Here we provide evidence that a HC diet restores the impaired intestinal barrier function in prediabetic DP-BB rats as reflected by increased ileal TEER, reduced serum zonulin levels and a reduced urinary lactulose:mannitol ratio. Interestingly, we observed an association between prediabetic intestinal permeability and diabetes outcome. Rats with a low prediabetic intestinal permeability developed autoimmune diabetes later or were protected against the disease.
Tight junctions in gut epithelia are pivotal for maintaining intestinal barrier function. Myosin IXb is an adaptor protein involved in linking the cytoskeleton with tight junctions, whereas claudins and occludin are involved in the architecture of tight junctions itself [27
]. High levels of myosin IXb and claudin-2, and low levels of claudin-1 and occludin will result in the opening of tight junctions and a subsequent increase in intestinal permeability [27
]. Therefore, our results for tight junction proteins may explain why, at 65 days of age, the intestinal barrier function of DP-BB rats on the standard diet is reduced compared with that of DR-BB rats. Interestingly, Watts et al. [14
] demonstrated that intestinal permeability of DP-BB rats increases from the age of 50 days, which correlates with the mRNA expression pattern of tight junction proteins shown in the current report.
Besides tight junctions, mucins are also important for maintaining intestinal barrier function. It was shown by Courtois et al. that a HC diet increased intestinal mucin levels and subsequently improved intestinal barrier function in BB rats as demonstrated by reduced uptake of FITC-labelled dextran across the gut epithelium [7
]. These results support those presented in this paper.
Here we show that in DP-BB rats, as opposed to DR-BB rats, the expression of Cldn1 strongly declined over time, leading to very low levels after the age of 50 days. The mRNA expression levels of Myo9b, Ocln and Cldn2 showed no significant changes over time, but Myo9b and Cldn2 mRNA expression levels were increased as compared with DR-BB rats.
In DR-BB and DP-BB rats of 34 and 41 days of age, Neu et al. found reduced ileal claudin-1 protein levels as compared with Wistar rats, but observed no differences between DP-BB and DR-BB rats [20
]. Interestingly, like Neu et al., we also found no difference in Cldn1
mRNA expression between DP-BB and DR-BB rats below the age of 50 days. However, we did observe a difference between these two rat groups above the age of 50 days, a difference caused by the decline of Cldn1
mRNA expression in DP-BB rats.
Interestingly, in DP-BB rats, the HC diet seemed to normalise tight junction mRNA expression levels towards levels observed in DR-BB rats. Therefore, the observed patterns of decreased Myo9b and Cldn2 expression and increased Cldn1 expression in DP-BB rats on a HC diet fits well with the hypothesis that the HC diet has a beneficial effect on gut permeability through modification of tight junction functionality.
A proinflammatory intestinal cytokine environment affects tight junction functionality and subsequently increases intestinal permeability [12
]. Here, the HC diet increased the anti-inflammatory cytokines IL-10 and TGF-ß, whereas TNF-α and IFN-γ levels were not affected (data not shown). The HC diet therefore creates an anti-inflammatory cytokine milieu in the small intestine, which might lead to improved intestinal barrier function.
In rats fed the standard diet and in DP-BB rats on a HC diet, the animals that did not develop diabetes had no higher ileal Cldn1
expression than the diabetic rats. This result was not expected, but might be explained by ageing. As shown in Figs and , and in ESM Fig. 1
expression declined with age in the DP-BB rats, but not in the DR-BB rats. The non-diabetic DP-BB rats were on average 50 days older than the diabetic rats. Therefore, the comparable mRNA expression levels of Cldn1
in diabetic and non-diabetic rats might have been due to ageing. Unfortunately, we did not kill non-diabetic DP-BB rats between 70 and 100 days of age.
Previous research by our group and others has shown that the HC diet affects autoimmune diabetes pathogenesis mainly between 30 and 70 days of age [1
]. DP-BB rats that were fed a cereal-based diet until 60 to 70 days and then switched to the HC diet were not protected against autoimmune diabetes development, suggesting a crucial window [1
]. Therefore, prevention of exposure to diabetogenic triggers in this window can affect diabetes outcome. The results presented here show that the decline of intestinal barrier function with age in DP-BB rats is delayed, but not completely prevented by the HC diet. However, this delay might be enough to prevent exposure to diabetogenic triggers in the diabetes-susceptible window and subsequently prevent or delay autoimmune diabetes onset.
Food components and intestinal bacteria can affect the integrity of the intestinal barrier [8
]. Recently, Mojibian et al. demonstrated that ~50% of established type 1 diabetes patients have T cell responses against wheat polypeptides [30
]. These results indicate that wheat might be a major dietary antigen capable of inducing type 1 diabetes. An important question raised by the Mojibian study is why in ~50% of type 1 diabetes patients a wheat polypeptide-specific response is found. Interestingly, studies by our group and others have shown that also ~50% of type 1 diabetes patients have intestinal barrier defects [13
]. Although Mojibian et al. did not investigate whether the wheat-specific T cell responses correlated with impaired intestinal barrier function, it is reasonable to hypothesise that impaired intestinal barrier function leads to an increased passage of intestinal diabetogenic antigens (e.g. wheat peptides, bacterial agents) that induce the autoimmune cascade typical of type 1 diabetes [11
]. To prove this hypothesis, further investigation of the relationship between intestinal barrier function and immune responses against intestinal antigens and beta cells in type 1 diabetes patients will be required.
In summary, the results presented here show that in DP-BB rats the level of prediabetic gut permeability is associated with autoimmune diabetes development later in life and that improvement of this intestinal barrier function might contribute to the prevention of autoimmune diabetes by dietary intervention with the HC diet. Changes to the local intestinal cytokine profile, direct effects on tight junctions and reduced zonulin production might be important mechanisms for this effect. Taken together, these results suggest that a HC-based diet probably prevents diabetes development in the DP-BB rat by: (1) improving intestinal barrier function thereby contributing to no or less induction of the autoimmune cascade responsible for autoimmune diabetes development [1
]; (2) skewing the autoreactive T cells to a less pathogenic phenotype [1
]; and (3) the induction of islet neogenesis [1
Modification of intestinal permeability either directly by tight junction modulators or indirectly via environmental factors like bioactive peptides, prebiotics or probiotics [8
] could be a promising approach to the development of a whole new field of therapeutic strategies to prevent or treat autoimmune diabetes.