This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1. Metabolic perturbations of lactate dehydrogenase C knockout. Sperm produce high levels of ATP, 70% of which is utilized by axonemal dynein to drive sperm motility. Glycolysis was shown to be essential for ATP production in sperm by using gene targeting to disrupt some of these genes. The gene for the glycolytic enzyme lactate dehydrogenase C (Ldhc) also is expressed predominantly in spermatogenic cells, and LDHC is abundant in sperm. We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect. Project 2. Characterization of inositol phosphate metabolism by NMR. Inositol 1,4,5-trisphosphate (IP3) is the intracellular messenger that links receptor activation to Ca ion mobilization, and has been intensely studied over several decades. The physiological roles of other inositol phosphates have received less attention, but have been of increasing interest in recent years as additional physiological roles have been identified. In general, the inositol phosphates can have multiple positional isomers. These can be distinguished by NMR, but are more difficult to identify using other methods. We have recently worked with the Shears group (LST) in order to identify inositol phosphate metabolites formed under various conditions.

Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2013
Total Cost
$356,687
Indirect Cost
City
State
Country
Zip Code
Wang, Huanchen; DeRose, Eugene F; London, Robert E et al. (2014) IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family. Nat Commun 5:4178
Odet, Fanny; Gabel, Scott; London, Robert E et al. (2013) Glycolysis and mitochondrial respiration in mouse LDHC-null sperm. Biol Reprod 88:95
Bonini, Marcelo G; Gabel, Scott A; Ranguelova, Kalina et al. (2009) Direct magnetic resonance evidence for peroxymonocarbonate involvement in the cu,zn-superoxide dismutase peroxidase catalytic cycle. J Biol Chem 284:14618-27
Gabel, Scott A; London, Robert E (2008) Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry. J Biol Inorg Chem 13:207-17