Gut Microbiota And Obesity

Introduction The total genome of gut microbiota is 100 times greater than the human. Doubtlessly, intestinal flora carries out enormous diversities of metabolic and biochemical functions (1). Although, probiotic bacteria benefit the host, some members of gut flora contribute to obesity, depending on their composition.

Why do microbiota compositions differ in obese and lean host bodies?

Figure 1 Intestinal microbiota phylum composition in Standard-Chow-Diet(fat: 13% energy ) and High-Fat-Diet(fat: 45% energy ) (7) The composition of gut microbiota is easily changed by different host conditions; intestinal pH, host adiposity, diet and intestinal transit time. The composition was different in obese and lean mice. According to 16S rRNA gene sequencing of cecal DNA, leptin-deficient (genetically-obese) mice had much higher Firmicutes: Bacteroidetes (F:B) than the wild-type mice under the same diet (5). The same trend was observed in the human body (29). When wild-type mice changed feeding type from a standard-chow-diet to high-fat-diet, they showed weight/fat gain and changed from low F:B to high F:B [Figure 1](7). Then, is high F:B affected by host adiposity or diet? RELM? is a gene inducing insulin resistance (6). Unlike wild-type mice, RELM? knock-out mice (7) remained relatively lean, however showing similar changes in micro-ecology [Figure 1]. This shows diet influences gut microbiota rather than host adiposity, (7). Calorie intake also changes the composition under the same diet. Furthermore after the transplantation of microecology from obese mice ('obese-microbiota') to healthy germ-free mice, significant weight gain was shown under a low-fat-diet. (30) Then, how microbiota influences the host body?

Mechanism 1: Higher Efficiency of Energy Harvest Firmiticutes are major commensals metabolising indigestible polysaccharide into short-chain-fatty-acids (SCFAs). SCFAs absorbed from intestines are utilised for lipogenesis, gluconeogenesis or directly consumed for generating ATP. High Firmicutes: Bacteroidetes in 'obese-microbiota' harvests greater extra energy from the same fibre quantity. In fact, SCFAs can biochemically prevent insulin resistance (2); but their extra calorie-input is the issue. However, it is difficult to consume high enough fibre to cause high enough calorie harvest inducing obesity, as fibre intake discourages overeating (28). Hence SCFAs' contribution to obesity is controversial.

Mechanism 2: Influence on host genes by intestinal flora

Figure 2: Relative Fasting-induced adipose factor (Fiaf) in different diet and different microecology (left). Weight changes under western-diet in different microecology (Right) (31)

Diet Fat % in total calorie Intestinal microbiota Fiaf Weight increase Western-diet

41% Germ-free (GF) High Small Conventionalised(CVN) Low Large Low-fat

5% Germ-free High None Conventionalised Low None Low-fat standard-chow N/A 'Lean-microbiota' N/A None 'Obese-microbiota' N/A Large Table 1: Summary of impact of diet and microbiota on Fiaf expression and weight change (31). Impact of different microbiota on weight under the same diet (30) [Grey row] Although unexplored, the presence of gut microbiota is known to suppress several genes in epithelial cells. One of them is Fasting-induced adipose-factor (Fiaf) inhibiting the lipoprotein lipase (LPL). LPL hydrolyses triglycerides allowing fat accumulation. Suppressing Fiaf promotes LPL activity, therefore encourages fat storing. In conventionalised mice (CVN) on a western-diet (WD), there were lower Fiaf expression and greater weight gain [figure 2 left, Table 2] than in germ-free mice (GF). On a low-fat-diet (LF) small weight difference were shown, however with the lower Fiaf level in CVN than GF (31)[Figure 2 left, Table 2]. In different experiment, there was significant weight gain after transplanting 'obese-microbiota' to healthy mice under the same diet (30). These results can be interpreted as: . No effect from Fiaf. The weight gain in CVN/WD is due to shifts in microecology from 'conventionalised-microbiota' to 'obese-microbiota' following 8 weeks of western-diet. . Fiaf levels effect body weights only in high enough blood triglyceride levels, but 'obese-microbiota' can affect regardless of plasma conditions.

Mechanism 3: Endotoxemia Endotoxemia is a major but controversial mechanism, linking gut microbiota to diet-induced-obesity. Then, what is endotoxemia?

Figure 3: Cascade signalling initiated by LPS to low-grade inflammation (3) There are two types of bacteria; Gram-negative with lipopolysaccharide (LPS), and Gram-positive with no LPS. LPS from Gram-negative microbiota is recognised by epithelial toll-like-receptor4 (TLR4), activating the myeloid-differentiation-factor88. This stimulates nuclear factors, NF-?B, promoting transcri ptions of pro-inflammatory molecules; cytokines IFN-? and TNF-? (3) [Figure 3]. Free LPS is absorbed into intestinal capillaries and activates TLR4 in other cells including adipocytes and macrophages (8). This is low-grade inflammation shown in obesity and insulin resistance, implicating LPS in obesity (9). Therefore, endotoxemia is high LPS level with harmful impacts on metabolisms.

Mechanism underlying malign obesity derived from LPS The pro-inflammatory molecules produced from TLR4 signalling help macrophages infiltrate adipose tissues. Some macrophages trans-differentiate into bone-marrow-progenitor-derived adipocytes, increasing adipocytes number. This unique adipocyte has decreased leptin-expression, and increased cytokines-expression. Low-leptin levels lead to low post-meal satiety, encouraging hyperphagia. Some macrophages hydrolyse and release lipids, increasing plasma free-fatty-acids levels, hence stimulating hyperinsulinemia and insulin resistance (10-12). Additionally TNF-? maintains inflammations and stimulates phosphorylation insulin-receptor-substrate-1, interrupting insulin receptor activation (13-14). Hypothalamic insulin resistance also induces hyperphagia (15), thus high-calorie-diet increases LPS. Endotoxemia also promotes lipogenesis, stimulating fat accumulation (33, 34). These mechanisms are positive-feedback in pathophysiology of obesity.

High-fat-diet and intestinal LPS

Figure 4 Re-categorisation of the composition in Figure 1 in terms of Gram-positive and Gram-negative (7, 32)

Figure 5: The experiment was undertaken using control-fed-mice (CT) and high-fat-diet (HF). A: Endotoxin level in 24 hrs period B: Change in microbiota species C: Change in LPS level upon oil, water or LPS intake D: Endotoxin level comparision in different diets, modified after (16) Another experiment was undertaken using healthy mice having Bacteroides (Gram-negative) and E.rectale/ C.coccoides (Gram-positive) as dominant microbiota. Under 4 weeks high-fat-diet (HF) (72% fat, <1% carbohydrate), they had 270% higher plasma LPS level [Figure 5, D]. LPS level positively correlated with fat gain regardless of food intake(16). However Gram-negative Bacteroides number was shrunken in mice having high LPS level under HF[Figure 5, B]. Furthermore, Gram-negative: Gram-positive composition decreased after a high-fat-diet in different experiments [Figure 4]. The authors simply suggested the higher LPS level with reduced Gram-negative number is due to: . fewer Gram-positive bacteria (16) . fewer Bifidobacteria which led to mucosal barrier dysfunction, encouraging more LPS absorption (17). However, considering the huge reduction in Gram-negative population and proportion after HF [Figure 5, B] and considering the Bifidobacterium% (phylum Actinobacteria) in total communities [Figure 1], other factors should be considered.

Do bile acids increase the death rate of gut microbiota? Figure 6: Structure of lipopolysaccharide with Lipid A on Gram-negative bacteria (35) LPS is normally released when Gram-negative bacteria die (18). Bile acids increase bioactivity of lipid A [figure 6] (22) and solubilise bacterial membranes, (20) destroying Gram-negative bacteria and releasing LPS in the intestine. The bile secretion explains a sharp increase in endotoxin in control mice (CT) while feeding (Dark) [Figure 5, A], as well as the increase in LPS after oil consumption [Figure 5, C]. However, [Figure 5 A, HF] challenges the bile acids principle. . Little fluctuation in endotoxin level [Figure 5, A] despite: - deteriorated mucosal barrier function, from reduced Bifidobacterium number. - a large quantity of fat consumption. . Higher endotoxin level despite less bile secretion. -In a high-fat/low-carbohydrate-diet used for HF, less bile is secreted than in a normal diet (24).

Although being unclear, these may be explained: In HF(72% fat, <1% carbohydrate), less bile secreted mostly used to emulsify the fat ingested, so Gram-negative bacteria are 'hidden' from bile acids so destroyed slowly but constantly. Moreover, large amounts of lipid absorption through the thinner mucus layer also constantly high LPS absorption. The total number of microbiota in CT was roughly 5 times higher than that in HF. [Figure 5, B] (?log10(bacterial cells/g)?0.65), indicating higher 'death rate' than 'birth rate' of microbiota. Assuming no chance influenced, what is causing the total number decrease even with the lower bile (killing factor) concentration? HF(72% fat, <1% carbohydrate diet) has no fibre, so constipation makes bile acid remain in intestines longer(26). The time damaging Gram-negative and Bifidobacteria is longer, so higher 'death rate' and more effective and continuous LPS absorptions are achieved. With some carbohydrate in diet, higher fat intake stimulates more bile secretion (23). High-fat/low-fibre/simple-carbohydrate is an optimised diet for Gram-negative bacteria to release LPS, leading to endotoxemia-induced-obesity. (25, 27)

Conclusion Obese people are blamed a lot; in fact their overeating can be induced by a biological feedback from gut microbiota! Short-Chain-Fatty-Acids and Lipopolysaccharide produced from intestinal symbionts, and their effects on many host's genes encourage weight gain. However, these cannot fully explain the weight gain after 'obese-microbiota transplantation' under the same diet. The species-specific studies will be able to illuminate how each mechanism contributes, hence finding a better way of curing of obesity and other metabolic diseases.

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