SUMMARY Regulation of intestinal dietary fat absorption is critical to maintaining energy balance. While intestinal microbiota clearly impact the host’s energy balance, their role in intestinal absorption and extra-intestinal metabolism of dietary fat is less clear. Using in vivo imaging of fluorescent fatty acid (FA) analogs delivered to gnotobiotic zebrafish hosts, we reveal that microbiota stimulate FA uptake and lipid droplet (LD) formation in the intestinal epithelium and liver. Microbiota increase epithelial LD number in a diet-dependent manner. The presence of food led to the intestinal enrichment of bacteria from the phylum Firmicutes. Diet-enriched Firmicutes and their products were sufficient to increase epithelial LD number, whereas LD size was increased by other bacterial types. Thus, different members of the intestinal microbiota promote FA absorption via distinct mechanisms. Diet-induced alterations in microbiota composition might influence fat absorption, providing mechanistic insight into how microbiota-diet interactions regulate host energy balance.

Lipids are essential for cellular function as sources of fuel, critical signaling molecules and membrane components. Deficiencies in lipid processing and transport underlie many metabolic diseases. To better understand metabolic function as it relates to disease etiology, a whole animal approach is advantageous, one in which multiple organs and cell types can be assessed simultaneously in vivo. Towards this end, we have developed an assay to visualize fatty acid (FA) metabolism in larval zebrafish (Danio rerio). The method utilizes egg yolk liposomes to deliver different chain length FA analogs (BODIPY-FL) to six day-old larvae. Following liposome incubation, larvae accumulate the analogs throughout their digestive organs, providing a comprehensive readout of organ structure and physiology. Using this assay we have observed that different chain length FAs are differentially transported and metabolized by the larval digestive system. We show that this assay can also reveal structural and metabolic defects in digestive mutants. Because this labeling technique can be used to investigate digestive organ morphology and function, we foresee its application in diverse studies of organ development and physiology.

Lipids serve essential functions in cells as signaling molecules, membrane components, and sources of energy. Defects in lipid metabolism are implicated in a number of pandemic human diseases, including diabetes, obesity, and hypercholesterolemia. approaches for disease prevention and treatment. Numerous studies have shown that the zebrafish is an excellent model for vertebrate lipid metabolism. In this chapter, we review studies that employ zebrafish to better understand lipid signaling and metabolism.

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.

The human monocytic leukemia zinc finger (MOZ) protein is an essential transcriptional coactivator and histone acetyltransferase (HAT) that plays a primary role in the differentiation of erythroid and myeloid cells and is required to maintain hematopoietic stem cells. Chromosomal translocations involving the HAT-encoded region are also associated with acute myeloid leukemia. Here we present the x-ray crystal structure of the MOZ HAT domain and related biochemical studies. We find that the HAT domain contains a central region that is structurally and functionally conserved with the yeast MYST HAT protein Esa1, but contains more divergent N-and C-terminal regions harboring a TFIIIA-type zinc finger and helix-turn-helix DNA-binding motifs. Solution DNA-binding and acetyltransferase activity assays, in concert with mutagenesis, confirm that the MOZ HAT domain binds strongly to DNA through the zinc finger and helix-turn-helix motifs and that DNA binding and catalysis are not mutually exclusive. Consistent with the DNA-binding properties of MOZ, we also show that MOZ is able to acetylate nucleosomes and free histones equally well, whereas other HATs prefer free histones. Our results reveal, for the first time, that enzymatic and DNA-targeting activities can be contained within the same chromatin regulatory domain.The eukaryotic genome is packaged into chromatin, the highly organized DNA⅐protein complex that not only serves as a structural element in preserving genetic information but also as a dynamic scaffold from which nuclear processes occur such as transcription, replication, DNA repair, mitosis, and apoptosis (1, 2). The fundamental unit of chromatin is the nucleosome, consisting of 145-147 bp of DNA wrapped around an octameric histone core containing two molecules each of histone proteins H2A, H2B, H3, and H4. There are at least four types of protein domains that regulate DNA processes through chromatin modification. These include (a) enzymatic domains that either use ATP to translocate the DNA relative to the histone core proteins (3) or post-translationally modify the histone proteins (4, 5) and (b) non-enzymatic domains that recognize chromatin, either through interactions with unmodified or modified N-terminal histone tails or the DNA, or histone chaperone proteins that deposit histones or replace variant histones into chromatin. Many chromatin regulatory proteins often contain both an enzymatic and chromatin recognition domain, although, to date, there have been no reports of a single domain harboring both activities.Chromatin recognition domains that target histones include bromodomains (6, 7), which recognize specific acetyllysine modifications, chromodomains (8, 9), and tudor (10) domains, which bind specific methyllysine modifications, 14-3-3 domains, which recognize phosphoserine modifications (11), and SANT domains (12), which recognize unmodified histones. The SLIDE (12) and SWIRM (13, 14) domains are chromatin recognition modules that can target the DNA within nucleosomes.Among the enzymes that medi...

The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys 16 to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a "primitive" model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-Å crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional "bent helix" proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.Sulfolobus solfataricus, a thermoacidophile, is a member of the archaeal domain of life, and is likely to have diverged from bacteria and eukaryotes early during evolution. Despite its lack of a nucleus or other organelles, archaeal DNA replication and chromatin regulation seem to more closely resemble eukaryotes than bacteria (1, 2). Sulfolobus belongs to the phylum Crenarchaeota, which lacks histones, and instead uses two analogous chromatin proteins: Sul7d and ALBA 3 (acetylation lowers binding affinity). Both proteins have been shown to undergo post-translational modification in Sulfolobus. Sul7d is monomethylated (3) and ALBA is acetylated (4, 5). The acetylation of ALBA by protein acetyltransferase (PAT) on Lys 16 has been shown to reduce DNA-binding affinity, and deacetylation of ALBA by archaeal Sir2 deacetylase has been shown to repress transcription in what appears to be a primitive form of chromatin regulation by reversible post-translational modification (4, 5). PAT is also likely to regulate other proteins in Sulfolobus. Based on its homology to PAT from Salmonella enterica, PAT from Sulfolobus may also play a role in metabolism by regulating the activity of acetyl-coenzyme A synthetase (6).There are at least four families of histone acetyltransferases (HATs) in eukaryotes: the Gcn5/PCAF family that also shows sequence and structural homology to the GNAT (Gcn5-related acetyltransferase) superfamily, which includes many small molecule acetyltransferases such as antibiotic acetyltransferases (aminoglycoside N-acetyltransferases) and serotonin N-acetyltransferase; the MYST family, named from the founding members of MOZ, Ybf2/Sas3, Sas2, and Tip60; the metazoan-specific transcriptional coactivators p300 and CREB-binding protein; and the recently characterized fungal-s...

Many fundamental questions remain regarding the cellular and molecular mechanisms of digestive lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in mice, yet each approach possesses certain limitations. The zebrafish (Danio rerio) offers an excellent model system in which to study lipid metabolism in vivo, owing to its small size, genetic tractability and optical clarity. Fluorescent lipid dyes and optical reporters of lipid-modifying enzymes are now being used in live zebrafish to generate visible readouts of digestive physiology. Here we review recent advances in visualizing intestinal lipid metabolism in live larval zebrafish.

Lipids serve essential functions in cells as signaling molecules, membrane components, and sources of energy. Defects in lipid metabolism are implicated in a number of pandemic human diseases, including diabetes, obesity, and hypercholesterolemia. Many aspects of how fatty acids and cholesterol are absorbed and processed by intestinal cells remain unclear and present a hurdle to developing approaches for disease prevention and treatment. Numerous studies have shown that the zebrafish is an excellent model for vertebrate lipid metabolism. In this chapter, we review commercially available fluorescent lipids that can be deployed in live zebrafish to better understand lipid signaling and metabolism. In this chapter, we present criteria one should consider when selecting specific fluorescent lipids for the study of digestive physiology or lipid metabolism in larval zebrafish.