| 1. |
- Ortsäter, Henrik, et al.
(författare)
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Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate
- 2007
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 7:17, s. 3105-3115
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.
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| 2. |
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| 3. |
- Sol, E-ri M, et al.
(författare)
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Role of MAPK in apolipoprotein CIII-induced apoptosis in INS-1E cells
- 2009
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Ingår i: Lipids in Health and Disease. - 1476-511X. ; 8, s. 3-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: 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 beta-cell apoptosis. The present study was undertaken to investigate apoptotic mechanisms induced by the apolipoprotein. RESULTS: ApoCIII (10 microg/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 microM) or ERK1/2 inhibitor PD98059 (100 microM) 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. CONCLUSION: It is suggested that elevated circulating apoCIII levels may contribute to beta-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 beta-cell but also for cardiovascular protection.
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| 4. |
- Sundsten, Tea, et al.
(författare)
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Plasma proteome changes in subjects with Type 2 diabetes mellitus with a low or high early insulin response
- 2008
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Ingår i: Clinical Science. - 0143-5221 .- 1470-8736. ; 114:7, s. 499-507
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Tidskriftsartikel (refereegranskat)abstract
- Circulating proteins contribute to the pathogenesis of T2DM (Type 2 diabetes mellitus) in various ways. The aim of the present study was to investigate variations in plasma protein levels in subjects with T2DM and differences in beta-cell function, characterized by the EIR (early insulin response), and to compare these protein levels with those observed in individuals with NGT (normal glucose tolerance). Ten subjects with NGT+high EIR, ten with T2DM+high EIR, and ten with T2DM+low EIR were selected from the community-based ULSAM (Uppsala Longitudinal Study of Adult Men) cohort. Plasma protein profiling was performed using SELDI-TOF (surface-enhanced laser-desorption ionization-time-of-flight) MS. In total, nine plasma proteins differed between the three study groups (P<0.05, as determined by ANOVA). The levels of two forms of transthyretin, haemoglobin alpha-chain and haemoglobin beta-chain were decreased in plasma from subjects with T2DM compared with subjects with NGT, irrespective of the EIR of the subjects. Apolipoprotein H was decreased in plasma from individuals with T2DM+high EIR compared with subjects with NGT. Four additional unidentified plasma proteins also varied in different ways between the experimental groups. In conclusion, the proteins detected in the present study may be related to the development of beta-cell dysfunction.
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| 5. |
- Sundsten, Tea, 1975-
(författare)
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Protein Profiling and Type 2 Diabetes
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
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| 7. |
- Sundsten, Tea, et al.
(författare)
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Proteomics in diabetes research
- 2009
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Ingår i: Molecular and Cellular Endocrinology. - : Elsevier BV. - 0303-7207 .- 1872-8057. ; 297:1-2, s. 93-103
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Forskningsöversikt (refereegranskat)abstract
- Both type 1 and type 2 diabetes mellitus are heterogeneous diseases with alterations in many genes and their products. Not all transcriptional alterations lead to protein changes, which makes it very important to, in conjunction with mRNA expression studies, also address changes in cellular protein levels. Various proteomic techniques are available for measuring many protein changes simultaneously. Many proteomic studies have been performed in the context of diabetes research, with the aims of both describing the healthy tissue and to unravel the complex pathophysiology behind the disease. In addition, effects on proteins induced by different treatments have also been investigated using proteomic approaches. In this paper the field of diabetes proteomics today will be reviewed. Findings from proteomic studies investigating pancreatic islets and beta-cells as well as serum, fat, skeletal muscle and liver are described.
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| 8. |
- Sundsten, Tea, et al.
(författare)
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Serum protein patterns in newly diagnosed type 2 diabetes mellitus--influence of diabetic environment and family history of diabetes.
- 2008
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Ingår i: Diabetes/Metabolism Research Reviews. - : Wiley. - 1520-7552 .- 1520-7560. ; 24:2, s. 148-154
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Individuals with normal glucose tolerance (NGT) and type 2 diabetes mellitus (T2DM) represent heterogeneous groups with differences in beta-cell function and genetic background. The aim of the present study was to compare serum protein profiles of NGT and T2DM individuals and determine the influence of the genetic background versus diabetic environment on differentially displayed proteins. METHODS: Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to compare serum protein profiles of NGT persons and T2DM patients. All participants were from the Stockholm Diabetes Prevention Program (SDPP) cohort. They were selected to have high or low beta-cell function (HOMA-beta) and family history of type 2 diabetes (FHD) or not. RESULTS: Eight proteins were found to be elevated and five lowered (p<0.05) in serum of T2DM patients. In a second comparison, the NGT and T2DM groups were divided into persons with FHD and low HOMA-beta and those without FHD and high HOMA-beta. Three proteins were rediscovered and interpreted to be different due to genetic background. Two of these were identified as apolipoprotein C3 (apoC3) and albumin. Ten proteins were interpreted to be not related to FHD, and one of these was identified as transthyretin. CONCLUSIONS: Using the SELDI-technique, serum protein profiles of NGT and T2DM persons with differences in beta-cell function and FHD were compared. The diabetic environment had a major influence on most of these proteins, while FHD was an important factor for apoC3 and albumin.
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| 9. |
- Sundsten, Tea, et al.
(författare)
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The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes
- 2006
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Ingår i: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 4, s. 22-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
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