Identification of functional insulin receptors on membranes from an insulin-producing cell line (RINm5F).
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ABSTRACT:
SUBMITTER: Gazzano H
PROVIDER: S-EPMC1144788 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
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Project description:1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.
| S-EPMC1154230 | biostudies-other
Project description:Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H2O2) across cellular membranes. H2O2 serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic β-cells, H2O2 has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H2O2 homeostasis in pancreatic β-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H2O2 specific biosensor HyPer together with endogenous generation of H2O2 using d-amino acid oxidase, live cell imaging revealed enhanced H2O2 flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H2O2 sensor HyPer7.2 clearly unveiled AQP8 as a H2O2 transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H2O2 pore in insulin-producing RINm5F cells involved in the transport of H2O2 through the mitochondria and cell membrane and may help to explain the H2O2 transport and toxicity in pancreatic β-cells.
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Project description:The regulation of intracellular pH (pHi) and its role in the insulin-secretory process were evaluated, by using the clonal insulin-secreting cell line RINm5F. Glyceraldehyde, lactate and dihydroxyacetone decreased pHi, but only the first two released insulin. In the presence of extracellular Na+ the cells counteracted the acidification. Blocking the Na+/H+ exchange in acidic cells resulted in a drastic further lowering of pHi, an effect not obtained under basal conditions. Whereas glyceraldehyde depolarized the cells, lactate was without effect. Dihydroxyacetone hyperpolarized the cells in the presence of extracellular Na+, but this effect disappeared when Na+ was excluded from the medium. Stimulation with glyceraldehyde resulted in increased free cytoplasmic Ca2+ concentration ([Ca2+]i). Dihydroxyacetone and lactate had no effect on [Ca2+]i in the presence of Na+, but lactate induced a decrease in [Ca2+]i in Na(+)-deficient medium. In RINm5F cells the activity of the Na+/H+ antiport could not be augmented by activation of protein kinase C (PKC). Hence, in insulin-secreting cells a PKC-insensitive Na+/H+ antiport is the major mechanism restoring a decrease in pHi. Acidification itself does not affect membrane potential, but may directly interact with the mechanisms regulating exocytosis.
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Project description:Lectin-glycan interactions play important roles in many biological systems, but the nature of glycoprotein counter-receptors expressed on cell membranes is often poorly understood. To help overcome this problem, we developed a method based on proximity labeling technology. Using a peroxidase-coupled lectin, addition of H2O2 and tyramide-biotin substrates leads to generation of short-range biotin radicals that biotinylate proteins in the immediate vicinity of the bound lectin, which can subsequently be identified. As a proof-of-principle, sialoadhesin-horseradish peroxidase-human IgG1 Fc recombinant protein constructs were precomplexed with anti-Fc antibodies, bound to human erythrocytes and reacted with H2O2 and tyramide-SS-biotin. The erythrocyte membrane protein with strongest biotinylation was identified as glycophorin A, in agreement with early studies using lectin overlay and reglycosylation approaches. As a further test of the method, the plant lectin MAL II was conjugated with horseradish peroxidase and used in proximity labeling of human erythrocytes. Glycophorin A was again selectively labeled, which is consistent with previous reports that MAL II has high affinity for glycophorin. This method could be applied to other lectins to identify their membrane counter-receptors.
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Project description:Diabetes mellitus (DM) is one of the most prevalent feline endocrinopathies, affecting up to 1% of pet cats. De novo generation of functional insulin producing cell (IPC) clusters via transdifferentiation of feline adipose-derived multipotent stromal cells (ASCs) may not only provide a viable, functional cell therapy for feline DM, but may also serve as a platform for developing a comparable human treatment given feline and human DM similarities. Cells were induced to form IPCs with a novel, three-stage culture process with stromal or differentiation medium under static and dynamic conditions. Clusters were evaluated for intracellular zinc, viability, intracellular insulin, glucagon, and somatostatin, ultrastructure, glucose stimulated insulin secretion in the presence or absence of theophylline, and protein and gene expression. Isolated cells were multipotent, and cell clusters cultured in both media had robust cell viability. Those cultured in differentiation medium contained zinc and mono- or polyhormonal α-, β-, and δ-like cells based on immunohistochemical labeling and Mallory-Heidenhan Azan-Gomori's staining. Ultrastructurally, cell clusters cultured in differentiation medium contained insulin granules within vesicles, and clusters had a concentration-dependent insulin response to glucose in the presence and absence of theophylline which increased both insulin secretion and intracellular content. Expression of NK6.1, Pax6, Isl1, Glut2, RAB3A, glucagon, insulin, and somatostatin increased with differentiation stage for both sexes, and expression of nestin at stages 1 and 2 and Neurod1 at stage 2 was higher in cells from female donors. The cluster insulin secretion responses and endocrine and oncogene gene expression profiles were inconsistent with insulinoma characteristics. A total of 180 proteins were upregulated in differentiated clusters, and the majority were associated with biological regulation, metabolic processes, or stimulus response. Dynamic culture of IPC clusters resulted in clusters composed of cells primarily expressing insulin that released higher insulin with glucose stimulation than those in static culture. Collectively, the results of this study support generation of functional IPC clusters using feline ASCs isolated from tissues removed during routine sterilization. Further, cluster functionality is enhanced with dynamic, motion-driven shear stress. This work establishes a foundation for development of strategies for IPC therapy for short or long-term diabetes treatment and may represent an option to study prevention and treatment of diabetes across species.
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Project description:Glucose metabolism and insulin release were studied in isolated rat islets and in an insulin-producing rat cell-line (RINm5F). Intact islets displayed two components of glucose utilization, with glucose stimulation of insulin release being associated with the high-Km component (reflecting glucokinase-like activity). Glucose failed to stimulate insulin release from RINm5F cells, which only displayed a single low-Km component of glucose utilization. Only low-Km (hexokinase-like) glucose-phosphorylating activity was found for disrupted RINm5F cells. These changes in glucose metabolism may contribute towards the failure of glucose to stimulate insulin release from RINm5F cells.
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Project description:Ryanodine receptors (RyR) are Ca(2+) channels that mediate Ca(2+) release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca(2+) entry that was independent of store-operated Ca(2+) entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (gamma(K) = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic beta-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP(3) receptors (IP(3)R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP(3)R3, we showed that RyR2 mediates both the Ca(2+) entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic beta-cells, where they mediate Ca(2+) entry.
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Project description:Aims/hypothesisEndothelial cells (ECs) play an essential role in pancreatic organogenesis. We hypothesise that effective in vitro interactions between human microvascular endothelial cells (HMECs) and human pluripotent stem cells (hPSCs) results in the generation of functional pancreatic beta cells.MethodsEmbryoid bodies (EBs) derived from hPSCs were cultured alone (controls) or with ECs in collagen gels. Subsequently, cells were analysed for pancreatic beta cell markers, and then isolated and expanded. Insulin secretion in response to glucose was evaluated in vitro by static and dynamic (perifusion) assays, and in vivo by EB transplantation into immunodeficient mice.ResultsCo-cultured EBs had a higher expression of mature beta cells markers and enhanced insulin secretion in vitro, compared with controls. In mice, transplanted EBs had higher levels of human C-peptide secretion with a significant reduction in hyperglycaemia after the selective destruction of native pancreatic beta cells. In addition, there was significant in vitro upregulation of bone morphogenetic proteins 2 and 4 (BMP-2, 4) in co-cultured cells, compared with controls.Conclusions/interpretationECs provide essential signalling in vitro, such as activation of the BMP pathway, for derivation of functional insulin-producing beta cells from hPSCs.
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Project description:Stem cell-derived insulin-producing beta cells (SC-β) offer an inexhaustible supply of functional β cells for cell replacement therapies and disease modeling for diabetes. While successful directed differentiation protocols for this cell type have been described, the mechanisms controlling its differentiation and function are not fully understood. Here we report that the Hippo pathway controls the proliferation and specification of pancreatic progenitors into the endocrine lineage. Downregulation of YAP, an effector of the pathway, enhances endocrine progenitor differentiation and the generation of SC-β cells with improved insulin secretion. A chemical inhibitor of YAP acts as an inducer of endocrine differentiation and reduces the presence of proliferative progenitor cells. Conversely, sustained activation of YAP results in impaired differentiation, blunted glucose-stimulated insulin secretion, and increased proliferation of SC-β cells. Together these results support a role for YAP in controlling the self-renewal and differentiation balance of pancreatic progenitors and limiting endocrine differentiation in vitro.
| S-EPMC6443737 | biostudies-literature
Project description:Aims/hypothesisBeta 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.MethodsMIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]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.ResultsInsulin 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 [Ca(2+)]c response.Conclusions/interpretationWe 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 [Ca(2+)]c.
| S-EPMC3671110 | biostudies-literature