Mild hypothermia during the reperfusion phase protects mitochondrial bioenergetics against ischemia-reperfusion injury in an animal model of ex-vivo liver transplantation-an experimental study.

The organ preservation paradigm has changed following the development of new ways to preserve organs. The use of machine perfusion to preserve organs appears to have several advantages compared with conventional static cold storage. For liver transplants, the temperature control provided by machine perfusion improves organ preservation. In this experimental study, we measured the effects of different temperatures on mitochondrial bioenergetics during the reperfusion phase. An experimental model of ex-vivo liver transplantation was developed in Wistar rats (Rattus norvegicus). After total hepatectomy, cold static preservation occurred at 4ºC and reperfusion was performed at 37ºC and 32ºC using a Langendorff system. We measured parameters associated with mitochondrial bioenergetics in the livers. Compared with the livers that underwent normothermic reperfusion, mild hypothermia during reperfusion caused significant increases in the mitochondrial membrane potential, the adenosine triphosphate content, and mitochondrial respiration, and a significant reduction in the lag phase (all P < 0.001). Mild hypothermia during reperfusion reduced the effect of ischemia-reperfusion injury on mitochondrial activity in liver tissue and promoted an increase in bioenergetic availability compared with normothermic reperfusion.

Mitohormesis and metabolic health: The interplay between ROS, cAMP and sirtuins.

The key role of mitochondria in oxidative metabolism and redox homeostasis explains the link between mitochondrial dysfunction and the development of metabolic disorders. Mitochondria's highly dynamic nature, based on alterations in biogenesis, mitophagy, fusion and fission, allows adjusting sequential redox reactions of the electron transport chain (ETC) and dissipation of the membrane potential by ATP synthase, to different environmental cues. With reactive oxygen species being an inevitable by-product of oxidative phosphorylation (OXPHOS), alterations on mitochondrial oxidative rate with a consequent excessive load of reactive oxygen species have been traditionally associated with pathological conditions. However, reactive oxygen species have also been suggested as promoters of mitohormesis, a process in which low, non-cytotoxic concentrations of reactive oxygen species promote mitochondrial homeostasis. Therefore, signaling systems involved in the regulation of mitochondrial homeostasis are attractive candidates for drug development for metabolic diseases triggered by mitochondrial dysfunction. Reversible phosphorylation downstream the cyclic AMP (cAMP) signaling cascade and deacetylation mediated by sirtuins are recognized as major mitochondrial regulators.

Evaluation of bioenergetic and mitochondrial function in liver transplantation.

BACKGROUND/AIMS: We measured changes in mitochondrial function and bioenergetics that occur during ischemia/ reperfusion in fresh liver samples of patients undergoing liver transplantation. These variations correlated with markers of liver function and clinical outcome. Ischemia/reperfusion injury related to liver transplantation affects mitochondrial function and bioenergetics. Experimental studies were conducted to identify the role of bioenergetics and mitochondrial dysfunction. To the best of our knowledge, no investigation of these two factors' impacts on liver transplantation has been performed. METHODS: This was a prospective study of 28 patients who underwent liver transplantation. We measured parameters of mitochondrial function and bioenergetics in biopsies performed during the procedure. RESULTS: We observed a statistically significant reduction in mitochondrial membrane potential, an increase in lag phase, and decreases in mitochondrial respiration and adenosine triphosphate content (P<0.010). Higher postoperative aminotransferase peaks correlated with worse mitochondrial function; mitochondrial respiration correlated with arterial lactate (P<0.010). CONCLUSION: There is a relationship between mitochondrial function and ischemia/reperfusion injury. The future use of these clinical markers as prognostic factors may allow early identification of post-transplant liver failure and may indicate the need to perform a new transplant.

Mitochondrial Membrane Potential (ΔΨ) Fluctuations Associated with the Metabolic States of Mitochondria.

The proton electrochemical gradient generated by the respiratory chain activity accounts for over 90% of the available respiratory energy, and, as such, its evaluation and accurate measurement regarding total values and fluctuations are an invaluable component of the understanding of mitochondrial function. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulation and transport is known as the mitochondrial membrane potential, or ΔΨ, and is reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure ΔΨ, ranging from fluorometric evaluations to electrochemical probes. Here, we will expose a particular method for ΔΨ evaluation, which is dependent on the movement of a particular ion, tetraphenylphosphonium (TPP+) with a selective electrode. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane is a sensitive, immediate, accurate, and simple method of evaluation of ΔΨ in isolated, respiring mitochondria.

Indirubin and NAD+ prevent mitochondrial ischaemia/reperfusion damage in fatty livers.

BACKGROUND: Fatty livers are considerably more susceptible to acute stressors, such as ischaemia/reperfusion (I/R). As the incidence of I/R is high due to surgical events and some pathologies, there is an urgent need to find strategies against I/R injury (I/RI) in fatty livers. We postulate that an acute pretreatment with indirubin-3'-oxime (Ind) or NAD+ prevents mitochondrial dysfunction associated with warm I/RI in fatty livers. MATERIALS AND METHODS: Zucker fatty rats were subjected to warm ischaemia and 12 hours of reperfusion. Ind or NAD+ was administered in the hepatic artery 30 minutes before ischaemia. Hepatic mitochondrial isolation was performed, and functional assays as well as molecular analysis were performed. RESULTS: Pretreatment decreased markers of liver injury while preserving mitochondrial cytochrome c content, which is related to the prevention of calcium-induced mitochondrial permeability transition (mPT), the decline in mitochondrial respiratory state 3 and ATP content. The generation of reactive oxygen species (ROS) was also diminished. Inhibition of GSK-3ß by Ind resulted in the prevention of cyclophilin-D (CypD) phosphorylation, unabling it to bind to the adenine nucleotide translocator (ANT), thus, preventing mPT induction. Furthermore, deacetylation of CypD at Lys residue by sirtuin 3 (SIRT3) caused its dissociation from ANT, contributing to an increase in mPT threshold in NAD+ -pretreated animals. CONCLUSIONS: Pretreatment with Ind or NAD+ protects fatty livers by maintaining mitochondrial calcium homoeostasis, thus, preserving mitochondrial function and energetic balance. As such, CypD might be a new protective target against I/RI in fatty livers.

Recent insights into mitochondrial targeting strategies in liver transplantation.

Ischemia/reperfusion (I/R) injury in liver transplantation can disrupt the normal activity of mitochondria in the hepatic parenchyma. This potential dysfunction of mitochondria after I/R injury could be responsible for the initial poor graft function or primary nonfunction observed after liver transplantation. Thus, determining the mechanisms that lead to human hepatic mitochondrial dysfunction might contribute to improving the outcome of liver transplantation. Furthermore, early identification of novel prognostic factors involved in I/R injury could serve as a key endpoint to predict the outcome of liver grafts and also to promote the early adoption of novel strategies that protect against I/R injury. Here, we briefly review recent advances in the study of mitochondrial dysfunction and I/R injury, particularly in relation to liver transplantation. Next, we highlight various pharmacological therapeutic strategies that could be applied, and discuss their relationship to relevant mitochondrion-related processes and targets. Lastly, we note that although considerable progress has been made in our understanding of I/R injury and mitochondrial dysfunction, further investigation is required to elucidate the cellular and molecular mechanisms underlying these processes, thereby identifying biomarkers that can help in evaluating donor organs.

GSK3ß and VDAC Involvement in ER Stress and Apoptosis Modulation during Orthotopic Liver Transplantation.

We investigated the involvement of glycogen synthase kinase-3ß (GSK3ß) and the voltage-dependent anion channel (VDAC) in livers subjected to cold ischemia-reperfusion injury (I/R) associated with orthotopic liver transplantation (OLT). Rat livers were preserved in University of Wisconsin (UW) and Institute Georges Lopez (IGL-1) solution, the latter enriched or not with trimetazidine, and then subjected to OLT. Transaminase (ALT) and HMGB1 protein levels, glutamate dehydrogenase (GLDH), and oxidative stress (MDA) were measured. The AKT protein kinase and its direct substrates, GSK3ß and VDAC, as well as caspases 3, 9, and cytochrome C and reticulum endoplasmic stress-related proteins (GRP78, pPERK, ATF4, and CHOP), were determined by Western blot. IGL-1+TMZ significantly reduced liver injury. We also observed a significant phosphorylation of AKT, which in turn induced the phosphorylation and inhibition of GSK3ß. In addition, TMZ protected the mitochondria since, in comparison with IGL-1 alone, we found reductions in VDAC phosphorylation, apoptosis, and GLDH release. All these results were correlated with decreased ER stress. Addition of TMZ to IGL-1 solution increased the tolerance of the liver graft to I/R injury through inhibition of GSK3ß and VDAC, contributing to ER stress reduction and cell death prevention.

Hepatic and skeletal muscle mitochondrial toxicity of chitosan oligosaccharides of normal and diabetic rats.

Diabetes and associated conditions are now considered a worldwide epidemic, with increasing costs and burdens with no cure yet developed. The chitin-derived glucosamine biopolymer chitosan has shown promising results when supplied to diabetic patients. However, no study has investigated the possible toxic side effects of chitosan treatments, in particular when regarding the most important bioenergetic organelle, mitochondria. As such, we aimed to understand if supplementation of chitosan to the diet of normal and diabetic rats could compromise mitochondrial function on two of the major organs involved in diabetes, obesity, and metabolic regulation, the liver and skeletal muscle. We supplemented the drinking water of normal Wistar and diabetic Goto-Kakizaki rats with 0.5% chitosan for 6 weeks. We show here that, in terms of hepatic bioenergetics, chitosan was relatively inert and had no major side effects. However, regarding skeletal muscle bioenergetics, chitosan significantly affected various bioenergetic parameters. As such, we conclude that chitosan, at the tested doses, is relatively safe for treatment of diabetic situations. Nonetheless, the potential for adverse toxicological side effects appears to be present, which might be relevant if higher doses are utilized.

The bile acid chenodeoxycholic acid directly modulates metabolic pathways in white adipose tissue in vitro: insight into how bile acids decrease obesity.

Obesity is a worldwide epidemic, and associated pathologies, including type 2 diabetes and cardiovascular alterations, are increasingly escalating morbidity and mortality. Despite intensive study, no effective simple treatment for these conditions exists. As such, the need for go-to drugs is serious. Bile acids (BAs) present the possibility of reversing these problems, as various in vivo studies and clinical trials have shown significant effects with regard to weight and obesity reduction, insulin sensitivity restoration and cardiovascular improvements. However, the mechanism of action of BA-induced metabolic improvement has yet to be fully established. The currently most accepted model involves non-shivering thermogenesis for energy waste, but this is disputed. As such, we propose to determine whether the BA chenodeoxycholic acid (CDCA) can exert anti-obesogenic effects in vitro, independent of thermogenic brown adipose tissue activation. By exposing differentiated 3 T3-L1 adipocytes to high glucose and CDCA, we demonstrate that this BA has anti-obesity effects in vitro. Nuclear magnetic resonance spectroscopic analysis of metabolic pathways clearly indicates an improvement in metabolic status, as these cells become more oxidative rather than glycolytic, which may be associated with an increase in fatty acid oxidation. Our work demonstrates that CDCA-induced metabolic alterations occur in white and brown adipocytes and are not totally dependent on endocrine/nervous system signaling, as thought until now. Furthermore, future exploration of the mechanisms behind these effects will undoubtedly reveal interesting targets for clinical modulation.

Low-dose, subchronic exposure to silver nanoparticles causes mitochondrial alterations in Sprague-Dawley rats.

AIM: Nanoparticles (NPs) have increasingly been studied due to their probable harmful effects to both humans and the environment. However, despite several indications of possible harmful effects, no long-term studies using a low dose of silver nanoparticles (AgNP) have been conducted in vivo. RESULTS: Our data demonstrate that the prolonged exposure to a very low dose of AgNP was sufficient to cause alterations in hepatic mitochondrial function. Mitochondrial function compromised by AgNPs is recovered by pretreatment with the antioxidant N-acetylcysteine, which highlights the crucial role of oxidative stress in AgNPs' toxicity. CONCLUSION: Our data show for the first time that even a very low dose of AgNP can cause harmful effects on mitochondrial function, thus compromising the normal function of the organ.

PPARα Agonist WY-14643 Induces SIRT1 Activity in Rat Fatty Liver Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) remains a frequent complication in surgery, especially in case of steatotic livers that present decreased tolerance towards IRI. Apart from its major role in metabolism, activation of peroxisome proliferator-activated receptor α (PPARα) has been related with positive effects on IRI. In addition, the deacetylase enzyme sirtuin 1 (SIRT1) has recently emerged as a promising target for preventing IRI, through its interaction with stress-related mechanisms, such as endoplasmic reticulum stress (ERS). Taking this into account, this study aims to explore whether PPARα agonist WY-14643 could protect steatotic livers against IRI through sirtuins and ERS signaling pathway. Obese Zucker rats were pretreated or not pretreated with WY-14643 (10 mg/kg intravenously) and then submitted to partial (70%) hepatic ischemia (1 hour) followed by 24 hours of reperfusion. Liver injury (ALT levels), lipid peroxidation (MDA), SIRT1 activity, and the protein expression of SIRT1 and SIRT3 and ERS parameters (IRE1α, peIF2, caspase 12, and CHOP) were evaluated. Treatment with WY-14643 reduced liver injury in fatty livers, enhanced SIRT1 activity, and prevented ERS. Together, our results indicated that PPARα agonist WY-14643 may exert its protective effect in fatty livers, at least in part, via SIRT1 induction and ERS prevention.

Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation.

AIM: To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. METHODS: Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4 °C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD(+), a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. RESULTS: The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 ± 133.44 vs 206 ± 33.61, P < 0.05) and aspartate aminotransferase levels (893.57 ± 397.69 vs 500.85 ± 118.07, P < 0.05). The lessened hepatic injury in case of losartan was associated with enhanced SIRT1 protein expression and activity (5.27 ± 0.32 vs 6.08 ± 0.30, P < 0.05). This was concomitant with increased levels of NAD(+) (0.87 ± 0.22 vs 1.195 ± 0.144, P < 0.05) the co-factor necessary for SIRT1 activity, as well as with decreases in ac-FoxO1 expression. Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. CONCLUSION: The present study reports that losartan induces SIRT1 expression and activity, and that it reduces hepatic injury in a ROLT model.

Sirtuin 1 in rat orthotopic liver transplantation: an IGL-1 preservation solution approach.

AIM: To investigate the possible involvement of Sirtuin 1 (SIRT1) in rat orthotopic liver transplantation (OLT), when Institute Georges Lopez 1 (IGL-1) preservation solution is enriched with trimetazidine (TMZ). METHODS: Male Sprague-Dawley rats were used as donors and recipients. Livers were stored in IGL-1 preservation solution for 8h at 4 °C, and then underwent OLT according to Kamada's cuff technique without arterialization. In another group, livers were stored in IGL-1 preservation solution supplemented with TMZ, at 10(-6) mol/L, for 8 h at 4 °C and then underwent OLT. Rats were sacrificed 24 h after reperfusion, and liver and plasma samples were collected. Liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity) oxidative stress (malondialdehyde levels), and nicotinamide adenine dinucleotide (NAD(+)), the co-factor necessary for SIRT1 activity, were determined by biochemical methods. SIRT1 and its substrates (ac-FoxO1, ac-p53), the precursor of NAD(+), nicotinamide phosphoribosyltransferase (NAMPT), as well as the phosphorylation of adenosine monophosphate activated protein kinase (AMPK), p-mTOR, p-p70S6K (direct substrate of mTOR), autophagy parameters (beclin-1, LC3B) and MAP kinases (p-p38 and p-ERK) were determined by Western blot. RESULTS: Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preserved in IGL-1 solution alone. In addition, livers preserved in IGL-1 + TMZ presented reduced levels of oxidative stress. This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity, as indicated by decreased acetylation of p53 and FoxO1. The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD(+)/NADH levels. Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of mTOR and its direct substrate (p-p70S6K). As a consequence, autophagy mediators (beclin-1 and LC3B) were over-expressed. Furthermore, MAP kinases were regulated in livers preserved with IGL-1 + TMZ, as they were characterized by enhanced p-ERK and decreased p-p38 protein expression. CONCLUSION: Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT, through SIRT1 up-regulation.

Berberine reverts hepatic mitochondrial dysfunction in high-fat fed rats: a possible role for SirT3 activation.

Berberine is an isoquinoline alkaloid with anti-diabetic properties. Despite the central role of liver and thus hepatic mitochondria in whole-body metabolism, berberine effects on hepatic mitochondrial function in an obesity model are still unknown. Here, we demonstrate that berberine treatment recovers mitochondrial efficiency when altered by a high-fat feeding. Mitochondria isolated from the liver of high-fat fed rats exhibited decreased capacity to accumulate calcium and impaired oxidative phosphorylation (OXPHOS) capacity, as shown by impaired mitochondrial membrane potential, oxygen consumption and cellular ATP levels. Interestingly, the recovery of mitochondrial function by berberine was associated with an increased activity of the mitochondrial sirtuin 3 (SirT3). In conclusion, berberine potent protective effects against metabolic syndrome may rely on increasing mitochondrial SirT3 activity, normalizing mitochondrial function and preventing a state of energetic deficit caused by impaired OXPHOS.

Uncovering the beginning of diabetes: the cellular redox status and oxidative stress as starting players in hyperglycemic damage.

Early hyperglycemic insult can lead to permanent, cumulative damage that might be one of the earliest causes for a pre-diabetic situation. Despite this, the early phases of hyperglycemic exposure have been poorly studied. We have previously demonstrated that mitochondrial injury takes place early on upon hyperglycemic exposure. In this work, we demonstrate that just 1 h of hyperglycemic exposure is sufficient to induce increased mitochondrial membrane potential and generation. This is accompanied (and probably caused) by a decrease in the cells' NAD(+)/NADH ratio. Furthermore, we show that the modulation of the activity of parallel pathways to glycolysis can alter the effects of hyperglycemic exposure. Activation of the pentose phosphate pathway leads to diminished effects of glucose on the above parameters, either by removing glucose from glycolysis or by NADPH generation. We also demonstrate that the hexosamine pathway inhibition also leads to a decreased effect of excess glucose. So, this work demonstrates the need for increased focus of study on the reductive status of the cell as one of the most important hallmarks of initial hyperglycemic damage.

The NAD ratio redox paradox: why does too much reductive power cause oxidative stress?

The reductive power provided by nicotinamide adenine dinucleotides is invaluable for several cellular processes. It drives metabolic reactions, enzymatic activity, regulates genetic expression and allows for the maintenance of a normal cell redox status. Therefore, the balance between the oxidized (NAD(+)) and the reduced (NADH) forms is critical for the cell's proper function and ultimately, for its survival. Being intimately associated with the cells' metabolism, it is expected that alterations to the NAD(+)/NADH ratio are to be found in situations of metabolic diseases, as is the case of diabetes. NAD(+) is a necessary cofactor for several enzymes' activity, many of which are related to metabolism. Therefore, a decrease in the NAD(+)/NADH ratio causes these enzymes to decrease in activity (reductive stress), resulting in an altered metabolic situation that might be the first insult toward several pathologies, such as diabetes. Here, we review the importance of nicotinamide adenine dinucleotides in the liver cell and its fluctuations in a state of type 2 diabetes mellitus.

Hepatic FXR: key regulator of whole-body energy metabolism.

The farnesoid X receptor (FXR) is a nuclear receptor whose activation leads to alterations in pathways involved in energy metabolism. For example, it serves as a bile acid receptor in tissues such as the liver, and as an energy metabolism regulator in liver, muscle and adipose tissue. However, the effects of FXR activation are not exclusive to the tissue where it is present, because receptor crosstalk affects tissues throughout the body. It has been demonstrated that FXR regulates the metabolism of not just bile acids, but also of fats and hydrocarbon metabolites. FXR is currently under study as a therapeutic target for the treatment of diseases of excess, such as diabetes. Here we review the effects of FXR activation in the response of an organism to excess energy.

Exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and tetraethyl lead affects lung mitochondria bioenergetics.

Environmental pollutants such as TCDD and tetraethyl lead are extremely toxic and related with pulmonary disease development. Lung mitochondria are primary cellular targets for dioxins exposure-induced toxicity. TCDD showed a delay in the repolarization after a phosphorylative cycle and a decrease on state 3 respiration, suggesting alterations at the phosphorylative system level. The ATPase activity showed no differences between control and lung mitochondria incubated with TCDD, implying alterations in other components of the phosphorylative system. Tetraethyl lead also showed a delay in the repolarization after a phosphorylative cycle and a decrease on RCR. These data suggest that lung mitochondria incubated with TCDD and tetraethyl lead showed impaired mitochondrial function, reflecting the loss of oxidative phosphorylation capacity.

Estimating gluconeogenesis by NMR isotopomer distribution analysis of [13C]bicarbonate and [1-13C]lactate.

The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 +/- 3% for [13C]bicarbonate perfusion and 91 +/- 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 +/- 1% and 99 +/- 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output.

Diabetes and mitochondrial oxidative stress: a study using heart mitochondria from the diabetic Goto-Kakizaki rat.

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe2+ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O2 consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O2 consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determining alpha-tocopherol, GSH and coenzyme Q content. Although no difference of alpha-tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q9, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.