Adenosine 3\u27, 5\u27-cyclic monophosphate activation of islet chloride channels

The objective of this thesis was to understand the regulation of islet Cl⁻ current by cAMP. This current, known as Icl,islet flew is the first Cl⁻ channel current characterized in pancreatic �� cells. Icl,islet has been hypothesized to modulate insulin secretion through changes in islet electrical activity. Both 5 ��M forskolin and 100 ��M IBMX (3-isobutyl-1-methylxanthine), agents that increase intracellular cAMP, were shown to activate an outwardly-rectifying ionic current in HIT cells that closely resembled Icl,islet. The current was blocked when iodide was substituted for external Cl⁻ or when the Cl⁻ channel blocker niflumic acid was applied to cells. In contrast, removal of [Na⁺]O did not inhibit the current. In many cells, Cl⁻ current activated and then spontaneously deactivated following cAMP stimulation, suggesting the possibility that the channel desensitizes to [cAMP]i. Exposing cells to multiple cAMP activators revealed that Cl⁻ current declined because it became refractory to increased [cAMP]i. The implication of these results to islet physiology is discussed

What Is the Metabolic Amplification of Insulin Secretion and Is It (Still) Relevant?

The pancreatic beta-cell transduces the availability of nutrients into the secretion of insulin. While this process is extensively modified by hormones and neurotransmitters, it is the availability of nutrients, above all glucose, which sets the process of insulin synthesis and secretion in motion. The central role of the mitochondria in this process was identified decades ago, but how changes in mitochondrial activity are coupled to the exocytosis of insulin granules is still incompletely understood. The identification of ATP-sensitive K+-channels provided the link between the level of adenine nucleotides and the electrical activity of the beta cell, but the depolarization-induced Ca2+-influx into the beta cells, although necessary for stimulated secretion, is not sufficient to generate the secretion pattern as produced by glucose and other nutrient secretagogues. The metabolic amplification of insulin secretion is thus the sequence of events that enables the secretory response to a nutrient secretagogue to exceed the secretory response to a purely depolarizing stimulus and is thus of prime importance. Since the cataplerotic export of mitochondrial metabolites is involved in this signaling, an orienting overview on the topic of nutrient secretagogues beyond glucose is included. Their judicious use may help to define better the nature of the signals and their mechanism of action

An emerging role for NAADP-mediated Ca2+ signaling in the pancreatic beta-cell

Several recent reports, including one in this journal, have reignited the debate about whether the calcium-mobilizing messenger, nicotinic adenine nucleotide diphosphate (NAADP) plays a central role in the regulation of calcium signalling in pancreatic β-cell. These studies have highlighted a role for NAADP-induced Ca(2+) mobilization not only in mediating the effects of the incretin, GLP-1 and the autocrine proliferative effects of insulin, but also possibly a fundamental role in glucose-mediated insulin secretion in the pancreatic β-cell

The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR Review 19.

Acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC) are both members of the ENaC/degenerin family of amiloride-sensitive Na(+) channels. ASICs act as proton sensors in the nervous system where they contribute, besides other roles, to fear behaviour, learning and pain sensation. ENaC mediates Na(+) reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction, here we will review selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signalling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone-dependent ENaC regulation and the evidence for an aldosterone-independent control of ENaC activity, such as regulation by dietary K(+) . We then provide an overview of the regulation of ENaC by proteases, a topic of increasing interest over the past few years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles

Fatty acid required for glucose-induced change in beta cell plasma membrane potential leading to insulin secretion

The pancreatic β−cell secretes insulin through a combination of triggering and amplification pathways in response to glucose. Glucose metabolism is known to increase intracellular calcium and trigger insulin secretion from the β−cell while fatty acid (FA), an essential component of glucose stimulated insulin secretion (GSIS), is known to amplify secretion through varied mechanisms. Orlistat, a lipase inhibitor, blocks GSIS although the mechanism has not been clearly elucidated. We show that orlistat can also inhibit basal insulin secretion. FA can prevent/reverse orlistat-induced inhibition of secretion but fails to amplify GSIS after orlistat treatment. Here we test the hypothesis that FA is required to maintain normal plasma membrane potential and calcium influx in the β−cell. Clonal pancreatic β−cells (INS-1 832/13) were cultured in RPMI media containing 11 mM glucose and 10% FBS. Insulin secretion was measured over 2 hours with and without FA and orlistat (200 µM) using homogeneous time resolved fluorescence insulin assay (HTRF, Cisbio) and reported as ng/million cells. Intracellular calcium was measured in single cells using fura-2 AM. Single cell membrane potential was measured with virally expressed ARC Lite protein (Montana molecular). High glucose (8 mM) stimulated insulin secretion at least 3-fold over basal glucose (2 mM) and addition of FA enhanced GSIS. Orlistat almost completely abolished GSIS. This inhibition was mostly prevented in the presence of FA. Orlistat blocked calcium influx required for triggering insulin release while addition of FA recovered normal calcium homeostasis. The monoacylglycerol lipase inhibitor JZL 184 reduced both calcium influx and the increase in plasma membrane potential induced by KCl. FA recovered plasma membrane potential inhibited by JZL 184. Our results suggest that reducing β−cell intracellular FA availability by lipase inhibition blocks GSIS by preventing the glucose-induced rise in plasma membrane potential required to induce β−cell calcium influx through voltage dependent calcium channels (VDCC). FA is thus required to maintain normal nutrient metabolic coupling to insulin secretion. Whether the effect of FA to modulate plasma membrane potential plays a role in β−cell insulin hypersecretion resulting from excess nutrients requires further investigation

Stimulatory effect of endogenous and exogenous growth hormone secretagogues on isolated porcine somatotropes

This dissertation is an effort to further define sites and cellular mechanisms of action of exogenous and endogenous GH secretagogues (GHS) in porcine somatotropes.;The first study looked at the ability of benzolactam GHS - L-692,585 (L-585) to stimulate Ca2+ transients and GH secretion from cultured porcine somatotropes. Perfusion application of L-585 dose dependently increased intracellular calcium ([Ca2+]i) in somatotropes. Using reverse hemolytic plaque assay (RHPA) it was shown that incubation of cultured cells with L-585 had a stimulatory effect of L-585 on GH secretion. With concurrent [Ca2+]i measurements and RHPA and, it was shown that the increase in [Ca2+]i evoked by L-585 coincides with GH secretion.;The second study using atomic force microscopy demonstrated for the first time the presence of the fusion pores at the plasma membrane of isolated porcine somatotropes. In the resting somatotropes were found \u27pits\u27 containing several \u27depressions\u27, or fusion pores, with diameter of 100--200 nm. After stimulation of secretion with L-585 \u27depressions\u27 enlargement for about 40% was observed. That \u27depressions\u27 are structures at plasma membrane of somatotropes involved in hormone secretion was confirmed by using gold-tagged GH-antibody. It is proposed that fusion pores may be common structures for all secretory cells, and that they are the sites where following stimulation membrane-bound secretory vesicles transiently dock and fuse to release vesicular content.;The third study investigated the effects of an endogenous GHS - ghrelin on [Ca2+]i in cultured porcine somatotropes. The application of ghrelin resulted with changes in [Ca2+] i similar to those induced by L-585. Our experiments have shown that rapid increase in [Ca2+]i induced both by L-585 and ghrelin originated from the intracellular stores, while the plateau phase resulted from somatotropes depolarization and extracellular calcium entry. Simultaneous application or application in succession of L-585 and ghrelin has revealed antagonistic effects between these two agonists that may result from competition at the receptor level and/or downstream from GHS-R.;Collectively, the results from these studies indicate that stimulatory effect of ghrelin and synthetic GHS on [Ca2+]i in cultured porcine somatotropes closely parallel each other, in a manner that is consistent with an increase of GH secretion

An insulin hypersecretion phenotype precedes pancreatic β cell failure in MODY3 patient-specific cells

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C β cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 β cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 β cells. Our findings identify a pathogenic mechanism leading to β cell failure in MODY3.Peer reviewe

Sweet Taste Signaling: The Core Pathways and Regulatory Mechanisms

Sweet taste, a proxy for sugar-derived calories, is an important driver of food intake, and animals have evolved robust molecular and cellular machinery for sweet taste signaling. The overconsumption of sugar-derived calories is a major driver of obesity and other metabolic diseases. A fine-grained appreciation of the dynamic regulation of sweet taste signaling mechanisms will be required for designing novel noncaloric sweeteners with better hedonic and metabolic profiles and improved consumer acceptance. Sweet taste receptor cells express at least two signaling pathways, one mediated by a heterodimeric G-protein coupled receptor encoded by taste 1 receptor members 2 and 3 (TAS1R2 + TAS1R3) genes and another by glucose transporters and the ATP-gated potassium (KATP) channel. Despite these important discoveries, we do not fully understand the mechanisms regulating sweet taste signaling. We will introduce the core components of the above sweet taste signaling pathways and the rationale for having multiple pathways for detecting sweet tastants. We will then highlight the roles of key regulators of the sweet taste signaling pathways, including downstream signal transduction pathway components expressed in sweet taste receptor cells and hormones and other signaling molecules such as leptin and endocannabinoids