| 1. |
- Åkerblom, Björn, 1976-
(författare)
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Frk/Shb Signalling in Pancreatic Beta-cells : Roles in Islet Function, Beta-cell Development and Survival as Implicated in Mouse Knockout Models
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The adaptor protein Shb and the non-receptor tyrosine kinase Frk have been implicated in intracellular signalling in insulin-producing beta cells. In this thesis, knockout mice are used to further elucidate the role of Shb and Frk for beta cell number, cytokine-induced cell death, and glucose homeostasis. In addition, the effect of Shb deficiency upon tumour growth is studied in a mouse model of endogenous tumourigenesis. Previously, overexpression of Frk has been associated with increased beta cell replication, and increased susceptibility to cytokine induced beta cell destruction. To test whether Frk has a non-redundant role in regulating beta cell mass, beta cell number in Frk-/- mice was assessed at different stages of life. The results showed that Frk is involved in regulating beta cell number during embryonal and early postnatal life, but is probably redundant in the adult. An earlier study had suggested that Shb participates in cytokine-induced beta cell death, a model of autoimmune diabetes. To test this further, Shb-/- islets were exposed to cytokines, or to an ER-stress inducing agent. Shb knockout islets exhibited decreased cell death, and this effect appeared to be independent of NO, JNK, p38 MAP kinase, FAK and c-Abl, but may involve an augmented induction of Hsp70. Furthermore, glucose homeostasis in Shb-/- mice was impaired, with elevated basal blood sugar concentration and reduced glucose-induced insulin secretion. Previously Shb deficient mice had showed an impaired ability to sustain growth of implanted tumour cells, due to reduced angiogenesis. Tumour growth and angiogenesis were here assessed in an inheritable tumour model. Shb deficient mice exhibited fewer tumours, and reduced vessel density in small tumours, indicating impaired angiogenesis. However, a few large tumours developed in Shb-/- mice, suggesting that tumours can escape the angiogenic restriction caused by the absence of Shb.
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| 2. |
- Nagorny, Cecilia, et al.
(författare)
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Distribution of melatonin receptors in murine pancreatic islets.
- 2011
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Ingår i: Journal of Pineal Research. - 1600-079X. ; 50, s. 412-417
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Tidskriftsartikel (refereegranskat)abstract
- Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.
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| 3. |
- Mulder, Hindrik
(författare)
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Transcribing β-cell mitochondria in health and disease
- 2017
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Ingår i: Molecular Metabolism. - : Elsevier BV. - 2212-8778. ; 6:9, s. 1040-1051
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Forskningsöversikt (refereegranskat)abstract
- Background: The recent genome-wide association studies (GWAS) of Type 2 Diabetes (T2D) have identified the pancreatic β-cell as the culprit in the pathogenesis of the disease. Mitochondrial metabolism plays a crucial role in the processes controlling release of insulin and β-cell mass. This notion implies that mechanisms controlling mitochondrial function have the potential to play a decisive pathogenetic role in T2D. Scope of the review: This article reviews studies demonstrating that there is indeed mitochondrial dysfunction in islets in T2D, and that GWAS have identified a variant in the gene encoding transcription factor B1 mitochondrial (TFB1M), predisposing to T2D due to mitochondrial dysfunction and impaired insulin secretion. Mechanistic studies of the nature of this pathogenetic link, as well as of other mitochondrial transcription factors, are described. Major conclusions: Based on this, it is argued that transcription and translation in mitochondria are critical processes determining mitochondrial function in β-cells in health and disease.
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| 4. |
- Schvartz, D., et al.
(författare)
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The Human Diabetes Proteome Project (HDPP) : The 2014 update
- 2015
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Ingår i: Translational Proteomics. - : Elsevier BV. - 2212-9626. ; 8-9, s. 1-7
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Tidskriftsartikel (refereegranskat)abstract
- Diabetes is an increasing worldwide problem leading to major associated health issues and increased health care costs. In 2012, 9.3% of the American population was affected by diabetes, according to the American Diabetes Association, with 1.7 million of new cases since during the year (www.diabetes.org). Proteome initiatives can provide a deeper understanding of the biology of this disease and help develop more effective treatments. The collaborative effort of the Human Diabetes Proteome Project (HDPP) brings together a wide variety of complementary resources to increase the existing knowledge about both type 1 and type 2 diabetes and their related complications. The goals are to identify proteins and protein isoforms associated with the pathology and to characterize underlying disease-related pathways and mechanisms. Moreover, a considerable effort is being made on data integration and network biology. Sharing these data with the scientific community will be an important part of the consortium. Here we report on: the content of the HDPP session held at the 12th HUPO meeting in Yokohama; recent achievements of the consortium; discussions of several HDPP workshops; as well as future HDPP directions as discussed at the 13th HUPO congress in Madrid, with a special attention given to the lists of prioritized, diabetes-related proteins and the proteomic means to study them.
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| 5. |
- Blanchi, Bruno, et al.
(författare)
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EndoC-βH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion
- 2023
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Ingår i: Molecular Metabolism. - 2212-8778. ; 76
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family. Methods: EndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays. Results: Ready to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation. Conclusions: EndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.
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| 6. |
- Cataldo, Luis Rodrigo, et al.
(författare)
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The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells
- 2021
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Ingår i: Metabolism: Clinical and Experimental. - : Elsevier BV. - 1532-8600.
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Tidskriftsartikel (refereegranskat)abstract
- The amplification of glucose-stimulated insulin secretion (GSIS) through incretin signaling is critical for maintaining physiological glucose levels. Incretins, like glucagon-like peptide 1 (GLP1), are a target of type 2 diabetes drugs aiming to enhance insulin secretion. Here we show that the protein phosphatase 1 inhibitor protein 1A (PPP1R1A), is expressed in β-cells and that its expression is reduced in dysfunctional β-cells lacking MafA and upon acute MafA knock down. MafA is a central regulator of GSIS and β-cell function. We observed a strong correlation of MAFA and PPP1R1A mRNA levels in human islets, moreover, PPP1R1A mRNA levels were reduced in type 2 diabetic islets and positively correlated with GLP1-mediated GSIS amplification. PPP1R1A silencing in β-cell lines impaired GSIS amplification, PKA-target protein phosphorylation, mitochondrial coupling efficiency and also the expression of critical β-cell marker genes like MafA, Pdx1, NeuroD1 and Pax6. Our results demonstrate that the β-cell transcription factor MafA is required for PPP1R1A expression and that reduced β-cell PPP1R1A levels impaired β-cell function and contributed to β-cell dedifferentiation during type 2 diabetes. Loss of PPP1R1A in type 2 diabetic β-cells may explains the unresponsiveness of type 2 diabetic patients to GLP1R-based treatments.
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| 7. |
- Huang, Mi, et al.
(författare)
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Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function
- 2023
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Ingår i: eLife. - 2050-084X.
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Tidskriftsartikel (refereegranskat)abstract
- Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.
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| 8. |
- Ofori, Jones K., et al.
(författare)
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Elevated miR-130a/miR130b/miR-152 expression reduces intracellular ATP levels in the pancreatic beta cell
- 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
- MicroRNAs have emerged as important players of gene regulation with significant impact in diverse disease processes. In type-2 diabetes, in which impaired insulin secretion is a major factor in disease progression, dysregulated microRNA expression in the insulin-secreting pancreatic beta cell has been widely-implicated. Here, we show that miR-130a-3p, miR-130b-3p, and miR-152-3p levels are elevated in the pancreatic islets of hyperglycaemic donors, corroborating previous findings about their upregulation in the islets of type-2 diabetes model Goto-Kakizaki rats. We demonstrated negative regulatory effects of the three microRNAs on pyruvate dehydrogenase E1 alpha (PDHA1) and on glucokinase (GCK) proteins, which are both involved in ATP production. Consequently, we found both proteins to be downregulated in the Goto-Kakizaki rat islets, while GCK mRNA expression showed reduced trend in the islets of type-2 diabetes donors. Overexpression of any of the three microRNAs in the insulin-secreting INS-1 832/13 cell line resulted in altered dynamics of intracellular ATP/ADP ratio ultimately perturbing fundamental ATP-requiring beta cell processes such as glucose-stimulated insulin secretion, insulin biosynthesis and processing. The data further strengthen the wide-ranging influence of microRNAs in pancreatic beta cell function, and hence their potential as therapeutic targets in type-2 diabetes.
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| 9. |
- Santos, Laila R.B., et al.
(författare)
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NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells
- 2017
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Ingår i: Molecular Metabolism. - : Elsevier BV. - 2212-8778. ; 6:6, s. 535-547
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The glucose stimulation of insulin secretion (GSIS) by pancreatic β-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and δpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets. Methods: Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures. Results: NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in β-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. Conclusion: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.
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| 10. |
- Schultz, Julia, et al.
(författare)
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Precise expression of Fis1 is important for glucose responsiveness of beta cells
- 2016
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Ingår i: Journal of Endocrinology. - 0022-0795. ; 230:1, s. 81-91
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucosestimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.
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