Physiological And Metabolic Magnetic Resonance Studies

Environmental Health Sciences

Investigators

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Abstract

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: [unreadable] [unreadable] Project 1: Characterization of the interaction of borate with biological molecules is of fundamental importance for understanding the physiological and toxicological properties of boron, as well as for the development of boronate ligands. During the past year, we explored the extent to which borate can act as a phosphate mimic. Phosphate mimicry by oxyanions such as vanadate, molybdate, or tungstate have provided fundamental insight into the mechanisms of phosphotransfer, and also are important for understanding their toxicological and pharmacological properties. Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural analogy of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl and other ligands, suggested that phosphate-ester recognition sites on proteins might exhibit significant affinity for non-enzymatically formed borate esters. Boron-11 NMR studies and activity measurements on ribonuclease A in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A3-deoxycytidine-2-borate ternary complex that mimics the complex formed between RNase A and a 2-cytidine monophosphate (2CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2-deoxycytidine mixture, demonstrating the critical importance of the 2-hydroxyl group for complex formation. Titration of the ternary complex with 2-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant for RNase A by 2CMP. Although increased ternary complex concentration is observed with increasing pH, the concentration of ternary complex does not increase as rapidly as the concentrations of borate and borate monoesters in solution. This result indicates that the affinity of the enzyme for the borate ester decreases with increasing pH, similar to the pH dependence observed for the binding of phosphate ester inhibitors such as 2- or 3-CMP. RNase A binding of a cyclic cytidine-2,3-borate ester, which is a structural analog of the cytidine-2,3-cyclic phosphate substrate, could also be demonstrated. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters can support enzyme catalysis.[unreadable] [unreadable] [unreadable] Project 2. [unreadable] In order to study effects of gender on cardiovascular disease, we have utilized an isolated mouse heart model due to the availability of transgenic strains and to the size compatibility of the mouse heart model with available NMR probes. Our previous studies have suggested that the protection seen in females may be related to differences in substrate selection by the heart. We found that glucose-dependent inhibitory polypeptide (GIP) protected male hearts. GIP treatment also activated many genes associated with metabolism. Additionally, using 13C-labeled substrates, we found that the relative glucose/fatty acid precursor incorporation into the acetyl-CoA pool in males was lower than in females, and that GIP treatment abolished the difference seen between genders. We are now trying to measure the activity of pyruvate dehydrogenase (PDH). If there is a difference in substrate utilization, then we might expect this variation in substrate selection to occur through PDH activity. In another study, we looked at Thioredoxin-Interacting Protein (Txnip) knock out mice. Txnip is typically thought to be involved in redox regulation. Using 31P NMR, we monitored glucose uptake in the heart using the glucose analog 2-deoxyglucose. We found that the Txnip-KO mice exhibited dramatically increased glucose uptake compared to controls. This finding suggests that Txnip may play an important role in metabolic regulation of the heart. We also have looked at β-arrestin knock out mice. β-arrestin is a scaffolding protein involved in G-protein coupling signaling. We hypothesized that the loss of this protein could potentially disrupt the pathway through which the protective effect of preconditioning occurs. Instead, we found that protection from preconditioning was still present, though possibly at an attenuated level.