Differential regulation of Kidins220 isoforms in Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.

Author Correction: Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

The original version of this Article contained an error in the spelling of the author Álvaro Sebastián-Serrano, which was incorrectly given as Álvaro Sebastián Serrano. This has now been corrected in both the PDF and HTML versions of the Article.

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.

Insulin Regulates Astrocytic Glucose Handling Through Cooperation With IGF-I.

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes.

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.

Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine 1412.

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.

Methazolamide is a new hepatic insulin sensitizer that lowers blood glucose in vivo.

We previously used Gene Expression Signature technology to identify methazolamide (MTZ) and related compounds with insulin sensitizing activity in vitro. The effects of these compounds were investigated in diabetic db/db mice, insulin-resistant diet-induced obese (DIO) mice, and rats with streptozotocin (STZ)-induced diabetes. MTZ reduced fasting blood glucose and HbA(1c) levels in db/db mice, improved glucose tolerance in DIO mice, and enhanced the glucose-lowering effects of exogenous insulin administration in rats with STZ-induced diabetes. Hyperinsulinemic-euglycemic clamps in DIO mice revealed that MTZ increased glucose infusion rate and suppressed endogenous glucose production. Whole-body or cellular oxygen consumption rate was not altered, suggesting MTZ may inhibit glucose production by different mechanism(s) to metformin. In support of this, MTZ enhanced the glucose-lowering effects of metformin in db/db mice. MTZ is known to be a carbonic anhydrase inhibitor (CAI); however, CAIs acetazolamide, ethoxyzolamide, dichlorphenamide, chlorthalidone, and furosemide were not effective in vivo. Our results demonstrate that MTZ acts as an insulin sensitizer that suppresses hepatic glucose production in vivo. The antidiabetic effect of MTZ does not appear to be a function of its known activity as a CAI. The additive glucose-lowering effect of MTZ together with metformin highlights the potential utility for the management of type 2 diabetes.

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.

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)

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.

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