Wednesday, November 16, 2011

Intestinal Bacteria and Obesity

Some important biochemical interactions and responses to obesity have previously been discussed here.

In recent years explanations for the sudden rise in obesity have ranged from a further unbalanced internal biological energy balance to environmental pollution. Another accompanying explanation that is gaining support is that the type of bacteria residing in an individual’s intestinal tract is important relative to what foods an individual consumes. There is widespread belief that particular bacteria types drive certain metabolic rates and processes that have a significant effect on weight loss vs. weight retention.

The digestive process can be broken down into three stages after chewing. First, the food enters the stomach and is rendered into chyme by hydrochloric acid. Second, the chyme goes into the small intestine where a vast majority of the nutrient absorption occurs through osmosis, active transport and diffusion to nearby capillaries and eventual transport to the blood stream. Third, the indigestible and unabsorbed material passes through the large intestine where some of the indigestible material is processed (usually fermentation) by appropriate intestinal bacteria, water is reabsorbed and remaining material is packaged for excretion. It is this third element that is of particular interest here.

The human intestinal “metagenome” consists of trillions of microbes that provide enhanced metabolic capabilities due to absent enzyme inclusion (polysaccharide metabolization), protection against pathogens (indirect mucosal defense and luminal colonization competition), immune system support and aids gastrointestinal development and maintenance through interaction with epithelial cells.1-5 The two major elements which drive the specific populations of the metagenome in a given individual are genetics and diet. At the moment there is little that can be done regarding genetics, but the influence of diet is prevalent and that influence begins as early as infancy.1 In fact there is reason to believe that this “metagenome” is most influenced within the first few years of life and can have significant effect on immunity development.6,7

A vast majority of intestinal bacteria belong to one of two phylum of bacteria: Firmicutes and Bacteroidetes. Among these two phylum the bacteria in the intestinal with the largest populations are thought to be (in no particular order) genera Bacteroides (bact.), Clostridium (firm.), Bifidobacterium (bact.), Peptostreptococcus (firm.) and Ruminococcus (firm.) with minor populations of Escherichia (proteo), Lactobacillus (firm), Enterobacter (proteo) and Enterococcus (firm) with various methanogens.3,8,9. The parentheses identify the phylum type for the particular bacteria. It must be emphasized that specifics regarding exact populations are still far and few between relative to the specific genus which make up the Firmicutes and Bacteroidetes phylums for they contain 250 and 20 genera respectively;10 however, it is thought that Ruminococcus makes up a significant percentage of the Firmicutes phylum. On a side note Firmicutes bacteria are typically gram-positive (outside a very small few which have pseudo membrane walls) and Bacteroidetes bacteria are typically gram-negative.

Not surprisingly various intestinal bacteria populations are not evenly distributed throughout the digestive system, but each specific bacteria group has some environmental niche, notable is that higher bacterial populations are found in the lower portion of the intestinal tract vs. the upper portion. Also the upper portion has a large percentage of aerobic bacteria vs. the lower portion having a large percentage of anaerobic bacteria with the terminal ileum as the transition zone.7,11

The principle reason why intestinal bacteria have perked interest in the obesity ‘epidemic’ originated from an experiment in mice which demonstrated that intestinal bacteria play an important role in energy metabolism and weight changes. The study involved using a set of control mice and axenic mice (note that axenic mice are mice without any significant amounts of intestinal bacteria i.e. germ-free mice). Under normal conditions the axenic mice, controlled for age and background, weighted about 40% less than the control mice. However, after colonizing intestinal microflora (from the distal section) derived from the control mice within the axenic mice, the weight of the axenic mice increased by 60% over a short period of time.12 The inclusion of the microflora is thought to influence weight gain through three mechanisms: increases in intestinal glucose absorption, energy extraction from indigestible foods and concomitant higher glycemia and insulinemia.12,13

Changes in the suggested mechanisms from above are though to occur through influence on the action of two signaling proteins: carbohydrate response element-binding protein (ChREBP) and liver sterol response element-binding protein type-1 (SREBP-1) which in turn influence intestinal fasting-induced adipocyte factor [Fiaf; a.k.a. (angiopoietin-like protein 4)].14 When Fiaf is expressed it inhibits lipoprotein lipase activity, which increases the probability that fatty acids are released from triacylglycerols; these fatty acids can then be absorbed by muscles and adipose tissues to be used as energy (basically the fatty acids are consumed). If Fiaf is not expressed then lipoprotein lipase activity increases, increasing the probability of more fat synthesis. Germ-free mice seem to avoid obesity due to excess food consumption, commonly called diet-induced obesity, through three independent mechanisms: increased levels of Fiaf, increased levels of adenosine monophosphate activated protein kinase and reduced food consumption.14

Since the original study more studies have demonstrated differing intestinal bacteria populations in individuals of various weights. Most studies have developed support for a similar pattern between the obese and the non-obese in that more obese mice have a higher population of Firmicutes over Bacteroidetes.15-18 However, other studies have demonstrated no changes with populations in these bacteria or even the reverse with Bacteroidetes at higher population than Firmicutes.19,20 Thus the principle question becomes: do bacteria x protect against obesity in some way or are they simply preferentially selected in non-obese individuals vs. bacteria x which are preferentially selected in obese individuals?

Associate these elements with the fact that the Firmicutes/Bacteroidetes ratio drops when obese individuals lose weight (assuming no dramatic increase in fiber consumption) and Firmicutes population could be tied to fat, possibly through lipid production and storage. One study did demonstrate specific enzymatic activity in obese individuals associated with gram positive bacteria (Firmicutes) over gram negative bacteria (Bacteroidetes).21,22

The problem with fully determining the role of the Firmicutes/Bacteroidetes relationship is the contrasting results. For example some studies report that Bacteroidetes population increases from 3% to 15% with a hypocaloric diet in obese individuals where the Firmicutes population does not undergo significant changes.13,19 If this case is accurate it indicates that Firmicutes growth is not augmented by increased calories/fat, but instead Bacteroidetes growth is inhibited by those elements in some way. However, others report a decrease in Firmicutes population with weight loss and a decrease in Bacteroidetes (50% reduction) in obese individuals vs. non-obese.19

The issue with the Firmicutes/Bacteroidetes ratio may not be the change in the ratio, but instead the change in absolute population. For example in obese individuals what drives the change in the ratio, a decrease in Bacteroidetes population, an increase in Firmicutes population or do both change in general consort with each other? For example if an increase in the Firmicutes population is the dominating factor then it could be possible that Firmicutes responds to non-insoluble fiber elements. However, if a decrease in the Bacteroidetes population is the dominating factor then it could be possible that Bacteroidetes reduces fat absorption.

Other results have shown that axenic mice gain more weight when colonized with microbiota from obese mice opposed to lean mice.15 This result leads to the question of whether Firmicutes are able to extract more energy from a conventional diet over Bacteroidetes or do Firmicutes drive greater amounts of fat storage over Bacteroidetes? The second possibility sees support in that decreases in Bifidobacterium in mice fed a high fat diet also correlated to an increase in lipid polysaccharide (LPS) concentrations.23

The ‘battle’ between Firmicutes and Bacteroidetes begins at birth. The most influential element in early childhood appears to be the duration of time an infant spends consuming breast milk over solid foods and formulas.1 Based on comparisons of Firmicutes and Bacteroidetes populations between infants who consume breast milk and infants who consume formula, infants that consume breast milk longer have lower Firmicutes/Bacteroidetes ratios and seem to have lower probabilities for future obesity1,24-26 (E/A, Gillman et al. 2001, Kalies et al. 2005, Mayer-Davis et al. 2006). Examination of different populations of infants between Africa and Europe supported this conclusion of higher Bacteroidetes populations and lower Firmicutes populations in children breastfeed longer. The rationality behind the difference between African and European children is that Africa infants had to be breastfeed for additional time due to financial limitation or resource availability regarding formula.

One of the major reasons for this developmental difference seems to be the population growth of Lactobacilli and Bifidobacteria in breastfeed infants vs. formula feed infants, which fail to develop these two types of bacteria in significant proportions.27-29 The colonization of Bifidobacteria is thought to be especially important in the maturation of the intestinal lining and localized lymphoid tissue and delayed Bifidobacterial colonization increases the probability of a variety of gastrointestinal and/or allergic conditions.30-32

Originally it was thought that the bacteria present in breast milk was from skin contaminates, but recent testing has developed support for the idea that the bacteria is, not surprisingly, derived from the maternal intestine and follows the entero-mammary pathway to the mammary gland.33 Also no Bifidobacteria has ever been isolated from skin samples from women who have Bifidobacteria in their breast milk.30 The derivation of these bacterium from the mother’s own intestinal system may provide insight into why obese mothers have children that are pre-disposed to becoming obese and why fit mothers have children that have resistance against obesity as those bacteria populations heavily influence the populations in the infants.

Another important association between intestinal bacteria and obesity is the role of interspecies hydrogen transfer from hydrogen producing bacterium to hydrogen consuming methanogens. Non-obese individuals have very small methanogen-based intestinal populations whereas obese individuals have larger populations.10 This population shift has also been associated with genetically homogeneous obese mice (ob+/ob+) over heterogeneous mice (ob+/ob-) and homogeneous non-obese (ob-/ob-).15 The association with genetically obese mice over mice that have become obese through food consumption supports the notion that methanogen population influences weight over methanogen bacteria being selected for based on weight. Basically the methanogen population of bacteria expands first before one gains significant weight. The importance behind this relationship is best demonstrated by understanding the biochemical process involved in the formation of fatty acids in the body.

Methanogens like Methanobrevibacter smithii enhance fermentation efficiency by removing excess free hydrogen and formate in the colon. A reduced concentration of hydrogen leads to an increased rate of conversion of insoluble fibers into short-chain fatty acids.10 Proprionate, acetate, butyrate and formate are the most common SCFAs formed and absorbed across the intestinal epithelium providing a significant portion of the energy for intestinal epithelial cells promoting survival, differentiation and proliferation ensuring effective stomach lining.3,10,34 Butyric acid is also utilized by the colonocytes.35 Formate also can be directly used by hydrogenotrophic methanogens and propionate and lactate can be fermented to acetate and H2.10

The Methanobrevibacter smithii population in non-obese individuals is very small on an absolute level whereas the population in obese individuals is much higher (gastric). This result is supported by metagenomic study which identified more Archaea gene fragments in ob+/ob+ mice over leaner heterogeneous ob+/- or ob-/ob- mice.15 Overall the population of Archaea bacteria in the gut, largely associated to Methanobrevibacter smithii, is tied to obesity with the key factor being availability of free hydrogen. If there is a lot of free hydrogen then there is a higher probability for a lot of Archaea, otherwise there is a very low population of Archaea because there is a limited ‘food source’.

Interestingly anorexic individuals also see an increase in Methanogen bacteria (Methanobrevibacter) over non-obese healthy individuals.21 This increase in anorexic individuals seems to make sense as fermentation rates probably increase in effort to maximize energy optimization from food intake due to reduced food consumption. Increased fermentation rates would increase H2 concentrations resulting in increased Methanogen populations.

Other investigators have looked at how receptor interaction with intestinal microbes influences weight. A promising avenue of research is Toll-like receptor (TLR) 5, a transmembrane protein expressed in the intestinal mucosa that recognizes bacterial flagellin.35 Analysis of TLR5 knockout mice vs. controls demonstrates a 20% greater body mass in the knockouts, a weight which corresponds to an increase in visceral fat.35 This additional body mass is thought to occur through greater food consumption (knockout mice consume 10% more food than controls), which seems to lead to greater fat deposit formation. However, despite this increased food consumption there were no significant changes in short-chain fatty acid concentrations between knockouts and controls.35 Also due to mixed results it is difficult to draw any conclusions regarding differing influences on orexigenic or anorexic hormones between knockouts and control.35

Elimination of intestinal bacteria through broad spectrum antibiotic treatment supported the contention that intestinal bacteria and TLR5 had an interactive relationship in controlling an individual’s weight as germfree TLR5 knockout mice did not suffer from the same weight gain as their TLR5 knockout non-germ free kin.35 The implantation of the microbiota from a TLR5 knockout mouse into a previously germ-free non-knockout mouse lead to the development of a similar phenotype to the TLR5 knockout in the germ-free mouse.35 This result suggests that there are certain bacteria that interact with TLR5 because despite the non-knockouts having the necessary receptors they still develop attributes similar the knockouts, thus the microbiota of the knockouts do not appear to have the required bacteria for activation. This lacking makes sense because without TLR5 receptors it stands to reason that bacteria, which activate TLR5, would be selected against.

Based on the information above it appears that activation of TLR5 somehow reduces weight gain. This result occurs either through interaction between TLR5 and orexigenic and anorexic hormones (which would influence appetite) or involves the reduction in fat deposit synthesis from soluble elements. Due to the results from germfree knockouts and the mixed hormone results, the second possibility seems viable. For example interaction between Bacteroidetes and TLR5 could lead to the inhibition of lipoprotein lipase activity (possibility through increased expression of Fiaf). This action would result in less fat storage and less overall weight gain.

If the above contention were true this action of changing Fiaf expression probably has a positive feedback effect in lean individuals and a negative feedback effect in obese individuals. For example as individuals lose weight the Bacteroidetes population increases which would lead to more TLR5 activation and less fat storage. However, as individuals gain weight Bacteroidetes population decreases which would lead to less TLR5 activation and increase the probability for greater fat storage.

One of the big remaining questions is how do the populations of Bacteroidetes and Firmicutes change to influence weight changes? One possibility is that while both Bacteroidetes and Firmicutes assist in fermentation perhaps Bacteroidetes are more responsive to complex sugars and other complex carbohydrates and Firmicutes are more responsive to simple sugars. Usually obese individuals consume lots of fat and simple sugars which are converted more easily to fat. The consumption of these types of foods select for Firmicutes over Bacteroidetes. When an individual loses weight it typically involves changes in the diet largely a reduction the amount of simple sugars and fats. This change could lead to a reduction in Firmicutes and due to less competition from the Firmicutes a corresponding increase in Bacteroidetes.

Another possibility for the increase in Bacteroidetes is that weight loss (excluding surgical intervention) involves a large amount of exercise. This additional exercise would lead to larger demands for energy consumption both in currently stored fat and newly consumed food. Such a change should reduce the amount of fat storage possibly involving the increased expression of TLR5 which could increase the population of Bacteroidetes if Bacteroidetes do indeed activate TLR5.

Overall it certainly appears that Firmicutes and Bacteroidetes play an important role in controlling weight. This influence seems to stem from two different mechanisms: overall food consumption and the extraction of energy from that food and probability of fat storage vs. fat consumption. While the exact mechanisms have not been discovered, Bacteroidetes appears to favor lean bodies and Firmicutes appears to favor obese bodies. Whether or not there is an evolutionary element is unclear. Beastfeeding also appears to be an important early element in driving either a lean or obese future. Due to potential feedback elements associated with fat content and intestinal bacteria populations like Firmicutes doping individuals with Bacteroidetes like Bifidobacteria may seem like a good idea, but the best option for weight loss involves the old stable tactics of high quality diet with insoluble fibers and exercise.


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