Mechanistic Study Of The Effects Of NAD~+ Administration On Energy Metabolism And Antioxidative Capacity

NAD~+is a coenzyme commonly found in all living cells and plays an important role in various biological processes,such as energy metabolism,immunological functions,mitochondrial functions and aging.Studies have shown that intracellular NAD~+levels decline during the aging process,which may lead to mitochondrial and nuclear dysfunction,resulting in aging-related disorders.Supplementation with NAD~+and its precursors,such as nictinamide and nicotinamide ribose,can dramatically improve aging-related functional defects,ameliorating age-related diseases,including neurodegenerative diseases.However,there has been relatively few studies on the mechanisms underlying the beneficial effects of NAD~+.For example:1)Energy metabolism is a key regulator of cell function and death,and NAD~+supplementation can attenuate cellular ATP depletion in multiple injury models.What is the exact mechanism underlying the effects of NAD~+on intracellular ATP?How does extracellular NAD~+enter the cells?2)Oxidative stress is considered to be a major culprit of many diseases,such as cancer,diabetes,neurodegenerative diseases and aging.NAD~+treatment has been reported to reduce reactive oxygen species in tissues and cells.What is the molecular mechanism underlying the effects of NAD~+on the antioxidant capacity of the cells?Studies on these issues will help reveal the molecular mechanisms of the protective effects of NAD~+and provide a theoretical basis for the clinical application of NAD~+.In order to solve the first problem,we used the immune cells in the brain-microglia as a cellular model,and extracellularly applied NAD~+.We found that extracellular application of NAD~+significantly increased intracellular and extracellular AMP,ADP and ATP levels,but did not change the intracellular AMP/ATP ratio,ADP/ATP ratio and NAD~+/NADH ratio.Moreover,NAD~+treatment did not affect the glycolytic rate and mitochondrial activity.Further mechanistic studies suggested that extracellular degradation to adenosine was the main mechanism by which NAD~+increases cellular adenylate pool:1)Extracellularly applied NAD~+significantly increased both intracellular and extracellular adenosine levels,and extracellularly applied adenosine also increased intracellular AMP,ADP and ATP levels;2)Dipyridamole(DPR),an inhibitor of ENT(equilibrative nucleoside transporters)-the main transporter by which extracellular adenosine is transported into the cells,blocked NAD~+-induced increases in intracellular AMP,ADP and ATP levels;3)Inhibition of adenosine kinase by 5-Iodotubercidin(5-ITU)effectively blocked NAD~+-induced increases in intracellular AMP,ADP and ATP levels.Based on these data,we explored the role of AMPK,a key energy sensor,in NAD~+-induced increase in ATP levels:1)Extracellular application of NAD~+significantly increased the phosphorylation of AMPK and its substrate Acetyl-Co A carboxylase(ACC);2)5-ITU blocked the NAD~+-induced increases in AMPK phosphorylation;3)Inhibitor and small interfering RNA(si RNA)of AMPK effectively blocked NAD~+-caused increase in intracellular ATP;4)Extracellular application of AMP also significantly increased the intracellular AMP,ADP and ATP levels,and increase AMPK activity without affecting AMP/ATP ratio.Based on these results,we revealed the mechanisms how NAD~+affected cellular enenergy levels:extracellularly applied NAD~+does not affect cellular ATP levels by improving glycolysis or oxidative phosphorylation,but by degradation extracellularly into adenosine,which enters the cells via ENT and is then rapidly converted into AMP by adenosine kinase,thus activating AMPK,resulting in enhanced ATP production.Our findings have also suggested that NAD~+administration in various disease and aging models may also produce its effects by affecting the microglia that are not under pathological insults.In the second part,we used the neurotoxic agent rotenone to treat PC12 cells to establish a cellular model of Parkinson's disease,and explored how NAD~+affected the antioxidant capacity of PC12 cells.A previous study from our lab has shown that extracellularly applied NAD~+can reduce rotenone-induced cell death of PC12 cells.In this study,we focused on the effects of NAD~+on the glutathione system,the main antioxidant system in neurons.We have obtained the following findings:1)Extracellularly applied NAD~+rescued the rise in reactive oxygen species and glutathione depletion caused by rotenone;2)NAD~+treatment increased the m RNA and protein levels of?-glutamylcysteine ligase(GCL),and increased Nrf2 expression and nuclear translocation;3)Inhibition and silencing of SIRT2,a NAD~+dependent deacetylase,effectively blocked NAD~+-caused increase in glutathione synthesis,GCL expression and Nrf2 expression;4)U0126,an inhibitor of the MEK/ERK pathway effectively reversed the increase in GSH,GSH/GSSG ratio,GCL expression,Nrf2 nuclear translocation and expression caused by NAD~+;5)NAD~+treatment increased ERK phosphorylation,which was blocked by SIRT2 inhibition and si RNA.Based on these results,we obtained a new insight into the mechanisms underlying the effects of NAD~+treatment on the antioxidant capacity of the cells:extracellularly applied NAD~+increases SIRT2 activity,leading to ERK activation and thus promoting Nrf2 nuclear translocation,resulting in enhanced GCL expression and glutathione synthesis.These findings are critical for understanding the protective effects of NAD~+against oxidative damage,which is essential for understanding the profound beneficial effects of NAD~+administration in numerous models of diseases and aging.In conclusion,the present project has investigated the effects and mechanisms of extracellular applied NAD~+on cellular energy metabolism and antioxidant capacity.These studies demonstrate that extracellularly applied NAD~+increases ATP synthesis by extracellular degradation to adenosine,and NAD~+activates Nrf2 by activating the SIRT2-ERK pathway,thereby regulating glutathione synthesis.These findings significantly advances our understanding of the role and mechanisms of the protective effects of NAD~+in neuroprotection and provide a theoretical basis for the clinical application of NAD~+.