| 1. |
- Berggård, Tord, et al.
(författare)
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140 mouse brain proteins identified by Ca2+-calmodulin affinity chromatography and tandem mass spectrometry.
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:3, s. 669-687
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.
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| 2. |
- Björkqvist, Maria, et al.
(författare)
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The R6/2 transgenic mouse model of Huntington's disease develops diabetes due to deficient {beta}-cell mass and exocytosis.
- 2005
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 14:5, s. 565-574
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Tidskriftsartikel (refereegranskat)abstract
- Diabetes frequently develops in Huntington's disease (HD) patients and in transgenic mouse models of HD such as the R6/2 mouse. The underlying mechanisms have not been clarified. Elucidating the pathogenesis of diabetes in HD would improve our understanding of the molecular mechanisms involved in HD neuropathology. With this aim, we examined our colony of R6/2 mice with respect to glucose homeostasis and islet function. At week 12, corresponding to end-stage HD, R6/2 mice were hyperglycemic and hypoinsulinemic and failed to release insulin in an intravenous glucose tolerance test. In vitro, basal and glucose-stimulated insulin secretion was markedly reduced. Islet nuclear huntingtin inclusions increased dramatically over time, predominantly in ß-cells. ß-cell mass failed to increase normally with age in R6/2 mice. Hence, at week 12, ß-cell mass and pancreatic insulin content in R6/2 mice were 35±5 and 16±3% of that in wild-type mice, respectively. The normally occurring replicating cells were largely absent in R6/2 islets, while no abnormal cell death could be detected. Single cell patch-clamp experiments revealed unaltered electrical activity in R6/2 ß-cells. However, exocytosis was virtually abolished in ß- but not in {alpha}-cells. The blunting of exocytosis could be attributed to a 96% reduction in the number of insulin-containing secretory vesicles. Thus, diabetes in R6/2 mice is caused by a combination of deficient ß-cell mass and disrupted exocytosis.
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| 3. |
- Fex, Malin, et al.
(författare)
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A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis.
- 2009
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 52, s. 271-280
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. METHODS: To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. RESULTS: We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. CONCLUSIONS/INTERPRETATION: Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion.
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| 4. |
- Fex, Malin, et al.
(författare)
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{beta}-Cell Lipases and Insulin Secretion.
- 2006
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Ingår i: Diabetes. - 1939-327X. ; 55:Suppl 2, s. 24-31
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Tidskriftsartikel (refereegranskat)abstract
- Lipids have been implicated in ß-cell stimulus-secretion coupling. Thus, lipases in ß-cells would be required to generate coupling factors from intracellular lipids. Indeed, we found that glucose stimulates lipolysis in rodent islets and clonal ß-cells. Lipolysis and diglyceride lipase activity in islets are abolished by orlistat, a pan-lipase inhibitor. Moreover, orlistat dose-dependently inhibits glucose- and forskolin-stimulated insulin secretion, while leaving glucose oxidation and the rise in ATP-to-ADP ratio intact. In an effort to identify ß-cell lipase(s), we found that hormone-sensitive lipase (HSL), the rate-limiting enzyme for acylglyceride hydrolysis in adipocytes, is active in rodent ß-cells. To further address the role of HSL, a global and ß-cell–specific inactivation, respectively, of the lipase has been created in mice. Whereas our line of HSL null mice is moderately glucose intolerant due to reduced peripheral insulin sensitivity, it exhibits normal islet metabolism and insulin secretion. Preliminary analysis of the ß-cell–specific HSL knockout has revealed no evidence for disturbed islet function. Thus, studies of ours and others indicate that there is a complex lipid regulatory component in ß-cell stimulus-secretion coupling. The role of HSL and other lipases needs to be further clarified to provide a balanced view of the role of lipids and lipolysis in ß-cells.
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| 5. |
- Fex, Malin, et al.
(författare)
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Enhanced mitochondrial metabolism may account for the adaptation to insulin resistance in islets from C57BL/6J mice fed a high-fat diet.
- 2007
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 50:1, s. 74-83
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Tidskriftsartikel (refereegranskat)abstract
- Aim/hypothesis Hyperinsulinaemia maintains euglycaemia in insulin-resistant states. The precise cellular mechanisms by which the beta cells adapt are still unresolved. A peripherally derived cue, such as increased circulating fatty acids, may instruct the beta cell to initiate an adaptive programme to maintain glucose homeostasis. When this fails, type 2 diabetes ensues. Because mitochondria play a key role in beta cell pathophysiology, we tested the hypothesis that mitochondrial metabolism is critical for beta cell adaptation to insulin resistance. Methods C57BL/6J mice were given high-fat (HF) diet for 12 weeks. We then analysed islet hormone secretion, metabolism in vivo and in vitro, and beta cell morphology. Results HF diet resulted in insulin resistance and glucose intolerance but not frank diabetes. Basal insulin secretion was elevated in isolated islets from HF mice with almost no additional response provoked by high glucose. In contrast, a strong secretory response was seen when islets from HF mice were stimulated with fuels that require mitochondrial metabolism, such as glutamate, glutamine, alpha-ketoisocaproic acid and succinate. Moreover, while glucose oxidation was impaired in islets from HF mice, oxidation of glutamine and palmitate was enhanced. Ultrastructural analysis of islets in HF mice revealed an accumulation of lipid droplets in beta cells and a twofold increase in mitochondrial area. Conclusions/interpretation We propose that beta cells exposed to increased lipid flux in insulin resistance respond by increasing mitochondrial volume. This expansion is associated with enhanced mitochondrial metabolism as a means of beta cell compensation. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00125-006-0464-4 and is accessible to authorised users.
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| 6. |
- Fex, Malin, et al.
(författare)
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Lipases in the pancreatic beta-cell: implications for insulin secretion.
- 2008
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Ingår i: Biochemical Society Transactions. - 0300-5127. ; 36:Pt 5, s. 885-890
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Tidskriftsartikel (refereegranskat)abstract
- Lipids have been implicated in beta-cell stimulus-secretion coupling. In such a role, lipases in beta-cells would be required to generate lipid coupling factors. We have shown previously that glucose stimulates lipolysis in rodent islets. In addition, lipolysis and diacylglycerol lipase activity in islets are abolished by orlistat, an irreversible lipase inhibitor with a broad specificity for substrates. Moreover, orlistat dose-dependently inhibits glucose- and forskolin-stimulated insulin secretion, while leaving glucose oxidation and the rise in the ATP/ADP ratio intact. In an effort to identify beta-cell lipase(s), we found that HSL (hormone-sensitive lipase), the rate-limiting enzyme for acylglycerol hydrolysis in adipocytes, is expressed in rodent beta-cells. To resolve the role of this lipase, we have created global and beta-cell-specific knockout mice. Although our line of global HSL-knockout mice is moderately glucose-intolerant owing to reduced peripheral insulin sensitivity and exhibits normal islet metabolism and insulin secretion, other HSL-knockout lines have displayed impaired insulin secretion under certain conditions. In contrast, beta-cell-specific HSL-knockout mice, which are less prone to genetic redundancy, are hyperglycaemic, presumably caused by a perturbation of first-phase insulin secretion. Thus studies by us and others demonstrate that lipases, such as HSL, play a regulatory role in beta-cell stimulus-secretion coupling.
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| 7. |
- Fex, Malin
(författare)
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Lipid metabolism in the pancreatic beta-cell. Implications for insulin secretion.
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We have investigated the role of lipid metabolism with regard to beta-cell function and insulin secretion. Lipids are known to play a crucial functional role in the pancreatic beta-cell, where they are essential for adequate hormone release, but may also exert a long-term toxic effect, leading to beta-cell dysfunction. Our studies in mice, where insulin resistance and glucose intolerance was induced by high fat diet, showed that beta-cells compensate by increasing mitochondrial mass and hence shift oxidation from glucose to other fuels, such as amino acids and free fatty acids. This process is likely a means to maintain euglycemia, and if it fails diabetes will evolve. We have studied the consequences of a targeted inactivation of a key enzyme in lipolysis: hormone sensitive lipase (HSL). To this end, both a global knock out of HSL and a beta-cell specific KO of the enzyme (beta-HSL KO) were created. In theory, disruption of HSL could cause an accumulation of lipids, inducing cellular toxicity, as well as having an effect on overall energy homeostasis. Additionally, insulin secretion from beta-cells could be compromised due to abrogation of an essential lipid signal normally provided by HSL. In the global KO of HSL, we found that ablation of HSL causes insulin resistance in skeletal muscle, adipose tissue and liver; accumulation of diglyceride was observed in adipose tissue. However, lack of HSL did not have an effect on insulin secretion in the global KO mouse of HSL. This may be due to compensatory mechanisms. By contrast, in our beta-HSL KO, an ablation of the protein made a strong impact on insulin secretion both in vivo and in vitro. beta-HSL KO mice were hyperglycemic, and the first phase of insulin secretion was selectively affected. Furthermore, an increase in adipose mass in beta-HSL KO mice, accompanied by a rise in plasma leptin levels, as well as increased peripheral insulin sensitivity, indicates crosstalk between tissues involved in metabolic control. We hereby conclude that HSL is a key enzyme in overall glucose homeostasis. Its pivotal role may be attributed to the provision of lipid-derived signals essential for control of insulin release.
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| 8. |
- Fex, Malin, et al.
(författare)
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Rat insulin promoter 2-Cre recombinase mice bred onto a pure C57BL/6J background exhibit unaltered glucose tolerance
- 2007
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Ingår i: Journal of Endocrinology. - 1479-6805. ; 194:3, s. 551-555
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Tidskriftsartikel (refereegranskat)abstract
- beta-Cell-specific gene targeting is a widely used tool when studying genes involved in beta-cell function. For this purpose, several conditional beta-cell knockouts have been generated using the rat insulin promoter 2-Cre recombinase (RIP2-Cre) mouse. However, it was recently observed that expression of Cre alone in P-cells may affect whole body glucose homeostasis. Therefore, we investigated glucose homeostasis, insulin secretion, and beta-cell mass in our line of RIP2-Cre mice bred onto the C57BL/6J genetic background. We used 12- and 28-week-old female RIP2-Cre mice for analyses of insulin secretion in vitro, glucose homeostasis in vivo and beta-cell mass. Our mouse line has been backcrossed for 14 generations to yield a near 100% pure C57BL/6J background. We found that fasting plasma glucose and insulin levels were similar in both genotypes. An i.v. glucose tolerance test revealed no differences in glucose clearance and insulin secretion between 12-week-old RIP2-Cre and WT mice. Moreover, insulin secretion hi vitro in islets isolated from 28-week-old RIP2-Cre mice and controls was similar. In addition, beta-cell mass was not different between the two genotypes at 28 weeks of age. In our experiments, we observed no differences in glucose tolerance, insulin secretion in vivo and in vitro, or in beta-cell mass between the genotypes. As our RIP2-Cre mice are on a near 100% pure genetic background (C57BL/6J), we suggest that the perturbations in glucose homeostasis previously reported in RIP2-Cre mouse lines can be accounted for by differences in genetic background.
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| 9. |
- Lyssenko, Valeriya, et al.
(författare)
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Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired early insulin secretion.
- 2009
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 41:1, s. 82-88
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Tidskriftsartikel (refereegranskat)abstract
- Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in beta cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal beta cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on beta cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.
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| 10. |
- Resjö, Svante, et al.
(författare)
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Proteomic studies in animal models of diabetes
- 2008
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Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 2:5, s. 654-669
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Forskningsöversikt (refereegranskat)abstract
- The aim of this review is to provide an overview of proteomic studies in animal models of diabetes and to give some insight into the different methods available today in the rapidly developing field of proteomics. A summary of 31 papers published between 1997 and 2007 is presented. For instance, proteomics has been used to study the development of both type 1 and type 2 diabetes, diabetic complications in tissues like heart, kidney and retina and changes after treatment with anti-diabetic drugs like peroxisome proliferator-activated receptors agonists. Together, these studies give a good overview of a number of experimental approaches. Proteomics holds the promise of providing major contributions to the field of diabetes research. However, to achieve this, a number of issues need to be resolved. Appropriate data representation to facilitate data comparison, exchange, and verification is required, as well as improved statistical assessment of proteomic experiments. In addition, it is important to follow up the results with functional studies to be able to make biologically relevant conclusions. The potential of proteomics to dissect complex human disorders is now beginning to be realized. In the future, this will result in new important information concerning diabetes.
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