Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells.
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ABSTRACT:
SUBMITTER: Cantrell Stanford J
PROVIDER: S-EPMC3339968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aims/hypothesisRho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.MethodsCo-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).ResultsWestern blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.Conclusions/interpretationOur data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.
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Project description:Pancreatic β-cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the β cell, and here, we characterize its role in the regulation of β-cell function. Pim3 expression in the β-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the β cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3-/- mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3-/- mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3-/- mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3-/- islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating β-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3-/- islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the β cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.
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Project description:Brain-selective kinase 2 (BRSK2) has been shown to play an essential role in neuronal polarization. In the present study, we show that BRSK2 is also abundantly expressed in pancreatic islets and MIN6 β-cell line. Yeast two-hybrid screening, GST fusion protein pull-down, and co-immunoprecipitation assays reveal that BRSK2 interacts with CDK-related protein kinase PCTAIRE1, a kinase involved in neurite outgrowth and neurotransmitter release. In MIN6 cells, BRSK2 co-localizes with PCTAIRE1 in the cytoplasm and phosphorylates one of its serine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2 reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Conversely, knockdown of BRSK2 by siRNA increases serum insulin levels in mice. Our results reveal a novel function of BRSK2 in the regulation of GSIS in β-cells via a PCTAIRE1-dependent mechanism and suggest that BRSK2 is an attractive target for developing novel diabetic drugs.
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Project description:The construction and characterization of a core kinetic model of the glucose-stimulated insulin secretion system (GSIS) in pancreatic beta cells is described. The model consists of 44 enzymatic reactions, 59 metabolic state variables, and 272 parameters. It integrates five subsystems: glycolysis, the TCA cycle, the respiratory chain, NADH shuttles, and the pyruvate cycle. It also takes into account compartmentalization of the reactions in the cytoplasm and mitochondrial matrix. The model shows expected behavior in its outputs, including the response of ATP production to starting glucose concentration and the induction of oscillations of metabolite concentrations in the glycolytic pathway and in ATP and ADP concentrations. Identification of choke points and parameter sensitivity analysis indicate that the glycolytic pathway, and to a lesser extent the TCA cycle, are critical to the proper behavior of the system, while parameters in other components such as the respiratory chain are less critical. Notably, however, sensitivity analysis identifies the first reactions of nonglycolytic pathways as being important for the behavior of the system. The model is robust to deletion of malic enzyme activity, which is absent in mouse pancreatic beta cells. The model represents a step toward the construction of a model with species-specific parameters that can be used to understand mouse models of diabetes and the relationship of these mouse models to the human disease state.
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Project description:Previous studies reported that secreted frizzled-related protein-5 (Sfrp5) decreases beta cell proliferation and increases fasting insulin levels, but studies on direct effects of Sfrp5 on insulin secretion and its underlying mechanisms are missing. This study examined effects of Sfrp5 on (i) beta cell viability and proliferation, (ii) basal and glucose-stimulated insulin secretion and (iii) canonical and non-canonical Wnt signalling pathways. We incubated rat INS-1E cells with 0.1, 1 or 5 μg/ml recombinant Sfrp5 for 24h. We measured basal and glucose-stimulated insulin secretion at glucose concentrations of 2.5 and 20 mmol/l. Phosphorylated and total protein content as well as mRNA levels of markers of cell proliferation, canonical and non-canonical Wnt signalling pathways were examined using Western blotting and real-time PCR. Differences between treatments were analysed by repeated measurement one-way ANOVA or Friedman's test followed by correction for multiple testing using the Benjamini-Hochberg procedure. At 5 μg/ml, Sfrp5 reduced mRNA levels of cyclin-B1 by 25% (p<0.05). At 1 and 5 μg/ml, Sfrp5 increased glucose-stimulated insulin secretion by 24% and by 34% (both p<0.05), respectively, but had no impact on basal insulin secretion. Sfrp5 reduced the phosphorylation of the splicing forms p46 and p54 of JNK by 39% (p<0.01) and 49% (p<0.05), respectively. In conclusion, Sfrp5 reduced markers of cell proliferation, but increased in parallel dose-dependently glucose-stimulated insulin secretion in INS-1E cells. This effect is likely mediated by reduced JNK activity, an important component of the non-canonical Wnt signalling pathway.
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Project description:The pancreas plays a critical role in maintaining glucose homeostasis through the secretion of hormones from the islets of Langerhans. Glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell is the main mechanism for reducing elevated plasma glucose. Here we present a systematic modeling workflow for the development of kinetic pathway models using the Systems Biology Markup Language (SBML). Steps include retrieval of information from databases, curation of experimental and clinical data for model calibration and validation, integration of heterogeneous data including absolute and relative measurements, unit normalization, data normalization, and model annotation. An important factor was the reproducibility and exchangeability of the model, which allowed the use of various existing tools. The workflow was applied to construct a novel data-driven kinetic model of GSIS in the pancreatic β-cell based on experimental and clinical data from 39 studies spanning 50 years of pancreatic, islet, and β-cell research in humans, rats, mice, and cell lines. The model consists of detailed glycolysis and phenomenological equations for insulin secretion coupled to cellular energy state, ATP dynamics and (ATP/ADP ratio). Key findings of our work are that in GSIS there is a glucose-dependent increase in almost all intermediates of glycolysis. This increase in glycolytic metabolites is accompanied by an increase in energy metabolites, especially ATP and NADH. One of the few decreasing metabolites is ADP, which, in combination with the increase in ATP, results in a large increase in ATP/ADP ratios in the β-cell with increasing glucose. Insulin secretion is dependent on ATP/ADP, resulting in glucose-stimulated insulin secretion. The observed glucose-dependent increase in glycolytic intermediates and the resulting change in ATP/ADP ratios and insulin secretion is a robust phenomenon observed across data sets, experimental systems and species. Model predictions of the glucose-dependent response of glycolytic intermediates and biphasic insulin secretion are in good agreement with experimental measurements. Our model predicts that factors affecting ATP consumption, ATP formation, hexokinase, phosphofructokinase, and ATP/ADP-dependent insulin secretion have a major effect on GSIS. In conclusion, we have developed and applied a systematic modeling workflow for pathway models that allowed us to gain insight into key mechanisms in GSIS in the pancreatic β-cell.
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