microRNAs-mediated regulation of insulin signaling in white adipose tissue during aging: Role of caloric restriction.

Caloric restriction is a non-pharmacological intervention known to ameliorate the metabolic defects associated with aging, including insulin resistance. The levels of miRNA expression may represent a predictive tool for aging-related alterations. In order to investigate the role of miRNAs underlying insulin resistance in adipose tissue during the early stages of aging, 3- and 12-month-old male animals fed ad libitum, and 12-month-old male animals fed with a 20% caloric restricted diet were used. In this work we demonstrate that specific miRNAs may contribute to the impaired insulin-stimulated glucose metabolism specifically in the subcutaneous white adipose tissue, through the regulation of target genes implicated in the insulin signaling cascade. Moreover, the expression of these miRNAs is modified by caloric restriction in middle-aged animals, in accordance with the improvement of the metabolic state. Overall, our work demonstrates that alterations in posttranscriptional gene expression because of miRNAs dysregulation might represent an endogenous mechanism by which insulin response in the subcutaneous fat depot is already affected at middle age. Importantly, caloric restriction could prevent this modulation, demonstrating that certain miRNAs could constitute potential biomarkers of age-related metabolic alterations.

Novel Insights in the Physiopathology and Management of Obesity-Related Kidney Disease.

Obesity is recognized as an independent risk factor for the development of kidney disease, which has led to the designation of obesity-related glomerulopathy (ORG). Common renal features observed in this condition include glomerular hypertrophy, glomerulosclerosis, haemodynamic changes and glomerular filtration barrier defects. Additionally, and although less studied, obesity-related kidney disease also involves alterations in renal tubules, including tubule hypertrophy, lipid deposition and tubulointerstitial fibrosis. Although not completely understood, the harmful effects of obesity on the kidney may be mediated by different mechanisms, with alterations in adipose tissue probably playing an important role. An increase in visceral adipose tissue has classically been associated with the development of kidney damage, however, recent studies point to adipose tissue surrounding the kidney, and specifically to the fat within the renal sinus, as potentially involved in the development of ORG. In addition, new strategies for the treatment of patients with obesity-related kidney disease are focusing on the management of obesity. In this regard, some non-invasive options, such as glucagon-like peptide-1 (GLP-1) receptor agonists or sodium-glucose cotransporter-2 (SGLT2) inhibitors, are being considered for application in the clinic, not only for patients with diabetic kidney disease but as a novel pharmacological strategy for patients with ORG. In addition, bariatric surgery stands as one of the most effective options, not only for weight loss but also for the improvement of kidney outcomes in obese patients with chronic kidney disease.

Tackling the effects of extracellular vesicles in fibrosis.

Fibrosis is a physiological process of tissue repair that turns into pathological when becomes chronic, damaging the functional structure of the tissue. In this review we outline the current status of extracellular vesicles as modulators of the fibrotic process at different levels. In adipose tissue, extracellular vesicles mediate the intercellular communication not only between adipocytes, but also between adipocytes and other cells of the stromal vascular fraction. Thus, they could be altering essential processes for the functionality of adipose tissue, such as adipocyte hypertrophy/hyperplasia, tissue plasticity, adipogenesis and/or inflammation, and ultimately trigger fibrosis. This process is particularly important in obesity, and may eventually, influence the development of obesity-associated alterations. In this regard, obesity is now recognized as an independent risk factor for the development of chronic kidney disease, although the role of extracellular vesicles in this connection has not been explored so far. Nonetheless, the role of extracellular vesicles in the onset and progression of renal fibrosis has been highlighted due to the critical role of fibrosis as a common feature of kidney diseases. In fact, the content of extracellular vesicles disturbs cellular signaling cascades involved in fibrosis in virtually all types of renal cells. What is certain is that the study of extracellular vesicles is complex, as their isolation and manipulation is still difficult to reproduce, which complicates the overview of their physiopathological effects. Nevertheless, new strategies have been developed to exploit the potential of extracellular vesicles and their cargo, both as biomarkers and as therapeutic tools to prevent the progression of fibrosis towards an irreversible event.

Lipidomic and Metabolomic Signature of Progression of Chronic Kidney Disease in Patients with Severe Obesity.

Severe obesity is a major risk for chronic kidney disease (CKD). Early detection and careful monitoring of renal function are critical for the prevention of CKD during obesity, since biopsies are not performed in patients with CKD and diagnosis is dependent on the assessment of clinical parameters. To explore whether distinct lipid and metabolic signatures in obesity may signify early stages of pathogenesis toward CKD, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-high resolution accurate mass-mass spectrometry (GC-HRAM-MS) analyses were performed in the serum and the urine of severely obese patients with and without CKD. Moreover, the impact of bariatric surgery (BS) in lipid and metabolic signature was also studied, through LC-MS and GC-HRAM-MS analyses in the serum and urine of patients with severe obesity and CKD before and after undergoing BS. Regarding patients with severe obesity and CKD compared to severely obese patients without CKD, serum lipidome analysis revealed significant differences in lipid signature. Furthermore, serum metabolomics profile revealed significant changes in specific amino acids, with isoleucine and tyrosine, increased in CKD patients compared with patients without CKD. LC-MS and GC-HRAM-MS analysis in serum of patients with severe obesity and CKD after BS showed downregulation of levels of triglycerides (TGs) and diglycerides (DGs) as well as a decrease in branched-chain amino acid (BCAA), lysine, threonine, proline, and serine. In addition, BS removed most of the correlations in CKD patients against biochemical parameters related to kidney dysfunction. Concerning urine analysis, hippuric acid, valine and glutamine were significantly decreased in urine from CKD patients after surgery. Interestingly, bariatric surgery did not restore all the lipid species, some of them decreased, hence drawing attention to them as potential targets for early diagnosis or therapeutic intervention. Results obtained in this study would justify the use of comprehensive mass spectrometry-based lipidomics to measure other lipids aside from conventional lipid profiles and to validate possible early markers of risk of CKD in patients with severe obesity.

Renoprotective role of bariatric surgery in patients with established chronic kidney disease.

BACKGROUND: Bariatric surgery (BS) has been postulated as the most effective measure for weight reduction. Weight loss improves metabolic parameters and exerts changes in renal function that lead to the amelioration of absolute or relative glomerular hyperfiltration, a condition that may be renoprotective in the long term. However, few studies have demonstrated the influence of BS in patients with severe obesity and chronic kidney disease (CKD). Our objective was to analyse the evolution of renal function, adipose tissue-derived molecules and inflammatory parameters in patients with CKD after BS. METHODS: This is an observational and prospective study. Thirty patients were screened and 12 were included between January 2016 and January 2018 with a 24-month follow-up. Glomerular filtration rate (GFR) was determined by plasma iohexol clearance. Adipokines, cytokines, circulating hormones and fibrotic parameters were evaluated before and 12 months after BS using the Bioplex system. RESULTS: The mean age was 50.6 years and 58.3% were males. Seven patients had a body mass index >40 kg/m2 and 66.7% were diabetic. Twenty-four months following BS there was a significant decrease in body weight (36.4%). Proteinuria decreased by 63.7 ± 28.2%. Measured GFR significantly diminished from before surgery to Month 24 after surgery (94 ± 44 to 79 ± 44 mL/min, P = 0.03). There was a significant decrease in adipocyte-derived molecules (leptin and vifastin) as well as in pro-inflammatory cytokines [interleukin (IL)-1ß, tumour necrosis factor α, IL-6 and monocyte chemoattractant protein-1] and other circulating factors (vascular endothelial growth factor and transforming growth factor ß isoforms). CONCLUSIONS: BS is an effective option to prevent kidney damage in obese subjects with CKD due to the improvement of glomerular hyperfiltration, adipocyte cytokines metabolic and inflammatory parameters.

Cardiac GRK2 Protein Levels Show Sexual Dimorphism during Aging and Are Regulated by Ovarian Hormones.

Cardiovascular disease (CVD) risk shows a clear sexual dimorphism with age, with a lower incidence in young women compared to age-matched men. However, this protection is lost after menopause. We demonstrate that sex-biased sensitivity to the development of CVD with age runs in parallel with changes in G protein-coupled receptor kinase 2 (GRK2) protein levels in the murine heart and that mitochondrial fusion markers, related to mitochondrial functionality and cardiac health, inversely correlate with GRK2. Young female mice display lower amounts of cardiac GRK2 protein compared to age-matched males, whereas GRK2 is upregulated with age specifically in female hearts. Such an increase in GRK2 seems to be specific to the cardiac muscle since a different pattern is found in the skeletal muscles of aging females. Changes in the cardiac GRK2 protein do not seem to rely on transcriptional modulation since adrbk1 mRNA does not change with age and no differences are found between sexes. Global changes in proteasomal or autophagic machinery (known regulators of GRK2 dosage) do not seem to correlate with the observed GRK2 dynamics. Interestingly, cardiac GRK2 upregulation in aging females is recapitulated by ovariectomy and can be partially reversed by estrogen supplementation, while this does not occur in the skeletal muscle. Our data indicate an unforeseen role for ovarian hormones in the regulation of GRK2 protein levels in the cardiac muscle which correlates with the sex-dependent dynamics of CVD risk, and might have interesting therapeutic applications, particularly for post-menopausal women.

From Obesity to Chronic Kidney Disease: How Can Adipose Tissue Affect Renal Function?

Obesity is directly associated with an increased risk of developing CKD, regardless of other comorbid conditions. Although the molecular mechanisms that link both diseases are not well established, the role of adipose tissue (AT) is becoming increasingly important in obesity-associated kidney damage. In the context of obesity, lipotoxicity and the alteration of AT secretion profile promote inflammation, oxidative stress, and fibrosis in the kidney, which ultimately leads to impaired renal function. Different studies have highlighted the importance of body weight loss in the improvement of renal function markers. In this regard, bariatric surgery, rather than low-calorie diets, has been accepted as the most effective option to lose weight. In fact, a significant reduction in proteinuria and hyperfiltration has been observed in association with surgically induced weight loss. Detection of early signs of kidney dysfunction in patients with obesity has not been accomplished yet, though. Therefore, understanding the harmful effects within the adipo-renal axis is essential to prevent the progression to the irreversible renal insufficiency. MicroRNAs have recently been described as important modulators of normal kidney function. Some of these microRNAs could be potential early markers of kidney damage, which would help with the diagnosis and the prevention of CKD.

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

BACKGROUND: Insulin secretion from the pancreatic ß-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 ß-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 ß-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 ß-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 ß-cell insulin secretion specifically during the early phase, an effect that may carry interesting therapeutic applications.

Myeloid GRK2 Regulates Obesity-Induced Endothelial Dysfunction by Modulating Inflammatory Responses in Perivascular Adipose Tissue.

Perivascular adipose tissue (PVAT) is increasingly being regarded as an important endocrine organ that directly impacts vessel function, structure, and contractility in obesity-associated diseases. We uncover here a role for myeloid G protein-coupled receptor kinase 2 (GRK2) in the modulation of PVAT-dependent vasodilation responses. GRK2 expression positively correlates with myeloid- (CD68) and lymphoid-specific (CD3, CD4, and CD8) markers and with leptin in PVAT from patients with abdominal aortic aneurysms. Using mice hemizygous for GRK2 in the myeloid lineage (LysM-GRK2+/-), we found that GRK2 deficiency in myeloid cells allows animals to preserve the endothelium-dependent acetylcholine or insulin-induced relaxation, which is otherwise impaired by PVAT, in arteries of animals fed a high fat diet (HFD). Downregulation of GRK2 in myeloid cells attenuates HFD-dependent infiltration of macrophages and T lymphocytes in PVAT, as well as the induction of tumor necrosis factor-α (TNFα) and NADPH oxidase (Nox)1 expression, whereas blocking TNFα or Nox pathways by pharmacological means can rescue the impaired vasodilator responses to insulin in arteries with PVAT from HFD-fed animals. Our results suggest that myeloid GRK2 could be a potential therapeutic target in the development of endothelial dysfunction induced by PVAT in the context of obesity.

GRK2 levels in myeloid cells modulate adipose-liver crosstalk in high fat diet-induced obesity.

Macrophages are key effector cells in obesity-associated inflammation. G protein-coupled receptor kinase 2 (GRK2) is highly expressed in different immune cell types. Using LysM-GRK2+/- mice, we uncover that a reduction of GRK2 levels in myeloid cells prevents the development of glucose intolerance and hyperglycemia after a high fat diet (HFD) through modulation of the macrophage pro-inflammatory profile. Low levels of myeloid GRK2 confer protection against hepatic insulin resistance, steatosis and inflammation. In adipose tissue, pro-inflammatory cytokines are reduced and insulin signaling is preserved. Macrophages from LysM-GRK2+/- mice secrete less pro-inflammatory cytokines when stimulated with lipopolysaccharide (LPS) and their conditioned media has a reduced pathological influence in cultured adipocytes or naïve bone marrow-derived macrophages. Our data indicate that reducing GRK2 levels in myeloid cells, by attenuating pro-inflammatory features of macrophages, has a relevant impact in adipose-liver crosstalk, thus preventing high fat diet-induced metabolic alterations.

Autophagy mediates hepatic GRK2 degradation to facilitate glucagon-induced metabolic adaptation to fasting.

The liver plays a key role during fasting to maintain energy homeostasis and euglycemia via metabolic processes mainly orchestrated by the insulin/glucagon ratio. We report here that fasting or calorie restriction protocols in C57BL6 mice promote a marked decrease in the hepatic protein levels of G protein-coupled receptor kinase 2 (GRK2), an important negative modulator of both G protein-coupled receptors (GPCRs) and insulin signaling. Such downregulation of GRK2 levels is liver-specific and can be rapidly reversed by refeeding. We find that autophagy, and not the proteasome, represents the main mechanism implicated in fasting-induced GRK2 degradation in the liver in vivo. Reducing GRK2 levels in murine primary hepatocytes facilitates glucagon-induced glucose production and enhances the expression of the key gluconeogenic enzyme Pck1. Conversely, preventing full downregulation of hepatic GRK2 during fasting using adenovirus-driven overexpression of this kinase in the liver leads to glycogen accumulation, decreased glycemia, and hampered glucagon-induced gluconeogenesis, thus preventing a proper and complete adaptation to nutrient deprivation. Overall, our data indicate that physiological fasting-induced downregulation of GRK2 in the liver is key for allowing complete glucagon-mediated responses and efficient metabolic adaptation to fasting in vivo.

Involvement of G protein-coupled receptor kinase 2 (GRK2) in the development of non-alcoholic steatosis and steatohepatitis in mice and humans.

Insulin resistance (IR) and obesity are important risk factors for non-alcoholic fatty liver disease (NAFLD). G protein-coupled receptor kinase 2 (GRK2) is involved in the development of IR and obesity in vivo. However, its possible contribution to NAFLD and/or non-alcoholic steatohepatitis (NASH) independently of its role on IR or fat mass accretion has not been explored. Here, we used wild-type (WT) or GRK2 hemizygous (GRK2±) mice fed a high-fat diet (HFD) or a methionine and choline-deficient diet (MCD) as a model of NASH independent of adiposity and IR. GRK2± mice were protected from HFD-induced NAFLD. Moreover, MCD feeding caused an increased in triglyceride content and liver-to-body weight ratio in WT mice, features that were attenuated in GRK2± mice. According to their NAFLD activity score, MCD-fed GRK2± mice were diagnosed with simple steatosis and not overt NASH. They also showed reduced expression of lipogenic and lipid-uptake markers and less signs of inflammation in the liver. GRK2± mice preserved hepatic protective mechanisms as enhanced autophagy and mitochondrial fusion and biogenesis, together with reduced endoplasmic reticulum stress. GRK2 protein was increased in MCD-fed WT but not in GRK2± mice, and enhanced GRK2 expression potentiated palmitic acid-triggered lipid accumulation in human hepatocytes directly relating GRK2 levels to steatosis. GRK2 protein and mRNA levels were increased in human liver biopsies from simple steatosis or NASH patients in two different human cohorts. Our results describe a functional relationship between GRK2 levels and hepatic lipid accumulation and implicate GRK2 in the establishment and/or development of NASH.

G protein-coupled receptor kinase 2 (GRK2) as an integrative signalling node in the regulation of cardiovascular function and metabolic homeostasis.

G protein-coupled receptor kinase 2 (GRK2) is emerging as a pivotal signalling hub able to integrate different transduction cascades. This ability appears to underlie its central role in different physiological and pathological conditions. Key mediators of cardiovascular function (such as catecholamines or angiotensin II) and components of the systemic milieu altered in insulin resistance conditions converge in increasing GRK2 levels in diverse cardiovascular cell types. In turn, GRK2 would simultaneously modulate several cardiovascular regulatory pathways, including GPCR and insulin signalling cascades, NO bioavailability and mitochondrial function. This fact can help explain the contribution of increased GRK2 levels to maladaptive cardiovascular function and remodeling. It also unveils GRK2 as a link between cardiovascular pathologies and co-morbidities such as obesity or type 2 diabetes. On the other hand, enhanced GRK2 expression, as observed in adipose tissues, liver or skeletal muscle during insulin resistance-related pathologies, could modify the orchestration of GPCR and insulin signalling in these crucial metabolic organs, and contribute to key features of the obese and insulin-resistant phenotype.

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.

Angiopoietin-like protein 8 (ANGPTL8) in pregnancy: a brown adipose tissue-derived endocrine factor with a potential role in fetal growth.

Angiopoietin-like protein 8 (ANGPTL8), a protein implicated in lipid and glucose homeostasis, is present only in mammals, suggesting that it is involved in processes unique to these vertebrates such as pregnancy and homeothermy. We explored the role of ANGPTL8 in maternal-fetal crosstalk and its relationship with newborn adiposity. In a longitudinal analysis of healthy pregnant women, ANGPTL8 levels decreased progressively during pregnancy although remained higher than levels in the postpartum period. In a cross-sectional observational study of women with or without gestational diabetes mellitus (GDM), and their offspring, ANGPTL8 levels were higher in venous cord blood than those in maternal blood and were significantly lower in GDM patients than those in healthy women. Infants small for gestational age and with low-fat mass had the highest ANGPTL8 cord blood levels. Studies in vitro revealed that ANGPTL8 was secreted by brown adipocytes and its expression was increased in experimental models of white-to-brown fat conversion. In addition, ANGPTL8 induced the expression of markers of brown adipocytes. The high levels of ANGPTL8 found in fetal life together with its relationship with newborn adiposity and brown adipose tissue point to ANGPTL8 as a potential new player in the modulation of the thermogenic machinery during the fetal-neonatal transition.

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. In particular, 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 hub.

Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2.

Insulin resistance is a common feature of obesity and predisposes individuals to various prevalent pathological conditions. G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor kinase 2 (GRK2) integrates several signal transduction pathways and is emerging as a physiologically relevant inhibitor of insulin signaling. GRK2 abundance is increased in humans with metabolic syndrome and in different murine models of insulin resistance. To support GRK2 as a potential drug target in type 2 diabetes and obesity, we investigated whether lowering GRK2 abundance reversed an ongoing systemic insulin-resistant phenotype, using a mouse model of tamoxifen-induced GRK2 ablation after high-fat diet-dependent obesity and insulin resistance. Tamoxifen-triggered GRK2 deletion impeded further body weight gain, normalized fasting glycemia, improved glucose tolerance, and was associated with preserved insulin sensitivity in skeletal muscle and liver, thereby maintaining whole-body glucose homeostasis. Moreover, when continued to be fed a high-fat diet, these animals displayed reduced fat mass and smaller adipocytes, were resistant to the development of liver steatosis, and showed reduced expression of proinflammatory markers in the liver. Our results indicate that GRK2 acts as a hub to control metabolic functions in different tissues, which is key to controlling insulin resistance development in vivo. These data suggest that inhibiting GRK2 could reverse an established insulin-resistant and obese phenotype, thereby putting forward this enzyme as a potential therapeutic target linking glucose homeostasis and regulation of adiposity.

Downregulation of G protein-coupled receptor kinase 2 levels enhances cardiac insulin sensitivity and switches on cardioprotective gene expression patterns.

G protein-coupled receptor kinase 2 (GRK2) has recently emerged as a negative modulator of insulin signaling. GRK2 downregulation improves insulin sensitivity and prevents systemic insulin resistance. Cardiac GRK2 levels are increased in human heart failure, while genetically inhibiting GRK2 leads to cardioprotection in mice. However, the molecular basis underlying the deleterious effects of GRK2 up-regulation and the beneficial effects of its inhibition in the heart are not fully understood. Therefore, we have explored the interconnections among a systemic insulin resistant status, GRK2 dosage and cardiac insulin sensitivity in adult (9 month-old) animals. GRK2(+/-) mice display enhanced cardiac insulin sensitivity and mild heart hypertrophy with preserved systolic function. Cardiac gene expression is reprogrammed in these animals, with increased expression of genes related to physiological hypertrophy, while the expression of genes related to pathological hypertrophy or to diabetes/obesity co-morbidities is repressed. Notably, we find that cardiac GRK2 levels increase in situations where insulin resistance develops, such as in ob/ob mice or after high fat diet feeding. Our data suggest that GRK2 downregulation/inhibition can help maintain cardiac function in the face of co-morbidities such as insulin resistance, diabetes or obesity by sustaining insulin sensitivity and promoting a gene expression reprogramming that confers cardioprotection.

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

G 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.