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Additional file 1: Supplementary Figure 1. Control of specificity of GRK2 ‘PF2’ antibody in immunohistochemistry and islets detection in WT and GRK2+/- pancreatic sections. A) Representative photomicrographs showing the immunohistochemical staining of serial pancreatic sections obtained from Tamoxifen-inducible GRK2-/- mice (Tx- GRK2-/-, Vila-Bedmar et al., 2015), WT and GRK2+/- mice using the ‘PF2’ antibody against GRK2, counterstained with hematoxylin (magnification 40x; image size adjusted to the islet area) or B) pancreas from WT and GRK2+/- mice using a 4x magnification and insulin as an islet marker (scale bar, 0.5 mm). Arrows indicate the location of the islets. Incubations without primary antibody were performed as a negative control.

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-04-2018, Esta tesis tiene embargado el acceso al texto completo hasta el 27-04-2021, G 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.

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017., Non-alcoholic fatty liver disease (NAFLD) is an extremely prevalent hepatic pathology that affects about one quarter of adults in the developed world. This disease spectrum includes diff erent conditions ranging from simple steatosis, to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Insulin resistance and obesity are two well-established pathogenic factors associated to NAFLD, and in fact NAFLD is a very common condition among patients with type 2 diabetes. G protein-coupled receptor kinase 2 (GRK2), classically known as a regulator of G protein-coupled receptors, has been described to play a relevant role in the development of IR and obesity in vivo. However, the eff ect of GRK2 in the development of NASH had not been addressed so far. Here we investigate the contribution of GRK2 to NASH in mice subjected to a methionine and choline-deficient diet (MCD), a well-stablished model of NASH that is independent of fat mass accretion. [Results]: We analyzed a variety of metabolic parameters in WT and GRK2+/- mice fed a MCD diet for four weeks. MCD induced similar alterations and a comparable elevation in plasma transaminase activity in WT and GRK2+/- mice. However, steatosis and other negative effects of this diet in the liver were partially alleviated in GRK2 +/- animals. For instance, the increase in hepatic triglyceride accumulation? and in the liver to body weight ratio induced by MCD were significantly lower in GRK2+/- mice. Furthermore, pathophysiological examination revealed signifi cantly lower NAFLD activity score (NAS) in GRK2+/- mice which were protected from overt NASH. GRK2+/- mice also presented protection from some deleterious effects of the MCD in the liver, such as reduced expression of markers of endoplasmic reticulum stress and inflammation. Moreover, GRK2+/- mice retained certain hepatic protective mechanisms after the MCD as revealed using markers of autophagy and mitochondrial fusion processes. We also found that MCD feeding induced an increase in GRK2 protein levels in WT but not in GRK2+/- livers, similar to what is observed in hepatic tissue of mice fed with a high fat diet. Interestingly, a similar increase in GRK2 protein was also observed in human hepatic samples of NAFLD patients by immunohistochemistry. In the same line, we found that Huh7 cells overexpressing GRK2 accumulated more lipids when exposed to palmitic acid compared to controls. [Conclusion]: Our results uncover a role for GRK2 in the regulation of hepatic lipid metabolism, and provide a proof of concept that reducing the levels of this kinase can impair the development of NASH.

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.

Additional file 1: Figure S1. A) Weight gain induced by 30 weeks of HFD feeding in WT and GRK2+/− genotypes expressed as fold-increase over control SD-fed mice (N = 5–7). Data are mean ± SEM. ++p

Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.-- et al., [Background]: Insulin resistance (IR) and obesity are major health problems and important risk factors for the development of non-alcoholic fatty liver disease (NAFLD), a disease spectrum that includes hepatic steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. G proteincoupled receptor kinase 2 (GRK2), fi rst identifi ed as a G protein-coupled receptor regulator, has been recently described to play a relevant role in IR and obesity in vivo. GRK2 hemizygous (GRK2+/-) mice are protected against high fat diet (HFD)-induced systemic and hepatic insulin resistance and obesity. However, the effect of GRK2 in the development of HFD-induced NAFLD has not been studied. [Materials and methods]: Given the lack of a potent and specific GRK2 inhibitor, we used a global tamoxifen (Tx)-inducible murine model in which GRK2 levels are decreased once HFD-induced IR has been established. Also, a long term chronic state of IR and obesity was triggered by feeding wild type (WT) and GRK2+/- mice a 32 week-long HFD. To discriminate the effects of the differential body weight gain, we fed mice with a methionine-choline defi cient diet (MCD), which induces NAFLD independently of fat mass accretion. [Results]: Systemic insulin sensitivity was preserved after decreasing GRK2 levels in the middle of a high fat feeding using tamoxifen, and accordingly enhanced insulin signaling was observed in the liver of HFD-fed Tx-induced GRK2-/- mice. Moreover, hepatic steatosis, a hallmark of NAFLD, was decreased in these mice. Also, different signs of infl ammation present in WT mice were absent in Tx-induced GRK2-/- animals. Nevertheless, after a long term HFD we found no differences in steatosis between WT and GRK2+/- mice, despite the decrease in body weight gain and liver weight. Finally, we found an increase in GRK2 protein levels in the liver of mice fed a MCD, a well established model of NASH, similar to what is detected during HFD feeding in WT animals, and we are further characterizing this model. [Conclusion]: Taken together, these results suggest a role for GRK2 in the establishment and development of NAFLD.