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    • PKC does not however directly

    2022-04-09

    PKC does not, however, directly stimulate secretion by initiating calcium influx into the cell [51]. Work from multiple groups measuring calcium currents has shown that PMA alone or coupled with glucose does not modulate intracellular calcium influx [34], [51], [52], [53]. Instead of contributing to calcium influx, the application of PMA shifts the calcium sensitivity of exocytosis to lower calcium concentrations. This was originally shown in chromaffin AMG 487 [54], [55] and then in beta cells [56], [57], [58], [59] using a variety of cracked or patched cell systems where the calcium concentration can be clamped. In pioneering work from Zawalich et al., it was shown that the combination of a calcium ionophore and PMA could reproduce the classic biphasic insulin release pattern [36]. Calcium ionophores produced the initial spike in insulin release, and PMA treatment supported the second phase of insulin release in the continued presence of the ionophore. Indeed, PKC activation alone is not required for the first phase of insulin secretion after glucose stimulus [60], [61]. Together, these early studies suggested PKC potentiates insulin released in response to glucose stimulation, but PKC itself cannot initiate release [62]. PKC likely increases the calcium sensitivity of the exocytotic machinery, thereby potentiating the overall amount of insulin secreted in response to a given calcium stimulus. Aside from studies in model systems of insulin secretion, there is evidence that PKC dysfunction could play a role in diabetes. Aberrant PKC activity, perhaps through misregulation of PKC isozymes, might underlie the diabetic phenotype in Goto-Kakizaki rats, a common animal model for type 2 diabetes [63], [64], [65]. Furthermore, PKC-ε knockout in a mouse model prevented glucose intolerance and inhibition of PKC-ε increased insulin secretion in diabetic mice, suggesting a role of this isozyme in disease [66]. In new work, the immediately-releasable pool of vesicles, composed of vesicles tightly associated with calcium channels, was observed to be absent in insulin-secreting cells from human donors with type II diabetes [67]. As discussed below, interaction between L-type calcium channels and insulin granules may be modulated by PKC. A word of caution before we consider how PKC enhances insulin release. Although a useful pharmacologic tool, the broad activity of phorbol esters likely has contributed to significant confusion in the field. It was noted at least as early as 1985 by Tamagawa et al. that other targets of PMA could be contributing to the drug’s effects on insulin release [59], [68]. For example, munc13, an essential factor in vesicle priming and exocytosis, is a phorbol ester receptor [69], [70]. Studies have identified munc13 as being crucial for the second phase of insulin release, the phase in which PKC was originally postulated to act [36], [71], [72]. Though more recent work in neurons has developed an integrated model in which both PKC-dependent (possibly via munc18) and PKC-independent (possibly via munc13) pathways are necessary for exocytosis [73], [74], the wide-ranging targets of PMA make deconvoluting the specific effects of PKC challenging.
    How does PKC globally enhance insulin release? Consistent with its ubiquitous activity, PKC likely acts on many targets to regulate insulin release. Here, we distinguish between global cell-wide PKC effects (Fig. 1A) AMG 487 and local effects on protein targets directly involved in exocytosis of dense core vesicles (Fig. 1B). First, we will cover PKC modulation at global sites: One global effect of PKC activation is actin rearrangements that allow vesicles to move towards the plasma membrane. Many secretory cells have a ∼100nm-thick dense cortical network of actin near the plasma membrane that is thought to act as a physical barrier to vesicles [75]. PKC-induced reorganization of this actin layer has been observed in multiple systems: beta cells, chromaffin cells, neuronal systems, and others [47], [53], [76], [77], [78], [79]. Rearrangement of the actin cortex has been interpreted as having two effects. First, vesicle pools near the plasma membrane are larger (in the case of pre-stimulus PKC activation), which accounts for increased initial rates of exocytosis from the readily-releasable pool (RRP) [53], [80]. The second effect is to enhance RRP refilling after initial stimulation to potentiate sustained release or cell adaptation [53], [81]. The sustained phase of insulin release must include the recruitment of additional insulin granules from the reserve pool to the plasma membrane, consequently it seems likely PKC-mediated cortical actin rearrangement could play a prominent role in potentiating sustained insulin release.