The gut-liver axis in nonalcoholic fatty liver disease: Another pathway to insulin resistance?

Nonalcoholic fatty liver disease (NAFLD), the hepatic component of the metabolic syndrome (MS), is a complex trait resulting from the interaction between multiple genes and social, behavioral, and environmental factors. Steatosis, the histological hallmark of NAFLD, is thought to be a relatively benign state, but predisposes the liver to necroinflammatory changes and fibrosis, leading to nonalcoholic steatohepatitis (NASH).

⁵¹Cr-EDTA, chromium-51 ethylenediamine tetraacetate; Fiaf, fasting-induced adipocyte factor; LPS, lipopolysaccharide; MS, metabolic syndrome; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPAR-γ2, peroxisome proliferator-activated receptor gamma 2; SIBO, small intestinal bacterial overgrowth; TJ, tight junction; TLR4, Toll-like receptor-4; TNF-α, tumor necrosis factor-α; ZO-1, zonula occludens-1.

The concept that gut microbiota, i.e., the billions of bacteria resident within the human gastrointestinal tract, can be involved in the pathogenesis of NAFLD/NASH has repeatedly resurged. The gut harbors 15,000-35,000 species of bacteria, whose concentration and type are mainly influenced by host genotype and nutrient availability.1 It has been proposed that gut microbiota is able to promote steatohepatitis by enhancing hepatic exposure to endotoxins. Possible mechanisms include bacterial overgrowth, release of the lipopolysaccharide (LPS) constituent of the gram-negative bacteria, and impaired intestinal barrier integrity, resulting in increased endotoxin absorption.2

As a matter of fact, NASH was encountered as a common complication of jejunoileal bypass surgery for morbid obesity during the 1980s and could be reversed by treatment with metronidazole. Various rat models of small intestinal bacterial overgrowth (SIBO) have been associated with liver lesions similar to NASH.2 In genetically obese rats, a disruption of the intestinal barrier leads to systemic endotoxemia and favors NASH, via release of tumor necrosis factor-alpha (TNF-α).3 In humans, the data available to date are inconclusive regarding a potential role for SIBO in NASH development and disease progression. SIBO is generally highly prevalent in patients with NAFLD4–6 and has been associated with severity of steatosis, but not with necroinflammatory activity or fibrosis. Moreover, significant alterations of intestinal permeability in NAFLD/NASH have never been described so far.

The report by Miele and coworkers7 in this issue of HEPATOLOGY adds importantly to this body of information. In this proof-of-concept study, the authors were able to demonstrate both the presence of SIBO (by glucose breath test) and of increased intestinal permeability (by the ⁵¹Cr-EDTA [ethylenediamine tetraacetate] excretion test) in patients with NAFLD, providing the first demonstration of gut leakiness in NAFLD. The association between increased excretion of ⁵¹Cr-EDTA and decreased zonula occludens-1 (ZO-1) expression in duodenal specimens is consistent with the putative role of tight junction (TJ) disruption in the increased gut permeability.

The sealing properties of the intestinal mucosa require an intact epithelium as well as efficient apical cell junctional complexes. TJ function is strictly related to the expression, localization, and integrity of a variety of proteins, such as occludin and ZO-1; substantial changes in expression/distribution of the TJ proteins, leading to a consistent leakage of bacterial endotoxins into the portal blood circulation, had been previously demonstrated in genetically obese mice.3

In the present study, the correlation between gut leakiness and SIBO suggests a causative role of quantitative alterations of gut microbiota. However, in insulin-resistant states, hyperinsulinemia and high circulating levels of inflammatory cytokines may per se contribute to enhanced intestinal permeability.8 Although endotoxemia has not been determined in this study, the lack of association of SIBO and/or intestinal permeability with necroinflammatory activity and fibrosis, which are distinctive features of steatohepatitis, does not support a role of gut bacteria in the development of NASH.

Most intriguing is the close association between SIBO, degree of hepatic steatosis, and increased prevalence of MS. Of note, increased intestinal permeability was associated with the same parameters, although to a lesser extent. A possible explanation is that components of MS, such as obesity and diabetes, may predispose an individual to SIBO due to intestinal dysmotility and stasis.9 However, the patients with NAFLD enrolled were only slightly overweight, and the presence of SIBO had no correlation with fasting glucose, although data on the prevalence of diabetes were not reported.

Recently, gut microbiota has been shown to affect fat storage and energy harvesting, playing a direct role in the development of insulin resistance and of the related metabolic diseases. The hypothesis is that, depending on specific dietary conditions, intestinal microflora changes, which leads to increased intestinal permeability and consequent endotoxemia and triggers inflammation and metabolic disorders. The cross-sectional nature of the study by Miele et al.7 does not allow conclusions regarding the underlying mechanisms; nevertheless, their data suggest a potential relationship between gut microbiota and clinical (MS) and histological (hepatic steatosis) evidence of insulin resistance in patients with NAFLD.

Gut microbiota can influence the host metabolism in several ways, some of which do not require an altered intestinal permeability whereas some others do. Among the former category are regulation of energy extraction from nutrients and modulation of genes involved in substrates metabolism. An altered gut permeability is conversely involved in promoting inflammatory mechanisms, not necessarily leading to local liver damage, but possibly contributing to the development of hepatic insulin resistance and to subclinical systemic inflammation.

Backhed et al.10 found that conventionally reared mice had a 40% higher body fat content than germ-free mice and showed that microbiota promoted absorption of monosaccharides from the gut and induced hepatic lipogenesis in the host, through suppression of lipoprotein lipase inhibitor Fiaf (fasting-induced adipocyte factor). Composition of cecal microbiota is also important, because obese and insulin-resistant ob/ob mice have a higher ratio of Firmicutes/Bacteroidetes than do lean controls,11 resulting in increased production of short-chain fatty acids by fermentation of dietary fibers.12 In humans, the fecal Firmicutes/Bacteroidetes ratio in obese individuals decreased after consumption of low-calorie diets, providing an association between gut microbiota profile and weight management.13 The composition of the gut microbiota is established within the first year of life and is determined by host genotype, but dietary factors can lead to transient as well as long-term changes.1 In animal models, high-fat feeding promotes endotoxemia and affects bacterial populations, favoring an increase in the ratio of gram-negative to gram-positive bacteria,14 whereas moderate fructose consumption leads to enhanced intestinal translocation of bacterial LPS, induction of TNF-α, and liver steatosis.15 On the other hand, in healthy men, plasma LPS concentration is associated with energy intake, independent of diet composition, suggesting that various high-energy diets may result in metabolic endotoxemia in humans.16 Nutrients can also directly affect the expression of genetic information through epigenetic mechanisms. For example, total and saturated dietary fats can influence the Pro12Ala variant of peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2), whereas a low-fat diet can modulate the adiponectin gene expression.17

Modification of gut microbiota by dietary habits is also capable of inducing inflammatory mechanisms. In the gut, Toll-like receptors on innate immune cells react to bacterial infections by recognizing LPS; importantly, nutritional fatty acids trigger inflammatory response via the Toll-like receptor-4 (TLR4) signaling in the adipocytes and macrophages,18 highlighting once again the interconnection between gut microbiota and nutrients. Endogenous LPS is transported from the intestine toward target tissues by a mechanism facilitated by chylomicrons synthesized in response to a high-fat diet and triggers the secretion of proinflammatory cytokines when it binds to TLR4.18 Hence, high-fat feeding may induce chronic metabolic endotoxemia and lead to obesity, insulin resistance, and diabetes.14 Further, a high-fat diet strongly increases the intestinal permeability and reduces the expression of genes coding for TJ proteins ZO-1 and occludin.19 Taken together, these observations suggest the existence of strain-specific and genetically determined selection of gut-microbial metabolism, and strongly supports the idea that complex metabolic diseases are a product of multiple perturbations under the influence of a triggering factor, in this case, a dietary change. In this setting, Miele et al.7 collected only partial data about dietary habits and found no significant difference between patients with NAFLD and control subjects (healthy and celiac). Nevertheless, the influence of dietary composition and genetic makeup on SIBO remains an intriguing matter for further studies.

These new findings are of interest also for their therapeutic implications. Although it is clearly no substitute for lifestyle therapy, manipulation of gut flora by antibiotics, prebiotics, and probiotics could be an additional tool to counteract the impact of unbalanced diets on metabolic diseases. In animal models, treatment with prebiotic dietary fibers (oligofructose) reduced metabolic endotoxemia, adipose tissue inflammation, and oxidative stress and lead to an improvement in glucose tolerance and to reduction of body weight.20 In ob/ob mice, modification of gut microbiota by probiotics ameliorated hepatic insulin sensitivity, hepatic steatosis, and inflammation;1 similarly, broad-range antibiotics improved glucose tolerance, hepatic insulin resistance, and steatosis by modulating the hepatic and intestinal expression of genes involved in inflammation and metabolism.22

In conclusion, the study by Miele and colleagues raises the possibility that gut microbiota and intestine permeability are important mediators of diet-induced metabolic disturbances in NAFLD. Certainly, a number of questions remain to be answered. First, what is the relative contribution of gut microbiota versus other mediators (i.e., gut-derived hormones and peptides) in inducing hepatic steatosis and what are the mechanisms linking gut microbiota composition and insulin resistance. Also, the environmental and genetic factors modulating the individual gut microbiota must be further elucidated, because changes in intestinal bacterial flora may be either the cause or the result of metabolic disturbances in NAFLD. A better knowledge of the gut-liver axis will provide interesting opportunities for the management and prevention of NAFLD.