| 1. |
- Barghouth, Mohammad, et al.
(författare)
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The T-type calcium channel CaV3.2 regulates insulin secretion in the pancreatic β-cell
- 2022
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Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160. ; 108
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated Ca2+ (CaV) channel dysfunction leads to impaired glucose-stimulated insulin secretion in pancreatic β-cells and contributes to the development of type-2 diabetes (T2D). The role of the low-voltage gated T-type CaV channels in β-cells remains obscure. Here we have measured the global expression of T-type CaV3.2 channels in human islets and found that gene expression of CACNA1H, encoding CaV3.2, is negatively correlated with HbA1c in human donors, and positively correlated with islet insulin gene expression as well as secretion capacity in isolated human islets. Silencing or pharmacological blockade of CaV3.2 attenuates glucose-stimulated cytosolic Ca2+ signaling, membrane potential, and insulin release. Moreover, the endoplasmic reticulum (ER) Ca2+ store depletion is also impaired in CaV3.2-silenced β-cells. The linkage between T-type (CaV3.2) and L-type CaV channels is further identified by the finding that the intracellular Ca2+ signaling conducted by CaV3.2 is highly dependent on the activation of L-type CaV channels. In addition, CACNA1H expression is significantly associated with the islet predominant L-type CACNA1C (CaV1.2) and CACNA1D (CaV1.3) genes in human pancreatic islets. In conclusion, our data suggest the essential functions of the T-type CaV3.2 subunit as a mediator of β-cell Ca2+ signaling and membrane potential needed for insulin secretion, and in connection with L-type CaV channels.
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| 2. |
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| 3. |
- Cowan, Elaine, et al.
(författare)
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MicroRNAs in Type 2 Diabetes : Focus on MicroRNA Profiling in Islets of Langerhans
- 2022
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Ingår i: Type-1 Diabetes : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029. - 9781071628065 - 9781071628072 ; :2592, s. 113-142
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Bokkapitel (refereegranskat)abstract
- Differential expression of microRNAs (miRNAs) is observed in many diseases including type 2 diabetes (T2D). Insulin secretion from pancreatic beta cells is central for the regulation of blood glucose levels and failure to release enough insulin results in hyperglycemia and T2D. The importance in T2D pathogenesis of single miRNAs in beta cells has been described; however, to get the full picture, high-throughput miRNA sequencing is necessary. Here we describe a method using small RNA sequencing, from sample preparation to expression analysis using bioinformatic tools. In the end, a tutorial on differential expression analysis is presented in R using publicly available data.
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| 4. |
- Dos Santos, Cristiane, et al.
(författare)
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Glucocorticoids and glucolipotoxicity alter the DNA methylome and function of human EndoC-βH1 cells
- 2022
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Ingår i: Life Sciences. - : Elsevier BV. - 1879-0631 .- 0024-3205. ; 307
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Synthetic glucocorticoids, including dexamethasone (DEX), are clinically prescribed due to their immunoregulatory properties. In excess they can perturb glucose homeostasis, with individuals predisposed to glucose intolerance more sensitive to these negative effects. While DEX is known to negatively impact β-cell function, it is unclear how. Hence, our aim was to investigate the effect of DEX on β-cell function, both alone and in combination with a diabetogenic milieu in the form of elevated glucose and palmitate.MAIN METHODS: Human pancreatic EndoC-βH1 cells were cultured in the presence of high glucose and palmitate (glucolipotoxicity) and/or a pharmacological concentration of DEX, before functional and molecular analyses.KEY FINDINGS: Either treatment alone resulted in reduced insulin content and secretion, while the combination of DEX and glucolipotoxicity promoted a strong synergistic effect. These effects were associated with reduced insulin biosynthesis, likely due to downregulation of PDX1, MAFA, and the proinsulin converting enzymes, as well as reduced ATP response upon glucose stimulation. Genome-wide DNA methylation analysis found changes on PDE4D, MBNL1 and TMEM178B, all implicated in β-cell function, after all three treatments. DEX alone caused very strong demethylation of the glucocorticoid-regulated gene ZBTB16, also known to influence the β-cell, while the combined treatment caused altered methylation of many known β-cell regulators and diabetes candidate genes.SIGNIFICANCE: DEX treatment and glucolipotoxic conditions separately alter the β-cell epigenome and function. The combination of both treatments exacerbates these changes, showing that caution is needed when prescribing potent glucocorticoids in patients with dysregulated metabolism.
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| 5. |
- Karagiannopoulos, Alexandros, et al.
(författare)
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Human pancreatic islet miRNA-mRNA networks of altered miRNAs due to glycemic status
- 2022
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 25:4
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression via mRNA targeting, playing important roles in the pancreatic islets. We aimed to identify molecular pathways and genomic regulatory regions associated with altered miRNA expression due to glycemic status, which could contribute to the development of type 2 diabetes (T2D). To this end, miRNAs were identified by a combination of differential miRNA expression and correlation analysis in human islet samples from donors with normal and elevated blood glucose levels. Analysis and clustering of highly correlated, experimentally validated gene targets of these miRNAs revealed two islet-specific clusters, which were associated with key aspects of islet functions and included a high number of T2D-related genes. Finally, cis-eQTLs and public GWAS data integration uncovered suggestive genomic signals of association with insulin secretion and T2D. The miRNA-driven network-based approach presented in this study contributes to a better understanding of impaired insulin secretion in T2D pathogenesis.
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| 6. |
- Ofori, Jones, et al.
(författare)
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Human Islet MicroRNA-200c Is Elevated in Type 2 Diabetes and Targets the Transcription Factor ETV5 to Reduce Insulin Secretion
- 2022
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 71:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are part of deregulated insulin secretion in type 2 diabetes (T2D) development. Rodent models have suggested miR-200c to be involved, but the role and potential as therapeutic target of this miRNA in human islets are not clear. Here we report increased expression of miR-200c in islets from T2D as compared with nondiabetic (ND) donors and display results showing reduced glucose-stimulated insulin secretion in EndoC-βH1 cells overexpressing miR-200c. We identify transcription factor ETV5 as the top rank target of miR-200c in human islets using TargetScan in combination with Pearson correlation analysis of miR-200c and mRNA expression data from the same human donors. Among other targets were JAZF1, as earlier shown in miR-200 knockout mice. Accordingly, linear model analysis of ETV5 and JAZF1 gene expression showed reduced expression of both genes in islets from human T2D donors. Western blot analysis confirmed the reduced expression of ETV5 on the protein level in EndoC-βH1 cells overexpressing miR-200c, and luciferase assay validated ETV5 as a direct target of miR-200c. Finally, LNA knockdown of miR-200c increased glucose-stimulated insulin secretion in islets from T2D donors approximately threefold. Our data reveal a vital role of the miR-200c–ETV5 axis in β-cell dysfunction and pathophysiology of T2D.
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| 7. |
- Ofori, Jones K, et al.
(författare)
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The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion
- 2022
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Ingår i: Acta Physiologica. - : Wiley. - 1748-1708 .- 1748-1716. ; 236:1
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Tidskriftsartikel (refereegranskat)abstract
- Aim SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type 2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin-secreting cells. Methods Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-beta H1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch-clamp was used for single-cell electrophysiology. Confocal microscopy was used to determine intracellular localization. Results Human islet expression of the transcription factor PDX1 was positively correlated with SYT11 (p = 2.4e(-10)) and SYT13 (p < 2.2e(-16)). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K+. Interestingly, the cAMP-raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. Conclusion SYT11 and SYT13 have similar localization and transcriptional regulation, but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease.
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| 8. |
- Verma, Gaurav, et al.
(författare)
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Ribosomal biogenesis regulator DIMT1 controls β-cell protein synthesis, mitochondrial function, and insulin secretion
- 2022
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Ingår i: The Journal of biological chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 298:3
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Tidskriftsartikel (refereegranskat)abstract
- We previously reported that loss of mitochondrial transcription factor B1 (TFB1M) leads to mitochondrial dysfunction and is involved in the pathogenesis of type 2 diabetes (T2D). Whether defects in ribosomal processing impact mitochondrial function and could play a pathogenetic role in β-cells and T2D is not known. To this end, we explored expression and the functional role of dimethyladenosine transferase 1 homolog (DIMT1), a homolog of TFB1M and a ribosomal RNA (rRNA) methyltransferase implicated in the control of rRNA. Expression of DIMT1 was increased in human islets from T2D donors and correlated positively with expression of insulin mRNA, but negatively with insulin secretion. We show that silencing of DIMT1 in insulin-secreting cells impacted mitochondrial function, leading to lower expression of mitochondrial OXPHOS proteins, reduced oxygen consumption rate, dissipated mitochondrial membrane potential, and a slower rate of ATP production. In addition, the rate of protein synthesis was retarded upon DIMT1-deficiency. Consequently, we found that DIMT1 deficiency led to perturbed insulin secretion in rodent cell lines and islets, as well as in a human β-cell line. We observed defects in rRNA processing and reduced interactions between NIN1 (RPN12) binding protein 1 homolog (NOB-1) and Pescadillo ribosomal biogenesis factor 1 (PES-1), critical ribosomal subunit RNA proteins, the dysfunction of which may play a part in disturbing protein synthesis in β-cells. In conclusion, DIMT1 deficiency perturbs protein synthesis, resulting in mitochondrial dysfunction and disrupted insulin secretion, both potential pathogenetic processes in T2D.
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| 9. |
- Wu, Rui, et al.
(författare)
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The Calcium Channel Subunit Gamma-4 as a Novel Regulator of MafA in Pancreatic Beta-Cell Controls Glucose Homeostasis
- 2022
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Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) are high-risk factors of diabetes development and may be caused by defective insulin secretion in pancreatic beta-cells. Glucose-stimulated insulin secretion is mediated by voltage-gated Ca2+ (CaV) channels in which the gamma-4 subunit (CaVγ4) is required for the beta-cell to maintain its differentiated state. We here aim to explore the involvement of CaVγ4 in controlling glucose homeostasis by employing the CaVγ4-/- mice to study in vivo glucose-metabolism-related phenotypes and glucose-stimulated insulin secretion, and to investigate the underlying mechanisms. We show that CaVγ4-/- mice exhibit perturbed glucose homeostasis, including IFG and IGT. Glucose-stimulated insulin secretion is blunted in CaVγ4-/- mouse islets. Remarkably, CaVγ4 deletion results in reduced expression of the transcription factor essential for beta-cell maturation, MafA, on both mRNA and protein levels in islets from human donors and CaVγ4-/- mice, as well as in INS-1 832/13 cells. Moreover, we prove that CaMKII is responsible for mediating this regulatory pathway linked between CaVγ4 and MafA, which is further confirmed by human islet RNA-seq data. We demonstrate that CaVγ4 is a key player in preserving normal blood glucose homeostasis, which sheds light on CaVγ4 as a novel target for the treatment of prediabetes through correcting the impaired metabolic status.
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| 10. |
- Ye, Yingying, et al.
(författare)
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A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic beta-cells
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-induced insulin secretion depends on beta-cell electrical activity. Inhibition of ATP-regulated potassium (K-ATP) channels is a key event in this process. However, K-ATP channel closure alone is not sufficient to induce beta-cell electrical activity; activation of a depolarizing membrane current is also required. Here we examine the role of the mechanosensor ion channel PIEZO1 in this process. Yoda1, a specific PIEZO1 agonist, activates a small membrane current and thereby triggers beta-cell electrical activity with resultant stimulation of Ca2+-influx and insulin secretion. Conversely, the PIEZO1 antagonist GsMTx4 reduces glucose-induced Ca2+-signaling, electrical activity and insulin secretion. Yet, PIEZO1 expression is elevated in islets from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db mouse), in which insulin secretion is reduced. This paradox is resolved by our finding that PIEZO1 translocates from the plasmalemma into the nucleus (where it cannot influence the membrane potential of the beta-cell) under experimental conditions emulating T2D (high glucose culture). beta-cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced glucose-induced insulin secretion, beta-cell electrical activity and Ca2+ elevation in vitro. These results implicate mechanotransduction and activation of PIEZO1, via intracellular accumulation of glucose metabolites, as an important physiological regulator of insulin secretion. Insulin secretion depends on action potential firing in pancreatic islet beta-cells, but the underlying mechanism is unclear. Here, the authors show that activation of the mechanosensor ion channel PIEZO1 plays a central role in beta-cell electrical activity and insulin release.
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