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.

GRK2 contribution to the regulation of energy expenditure and brown fat function.

Obesity is a major health problem and an important risk factor for the development of multiple disorders. Previous studies in our laboratory have revealed that down-regulation of GRK2 decreases age-related adiposity, but the physiological and molecular mechanisms underlying this outcome remain unclear. We evaluate whether the lean phenotype results from a direct effect of GRK2 on energy homeostasis. The study of white adipose tissue (WAT) in wild-type (WT) and GRK2(+/-) littermates showed a reduced expression of lipogenic enzymes and enhanced lipolytic rate in adult GRK2(+/-) mice. Moreover, hemizygous mice display higher energy expenditure and lower respiratory exchange ratio. Analysis of brown adipose tissue (BAT) from adult GRK2(+/-) mice showed a less deteriorated morphology associated with age compared to WT, which is correlated with a higher basal core temperature. BAT from young GRK2(+/-) mice showed an increase in gene expression of thermogenesis-related genes. Accordingly, hemizygous mice displayed better thermogenic capacity and exhibited a more oxidative phenotype in both BAT and WAT than WT littermates. Overexpression of GRK2 in brown adipocytes corroborated the negative effect of this kinase in BAT function and differentiation. Collectively, our data point to GRK2 inhibition as a potential tool for the enhancement of brown fat activity, which may have important therapeutic implications for the treatment of obesity and associated metabolic disorders.

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance.

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.

G protein-coupled receptor kinase 2 plays a relevant role in insulin resistance and obesity.

OBJECTIVE: Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein-coupled receptor kinase 2 (GRK2), first identified as a G protein-coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS: We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS: Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS: Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.

Molecular mechanisms involved in obesity-associated insulin resistance: therapeutical approach.

Insulin resistance is an important contributor to the pathogenesis of T2D and obesity is a risk factor for its development. It has been demonstrated that these obesity-related metabolic disorders are associated with a state of chronic low-intensity inflammation. Several mediators released from adipocytes and macrophages, such as the pro-inflammatory cytokines TNF-alpha and IL-6, have been suggested to impair insulin action in peripheral tissues, including fat and skeletal muscle. Such insulin resistance can initially be compensated by increased insulin secretion, but the prolonged presence of the hormone is detrimental for insulin sensitivity. Stress and pro-inflammatory kinases as well as more recent players, phosphatases, seem to be involved in the molecular mechanisms by which pro-inflammatory cytokines and hyperinsulinemia disrupt insulin signalling at the level of IRSs. Pharmacological approaches, such as treatment with PPAR and LXR agonists, overcome such insulin resistance, exerting anti-inflammatory properties as well as controlling the expression of cytokines with tissular specificity.

c-Jun N-terminal kinase 1/2 activation by tumor necrosis factor-alpha induces insulin resistance in human visceral but not subcutaneous adipocytes: reversal by liver X receptor agonists.

AIMS: Obesity is associated with a chronic systemic low-grade inflammatory state. Markers of inflammation such as TNF-alpha are linked with increased risk for insulin resistance and type 2 diabetes. The objective of the present study was to dissect the molecular mechanisms that may regulate TNF-alpha-induced insulin resistance in human adipose tissue. METHODS: We analyzed the impact of TNF-alpha on glucose uptake and insulin action in human visceral and sc adipocytes. The contribution of different intracellular signaling pathways on metabolic effects of TNF-alpha and the reversal of some of these effects with nuclear receptor agonists were also studied. RESULTS: TNF-alpha per se increased glucose transporter-4 translocation to the plasma membrane and glucose uptake by activating the AMP-activated protein kinase/AS160 pathway in both visceral and sc adipocytes. Nevertheless, this cytokine induced an insulin-resistant state in visceral adipocytes by impairing insulin-stimulated glucose uptake and insulin signaling at the insulin receptor substrate (IRS)-1/AKT level. Activation of c-Jun N-terminal kinase (JNK) 1/2 seems to be involved in TNF-alpha-induced insulin resistance, causing phosphorylation of IRS1 at the Ser312 residue. Accordingly, silencing JNK1/2 with either small interfering RNA or chemical inhibitors impaired serine phosphorylation of IRS1, restored downstream insulin signaling, and normalized insulin-induced glucose uptake in the presence of TNF-alpha. Furthermore, TNF-alpha increased the secretion of other proinflammatory cytokines such as IL-6. Pharmacological treatment of adipocytes with liver X receptor agonists reestablished insulin sensitivity by impairing TNF-alpha induction of JNK1/2, phosphorylation of IRS1 (Ser312), and stabilizing IL-6 secretion. CONCLUSIONS: TNF-alpha induces insulin resistance on glucose uptake in human visceral but not sc adipocytes, suggesting depot-specific effects of TNF-alpha on glucose uptake. Activation of JNK1/2 appears to be involved in serine phosphorylation of IRS1 and subsequently insulin resistance on glucose uptake, a state that can be reversed by liver X receptor agonists.

Resistencia a insulina en el músculo esquelético: conexión con la obesidad / Obesity-Associated Insulin resistance in skeletal muscle

Entre las complicaciones asociadas a la Obesidad, tiene una especial relevancia el desarrollo de resistencia a la insulina, siendo el primer eslabón de una amplia patología conocida como diabetes tipo 2. La Obesidad se considera como un estado crónico de inflamación de baja intensidad, como indican los niveles circulantes elevados de moléculas proinflamatorias. Se ha propuesto al TNFα como el nexo de unión entre adiposidad y desarrollo de resistencia a insulina ya que la mayoría de los pacientes con diabetes tipo 2 son obesos y tienen aumentada la expresión de TNFα en sus adipocitos, y los animales obesos deleccionados para la función del TNFα o su receptor no desarrollan resistencia a insulina. Las citocinas proinflamatorias producidas por los adipocitos y/o macrófagos activan quinasas de estrés, proinflamatorias y factores de transcripción que actúan sobre los tejidos periféricos (entre ellos el músculo y el propio tejido adiposo) produciendo resistencia a la acción de la insulina, que es un defecto en la señalización a varios niveles. En concreto, el TNFα activa la quinasa p38MAPK que fosforila en residuos de serina a los IRSs, bloqueando su fosforilación en tirosina en respuesta a la insulina, tanto en adipocitos marrones como en miocitos. Muy recientemente hemos observado que la fosfatasa PTP1B también está implicada en la resistencia a insulina por TNFα en ambos modelos. En la clínica se está utilizando actualmente el tratamiento con tiazolidindionas en pacientes con diabetes tipo 2. Otros agonistas de receptores nucleares empiezan a aparecer en la bibliografía como potenciales sensibilizadores a acción de la insulina, entre ellos el LXR, que puede antagonizar la señalización proinflamatoria en los propios adipocitos y/o en el músculo (AU)

Insulin resistance is an important contributor to the pathogenesis of type 2 diabetes and obesity is a risk factor for its development, due in part to the fact that adipose tissue secretes proteins called adipokines that may influence insulin sensitivity. Among these molecules, TNFα has been proposed as a link between obesity and insulin resistance because TNFα is overexpressed in adipose tissues of obese animals and humans, and obese mice lacking either TNFα or its receptor show protection for developing insulin resistance. The direct exposure to TNFα induced a state of insulin resistance on glucose uptake in myocytes and brown adipocytes, due to the activation of pro-inflammatory pathways that impair insulin-signaling at the level of the IRS proteins. In this regard the residue Ser307 in IRS-1 has been identified as a site for TNFα- inhibitory effects in myotubes, with being p38MAPK and IKK involved in the phosphorylation of this residue. Conversely, serine phosphorylation of IRS-2 mediated by TNFα activation of MAPKs was the mechanism found in brown adipocytes. The phosphatase PTP1B acts as a physiological negative regulator of insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1B expression is increased in muscle and white adipose tissue of obese and diabetic humans and rodents. Moreover, up-regulation of PTP1B expression has recently been found in cells treated with TNFα. Accordingly, myocytes and primary brown adipocytes deficient on PTP1B are protected against insulin resistance by this cytokine. Furthermore, down-regulation of PTP1B activity is also possible by the use of pharmacological agonists of nuclear receptors that restored insulin sensitivity in the presence of TNFα. In conclusion, the lack of PTP1B in muscle and brown adipocytes increase insulin sensitivity and glucose uptake and could confer protection against insulin resistance induced by adipokines (AU)

Dual role of interleukin-6 in regulating insulin sensitivity in murine skeletal muscle.

OBJECTIVE: Cytokines are elevated in various insulin-resistant states, including type 2 diabetes and obesity, although the contribution of interleukin-6 (IL-6) in the induction of these diseases is controversial. RESEARCH DESIGN AND METHODS: We analyzed the impact of IL-6 on insulin action in murine primary myocytes, skeletal muscle cell lines, and mice (wild type and protein-tyrosine phosphatase 1B [PTP1B] deficient). RESULTS: IL-6 per se increased glucose uptake by activating serine/threonine protein kinase 11 (LKB1)/AMP-activated protein kinase/protein kinase B substrate of 160 kDa (AS160) pathway. A dual effect on insulin action was observed when myotubes and mice were exposed to this cytokine: additive with short-term insulin (increased glucose uptake and systemic insulin sensitivity) but chronic exposure produced insulin resistance (impaired GLUT4 translocation to plasma membrane and defects in insulin signaling at the insulin receptor substrate 1 [IRS-1] level). Three mechanisms seem to operate in IL-6-induced insulin resistance: activation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2), accumulation of suppressor of cytokine signaling 3 (socs3) mRNA, and an increase in PTP1B activity. Accordingly, silencing JNK1/2 with either small interfering RNA or chemical inhibitors impaired phosphorylation of IRS-1 (Ser307), restored insulin signaling, and normalized insulin-induced glucose uptake in myotubes. When using a pharmacological approach, liver X receptor agonists overcome IL-6-induced insulin resistance by producing downregulation of socs3 and ptp1b gene expression. Finally, the lack of PTP1B confers protection against IL-6-induced insulin resistance in skeletal muscle in vitro and in vivo, in agreement with the protection against the IL-6 hyperglycemic effect observed on glucose and insulin tolerance tests in adult male mice. CONCLUSIONS: These findings indicate the important role of IL-6 in the pathogenesis of insulin resistance and further implicate PTP1B as a potential therapeutic target in the treatment of type 2 diabetes.

Insulin resistance associated to obesity: the link TNF-alpha.

Adipose tissue secretes proteins which may influence insulin sensitivity. Among them, tumour necrosis factor (TNF)-alpha has been proposed as a link between obesity and insulin resistance because TNF-alpha is overexpressed in adipose tissue from obese animals and humans, and obese mice lacking either TNF-alpha or its receptor show protection against developing insulin resistance. The activation of proinflammatory pathways after exposure to TNF-alpha induces a state of insulin resistance in terms of glucose uptake in myocytes and adipocytes that impair insulin signalling at the level of the insulin receptor substrate (IRS) proteins. The mechanism found in brown adipocytes involves Ser phosphorylation of IRS-2 mediated by TNF-alpha activation of MAPKs. The Ser307 residue in IRS-1 has been identified as a site for the inhibitory effects of TNF-alpha in myotubes, with p38 mitogen-activated protein kinase (MAPK) and inhibitor kB kinase being involved in the phosphorylation of this residue. Moreover, up-regulation of protein-tyrosine phosphatase (PTP)1B expression was recently found in cells and animals treated with TNF-alpha. PTP1B acts as a physiological negative regulator of insulin signalling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1B expression is increased in peripheral tissues from obese and diabetic humans and rodents. Accordingly, down-regulation of PTP1B activity by treatment with pharmacological agonists of nuclear receptors restores insulin sensitivity in the presence of TNF-alpha. Furthermore, mice and cells deficient in PTP1B are protected against insulin resistance induced by this cytokine. In conclusion, the absence or inhibition of PTP1B in insulin-target tissues could confer protection against insulin resistance induced by cytokines.

Hyperinsulinemia induces insulin resistance on glucose and lipid metabolism in a human adipocytic cell line: paracrine interaction with myocytes.

CONTEXT: Adipocytes release a variety of factors which deregulation could provide the basis for complications such as insulin resistance, an early defect on the onset of type 2 diabetes. Such insulin resistance can initially be overcome by compensatory hyperinsulinemia, but the prolonged presence of the hormone can be detrimental for insulin sensitivity. OBJECTIVE: The objective of the study was to dissect the molecular mechanisms that may regulate hyperinsulinemia-induced insulin resistance in a human liposarcoma cell line and its paracrine interactions with a human rhabdomyosarcoma cell line. DESIGNS: We studied glucose uptake, lipolysis, insulin signaling, and secretion pattern at different days of adipocyte differentiation in the presence of insulin. RESULTS: Adipocytes differentiated for 14 d gain insulin sensitivity on glucose uptake and inhibition of lipolysis, but prolonged cultures develop an insulin-resistant state characterized by an increase in phosphatase and tensin homolog-deleted on chromosome 10 expression and defects in insulin signaling at the insulin receptor substrate-1/AKT level. The secretion pattern of nonesterified fatty acids, IL-6, adiponectin, leptin, and monocyte chemotactic protein-1 was in keeping with the changes in insulin sensitivity during differentiation. An inverse biphasic response was also observed in human myocytes when they were cultured with various adipocyte-conditioned media, although insulin resistance was detected earlier than in adipocytes. This behavior mimics hyperinsulinemia because insulin action was restored when adipocytes were cultured in the absence of the hormone. Pharmacological treatment of adipocytes with a liver X receptor agonist reestablishes insulin-stimulated glucose uptake, whereas treatment with a peroxisome proliferator-activated receptor-gamma agonist restored the antilipolytic action of insulin. CONCLUSIONS: Hyperinsulinemia deregulates adipocyte secretion pattern, producing insulin resistance in adipocytes and myocytes, a situation that can be ameliorated with nuclear receptor agonists.

Nuclear exclusion of forkhead box O and Elk1 and activation of nuclear factor-kappaB are required for C2C12-RasV12C40 myoblast differentiation.

Activating ras point mutations are frequently found in skeletal muscle tumors such as rhabdomyosarcomas. In this study we investigated the impact of two different H-ras mutants in skeletal muscle differentiation: RasV12, a constitutively active form, and RasV12C40, a mutant deficient in Raf1 activation. Stably transfected C2C12-RasV12 myoblasts actively proliferated as indicated by the sustained expression of proliferating cell nuclear antigen and retinoblastoma at the hyperphosphorylated state and failed to express differentiation markers. This differentiation-defective phenotype was a consequence of the chronic p44/p42MAPK phosphorylation and the inability of the cells to activate AKT. Moreover, we observed that p44/p42MAPK activation in C2C12-RasV12 myoblasts phosphorylated the ETS-like transcription factor (ELK) 1, which translocates to the nuclei and seemed to be involved in maintaining myoblast proliferation. C2C12-RasV12C40 myoblasts cultured in low serum repressed phosphorylation of p44/p42MAPK and ELK1, resulting in cell cycle arrest and myogenic differentiation. Under this condition, activation of AKT, p70S6K, and p38MAPK was produced, leading to formation of myotubes in 3 d, 1 d earlier than in control C2C12-AU5 cells. Moreover, the expression of muscle-specific proteins, mainly the terminal differentiation markers caveolin-3 and myosin heavy chain, also occurred 1 d earlier than in control cells. Furthermore, AKT activation produced phosphorylation of Forkhead box O that led to nuclear exclusion and inactivation, allowing myogenesis. In addition, we found an induction of nuclear factor-kappaB activity in the nucleus in C2C12-RasV12C40 myotubes attributed to p38MAPK activation. Accordingly, muscle differentiation is associated with a pattern of transcription factors that involves nuclear exclusion ELK1 and Forkhead box O and the increase in nuclear factor-kappaB DNA binding.

Contribution of TNF-alpha to Obesity-Associated Insulin Resistance

Entre las complicaciones asociadas a la Obesidad, tiene una especial relevanciael desarrollo de resistencia a la insulina, siendo el primer eslabón de unaamplia patología conocida como diabetes tipo 2. La Obesidad se considera comoun estado crónico de inflamación de baja intensidad, como indican los nivelescirculantes elevados de moléculas proinflamatorias. Se ha propuesto al TNF-alfacomo el nexo de unión entre adiposidad y desarrollo de resistencia a insulina yaque la mayoría de los pacientes con diabetes tipo 2 son obesos y tienen aumentadala expresión de TNF-alfa en sus adipocitos, y los animales obesos deleccionados parala función del TNF-alfa o su receptor no desarrollan resistencia a insulina. Las citocinasproinflamatorias producidas por los adipocitos y/o macrófagos activan quinasasde estrés, proinflamatorias y factores de transcripción que actúan sobre lostejidos periféricos (entre ellos el músculo, así como el propio tejido adiposo) produciendoresistencia a la acción de la insulina, que es un defecto en la señalizacióna varios niveles. En concreto, el TNF-alfa activa la quinasa p38MAPK que fosforilaen residuos de serina a los IRSs, bloqueando su fosforilación en tirosina en respuestaa la insulina, tanto en adipocitos marrones como en miocitos. Muy recientementehemos observado que la fosfatasa PTP1B también está implicada en laresistencia a insulina por TNF-alfa en ambos modelos. En la Clínica se está utilizandoactualmente el tratamiento con tiazolidindionas en pacientes con diabetes tipo2. Otros agonistas de receptores nucleares empiezan a aparecer en la bibliografíacomo potenciales sensibilizadores a acción de la insulina, entre ellos el LXR, quepuede antagonizar la señalización proinflamatoria tanto en los propios adipocitoscomo en el músculo

Insulin resistance is an important contributor to the pathogenesis of type 2diabetes, and obesity is a risk factor for its development, due in part to the factthat adipose tissue secretes proteins called adipokines, that may influence insulinsensitivity. Among these molecules, TNF-alpha has been proposed as a link betweenobesity and insulin resistance because TNF-alpha is overexpressed in adipose tissuesof obese animals and humans, and obese mice lacking either TNF-alpha or its receptorshow protection for developing insulin resistance. The direct exposure to TNF-alphainduced a state of insulin resistance on glucose uptake in myocytes and brownadipocytes, due to the activation of pro-inflammatory pathways that impair insulin-signaling at the level of the IRS proteins. In this regard the residue Ser307 inIRS-1 has been identified as a site for TNF-alpha-inhibitory effects in myotubes, with p38MAPK and IKK being involved in the phosphorylation of this residue. Conversely,serine phosphorylation of IRS-2 mediated by TNF-alpha activation of MAPKs wasthe mechanism found in brown adipocytes. The phosphatase PTP1B acts as aphysiological negative regulator of insulin signaling by dephosphorylating the phosphotyrosineresidues of the insulin receptor and IRS-1, and PTP1B expression isincreased in muscle and white adipose tissue of obese and diabetic humans androdents. Moreover, up-regulation of PTP1B expression was recently found in cellstreated with TNF-alpha. Accordingly, myocytes and primary brown adipocytes deficienton PTP1B are protected against insulin resistance by this cytokine. Furthermore,down-regulation of PTP1B activity is also possible by the use of pharmacologicalagonists of nuclear receptors that restore insulin sensitivity in the presenceof TNF-alpha. In conclusion, the lack of PTP1B in muscle and brown adipocytesincreases insulin sensitivity and glucose uptake and could confer protection againstinsulin resistance induced by adipokines

Protein-tyrosine phosphatase 1B-deficient myocytes show increased insulin sensitivity and protection against tumor necrosis factor-alpha-induced insulin resistance.

Protein-tyrosine phosphatase (PTP)1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. In this study, we have assessed the role of PTP1B in the insulin sensitivity of skeletal muscle under physiological and insulin-resistant conditions. Immortalized myocytes have been generated from PTP1B-deficient and wild-type neonatal mice. PTP1B(-/-) myocytes showed enhanced insulin-dependent activation of insulin receptor autophosphorylation and downstream signaling (tyrosine phosphorylation of insulin receptor substrate [IRS]-1 and IRS-2, activation of phosphatidylinositol 3-kinase, and serine phosphorylation of AKT), compared with wild-type cells. Accordingly, PTP1B(-/-) myocytes displayed higher insulin-dependent stimulation of glucose uptake and GLUT4 translocation to the plasma membrane than wild-type cells. Treatment with tumor necrosis factor-alpha (TNF-alpha) induced insulin resistance on glucose uptake, impaired insulin signaling, and increased PTP1B activity in wild-type cells. Conversely, the lack of PTP1B confers protection against insulin resistance by TNF-alpha in myocyte cell lines and in adult male mice. Wild-type mice treated with TNF-alpha developed a pronounced hyperglycemia along the glucose tolerance test, accompanied by an impaired insulin signaling and increased PTP1B activity in muscle. However, mice lacking PTP1B maintained a rapid clearance of glucose and insulin sensitivity and displayed normal muscle insulin signaling regardless the presence of TNF-alpha.