK2 se incrementan en el hígado en diferentes modelos murinos de esta patología y demostramos que los ratones con niveles reducidos de GRK2 (animales GRK2 +/-) están protegidos frente al desarrollo de NAFLD inducida por una dieta alta en grasa (HFD). Además, mostramos que una reducción genética inducible en los niveles de GRK2 no sólo previene sino que revierte el desarrollo de obesidad, IR y NAFLD. Los ratones Tam-GRK2-/- (inducidos por tamoxifeno) en los que se reducen los niveles de la quinasa cuando los animales ya han desarrollado un fenotipo obeso y resistente a la insulina por la dieta dejan de ganar peso a pesar de continuar con la HFD, tienen menor acumulación de grasa y una mayor expresión de marcadores de termogénesis en el tejido adiposo marrón. Además, estos animales Tam-GRK2-/- no desarrollan NAFLD tras la HFD mostrando una menor acumulación de lípidos y menos marcadores de inflamación en el hígado en comparación con animales control. Por otro lado, se utilizó una dieta deficiente en metionina y colina (MCD) como modelo de esteatohepatitis no alcohólica (NASH) independiente de obesidad e IR. Un análisis anatomopatológico ciego reveló que los ratones GRK2+/- están protegidos frente al desarrollo de NASH, al contrario que los animales silvestres (WT) que sí desarrollan esta patología tras la MCD. En ratones GRK2+/-, la MCD causa menor acumulación de triglicéridos, inflamación y estrés de retículo en el hígado comparado con animales WT. Además, estos animales GRK2 +/- conservan mecanismos de protección frente a NASH que se pierden en ratones WT, como indican los marcadores de autofagia o de biogénesis y fusión mitocondrial. Los niveles de proteína y mRNA de GRK2 se encuentran aumentados en biopsias hepáticas de pacientes diagnosticados con esteatosis simple o NASH en comparación con individuos con histología hepática normal. Además, la sobreexpresión de GRK2 en una línea de células hepáticas humanas provoca per se una mayor esteatosis inducida por ácido palmítico estableciendo así no sólo un paralelismo sino una relación causa-efecto entre los niveles de GRK2 y la acumulación de lípidos intracelulares en hepatocitos. El ayuno, que induce un estado de esteatosis hepática fisiológica, provoca la acumulación de grasa en hígado además de una importante reducción en los niveles de GRK2 en la misma medida en ratones WT y GRK2+/-. Los ratones GRK2 +/- tienen niveles más altos de ácidos grasos libres circulantes así como de glucosa en ayunas. El silenciamiento de GRK2 en hepatocitos primarios de ratón provoca una mayor producción de glucosa a partir de piruvato y un aumento en la expresión de la enzima gluconeogénica PEPCK inducidas por glucagón, lo que demuestra que GRK2 puede regular las acciones del glucagón. Nuestros resultados identifican a GRK2 como un nodo señalizador al ser capaz de regular tanto la cascada de la insulina como GPCRs importantes para el control del metabolismo como el receptor de glucagón. Nuestros resultados consolidan esta quinasa como una posible diana terapéutica para una posible intervención clínica en enfermedades con un componente metabólico como IR, obesidad y NAFLD

Obesity-induced cardiac lipid accumulation in adult mice is modulated by G protein-coupled receptor kinase 2 levels

[Background] The leading cause of death among the obese population is heart failure and stroke prompted by structural and functional changes in the heart. The molecular mechanisms that underlie obesity-related cardiac remodeling are complex, and include hemodynamic and metabolic alterations that ultimately affect the functionality of the myocardium. G protein-coupled receptor kinase 2 (GRK2) is an ubiquitous kinase able to desensitize the active form of several G protein-coupled receptors (GPCR) and is known to play an important role in cardiac GPCR modulation. GRK2 has also been recently identified as a negative modulator of insulin signaling and systemic insulin resistance.[Methods] We investigated the effects elicited by GRK2 downregulation in obesity-related cardiac remodeling. For this aim, we used 9 month-old wild type (WT) and GRK2+/− mice, which display circa 50% lower levels of this kinase, fed with either a standard or a high fat diet (HFD) for 30 weeks. In these mice we studied different parameters related to cardiac growth and lipid accumulation.[Results] We find that GRK2+/− mice are protected from obesity-promoted cardiac and cardiomyocyte hypertrophy and fibrosis. Moreover, the marked intracellular lipid accumulation caused by a HFD in the heart is not observed in these mice. Interestingly, HFD significantly increases cardiac GRK2 levels in WT but not in GRK2+/− mice, suggesting that the beneficial phenotype observed in hemizygous animals correlates with the maintenance of GRK2 levels below a pathological threshold. Low GRK2 protein levels are able to keep the PKA/CREB pathway active and to prevent HFD-induced downregulation of key fatty acid metabolism modulators such as Peroxisome proliferator-activated receptor gamma co-activators (PGC1), thus preserving the expression of cardioprotective proteins such as mitochondrial fusion markers mitofusin MFN1 and OPA1.[Conclusions] Our data further define the cellular processes and molecular mechanisms by which GRK2 down-regulation is cardioprotective during diet-induced obesity, reinforcing the protective effect of maintaining low levels of GRK2 under nutritional stress, and showing a role for this kinase in obesity-induced cardiac remodeling and steatosis.This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO, Grant SAF2014-55511-R to F. M and C. M); Fundación Ramón Areces (to C. M.); Comunidad de Madrid Grant S2010/BMD-2332 (Inter-Disciplinary Research Network); Ministerio Sanidad y Consumo-Instituto Carlos III Cardiovascular Network (Grant RD12/0042/0012 to F. M. and RD12/0042/0033 (to V. C.); and PI15/01060 (to V. C.). R. V. B. is recipient of a postdoctoral contract by the MINECO. We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Sex differences in high fat diet-induced metabolic alterations correlate with changes in the modulation of GRK2 levels

A differential sex-related sensitivity has been reported in obesity and insulin resistance-related cardio-metabolic diseases, with a lower incidence of these pathologies being observed in young females when compared to age-matched males. However, such relative protection is lost with age. The mechanisms underlying such sex and age-related changes in the susceptibility to diabetes and obesity are not fully understood. Herein, we report that the relative protection that is displayed by young female mice, as compared to male littermates, against some of the metabolic alterations that are induced by feeding a high fat diet (HFD), correlates with a lower upregulation of the protein levels of G protein-coupled receptor kinase (GRK2), which is a key regulator of both insulin and G protein-coupled receptor signaling, in the liver and adipose tissue. Interestingly, when the HFD is initiated in middle-aged (32 weeks) female mice, these animals are no longer protected and display a more overt obese and insulin-resistant phenotype, along with a more evident increase in the GRK2 protein levels in metabolically relevant tissues in such conditions. Our data suggest that GRK2 dosage might be involved in the sex and age-biased sensitivity to insulin resistance-related pathologies.Agencia Estatal de Investigación of Spain (grant SAF2014-55511-R and SAF2017-84125-R to F.M.J. and C.M.), CIBERCV-Instituto de Salud Carlos III, Spain (grant CB16/11/00278 to F.M., co-funded with European Regional Development Fund-FEDER contribution), Programa de Actividades en Biomedicina de la Comunidad de Madrid (grant B2017/BMD-3671-INFLAMUNE to F.M.J.) and Fundación Ramón Areces.Peer reviewe

G protein-coupled receptor kinase 2 plays a role in the development of non-alcoholic steatohepatitis

Resumen del póster presentado al XXXIX Congreso anual de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca, del 5 al 8 de septiembre 2016.[Introduction]: Insulin resistance (IR) and obesity are major health problems and important risk factors for the development of non-alcoholic fatty liver disease, a disease spectrum that may include hepatic steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. G protein-coupled receptor kinase 2 (GRK2), first identified as a regulator of G protein-coupled receptors (GPCRs), has been described to play a relevant role in the development of IR and obesity in vivo. However, the effect of GRK2 in the development of NASH had not been addressed so far. Since the deletion of GRK2 prevents excessive body weight gain, we fed WT and GRK2 global hemizygous mice (GRK2+/-) with a methionine and choline-deficient diet (MCD), a well stablished model of NASH that is independent of fat mass accretion. [Results]: Even though the MCD diet induced similar metabolic alterations and a comparable elevation in plasma transaminase activity in WT and GRK2+/- mice, other negative effects of the MCD were partially alleviated in GRK2 +/- animals. The increase in hepatic triglyceride content caused by this diet was significantly lower in GRK2+/- mice and, interestingly, MCD feeding induced an increase in GRK2 protein levels in WT but not in GRK2+/- livers. Moreover, GRK2+/- mice presented protection from some deleterious effects of MCD in the liver as indicated by reduced markers of endoplasmic reticulum stress, and conversely maintained some hepatic protective mechanisms such as autophagy or mitochondrial fusion. Accordingly, these results provide a link between GRK2 levels and hepatic lipid homeostasis leading to NASH. [Conclusion]: Taken together, these results suggest a role for GRK2 in the establishment and/or development of NASH.Peer reviewe