Open Access
Vrablik, Tracy Lynn
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
February 21, 2011
Committee Members:
  • Wendy Hanna Rose, Dissertation Advisor
  • Wendy Hanna Rose, Committee Chair
  • Melissa Rolls, Committee Member
  • Zhi Chun Lai, Committee Member
  • Troy Ott, Committee Member
  • Squire J Booker, Committee Member
  • NAD+ metabolism
  • niacin
  • development
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. While NAD+ biosynthesis has been intensely studied, little analysis has been done in developmental models. I have uncovered novel developmental roles for a nicotinamidase, the first enzyme in the NAD+ salvage pathway of invertebrates. Nicotinamidase activity hydrolyzes nicotinamide (NAM) and shuttles it into the NAD+ salvage pathway, thereby lowering NAM levels and replenishing levels of NAD+. Mutations in the C. 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. Through pharmacological analysis of the reproductive defects I determined that regulation of both substrate (NAM) and product (NA/NAD+) level is key to the biological activity of PNC-1. Interestingly, these metabolites function separately, as exogenous NAM in wild-type animals induces the uv1 cell necrosis and egg-laying defects, but not the gonad timing defect. Gonad timing in pnc-1 mutants is rescued by restoring NAD+ salvage. I propose that our C. elegans model is highly relevant to vertebrates. I demonstrate that nicotinamide phosphoribosyltransferase (Nampt), the equivalent enzyme in nicotinamide recycling to NAD+ in vertebrates, can functionally substitute for PNC-1. Nampt is secreted and I identified a secreted isoform of PNC-1 and confirmed its extracellular localization and function in vivo, suggesting a conserved role for extracellular NAD+ synthesis. The NAM-NAD+ salvage pathway is an important myogenic regulator in vertebrates and I uncovered a similar role in muscle development and function in our C. elegans model. C. elegans pnc-1 males have a spicule muscle defect, which prevents them from successfully mating. In collaboration with my colleague Wenqing Wang, we demonstrate muscle-cell-type specificity to perturbations of NAD production and/or NAM levels. In male spicule muscles the role of PNC-1 is to promote NAD+ biosynthesis during development, while in vulval muscles PNC-1 prevents accumulation of NAM. Therefore I postulate an evolutionarily conserved role for NAM to NAD+ salvage pathway modulation of NAM and NAD+ levels in the developmental regulation of multiple tissues. To test our model of metabolite function in development, I quantified the physiological levels of NAM and NAD+ in C. elegans and how these levels respond to genetic and pharmacological perturbations. I found that physiological levels of NAM were elevated in pnc-1 mutants as expected, however NAD+ levels were resistant to perturbation. I hypothesize that organisms maintain NAD+ levels via endocrine or other regulatory mechanisms that modulate NAD+ synthesis and utilization. To provide phylogenetic context to these metabolite levels, I quantified the levels of these metabolites in multiple systems. Organisms that employ nicotinamidases have lower physiological levels of NAM than tissues and cells that salvage NAM via Nampt. To better understand this phenomenon, I investigated the steady state kinetics of C. elegans nicotinamidases and found that the Km and kcat are similar to the yeast Pnc1p. As Nampt is reported to have a much lower kcat than nicotinamidases, I propose the differences in NAM levels result from the catalytic rates of these NAD+ salvage enzyme. In summary, this work has established a new metazoan model to investigate the intricacies of NAD+ salvage regulation of metabolites on higher biological processes. I have demonstrated that this system is likely relevant to vertebrates and amenable to a wide variety of biochemical and genetic analysis. This characterization of pnc-1 in development has given surprising insight into how NAD+ metabolism, through modulation of NAM and NAD+, regulates broader physiological processes.