David Mears

Regulation of Insulin Secretion in Islets of Langerhans by Ca2+Channels

Insulin secretion from β-cells of the pancreatic islets of Langerhans is triggered by Ca2+ influx through voltage-dependent Ca2+ channels. Electrophysiological and molecular studies indicate that β-cells express several subtypes of these channels. This review discusses their roles in regulating insulin secretion, focusing on recent studies using β-cells, exogenous expression systems, and Ca2+ channel knockout mice. These investigations reveal that L-type Ca2+ channels in the β-cell physically interact with the secretory apparatus by binding to synaptic proteins on the plasma membrane and insulin granule. As a result, Ca2+ influx through L-type channels efficiently and rapidly stimulates release of a pool of insulin granules in close contact with the channels. Thus, L-type Ca2+ channel activity is preferentially coupled to exocytosis in the β-cell, and plays a critical role in regulating the dynamics of insulin secretion. Non-L-type channels carry a significant portion of the total voltage-dependent Ca2+ current in β-cells and cell lines from some species, but nevertheless account for only a small fraction of insulin secretion. These channels may regulate exocytosis indirectly by affecting membrane potential or second messenger signaling pathways. Finally, voltage-independent Ca2+ entry pathways and their potential roles in β-cell function are discussed. The emerging picture is that Ca2+ channels regulate insulin secretion at multiple sites in the stimulus-secretion coupling pathway, with the specific role of each channel determined by its biophysical and structural properties.

Role of the M3 muscarinic acetylcholine receptor in β‐cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose‐stimulated insulin secretion from pancreatic β‐cells. Studies with isolated islets prepared from whole body M3 muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose‐dependent insulin secretion is exclusively mediated by the M3 muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M3 receptors only in pancreatic β‐cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M3 receptors in pancreatic β‐cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet‐induced glucose intolerance and hyperglycaemia. These findings indicate that β‐cell M3 muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through β‐cell M3 muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Ion channel formation by Alzheimer's disease amyloid β-peptide (Aβ40) in unilamellar liposomes is determined by anionic phospholipids

Abstract Incorporation of Alzheimers disease amyloid β-proteins (AβPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide–membrane interactions involved in channel formation, we used cation reporter dyes to measure AβP-induced influx of Na + , Ca 2+ , and K + into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Aβ40, but not Aβ40-1 or Aβ28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Aβ40-induced changes in cation concentration, which we attribute to ion entry through Aβ40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AβP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Aβ40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AβP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AβP channels.

Network science and the human brain: Using graph theory to understand the brain and one of its hubs, the amygdala, in health and disease.

Over the past 15 years, the emerging field of network science has revealed the key features of brain networks, which include small‐world topology, the presence of highly connected hubs, and hierarchical modularity. The value of network studies of the brain is underscored by the range of network alterations that have been identified in neurological and psychiatric disorders, including epilepsy, depression, Alzheimers disease, schizophrenia, and many others. Here we briefly summarize the concepts of graph theory that are used to quantify network properties and describe common experimental approaches for analysis of brain networks of structural and functional connectivity. These range from tract tracing to functional magnetic resonance imaging, diffusion tensor imaging, electroencephalography, and magnetoencephalography. We then summarize the major findings from the application of graph theory to nervous systems ranging from Caenorhabditis elegans to more complex primate brains, including man. Focusing, then, on studies involving the amygdala, a brain region that has attracted intense interest as a center for emotional processing, fear, and motivation, we discuss the features of the amygdala in brain networks for fear conditioning and emotional perception. Finally, to highlight the utility of graph theory for studying dysfunction of the amygdala in mental illness, we review data with regard to changes in the hub properties of the amygdala in brain networks of patients with depression. We suggest that network studies of the human brain may serve to focus attention on regions and connections that act as principal drivers and controllers of brain function in health and disease.†Published 2016

Muscarinic Agonists Activate Ca2+ Store-operated and -independent Ionic Currents in Insulin-secreting HIT-T15 Cells and Mouse Pancreatic β-Cells

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic β-cells by depolarizing the membrane to enhance voltage-gated Ca2+ influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal β-cell line (HIT-T15) and mouse pancreatic β-cells. In whole-cell recordings, the muscarinic agonist carbachol (CCh) dose-dependently and reversibly activated a voltage-independent, nonselective current (whole-cell conductance 24 pS/pF, reversal potential ~-15 mV). The current, which we refer to as Imusc, was blocked by atropine, a muscarinic receptor antagonist, and SKF 96365, a nonspecific ion channel blocker. The magnitude of the current decreased by 52% when extracellular Na+ was removed, but was not affected by changes in extracellular Ca2+, confirming that Imusc is a nonselective current. To determine if Imusc activates following release of Ca2+ from an intracellular store, we blocked intracellular IP3 receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca2+ store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential (~0 mV) than the current activated by CCh under control conditions. This result indicates that heparin had blocked a component of Imusc, which likely activates following release of stored Ca2+. Depleting IP3-sensitive calcium stores with thapsigargin also activated a non-selective, SKF 96365-blockable current in HIT cells. The properties of this putative store-operated current were similar to the component of Imusc that was blocked by heparin, being voltage-independent and reversing near −30 mV. We conclude that Imusc consists of store-operated and store-independent components, both of which may contribute to the depolarizing action of muscarinic agonists on pancreatic β-cells.

In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components.

Tungsten-alloy has carcinogenic potential as demonstrated by cancer development in rats with intramuscular implanted tungsten-alloy pellets. This suggests a potential involvement of epigenetic events previously implicated as environmental triggers of cancer. Here, we tested metal induced cytotoxicity and epigenetic modifications including H3 acetylation, H3-Ser10 phosphorylation and H3-K4 trimethylation. We exposed human embryonic kidney (HEK293), human neuroepithelioma (SKNMC), and mouse myoblast (C2C12) cultures for 1-day and hippocampal primary neuronal cultures for 1-week to 50-200 μg/ml of tungsten-alloy (91% tungsten/6% nickel/3% cobalt), tungsten, nickel, and cobalt. We also examined the potential role of intracellular calcium in metal mediated histone modifications by addition of calcium channel blockers/chelators to the metal solutions. Tungsten and its alloy showed cytotoxicity at concentrations > 50 μg/ml, while we found significant toxicity with cobalt and nickel for most tested concentrations. Diverse cell-specific toxic effects were observed, with C2C12 being relatively resistant to tungsten-alloy mediated toxic impact. Tungsten-alloy, but not tungsten, caused almost complete dephosphorylation of H3-Ser10 in C2C12 and hippocampal primary neuronal cultures with H3-hypoacetylation in C2C12. Dramatic H3-Ser10 dephosphorylation was found in all cobalt treated cultures with a decrease in H3 pan-acetylation in C2C12, SKNMC and HEK293. Trimethylation of H3-K4 was not affected. Both tungsten-alloy and cobalt mediated H3-Ser10 dephosphorylation were reversed with BAPTA-AM, highlighting the role of intracellular calcium, confirmed with 2-photon calcium imaging. In summary, our results for the first time reveal epigenetic modifications triggered by tungsten-alloy exposure in C2C12 and hippocampal primary neuronal cultures suggesting the underlying synergistic effects of tungsten, nickel and cobalt mediated by changes in intracellular calcium homeostasis and buffering.

The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic β-cells.

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca2+. The contribution of intracellular Ca2+ stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP3 receptors and defects in IP3-dependent Ca2+ signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca2+ stores in the β-cell, we measured the effects of Ca2+ mobilizing agents on electrical activity, intracellular Ca2+ and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca2+ mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca2+ stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.

Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cells

We have investigated the effects of loperamide on intracellular Ca2+ stores and membrane K+ channels in insulin‐secreting hamster insulinoma (HIT‐T15) cells. In cell‐attached patch‐clamp mode, loperamide (3–250 μM) activated large single‐channel currents. The loperamide‐activated currents were tentatively identified as Ca2+‐activated K+ channel (KCa) currents based on their single‐channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single‐channel currents with a conductance (32 pS) indicative of adenosine triphosphate‐sensitive K+ channels (KATP channels) were also recorded, but were insensitive to loperamide. Surprisingly, the loperamide‐activated currents persisted in the absence of extracellular Ca2+. Yet under these conditions, we still measured loperamide‐induced Ca2+ increases. These effects are dose dependent. Loperamide had no effects in the inside‐out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca2+ from intracellular stores. Carbachol (100 μM), an agonist of muscarinic receptors, which mediates IP3‐dependent intracellular Ca2+ release, enhanced the effects of loperamide on KCa channels. Both the putative KCa currents and Ca2+ signals induced by loperamide (with ‘0’ [Ca2+]o) were abolished when the intracellular Ca2+ stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca2+‐ATPase inhibitor that blocks reuptake of calcium. These data indicate that loperamide in insulin‐secreting β‐cells evokes intracellular Ca2+ release from IP3‐gated stores and activates membrane currents that appear to be carried by KCa, rather than KATP channels.