Metagenomic Phenotyping of Chemical-Induced Metabolic Disease Models

Abstract

Background: The global burden of chronic metabolic disease is on the rise, and is attributed to our lifestyle-related choices (e.g., high-calorie diet). Gut microbial dysbiosis that includes altered microbial abundance, metabolic functions and decreased diversity has been attributed to the intestinal-level trigger for non-communicable metabolic disease. Chemically induced in vivo animal models were considered as the gold standard for studying the mechanistic aspects of disease pathogenesis. For this purpose, several chemical-induced animal models have been well established to study metabolic diseases. In line, streptozotocin (STZ) induced diabetes models, acetaminophen (APAP) induced hepatic injury models and dextran sulfate sodium (DSS) induced colitis models were well established. These chemical models were orally gavaged to induce tissue-specific injury, thereby triggering metabolic complications. Despite the well-established role of gut microbiota in triggering metabolic diseases, it remains inconclusive whether beyond direct tissue damage these chemical models also negatively impacts the gut microbiota. Therefore, we hypothesized that the metabolic disease-causing role of these chemicals are in part attributed to the negative impact on the gut microbiota. Methodology: We examined the microbiota-modulating effects of STZ, APAP and DSS using an in vitro pseudo-colonic model (AMMR). For this purpose, we anaerobically cultured gut microbes using a proprietary in vitro pseudo-colon model for 24h and growth was monitored periodically. The gut microbial inoculum was collected from individuals without metabolic diseases. After incubation, the samples were removed, DNA extracted and subjected to 16s rRNA sequencing of the V3-V4 hypervariable regions. The sequenced data was analysed using the QIIME2 pipeline and metagenomic annotations were done based on SILVA database. PICRUSt was used for microbial functional data analysis. 2 Results: The data showed distinct shifts in gut microbial abundance due to the treatments relative to untreated control. The Firmicutes-to-Bacteroidetes ratio was increased in all groups in comparison to control. A high abundance of Proteobacteria was observed in STZ group, which was consistent with clinical data showing increased Proteobacteria in diabetic patients. An increase in Actinobacteria and Roseburia, and a decrease in Akkermansia in APAP group was clinically associated with hepatic injury. An increase in Bifidobacterium, Faecalibacterium, and a decrease in Dubosiella in DSS group. The taurine & hypotaurine metabolism, fatty acid degradation, D-alanine metabolism, sphingolipid metabolism, and thiamine metabolism microbial functions were significantly induced upon treatment of STZ in comparison to control. The upregulation of fatty acid biosynthesis, butanoate metabolism, glutamine and glutamate metabolism, along with a decrease in krebs cycle, lipoic acid synthesis in APAP group. The functional activity of fatty acid degradation and biosynthesis, arginine and proline metabolism, bacterial chemotaxis, and ketone bodies were enhanced in DSS group compared to control. Conclusion: The data from these sets of experiments performed in a strictly anaerobic pseudo-colon system suggest that STZ, APAP and DSS induce dramatic shifts in the gut microbial population and their metabolic functions. Therefore, the metabolic disease-causing potentials of these experimental models were also associated with triggering gut microbial dysbiosis. Keywords: Streptozotocin, Acetaminophen, Dextran sulfate sodium, Diabetes, Drug induced liver injury, Ulcerative colitis, AMMR, Gut microbiome, Metabolome