Tributyrin supplementation protects mice from acute ethanol-induced gut injury.

EXCESSIVE ALCOHOL CONSUMPTION is associated with liver injury. Alcoholic liver disease is characterized by fatty liver, hepatitis, fibrosis, and cirrhosis. There is growing appreciation that interorgan crosstalk contributes to ethanol (EtOH)-induced liver injury. Impairment of intestinal barrier function during heavy EtOH exposure is associated with progression of EtOH-induced liver injury in humans and rodents (Rao, 2009). The epithelial barrier is controlled by a variety of specific proteins and intercellular junctional complexes (e.g., occludins, zonula occludens, adherens junctions), together forming a complex called the tight junction (TJ; Rao, 2009). Intact TJs prevent diffusion of macromolecules, such as endotoxin, from the intestine into the lymphatic system and blood stream. Mechanisms by which EtOH impairs the epithelial barrier are not completely understood, but involve complex interactions between reactive EtOH metabolites, changes in intestinal epithelial cell function, as well as shifts in the gut microbiota (Bull-Otterson et al., 2013; Mutlu et al., 2009). EtOH metabolism, via the alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) pathways, generates acetaldehyde. Both ADH and CYP2E1 are expressed in liver and intestine, and ADH is also expressed in commensal gut microbiota (Bergheim et al., 2005; Visapaa et al., 2002). While ingested EtOH is primarily absorbed in the proximal small intestine, the distal intestine is exposed to EtOH on the basolateral surface via the blood. EtOH concentration in the colonic lumen is equivalent to that in blood (Halsted et al., 1973). Acetaldehyde increases gut permeability to gut-derived endotoxin by altering intestinal epithelial integrity (Visapaa et al., 2002). Acetaldehyde is further metabolized to acetate by aldehyde dehydrogenase (Geokas et al., 1981). In humans, 70 to 80% of oxidized EtOH appears as free acetate in the hepatic vein (Lundquist et al., 1962). EtOH-derived acetate affects peripheral tissues, for example, acetate has marked effects on central nervous system function (Israel et al., 1994), but it is not clear if acetate also impacts intestinal epithelial integrity. The gut microbiota is comprised of trillions of commensal bacteria dominated by nearly 800 different species (Hooper et al., 2002). One of the best characterized beneficial functions of gut microbiota is the ability to ferment long-chain polysaccharides that escape human digestion yielding short-chain fatty acids (SCFA). The predominant SCFA produced are acetate, propionate, and butyrate (Canani et al., 2011; Velazquez et al., 1996). Although butyrate is the least abundant SCFA, it serves many biological roles. Butyrate induces epithelial cell proliferation in normal intestinal tissue, but decreases cell proliferation, increases apoptosis, and stimulates cell differentiation in colon cancer cells (Canani et al., 2011). Butyrate stimulates water and electrolyte absorption and inhibits prosecretory action of several cAMP-generating secretagogues, making butyrate beneficial in treating diarrheal disorders (Binder, 2010). Butyrate is the primary fuel source for colonocytes and improves gut barrier function (Canani et al., 2011; Ploger et al., 2012). Butyrate inhibits activation of transcription factor NF-κB, decreasing expression of inflammatory cytokines by colonic epithelial cells (Inan et al., 2000). Absence of intestinal butyrate is associated with apoptosis, inflammation and mucosal atrophy (Hass et al., 1997; Thangaraju et al., 2008, 2009). Higher acetate:butyrate ratios are associated with colonic pathology, including colon polyps and tumors (Weaver et al., 1988). Multiple concentration-dependent processes regulate intestinal transport of SCFA (Thibault et al., 2010). Mono-carboxylate transporters (MCT), expressed in both apical and basolateral membranes of intestinal epithelial cells, play an important role in absorption of SCFA into the enterocytes from the gut lumen and systemic circulation (Shimoyama et al., 2007). Arterial plasma acetate levels are elevated as much as 20-fold following high-dose EtOH consumption (Lundquist et al., 1962). Therefore, acetate transport from systemic circulation into the gut lumen via MCTs during EtOH consumption is highly likely. Butyrate also interacts with several nutrient sensing G protein–coupled receptors, identified as SCFA receptors (Brown et al., 2003). GPR109A is abundantly expressed in the lumen-facing apical membrane of colonocytes (Thangaraju et al., 2009) and serves as a luminal SCFA sensor (Borthakur et al., 2012). Thus, effects of SCFA are mediated both via activation of these receptors and/or transport into the cell (Brown et al., 2003). When mice are chronically exposed to high concentrations of EtOH (40 to 60% kcal), gut dysbiosis results (Bull-Otterson et al., 2013; Mutlu et al., 2009; Yan et al., 2011). Acute or binge exposure to EtOH also causes gut dysbiosis, resulting in higher intestinal ratios of Escherichia coli to Lactobacilli, increased plasma endotoxin levels and histological and ultrastructure changes in the intestinal mucosa and epithelia (Zhou et al., 2013). These gut integrity changes are associated with liver injury after acute and chronic EtOH exposure (Bertola et al., 2013; Wang et al., 2012; Zhou et al., 2013). Probiotic supernatant protects against negative effects of acute EtOH exposure on markers of intestinal permeability, endotoxemia, and liver injury (Wang et al., 2012). Similarly, prebiotic and probiotic administration minimizes chronic EtOH-induced changes in gut microbiota (Bull-Otterson et al., 2013; Mutlu et al., 2009) and oral administration of broad-spectrum antibiotics reduces endotoxemia and EtOH-induced liver injury severity in rats (Adachi et al., 1995). One consequence of gut dysbiosis following EtOH exposure is a skewing of intestinal SCFA concentrations, characterized by higher acetate and lower butyrate concentrations (Xie et al., 2013). Therefore, we hypothesized that the combination of EtOH-induced gut dysbiosis, resulting in lower luminal butyrate concentrations, as well as increased acetate concentrations resulting from EtOH metabolism would negatively alter the acetate:butyrate ratio in the intestinal lumen. Given the important role of butyrate in maintaining gut health and integrity, the purpose of this study was to investigate the effcacy of pharmacologic manipulation of luminal SCFA with the pro-drug tributyrin (glyceryl tributyrate) during EtOH exposure in protecting gut integrity and mitigating EtOH-induced release of liver enzymes, hepatic steatosis, and expression of inflammatory cytokines and chemokines in liver.