Mass spectrometry for measuring protein secretion from insulin sensitive tissues during obesity

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Copyright: Wu, Lindsay Edward
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Abstract
Due to the accessibility of the serum relative to other areas of the body, secretory proteins afford the best opportunity for diagnosis and treatment of disease. Identification of secretory proteins from cell culture conditioned media samples via a mass spectrometry approach has been attempted in the past. Contamination of conditioned media with non-secretory proteins released by necrotic cells dilutes the detection of low-abundance secretory proteins and has thwarted these previous attempts. To circumvent this problem, a lectin affinity chromatography approach was used to exploit N-glycosylation, a post-translational modification common to secretory proteins but largely absent from non-secretory proteins. This approach was used to survey the secretome of multiple insulin sensitive tissues with a focus on adipocytes, which play an integral role in the endocrine system. The anti-angiogenic molecule pigment epithelial derived factor was identified as an adipocyte secretory factor, and an important role for this protein in obesity induced insulin resistance was ascertained. Lectin affinity chromatography was combined with partial metabolic labelling and mass spectrometry to quantitatively measure protein secretion from tissue explants and primary cultures. This allowed a direct comparison of the secretory abilities of visceral and subcutaneous adipose tissue and their constituent cell types, including endothelial cells, for which little is known of their secretory repertoire. Intrinsic differences in secretory ability were observed between visceral and subcutaneous derived samples, which may help explain the metabolically deleterious effects of visceral, but not subcutaneous, adipose tissue. Finally in this investigation, conditioned media experiments were used in combination with quantitative mass spectrometry to identify a role for hypoxia-mediated secretion of fatty acid binding protein 4 in the adipocyte – β cell axis. This observation fills an important gap in knowledge regarding insulin measurements during obesity, as it indicates the elevated levels of insulin observed during obesity are not simply due to compensation for insulin resistance. This work has uncovered yet another important endocrine role for adipose tissue. From this work it is becoming clear that insulin levels should not be taken at face value, and are the result of an inter-organ communication network.
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Author(s)
Wu, Lindsay Edward
Supervisor(s)
James, David
Guilhaus, Michael
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Publication Year
2010
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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