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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., This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU) grants to T.I. (SAF2017‐88885‐R), J.J.L. (SAF2015‐65371‐R) and M.R.C. (SAF2017‐88881‐R); by B2017/BMD‐3700 NEUROMETAB‐CM (Comunidad de Madrid, Madrid, Spain) to T.I. and institutional grants to CBMSO from Fundación Ramón Areces and Fundación Banco de Santander to J.J.L. T.I. and J.J.L. are also funded by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) and CIBERNED cooperative project 2015‐2/06 (Instituto de Salud Carlos III, Spain). GS work was supported by the Wellcome Trust Senior Investigator Award (107116/Z/15/Z), the European Union's Horizon 2020 Research and Innovation programme under grant agreement 739572 and a UK Dementia Research Institute Foundation award. A.S.‐S. was funded by a contract from CIBERNED‐2015‐2/06.

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-¿B/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-¿B/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., This work was supported by grants SAF2014-52737-P to T.I., SAF2013-45258-P to M.R. C., BFU2016-77885-P to F.H., SAF2014-54070-JIN to A.M. from Ministerio de Economía, Industria y Competitividad (Spain). It was also funded by P2010/BMD-2332 (Neurodegmodels) from Comunidad de Madrid to T.I., F.H. and J.A.) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, Spain) to T.I., F.H., J.A., and I.F.). J.P.-U. is a recipient of a predoctoral contract from SAF2014-52737-P; L.G.-G. was funded by a contract from CIBERNED; A.G.-M. and A.S.S. were funded by contracts from CIBERNED cooperative projects 2013/07 and CIBERNED 2015-2/06, respectively. A.M., A.D.P. is a recipient of a Juan de la Cierva formación fellowship (Ministerio de Economía, Industria y Competitividad, Spain) associated to CIBERNED. J.J.-A. was funded by a predoctoral contract from CSIC (JAEPredoc program) and by CIBERNED. The cost of this publication has been paid in part by FEDER (European Funds for Regional Development) funds.

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 HbA1c 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.

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.

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.

Insulin resistance is an important contributor to the pathogenesis of type 2 diabetes, and obesity is a risk factor for its development, in part because adipose tissue secretes proteins, called adipokines, that may influence insulin sensitivity. Among these molecules, tumor necrosis factor (TNF)-alpha has been proposed as a link between obesity and insulin resistance because TNF-alpha is overexpressed in adipose tissues of obese animals and humans, and obese mice lacking either TNF-alpha or its receptor show protection against developing insulin resistance. Direct exposure to TNF-alpha induces a state of insulin resistance in terms of glucose uptake in myocytes and brown adipocytes because of the activation of proinflammatory pathways that impair insulin signaling at the level of the insulin receptor substrate (IRS) proteins. In this regard, the Ser(307) 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 and inhibitor kB kinase being involved in the phosphorylation of this residue. Conversely, Ser phosphorylation of IRS-2 mediated by TNF-alpha activation of mitogen-activated protein kinase was the mechanism found in brown adipocytes. Protein-Tyr phosphatase (PTP)1B 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 was recently found in cells treated with TNF-alpha Accordingly, myocytes and primary brown adipocytes deficient in PTP1B are protected against insulin resistance induced by this cytokine. Furthermore, down-regulation of PTP1B activity is possible by the use of pharmacological agonists of nuclear receptors that restore insulin sensitivity in the presence of TNF-alpha. In conclusion, the lack of PTP1B in muscle and brown adipocytes increases insulin sensitivity and glucose uptake and could confer protection against insulin resistance induced by adipokines.

This is a pre-copyedited, author-produced pdf of an article accepted for publication in Journal of Molecular Cell Biology following peer review. The version of record Journal of Molecular Cell Biology 6.4 (2014) is available online at: http://dx.doi.org/10.1093/jmcb/mju025, 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., This work was supported by Grants BFU2008-04043 (to M.L. and S.F.-V.), SAF2012-3618 (to S.F.-V.), and SAF2011-23800 (to F.M.) from Ministerio de Economía y Competitividad, Spain; S2010/BMD-2332 (INDISNET) from Comunidad de Madrid, Spain (to F.M.); CIBER de Diabetes y Enfermedades Metabólicas Asociadas and The Cardiovascular Network (RD06- 0014/0037 and RD12/0042/0012) from Ministerio Sanidad y Consumo-Instituto Carlos III, Spain (to F.M.); UAM-Banco de Santander (to C.M.); BFU2010-14884 (to M.R.-G.). S.F.-V. is recipient of a ‘Miguel Servet’ tenure track program (CP10/00438) co-financed by the European Regional Development Fund (ERDF). We also acknowledge the support of COST Action BM0602 from the European Commission (to M.L.) and institutional support from Fundación Ramón Areces

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. © FASEB.

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.