Proteomic and phosphoproteomic analysis of the effect of exendin-4 treatment on the rat pancreatic beta cell line ins-1

Diabetes is a major worldwide health problem, and the incidence is increasing. Although the etiology of types 1 and 2 diabetes is different, the developmental complications of both types involve destruction of pancreatic beta cells. Enhancing or inducing pancreatic beta cell proliferation is thus a focus of much research. Exendin-4, a GLP-1 analogue, was introduced into the clinic in 2005 as an inducer of insulin secretion, but also has proliferative effects on pancreatic beta cells in vitro. These effects have been characterized in targeted studies, identifying specific proteins in particular signaling pathways. In our study, we aimed to carry out global proteomic and phosphoproteomic relative quantitation screening to identify the proteins and their phosphorylation sites regulated by exendin-4 treatment in rat ins-1 cells, compared to their untreated counterparts. We were able to quantify 3766 proteins among which 667 proteins were significantly regulated. In addition, we quantified 2170 phosphosites, 400 of which were significantly regulated. The significantly regulated proteins and phosphosites after exendin-4 treatment pointed to a protective role for this peptide in maintaining cell survival and inhibiting apoptosis due to the stress induced by the serum starvation, which is in agreement of previous reports. This thesis thus provides the first global proteomic analysis of the effects of exendin-4 on the rat pancreatic beta cells. Further studies employing different culture conditions and knockdown or overexpression of candidate proteins are now required to give deeper insight into the pathways involved.Open Acces

Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151

Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151