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IntroductionDuring thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called “browning” and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis.MethodsWild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the β-adrenergic receptor agonist CL316,243.ResultsTrpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation.DiscussionOur data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

Sirtuin 6 (SIRT6) has a critical role in cutaneous Squamous Cell Carcinoma (cSCC): SIRT6 silencing in skin SCC cells has pro-differentiating effects and SIRT6 deletion abrogated DMBA-TPA-induced skin tumorigenesis in mice. On the other hand, SIRT6 acts as tumor suppressor in SCC by enhancing glycolysis in tumor propagating cells. Herein, pharmacological modulation of SIRT6 deacetylase activity was investigated in cSCC, with S6 (inhibitor) or MDL-800 (activator). In cSCC cells, S6 recreated the pro-differentiating effects of SIRT6 silencing, as the levels of Keratin 1, Keratin 10 and Loricrin were upregulated compared to controls. Next, the effects of SIRT6 pharmacological modulation were evaluated in a DMBA-TPA-induced skin cancer mouse model. Mice treated with the inhibitor S6 in a preventive approach, i.e. at the beginning of the promotion stage, presented reduced number and size of papillomas, compared to the controls. The epidermal hyperproliferation marker Keratin 6 and the cSCC marker Keratin 8 were less abundant when SIRT6 was inhibited. In S6-treated lesions, the Epithelial-Mesenchymal Transition (EMT) markers Zeb1 and Vimentin were less expressed compared to untreated lesions. In a therapeutic approach, i.e. treatment starting after papilloma appearance, the S6 group presented reduced papillomas (number and size), whereas MDL-800-treated mice displayed an opposite trend. In S6-treated lesions, Keratin 6 and Keratin 8 were less expressed, EMT was less advanced, with a higher E-cadherin/Vimentin ratio, indicating a delayed carcinogenesis when SIRT6 was inhibited. Our results confirm that SIRT6 plays a role in skin carcinogenesis and suggest SIRT6 pharmacological inhibition as a promising strategy in cSCC.

Sirtuin 6 (SIRT6) is a member of the mammalian NAD+-dependent deac(et)ylase sirtuin family. SIRT6’s anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD+ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca2+ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD+ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD+ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT “browning” were enhanced in Cd38−/− mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38−/− mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38−/− mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38−/− animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38−/− mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38−/− mice, which might contribute to enhanced BAT activation in Cd38−/− mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

The binding of SARS-CoV-2 spikes to the cell receptor angiotensin-converting enzyme 2 (ACE2) is a crucial target both in the prevention and in the therapy of COVID-19. We explored the involvement of oxidoreductive mechanisms by investigating the effects of oxidants and antioxidants on virus uptake by ACE2-expressing cells of human origin (ACE2-HEK293). The cell uptake of pseudoviruses carrying the envelope of either Delta or Omicron variants of SARS-CoV-2 was evaluated by means of a cytofluorimetric approach. The thiol N-acetyl-L-cysteine (NAC) inhibited the uptake of both variants in a reproducible and dose-dependent fashion. Ascorbic acid showed modest effects. In contrast, neither hydrogen peroxide (H2O2) nor a system-generating reactive oxygen species (ROS), which play an important role in the intracellular alterations produced by SARS-CoV-2, were able to affect the ability of either Delta or Omicron SARS-CoV-2 pseudoviruses to be internalized into ACE2-expressing cells. In addition, neither H2O2 nor the ROS generating system interfered with the ability of NAC to inhibit that mechanism. Moreover, based on previous studies, a preventive pharmacological approach with NAC would have the advantage of decreasing the risk of developing COVID-19, irrespective of its variants, and at the same time other respiratory viral infections and associated comorbidities.

Since 2020, the COVID-19 pandemic represented an important worldwide burden. Well-structured surveillance by reliable and timely genomic data collection is crucial. In this study, a genomic monitoring analysis of all SARS-CoV-2 positive samples retrieved at the Fondazione Policlinico Universitario Campus Bio-Medico, in Rome, Italy, between December 2021 and June 2022, was performed. Two hundred and seventy-four SARS-CoV-2-positive samples were submitted to viral genomic sequencing by Illumina MiSeqII. Consensus sequences were generated by de novo assembling using the iVar tool and deposited on the GISAID database. Lineage assignment was performed using the Pangolin lineage classification. Sequences were aligned using ViralMSA and maximum-likelihood phylogenetic analysis was performed by IQ-TREE2. TreeTime tool was used to obtain dated trees. Our genomic monitoring revealed that starting from December 2021, all Omicron sub-lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) were circulating, although BA.1 was still the one with the highest prevalence thought time in this early period. Phylogeny revealed that Omicron isolates were scattered throughout the trees, suggesting multiple independent viral introductions following national and international human mobility. This data represents a sort of thermometer of what happened from July 2021 to June 2022 in Italy. Genomic monitoring of the circulating variants should be encouraged considering that SARS-CoV-2 variants or sub-variants emerged stochastically and unexpectedly.

ADP-ribosyl cyclases (ADPRCs) catalyze the synthesis of the Ca2+-active second messengers Cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from NAD+ as well as nicotinic acid adenine dinucleotide phosphate (NAADP+) from NADP+. The best characterized ADPRC in mammals is CD38, a single-pass transmembrane protein with two opposite membrane orientations. The first identified form, type II CD38, is a glycosylated ectoenzyme, while type III CD38 has its active site in the cytosol. The ectoenzymatic nature of type II CD38 raised long ago the question of a topological paradox concerning the access of the intracellular NAD+ substrate to the extracellular active site and of extracellular cADPR product to its intracellular receptors, ryanodine (RyR) channels. Two different transporters, equilibrative connexin 43 (Cx43) hemichannels for NAD+ and concentrative nucleoside transporters (CNTs) for cADPR, proved to mediate cell-autonomous trafficking of both nucleotides. Here, we discussed how type II CD38, Cx43 and CNTs also play a role in mediating several paracrine processes where an ADPRC+ cell supplies a neighboring CNT-and RyR-expressing cell with cADPR. Recently, type II CD38 was shown to start an ectoenzymatic sequence of reactions from NAD+/ADPR to the strong immunosuppressant adenosine; this paracrine effect represents a major mechanism of acquired resistance of several tumors to immune checkpoint therapy.

Nicotinamide adenine dinucleotide (NAD+) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD+ degrading enzymes. Among these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca2+-mobilizing second messenger, starting from NAD+, and as the major NAD+-consumer, to be inhibited to increase NAD+ levels. Indeed, the regulation of NAD+ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.

Adriana Albini,1,* Arianna Pagani,2,3,* Laura Pulze,2 Antonino Bruno,3 Elisa Principi,3 Terenzio Congiu,4 Elisabetta Gini,2,4 Annalisa Grimaldi,2 Barbara Bassani,2,3 Silvio De Flora,5 Magda de Eguileor,2 Douglas M Noonan2,3 1Laboratory of Translational Research, IRCCS Arcispedale Santa Maria Nuova, Reggio Emilia, 2Department of Biotechnologies and Life Sciences, University of Insubria, Varese, 3Scientific and Technology Park, IRCCS MultiMedica, Milano, 4Department of Surgical and Morphological Sciences, University of Insubria, Varese, 5Department of Health Sciences, University of Genoa, Genoa, Italy *These authors contributed equally to this work Abstract: Carbon nanotubes (CNTs) have been extensively investigated and employed for industrial use because of their peculiar physical properties, which make them ideal for many industrial applications. However, rapid growth of CNT employment raises concerns about the potential risks and toxicities for public health, environment, and workers associated with the manufacture and use of these new materials. Here we investigate the main routes of entry following environmental exposure to multi-wall CNTs (MWCNTs; currently the most widely used in industry). We developed a novel murine model that could represent a surrogate of a workplace exposure to MWCNTs. We traced the localization of MWCNTs and their possible role in inducing an innate immune response, inflammation, macrophage recruitment, and inflammatory conditions. Following environmental exposure of CD1 mice, we observed that MWCNTs rapidly enter and disseminate in the organism, initially accumulating in lungs and brain and later reaching the liver and kidney via the bloodstream. Since recent experimental studies show that CNTs are associated with the aggregation process of proteins associated with neurodegenerative diseases, we investigated whether MWCNTs are able to induce amyloid fibril production and accumulation. Amyloid deposits in spatial association with macrophages and MWCNT aggregates were found in the brain, liver, lungs, and kidneys of exposed animals. Our data suggest that accumulation of MWCNTs in different organs is associated with inflammation and amyloid accumulation. In the brain, where we observed rapid accumulation and amyloid fibril deposition, exposure to MWCNTs might enhance progression of neurodegenerative and other amyloid-related diseases. Our data highlight the conclusion that, in a novel rodent model of exposure, MWCNTs may induce macrophage recruitment, activation, and amyloid deposition, causing potential damage to several organs. Keywords: nanoparticles, animal model, environmental exposure, inflammation, amyloid fibrils, macrophages

Cigarette smoke (CS) and ethanol (EtOH) are known to synergize in the causation of cancers of the upper aerodigestive tract and of the liver. Little is known about possible interactions between these agents in other organs. These premises prompted us to evaluate the clastogenic effects resulting from the inhalation for 3 weeks of mainstream CS and oral administration of EtOH, which were tested either individually or in combination in cells of adult BDF1 mice and their fetuses. CS exerted clastogenic effects in haematopoietic cells of adult male mice by increasing the frequency of micronucleated erythroid cells both in bone marrow and in peripheral blood as well as the frequency of micronucleated and polynucleated pulmonary alveolar macrophages. Likewise, exposure to CS of pregnant mice resulted in a clastogenic damage in maternal bone marrow cells and in the liver and peripheral blood of their fetuses. Under all experimental conditions, EtOH was consistently devoid of clastogenic effects when given alone. In adult mice, EtOH exhibited a mild stimulating effect on the clastogenicity of CS in haematopoietic cells, while an opposite effect was observed in the respiratory tract, where EtOH attenuated the cytogenetic alterations induced by CS in pulmonary alveolar macrophages. At variance with the mild synergism observed in haematopoietic cells of adult mice, EtOH inhibited the clastogenicity of CS in the liver and peripheral blood cells of transplacentally exposed fetuses. Therefore, the effects of EtOH in CS-exposed mice show different trends depending both on the life stage and on the cells analyzed.

In recent years, Abscisic Acid (ABA) has been demonstrated to be involved in the regulation of glucose homeostasis in mammals as an endogenous hormone, by stimulating both insulin release and peripheral glucose uptake. In addition, ABA is released by glucose- or GLP-1-stimulated β-pancreatic cells. Here we investigated whether ABA can stimulate GLP-1 release. The human enteroendocrine L cell line hNCI-H716 was used to explore whether ABA stimulates in vitro GLP-1 secretion and/or transcription. ABA induced GLP-1 release in hNCI-H716 cells, through a cAMP/PKA-dependent mechanism. ABA also enhanced GLP-1 transcription. In addition, oral administration of ABA significantly increased plasma GLP-1 and insulin levels in rats. In conclusion, ABA can stimulate GLP-1 release: this result and the previous observation that GLP-1 stimulates ABA release from β -cells, suggest a positive feed-back mechanism between ABA and GLP-1, regulating glucose homeostasis. Type 2 diabetes treatments targeting the GLP-1 axis by either inhibiting its rapid clearance by dipeptidyl-peptidase IV or using GLP-1 mimetics are currently used. Moreover, the development of treatments aimed at stimulating GLP-1 release from L cells has been considered as an alternative approach. Accordingly, our finding that ABA increases GLP-1 release in vitro and in vivo may suggest ABA and/or ABA analogs as potential anti-diabetic treatments.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients. [ABSTRACT FROM AUTHOR]

We stratified the Italian population according to age and gender in order to evaluate mortality trends over more than one century. Data covering the 1901-2008 period were used to study the yearly variations in mortality. Fluctuations in age-adjusted mortality curves were analyzed by Join Point Regression Models, identifying Join Points and Annual Percent Changes. A consistent decline in all-cause mortality occurred across the whole period, the most striking variations being observed in the 0-49 years population. In 1901, other and undefined diseases were the main causes of death, followed by infectious, digestive, and respiratory diseases in the 0-49 years population and by respiratory, cardiovascular, and cerebrovascular diseases in the ≥ 50 years population groups. In 2008 the main causes of death were accidents (males) and tumors (females) in the 0-49 age class, tumors in the 50-69 age class (both genders), and tumors (males) and cardiovascular diseases (females) in the elderly. The results highlight the interplay between age and gender in affecting mortality trends and reflect the dramatic progress in nutritional, lifestyle, socioeconomic, medical, and hygienic conditions.