Accelerated Generation of Extra-Islet Insulin-Producing Cells in Diabetic Rats, Treated with Sodium Phthalhydrazide

Abstract

β-cells dysfunction plays an important role in the pathogenesis of type 2 diabetes (T2D), partially may be compensated by the generation of extra-islet insulin-producing cells (IPCs) in pancreatic acini and ducts. Pdx1 expression and inflammatory level are suggested to be involved in the generation of extra-islet IPCs, but the exact reasons and mechanisms of it are unclear. Macrophages are key inflammatory mediators in T2D. We studied changes in mass and characteristics of extra-islet IPCs in rats with a streptozotocin-nicotinamide model of T2D and after i.m. administration of 20 daily doses of 2 mg/kg b.w. sodium aminophthalhydrazide (APH). Previously, we found that APH modulates macrophage production and increases the proliferative activity of pancreatic β-cells. Expressions of insulin and Pdx1, as well as F4/80 (macrophage marker), were detected at the protein level by immunohistochemistry analysis, the concentration of pro- and anti-inflammatory cytokines in blood and pancreas-by ELISA. Diabetic rats treated with APH showed an increasing mass of extra-islet IPCs and the content of insulin in them. The presence of Pdx1⁺ cells in the exocrine pancreas also increased. F4/80⁺ cell reduction was accompanied by increasing TGF-β1 content. Interestingly, during the development of diabetes, the mass of β-cells decreased faster than the mass of extra-islet IPCs, and extra-islet IPCs reacted to experimental T2D differently depending on their acinar or ductal location.

Keywords: extra-islet insulin-producing cell; immunomodulators; macrophage; macrophage plasticity; sodium aminophthalhydrazide; streptozotocin-nicotinamide-induced diabetes; type 2 diabetes mellitus.

Figure 1

The effects of 5-amino-2.3-dihydrophthalazine-1.4-dione salts (APH) treatment on rats with experimental type 2 diabetes (T2D). The fasting blood glucose level (a), concentration of glycated hemoglobin (b), mean of HOMA-IR index (d), and quantity of white blood cells (WBC) (f) showed a decrease in APH-treated T2D rats compared to rats with T2D60; conversely, plasma insulin level (c) increased. Oral glucose tolerance test (OGTT) (e) showed no difference between blood glucose concentration before and 120 min after 1 g oral glucose load in APH-treated diabetic animals. ND—non-diabetic rats, T2D30—rats with T2D lasts 30 days, T2D60—rats with T2D lasts 60 days, T2D60 + APH—rats with T2D lasts 60 days, treated with APH; n = 10 in each group. Data are mean ± SEM. (a–e): *—p < 0.05 in comparison with ND; **—p < 0.05 in comparison with T2D60+APH; (f): *—p < 0.05 in comparison with baseline (t = 0 min).

Figure 2

Extra-islet insulin-producing cells (IPCs) in healthy, diabetic, and APH-treated diabetic rats. APH promotes an increase in the number of extra-islet IPCs (a) and the optical density of insulin in them (in conventional units—c.u.) (d) in both acini and ducts. Areas of acinar IPCs are bigger than ductal IPCs, as in the case of solitary located IPCs (b), as in small groups of IPCs (c,e). (e–i)—immunolocalization of insulin in the pancreas of ND (f), T2D30 (g), T2D60 (h), and APH-treated T2D60 (i) rats (brown), light microscopy. e(a)—IPCs in acini, e(b)—IPCs in ducts, e(c)—in acini, enlarged. ND—non-diabetic rats, T2D30—rats with T2D lasts 30 days, T2D60—rats with T2D lasts 60 days, T2D60 + APH—rats with T2D lasts 60 days, treated with APH; n = 10 in each group. Data are mean ± SEM. *—p < 0.05 in comparison with ND; **—p < 0.05 in comparison with T2D60 + APH; ^—p < 0.05 in comparison with T2D30; ⎴ —p < 0.05 between acinar and ductal IPCs.

Figure 3

APH reduces the number of macrophages (F4/80+ cells) in the pancreas of T2D rats, changes cytokine concentration in blood and pancreas of T2D rats, and stimulates the expression of Pdx1 in the exocrine pancreas of T2D rats. (a1)—total amount of F4/80-positive (⁺) cells in exocrine pancreas, in particular: (a2)—in acini, (a3)—in ducts; (a4–a7)—immunohistochemistry for macrophage marker F4/80 in rat pancreas, light microscopy. Concentration of cytokines: (b1)—in blood, (b2)—in pancreas. Quantity of Pdx1-positive (⁺) cells: (c1)—in acini, (c2)—in ducts; (c3)—in islets. n = 10 in each group. (c4–c7)—immunofluorescence staining of the rat pancreas for Pdx1, confocal laser microscopy; in the pancreas of ND (c4), T2D30 (c5), and T2D60 (c6) rats, Pdx1⁺ cells are mainly localized in islets, in APH-treated T2D rats the presence of Pdx1⁺ cells in exocrine acinar (c7, surrounded by a white frame) increases. Data are mean ± SEM. *—p < 0.05 in comparison with ND; **—p < 0.05 in comparison with T2D60 + APH; ^—p < 0.05 in comparison with T2D30.