Cell surface proteins in the adipose tissue
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1 Research Collection Doctoral Thesis Cell surface proteins in the adipose tissue Author(s): Möst, Hansjörg Publication Date: 2012 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library
2 DISS. ETH NO CELL SURFACE PROTEINS IN THE ADIPOSE TISSUE A dissertation submitted to ETH ZURICH for the degree of DOCTOR OF SCIENCES presented by HANSJÖRG MÖST Dipl. Biotech., Ecole Supérieure de Biotechnologie Strasbourg born May 29 th 1982 citizen of Germany accepted on the recommendation of Prof. Dr. Christian Wolfrum Dr. Bernd Wollscheid Prof. Dr. Ruedi Aebersold Prof. Dr. Christoph Meier 2012
3 Abstract Abstract In the 21 st century obesity is recognized as one of the most serious health problems increasing the risks for secondary disorders like type 2 diabetes, atherosclerosis and cardiovascular diseases. Maintenance of normal adipose tissue function is important for whole body metabolism. Increased triglyceride accumulation in the adipocytes caused by a misbalance between energy intake and energy consumption, results in increased adipocyte size, leading to excess adipose tissue, and to increased body weight and obesity. It is well established that larger adipocytes possess several malfunctions like for example decreased insulin sensitivity, contributing to overall decrease in insulin sensitivity and eventually insulin resistance, as a key factor for the development of type 2 diabetes. An underlying molecular cause for dysfunctional adipocyte behavior is that these cells are not able to properly integrate and respond to signals from their environment anymore. Cell surface proteins (referred to as surfaceome) are key players in the cellular interface to the microenvironment, which both enable and limit the adipocyte signaling capacity. Therefore, quantitative changes in the adipocyte surfaceome are suspected to be responsible for the dysfunctional phenotype of adipocytes in the context of obesity. Here, I established and quantitatively compared surfaceome maps of primary adipocytes derived from different mouse models of metabolic disorders. By using the Cell Surface Capturing (CSC) technology I succeeded in the identification and label-free quantification of hundreds of cell surface proteins including detection of clinically interesting obesity-induced surfaceome changes. The differentially regulated cell surface proteins detected in the proteomic surfaceome screen were functionally investigated individually in RNA interference (RNAi) experiments. Adiponectin secretion and lipolytic activity assays of adipocytes upon RNAi treatment demonstrated the influence of certain cell surface proteins on these adipocyte functions. These results indicate that orchestrating their activity could improve or prevent adipocyte malfunction. In the second part of the presented work, cell surface proteins were investigated as potential novel markers for adipocyte progenitor identification and isolation. The mature white and brown adipocytes are derived from adipocyte progenitors which reside in the white and brown adipose tissue (WAT and BAT) depots. These preadipocytes are located in the stromal-vascular fraction (SVF) of the adipose tissue which is a heterogeneous cell population including mesenchymal stem cell, macrophages, T-cells and endothelial cells but is depleted from mature adipocytes. Due to the cellular heterogeneity of the adipose tissue depots and iii
4 Abstract the current lack of appropriate cell surface marker proteins, these progenitor cells remain largely uncharacterized. In order to find new marker candidates, the cell surface proteome landscape of the heterogeneous SVF of different adipose tissue depots was investigated. By a discovery driven mass spectrometry approach over 200 cell surface proteins were identified serving as novel marker candidates for adipocyte progenitors. Four cell surface proteins of the dataset, namely OX-2 membrane glycoprotein, melanoma cell adhesion molecule, fatty acid translocase and alanyl aminopeptidase were tested for their isolation efficiency for adipocyte progenitors from the SVF of brown adipose tissue in an in vitro differentiation assay system. The subsequent functional screening experiments upon progenitor cell isolation using these markers showed evidence for alanyl aminopeptidase as a promising cell surface marker for brown and white adipocyte progenitor cells. Together, my research led to generation of clinically useful surfaceome maps of adipocytes in the context of obesity, and to the discovery of alanyl aminopeptidase as a new marker for the isolation of adipocyte progenitors facilitating their future characterization. iv
5 Zusammenfassung Zusammenfassung Adipositas erhöht das Risiko für Folgeerkrankungen wie Typ-2-Diabetes, Arteriosklerose und Herz-Kreislauf-Erkrankungen und ist eines der gravierendsten Gesundheitsprobleme im 21. Jahrhundert. Das Fettgewebe besitzt eine Schlüsselrolle in der Regulation des Stoffwechsels im ganzen Körper und es ist deshalb wichtig seine einwandfreie Funktionsweise aufrechtzuerhalten. Überschüssiges Fettgewebe kann zur fehlerhaften Adipozytenfunktion führen wie zum Beispiel der Insulin-Resistenz welche wiederum zur Entwicklung von Typ-2- Diabetes beiträgt. Mit anderen Worten dysfunktionale Adipozyten sind nicht mehr in der Lage, Signale aus ihrer Umgebung richtig zu integrieren und entsprechend darauf zu reagieren. Wichtigster Bestandteil für die Integration von Signalen aus der Umgebung sind Zelloberflächenproteine. Aus diesem Grund hatten wir die Vermutung, dass veränderte Expressionsmuster der Oberflächenproteine verantwortlich sind für den dysfunktionalen Phänotyp des Adipozyten. In der vorliegenden Arbeit haben wir die Expressionsmuster von Zelloberflächenproteinen von primären Adipozyten aus verschiedenen Mausmodellen für Stoffwechselerkrankungen bestimmt. Per massenspektrometrischer Analyse konnten wir hunderte Oberflächenproteine quantifizieren und haben tatsächlich Veränderungen in den verwendeten Mausmodellen beobachtet. Den Einfluss der veränderten Oberflächenproteine auf zwei wichtige Adipozytenfunktionen nämlich die Sekretion von Adiponectin und die Mobilisierung von Triglyceriden haben wir in funktionellen RNA-Interferenz Experimenten überprüft und teilweise bestätigt. Die gefundenen Proteine tragen somit einen Teil zur Fehlfunktion der Adipozyten bei und eine Einflussnahme auf die Aktivität dieser Proteine könnte die Adipozytenfehlfunktion verbessern oder sogar verhindern. Im zweiten Teil der Arbeit haben wir nach Markern für die Isolation von Adipozytenvorläufer gesucht. Die Vorläuferzellen für Adipozyten befinden sich in der stromalen vaskulären Fraktion des Fettgewebes welche viele verschiedene Zelltypen beinhaltet wie mesenchymale Stammzellen, Endothelzellen, Makrophagen und T-Zellen. Die Bildung von reifen Adipozyten hängt von diesen Vorläuferzellen ab. Aufgrund der Zellheterogenität und dem Mangel an vernünftigen Markern sind diese Vorläuferzellen nur mangelhaft charakterisiert. Um neue Markermoleküle zu finden, haben wir die Zusammensetzung der Oberflächenproteine von der stromalen vaskulären Fraktion von verschiedenen Fettdepots untersucht. Mit Hilfe eines massenspektrometrischen Ansatzes konnten wir über 200 Oberflächenproteine identifizieren, welche auch als potentielle Marker v
6 Zusammenfassung in Betracht kommen. Vier von den identifizierten Proteinen haben wir für die Isolation von Adipozytenvorläufern getestet. Die Alanyl Aminopeptidase einer von den Markerkandidaten war in der Lage, Adipozytenvorläuferzellen erfolgreich anzureichern. Die Quintessenz meiner Arbeit ist erstens die Aufklärung von klinisch relevanten Veränderungen von Zelloberflächenproteine in dysfunktionalen Adipozyten im Kontext der Adipositas und zweitens die Entdeckung eines Markers nämlich der Alanyl Aminopeptidase für die Isolation von Adipozyten-Vorläuferzellen aus der stromalen vaskulären Fraktion des Fettgewebes. vi
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