Implication of AMPK in glucose-evoked modulation of Na,K-ATPase

Background and aims: Na,K-ATPase is an integral membrane protein that maintains the gradients of Na+ and K+, using the energy of ATP hydrolysis, maintaining the ionic gradients that allow electrical activity to occur. It has been demonstrated that, in pancreatic β-cells, Na,K-ATPase is regulated by glucose and that this phenomenon is impaired in glucose intolerant subjects. However, the mechanism underlying glucose-induced modulation of Na,K-ATPase is still unclear. The AMP-activated protein kinase (AMPK) is a molecular key player in energy homeostasis, providing exquisite sensitivity to small changes in intracellular AMP levels and thus to intracellular [ATP]/[ADP] ratio, that is known to activate protein regulatory pathways. Since in pancreatic β-cell, glucose has marked effects on oxidative metabolism and total intracellular ATP and AMP levels, the involvement of AMPK in the cascade of events regulating Na,K-ATPase regulation in pancreatic β-cells was postulated. The aim of this work was to evaluate the putative role of AMPK in the glucose-evoked regulation of Na,K-ATPase activity in the pancreatic β-cell. Materials and methods: Pancreatic -cells from normal (control) or glucose-intolerant Wistar rats (GIR) were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2.1mM glucose to reach basal activity. Afterwards cells were challenged to 8.4mM glucose for 20min, in the presence or absence of AMPK agonists (AICAR) and antagonists (compound C; CC). ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain. Results: In basal conditions the activity of Na,K-ATPase from normal and GIR pancreatic β-cell was similar (0.184±0.030 and 0.186±0.020 molPi/min/mgProt, respectively). Challenging the control β-cells with glucose 8.4mM evoked a 62% reduction of Na,K-ATPase activity whereas in GIR β-cells a significantly lower inhibition (40%) was observed. The addition of AICAR 1mM abolished glucose-induced Na,K-ATPase inhibition (0,166±0.011 molPi/min/mg). In control β-cell, the addition of CC 10 μM had no effect on glucose-induced inhibition of Na,K-ATPase. In the contrary, in GIR β-cells it significantly potentiated glucose-evoked inhibition of Na,K-ATPase reaching values similar to that observed in the controls (66%). Conclusions: The AMPK agonist AICAR counteracts the inhibitory action of glucose on Na,K-ATPase of control β-cells whereas CC amplified the glucose-induced inhibition of Na,K-ATPase in GIR β-cells. These results suggest that AMPK plays a central role in the cascade of events underlying glucose-induced modulation of Na,K-ATPase and that the defect must be upstream of AMPK. Finally, abnormal glucose-induced regulation of Na,K-ATPase occurs prior to overt type 2 diabetes and might be a feature in the disease development

Abnormal regulation of Na,K-ATPase in Glucose Intolerant Rats.

Introduction: Glucose is the most important physiological insulin secretagogue. However, the mechanisms underlying glucose-induced insulin release are not fully understood. The role of electrogenic systems such as ionic pumps, to these events remains essentially uninvestigated. Na,K-ATPase, responsible for maintaining Na+ and K+ gradients across the plasma membrane and generates a net outward current, thus changes in its activity may contribute to the early ionic events regulating insulin secretion (Therien and Blostein, 2000). Objective: The aim of this work was to evaluate the regulation of Na,K-ATPase activity by glucose in intact -cells of normal and glucose intolerant (GI) rats and its putative contribution to the regulation of insulin secretion. Material and Methods: Pancreatic -cells, from normal or control or GI rats, were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2mM glucose to reach basal. Afterwards cells were challenged with glucose in the interval 0-11mM for 60min, for dose-dependence evaluation, or with 8mM glucose for 5-120min, for time-dependence evaluation. ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain (Costa et al., 2009). Results: In β-cells from normal rats, glucose induced a bimodal regulation of Na,K-ATPase. In the absence of glucose, Na,K-ATPase activity was 0.056±0.015 U/mg. Stimulation with 2mM glucose induced an increase of Na,K-ATPase activity of ~4 fold whereas for [glucose] above 2mM it was observed a significant inhibition of Na,K-ATPase activity (0.061±0.013, 0.080±0.009 and 0.064±0.005 U/mg for 5.6, 8.4 and 11mM glucose, respectively, compared to 0.188±0.035 U/mg observed in 2mM G; n=3-8). β-cells from GI rats does not present this profile; in the absence of glucose, Na,K-ATPase activity was 0.202±0.036 U/mg and no significant differences from this value were observed with the other glucose concentration tested. Addicionally, in β-cells from normal rats, glucose (8mM) induced a time-dependent inhibition, with a biphasic profile, of Na,K-ATPase - it was observed a decrease in the pump activity between 0 and 20min stimulation where it reached a minimum value (77%). For incubation periods over 20min, the pump activity slowly and partially recovered (54%, 55% and 52%, for 30, 60 and 120min, respectively; n=7). In β-cells from GI animals, an less accentuated decrease of Na,K-ATPase activity between 0 ans 20min was also observed (34%), and is not observed further recover in activity. Conclusions: This work demonstrates there Na,K-ATPase is strictly regulated by glucose in pancreatic β-cell. This regulation is unpaired in GI animals. Na,K-ATPase contribution to glucose-induced ionic events and insulin secretion might be relevant and must be explored as a possible therapeutic target in TD2 . 1. Therien AG, Blostein R (2000) Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279:C541-C566 2. Costa AR, Real J, Antunes CM, Cruz-Morais J (2009) A new approach for determination of Na,K-ATPase activity: application to intact pancreatic beta-cells. In Vitro Cell Dev Biol Ani

Estudo para o desenvolvimento de métodos para quantificação de Phl p5 em amostras de ar exterior colhidas na cidade de Évora

Estudo para o desenvolvimento de métodos para quantificação de Phl p5 em amostras de ar exterior colhidas na cidade de Évora R Ribeiro1, CM Antunes1,4 & R Brandão2,3 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM); 3Departamento Biologia, Universidade de Évora, Largo dos Colegiais 2, 7000 Évora UE; 4Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra; [email protected] As doenças alérgico-respiratórias decorrentes da exposição a pólenes na atmosfera constituem na actualidade um problema de saúde pública, afectando cerca de 40% da população europeia. Em Portugal, os pólenes das gramíneas são uma das principais fontes de aeroalergénios atmosféricos, sendo o Phl p5 o principal alergénio da família Phleum pratense. e responsável por 80 a 90% das rinites e asma brônquica que atingem das crianças e adolescentes e 40 a 50% dos adultos já asmáticos. Assim, a realização de estudos para o desenvolvimento de metodologias que permitam prever o potencial alergénico da atmosfera e assim contribuir para desenvolver estratégias de prevenção da patologia alérgica são muito relevantes. Neste trabalho pretende-se desenvolver um teste imunoquímico para detecção e quantificação do alergénio Phl p5. Numa plataforma meteorológica situada no centro da cidade de Évora (Colégio Luís António Verney, Fase III), foram monitorizados em simultâneo e diariamente o conteúdo polínico atmosférico de Poaceae e o aeroalergénio Phl p5. Para a contagem dos primeiros, utilizou-se um colector volumétrico do tipo “Hirst” (Burkard Seven Day Recording Volumetric Spore Trap) e os segundos foram monitorizados mediante a utilização de um colector de impacto de médio volume (CHEMVOL) com filtros de poli-uretano, entre os dias 30 de Abril e 8 de Julho de 2009. Após extracção, a quantificação dos aeroalergénios efectuou-se mediante uma técnica de ELISA tipo “sandwich”, utilizando anticorpos específicos. Durante a época polínica de 2009 observaram-se níveis elevados de pólenes de gramíneas (entre 2 de Maio e 1 de Junho de 2009). Observou-se ainda que o conteúdo de alergénio Phl p5 estava directamente correlacionado com as contagens polínicas de pólenes de Poaceae. Estes resultados sugerem que a quantificação directa dos aeroalergénios pode contribuir, em conjunto com as contagens polínicas, para definir com melhor precisão do risco alérgico. Este estudo prosseguirá na época polínica de 2010 com vista à certificação do método. Agradecimentos: Este trabalho realiza-se no âmbito do projecto europeu HIALINE (“grant agreement” 20081107 II.5.1)

Estudo para o desenvolvimento de métodos para quantificação de Ole e1 em amostras de ar exterior colhidas na cidade de Évora

Estudo para o desenvolvimento de métodos para quantificação de Ole e1 em amostras de ar exterior colhidas na cidade de Évora. R Ferro1*, CM Antunes1,4 & R Brandão2,3 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM); 3Departamento Biologia, Universidade de Évora, Largo dos Colegiais 2, 7000 Évora UE; 4Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra; [email protected] As doenças alérgico-respiratórias decorrentes da exposição a pólenes na atmosfera representam um dos principais problemas clínicos da actualidade, afectando cerca de 40% da população europeia. Em Portugal, a polinização da oliveira é seguramente uma das principais fontes de aeroalergénios atmosféricos e consequentemente uma das maiores causas de alergia respiratória, sendo a Ole e1 a glicoproteína mais abundante do grão de pólen e o alergénio que mais significativamente contribui para o grau de alergenicidade deste pólen. Assim, o desenvolvimento de metodologias que permitam prever o potencial alergénico da atmosfera são da maior relevância já que poderão contribuir para desenvolver estratégias de prevenção da patologia alérgica. Neste trabalho pretende-se desenvolver e optimizar testes imunoquímicos para detecção e quantificação de Ole e1. Numa plataforma meteorológica situada no centro da cidade de Évora (Colégio Luís António Verney - Fase III), foram monitorizados em simultâneo e diariamente o conteúdo polínico atmosférico da Olea europaea e do aeroalergeno Ole e1. Para a contagem do primeiro, utilizou-se um colector volumétrico do tipo “Hirst” (Burkard Seven Day Recording Volumetric Spore Trap) e o segundo foi monitorizado mediante a utilização de um colector de impacto de médio volume (CHEMVOL) com filtros de poli-uretano, entre os dias 30 de Abril e 8 de Julho de 2009. Após extracção, a quantificação do aeroalergénio efectuou-se mediante uma técnica de ELISA tipo “sandwich”, utilizando um anticorpo específico. Durante a época polínica de 2009, observaram-se níveis elevados de pólen de oliveira entre 2 de Maio e 30 de Maio. Observou-se ainda que o conteúdo do alergénio Ole e1 estava directamente correlacionado com as contagens polínicas de Oleaceae. Estes resultados sugerem que a quantificação directa dos aeroalergénios pode contribuir, em conjunto com as contagens polínicas, para definir com melhor precisão o risco alérgico. Este estudo continuará na época polínica de 2010 com vista à certificação do método. Agradecimentos: Este trabalho realiza-se no âmbito do projecto europeu HIALINE (“grant agreement” 20081107 II.5.1)

Glucose-evoked Na+,K+-ATPase modulation in pancreatic ß-cells from normal and impaired glucose tolerance: role of AMPK

Na+,K+-ATPase is regulated by glucose in pancreatic ß-cells, a process that is altered in glucose impaired tolerance. Although AMP dependent protein kinase (AMPK), a metabolic sensor, is believed to be central in the signal transduction cascade underlying the Na+,K+-ATPase regulation in pancreatic β-cells, its role remains unknown. The aim of this work was to clarify the role of AMPK in glucoseevoked inhibition of Na+,K+-ATPase and to evaluate whether AMPK is differently regulated in pancreatic β-cells from subjects with normal and impaired glucose tolerance. Pancreatic β-cells or islets from normal (control) or glucose-intolerant Wistar rats (GIR) were isolated and cultured. After a pre-incubation (30min) with 2.1mM glucose (G2), batches were challenged for 20min with 2.1 or 8.4mM glucose (G8) in the presence or absence of AMPK agonist (AICAR, 1mM) and antagonist (Compound C (CC), 10μM). Na+,K+-ATPase activity was assessed by quantification of Pi, in the absence and in presence of 1mM ouabain. Phosphorylation levels of α1 subunit of Na+,K+-ATPase- (Ser-23) and αAMPK-(Thr-172) was evaluated by Western blot (WB). In G2 Na+,K+-ATPase activity from normal and GIR β-cell was similar (0.184±0.030 and 0.186±0.020 μmolPi/min/mgProt, respectively). Challenging the β-cells with G8 evoked a lower inhibition of Na+,K+- ATPase activity in GIR (40%) compared to controls (62%). In control β-cell, AICAR abolished glucoseinduced Na+,K+-ATPase inhibition (0,166±0.011 μmolPi/min/mg) whereas CC had no effect. In the contrast, CC significantly potentiated glucose-evoked inhibition of Na+,K+-ATPase in GIR β-cells, reaching values similar to the controls (66%), For both GIR and control islets, G8 induced a 50% decrease of AMPK phosphorylation level compared to G2. CC mimicked the effect of G8, but was less efficient in GIR. Concomitantly, α1-Na+,K+-ATPase-(Ser-23) phosphorylation level was increased upon G8 or CC stimulation, compared to G2 or AICAR. These results suggest that AMPK plays a key role in the signaling mechanism underlying glucoseinduced modulation of the pump, a process dependent on phosphorylation cascades, and that the defect in GIR must be upstream of AMPK. Glucose-induced inhibition of Na+,K+-ATPase may result from AMPK inhibition by the fuel metabolism and subsequent activation of PKC, known to phosphorylate α1-Na+,K+-ATPase-(Ser-23). This mechanism is impaired in GIR, thus potentially contributing to the impaired glucose-induced insulin secretion in IGT. Occurring prior to overt type 2 diabetes, this might be a feature in the disease development

Alterações na modulação da Na,K-ATPase pela glicose em célula β-pancreática de animais intolerantes à glicose.

Alterações na modulação da Na,K-ATPase pela glicose em célula β-pancreática de animais intolerantes à glicose AR Costa1,2, CM Antunes1,3, J Cruz-Morais1,2 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Largo dos Colegiais 2, 7000 Évora; 3Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra;[email protected] A diabetes tipo 2 é uma síndrome metabólica, que se caracteriza por alterações da secreção ou acção da insulina, cujas bases bioquímicas não estão completamente esclarecidas. Na célula-, a resposta fisiológica à glicose envolve a despolarização, resultante principalmente do fecho de canais de K+ sensíveis ao ATP, seguido do influxo de Ca2+ por canais sensíveis à voltagem tipo-L e consequente aumento da concentração intracelular de Ca2+, necessário à exocitose da insulina, hormona responsável pela homeostasia da glicose. A Na,K-ATPase (ou bomba de sódio), é o sistema de transporte activo responsável por manter o gradiente transmembranar de Na+ e K+. Estudos recentes têm sugerindo que a inibição da Na,K-ATPase possa contribuir para a despolarização induzida por glicose. Como o acoplamento estímulo-secreção se encontra alterado em célula- de animais diabéticos, proposemo-nos estudar a participação da Na,K-ATPase neste mecanismo utilizando um modelo de pré-diabetes que se caracteriza essencialmente por apresentar alterações na curva de tolerância à glicose. Para isso, desenvolveu-se um método que permite avaliar a actividade da bomba em células β-pancreáticas primárias, mantidas em culturas aderentes, que preservam os processos de transdução de sinal. Com este método observámos que a glicose inibe a Na,K-ATPase em animais normais, estando este processo significativamente atenuado em animais pré-diabéticos. Observámos ainda que a secreção de insulina estimulada por glicose, nas células de pré-diabéticos, é cerca de 4 vezes menor relativamente aos controlos. Estes dados permitem-nos sugerir que a regulação da bomba por glicose se encontra modificada em animais pré-diabéticos, podendo contribuir para as alterações dos mecanismos iónicos responsáveis pelo acoplamento estímulo-secreção, nomeadamente na cinética da despolarização, comprometendo a secreção de insulina. O trabalho prossegue no sentido de esclarecer os mecanismos através dos quais a glicose induz inibição na Na,K-ATPase, bem como no levantamento dos factores que podem contribuir para as diferenças observadas entre os animais normais e pré-diabéticos, nomeadamente ao nível da expressão isoenzimática

Comparison between Airborne Pollen and Aeroallergen Quantification with the ChemVol Impact Sampler. Olive pollen vs Ole e 1

Comparison between Airborne Pollen and Aeroallergen Quantification with the ChemVol Impact Sampler. Olive pollen vs Ole e 1. Torres M.C.1, C. Antunes2, M.J. Velasco1, R. Ferro2, H. García-Mozo1, R. Ribeiro2, R.Brandao3, Galán, C1 and the HIALINE team4 1Department of Botany, Ecology and Plant Physiology, University of Córdoba 2Department of Chemistry, University of Évora, Portugal 3Department of Biology, University of Évora, Portugal 4J.T.M. Buters, Germany, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C. Antunes, Portugal, G. Reese, Germany, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy Nowadays, pollinosis is affecting a large percentage of population in the countries with a western life style. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with an allergen load. Olive pollen is recognized as one of the main causes of allergic disease in the Mediterranean area. In this study we have tried to compare olive pollen count in the air and Ole e 1 as major allergen of this species, at two different localities in South of Europe: Evora (Portugal) and Córdoba (Spain). At each location both samplers were placed side-by-side. Pollen grains have been sampled using a volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm were used for detecting aeroallergens. Ole e 1 major allergen was determined using allergen specific ELISA´s. Similar behaviour between pollen and the total allergenic load was observed during the pollen season. Nevertheless, at some occasions, during the previous and later period of the pollen season, airborne allergenic load was detected in South Spain, due to the contributions from other Oleaceae species. For this reason the use of these two different methodologies allow a better understanding of the allergenic load in the atmosphere. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 20081107

Alterações na actividade enzimática da amílase salivar em indivíduos obesos

Nos últimos anos o estudo da composição proteica da saliva ganhou interesse pelo seu potencial no diagnóstico não invasivo e compreensão de diversas doenças, quer orais, quer sistémicas. Para além disso, a interacção deste fluido com os constituintes dos alimentos tornam a sua análise importante para a compreensão das variações a nível da percepção dos alimentos e escolhas alimentares. Há, contudo, poucos estudos que comparem a composição proteica da saliva de indivíduos obesos com indivíduos normoponderais. Uma das proteínas mais abundante na saliva é a α-amilase. Esta proteína é responsável pelo início da digestão de hidratos de carbono na boca influenciando a percepção dos alimentos. Para além disso, os seus níveis são indicadores do funcionamento das glândulas salivares, tendo ainda sido sugerida como potencial marcador de condições de stress e de actividade do sistema nervoso simpático. O presente trabalho teve como objectivo comparar os níveis de α-amilase salivar entre indivíduos obesos (IMC>30; N=10) e normoponderais (IMC<25; N=10) do sexo feminino. Foram feitas recolhas de saliva mista, na ausência de estimulação e sempre à mesma hora do dia. Para determinar a actividade enzimática da α-amilase utilizou-se um kit colorimétrico (Sentinel Diagnostics). Foi observada uma maior actividade enzimática de α-amilase nos indivíduos obesos comparativamente aos normoponderais (243,2 X 103 ± 37,7 X 103 e 106,1 X 103 ± 44,2 X 103 U/L, respectivamente) A maior actividade enzimática da α-amilase poderá contribuir para alterações na percepção dos alimentos, nomeadamente no que diz respeito ao gosto doce dos hidratos de carbono. Uma diferente sensibilidade gustativa poderá ter influências nas escolhas alimentares. Estes resultados reforçam a importância de estudos mais aprofundados acerca da função salivar e percepção gustativa na obesidade

Changes associated with Na,K-ATPase in brain, kidney, heart and liver of the spontaneously diabetic Goto-Kakizaki rat

Alterations in Na,K-ATPase activity, in isoenzyme expression and/or number of units of the pump present in the plasma membrane have been associated with diabetes. These changes were described in different organs and tissues such as brain, heart, kidney, among others, and may result from altered insulin levels. However, the vast majority of studies were conducted in animal models of chemically induced diabetes, which are not consensual models for type 2 diabetes (T2D). The major goal of this work was to investigate putative modifications in Na,K-ATPase enzymatic activity or expression in brain, kidney, heart and liver in T2D. The Goto-Kakizaky rat (GK) strain was used as a model of spontaneously developed T2D, and Wistar rats as controls. Na,K-ATPase activity was assessed by the hydrolysis of ATP (Pi formed in the presence/absence of ouabain was measured using a colorimetric assay) and the isoenzymatic expression by Westernblot. A decrease in Na,K-ATPase activity in renal and cardiac tissues from GK comparatively to controls (55.7% and 77.5%, respectively) was observed. The pump activity was similar in liver and brain tissues. In renal tissue, expression of α1-Na,K-ATPase was similar between GK and controls but α2- was 2.3x higher and α3- was detected only in GK. Contrastingly a decreased expression of α1- (49.5%) and α2-isoforms (67.6%) was found in cardiac tissue. Despite similar Na,K-ATPase activity in liver and brain tissues, α1-isoform expression was decreased (33.9%) in the liver from GK while in the brain an increase of α1-isoform (~2x) together with a decrease of α2-isoform (14.0%) expressions were observed. It is unclear, except maybe for cardiac tissue where lower expression is potentially underlying the diminished pump activity in GK, whether the changes in isoenzyme expression is a key factor for differential Na,K-ATPase activity. Being responsive to complex regulation, other regulatory mechanisms may contribute to the impaired activity observed in kidney and heart. These results have uncover changes in Na,K-ATPase activity and/or enzymatic expression in GK brain, kidney, heart and liver that may contribute to the undesirable conditions associated with T2D. This work highlight the relevance of further investigation about Na,K-ATPase regulation and role in physiopathology of T2D

A new approach for determination of Na,K-ATPase activity: application to intact pancreatic ß-cells

It has been postulated that a decrease in Na,KATPase-mediated ion gradients may be a contributing mechanism to insulin secretion. However, the precise role of the Na,K-ATPase in pancreatic β-cell membrane depolarization and insulin secretion signalling have been difficult to evaluate, mostly because data reporting changes in enzymatic activity have been obtained in cell homogenates or membrane preparations, lacking intact intracellular signalling pathways. The aim of this work was to develop a method to characterize Na,K-ATPase activity in intact pancreatic β-cells that will allow the investigation of putative Na,K-ATPase activity regulation by glucose and its possible role in insulin secretion signalling. This work demonstrates for the first time that it is possible to determine Na,K-ATPase activity in intact pancreatic βcells and that this is a suitable method for the study of the mechanisms involved in the Na,K-ATPase regulation and eventually its relevance for insulin secretion signalling