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.

Maladaptive signaling by pro-inflammatory cytokines (PICs) such as Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL1β), and Interferon ɣ (IFNɣ), can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed: Cre-Enabled and Tetracycline-Inducible transgenic system for conditional, tissue-specific expression of Pro-Inflammatory Cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of TNFα, IL1β, IFNɣ, and an H2B-GFP fluorescent reporter gene in a dose dependent manner. Furthermore, cytokine mis-expression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction (qRT-PCR) demonstrated increased expression levels of IFNɣ, IL1β, and TNFα mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both TNF-α immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species (ROS)- mediated stress and endoplasmic reticulum (ER) stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis, and therapy development of diseases with an underlying inflammatory etiology.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in adults. NAFLD progresses from benign liver fat accumulation to liver inflammation and cirrhosis, and ultimately leads to liver failure. Although several rodent models have been established for studying NAFLD, they have limitations that include cost, speed of disease development, key dissimilarities, and poor amenability to pharmacological screens. Here, we present a novel 2-hit zebrafish model to replicate aspects of NAFLD pathogenesis. We fed zebrafish larvae a high-fat diet (HFD) to drive liver fat accumulation (first hit). Next, we exacerbated liver-specific inflammation using a transgenic line (fabp10-CETI-PIC3) that induces the expression of proinflammatory cytokines following induction with doxycycline (second hit). These hits promoted fat accumulation and liver inflammation, as demonstrated by the high expression of inflammatory cytokines, macrophage infiltration, stress induction, and hepatic lipid droplet accumulation. Furthermore, zebrafish in this paradigm showed deranged glucose metabolism. To validate a small-molecule screening approach, we treated HFD-fed fish with pioglitazone, a drug shown to be beneficial for NAFLD in humans, and measured a sharp reduction in liver lipid accumulation. These results demonstrate new utility for zebrafish in modeling early NAFLD pathogenesis and demonstrate their feasibility for in vivo screening of new pharmacological interventions.

Islet β-cell inflammation, dysfunction, and death are characteristic of diabetes. 12/15-Lipoxygenase (12/15-LOX) produces 12-HETE and is found activated under conditions of insulin resistance and systemic inflammation. 12-HETE acts as an inflammatory mediator that promotes β-cell dysfunction and death through oxidative stress. Since 12-HETE has been shown to bind the G-protein coupled receptor 31 (GPR31), we hypothesized that this binding activates pathways leading to β-cell dysfunction. To test our hypothesis, we screened primary islets and β-cell lines for the presence of GPR31 and studied islets and glucose homeostasis in Gpr31b-/- mice. We observed that GPR31 is present in human and mouse islets, and the mouse β cell-derived cell line (βTC3). Consistent with a role in inflammation, GPR31 expression was increased upon stimulation with a cocktail of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β). We also created a germline knockout of the gene encoding GPR31 (Gpr31b) in mice. Similar to mice with deletion of the gene for 12/15-LOX (Alox15-/-), Gpr31b-/- mice were viable and healthy, with body weight and glucose tolerance indistinguishable from control littermates. To mimic conditions of insulin resistance in vitro, we treated isolated islets Gpr31-/- and control littermates with 10 µM S961, an insulin receptor inhibitor. Treated control islets showed an expected decrease in β-cell identity genes (Ins1/2, Pdx1), genes encoding antioxidant enzymes (Gpx1), and an increase in genes associated with ER stress (Chop, sXbp1). In contrast, Gpr31b-/- islets expression of these genes was resistant to S961. Compared to control littermates, Gpr31-/- mice were resistant to glucose intolerance induced by the β-cell inflammatory toxin streptozotocin. Together these data support the hypothesis that the major product of 12/15-LOX, 12-HETE, acts via GPR31 in promoting β-cell dysfunction in the setting of insulin resistance and inflammation. Disclosure M. Hernandez-Perez: None. I. Haider: None. R.M. Anderson: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Funding JDRF (3-PDF-2019-750-A-N); National Institutes of Health (T32DK064466, R01DK105588, (P30DK097512)

The spectrum of nonalcoholic liver disease (NAFLD) ranges from simple steatosis to steatohepatitis with the latter having the potential to irreversibly progress to cirrhosis. Compared with the general population, NAFLD is more frequently seen in patients with type 2 diabetes (T2D), and conversely, NAFLD is associated with increased incidence of T2D. Existing rodent models of NAFLD focus narrowly on certain stages of liver damage and insufficiently encompass all metabolic changes, limiting their utility. The aims of this study were to develop a new model of NAFLD in young zebrafish and use it to probe the associations between liver inflammation and pancreatic islet function. We examined hepatopancreatic inflammation under three conditions: (1) in a zebrafish line (fabp10-CETI-PIC3) in which liver-specific expression of pro-inflammatory cytokines IL-1β, TNFα, and IFN-γ was induced by doxycycline treatment (M1); (2) in wild-type zebrafish fed a high-fat diet (M2); and (3) in a combination of these models (M3). All three conditions showed increased expression of inflammatory cytokines compared to Tg(fabp10:cre) control larvae, and when compared to controls, all three showed increased macrophage infiltration in the liver. Strikingly, staining with CellRox Deep Red to assess reactive oxygen species (ROS)-mediated stress in the liver revealed a 2-fold increase in the M3 combination model, but not in either M1 or M2. Further, although all models showed hyperglycemia, decreased insulin transcription without β cell death, and ROS accumulation in the pancreas, only the M3 condition demonstrated a significant increase in macrophage infiltration of the pancreatic islets as compared to controls. In sum, our novel zebrafish model of liver steatosis and steatohepatitis is a promising model in which to study the intricate associations between NAFLD and T2D, and may serve as an amenable platform for screening novel pharmaceutical interventions. Disclosure S. Ibrahim: None. I. Haider: None. A. Kulkarni: None. I. Doycheva: None. R. G. Mirmira: Advisory Panel; Self; Hibercell Inc., Sigilon Therapeutics, Inc., Veralox Therapeutics, Employee; Spouse/Partner; Beta Bionics, Inc. E. K. Sims: None. R. Anderson: None.