Alessio Menga

ATP-citrate lyase is essential for macrophage inflammatory response.

Growing evidence suggests that energy metabolism and inflammation are closely linked and that cross-talk between these processes is fundamental to the pathogenesis of many human diseases. However, the molecular mechanisms underlying these observations are still poorly understood. Here we describe the key role of ATP-citrate lyase (ACLY) in inflammation. We find that ACLY mRNA and protein levels markedly and quickly increase in activated macrophages. Importantly, ACLY activity inhibition as well as ACLY gene silencing lead to reduced nitric oxide, reactive oxygen species and prostaglandin E2 inflammatory mediators. In conclusion, we present a direct role for ACLY in macrophage inflammatory metabolism.

Elements in support of the ‘non-identity’ of the PGRMC1 protein with the σ2 receptor

σ2 Receptor subtype is overexpressed in a variety of human tumors, with σ2 agonists showing antiproliferative effects towards tumor cells through multiple pathways that depend both on the tumor cell type and on the molecule type. Therefore, σ2 receptor is an intriguing target for tumor diagnosis and treatment despite the fact that that it has not yet been cloned. One of the last attempts to characterize σ2 receptors led to identify it as the progesterone receptor membrane component 1 (PGRMC1). Although still controversial, such identity appears to have been accepted. We the aim of contributing to solve this controversy, in this work we stably silenced or overexpressed PGRMC1 protein in human MCF7 adenocarcinoma cells. Western blotting analyses were performed to quantify the presence of PGRMC1 protein on each of the three MCF7 cell lines variants, while scatchard analyses with radioligand were performed in order to determine the expression of the σ2 receptors. In order to correlate the antiproliferative effect of σ2 receptor agonist with PGRMC1 density, some σ2 ligands were administered to each of the three MCF7 cells variants. The results suggested that PGRMC1 and σ2 receptors are two different molecular entities.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

A key role of the mitochondrial citrate carrier (SLC25A1) in TNFα- and IFNγ-triggered inflammation.

The chronic induction of inflammation underlies multiple pathological conditions, including metabolic, autoimmune disorders and cancer. The mitochondrial citrate carrier (CIC), encoded by the SLC25A1 gene, promotes the export of citrate from the mitochondria to the cytoplasm, a process that profoundly influences energy balance in the cells. We have previously shown that SLC25A1 is a target gene for lipopolysaccharide signaling and promotes the production of inflammatory mediators. We now demonstrate that SLC25A1 is induced at the transcriptional level by two key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), and such induction involves the activity of the nuclear factor kappa B and STAT1 transcription factors. By studying the down-stream events following SLC25A1 activation during signals that mimic inflammation, we demonstrate that CIC is required for regulating the levels of nitric oxide and of prostaglandins by TNFα or IFNγ. Importantly, we show that the citrate exported from mitochondria via CIC and its downstream metabolic intermediate, acetyl-coenzyme A, are necessary for TNFα or IFNγ to induce nitric oxide and prostaglandin production. These findings provide the first line of evidence that the citrate export pathway, via CIC, is central for cytokine-induced inflammatory signals and shed new light on the relationship between energy metabolism and inflammation.

MEF2C exon α: Role in gene activation and differentiation

Myocyte enhancer factor 2C (MEF2C) belongs to the MEF2 transcription factors. All products of MEF2 genes have a common amino-terminal DNA binding and dimerization domain. All four vertebrate MEF2 gene transcripts are also alternatively spliced. In the present study we identify two novel MEF2C splice variants, named VP and VP2. These variants are generated by the skipping of exon α. The identified α- variants are ubiquitously expressed, although at very low levels compared to the α+ variants. The existence of MEF2C α- variants gave us the opportunity to study for the first time the function of exon α. Transactivation experiments show that the presence of exon α induces a reduction of transcription levels. Moreover, α- variants are significantly expressed during neuronal cell differentiation, indicating a putative role of these variants in development.

Insight into mechanism of in vitro insulin secretion increase induced by antipsychotic clozapine: Role of FOXA1 and mitochondrial citrate carrier

The use of clozapine and other antipsychotic drugs is known to be associated with a number of adverse metabolic side effects, including diabetes mellitus. These side effects could be, at least in part, the result of impaired islet cell function and abnormal insulin secretion, although the underlying mechanisms are unknown. The aim of this study is the identification of targets for clozapine related to the abnormal insulin secretion. We identify a specific activation of the transcriptional factor FOXA1, but not FOXA2 and FOXA3, by clozapine in HepG2 cells. Clozapine enhances FOXA1 DNA-binding and its transcriptional activity, increasing mitochondrial citrate carrier gene expression, which contains a FOXA1 site in its promoter. Haloperidol, a conventional antipsychotic drug, does not determine any increase of FOXA1 gene expression. We also demonstrate that clozapine upregulates FOXA1 and CIC gene expression in INS-1 cells only at basal glucose concentration. In addition, we find that abnormal insulin secretion in basal glucose conditions could be completely abolished by FOXA1 silencing in INS-1 cells treated with clozapine. The identification of FOXA1 as a novel target for clozapine may shed more light to understand molecular mechanism of abnormal insulin secretion during clozapine treatment.

The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

Highlights • SLC25A12 gene expression is regulated in SH-SY5Y cells through CREB.• SLC25A12 gene is up-regulated in SH-SY5Y cells under conditions of differentiation.• Ca2+ interaction with CREB further enhances SLC25A12 gene expression.• SLC25A12 gene is down-regulated in neuroinflammation.• SLC25A12 gene is up-regulated in SH-SY5Y cells by neuregulin-1.

Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels

The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS‐activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.

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.

Metabolism and TAM functions—it takes two to tango

From the evidence on clinical studies and experimental mouse models we now know that tumor‐associated macrophages (TAMs) sustain tumor development in many different ways. They play a role in angiogenesis, tumor cell invasion, and metastasis formation. Additionally, TAMs interfere with natural killer and T‐cell antitumoral activities, producing an immune‐suppressive environment that protects tumor cell growth. This indicates that the tumoricidal activity of macrophages within the tumor microenviroment is lost due to an imbalance of the regulatory mechanisms underpinning these cells’ function. Since metabolism is emerging as a major modulator of macrophage function, metabolic changes in response to signals coming from cancer or other immune cells might promote this imbalance, enhancing the tumorigenic activities of TAMs. In this review we describe the novel, most recent findings on how metabolism shapes TAM functions or conversely, how TAMs influence the activity of other cells through metabolic mechanisms. The complete elucidation of the metabolic switches between pro‐ and antitumoral properties of macrophages, now still in its infancy, is destined to provide scientists with new instruments not only to understand but also to combat cancer.