Endoplasmic Reticulum Lipid Flux Influences Enterocyte Nuclear Morphology and Lipid-dependent Transcriptional Responses.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block ß-lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent ß-lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

NIAID's Comprehensive Support Mechanisms for Antibiotic Development.

The National Institute of Allergy and Infectious Diseases (NIAID) recognizes the continuing threat of antimicrobial resistance and the need to develop new therapeutics and strategies to combat multidrug resistant organisms. NIAID leverages multiple mechanisms to help support antibiotic developers struggling in the "valley of death" of preclinical antibiotic development. The Division of Microbiology and Infectious Diseases' (DMID) preclinical services are a comprehensive set of services to facilitate efforts to develop vaccines, diagnostics, and therapeutics for a broad array of bacterial, viral, fungal, and parasitic pathogens. These services are available to investigators worldwide at no charge.

Studying Lipid Metabolism and Transport During Zebrafish Development.

The zebrafish model facilitates the study of lipid metabolism and transport during development. Here, we outline methods to introduce traceable fluorescent or radiolabeled fatty acids into zebrafish embryos and larvae at various developmental stages. Labeled fatty acids can be injected into the large yolk cell prior to the development of digestive organs when the larvae is entirely dependent on the yolk for its nutrition (lecithotrophic state). Once zebrafish are able to consume exogenous food, labeled fatty acids can be incorporated into their food. Our group and others have demonstrated that the transport and processing of these injected or ingested fatty acid analogs can be followed through microscopy and/or biochemical analysis. These techniques can be easily combined with targeted antisense approaches, transgenics, or drug treatments (see Note 1 ), allowing studies of lipid cell biology and metabolism that are exceedingly difficult or impossible in mammals.

Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake.

Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4-6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and serves as a springboard for future investigations to elucidate their roles in development and disease in the larval zebrafish model.

We see the light: chemical-genetic protein regulation.

The challenge of studying complex protein networks in whole animals has driven the development of new methods for manipulating protein function with spatial and temporal precision. A novel combination of chemical and genetic protein regulation (Rodriguez and Wolfgang, in this issue of Chemistry & Biology) achieves levels of control that will revolutionize the study of protein function.