Cecilia Fuentes

Calcium sensing receptor activation elevates proinflammatory factor expression in human adipose cells and adipose tissue.

The proinflammatory status of adipose tissue has been linked to the metabolic and cardiovascular consequences of obesity. Human adipose cells express the calcium sensing receptor (CaSR), and its expression is elevated in inflammatory states, such as that associated with obesity. Given the CaSRs association with inflammation in other tissues, we evaluated its role elevating the adipose expression of inflammatory factors. The CaSR activation by the calcimimatic cinacalcet (5μM) in adipose tissue and in vitro cultured LS14 adipose cells elicited an elevation in the expression of the proinflammatory cytokines IL6, IL1β, TNFα, and the chemoattractant CCL2. This was in part reverted by SN50, an inhibitor of the inflammatory mediator nuclear factor kappa B (NFκB). Our observations suggest that CaSR activation elevates cytokine and chemokine production, partially mediated by NFκB. These findings support the relevance of the CaSR in the pathophysiology of obesity-induced adipose tissue dysfunction, with an interesting potential for pharmacological manipulation.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line ☆

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

Preadipocyte proliferation is elevated by calcium sensing receptor activation

Obesity is a major worldwide problem, despite considerable efforts against it. While excess body fat defines obesity, adipose tissue quality and functionality are key to whether cardiovascular and metabolic comorbidities develop. Adipose tissue cellular composition can vary considerably, and excess adipocyte progenitors (preadipocytes) is associated with obesity. We have proposed that calcium sensing receptor (CaSR) activation in adipose tissue leads to dysfunction. This study evaluated whether CaSR activation elevates preadipocyte proliferation. Human LS14 preadipocytes were exposed to CaSR activators cinacalcet (2 µM), GdCl3 (5 µM) and spermine (1 µM), and cell viability was evaluated after 72h. CaSR activators elevated proliferation by 19-24%, and CaSR silencing (siRNA) abolished the effect. Cinacalcet elevated phospho-ERK1/2 content, and upstream inhibition of ERK1/2 phosphorylation reverted cinacalcet-induced proliferation. Cinacalcet also elevated expression of the proinflammatory factors IL1β, IL6 and CCL2. The results suggest that CaSR induces preadipocyte proliferation, partly through ERK1/2 activation. Considering reported proinflammatory and adipogenic CaSR effects, excess preadipocyte proliferation further supports the dysfunctional effect of CaSR in obesity.

Calcium sensing receptor effects in adipocytes and liver cells: Implications for an adipose-hepatic crosstalk.

The calcium sensing receptor (CaSR) is expressed in human adipose cells, and its activation may associate with adipose tissue (AT) dysfunction. We evaluated whether CaSR stimulation influences adipocyte triglyceride (TG) and fatty acid binding protein 4 (aP2) content, and hepatocyte TGs and proinflammatory cytokine expression. The effect of the calcimimetic cinacalcet on TGs (fluorimetry), lipogenic genes (qPCR) and aP2 (immunoblot) was evaluated in LS14 adipocytes or AT. In the human HepG2 hepatic cell line, we assessed CaSR expression and cinacalcet effect on TGs and lipogenic and proinflammatory genes. CaSR activation decreased adipocyte TG content by 20% and the expression of GPD and LPL by 34% and 20%, respectively. Cinacalcet increased aP2 protein expression by 60%. CaSR expression was shown in HepG2 cells and human liver samples. Cinacalcet-treated HepG2 cells in the presence of oleic acid exhibited a19% increased TG content. No changes were observed in the expression of lipogenic genes in HepG2 cells, however there was a 50%-300% elevation in the expression of proinflammatory cytokines. CaSR activation in adipocytes may associate with decreased TG storage ability and increased aP2. Hepatic CaSR stimulation may elevate steatosis and proinflammatory factors. We propose that CaSR may contribute to obesity-associated hepatic metabolic consequences.

Calcium-sensing receptor activates the NLRP3 inflammasome in LS14 preadipocytes mediated by ERK1/2 signaling

The study of the mechanisms that trigger inflammation in adipose tissue is key to understanding and preventing the cardiometabolic consequences of obesity. We have proposed a model where activation of the G protein‐coupled calcium sensing receptor (CaSR) leads to inflammation and dysfunction in adipose cells. Upon activation, CaSR can mediate the expression and secretion of proinflammatory factors in human preadipocytes, adipocytes, and adipose tissue explants. One possible pathway involved in CaSR‐induced inflammation is the activation of the NLR family, pyrin domain‐containing 3 (NLRP3) inflammasome, that promotes maturation and secretion of interleukin (IL)‐1β. The present work aimed to study whether CaSR mediates the activation of NLRP3 inflammasome in the human adipose cell model LS14. We assessed NLRP3 inflammasome priming and assembly after cinacalcet‐induced CaSR activation and evaluated if this activation is mediated by downstream ERK1/2 signaling in LS14 preadipocytes. Exposure to 2 μM cinacalcet elevated mRNA expression of NLRP3, CASP‐1, and IL‐1β, as well as an increase in pro‐IL‐1β protein. In addition, CaSR activation triggered NLRP3 inflammasome assembly, as evidenced by a 25% increase in caspase‐1 activity and 63% IL‐1β secretion. CaSR silencing (siRNA) abolished the effect. Upstream ERK pathway inhibition decreased cinacalcet‐dependent activation of NLRP3 inflammasome. We propose CaSR‐dependent NLRP3 inflammasome activation in preadipocytes through ERK signaling as a novel mechanism for the development of adipose dysfunction, that may favor the cardiovascular and metabolic consequences of obesity. To the best of our knowledge, this is the first report linking the inflammatory effect of CaSR to NLRP3 inflammasome induction in adipose cells.

Autophagy mediates calcium-sensing receptor-induced TNFα production in human preadipocytes

Obesity is a major current public health problem worldwide due to the severe co-morbid conditions that this disease entails. The development of obesity-related cardiometabolic disorders is in direct association with adipose tissue inflammation that leads to its functional impairment. Activation of the Calcium-Sensing Receptor (CaSR) in adipose tissue contributes to inflammation and adipose dysfunction. Autophagy, a process of cell component degradation, is closely related to inflammation in many diseases, however, whether autophagy is associated with CaSR-induced inflammation remains unknown. Using LS14 and SW872 preadipose cell lines as well as primary human preadipocytes, we show that CaSR activation with the allosteric activator cinacalcet induces autophagosome formation. Cinacalcet-induced LC3II content elevation was precluded by knockdown of the CaSR and enhanced by CaSR overexpression, indicating a specific effect. Autophagy inhibition using 3-methyladenine prevented CaSR-induced TNFα production, indicating that autophagy contributes to CaSR-induced inflammation in human preadipocytes. Our results suggest that modulation of CaSR-induced autophagy is an attractive target in obese inflamed adipose tissue, to prevent the development of diseases triggered by adipose dysfunction. We describe a novel mechanism and possible new target to modulate and prevent adipose inflammation and hence the resulting disease-generating adipose tissue dysfunction.

Cytokine expression in SAT and VAT from PCOS patients and control women; possible proinflammatory effect of testosterone depending on BMI - a preliminary report

Nicolas Crisosto DOI: 10.3252/pso.eu.17ece.2015 Reproduction, endocrine disruptors and signalling (1)Laboratory of Endocrinology and Metabolism, Faculty of Medicine, West Division, University of Chile (2) Laboratory of Nutritional Genomics, Department of Nutrition, Faculty of Medicine, University of Chile.(3) Basic Nutrition and Genetic Epidemiology Laboratory Institute of Nutrition and Food Technology (INTA), University of Chile. (4) Surgery Unit, Hospital San Juan de Dios, (5) Surgery Unit, Clinica Las Condes. FONDECYT Iniciacion 11130126 FONDECYT 1110864