Emerging roles for specific fatty acids in developmental processes.

Animals synthesize a vast range of fatty acids serving diverse cellular functions. The roles of specific fatty acids in early development are just beginning to be characterized. In the March 15, 2012, issue of Genes & Development, Kniazeva and colleagues (pp. 554-566) describe how the particular combination of a branched chain fatty acid and an acyl-CoA synthetase is required for critical cellular processes during early embryogenesis in Caenorhabditis elegans.

Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein.

Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified Caenorhabditis elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols is rich in fatty acid species obtained from the dietary Escherichia coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a very similar proteome in both strains, except that the most abundant protein in the C. elegans lipid droplet proteome, MDT-28, is relatively less abundant in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans. Finally, we confirmed the localization of one of the newly identified lipid droplet proteins, ACS-4. We found that ACS-4 localizes to the surface of lipid droplets in the C. elegans intestine and skin. This study bolsters C. elegans as a model to study the dynamics and functions of lipid droplets in a multicellular organism.

Polyunsaturated fatty acid derived signaling in reproduction and development: insights from Caenorhabditis elegans and Drosophila melanogaster.

Polyunsaturated fatty acids (PUFAs) exhibit a diverse range of critical functions in biological systems. PUFAs modulate the biophysical properties of membranes and, along with their derivatives, the eicosanoids and endocannabinoids, form a wide array potent lipid signaling molecules. Much of our early understanding of PUFAs and PUFA-derived signaling stems from work in mammals; however, technological advances have made comprehensive lipid analysis possible in small genetic models such as Caenorhabditis elegans and Drosophila melanogaster. These models have a number of advantages, such as simple anatomy and genome-wide genetic screening techniques, which can broaden our understanding of fatty-acid-derived signaling in biological systems. Here we review what is known about PUFAs, eicosanoids, and endocannabinoids in the development and reproduction of C. elegans and D. melanogaster. Fatty acid signaling appears to be fundamental for multicellular organisms, and simple invertebrates often employ functionally similar pathways. In particular, studies in C. elegans and Drosophila are providing insight into the roles of PUFAs and PUFA-derived signaling in early developmental processes, such as meiosis, fertilization, and early embryonic cleavage.

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans.

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD(+) after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD(+) salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD(+) salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD(+) salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD(+) production or NAM levels. Active NAD(+) biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD(+) salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD(+) biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types.

Nicotinamidase modulation of NAD+ biosynthesis and nicotinamide levels separately affect reproductive development and cell survival in C. elegans.

Nicotinamide adenine dinucleotide (NAD(+)) is a central molecule in cellular metabolism and an obligate co-substrate for NAD(+)-consuming enzymes, which regulate key biological processes such as longevity and stress responses. Although NAD(+) biosynthesis has been intensely studied, little analysis has been done in developmental models. We have uncovered novel developmental roles for a nicotinamidase (PNC), the first enzyme in the NAD(+) salvage pathway of invertebrates. Mutations in the Caenorhabditis elegans nicotinamidase PNC-1 cause developmental and functional defects in the reproductive system; the development of the gonad is delayed, four uterine cells die by necrosis and the mutant animals are egg-laying defective. The temporal delay in gonad development results from depletion of the salvage pathway product NAD(+), whereas the uv1 cell necrosis and egg-laying defects result from accumulation of the substrate nicotinamide. Thus, regulation of both substrate and product level is key to the biological activity of PNC-1. We also find that diet probably affects the levels of these metabolites, as it affects phenotypes. Finally, we identified a secreted isoform of PNC-1 and confirmed its extracellular localization and functional activity in vivo. We demonstrate that nicotinamide phosphoribosyltransferase (Nampt), the equivalent enzyme in nicotinamide recycling to NAD(+) in vertebrates, can functionally substitute for PNC-1. As Nampt is also secreted, we postulate an evolutionarily conserved extracellular role for NAD(+) biosynthetic enzymes during development and physiology.

Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 µM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid (18)O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.

Dietary supplementation of polyunsaturated fatty acids in Caenorhabditis elegans.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acid sodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.