Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell.
It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage‐sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta‐cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin‐containing secretory granules. Both glucose and 2‐ketoisocaproate (KIC) raised [Ca2+]i in a dose‐dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell‐to‐cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage‐sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.
RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.
The alpha‐2,8‐linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA‐NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre‐existing PSA from the plasma membrane and the re‐expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA‐NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA‐NCAM expression and insulin secretion to control, non‐stimulated levels. Finally, subcellular fractionation of an insulin‐secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA‐NCAM. These results suggest that PSA‐NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA‐NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.
Abstract. The capacity for long-distance migration of the oligodendrocyte precursor cell, oligondendrocytetype 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-mehtyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca 2÷ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg 2÷. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.
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