Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Cytoskeletal and scaffolding proteins as structural and functional determinants of TRP channels

Transient receptor potential (TRP) channels are six transmembrane-spanning proteins, with variable selectivity for cations, that play a relevant role in intracellular Ca^2 + homeostasis. There is a large body of evidence that shows association of TRP channels with the actin cytoskeleton or even the microtubules and demonstrating the functional importance of this interaction for TRP channel function. Conversely, cation currents through TRP channels have also been found to modulate cytoskeleton rearrangements. The interplay between TRP channels and the cytoskeleton has been demonstrated to be essential for full activation of a variety of cellular functions. Furthermore, TRP channels have been reported to take part of macromolecular complexes including different signal transduction proteins. Scaffolding proteins play a relevant role in the association of TRP proteins with other signaling molecules into specific microdomains. Especially relevant are the roles of the Homer family members for the regulation of TRPC channel gating in mammals and INAD in the modulation of Drosophila TRP channels. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.     

Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.     

Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling

Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca²⁺]_i) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca²⁺]_i transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca²⁺ dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca²⁺]_i was increased, the systolic [Ca²⁺]_i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca²⁺ load was reduced. These effects were correlated to a decrease in the Na⁺/Ca²⁺ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca²⁺]_i and global [Ca²⁺]_i transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca²⁺]_i handling play an essential role in postconditioning action of Ucn.     

Downregulation of 14-3-3 Proteins in a Kainic Acid-Induced Neurotoxicity Model

The 14-3-3 proteins are among the most abundant proteins expressed in the brain, comprising about 1% of the total amount of soluble brain proteins. Through phosphoserine- and phosphothreonine-binding motifs, 14-3-3 proteins regulate many signaling proteins and cellular processes including cell death. In the present study, we utilized a well-known kainic acid (KA)-induced excitotoxicity rat model and examined the expression of 14-3-3 and its isoforms in the frontal cortex of KA-treated and control animals. Among the different 14-3-3 isoforms, abundant levels of eta and tau were detected in the frontal cortex, followed by sigma, epsilon, and gamma, while the expression levels of alpha/beta and zeta/delta isoforms were low. Compared to the control animals, KA treatment induced a significant downregulation of the overall 14-3-3 protein level as well as the levels of the abundant isoforms eta, tau, epsilon, and gamma. We also investigated two 14-3-3-interacting proteins that are involved in the cell death process: Bcl-2-associated X (BAX) and extracellular signal-regulated kinase (ERK). Both BAX and phosphorylated ERK showed increased levels following KA treatment. Together, these findings demonstrate an abundance of several 14-3-3 isoforms in the frontal cortex and that KA treatment can cause a downregulation of 14-3-3 expression and an upregulation of 14-3-3-interacting proteins BAX and phospho-ERK. Thus, downregulation of 14-3-3 proteins could be one of the early molecular events associated with excitotoxicity. This could lead to subsequent upregulation of 14-3-3-binding proteins such as BAX and phospho-ERK that contribute to further downstream apoptosis processes, eventually leading to cell death. Maintaining sufficient levels of 14-3-3 expression and function may become a target of therapeutic intervention for excitotoxicity-induced neurodegeneration.     

Complex regulation of store-operated Ca2+ entry pathway by PKC-{varepsilon} in vascular SMCs

The role of PKC in the regulation of store-operated Ca²⁺ entry (SOCE) is rather controversial. Here, we used Ca²⁺-imaging, biochemical, pharmacological, and molecular techniques to test if Ca²⁺-independent PLA₂β (iPLA₂β), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂β and Ca²⁺ entry that were absent in SMCs in which iPLA₂β was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMCs, which correlates with the expression of the specific PKC- isoform. Molecular downregulation of PKC- impaired TG- and OAG-induced Ca²⁺ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs. protein kinase C-; Ca²⁺-independent phospholipase A₂; diacylglycerol; smooth muscle cells     

Association analysis of urotensin II gene (UTS2) and flanking regions with biochemical parameters related to insulin resistance

Background

Urotensin II (UII) is a potent vasoconstrictor peptide, which signals through a G-protein coupled receptor (GPCR) known as GPR14 or urotensin receptor (UTR). UII exerts a broad spectrum of actions in several systems such as vascular cell, heart muscle or pancreas, where it inhibits insulin release.

Objective

Given the reported role of UII in insulin secretion, we have performed a genetic association analysis of the UTS2 gene and flanking regions with biochemical parameters related to insulin resistance (fasting glucose, glucose 2 hours after a glucose overload, fasting insulin and insulin resistance estimated as HOMA).

Results and Conclusions

We have identified several polymorphisms associated with the analysed clinical traits, not only at the UTS2 gene, but also in thePER3 gene, located upstream from UTS2. Our results are compatible with a role for UII in glucose homeostasis and diabetes although we cannot rule out the possibility that PER3 gene may underlie the reported associations.     

Differential segmental activation of Ca2+ -dependent CI-and K+ channels in pulmonary arterial myocytes

Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca2+ -dependent CI- (CICa) and K+ (KCa) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca2+] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca2+ oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of KCa and CICa conductances. Resistance arteries contained a more homogeneous cell population with predominance of CICa channel activation. The relation between KCa and CICa conductances in a given conduit myocyte determines the size of the V(m)change in response to a rise of cytosolic [Ca2+]. Conduit myocytes tend to hyperpolarize towards the K+ equilibrium potential (approximately - 90 m V). In resistance myocytes, release of Ca2+ from stores activates CI Cachannels and brings Vm to a value close to the chloride equilibrium potential (approximately - 20 or - 30 m V) thus favouring opening of Ca2+ channels and Ca2+ influx. In resistance vessels CICachannels contribute to link agonist-induced Ca2+ release from stores and membrane depolarization, thus permitting protracted vasoconstriction.