La fenotipizzazione di modelli murini tramite metodiche di imaging PET e CT rivela l’importanza di nuovi processi patofisiologici alla base della disregolazione metabolica associata ad obesità.

Lo studio e la caratterizzazione dei processi patogenetici alla base dell’obesita’ si basa sempre di piu’ sull’impiego di modelli animali di malattia, quali topi geneticamente modificati per geni coinvolti in specifiche vie metaboliche. In questo contesto, le metodiche di imaging molecolare offrono un importante vantaggio, quello della visualizzazione e quantificazione del processo metabolico attraverso delle immagini, senza quindi la necessita’ di sacrificare l’animale. In questo lavoro di tesi viene mostrato come apparecchiature di imaging PET (tomografia ad emissione di positroni) e TAC (tomografia assiale computerizzata) per piccoli roditori, siano funzionali per lo studio del fenotipo di modelli animali di obesita’, in quanto capaci di fornire informazioni di natura metabolica (con la PET), o anatomica (per quanto riguarda la TAC), in modo non invasivo. Queste metodiche di imaging, in associazione ad altre metodiche convenzionali, hanno permesso di ottenere nuove informazioni sui meccanismi di funzionamento di sistemi patofisiologici coinvolti nel controllo dell’omeostasi energetica, e quindi del controllo del peso corporeo. Il primo progetto di questo dottorato di ricerca ha riguardato lo studio dei meccanismi di controllo del metabolismo energetico da parte del sistema endocannabinoide. In particolare e’ stato valutato il ruolo svolto dal recettore CB1 (il principale recettore coinvolto nella regolazione del metabolismo energetico da parte degli endocannabinoidi) espresso in alcune popolazioni cellulari neuronali. Il sistema endocannabinoide e’ un sistema fisiologico composto da molecole lipidiche endogene, gli endocannabinoidi, che durante l’obesita’ va in contro ad una patologica overattivazione e in questo modo contribuisce all’accumulo della massa grassa e alle alterazioni metaboliche ad essa conseguenti negli individui obesi. Gli endocannabinoidi hanno la proprieta’ di regolare il metabolismo energetico agendo sia a livello del sistema nervoso centrale, che in tessuti periferici come il tessuto adiposo, il fegato, il pancreas e il muscolo scheletrico A partire dallo studio del fenotipo di topi con delezione completa (in tutto l’organismo) di CB1, e di topi conditional knockout che presentano delezione del recettore esclusivamente in alcune popolazioni cellulari neuronali, si e’ cercato di capire quali siano i meccanismi alla base della capacita’ degli endocannabinoidi di regolare l’omeostasi energetica. Inoltre, confrontando il fenotipo dei due diversi modelli animali, e’ stato valutato se l’azione degli endocannabinoidi a livello dei neuroni ipotalamici e’ importante nel controllare il bilancio energetico e conseguentemente il peso corporeo durante l’obesita’. E’ stato notato come il sistema endocannabinoide possa agire attraverso il recettore CB1 neuronale e determinare un profondo effetto sul metabolismo generale, in termini di controllo dell’omeostasi energetica e di regolazione del profilo metabolico. La capacita’ del recettore CB1 neuronale di mediare questi effetti e’ strettamente dipendente dalla sua capacita’ di regolare l’attivita’ nervosa simpatica sugli organi periferici; in questo senso il tessuto adiposo bruno (BAT) sembra essere fortemente influenzato da questo sistema di controllo che origina dal recettore CB1 neuronale e che ha come importante mediatore il sistema nervoso simpatico (SNS). Questi risultati, che mostrano una stretta relazione esistente tra sistema endocannabinoide e termogenesi del BAT, evidenziano come il BAT possa avere un ruolo importante nel controllare la massa grassa ed il peso corporeo degli individui obesi. Il secondo progetto del dottorato ha riguardato lo studio della relazione esistente tra tessuto adiposo bruno, sensibilita’ insulinica ed obesita’. Il tessuto adiposo bruno e’ un tessuto che recentemente e’ emerso come importante sito di controllo del metabolismo, infatti l’attivita’ funzionale di questo tessuto sembra contribuire a ridurre la capacita’ di accumulare l’energia contenuta nel cibo e quindi contrastare l’accumulo di massa grassa e peso corporeo in seguito ad alimentazione ipercalorica. Un aspetto ancora sconosciuto di questo tessuto e’ la sua capacita’ di rispondere alla azione dell’insulina. Dati preliminari ottenuti in vitro, sembrerebbero evidenziare che il tessuto adiposo bruno ha una forte capacita’ di rispondere alla azione dell’ormone, e che in condizioni di obesita’ la sensibilita’ insulinica in questo tessuto puo’ risultare compromessa. Alla luce della potenziale importanza di questo tessuto nel controllo dell’azione dell’insulina, in questo lavoro si e’ voluto valutare, in vivo, la sensibilita’ insulinica del tessuto in condizioni fisiologiche e di obesita’ indotta da dieta. A questo scopo, attraverso imaging molecolare PET e TAC, e’ stata studiata la capacita’ dell’insulina di stimolare l’uptake di glucosio nel tessuto adiposo bruno di topi normali (magri, alimentati con dieta alimentare standard) ed in topi con obesita’ indotta da dieta. E’ stato dimostrato come l’insulina possa agire in modo significativo nel regolare l’uptake di glucosio nel BAT e come l’obesita’ indotta da dieta sia caratterizzata da una ridotta funzionalita’ insulinica in questo tessuto. Recenti evidenze sperimentali, sebbene preliminari, indicano come l’insulina possa stimolare allo stesso livello del muscolo scheletrico, l’uptake di glucosio nel BAT di esseri umani adulti sani (non obesi). Alla luce di queste evidenze e dei dati presentati in questo lavoro di tesi, e’ possibile quindi che il BAT degli esseri umani vada incontro ad una alterata funzionalita’ insulinica in seguito ad obesita’. Questo tessuto, quindi, potrebbe rappresentare un nuovo target per controllare deregolazioni metaboliche che si originano in seguito ad obesita’, come la resistenza insulinica.Emerging molecular imaging techniques in small animals allow a non invasive study of animal models of diseases, providing in vivo information on metabolic pathways. The metabolic process can be visualized and quantified by images, avoiding animal sacrifice which is often mandatory with conventional techniques In this work we show how small animals Positron Emission Tomography (PET) and Computed Tomography (CT) usefully provide metabolic and anatomic information respectively, in a non invasive fashion. Toghether with other conventional approaches, these molecular imaging techniques allowed, in the present work, to collect information on the pathofisiological mechanisms underlying energy metabolism and consequently body weight control in obesity. The first project of the PhD focused on the comprehension of the mechanism by which endocannabinoid system is able to control energy balance at different anatomical sites. The endocannabinoid system (ECS) is a physiological system composed by endogenous lipid molecules regulating energy metabolism acting on the cannabinoid receptor type 1 (CB1). This receptor is expressed in different anatomical sites as central and peripheral nervous system, adipose tissue, liver, skeletal muscle. The ECS is pathologically over-activated during obesity and this overactivation is known to contribute to fat mass and body weight accumulation in obese individuals. The mechanisms and the major anatomical sites underlying this control, are, however, not fully elucidated yet. We focused on the role played by CB1 receptor expressed in some neuronal population. To this aim, the phenotype of conditional knockout for CB1 receptor in some neuronal populations was studied, and compared with the phentype of global CB1 receptor knockout mice (mice having a whole body deletion of the receptor). We found that neuronal CB1 receptor has a deep influence in the ability of ECS to control energy balance and the plasma metabolic profile during obesity. This property of neuronal CB1 receptor is strictly linked with a modulation of the peripheral sympathetic tone which finally influences the metabolic activity of peripheral organs. This loop seems to be particularly effective in modulating brown adipose tissue (BAT) activity, which apparently underlyes most of the changes in whole body energy metabolism observed in the conditional mutant mice for neuronal CB1. These results, which show a strict functional relationship between ECS and BAT thermogenic activity, highlight BAT as a key organ able to control fat mass and body weight gain in obese individuals. The second project of this PhD thesis focused on the relationship between BAT, insulin sensitivity and obesity. BAT is an insulin sensitive organ having a very high uptake of glucose per gram of tissue. The role of insulin on glucose uptake in BAT is still poorly understood, and it is not clear whether BAT insulin function is compromised with obesity. Using a small animal PET/CT imaging approach we analysed in vivo glucose uptake in the BAT of lean and diet-induced obese mice in basal condition and after insulin stimulation. All these findings allowed us to demonstrate that diet induced obesity is associated with an altered (reduced) insulin function in the BAT. Recent experimental evidences indicate that BAT is an important organ in insulin induced glucose clearance in humans; thus, the data obtained in this PhD project, which indicate a compromised insulin function in the BAT of obese individuals, highlight this tissue as a new target to control insulin resistance in obesity

New evidence of MIS 3 relative sea level changes from the Messina Strait, Calabria (Italy)

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Antonioli, F., Calcagnile, L., Ferranti, L., Mastronuzzi, G., Monaco, C., Orru, P., Quarta, G., Pepe, F., Scardino, G., Scicchitano, G., Stocchi, P., & Taviani, M. New evidence of MIS 3 relative sea level changes from the Messina Strait, Calabria (Italy). Water, 13(19), (2021): 2647, https://doi.org/10.3390/w13192647.Investigation of sea-level positions during the highly-dynamic Marine Isotope Stage 3 (MIS 3: 29–61 kyrs BP) proves difficult because: (i) in stable and subsiding areas, coeval coastal sediments are currently submerged at depths of few to several tens of meters below the present sea level; (ii) in uplifting areas, the preservation of geomorphic features and sedimentary records is limited due to the erosion occurred during the Last Glacial Maximum (LGM) with sea level at a depth of −130 m, followed by marine transgression that determined the development of ravinement surfaces. This study discusses previous research in the Mediterranean and elsewhere, and describes new fossiliferous marine deposits overlaying the metamorphic bedrock at Cannitello (Calabria, Italy). Radiocarbon ages of marine shells (about 43 kyrs cal BP) indicate that these deposits, presently between 28 and 30 m above sea level, formed during MIS 3.1. Elevation correction of the Cannitello outcrops (considered in an intermediate-to-far-field position with respect to the ice sheet) with the local vertical tectonic rate and Glacial Isostatic Adjustment (GIA) rate allows the proposal of a revision of the eustatic depth for this highstand. Our results are consistent with recently proposed estimates based on a novel ice sheet modelling technique.This research received no external funding

Lack of Hypophagia in CB1 Null Mice is Associated to Decreased Hypothalamic POMC and CART Expression

Background: Cumulative data indicate that the endocannabinoid system plays a major role in feeding behavior and energy balance. Genetic silencing of cannabinoid receptor type 1 (CB1) reduces body weight gain, independently of food intake. Methods: In this work, we investigated whether the hypothalamic neuropeptide expression pattern supports the absence of the anorexigenic response observed under constitutive CB1 ablation, by using neuronal CB1 conditional null mice (CamK-CB1-KO) and whole body CB1 null mice (CB1-KO). Results: Our data showed that both CB1 null models display a marked decrease in proopiomelanocortin (POMC) and cocaine-amphetamine-regulated transcript (CART) expression in the arcuate nucleus of the hypothalamus (ARC). Conclusions: This evidence suggests that a lack of hypophagia is associated with the suppression of ARC anorexigenic neuropeptides and that behavioral changes in food intake (or lack thereof) after constitutive CB1 ablation are likely mediated by impaired melanocortin and CART signaling in the hypothalamus.The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 281854 of the ObERStress European Research Council Project (Dr López) and 245009 of the Neurofast project (Drs Nogueiras, Diéguez, and López); Xunta de Galicia (Dr Nogueiras: EM 2012/039 and 2012-CP069; Dr López: 2012-CP070); the Frank Mohn Foundation, Bergen (Dr Fernø); Instituto de Salud Carlos III (ISCIII; Dr López: PI12/01814 and PIE13/00024); and MINECO, co-funded by the FEDER Program of EU (Dr Nogueiras: RyC-2008-02219 and BFU2012-35255; Dr Diéguez: BFU2011-29102). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIIIS

Nat Metab

Hypothalamic AgRP and POMC neurons are conventionally viewed as the yin and yang of the body’s energy status, since they act in an opposite manner to modulate appetite and systemic energy metabolism. However, although AgRP neurons’ functions are comparatively well understood, a unifying theory of how POMC neuronal cells operate has remained elusive, probably due to their high level of heterogeneity, which suggests that their physiological roles might be more complex than initially thought. In this Perspective, we propose a conceptual framework that integrates POMC neuronal heterogeneity with appetite regulation, whole-body metabolic physiology and the development of obesity. We highlight emerging evidence indicating that POMC neurons respond to distinct combinations of interoceptive signals and food-related cues to fine-tune divergent metabolic pathways and behaviours necessary for survival. The new framework we propose reflects the high degree of developmental plasticity of this neuronal population and may enable progress towards understanding of both the aetiology and treatment of metabolic disorders.Bordeaux Region Aquitaine Initiative for NeuroscienceInnovations instrumentales et procédurales en psychopathologie expérimentale chez le rongeurLa signalisation des acides biliaires dans le cerveau et son rôle dans le contrôle métaboliqueRôle du récepteur aux cannabinoïdes de type 1 mitochondriale dans les circuits hypothalamiques et son interaction avec la voie mTORC1 dans l'obésité.Rôle de Tbx3 dans la détermination de l'identité fonctionnelle des neurones POMC dans l'obésitéEuropean Union Seventh Framework Programme FP7/2007-201

CB1 and GLP-1 Receptors Cross Talk Provides New Therapies for Obesity

Bordeaux Region Aquitaine Initiative for NeuroscienceInnovations instrumentales et procédurales en psychopathologie expérimentale chez le rongeurLa signalisation des acides biliaires dans le cerveau et son rôle dans le contrôle métaboliqueRôle du récepteur aux cannabinoïdes de type 1 mitochondriale dans les circuits hypothalamiques et son interaction avec la voie mTORC1 dans l'obésité

Functional heterogeneity of POMC neurons relies on mTORC1 signaling.

Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior

Il fenomeno delle dipendenze patologiche nella Provincia di Ragusa. Anno 2005. I Rapporto

Report on the state of legal and illegal substances use in the territory of Ragusa ProvinceIl Report analizza il fenomeno delle dipendenze nel territorio della Provincia di Ragusa. La descrizione del fenomeno si sviluppa intorno all\u27analisi degli indicatori individuati dall\u27Osservatorio Europeo delle Dipendenze di Lisbona (OEDT): 1-uso di sostanze nella popolazione generale (questo indicatore va a rilevare i comportamenti nei confronti di alcol e sostanze psicoattive da parte della popolazione generale); 2-prevalenza d\u27uso problematico delle sostanze psicoattive; 3-domanda di trattamento degli utilizzatori di sostanze; 4-mortalit? degli utilizzatori di sostanze; 5-malattie infettive. Altri due importanti indicatori che si stanno sviluppando, e che vengono qui illustrati, sono l\u27analisi delle Schede di Dimissione Ospedaliera (SDO) e gli indicatori relativi alle conseguenza sociali dell\u27uso di droghe (criminalit? droga correlata). Inoltre sono state applicate diverse metodologie standard di stima sia per quantificare la quota parte sconosciuta di utilizzatori di sostanze che non afferiscono ai servizi, sia per identificarne alcune caratteristiche

CB1 Signaling in Forebrain and Sympathetic Neurons Is a Key Determinant of Endocannabinoid Actions on Energy Balance

SummaryThe endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB1) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB1 modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB1 receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB1 expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB1 signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography–mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance

Preclinical evaluation of KIT/PDGFRA and mTOR inhibitors in gastrointestinal stromal tumors using small animal FDG PET

<p>Abstract</p> <p>Background</p> <p>Primary and secondary drug resistance to imatinib and sunitinib in patients with gastrointestinal stromal tumors (GISTs) has led to a pressing need for new therapeutic strategies such as drug combinations. Most GISTs are caused by mutations in the KIT receptor, leading to upregulated KIT tyrosine kinase activity. Imatinib and nilotinib directly inhibit the kinase activity of KIT, while RAD001 (everolimus) inhibits mTOR. We report a preclinical study on drug combinations in a xenograft model of GIST in which effects on tumor dimensions and metabolic activity were assessed by small animal PET imaging.</p> <p>Methods</p> <p>Rag2-/-; γcommon -/- male mice were injected s.c. into the right leg with GIST 882. The animals were randomized into 6 groups of 6 animals each for different treatment regimens: No therapy (control), imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, everolimus (10 mg/kg/d.) by oral gavage, everolimus (10 mg/kg/d.) + imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, nilotinib (75 mg/kg/d.) by oral gavage, nilotinib (75 mg/kg/d.) + imatinib (150 mg/kg b.i.d) by oral gavage for 6 days, then once/day for another 7 days. Tumor growth control was evaluated by measuring tumor volume (cm³). Small animal PET (GE Explore tomography) was used to evaluate tumor metabolism and performed in one animal per group at base-line then after 4 and 13 days of treatment.</p> <p>Results</p> <p>After a median latency time of 31 days, tumors grew in all animals (volume 0,06-0,15 cm³) and the treatments began at day 38 after cell injection. Tumor volume control (cm3) after 13 days of treatment was > 0.5 for imatinib alone and nilotinib alone, and < 0.5 for the 2 combinations of drugs and for everolimus alone. The baseline FDG uptake was positive in all animals. FDG/SUV/TBR was strongly reduced over time by everolimus both as a single agent and in combination with imatinib respectively: 3.1 vs. 2.3 vs. 1.9 and 2.5 vs 2.3 vs 0.</p> <p>Conclusions</p> <p>As single agents, all drugs showed an anti-tumor effect in GIST xenografts but everolimus was superior. The everolimus plus imatinib combination appeared to be the most active regimen both in terms of inhibiting tumor growth and tumor metabolism. The integration of everolimus in GIST treatment merits further investigation.</p