Year: 2011
Type: dissertation
Abstract: Obesity and its co-morbidities have developed into a major health threat.Due to the infiltration of macrophages, obese adipose tissue is characterized by chronic low-grade inflammation.Suganami, et al. (1) suggested the existence of a paracrine loop between adipocytes and macrophages, which aggravates coculture-induced changes.Moreover, free fatty acids and TNFα were identified as major components of this vicious cycle.The aim of this master thesis was to use an in vitro coculture system comprising 3T3-L1 adipocytes and RAW264.7 macrophages in order to study the cell-cell communication.This coculture system was established successfully for multiple well formats and up-scaled for protein harvest.A particular emphasis lay on enabling a proteome-wide detection of intra-cellular communication by mass spectrometry.Moreover, 24 hours of incubation proved to be optimal for MS analysis.Furthermore, biomarkers including Adiponectin, TNFα, MCP-1, IL-6, Cox-2 and FFAs were used to study the adipocyte-macrophage communication on the RNA expression and metabolic level.In Addition, the phosphorylation state of JNK, an inflammatory key molecule, and its target molecules including cJUN and IRS-1 were studied by western blotting.Based on the performed experiments, it was proven that coculture of adipocytes and macrophages resulted in major changes concerning RNA expression and protein levels.A set of biomarkers was established to study coculture-induced effects.Furthermore, phosphorylation of JNK and cJun, was induced under cocultural conditions.In a SDS-Page-based approach on average 235 proteins were identified per slice.Several important proteins including LPL, perilipin, Cypf2f2 and TLR2 and various metabolic pathways (TCA cycle, pentose phosphate pathways, oxidative phosphorylation) were identified.Coculture experiments in an up-scaled system were successfully combined with stable isotope labeling with amino acids in cell culture (SILAC).Thereby, three glycolytic enzymes were found up-regulated in RAW264.7 macrophages after 8 hours of coculture.
Source: MPG.PuRe (Max Planck Society)
Author: Annabell Witzke
Language: English
Subfield: Endocrinology, Diabetes and Metabolism
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W647232244