Emy Luiza Ishii-Iwamoto

Effects of four monoterpenes on germination, primary root growth, and mitochondrial respiration of maize

The effects of four monoterpenes—camphor, eucaliptol, limonene, and α-pinene—on the oxidative metabolism of mitochondria isolated from maize root (Zea mays), on maize seed germination, and on primary root growth were investigated. The effects of individual monoterpenes on respiration were variable. α-Pinene concentrations of 0.05–1.0 mM stimulated respiration with a mixture of substrates composed of NADH, L-malate, succinate, and L-glutamate, and in the absence of exogenously added ADP (basal respiration). However, at concentrations higher than 1.0 mM, α-pinene inhibited respiration both in the absence (basal respiration) and presence of ADP (coupled respiration). Limonene at 0.1 mM or above stimulated basal respiration and inhibited in parallel the coupled respiration. Similar effects were promoted by eucaliptol, but at a higher concentration range (1.0 mM or above). Camphor was less active. At 10 mM concentration, it caused stimulation of basal respiration but did not affect coupled respiration. In the concentration range 0.1–10.0 mM, limonene was inactive on seed germination and primary root growth. Camphor and eucaliptol did not inhibit germination but reduced fresh and/or dry weight of roots at 5.0 mM and above. α-Pinene inhibited both seed germination and fresh weight of primary roots at 10.0 mM concentration. The results indicate that intact seeds and primary roots are less sensitive than isolated mitochondria. The relatively more lipophilic monoterpenes α-pinene and limonene had less activity than the more water-soluble oxygenated monoterpenes camphor and eucaliptol in inhibiting seed germination and/or primary root growth, despite the fact that they had a higher activity on the oxidative metabolism of isolated mitochondria. The findings suggest that the solubility of monoterpenes may be the major factor implicated in these differences.

Toxicological studies on Stryphnodendron adstringens

This study was carried out to determine the acute toxicity of total barbatimão extract (LD(50)) after oral administration to mice, and its effect on certain biochemical parameters in plasma of rats after 30 days of administration. The LD(50) value of the extract was 2699 mg/kg. A daily oral administration of extracts at 800 and 1600 mg/kg doses for 30 days caused a decrease in body weight, thymic involution, and an increase of plasma glucose and aspartate aminotransferase levels in the animals. The results showed that the extract administered in a prolonged period produced toxic effects in the experimental animals.

Gluconeogenesis in the liver of arthritic rats.

The gluconeogenic response in the liver from rats with chronic arthritis to various substrates and the effects of glucagon were investigated. The experimental technique used was the isolated liver perfusion. Hepatic gluconeogenesis in arthritic rats was generally lower than in normal rats. The difference between normal and arthritic rats depended on the gluconeogenic substrate. In the absence of glucagon the following sequence of decreasing differences was found: alanine (−71·8 per cent)∽glutamine (−71·7 per cent)>pyruvate (−60 per cent)>lactate+pyruvate (−44·9 per cent)>xylitol (n.s.=non‐significant)∽glycerol (n.s.). For most substrates glucagon increased hepatic gluconeogenesis in both normal and arthritic rats. The difference between normal and arthritic rats, however, tended to diminish, as revealed by the data of the following sequence: alanine (−48·9 per cent)∽pyruvate (−47·6 per cent)>glutamine (−33·8 per cent)>glycerol (n.s.)∽lactate+pyruvate (n.s.)∽xylitol (n.s.). The causes for the reduced hepatic gluconeogenesis in arthritic rats are probably related to: (a) lower activities of key enzymes catalyzing most probably steps preceding phosphoenolpyruvate (e.g. phosphoenolpyruvate carboxykinase, pyruvate carboxylase, etc.); (b) a reduced availability of reducing equivalents in the cytosol; (c) specific differences in the situations induced by hormones or by the individual substrates. Since glycaemia is almost normal in chronically arthritic rats, it seems that lower gluconeogenesis is actually adapted to the specific needs of these animals. Copyright

The metabolism of fructose in the bivascularly perfused rat liver

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.

Metabolic effects of silibinin in the rat liver.

The flavonolignan silibinin, which is a mixture of two diastereoisomers, silybin A and silybin B, is a component of the extract obtained from the fruit and seeds of the variegated milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin. Among the therapeutic properties credited to silibinin, its antihyperglycaemic action has been extensively explored. Silibinin is structurally related to the flavonoids quercetin and fisetin, which have been previously demonstrated to be very active on liver metabolic processes related to glycaemic regulation. The aim of the present work was to investigate the effects of silibinin on metabolic pathways responsible for the maintenance of glycaemia, particularly glycogenolysis and gluconeogenesis, in the perfused rat liver. The activities of some key enzymes in these pathways and on parameters of energy metabolism in isolated mitochondria were also examined. At a concentration range of 50-300μM, silibinin inhibited gluconeogenesis in the fasted condition and inhibited glycogenolysis and glycolysis in the fed condition. The mechanisms by which silibinin exerted these actions were multiple and complex. It inhibited the activity of glucose 6-phosphatase, inhibited the pyruvate carrier, and reduced the efficiency of mitochondrial energy transduction. It can also act by reducing the supply of NADH for gluconeogenesis and mitochondria through its pro-oxidative actions. In general, the effects and the potency of silibinin were similar to those of quercetin and fisetin. However, silibinin exerted some distinct effects such as the inhibitory effect on oxygen consumption in the fed condition and a change in the energy status of the perfused livers. It can be concluded that the effects of silibinin on liver glucose metabolism may explain its antihyperglycaemic property. However, this effect was, in part, secondary to impairment in cellular energy metabolism, a finding that should be considered in its therapeutic usage.

The urea cycle in the liver of arthritic rats.

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (−32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.

Effects of rutin and quercetin on mitochondrial metabolism and on ATP levels in germinating tissues of Glycine max

The effects of quercetin and its glycoside rutin, flavonoid compounds widely distributed in higher plants, on mitochondria isolated from soybean hypocotyls and on ATP levels of soybean embryonic axes were, investigated. Quercetin at 0.5 mM, and lower, inhibited respiration with a mixture of substrates composed of NADH, L-malate, succinate and L-glutamate, both in the absence or in the presence of exogenously added ADP. At concentrations higher than 0.5 mM, the effects of quercetin on basal respiration were shifted toward stimulation. Respiration of uncoupled mitochondria was inhibited by quercetin, irrespective of the nature of the substrates. Swelling driven by phosphate uptake was also inhibited by quercetin. Rutin had no effect on isolated mitochondria. The ATP levels of embryonic axes decreased in the presence of either quercetin or rutin. These results indicate that quercetin impairs the respiratory activity of soybean hypocotyl mitochondria by at least three mechanisms: (a) it inhibits substrate oxidation, probably by a direct action on the electron transfer chain; (b) it inhibits phosphate uptake; and (c) it possibly acts as an uncoupler of oxidative phosphorylation at concentrations higher than 0.5 mM. It was concluded that the action of quercetin on energy metabolism in mitochondria might be the cause of the reduction of the ATP levels in embryonic axes.

Changes in energy metabolism and antioxidant defense systems during seed germination of the weed species Ipomoea triloba L. and the responses to allelochemicals.

The relationships between changes in energy metabolism and the antioxidant defense system in the weed species Ipomoea triloba L. during seed germination and early seedling growth were investigated. The effects of some common allelochemicals on these parameters also were studied. Respiratory activity and the activities of alcohol dehydrogenase, superoxide dismutase, catalase, guaicol peroxidase, ascorbate peroxidase, glutathione reductase, and lipoxygenase were measured. Mitochondrial oxidative phosphorylation resumed shortly after the seed imbibition period, as indicated by considerable KCN-sensitive respiratory activity in embryos of I. triloba. The occurrence of superoxide dismutase, catalase, guaicol peroxidase, and lipoxygenase activities in the embryos, along with significant KCN-insensitive respiration, suggest that production of reactive oxygen species (ROS) is initiated as soon as mitochondrial respiration is resumed. All assayed antioxidant enzymes were present in the embryos except ascorbate peroxidase, which appeared only in primary roots. The activities of antioxidant enzymes increased after completion of germination, especially in primary roots. Superoxide dismutase, catalase, and guaicol peroxidase probably were the crucial enzymes involved in the neutralization of ROS, since they had higher levels of activity compared with other enzymes, such as ascorbate peroxidase and glutathione reductase. When seeds were grown in the presence of α-pinene, coumarin, quercetin, and ferulic acid, there was an additional increase in activities of antioxidant enzymes, as well as increases in lipoxygenase activity and KCN-insensitive respiration, suggesting a further increase in ROS generation. The antioxidant defense system of I. triloba was not effective in preventing lipid peroxidation caused by α-pinene. The data indicate that during seed germination and initial growth of I. triloba, a period when antioxidant enzyme activity increases to counteract the harmful ROS effects produced during mitochondrial metabolism resumption, the presence of allelochemicals, which cause further oxidative stress, may leave the seeds/seedlings more vulnerable to cellular dysfunction and cell death.

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.

Glycogen levels and glycogen catabolism in livers from arthritic rats

Hepatic glycogen catabolism and glycogen levels in rats with chronic arthritis were investigated. At 9:00 a.m., the hepatic glycogen contents of ad libitum fed arthritic and normal rats were 225.5 ± 17.7 and 332.1 ± 28.6 μmol glucosyl units × (g liver)–1, respectively. Food intake of arthritic and normal rats was equal to 100.1 ± 6.7 and 105.0 ± 3.1 mg × (g body w)–1 × (per 24 h)–1, respectively. In isolated perfused livers from normal and arthritic rats the rates of glucose, lactate and pyruvate release were the same when substrate- and hormone-free perfusion was performed. During an infusion period of 20 min glucagon caused an increment in glucose release of 35.3 ± 4.7 μmol × (g liver)–1 in livers from arthritic rats; in the normal condition the corresponding increment was 69.6 ± 5.7 μmol × (g liver)–1. Lactate and pyruvate productions (indicators of glycolysis) were diminished by glucagon in livers from normal rats; in the arthritic condition an initial stimulation was found, followed by a slow decay, which did not result in significant inhibition at the end of the glucagon infusion period (20 min). The actions of cAMP and dibutyryl-cAMP were similar to those of glucagon. It was concluded that livers from arthritic rats show an impaired capacity of releasing glucose under the stimulus of glucagon. This can be partly due to the lower glycogen levels and partly to a smaller capacity of inhibiting glycolysis. Reduction in glycogen levels was not associated with reduction in food intake or failure in the energetic state of the hepatic cells. These changes in glycogen metabolism may be related to reduced gluconeogenic capacity of the livers and/or to production of inflammatory mediators observed in the arthritis disease.