Cross-talk tra cellula adiposa e cellula neoplastica modelli di cocoltura

White adipose tissue (WAT) is considered as a complex organ with a central role in accumulation of lipids, and an important endocrine function with the secretion of several peptides as adipokines (leptin, adiponectin), cytokines and chemokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6). Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries. The number of cancer cases is expected to rise due to the increasing aging population.Epidemiological studies have shown that an increased risk of several cancers, including colon cancer, endometrial, breast, kidney, esophagus, pancreas, gallbladder, liver, and hematological malignancies, is associated with obesity. Moreover, this condition can lead to a reduction in the expected results from treatment, to a worse prognosis and to an increase of the cancer-associated mortality. Several studies have shown that in the white adipose tissue of obese subjects there is a decrease in the maturation of preadipocytes to adipocytes, as well as an imbalance between leptin and adiponectin; in addition, obesity is associated with hyperinsulinemia, hyperglycemia, insulin resistance, aberrant glucose metabolism, chronic inflammation and production of high levels of IGF-1, important risk factor for cancer.Many studies have highlighted the complexity of the tumors and of their microenvironment. Tumor microenvironment is constituted by several different types of cells as immune system cells, cells of the vascular and lymphatic system (endothelial cells), fibroblasts, pericytes, adipocytes and stromal cells derived from adipose tissue. The role of adipose tissue, and more specifically of adipocytes, in cancer initiation, growth and metastatization is a relatively new area of investigation.In tumors growing in a microenvironment dominated by adipocytes, it was observed that adipocytes disappear with an accumulation of fibroblast-like cells and subsequent formation of a desmoplastic stroma. Histological observations in some types of cancer, confirm that adipocytes localized at the tumor invasive front, become smaller and the number of fibroblast-like cells increases. It has been hypothesized that fibroblast-like cells could derived from dedifferentiation of adipocytes. In a previous in vitro study with 3T3-L1 cells differentiated to adipocytes, adipocytes promoted the growth, proliferation and survival of human breast cancer cells. However the role of adipocytes in the tumor microenvironment is only incompletely known; in particular in some types of tumors, as melanoma and pancreatic cancer, the role of adipocytes in cancer proliferation and invasiveness is not known. The main focus of this work was to study the interaction between adipocytes and cancer through a co-culture in vitro model. In particular, the study focused on the interaction between adipocytes and human pancreatic cancer cells and human melanoma cells, using a co-culture system between murine fibroblasts 3T3-L1 cell line differentiated to adipocytes and MiaPaca2 and A375 cell line respectively. Adipocytes co-cultured with both types of cells (human pancreatic cancer MiaPaca2 and human melanoma A375 cells) show a progressive loss of lipid content with more centralized nuclei and an elongated shape, similar to the fibroblasts morphology. Moreover, in both co-culture models, dedifferentiated adipocytes loste the adipocyte gene expression profile and acquire a gene profile of reprogramming cells. Finally, MiaPaca2 cells in co-culture showed an up-regulation of Wnt5a and greater activation of STAT3 compared to control; 3T3-L1 cells in co-culture had a greater ability to bind both c-Jun and AP-1, two proteins activated by the Wnt5a pathway; A375 cells in co-culture showed an increased migratory capacity compared to controls and a greater expression of \u3b2-catenin and LEF1. The plasticity of AT and the existence of dedifferentiation phenomena could bring new light into the complex relation between obesity, AT dysfunction and increased cancer risk

Relationship between lipid droplets size and integrated optical density

Lipid accumulation is largely investigated due to its role in many human diseases. The attention is mainly focused on the lipid droplets (LDs), spherical cytoplasmic organelles which are devoted to the storage of the lipids. The amount of lipid content is often evaluated by measuring LDs size and/or the integrated optical density (IOD) in cultured cells. Both evaluations are directly associated to the lipid content and therefore they are correlated to each other, but a lack of theoretical relationship between size and IOD was observed in literature. Here we investigated the size-IOD relationship of LDs observed in microscopical images of cultured cells. The experimental data were obtained from immature and differentiated 3T3-L1 murine cells, which have been extensively used in studies on adipogenesis. A simple model based on the spherical shape of the LDs and the Lambert-Beer law, which describes the light absorption by an optical thick material, leads to a mathematical relationship. Despite only light rays\u2019 absorption was considered in the model, neglecting their scattering, a very good agreement between the theoretical curve and the experimental data was found. Moreover, a computational simulation corroborates the model indicating the validity of the mathematically theoretical relationship between size and IOD. The theoretical model could be used to calculate the absorption coefficient in the LDs population and it could be applied to seek for morphologically and functionally LDs subpopulations. The identification of LDs dynamic by measuring size and IOD could be related to different pathophysiological conditions and useful for understand cellular lipid-associated diseases

Myocardial fibrosis and steatosis in patients with aortic stenosis: roles of myostatin and ceramides

Aortic stenosis (AS) involves progressive valve obstruction and a remodeling response of the left ventriculum (LV) with systolic and diastolic dysfunction. The roles of interstitial fibrosis and myocardial steatosis in LV dysfunction in AS have not been completely characterized. We enrolled 31 patients (19 women and 12 men) with severe AS undergoing elective aortic valve replacement. The subjects were clinically evaluated, and transthoracic echocardiography was performed pre-surgery. LV septal biopsies were obtained to assess fibrosis and apoptosis and fat deposition in myocytes (perilipin 5 (PLIN5)), or in the form of adipocytes within the heart (perilipin 1 (PLIN1)), the presence of ceramides and myostatin were assessed via immunohistochemistry. After BMI adjustment, we found a positive association between fibrosis and apoptotic cardiomyocytes, as well as fibrosis and the area covered by PLIN5. Apoptosis and PLIN5 were also significantly interrelated. LV fibrosis increased with a higher medium gradient (MG) and peak gradient (PG). Ceramides and myostatin levels were higher in patients within the higher MG and PG tertiles. In the linear regression analysis, increased fibrosis correlated with increased apoptosis and myostatin, independent from confounding factors. After adjustment for age and BMI, we found a positive relationship between PLIN5 and E/A and a negative correlation between septal S', global longitudinal strain (GLS), and fibrosis. Myostatin was inversely correlated with GLS and ejection fraction. Fibrosis and myocardial steatosis altogether contribute to ventricular dysfunction in severe AS. The association of myostatin and fibrosis with systolic dysfunction, as well as between myocardial steatosis and diastolic dysfunction, highlights potential therapeutic targets

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling

Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment

A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment

Models of lipid droplets growth and fission in adipocyte cells

Lipid droplets (LD) are spherical cellular inclusion devoted to lipids storage. It is well known that excessive accumulation of lipids leads to several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis and atherosclerosis. LDs' size range from fraction to one hundred of micrometers in adipocytes and is related to the lipid content, but their growth is still a puzzling question. It has been suggested that LDs can grow in size due to the fusion process by which a larger LD is obtained by the merging of two smaller LDs, but these events seems to be rare and difficult to be observed. Many other processes are thought to be involved in the number and growth of LDs, like the de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets. Moreover the number and size of LDs are influenced by the catabolism and the absorption or interaction with other organelles. The comprehension of these processes could help in the confinement of the pathologies related to lipid accumulation. In this study the LDs' size distribution, number and the total volume of immature (n=12), mature (n=12, 10-days differentiated) and lipolytic (n=12) 3T3-L1 adipocytes were considered. More than 11,000 LDs were measured in the 36 cells after Oil Red O staining. In a previous work Monte Carlo simulations were used to mimic the fusion process alone between LDs. We found that, considering the fusion as the only process acting on the LDs, the size distribution in mature adipocytes can be obtained with numerical simulation starting from the size distribution in immature cells provided a very high rate of fusion events. In this paper Monte Carlo simulations were developed to mimic the interaction between LDs taking into account many other processes in addition to fusion (de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets) in order to reproduce the LDs growth and we also simulated the catabolism (fission and the decrease through neutral lipid exit from pre-existing droplets) to reproduce their size reduction observed in lipolytic conditions. The results suggest that each single process, considered alone, can not be considered the only responsible for the size variation observed, but more than one of them, playing together, can quite well reproduce the experimental data

Simulating the dynamics of lipid droplets in adipocyte differentiation

Background Lipid droplets are cellular organelles that regulate the storage and hydrolysis of neutral lipids. The dynamic of lipid droplets (LDs), during the differentiation process from fibroblast-like cells into adipocyte, is strictly related to the lipid storage in cells. The number and size of the LDs depends on the lipidic or lipolytic stimulations to which the cells are exposed. Method Here, we propose a computational approach to study the processes regulating the LDs' number and growth/reduction in size using Monte Carlo simulations. The number and size of LDs are measured before and after experimental treatment in 3T3-L1 cell cultures. The algorithms simulating the evolution from basal to differentiate (lipidic or lipolytic) conditions are here detailed step by step. The algorithms can mimic thousand interacting events between LDs or squeezing/enlargement events of a single LD in a very brief computational time, from seconds up to few minutes. Results The main processes regulating the interactions between LDs are here presented, and for each of them, all the needed information to re-write the computational routine are provided. More specifically, the results obtained, analyzing the fusion process between LDs, are here presented. Conclusions Here, we would like to supply the basis to explore the dynamics of lipid storage in cells with a computational approach and to encourage the applications of numerical simulation to cell studies

Phenotypic Shift of Adipocytes by Cholecalciferol and 1\u3b1,25 Dihydroxycholecalciferol in Relation to Inflammatory Status and Calcium Content

Recent experimental data seem to suggest a relevant role for 1,25[OH]2cholecalciferol (1,25[OH]2D3) in adipocyte physiology and pathophysiology, with some studies showing adipogenic and pro-inflammatory properties, and others lipolytic and anti-inflammatory functions. Moreover, to our knowledge, the role of cholecalciferol (D3) in adipocytes function is still not known. Therefore, the aim of this study was to investigate in vitro the effects of 1,25[OH]2D3, as well as of D3, in 3T3-L1 adipocytes in basal and inflammatory conditions, testing the effects of different calcium concentrations in adipocytes culture medium. In 3T3-L1 adipocytes, CYP27A1 and CYP27B1 mRNA were detected in basal conditions and induced after D3 treatment. Pre-treatment of 3T3-L1 adipocytes not only with 1,25[OH]2D3, but also with D3 before inflammatory stimulation, significantly prevented the increase in gene expression and protein secretion of IL-6 and TNF-\u3b1, and significantly increased IL-10 mRNA and protein production compared with adipocytes treated only with lipopolysaccharide (LPS). Biological effects of D3 were still present after inhibition of P450 activity with ketokonazole. LPS determined a decrease in cell area compared with controls, paralleled by a significant increase in optical density (OD) of lipid droplets, whereas 1,25[OH]2D3 and D3 alone significantly increased adipocytes area and decreased OD. Pretreatment with both forms of vitamin D preserved cells from the reduction in their area observed after LPS treatment. LPS decreased more the area of cells grown in a high calcium medium than of adipocytes grown in a low calcium medium. In the presence of a high calcium medium, 1,25(OH)2D3 treatment preserved cell area, maintaining its anti-inflammatory and adipogenic properties. In conclusion our results show that D3, besides 1,25[OH]2D3, presents anti-inflammatory effects on 3T3-L1, as well as that adipocytes have the enzymatic pathways necessary to locally regulate the production of active forms of vitamin D, capable of influencing adipocyte phenotype and function

In vitro model of chronological aging of adipocytes: Interrelationships with hypoxia and oxidation

Aging is a physiological process characterized by an age-progressive decline in intrinsic physiological functions, with an increased risk of developing chronic metabolic conditions, such as insulin resistance and diabetes. Furthermore, from a physiopathological point of view, several authors describe an association between oxidative stress, hypoxia and these metabolic conditions. It had been suggested that adipose tissue (AT) dysfunction, senescent cell accumulation and proinflammatory pathways may be involved in this processes. The purpose of this study was to develop an in vitro model to study the progressive morphological and functional changes of adipocytes with aging, in standard culture conditions and after severe hypoxia and hydrogen peroxide treatment. We evaluated the degree of apoptosis and intracellular reactive oxygen species (ROS) accumulation as well as the gene expression profile of aging adipocytes. Our results show that aged adipocytes become senescent, undergo apoptosis, accumulate ROS, and present an inflammatory profile with an increase in mRNA expression level of key proteins related to the remodeling of the extracellular matrix (ECM). Aged adipocytes present increased levels of p53, p21 and p16, key regulators of senescence, and a decrease in SIRT-1 protein compared to younger cells. Moreover, adipocytes aged in hypoxia or in oxidative stress conditions represent a model of accelerated aging with a decrease in their area, a greater proportion of apoptotic and of intracellular ROS accumulation compared to controls. This study characterizes the progressive morphological and functional changes in aging adipocytes during prolonged cell cultures and explores the addictive effects of hypoxia and oxidation, given at different stages of cellular maturation and senescence