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: Biomechanical overload induces cardiac hypertrophy and heart failure, and reactive oxygen species (ROS) play a role in both processes. Thioredoxin-Interacting Protein (Txnip) is encoded by a mechanically-regulated gene that controls cell growth and apoptosis in part through interaction with the endogenous dithiol antioxidant thioredoxin. Here we show that Txnip is a critical regulator of the cardiac response to pressure overload. We generated inducible cardiomyocyte-specific and systemic Txnip-null mice (Txnip-KO) using Flp/frt and Cre/loxP technologies. Compared with littermate controls, Txnip-KO hearts had attenuated cardiac hypertrophy and preserved left ventricular (LV) contractile reserve through 4 weeks of pressure overload; however, the beneficial effects were not sustained and Txnip deletion ultimately led to maladaptive LV remodeling at 8 weeks of pressure overload. Interestingly, these effects of Txnip deletion on cardiac performance were not accompanied by global changes in thioredoxin activity or ROS. Phosphorus-31 NMR studies were used to follow the time-dependent phosphorylation of 2-deoxyglucose, and revealed that Txnip-KO hearts had a robust increase in myocardial glucose uptake. Thus, deletion of Txnip plays an unanticipated role in myocardial energy homeostasis rather than redox regulation. These results support the emerging concept that the function of Txnip is not as a simple thioredoxin inhibitor but as a metabolic control protein. (Circ Res. 2007;101:1328-1338.)? ? Project 2. Cu, Zn-superoxide dismutase (SOD1) is a copper and zinc-dependent enzyme whose main function is believed to be the scavenging and detoxification of superoxide radicals. Nevertheless, the last 10 years have seen a rapid accumulation of evidence indicating that SOD1 may also act as a peroxidase, an alternative function that has been implicated in the onset and progression of familial amyotrophic lateral sclerosis (FALS). Although SOD1 peroxidase activity and its dependence on bicarbonate have been well described, the molecular basis of the SOD1 peroxidase cycle remains obscure since none of the proposed catalytic intermediates have so far been identified. In view of recent observations, we hypothesized that the SOD1 peroxidase cycle relies on two steps: (1) reduction of SOD-Cu(II) by hydrogen peroxide followed by (2) oxidation of SOD-Cu(I) by peroxymonocarbonate, the product of the spontaneous reaction of bicarbonate with hydrogen peroxide, to produce SOD-Cu(II) and carbonate radical anion. This hypothesis has been investigated through electron paramagnetic resonance and nuclear magnetic resonance to provide direct evidence for a peroxycarbonate-driven, SOD1-catalyzed carbonate radical production. Preliminary results indicate that peroxymonocarbonate (HCO4-) is a key intermediate in the SOD1 peroxidase cycle and identify this species as the precursor of carbonate radical anions.? ? Project 3. The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between lactate and pyruvate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. Disruption of ldhc severely impairs fertility in male ldhc-/- mice. We have initiated studies of the metabolism of 1-13C-D-glucose in spermatozoa derived from LDHC-KO mice. Glucose uptake/metabolism was severely impaired in spermatozoa derived from the ldhc -/- mice, although they were able to utilize pyruvate. Several other labeled metabolites have been identified, including glycerol and acetate. We are currently studying extracts derived from these cells in order to further characterize the metabolic perturbation, and ultimately the basis for impaired fertility.
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