Expression pattern of 12-lipoxygenase in human islets with type 1 diabetes and type 2 diabetes

Abstract

Context: Inflammation in the pancreas can cause β-cell stress, leading to diabetes development. Access to human pancreas tissues via the Network for Pancreatic Organ Donors with Diabetes (nPOD) has allowed characterization of pathways leading to this inflammation.

Objective: 12-Lipoxygenase (12-LO) induces inflammation and has been implicated in diabetes development. Our goal was to determine expression of 12-LO in human islets from control, autoantibody-positive, type 1 diabetic, and type 2 diabetic nPOD pancreas donors.

Design: Pancreas tissues from nPOD donors were examined by immunohistochemistry and immunofluorescence for islet expression of 12-LO in different subsets of islet cells.

Participants: Donor pancreas samples were obtained from nPOD based on disease status (control, n = 7; autoantibody-positive, n = 8; type 1 diabetic, n = 17; or type 2 diabetic donors, n = 15).

Main outcome measure: Determination of 12-LO expression within human islets served as the main outcome measure, including distinguishing which types of islet cells expressed 12-LO.

Results: Islets from control participants (nondiabetic) lacked islet expression of 12-LO. Of donors in the other groups, 25% to 37% expressed islet 12-LO with a clear inverse relation between the numbers of β-cells and 12-LO(+) cells within islets of 12-LO(+) cases. 12-LO expression was not seen within macrophages, endothelial cells, α-cells, or β-cells, but only within cells expressing low levels of pancreatic polypeptide (PP) and increased levels of vimentin.

Conclusions: 12-LO expression colocalizes within a specific type of islet PP(+) cell under prediabetic and diabetic conditions. The costaining of PP and vimentin suggests that 12-LO participates in the process leading to β-cell dedifferentiation in the islet.

Figure 1.

12-LO protein expression in human pancreatic islets. Immunofluorescence was used to determine the expression of 12-LO (red) and insulin (green) in nondiabetic nPOD6112 (A), AAb⁺ nPOD6023 (B), TID nPOD6038 (C), and T2D nPOD6273 (D) pancreatic sections. Arrows indicate islets. Scale bars correspond to 50 μm.

Figure 2.

Morphometric analyses of islets in 12-LO⁺ and 12-LO⁻ cases. Islet area, β-cell area, and 12-LO⁺ area were determined as in Materials and Methods in 12-LO⁺ and 12-LO⁻ cases from nondiabetic (non-DM), AAb⁺, T1D, and T2D subjects. 12-LO expression was not seen in any nondiabetic donors. Data are means ± SEM (n = 3–4). A and B, islet area (A) and β-cell area (B) were compared using 2 statistics: a, one-way ANOVA with Dunnett posttest in comparison with nondiabetic, ***, P < .001; b, Student t tests between 12-LO⁺ and 12-LO⁻ donors of each diabetic status, #, P < .05. C–E, 12-LO⁺ areas were expressed in relation to pancreatic area (C), islet area (D), and β-cell area (E) in 12-LO⁺ subjects. A Student t test was use for comparison between the indicated groups: *, P < .05; **, P < .01.

Figure 3.

12-LO⁺ cells in islets do not colocalize with insulin, glucagon, CD31, or MAC-2 in an AAb⁺ case. Immunofluorescence was used to analyze the expression of 12-LO (red) and insulin (green, A), glucagon (green, B), CD31 (green, C), and Mac2 (green, D) in AAb⁺ nPOD6023. Scale bars correspond to 50 μm.

Figure 4.

Islets with 12-LO⁺ cells are enriched with PP staining. Immunofluorescent analysis of AAb⁺ nPOD6023 for 12-LO (red) and PP (green) is shown. An Axiophot epifluorescent microscope (A and B) or an LSM510 confocal microscope (C–E) was used to capture images in the nonuncinate (A, B, C, and E) and uncinate (D) areas of the pancreas. The arrows indicate cells strongly positive for PP staining in E. Scale bars correspond to 50 μm.

Figure 5.

Quantification of PP intensity in islets from 12-LO⁺ and 12-LO⁻ donors. The intensity of PP immunostaining within islets was quantified as in Materials and Methods in the uncinate (A) and nonuncinate (B) regions of the pancreas. Donors with AAb⁺, T1D, and T2D were separated based on the positivity of 12-LO in islets. We did not identify subjects with 12-LO⁺ cells in nondiabetic (non-DM) donors. Values are expressed as taking the average intensity of PP staining in the uncinate region of nondiabetic donors as 1. Data are means ± SEM (n = 3–4). Two statistical comparisons were performed: a, one-way ANOVA with Dunnett posttest in comparison with nondiabetic, *, P < .05; ***, P < .001; b, Student t tests between 12-LO⁺ and 12-LO⁻ donors of each diabetic status, #, P < .05; ##, P < .001; ####, P < .0001.

Figure 6.

Vimentin expression in islets of 12-LO⁺ donors. Immunofluorescence was used to determine the expression of vimentin (red) and PP (green) in nondiabetic nPOD6013 (A), AAb⁺ nPOD6023 (B), T1D nPOD6038 (C), and T2D nPOD6157 (D) pancreas sections. Scale bars correspond to 50 μm.