Effects of high-fat,low-cholesterol diets on hepatic lipid peroxidation and antioxidants in apolipoprotein E-deficient mice

The present study describes the effects of several high-fat low-cholesterol antiatherogenic diets on the hepatic lipid peroxidation and hepatic antioxidant systems in apolipoprotein E-deficient mice. Eighty mice were distributed into five groups and fed with regular mouse chow or chow supplemented with coconut, palm, olive and sunflower seed oils. After ten weeks, they were sacrificed and the livers were removed so that lipid peroxidation and -tocopherol concentrations, and superoxide dismutase, glutathione peroxidase and glutathione reductase activities could be measured. The size of the atherosclerotic lesions in the aortas was also measured. Results showed that the diets supplemented with olive oil, palm oil or sunflower seed oil significantly decreased the size of the lesion. However, there was an association between those mice that were on diets supplemented with palm or coconut oils and a significant increase in hepatic lipid peroxidation. This association was not found in animals fed with olive or sunflower seed oils, the diets with the highest content of vitamin E. The dietary content of vitamin E was significantly correlated (r = 0.98; p < 0.05) with the hepatic concentration of this compound. Our study suggests that the high content of vitamin E in olive oil or sunflower seed oil may protect from the undesirable hepatotoxic effects of high-fat diets in apo E-deficient mice and that this should be taken into account when these diets are used to prevent atherosclerosis.     

The continuous administration of aspirin attenuates atherosclerosis in apolipoprotein E-deficient mice

Aspirin reduces the incidence of thrombotic occlusive events. Classically this has been thought to be due to the platelet inhibitory action of aspirin but it has recently been shown that inflammation plays a predominant role in the initiation and progression of lesions in atherosclerosis. In humans, treatment with aspirin reduces cardiovascular risk and slows carotid plaque growth in a dose-dependent fashion. We have explored this issue in Apo E-deficient mice on a high-fat, high cholesterol diet which provided these animals with a continuous administration of 500 microg/day of acetylsalicylic acid in the drinking water. After 10 weeks of treatment, the size of the atherosclerotic lesion at the aortic sinus had reduced by 35%. At the end of the trial there were no significant changes in either plasma lipids or in the quantitative distribution among lipoproteins. Likewise, the total antioxidant status and the resistance of plasma to oxidation in vitro was similar and there was no change in the distribution of iron deposits and in the relative composition of plasma pro-oxidants and antioxidants, or in the concentration of plasma in ferritin. Therefore, it is our hypothesis that the antiinflammatory effect is responsible for the reduction in lesion size. We propose that antiinflammatory molecules which do not cause gastrointestinal complications should be tested in humans to determine long-term efficacy in the attenuation of atherosclerosis.     

Continuous administration of polyphenols from aqueous rooibos (Aspalathus linearis) extract ameliorates dietary-induced metabolic disturbances in hyperlipidemic mice

The incidence of obesity and related metabolic diseases is increasing globally. Current medical treatments often fail to halt the progress of such disturbances, and plant-derived polyphenols are increasingly being investigated as a possible way to provide safe and effective complementary therapy. Rooibos (Aspalathus linearis) is a rich source of polyphenols without caloric and/or stimulant components. We have tentatively characterized 25 phenolic compounds in rooibos extract and studied the effects of continuous aqueous rooibos extract consumption in mice. The effects of this extract, which contained 25% w/w of total polyphenol content, were negligible in animals with no metabolic disturbance but were significant in hyperlipemic mice, especially in those in which energy intake was increased via a Western-type diet that increased the risk of developing metabolic complications. In these mice, we found hypolipemiant activity when given rooibos extract, with significant reductions in serum cholesterol, triglyceride and free fatty acid concentrations. Additionally, we found changes in adipocyte size and number as well as complete prevention of dietary-induced hepatic steatosis. These effects were not related to changes in insulin resistance. Among other possible mechanisms, we present data indicating that the activation of AMP-activated protein kinase (AMPK) and the resulting regulation of cellular energy homeostasis may play a significant role in these effects of rooibos extract. Our findings suggest that adding polyphenols to the daily diet is likely to help in the overall management of metabolic diseases.     

Changes in the expression of genes related to apoptosis and fibrosis pathways in CCl4-treated rats

Chronic liver diseases are accompanied by changes in the biochemical pathways related to the regulation of apoptosis and extra-cellular matrix deposition. The present study was designed to investigate, using low density arrays, changes in the hepatic gene expression together with hepatic biochemical and histological alterations in rats that had liver impairment induced by chronic exposure to CCl₄. Further, we examined the possible recovery of genetic and pathological changes following the cessation of the hepatotoxic injury. Experimental fibrosis was induced in male Wistar rats by CCl₄ administration. Animals were subdivided into two groups. One group was given CCl₄ and animals were killed at 8 and 12 weeks of treatment. The other group was treated with CCl₄ for 6 weeks, the CCl₄ was then stopped and, subsequently, subgroups of animals were killed after 1 and 2 weeks of recovery. CCl₄ administration over 12 weeks was associated with significant changes in B-cell leukemia/lymphoma 2, procollagen type I α 2, matrix metalloproteinases 3 and 8, tissue inhibitors of metalloproteinases 1, 2, and 3 and the inhibitor of apoptosis 4 gene expressions. Recovery after CCl₄ cessation was associated with changes in procollagen type I α 2, matrix metalloproteinase 7, tissue inhibitors of metalloproteinases 1 and 2, inhibitor of apoptosis 4, and survivin gene expressions. This study shows an association between changes in the expression of several genes regulating hepatic cell apoptosis, the fibrosis process, and the recovery of the hepatic function after removal of the toxic injury.     

Metformin targets histone acetylation in cancer-prone epithelial cells

The usage of metabolic intermediates as substrates for chromatin-modifying enzymes provides a direct link between the metabolic state of the cell and epigenetics. Because this metabolism-epigenetics axis can regulate not only normal but also diseased states, it is reasonable to suggest that manipulating the epigenome via metabolic interventions may improve the clinical manifestation of age-related diseases including cancer. Using a model of BRCA1 haploinsufficiency-driven accelerated geroncogenesis, we recently tested the hypothesis that: 1.) metabolic rewiring of the mitochondrial biosynthetic nodes that overproduce epigenetic metabolites such as acetyl-CoA should promote cancer-related acetylation of histone H3 marks; 2.) metformin-induced restriction of mitochondrial biosynthetic capacity should manifest in the epigenetic regulation of histone acetylation. We now provide one of the first examples of how metformin-driven metabolic shifts such as reduction of the 2-carbon epigenetic substrate acetyl-CoA is sufficient to correct specific histone H3 acetylation marks in cancer-prone human epithelial cells. The ability of metformin to regulate mitonuclear communication and modulate the epigenetic landscape in genomically unstable pre-cancerous cells might guide the development of new metabolo-epigenetic strategies for cancer prevention and therapy.     

Gerometabolites: The pseudohypoxic aging side of cancer oncometabolites

Oncometabolites are defined as small-molecule components (or enantiomers) of normal metabolism whose accumulation causes signaling dysregulation to establish a milieu that initiates carcinogenesis. In a similar manner, we propose the term “gerometabolites” to refer to small-molecule components of normal metabolism whose depletion causes signaling dysregulation to establish a milieu that drives aging. In an investigation of the pathogenic activities of the currently recognized oncometabolites R(-)-2-hydroxyglutarate (2-HG), fumarate, and succinate, which accumulate due to mutations in isocitrate dehydrogenases (IDH), fumarate hydratase (FH), and succinate dehydrogenase (SDH), respectively, we illustrate the fact that metabolic pseudohypoxia, the accumulation of hypoxia-inducible factor (HIFα) under normoxic conditions, and the subsequent Warburg-like reprogramming that shifts glucose metabolism from the oxidative pathway to aerobic glycolysis are the same mechanisms through which the decline of the “gerometabolite” nicotinamide adenine dinucleotide (NAD)⁺ reversibly disrupts nuclear–mitochondrial communication and contributes to the decline in mitochondrial function with age. From an evolutionary perspective, it is reasonable to view NAD⁺-driven mitochondrial homeostasis as a conserved response to changes in energy supplies and oxygen levels. Similarly, the natural ability of 2-HG to significantly alter epigenetics might reflect an evolutionarily ancient role of certain metabolites to signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency. However, when chronically altered, these responses become conserved causes of aging and cancer. Because HIFα-driven pseudohypoxia might drive the overproduction of 2-HG, the intriguing possibility exists that the decline of gerometabolites such as NAD⁺ could promote the chronic accumulation of oncometabolites in normal cells during aging. If the sole activation of a Warburg-like metabolic reprogramming in normal tissues might be able to significantly increase the endogenous production of bona fide etiological determinants in cancer, such as oncometabolites, this undesirable trade-off between mitochondrial dysfunction and activation of oncometabolites production might then pave the way for the epigenetic initiation of carcinogenesis in a strictly metabolic-dependent manner. Perhaps it is time to definitely adopt the view that aging and aging diseases including cancer are governed by a pivotal regulatory role of metabolic reprogramming in cell fate decisions.     

Turpentine-induced inflammation reduces the hepatic expression of the multiple drug resistance gene, the plasma cholesterol concentration and the development of atherosclerosis in apolipoprotein E deficient mice

We aimed to investigate the effect of turpentine-induced inflammation in an atherosclerosis-prone murine model. We have induced a chronic aseptic inflammation in apolipoprotein E-deficient mice, with or without a dietary supplement of aspirin (n = 10, each), by the injection of a mixture (1:1) of turpentine and olive oil in the hind limb twice weekly for a period of 12 weeks. Control animals were injected with olive oil alone (n = 10). The control mice did show any alteration neither in plasma nor at the site of injection. Turpentine-treated mice showed a significant increase in plasma TNF-alpha and SAA concentrations which indicated a systemic inflammatory response that was not substantially affected by aspirin. Also, turpentine injections significantly reduced the plasma cholesterol concentration, probably decreasing intestinal cholesterol re-absorption, and attenuated the size of atherosclerotic lesion. Both effects were minimally influenced by aspirin. The burden of atherosclerosis correlated with plasma lipid levels but not with plasma inflammatory markers. Finally, there was a concomitant decrease in the expression of the hepatic mdr1b gene that correlated with the decrease in plasma cholesterol concentration. Therefore, we conclude that mdr1 is an additional factor to consider in the complexity of alterations in cholesterol metabolism that occur in this model.     

The value of apolipoprotein E knockout mice for studying the effects of dietary fat and cholesterol on atherogenesis

The ability of the apolipoprotein E-deficient mouse to develop spontaneous atherosclerosis, which resembles the human process, is an excellent model in which to assess the impact of dietary factors. This review discusses the role of several nutrients in the development of atherosclerosis and the mechanisms through which they act.     

The role of combined assessment of defense against oxidative stress and inflammation in the evaluation of peripheral arterial disease

Atherosclerosis in symptomatic peripheral arterial disease affects wide portions of numerous arteries in lower extremities. The resulting active inflammation in a considerable amount of arterial tissue facilitates systemic detection via measurement of inflammation-related variables. We reasoned that the combined assessment of defense against oxidative stress, in the form of paraoxonase-1 (PON1), and monocyte migration measured as circulating (C-C motif) ligand 2 (CCL2), may play a role in the evaluation of these patients. Plasma CCL2 and serum PON1-related variables, assessed by their interaction with functional genetic variants, were measured in a cross-sectional study in patients with symptomatic PAD. We found that PON1 activity and concentration were significantly lower and CCL2 concentration higher in PAD patients compared to controls, that the combination of plasma CCL2 and PON1- related values, especially PON1 concentration differentiated, almost perfectly, controls from patients and that the expression of CCL2 and PON1 generally co-localized in the atherosclerotic lesion. Since no association with genetic variants was found, such a relationship is probably the result of the disease. Our data suggest a coordinated role between CCL2 and PON1 that may be detected in blood with simple measurements and may represent an indicator of the extent of atherosclerosis.