Characterisation of epicardial adipose tissue and myocardial fat infiltration in humans

L\u2019obesit\ue0 ed il diabete mellito di tipo 2 sono caratterizzati da un basso grado di infiammazione sistemica, aumento di dimensione del tessuto adiposo (TA), rilascio incontrollato di acidi grassi liberi nella circolazione; sono entrambi fortemente correlati con patologie metaboliche e cardiovascolari, come la dislipidemia, la patologia coronarica, l\u2019ipertensione e l\u2019infarto miocardico. Il TA bianco \ue8 stato accettato come essere molto pi\uf9 di un semplice deposito statico di energia. Le sue capacit\ue0 di regolare meccanismi omeostatici e metabolici dell\u2019intero organismo sono stati evidenziati negli ultimi 20 anni; molti studi hanno proposto il TA come un organo endocrino, secernente ormoni, adipochine ed altre sostanze biologicamente attive localmente o a livello sistemico. I depositi di TA non sono uniformi; le loro caratteristiche cambiano in diverse aree corporee, partecipando in modo diverso alla possibile patogenesi delle diverse patologie. Il TA epicardico (EAT) \ue8 un particolare deposito di TA, che \ue8 stato recentemente al centro di molti studi. Si trova localizzato principalmente sulla parete destra libera del cuore e circonda le coronarie, entrando a diretto contatto con il miocardio. Si \ue8 visto che EAT secerne molte differenti adipochine e si suppone quindi abbia un ruolo nella generazione e progressione della patologia coronarica. Lo scopo di questa tesi \ue8 quello di meglio caratterizzare EAT e lo strato di miocardio ad esso sottostante. Nell\u2019articolo 1 abbiamo analizzato le dimensioni cellulari adipocitarie e la secrezione di adiponectina confrontando EAT, il TA viscerale (VAT) e sottocutaneo (SAT). EAT \ue8 risultato avere cellule pi\uf9 piccole con ridotta secrezione di adiponectina. La dimensione adipocitaria sia in EAT che SAT \ue8 positivamente correlata con l\u2019insulino resistenza, e mostra associazione negativa con l\u2019espressione locale di adiponectina. I soggetti affetti da patologia cardiovascolare presentano inoltre adipociti significativamente pi\uf9 grandi in EAT. Nell\u2019articolo 2 abbiamo focalizzato la nostra attenzione sugli effetti della patologia diabetica su EAT, mostrando maggiore espressione genica di MCP-1, CD-68 e ridotta di adiponectina, cos\uec come dimensioni adipocitarie maggiori in soggetti diabetici rispetto ai non diabetici. In alcuni dati ancora non pubblicati abbiamo inoltre valutato le caratteristiche immunoistochimiche di infiltrazione lipidica e macrofagica del miocardio della stessa popolazione di pazienti. Alla tesi \ue8 stato inoltre aggiunto come articolo 3 il risultato di uno studio condotto nel corso di un\u2019esperienza all\u2019estero della durata di un anno presso il Lipid Laboratory KI di Stoccolma Svezia. Lasciando il soggetto di EAT, il lavoro si concentra su TA e gli aspetti genetici dell\u2019obesit\ue0. Niemann-Pick C1 (NPC-1) \ue8 stato recentemente implicato come gene di suscettibilit\ue0 per l\u2019obesit\ue0 attraverso uno studio genome wide. Abbiamo approfondito quindi il rapporto tra NPC-1 e l\u2019obesit\ue0 nell\u2019uomo. Le analisi mRNA hanno mostrato aumentata espressione nel paziente obeso sia in SAT che in VAT e riduzione in seguito a calo di peso. L\u2019espressione genica di NPC-1 era maggiore nelle cellule adipocitarie isolate, rispetto al tessuto in toto, sia in SAT che in VAT, ma non era modificata nel corso della differenziazione adipocitaria. L\u2019espressione proteica di NPC-1 rispecchiava l\u2019espressione genica.Obesity and type 2 diabetes are characterized by mild systemic inflammation, enlargement of fat depots, and uncontrolled release of free fatty acids into the circulation; they are both strongly associated with metabolic and cardiovascular disorders, such as dyslipidemia, coronary heart disease, high blood pressure and myocardial infarct. White adipose tissue has been widely accepted to be much more than a static fuel storage organ. Its capability of regulating homeostatic and metabolic mechanisms has been underlined during the last 20 years; several studies have proposed adipose tissue as an endocrine organ, secreting hormones, adipokines and other biologically active agents acting locally or in a systemic manner. Adipose tissue depots are not uniform; their characteristics change in different areas of the body, displaying distinct structural and functional properties and having different putative roles in pathologies. Epicardial adipose tissue (EAT) is a peculiar adipose tissue depot, which has recently been the center of many studies. It\u2019s located predominantly on the right free wall of the heart, surrounding coronary arteries and being directly in contact with the myocardial layer. EAT has been shown to secrete many different adipokines and is supposed to have a role in the generation and progression of coronary artery diseases. The aim of this thesis was to better characterize EAT and the underlying myocardial layer. In paper 1 we investigated adipocyte cell size and adiponectin secretion comparing EAT, visceral AT (VAT) and subcutaneous AT (SAT). EAT resulted to have smaller adipocytes and lower adiponectin secretion levels. Adipocyte size, both in EAT and in SAT, is positively related with insulin resistance, shows negative association with local adiponectin gene expression, and bigger in subjects with coronary artery disease. Adiponectin gene expression is significantly lower in EAT than in SAT. In paper 2 we focused our attention on the effect of diabetic state on EAT, showing higher MCP-1, CD-68, lower adiponectin level, and bigger adipocytes in subjects with than those without diabetes. We also analyzed thorough immunohistochemistry and present as unpublished data, the characteristics of fat and macrophagic infiltration of the myocardium of the same cohort of patients. Additionally, the result of a one year internship conducted at Lipid Laboratory in KI Sweden, has been included in the thesis, as paper 3. Leaving the subject of EAT, the paper is focused on adipose tissue and it\u2019s molecular and genetic aspects in obesity. Since the gene Niemann-Pick C1 (NPC-1) has recently being implicated in susceptibility to obesity, through a genome wide association study, we dig into the relationship between NPC-1 and obesity in humans. The analysis of NPC-1 mRNA ad protein in obesity, showed that NPC1 mRNA was significantly increased in obese individuals in SAT and VAT and down-regulated by weight loss. NPC-1 mRNA was enriched in isolated fat cells of WAT, in SAT versus VAT, but not modified during adipocyte differentiation. NPC-1 protein mirrored expression of mRNA in lean and obese individual

NPC1 in human white adipose tissue and obesity

Abstract Background Genetic studies have implicated the NPC1 gene (Niemann Pick type C1) in susceptibility to obesity. Methods To assess the potential function of NPC1 in obesity, we determined its expression in abdominal white adipose tissue (WAT) in relation to obesity. NPC1 mRNA was measured by RT-qPCR in lean and obese individuals, paired samples of subcutaneous (sc) and omental (om) WAT, before and after weight loss, in isolated adipocytes and intact adipose pieces, and in primary adipocyte cultures during adipocyte differentiation. NPC1 protein was examined in isolated adipocytes. Results NPC1 mRNA was significantly increased in obese individuals in scWAT and omWAT and downregulated by weight loss. NPC1 mRNA was enriched in isolated fat cells of WAT, in scWAT versus omWAT but not modified during adipocyte differentiation. NPC1 protein mirrored expression of mRNA in lean and obese individuals. Conclusions NPC1 is highly expressed in human WAT adipocytes with increased levels in obese. These results suggest that NPC1 may play a role in adipocyte processes underlying obesity.</p

LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

OBJECTIVE: In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times. METHODS: 3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 \u3bcg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed. RESULTS: Cells activated by increasing doses of LPS upregulated TNF-\u3b1 expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1\u3b1. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-\u3b1 and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 \ub0C. The chemokines MIP-1\u3b1/CCL3 and MIP-1\u3b2/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments. CONCLUSION: These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte

Inflammatory profile in subcutaneous and epicardial adipose tissue in men with and without diabetes

In recent years, evidence has emerged indicating that insulin resistance and diabetes mellitus type 2 are associated with inflammation of adipose tissue (AT). Interest has been focused on epicardial AT (EAT) because of its possible involvement with atherosclerosis and cardiovascular diseases. The aim of this study was to characterize adipocyte size and inflammatory profile in subcutaneous (SAT) and EAT among subjects with or without diabetes. Biopsies were collected from SAT and EAT in 34 men undergoing elective cardiac surgery. Weight, height, body mass index, waist circumference, as well as serum levels of glucose, insulin, lipids, adiponectin, and leptin were determined in all subjects. Adiponectin, MCP-1, and CD68 mRNA levels present within cells from AT biopsies were determined by real-time polymerase chain reaction. Adipocyte size was determined by optic microscopy and morphometry. Regarding the experimental group as a whole, gene-expression levels within EAT were significantly lower for adiponectin and higher, albeit not significantly, for MCP-1, when compared with that of SAT. In addition, adipocytes in EAT were significantly smaller than those in SAT. Subjects with diabetes showed lower adiponectin gene-expression levels in both SAT and EAT when compared with subjects without diabetes. By contrast, MCP-1 and CD68 gene-expression levels were higher in both tissue types of diabetic subjects. Adipocyte size in EAT was significantly larger in diabetic subjects than in nondiabetic subjects. Our data revealed a predominantly inflammatory profile in both SAT and EAT in subjects with diabetes in comparison with those without diabetes. \ua9 Springer 2013

Heart fat infiltration in subjects with and without coronary artery disease

Context: Fat may accumulate around the heart in epicardial adipose tissue or inside the heart as lipid droplets (LDs). Objective: To compare myocardial steatosis between subjects with and without coronary artery disease (CAD and non-CAD) and to identify which cells contain LDs. Design: Body mass index, waist circumference, glucose, insulin, homeostasis model assessment index, leptin, adiponectin, and high-sensitivity C-reactive protein were evaluated in CAD and non-CAD subjects. Biopsies were collected from right atrial myocardium. Immunohistochemistry for perilipin (PLIN) 1 and 2 was used to characterize LDs and their localization in adipocytes or myocardial cells, respectively. Cardiomyocytes apoptosis and hypoxia inducible factor 1 alpha were obtained in a subgroup of subjects. Setting: The study took place in a hospital. Patients: Male subjects consecutively undergoing elective cardiac surgery either for coronary bypass grafting (CAD, n = 23) or for valve replacement (non-CAD, n = 18). Main Outcomes and Measures: The study was designed to compare myocardial steatosis between subjects with and without coronary artery disease. Results: PLIN1 and PLIN2 resulted significantly higher in CAD than in non-CAD subjects, as did apoptosis. PLIN1 was positively associated with circulating leptin, high-sensitivity C-reactive protein, and apoptosis, and negatively with adiponectin. PLIN2 was positively associated with body mass index, waist circumference, and leptin and negatively with adiponectin. After taking into account the absence/presence of hypertension, diabetes, and CAD/non-CAD, adiponectin was negatively associated with PLIN1 (r2 = 0.532); waist circumference and adiponectin were associated with PLIN2 (r2 = 0.399). Conclusions: Myocardial steatosis is greater in CAD than non-CAD subjects, depending on both metabolically active adipocytes interspersed among cardiomyocytes and higher fat deposition inside cardiomyocytes; serum adiponectin and waist circumference are independent predictors of myocardial steatosis