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Numerous nutritional approaches aimed at reducing body weight have been devel- oped as a strategy to reduce obesity. Most of these interventions rely on reducing caloric intake or limiting calories access to a few hours per day. In this work, we analyzed the effects of the extended (24 hours/day) or restricted (1 hour/day) access to a cafeteria-style (CAF) diet, on rat body weight and hepatic lipid metabolism, with respect to control rats (CTR) fed with a standard chow diet. The body weight gain of restricted-fed rats was not different from CTR, despite the slightly higher total caloric intake, but resulted significantly lower than extended-fed rats, which showed a CAF diet-induced obesity and a dramatically higher total caloric intake. However, both CAF-fed groups of rats showed, compared to CTR, unhealthy serum and hepatic parameters such as higher serum glucose level, lower HDL values, and increased hepatic triacylglycerol and cholesterol amount. The hepatic expression and activity of key enzymes of fatty acid synthesis, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), was similarly reduced in both CAF-fed groups of rats with respect to CTR. Anyway, while in extended-fed rats this reduction was associ- ated to a long-term mechanism involving sterol regulatory element-binding protein-1 (SREBP-1), in restricted-fed animals a short-term mechanism based on PKA and AMPK activation occurred in the liver. Furthermore, hepatic fatty acid oxidation (FAO) and oxidative stress resulted significantly increased in extended, but not in restricted-fed rats, as compared to CTR. Overall, these results demonstrate that al- though limiting the total caloric intake might successfully fight obesity development, the nutritional content of the diet is the major determinant for the health status.

Hepatic de novo lipogenesis (DNL), the process by which carbohydrates are converted into lipids, is strictly controlled by nutritional and hormonal status. 3,5-Diiodo-L-thyronine (T2), a product of the 3,5,3'-triiodo-L-thyronine (T3) peripheral metabolism, has been shown to mimic some T3 effects on lipid metabolism by a short-term mechanism independent of protein synthesis. Here, we report that T2, administered for 1 week to hypothyroid rats, increases total fatty acid synthesis from acetate in isolated hepatocytes. Studies carried out on liver subcellular fractions demonstrated that T2 not only increases the activity and the expression of acetyl-CoA carboxylase and fatty acid synthase but also of other proteins linked to DNL such as the mitochondrial citrate carrier and the cytosolic ATP citrate lyase. Parallelly, T2 stimulates the activities of enzymes supplying cytosolic NADPH needed for the reductive steps of DNL. With respect to both euthyroid and hypothyroid rats, T2 administration decreases the hepatic mRNA level of SREBP-1, a transcription factor which represents a master regulator of DNL. However, when compared to hypothyroid rats T2 significantly increases, without bringing to the euthyroid value, the content of both mature (nSREBP-1), and precursor (pSREBP-1) forms of the SREBP-1 protein as well as their ratio. Moreover, T2 administration strongly augmented the nuclear content of ChREBP, another crucial transcription factor involved in the regulation of lipogenic genes. Based on these results, we can conclude that in the liver of hypothyroid rats the transcriptional activation by T2 of DNL genes could depend, at least in part, on SREBP-1- and ChREBP-dependent mechanisms. © 2019 IUBMB Life, 2019.

In recent years, lipid metabolism has gained greater attention in several diseases including cancer. Dysregulation of fatty acid metabolism is a key component in breast cancer malignant transformation. In particular, de novo lipogenesis provides the substrate required by the proliferating tumor cells to maintain their membrane composition and energetic functions during enhanced growth. However, it appears that not all breast cancer subtypes depend on de novo lipogenesis for fatty acid replenishment. Indeed, while breast cancer luminal subtypes rely on de novo lipogenesis, the basal-like receptor-negative subtype overexpresses genes involved in the utilization of exogenous-derived fatty acids, in the synthesis of triacylglycerols and lipid droplets, and fatty acid oxidation. These metabolic differences are specifically associated with genomic and proteomic changes that can perturb lipogenic enzymes and related pathways. This behavior is further supported by the observation that breast cancer patients can be stratified according to their molecular profiles. Moreover, the discovery that extracellular vesicles act as a vehicle of metabolic enzymes and oncometabolites may provide the opportunity to noninvasively define tumor metabolic signature. Here, we focus on de novo lipogenesis and the specific differences exhibited by breast cancer subtypes and examine the functional contribution of lipogenic enzymes and associated transcription factors in the regulation of tumorigenic processes.

The pathogenesis of cancer involves multiple molecular alterations at the level of genome, epigenome, and stromal environment, resulting in several deregulated signal transduction pathways. Metabolites are not only end products of gene and protein expression but also a consequence of the mutual relationship between the genome and the internal environment. Considering that metabolites serve as a comprehensive chemical fingerprint of cell metabolism, metabolomics is emerging as the method able to discover metabolite biomarkers that can be developed for early cancer detection, prognosis, and response to treatment. Urine represents a noninvasive source, available and rich in metabolites, useful for cancer diagnosis, prognosis, and treatment monitoring. In this chapter, we reported the main published evidences on urinary metabolic biomarkers in the studied cancers related to hepatopancreatic and urinary tract with the aim at discussing their promising role in clinical practice.

Diet is a major driver of gut microbiota variation and plays a role in metabolic disorders, including metabolic syndrome (MS). Mycorrhized foods from symbiotic agriculture (SA) exhibit improved nutritional properties, but potential benefits have never been investigated in humans. We conducted a pilot interventional study on 60 adults with ≥ 1 risk factors for MS, of whom 33 consumed SA-derived fresh foods and 27 received probiotics over 30 days, with a 15-day follow-up. Stool, urine and blood were collected over time to explore changes in gut microbiota, metabolome, and biochemical, inflammatory and immunologic parameters, previous dietary habits were investigated through a validated food-frequency questionnaire. The baseline microbiota showed alterations typical of metabolic disorders, mainly an increase in Coriobacteriaceae and a decrease in health-associated taxa, which were partly reversed after the SA-based diet. Improvements were observed in metabolome, MS presence (two out of six subjects no longer had MS) or components. Changes were more pronounced with less healthy baseline diets. Probiotics had a marginal, not entirely favorable, effect, although one out of three subjects no longer suffered from MS. These findings suggest that improved dietary patterns can modulate the host microbiota and metabolome, counteracting the risk of developing MS.

Long-term high-fat diet (HFD) consumption can cause weight gain and obesity, two conditions often associated with hepatic non-alcoholic fatty liver and oxidative stress. Oleoylethanolamide (OEA), a lipid compound produced by the intestine from oleic acid, has been associated with different beneficial effects in diet-induced obesity and hepatic steatosis. However, the role of OEA on hepatic oxidative stress has not been fully elucidated. In this study, we used a model of diet-induced obesity to study the possible antioxidant effect of OEA in the liver. In this model rats with free access to an HFD for 77 days developed obesity, steatosis, and hepatic oxidative stress, as compared to rats consuming a low-fat diet for the same period. Several parameters associated with oxidative stress were then measured after two weeks of OEA administration to diet-induced obese rats. We showed that OEA reduced, compared to HFD-fed rats, obesity, steatosis, and the plasma level of triacylglycerols and transaminases. Moreover, OEA decreased the amount of malondialdehyde and carbonylated proteins and restored the activity of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, which decreased in the liver of HFD-fed rats. OEA had also an improving effect on parameters linked to endoplasmic reticulum stress, thus demonstrating a role in the homeostatic control of protein folding. Finally, we reported that OEA differently regulated the expression of two transcription factors involved in the control of lipid metabolism and antioxidant genes, namely nuclear factor erythroid-derived 2-related factor 1 (Nrf1) and Nrf2, thus suggesting, for the first time, new targets of the protective effect of OEA in the liver.

Binge eating disorder (BED) is the most frequent eating disorder, for which current pharmacotherapies show poor response rates and safety concerns, thus highlighting the need for novel treatment options. The lipid-derived messenger oleoylethanolamide (OEA) acts as a satiety signal inhibiting food intake through the involvement of central noradrenergic and oxytocinergic neurons. We investigated the anti-binge effects of OEA in a rat model of binge-like eating, in which, after cycles of intermittent food restrictions/refeeding and palatable food consumptions, female rats show a binge-like intake of palatable food, following a 15-min exposure to their sight and smell (“frustration stress”). Systemically administered OEA dose-dependently (2.5, 5, and 10 mg kg−1) prevented binge-like eating. This behavioral effect was associated with a decreased activation (measured by mapping the expression of c-fos, an early gene widely used as a marker of cellular activation) of brain areas responding to stress (such as the nucleus accumbens and amygdala) and to a stimulation of areas involved in the control of food intake, such as the VTA and the PVN. These effects were paralleled, also, to the modulation of monoamine transmission in key brain areas involved in both homeostatic and hedonic control of eating. In particular, a decreased dopaminergic response to stress was observed by measuring dopamine extracellular concentrations in microdialysates from the nucleus accumbens shell, whereas an increased serotonergic and noradrenergic tone was detected in tissue homogenates of selected brain areas. Finally, a decrease in corticotropin-releasing factor (CRF) mRNA levels was induced by OEA in the central amygdala, while an increase in oxytocin mRNA levels was induced in the PVN. The restoration of a normal oxytocin receptor density in the striatum paralleled the oxytocinergic stimulation produced by OEA. In conclusion, we provide evidence suggesting that OEA might represent a novel potential pharmacological target for the treatment of binge-like eating behavior.

The application of non-targeted serum metabolomics profiling represents a noninvasive tool to identify new clinical biomarkers and to provide early diagnostic differentiation, and insight into the pathological mechanisms underlying hepatocellular carcinoma (HCC) progression. In this study, we used proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy and multivariate data analysis to profile the serum metabolome of 64 HCC patients, in early (n = 28) and advanced (n = 36) disease stages. We found that 1H-NMR metabolomics profiling could discriminate early from advanced HCC patients with a cross-validated accuracy close to 100%. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed significant changes in serum glucose, lactate, lipids and some amino acids, such as alanine, glutamine, 1-methylhistidine, lysine and valine levels between advanced and early HCC patients. Moreover, in early HCC patients, Kaplan&ndash, Meier analysis highlighted the serum tyrosine level as a predictor for overall survival (OS). Overall, our analysis identified a set of metabolites with possible clinical and biological implication in HCC pathophysiology.

Resveratrol, a naturally occurring phytoalexin, has long been known to play an important regulatory role in key functions in cell physiology. This multifunctional role of resveratrol is explained by its ability to interact with several targets of various cell pathways. In the recent past, synthetic chemical modifications have been made in an attempt to enhance the biological effects of resveratrol, including its anti-cancer properties. In this study, we investigated the molecular mechanisms of action of novel trans-restricted analogues of resveratrol in which the C-C double bond of the natural derivative has been replaced by diaryl-substituted imidazole analogues. In ovarian cancer models, the results of in vitro screening revealed that the resveratrol analogues exhibited enhanced anti-proliferative properties compared with resveratrol. We found that the resveratrol analogues also significantly inhibited Akt and MAPK signalling and reduced the migration of IL-6 and EGF-treated cells. Finally, in ascite-derived cancer cells, we demonstrated that the resveratrol analogues reduced the expression of epithelial mesenchymal transition (EMT) markers. Collectively, these findings indicate the enhanced anti-cancer properties of the resveratrol analogues.

The activities of lipogenic enzymes appear to fluctuate with changes in the level and type of dietary fats. Polyunsaturated fatty acids (PUFAs) are known to induce on hepatic de novo lipogenesis (DNL) the highest inhibitory effect, which occurs through a long-term adaptation. Data on the acute effects of dietary fatty acids on DNL are lacking. In this study with rats, the acute 1-day effect of high-fat (15 % w/w) diets (HFDs) enriched in saturated fatty acids (SFAs) or unsaturated fatty acids (UFAs), i.e., monounsaturated (MUFA) and PUFA, of the ω-6 and ω-3 series on DNL and plasma lipid level was investigated; a comparison with a longer time feeding (21 days) was routinely carried out. After 1-day HFD administration UFA, when compared to SFA, reduced plasma triacylglycerol (TAG) level and the activities of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), a decreased activity of the citrate carrier (CIC), a mitochondrial protein linked to lipogenesis, was also detected. In this respect, ω-3 PUFA was the most effective. On the other hand, PUFA maintained the effects at longer times, and the acute inhibition induced by MUFA feeding on DNL enzyme and CIC activities was almost nullified at 21 days. Mitochondrial fatty acid composition was slightly but significantly changed both at short- and long-term treatment, whereas the early changes in mitochondrial phospholipid composition vanished in long-term experiments. Our results suggest that in the early phase of administration, UFA coordinately reduced both the activities of de novo lipogenic enzymes and of CIC. ω-3 PUFA showed the greatest effect.

Oleoylethanolamide (OEA) is a naturally occurring bioactive lipid belonging to the family of N-acylethanolamides. A variety of beneficial effects have been attributed to OEA, although the greater interest is due to its potential role in the treatment of obesity, fatty liver, and eating-related disorders. To better clarify the mechanism of the antiadipogenic effect of OEA in the liver, using a lipidomic study performed by 1H-NMR, LC-MS/MS and thin-layer chromatography analyses we evaluated the whole lipid composition of rat liver, following a two-week daily treatment of OEA (10 mg kg&minus, 1 i.p.). We found that OEA induced a significant reduction in hepatic triacylglycerol (TAG) content and significant changes in sphingolipid composition and ceramidase activity. We associated the antiadipogenic effect of OEA to decreased activity and expression of key enzymes involved in fatty acid and TAG syntheses, such as acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase 1. Moreover, we found that both SREBP-1 and PPAR&gamma, protein expression were significantly reduced in the liver of OEA-treated rats. Our findings add significant and important insights into the molecular mechanism of OEA on hepatic adipogenesis, and suggest a possible link between the OEA-induced changes in sphingolipid metabolism and suppression of hepatic TAG level.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia associated with alterations in carbohydrate, lipid, and protein metabolism. The prognosis of T2DM patients is highly dependent on the development of complications, and therefore the identification of biomarkers of T2DM progression, with minimally invasive techniques, is a huge need. In the present study, we applied a 1H-Nuclear Magnetic Resonance (1H-NMR)-based metabolomic approach coupled with multivariate data analysis to identify serum metabolite profiles associated with T2DM development and progression. To perform this, we compared the serum metabolome of non-diabetic subjects, treatment-na&iuml, ve non-complicated T2DM patients, and T2DM patients with complications in insulin monotherapy. Our analysis revealed a significant reduction of alanine, glutamine, glutamate, leucine, lysine, methionine, tyrosine, and phenylalanine in T2DM patients with respect to non-diabetic subjects. Moreover, isoleucine, leucine, lysine, tyrosine, and valine levels distinguished complicated patients from patients without complications. Overall, the metabolic pathway analysis suggested that branched-chain amino acid (BCAA) metabolism is significantly compromised in T2DM patients with complications, while perturbation in the metabolism of gluconeogenic amino acids other than BCAAs characterizes both early and advanced T2DM stages. In conclusion, we identified a metabolic serum signature associated with T2DM stages. These data could be integrated with clinical characteristics to build a composite T2DM/complications risk score to be validated in a prospective cohort.

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International audience; Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins at the injury-site of rat sciatic nerve at 20 days after damage. By a label-free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signaling and remodeling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells at the injury site in the sciatic nerve. This further supports a specific local regulation of lipid metabolism in peripheral nerve after damage.

Chitosan nanoparticles (CS NPs) have been widely exploited for the delivery of various types of drugs due to their biocompatibility, availability, ease of functionalization and other advantages. Nevertheless, despite their wide use, their mechanism of action is not very clear and many aspects still need to be investigated in detail, with only a few studies having studied the behavior of this polymer. We prepared CS NPs encapsulating dopamine (DA) and studied the generation of reactive oxygen species (ROS) and the antioxidant effect of the neurotransmitter in detail. Encapsulation of the drug and its subsequent sustained release significantly reduced the oxidation rate in vitro, thus potentially exerting neuroprotective effects. ROS production in SH-SY5Y cells was investigated through a H2O2 assay, while a deeper study of the enzymatic activity allowed us to determine the significant contribution of both GPx and SOD enzymes in preventing oxidative stress.

Growing evidence supports the pivotal role played by oxidative stress in tissue injury development, thus resulting in several pathologies including cardiovascular, renal, neuropsychiatric, and neurodegenerative disorders, all characterized by an altered oxidative status. Reactive oxygen and nitrogen species and lipid peroxidation-derived reactive aldehydes including acrolein, malondialdehyde, and 4-hydroxy-2-nonenal, among others, are the main responsible for cellular and tissue damages occurring in redox-dependent processes. In this scenario, a link between the endocannabinoid system (ECS) and redox homeostasis impairment appears to be crucial. Anandamide and 2-arachidonoylglycerol, the best characterized endocannabinoids, are able to modulate the activity of several antioxidant enzymes through targeting the cannabinoid receptors type 1 and 2 as well as additional receptors such as the transient receptor potential vanilloid 1, the peroxisome proliferator-activated receptor alpha, and the orphan G protein-coupled receptors 18 and 55. Moreover, the endocannabinoids lipid analogues N-acylethanolamines showed to protect cell damage and death from reactive aldehydes-induced oxidative stress by restoring the intracellular oxidants-antioxidants balance. In this review, we will provide a better understanding of the main mechanisms triggered by the cross-talk between the oxidative stress and the ECS, focusing also on the enzymatic and non-enzymatic antioxidants as scavengers of reactive aldehydes and their toxic bioactive adducts.

International audience; No abstract available

l-Carnitine is an amino acid derivative widely known for its involvement in the transport of long-chain fatty acids into the mitochondrial matrix, where fatty acid oxidation occurs. Moreover, l-Carnitine protects the cell from acyl-CoA accretion through the generation of acylcarnitines. Circulating carnitine is mainly supplied by animal-based food products and to a lesser extent by endogenous biosynthesis in the liver and kidney. Human muscle contains high amounts of carnitine but it depends on the uptake of this compound from the bloodstream, due to muscle inability to synthesize carnitine. Mitochondrial fatty acid oxidation represents an important energy source for muscle metabolism particularly during physical exercise. However, especially during high-intensity exercise, this process seems to be limited by the mitochondrial availability of free l-carnitine. Hence, fatty acid oxidation rapidly declines, increasing exercise intensity from moderate to high. Considering the important role of fatty acids in muscle bioenergetics, and the limiting effect of free carnitine in fatty acid oxidation during endurance exercise, l-carnitine supplementation has been hypothesized to improve exercise performance. So far, the question of the role of l-carnitine supplementation on muscle performance has not definitively been clarified. Differences in exercise intensity, training or conditioning of the subjects, amount of l-carnitine administered, route and timing of administration relative to the exercise led to different experimental results. In this review, we will describe the role of l-carnitine in muscle energetics and the main causes that led to conflicting data on the use of l-carnitine as a supplement.

Cholestasis is one of the major causes of liver diseases. A chronic accumulation of toxic bile acids in the liver, which occurs in this condition, can induce fibrosis and cirrhosis. Inflammation is a fundamental component of acute and chronic cholestatic liver injury.Platelet-activating factor (PAF) is a proinflammatory lipid which may be generated by two independent pathways called the de novo and remodeling pathway being the last responsible for the synthesis of PAF during inflammation. In recent years a key role in PAF remodeling has been attributed to lysophosphatidylcholine acyltransferase (LPCAT) enzymes. Although the knowledge on their characteristic is growing, the exact mechanism of LPCAT in pathological conditions remains still unknown.Here, we reported that the level of lyso-PAF and PAF significantly increased in the liver of cirrhotic vs. control rats together with a significant decrease in both mRNA abundance and protein level of both LPCAT1 and LPCAT2. Acyltransferase activities of both LPCAT1 and LPCAT2 were parallel decreased in the liver of cirrhotic animals. Interestingly, treatment with silybin strongly decreased the level of both pro-inflammatory lipids and restored the activity and expression of both LPCAT1 and LPCAT2 of cirrhotic liver. Silybin effect was specific for LPCAT1 and LPCAT2 since it did not affect LPCAT3 mRNA abundance of cirrhotic liver.

4-hydroxy-2-nonenal (HNE) is considered to be a strong marker of oxidative stress; the interaction between HNE and cellular proteins leads to the formation of HNE-protein adducts able to alter cellular homeostasis and cause the development of a pathological state. By virtue of its high lipid concentration, oxygen utilization, and the presence of metal ions participating to redox reactions, the brain is highly susceptible to the formation of free radicals and HNE-related compounds. A variety of neuropsychiatric disorders have been associated with elevations of HNE concentration. For example, increased levels of HNE were found in the cortex of bipolar and schizophrenic patients, while HNE plasma concentrations resulted high in patients with major depression. On the same line, high brain concentrations of HNE were found associated with Huntington's inclusions. The incidence of high HNE levels is relevant also in the brain and cerebrospinal fluid of patients suffering from Parkinson's disease. Intriguingly, in this case the increase of HNE was associated with an accumulation of iron in the substantia nigra, a brain region highly affected by the pathology. In the present review we recapitulate the findings supporting the role of HNE in the pathogenesis of different neuropsychiatric disorders to highlight the pathogenic mechanisms ascribed to HNE accumulation. The aim of this review is to offer novel perspectives both for the understanding of etiopathogenetic mechanisms that remain still unclear and for the identification of new useful biological markers. We conclude suggesting that targeting HNE-driven cellular processes may represent a new more efficacious therapeutical intervention.

The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism.

Citrate carrier (CiC), an integral protein of the mitochondrial inner membrane, plays an important role in hepatic intermediary metabolism, supplying the cytosol with acetyl-coenzyme A for fatty acid and cholesterol synthesis. Here, the effect of streptozotocin-induced diabetes on CiC activity and expression in rat liver was investigated. The rate of citrate transport was reduced by about 35% in mitochondria from diabetic vs. control rats. Kinetic studies in mitochondria from diabetic rats showed a reduction in maximum velocity and almost unchanged Michaelis-Menten constant of the CiC protein. Mitochondrial phospholipid amount was not significantly affected, whereas an increase in the cholesterol content and in the cholesterol/phospholipid ratio was observed. To thoroughly investigate the mechanism responsible for the reduced CiC activity in the diabetic state, molecular studies were performed. Ribonuclease protection assays and Western blotting analysis indicated that both hepatic CiC mRNA accumulation and protein level decreased similarly to the CiC activity. The reduced mRNA level and the lower content of the mitochondrial CiC protein, might account for the decline of CiC activity in diabetic animals. To discriminate between the role played by hyperglycemia from that of hypoinsulinemia in the reduction of CiC activity and expression, studies were conducted administrating phlorizin or insulin to streptozotocin-diabetic rats. Our data indicated that both insulin and glucose affect CiC activity and expression in diabetic rats, although they act at different regulatory steps.

The human brain is the most cholesterol-rich organ harboring 25% of the total cholesterol pool of the whole body. Cholesterol present in the central nervous system (CNS) comes, almost entirely, from the endogenous synthesis, being circulating cholesterol unable to cross the blood-brain barrier (BBB). Astrocytes seem to be more active than neurons in this process. Neurons mostly depend on cholesterol delivery from nearby cells for axonal regeneration, neurite extension and synaptogenesis. Within the brain, cholesterol is transported by HDL-like lipoproteins associated to apoE which represents the main apolipoprotein in the CNS. Although CNS cholesterol content is largely independent of dietary intake or hepatic synthesis, a relationship between plasma cholesterol level and neurodegenerative disorders, such as Alzheimer's disease (AD), has often been reported. To this regard, alterations of cholesterol metabolism were suggested to be implicated in the etiology of AD and amyloid production in the brain. Therefore a special attention was dedicated to the study of the main factors controlling cholesterol metabolism in the brain. Brain cholesterol levels are tightly controlled: its excessive amount can be reduced through the conversion into the oxidized form of 24-S-hydroxycholesetrol (24-OH-C), which can reach the blood stream. In fact, the BBB is permeable to 24-OH-C as well as to 27-OH-C, another oxidized form of cholesterol mainly synthesized by non- neural cells. In this review, we summarize the main mechanisms regulating cholesterol homeostasis and review the recent advances on the role played by cholesterol and cholesterol oxidized products in AD. Moreover, we delineate possible pharmacological strategies to control AD progression by affecting cholesterol homeostasis.

Background Conjugated linoleic acids (CLAs) exert numerous effects in animal models as well as in humans. Among other things, CLAs decrease plasma lipid levels and bring about hepatic steatosis. The latter effects are attributed to an agonistic action of CLAs on the peroxisome-proliferator-activated receptor family primarily responsible for activating genes involved in lipid metabolism and are related to changes in mRNA levels. Such changes are not necessarily reflected in changes in activity of controlling enzymes.

Background Erythrocyte membrane modifications in patients with cholestasis are supposed to reflect those of hepatocytes. Methods Erythrocyte membrane composition (cholesterol, phospholipids, fatty acids, protein sulphydrils and carbonyls) was assessed and related to the stage of liver disease in patients with primary biliary cirrhosis before and after 1 year of ursodeoxycholate treatment. Results Compared with controls, patients showed lower levels of protein sulphydrils (28·9 ± 7·1 vs. 65·6 ± 1·8 nmol mg−1 prot) and accumulation of carbonyls (4·7 ± 1·7 vs. 1·4 ± 0·1 nmol mg−1 prot). Phosphatidylethanolamine level was lower in stage III–IV cirrhosis while phosphatidylcholine and cholesterol levels were higher; as a consequence the phosphatidylcholine/sphingomyelin ratio was higher than in controls (4·25 ± 0·55 in the I–II stage and 2·89 ± 0·44 in the stage III–IV vs. 1·61 ± 0·30). These changes were particularly evident in patients with more advanced stages of liver disease. Protein sulphydrils and carbonyls, phosphatidylethanolamine and cholesterol levels correlated (P

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4(+) T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4(+) T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

The accumulation of toxic hydrophobic bile acids in hepatocytes, observed during chronic cholestasis, induces substantial modification in the redox state and in mitochondrial functions. Recent reports have suggested a significant role of impaired lipid metabolism in the progression of chronic cholestasis. In this work we report that changes observed in the expression of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase were associated with a decrease in the activity of citrate carrier (CIC), a protein of the inner mitochondrial membrane closely related to hepatic lipogenesis. We also verified that the impairment of citrate transport was dependent on modification of the phospholipid composition of the mitochondrial membrane and on cardiolipin oxidation. Silybin, an extract of silymarin with antioxidant and anti-inflammatory properties, prevented mitochondrial reactive oxygen species (ROS) production, cardiolipin oxidation, and CIC failure in cirrhotic livers but did not affect the expression of lipogenic enzymes. Moreover, supplementation of silybin was also associated with mitochondrial biogenesis. In conclusion, we demonstrate that chronic cholestasis induces cardiolipin oxidation that in turn impairs mitochondrial function and further promotes ROS production. The capacity of silybin to limit mitochondrial failure is part of its hepatoprotective property.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only Vmax decreased, whereas Km was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.

An increased mitochondrial proton leak occurs in aging, but the origin of such modification remains unclear. This study defined the cause of mitochondrial uncoupling in mitotic (liver) and postmitotic (heart) rat tissues during aging and its effects on energy homeostasis and free radical production. Proton leak in old heart mitochondria was dependent on uncoupling proteins' upregulation, whereas it was caused by alterations in the mitochondrial membrane composition in old liver. ATP homeostasis was impaired in both tissues from old animals and was associated to disrupted F0F1-ATPase activity. H2O2 production rate and 4-hydroxy-2-nonenalprotein adducts were higher in old liver mitochondria compared with young liver mitochondria, but they were similar in heart mitochondria from both groups. Moreover, key mitochondrial biogenesis regulators were upregulated in old liver but downregulated in old heart. In conclusion, uncoupling proteins mediate proton leak and avoid oxidative damage in heart, acting as a protective mechanism. This does not occur in liver, where ATP depletion and oxidative stress may stimulate mitochondrial biogenesis and eliminate damaged cells.

The role played by oxidative stress in amiodarone-induced mitochondrial toxicity is debated. Dronedarone shows pharmacological properties similar to those of amiodarone but several differences in terms of toxicity. In this study, we analyzed the effects of the two drugs on liver mitochondrial function by administering an equivalent human dose to a rat model. Amiodarone increased mitochondrial H(2)O(2) synthesis, which in turn induced cardiolipin peroxidation. Moreover, amiodarone inhibited Complex I activity and uncoupled oxidative phosphorylation, leading to a reduction in the hepatic ATP content. We also observed a modification of membrane phospholipid composition after amiodarone administration. N-acetylcysteine completely prevented such effects. Although dronedarone shares with amiodarone the capacity to induce uncoupling of oxidative phosphorylation, it did not show any of the oxidative effects and did not impair mitochondrial bioenergetics. Our data provide important insights into the mechanism of mitochondrial toxicity induced by amiodarone. These results may greatly influence the clinical application and toxicity management of these two antiarrhythmic drugs.

There is growing evidence that mitochondrial dysfunction, and more specifically fatty acid β-oxidation impairment, is involved in the pathophysiology of non-alcoholic steatohepatitis (NASH). The goal of the present study was to achieve more understanding on the modification/s of carnitinepalmitoyltransferase-I (CPT-I), the rate-limiting enzyme of the mitochondrial fatty acid β-oxidation, during steatohepatitis. A high fat/methionine-choline deficient (MCD) diet, administered for 4 weeks, was used to induce NASH in rats.We demonstrated that CPT-I activity decreased, to the same extent, both in isolated liver mitochondria and in digitonin-permeabilized hepatocytes from MCD-diet fed rats.At the same time, the rate of total fatty acid oxidation to CO(2) and ketone bodies, measured in isolated hepatocytes, was significantly lowered in treated animals when compared to controls. Finally, an increase in CPT-I mRNA abundance and protein content, together with a high level of CPT-I protein oxidation was observed in treated rats. A posttranslational modification of rat CPT-I during steatohepatitis has been here discussed.

Mitochondrial dysfunction and oxidative stress are determinant events in the pathogenesis of nonalcoholic steatohepatitis. Silybin has shown antioxidant, anti-inflammatory, and antifibrotic effects in chronic liver disease. We aimed to study the effect of the silybin-phospholipid complex (SILIPHOS) on liver redox balance and mitochondrial function in a dietary model of nonalcoholic steatohepatitis. To accomplish this, glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H(2)O(2) production rate were evaluated in isolated liver mitochondria from rats fed a methionine- and choline-deficient (MCD) diet and the MCD diet plus SILIPHOS for 7 and 14 weeks. Oxidative proteins, hydroxynonenal (HNE)- and malondialdehyde (MDA)-protein adducts, and mitochondrial membrane lipid composition were also measured. Treatment with SILIPHOS limited glutathione depletion and mitochondrial H(2)O(2) production. Moreover, SILIPHOS preserved mitochondrial bioenergetics and prevented mitochondrial proton leak and ATP reduction. Finally, SILIPHOS limited the formation of HNE- and MDA-protein adducts. In conclusion, SILIPHOS is effective in preventing severe oxidative stress and preserving hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis induced by the MCD diet. The modifications of mitochondrial membrane fatty acid composition induced by the MCD diet are partially prevented by SILIPHOS, conferring anti-inflammatory and antifibrotic effects. The increased vulnerability of lipid membranes to oxidative damage is limited by SILIPHOS through preserved mitochondrial function.

Background Mitochondrial dysfunction is considered a key player in non-alcoholic steatohepatitis (NASH) but no data are available on the mitochondrial function and ATP homeostasis in the liver during NASH progression. In the present paper we evaluated the hepatic mitochondrial respiratory chain activity and ATP synthesis in a rodent model of NASH development. Materials and methods Male Wistar rats fed a High Fat/Methionine-Choline Deficient (MCD) diet to induce NASH or a control diet (SHAM), and sacrificed after 3, 7 and 11 weeks. The oxidative phosphorylation, the F(0)F(1)ATPase (ATP synthase) and the ATP content were assessed in liver mitochondria. Results NASH mitochondria exhibited an increased rate of substrate oxidation at 3 weeks, which returned to below the normal level at 7 and 11 weeks, concomitantly with the coupling between the phosphorylation activity and the mitochondrial respiration (ADP/O). Uncoupling of NASH liver mitochondria did not allow the recovery of the maximal respiration rate at 7 and 11 weeks. The ATPase (ATP synthase) activity was similar in NASH and SHAM rats, but the mitochondrial ATP content was significantly lower in NASH livers. Conclusions The loss of hepatic ATP stores is not dependent on the F(0)F(1)-ATPase but resides in the respiratory chain. Dysfunction of both Complex I and II of the mitochondrial respiratory chain during NASH development implies a mitochondrial adaptive mechanism occurring in the early stages of NASH.

The effect of hypothyroidism on citrate carrier (CiC) activity has been investigated in rat-liver mitochondria. The rate of citrate transport was reduced by approximately 50% in mitochondria from hypothyroid as compared with euthyroid rats. In parallel, a decrease in the rate of de novo fatty acid synthesis was observed in the cytosol of the former animals. Kinetic analysis of citrate transport revealed that only the Vmax was reduced by hypothyroidism, while Km was almost unaffected. Hypothyroidism increased the mitochondrial percentage of phosphatidylcholine while decreased that of phosphatidylethanolamine; an altered fatty acid pattern but no significant difference in the sum of saturated and unsaturated fatty acids as well as in the unsaturation index was observed. The CiC Arrhenius plot did not show appreciable difference between the two groups of rats. However, Western blot analysis associated with mRNA quantitation indicated that both protein level and mRNA accumulation of hepatic CiC were noticeably decreased in hypothyroid state. Therefore, a reduced content of the carrier protein can represent a plausible mechanism to explain the decline in the CiC activity observed in rat liver mitochondria of hypothyroid rats.

Short-term effects of 3,5-l-diiodothyronine (T2) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T3 was routinely carried out. After T2 addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-14C]acetate into these lipid fractions, was observed. The T2 dose-dependent effect on both metabolic pathways, already detectable at 10−8-10−9m, reached a 2-fold stimulation at 10−5m T2. At this concentration, the stimulatory effect was evident within 1 h of T2 addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T2 stimulation of lipogenesis was also confirmed by incubating hepatocytes with [3H]H2O, used as an independent index of lipogenic activity. The effects of T2 are rather specific as 3,3′,5,5′-tetraiodo-d-thyronine and 3,5-diiodo-l-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T2 addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T2 with those seen in the presence of T3, it appeared that T2 was able to mimic T3 effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T2 stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.

In the present study the effects of some C18fatty acids on hepatic fatty acid metabolism have been compared. Male rats were fed cholesterol-free diets containing either C18:0, C18:1cisor C18:1transisomers as the variables. In accordance with previous work, oleic acid in the diet caused an increase in cholesterol concentration in the liver and in the lipoprotein fraction of density (d; kg/l)18:1transisomers diet induced a decrease in the amount of cholesterol in total plasma as well as in the 1·019

Starvation has been associated with a reduced citrate carrier (CTP) activity in rat liver mitochondria. In the present study the molecular mechanism responsible for this reduction was investigated. Northern blot analysis performed with hepatic total RNA showed a decrease of about 40% in the CTP mRNA abundance in starved rats, when compared to fed animals. Nuclear run-on assay did not reveal any appreciable difference in the rate of CTP mRNA synthesis between the two groups of animals, while the apparent half-life of CTP mRNA in hepatocytes from fed and starved rats was 11 and 6 h, respectively. Therefore, these results suggest that in starved rats the regulation of hepatic CTP expression occurs at posttranscriptional level. Moreover, the reduced CTP activity in starved animals gradually increased by refeeding. The carrier activity reached fed rat values 6–9 h following refeeding. Interestingly, the accumulation of CTP mRNA raised in parallel with the transport activity.

After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.

The short-term effect of dexamethasone, a synthetic glucocorticoid, on both fatty acid and cholesterol synthesis has been investigated in rat hepatocyte cultures. Within 4h following hormone addition to the cultures, a noticeable stimulation of labelled acetate incorporation into fatty acids was observed. Similar behaviour was noticed when [3H]H2O was used as an independent index of the lipogenic activity. In the same cultures, however, cholesterol synthesis from both [14C]acetate or [3H]H2O was significantly reduced by dexamethasone addition. In these conditions, no significative variation of cholesterol synthesis starting from labelled mevalonate was observed.

Extra virgin olive oil (EVOO) significantly contributes to human health. Bioactive components of EVOO are oleic acid (OA) and phenolic compounds with high antioxidant activity. In primary cultures of rat hepatocytes, hydroxytyrosol (HTyr), tyrosol, and oleuropein, the main EVOO phenols, significantly reduce both de novo lipogenesis (DNL) and cholesterol synthesis by affecting the activities of acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methyl glutaryl-CoA reductase (HMGCR), key enzymes of DNL and cholesterol synthesis, respectively. The same inhibitory effect on lipid synthesis in hepatocytes was reported by EVOO extracts with high phenol content. Findings in experimental animals and humans highlight hypolipidemic and antisteatotic actions of EVOO phenols in nonalcoholic fatty liver disease, the most common chronic liver disease in western countries. Moreover, HTyr and OA, the latter representing the main fatty acid in EVOO, decrease fatty acid and cholesterol synthesis in a glioblastoma cell line model by inhibiting ACC and HMGCR activity and expression.