A proteomics-based approach to investigate proteins associated with adipogenesis
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. Adipose tissue has recently been shown to play an important role in the regulation of energy metabolism. Malfunction of adipose tissue is one of the major causes-of insulin resistance and its associated complications, such as type 2 diabetes and cardiovascular disease. Studies of adipocytes are therefore important. In this study, a differential proteome mapping strategy was used to identify intracellular proteins whose expression was substantially altered during conversion of mouse 3T3-L1 preadipocytes to adipocytes. Two-dimensional gel electrophoresis analysis identified ten proteins, which were induced following hormone-insulin evoked differentiation. Nine of the ten proteins were found to have up-regulated expression or were predominantly expressed in adipocytes, and one of these was a novel protein. One of the ten proteins was diminished and identified as an ?2 macroglobulin-like protein fragment. The novel protein and the diminished protein were then further investigated in this study. The first protein chosen for further study was the novel protein. We called it Fat tissue-specific Low molecular weight Protein (FALP). It was preferentially expressed in adipocytes but not in preadipocytes. The falp cDNA was cloned both from mouse (accession number AY079153) and two isoforms from human (accession numbers AY078152 and AF4835) using degenerate PCR. Northern blot analysis showed that the falp gene was predominantly expressed in brown and white fat tissue. Human homologues of mouse FALP were found to exist as two alternatively spliced isoforms, which shared the same NH2-terminus but with different COOH-termini. A sequence homology search revealed that FALP did not share sequence identity with any genes of known function. Both human and mouse FALP contained a conserved putative transmembrane domain. Immunocytochemistry analyses suggested that FALP localized at mitochondria. It interacted with glutaryl CoA dehydrogenase as demonstrated by in vitro pull-down and in vivo co-immunoprecipitation. These data suggested FALP might be involved in fatty acid metabolism during adipogenesis. The second protein studied was the ?2 macroglobulin-like protein fragment. It was found to be present in very high abundance in 3T3-L1 preadipocytes. It was dramatically decreased after hormone-insulin induced differentiation of preadipocytes to adipocytes. Analysis by metabolic radiolabelling with [35S] methionine indicated that the intracellular accumulation of the ?2 macroglobulin fragment was derived from the extracellular medium and not by de novo synthesis. A similar phenomenon also occurred in primary preadipocytes (stromal-vascular cells) and adipocytes isolated from mouse peri-uterine fat pads. Incubation of 3T3-L1 preadipocytes with an anti-?2 macroglobulin neutralising antibody caused depletion of the intracellular ?2 macroglobulin fragment, and also enhanced spontaneous adipocyte differentiation. These results suggest that: 1. Intracellular accumulation of the ?2 macroglobulin fragment could inhibit adipocyte differentiation, and 2. Differentiation can be induced, at least in part by suppressing the activity of intracellular ?2 macroglobulin in 3T3-L1 preadipocytes. In summary, the results of this work have identified two new regulatory mechanisms related to the conversion of preadipocytes into adipocytes, and the regulation of adipocyte metabolism.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/2004