Ewa Szalowska

Characterization of the Human Visceral Adipose Tissue Secretome

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48–114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing l-[13C6,15N2]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with ≥99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.

Resistin Is More Abundant in Liver Than Adipose Tissue and Is Not Up-Regulated by Lipopolysaccharide

CONTEXT Resistin is an adipokine correlated with inflammatory markers and is predictive for cardiovascular diseases. There is evidence that serum resistin levels are elevated in obese patients; however, the role of resistin in insulin resistance and type 2 diabetes remains controversial. OBJECTIVE We addressed the question of whether inflammation may induce expression of resistin in organs involved in regulation of total body energy metabolism, such as liver and adipose tissue (AT). METHODS Human liver tissue, sc AT, and omentum were cultured in the absence/presence of lipopolysaccharide (LPS). The resistin and cytokine mRNA and protein expression levels were determined by real-time PCR, ELISA, and Multiplex Technology, respectively. The localization of resistin in human liver was analyzed by immunohistochemistry. RESULTS Resistin gene and protein expression was significantly higher in liver than in AT. Exposure of human AT and liver tissue in culture to LPS did not alter resistin concentration; however, concentrations of IL-1beta, IL-6, and TNFalpha were significantly increased in these tissues. In liver, resistin colocalizes with markers for Kupffer cells, for a subset of endothelial and fibroblast-like cells. CONCLUSIONS High level of resistin gene and protein expression in liver compared to AT implies that resistin should not be considered only as an adipokine in humans. LPS-induced inflammation does not affect resistin protein synthesis in human liver and AT. This suggests that elevated serum resistin levels are not indicative for inflammation of AT or liver in a manner similar to known inflammatory markers such as IL-1beta, IL-6, or TNFalpha.

Fractional Factorial Design for Optimization of the SELDI Protocol for Human Adipose Tissue Culture Media

The early factors inducing insulin resistance are not known. Therefore, we are interested in studying the secretome of the human visceral adipose tissue as a potential source of unknown peptides and proteins inducing insulin resistance. Surface‐enhanced laser desorption/ionization time‐of‐flight (SELDI‐TOF) mass spectrometry is a high‐throughput proteomics technology to generate peptide and protein profiles (MS spectra). To obtain good quality and reproducible data from SELDI‐TOF, many factors in the sample pretreatment and SELDI protocol should be optimized. To identify the optimal combination of factors resulting in the best and the most reproducible spectra, we designed an experiment where factors were varied systematically according to a fractional factorial design. In this study, seven protein chip preparation protocol factors were tested in 32 experiments. The main effects of these factors and their interactions contributing to the best quality spectra were identified by ANOVA. To assess the reproducibility, in a subsequent experiment the eight protocols generating the highest quality spectra were applied to samples in quadruplicates on different chips. This approach resulted in the development of an improved chip protocol, yielding higher quality peaks and more reproducible spectra.

Comparative analysis of the human hepatic and adipose tissue transcriptomes during LPS-induced inflammation leads to the identification of differential biological pathways and candidate biomarkers

BackgroundInsulin resistance (IR) is accompanied by chronic low grade systemic inflammation, obesity, and deregulation of total body energy homeostasis. We induced inflammation in adipose and liver tissues in vitro in order to mimic inflammation in vivo with the aim to identify tissue-specific processes implicated in IR and to find biomarkers indicative for tissue-specific IR.MethodsHuman adipose and liver tissues were cultured in the absence or presence of LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for secretome were performed using publicly available bioinformatics tools (DAVID, STRING, SecretomeP). The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome.ResultsLPS treatment significantly affected 667 and 483 genes in adipose and liver tissues respectively. The GO analysis revealed that during inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes and the remaining genes were the differential candidate biomarkers indicative for inflamed adipose or liver tissue. The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data.ConclusionsThe higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver tissue, suggests that adipose tissue is the major organ contributing to the development of systemic inflammation observed in IR. The identified tissue-specific functional clusters and biomarkers might be used in a strategy for the development of tissue-targeted treatment of insulin resistance in patients.

Sub-chronic administration of stable GIP analog in mice decreases serum LPL activity and body weight

GIP receptor knockout mice were shown to be protected from the development of obesity on a high fat diet, suggesting a role of GIP in the development of obesity. In our study we aimed to test the hypothesis if excess of GIP could accelerate development of obesity and to identify GIP gene targets in adipose tissue. Therefore, mice were kept on a chow or a high fat diet and during the last 2 weeks D-Ala(2)-GIP or PBS injections were performed. Afterwards, serum LPL activity and several biochemical parameters (TG, FFA, cholesterol, glucose, insulin, resistin, IL-6, IL-1β, TNFα, GIP) were measured. Fat tissue was isolated and QPCR was performed for a set of genes involved in energy metabolism and inflammation. A DNA-microarray was used to identify GIP gene targets in adipose tissue of the chow diet group. We found that the D-Ala(2)-GIP injections caused a significant decrease in both body weight and LPL activity compared to controls. Serum biochemical parameters were not affected by D-Ala(2)-GIP, with an exception for resistin and insulin. The set of inflammatory genes were significantly decreased in adipose tissue in the D-Ala(2)-GIP injected animals on a chow diet. A DNA-microarray revealed that APO-genes and CYP-genes were affected by D-Ala(2)-GIP treatment in adipose tissue. These results suggest that the body weight-reducing effect of D-Ala(2)-GIP may be explained by lower LPL activity and insulin serum level. Moreover, the identified GIP candidate gene targets in adipose tissue link GIP action to lipid metabolism exerted by APO and CYP genes.

TUB gene expression in hypothalamus and adipose tissue and its association with obesity in humans

Background/Objectives:Mutations in the Tubby gene (TUB) cause late-onset obesity and insulin resistance in mice and syndromic obesity in humans. Although TUB gene function has not yet been fully elucidated, studies in rodents indicate that TUB is involved in the hypothalamic pathways regulating food intake and adiposity. Aside from the function in central nervous system, TUB has also been implicated in energy metabolism in adipose tissue in rodents. We aimed to determine the expression and distribution patterns of TUB in man as well as its potential association with obesity.Subjects/Methods:In situ hybridization was used to localize the hypothalamic regions and cells expressing TUB mRNA. Using RT–PCR, we determined the mRNA expression level of the two TUB gene alternative splicing isoforms, the short and the long transcript variants, in the hypothalami of 12 obese and 12 normal-weight subjects, and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissues from 53 severely obese and 24 non-obese control subjects, and correlated TUB expression with parameters of obesity and metabolic health.Results:Expression of both TUB transcripts was detected in the hypothalamus, whereas only the short TUB isoform was found in both VAT and SAT. TUB mRNA was detected in several hypothalamic regions involved in body weight regulation, including the nucleus basalis of Meynert and the paraventricular, supraoptic and tuberomammillary nuclei. We found no difference in the hypothalamic TUB expression between obese and control groups, whereas the level of TUB mRNA was significantly lower in adipose tissue of obese subjects as compared to controls. Also, TUB expression was negatively correlated with indices of body weight and obesity in a fat-depot-specific manner.Conclusions:Our results indicate high expression of TUB in the hypothalamus, especially in areas involved in body weight regulation, and the correlation between TUB expression in adipose tissue and obesity. These findings suggest a role for TUB in human obesity.