ACLY as a modulator of liver cell functions and its role in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND: Non-alcoholic Fatty Liver Disease (NAFLD), now better known as Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and its progression to Nonalcoholic Steatohepatitis (NASH), more recently referred to as Metabolic (Dysfunction)-Associated Steatohepatitis (MASH) are the most common causes of liver failure and chronic liver damage. The new names emphasize the metabolic involvement both in relation to liver function and pathological features with extrahepatic manifestations. This study aims to explore the role of the immunometabolic enzyme ATP citrate lyase (ACLY), with a critical function in lipogenesis, carbohydrate metabolism, gene expression and inflammation. METHODS: ACLY function was investigated in TNFα-triggered human hepatocytes and in PBMC-derived macrophages from MASH patients. Evaluation of expression levels was carried out by western blotting and/or RT-qPCR. In the presence or absence of ACLY inhibitors, ROS, lipid peroxidation and GSSG oxidative stress biomarkers were quantified. Chromatin immunoprecipitation (ChIP), transient transfections, immunocytochemistry, histone acetylation quantitation were used to investigate ACLY function in gene expression reprogramming. IL-6 and IL-1ß were quantified by Lumit immunoassays. RESULTS: Mechanistically, ACLY inhibition reverted lipid accumulation and oxidative damage while reduced secretion of inflammatory cytokines in TNFα-triggered human hepatocytes. These effects impacted not only on lipid metabolism but also on other crucial features of liver function such as redox status and production of inflammatory mediators. Moreover, ACLY mRNA levels together with those of malic enzyme 1 (ME1) increased in human PBMC-derived macrophages from MASH patients when compared to age-matched healthy controls. Remarkably, a combination of hydroxycitrate (HCA), the natural ACLY inhibitor, with red wine powder (RWP) significantly lowered ACLY and ME1 mRNA amount as well as IL-6 and IL-1ß production in macrophages from subjects with MASH. CONCLUSION: Collectively, our findings for the first time highlight a broad spectrum of ACLY functions in liver as well as in the pathogenesis of MASH and its diagnostic and therapeutic potential value.

New Insights into NF-κB Signaling in Innate Immunity: Focus on Immunometabolic Crosstalks.

The nuclear factor kappa B (NF-κB) is a family of transcription factors that, beyond their numberless functions in various cell processes, play a pivotal role in regulating immune cell activation. Two main pathways-canonical and non-canonical-are responsible for NF-κB activation and heterodimer translocation into the nucleus. A complex crosstalk between NF-κB signaling and metabolism is emerging in innate immunity. Metabolic enzymes and metabolites regulate NF-κB activity in many cases through post-translational modifications such as acetylation and phosphorylation. On the other hand, NF-κB affects immunometabolic pathways, including the citrate pathway, thereby building an intricate network. In this review, the emerging findings about NF-κB function in innate immunity and the interplay between NF-κB and immunometabolism have been discussed. These outcomes allow for a deeper comprehension of the molecular mechanisms underlying NF-κB function in innate immune cells. Moreover, the new insights are important in order to perceive NF-κB signaling as a potential therapeutic target for inflammatory/immune chronic diseases.

Immunometabolism Modulation by Extracts from Pistachio Stalks Formulated in Phospholipid Vesicles.

Several studies have demonstrated the effectiveness of plant extracts against various diseases, especially skin disorders; namely, they exhibit overall protective effects. The Pistachio (Pistacia vera L.) is known for having bioactive compounds that can effectively contribute to a person's healthy status. However, these benefits may be limited by the toxicity and low bioavailability often inherent in bioactive compounds. To overcome these problems, delivery systems, such as phospholipid vesicles, can be employed. In this study, an essential oil and a hydrolate were produced from P. vera stalks, which are usually discarded as waste. The extracts were characterized by liquid and gas chromatography coupled with mass spectrometry and formulated in phospholipid vesicles intended for skin application. Liposomes and transfersomes showed small size (<100 nm), negative charge (approximately -15 mV), and a longer storage stability for the latter. The entrapment efficiency was determined via the quantification of the major compounds identified in the extracts and was >80%. The immune-modulating activity of the extracts was assayed in macrophage cell cultures. Most interestingly, the formulation in transfersomes abolished the cytotoxicity of the essential oil while increasing its ability to inhibit inflammatory mediators via the immunometabolic citrate pathway.

Mitochondrial Role in Intrinsic Apoptosis Induced by a New Synthesized Chalcone in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fourth cause of cancer-related deaths worldwide. Presently, a few drugs are available for HCC treatment and prevention, including both natural and synthetic compounds. In this study, a new chalcone, (E)-1-(2,4,6-triethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (ETTC), was synthesized and its effects and mechanisms of action over human hepatoma cells were investigated. Cytotoxic activity was revealed in HCC cells, while no effects were observed in normal hepatocytes. In HCC cells, ETTC caused subG1 cell cycle arrest and apoptosis, characterized by nuclear fragmentation. The activation of caspases 3/7 and 9, the increase in pro-apoptotic BAX, and the decrease in anti-apoptotic BCL-2 suggest the activation of the intrinsic pathway of apoptosis. ETTC mitochondrial targeting is confirmed by the reduction in mitochondrial membrane potential and Complex I activity together with levels of superoxide anion increasing. Our outcomes prove the potential mitochondria-mediated antitumor effect of newly synthesized chalcone ETTC in HCC.

PPAR Alpha as a Metabolic Modulator of the Liver: Role in the Pathogenesis of Nonalcoholic Steatohepatitis (NASH).

The strong relationship between metabolic alterations and non-alcoholic steatohepatitis (NASH) suggests a pathogenic interplay. However, many aspects have not yet been fully clarified. Nowadays, NASH is becoming the main cause of liver-associated morbidity and mortality. Therefore, an effort to understand the mechanisms underlying the pathogenesis of NASH is critical. Among the nuclear receptor transcription factors, peroxisome-proliferator-activated receptor alpha (PPARα) is highly expressed in the liver, where it works as a pivotal transcriptional regulator of the intermediary metabolism. In this context, PPARα's function in regulating the lipid metabolism is essential for proper liver functioning. Here, we review metabolic liver genes under the control of PPARα and discuss how this aspect can impact the inflammatory condition and pathogenesis of NASH.

Mitochondria-Mediated Apoptosis of HCC Cells Triggered by Knockdown of Glutamate Dehydrogenase 1: Perspective for Its Inhibition through Quercetin and Permethylated Anigopreissin A.

Metabolic reprogramming is a hallmark of cancer cells required to ensure high energy needs and the maintenance of redox balance. A relevant metabolic change of cancer cell bioenergetics is the increase in glutamine metabolism. Hepatocellular carcinoma (HCC), one of the most lethal cancer and which requires the continuous development of new therapeutic strategies, shows an up-regulation of human glutamate dehydrogenase 1 (hGDH1). GDH1 function may be relevant in cancer cells (or HCC) to drive the glutamine catabolism from L-glutamate towards the synthesis of α-ketoglutarate (α-KG), thus supplying key tricarboxylic acid cycle (TCA cycle) metabolites. Here, the effects of hGLUD1 gene silencing (siGLUD1) and GDH1 inhibition were evaluated. Our results demonstrate that siGLUD1 in HepG2 cells induces a significant reduction in cell proliferation (58.8% ± 10.63%), a decrease in BCL2 expression levels, mitochondrial mass (75% ± 5.89%), mitochondrial membrane potential (30% ± 7.06%), and a significant increase in mitochondrial superoxide anion (25% ± 6.55%) compared to control/untreated cells. The inhibition strategy leads us to identify two possible inhibitors of hGDH1: quercetin and Permethylated Anigopreissin A (PAA). These findings suggest that hGDH1 could be a potential candidate target to impair the metabolic reprogramming of HCC cells.

ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation.

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell's function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol-nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including SLC25A1-which encodes the mitochondrial citrate carrier-and ACLY, thus promoting the existence of a proinflammatory loop involving SLC25A1 and ACLY genes.

Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target.

In order to meet the high energy demand, a metabolic reprogramming occurs in cancer cells. Its role is crucial in promoting tumor survival. Among the substrates in demand, oxygen is fundamental for bioenergetics. Nevertheless, tumor microenvironment is frequently characterized by low-oxygen conditions. Hypoxia-inducible factor 1 (HIF-1) is a pivotal modulator of the metabolic reprogramming which takes place in hypoxic cancer cells. In the hub of cellular bioenergetics, mitochondria are key players in regulating cellular energy. Therefore, a close crosstalk between mitochondria and HIF-1 underlies the metabolic and functional changes of cancer cells. Noteworthy, HIF-1 represents a promising target for novel cancer therapeutics. In this review, we summarize the molecular mechanisms underlying the interplay between HIF-1 and energetic metabolism, with a focus on mitochondria, of hypoxic cancer cells.

Phenolic Compounds of Red Wine Aglianico del Vulture Modulate the Functional Activity of Macrophages via Inhibition of NF-κB and the Citrate Pathway.

Phenolic compounds of red wine powder (RWP) extracted from the Italian red wine Aglianico del Vulture have been investigated for the potential immunomodulatory and anti-inflammatory capacity on human macrophages. These compounds reduce the secretion of IL-1ß, IL-6, and TNF-α proinflammatory cytokines and increase the release of IL-10 anti-inflammatory cytokine induced by lipopolysaccharide (LPS). In addition, RWP restores Annexin A1 levels, thus involving activation of proresolutive pathways. Noteworthy, RWP lowers NF-κB protein levels, promoter activity, and nuclear translocation. As a consequence of NF-κB inhibition, reduced promoter activities of SLC25A1-encoding the mitochondrial citrate carrier (CIC)-and ATP citrate lyase (ACLY) metabolic genes have been observed. CIC, ACLY, and citrate are components of the citrate pathway: in LPS-activated macrophages, the mitochondrial citrate is exported by CIC into the cytosol where it is cleaved by ACLY in oxaloacetate and acetyl-CoA, precursors for ROS, NO·, and PGE2 inflammatory mediators. We identify the citrate pathway as a RWP target in carrying out its anti-inflammatory activity since RWP reduces CIC and ACLY protein levels, ACLY enzymatic activity, the cytosolic citrate concentration, and in turn ROS, NO·, PGE2, and histone acetylation levels. Overall findings suggest that RWP potentially restores macrophage homeostasis by suppressing inflammatory pathways and activating proresolutive processes.

Pistacia lentiscus Hydrosol: Untargeted Metabolomic Analysis and Anti-Inflammatory Activity Mediated by NF-κB and the Citrate Pathway.

Pistacia lentiscus shows a long range of biological activities, and it has been used in traditional medicine for treatment of various kinds of diseases. Moreover, related essential oil keeps important health-promoting properties. However, less is known about P. lentiscus hydrosol, a main by-product of essential oil production, usually used for steam distillation itself or discarded. In this work, by using ultra-high-resolution ESI(+)-FT-ICR mass spectrometry, a direct identification of four main classes of metabolites of P. lentiscus hydrosol (i.e., terpenes, amino acids, peptides, and condensed heterocycles) was obtained. Remarkably, P. lentiscus hydrosol exhibited an anti-inflammatory activity by suppressing the secretion of IL-1ß, IL-6, and TNF-α proinflammatory cytokines in lipopolysaccharide- (LPS-) activated primary human monocytes. In LPS-triggered U937 cells, it inhibited NF-κB, a key transcription factor in inflammatory cascade, regulating the expression of both the mitochondrial citrate carrier and the ATP citrate lyase genes. These two main components of the citrate pathway were downregulated by P. lentiscus hydrosol. Therefore, the levels of ROS, NO, and PGE2, the inflammatory mediators downstream the citrate pathway, were reduced. Results shed light on metabolic profile and anti-inflammatory properties of P. lentiscus hydrosol, suggesting its potential as a therapeutic agent.

Liposome-Mediated Inhibition of Inflammation by Hydroxycitrate.

Hydroxycitrate (HCA), a main organic acid component of the fruit rind of Garcinia cambogia, is a natural citrate analog that can inhibit the ATP citrate lyase (ACLY) enzyme with a consequent reduction of inflammatory mediators (i.e., nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2)) levels. Therefore, HCA has been proposed as a novel means to prevent, treat, and ameliorate conditions involving inflammation. However, HCA presents a low membrane permeability, and a large quantity is required to have a biological effect. To overcome this problem, HCA was formulated in liposomes in this work, and the enhancement of HCA cell availability along with the reduction in the amount required to downregulate NO, ROS, and PGE2 in macrophages were assessed. The liposomes were small in size (~60 nm), monodispersed, negatively charged (-50 mV), and stable on storage. The in vitro results showed that the liposomal encapsulation increased by approximately 4 times the intracellular accumulation of HCA in macrophages, and reduced by 10 times the amount of HCA required to abolish LPS-induced NO, ROS, and PGE2 increase. This suggests that liposomal HCA can be exploited to target the citrate pathway involved in inflammatory processes.

New synthesized polyoxygenated diarylheptanoids suppress lipopolysaccharide-induced neuroinflammation.

In neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease, Parkinson's disease and multiple sclerosis, neuroinflammation induced by the microglial activation plays a crucial role. In effort to develop effective anti-neuroinflammatory compounds, different new linear polyoxygenated diarylheptanoids were synthesized. In LPS-triggered BV-2 microglial cells their ability to reduce the concentration of IL-6 and TNF-α pro-inflammatory cytokines was evaluated. Moreover, their effect on NF-κB and ATP citrate lyase (ACLY), a recently emerged target of metabolic reprogramming in inflammation, was assessed. Finally, we turned our attention to inflammatory mediators derived from the cleavage of citrate catalyzed by ACLY: prostaglandin E2, nitric oxide and reactive oxygen species. All compounds showed null or minimal cytotoxicity; most of them had a great anti-neuroinflammatory activity. Diarylheptanoids 6b and 6c, bearing a halide atom and benzyl ether protective groups, exhibited the best effect since they blocked the secretion of all inflammatory mediators analyzed and reduced NF-κB and ACLY protein levels.

TCA Cycle Rewiring as Emerging Metabolic Signature of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy. Despite progress in treatment, HCC is still one of the most lethal cancers. Therefore, deepening molecular mechanisms underlying HCC pathogenesis and development is required to uncover new therapeutic strategies. Metabolic reprogramming is emerging as a critical player in promoting tumor survival and proliferation to sustain increased metabolic needs of cancer cells. Among the metabolic pathways, the tricarboxylic acid (TCA) cycle is a primary route for bioenergetic, biosynthetic, and redox balance requirements of cells. In recent years, a large amount of evidence has highlighted the relevance of the TCA cycle rewiring in a variety of cancers. Indeed, aberrant gene expression of several key enzymes and changes in levels of critical metabolites have been observed in many solid human tumors. In this review, we summarize the role of the TCA cycle rewiring in HCC by reporting gene expression and activity dysregulation of enzymes relating not only to the TCA cycle but also to glutamine metabolism, malate/aspartate, and citrate/pyruvate shuttles. Regarding the transcriptional regulation, we focus on the link between NF-κB-HIF1 transcriptional factors and TCA cycle reprogramming. Finally, the potential of metabolic targets for new HCC treatments has been explored.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

Cystic Hygroma: A Preliminary Genetic Study and a Short Review from the Literature.

BACKGROUND: The objective of this study is to examine the hypothesis that cystic hygroma (CH) with normal karyotype can manifest as a Mendelian inherited trait, and that a genetic similitude with hereditary lymphedema exists. To reach this goal, we investigated the prevalence of genetic variants in angiogenesis and lymphangiogenesis genes in a cohort of euploid fetuses with CH that almost resolved before delivery. A short review of cases from literature is also reported. METHODS AND RESULTS: Five fetuses were screened using a next-generation sequencing approach by targeting 33 genes known to be associated with vascular and lymphatic malformations. The genetic evaluation revealed two novel variants in KDR and KRIT1 genes. CONCLUSION: A review of the literature to date revealed that an association exists between CH and hereditary lymphedema and, similar to lymphedema, CH can be inherited in autosomal recessive and autosomal dominant manner, with the latter most likely associated with a better prognosis. About KDR and KRIT1 genes, no other similar associations are reported in the literature and caution is needed in their interpretation. In conclusion, we thought that a genetic test for the outcome of familial CH could be of enormous prognostic value.

Epigenetic upregulation and functional role of the mitochondrial aspartate/glutamate carrier isoform 1 in hepatocellular carcinoma.

Metabolic reprogramming is a common hallmark of cancer cells. Although some biochemical features have been clarified, there is still much to learn about cancer cell metabolism and its regulation. Aspartate-glutamate carrier isoform 1 (AGC1), encoded by SLC25A12 gene, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate plus a proton across the mitochondrial membrane, so supplying aspartate to the cytosol. SLC25A12, expressed in brain, heart, and skeletal muscle, is silenced in normal liver. Here, we demonstrate that SLC25A12 gene is reactivated in hepatocellular carcinoma (HCC) HepG2 cell line through histone acetylation and CREB recruitment. Furthermore, SLC25A12 knockdown by small interfering RNA, impairs HepG2 cell proliferation by inducing cell cycle arrest. AGC1 sustains HCC cell growth by supplying cytosolic aspartate for nucleotide biosynthesis. In addition, SLC25A12-silenced HCC cells show a strong reduction of cell migration. Overall, we have provided evidence for molecular mechanisms controlling SLC25A12 gene expression in liver and pointing to an important role for AGC1 in HCC.

New Insights into Behçet's Syndrome Metabolic Reprogramming: Citrate Pathway Dysregulation.

To date, a major research effort on Behçet's syndrome (BS) has been concentrated on immunological aspects. Little is known about the metabolic reprogramming in BS. Citrate is an intermediary metabolite synthesized in mitochondria, and when transported into the cytosol by the mitochondrial citrate carrier-SLC25A1-encoded protein-it is cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase (ACLY). In induced macrophages, mitochondrial citrate is necessary for the production of inflammatory mediators. The aim of our study was to evaluate SLC25A1 and ACLY expression levels in BS patients. Following a power analysis undertaken on few random samples, the number of enrolled patients was set. Thirty-nine consecutive BS patients fulfilling ISG criteria, and 21 healthy controls suitable for age and sex were recruited. BS patients were divided into two groups according to the presence (active) or absence (inactive) of clinical manifestations. Real-time PCR experiments were performed on PBMCs to quantify SLC25A1 and ACLY mRNA levels. Data processing through the Kruskal-Wallis test and Dunn's multiple comparison test as post hoc showed higher SLC25A1 and ACLY mRNA levels in BS patients compared to those in healthy controls. Therefore, SLC25A1 and ACLY upregulation suggests that metabolic reprogramming in BS involves the citrate pathway dysregulation.

New diphenylmethane derivatives as peroxisome proliferator-activated receptor alpha/gamma dual agonists endowed with anti-proliferative effects and mitochondrial activity.

We screened a short series of new chiral diphenylmethane derivatives and identified potent dual PPARα/γ partial agonists. As both enantiomers of the most active compound 1 displayed an unexpected similar transactivation activity, we performed docking experiments to provide a molecular understanding of their similar partial agonism. We also evaluated the ability of both enantiomers of 1 and racemic 2 to inhibit colorectal cancer cells proliferation: (S)-1 displayed a more robust activity due, at least in part, to a partial inhibition of the Wnt/ß-catenin signalling pathway that is upregulated in the majority of colorectal cancers. Finally, we investigated the effects of (R)-1, (S)-1 and (R,S)-2 on mitochondrial function and demonstrated that they activate the carnitine shuttle system through upregulation of carnitine/acylcarnitine carrier (CAC) and carnitine-palmitoyl-transferase 1 (CPT1) genes. Consistent with the notion that these are PPARα target genes, we tested and found that PPARα itself is regulated by a positive loop. Moreover, these compounds induced a significant mitochondrial biogenesis. In conclusion, we identified a new series of dual PPARα/γ agonists endowed with novel anti-proliferative properties associated with a strong activation of mitochondrial functions and biogenesis, a potential therapeutic target of the treatment of insulin resistance.

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines.

Significant metabolic changes occur in the shift from resting to activated cellular status in inflammation. Thus, changes in expression of a large number of genes and extensive metabolic reprogramming gives rise to acquisition of new functions (e.g. production of cytokines, intermediates for biosynthesis, lipid mediators, PGE, ROS and NO). In this context, mitochondrial carriers, which catalyse the transport of solute across mitochondrial membrane, change their expression to transport mitochondrially produced molecules, among which citrate and succinate, to be used as intracellular signalling molecules in inflammation. This review summarises the mitochondrial carriers studied so far that are, directly or indirectly, involved in inflammation.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.