Increased Energy Harvest from the Diet

Nutrient absorption and gut motility can be modulated by short chain fatty acids (SCFAs), the major end products of bacterial fermentation. SCFAs (propionate, acetate, and butyrate) represent more than 60% of energy content of carbohydrates from the diet and are ligands for Gpr41 and Gpr43, 2 G protein–coupled receptors that induce intestinal secretion of peptide YY (PYY) and leptin.

Gpr41 functional deletion was related with a reduction in PYY expression, a faster intestinal transit rate, and a reduction of energy uptake from the diet. Consistently, Grp43-deficient mice showed lower total body fat and improved insulin sensitivity; moreover, GPR43 inhibition was associated with higher energy expenditure accompanied by higher core body temperature and increased food intake.

Collectively, these findings disclose the pivotal role for Gpr41 and Gpr43 in mediating microbiota regulation of energy harvest from the diet.

Regulation of Host Energy Storage

In conventionalized mice, microbiota promotes absorption of monosaccharides from the gut lumen. Increased carbohydrate availability promotes de novo lipogenesis in the liver and the adipose tissue by stimulating carbohydrate response element binding protein–mediated and sterol response element binding protein 1–mediated transcription of genes encoding 2 rate-limiting lipogenetic enzymes: acetyl-CoA carboxylase 1 and fatty acid synthase. This mechanism leads to an accumulation of triglycerides in the liver and in adipose tissue.

Fasting-induced adipose factor (Fiaf), also called angiopoietin-like protein 4, is an inhibitor of adipose lipoprotein lipase produced by enterocytes, hepatocytes, skeletal myocytes, and adipocytes in response to fasting, peroxisome proliferator-activated receptor-γ activation, and inflammatory prostaglandins, PGD ₂ and PGJ ₂ . Fiaf also modulates fatty acid oxidation in skeletal muscle and in adipocytes, increasing the nuclear transcription factor peroxisomal proliferator-activated receptor coactivator 1α, a coactivator of genes encoding key enzymes involved in mitochondrial fatty acid oxidation.

Gut microbiota affect storage of circulating triglycerides into adipocytes by regulating intestinal secretion of Fiaf: conventionalization of germ-free mice suppressed intestinal expression of Fiaf in differentiated villous epithelial cells in the ileum; consistently, germ-free Fiaf-KO mice fed a high-fat/high-carbohydrate diet were not protected against diet-induced obesity. Specific microbiota has different effects on expression of Fiaf: mice fed a high-fat diet supplemented with Lactobacillus paracasei showed increased levels of Fiaf and displayed significantly less body fat and reduced triglyceride levels. In coculture experiments, Lactobacillus also induced Fiaf gene expression. These data suggest that modulation of Fiaf through manipulating gut flora could be an important therapeutic target.

Microbiota may regulate the fatty acid metabolism also by affecting adenosine AMP–AMP (AMPK) activation. AMPK stimulates fatty acid oxidative pathways in the liver and the skeletal muscle through activation of mitochondrial enzymes, such as acetyl-CoA carboxylase and carnitine palmitoyltransferase I, and reduces hepatic glycogen-synthase activity and glycogen stores, improving hepatic and muscle insulin sensitivity.

Gut flora may have an inhibitory effect on AMPK-regulated fatty acid oxidation, because germ-free mice present a persistent activation of hepatic and muscle AMPK, whereas AMPK activity and related metabolic pathways were suppressed in conventionalized mice.

Regulation of Chronic Low-grade Endotoxinemia and Host Inflammatory Response

Chronic activation of the immune system is linked to the development of obesity and T2DM; TLR4-activated inflammatory pathway has been specifically connected with the low-grade chronic inflammation, which characterizes obesity-related disorders.

Gram-negative microbiota may affect host metabolism through lipopolysaccharide (LPS), which binds the complex of CD14 and TLR4 at the surface of innate immune cells, activating inflammatory pathways implicated in the pathogenesis of obesity, insulin-resistance, and T2DM.

Beside LPS, free fatty acid and products from dying cell can bind TLR4 and stimulate inflammatory response in cell expressing TLR4 (gut immune cells, adipocytes, endothelial cells, tissue macrophages, hepatocytes, and hepatic Kupffer and stellate cells). The hepatic Kupffer cells may have an independent role in this contest: in mice, high-fat diet promotes the activation of Kupffer cells, resulting in insulin resistance and glucose intolerance, whereas selective depletion of these cells restores hepatic insulin sensitivity and improves whole-body and hepatic fat accumulation, without affecting adipose tissue macrophages.

Metabolic endotoxiemia is also associated with nonalcoholic steatohepatitis, through hepatic inflammasome activation: a recent study reported, in a mouse model of nonalcoholic steatohepatitis, saturated fatty acids upregulation of the inflammasome that led to sensitization to LPS-induced inflammasome activation. LPS administration modifies the gut microbiota composition (reduction of Bifidobacteria and Eubacteria spp) and determines metabolic effects, such as systemic insulin resistance, increased plasma and hepatic triglyceride content, and reduction of high-density lipoprotein levels ; mice fed a high-fat diet shown the same change in microbiota, associated with a low-grade elevation in circulating LPS levels (metabolic endotoxemia). Consistently, LPS receptor deletion or changes of gut microbiota composition induced by antibiotic administration prevented the metabolic alteration of a high-fat diet.

Modification in gut microbiota composition results in change of metabolic endotoxiemia level: prebiotic fermentable oligofructose (OFS) administration increased the intestinal proportion of Lactobacilli and Bifidobacteria in ob/ob mice, restored normal intestinal permeability through stimulation of epithelial tight-junction proteins, and reduced systemic endotoxiemia, in association with enhanced intestinal glucagon-like peptide (GLP)-2 levels.

Gut microbiota modulates the gut-derived peptide secretion, promoting L-cell differentiation in the proximal colon of rats and increasing GLP-1 secretion in response to a meal in healthy humans ; deletion of GLP-1 abolished the beneficial effects of prebiotics on weight gain, glucose metabolism, and inflammatory pathway activation. Furthermore, gut microbiota may modulate gut barrier integrity and endotoxinemia through GLP-2, a 33-amino acid peptide with known intestinotrophic properties, which is cosecreted with GLP-1 by enteroendocrine L cells.

Ob/ob mice treated with prebiotic plus carbohydrates diet presented an increased circulating GLP-1 and GLP-2, which were associated with an altered gut flora composition (increased proportion of Lactobacilli and Bifidobacteria ), restored tight junction integrity and intestinal barrier function, and lowered endotoxinemia. Administration of a GLP-2 antagonist prevented these effects, which were mimicked by the administration of a GLP-2 agonist, suggesting that GLP-2 could mediate the effects of prebiotics.

Microbiota, such as Bifidobacterium and Lactobacillus , may exert an anti-inflammatory effect through the synthesis of bioactive isomers of conjugated linoleic acid, which shows antidiabetic, antiatherosclerotic, hypocholesterolemic, hypotriglyceridemic, and immunomodulatory activity.

In different mammalian models, dietary supplementation of linoleic acid plus Bifidobacterium breve altered the profile of polyunsaturated fatty acid composition, resulting in higher intestinal, hepatic, and adipose tissue content of c9,t11 conjugated linoleic acid; the animals also present a higher adipose tissue concentrations of eicosapentaenoic acid and docosahexaenoic acid, 2 omega-3 polynsatured fatty acids with anti-inflammatory and lipid-lowering properties. These changes were associated with a reduced expression of proinflammatory cytokines, such as tumor necrosis factor α, interleukin-6, interleukin-1β, and interleukin-8, accompanied with a higher anti-inflammatory interleukin-10 secretion.

Finally, SCFAs elevation also could result in a reduction of the inflammation and an improvement of insulin sensitivity. Butyrate shows anti-inflammatory properties that could improve epithelial permeability. Acetate raised plasma PYY and GLP-1 and suppressed proinflammatory cytokines.

Collectively, these data suggest that endotoxinemia is involved in the pathogenesis of obesity-related diseases, is affected by dietary nutrient composition, and may be modulated by manipulation of gut microbiota composition.

The Role of Vitamin D

Vitamin D deficiency has been associated with allergic diseases development and increased body mass index.

Vitamin D plays a role in immunomodulation and a decreased vitamin D uptake has been correlated with a change in fecal microbiota composition in one study, although this association needs to be confirmed in larger cohorts.

Mice lacking the vitamin D receptor present chronic, low-grade inflammation in the gastrointestinal tract and the absence of the vitamin D receptor results in enhanced inflammation in response to normally nonpathogenic bacterial flora. Moreover, intestinal vitamin D receptor has also been shown to negatively regulate bacterial-induced intestinal nuclear factor κB activation and to attenuate response to infection, suggesting that the vitamin D may affect the impact of intestinal flora on inflammatory disorders.

Probiotics

Probiotics are food supplements that contain living bacteria, such as Bifidobacteria, Lactobacilli, Streptococci , and nonpathogenic strains of Escherichia coli . When administered, they confer beneficial effects to the host because of changes in the gut microbiota that are transient and diminish gradually with time after cessation. Different studies suggest that probiotics influence the intestinal lumen rather than the gut-epithelium, possibly explaining the transient effect of probiotics. This thesis was tested by Goossens and colleagues : they compared the effects of consuming Lactobacillus plantarum on the microbial colonization of feces and biopsies from the ascending colon and rectum. Within fecal samples, the amount of Lactobacilli was significantly increased. The biopsies did not, however, confirm a growth of Lactobacilli. Recently, van Baarlen and colleagues described changes in the expression of up to thousands of genes in duodenal biopsies after administration of 3 types of Lactobacilli. Alterations in the gut microbiota as a result of probiotics are commonly observed but evidence showing that probiotica administration directly affects inflammatory state has only recently been demonstrated in humans. In contrast, studies on the effects of probiotics on characteristics of T2DM are mostly performed in animal models, reporting beneficial effects by various strains of Lactobacilli on characteristics of T2DM. Both antidiabetic and anti-inflammatory effects of Lactobacillus casei in diet-induced obese mice were recently described. In addition, diet-induced obese mice showed a reduction in body weight gain after they were supplemented with Lactobacillus rhamnosus PL60 plus an adequate diet. In the same way, Kang and colleagues studied the effects of Lactobacillus gasseri BNR17 on diet-induced overweight rats; they found that the percent increase in body weight and fat pad mass was significantly lower in the BNR17 group. Although these animal findings are interesting, the relevance of lactobacilli supplementation for the control of adiposity is a matter of debate. To clarify the effect of Lactobacillus-containing probiotics on weight, Million and colleagues performed a meta-analysis of clinical studies and experimental models. They included 17 RCTs in humans, 51 studies on farm animals, and 14 experimental models and they concluded that different Lactobacillus species are associated different effects on weight change that are host-specific. In particular, Lactobacillus fermentum and Lactobacillus ingluviei were associated with weight gain in animals; Lactobacillus plantarum was associated with weight loss in animals and Lactobacillus gasseri was associated with weight loss both in obese humans and in animals.

Prebiotics

Prebiotics (mostly oligosaccharides) are nondigestible but fermentable food ingredients that selectively stimulate the growth or activity of one or multiple gut microbes that are beneficial to their human hosts. The beneficial metabolic effects of prebiotics are in part mediated by a reduction in metabolic endotoxiemia. In physiologic situations, Bifidobacteria are capable of lowering LPS levels. The number of Bifidobacteria was inversely correlated with the development of fat mass, glucose intolerance, and LPS level. High-fat diets promote the growth of LPS-producing gut microbiota and subsequently restrict the amount of Bifidobacteria . Bifidobacterium spp and Lactobacillus spp are sensitive to the administration of certain prebiotics. Prebiotics containing OFS specifically stimulate the growth of these intestinal bacteria. OFS administration completely restored Bifidobacteria spp and normalized plasma endotoxin levels, leading to improved glucose tolerance, increased satiety, and weight loss in human subjects. Besides modulating endotoxemia, OFS can alter metabolism in various other manners. Cani and colleagues showed that effects of OFS were mediated via a GLP1-dependent pathway. High-fat–fed diabetic mice on OFS treatment demonstrated improved glucose tolerance, diminished body weight, and decreased endogenous glucose production. Either adding the GLP-1 receptor antagonist exendin 9–39 or using GLP-1 knockout mice resulted in a complete lack of the OFS-mediated beneficial effects, thus showing the causal role of GLP-1 in this pathway in animals. Attempts to translate these findings to human subjects are ambiguous, showing that OFS tends to dose dependently decrease energy intake and increase PYY plasma concentrations, but reported effects on satiety are conflicting. Everard and colleagues found that in ob/ob mice, prebiotic feeding decreased Firmicutes and increased Bacteroidetes phyla, improved glucose tolerance, increased L-cell number and associated parameters (intestinal proglucagon mRNA expression and plasma GLP-1 levels), and reduced fat-mass development, oxidative stress, and low-grade inflammation. In high-fat–fed mice, prebiotic treatment improved leptin sensitivity as well as metabolic parameters. Furthermore, OFS fermentation directly affects SFCA butyrate synthesis from extracellular acetate and lactate, implicating the therapeutic potential of prebiotics. In addition, insulin-type fructance decreased the activity of the endocannabinoid system (by reducing the expression of cannabinoid receptor 1, restoring the expression of anandamide-degrading enzyme, and decreasing anandamide levels in the intestinal and adipose tissues), a phenomenon that contributes to an improvement barrier function of the gut and adipogenesis. Finally, insulin-type fructan prebiotics counteract the overespression of GPR43 in the adipose tissue, which is related to a decrease rate of differentiation and a reduce adipocyte size. Thus, available evidence supports the hypothesis that prebiotics can influence metabolic disturbances. The beneficial effect on clinical endpoints in metabolic disturbances remains to be demonstrated in large prospective randomized controlled trials.