D-galactose-induced aging in rats - The effect of metformin on bioenergetics of brain, skeletal muscle and liver.

Chronic D-galactose administration induces accelerated aging in rodents. The aim of the study was to find by in vivo31P MRS suitable markers of early stages of brain degeneration on this metabolic model in rats. Additionally, we studied the therapeutic effect of antidiabetic drug metformin. The study has been extended by in vitro determination of mitochondrial function in brain, skeletal muscle and liver mitochondria, oxidative stress parameter thiobarbituric acid reactive substances (TBARS), and lipophilic antioxidants levels. In vivo31P MRS revealed lower intracellular pH (pHi) and lower inorganic phosphate to ATP ratio (Pi/ATP), with higher index of oxidative phosphorylation - phosphocreatine (PCr) to Pi ratio - in brain of rats chronically administered with D-galactose. The function of brain mitochondria was not affected. Administration of metformin diminished changes in brain pHi and plasma TBARS. The function of skeletal muscle mitochondria and their coenzyme Q (CoQ) content were considerably reduced after D-galactose administration. Metformin administered simultaneously with D-galactose did not prevent these changes. The results of in vivo31P MRS revealed evidence of early stage of neurodegeneration that may indicate pre-inflammation. Our data show different susceptibility of brain, skeletal muscle, and liver to the chronic exposure to D-galactose and metformin. The D-galactose model presented in the literature as a model for "age-related dementia" had much more devastating effects on skeletal muscle than on the brain.

Platelet mitochondrial respiration and coenzyme Q10 could be used as new diagnostic strategy for mitochondrial dysfunction in rheumatoid diseases.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory autoimunne disorder affecting both small and large synovial joints, leading to their destruction. Platelet biomarkers are involved in inflammation in RA patients. Increased circulating platelet counts in RA patients may contribute to platelet hyperactivity and thrombosis. In this pilot study we evaluated platelet mitochondrial bioenergy function, CoQ10 levels and oxidative stress in RA patients. METHODS: Twenty-one RA patients and 19 healthy volunteers participated in the study. High resolution respirometry (HRR) was used for analysis of platelet mitochondrial bioenergetics. CoQ10 was determined by HPLC method; TBARS were detected spectrophotometrically. RESULTS: Slight dysfunction in platelet mitochondrial respiration and reduced platelet CoQ10 levels were observed in RA patients compared with normal controls. CONCLUSIONS: The observed decrease in platelet CoQ10 levels may lead to platelet mitochondrial dysfunction in RA diseases. Determination of platelet mitochondrial function and platelet CoQ10 levels could be used as new diagnostic strategies for mitochondrial bioenergetics in rheumatoid diseases.

Platelet Mitochondrial Respiration, Endogenous Coenzyme Q10 and Oxidative Stress in Patients with Chronic Kidney Disease.

Chronic kidney disease (CKD) is characterized by a progressive loss of renal function and a decrease of glomerular filtration rate. Reduced mitochondrial function, coenzyme Q10 (CoQ10), and increased oxidative stress in patients with CKD contribute to the disease progression. We tested whether CoQ10 levels, oxidative stress and platelet mitochondrial bioenergetic function differ between groups of CKD patients. METHODS: Twenty-seven CKD patients were enrolled in this trial, 17 patients had arterial hypertension (AH) and 10 patients had arterial hypertension and diabetes mellitus (AH and DM). The control group consisted of 12 volunteers. A high-resolution respirometry (HRR) method was used for the analysis of mitochondrial bioenergetics in platelets, and an HPLC method with UV detection was used for CoQ10 determination in platelets, blood, and plasma. Oxidative stress was determined as thiobarbituric acid reactive substances (TBARS). RESULTS: Platelets mitochondrial respiration showed slight, not significant differences between the groups of CKD patients and control subjects. The oxygen consumption by intact platelets positively correlated with the concentration of CoQ10 in the platelets of CKD patients. CONCLUSION: A decreased concentration of CoQ10 and oxidative stress could contribute to the progression of renal dysfunction in CKD patients. The parameters of platelet respiration assessed by high-resolution respirometry can be used only as a weak biological marker for mitochondrial diagnosis and therapy monitoring in CKD patients.

A new insight into the molecular hydrogen effect on coenzyme Q and mitochondrial function of rats.

Mitochondria are the major source of cellular energy metabolism. In the cardiac cells, mitochondria produce by way of the oxidative phosphorylation more than 90% of the energy supply in the form of ATP, which is utilized in many ATP-dependent processes, like cycling of the contractile proteins or maintaining ion gradients. Reactive oxygen species (ROS) are by-products of cellular metabolism and their levels are controlled by intracellular antioxidant systems. Imbalance between ROS and the antioxidant defense leads to oxidative stress and oxidative changes to cellular biomolecules. Molecular hydrogen (H2) has been proved as beneficial in the prevention and therapy of various diseases including cardiovascular disorders. It selectively scavenges hydroxyl radical and peroxynitrite, reduces oxidative stress, and has anti-inflammatory and anti-apoptotic effects. The effect of H2 on the myocardial mitochondrial function and coenzyme Q levels is not well known. In this paper, we demonstrated that consumption of H2-rich water (HRW) resulted in stimulated rat cardiac mitochondrial electron respiratory chain function and increased levels of ATP production by Complex I and Complex II substrates. Similarly, coenzyme Q9 levels in the rat plasma, myocardial tissue, and mitochondria were increased and malondialdehyde level in plasma was reduced after HRW administration. Based on obtained data, we hypothesize a new metabolic pathway of the H2 effect in mitochondria on the Q-cycle and in mitochondrial respiratory chain function. The Q-cycle contains three coenzyme Q forms: coenzyme Q in oxidized form (ubiquinone), radical form (semiquinone), or reduced form (ubiquinol). H2 may be a donor of both electron and proton in the Q-cycle and thus we can suppose stimulation of coenzyme Q production. When ubiquinone is reduced to ubiquinol, lipid peroxidation is reduced. Increased CoQ9 concentration can stimulate electron transport from Complex I and Complex II to Complex III and increase ATP production via mitochondrial oxidative phosphorylation. Our results indicate that H2 may function to prevent/treat disease states with disrupted myocardial mitochondrial function.

Platelets mitochondrial function depends on CoQ10 concentration in winter, not in spring season.

Seasonal variations in temperature may influence the physiological and pathological metabolic pathways, concentrations of antioxidants, degree of oxidative stress and mitochondrial function. The aim of this study was to evaluate platelet mitochondrial function in human subjects during seasonal variations in temperature. Two groups of healthy young subjects were enroled in the study. Winter group, mean outside temperature was 4.77°C and Spring group, mean outside temperature was 24.32°C. High-resolution respirometry method was used for determination of mitochondrial respiration and oxidative phosphorylation in platelets. Concentrations of coenzyme Q10 (CoQ10) and tocopherols were determined in platelets, blood and plasma. Our data showed slightly (not significantly) reduced respiration in intact platelets, basal and ADP-stimulated mitochondrial respiration at Complex I, as well as CoQ10-TOTAL and α-tocopherol concentrations in winter. The concentration of γ-tocopherol was higher in winter. Platelet mitochondrial ATP production depended on platelet CoQ10-TOTAL concentration in winter, not in spring. We conclude that seasonal temperature participates in the mechanism of platelet mitochondrial respiratory chain function and oxidative phosphorylation that depends on their CoQ10-TOTAL concentration at lower winter outside temperature. CoQ10 supplementation may improve platelets mitochondrial ATP production at winter season. High-resolution respirometry offers sensitive method for detection of changes of platelets mitochondrial respiratory function.

Rooibos tea (Aspalathus linearis) ameliorates the CCl4-induced injury to mitochondrial respiratory function and energy production in rat liver.

The rooibos tea (RT) is a source of valuable dietary dihydrochalcones  aspalathin, and nothofagin and other polyphenols. Many in vitro and in vivo studies have shown that RT flavonoids have strong antioxidant effect and significantly reduce oxidative stress. We investigated the antioxidant activity and protective effect of an aqueous extract of RT on the liver mitochondria oxidative phosphorylation in rats with carbon tetrachloride-induced (CCl4-induced) liver damage. Mitochondrial respiration and ATP production was determined amperometrically using a Clark-type oxygen electrode. We found significantly decreased parameters of oxidative phosphorylation in the group having received CCl4 for 10 weeks. Simultaneous administration of RT increased oxygen uptake stimulated with ADP, and the rate of ATP generation in the mitochondria of rats, both having been impaired in rats treated with CCl4 only. Treatment with RT significantly decreased CCl4-induced elevated enzyme levels, improved capacity of the respiratory chain and energy production, presumably due to its potent and direct antioxidant activity, including inhibition of mitochondrial lipid peroxidation. Improved histological features support the view of antioxidant and membrane-stabilizing activity of RT. This fact may play a significant role in the protection of the liver from injury caused by known toxins, and from subsequent development of steatosis and fibrosis..

Neuronal marker recovery after Simvastatin treatment in dementia in the rat brain: in vivo magnetic resonance study.

The aim of study was to search for new biomarkers with a magnetic resonance technique to identify the early stages of dementia, induced by D-galactose, and evaluate Simvastatin therapy. Localized proton magnetic resonance spectroscopy measurements showed a significant decrease in the concentration of N-acetylaspartate+N-acetylaspartylglutamate and myo-inositol in the D-galactose group compared to the control group, and, conversely, an increase of N-acetylaspartate+N-acetylaspartylglutamate in the D-galactose/Simvastatin group. Using a saturation transfer experiment, with phosphorus magnetic resonance spectroscopy, we observed a significant elevation of the forward rate constant of the creatine kinase reaction in the brains of the D-galactose group compared to controls, and subsequently, a significant reduction of this reaction in the D-galactose/Simvastatin group. Spatial learning and memory were evaluated using the modified Morris water maze test. The dynamics of the learning process represented by the learning index revealed a significant reduction in learning in the D-galactose group, but the deficits as a consequence of the D-galactose effects were recovered in the D-galactose/Simvastatin group, in which the learning dynamics resembled those of the control group. By determining the thiobarbituric acid reactive substances and total coenzyme Q9 in plasma, we have shown that long-term administration of D-galactose created conditions for oxidative stress, and that the administration of Simvastatin decreased oxidative stress in plasma. Volumetry analyses from the hippocampal area show a reduction in the segmented area in the D-galactose group, compared with the control group, and an enlarged area in the hippocampus in the d-galactose/Simvastatin group.

Effect of PUFA-rich plant oil on risk factors of STZ-induced diabetes in Wistar rats.

OBJECTIVE: This study has been focused on the effect of an n-6 polyunsaturated fatty acid (PUFA) rich plant oil on oxidation and glycooxidation stress markers as well as on antioxidant enzyme activities in male Wistar rats with streptozotocin-induced diabetes. METHODS: The non-diabetic and diabetic groups of Wistar rats were administered plant oil at concentrations of 100 and 500 mg/kg body weight and controls without plant oil. The parameters of glycaemic control, lipid profile, total antioxidant status, antioxidant enzyme activities, together with oxidative and glycooxidative stress markers were measured in the blood. RESULTS: The intake of the plant oil did not significantly influence the parameters of glycaemic control and significantly increased the levels of all lipid profile parameters in the diabetic rats. Plant oil administration significantly decreased the total antioxidant status and glutathione peroxidase activity and the activity of Cu/Zn superoxide dismutase was significantly increased. The plant oil also increased the levels of lipoperoxides and advanced the glycation end products. DISCUSSION: These results suggest that the plant oil with high concentrations of n-6 PUFA - linoleic acid, acts prooxidatively when administered to the rats.

Effects of coenzyme Q and creatine supplementation on brain energy metabolism in rats exposed to chronic cerebral hypoperfusion.

It is known that oxidative stress and mitochondrial dysfunction both play an important role in animal models of brain ischemia. The present study was undertaken to test whether oral supplementation of coenzyme Q10 (ubiquinone) or creatine citrate could protect against brain ischemia-induced mitochondrial damage in the rats model. Brain ischemia was induced for 50 minutes with three-vessel occlusion (3-VO). Coenzyme Q10 was administered for 30 days before the ischemic event and coenzyme Q10 or creatine citrate for 30 days post-ischemia. Moreover, the concentrations of coenzyme Q10 and α-, γ- tocopherols as well as the formation of thiobarbituric acid reactive substances (TBARS) were measured in brain mitochondria and in plasma. Transient hypoperfusion revealed significant impairment in brain energy metabolism as detected by mitochondrial oxidative phosphorylation as well as decreased concentrations of brain and plasma endogenous antioxidants and increased formation of TBARS in plasma. When compared with the ischemic group, supplementation of coenzyme Q10 was ineffective as a preventive agent. However, the positive effect of therapeutic coenzyme Q10 supplementation was supported by the oxygen consumption values (p < 0.05) and ATP production (p < 0.05) in brain mitochondria, as well as by increased concentration of coenzyme Q9 (p < 0.05) and concentration of α-tocopherol (p < 0.05) in brain mitochondria and by increased concentration of α-tocopherol (p < 0.05) and γ-tocopherol in plasma. This suggests that coenzyme Q10 therapy involves resistance to oxidative stress and improved brain bioenergetics, when supplemented during reperfusion after ischemic brain injury.

Mitochondrial function in heart and kidney of spontaneously hypertensive rats: influence of captopril treatment.

Effect of captopril treatment on capability of heart and kidney mitochondria to produce ATP was investigated in spontaneously hypertensive rats (SHR). Heart mitochondria from SHR responded to hypertension with tendency to compensate the elevated energy demands of cardiac cells by moderate increase in mitochondrial Mg2+-ATPase activity, membrane fluidity (MF) and in majority of functional parameters of the mitochondria (p>0.05). Significant increase exhibited only the oxygen consumption (QO2; p<0.01-0.001) and oxidative phosphorylation rate (OPR; p<0.003) with glutamate+malate (GLUT+MAL) as substrates. Lowering the blood pressure (p<0.02) captopril also eliminated the above compensatory response and impaired the oxidative ATP production by decreasing OPR (p<0.001). Kidney mitochondria of SHR experienced serious disarrangement in parameters of oxidative ATP production: increase in Mg2+-ATPase activity (p<0.05) but, also scattered QO2 values (p<0.03-0.01) leading to decrease in OPR and the ADP:O (p<0.05-0.01) values with both GLUT+MAL and succinate as substrates. Captopril treatment does not alleviated but even worsened the above alterations. Mg2+-ATPase became also decreased and the depression of ADP:O became aggravated (p<0.0001).

In vivo and in vitro assessment of brain bioenergetics in aging rats.

Brain energy disorders can be present in aged men and animals. To this respect, the mitochondrial and free radical theory of aging postulates that age-associated brain energy disorders are caused by an imbalance between pro- and anti-oxidants that can result in oxidative stress. Our study was designed to investigate brain energy metabolism and the activity of endogenous antioxidants during their lifespan in male Wistar rats. In vivo brain bioenergetics were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and in vitro by polarographic analysis of mitochondrial oxidative phosphorylation. When compared to the young controls, a significant decrease of age-dependent mitochondrial respiration and adenosine-3-phosphate (ATP) production measured in vitro correlated with significant reduction of forward creatine kinase reaction (kfor) and with an increase in phosphocreatine (PCr)/ATP, PCr/Pi and PME/ATP ratio measured in vivo. The levels of enzymatic antioxidants catalase, GPx and GST significantly decreased in the brain tissue as well as in the peripheral blood of aged rats. We suppose that mitochondrial dysfunction and oxidative inactivation of endogenous enzymes may participate in age-related disorders of brain energy metabolism.

Effect of poly(amido)amine (PAMAM) G4 dendrimer on heart and liver mitochondria in an animal model of diabetes.

Diabetes-induced injury related to hyperglycaemia is associated with impaired function of mitochondria. Regardless of their cytotoxicity, PAMAM [poly(amido)amine] G4 dendrimers lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia in experimental diabetes. In the present study, we aimed at verifying whether such modulatory effects of PAMAM G4 (0.5 micromol/kg of body weight daily for 60 days) may contribute to improved respiration in heart and liver mitochondria from streptozotocin-diabetic rats. PAMAM G4 alleviated long-term markers of hyperglycaemia and reduced blood and tissue lipophilic antioxidants in diabetic animals, but did not restore mitochondrial function. In hearts, but not livers, dendrimers further reduced respiratory function and oxidative phosphorylation. Thus ameliorating effects of PAMAM G4 on glycation and glycoxidation in experimental diabetes are not sufficient to restore the impaired mitochondrial function in diabetes.

Influence of pyridoxylidene aminoguanidine on biomarkers of the oxidative stress and selected metabolic parameters of rats with diabetes mellitus.

Oxidative damage is considered to play an important role in the pathogenesis of several diseases, such as diabetes mellitus (DM), atherosclerosis, cardiovascular complications and chronic renal failure. DM is associated with the oxidative stress and formation of advanced glycation end products (AGEs). Different drugs inhibit oxidative stress and formation of advanced glycation end products. Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of DM. Recent reports show a pro-oxidant activity of AG. Therefore we examined the effect of structural analogue of AG, its Schiff base with pyridoxal-pyridoxylidene aminoguanidine (PAG) on the level of selected markers of oxidative stress. We found that PAG decreased total damage to DNA in controls as well as in diabetic group of rats. However, we also found that PAG supplementation increases susceptibility of lipoproteins to oxidation and formation of conjugated dienes in both, diabetic as well as control animals. Its administration to diabetic rats decreases antioxidant capacity of plasma. Therefore, it is necessary to search for other structural modifications of AG that would combine its higher anti-diabetic activity with less toxicity.

PAMAM G4 dendrimers lower high glucose but do not improve reduced survival in diabetic rats.

For nearly a decade poly(amidoamine) (PAMAM) dendrimers G4 were claimed unnegligible cytotoxic agents. Here we monitored whether in vivo cytotoxic effect of PAMAM G4 (0.5 micromol kg(-1) day(-1)) may be compromised by its ameliorating effect on severe hyperglycaemia in chronic streptozotocin-diabetic Wistar rats. PAMAM G4 significantly reduced the 60-day overall survival in long-term experimental diabetes: treated animals were 6.7 times more likely to die than control animals (p<0.025). PAMAM G4 significantly reduced numerous biochemical parameters in blood, including glucose, glycated haemoglobin or protein oxidation, cholesterol and triglycerides, but apparently unchanged plasma insulin peptide C. Terminal blood glucose in PAMAM-treated animals was significantly higher in survivors, pointing to the possible preventive role of glycation in reducing of PAMAM G4 cytotoxicity. Our results provide the first in vivo evidence that PAMAM G4 is able to lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia. Nevertheless, this beneficial influence cannot override PAMAM G4 cytotoxic effects in the increased mortality of streptozotocin-diabetic rats.

Study of the oxidative stress in a rat model of chronic brain hypoperfusion.

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k(for), was studied by saturation transfer (31)P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k(for) in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of alpha-phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (.OH) trapped in DMSO as the .CH(3) adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.