| 1. |
- Amisten, Stefan, et al.
(författare)
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A comparative analysis of human and mouse islet G-protein coupled receptor expression
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- G-protein coupled receptors (GPCRs) are essential for islet function, but most studies use rodent islets due to limited human islet availability. We have systematically compared the GPCR mRNA expression in human and mouse islets to determine to what extent mouse islets can be used as surrogates for human islets to study islet GPCR function, and we have identified species-specific expression of several GPCRs. The A 3 receptor (ADORA3) was expressed only in mouse islets and the A 3 agonist MRS 5698 inhibited glucose-induced insulin secretion from mouse islets, with no effect on human islets. Similarly, mRNAs encoding the galanin receptors GAL 1 (GALR1), GAL 2 (GALR2) and GAL 3 GALR3) were abundantly expressed in mouse islets but present only at low levels in human islets, so that it reads (GALR3) and galanin inhibited insulin secretion only from mouse islets. Conversely, the sst1 receptor (SSTR1) was abundant only in human islets and its selective activation by CH 275 inhibited insulin secretion from human islets, with no effect on mouse islets. Our comprehensive human and mouse islet GPCR atlas has demonstrated that species differences do exist in islet GPCR expression and function, which are likely to impact on the translatability of mouse studies to the human context.
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| 2. |
- Amisten, Stefan, et al.
(författare)
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Activation of imidazoline receptor I-2, and improved pancreatic beta-cell function in human islets
- 2018
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Ingår i: Journal of Diabetes and Its Complications. - : Elsevier BV. - 1056-8727. ; 32:9, s. 813-818
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Tidskriftsartikel (refereegranskat)abstract
- Aim: The impact of BL11282, an imidazoline receptor (NISCH) agonist, on potentiation of glucose-stimulated insulin secretion (GSIS) from isolated human non-diabetic (ND) and type 2 diabetic (T2D) islets was investigated. Methods: Analysis of mRNA was performed by RNA-sequencing and qPCR Insulin and cAMP by RIA and EUSA respectively. Results: RNA-sequencing data revealed that NISCH is highly expressed in fat tissues, islets, liver and muscles, with eight detectable splice variants of transcripts in islets. NISCH had a positive correlation with GLP-1 (GLP1R) and GIP (GIPR) receptor transcripts. The expression of NISCH was confirmed by qPCR in human islets. NISCH and GLP1R were comparably higher expressed in mouse islets compared to human islets. GSIS was dose-dependently potentiated by BL11282 from incubated islets of ND and T2D human islet donors. The insulinotropic action of BL11282 was associated with increased cAMP. While the harmful effect of high glucose on reductive capacity of islet cells was enhanced by glibenclamide during long-term culture, it was counteracted by BL11282 or Bt2-cAMP. BL11282 also increased proliferation of INS-1 cells during long-time culture. Conclusion: Our data suggest that BL11282 potentiates GSIS by an action involving cAMP/PKA system and BL11282 could be an attractive insulinotropic and beta-cell protective agent. (C) 2018 Elsevier Inc. All rights reserved.
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| 5. |
- Amisten, Stefan, et al.
(författare)
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ADP mediates inhibition of insulin secretion by activation of P2Y13 receptors in mice.
- 2010
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; Jul 1, s. 1927-1934
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESES: To investigate the effects of extracellular purines on insulin secretion from mouse pancreatic islets. METHODS: Mouse islets and beta cells were isolated and examined with mRNA real-time quantification, cAMP quantification and insulin and glucagon secretion. ATP release was measured in MIN6c4 cells. Insulin and glucagon secretion were measured in vivo after glucose injection. RESULTS: Enzymatic removal of extracellular ATP at low glucose levels increased the secretion of both insulin and glucagon, while at high glucose levels insulin secretion was reduced and glucagon secretion was stimulated, indicating an autocrine effect of purines. In MIN6c4 cells it was shown that glucose does induce release of ATP into the extracellular space. Quantitative real-time PCR demonstrated the expression of the ADP receptors P2Y(1) and P2Y(13) in both intact mouse pancreatic islets and isolated beta cells. The stable ADP analogue 2-MeSADP had no effect on insulin secretion. However, co-incubation with the P2Y(1) antagonist MRS2179 inhibited insulin secretion, while co-incubation with the P2Y(13) antagonist MRS2211 stimulated insulin secretion, indicating that ADP acting via P2Y(1) stimulates insulin secretion, while signalling via P2Y(13) inhibits the secretion of insulin. P2Y(13) antagonism through MRS2211 per se increased the secretion of both insulin and glucagon at intermediate (8.3 mmol/l) and high (20 mmol/l) glucose levels, confirming an autocrine role for ADP. Administration of MRS2211 during glucose injection in vivo resulted in both increased secretion of insulin and reduced glucose levels. CONCLUSIONS/INTERPRETATION: In conclusion, ADP acting on the P2Y(13) receptors inhibits insulin release. An antagonist to P2Y(13) increases insulin release and could be evaluated for the treatment of diabetes.
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| 6. |
- Amisten, Stefan, et al.
(författare)
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An atlas and functional analysis of G-protein coupled receptors in human islets of Langerhans
- 2013
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Ingår i: Pharmacology and Therapeutics. - : Elsevier BV. - 0163-7258. ; 139:3, s. 359-391
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Forskningsöversikt (refereegranskat)abstract
- G-protein coupled receptors (GPCRs) regulate hormone secretion from islets of Langerhans, and recently developed therapies for type-2 diabetes target islet GLP-1 receptors. However, the total number of GPCRs expressed by human islets, as well as their function and interactions with drugs, is poorly understood. In this review we have constructed an atlas of all GPCRs expressed by human islets: the 'islet GPCRome'. We have used this atlas to describe how islet GPCRs interact with their endogenous ligands, regulate islet hormone secretion, and interact with drugs known to target GPCRs, with a focus on drug/receptor interactions that may affect insulin secretion. The islet GPCRome consists of 293 GPCRs, a majority of which have unknown effects on insulin, glucagon and somatostatin secretion. The islet GPCRs are activated by 271 different endogenous ligands, at least 131 of which are present in islet cells. A large signalling redundancy was also found, with 119 ligands activating more than one islet receptor. Islet GPCRs are also the targets of a large number of clinically used drugs, and based on their coupling characteristics and effects on receptor signalling we identified 107 drugs predicted to stimulate and 184 drugs predicted to inhibit insulin secretion. The islet GPCRome highlights knowledge gaps in the current understanding of islet GPCR function, and identifies GPCR/ligand/drug interactions that might affect insulin secretion, which are important for understanding the metabolic side effects of drugs. This approach may aid in the design of new safer therapeutic agents with fewer detrimental effects on islet hormone secretion. (C) 2013 Elsevier Inc. All rights reserved.
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| 7. |
- Amisten, Stefan, et al.
(författare)
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An atlas of G-protein coupled receptor expression and function in human subcutaneous adipose tissue.
- 2015
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Ingår i: Pharmacology and Therapeutics. - : Elsevier BV. - 0163-7258. ; 146:Sep 19, s. 61-93
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Forskningsöversikt (refereegranskat)abstract
- G-protein coupled receptors (GPCRs) are involved in the regulation of adipose tissue function, but the total number of GPCRs expressed by human subcutaneous adipose tissue, as well as their function and interactions with drugs, is poorly understood. We have constructed an atlas of all GPCRs expressed by human subcutaneous adipose tissue: the 'adipose tissue GPCRome', to support the exploration of novel control nodes in metabolic and endocrine functions. This atlas describes how adipose tissue GPCRs regulate lipolysis, insulin resistance and adiponectin and leptin secretion. We also discuss how adipose tissue GPCRs interact with their endogenous ligands and with GPCR-targeting drugs, with a focus on how drug/receptor interactions may affect lipolysis, and present a model predicting how GPCRs with unknown effects on lipolysis might modulate cAMP-regulated lipolysis. Subcutaneous adipose tissue expresses 163 GPCRs, a majority of which have unknown effects on lipolysis, insulin resistance and adiponectin and leptin secretion. These GPCRs are activated by 180 different endogenous ligands, and are the targets of a large number of clinically used drugs. We identified 119 drugs, acting on 23 GPCRs, that are predicted to stimulate lipolysis and 173 drugs, acting on 25 GPCRs, that are predicted to inhibit lipolysis. This atlas highlights knowledge gaps in the current understanding of adipose tissue GPCR function, and identifies GPCR/ligand/drug interactions that might affect lipolysis, which is important for understanding and predicting metabolic side effects of drugs. This approach may aid in the design of new, safer therapeutic agents, with fewer undesired effects on lipid homeostasis.
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| 8. |
- Amisten, Stefan, et al.
(författare)
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Anti-diabetic action of all- trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic beta-cells
- 2017
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Ingår i: Endocrine Journal. - 0918-8959. ; 64:3, s. 325-338
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Tidskriftsartikel (refereegranskat)abstract
- Pancreatic islets express high levels of the orphan G-protein coupled receptor C5C (GPRC5C), the function of which remains to be established. Here we have examined the role of GPRC5C in the regulation of insulin secretion and beta-cell survival and proliferation using human and mouse pancreatic islets. The expression of GPRC5C was analysed by RNA-sequencing, qPCR, western blotting and confocal microscopy. Insulin secretion and cell viability were determined by RIA and MTS assays, respectively. GPRC5C mRNA expression and protein level were reduced in the islets from type-2 diabetic donors. RNA sequencing in human islets revealed GPRC5C expression correlated with the expression of genes controlling apoptosis, cell survival and proliferation. A reduction in Gprc5c mRNA and protein expression was observed in islets isolated from old mice (>46 weeks of age) compared to that in islets from newborn (<3 weeks) mice. Down-regulation of Gprc5c led to both moderately reduced glucose-stimulated insulin release and also reduced cAMP content in mouse islets. Potentiation of glucose-stimulated insulin secretion concomitant with enhanced islet cAMP level by all-trans retinoic acid (ATRA) was attenuated upon Gprc5c-KD. ATRA also increased [Ca+2](i) in Huh7-cells. Gprc5c over expression in Huh7 cells was associated with increased ERK1/2 activity. Gprc5c-KD in clonal MIN6c4 cells reduced cell proliferation and in murine islets increased apoptosis and the sensitivity of primary islet cells to a cocktail of pro-apoptotic cytokines. Our results demonstrate that agents activating GPRC5C represent a novel modality for the treatment and/or prevention of diabetes by restoring and/or maintaining functional beta-cell mass.
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| 9. |
- Amisten, Stefan, et al.
(författare)
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Gene expression profiling for the identification of G-protein coupled receptors in human platelets
- 2008
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Ingår i: Thrombosis Research. - : Elsevier BV. - 1879-2472 .- 0049-3848. ; 122:1, s. 47-57
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION AND MATERIALS AND METHODS: G-protein coupled receptors (GPCRs) play an important role in platelet aggregation. To identify new platelet GPCRs, a platelet gene expression profile was generated and validated using quantitative real-time PCR. RESULTS: In total, mRNA of 28 GPCR genes was detected in human platelets. The 12 most abundant platelet GPCR transcripts were: thrombin receptor PAR1 (1865+/-178%), ADP receptor P2Y(12) (459+/-88%), succinate receptor 1 (257+/-48%), ADP receptor P2Y(1) (100%), orphan P2RY(10) (68.2+/-3.3%), lysophosphatidic acid receptors GPR23 (48.2+/-11%) and GPR92 (26.1+/-3.3%), adrenergic receptor alpha(2A) (18.4+/-4.4%), orphan EBI2 (6.31+/-0.42), adenosine receptors A(2A) (2.94+/-0.24%) and A(2B) (2.88+/-0.16%) and lysophosphatidic acid receptor LPA(1) (2.59+/-0.39%) (% relative to the chosen calibrator P2Y(1)). A surprising G-protein coupled receptor redundancy was found: two ADP receptors (P2Y(1) and P2Y(12)), three adenosine receptors (A(2A), A(2B), and A(1)), four lysophosphatidic acid receptors (LPA(1), LPA(3), GPR23 and GPR92), two l-glutamate receptors (mGlu(3) and mGlu(4)) and two serotonin receptors (5-HT(1F) and 5-HT(4)). The adenosine receptor A(2B) gene expression was validated with protein expression and functional studies. Western blot confirmed A(2B) receptor protein expression and platelet flow cytometry demonstrated inhibition of the effect of NECA by the adenosine A(2B)-antagonist MRS1754. CONCLUSIONS: We have detected several GPCRs not previously known to be expressed in platelets, including a functional adenosine A(2B) receptor. The findings could improve our understanding of platelet aggregation and provide new targets for drug development.
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| 10. |
- Amisten, Stefan
(författare)
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Genetic studies on cardiovascular disease - identification of novel drug targets
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis was to identify novel potential drug targets of cardiovascular disease with a focus on G-protein coupled receptors. We identified a common genetic variant of the ATP receptor P2Y11, where an alanine is substituted with a threonine at position 87 of the P2Y11 receptor that increases the risk of developing acute myocardial infarction. Also, the Thr-87 variant of P2Y11 is associated with elevated serum concentrations of C-reactive protein. In CHO cells, ATP?S stimulated recombinant P2Y11 Thr-87 displayed a reduced cAMP response compared to the P2Y11 Ala-87 variant. We hypothesize that a loss of a protective mechanism mediated by P2Y11 cAMP signaling is involved in the increased risk of myocardial infarction seen in the AMI case-control study. Polymorphisms forming a H2 haplotype of the ADP receptor P2Y12 have been linked to an increased platelet response to ADP and increased risks of atherosclerosis and peripheral arterial disease. We show that a polymorphism of the P2Y13 gene, causing a methionine to threonine substitution at position 158 of the P2Y13 receptor, is in complete linkage disequilibrium with the P2Y12 H2 haplotype. However, in three separate populations, we found no association between the P2Y12 H2 haplotype/P2Y13 Thr-158 polymorphisms and myocardial infarction, type 2 diabetes or any examined cardiovascular risk factor. G-protein coupled receptors are important regulators of platelet activation. Using microarray, we identified several G-protein coupled receptors in platelets whose expression in platelets has not previously been known. To illustrate our findings, we demonstrate for the first time adenosine A2B receptor mediated inhibition of platelet activation. Finally, using a unique in vitro model, we examined the molecular response to the implantation of bare metal and drug-eluting cardiovascular stents into human arteries. We found several differentially expressed genes involved in the regulation of inflammation, endothelial cell function, platelet activation and cellular proliferation. We hope that our model will increase the understanding of the processes involved in restenosis and late stent thrombosis associated with bare metal and drug-eluting stents and that our model may be useful in the development of next generation drug eluting stents.
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