There is currently intense interest in understanding the gut microbiota’s contributions to vertebrate nutrient metabolism and energy balance (Musso et al., 2011
). Although microbial contributions to degradation of complex dietary carbohydrates have been studied extensively (Flint et al., 2012
), the impact of the microbiota on dietary lipid metabolism has received relatively little attention. Previous investigations of dietary lipid metabolism in gnotobiotic mammals evaluated serum lipid metabolites (Bäckhed et al., 2004
; Bäckhed et al., 2007
; Martin et al., 2009
), which do not distinguish between exogenous and endogenous lipid sources, and serum chylomicrons (Velagapudi et al., 2010
) or fecal crude fat (Yoshida et al., 1968
; Rabot et al., 2010
), which do not do not distinguish between dietary and microbe-produced lipids. Here, we used an in vivo
imaging strategy in transparent zebrafish larvae to uncover a role for the microbiota in stimulating dietary FA absorption in the intestinal epithelium and extra-intestinal tissues. Our results identify the gut microbial community as a target for controlling dietary fat absorption and energy balance.
Fatty acid absorption, intracellular LD assembly in enterocytes, and subsequent secretion as chylomicrons and FFA have been extensively studied (Iqbal and Hussain, 2009
). However, our mechanistic understanding of these physiologic processes in vivo
remains incomplete, which poses challenges for understanding how they are regulated by environmental factors such as the microbiota. Our data reveal that colonization with a microbiota promotes epithelial absorption of FAs, resulting in accumulation of LDs in enterocytes and increased accumulation of dietary FAs in extra-intestinal tissues. We propose four nonexclusive mechanisms by which microbes might stimulate FA absorption and LD accumulation in enterocytes. First, microbes might increase bioavailability of FAs by modifying the production or composition of bile salts (Swann et al., 2011
). Second, microbes could directly contribute to luminal lipolytic activity that promotes FA availability for potential absorption in the intestinal epithelium (Ringø et al., 1995
). Third, microbes might enhance FA absorption indirectly by evoking physiologic responses in the intestinal epithelium that stimulate its inherent absorptive capabilities. Finally, the microbiota might reduce rates of fatty acid oxidation in intestinal epithelial cells permitting increased storage of FA in LDs. Identification of the underlying mechanisms could facilitate microbe-based strategies for controlling dietary fat absorption.
Our results show that the presence of a microbiota promotes two distinct phenotypes of LD formation within the enterocyte: increased LD number and increased LD size. The majority of enterocyte LDs evaluated in this study presumably represent temporary storage LDs that occur after high-fat feeding (Glatz et al., 2010
). While enterocyte LD size and number reflect distinct and quantifiable aspects of FA absorption, they likely share common cell biological processes. Previous genetic analyses of LD formation have established that these dynamic organelles are under complex regulatory control and functionally linked to other cellular organelles and pathways (Guo et al., 2008
; Beller et al., 2008
). However, we anticipate that at least a subset of the ‘small LDs’ enumerated in this study (<0.55 μm2
) represent chylomicrons, as this small size range is inclusive of the predicted chylomicron size in teleosts (Sire et al., 1981
). Investigation of the mechanisms underlying the microbial and dietary regulation of these distinct lipid-rich organelles could provide insights into enterocyte lipid metabolism.
In addition to revealing a role for the microbiota in stimulating FA absorption, our data show that this host response to the microbiota is influenced by diet history. Although enterocyte LD size was increased by the microbiota regardless of diet history, consistent increases in enterocyte LD number were only observed in fed animals. These results are surprising since our data were collected at 6 dpf only ~1 day after they normally begin feeding. This suggests that the observed effects of starvation on zebrafish at 6 dpf might be determined primarily by diet-dependent effects on the microbiota rather than direct effects of diet on the host. Consistent with this notion, we discovered that the presence of diet results in alterations in the zebrafish gut microbiota including enrichment of Firmicutes bacteria. Furthermore, we find that monoassociation of GF zebrafish with a representative Firmicutes strain induced increased LD number, whereas two other non-Firmicutes strains induced increased LD size. Although it remains unknown if these findings are generalizable to other members of their respective phyla, these data are consistent with our observations that the intestines of fed CONVD animals enriched with Firmicutes display increased LD number while all CONVD animals display increased LD size regardless of diet history. Based on these results, we propose two distinct mechanisms to explain the observed diet-dependent interactions between gut microbial ecology and host FA absorption. First, Firmicutes are enriched in the intestines of fed animals, where they enhance the ability of host enterocytes to absorb FAs. Second, non-Firmicutes bacteria that colonize the gut irrespective of dietary status induce increased accumulation of large LDs within host enterocytes. In animals that are fed, these two bacterial signals combine to stimulate FA absorption through increases in both enterocyte LD size and number () and increased export to extra-intestinal tissues.
Our results provide insight into the impact of diet on the zebrafish gut microbiota, and the relationship between the microbiota of the zebrafish gut and the surrounding water. A frequently observed pattern in humans, mice, and pythons is that the relative abundance of Firmicutes in the gut is positively correlated with dietary caloric intake. Our evaluation of gut bacterial communities in fed and starved zebrafish revealed that this ecological principle also applies to bony fishes. Strikingly, diet-dependent enrichment of Firmicutes bacteria in the gut but not in the surrounding water provides strong evidence that the presence of diet exerts different selective pressures on bacteria in the zebrafish gut versus the surrounding water. Future longitudinal analyses of the zebrafish gut and surrounding water could help resolve temporal relationships between the respective diet-induced alterations in microbial community assembly and maintenance in these different habitats.
The mechanisms that promote Firmicutes abundance in nutrient-rich environments remain unresolved. Our monoassociation results suggest that the diet-dependent enrichment of Firmicutes may be due, at least in part, to an autonomous bacterial requirement for diet-derived nutrients to allow colonization. In contrast, the two non-Firmicutes bacterial strains tested here colonized in both starved and fed conditions, suggesting that nutritional niches provided by the host are sufficient for these microorganisms to colonize the zebrafish gut. It will be interesting to determine if diet-dependent enrichment of Firmicutes in the context of a more complex microbial community is also mediated in part by inter-microbe competitions. Furthermore, it will be important to determine whether the impact of diet on bacterial colonization of the gut is mediated by permitting initial gut colonization and/or maintenance of colonization over time. Firmicutes-enriched communities arising from genetic or diet-induced obesity have been shown to promote positive energy balance (Turnbaugh et al., 2006
; Turnbaugh et al., 2008
). Therefore, identification of the mechanisms underlying diet-dependent enrichment of Firmicutes could lead to new approaches for controlling energy balance in humans and other animals.