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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.

It is widely accepted that chronic stress may alter the homeostatic mechanisms of body weight control. In this study, we followed the metabolic changes occurring in mice when chronic stress caused by psychosocial defeat (CPD) is associated with ad libitum exposure to a palatable high-fat diet (HFD). In this model, CPD mice consumed more HFD than unstressed (Un) mice without gaining body weight. We focused on metabolic processes involved in weight control, such as de novo lipogenesis (DNL), fatty acid β-oxidation (FAO), and thermogenesis. The activity and expression of DNL enzymes were reduced in the liver and white adipose tissue of mice consuming the HFD. Such effects were particularly evident in stressed mice. In both CPD and Un mice, HFD consumption increased the hepatic expression of the mitochondrial FAO enzyme carnitine palmitoyltransferase-1. In the liver of mice consuming the HFD, stress exposure prevented accumulation of triacylglycerols; however, accumulation of triacylglycerols was observed in Un mice under the same dietary regimen. In brown adipose tissue, stress increased the expression of uncoupling protein-1, which is involved in energy dissipation, both in HFD and control diet-fed mice. We consider increased FAO and energy dissipation responsible for the antiobesity effect seen in CPD/HFD mice. However, CPD associated with HFD induced hepatic oxidative stress.-Giudetti, A. M., Testini, M., Vergara, D., Priore, P., Damiano, F., Gallelli, C. A., Romano, A., Villani, R., Cassano, T., Siculella, L., Gnoni, G. V., Moles, A., Coccurello, R., Gaetani, S. Chronic psychosocial defeat differently affects lipid metabolism in liver and white adipose tissue and induces hepatic oxidative stress in mice fed a high-fat diet.

Brain fatty acid (FA) metabolism deserves a close attention not only for its energetic aspects but also because FAs and their metabolites/derivatives are able to influence many neural functions, contributing to brain pathologies or representing potential targets for pharmacological and/or nutritional interventions. Glucose is the preferred energy substrate for the brain, whereas the role of FAs is more marginal. In conditions of decreased glucose supply, ketone bodies, mainly formed by FA oxidation, are the alternative main energy source. Ketogenic diets or medium-chain fatty acid supplementations were shown to produce therapeutic effects in several brain pathologies. Moreover, the positive effects exerted on brain functions by short-chain FAs and the consideration that they can be produced by intestinal flora metabolism contributed to the better understanding of the link between "gut-health" and "brain-health". Finally, attention was paid also to the regulatory role of essential polyunsaturated FAs and their derivatives on brain homeostasis.

Although H2O2 is traditionally known to have cytotoxic effects, recent studies argue about its regulatory role on lipid metabolism. However, the mechanism underlying the induction of lipid biosynthesis by oxidative stress still remains unknown. To shed light on this aspect we investigated the H2O2-induced lipogenesis in rat liver BRL-3A cells. We found that a short-term exposition of cells to 35μM H2O2 didn't cause any significant sign of cell damage measured by following diene formation and lactate dehydrogenase release from cells. However, in this stressful condition, a significant increase of [1-(14)C]acetate incorporation into fatty acids and cholesterol, associated to an increase in the activity and expression of key enzymes of fatty acid and cholesterol synthesis, were measured. mRNA and protein contents of the transcription factors SREBP-1 and SREBP-2, involved in the activation of lipid synthesis, increased as well. The analysis of molecular mechanism of SREBP-1 activation revealed, in treated compared to control cells, a higher SREBP-1a mRNA translation involving an internal ribosome entry side (IRES), present in the leader region of its mRNA. Longer exposition to the pro-oxidant induced a progressive loss of cell viability together with an increase of cell triacylglycerol content.

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.

The mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment. These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein.

The properties of fatty acid chain elongation synthesis have been investigated in liver mitochondria of the European eel (Anguilla anguilla). The incorporation of [1-(14)C]acetyl-CoA into fatty acids shows a specific activity of 0.43+/-0.05 nmol/min x mg protein (n=6), which is more than twice higher than that previously reported in rat liver mitochondria. Label incorporation into fatty acids was, in mitochondria disrupted by freezing and thawing, much higher than in intact organelles thus suggesting a probable localization of this pathway inside mitochondria. Only a negligible acetyl-CoA incorporation into fatty acids occurs in the absence of ATP, Mg2+ or reduced pyridine nucleotides; NADH alone seems to be as effective as NADH + NADPH as a hydrogen donor for the reducing steps. CoASH, without effect up to 10 microM, showed a strong inhibition at higher concentrations. From the ratio of total radioactivity and radioactivity in carboxyl carbon it can be inferred that in eel-liver mitochondria only chain elongation of preexisting fatty acids occurs. A significant fatty acid chain elongation activity is also present when, instead of acetyl-CoA, [2-(14)C]malonyl-CoA is used as a carbon unit donor. Moreover, the synthesized fatty acids were actively incorporated into phopholipids, mainly phosphatidylcholine, phosphatidylethanolamine and sphyngomyelin.

The tricarboxylate carrier was purified to homogeneity from liver mitochondria of European eel at the silver and the yellow stage and functionally reconstituted into liposomes. Unexpectedly, the molecular activity of the tricarboxylate carrier obtained from silver eel was about twofold higher than that of the same protein from yellow eel, although eels at the silver stage stop feeding. Parallel changes were found in the activities of the lipogenic enzymes in silver eels. This suggests a functional coordination between all these proteins sequentially involved in hepatic lipogenesis. Cardiolipin added to proteoliposomes strongly stimulated the activity of the purified tricarboxylate carrier from yellow eels, whereas it slightly reduced the activity of the same protein from silver eels. The higher activity of the tricarboxylate carrier from silver eels could therefore be ascribed, at least in part, to a different composition of the lipid domain surrounding the carrier protein, possibly in response to the hormonal alterations accompanying metamorphosis from yellow to silver stage.

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.

Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.

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.

The activity of the tricarboxylate (citrate) carrier has been assayed in intact liver mitochondria from yellow eel (Anguilla anguilla) and compared to that from rat. The eel-citrate carrier specific activity was approximately 1.7-fold higher than that assayed in rat-liver mitochondria. The content of the main mitochondrial phospholipids, phosphatidylethanolamine and phosphatidylcholine, did not show a significant difference between the two species, while in eel a higher cardiolipin level was observed. Fatty acid composition of eel-liver mitochondrial phospholipids was characterised by a large amount of unsaturated fatty acids, dominated by octadecaenoic acid (C(18:1) (n-9)) and docosahexaenoic acid (C(22:6) (n-3)). The cardiolipin fatty acid pattern of eel-liver mitochondria showed, with respect to the rat, a higher C(20:5) (n-3) and C(22:6) (n-3) content and a lower amount of C(18:2) (n-6) and C(20:4) (n-6). A noticeable activity of lipogenic enzymes was also detected in eel liver cytosol. The results of this study suggest that the remarkable activity of the citrate carrier in eel-liver mitochondria can most likely be ascribed to a considerable cardiolipin level. A covariance of citrate carrier and lipogenic enzyme activities was observed.

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.

The tricarboxylate carrier (TCC), an integral protein of the mitochondrial inner membrane, transports mitochondrial acetyl-CoA into the cytosol, where lipogenesis occurs. We investigated in rat liver mitochondria the effect of diets enriched with saturated fatty acids (beef tallow, BT), monounsaturated fatty acids (olive oil, OO) or n−3 polyunsaturated fatty acids (fish oil, FO), respectively, on the activity and expression of TCC. TCC activity decreased, in parallel with TCC mRNA abundance, only upon FO-feeding. The TCC transcription rate, mRNA turnover and RNA processing indicated that FO administration regulates TCC gene at transcriptional and post-transcriptional steps, whereas BT- and OO-feeding do not seem to affect either TCC activity or gene expression.