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We investigated the extent to which adherence to the Mediterranean diet (MD) in combination with Mediterranean lifestyle factors influenced students’ perceptions of subjective well-being (SWB) and distress. 939 undergraduates completed a survey to assess sociodemographic and lifestyle characteristics, including adherence to the MD, depression, anxiety, stress, and SWB. Data were analysed with correlation, logistic, and multiple linear regression models. Higher adherence to MD correlated with better SWB. Fruit, red meat, sweet and caffeinated beverages contributed significantly. However, it was the combination of adherence to MD with other factors, including quality of social relationships, income, smoking, sleep, and physical activity that better predicted SWB. Our results confirm the positive influence of MD on SWB. However, they also suggest the need to consider perceptions of well-being by a more holistic approach that considers physical and social factors simultaneously to improve the development of more effective educational and motivational programmes.

Time spent outdoors and physical activity (PA) promote mental health. To confirm this relationship in the aftermath of COVID-19 lockdowns, we explored individual levels of anxiety, depression, stress and subjective well-being (SWB) in a cohort of academic students and staff members and tested their association with sport practice, PA at leisure time and time spent outdoors. Our cross-sectional study collected data during the COVID-19 outbreak (April–May 2021) on 939 students and on 238 employees, who completed an online survey on sociodemographic and lifestyle features, depression, anxiety, stress, and SWB. Results showed that the students exhibited higher levels of anxiety, depression, and stress, and lower levels of SWB (p < 0.001 for all domains) compared to the staff members. Correlation analysis confirmed that PA and time spent in nature were associated to high mental health scores among staff and, more consistently, among students. Finally, mediation analyses indicated that the time spent in nature, social relationships, and levels of energy play a mediator role in the relationship between sport practice and SWB. Our evidence reinforces the protective role of time spent in nature in improving mental health, and provides support for policymakers to make appropriate choices for a better management of COVID-19 pandemic consequences, P.P. was funded by Life Quality Research Centre—UIDP/04748/2020, a program financially supported by FCT—Fundação para a Ciência e Tecnolo-gia/Ministério da Educação e Ciência.

Simple Summary: The aim of this study was to identify new and innovative strategies to improve the tissue-regeneration process. Concentrated growth factor (CGF) is an autologous biomaterial rich in growth factors and multipotent stem cells. The purpose of our study was to evaluate the osteogenic differentiation of CGF primary cells in the presence of a hydroxyapatite–silicon scaffold, which represents a very interesting material in the field of bone reconstructive surgery. Our findings showed that the hydroxyapatite–silicon scaffold provided support to primary CGF cells by enhancing osteogenic differentiation. These data suggest interesting perspectives in the use of CGF together with scaffolds in the field of regenerative medicine. Abstract: The application of scaffolding materials together with stem cell technologies plays a key role in tissue regeneration. Therefore, in this study, CGF (concentrated growth factor), which represents an autologous and biocompatible blood-derived product rich in growth factors and multipotent stem cells, was used together with a hydroxyapatite and silicon (HA-Si) scaffold, which represents a very interesting material in the field of bone reconstructive surgery. The aim of this work was to evaluate the potential osteogenic differentiation of CGF primary cells induced by HASi scaffolds. The cellular viability of CGF primary cells cultured on HA-Si scaffolds and their structural characterization were performed by MTT assay and SEM analysis, respectively. Moreover, the matrix mineralization of CGF primary cells on the HA-Si scaffold was evaluated through Alizarin red staining. The expression of osteogenic differentiation markers was investigated through mRNA quantification by real-time PCR. We found that the HA-Si scaffold was not cytotoxic for CGF primary cells, allowing their growth and proliferation. Furthermore, the HASi scaffold was able to induce increased levels of osteogenic markers, decreased levels of stemness markers in these cells, and the formation of a mineralized matrix. In conclusion, our results suggest that HA-Si scaffolds can be used as a biomaterial support for CGF application in the field of tissue regeneration.

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.

Spiramycin is a macrolide antibiotic and antiparasitic that is used to treat toxoplasmosis and various other infections of soft tissues. In the current study, we evaluated the effects of α-cyclodextrin, β-cyclodextrin, or methyl-β-cyclodextrin supplementation to a synthetic culture medium on biomass and spiramycin production by Streptomyces ambofaciens ATCC 23877. We found a high stimulatory effect on spiramycin production when the culture medium was supplemented with 0.5% (w/v) methyl-β-cyclodextrin, whereas α-cyclodextrin or β-cyclodextrin weakly enhanced antibiotic yields. As the stimulation of antibiotic production could be because of spiramycin complexation with cyclodextrins with effects on antibiotic stability and/or efflux, we analyzed the possible formation of complexes by physical–chemical methods. The results of Job plot experiment highlighted the formation of a nonhost@guest complex methyl-β-cyclodextrin@spiramycin I in the stoichiometric ratio of 3:1 while they excluded the formation of complex between spiramycin I and α- or β-cyclodextrin. Fourier-transform infrared spectroscopy measurements were then carried out to characterize the methyl-β-cyclodextrin@spiramycin I complex and individuate the chemical groups involved in the binding mechanism. These findings may help to improve the spiramycin fermentation process, providing at the same time a new device for better delivery of the antibiotic at the site of infection by methyl-β-cyclodextrin complexation, as it has been well-documented for other bioactive molecules.

Mitochondria are fundamental organelles producing energy and reactive oxygen species (ROS); their impaired functions play a key role in endothelial dysfunction. Hydroxytyrosol (HT), a well-known olive oil antioxidant, exerts health benefits against vascular diseases by improving endothelial function. However, the HT role in mitochondrial oxidative stress in endothelial dysfunction is not clear yet. To investigate the HT effects on mitochondrial ROS production in the inflamed endothelium, we used an in vitro model of endothelial dysfunction represented by cultured endothelial cells, challenged with phorbol myristate acetate (PMA), an inflammatory, prooxidant, and proangiogenic agent. We found that the pretreatment of endothelial cells with HT (1–30 μmol/L) suppressed inflammatory angiogenesis, a crucial aspect of endothelial dysfunction. The HT inhibitory effect is related to reduced mitochondrial superoxide production and lipid peroxidation and to increased superoxide dismutase activity. HT, in a concentration-dependent manner, improved endothelial mitochondrial function by reverting the PMA-induced reduction of mitochondrial membrane potential, ATP synthesis, and ATP5β expression. In PMA-challenged endothelial cells, HT also promoted mitochondrial biogenesis through increased mitochondrial DNA content and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, nuclear respiratory factor-1, and mitochondrial transcription factor A. These results highlight that HT blunts endothelial dysfunction and pathological angiogenesis by ameliorating mitochondrial function, thus suggesting HT as a potential mitochondria-targeting antioxidant in the inflamed endothelium.

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.

A growing amount of evidence suggests the involvement of ER (endoplasmic reticulum) stress in lipid metabolism and in the development of some liver diseases such as steatosis. The transcription factor SREBP-1 (sterol-regulatory-element-binding protein 1) modulates the expression of several enzymes involved in lipid synthesis. Previously, we showed that ER stress increased the SREBP-1a protein level in HepG2 cells, by inducing a cap-independent translation of SREBP-1a mRNA, through an IRES (internal ribosome entry site), located in its leader region. In the present paper, we report that the hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) interacts with 5′-UTR (untranslated region) of SREBP-1a mRNA, as an ITAF (IRES trans-acting factor), regulating SREBP-1a expression in HepG2 cells and in primary rat hepatocytes. Overexpression of hnRNP A1 in HepG2 cells and in rat hepatocytes increased both the SREBP-1a IRES activity and SREBP-1a protein level. Knockdown of hnRNP A1 by small interfering RNA reduced either the SREBP-1a IRES activity or SREBP-1a protein level. hnRNP A1 mediates the increase of SREBP-1a protein level and SREBP-1a IRES activity in Hep G2 cells and in rat hepatocytes upon tunicamycin- and thapsigargin-induced ER stress. The induced ER stress triggered the cytosolic relocation of hnRNP A1 and caused the increase in hnRNP A1 bound to the SREBP-1a 5′-UTR. These data indicate that hnRNP A1 participates in the IRES-dependent translation of SREBP-1a mRNA through RNA–protein interaction. A different content of hnRNP A1 was found in the nuclei from high-fat-diet-fed mice liver compared with standard-diet-fed mice liver, suggesting an involvement of ER stress-mediated hnRNP A1 subcellular redistribution on the onset of metabolic disorders.

Alginate micro beads containing Lactobacillus kefiri (the principal bacteria present in the kefir probiotic drink) were produced by a novel technique based on dual aerosols spaying of alginate based solution and CaCl2 as cross linking agent. Carboxymethylcellulose (CMC) has been also added to the alginate in order to change the physic-chemical properties (viscosity and permeability) of the microbeads. Calcium alginate and CMC are biopolymers that can be used for developing oral drug-delivery systems. These biopolymers have been reported to show a pH-dependent swelling behaviour. Calcium alginate and CMC have also been known to possess an excellent mucoadhesive property. The loaded microbeads have been characterized in terms of morphology, chemical composition and stability in different conditions mimicking the gastric environment. In this study, we demonstrate the feasibility of a continuous fabrication of alginate microbeads in a range of 50–70 μm size, encapsulating L. kefiri as active ingredient. The technique involves the use of a double aerosols of alginate based solution and CaCl2 as crosslinking agent. Moreover, the encapsulation process was proved to be effective and not detrimental to bacteria viability. At the same time, it was verified the protective efficacy of the microcapsules against the gastric environment using both SGF pH 1.2 (fasted state) and pH 2.2 (feed state).

Citrate carrier (CiC), a mitochondrial inner membrane protein, is an essential component of the shuttle system which transports acetyl-CoA from mitochondria to the cytosol where lipogenesis occurs. CiC is regulated by SREBP-1, a transcription factor that controls the expression of several lipogenic genes. CiC is also implicated in cholesterol synthesis, glycolysis and gluconeogenesis, suggesting that besides SREBP-1 other transcription factors could modulate the expression of its gene. Here, we provide evidences demonstrating that CiC expression is regulated by peroxisome proliferator-activated receptor (PPAR) alpha and gamma in hepatocytes and adipocytes, respectively. CiC expression increased in rat BRL-3A hepatocytes treated with WY-14,643, agonist of PPARα, and in murine 3T3-L1 adipocytes treated with rosiglitazone, agonist of PPARγ. The overexpression of PPARα/RXRα and PPARγ/RXRα heterodimer enhanced CiC promoter activity in BRL-3A and 3T3-L1, respectively. Luciferase reporter gene and gel mobility shift assays indicated that a functional peroxisome proliferator-activated receptor response element (PPRE), identified in the CiC promoter, conferred responsiveness to activation by PPARs. The binding of PPRE of CiC promoter by PPARα and PPARγ in vivo was confirmed by ChIP assay in BRL-3A and 3T3-L1 cells, respectively.

Liver is an important target for thyroid hormone actions. T3 exerts its effects by two mechanisms: (i) Genomic actions consisting of T3 link to nuclear receptors that bind responsive elements in the promoter of target genes, (ii) non-genomic actions including integrin avb3 receptor-mediated MAPK/ERK and PI3K/Akt/mTOR-C1 activation. SREBP-1a, SREBP-1c, and SREBP-2 are transcription factors involved in the regulation of lipogenic genes. We show in Hep G2 cells that T3 determined a dose- and time-dependent increase in the level of the precursor form of SREBP-1 without affecting SREBP-1 mRNA abundance. T3 also induced phosphorylation of ERK1/2, Akt and of mTORC1 target S6K-P70, and the cytosol-to-membrane translocation of PKC-a. Modulation of SREBP-1 protein level by T3 was dependent on MAPK/ERK, PI3K/Akt/mTOR-C1 pathway activation since the MEK inhibitor PD98059 or the PI3K inhibitor LY294002 abolished the stimulatory effect of T3. Conversely, the effect of T3 on SREBP-1 level was enhanced by using rapamycin, mTOR-C1 inhibitor. These data suggest a negative control of mTOR-C1 target S6K-P70 on PI3K/Akt pathway. The effect of T3 on SREBP-1 content increased also by using PKC inhibitors. These inhibitors increased the action of T3 on Akt phosphorylation suggesting that conventional PKCs may work as negative regulators of the T3-dependent SREBP-1 increase. T3 effects were partially abrogated by tetrac, an inhibitor of the T3-avb3 receptor interaction and partially evoked by T3 analog T3-agarose. These findings support a model in which T3 activates intracellular signaling pathways which may be involved in the increment of SREBP-1 level through an IRES-mediated translation mechanism. J. Cell. Physiol. 227: 2388-2397, 2012.

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 citrate carrier (CiC), a nuclear-encoded protein located in the mitochondrial inner membrane, is a member of the mitochondrial carrier family. CiC plays an important role in hepatic lipogenesis, which is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid and cholesterol synthesis. In addition, CiC is a key component of the isocitrate-oxoglutarate and the citrate-malate shuttles. CiC has been purified from various species and its reconstituted function characterized as well as its cDNA isolated and sequenced. CiC mRNA and/or CiC protein levels are high in liver, pancreas, and kidney, but are low or absent in brain, heart, skeletal muscle, placenta, and lungs. A reduction of CiC activity was found in diabetic, hypothyroid, starved rats, and in rats fed on a polyunsaturated fatty acid (PUFA)-enriched diet. Molecular analysis suggested that the regulation of CiC activity occurs mainly through transcriptional and post-transcriptional mechanisms. This review begins with an assessment of the current understanding of CiC structural and biochemical characteristics, underlying the structure-function relationship. Emphasis will be placed on the molecular basis of the regulation of CiC activity in coordination with fatty acid synthesis.

Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-(14)C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced ( approximately 80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease characterized by accumulation of lipid droplets in hepatocytes. Enhanced release of non-esterified fatty acids from adipose tissue accounts for a remarkable fraction of accumulated lipids. However, the de novo lipogenesis (DNL) is also implicated in the etiology of the NAFLD. Sterol Regulatory Element-Binding Protein-1 (SREBP-1) is a transcription factor modulating the expression of several lipogenic enzymes. In the present study, in order to investigate the effect of lipid droplet accumulation on DNL, we used a cellular model of steatosis represented by HepG2 cells cultured in a medium supplemented with free oleic and palmitic fatty acids (FFAs). We report that FFA supplementation induces the expression of genes coding for enzymes involved in the DNL as well as for the transcription factor SREBP-1a. The SREBP-1a mRNA translation, dependent on an internal ribosome entry site (IRES), and the SREBP-1a proteolytic cleavage are activated by FFAs. Furthermore, FFA treatment enhances the expression and the nucleus-cytosolic shuttling of hnRNP A1, a trans-activating factor of SREBP-1a IRES. The binding of hnRNP A1 to the SREBP-1a IRES is also increased upon FFA supplementation. The relocation of hnRNP A1 and the consequent increase of SREBP-1a translation are dependent on the p38 MAPK signal pathway, which is activated by FFAs. By RNA interference approach, we demonstrate that hnRNP A1 is implicated in the FFA-induced expression of SREBP-1a and of its target genes as well as in the lipid accumulation in cells.

The tricarboxylate (citrate) carrier (TCC), a protein of the mitochondrial inner membrane, is an obligatory component of the shuttle system by which mitochondrial acetyl-CoA is transported into the cytosol, where lipogenesis occurs. The aim of this study was to investigate the molecular basis for the regulation of TCC gene expression by a high-fat, n-6 PUFA-enriched diet. Rats received for up to 4 weeks a diet enriched with 15% safflower oil (SO), which is high in linoleic acid (70.4%). We found a gradual decrease of TCC activity and a parallel decline in the abundance of TCC mRNA, the maximum effect occurring after 4 weeks of treatment. At this time, the estimated half-life of TCC mRNA was the same in the hepatocytes from rats on both diets, whereas the transcriptional rate of TCC mRNA, tested by nuclear run-on assay, was reduced by approximately 38% in the rats on the SO-enriched diet. The RNase protection assay showed that the ratio of mature to precursor RNA, measured in the nuclei, decreased with the change to the n-6 PUFA diet. These results suggest that administration of n-6 PUFAs to rats leads to changes not only in the transcriptional rate of the TCC gene but also in the processing of the nuclear precursor for TCC RNA.

Objective—Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial–leukocyte adhesion molecule expression and monocyte adhesion.Methods and Results—Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-κB, activator protein-1, and possibly GATA binding sites in mediating VCAM-1 transcriptional inhibition. The involvement of nuclear factor-κB and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays.Conclusions—Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

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.

Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (−25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (−49%) = oleuropein (−48%) > tyrosol (−30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, −41%, = oleuropein, −38%, > tyrosol, −17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

Thyroid hormone 3,5,3′-triiodo- l -thyronine (T3) is known to affect cell metabolism through both the genomic and non-genomic actions. Recently, we demonstrated in HepG2 cells that T3 controls the expression of SREBP-1, a transcription factor involved in the regulation of lipogenic genes. This occurs by activation of a cap-independent translation mechanism of its mRNA. Such a process is dependent on non-genomic activation of both MAPK/ERK and PI3K/Akt pathways. The physiological role of 3,5-diiodo- l -thyronine (T2), previously considered only as a T3 catabolite, is of growing interest. Evidences have been reported that T2 rapidly affects some metabolic pathways through non-genomic mechanisms. Here, we show that T2, unlike T3, determines the block of proteolytic cleavage of SREBP-1 in HepG2 cells, without affecting its expression at the transcriptional or translational level. Consequently, Fatty Acid Synthase expression is reduced. T2 effects depend on the concurrent activation of MAPKs ERK and p38, of Akt and PKC-δ pathways. Upon the activation of these signals, apoptosis of HepG2 cells seems to occur, starting at 12 h of T2 treatment. PKC-δ appears to act as a switch between p38 activation and Akt suppression, suggesting that this PKC may function as a controller in the balance of pro-apoptotic (p38) and anti-apoptotic (Akt) signals in HepG2 cells.

The tricarboxylate carrier of the inner membrane of eel liver mitochondria has been solubilized with Triton X-100 and partially purified by chromatography of the mitochondrial extract on dry hydroxyapatite. The purified fraction has been reconstituted into liposomes and functionally analyzed. The reconstituted carrier protein catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate uptake in liposomes. The substrate specificity and the inhibitor sensitivity of the tricarboxylate carrier are similar to those previously determined for the same protein of mammalian mitochondria.

The carnitine/acylcarnitine translocase (CACT), an integral protein of the mitochondrial inner membrane, belongs to the carnitine-dependent system of fatty acid transport into mitochondria, where beta-oxidation occurs. CACT exchanges cytosolic acylcarnitine or free carnitine for carnitine in the mitochondrial matrix. The object of this study was to investigate in rat liver the effect, if any, of diets enriched with saturated fatty acids (beef tallow, BT, the control), n-3 polyunsaturated fatty acids (PUFA) (fish oil, FO), n-6 PUFA (safflower oil, SO), and mono-unsaturated fatty acids (MUFA) (olive oil, OO) on the activity and expression of CACT. Translocase exchange rates increased, in parallel with CACT mRNA abundance, upon FO-feeding, whereas OO-dietary treatment induced a decrease in both CACT activity and expression. No changes were observed upon SO-feeding. Nuclear run-on assay revealed that FO-treatment increased the transcriptional rate of CACT mRNA. On the other hand, only in the nuclei of hepatocytes from OO-fed rats splicing of the last intron of CACT pre-mRNA and the rate of formation of the 3'-end were affected. Overall, these findings suggest that compared to the BT-enriched diet, the SO-enriched diet did not influence CACT activity and expression, whereas FO- and OO-feeding alters CACT activity in an opposite fashion, i.e. modulating its expression at transcriptional and post-transcriptional levels, respectively.

Citrate carrier (CiC), also known as tricarboxylate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. Here, we report the effect of streptozotocin-induced diabetes on the activity and expression of CiC in rat liver mitochondria. A significant reduction of CiC activity and a parallel decline in the abundance of CiC mRNA were found in liver from diabetic rats. Diabetes did not influence CiC mRNA stability, whereas nuclear run-on assay revealed that the transcriptional rate of CiC mRNA decreased, when compared to control, in the nuclei from diabetic rats. The ratio of mature to precursor CiC RNA decreased in diabetic animals, indicating that the splicing of CiC RNA was also affected. The 3′-end processing rate of CiC mRNA was not altered in diabetes. These results suggest that diabetes affects CiC expression at both transcriptional and posttranscriptional levels. In addition, by in vitro transfection experiments in rat hepatocytes cultured in the absence of insulin, a reduction of CiC promoter activity was observed, and this was ascribed to a decreased expression of sterol regulatory element-binding protein-1 transcriptional factor. Furthermore, the binding of sterol regulatory element-binding protein-1 to the CiC promoter was reduced in STZ-diabetic rats with respect to control ones, and it was restored to the control values after insulin treatment.

SREBPs (sterol-regulatory-element-binding proteins) are a family of transcription factors that modulate the expression of several enzymes implicated in endogenous cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In the present study, evidence for SREBP-1 regulation at the translational level is reported. Using several experimental approaches, we have demonstrated that the 5′-UTR (untranslated region) of the SREBP-1a mRNA contains an IRES (internal ribosome entry site). Transfection experiments with the SREBP-1a 5′-UTR inserted in a dicistronic reporter vector showed a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. Insertion of the SREBP-1c 5′-UTR in the same vector also stimulated the translation of the downstream cistron, but the observed effect can be ascribed, at least in part, to a cryptic promoter activity. Cellular stress conditions, such as serum starvation, caused an increase in the level of SREBP-1 precursor and mature form in both Hep G2 and HeLa cells, despite the overall reduction in protein synthesis, whereas mRNA levels for SREBP-1 were unaffected by serum starvation. Transfection experiments carried out with a dicistronic construct demonstrated that the cap-dependent translation was affected more than IRES-mediated translation by serum starvation. The thapsigargin- and tunicamycin-induced UPR (unfolded protein response) also increased SREBP-1 expression in Hep G2 cells, through the cap-independent translation mediated by IRES. Overall, these findings indicate that the presence of IRES in the SREBP-1a 5′-UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.

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.

The divergent transcription of two tRNA genes encoded in sunflower mitochondrial DNA, proposed as genes of different genetic origin, has been studied in detail. The transcription initiation site (TIS) for both transcript precursors has been identified by hybridization with in vitro 32 P-capped total RNAs and primer extension. The location of two TISs and the analysis of distribution of sequence elements (motifs) usually present in higher plant mitochondrial promoters led to the identification of two short regions (about 30–40 bp) which can be proposed as the promoters for the transcription of two genes. This conclusion is supported by the observation that within the short intergenic region included between the 5′ termini of two genes (1924 bp) the distribution of those specific motifs is unique around the TISs, although not identical for the two promoters. Based on specific experimental results the trnE promoter shows a higher efficiency in comparison with that of the trnH promoter. This result is in good agreement with its structure which strictly conforms to those described for mitochondrial genes of dicot plants. Instead the other promoter shows some divergences which could be responsible for its lower efficiency. The context in which trnH lies in the sunflower mitochondrial genome and other features described in the paper may suggest that, despite the high similarity with the chloroplast counterpart, the trnH gene could have a native origin.

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.

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 tricarboxylate carrier from eel (Anguilla anguilla) liver mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B. On SDS-polyacrylamide gel electrophoresis, the purified fraction showed a single polypeptide band with an apparent molecular mass of 30.4 kDa. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a very active 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. It was purified 641-fold with a recovery of 13.3% and a protein yield of 0.02% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate carrier purified from rat liver mitochondria. These studies provide the first information on the mitochondrial tricarboxylate transport protein of a fish.

Dialyzed rat liver cytosol possesses the ability to stimulate the incorporation of [35S]-methionine into mitochondrial proteins. The addition of dialyzed cytosol stimulates sevenfold the rate of protein synthesis by isolated mitochondria after 10 minutes incubation and the extent of this stimulation increases with the time. Various control experiments indicate that the effect is not artifactual nor does it appear to be brought about by GDP or GTP.

In higher plants the gene for cytochrome b (CYB gene) has been sequenced on mt DNA of maize (1) and wheat (2) (monocotyledonous plants) and oenothera (3) (dicotyledonous). The gene is continuous and codes for a protein of 388, 394, and 398 amino acids respectively.

Abstract —Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1α, IL-1β, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 μmol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 μg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-κB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment.