Fernanda M. Ferreira

Decreased susceptibility to lipid peroxidation of Goto-Kakizaki rats: Relationship to mitochondrial antioxidant capacity

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.

Higher efficiency of the liver phosphorylative system in diabetic Goto‐Kakizaki (GK) rats

Liver mitochondrial bioenergetics of Goto‐Kakizaki (GK) rats (a model of non‐insulin dependent diabetes mellitus) reveals a ΔΨ upon energization with succinate significantly increased relatively to control animals. The repolarization rate following ADP phosphorylation is also significantly increased in GK mitochondria in parallel with increased ATPase activity. The increase in the repolarization rate and ATPase activity is presumably related to an improved efficiency of F0F1‐ATPase, either from a better phosphorylative energy coupling or as a consequence of an enlarged number of catalytic units. Titrations with oligomycin indicate that diabetic GK liver mitochondria require excess oligomycin pulses to completely abolish phosphorylation, relative to control mitochondria. Therefore, accepting that the number of operational ATP synthase units is inversely proportional to the amount of added oligomycin, it is concluded that liver mitochondria of diabetic GK rats are provided with extra catalytic units relative to control mitochondria of normal rats. Other tissues (kidney, brain and skeletal muscle) were evaluated for the same bioenergetic parameters, confirming that this feature is exclusive to liver from diabetic GK rats.

Alterations of liver mitochondrial bioenergetics in diabetic Goto-Kakizaki rats

Respiratory indexes and the transmembrane electrical potential (delta psi) were evaluated in mitochondrial preparations from 6-month-old Goto-Kakizaki (GK) and Wistar rats in the presence of glutamate + malate and succinate. We found that in diabetic GK mitochondria, flavin adenine dinucleotide (FAD)-linked respiratory indexes (respiratory control ratio [RCR] and adenosine diphosphate [ADP] to oxygen ratio [ADP/O]) are increased and uncoupled respiration is largely enhanced, indicating increased respiratory chain activity in GK rats. Delta psi development in GK mitochondrial preparations, energized using glutamate + malate or succinate as substrates, and the repolarization rate upon phosphorylation of the added ADP were significantly higher in GK mitochondrial preparations. These results indicate an enhanced activity of the phosphorylation system, confirmed by evaluating delta psi development when the mitochondria are energized by adenosine triphosphate (ATP). Moreover, recovery of the potential upon a phosphorylative cycle is increased in GK mitochondria, reflecting a more efficient coupling between the phosphorylative and oxidative system. Contrasting with results obtained for alloxan- or streptozotocin-induced diabetic rats, this study clearly demonstrates no impairment of mitochondrial bioenergetics in diabetic GK rats. On the contrary, at this age, we observed a higher efficiency of the phosphorylation system as compared with Wistar rats.

Nerolidol effects on mitochondrial and cellular energetics

In the present work, we evaluated the potential toxic effects of nerolidol, a sesquiterpenoid common in plants essential oils, both on mitochondrial and cellular energetics. Samples of enriched natural extracts of nerolidol (a racemic mixture of cis and trans isomers) were tested on rat liver mitochondria and a decrease in phosphorylative system was observed but not in the mitochondrial respiratory chain activity, which reflects a direct effect on F1-ATPase. Hence, respiratory control ratio was also decreased. Cellular ATP/ADP levels were significantly decreased in a concentration-dependent manner, possibly due to the direct effect of nerolidol on F(0)F(1)-ATPsynthase. Nerolidol stimulates respiratory activity probably due to an unspecific effect, since it does not show any protonophoric effect. Furthermore, we observed that mitochondrial permeability transition was delayed in the presence of nerolidol, possibly due to its antioxidant activity and because this compound decreases mitochondrial transmembrane electric potential. Our results also show that, in human hepatocellular liver carcinoma cell line (HepG2), nerolidol both induces cell death and arrests cell growth, probably related with the observed lower bioenergetic efficiency.

Age-related alterations in liver mitochondrial bioenergetics of diabetic Goto-Kakizaki rats.

Abstract Type 2 diabetes mellitus is one of the most common chronic metabolic diseases in man. Due to long-term complications of the disease, severely decreasing the quality of life of diabetic patients, early interventions to obviate the risk of complications are of major importance. Therefore, diabetic animal models are of major importance in research for interventional treatment of type 2 diabetes.In this work we investigated the possible alterations in mitochondrial energetic metabolism of Goto-Kakizaki (GK) rats during the progression of the disease, since glucose metabolism is closely related to intracellular ATP content. For that reason, respiratory indexes (state 4, state 3, RCR and ADP/O) were evaluated either in the presence of NAD- or FAD-linked substrates (glutamate + malate and succinate, respectively) in mitochondrial preparations of GK and control rats with 8, 12, 26 and 52 weeks of age. Until the age of 1 year (52 weeks) we found no impairment of mitochondrial respiratory indexes both in the presence of glutamate + malate and succinate. In conclusion, this study indicates that GK rat is a good model for studying the initial events of diabetes, since it presents no impairment of liver mitochondrial functions during the first year of life, contrasting clearly with pharmacological induced diabetes.

Histological changes and impairment of liver mitochondrial bioenergetics after long-term treatment with α-naphthyl-isothiocyanate (ANIT)

This study was designed to evaluate the effects of long-term treatment with alpha-naphthyl-isothiocyanate (ANIT) on liver histology and at the mitochondrial bioenergetic level. Since, ANIT has been used as a cholestatic agent and it has been pointed out that an impairment of mitochondrial function is a cause of hepatocyte dysfunction leading to cholestatic liver injury, serum markers of liver injury were measured and liver sections were analyzed in ANIT-treated rats (i.p. 80 mg/kg/week x 16 weeks). Mitochondrial parameters such as transmembrane potential, respiration, calcium capacity, alterations in permeability transition susceptibility and ATPase activity were monitored. Histologically, the most important features were the marked ductular proliferation, proliferation of mast cells and the presence of iron deposits in ANIT-treated liver. Mitochondria isolated from ANIT-treated rats showed no alterations in state 4 respiration, respiratory control ratio and ADP/O ratio, while state 3 respiration was significantly decreased. No changes were observed on transmembrane potential, but the repolarization rate was decreased in treated rats. Consistently with these data, there was a significant decrease in the ATPase activity of treated mitochondria. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to mitochondrial permeability transition pore opening (lower calcium capacity). Since, human cholestatic liver disease progress slowly overtime, these data provide further insight into the role of mitochondrial dysfunction in the process.

Antioxidant capacity and toxicological evaluation of Pterospartum tridentatum flower extracts

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-O-glucuronide 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 μg.mL−1). 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.

Mitochondrial Bioenergetics, Diabetes, and Aging: Top-Down Analysis Using the Diabetic Goto-Kakizaki (GK) Rat as a Model

In the present study we investigated the changes in the oxidative phosphorylation system of liver mitochondria, isolated from diabetic Goto-Kakizaki (GK) and Wistar (control) rats with different ages (6, 12, 26, and 52 weeks). We used a kinetic approach known as “top-down” analysis, which conceptually divides the oxidative phosphorylation system into two subsystems: one producing the protonmotive force (Δp) and another that consumes Δp. The overall response of the Δp generators to Δp was obtained from an uncoupler titration of respiration rate versus Δp, while the overall response of Δp consumers to Δp was obtained from an inhibitor titration of respiration rate versus Δp. Our results showed that GK liver mitochondrial preparations presented an increase in Δp production and phosphorylative subsystems (using succinate as respiratory substrate). The alterations observed may suggest the existence of biochemical compensatory mechanisms to type 2 diabetes mellitus in GK rats during their first year of life, in order to reduce the injury associated with the disease. Furthermore, we observed that liver metabolic efficiency of mitochondrial respiration declined with age, this decrease in respiratory activity being visible both in control and diabetic rats.