Inhibition of O-acetylserine (thiol) lyase as a promising new mechanism of action for herbicides.

Enzymes of the sulfur assimilation pathway of plants have been identified as potential targets for herbicide development, given their crucial role in synthesizing amino acids, coenzymes, and various sulfated compounds. In this pathway, O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) catalyzes the synthesis of L-cysteine through the incorporation of sulfate into O-acetylserine (OAS). This study used an in silico approach to select seven inhibitors for OAS-TL. The in silico experiments revealed that S-benzyl-L-cysteine (SBC) had a better docking score (-7.0 kcal mol-1) than the substrate OAS (-6.6 kcal mol-1), indicating its suitable interaction with the active site of the enzyme. In vitro experiments showed that SBC is a non-competitive inhibitor of OAS-TL from Arabidopsis thaliana expressed heterologously in Escherichia coli, with a Kic of 4.29 mM and a Kiu of 5.12 mM. When added to the nutrient solution, SBC inhibited the growth of maize and morning glory weed plants due to the reduction of L-cysteine synthesis. Remarkably, morning glory was more sensitive than maize. As proof of its mechanism of action, L-cysteine supplementation to the nutrient solution mitigated the inhibitory effect of SBC on the growth of morning glory. Taken together, our data suggest that reduced L-cysteine synthesis is the primary cause of growth inhibition in maize and morning glory plants exposed to SBC. Furthermore, our findings indicate that inhibiting OAS-TL could potentially be a novel approach for herbicidal action.

The harmful acute effects of clomipramine in the rat liver: Impairments in mitochondrial bioenergetics.

Clomipramine, a tricyclic antidepressant used to treat depression and obsessive-compulsive disorder, has been linked to a few cases of acute hepatotoxicity. It is also recognized as a compound that hinders the functioning of mitochondria. Hence, the effects of clomipramine on mitochondria should endanger processes that are somewhat connected to energy metabolism in the liver. For this reason, the primary aim of this study was to examine how the effects of clomipramine on mitochondrial functions manifest in the intact liver. For this purpose, we used the isolated perfused rat liver, but also isolated hepatocytes and isolated mitochondria as experimental systems. According to the findings, clomipramine harmed metabolic processes and the cellular structure of the liver, especially the membrane structure. The considerable decrease in oxygen consumption in perfused livers strongly suggested that the mechanism of clomipramine toxicity involves the disruption of mitochondrial functions. Coherently, it could be observed that clomipramine inhibited both gluconeogenesis and ureagenesis, two processes that rely on ATP production within the mitochondria. Half-maximal inhibitory concentrations for gluconeogenesis and ureagenesis ranged from 36.87 µM to 59.64 µM. The levels of ATP as well as the ATP/ADP and ATP/AMP ratios were reduced, but distinctly, between the livers of fasted and fed rats. The results obtained from experiments conducted on isolated hepatocytes and isolated mitochondria unambiguously confirmed previous propositions about the effects of clomipramine on mitochondrial functions. These findings revealed at least three distinct mechanisms of action, including uncoupling of oxidative phosphorylation, inhibition of the FoF1-ATP synthase complex, and inhibition of mitochondrial electron flow. The elevation in activity of cytosolic and mitochondrial enzymes detected in the effluent perfusate from perfused livers, coupled with the increase in aminotransferase release and trypan blue uptake observed in isolated hepatocytes, provided further evidence of the hepatotoxicity of clomipramine. It can be concluded that impaired mitochondrial bioenergetics and cellular damage are important factors underlying the hepatotoxicity of clomipramine and that taking excessive amounts of clomipramine can lead to several risks including decreased ATP production, severe hypoglycemia, and potentially fatal outcomes.

Toluidine blue O directly and photodynamically impairs the bioenergetics of liver mitochondria: a potential mechanism of hepatotoxicity.

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 FoF1-ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40 µM. Additionally, TBO (40 µM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT.

The metabolic and toxic acute effects of phloretin in the rat liver.

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 FoF1-ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.

Inhibiting tricin biosynthesis improves maize lignocellulose saccharification.

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.

Morphogenic responses and biochemical alterations induced by the cover crop Urochloa ruziziensis and its component protodioscin in weed species.

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.

Cadmium uncouples mitochondrial oxidative phosphorylation and induces oxidative cellular stress in soybean roots.

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.

Kinetic mechanisms by which nickel alters the calcium (Ca2+) transport in intact rat liver.

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.

The photodynamic and intrinsic effects of Azure B on mitochondrial bioenergetics and the consequences of its intrinsic effects on hepatic energy metabolism.

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.

The photosensitiser azure A disrupts mitochondrial bioenergetics through intrinsic and photodynamic effects.

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 (0.540 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.

Association between metabolic syndrome, hepatic steatosis, and testosterone deficiency: evidences from studies with men and rodents.

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.

The photodynamic and direct actions of methylene blue on mitochondrial energy metabolism: A balance of the useful and harmful effects of this photosensitizer.

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.

Differential Effects of Exogenous Resveratrol on the Growth and Energy Metabolism of Zea mays and the Weed Ipomoea grandifolia.

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 Zea mays L. (corn) and in the weed species Ipomoea grandifolia (Dammer) O'Donell (morning glory). At a concentration range from 220 to 2200 µM resveratrol exerted a stimulus on Z. mays seedling growth that was more pronounced at low concentrations; in the weed species I. grandifolia, resveratrol exerted inhibitory action on seedling growth in all of the assayed concentration range. In I. grandifolia, resveratrol also inhibited the respiratory activity of the primary roots. In mitochondria isolated from Z. mays roots, resveratrol at concentrations above 440 µM inhibited the respiration coupled to ADP phosphorylation and the activities of NADH-oxidase, succinate-oxidase, and ATPsynthase. These effects were not reproduced in Z. mays grown in the presence of resveratrol as the respiratory activities of the roots were not affected. The finding that the resveratrol exerts beneficial effects on growth of Z. mays seedlings and inhibits the growth of I. grandifolia heightens the potential of resveratrol application for crop protection.

Cafeteria Diet Feeding in Young Rats Leads to Hepatic Steatosis and Increased Gluconeogenesis under Fatty Acids and Glucagon Influence.

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.

Sex differences in the development of hepatic steatosis in cafeteria diet-induced obesity in young mice.

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.

The acute effects of citrus flavanones on the metabolism of glycogen and monosaccharides in the isolated perfused rat liver.

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.

Molecular mechanisms of citrus flavanones on hepatic gluconeogenesis.

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.

Citrus flavanones affect hepatic fatty acid oxidation in rats by acting as prooxidant agents.

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.

Uncaria tomentosa exerts extensive anti-neoplastic effects against the Walker-256 tumour by modulating oxidative stress and not by alkaloid activity.

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.