| 1. |
- Kononenko, Olga, et al.
(författare)
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Opioid precursor protein isoform is targeted to the cell nuclei in the human brain
- 2017
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165 .- 1872-8006. ; 1861:2, s. 246-255
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus.RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.
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| 2. |
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| 3. |
- Abels, Mia, et al.
(författare)
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CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion
- 2016
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 59:9, s. 1928-1937
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Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart(-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. Methods CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca2+ oscillation patterns and exocytosis were studied in mouse islets. Results We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca2+ signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. Conclusions/interpretation We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.
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| 4. |
- Alenkvist, Ida, et al.
(författare)
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Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets
- 2014
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Ingår i: Journal of Endocrinology. - 0022-0795 .- 1479-6805. ; 223:3, s. 267-275
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Tidskriftsartikel (refereegranskat)abstract
- The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression of Shb in β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereas Shb deficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics in Shb-knockout mice. Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor was Shb deficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca(2) (+) handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in the Shb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion, Shb deficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.
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| 5. |
- Balboa, Diego, et al.
(författare)
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Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.
- 2022
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Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 40:7, s. 1042-1055
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.
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| 6. |
- Chowdhury, Azazul Islam, et al.
(författare)
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Functional differences between aggregated and dispersed insulin-producing cells
- 2013
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 56:7, s. 1557-1568
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Tidskriftsartikel (refereegranskat)abstract
- Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, alpha-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca2+ concentration ([Ca2+](c); microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca2+](c) response. We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca2+](c).
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| 7. |
- Dezaki, Katsuya, et al.
(författare)
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Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet beta-Cells
- 2011
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 60:9, s. 2315-2324
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE-Ghrelin reportedly restricts insulin release in islet beta-cells via the G alpha(i2) subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet beta-cells. RESEARCH DESIGN AND METHODS-Insulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP](i)) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single beta-cells, cytosolic protein kinase-A activity ([PKA](i)) and Ca(2+) concentration ([Ca(2+)](i)) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique. RESULTS-Ghrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP](i) elevation and [PKA](i) activation in MIN6 and rat beta-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K(+) (Kv) channel currents without altering Ca(2+) channel currents and attenuated glucose-induced [Ca(2+)](i) increases in rat beta-cells in a PKA-dependent manner. CONCLUSIONS-Ghrelin directly interacts with islet beta-cells to attenuate glucose-induced cAMP production and PKA activation, which lead In activation of Kv channels and suppression of glucose-induced [Ca(2+)](i) increase and insulin release.
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| 8. |
- Dyachok, Oleg, et al.
(författare)
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Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells
- 2004
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Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160 .- 1532-1991. ; 36:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- The effect of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibition on the cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in primary insulin-releasing pancreatic β-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca2+-deficient medium the individual primary β-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca2+]i followed by an explosive transient elevation. The [Ca2+]i transients were preferentially observed at low intracellular concentrations of the Ca2+ indicator fura-2 and were unaffected by pre-treatment with 100 μM ryanodine. Whereas 20 mM caffeine had no effect on basal [Ca2+]i or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca2+]i transients as well as inositol 1,4,5-trisphosphate-mediated [Ca2+]i transients in response to carbachol. In striking contrast to the primary β-cells, caffeine readily mobilized intracellular Ca2+ in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca2+ from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca2+-induced Ca2+ release (CICR). In primary pancreatic β-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal β-cells.
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| 9. |
- Dyachok, Oleg, et al.
(författare)
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Ca2+-induced Ca2+ Release via Inositol 1,4,5-trisphosphate Receptors Is Amplified by Protein Kinase A and Triggers Exocytosis in Pancreatic β-Cells
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:44, s. 45455-45461
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Tidskriftsartikel (refereegranskat)abstract
- Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+ release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+ spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+ concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+ spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]i spikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release.
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