Transgenic Overexpression of Galectin-3 in Pancreatic β Cells Attenuates Hyperglycemia in Mice: Synergistic Antidiabetic Effect With Exogenous IL-33

Abstract

Galectin-3 (Gal-3) has diverse roles in inflammatory and autoimmune diseases. There is evidence that Gal-3 plays a role in both type 1 and type 2 diabetes. While the role of Gal-3 expression in immune cells invading the pancreatic islets in the experimental model of type 1 diabetes mellitus has been already studied, the importance of the overexpression of Gal-3 in the target β cells is not defined. Therefore, we used multiple low doses of streptozotocin (MLD-STZ)-induced diabetes in C57Bl/6 mice to analyze the effect of transgenic (TG) overexpression of Gal-3 in β cells. Our results demonstrated that the overexpression of Gal-3 protected β cells from apoptosis and attenuated MLD-STZ-induced hyperglycemia, glycosuria, and ketonuria. The cellular analysis of pancreata and draining lymph nodes showed that Gal-3 overexpression significantly decreased the number of pro-inflammatory cells without affecting the presence of T-regulatory cells. As the application of exogenous interleukin 33 (IL-33) given from the beginning of MLD-STZ diabetes induction attenuates the development of disease, by increasing the presence of regulatory FoxP3⁺ ST2⁺ cells, we evaluated the potential synergistic effect of the exogenous IL-33 and TG overexpression of Gal-3 in β cells at the later stage of diabetogenesis. The addition of IL-33 potentiated the survival of β cells and attenuated diabetes even when administered later, after the onset of hyperglycemia (12-18 days), suggesting that protection from apoptosis and immunoregulation by IL-33 may attenuate type 1 diabetes.

Keywords: galectin-3; interleukin 33 (IL-33); regulatory T cells (T reg); type 1 diabetes mellitus (T1D); β cells.

FIGURE 1

Baseline analysis and islets biology in untreated mice. (A) Immunohistochemical expression of galectin-3+ cells in islets of untreated TG and WT mice. (B) Level of secreted galectin-3 in sera. (C) Representative dot plots of intracellular galectin-3 expression in Glut-2+ beta cells. (D) PCR confirmation of genotype. PCR reaction was performed using specific set of primers (Kapa Biosystems, USA), and the presence of a product was visualized on agarose gel. (E) Immunohistochemical expression of caspase-3+ cells in the islets of untreated TG and WT mice. (F) RT-PCR analysis of relative expression of Bcl-2 and Bax mRNA. (G) Annexin V/propidium iodide analysis of isolated islets. (H) Immunohistochemical expression of KI-67+ cells in islets of untreated TG and WT mice. The analysis was performed by a light microscope using a magnifying lens of 40 X. Data from two experiments with 5–7 mice per group are shown as mean ± SEM; by the Mann–Whitney U test and independent-sample Student’s t-test.

FIGURE 2

Transgenically enhanced Gal-3 expression on β-cells improves the parameters of glucoregulation in the MLD–STZ model of type 1 diabetes. Differences in the parameters of glucoregulation were observed after the administration of multiple low doses of streptozotocin. (A) There were no differences in body weight and (B) the body weight gain expressed as percentage of initial body weight. (C) On the 20th day of the experiment, glycemia was significantly lower in mice with transgenically enhanced galectin 3 expression on β-cells than in the control group. (D) Blood glucose level gain expressed as percentage of initial glycemia was significantly lower in mice with the transgenically enhanced galectin 3 expression on β-cells than in the control group. (E) On the 20th day of the experiment, glycosuria and (F) ketonuria were significantly lower in TG mice than in the WT mice. (G) The blood insulin level was significantly higher in TG mice, while the average glycemia was significantly lower than in the WT group. Data from two experiments with 5–7 mice per group are shown as mean ± SEM; by the Mann-Whitney U test and independent-sample Student’s t-test.

FIGURE 3

Transgenically enhanced Gal-3 expression in β-cells attenuates inflammation and insulitis. Differences in islet inflammation and insulitis severity were observed after the administration of multiple low doses of streptozotocin. (A) Representative photomicrographs of H&E staining. Average size of the mononuclear infiltrate expressed as a percentage of islet area. (B) Histology of the islet showed a significantly lower influx of mononuclear cells in TG mice in comparison with the WT group. (C) Immunohistochemical expression of caspase-3+ cells in islets was significantly lower in TG STZ mice. (D) Immunohistochemical expression of insulin + cells in islets were significantly higher in TG STZ mice. The analysis was performed by a light microscope using a magnifying lens of 40 X. Data from two individual experiments with at least 5–7 mice per group are shown as mean ± SD and compared by the paired t-test or Mann–Whitney test.

FIGURE 4

Transgenically enhanced Gal-3 expression on β-cells decreases the percentage of pro-inflammatory cells in pancreatic islets. Phenotypic characteristics of cells in pancreatic islets. Percentage of pro-inflammatory and regulatory cells. The percentages and representative dot and contour plots of (A) CD4⁺ cells, (B) CD4+CXCR3+ cells, (C) CD4+CCR6+ cells, (D) CD8⁺ cells, (E) CD8+CXCR3+ cells (F) CD4+FoxP3+ cells, and (G) CD4+FoxP3+ST2+. Data from two individual experiments with at least 5–7 mice per group are shown as mean ± SD and compared by the paired t-test or Mann–Whitney test.

FIGURE 5

Application of IL-33 (12–18 days) significantly increased the number of Foxp3+ cells in draining pancreatic lymph nodes of TG mice. Differences in number of regulatory and pro-inflammatory cells were observed after administration of multiple low doses of streptozotocin. The percentage, total number, and representative dot plots of (A) CD4⁺ cells, (B) CD4+IFN-γ+ cells, (C) CD4+Foxp3+ cells, (D) CD4+Foxp3+ST2+ cells, and (E) CD8⁺ cells. Data from two individual experiments with at least 5–7 mice per group are shown as mean ± SD and compared by the paired t-test or Mann–Whitney test.

FIGURE 6

Application of exogenous IL-33 on 12–18 days attenuates the development of hyperglycemia, glycosuria, ketonuria, and hyperglycemia during ipGTT. (A) There were no differences in body weight in between TG and WT mice. (B) Fasting glycemia was significantly lower in TG mice than in the WT mice. (C) Glycosuria was significantly lower in TG mice after the application of IL-33 than in the WT group. (D) Ketonuria was significantly lower in TG mice than in the WT mice. (E) Glycemia in ipGTT was significantly lower in TG mice than in the WT mice. Data from two individual experiments with at least 5–7 mice per group are shown as mean ± SD and compared by the paired t-test or Mann–Whitney test.

FIGURE 7

Combined effect of intracellular galectin-3 and IL-33 in STZ-induced diabetes. (A) Overexpression of intracellular galectin-3 on beta cells increases the resistance to apoptosis and STZ-induced release of autoantigen. (B) Exogenous IL-33 increases the number of regulatory T cells and reduces the number of effector CD4⁺ and CD8⁺ cells.