Role of the intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-prone rats

Abstract

Increased intestinal permeability has been observed in numerous human autoimmune diseases, including type-1 diabetes (T1D) and its' animal model, the BB-wor diabetic prone rat. We have recently described zonulin, a protein that regulates intercellular tight junctions. The objective of this study was to establish whether zonulin-dependent increased intestinal permeability plays a role in the pathogenesis of T1D. In the BB diabetic-prone rat model of T1D, intestinal intraluminal zonulin levels were elevated 35-fold compared to control BB diabetic-resistant rats. Zonulin up-regulation was coincident with decreased small intestinal transepithelial electrical resistance, and was followed by the production of autoantibodies against pancreatic beta cells, which preceded the onset of clinically evident T1D by approximately 25 days. In those diabetic prone rats that did not progress to diabetes, both intraluminal zonulin and transepithelial electrical resistance were similar to those detected in diabetic-resistant animal controls. Blockade of the zonulin receptor reduced the cumulative incidence of T1D by 70%, despite the persistence of intraluminal zonulin up-regulation. Moreover, treatment responders did not seroconvert to islet cell antibodies. Combined together, these findings suggest that the zonulin-induced loss in small intestinal barrier function is involved in the pathogenesis of T1D in the BB diabetic-prone animal model.

Figures

Fig. 1.

Zonulin levels and serum glucose levels in both BBDR (A) and BBDP (B) rats at increasing ages. BBDP rats showed an increase in both intraluminal (squares) and serum (circles) zonulin starting from the age 50 days group, whereas differences in serum glucose (triangles, dotted line) were detected only in animals >75 days old. No significant changes were observed in BBDR rats in either zonulin (both serum and luminal) or serum glucose levels at any age group. n = 3–6 for each group. *, P < 0.01 compared to BBDR animals; **, P < 0.005 compared to BBDR animals.

Fig. 2.

Intestinal resistance (TEER, Ω·cm2) in BBDP (filled bars) and BBDR (open bars) rats. No difference in TEER between BBDR and BBDP rats was observed at age 20 days, irrespective of the intestinal tract examined. By age 50 days, the TEER of the small intestine in BBDP animals was significantly lower in both the jejunum and ileum, whereas the colon showed no differences in TEER between the two groups. Significant differences in ileal TEER were observed also at age 75 days. n = 6 for each group. *, P < 0.05 compared to BBDR animals.

Fig. 3.

In vivo intestinal permeability and serum glucose levels in BBDP rats. (A) Untreated BBDP animals that evolved to T1D showed an increase in intestinal permeability as measure by LA/MA ratio (squares) that became statistically significant at age 44 days (P < 0.05–0.002 age 44–72 days compared to age 30 days). These permeability changes were followed by a significant increase in serum glucose levels (diamonds) starting ≈2 weeks after the increase in intestinal permeability (P < 0.05–0.0001 age 65–72 days compared to age 30 days). (B) Conversely, BBDP rats treated with FZI/0 and that did not develop T1D had no changes in either intestinal permeability or serum glucose levels. The FZI/0-treated animals that developed diabetes (n = 4) and the untreated animals that did not develop diabetes (n = 3) were eliminated from the final analysis. Therefore, n for treated group = 11; n for untreated group = 12.

Fig. 4.

In vivo effect of the zonulin inhibitor FZI/0 on TEER (A) and intestinal zonulin release (B) in BBDP rats. Untreated BBDP rats that developed T1D (filled bars) showed a significant decrease in ileal TEER and increase in intestinal luminal zonulin compared to BBDR animals (open bars) (A). FZI/0 treatment (dashed bars) in BBDP animals prevented the ileal TEER decrement (A) without affecting the intraluminal zonulin release (B). n = 7–15 for each group. *, P < 0.02.

Fig. 5.

ICA in both FZI/0-treated rats and untreated diabetic animals. No ICA were detected in non-T1D-treated animals (A), whereas untreated rats that developed diabetes showed the presence of the autoantibodies in their serum (B).