Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, results are sometimes controversial, since experimental conditions diverge, including age and strain of used animals. The purpose of this study was to evaluate the metabolic modifications in liver mitochondria, both in the presence of severe (STZ-treated rats) and mild hyperglycaemia [Goto-Kakizaki (GK) rats], when compared with control animals of similar age. Moreover, metabolic alterations were evaluated also at initial and advanced stages of the disease. We observed that both models of diabetes (type 1 and type 2) presented a decreased susceptibility of liver mitochondria to the induction of permeability transition (MPT). Apparently, there is a positive correlation between the severity of diabetes mellitus (and duration of the disease) and the decline in the susceptibility to MPT induction. We also found that liver mitochondria isolated from diabetic rats presented some metabolic adaptations, such as an increase in coenzyme Q and cardiolipin contents, that can be responsible for the observed decrease in the susceptibility to multiprotein pore (MPTP) opening.

Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, some of the results reported are controversial, since experimental conditions, such as aging, and/or strain of animals used were different. The purpose of this study was to evaluate the metabolic changes in liver mitochondria, both in the presence of severe hyperglycaemia (STZ-treated rats) and mild hyperglycaemia (Goto-Kakizaki (GK) rats). Moreover, metabolic alterations were evaluated both at initial and at advanced states of the disease. We observed that both models of type 1 and type 2 diabetes presented alterations on respiratory chain activity. Because of continual severe hyperglycaemia, 9 weeks after the induction of diabetes, the respiratory function declined in STZ-treated rats, as observed by membrane potential and respiratory ratios (RCR, P/O, and FCCP-stimulated respiration) assessment. In contrast, GK rats of 6 months age presented increased respiratory ratios. To localize which respiratory complexes are affected by diabetes, enzymatic respiratory chain activities were evaluated. We observed that succinate dehydrogenase and cytochrome c oxidase activities were significantly augmented both in STZ-treated rats and GK rats of 6 months age. Moreover, H(+)-ATPase activity was also significantly increased in STZ-treated rats with 3 weeks of diabetes and in GK rats of 6 months age as compared to controls. Therefore, these results clearly suggest that both animal models of diabetes present some metabolic adjustments in order to circumvent the deleterious effects promoted by the high glucose levels typical of the disease.

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The respiratory function and the antioxidant capacity of liver mitochondrial preparations isolated from Goto-Kakizaki non-insulin dependent diabetic rats and from Wistar control rats, with the age of 6 months, were compared. It was found that Goto-Kakizaki mitochondrial preparations presented a higher coupling between oxidative and phosphorylative systems, compared to non-diabetic preparations. Goto-Kakizaki mitochondria presented a lower susceptibility to lipid peroxidation induced by ADP/Fe2+, as evaluated by the formation of thiobarbituric acid substances. The decreased susceptibility to peroxidation in diabetic rats was correlated with an increase in mitochondrial vitamin E (alpha-tocopherol) content and GSH/GSSG ratio. Moreover, the glutathione reductase activity was significantly increased, whereas the glutathione peroxidase was decreased. Superoxide dismutase activity was unchanged in diabetic rats. Fatty acid analyses showed that the content in polyunsaturated fatty acids of Goto-Kakizaki mitochondrial membranes was significantly higher compared to controls. These results indicate that the lower susceptibility to lipid peroxidation of mitochondria from diabetic rats was related to their antioxidant defense systems, and may correspond to an adaptative response of the cells against oxidative stress in the early phase of diabetes.

Pterospartum tridentatum Willk. (prickled broom) is an autochthonous plant, common in Portuguese territory. The yellow flowers are used in traditional medicine, as a potential cure for all body illnesses, mainly to treat throat irritations or for diabetes, hypertension and hypercholesterolemia therapy. Despite its traditional use, no toxicological assessment has been performed to our knowledge. A high antioxidant activity of P. tridentatum flower water extract was found, in good agreement with its electrospray ionisation-mass spectroscopy (ESI-MS) spectrum which revealed the presence of several flavonoids, such as luteolin-O-(O-acetyl)-glucuronide, luteolin-Oglucuronide or isorhamnetin-O-hexoside. Mitocondrial respiratory rates (state 4, state 3 and FCCP-stimulated respiration) and respiratory indexes (respiratory control and P/O ratios) showed no consistent decrease of respiratory and phosphorylative efficiencies for the concentrations tested (up to 500 mg.mL 71 ). Cytotoxicity evaluation, using MTT assay, was in agreement with the previous results. In conclusion, for the concentration range commonly used, P. tridentatum flower usage can be regarded as harmless and credible.Keywords: antioxidant capacity; cytotoxicity; oxidative phosphorylation; phytotherapy; Pterospartum tridentatum (L.); Willk.; toxicologic evaluation Pterospartum tridentatum Willk. es una planta auto´ctona comu´n en el territorio portugue´s. Las flores amarillas se usan en medicina tradicional como cura potencial para todas las enfermedades, especialmente para tratamientos de irritacio´n de garganta o para tratamientos contra diabetes, hipertensio´n o hipercolesterolemia. A pesar de su uso tradicional, ninguna evaluacio´n toxicolo´gica se ha llevado a cabo. Se encontro´una alta actividad antioxidante de extracto acuoso de flor de P. Tridentatum, concordando con su espectro ESI-MS, el cual revelo´la presencia de varios flavonoides, como luteolina O-(O-acetil)-glucuro´nido, luteolina-Oglucuro´nido o isorramntina-O-hexo´sido. Las frecuencias respiratorias mitocondriales (estado 4, estado 3 y respiracio´n estimulada por FCCP) mostraron un descenso no consistente de eficiencia respiratoria y fosforilativa para las concentraciones analizadas (hasta 500 mg.mL -1 ). La evaluacio´n de citotoxicidad, usando ensayo MTT, coincidio´con los resultados previos. En conclusio´n, para las concentraciones comu´nmente empleadas, el uso de las flores de P. tridentatum se puede considerar inocuo y verosı´mil.Palabras claves: capacidad antioxidante; citotoxicidad; fosforilacio´n oxidativa; fitoterapia; Pterospartum tridentatum (L.) Willk; evaluacio´n toxicolo´gica Abbreviations: ESI, electrospray ionization; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HepG2, a human hepatoma cell line; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P/O ratio, ratio of ADP molecules phosphorylated to atoms of oxygen consumed; RCR, respiratory control ratio (state 3 respiratory rate/state 4 respiratory rate)