In 2008 the global prevalence of hypertension [high blood pressure (BP), systolic ≥140 mmHg and/or diastolic ≥90 mmHg] was around 40% in adults > 25 yrs of age, according to the 2013 WHO statistics. Hypertension is a major risk factor for myocardial infarction, heart failure and stroke. Currently, around 20% of the Canadian population is affected by hypertension. Hypertension is more closely associated with diabetes. More than two thirds of people with diabetes have hypertension, alongwith increased activity of the renin angiotensin aldosterone (RAAS) system. The RAAS plays a major role in maintaining fluid balance, vascular tone and BP. The components of the RAAS include the hormone renin, which cleaves angiotensinogen, a circulating inactive peptide into angiotensin I. Angiotensin converting enzyme (ACE) converts angiotensin I into the active peptide angiotensin II (Ang II). Ang II causes vasoconstriction, sodium reabsorption from the kidney tubules and also release of the hormone, aldosterone, from the adrenal cortex. The epidemic of hypertension, diabetes and obesity is widely attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. However, the underlying molecular mechanisms are far from clear. A high fructose diet increases BP in Sprague-Dawley (SD) rats; along with elevated plasma and aortic levels of methylglyoxal (MG). MG is a reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. Small amounts of MG are also formed during amino acid (threonine) and fatty acid metabolism. MG reacts with certain proteins to form irreversible advanced glycation end products (AGEs). MG has high affinity for arginine, lysine and cysteine. Plasma MG levels are increased in hypertensive rats and diabetic patients. However, it is not yet clear whether MG is the cause or effect of hypertension. Moreover, safe and specific MG scavengers are not available. The aim of the project was to determine the effect of MG and a high fructose diet on the RAAS and the BP in male SD rats. The hypothesis that L-arginine, and its inactive isomer D-arginine, can efficiently scavenge MG in vitro, was also tested. Male SD rats were treated with a continuous infusion of MG with a subcutaneous minipump for 4 weeks, or with a high fructose diet (60% of total calories) for 16 weeks. We also used isolated aortic rings from 12 week old normal male SD rats to study endothelial function. Organs / tissues, cultured human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) were used for molecular studies. HPLC, Western blotting and Q-PCR were used to measure MG, reduced glutathione (GSH), proteins and mRNA, respectively. siRNA for angiotensinogen and the receptor for advanced glycation endproducts (RAGE) were used to study mechanisms. MG treated rats developed a significant increase in BP and plasma levels of aldosterone, renin, angiotensin and catecholamines. MG level, and protein and mRNA for angiotensin, AT1 receptor, adrenergic α1D receptor and renin were significantly increased in the aorta and/or kidney of MG treated rats, a novel finding. Alagebrium, a MG scavenger and AGEs breaker, attenuated the above effects of MG. Treatment of cultured VSMCs with MG or high glucose (25mM) significantly increased cellular MG, and protein and mRNA for nuclear factor kappa B (NF-κB), angiotensin, AT1 and α1D receptors, which were prevented by inhibition of NF-κB, and by alagebrium. Silencing of mRNA for RAGE prevented the increase in NF-kB induced by MG. Silencing of mRNA for angiotensinogen prevented the increase in NF-κB, angiotensin, AT1 and α1D receptors’ protein. Fructose treated rats developed a significant increase in BP. MG level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of fructose and MG were attenuated by metformin, a MG scavenger and AGEs inhibitor. In experiments to test the MG scavenging action of arginine, both D-arginine and L-arginine prevented the attenuation of acetylcholine-induced endothelium-dependent vasorelaxation by MG and high glucose. However, the inhibitory effect of the NOS inhibitor, Nω-nitro-L-arginine methyl ester, on vasorelaxation was prevented only by L-arginine, but not by D-arginine. MG and high glucose increased protein expression of arginase, a novel finding, and also of NADPH oxidase 4 and NF-κB, and production of reactive oxygen species in HUVECs and VSMCs, which were attenuated by D- and L-arginine. However, D- and L-arginine did not attenuate MG and high glucose-induced increased arginase activity in VSMCs and the aorta. D- and L-arginine also attenuated the increased formation of the MG-specific AGE, Nε-carboxyethyl lysine, caused by MG and high glucose in VSMCs. In conclusion, MG activates NF-κB through RAGE and thereby increases renin angiotensin levels, a novel finding, and a probable mechanism of increase in BP. There is a strong association between elevated levels of MG, RAGE, NF-κB, mediators of the RAAS and BP in high fructose diet fed rats. Arginine attenuates the increased arginase expression, oxidative stress, endothelial dysfunction and AGEs formation induced by MG and high glucose, by an endothelial NOS independent mechanism.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A superativação do eixo ECA/AT1r está intimamente relacionada à síndrome metabólica e no organismo tem grande relação com o quadro de inflamação. A administração de frutose, seja por dieta ou pela água, tem sido usada como um modelo para a indução da superatividade desse eixo e para o estudo das vias inflamatórias relacionadas ao AT1r. Com isso, o objetivo deste trabalho foi avaliar se a administração de GW510156 poderia diminuir a superativação do eixo ECA/AT1r e consequentemente diminuir os danos causados pela dieta rica em frutose. Para isso foram utilizados camundongos machos C57Bl/6 que receberam uma dieta contendo 47% de frutose durante oito semanas ou uma dieta controle. Após oito semanas, os grupos foram redivididos aleatoriamente para o início da administração do GW501516 durante três semanas, totalizando quatro grupos experimentais. Os animais tratados apresentaram uma melhora da pressão arterial sistólica e também dos parâmetros urinários como proteinúria e ácido úrico. Houve ainda uma melhora dos triglicerídeo e ácido úrico plasmáticos. No tecido adiposo branco, o GW501516 foi capaz de diminuir a expressão dos componentes do eixo ECA/AT1r e também amenizou a inflamação causada pela dieta rica em frutose. No fígado, não houve alterações significativa do eixo, porém a fosforilação de JAK2 dependente de AT1r foi diminuída e consequentemente houve uma menor ativação das células estreladas no grupo que recebeu o GW501516. Além disso, as proteínas e genes relacionados à β-oxidação foram aumentados com o tratamento e aqueles relacionados à lipogênese de novo, diminuídos o que resultou em menor esteatose no parênquima hepático. Os rins apresentaram uma melhora da inflamação induzida pelo eixo, apesar de o eixo também não ter apresentado diferenças significativas com o tratamento. Também não foram encontradas diferenças significativas na expressão proteica e gênica das proteínas antioxidantes. Com esses resultados podemos concluir que a curta administração do GW501516 pôde aliviar os efeitos inflamatórios e a esteatose hepática causada pela dieta rica em frutose, podendo ser pensado como uma nova ferramenta terapêutica no tratamento da superativação do eixo ECA/AT1r. / High-activation of ACE/AT1r axis is closely linked to metabolix syndrome and low-grade inflammation state. Fructose administration in water or in diet has been proposed as a model to study the high-activity of this axis and AT1r-related inflammatory pathways. In this view, we aimed to evaluate if GW501516 administration could decrease the high-activation of ACE/AT1r axis and consequently fructose damage. To this males mice C57Bl/6 were fed a high-fructose diet (47%) during eight weeks or standard-chow diet. After eight weeks, the groups were randomly divided to start treatment with GW501516, totaling four experimental groups. Animals treated with GW501516 presented an improvement of systolic blood pressure and in urinary parameters, as proteinuria and uric acid. Also was verified an improvement in plasmatic triglycerides and uric acid. In white adipose tissue, GW501516 was able to decrease the components of this axis and improved inflammation as well. In liver, it was not found differences in axis, but JAK2 phosphorylation AT1r-dependent was decreased and consequently it was found a diminished activations of hepatic stellate cells. In addition, proteins and genes related to β-oxidation were increased with GW501516 and those related to lipogenesis de novo, were diminished, improving hepatic parenchyma. Kidneys presented an improvement of inflammation state, although it was not found differences in axis with treatment. Also, it was not found differences in gene and protein expression in relation to anti-oxidants proteins. These results show that short-administration of GW501516 could alleviate inflammatory effects and hepatic steatosis caused by high fructose diet, suggesting that GW501516 could be a new therapeutic option to treat the high activation of ACE/AT1r axis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A superativação do eixo ECA/AT1r está intimamente relacionada à síndrome metabólica e no organismo tem grande relação com o quadro de inflamação. A administração de frutose, seja por dieta ou pela água, tem sido usada como um modelo para a indução da superatividade desse eixo e para o estudo das vias inflamatórias relacionadas ao AT1r. Com isso, o objetivo deste trabalho foi avaliar se a administração de GW510156 poderia diminuir a superativação do eixo ECA/AT1r e consequentemente diminuir os danos causados pela dieta rica em frutose. Para isso foram utilizados camundongos machos C57Bl/6 que receberam uma dieta contendo 47% de frutose durante oito semanas ou uma dieta controle. Após oito semanas, os grupos foram redivididos aleatoriamente para o início da administração do GW501516 durante três semanas, totalizando quatro grupos experimentais. Os animais tratados apresentaram uma melhora da pressão arterial sistólica e também dos parâmetros urinários como proteinúria e ácido úrico. Houve ainda uma melhora dos triglicerídeo e ácido úrico plasmáticos. No tecido adiposo branco, o GW501516 foi capaz de diminuir a expressão dos componentes do eixo ECA/AT1r e também amenizou a inflamação causada pela dieta rica em frutose. No fígado, não houve alterações significativa do eixo, porém a fosforilação de JAK2 dependente de AT1r foi diminuída e consequentemente houve uma menor ativação das células estreladas no grupo que recebeu o GW501516. Além disso, as proteínas e genes relacionados à β-oxidação foram aumentados com o tratamento e aqueles relacionados à lipogênese de novo, diminuídos o que resultou em menor esteatose no parênquima hepático. Os rins apresentaram uma melhora da inflamação induzida pelo eixo, apesar de o eixo também não ter apresentado diferenças significativas com o tratamento. Também não foram encontradas diferenças significativas na expressão proteica e gênica das proteínas antioxidantes. Com esses resultados podemos concluir que a curta administração do GW501516 pôde aliviar os efeitos inflamatórios e a esteatose hepática causada pela dieta rica em frutose, podendo ser pensado como uma nova ferramenta terapêutica no tratamento da superativação do eixo ECA/AT1r. / High-activation of ACE/AT1r axis is closely linked to metabolix syndrome and low-grade inflammation state. Fructose administration in water or in diet has been proposed as a model to study the high-activity of this axis and AT1r-related inflammatory pathways. In this view, we aimed to evaluate if GW501516 administration could decrease the high-activation of ACE/AT1r axis and consequently fructose damage. To this males mice C57Bl/6 were fed a high-fructose diet (47%) during eight weeks or standard-chow diet. After eight weeks, the groups were randomly divided to start treatment with GW501516, totaling four experimental groups. Animals treated with GW501516 presented an improvement of systolic blood pressure and in urinary parameters, as proteinuria and uric acid. Also was verified an improvement in plasmatic triglycerides and uric acid. In white adipose tissue, GW501516 was able to decrease the components of this axis and improved inflammation as well. In liver, it was not found differences in axis, but JAK2 phosphorylation AT1r-dependent was decreased and consequently it was found a diminished activations of hepatic stellate cells. In addition, proteins and genes related to β-oxidation were increased with GW501516 and those related to lipogenesis de novo, were diminished, improving hepatic parenchyma. Kidneys presented an improvement of inflammation state, although it was not found differences in axis with treatment. Also, it was not found differences in gene and protein expression in relation to anti-oxidants proteins. These results show that short-administration of GW501516 could alleviate inflammatory effects and hepatic steatosis caused by high fructose diet, suggesting that GW501516 could be a new therapeutic option to treat the high activation of ACE/AT1r axis.