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Previous research has revealed that mitochondria are an important target for photodynamic therapy (PDT), which might be employed as a therapeutic approach for several malignancies, including hepatocellular carcinoma (HCC). In this study, we investigated both intrinsic toxicity and photodynamic effects of the photosensitizer (PS) aluminum chloride phthalocyanine (AlClPc) on mitochondrial functions. Several aspects of mitochondrial bioenergetics, structure, and oxidative state were investigated in the isolated mitochondria obtained from rat liver by differential centrifugation. Additionally, experiments were conducted to demonstrate the intrinsic and photodynamic effects of AlClPc on the viability of HepG2 cells. AlClPc interacted with mitochondria regardless of photostimulation; however, at the maximum utilized concentration (40 μM), photostimulation reduced its interaction with mitochondria. Although AlClPc hindered catalase (CAT) and glutathione reductase (GR) activities intrinsically, it had no discernable capacity to generate oxidative stress or impact bioenergetics in mitochondria without photostimulation, as one would anticipate from an ideal PS. When exposed to light, however, AlClPc had a substantially unfavorable influence on mitochondrial function, strengthening its intrinsic inhibitory action on CAT, producing oxidative stress, and jeopardizing mitochondrial bioenergetics. In terms of oxidative stress parameters, AlClPc induced lipid peroxidation and decreased the level of reduced glutathione (GSH) in mitochondria. Regarding bioenergetics, AlClPc promoted oxidative phosphorylation uncoupling and photodynamic inactivation of complex I, complex II, and the F o F 1 -ATP synthase complex, lowering mitochondrial ATP production. Lastly, AlClPc exhibited a concentration-dependent decrease in the viability of HepG2 cells, regardless of the presence or absence of photostimulation. While the harmful photodynamic effects of AlClPc on mitochondrial bioenergetics hold promise for treating HCC and other malignancies, the inherent toxic impacts on HepG2 cells underscore the need for caution in its application for this purpose., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 F o F 1 -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., Competing Interests: Declaration of Competing Interest The authors affirm that they have no known financial or interpersonal conflicts that would have seemed to influence the research that is the subject of this publication., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 o F 1 -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., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

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 o F 1 -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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In order to assist sustainable agriculture, new strategies and methods are being used based on the utilization of new natural molecules. These natural compounds can be used as potential natural crop protectors and growth promoters, and the elucidation of their modes/mechanisms of action can represent a big step towards cleaner agriculture free of agrochemicals. In the present paper, the mechanisms underlying the effects of exogenous resveratrol (R), a natural phytoalexin found in plants, on Lactuca sativa metabolism were investigated through physiological and metabolomic approaches. The results highlighted that R stimulates the growth of lettuce. A reduction of the O 2 ⋅- production in R-treated seedlings and an increase in the photosynthesis efficiency was observed, indicated by a higher Fv/Fm. The metabolomic analysis of lettuce seedlings treated with R identified 116 metabolites related to galactose, amino acids, sugar and nucleotide sugar, and ascorbate and aldarate metabolisms. Increased content of some polyamines and several metabolites was also observed, which may have contributed to scavenging free radicals and activating antioxidant enzymes, thus reducing oxidative damage and improving PSII protection in R-treated seedlings., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 F o F 1 -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., (Copyright © 2021. Published by Elsevier B.V.)

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 IC 50 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 IC 50 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., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

In the present work, the multiple-indicator dilution (MID) technique was used to investigate the kinetic mechanisms by which nickel (Ni 2+ ) affects the calcium (Ca 2+ ) transport in intact rat liver. 45 Ca 2+ and extra- and intracellular space indicators were injected in livers perfused with 1 mM Ni 2+ , and the outflow profiles were analyzed by a mathematical model. For comparative purposes, the effects of norepinephrine were measured. The influence of Ni 2+ on the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) in human hepatoma Huh7 cells and on liver glycogen catabolism, a biological response sensitive to cellular Ca 2+ , was also evaluated. The estimated transfer coefficients of 45 Ca 2+ transport indicated two mechanisms by which Ni 2+ increases the [Ca 2+ ] c in liver under steady-state conditions: (1) an increase in the net efflux of Ca 2+ from intracellular Ca 2+ stores due to a stimulus of Ca 2+ efflux to the cytosolic space along with a diminution of Ca 2+ re-entry into the cellular Ca 2+ stores; (2) a decrease in Ca 2+ efflux from the cytosolic space to vascular space, minimizing Ca 2+ loss. Glycogen catabolism activated by Ni 2+ was transient contrasting with the sustained activation induced by norepinephrine. Ni 2+ caused a partial reduction in the norepinephrine-induced stimulation in the [Ca 2+ ] c in Huh7 cells. Our data revealed that the kinetic parameters of Ca 2+ transport modified by Ni 2+ in intact liver are similar to those modified by norepinephrine in its first minutes of action, but the membrane receptors or Ca 2+ transporters affected by Ni 2+ seem to be distinct from those known to be modulated by norepinephrine., (© 2021. Society for Biological Inorganic Chemistry (SBIC).)

To date, synthetic herbicides are the main tools used for weed control, with consequent damage to both the environment and human health. In this respect, searching for new natural molecules and understanding their mode of action could represent an alternative strategy or support to traditional management methods for sustainable agriculture. Protodioscin is a natural molecule belonging to the class of steroid saponins, mainly produced by monocotyledons. In the present paper, protodioscin's phytotoxic potential was assessed to identify its target and the potential mode of action in the model plant Arabidopsis thaliana . The results highlighted that the root system was the main target of protodioscin, which caused a high inhibitory effect on the primary root length (ED 50 50 μM) with morphological alteration, accompanied by a significant increase in the lateral root number and root hair density. Through a pharmacological and microscopic approach, it was underlined that this saponin modified both auxin distribution and transport, causing an auxin accumulation in the region of root maturation and an alteration of proteins responsible for the auxin efflux (PIN2). In conclusion, the saponin protodioscin can modulate the root system of A. thaliana by interfering with the auxin transport (PAT).

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 F 1 F O -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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 F 1 F O -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

Studies of the phytotoxic effects between plants can be a crucial tool in the discovery of innovative compounds with herbicide potential. In this sense, we can highlight ruzigrass (Urochloa ruziziensis), which is traditionally used in the crop rotation system in order to reduce weed emergence. The aim of this work was to characterize the secondary metabolites of ruzigrass and to evaluate its phytotoxic effects. In total, eight compounds were isolated: friedelin, oleanolic acid, α-amyrin, 1-dehydrodiosgenone, sitosterol and stigmasterol glycosides, tricin and p-coumaric acid. Phytotoxic effects of the crude methanolic extract and fractions of ruzigrass were assessed using germination rate, initial seedling growth, and biomass of Bidens pilosa, Euphorbia heterophylla and Ipomoea grandifolia. Chemometric analysis discriminated the weed species into three groups, and B. pilosa was the most affected by fractions of ruzigrass. The phytotoxic activities of 1-dehydrodiosgenone, tricin, and p-coumaric acid are also reported, and p-coumaric acid and 1-dehydrodiosgenone were active against B. pilosa., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

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 F o F 1 -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., Competing Interests: Declaration of Competing Interest The authors have declared that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The exogenous application of plant hormones and their analogues has been exploited to improve crop performance in the field. Protodioscin is a saponin whose steroidal moiety has some similarities to plant steroidal hormones, brassinosteroids. To test the possibility that protodioscin acts as an agonist or antagonist of brassinosteroids or other plant growth regulators, we compared responses of the weed species Bidens pilosa L. to treatment with protodioscin, brassinosteroids, auxins (IAA) and abscisic acid (ABA). Seeds were germinated and grown in agar containing protodioscin, dioscin, brassinolides, IAA and ABA. Root apex respiratory activity was measured with an oxygen electrode. Malondialdehyde (MDA) and antioxidant enzymes activities were assessed. Protodioscin at 48-240 μm inhibited growth of B. pilosa seedlings. The steroidal hormone 24-epibrassinolide (0.1-5 μm) also inhibited growth of primary roots, but brassicasterol was inactive. IAA at higher concentrations (0.5-10.0 μm) strongly inhibited primary root length and fresh weight of stems. ABA inhibited all parameters of seedling growth and also seed germination. Respiratory activity of primary roots (KCN-sensitive and KCN-insensitive) was activated by protodioscin. IAA and ABA reduced KCN-insensitive respiration. The content of MDA in primary roots increased only after protodioscin treatment. All assayed compounds increased APx and POD activity, with 24-epibrassinolide being most active. The activity of CAT was stimulated by protodioscin and 24-epibrassinolide. The results revealed that protodioscin was toxic to B. pilosa through a mechanism not related to plant growth regulator signalling. Protodioscin caused a disturbance in mitochondrial respiratory activity, which could be related to overproduction of ROS and consequent cell membrane damage., (© 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

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 14 CO₂ production from [ 14 C] 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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective:  The aim of this work was to evaluate the changes caused by estrogen deficiency in lipid metabolism., Methods:  This study encompassed direct measurements of plasma biochemical analyses, liver lipid contents, and assessments of the mitochondrial β-oxidation capacity as well as an evaluation of the liver redox status in an animal model of estrogen deficiency., Results:  When compared with control mice, the livers of ovariectomized (OVX) mice presented considerable accretions in their lipid contents, which were accompanied by increased levels of lipid peroxidation in liver homogenates and mitochondria from OVX groups and decreased reduced glutathione (GSH) contents. In isolated mitochondria, estrogen deficiency inhibited mitochondrial β-oxidation of fatty acids irrespective of their chain length. The liver mitochondrial and peroxisomal H 2 O 2 generations in OVX mice were increased. Additionally, the activities of all antioxidant enzymes assessed were decreased., Conclusion:  These data provide one potential explanation for the increased susceptibility to metabolic diseases observed after menopause., Competing Interests: The authors declare that there are no conflicts of interest., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo , hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo . Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2α western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [ Gapdh ], glucose transporter 1 [ Glut1 ], pyruvate dehydrogenase kinase 1 [ Pdk1 ], and lactate dehydrogenase A [ Ldha ]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 ( Cpt1 ), apolipoprotein B ( Apob ), and acetyl-coenzyme A carboxylase 1 ( Acc1 ), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α ( Hnf4α ) and farnesoid X receptor ( Fxr ) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr . Conclusion : Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. ( Hepatology Communications 2018;2:299-312).

Senna obtusifolia L., a common weed in the tropical and subtropical regions of the world, is able to germinate under adverse environmental conditions, suggesting that this species has efficient stress-adaptation strategies. The aims of the present work were to examine the energy metabolism and the antioxidant defense system of the Senna obtusifolia L. during seed germination and initial growth, and the responses to allelochemical-induced stress. Respiratory activity, the activities of alcohol dehydrogenase (ADH), superoxide dismutase (SOD), catalase (CAT),guaicol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), lipoxygenase (LOX) and the content of malondialdehyde (MDA) and glutathione (GSSG and GSH) were measured. Shortly after seed imbibition, mitochondrial respiratory activity was active and the presence of SOD, CAT, GR and LOX activity in embryos, along with significant KCN-insensitive respiration, indicated that the production of reactive oxygen species (ROS) is initiated as soon as mitochondrial respiration resumes. Among the fourteen allelochemicals assayed, only coumarin significantly supressed the growth of S. obtusifolia seedlings. Although coumarin reduced the activities of CAT, POD and APX, the GSH, GSSG and MDA levels were not altered. Alpha-pinene, quercetin and ferulic acid did not modify the activity of the antioxidant enzymes or the contents of GSH, GSSH and MDA. Thus the antioxidant defense system of S. obstusifolia may be effective in counteracting the harmful effects of ROS generated during seed germination and initial growth in the presence of toxic allelochemicals.

Menopause is often followed by obesity and, related to this, non-alcoholic fatty liver disease (NAFLD). Two bile acid (BA) receptors, farnesoid X receptor (FXR) and G-protein-coupled receptor TGR5, have emerged as putative therapeutic targets for obesity and NAFLD., Aim of This Study: to evaluate the efficacy of selective agonists INT747/obeticholic acid (FXR) and INT777 (TGR5) as novel treatments for the metabolic effects of oestrogen deficiency. Ovariectomized (OVX) or sham-operated (SHAM) mice were fed a high-fat diet (HFD) for 5weeks. During the last 4weeks two groups of OVX and SHAM mice received either INT747- or INT777-supplemented HFD. OVX mice had significantly higher bodyweight gain than SHAM mice, which was attenuated by INT747- or INT777-treatment. No significant changes in food intake or physical activity were found. OVX mice had significantly lower energy expenditure than SHAM mice; INT747- and INT777-treated OVX mice had intermediate energy expenditure. Liver triglyceride and cholesterol content was significantly increased in OVX compared to SHAM mice, which was normalized by INT747- or INT777-treatment. Significant changes in metabolic gene expression were found in liver (Cpt1, Acox1), muscle (Ucp3, Pdk4, Cpt1, Acox1, Fasn, Fgf21), brown adipocytes (Dio2) and white adipocytes (c/EBPα, Pparγ, Adipoq). For the first time, expression of FXR and induction of its target gene Pltp1 was shown in skeletal muscle. BA receptor agonists are suitable therapeutics to correct postmenopausal metabolic changes in an OVX mouse model. Potential mechanisms include increased energy expenditure and changes in expression patterns of key metabolic genes in liver, muscle and adipose tissues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fipronil is an insecticide used to control pests in animals and plants that can causes hepatotoxicity in animals and humans, and it is hepatically metabolized to fipronil sulfone by cytochrome P-450. The present study aimed to characterize the effects of fipronil (10-50μM) on energy metabolism in isolated perfused rat livers. In fed animals, there was increased glucose and lactate release from glycogen catabolism, indicating the stimulation of glycogenolysis and glycolysis. In the livers of fasted animals, fipronil inhibited glucose and urea production from exogenous l-alanine, whereas ammonia and lactate production were increased. In addition, fipronil at 50μM concentration inhibited the oxygen uptake and increased the cytosolic NADH/NAD⁺ ratio under glycolytic conditions. The metabolic alterations were found both in livers from normal or proadifen-pretreated rats revealing that fipronil and its reactive metabolites contributed for the observed activity. The effects on oxygen uptake indicated that the possible mechanism of toxicity of fipronil involves impairment on mitochondrial respiratory activity, and therefore, interference with energy metabolism. The inhibitory effects on oxygen uptake observed at the highest concentration of 50μM was abolished by pretreatment of the rats with proadifen indicating that the metabolites of fipronil, including fipronil sulfone, acted predominantly as inhibitors of respiratory chain. The hepatoxicity of both the parent compound and its reactive metabolites was corroborated by the increase in the activity of lactate dehydrogenase in the effluent perfusate in livers from normal or proadifen-pretreated rats., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Vitex agnus-castus (VAC) is a plant that has recently been used to treat the symptoms of menopause, by its actions on the central nervous system. However, little is known about its actions on disturbances in lipid metabolism and nonalcoholic fat liver disease (NAFLD), frequently associated with menopause. Ovariectomized (OVX) rats exhibit increased adiposity and NAFLD 13 weeks after ovary removal and were used as animal models of estrogen deficiency. The rats were treated with crude extract (CE) and a butanolic fraction of VAC (ButF) and displayed the beneficial effects of a reduction in the adiposity index and a complete reversion of NAFLD. NAFLD reversion was accompanied by a general improvement in the liver redox status. The activities of some antioxidant enzymes were restored and the mitochondrial hydrogen peroxide production was significantly reduced in animals treated with CE and the ButF. It can be concluded that the CE and ButF from Vitex agnus-castus were effective in preventing NAFLD and oxidative stress, which are frequent causes of abnormal liver functions in the postmenopausal period.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Adjuvant-induced arthritis is an experimental immunopathology in rats that is often used as a model for studying autoimmune chronic inflammation and inflammatory cachexia. In these animals oxidative stress is quite pronounced in the articular inflammation sites. The purpose of this study was to evaluate oxidative stress in the liver of arthritic rats in which morphological and metabolic alterations have been reported to occur. Oxidative injury parameters, levels and production of reactive oxygen species (ROS), and antioxidant parameters were measured in the total liver homogenate and in subcellular fractions, namely cytosol, mitochondria, and peroxisomes. Arthritic rats presented higher levels of ROS than controls in the total homogenate (46% higher) and in all subcellular fractions (51, 38, and 55% higher for mitochondria, peroxisome, and cytosol, respectively). Arthritic rats also presented higher levels of protein carbonyl groups in the total homogenate (75%) and in all subcellular fractions (189, 227, and 260%, respectively, for mitochondria, peroxisomes, and cytosol). The TBARS levels of arthritic rats were more elevated in the total homogenate (36%), mitochondria (20%), and peroxisomes (16%). Arthritic rats also presented higher levels of NO markers in the peroxisomes (112%) and in the cytosol (35%). The catalase activity of all cell compartments was strongly diminished (between 77 and 87%) by arthritis, and glutathione peroxidase activities were diminished in the mitochondria (33.7%) and cytosol (41%). The cytosolic glucose-6-phosphate dehydrogenase activity, on the other hand, was increased (62.9%), the same happening with inducible peroxisomal NO synthase (119.3%). The superoxide dismutase and glutathione reductase activities were not affected. The GSH content was diminished by arthritis in all cellular compartments (50 to 59% diminution). The results reveal that the liver of rats with adjuvant-induced arthritis presents a pronounced oxidative stress and that, in consequence, injury to lipids and proteins is highly significant. The higher ROS content of the liver of arthritic rats seems to be the consequence of both a stimulated pro-oxidant system and a deficient antioxidant defense with a predominance of the latter as indicated by the strongly diminished activities of catalase and glutathione peroxidase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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

Estrogen deficiency accelerates the development of several disorders including visceral obesity and hepatic steatosis. The predisposing factors can be exacerbated by drugs that affect hepatic lipid metabolism. The aim of the present work was to determine if raloxifene, a selective estrogen receptor modulator (SERM) used extensively by postmenopausal women, affects hepatic fatty acid oxidation pathways. Fatty acids oxidation was measured in the livers, mitochondria and peroxisomes of ovariectomized (OVX) rats. Mitochondrial and peroxisomal β-oxidation was inhibited by raloxifene at a concentration range of 2.5-25 μM. In perfused livers, raloxifene reduced the ketogenesis from endogenous and exogenous fatty acids and increased the β-hydroxybutyrate/acetoacetate ratio. An increase in ¹⁴CO₂ production without a parallel increase in the oxygen consumption indicated that raloxifene caused a diversion of NADH from the mitochondrial respiratory chain to another oxidative reaction. It was found that raloxifene has a strong ability to react with H₂O₂ in the presence of peroxidase. It is likely that the generation of phenoxyl radical derivatives of raloxifene in intact livers led to the co-oxidation of NADH and a shift of the cellular redox state to an oxidised condition. This change can perturb other important liver metabolic processes dependent on cellular NADH/NAD⁺ ratio., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The purpose of the present work was to investigate Ca(2+) transport and distribution under the conditions of the intact rat liver in health and disease (adjuvant-induced arthritis). The multiple-indicator dilution technique was used with the simultaneous injection of (45)Ca(2+) and indicators into the portal vein under defined conditions and analysis of the outflow profiles by means of a space-distributed variable transit time model. The best description of the (45)Ca(2+) outflow profiles corresponds to a model that assumes rapid distribution of (45)Ca(2+) between the vascular space and the cell surface and a slower transfer into the hepatocytes. In kinetic terms two distinct cellular pools were distinguishable, the cytosol and the endoplasmic reticulum. The concentration of Ca(2+) in the cytosol was much lower than in the vascular space and in the endoplasmic reticulum. The most prominent modification observed in the livers of arthritic rats was the increased Ca(2+) concentration in the hormone-sensitive cellular pool. Furthermore, reduced rates of Ca(2+) influx and efflux between the hormone-sensitive cellular pool and the cytosolic space were also detected in combination with a significantly reduced expression of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA2) protein. All these observations mean that in livers from arthritic rats more time is required to replenish the hormone sensitive Ca(2+) stores., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

In this study, simvastatin, atorvastatin, ezetimibe, and ezetimibe + simvastatin combination were administered to arthritic rats, first to determine their effects on the inflammatory response, employing a low-dose adjuvant-induced arthritis model in rats. Arthritis was induced by the subcutaneous injection of a suspension of Mycobacterium tuberculosis (100 μg) in mineral oil [complete Freund's adjuvant used (CFA)] into the plantar surface of the hind paws. Simvastatin(40 mg/kg), atorvastatin(10 mg/kg), ezetimibe(10 mg/kg), ezetimibe(10 mg/kg) + simvastatin(20 mg/kg or 40 mg/kg) were given intragastrically and the treatment began on the day of CFA injection and continued daily up to the 28th day after arthritis induction. The ezetimibe + simvastatin combination was more effective in reducing the inflammatory response in arthritic rats than in atorvastatin, simvastatin, or ezetimibe monotherapy. The observed effect seems to be cholesterol-independent as there were no changes in plasma cholesterol levels. In spite of the benefits on joint lesions, treatment with ezetimibe + simvastatin combination caused a marked increment in liver, kidneys, spleen size, and plasma transaminases activities. Therefore, animals treated with the ezetimibe(10 mg/kg) + simvastatin(40 mg/kg) combination were also submitted to liver perfusion experiments. In this regard, ezetimibe + simvastatin did not improve the liver metabolic alterations seen in control arthritic rats, on the contrary, a worsening was observed in liver production of glucose from alanine, as well as in oxygen uptake. All of these metabolic changes appear to be induced by treatment with ezetimibe + simvastatin combination, as the same metabolic effects were observed in normal and treated arthritic animals., (© 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Française de Pharmacologie et de Thérapeutique.)

The aim of this work was to evaluate the effects of therapeutic doses of Cimicifuga racemosa on cardiovascular parameters and on liver lipid metabolism and redox status in an animal model of estrogen deficiency associated with hypertension, a condition that could make the liver more vulnerable to drug-induced injuries. Female Wistar rats were subjected to the surgical procedures of bilateral ovariectomy (OVX) and induction of renovascular hypertension (two-kidneys, one-clip; 2K1C). These animals (OVX + 2K1C) were treated with daily doses of a C. racemosa extract, using a dose that is similar to that recommended to postmenopausal women (0.6 mg/kg), over a period of 15 days. The results were compared to those of untreated OVX + 2K1C, OVX, and control rats. The treatment with C. racemosa caused a significant reduction in blood pressure. In the liver, treatment did not prevent the development of steatosis, and it reduced the mitochondrial and peroxisomal capacity to oxidize octanoyl-CoA compared to the untreated animals. In addition, C. racemosa caused numerous undesirable effects on the liver redox status: it increased the mitochondrial reactive oxygen species generation, an event that was not accompanied by an increase in the activity of superoxide dismutase, and it induced a decrease in peroxisomal catalase activity. Although the reduced glutathione content had not been affected, a phenomenon that probably reflected the restoration of glucose-6-phosphate dehydrogenase activity by C. racemosa, oxidative damage was evidenced by the elevated level of thiobarbituric acid-reactive substances found in the liver of treated animals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In the study, indomethacin, cyclophosphamide, and infliximab were administered to adjuvant-induced arthritic rats to determine if they were able to prevent the abnormalities caused by arthritis on hepatic metabolism. The drugs were administered to arthritic rats, and at the clinical onset of arthritis (day 14 after adjuvant injection), the livers were perfused to evaluate gluconeogenesis, ureagenesis, oxygen uptake, L: -lactate, pyruvate, and ammonia release from L: -alanine. The effects of the drugs on body weight gain and the signs of arthritis (paw edema, appearance of secondary lesions, and weights of lymphoid tissues) were also evaluated. Cyclophosphamide could completely prevent liver metabolic changes and the inflammatory response. Indomethacin restored ureagenesis, minimized the decrease in gluconeogenesis, and exerted a partially beneficial effect on inflammatory reactions. Infliximab did not improve arthritis-induced liver metabolic alterations or inflammatory responses. These results suggest the participation of prostaglandins, but not TNF-α, on arthritis-induced liver metabolic alterations.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tibolone is a synthetic steroid that has been extensively prescribed to treat climacteric symptoms and to prevent postmenopausal osteoporosis. Because menopause is a condition associated with increased incidence of metabolic disturbances and hepatic steatosis, the aim of this work was to evaluate the actions of tibolone on the liver. The effects of tibolone on glucose and fatty acid metabolism and on several parameters linked to mitochondrial energy metabolism, including the induction of cellular oxidative stress, were investigated in livers from female Wistar rats. Tibolone was assayed at concentrations ranging from 5 to 100 μM. In perfused livers, tibolone inhibited oxygen uptake, stimulated glycogenolysis and glycolysis, and inhibited gluconeogenesis from L-lactate and ketogenesis from exogenous octanotate. Tibolone also caused pronounced increases in both the cytosolic and mitochondrial NADH/NAD+ratios. In isolated mitochondria, tibolone inhibited oxygen uptake due to β-hydroxybutyrate and fatty acid oxidation without affecting the succinate oxidation. The inhibitory action of tibolone at complex I of the mitochondrial respiratory chain was suggested by the inhibition of the NADH-oxidase activity. Tibolone also induced oxidative stress in both perfused livers and isolated mitochondria, as indicated by the increased production of thiobarbituric acid reactive substances. These metabolic alterations may increase the risk of metabolic disturbances during tibolone administration, particularly in the postmenopausal condition., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2010 Wiley Periodicals, Inc.)

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., (2010 John Wiley & Sons, Ltd.)

This study investigated the effects of exogenously applied ethanol on Euphorbia heterophylla L., a troublesome weed in field and plantation crops. Ethanol at concentrations ranging from 0.25 to 1.5% caused a dose-dependent inhibition of germination and growth of E. heterophylla. Measurements of respiratory activity and alcohol dehydrogenase (E.C. 1.1.1.1) activity during seed imbibition and initial seedling growth revealed that ethanol induces a prolongation of hypoxic conditions in the growing tissues. In isolated mitochondria, ethanol inhibited the respiration coupled to ADP phosphorylation, an action that probably contributed to modifications observed in the respiratory activity of embryos. A comparison of the effects of methanol, ethanol, propanol and acetaldehyde on germination and growth of E. heterophylla indicates that alcohol dehydrogenase activity is required for the observed effects, with the conversion of ethanol to acetaldehyde playing a role in the ethanol-induced injuries.

The effects of zymosan on transport, distribution, and metabolism of octanoate in the perfused rat liver were investigated using the multiple-indicator dilution technique. Livers were perfused with 300 microM octanoate in the absence or in the presence of 100 microg/mL zymosan. Tracer amounts of [1-14C]octanoate, [3H] water, and [131I]albumin were injected into the portal vein, and the effluent perfusate was fractionated. The normalized dilution curves were analyzed by means of a space-distributed variable transit time model. Zymosan decreased the space into which octanoate undergoes flow-limited distribution, possibly the first cellular exchanging pool represented by plasma membranes and their adjacencies. However, the rate of transfer of octanoate from the plasma membrane into the rest of the cell was not modified as indicated by the similar values of the influx rates and also the net uptake of octanoate per unit of accessible cellular volume. However, when referred to the wet weight of the liver, the net uptake of octanoate was 37.5% reduced, a value corresponding to the diminution of the cellular accessible space. It can be concluded that an exclusion of a fraction of the liver parenchyma from the microcirculation is the main mechanism by which zymosan reduces the metabolism of exogenous octanoate.

The present study investigated arthritis induced by complete Freund adjuvant (AIA) in spontaneously hypertensive and normotensive rats (respectively, SHR and NTR rats). The inflammatory reaction was studied for 28 days by evaluating paw edema and secondary lesions found 10 days after complete Freund adjuvant (CFA) administration. The body weight of the animals and macroscopic alterations of several organs, including spleen, thymus, adrenal glands, and lymph nodes, were also analyzed. The results showed that the AIA manifestations were decreased in SHRs compared with NTRs. Moreover, this altered inflammatory response was not modified by surgical adrenalectomy.

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the plants of the Crotalaria species that causes cytotoxicity and genotoxicity in animals and humans, and it is hepatically metabolized to the alkylating agent dehydromonocrotaline by cytochrome P-450. The exact cellular and molecular mechanisms of MCT-induced tissue injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase at micromolar concentrations in isolated liver mitochondria, an effect associated with significantly reduced ATP synthesis. Impairment of energy metabolism is expected to lead to several alterations in cell metabolism. In this work, the action of different concentrations of monocrotaline (250, 500, and 750microM) on energy metabolism-linked parameters was investigated in isolated perfused rat livers. In the fed state, monocrotaline increased glycogenolysis and glycolysis, whereas in the livers of fasted rats, it decreased gluconeogenesis and urea synthesis from l-alanine. These metabolic alterations were only found in livers of phenobarbital-treated rats, indicating that active metabolites including dehydromonocrotaline were responsible for the observed activity. Our findings indicate that hepatic metabolic changes may be implicated, partly at least, in the hepatotoxicity of monocrotaline in animals and humans.

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease., ((c) 2008 John Wiley & Sons, Ltd.)