**7. Microbiomics**

*Metagenomics* is a broad term used for the study of genetic material collected from environmental samples including but not limited to soil, sediment and water and in particular relevance to obesity, the gut. The gut microbiota contains all the organisms within the gut and the gut microbiome contains the genome that is within these organisms. Measuring the microbiomic status of the gut environment allows for the study of the composition of all microbes in that ecosystem. Whilst the microbiota consists of all bacteria, fungi, archea, viruses and other microbes, most of the research effort in relation to gut microbiota has been focused on the bacteria portion. This is no surprise given the widespread dominance of bacteria in and on the human body and indeed throughout all ecosystems on Earth. Also no surprise given the location of this ecosystem is the findings over the last decade that the composition of the gut microbiota is altered with obesity and is impacted by the diet of the host. The relationship between the composition and activity of the resident gut microbiota and the effect that this has on the metabolism of the host is currently being delicately teased out and it is an area that has really benefitted from the modern sequencing methods and analytical techniques. By identifying the microbiome and comparing results to electronic databases, an understanding can be formed on the species of bacteria that compose a microbiota community. Like other"omes" mentioned throughout this discussion, modern advances in technology regarding sequencing has allowed further insight into the precise make‐up of the gut microbiota and how it is altered under different pathological conditions. At the forefront of these, analysis has been the use of highly conserved 16S sRNA genes as a molecular marker for microbial diversity (proxies for different species). With the development of sequencing technologies it has become possible to analyse all 16S rRNA genes in a sample and compare it with relevant databases that have been complied [53].

#### **7.1. Gut microbiota and obesity in humans**

The use of metabolomics and lipidomics is set to continue as they may prove useful in providing indications of metabolic health in the obese setting. Identification of metabolites as"biomarkers" may make it possible to identify individuals at risk of developing obesity, which could be useful as a way of tracking disease progression and provide a predictive tool for diseases associated with obesity. These measures could also theoretically be used to give an indication of the effectiveness of new weight‐loss therapies and their impact on metabolism. Studies have already taken place investigating bariatric surgery, in particular, Roux‐en‐Y gastric bypass surgery. How this type of surgery impacts the metabolome was not very well described so Arora et al. analysed the plasma metabolome and lipidome of morbidly obese individuals prior to and after surgery to describe the effects of surgery [52]. From 96 metabolites and 192 molecular lipid species compared, the factors that were most different at 42 days post‐surgery were decanoic acid and octanoic acid whose levels increased and the sphingomyelins 18:1, 21:0 and 18:1, 22:3 whose levels decreased [52]. Moving forward, it is likely that a greater understanding of fluxomics will be utilised. Fluxomics is an analytical method that describes the rates of metabolic reactions within a sample. This is important as metabolism is a dynamic process, and metabolomics and lipidomics analysis will only provide a snapshot or static picture of the metabolic reactions in the cell. It could be stated well that it is not the quantity of metabolites that is important in the obese setting but the rates at

*Metagenomics* is a broad term used for the study of genetic material collected from environmental samples including but not limited to soil, sediment and water and in particular relevance to obesity, the gut. The gut microbiota contains all the organisms within the gut and the gut microbiome contains the genome that is within these organisms. Measuring the microbiomic status of the gut environment allows for the study of the composition of all microbes in that ecosystem. Whilst the microbiota consists of all bacteria, fungi, archea, viruses and other microbes, most of the research effort in relation to gut microbiota has been focused on the bacteria portion. This is no surprise given the widespread dominance of bacteria in and on the human body and indeed throughout all ecosystems on Earth. Also no surprise given the location of this ecosystem is the findings over the last decade that the composition of the gut microbiota is altered with obesity and is impacted by the diet of the host. The relationship between the composition and activity of the resident gut microbiota and the effect that this has on the metabolism of the host is currently being delicately teased out and it is an area that has really benefitted from the modern sequencing methods and analytical techniques. By identifying the microbiome and comparing results to electronic databases, an understanding can be formed on the species of bacteria that compose a microbiota community. Like other"omes" mentioned throughout this discussion, modern advances in technology regarding sequencing has allowed further insight into the precise make‐up of the gut microbiota and how it is altered under different pathological conditions. At the forefront of these, analysis has been the use of highly conserved 16S sRNA genes as a molecular marker for microbial diversity (proxies for different species). With the development of sequencing technologies it

which they undergo metabolic reactions.

38 Adiposity - Omics and Molecular Understanding

**7. Microbiomics**

Human gut microbiota composition is formed in early life with colonisation occurring over the first 3–5 years until it reaches a more stable, adult‐like microbiota configuration [54]. However, in adulthood, alterations in this composition of bacteria can be observed in individuals with varying degrees of adiposity or metabolic health. Differences in the distal gut microbiota of obese versus lean humans has been demonstrated with the relative proportion of the phylum of bacteria named bacteroidetes found to be decreased in obese individuals compared to lean individuals, along with the finding that the bacteroidetes abundance increase as obese individuals lose weight [55]. These findings must be noted as somewhat controversial given other studies have shown no difference between obese and non‐obese groups in relation to the proportion of bacteroidetes nor any change in the proportion of bacteroidetes measured in faeces once obese individuals lose weight [56]. These observed differences could come down to the way in which the samples were obtained, the methods involved in measuring the bacteria or the differing diets that the obese individuals recruited to the study were consuming. In this latter study, it was observed that the phylum of bacteria called Firmicutes was reduced in faecal samples from obese subjects on weight‐loss diets, correlating changes in gut bacteria species to changes in adiposity [56]. It is difficult to distinguish whether changes in gut microbiota are just a response to obesity or weight loss are actually a causative factor.

One way to test whether human gut microbiota composition is causative in inducing an obesity‐associated phenotype is to take gut microbiota from humans and populate the gut of germ‐free mice (mice raised in an isolator so as to never be exposed to microorganisms) who have no gut microbiota. Although there are of course some species differences, on a whole, mice and humans share most of the same genes (~99%) and similar microbiota profiles making this type of experimental setup relevant for the evaluation of microbiota‐induced physiological effects on the host [57]. Utilising faecal samples obtained from adult human female twins that were discordant for obesity, Ridaura et al. were able to demonstrate that an obesity phenotype is transmissible from human to rodent [58]. In these studies, germ‐free mice were transplanted with the faecal matter of either the lean or obese human twin resulting in an increase in total body weight and fat mass in the mice receiving the microbiota from the obese twin. Intriguingly, if mice with the lean twin's microbiota and mice with the obese twin's microbiota were housed together (mice are coprophagic so will eat their cage mates' faeces), a recolonisation process occurred where bacteroidetes species from the lean microbiota invaded into the obese microbiota and this correlated with a protective metabolic phenotype where obesity‐related traits were no longer observed. This raises the possibility of a potential protective effect on obesity of these bacteroidetes species [58].

#### **7.2. Gut microbiota and obesity in animal models**

Other studies utilising germ‐free mice have demonstrated that microorganisms may have a role to play in dictating body weight. Germ‐free mice have less body fat than regularly raised mice despite the fact they actually eat more and take in more calories which should oppose this affect [59]. Further studies in germ‐free mice have demonstrated that an obese phenotype is transmissible from mouse to mouse via the gut microbiota. Germ‐free mice receiving microbiota transplants by oral gavage with microbiota either sourced from genetically obese *ob/ob* mice or lean mice have demonstrated that recipients of the *ob/ob* microbiota put on more body fat in the weeks after being inoculated as compared to those who received the "lean" microbiota transplant [60]. 16S‐rRNA‐gene‐sequence‐based analysis of samples obtained in this study demonstrated that the *ob/ob* donor microbiota samples had a greater abundance of firmicutes bacteria compared with that of the lean donor microbiota samples [60] indicating the possibility that enriching the gut with firmicutes may drive the body weight phenotype. Indeed a number of rodent studies have raised the possibility that the ratio of the phyla bacteroidetes to that of the phyla firmicutes is associated with obesity. In comparison with lean mice, *ob/ob* mice have a ~50% reduction in the abundance of bacteroidetes and an increase in firmicutes [61]. High‐fat feeding studies utilised to induce obesity via a dietary means also have found an increase in the firmicutes species [62] indicating that both obesity induced by hyperphagia (*ob/ob* mice) and obesity induced by energy dense food (high‐fat diet) is associated with an increase in firmicutes. As differences in gut microbiota communities may impact obesity and the metabolism of the host mouse, this should be considered a confounding factor when comparing different mouse strains, or the same mouse strain but from different mouse vendors or animal facilities as each facility is likely to harbour its own unique microbiota signature.

#### **7.3. How would the gut microbiota cause obesity or contribute to obesity progression?**

There are many possible reasons as to why the gut microbiota composition may impact the host and result in the development of obesity. First, certain types of gut microbiota may be more efficient in extracting energy out of food that passes through the gut and therefore make available more energy for the host in which they reside. Bomb calorimetry analysis measuring the energy content of faeces revealed that *ob/ob* mice have significantly less energy remaining in their faeces relative to their lean littermates suggestive that the microbiota within *ob/ob* mice are better at harvesting and extracting energy from the food within their digestive tract [60]. Other possibilities include the role of inflammation. Obesity has been associated with a type of chronic low‐grade inflammation and modification of the gut microbiota has been suggested to increase the leakiness of the gut barrier resulting in the increased appearance of microbial products such as lipopolysaccharide (LPS) (endotoxin) in the circulation [63, 64]. These products could potential cause peripheral tissue inflammation and impede normal metabolism. Yet another possibility is the fact that the gut microbiota is responsible for the production of metabolites such as short‐chain fatty acids (SCFAs) that are produced by the fermentation of dietary fibres by the bacteria in the gut [65]. SCFAs are organic acids with an aliphatic tail of less than six carbons and comprise of acetate, propionate, butyrate and valerate and are used by the host as an energy source. It is hypothesised that alterations in the production of these SCFAs with obesity could adversely affect satiety, hepatic glucose and lipid production as well as inflammatory processes and contribute to the progression of obesity and related conditions [66]. Thus, the microbiome composition can have an impact on another one of the"omes" the metabolome of the host and alter physiological processes via this mechanism. An interaction between the microbiome and the lipidome may also be at play. This is evident in a study of 893 individuals which identified 34 bacterial taxa associated with body mass index and blood lipids [67]. Cross‐validation analysis revealed that the microbiota present explains 4.5% of the variance in body mass index observed and 6% of the plasma triglycerides variance and this was independent of factors such as age and sex that were taken into consideration in the analysis [67].

The gut microbiota may play an important role in the balance between metabolic health and disease. If a characteristic microbiota signature can be described and confirmed in obesity (or in specific types of obesity) then the potential exists to modify this composition and improve health. Whilst the simplest solution to restore the composition of the microbiota would be a change in diet, other avenues such as prebiotic, probiotics and microbiota transplants are also being explored.
