Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

AMPA receptors regulate exocytosis and insulin release in pancreatic β cells

Ionotropic glutamate receptors (iGluRs) are expressed in islets and insulinoma cells and involved in insulin secretion. However, the exact roles that iGluRs play in β cells remain unclear. Here, we demonstrated that GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were expressed in mouse β cells. Glutamate application increased both cytosolic calcium and the number of docked insulin-containing granules, which resulted in augmentation of depolarization-induced exocytosis and high-glucose-stimulated insulin release. While glutamate application directly depolarized β cells, it also induced an enormous depolarization when K(ATP) channels were available. Glutamate application reduced the conductance of K(ATP) channels and increased voltage oscillations. Moreover, actions of AMPARs were absent in Kir6.2 knock-out mice. The effects of AMPARs on K(ATP) channels were mediated by cytosolic cGMP. Taken together, our experiments uncovered a novel mechanism by which AMPARs participate in insulin release.     

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic β cell death

There is a growing evidence of the role of autophagy in pancreatic β cell homeostasis. During development of type 2 diabetes, β cells are required to supply the increased demand of insulin. In such a stage, β cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, β cell death and overt diabetes. In this study, we used insulin secretion-deficient β cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived β cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased β cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient β cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in β cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.     

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic β cell death

There is a growing evidence of the role of autophagy in pancreatic β cell homeostasis. During development of type 2 diabetes, β cells are required to supply the increased demand of insulin. In such a stage, β cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, β cell death and overt diabetes. In this study, we used insulin secretion-deficient β cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived β cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased β cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient β cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in β cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.     

Gaegurin-6 stimulates insulin secretion through calcium influx in pancreatic β Rin5mf cells

Gaegurin-6, an antimicrobial peptide that belongs to the alpha-helix family, was isolated from the skin of Rana rugosa. Gaegurin-6 contains a hydrophobic motif at the N-terminus and a helical region at the C-terminus. Although gaegurin-6 has been implicated in cell signaling, the precise role in insulin secretion is currently unknown. We have attempted to determine whether gaegurin-6 affects insulin secretion and tried to elucidate the relationship between the structural motifs and biological activity. In this study, we have shown that gaegurin-6 stimulates insulin secretion and also increases the intracellular calcium concentration in pancreatic β Rin5mf cells. Moreover, a corollary study revealed that both the hydrophobicity of the N-terminus and the disulfide bridge of the C-terminus of gaegurin-6 are critical for its effects on insulin secretion. Membrane pore-forming ability is also observed in gaegurin-6, but not in the linear form or the N-terminus hydrophobic amino acid-deleted form. We further showed that these regions of gaegurin-6 are responsible for calcium influx in pancreatic β Rin5mf cells. Taken together, these results indicate that gaegurin-6 can affect insulin secretion in pancreatic β cells through the modulation of calcium influx.     

Oscillatory glucose flux in INS 1 pancreatic β cells: a self-referencing microbiosensor study

Signaling and insulin secretion in β cells have been reported to demonstrate oscillatory modes, with abnormal oscillations associated with type 2 diabetes. We investigated cellular glucose influx in β cells with a self-referencing (SR) microbiosensor based on nanomaterials with enhanced performance. Dose–response analyses with glucose and metabolic inhibition studies were used to study oscillatory patterns and transporter kinetics. For the first time, we report a stable and regular oscillatory uptake of glucose (averaged period 2.9 ± 0.6 min), which corresponds well with an oscillator model. This oscillatory behavior is part of the feedback control pathway involving oxygen, cytosolic Ca²⁺/ATP, and insulin secretion (periodicity approximately 3 min). Glucose stimulation experiments show that the net Michaelis–Menten constant (6.1 ± 1.5 mM) is in between GLUT2 and GLUT9. Phloretin inhibition experiments show an EC₅₀ value of 28 ± 1.6 μM phloretin for class I GLUT proteins and a concentration of 40 ± 0.6 μM phloretin caused maximum inhibition with residual nonoscillating flux, suggesting that the transporters not inhibited by phloretin are likely responsible for the remaining nonoscillatory uptake, and that impaired uptake via GLUT2 may be the cause of the oscillation loss in type 2 diabetes. Transporter studies using the SR microbiosensor will contribute to diabetes research and therapy development by exploring the nature of oscillatory transport mechanisms.     

ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3β in the signal network

HeLa cells treated with celastrol, a natural compound with inhibitive effect on proteasome, exhibited increase in apoptotic rate and characteristics of apoptosis. To clarify the signal network activated by celastrol to induce apoptosis, both the direct target proteins and undirect target proteins of celastrol were searched in the present study. Proteasome catalytic subunit β1 was predicted by computational analysis to be a possible direct target of celastrol and confirmed by checking direct effect of celastrol on the activity of recombinant human proteasome subunit β1 in vitro. Undirect target-related proteins of celastrol were searched using proteomic studies including two-dimensional electrophoresis (2-DE) analysis and iTRAQ-based LC-MS analysis. Possible target-related proteins of celastrol such as endoplasmic reticulum protein 29 (ERP29) and mitochondrial import receptor Tom22 (TOM22) were found by 2-DE analysis of total cellular protein expression profiles. Further study showed that celastrol induced ER stress and ER stress inhibitor could ameliorate cell death induced by celastrol. Celastrol induced translocation of Bax into the mitochondria, which might be related to the upregulation of BH-3-only proteins such as BIM and the increase in the expression level of TOM22. To further search possible target-related proteins of celastrol in ER and ER-related fractions, iTRAQ-based LC-MS method was use to analyze protein expression profiles of ER/microsomal vesicles-riched fraction of cells with or without celastrol treatment. Based on possible target-related proteins found in both 2-DE analysis and iTRAQ-based LC-MS analysis, protein–protein interaction (PPI) network was established using bioinformatic analysis. The important role of glycogen synthase kinase-3β (GSK3β) in the signal cascades of celastrol was suggested. Pretreatment of LiCL, an inhibitor of GSK3β, could significantly ameliorate apoptosis induced by celastrol. On the basis of the results of the present study, possible signal network of celastrol activated by celastrol leading to apoptosis was predicted.     

InsP₃receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2? entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP?Rs (InsP? receptors). The co-localization between Orai1 and IP?Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2? store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2? store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP?Rs and a basolateral pool that interacts with STIM1 following the Ca2? store depletion. Experiments on IP?R knockout animals demonstrated that the apical Orai1 localization does not require IP?Rs and that IP?Rs are not necessary for the activation of SOCE. However, the InsP?-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP?Rs could have an inhibitory effect on this Ca2? entry mechanism.     

Similar structural bases for the storage and release of secretory material in adreno-medullary and β pancreatic cells

Summary Both in normal pancreatic cells and in the adrenal medulla discontinuities have frequently been observed in the individual membranes surrounding secretion granules. In the cells the edges of these hiatuses are characteristically inverted in the manner of a scroll. It is suggested that intracytoplasmic release of secretory material may occur through these membrane perforations.     

Establishment of a pancreatic β cell proliferation model in vitro and a platform for diabetes drug screening

Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.     

Anlotinib exerts anti-cancer efficiency on lung cancer stem cells in vitro and in vivo through reducing NF-κB activity

Anlotinib is a multi-target tyrosine kinase inhibitor. Previous studies confirmed that anlotinib exerts anti-cancer efficiency. However, the functional roles of anlotinib on cancer stem cells (CSCs) are yet to be elucidated. In this study, lung CSCs were isolated and identified in vitro, and mouse xenografts were established in vivo. MTT assays, tumour sphere formation assays, TdT-mediated dUTP nick-end labelling (TUNEL) staining, Annexin V-FITC/PI staining, immunofluorescence analysis and Western blot were performed to investigate the anti-cancer effects of anlotinib on lung CSCs. The results showed that anlotinib inhibits the growth of lung CSCs in vitro and in vivo. In addition, anlotinib induced apoptosis of these cells along with down-regulated expression level of Bcl-2 whereas up-regulated Bax and cleaved caspase-3 expression. It also sensitized lung CSCs to the cytotoxicity of cisplatin and paclitaxel; the tumour sphere formation and expression levels of multiple stemness-associated markers, such as ALDH1 and CD133, were also decreased. Furthermore, the underlying mechanism indicated that anlotinib reduces the phosphorylated levels of NF-κB p65 and IκB-α in lung CSCs. Taken together, these findings suggested that anlotinib exerts potent anti-cancer effects against lung CSCs through apoptotic induction and stemness phenotypic attenuation. The mechanism could be associated with the suppression of NF-κB activity.     

PLCζ and its role as a trigger of development in vertebrates

A major unresolved issue in developmental biology is the precise mechanism whereby the sperm activates the oocyte. With the discovery that calcium signals are the primary trigger for oocyte activation, a key remaining question became the identification of the signaling protein that mediates such calcium signals at fertilization. A major step forward came in 2002 with the discovery of a sperm-specific mammalian phospholipase C called phospholipase C zeta (PLCζ), which had the expected properties of the mammalian oocyte activation factor and was subsequently identified in other vertebrate groups. Most recently, defects in PLCζ have been shown to be linked to certain types of male infertility in humans. Despite these advances, many questions remain about the precise mechanism of action of PLCζ and the extent of its role during oocyte activation in the vertebrate kingdom. In this review, we will look at the current state of understanding of PLCζ's mechanism of action and physiological role in mammals and other vertebrates, and identify areas of uncertainty that still remain to be resolved.     

Inhibition of histone acetyltransferase by naringenin and hesperetin suppresses Txnip expression and protects pancreatic β cells in diabetic mice

BackgroundThe damage of pancreatic β cells is a major pathogenesis of the development and progression of type 2 diabetes and there is still no effective therapy to protect pancreatic β cells clinically. In our previous study, we found that Quzhou Fructus Aurantii (QFA), which is rich in flavanones, had the protective effect of pancreatic β cells in diabetic mice. However, the underlying mechanism is still unclear.PurposeIn the current study, we administered naringenin and hesperetin, two major active components of QFA, to protect pancreatic β cells and to investigate the underlying molecular mechanism focusing on the epigenetic modifications.MethodsWe used diabetic db/db mouse and INS-1 pancreatic β cell line as in vivo and in vitro models to investigate the protective effect of naringenin and hesperetin on pancreatic β cells under high glucose environment and the related mechanism. The phenotypic changes were evaluatedby immunostaining and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-seq and ChIP-qPCR, flow cytometry and lentivirus infection.ResultsWe found that naringenin and hesperetin had an inhibitory effect on histone acetylation. We showed that naringenin and hesperetin protected pancreatic β cells in vivo and in vitro, and this effect was independent of their direct antioxidant capacity. The further study found that the inhibition of thioredoxin-interacting protein (Txnip) expression regulated by histone acetylation was critical for the protective role of naringenin and hesperetin. Mechanistically, the histone acetylation inhibition by naringenin and hesperetin was achieved through regulating AMPK-mediated p300 inactivation.ConclusionThese findings highlight flavanones and the phytomedicine rich in flavanones as important dietary supplements in protecting pancreatic β cells in advanced diabetes. In addition, targeting histone acetylation by phytomedicine is a potential strategy to delay the development and progression of diabetes.     

Bacteriophage lambda overcomes a perturbation in its host–viral genetic network through mutualism and evolution of life history traits*

An important driver of evolution in viruses is natural selection to optimize the use of their hosts’ genetic network. To learn how viruses respond to this pressure, we disrupted the genetic network of Escherichia coli to inhibit replication of its virus, bacteriophage lambda, and then observed how λ evolved to compensate. We deleted E. coli's dnaJ gene, which lambda uses to initiate DNA replication. Lambda partially restored its ability to reproduce with just two adaptive mutations associated with genes J and S. The location of the mutations was unexpected because they were not in genes that directly interact with DnaJ, rather they affected seemingly unrelated life history traits. A nonsynonymous J mutation increased lambda's adsorption rate and an S regulatory mutation delayed lysis timing. Lambda also recovered some of its reproductive potential through intracellular mutualism. This study offers two important lessons: first, viruses can rapidly adapt to disruptive changes in their host's genetic network. Second, organisms can employ mechanisms thought to operate at the population scale, such as evolution of life history traits and social interactions, in order to overcome hurdles at the molecular level. As life science research progresses and new fields become increasingly specialized, these results remind us of the importance of multiscale and interdisciplinary approaches to understand adaptation.     

SAD-A and AMPK kinases: The “yin and yang” regulators of mTORC1 signaling in pancreatic β cells

SAD-A kinase is a member of the AMPK-related family of kinases, which are under the control of LKB1 kinase. In the human kinome, SAD-A is most closely related to AMPK, a key energy sensor and master regulator of metabolism. In contrast to AMPK, little is known about the physiological function of the SAD-A kinase in metabolism. Recent studies using knockout mice have revealed a striking role of the SAD-A kinase in regulating dynamic functions of islet β cells, from glucose-stimulated insulin secretion (GSIS), islet β-cell size and mass, to GLP-1 response as the first tissue-specific effector of mTORC1 signaling. These studies suggest that SAD-A and AMPK kinase may function as the positive and negative regulators of mTORC1 signaling in islet β cells. Importantly, these findings have implicated SAD-A kinase as a novel drug target for the treatment of type 2 diabetes.     

Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic β cells: a simulation study

To clarify the mechanisms underlying the pancreatic β-cell response to varying glucose concentrations ([G]), electrophysiological findings were integrated into a mathematical cell model. The Ca(2+) dynamics of the endoplasmic reticulum (ER) were also improved. The model was validated by demonstrating quiescent potential, burst-interburst electrical events accompanied by Ca(2+) transients, and continuous firing of action potentials over [G] ranges of 0-6, 7-18, and >19 mM, respectively. These responses to glucose were completely reversible. The action potential, input impedance, and Ca(2+) transients were in good agreement with experimental measurements. The ionic mechanisms underlying the burst-interburst rhythm were investigated by lead potential analysis, which quantified the contributions of individual current components. This analysis demonstrated that slow potential changes during the interburst period were attributable to modifications of ion channels or transporters by intracellular ions and/or metabolites to different degrees depending on [G]. The predominant role of adenosine triphosphate-sensitive K(+) current in switching on and off the repetitive firing of action potentials at 8 mM [G] was taken over at a higher [G] by Ca(2+)- or Na(+)-dependent currents, which were generated by the plasma membrane Ca(2+) pump, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, and TRPM channel. Accumulation and release of Ca(2+) by the ER also had a strong influence on the slow electrical rhythm. We conclude that the present mathematical model is useful for quantifying the role of individual functional components in the whole cell responses based on experimental findings.