Galantamine Attenuates Type 1 Diabetes and Inhibits Anti-Insulin Antibodies in Nonobese Diabetic Mice

Abstract

Type 1 diabetes in mice is characterized by autoimmune destruction of insulin-producing pancreatic β-cells. Disease pathogenesis involves invasion of pancreatic islets by immune cells, including macrophages and T cells, and production of antibodies to self-antigens, including insulin. Activation of the inflammatory reflex, the neural circuit that inhibits inflammation, culminates on cholinergic receptor signals on immune cells to attenuate cytokine release and inhibit B-cell antibody production. Here, we show that galantamine, a centrally acting acetylcholinesterase inhibitor and an activator of the inflammatory reflex, attenuates murine experimental type 1 diabetes. Administration of galantamine to animals immunized with keyhole limpet hemocyanin (KLH) significantly suppressed splenocyte release of immunoglobulin G (IgG) and interleukin (IL)-4 and IL-6 during KLH challenge ex vivo. Administration of galantamine beginning at 1 month of age in nonobese diabetic (NOD) mice significantly delayed the onset of hyperglycemia, attenuated immune cell infiltration in pancreatic islets and decreased anti-insulin antibodies in serum. These observations indicate that galantamine attenuates experimental type 1 diabetes in mice and suggest that activation of the inflammatory reflex should be further studied as a potential therapeutic approach.

Figure 1

Galantamine alters antibody responses in immunized mice. KLH immunized mice were administered galantamine (4 mg/kg) or saline 4 h before death. Splenocytes were extracted, plated at 2 × 10⁵ in 96-well plates and incubated with indicated μg/well KLH. KLH-specific IgG (A) and total IgE (B) was determined by ELISA. Galantamine affected antibody levels (IgG, p < 0.05; IgE, p < 0.01 by two-way ANOVA; *p < 0.05 by Bonferroni posttest).

Figure 2

Galantamine alters cytokine responses in immunized mice. KLH immunized mice were administered galantamine (4 mg/kg) or saline 4 h before death. Splenocytes were extracted, plated at 2 × 10⁵ in 96-well plates and incubated with indicated μg/well KLH. Cytokines were determined on d 7 by Quansys multiplex ELISA. (A) Galantamine did not significantly affect IL-2. (B) IL-4 (p < 0.001) and IL-6 (p < 0.0001) were significantly different in galantamine-treated animals (two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, by Bonferroni posttest).

Figure 3

Galantamine administration reduces levels of circulating pathogenic anti-insulin antibodies. Serum from galantamine- or saline-administered was applied to ELISA plates coated with antigens and then probed with anti-mouse antibodies. Serum anti-insulin antibodies were reduced in animals administered galantamine (A) (p > 0.05), but other common NOD autoantibodies were not affected (B–E).

Figure 4

Galantamine delays the onset of hyperglycemia and diabetes. The 1 mg/kg galantamine (n = 11) or saline (n = 10) was administered intraperitoneally daily to NOD mice beginning at 5 wks of age. Blood glucose levels were measured weekly, with two successive weeks of blood glucose >199 mg/dL, indicating onset of diabetes. Administration of galantamine (A) significantly reduced the average blood glucose over time (p < 0.05, two-way ANOVA), and (B) significantly delayed onset of diabetes (p < 0.05, Mantel-Cox, results of two combined experiments).

Figure 5

Daily administration of galantamine decreases islet infiltration by immune cells. (A) Insulitis scoring scheme examplars. Islets with no infiltrating mononuclear cells were scored as 0, peri-islet inflammation only = 1, moderate intra-islet inflammation occupying <70% of the islet = 2, severe-complete intra-islet inflammation occupying >70% of the islet = 3. (B, C) Pancreata from 16-wk-old NOD mice injected intraperitoneally with saline or galantamine from 5 wks of age were isolated and frozen in OCT media and then sliced at 10 μm. At least 50 total islets from three disparate areas of each pancreas were scored blindly. Administration of galantamine (B) reduced the severity of insulitis in NOD mice (representative images), as well as (C) improved the overall level of insulitis in the pancreas (n = 5, χ² p < 0.0005).