Publications by authors named "Fabrizio Damiano"

41 Publications

IgM and IgG Profiles Reveal Peculiar Features of Humoral Immunity Response to SARS-CoV-2 Infection.

Int J Environ Res Public Health 2021 02 1;18(3). Epub 2021 Feb 1.

Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

The emergence of coronavirus disease 2019 (COVID-19) is globally a major healthcare threat. There is little information regarding the mechanisms and roles of the humoral response in SARS-CoV-2 infection. The aim of this study was to analyze the antibody levels (IgM and IgG) by chemiluminescence immunoassay in 54 subjects positive to SARS-CoV-2 swab test in relation to their clinical status (whether asymptomatic, pauci-symptomatic or with mild, sever or critical symptoms), the time from the symptom onset, sex, age, and comorbidities. Overall, the presence of comorbidities and the age of subjects were associated with their clinical status. The IgG concentrations were significantly higher in patients who developed critical and severe symptoms and seemed to be independent from age, sex and comorbidities. IgG titers peaked around day 60, and then began gradually to drop, decreasing by approximately 50% on the 180th day, while the IgM titers progressively decreased as early as the tenth day, but they could be detected even at later time points. Despite the small number of individuals, some peculiar characteristics of the humoral response in COVID-19 emerged. We observed a high inter-individual variability, an ephemeral IgG half-life in several patients, and a persistence of IgM in others.
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http://dx.doi.org/10.3390/ijerph18031318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908175PMC
February 2021

Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells.

Biology (Basel) 2020 Oct 30;9(11). Epub 2020 Oct 30.

Laboratory of Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.
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http://dx.doi.org/10.3390/biology9110370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693660PMC
October 2020

Decanoic Acid and Not Octanoic Acid Stimulates Fatty Acid Synthesis in U87MG Glioblastoma Cells: A Metabolomics Study.

Front Neurosci 2020 23;14:783. Epub 2020 Jul 23.

Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.

Medium-chain fatty acids (MCFA) are dietary components with a chain length ranging from 6 to 12 carbon atoms. MCFA can cross the blood-brain barrier and in the brain can be oxidized through mitochondrial β-oxidation. As components of ketogenic diets, MCFA have demonstrated beneficial effects on different brain diseases, such as traumatic brain injury, Alzheimer's disease, drug-resistant epilepsy, diabetes, and cancer. Despite the interest in MCFA effects, not much information is available about MCFA metabolism in the brain. In this study, with a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach, coupled with multivariate data analyses, we followed the metabolic changes of U87MG glioblastoma cells after the addition of octanoic (C8), or decanoic (C10) acids for 24 h. Our analysis highlighted significant differences in the metabolism of U87MG cells after the addition of C8 or C10 and identified several metabolites whose amount changed between the two groups of treated cells. Overall, metabolic pathway analyses suggested the citric acid cycle, Warburg effect, glutamine/glutamate metabolism, and ketone body metabolism as pathways influenced by C8 or C10 addition to U87MG cells. Our data demonstrated that, while C8 affected mitochondrial metabolism resulting in increased ketone body production, C10 mainly influenced cytosolic pathways by stimulating fatty acid synthesis. Moreover, glutamine might be the main substrate to support fatty acids synthesis in C10-treated cells. In conclusion, we identified a metabolic signature associated with C8 or C10 addition to U87MG cells that can be used to decipher metabolic responses of glioblastoma cells to MCFA treatment.
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http://dx.doi.org/10.3389/fnins.2020.00783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390945PMC
July 2020

In Steatotic Cells, ATP-Citrate Lyase mRNA Is Efficiently Translated through a Cap-Independent Mechanism, Contributing to the Stimulation of De Novo Lipogenesis.

Int J Mol Sci 2020 Feb 11;21(4). Epub 2020 Feb 11.

Laboratory of Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease in which excessive amount of lipids is accumulated as droplets in hepatocytes. Recently, cumulative evidences suggested that a sustained de novo lipogenesis can play an important role in NAFLD. Dysregulated expression of lipogenic genes, including ATP-citrate lyase (ACLY), has been found in liver diseases associated with lipid accumulation. ACLY is a ubiquitous cytosolic enzyme positioned at the intersection of nutrients catabolism and cholesterol and fatty acid biosyntheses. In the present study, the molecular mechanism of ACLY expression in a cell model of steatosis has been reported. We identified an internal ribosome entry site (IRES) in the 5' untranslated region of the ACLY mRNA, that can support an efficient mRNA translation through a Cap-independent mechanism. In steatotic HepG2 cells, ACLY expression was up-regulated through IRES-mediated translation. Since it has been demonstrated that lipid accumulation in cells induces endoplasmic reticulum (ER) stress, the involvement of this cellular pathway in the translational regulation of ACLY has been also evaluated. Our results showed that ACLY expression was increased in ER-stressed cells, through IRES-mediated translation of ACLY mRNA. A potential role of the Cap-independent translation of ACLY in NAFLD has been discussed.
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http://dx.doi.org/10.3390/ijms21041206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072811PMC
February 2020

Quercetin inhibition of SREBPs and ChREBP expression results in reduced cholesterol and fatty acid synthesis in C6 glioma cells.

Int J Biochem Cell Biol 2019 12 19;117:105618. Epub 2019 Sep 19.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari "Aldo Moro", 70124, Bari, Italy.

Quercetin (Que), a widely distributed flavonoid in the human diet, exerts neuroprotective action because of its property to antagonize oxidative stress. Here, we investigated the effects of Que on lipid synthesis in C6 glioma cells. A rapid Que-induced inhibition of cholesterol and, to a lesser extent, of fatty acid synthesis from [1-C]acetate was observed. The maximum decrease was detected at the level of palmitate, the end product of de novo fatty acid synthesis. The effect of Que on the enzyme activities of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), the two enzymes of this pathway, was investigated directly in situ in permeabilized C6 cells. An inhibitory effect on ACC1 was observed after 4 h of 25 μM Que treatment, while FAS activity was not affected. A reduction of polar lipid biosynthesis was also detected. A remarkable decrease of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) activity, regulatory enzyme of cholesterol synthesis, was evidenced. Expression studies demonstrated that Que acts at transcriptional level, by reducing the mRNA abundance and protein amount of ACC1 and HMGCR. Deepening the molecular mechanism, we found that Que decreased the expression of SREBP-1 and SREBP-2, transcriptional factors representing the main regulators of de novo fatty acid and cholesterol synthesis, respectively. Que also reduced the nuclear content of ChREBP, a glucose-induced transcription factor involved in the regulation of lipogenic genes. Our results represent the first evidence that a direct and rapid downregulatory effect of Que on cholesterol and de novo fatty acid synthesis is elicited in C6 cells.
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http://dx.doi.org/10.1016/j.biocel.2019.105618DOI Listing
December 2019

3,5-diiodo-L-thyronine increases de novo lipogenesis in liver from hypothyroid rats by SREBP-1 and ChREBP-mediated transcriptional mechanisms.

IUBMB Life 2019 07 1;71(7):863-872. Epub 2019 Feb 1.

Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100, Lecce, Italy.

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.
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http://dx.doi.org/10.1002/iub.2014DOI Listing
July 2019

Stimulatory Effects of Methyl-β-cyclodextrin on Spiramycin Production and Physical-Chemical Characterization of Nonhost@Guest Complexes.

ACS Omega 2018 Mar 1;3(3):2470-2478. Epub 2018 Mar 1.

Department of Biological and Environmental Sciences and Technologies, Department of Engineering for Innovation, and Department of Cultural Heritage, University of Salento, Via Monteroni, 73100 Lecce, Italy.

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 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.
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http://dx.doi.org/10.1021/acsomega.7b01766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130790PMC
March 2018

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.

FASEB J 2019 01 22;33(1):1428-1439. Epub 2018 Aug 22.

Department of Physiology and Pharmacology V. Erspamer, Sapienza University of Rome, Rome, Italy.

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.
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http://dx.doi.org/10.1096/fj.201801130RDOI Listing
January 2019

Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces.

Metab Eng 2018 07 23;48:254-268. Epub 2018 Jun 23.

Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. Electronic address:

Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production. By using integrated transcriptomic, proteomic and metabolite profiling, together with genetic complementation, we here show that most of these effects could be traced to the inability of the pirA-defective strain to modulate beta-oxidation pathway, leading to an unbalanced supply of precursor monomers for polyketide biosynthesis. Indeed, in silico protein-protein interaction modeling and in vitro experimental validation allowed us to demonstrate that PirA is a novel redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway.
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http://dx.doi.org/10.1016/j.ymben.2018.06.008DOI Listing
July 2018

Hydroxytyrosol Ameliorates Endothelial Function under Inflammatory Conditions by Preventing Mitochondrial Dysfunction.

Oxid Med Cell Longev 2018 18;2018:9086947. Epub 2018 Apr 18.

National Research Council-Institute of Clinical Physiology, Lecce, Italy.

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 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 mol/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 ATP5 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.
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http://dx.doi.org/10.1155/2018/9086947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932486PMC
October 2018

Oleic Acid and Hydroxytyrosol Inhibit Cholesterol and Fatty Acid Synthesis in C6 Glioma Cells.

Oxid Med Cell Longev 2017 24;2017:9076052. Epub 2017 Dec 24.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy.

Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA) and hydroxytyrosol (HTyr), two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS) and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR) were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 M OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.
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http://dx.doi.org/10.1155/2017/9076052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757140PMC
August 2018

Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Apr 16;1863(4):388-398. Epub 2018 Jan 16.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. Electronic address:

Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed.
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http://dx.doi.org/10.1016/j.bbalip.2018.01.006DOI Listing
April 2018

Time-Resolved Transcriptomics and Constraint-Based Modeling Identify System-Level Metabolic Features and Overexpression Targets to Increase Spiramycin Production in .

Front Microbiol 2017 12;8:835. Epub 2017 May 12.

Institute of Biomedical Technologies, National Research CouncilSegrate, Italy.

In this study we have applied an integrated system biology approach to characterize the metabolic landscape of and to identify a list of potential metabolic engineering targets for the overproduction of the secondary metabolites in this microorganism. We focused on an often overlooked growth period (i.e., post-first rapid growth phase) and, by integrating constraint-based metabolic modeling with time resolved RNA-seq data, we depicted the main effects of changes in gene expression on the overall metabolic reprogramming occurring in . Moreover, through metabolic modeling, we unraveled a set of candidate overexpression gene targets hypothetically leading to spiramycin overproduction. Model predictions were experimentally validated by genetic manipulation of the recently described ethylmalonyl-CoA metabolic node, providing evidence that spiramycin productivity may be increased by enhancing the carbon flow through this pathway. The goal was achieved by over-expressing the paralog in an engineered plasmid. This work embeds the first metabolic reconstruction of and the successful experimental validation of model predictions and demonstrates the validity and the importance of modeling tools for the overproduction of molecules with a biotechnological interest. Finally, the proposed metabolic reconstruction, which includes manually refined pathways for several secondary metabolites with antimicrobial activity, represents a solid platform for the future exploitation of biotechnological potential.
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http://dx.doi.org/10.3389/fmicb.2017.00835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427115PMC
May 2017

Encapsulation of Lactobacillus kefiri in alginate microbeads using a double novel aerosol technique.

Mater Sci Eng C Mater Biol Appl 2017 Aug 3;77:548-555. Epub 2017 Apr 3.

University of Salento, Department of Engineering for Innovation, Campus Ecotekne, Via Monteroni, Lecce, Italy.

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 CaCl 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 50-70μm size, encapsulating L. kefiri as active ingredient. The technique involves the use of a double aerosols of alginate based solution and CaCl 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 pH1.2 (fasted state) and pH2.2 (feed state).
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http://dx.doi.org/10.1016/j.msec.2017.04.010DOI Listing
August 2017

Action of Thyroid Hormones, T3 and T2, on Hepatic Fatty Acids: Differences in Metabolic Effects and Molecular Mechanisms.

Int J Mol Sci 2017 Mar 31;18(4). Epub 2017 Mar 31.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

The thyroid hormones (THs) 3,3',5,5'-tetraiodo-l-thyronine (T4) and 3,5,3'-triiodo-l-thyronine (T3) influence many metabolic pathways. The major physiological function of THs is to sustain basal energy expenditure, by acting primarily on carbohydrate and lipid catabolism. Beyond the mobilization and degradation of lipids, at the hepatic level THs stimulate the de novo fatty acid synthesis (de novo lipogenesis, DNL), through both the modulation of gene expression and the rapid activation of cell signalling pathways. 3,5-Diiodo-l-thyronine (T2), previously considered only a T3 catabolite, has been shown to mimic some of T3 effects on lipid catabolism. However, T2 action is more rapid than that of T3, and seems to be independent of protein synthesis. An inhibitory effect on DNL has been documented for T2. Here, we give an overview of the mechanisms of THs action on liver fatty acid metabolism, focusing on the different effects exerted by T2 and T3 on the regulation of the DNL. The inhibitory action on DNL exerted by T2 makes this compound a potential and attractive drug for the treatment of some metabolic diseases and cancer.
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http://dx.doi.org/10.3390/ijms18040744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412329PMC
March 2017

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria.

J Bioenerg Biomembr 2016 10 17;48(5):521-529. Epub 2016 Nov 17.

Center of Integrated Research, Campus Bio-Medico, University of Rome, Rome, 00100, Italy.

The role of 3,5-diiodo-L-thyronine (T), initially considered only a 3,3',5-triiodo-L-thyronine (T) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining HO release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T administration decreases mitochondrial oxidative stress as determined by lower HO production. We conclude that in rat liver mitochondria T acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T to reduce adiposity, dyslipidemia and to prevent liver steatosis.
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http://dx.doi.org/10.1007/s10863-016-9686-4DOI Listing
October 2016

Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia.

J Biol Chem 2016 09 30;291(38):19746-59. Epub 2016 Jul 30.

From the Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia.
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http://dx.doi.org/10.1074/jbc.M116.736876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025666PMC
September 2016

Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism.

Int J Mol Sci 2016 May 25;17(6). Epub 2016 May 25.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce 73100, Italy.

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.
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http://dx.doi.org/10.3390/ijms17060817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926351PMC
May 2016

Lipid accumulation stimulates the cap-independent translation of SREBP-1a mRNA by promoting hnRNP A1 binding to its 5'-UTR in a cellular model of hepatic steatosis.

Biochim Biophys Acta 2016 May 8;1861(5):471-81. Epub 2016 Feb 8.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.

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.
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http://dx.doi.org/10.1016/j.bbalip.2016.02.003DOI Listing
May 2016

Modulation of hepatic lipid metabolism by olive oil and its phenols in nonalcoholic fatty liver disease.

IUBMB Life 2015 Jan 28;67(1):9-17. Epub 2015 Jan 28.

Department of Biological and Environmental Sciences and Technologies, Laboratory of Biochemistry and Molecular Biology, University of Salento, Lecce, Italy.

Nonalcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in western countries, being considered the hepatic manifestation of metabolic syndrome. Cumulative lines of evidence suggest that olive oil, used as primary source of fat by Mediterranean populations, may play a key role in the observed health benefits on NAFLD. In this review, we summarize the state of the art of the knowledge on the protective role of both major and minor components of olive oil on lipid metabolism during NAFLD. In particular, the biochemical mechanisms responsible for the increase or decrease in hepatic lipid content are critically analyzed, taking into account that several studies have often provided different and/or conflicting results in animal models fed on olive oil-enriched diet. In addition, new findings that highlight the hypolipidemic and the antisteatotic actions of olive oil phenols are presented. As mitochondrial dysfunction plays a key role in the pathogenesis of NAFLD, the targeting of these organelles with olive oil phenols as a powerful therapeutic approach is also discussed.
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http://dx.doi.org/10.1002/iub.1340DOI Listing
January 2015

Expression of citrate carrier gene is activated by ER stress effectors XBP1 and ATF6α, binding to an UPRE in its promoter.

Biochim Biophys Acta 2015 Jan 27;1849(1):23-31. Epub 2014 Oct 27.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov. le Lecce-Monteroni, Lecce 73100, Italy.

The Unfolded Protein Response (UPR) is an intracellular signaling pathway which is activated when unfolded or misfolded proteins accumulate in the Endoplasmic Reticulum (ER), a condition commonly referred to as ER stress. It has been shown that lipid biosynthesis is increased in ER-stressed cells. The N(ε)-lysine acetylation of ER-resident proteins, including chaperones and enzymes involved in the post-translational protein modification and folding, occurs upon UPR activation. In both ER proteins acetylation and lipid synthesis, acetyl-CoA is the donor of acetyl group and it is transported from the cytosol into the ER. The cytosolic pool of acetyl-CoA is mainly derived from the activity of mitochondrial citrate carrier (CiC). Here, we have demonstrated that expression of CiC is activated in human HepG2 and rat BRL-3A cells during tunicamycin-induced ER stress. This occurs through the involvement of an ER stress responsive region identified within the human and rat CiC proximal promoter. A functional Unfolded Protein Response Element (UPRE) confers responsiveness to the promoter activation by UPR transducers ATF6α and XBP1. Overall, our data demonstrate that CiC expression is activated during ER stress through the binding of ATF6α and XBP1 to an UPRE element located in the proximal promoter of Cic gene. The role of ER stress-mediated induction of CiC expression has been discussed.
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http://dx.doi.org/10.1016/j.bbagrm.2014.10.004DOI Listing
January 2015

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.

Metab Eng 2014 Nov 19;26:1-16. Epub 2014 Aug 19.

Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce 73100, Italy. Electronic address:

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.
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http://dx.doi.org/10.1016/j.ymben.2014.08.001DOI Listing
November 2014

Differential effects of high-carbohydrate and high-fat diets on hepatic lipogenesis in rats.

Eur J Nutr 2014 Jun 7;53(4):1103-14. Epub 2013 Nov 7.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Provinciale Lecce-Monteroni, 73100, Lecce, Italy.

Purpose: Hepatic fatty acid synthesis is influenced by several nutritional and hormonal factors. In this study, we have investigated the effects of distinct experimental diets enriched in carbohydrate or in fat on hepatic lipogenesis.

Methods: Male Wistar rats were divided into four groups and fed distinct experimental diets enriched in carbohydrates (70% w/w) or in fat (20 and 35% w/w). Activity and expression of the mitochondrial citrate carrier and of the cytosolic enzymes acetyl-CoA carboxylase and fatty acid synthetase were analyzed through the study with assessments at 0, 1, 2, 4, and 6 weeks. Liver lipids and plasma levels of lipids, glucose, and insulin were assayed in parallel.

Results: Whereas the high-carbohydrate diet moderately stimulated hepatic lipogenesis, a strong inhibition of this anabolic pathway was found in animals fed high-fat diets. This inhibition was time-dependent and concentration-dependent. Moreover, whereas the high-carbohydrate diet induced an increase in plasma triglycerides, the high-fat diets determined an accumulation of triglycerides in liver. An increase in the plasmatic levels of glucose and insulin was observed in all cases.

Conclusions: The excess of sucrose in the diet is converted into fat that is distributed by bloodstream in the organism in the form of circulating triglycerides. On the other hand, a high amount of dietary fat caused a strong inhibition of lipogenesis and a concomitant increase in the level of hepatic lipids, thereby highlighting, in these conditions, the role of liver as a reservoir of exogenous fat.
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http://dx.doi.org/10.1007/s00394-013-0613-8DOI Listing
June 2014

3,5-Diiodo-l-thyronine induces SREBP-1 proteolytic cleavage block and apoptosis in human hepatoma (Hepg2) cells.

Biochim Biophys Acta 2013 Dec 13;1831(12):1679-89. Epub 2013 Aug 13.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Science and Technologies, University of Salento, Via Prov. le Lecce-Monteroni, Lecce 73100, Italy.

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 12h 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.
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http://dx.doi.org/10.1016/j.bbalip.2013.08.003DOI Listing
December 2013

Low level of hydrogen peroxide induces lipid synthesis in BRL-3A cells through a CAP-independent SREBP-1a activation.

Int J Biochem Cell Biol 2013 Jul 10;45(7):1419-26. Epub 2013 Apr 10.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy.

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.
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http://dx.doi.org/10.1016/j.biocel.2013.04.004DOI Listing
July 2013

hnRNP A1 mediates the activation of the IRES-dependent SREBP-1a mRNA translation in response to endoplasmic reticulum stress.

Biochem J 2013 Jan;449(2):543-53

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov. le Lecce-Monteroni, Lecce 73100, Italy.

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.
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http://dx.doi.org/10.1042/BJ20120906DOI Listing
January 2013

Citrate carrier promoter is target of peroxisome proliferator-activated receptor alpha and gamma in hepatocytes and adipocytes.

Int J Biochem Cell Biol 2012 Apr 10;44(4):659-68. Epub 2012 Jan 10.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Science and Technologies, University of Salento, Via Prov.le Lecce-Monteroni, Lecce 73100, Italy.

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 PPARγin vivo was confirmed by ChIP assay in BRL-3A and 3T3-L1 cells, respectively.
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http://dx.doi.org/10.1016/j.biocel.2012.01.003DOI Listing
April 2012

3,5,3'triiodo-L-thyronine induces SREBP-1 expression by non-genomic actions in human HEP G2 cells.

J Cell Physiol 2012 Jun;227(6):2388-97

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov.le Lecce-Monteroni, Lecce, Italy.

Liver is an important target for thyroid hormone actions. T(3) exerts its effects by two mechanisms: (i) Genomic actions consisting of T(3) link to nuclear receptors that bind responsive elements in the promoter of target genes, (ii) non-genomic actions including integrin αvb3 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 T(3) determined a dose- and time-dependent increase in the level of the precursor form of SREBP-1 without affecting SREBP-1 mRNA abundance. T(3) also induced phosphorylation of ERK1/2, Akt and of mTOR-C1 target S6K-P70, and the cytosol-to-membrane translocation of PKC-α. Modulation of SREBP-1 protein level by T(3) 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 T(3) . Conversely, the effect of T(3) 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 T(3) on SREBP-1 content increased also by using PKC inhibitors. These inhibitors increased the action of T(3) on Akt phosphorylation suggesting that conventional PKCs may work as negative regulators of the T(3) -dependent SREBP-1 increase. T(3) effects were partially abrogated by tetrac, an inhibitor of the T(3) -αvβ3 receptor interaction and partially evoked by T(3) analog T(3) -agarose. These findings support a model in which T(3) activates intracellular signaling pathways which may be involved in the increment of SREBP-1 level through an IRES-mediated translation mechanism.
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http://dx.doi.org/10.1002/jcp.22974DOI Listing
June 2012

Streptozotocin-induced diabetes affects in rat liver citrate carrier gene expression by transcriptional and posttranscriptional mechanisms.

Int J Biochem Cell Biol 2011 Nov 27;43(11):1621-9. Epub 2011 Jul 27.

Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Prov. Lecce-Monteroni, 73100 Lecce, Italy.

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.
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http://dx.doi.org/10.1016/j.biocel.2011.07.011DOI Listing
November 2011

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a 'rare' actinomycete.

Mol Microbiol 2010 Aug 1;77(3):716-29. Epub 2010 Jun 1.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Monteroni, 73100 Lecce, Italy.

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)-encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild-type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster-specific dpgA mRNA were stabilized during the idiophase in the wild-type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse-labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07240.xDOI Listing
August 2010