Hyperglycaemia caused by insulin deficiency is believed to play a major role in the de-velopment of neuropathy in diabetic patients. The clinical and pathological features of diabetic neuropathy vary considerably, although sensory and autonomic dysfunctions are the most common characteristics. Normalisation of the blood glucose level by ef-fective insulin treatment decreases the incidence of diabetic neuropathy in patients. However, intensive insulin therapy may result in more frequent hypoglycaemic epi-sodes than are provoked by less ambitious diabetes control. Neuropathy might also be induced by severe hypoglycaemia in diabetes or insulinoma. Accordingly, it seems that the diversity in clinical symptoms of diabetic neuropathy may be due to the combined effects of hyperglycaemia and hypoglycaemia. Based on that assumption, the general aim of this project was to study the relationship between severe hypoglycaemia and pe-ripheral neuropathy in diabetic rats. To understand how the development of neuropathy is related to glycaemic control, we needed to be aware of the glucose dynamics in the animal model that we used. The aim was to ascertain whether the diabetic rats were similar to type 1 diabetic patients with regard to such dynamics. To achieve that goal, we used a MiniMed continuous glucose monitoring system (CGMS®) to measure sub-cutaneous glucose in freely moving rats over a period of 72 hours. The glucose monitor worked well, and it showed that the insulin-treated diabetic BB/Wor rats with a hyper-glycaemic insulin regimen have a glycaemic status similar to that of type 1 diabetic patients with poor glycaemic control. The diabetic rats with a hypoglycaemic regimen generally had low blood glucose levels. Prolonged hypoglycaemia led to axonal de- and regeneration of large myelinated fibres in vagus nerve destined to the laryngeal muscle. Axonal de- and regeneration was also observed in the gastrocnemius and sural nerves, although the frequency of degeneration was much lower in the sural nerve. Small myelinated and unmyelinated nerve fibres were normal in these nerves. These results suggest that hypoglycaemia preferentially damages muscle-related nerve fibres. In contrast, in the diabetic rats exposed to pro-longed hyperglycaemia, only the sural nerve exhibited decreased myelinated fibre diameter in the absence of obvious axonal degeneration. In situ glucose measurements by microdialysis showed that the glucose concentrations in blood and subcutaneous tissue were similar in healthy, diabetic hyperglycaemic, and diabetic hypoglycaemic rats. In the healthy and hyperglycaemic animals, the lowest glucose level was found in the peripheral nerve. Moreover, in controls, the glucose level was lower in muscle than in blood. In hypoglycaemic rats, there were no signifi-cant differences in glucose concentrations between different tissues.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-01T12:26:27Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Doutorado. Tese de Lindalva Vieira. Ano 2015..pdf: 1577129 bytes, checksum: 08d435d57b1dfb816c8e6a8dcd551f50 (MD5) / Made available in DSpace on 2016-04-01T12:26:27Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Doutorado. Tese de Lindalva Vieira. Ano 2015..pdf: 1577129 bytes, checksum: 08d435d57b1dfb816c8e6a8dcd551f50 (MD5) Previous issue date: 2015-07-02 / CAPEs / A Diabetes Mellitus (DM) é um grave problema de saúde pública, sendo uma das maiores causas de morbimortalidade na grande maioria dos países. Diferentes órgãos são acometidos pela DM; contudo, as doenças cardiovasculares são as maiores responsáveis pela elevada mortalidade vinculada à diabetes. A metformina é atualmente o principal fármaco utilizado para o tratamento da DM do tipo 2 (DM2), embora os riscos-benefícios cardiovasculares dessa droga não estejam totalmente elucidados. O presente estudo teve como objetivo avaliar o potencial arritmogênico da metformina em corações e cardiomiócitos de ratos saudáveis. No tratamento por 10 semanas com metformina os parâmetros biométricos e a glicemia plasmática não foram modificados, mas foi constatado um prolongamento dos intervalos PR, QT e QTc no eletrocardiograma, e uma redução da corrente transitória de saída de K+, Ito.Quando os cardiomiócitos foram incubados por 24 h com metformina, a amplitude da corrente Ito foi reduzida, enquanto aumentou a duração do potencial de ação cardíaco (DPA). A exposição de cardiomiócitos isolados à metformina por um período de 30 min, não reduziu a amplitude da corrente Ito, comprovando que a redução da corrente Ito em cardiomiócitos tratados por 10 semanas e incubados por 24 h com metformina, não ocorreu por uma ligação direta da droga ao canal. A corrente de cálcio tipo L (ICa-L) e a dependência de voltagem da inativação e recuperação da inativação da corrente Ito não foram afetadas em ambas as situações experimentais. Os níveis de RNAm que codifica as subunidades KV4.2, KV4.3 e KChIP2 do canal Ito não foram alterados, sugerindo que a redução da corrente Ito não ocorreria por uma menor síntese proteica das subunidades que compõem o canal Ito. Propõe-se que, esta redução seria resultante de uma acelerada degradação dos canais ou de uma deficiência funcional destes na membrana celular. A partir dos resultados obtidos na presente tese, conclui-se que a metformina apresenta potenciais efeitos arritmogênicos sobre o coração de animais saudáveis, capazes de levar eventualmente à morte súbita. / Diabetes Mellitus (DM) is a major public health problem, and is one of the biggest causes of morbidity and mortality in most countries. Many different human organs are affected by the DM, but cardiovascular diseases are the leading causes of mortality in patients with DM. Even today, the leading drug used to treat type-2 diabetes is metformin. However, its cardiovascular risk-benefits are not entirely clear. This study aimed to determine the potential arrhythmogenic effect of metformin in cardiomyocytes from healthy animals. Therefore, a 10 week treatment with metformin was performed, and it was proved that biometric and metabolic parameters were not affected. However, a lengthening of the PR, QT and QTc interval was found on the electrocardiogram, as well as a reduction in the output of the transitory potassium Ito current. When cardiomyocytes were incubated for 24 h with metformin, the amplitude of the Ito current was reduced and duration of cardiac action potential (APD) increased. Exposure of cardiomyocytes treated with metformin over a period of 30 min did not reduce the amplitude of the current Ito, demonstrating that the reduction in Ito current cardiomyocytes treated for 10 weeks and incubated for 24 h with metformin, does not occur by direct binding of the drug to the canal. The L-type calcium current (ICa-L) and the kinetic properties of voltage-dependent inactivation and recovery from inactivation of Ito current remained unchanged in both experimental conditions. RNAm levels of KV4.2, KChIP2 4.3 and Kv subunits remained unchanged. This suggests that the reduction of the Ito current may not be caused by a decrease in the protein synthesis subunit which is part of the Ito channel.Therefore, this reduction may be caused by a main degradation of the channels or by a worse operating performance in the cell membrane. Experiments accomplished during this thesis try to demonstrate that metformin causes arrhythmogenic effects in the heart of healthy animals.