Aging and CR effects on the Plasma Membrane Redox System

National Institute On Aging

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Abstract

Data from our laboratory and others have demonstrated that the plasma membrane redox system (PMRS) is implicated in the maintenance of the antioxidant capacity during oxidative stress challenges induced by diet and in aging. The PMRS upregulation that occurs during caloric restriction (CR) decreases the levels of oxidative stress in aged membranes. CR extends life span of yeasts by decreasing NADH levels, which would connect this intervention to plasma membrane NADH-dependent dehydrogenases. CR modifies composition of fatty acid in the plasma membrane, resulting in decreased lipid peroxidation and related oxidative adducts. More importantly, the content of CoQ at the plasma membrane, which also declines with age, is enhanced by CR and when combined with PMRS activity provides protection to phospholipids and prevents/lowers the production of lipid peroxidation products. We are focusing our efforts on the generation of transgenic and knock out animals of the different dehydrogenases involved in this antioxidant system. We have successfully created NQO1 and cytochrome b5 reductase 3 (Cytb5r3) transgenic mice that overexpress each enzyme alone or combined. Moreover, we generated NQO1 and NRF2 KO animals. Longevity studies as well as short term interventions have been performed over the years to fully characterize these new mouse lines. The following section summarizes the key findings. A. In collaboration with the laboratory of Dr. Placido Navas we have shown that the Saccharomyces cerevisiae homolog of Cyt b5 reductase, NQR1, resides at the plasma membrane and when overexpressed extends both replicative and chronological lifespan. We demonstrated that NQR1 extends replicative lifespan in a sirtuin (SIR2)-dependent manner by shifting cells towards respiratory metabolism and decreasing the pyridine nucleotide pool without altering the NAD+/NADH ratio. Chronological lifespan extension, in contrast, occurs via a SIR2-independent decrease in ethanol production. We conclude that NQR1 is a key mediator of lifespan extension by CR through its effects on yeast metabolism and discuss how these findings could suggest a function for this protein in lifespan extension in mammals. B. Using global NQO1 overexpression model, we showed a shift in the physiology of mice on a high-fat diet towards that of mice on a standard diet, a phenomenon that was associated with the NQO1-mediated reduction in oxidative stress damage and prevention of the activation of the proinflammatory NF-B pathway. More recently, we reported another emerging role of NQO1 as an mRNA-binding protein. Using ribonucleoprotein immunoprecipitation (RIP) assays and microarray analysis, we identified a subset of mRNA targets of NQO1 in human hepatoma HepG2 cells. SERPINA1 mRNA, which encodes the serine protease inhibitor -1-antitrypsin A1AT, is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis, and hepatocellular carcinoma. Our results showed that although NQO1 can bind the 3-untranslated region (UTR) and the coding region of SERPINA1 mRNA, it did not affect SERPINA1 mRNA levels, but instead it enhanced the translation of SERPINA1 mRNA. We propose that this novel mechanism of action of NQO1 as RNA-binding protein may help to explain the pleiotropic biological effects of this enzyme. C. Chronic nutrient excess leads to metabolic disorders and insulin resistance, and activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. To ascertain whether the selective targeting of NQO1 leads to improvements in diet-induced metabolic dysfunction, we used genetic mouse models to explore the role of Nrf2 and its target gene NQO1 and showed that NQO1 was protective against diet-induced obesity (DIO) through the regulation of insulin sensitivity and lipid metabolism. The integration of physiological data, histological changes, and omics profiles indicates that under nutritional excess, NQO1 transgenesis confers healthful benefits through preservation of glucose homeostasis, insulin sensitivity, and lipid handling leading to enhanced health outcomes. D. NQO1, also known as NAD(P)H quinone oxidoreductase 1, is a multifunctional enzyme involved in cellular responses to oxidative stress, redox regulation, and in aging. Emerging roles of NQO1 include protection against diabetes and metabolic syndrome, enhanced generation of NAD+, reduced overall protein degradation, and binding of mRNA. NQO1 functions as an efficient intracellular generator of NAD+, an essential cofactor for NAD+-dependent enzymes, including members of the sirtuin (SIRT) deacetylase family and PARP involved in DNA repair, transcription, and modulation of chromatin structure. Studies from our lab have shown that NQO1 overexpression improves glucose and lipid metabolism and insulin sensitivity in high-fat diet-fed mice, suggesting a possible link between NQO1 expression and glucose metabolism in adipocytes, and is therefore seen as a possible target to combat obesity. Despite these advances, additional work is required to establish a relationship between NQO1 and activation of downstream targets implicated in adipogenesis. Using the will-established cellular model of adipocyte differentiation, we are examining the impact of NQO1 in modulating glucose metabolism during 3T3-L1 cell adipogenesis. Establishing the relationship between NQO1 and glucose metabolism shall provide insights on using NQO1 modulators as a tool to understand and counteract metabolic disorders and obesity-related diseases. We observed a reduction in NQO1 protein expression in the early stage of 3T3-L1 adipocyte differentiation that was accompanied by dynamic changes in the expression of SIRT 1-7 family members with concomitant temporal changes in the steady-state acetylation levels of target histone and nonhistone proteins. Pharmacological and siRNA-mediated reduction in NQO1 level had a negative impact on SIRT-dependent deacetylation of downstream targets and possibly their stability. We are currently investigating the relationship between NQO1 and cellular bioenergetics during adipogenesis using the Seahorse Analyzer platform. Plans are underway to assess the impact of inducible, tissue specific overexpression and knockout of NQO1 in transgenic mice maintained in our animal facility and their response to an obesogenic diet vis--vis body composition, glucose homeostasis, insulin sensitivity, and lipid handling through modulation of fat depot-specific signaling.