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Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the F

Elevar a produtividade e prolongar a vida útil dos frutos do morangueiro estão entre os principais objetivos dos produtores. A cultura do morango possui grande importância e a crescente competitividade no setor olerícola e o aumento da demanda pela cultura estimulou a procura dos agricultores por reguladores à base de giberelinas. Uma das funções da aplicação de fitormônios ou reguladores vegetais é promover precocidade ou atraso no florescimento e maturação dos pseudofrutos do morangueiro. Outra é obter aumentos no tamanho e na quantidade de pseudofrutos. A aplicação das giberelinas é realizada em diversas culturas, de acordo com o estágio de interesse do vegetal. Dessa forma, visando aumentar a produtividade, avaliamos os efeitos da aplicação foliar do fitorregulador Progibb 40%, em morango cultivado em sistema semi-hidropônico sobre: quantidade de flores e quantidade de pseudofrutos. Para o delineamento experimental utilizou-se blocos inteiramente casualizados. Foi realizado o plantio das mudas da variedade San Andreas e cultivadas em sistema semi-hidropônico, abastecidas via fertirrigação para o suprimento de água e nutrientes. A aplicação de fitorregulador Progibb 40% foi realizada 30 dias após o plantio das mudas, em dose de 25 Mm. A avaliação da quantidade de flores e pseudofrutos foi realizada em contagem manual, semanalmente. Os fatores temperatura e fotoperíodo e sua relação entre ambos ou com o ambiente, influenciaram no comportamento fisiológico da cultura. A aplicação do regulador proporcionou aumento no número de flores e de pseudofrutos, evidenciando uma eficiência para aumento de produtividade.

In the present work, the multiple-indicator dilution (MID) technique was used to investigate the kinetic mechanisms by which nickel (Ni2+) affects the calcium (Ca2+) transport in intact rat liver. 45Ca2+ and extra- and intracellular space indicators were injected in livers perfused with 1 mM Ni2+, and the outflow profiles were analyzed by a mathematical model. For comparative purposes, the effects of norepinephrine were measured. The influence of Ni2+ on the cytosolic Ca2+ concentration ([Ca2+]c) in human hepatoma Huh7 cells and on liver glycogen catabolism, a biological response sensitive to cellular Ca2+, was also evaluated. The estimated transfer coefficients of 45Ca2+ transport indicated two mechanisms by which Ni2+ increases the [Ca2+]c in liver under steady-state conditions: (1) an increase in the net efflux of Ca2+ from intracellular Ca2+ stores due to a stimulus of Ca2+ efflux to the cytosolic space along with a diminution of Ca2+ re-entry into the cellular Ca2+ stores; (2) a decrease in Ca2+ efflux from the cytosolic space to vascular space, minimizing Ca2+ loss. Glycogen catabolism activated by Ni2+ was transient contrasting with the sustained activation induced by norepinephrine. Ni2+ caused a partial reduction in the norepinephrine-induced stimulation in the [Ca2+]c in Huh7 cells. Our data revealed that the kinetic parameters of Ca2+ transport modified by Ni2+ in intact liver are similar to those modified by norepinephrine in its first minutes of action, but the membrane receptors or Ca2+ transporters affected by Ni2+ seem to be distinct from those known to be modulated by norepinephrine. Graphic abstract: [Figure not available: see fulltext.]

Urochloa ruziziensis, a cover plant used in no-till systems, can suppress weeds in the field through their chemical compounds, but the mode of action of these compounds is still unknown. The present study aimed to investigate the effects of a saponin-rich butanolic extract from U. ruziziensis straw (BfUr) and one of its components, protodioscin on an eudicot Ipomoea grandifolia and a monocot Digitaria insularis weed. The anatomy and the morphology of the root systems and several parameters related to energy metabolism and antioxidant defense systems were examined. The IC50 values for the root growth inhibition by BfUr were 108 μg mL−1 in D. insularis and 230 μg mL−1 in I. grandifolia. The corresponding values for protodioscin were 34 μg mL−1 and 54 μg mL−1. I. grandifolia exhibited higher ROS-induced peroxidative damage in its roots compared with D. insularis. In the roots of both weeds, the BfUr and protodioscin induced a reduction in the meristematic and elongation zones with a precocious appearance of lateral roots, particularly in I. grandifolia. The roots also exhibited features of advanced cell differentiation in the vascular cylinder. These alterations were similar to stress-induced morphogenic responses (SIMRs), which are plant adaptive strategies to survive in the presence of toxicants. At concentrations above their IC50 values, the BfUr or protodioscin strongly inhibited the development of both weeds. Such findings demonstrated that U. ruziziensis mulches may contribute to the use of natural and renewable weed control tools.

The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the F

The diversity of secondary compounds in the plant kingdom is huge. About 200,000 compounds are known, which are grouped into amines, non-protein amino acids, peptides, alkaloids, glucosinolates, cyanogenic glucosides, organic acids, terpenoids, quinones, polyacetylenes, and phenolics. The group of phenolic compounds consists of polyphenols, oligophenols and monophenols or simple phenolic compounds such as benzoic and cinnamic acids and their hydroxylated derivatives. Among the thousands of compounds present in ecological interactions, simple phenolic acids are the most abundant in soils, and many are described as allelochemicals. Given the physiological and biochemical importance of these compounds, we review their biosynthesis and metabolic actions in plants.

Lignin is a technological bottleneck to convert polysaccharides into fermentable sugars, and different strategies of genetic-based metabolic engineering have been applied to improve biomass saccharification. Using maize seedlings grown hydroponically for 24 h, we conducted a quick non-transgenic approach with five enzyme inhibitors of the lignin and tricin pathways. Two compounds [3,4-(methylenedioxy)cinnamic acid: MDCA and 2,4-pyridinedicarboxylic acid: PDCA] revealed interesting findings on root growth, lignin composition, and saccharification. By inhibiting hydroxycinnamoyl-CoA ligase, a key enzyme of phenylpropanoid pathway, MDCA decreased the lignin content and improved saccharification, but it decreased root growth. By inhibiting flavone synthase, a key enzyme of tricin biosynthesis, PDCA decreased total lignin content and improved saccharification without affecting root growth. PDCA was three-fold more effective than MDCA, suggesting that controlling lignin biosynthesis with enzymatic inhibitors may be an attractive strategy to improve biomass saccharification.

Cellulosic ethanol production will decrease our dependence on fossil fuels, positively impacting global warming, energy security, and urban pollution. In the last few years, our group has screened a few enzyme inhibitors of the phenylpropanoid pathway. We have shown that when some enzyme inhibitors are sprayed in young plants, they increase the lignocellulose saccharification in the long term at the workbench scale. Here, we screened five aromatic compounds for their ability to improve the saccharification of maize plants. Benzohydrazide increased saccharification in a broad range of concentrations in growth-room experiments, and it was selected for field-scale assays. At 20 g ha−1 (500 μM, 300 L ha−1), benzohydrazide increased by 33 and 46%, respectively, the saccharification of lignocellulose from maize leaves and stems. When the lignocellulose biomass of maize plants, sprayed with benzohydrazide or not, was submitted to hydrogen peroxide–acetic acid delignification pretreatment, benzohydrazide increased the saccharification by up to 76%. Benzohydrazide did not significantly affect any other biometric (length or fresh and dry weights) or biochemical (lignin, monolignols, structural hydroxycinnamates) parameters assessed. In brief, benzohydrazide could be used to improve saccharification in agroenergy crops.

In the present work, the multiple-indicator dilution (MID) technique was used to investigate the kinetic mechanisms by which nickel (Ni

Cadmium (Cd) inhibits soybean root growth, but its exact mode of action is still not completely understood. We evaluated the effects of Cd on growth, mitochondrial respiration, lipid peroxidation, total phenols, glutathione, and activities of lipoxygenase (LOX), superoxide dismutase (SOD), and catalase (CAT) in soybean roots. In primary roots, Cd stimulated KCN-insensitive respiration and KCN-SHAM-insensitive respiration, indicating the involvement of the alternative oxidase (AOX) pathway, while it decreased KCN-sensitive respiration, suggesting an inhibition of the cytochrome oxidase pathway (COX). In isolated mitochondria, Cd uncoupled the oxidative phosphorylation since it decreased state III respiration (coupled respiration) and ADP/O and respiratory control ratios, while it increased state IV respiration (depletion of exogenously added ADP). The uncoupling effect increased extramitochondrial LOX activity, lipid peroxidation, and oxidized and reduced glutathione, which induced an antioxidant response with enhanced SOD and CAT activities. In brief, our findings reveal that Cd acts as an uncoupler of the mitochondrial oxidative phosphorylation in soybean roots, disturbing cellular respiration and inducing oxidative cellular stress.

Titanium dioxide (TiO2) nanoparticles (NPs) are the most widely released nanomaterials in the environment and are considered an emerging contaminant. Although the phytotoxicity mechanism of TiO2 NPs on plants involves the elevated generation of reactive oxygen species (ROS), it is still not well established in soybean. Herein, we evaluated the effects of 250–1000 mg L−1 TiO2 NPs on seed germination, growth, content of ROS, lipid peroxidation, and activity of antioxidant enzymes in roots of soybean plants. Our data revealed that up to 1000 mg L−1 TiO2 NPs did not affect soybean seed germination. Transmission electron microscopy images and determinations of zeta potential and hydrodynamic diameter of a suspension of TiO2 NPs demonstrated that they form aggregates, favoring their adsorption to the root surface with consequent physical damage. The main deleterious effects noted on roots were reduced cell viability, reduced root hair number, striated aspect of root apexes, and reduced fresh and dry weights of roots. In disagreement with other studies, plant exposure to TiO2 NPs reduced the level of total ROS and lipid peroxidation, probably due to increased superoxide dismutase (SOD) activity. Altogether, our data suggest that the toxicity mechanism of TiO2 NPs on soybean roots involves physical damage resulting from their adsorption to the root surface, but not the generation of ROS.

Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.5 40 μM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO–ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.

Testosterone is the predominant androgen in men and the lack of it can be a trigger to the development of the metabolic syndrome. Here we review the relationship between testosterone deficiency, metabolic syndrome, and hepatic steatosis reported by studies with men and rodents. The prevalence of metabolic syndrome and testosterone deficiency is higher among older subjects. Low total and free testosterone levels were positively associated with disturbs on energy metabolism, changes in body fat distribution, and body composition. Studies reported visceral fat accumulation in men with hypogonadism and castrated rats. Despite some contradictions, the association between higher adiposity, low testosterone, and metabolic syndrome was a common point among the studies. Few studies evaluated the hepatic steatosis and found an association with hypogonadism. Most of the studies with rodents combined the castration with a high-fat diet to study metabolic disturbs. The importance of proper levels of testosterone for energy metabolism homeostasis in men was also underlined by studies that investigated the metabolic effects of testosterone replacement therapy and androgen deprivation therapy.

According to the literature, methylene blue (MB) is a photosensitizer (PS) with a high affinity for mitochondria. Therefore, several studies have explored this feature to evaluate its photodynamic effects on the mitochondrial apoptotic pathway under normoxic conditions. We are aware only of limited reports regarding MB's photodynamic effects on mitochondrial energy metabolism, especially under hypoxic conditions. Thus, the purposes of this study were to determine the direct and photodynamic acute effects of MB on the energy metabolism of rat liver mitochondria under hypoxic conditions and its direct acute effects on several parameters linked to energy metabolism in the isolated perfused rat liver. MB presented a high affinity for mitochondria, irrespective of photostimulation or proton gradient formation. Upon photostimulation, MB demonstrated high in vitro oxidizing species generation ability. Consequently, MB damaged the mitochondrial macromolecules, as could be evidenced by the elevated levels of lipid peroxidation and protein carbonyls. In addition to generating a pro-oxidant environment, MB also led to a deficient antioxidant defence system, as indicated by the reduced glutathione (GSH) depletion. Bioenergetically, MB caused uncoupling of oxidative phosphorylation and led to photodynamic inactivation of complex I, complex II, and F1FO-ATP synthase complex, thus decreasing mitochondrial ATP generation. Contrary to what is expected for an ideal PS, MB displayed appreciable dark toxicity on mitochondrial energy metabolism. The results indicated that MB acted via at least three mechanisms: direct damage to the inner mitochondrial membrane; uncoupling of oxidative phosphorylation; and inhibition of electron transfer. Confirming the impairment of mitochondrial energy metabolism, MB also strongly inhibited mitochondrial ATP production. In the perfused rat liver, MB stimulated oxygen consumption, decreased the ATP/ADP ratio, inhibited gluconeogenesis and ureogenesis, and stimulated glycogenolysis, glycolysis, and ammoniagenesis, fully corroborating its uncoupling action in intact cells, as well. It can be concluded that even under hypoxic conditions, MB is a PS with potential for photodynamic effect-induced mitochondrial dysfunction. However, MB disrupts the mitochondrial energy metabolism even in the dark, causing energy-linked liver metabolic changes that could be harmful in specific circumstances.

An increase in crop competitiveness relative to weed interference has the potential to reduce crop yield losses. In this study, the effects of phytoalexin resveratrol were examined in

The present study was planned to improve our understanding about sex differences in the development of hepatic steatosis in cafeteria diet-induced obesity in young mice. Female (FCaf) and male (MCaf) mice fed a cafeteria diet had similar body weight gain and adiposity index, but FCaf had a more extensive steatosis than MCaf. FCaf livers exhibited a higher non-alcoholic fatty liver disease activity score, elevated lipid percentage area (+34%) in Sudan III staining and increased TG content (+25%) compared to MCaf. Steatosis in FCaf was not correlated with changes in the transcript levels of lipid metabolism-related genes, but a reduced VLDL release rate was observed. Signs of oxidative stress were found in FCaf livers, as elevated malondialdehyde content (+110%), reduced catalase activity (−36%) and increased Nrf2 and Hif1a mRNA expression compared to MCaf. Interestingly, fibroblast growth factor 21 (Fgf21) mRNA expression was found to be exclusively induced in MCaf, which also exhibited higher FGF21 serum levels (+416%) and hepatic protein abundance (+163%) than FCaf. Moreover, cafeteria diet increased Fgfr1, Fsp27 and Ucp1 mRNA expression in brown adipose tissue of males (MCaf), but not females (FCaf). FGF21 hepatic production by male mice seems to be part of a complex network of responses to the nutritional stress of the cafeteria diet, probably related to the unfolded protein response activation. Although aimed at the restoration of hepatic metabolic homeostasis, the branch involving Fgf21 upregulation seems to be impaired in females, rendering them incapable of reducing the hepatic lipid content and cellular oxidative stress.

Background The present study aimed to characterize the intrinsic and photodynamic effects of azure B (AB) on mitochondrial bioenergetics, as well as the consequences of its intrinsic effects on hepatic energy metabolism. Methods Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Results AB interacted with mitochondria regardless of photostimulation, but its binding degree was reduced by mitochondrial energization. Under photostimulation, AB caused lipid peroxidation and protein carbonylation and decreased the content of reduced glutathione (GSH) in mitochondria. AB impaired mitochondrial bioenergetics in at least three distinct ways: (1) uncoupling of oxidative phosphorylation; (2) photoinactivation of complexes I and II; and (3) photoinactivation of the FoF1–ATP synthase complex. Without photostimulation, AB also demonstrated mitochondrial toxicity, which was characterized by the induction of lipid peroxidation, loss of inner mitochondrial membrane integrity, and uncoupling of oxidative phosphorylation. The perfused rat liver experiments showed that mitochondria were one of the major targets of AB, even in intact cells. AB inhibited gluconeogenesis and ureagenesis, two biosynthetic pathways strictly dependent on intramitochondrially generated ATP. Contrariwise, AB stimulated glycogenolysis and glycolysis, which are required compensatory pathways for the inhibited oxidative phosphorylation. Similarly, AB reduced the cellular ATP content and the ATP/ADP and ATP/AMP ratios. Conclusions Although the properties and severe photodynamic effects of AB on rat liver mitochondria might suggest its usefulness in PDT treatment of liver tumors, this possibility should be considered with precaution given the toxic intrinsic effects of AB on mitochondrial bioenergetics and energy-linked hepatic metabolism.

Made available in DSpace on 2020-12-12T02:34:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans. In a previous study using isolated rat liver mitochondria, we observed that IMD inhibited the activity of FoF1-ATP synthase. The aim of this study was to evaluate the effects of IMD on rat isolated hepatocytes and perfused rat liver, besides the influence of its biotransformation on the toxicological potential. For the latter goal, rats were pretreated with dexamethasone or phenobarbital, two classical cytochrome P-450 stimulators, before hepatocytes isolation or liver perfusion. IMD (150 and 200 μM) reduced state 3 mitochondrial respiration in digitonin-permeabilized cells that were energized with glutamate plus malate but did not dissipate the mitochondrial membrane potential. In intact (non-permeabilized) hepatocytes, the intracellular ATP concentration and cell viability were reduced when high IMD concentrations were used (1.5–3.0 mM), and only in cells isolated from dexamethasone-pretreated rats, revealing that IMD biotransformation increases its toxicity and that IMD itself affects isolated mitochondria or mitochondria in permeabilized hepatocytes in concentrations that do not affect mitochondrial function in intact hepatocytes. Coherently, in the prefused liver, IMD (150 and 250 μM) inhibited gluconeogenesis from alanine, but without affecting oxygen consumption and urea production, indicating that such effect was not of mitochondrial origin. The gluconeogenesis inhibition was incomplete and occurred only when the rats were pretreated with phenobarbital, signs that IMD biotransformation was involved in the observed effect. Our findings reveal that changes in hepatic energy metabolism may be acutely implicated in the hepatotoxicity of IMD only when animals and humans are exposed to high levels of this compound, and that IMD metabolites seem to be the main cause for its toxicity. College of Agricultural and Technological Sciences São Paulo State University (Unesp) Department of Biochemistry Maringá State University (UEM) School of Veterinary Medicine São Paulo State University (Unesp) College of Agricultural and Technological Sciences São Paulo State University (Unesp) School of Veterinary Medicine São Paulo State University (Unesp) FAPESP: 2015/19549-8

Female mice fed a cafeteria diet (FCaf) develop higher liver steatosis and oxidative stress than males (MCaf) as a consequence of unresolved ER stress. Here, we investigated whether mitochondria play a role in this sex difference. The isolated mitochondria from FCaf showed more signs of oxidative stress than those of MCaf, correlated with a reduced content of GSH, increased amount of reactive oxygen species (ROS), and lower activities of enzymes involved in ROS neutralisation. Mitochondria from FCaf and MCaf livers exhibited lower rates of succinate-driven state III respiration and reduced ATPase activity in intact coupled mitochondria compared to their controls fed a standard diet (FC and MC), with no differences between the sexes. Fatty acid oxidation in mitochondria and peroxisomes was higher in MCaf and FCaf compared to their respective controls. In the intact perfused liver, there was no difference between sex or diet regarding the fatty acid oxidation rate. These results indicated that cafeteria diet did not affect mitochondrial energy metabolism, even in FCaf livers, which have higher steatosis and cellular oxidative stress. Nevertheless, the increase in mitochondrial ROS generation associated with a decrease in the antioxidant defence capacity, probably contributes to inducing or reinforcing the ER stress in FCaf livers.

Gluconeogenesis overstimulation due to hepatic insulin resistance is the best-known mechanism behind elevated glycemia in obese subjects with hepatic steatosis. This suggests that glucose production in fatty livers may differ from that of healthy livers, also in response to other gluconeogenic determinant factors, such as the type of substrate and modulators. Thus, the aim of this study was to investigate the effects of these factors on hepatic gluconeogenesis in cafeteria diet-induced obese adult rats submitted to a cafeteria diet at a young age. The livers of the cafeteria group exhibited higher gluconeogenesis rates when glycerol was the substrate, but lower rates were found when lactate and pyruvate were the substrates. Stearate or glucagon caused higher stimulations in gluconeogenesis in cafeteria group livers, irrespective of the gluconeogenic substrates. An increased mitochondrial NADH/NAD+ ratio and a reduced rate of 14CO2 production from [14C] fatty acids suggested restriction of the citric acid cycle. The higher glycogen and lipid levels were possibly the cause for the reduced cellular and vascular spaces found in cafeteria group livers, likely contributing to oxygen consumption restriction. In conclusion, specific substrates and gluconeogenic modulators contribute to a higher stimulation of gluconeogenesis in livers from the cafeteria group.

Shikimic acid is the starting material for the synthesis of the antiviral oseltamivir phosphate, a front-line combatant against the human influenza A and B viruses. Nowadays, the demand for shikimic acid is met by seeds of Chinese star anise (Illicium verum), which grows exclusively in southern China. In the current work, the Calophyllum brasiliense plant is suggested as an alternative source of natural shikimic acid, which was extracted, purified and quantified by high performance liquid chromatography. The presence of shikimic acid in the extract of C. brasiliense was confirmed by nuclear magnetic resonance and mass spectrometry analysis. By using a simple and rapid extraction procedure, we showed that the content of shikimic acid in C. brasiliense leaves is 3.79%. Because C. brasiliense has some additional advantages over Chinese star anise, such as geographical distribution, ecological plasticity, natural regeneration, and versatility regarding large-scale cultivation, it may be a viable and promising source of shikimic acid, with potential industrial uses.

Citrus flavanones are often linked to their antihyperglycemic properties. This effect may be in part due to the inhibition of hepatic gluconeogenesis through different mechanisms. One of the possible mechanisms appears to be impairment of oxidative phosphorylation, which may also interfere with glycogen metabolism. Based on these facts, the purpose of the present study was to investigate the effects of three citrus flavanones on glycogenolysis in the isolated perfused rat liver. Hesperidin, hesperetin, and naringenin stimulated glycogenolysis and glycolysis from glycogen with concomitant changes in oxygen uptake. At higher concentrations (300 μM), hesperetin and naringenin clearly altered fructose and glucose metabolism, whereas hesperidin exerted little to no effects. In subcellular fractions hesperetin and naringenin inhibited the activity of glucose 6‐phosphatase and glucokinase and the mitochondrial respiration linked to ADP phosphorylation. Hesperetin and naringenin also inhibited the transport of glucose into the cell. At a concentration of 300 μM, the glucose influx rate inhibition was 83% and 43% for hesperetin and naringenin, respectively. Hesperidin was the less active among the assayed citrus flavanones, indicating that the rutinoside moiety noticeably decrease the activity of these compounds. The effects on glycogenolysis and fructolysis were mainly consequence of an impairment on mitochondrial energy metabolism. The increased glucose release, due to the higher glycogenolysis, together with glucose transport inhibition is the opposite of what is expected for antihyperglycemic agents.

Fisetin is a flavonoid dietary ingredient found in the smoke tree (Cotinus coggyria) and in several fruits and vegetables. The effects of fisetin on glucose metabolism in the isolated perfused rat liver and some glucose-regulating enzymatic activities were investigated. Fisetin inhibited glucose, lactate, and pyruvate release from endogenous glycogen. Maximal inhibitions of glycogenolysis (49%) and glycolysis (59%) were obtained with the concentration of 200 microM. The glycogenolytic effects of glucagon and dinitrophenol were suppressed by fisetin 300 microM. No significant changes in the cellular contents of AMP, ADP, and ATP were found. Fisetin increased the cellular content of glucose 6-phosphate and inhibited the glucose 6-phosphatase activity. Gluconeogenesis from lactate and pyruvate or fructose was inhibited by fisetin 300 microM. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC(50) = 163.10 +/- 12.28 microM); no such effect was observed in freeze-thawing disrupted mitochondria. It was concluded that fisetin inhibits glucose release from the livers in both fed and fasted conditions. The inhibition of pyruvate transport into the mitochondria and the reduction of the cytosolic NADH-NAD(+) potential redox could be the causes of the gluconeogenesis inhibition. Fisetin could also prevent hyperglycemia by decreasing glycogen breakdown or blocking the glycogenolytic action of hormones.

The action of carbenoxolone on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In perfused livers, carbenoxolone (200-300 microM) increased oxygen consumption, glucose production and glycolysis from endogenous glycogen. Gluconeogenesis from lactate or fructose, an energy-dependent process, was inhibited. This effect was already evident at a concentration of 25 microM. The cellular ATP levels and the adenine nucleotide content were decreased by carbenoxolone, whereas the AMP levels were increased. In isolated mitochondria, carbenoxolone stimulated state IV respiration and decreased the respiratory coefficient with the substrates beta-hydroxybutyrate and succinate. The ATPase of intact mitochondria was stimulated, the ATPase of uncoupled mitochondria was inhibited, and the ATPase of disrupted mitochondria was not altered by carbenoxolone. These results indicate that carbenoxolone acts as an uncoupler of oxidative phosphorylation and, possibly, as an inhibitor of the ATP/ADP exchange system. The inhibitory action of carbenoxolone on mitochondrial energy metabolism could be contributing to induce the mitochondrial permeability transition (MPT), a key phenomenon in apoptosis. The results of the present study can explain, partly at least, the in vivo hepatotoxic actions of carbenoxolone that were found in a previous clinical evaluation.

Citrus flavonoids have a wide range of biological activities and positive health effects on mammalian cells because of their antioxidant properties. However, they also act as prooxidants and thus may interfere with metabolic pathways. The purpose of this work was to evaluate the effects of three citrus flavanones, hesperidin, hesperetin, and naringenin, on several parameters linked to fatty acid oxidation in mitochondria, peroxisomes, and perfused livers of rats. When exogenous octanoate was used as substrate, hesperetin and naringenin reduced the mitochondrial NADH/NAD+ratio and stimulated the citric acid cycle without significant changes on oxygen uptake or ketogenesis. When fatty acid oxidation from endogenous sources was evaluated, hesperetin and naringenin strongly reduced the mitochondrial NADH/NAD+ratio. They also inhibited both oxygen uptake and ketogenesis and stimulated the citric acid cycle. Hesperidin, on the other hand, had little to no effect on these parameters. These results confirm the hypothesis that citrus flavanones are able to induce a more oxidised state in liver cells, altering parameters related to hepatic fatty acid oxidation. The prooxidant effect is most likely a consequence of the ability of these substances to oxidise NADH upon production of phenoxyl radicals in the presence of peroxidases and hydrogen peroxide.

It is well known that hyperglycaemia is the initiating cause of tissue damage associated with type 2 diabetes mellitus and that enhanced hepatic gluconeogenesis may account for the increase in blood glucose levels. The purpose of this work was to investigate the possible actions and mechanisms of three related citrus flavanones, namely hesperidin, hesperetin and naringenin, on hepatic gluconeogenesis and related parameters using isolated perfused rat liver. Hesperetin and naringenin (but not hesperidin) inhibited gluconeogenesis from lactate plus pyruvate, alanine and dihydroxyacetone. The inhibitory effects of these flavanones on gluconeogenesis from lactate and pyruvate (hesperetin IC50 75.6 μM; naringenin IC50 85.5 μM) as well as from alanine were considerably more pronounced than those from dihydroxyacetone. The main cause of gluconeogenesis inhibition is the reduction of pyruvate carboxylation by hesperetin (IC50 134.2 μM) and naringenin (IC50 143.5 μM) via inhibition of pyruvate transport into the mitochondria. Secondary causes are likely inhibition of energy metabolism, diversion of glucose 6-phosphate for glucuronidation reactions and oxidation of NADH by flavanone phenoxyl radicals. The influence of the structural differences between hesperetin and naringenin on their metabolic effects was negligible. Analytical evidence indicated that the presence of a rutinoside moiety in hesperidin noticeably decreases its metabolic effects, confirming that hesperetin and naringenin interact with intracellular enzymes and mitochondrial or cellular membranes better than hesperidin. Thus, the inhibition of the gluconeogenic pathway by citrus flavanones, which was similar to that of the drug metformin, may represent an attractive novel treatment strategy for type 2 diabetes.

This study aimed to compare the anti-neoplastic effects of an Uncaria tomentosa (UT) brute hydroethanolic (BHE) extract with those of two fractions derived from it. These fractions are choroformic (CHCl3) and n-butanolic (BuOH), rich in pentacyclic oxindole alkaloids (POA) and antioxidant substances, respectively. The cancer model was the subcutaneous inoculation of Walker-256 tumour cells in the pelvic limb of male Wistar rat. Subsequently to the inoculation, gavage with BHE extract (50 mg.kg(-1)) or its fractions (as per the yield of the fractioning process) or vehicle (Control) was performed during 14 days. Baseline values, corresponding to individuals without tumour or treatment with UT, were also included. After treatment, tumour volume and mass, plasma biochemistry, oxidative stress in liver and tumour, TNF-α level in liver and tumour homogenates, and survival rates were analysed. Both the BHE extract and its BuOH fraction successfully reduced tumour weight and volume, and modulated anti-oxidant systems. The hepatic TNF-α level indicated a greater effect from the BHE extract as compared to its BuOH fraction. Importantly, both the BHE extract and its BuOH fraction increased the survival time of the tumour-bearing animals. Inversely, the CHCl3 fraction was ineffective. These data represent an in vivo demonstration of the importance of the modulation of oxidative stress as part of the anti-neoplastic activity of UT, as well as constitute evidence of the lack of activity of isolated POAs in the primary tumour of this tumour lineage. These effects are possibly resulting from a synergic combination of substances, most of them with antioxidant properties.

Most studies using a hypercaloric diet to induce obesity have focused on the metabolism of fat and carbohydrates. Less concern has been given to the metabolism of amino acids, despite evidence of modifications in nitrogen metabolism during obesity. The aim of this study was to evaluate amino acid metabolism in livers from cafeteria diet-induced obese rats. Blood parameters were analysed, and histological sections of livers were stained with Sudan III. The enzymatic activities of some enzymes were determined in liver homogenates. Gluconeogenesis, ureagenesis, and oxygen consumption were evaluated in rat livers perfused with glutamine, alanine, or ammonium chloride. Compared to control rats, cafeteria-fed rats demonstrated higher levels of triacylglycerol and glucose in the blood and greater accumulation of fat in livers. Gluconeogenesis and urea production in livers perfused with glutamine and alanine at higher concentrations showed a substantial reduction in cafeteria-fed rats. However, no significant difference was observed among groups perfused with ammonium chloride. The activities of the enzymes alanine aminotransferase, glutaminase, and aspartate aminotransferase in the livers were reduced in cafeteria-fed rats. Taken together, these data are consistent with the hypothesis that livers from cafeteria diet-induced obese rats exhibit a limitation in their maximal capacity to metabolise glutamine and alanine to glucose, ammonia, and urea, not because of an impairment in gluconeogenesis and/or ureagenesis, but rather due to a depression in the activities of enzymes that catalyse the initial steps of amino acid metabolism.

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.

The purpose of this work was to evaluate if the fat liver accumulation interferes with intracellular calcium fluxes and the liver glycogenolytic response to a calcium-mobilizing α(1)-adrenergic agonist, phenylephrine. The animal model of monosodium L-glutamate (MSG)-induced obesity was used. The adult rats develop obesity and steatosis. Calcium fluxes were evaluated through measuring the (45)Ca(2+) uptake by liver microsomes, inside-out plasma membrane, and mitochondria. In the liver, assessments were performed on the calcium-dependent glycogenolytic response to phenylephrine and the glycogen contents. The Ca(2+) uptake by microsomes and plasma membrane vesicles was reduced in livers from obese rats as a result of reduction in the Ca(2+)-ATPase activities. In addition, the plasma membrane Na(+)/K(+)-ATPase was reduced. All these matched effects could contribute to elevated resting intracellular calcium levels in the hepatocytes. Livers from obese rats, albeit smaller and with similar glycogen contents to those of control rats, released higher amounts of glucose in response to phenylephrine infusion, which corroborates these observations. Mitochondria from obese rats exhibited a higher capacity of retaining calcium, a phenomenon that could be attributed to a minor susceptibility of the mitochondrial permeability transition pore opening.

Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinones and to oxidize NADH in a variety of mammalian cells. The purpose of this study was to investigate whether fisetin affects the liver energy metabolism and the mitochondrial NADH to NAD+ ratio. The action of fisetin on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In isolated mitochondria, fisetin decreased the respiratory control and ADP/O ratios with the substrates α-ketoglutarate and succinate. In the presence of ADP, respiration of isolated mitochondria was inhibited with both substrates, indicating an inhibitory action on the ATP-synthase. The stimulation of the ATPase activity of coupled mitochondria and the inhibition of NADH-oxidase activity pointed toward a possible uncoupling action and the interference of fisetin with mitochondrial energy transduction mechanisms. In livers from fasted rats, fisetin inhibited ketogenesis from endogenous sources. The β-hydroxybutyrate/ acetoacetate ratio, which reflects the mitochondrial NADH/NAD+ redox ratio, was also decreased. In addition, fisetin (200 μM) increased the production of (14)CO2 from exogenous oleate. The results of this investigation suggest that fisetin causes a shift in the mitochondrial redox potential toward a more oxidized state with a clear predominance of its prooxidant activity.

Although metformin has been used to treat type 2 diabetes for several decades, the mechanism of its action on glucose metabolism remains controversial. To further assess the effect of metformin on glucose metabolism this work was undertaken to investigate the acute actions of metformin on glycogenolysis, glycolysis, gluconeogenesis, and ureogenesis in perfused rat livers. Metformin (5 mM) inhibited oxygen consumption and increased glycolysis and glycogenolysis in livers from fed rats. In perfused livers of fasted rats, the drug (concentrations higher than 1.0 mM) inhibited oxygen consumption and glucose production from lactate and pyruvate. Gluconeogenesis and ureogenesis from alanine were also inhibited. The cellular levels of ATP were decreased by metformin whereas the AMP levels of livers from fasted rats were increased. Taken together our results indicate that the energy status of the cell is probably compromised by metformin. The antihyperglycemic effect of metformin seems to be the result of a reduced oxidative phosphorylation without direct inhibition of key enzymatic activities of the gluconeogenic pathway. The AMP-activated protein kinase cascade could also be a probable target for metformin, which switches on catabolic pathways such as glycogenolysis and glycolysis, while switches off ATP consuming processes.

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.