The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

BACKGROUND: The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. CONCLUSION: Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed

Role of MAPK in apolipoprotein CIII-induced apoptosis in INS-1E cells

<p>Abstract</p> <p>Background</p> <p>Individuals with type 2 diabetes mellitus (T2DM) have elevated levels of circulating apolipoprotein CIII (apoCIII). ApoCIII plays an important role for plasma triglyceride levels and elevated levels of the apolipoprotein have been connected with dyslipidemia in T2DM subjects. In addition, apoCIII has been linked to enhanced β-cell apoptosis. The present study was undertaken to investigate apoptotic mechanisms induced by the apolipoprotein.</p> <p>Results</p> <p>ApoCIII (10 μg/ml) enhanced apoptosis 2-fold in insulin-producing INS-1E cells after 24 hours exposure to the apolipoprotein. At this time point phosphorylation of mitogen activated protein kinase (MAPK) p38 had doubled but ERK1/2 and JNK were not activated. Instead, ERK1/2 showed rapid and transient phosphorylation (2-fold after 0.5 hour). No JNK phosphorylation was observed. In support of a role of activation of not only p38 but also ERK1/2 in apoCIII-induced apoptosis, inclusion of p38 inhibitor SB203580 (10 μM) or ERK1/2 inhibitor PD98059 (100 μM) normalized apoptosis. Whereas influx of Ca²⁺was linked to apoCIII-induced ERK1/2 activation, pro-apoptotic protein CHOP/GADD of the unfolded protein response (UPR) was not affected by apoCIII.</p> <p>Conclusion</p> <p>It is suggested that elevated circulating apoCIII levels may contribute to β-cell apoptosis via activation of p38 and ERK1/2 in individuals with T2DM. Therapies aiming at normalizing levels of apoCIII could be beneficial not only for the function of the β-cell but also for cardiovascular protection.</p

Protein Profiling and Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously. In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment. When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function