**5. Gut microbiota profile in Type 2 diabetes mellitus**

The gut microbiome was first defined scientifically in 2001 as "an ecological community of commensal, symbiotic and pathogenic microorganisms that collectively share our body space" [92]. Approximately 100 trillion microbes are found in the human gastrointestinal tract (GIT) and strongly influence the health status of individuals either directly or indirectly [93–97]. The primary reason for the pathophysiological effect of the gut microbiome on human physiology has been attributed to the disruption of the stable communities of gut microbes through medication, diet and lifestyle. A normal, healthy gut microbiome profile is termed eubiosis and abnormal gut microbiome composition is called dysbiosis [98–106]. Eubiosis typically refers to an ideal bacterial population comprising 95% of Bacteroidetes and 5% Firmicutes producing abundant microbial metabolites like short-chain fatty acids (SCFAs), branched-chain amino acids (BCAAs) and impacting lipid metabolism. SCFAs like butyrate, acetate and propionate are produced by the anaerobic fermentation of non-digestible carbohydrates (dietary fiber*)* and promote gut integrity and protect gut epithelial lining by forming tight junctions and preventing gut permeability [107]. These microbial secondary metabolites act as central components in microbe to host signaling pathways activation. Much of the specific microbiota involved in the production of these important secondary metabolites are reduced in T2DM patients.

Substantial data from human studies support the possibility that dysbiosis triggers obesity, inflammation, insulin resistance and T2DM [108–111]. Association of dysbiosis is also attributed to the pathogenesis of intestinal tissue. Intestinal disorders attributed to dysbiosis include inflammatory bowel disease, irritable bowel syndrome (IBD) and coeliac disease [102, 111, 112]. Whereas metabolic syndrome, obesity, and cardiovascular complications are attributed as extra-intestinal effects of dysbiosis. Dysbiosis has also been attributed not only to the initiation of the T2DM in humans (a condition known as prediabetes) but also during the progression and subsequent secondary complications of T2DM with several lines of evidence suggesting that manipulation of the gut microbiome helps to minimize or alleviate the T2DM conditions [98, 113–121].

The role of gut microbiota in health and disease and specifically the pathogenesis of T2DM has been experimentally investigated mainly by using rodent models as a limited amount of experimental data can be generated through human studies. Keeping in mind that the rodents and human physiology are not exactly similar and certain physiological differences exist. The non-human primates seem to be a much more appropriate animal model to study different aspects of primate physiology including the gut microbiome and its interaction with metabolic dysregulation [122–127]. Nonetheless, the current understanding of the role of the gut microbiome in the context of metabolic syndrome or pathogenesis of diabetes mellitus has

primarily originated from the data on rodent and human studies [94, 97, 116, 128–133]. Interestingly efforts have been made in the past to characterize the gut microbiome in normal and diabetic individuals as well as some therapeutic approaches have been adopted [95, 98, 113, 116, 117, 120, 121, 134].

The attempts to characterize the normal human gut microbiome revealed four primary phyla which are responsible for the physiological role of gut in metabolic modulation [128, 132, 133, 135–141]. These four specific phyla/families of microbes present in the gut include Bacteroidetes (Bacteroidota), Firmicutes (Bacillota), Proteobacteria (Pseudomonadota) and actinobacteria (Actinomycetota) [95, 142]. The specific proportion for each of these phyla in normal physiological and homeostatic conditions indicates that the largest group of microbes is the Firmicutes which make up to 64% of the total gut microbiota. Followed by the Bacteroidetes, which make up the second-largest group, contributing up to 23% of the total gut microbiota. Proteobacteria and actinobacteria contribute the rest with 8% and 3% respectively. These specific percentage contributions of each phylum are extremely important physiologically. Increased prevalence of pro-inflammatory conditions such as obesity, T2DM, arthritis and even cancer have been attributed to the disruption of these specific percentage contributions of each phylum [132, 143]. Human and animal data have highlighted the unique compositional changes in the microbiota profiles at the phylum level in T2DM conditions [113, 128]. T2DM patients exhibit increased membrane transport of sugars, BCAA transportation, methane metabolism and sulfate reduction [128]. These patients also have reduced butyrate biosynthesis and cofactors/vitamins metabolism.

Although a certain level of discrepancy does exist in terms of phyla composition data between different T2DM patients which has been attributed to the specific geographical location, culture-specific diet and medication use [144]. Numerous independent research groups have reported widely contrasting microbiota findings in the context of phyla composition in T2DM patients [113, 114, 117, 119, 128, 145, 146]. It seems highly unlikely that a single microbe species can play a significant or dominant role in determining the risk of T2DM. The conflicting data from several independent groups also have some interesting similarities. Specifically, it was a common observation among T2DM patients that butyrate-producing microbes were particularly depleted [117, 128]. As human microbiome is comprised mainly of Bacteroidetes and Firmicutes with a specific ratio (B/F > 1) and obesity has been shown to impact this ratio and result in the increased prevalence of Firmicutes to that of Bacteroidetes [109, 147–149]. Implicating that a disrupted B/F ratio can contribute to obesity in humans. Similarly increased concentration of Bacteroidetes and Proteobacteria with a significant decline in Firmicutes has been reported in T2DM patients [113] T2DM also demonstrates an increase in pathogenic microbial species like *Clostridium symbiosum*, *Clostridium ramosum*, and *Escherichia coli* resulting in systemic inflammation [119, 128].

Insulin resistance has also been attributed to disrupted Bacteroidetes and Firmicutes (B/F) ratio. An altered B/F ratio impacts intestinal permeability and lipopolysaccharide (LPS) from proteobacteria are translocated from inside the gut. LPS translocation activates immune response through interleukin-1 (IL-1), tumor necrosis factor (TNF), Jun N-terminal kinases (JNK) and IkB kinase (IKK). LPS-induced activation of JNK and IKK results in the phosphorylation of insulin receptor substrate (IRS) which fails to activate downstream effector molecules like PI3K and AKT thus rendering the insulin signaling cascade ineffective [150, 151]. IKK also activates the nuclear translocation of nuclear factor kappa B (NF-kB). NF-kB, a transcription

factor, induces the expression of several genes involved in inflammatory and apoptotic responses [152–155]. The inflammatory state also called metabolic endotoxemia is accompanied by insulin resistance and obesity.
