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 µg/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-α 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/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α 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 °C. The chemokines MIP-1α/CCL3 and MIP-1ß/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.

Morphological and Functional Changes in the Peritumoral Adipose Tissue of Colorectal Cancer Patients.

OBJECTIVE: The role of peritumoral adipose tissue (AT) has not been extensively studied in colorectal cancer (CRC). METHODS: This study was conducted in 20 male subjects undergoing elective surgery for CRC. The differences between the peritumoral visceral adipose tissue (P-VAT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) of the patients were described via immunohistochemistry and molecular biology analyses. The interactions between adipocytes and a colon cancer cell line were also investigated by using an in vitro coculture system. RESULTS: The analyses revealed that adipocytes near the tumor were significantly smaller than the adipocytes from other sites. The P-VAT was preferentially infiltrated by a CD68+/CD163+/IDO- macrophage subset with a prevalent reparative inflammatory response, while the macrophages identified in VAT and SAT mainly presented inflammatory features. Furthermore, the P-VAT presented a higher expression of adiponectin compared with other sites. Morphological analysis in vitro showed that after a few days of coculture, 3T3-L1 adipocytes were reduced in number and size with an increase in lipolysis rate and dedifferentiation phenomena. CONCLUSIONS: This study reveals important morphological and functional changes in the AT surrounding the tumor as an increase in lipolysis and in adiponectin-producing adipocytes, preferentially infiltrated by a macrophage subset, with prevalent reparative inflammatory response.

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.

Iron primes 3T3-L1 adipocytes to a TLR4-mediated inflammatory response.

OBJECTIVE: Iron participates in several mechanisms involving inflammation and innate immunity, yet the dysregulation of its homeostasis is a major cause of metabolic syndrome. Adipocytes should play a major role in iron metabolism, as an impairment in iron turnover is closely related to insulin resistance, obesity, and type 2 diabetes. The aim of this study was to investigate the role of iron in an in vitro-inflamed adipocyte model. METHODS: Gene expression of tumor necrosis factor-α, interleukin-6, inflammatory chemokines (CCL3, CCL4, and CXCL12), and molecules involved in iron metabolism were evaluated in an in vitro mouse 3T3-L1 cell model. Cells underwent treatment with FeSO4 heptahydrate and lipopolysaccharide (LPS) stimulation. Toll-like receptor 4 (TLR4) membrane expression, lipid droplet immunohystochemistry, and lipolysis were also evaluated. RESULTS: Iron sulphate heptahydrate elicited gene expression of hepcidin, hemojuvelin, and ferroportin at different time courses. Additionally, it activated lipolysis but did not trigger any adipokine gene expression. When cells treated with physiological doses of iron were also stimulated with LPS, an enhancement in the LPS-induced gene expression of cytokines and chemokines was observed. The enhancement occurred with different patterns depending on different time courses and investigated genes, showing its maximal effect for IL-6 gene expression. CONCLUSIONS: FeSO4 heptahydrate at a relatively physiological dose, induced gene expression of iron modulatory proteins and also enhanced RNA transcripts of several inflammatory cytokines and chemokines through a priming/synergistic mechanism involving membrane TLR4.

Phenotypic shift of adipocytes by cholecalciferol and 1α,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-α, 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.

Effect of high-frequency electromagnetic fields on trophoblastic connexins.

Connexins (Cx) are membrane proteins able to influence trophoblast functions. Here we investigated the effect of high-frequency electromagnetic fields (HF-EMF) on Cx expression and localization in extravillous trophoblast cell line HTR-8/SVneo. We also analysed cell ultrastructural changes induced by HF-EMF exposure. Samples were exposed to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1h: SAR of 2 W/kg). Cx mRNA expression was assessed through semi-quantitative RT-PCR, protein expression by Western blotting, protein localization by indirect immunofluorescence, cell ultrastructure using electron microscopy. HF-EMF exposure significantly and selectively increased Cx40 and Cx43, without altering protein expression. Nevertheless, Cx40 and Cx43 lost their punctuate fluorescence within the cell membrane, becoming diffuse after HF-EMF exposure. Electron microscopy evidenced a sharp decrease in intercellular gap junction-like structures. This study is the first to indicate that exposure of extravillous trophoblast to GSM-217 Hz signals can modify Cx gene expression, Cx protein localization and cellular ultrastructure.