A 12‐lipoxygenase‐Gpr31 signaling axis is required for pancreatic organogenesis in the zebrafish

Abstract

12-Lipoxygenase (12-LOX) is a key enzyme in arachidonic acid metabolism, and alongside its major product, 12-HETE, plays a key role in promoting inflammatory signaling during diabetes pathogenesis. Although 12-LOX is a proposed therapeutic target to protect pancreatic islets in the setting of diabetes, little is known about the consequences of blocking its enzymatic activity during embryonic development. Here, we have leveraged the strengths of the zebrafish-genetic manipulation and pharmacologic inhibition-to interrogate the role of 12-LOX in pancreatic development. Lipidomics analysis during zebrafish development demonstrated that 12-LOX-generated metabolites of arachidonic acid increase sharply during organogenesis stages, and that this increase is blocked by morpholino-directed depletion of 12-LOX. Furthermore, we found that either depletion or inhibition of 12-LOX impairs both exocrine pancreas growth and unexpectedly, the generation of insulinproducing β cells. We demonstrate that morpholino-mediated knockdown of GPR31, a purported G-protein-coupled receptor for 12-HETE, largely phenocopies both the depletion and the inhibition of 12-LOX. Moreover, we show that loss of GPR31 impairs pancreatic bud fusion and pancreatic duct morphogenesis. Together, these data provide new insight into the requirement of 12-LOX in pancreatic organogenesis and islet formation, and additionally provide evidence that its effects are mediated via a signaling axis that includes the 12-HETE receptor GPR31.

12-Lipoxygenase Governs the Innate Immune Pathogenesis of Islet Inflammation and Autoimmune Diabetes

“…This finding is especially relevant as 12-LOX is an enzyme with no known transcriptional function per se. It remains possible that this transcriptional effect is the result of the downstream activity of GPR31, a receptor for 12-HETE [25,42]. Further studies of Gpr31 mutant zebrafish and mice will be required to address this issue.…”

“…Previous studies in mouse models of T1D demonstrated that global deletion or chemical inhibition of the enzyme 12-LOX reduces the early infiltration of macrophages into islets, preserves β -cell mass, and prevents hyperglycemia [18,19]. We recently demonstrated the presence of a zebrafish 12-LOX ortholog encoded by alox12, which exhibits similar catalytic activity and product profiles (including the production of 12-HETE) to the mouse and human enzymes [25]. To test a role for 12-LOX in β -cell injury mediated by macrophages, we utilized the zebrafish β -cell injury model in the transgenic line Tg(ins:NTR) following depletion of 12-LOX by injection of a translation-blocking antisense morpholino (alox12 MO) [25].…”

“…The alox12 morpholino (MO) targeting the translational start was described previously [25]. A cxcr3.2-containing expression vector was generated by inserting the coding sequence of D. All mouse experiments were performed under pathogen-free conditions through protocols approved by the Indiana University, University of Chicago, and Eastern Virginia Medical School Institutional Animal Care and Use Committees.…”

“…We recently demonstrated the presence of a zebrafish 12-LOX ortholog encoded by alox12, which exhibits similar catalytic activity and product profiles (including the production of 12-HETE) to the mouse and human enzymes [25]. To test a role for 12-LOX in β -cell injury mediated by macrophages, we utilized the zebrafish β -cell injury model in the transgenic line Tg(ins:NTR) following depletion of 12-LOX by injection of a translation-blocking antisense morpholino (alox12 MO) [25]. With MTZ treatment there was a time-dependent reduction in β -cell number, with only 6% of β cells remaining at 24 h (Fig.…”

“…The alox12 morpholino (MO) targeting the translational start was described previously [25]. A cxcr3.2-containing expression vector was generated by inserting the coding sequence of D. rerio cxcr3.2 gene under control of the D. rerio macrophage-specific mpeg1 promoter (mpeg1:cxcr3.2).…”