Clairce Luzia Salgueiro-Pagadigorria

Liver mitochondrial function and redox status in an experimental model of non-alcoholic fatty liver disease induced by monosodium l-glutamate in rats

The purpose of this work was to determine if mitochondrial dysfunction is involved in the development of non-alcoholic fatty liver disease (NAFLD). Using a model of obesity induced by the neonatal treatment of rats with monosodium L-glutamate (MSG), several parameters of liver mitochondrial function and their impact on liver redox status were evaluated. Specifically, fatty acid β-oxidation, oxidative phosphorylation and Ca(2+)-induced mitochondrial permeability transition were assessed in isolated liver mitochondria, and reduced glutathione (GSH), linked thiol contents and the activities of several enzymes involved in the control of redox status were measured in the liver homogenate. Our results demonstrate that liver mitochondria from MSG-obese rats exhibit a higher β-oxidation capacity and an increased capacity for oxidising succinate, without loss in the efficiency of oxidative phosphorylation. Also, liver mitochondria from obese rats were less susceptible to the permeability transition pore (PTP) opening induced by 1.0 μM CaCl(2). Cellular levels of GSH were unaffected in the livers from the MSG-obese rats, whereas reduced linked thiol contents were increased. The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione peroxidase were increased, while catalase activity was unaffected and superoxide dismutase activity was reduced in the livers from the MSG-obese rats. In this model of obesity, liver fat accumulation is not a consequence of mitochondrial dysfunction. The enhanced glucose-6-phosphate dehydrogenase activity observed in the livers of MSG-obese rats could be associated with liver fat accumulation and likely plays a central role in the mitochondrial defence against oxidative stress.

Cimicifuga racemosa impairs fatty acid β-oxidation and induces oxidative stress in livers of ovariectomized rats with renovascular hypertension.

The aim of this work was to evaluate the effects of therapeutic doses of Cimicifuga racemosa on cardiovascular parameters and on liver lipid metabolism and redox status in an animal model of estrogen deficiency associated with hypertension, a condition that could make the liver more vulnerable to drug-induced injuries. Female Wistar rats were subjected to the surgical procedures of bilateral ovariectomy (OVX) and induction of renovascular hypertension (two-kidneys, one-clip; 2K1C). These animals (OVX + 2K1C) were treated with daily doses of a C. racemosa extract, using a dose that is similar to that recommended to postmenopausal women (0.6 mg/kg), over a period of 15 days. The results were compared to those of untreated OVX + 2K1C, OVX, and control rats. The treatment with C. racemosa caused a significant reduction in blood pressure. In the liver, treatment did not prevent the development of steatosis, and it reduced the mitochondrial and peroxisomal capacity to oxidize octanoyl-CoA compared to the untreated animals. In addition, C. racemosa caused numerous undesirable effects on the liver redox status: it increased the mitochondrial reactive oxygen species generation, an event that was not accompanied by an increase in the activity of superoxide dismutase, and it induced a decrease in peroxisomal catalase activity. Although the reduced glutathione content had not been affected, a phenomenon that probably reflected the restoration of glucose-6-phosphate dehydrogenase activity by C. racemosa, oxidative damage was evidenced by the elevated level of thiobarbituric acid-reactive substances found in the liver of treated animals.

Effects of fusaric acid on rat liver mitochondria

The effects of fusaric acid on hepatic energy metabolism were measured. Three experimental systems were employed: (a) Intact rat liver mitochondria; (b) freeze-thawing disrupted mitochondria; and (c) the isolated perfused rat liver. Fusaric acid affects mitochondrial energy metabolism by at least three modes of action: (1) Inhibition of succinate-dehydrogenase (in the 10(-3)-10(-2) M range); (2) inhibition of oxidative phosphorylation (in the 10(-5)-10(-4) M range); and (3) inhibition of alpha-ketoglutarate-dehydrogenase (in the 10(-5)-10(-4) M range). The inhibition of oxidative phosphorylation seems to be the result of a direct action on the ATP-synthase/ATPase without significant inhibition of the ATP/ADP exchange. In the isolated perfused rat liver, fusaric acid inhibits oxygen uptake and gluconeogenesis from pyruvate, the latter being strictly dependent on intramitochondrially generated ATP. The effects of fusaric acid on rat liver mitochondria are similar to those reported previously for maize root mitochondria. However, except for the action on succinate-dehydrogenase, rat liver mitochondria are approximately two orders of magnitude more sensitive than maize root mitochondria.

Effects of the nonsteroidal anti-inflammatory drug piroxicam on energy metabolism in the perfused rat liver

1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the metabolism of the isolated perfused rat liver were investigated. The main purpose was to verify if piroxicam is also active on glycogenolysis and energy metabolism, as demonstrated for several carboxylic nonsteroidal anti-inflammatories. 2. Piroxicam increased oxygen consumption in livers from both fed and fasted rats. 3. Piroxicam increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). 4. Gluconeogenesis from lactate plus pyruvate was inhibited. 5. The action of piroxicam on oxygen consumption was blocked by antimycin A, but not by atractyloside. 6. The action of piroxicam in the perfused rat liver metabolism seems to be a consequence of its action on mitochondria. 7. It can be concluded that inhibition of energy metabolism and stimulation of glycogenolysis are not specific properties of carboxylic nonsteroidal anti-inflammatory drugs.

Melatonin protects female rats against steatosis and liver oxidative stress induced by oestrogen deficiency.

AIMS Melatonin has been shown to protect cells against oxidative and inflammatory damage via endocrine, paracrine and autocrine actions. Postmenopausal condition is associated with a high incidence of many features of metabolic syndrome including obesity, steatosis and liver oxidative injuries. The aim of this work was to investigate whether treatment with melatonin improves metabolic disturbances associated with oestrogen deficiency in ovariectomised (OVX) rats. MAIN METHODS OVX and control (CON) female rats were treated with melatonin (10mg/kg×day for 3weeks, p.o.). Body weight gain, adiposity index, plasma biochemical parameters, liver lipid content, hepatic mitochondrial respiration, fatty acid oxidation and mitochondrial H2O2 generation and the activity of the most important enzymatic and non-enzymatic reactive oxygen species (ROS) scavenger systems were measured. KEY FINDINGS In OVX rats, melatonin suppressed lipid accumulation and cellular oxidative stress in the liver. There was a reduction in the levels of carbonylated proteins in the mitochondria and cytosol, reduction in the malondialdehyde contents in the liver homogenates, stimulation of cytosolic glutathione peroxidase and glutathione reductase activities and restoration of reduced glutathione contents to normal levels. SIGNIFICANCE Exogenous melatonin protects the liver of OVX rats against steatosis and cellular oxidative stress, possibly via activation of antioxidant enzymes related to glutathione metabolism and by a direct radical scavenging activity.

Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant‐induced arthritic rats to Ca2+ addition was investigated. The respiratory activity, the Ca2+‐induced osmotic swelling and the electrophoretic 45Ca2+ uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well‐known inhibitor of the mitochondrial PTP. The Ca2+‐induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca2+ concentration. Whereas in the normal condition, the Ca2+‐induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 µM Ca2+ concentration, in the arthritic condition, these events occurred at 1.0 µM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate 45Ca2+. All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca2+‐induced MPT may be an important factor in the pathogenesis of the arthritis disease. Copyright

Effects of norepinephrine on the metabolism of fatty acids with different chain lengths in the perfused rat liver.

The effects of norepinephrine on ketogenesis in isolated hepatocytes have been reported as ranging from stimulation to inhibition. The present work was planned with the aim of clarifying these discrepancies. The experimental system was the once-through perfused liver from fasted and fed rats. Fatty acids with chain lengths varying from 8-18 were infused. The effects of norepinephrine depended on the metabolic state of the rat and on the nature of the fatty acid. Norepinephrine clearly inhibited ketogenesis from long-chain fatty acids (stearate > palmitate > oleate), but had little effect on ketogenesis from medium-chain fatty acids (octanoate and laureate). With palmitate the decrease in oxygen uptake was restricted to the substrate stimulated portion; with stearate, the decrease exceeded the substrate stimulated portion; with oleate, oxygen uptake was transiently inhibited. Withdrawal of Ca2+ attenuated the inhibitory effects. 14CO2 production from [1-14C]oleate was inhibited. Net uptake of the fatty acids was not affected by norepinephrine. In livers from fed rats, oxygen uptake and ketogenesis from stearate were only transiently inhibited. The conclusions are: (a) in the fasted state norepinephrine reduces ketogenesis and respiration by means of a Ca2+-dependent mechanism; (b) the degree of inhibition varies with the chain length and the degree of saturation of the fatty acids; (c) norepinephrine favours esterification of the activated long-chain fatty acids in detriment to oxidation; (d) in the fed state the stimulatory action of norepinephrine on glycogen catabolism induces conditions which are able to reverse inhibition of ketogenesis and oxygen uptake.

Beneficial Effects of Tibolone on Blood Pressure and Liver Redox Status in Ovariectomized Rats With Renovascular Hypertension

Estrogen deficiency is associated with aging and increases the incidence of metabolic syndrome and hypertension. In this study, the effects of tibolone, a synthetic steroid, on the cardiovascular system, liver lipid metabolism, and redox status were evaluated, in ovariectomized (OVX) rats with renovascular hypertension (two-kidneys, one-clip, OVX + 2K1C). This study encompassed direct measurements of mean arterial pressure , plasma biochemical analysis, liver lipid contents, and assessments of the mitochondrial and peroxisomal β-oxidation capacities. Additionally, the liver redox status was assayed. Tibolone significantly reduced the mean arterial pressure of OVX + 2K1C rats, albeit reducing total and high-density lipoprotein (HDL) cholesterol levels. In the liver, although exerting an undesirable inhibition of mitochondrial and peroxisomal β-oxidation, tibolone reversed steatosis. Tibolone also improved the liver redox status: the reduced glutathione contents and the activity of glucose-6-phosphate dehydrogenase were restored by this compound, which also reduced the levels of thiobarbituric acid-reactive substances and the generation of mitochondrial reactive oxygen species. So, tibolone reversed the main alterations caused by hypertension and estrogen deficiency.

Tibolone impairs glucose and fatty acid metabolism and induces oxidative stress in livers from female rats.

Tibolone is a synthetic steroid that has been extensively prescribed to treat climacteric symptoms and to prevent postmenopausal osteoporosis. Because menopause is a condition associated with increased incidence of metabolic disturbances and hepatic steatosis, the aim of this work was to evaluate the actions of tibolone on the liver. The effects of tibolone on glucose and fatty acid metabolism and on several parameters linked to mitochondrial energy metabolism, including the induction of cellular oxidative stress, were investigated in livers from female Wistar rats. Tibolone was assayed at concentrations ranging from 5 to 100 μM. In perfused livers, tibolone inhibited oxygen uptake, stimulated glycogenolysis and glycolysis, and inhibited gluconeogenesis from L-lactate and ketogenesis from exogenous octanotate. Tibolone also caused pronounced increases in both the cytosolic and mitochondrial NADH/NAD+ratios. In isolated mitochondria, tibolone inhibited oxygen uptake due to β-hydroxybutyrate and fatty acid oxidation without affecting the succinate oxidation. The inhibitory action of tibolone at complex I of the mitochondrial respiratory chain was suggested by the inhibition of the NADH-oxidase activity. Tibolone also induced oxidative stress in both perfused livers and isolated mitochondria, as indicated by the increased production of thiobarbituric acid reactive substances. These metabolic alterations may increase the risk of metabolic disturbances during tibolone administration, particularly in the postmenopausal condition.

Instant coffee extract with high chlorogenic acids content inhibits hepatic G‐6‐Pase in vitro, but does not reduce the glycaemia

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5‐caffeoylquinic acid (5‐CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose‐6‐phosphatase (G‐6‐Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G‐6‐Pase activity and liver glucose output induced by 5‐CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G‐6‐Pase activity of the hepatocyte microsomal fraction in a dose‐dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5‐CQA (1 mM) reduced (p < 0.05) the activity of microsomal G‐6‐Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G‐6‐Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G‐6‐Pase and reduce the liver glucose output. Copyright