A Long-term Estrogen Deficiency in Ovariectomized Mice is Associated with Disturbances in Fatty Acid Oxidation and Oxidative Stress.

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 ß-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 ß-oxidation of fatty acids irrespective of their chain length. The liver mitochondrial and peroxisomal H2O2 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.

OBJETIVO: O objetivo desse trabalho foi avaliar as alterações causadas pela deficiência estrogênica no metabolismo de lipídeos. MéTODOS: Este estudo abrangeu análises bioquímicas plasmáticas, verificação de conteúdo lipídico do fígado e avaliações da capacidade de ß-oxidação mitocondrial e do estado redox do fígado em um modelo animal de deficiência estrogênica. RESULTADOS: Os fígados das camundongas ovariectomizadas (OVXs) apresentaram acréscimos consideráveis no conteúdo de lipídeos, que foram acompanhados por aumento de peroxidação lipídica em homogenatos e mitocôndrias de fígado e diminuição do conteúdo de glutationa reduzida (GSH) quando comparadas as camundongas do grupo controle. Nas mitocôndrias isoladas, a deficiência estrogênica causou a inibição da ß-oxidação mitocondrial independentemente do comprimento da cadeia dos ácidos graxos. A geração mitocondrial e peroxissomal de H2O2 apresentou-se aumentada em camundongas OVXs. Além disso, as atividades de todas as enzimas antioxidantes avaliadas foram diminuídas. CONCLUSãO: Esses dados fornecem uma explicação potencial para o aumento da suscetibilidade às doenças metabólicas observadas após a menopausa.

A long-term estrogen deficiency in ovariectomized mice is associated with disturbances in fatty acid oxidation and oxidative stress / Deficiência de estrogênio em longo prazo em camundongas ovarietcomizadas está associada a distúrbios na oxidação de ácidos graxos e estresse oxidativo

Abstract 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 andmitochondria 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 H2O2 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.

Resumo Objetivo O objetivo desse trabalho foi avaliar as alterações causadas pela deficiência estrogênica no metabolismo de lipídeos. Métodos Este estudo abrangeu análises bioquímicas plasmáticas, verificação de conteúdo lipídico do fígado e avaliações da capacidade de β-oxidação mitocondrial e do estado redox do fígado em um modelo animal de deficiência estrogênica. Resultados Os fígados das camundongas ovariectomizadas (OVXs) apresentaram acréscimos consideráveis no conteúdo de lipídeos, que foram acompanhados por aumento de peroxidação lipídica em homogenatos e mitocôndrias de fígado e diminuição do conteúdo de glutationa reduzida (GSH) quando comparadas as camundongas do grupo controle. Nas mitocôndrias isoladas, a deficiência estrogênica causou a inibição da β-oxidação mitocondrial independentemente do comprimento da cadeia dos ácidos graxos. A geração mitocondrial e peroxissomal de H2O2 apresentou-se aumentada em camundongas OVXs. Além disso, as atividades de todas as enzimas antioxidantes avaliadas foram diminuídas. Conclusão Esses dados fornecem uma explicação potencial para o aumento da suscetibilidade às doenças metabólicas observadas após a menopausa.

Analytical methods for evaluation of the fatty acid metabolism in rat liver / Métodos analíticos para avaliação do metabolismo em ratos hepáticos

The liver is an essential organ for body energy homeostasis, controlling the biosynthesis, uptake and the disposal of carbohydrates and lipids. The hepatic steatosis is a common condition frequently associated with metabolic diseases and is characterized by the excessive accumulation of triglycerides in the liver. In recent years, many efforts have been devoted to prevent and treat the hepatic steatosis, but it remains being pointed out as the major cause for chronic hepatic diseases in Western countries. A considerable part of the knowledge about the physiopathology of hepatic steatosis, the effects of diets and drugs on the metabolic capacity of the liver to metabolize fatty acids, as well as the potential therapeutic approaches for hepatic steatosis derived from experimental animal models using rodents. Here, in this article, we present the details of some of the most common techniques used to evaluate fatty acid metabolism in liver of rats, including quantification of total lipid content, measurement of fatty acid oxidation in isolated subcellular fractions and procedures to measure the activities of important lipogenic enzymes. Classical protocols previously described to be performed using samples from other tissues were adapted to liver samples and different techniques with equivalent aims were compared. The principles and the advantages in terms of reliability and costs were discussed and the procedures here described can be applied for a low-cost broad evaluation of the fatty acid metabolism in liver of rats submitted to different experimental conditions.

O fígado é um órgão essencial para a homeostase energética, controlando a biossíntese, a captação e a eliminação de carboidratos e lipídios. A esteatose hepática é uma condição frequentemente associada a doenças metabólicas e é caracterizada pelo acúmulo excessivo de triacilgliceróis no fígado. Nos últimos anos, muitos esforços têm sido dedicados para prevenir e tratar a esteatose hepática, mas essa condição continua sendo apontada como a principal causa de doenças hepáticas crônicas em países ocidentais. Uma parte considerável do conhecimento sobre a fisiopatologia da esteatose hepática, sobre os efeitos de dietas e drogas na capacidade metabólica do fígado em metabolizar ácidos graxos, bem como sobre as possíveis abordagens terapêuticas para a esteatose hepática, derivam de estudos com modelos animais experimentais usando roedores. Neste artigo, apresentamos os detalhes de algumas das técnicas que podem ser usadas para avaliar o metabolismo de ácidos graxos no fígado de ratos, incluindo a quantificação do conteúdo lipídico total, medida da oxidação de ácidos graxos em frações subcelulares isoladas e procedimentos para medir as atividades de importantes enzimas lipogênicas. Protocolos clássicos previamente descritos para serem realizados utilizando amostras de outros tecidos foram adaptados para amostras de fígado e diferentes técnicas com objetivos equivalentes foram comparadas. Os princípios e as vantagens em termos de confiabilidade e custos foram discutidos e os procedimentos aqui descritos podem ser aplicados para uma avaliação ampla e de baixo custo do metabolismo de ácidos graxos no fígado de ratos submetidos a diferentes condições experimentais.

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

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

Vitex agnus-castus L. (Verbenaceae) Improves the Liver Lipid Metabolism and Redox State of Ovariectomized Rats.

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.

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

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

Beneficial effects of tibolone on blood pressure and liver redox status in ovariectomized rats with renovascular hypertension.

Estrogen deficiency is associated with aging and increases the incidence of metabolic syndrome and hypertension. In this study, the effects of tibolone, a synthetic steroid, on the cardiovascular system, liver lipid metabolism, and redox status were evaluated, in ovariectomized (OVX) rats with renovascular hypertension (two-kidneys, one-clip, OVX + 2K1C). This study encompassed direct measurements of mean arterial pressure , plasma biochemical analysis, liver lipid contents, and assessments of the mitochondrial and peroxisomal ß-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 ß-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.

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

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

Changes in calcium fluxes in mitochondria, microsomes, and plasma membrane vesicles of livers from monosodium L-glutamate-obese rats.

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.

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

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to 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.

Chlorogenic acid reduces the plasma glucose peak in the oral glucose tolerance test: effects on hepatic glucose release and glycaemia.

The effects of chlorogenic acid (CA) on hepatic glucose output, blood glucose levels and on glucose tolerance were analysed. Hepatic uptake of CA and its effects on hepatic catabolism of L-alanine and glucose-6-phosphatase (G-6-Pase) activity were also evaluated. CA (1 mM) inhibited about 40% of G-6-Pase activity (p < 0.05) in the microsomal fraction of hepatocytes, but no effect was observed on production of glucose from gluconeogenesis or on L-alanine catabolism, at various concentrations of CA (0.33, 0.5 and 1 mM), in liver perfusion experiments. Since there were indications of a lack of uptake of CA by the liver, it is possible that this compound did not reach sufficiently high intracellular levels to inhibit the target enzyme. Accordingly, intravenous administration of CA also failed to provoke a reduction in blood glucose levels. However, CA did promote a significant reduction (p < 0.05) in the plasma glucose peak at 10 and 15 min during the oral glucose tolerance test, probably by attenuating intestinal glucose absorption, suggesting a possible role for it as a glycaemic index lowering agent and highlighting it as a compound of interest for reducing the risk of developing type 2 diabetes.

Naproxen affects Ca(2+) fluxes in mitochondria, microsomes and plasma membrane vesicles.

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca(2+) ions, including the metabolic responses of the liver to Ca(2+)-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca(2+) fluxes. The effects of naproxen on 45Ca(2+) fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45Ca(2+) and inhibited also ATP-dependent 45Ca(2+) uptake by microsomes. Naproxen did not modify 45Ca(2+) uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca(2+) efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca(2+) ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca(2+) ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca(2+)-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca(2+) through a direct action on the plasma membrane Ca(2+) influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.