**2.5 Preventive effect of FBRA on spontaneous type 1 diabetes in NOD female mice**

Based on the previous findings of anti-oxidative and anti-inflammatory effect *in vivo*, and the presence of antidiabetic components in FBRA, we planned to examine suppressive effects of FBRA on autoimmune-mediated type 1 diabetes. By using spontaneously occurring model in non-obese diabetic (NOD) female mice, we have recently reported that dietary administration of 0.5% FBRA delayed the spontaneous onset of diabetes [36]. How to estimate the *in vivo* effect, its possible mechanisms and the limit of this study are introduced below.

Diabetes in NOD mice shares many features with human type 1 diabetes [61]. In this model mice, autoreactive T cells are primed in the pancreatic lymph nodes and a disequilibrium between regulatory and effector T cells occurred at around 12 weeks of age triggers β cell destruction, resulting in diabetes onset [61]. Cyclophosphamide, an immune system disturbing agent, has often used to promote an onset of diabetes in immunological studies, whereas a spontaneous onset model is often used to examine effects of food-derived components or probiotics [13, 62]. While genetic and immunologic factors are important factors in the pathogenesis of type 1 diabetes, environmental factors such as diet and microbiota can also correlate to it [62, 63]. As mentioned above, certain rice bran components such as plant polyphenols and antioxidants have been shown to be anti-diabetic in mice models and in cultured cells. Those components have worked through improved viability or decreased apoptosis of β cells in the pancreas, or through regulating expression of related genes [12–15, 19–26].

Dietary concentration of FBRA was set to 0.5% based on daily food intake of NOD mice and our preliminary result, in which intragastric administration of FBRA at 0.33 mg/g body weight/day could delay an appearance of diabetes in NOD mice. Dose-dependent effect of FBRA was examined at the concentration of 0.25% – 1.0%. The highest concentration 5% was selected since the other animal studies have been using 5% or 10% FBRA containing diet with no harmful effect.

The criteria for diabetes onset and severity of insulitis in NOD mice were decided according to previous studies. Glucosuria was weekly monitored with test paper, and the onset of diabetes was further confirmed by measuring blood glucose levels as described by Lian et al. [64]. Mice showing 2.5 mg/ml or higher blood glucose were diagnosed as diabetic. To compare insulitis levels among the groups, pancreas was resected at the end of the experimental periods and HE-stained. Level of insulitis was assessed based on the level of lymphocyte infiltration, and the islets were graded scores 0, 1, 2, 3 or 4 as described by Serreze et al. [65]. The insulitis score of each mouse was calculated as follows: accumulated score of observed islets/ number of observed islets.

Dietary administration of 0.5% FBRA significantly delayed the appearance of diabetes in mice and lowered the level of insulitis score. Glucosuria and hyperglycemia appeared at around 20 week of age and the ratio of diabetic mice increased age-dependently in control diet-fed mice, but the ratio did not increase in the 0.5% FBRA -fed group. The percentage of diabetic mice was significantly lower at 24 weeks of age as compared to the control group (p = 0.01, log rank test). On HE-stained sections of mice pancreas, lymphocyte infiltration into pancreatic islets has already been observed at the age of 12 weeks. However, the 0.5% FBRA-fed group frequently had small intact islets and the ratio of intact islets was significantly higher than that of control-diet-fed mice (**Figure 2**, excerpted from Kataoka et al. [36]). Insulitis score of FBRA-fed group was also significantly lower compared to the control diet group. From these results, the suppressive effects of dietary FBRA is probably achieved by maintaining the number of intact islets (**Figure 3**).

#### **Figure 2.**

*Suppressive effect of FBRA on insulitis in NOD female mice. (a) Insulitis score and percentage of intact islets in NOD mice fed with control diet or FBRA-containing diet at 30 weeks of age (n = 7). Asterisk means a statistical difference between the two groups (Mann–Whitney's U test, P < 0.05). (b) Representative insulitis in HE-stained pancreatic section of NOD mice fed control diet or 0.5% FBRA-containing diet at the age of 30 weeks (HE stain, ×50). Small intact islets often observed in 0.5% FBRA-fed mice are shown with white arrows. (excerpeted from Kataoka K et al. [36], Journal of Functional Foods. 2021; 78: 104356, Figure 1, with slight modification).*

Possible targets of dietary FBRA in this type 1 diabetes model include: 1) isletspecific T lymphocyte activation; 2) islet-targeting lymphocyte infiltration; 3) cytokine-mediated inflammation or ROS production; 4) regeneration of damaged islets or apoptotic cell death of damaged islets. An Inflammatory cytokine IFNγ, released from activated T cells, has an important role as a trigger of inflammation and β-cell dysfunction in autoimmune-mediated insulitis [62, 66, 67].

However, in our experiment, the percentage and number of CD4+ and CD4+ IFNγ<sup>+</sup> T cells in the spleens and pancreatic lymph nodes at 12 weeks of age were not significantly different between control diet-fed and 0.5% FBRA-fed mice. Additionally, in adoptive transfer experiments, recipient mice who received a T cell fraction from spleen of 0.5% FBRA-treated NOD mice, could not keep the ratio of intact islets and rather increased the ratio of severely damaged islets, while the number and ratio of intact islets tended to increase in 0.5%FBRA-treated recipient mice who received a T cell fraction derived from control diet-fed donor mice. These results supported that FBRA or its components might suppress the onset of diabetes through keeping an enough number of intact islets in pancreas of NOD mice, not through inhibiting the step of islet-specific T lymphocyte activation.

Pancreas has been known to have regenerative potential for autoimmune- or other factor-mediated damage to islet β cells even in adult rodents [68–70]. Pdx1 and related molecules Foxo1, Reg2, Pdcd4 have important roles in islet function and the fate of injured islet cells [15, 71–73]. Pdx1 and Foxo1 are involved in pancreatic

*Fermented Brown Rice as a Functional Food DOI: http://dx.doi.org/10.5772/intechopen.98840*

**Figure 3.**

*Hypothetical schema of suppressive effect of FBRA on type 1 diabetes in NOD mice.*

development and β cell functional regulation through changes of their intracellular translocation [71]. The inflammatory cytokine IFNγ is reported to decrease nuclear localization of Pdx1 and to trigger β cell dysfunction [67]. Oppositely, phenolic compounds in plant-derived food have recently been reported to show anti-diabetic actions via various mechanisms, including increased expression of pdx1 or restoration of nuclear localization of Pdx1 [13, 15, 67].

Then, we examined effects of FBRA on expression of Pdx1 and related molecules. In our study, mRNA levels of *Pdx1* and related molecule genes were similar in whole pancreases of 19- and 22- week-old mice between the control diet-fed group and the 0.5% FBRA-fed group. However, immuno-histochemical analyses of pancreatic sections showed a tendency for more Pdx1 in the cell nuclei in the 0.5% FBRA-fed group. Then, intracellular localization of Pdx1/Foxo1 and their phosphorylation level at appropriate ages should be further examined in NOD mice with/without 0.5% FBRA treatment.

Our study showed that consumption of FBRA throughout the experimental period suppressed the spontaneously occurring diabetes in female NOD mouse with 0.5% dietary concentration as optimal. But several limitations are still present. At the first, ameliorating effects after onset of type 1 diabetes should be examined in animal model, because autoimmune-mediated diabetes suddenly occur and is generally found as glucosuria. The second is which component (s) and how the component(s) work against pathogenesis of type 1 diabetes. Although FBRA is commercially available processed food and no harmful phenomenon has not been observed in human including clinical trial Volunteers [37], to clarify suppressive mechanisms of FBRA including optimum dose of active component(s) is very important for using it as functional food. Finally, the suppressive effect of FBRA on type 1 diabetes was observed only at lower concentrations, but not at

higher concentrations (1% and 5%). At lower dietary concentration, anti-diabetic components in FBRA probably suppressed the development of diabetes through enhanced β cell viability and proliferation, but at higher dietary concentration, the other functional effect of FBRA might appear. For example, 5% dietary FBRA could increase resident *Lactobacillus* species in rat intestine [60], and certain *Lactobacillus* species has been reported to activate Th1 immunity [74–76].
