A variety of genetic and feeding models of diabetes have been established, nevertheless, the majority of them have shown an inconsistency in pathological defects compared to human disease. Therefore, among creatures, zebrafish gained a growing platform for developmental research on diseases modeling, based on high similarity of digestive tract and comparable microbial and gut colonization with human [28–30]. Although, adult zebrafish has not been generally applied as an experimental model to study diabetes; here, we have well established it by overfeeding at adult stage, to study the effects of probiotic supplements on T2DM in vivo model.
To generate this model, we used a gradient hyper-glucose accumulation methodology. Physiologically, adult zebrafish absorb molecules from water to make a hyperosmotical internal environment, therefore, immersing them in a glucose containing solution (starting from 50 mM) and rising to final 200 mM could be beneficial . According to our findings, this protocol increases blood glucose up to 300 mg/dL in T2DM Zebrafish which is in accordance to Gleeson et al. who showed a up to 400 mg/dL increase in blood glucose of adult zebrafish immersed in a 1% glucose solution . Moreover, this method provides micro-environment stability and prevents fish fatality.
In the biometric result, probiotic-supplemented diet improved fish weight compared with the standard diet in both T2DM and HC groups similar to observations of Valcarce et al. . Although, we did not expect to see significant changes on the length of the fish during the treatment periods, our data indicated a slight increase in the length and calculated body mass index (BMI) of HC-P group. This finding remarkably points out to the strong value of probiotic and contributes with the physiological conditions which enhances effective fish development and growth [14, 19].
Our histological analysis clearly showed some visible changes in the villus length and width during the probiotic treatment. Since the T2DM fish groups were under harsh condition due to high concentration of glucose, their homeostasis tries to adapt the condition to survive by extension of the villus diameter to increase the absorption. Additionally, result of intestinal staining specified hyperplasia in goblet cells located in microvillis’ in the T2DM group. As, one of the primary characteristic sign of obesity is hyperplasia and hypertrophy; administration of probiotic had such an ability to prevent goblet cells disruption due to high glucose in digestion system of our zebrafish diabetic model [33, 34] .
Alterations in the intestinal homoeostasis may play a major role in the development of systemic inflammatory diseases including diabetes [35, 36]. One of the main challenges in T2DM patients is blood glucose management. In parallel with previous research, we showed that the consumption of probiotic based foods had significantly decreased blood glucose [37, 38]. Moreover, we identified the blood glucose level slightly lower in the HC-P group than the others. Since probiotic can strongly affect the growth, development and immune system improvement; thus, current results specified the probiotic Lactobacillus rhamnosus capability to improve tolerance in high glucose concentration .
Recently, the contribution of the mucosal immune system and the gut microbiome in metabolic disease including T2DM has been highly concerned [35, 39]. Our findings showed that relative mRNA expression levels of IL1-β and TNF-α were down-regulated in fish with probiotic supplementation despite the induction of innate immune-related cytokine genes by probiotic Chromobacterium aquaticum reported by Yi et al. . This apparent inconsistency in cytokine profile was seen among the genus of Lactobacillus whereas L. sakei induced pro-inflammatory cytokines including IL-1𝛽 and TNF-𝛼; and L. johnsonii promoted the production of TGF-𝛽 in cellular models .
Moreover, there was a robust positive correlation between both IL1-β and TNF-α and blood glucose levels (Fig. 4C & D). Delgadillo-Silva et al. showed that altered composition of the gut microbiome stimulate the intestinal residing innate and adaptive immune cells and induce a cytokine-mediated inflammation which is accompanied by hyperglycemia . TNF as an inhibitor of insulin signaling is a major contributor towards obesity-related diseases. Indeed, it has been demonstrated that cytokines such as IL1-β and TNF-α may play role in the inflammatory destruction of insulin-producing β-cells in human T2DM [36, 43, 44]. Due to their synergistic effect, pharmacological blockage of mentioned cytokines has been clinically modulating inflammatory diseases; however, a therapeutic gap for managing islet inflammation and cytokine production in T2DM is present .
Finally, we assumed that probiotic bacteria, like L. rhamnosus, through their pathogen associated molecular pattern signaling pathway and bioactive components might reduce the immune-cell infiltration, decreased pro-inflammatory cytokines and ameliorate the hyperglycemic phenotype n fish models. Consumption of sufficient amount of probiotic could be a therapeutic goal for weight reduction to control T2DM . We might highlighte these results as one of the valuable efficiency of probiotics not only in blood glucose management in T2DM patients but also as an advantage of probiotic supplement for overall health improvement