Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion

Abstract

Extracellular ATP released from pancreatic ␤-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic ␤-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulinsecreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic ␤-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors. ectonucleotide triphosphate diphosphohydrolase 3; adenosine 5=-triphosphate; insulin secretion; islets IN PANCREATIC ␤-CELLS, ATP and related nucleotides are coreleased with insulin from the large, dense core insulin-containing granules in response to physiological stimuli that trigger insulin secretion (9, 23). Once released into the extracellular space, ATP and products of its metabolism, in turn, modulate insulin secretion in the autocrine manner (18).ATP increases insulin secretion in pancreatic ␤-cells via activation of a family of metabotropic P2Y and ionotropic P2X receptors located on the plasma membrane. Purinergic P2Y receptors have long been recognized as important regulators of insulin secretion (18,25). Activation of P2Y receptors amplifies glucose-induced insulin secretion via induction of Ca 2ϩ release from the inositol trisphosphate-sensitive intracellular stores and via generation of bioactive lipids such as diacylgycerol (8,31,34). Pancreatic ␤-cells were also reported to express ionotropic P2X receptors, including P2X1 and P2X3 subtypes (11,29). Activation of P2X receptors results in activation of Ca 2ϩ flux through the plasma membrane and stimulation of insulin secretion independently of ambient glucose levels.In addition to P2 receptors, pancreatic ␤-cells are also equipped with purinergic P1 receptors that are preferentially activated by adenosine, a product of ATP degradation (10, 28). Adenosine inhibits glucose-induced insulin secretion via activation of A1 adenosine receptor coupled to inhibition of...

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

“…We show here that ATP stimulates depolarisation-evoked exocytosis without affecting Ca 2+ currents in human beta cells. We found no evidence for a negative role of ATP in insulin secretion from human islets, consistent with the potentiation of insulin secretion from human islets following block of extracellular ATP degradation [11,33].…”

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

“…There is very limited information about NTPDase3 protein distribution in humans, but the NIH Genotype-Tissue Expression dataset shows that in addition to the pancreas, NTPDase3 mRNA is primarily expressed in the brain, salivary gland, and urinary bladder. Of particular interest to the current study, NTPDase3 is detectable by immunohistochemistry in pancreatic islets of both humans and rodents, and recent work has suggested activity in regulating insulin secretion (Lavoie et al, 2010;Petit et al, 2009;Syed et al, 2013). Our work shows that NTPDase3 is a cell-surface biomarker of adult human b cells and that the antibody directed to this protein could be developed for multiple applications including b cell sorting, in vivo imaging, and targeting.…”

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain

“…For example, one of thes NTPDase3/CD39L3's major biological functions appears to be in regulating insulin secretion from pancreatic β cells [25,26]. A better understanding of the molecular mechanism governing NTPDase activity should facilitate drugdesigning efforts on agonists and antagonists of NTPDase3/ CD39L3 protein for potential clinical therapeutic application.…”

“…Nglycan modification is also required for NTPDase 2 for trafficking and folding [24]; mutating Asn443 in ACR5 retains the protein in ER. NTPDase3/CD39L3, the dominantly expressed NTPDase in pancreatic islet cells [25,26], requires N-glycan for its folding and activity [27]. Mutating the highly conserved Asn81 N-linked site across all cell surface membrane NTPDases retains the cell surface expression, while these proteins exhibit significant loss of NTPDase activity [28].…”