Cell Life versus cell longevity: the mysteries surrounding the NAD+ precursor nicotinamide

Abstract

Nicotinamide, the amide form of niacin (vitamin B(3)), is the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD(+)) and plays a significant role during the enhancement of cell survival as well as cell longevity. Yet, these abilities of nicotinamide appear to be diametrically opposed. Here we describe the development of nicotinamide as a novel agent that is critical for modulating cellular metabolism, plasticity, longevity, and inflammatory microglial function as well as for influencing cellular life span. The capacity of nicotinamide to govern not only intrinsic cellular integrity, but also extrinsic cellular inflammation rests with the modulation of a host of cellular targets that involve mitochondrial membrane potential, poly(ADP-ribose) polymerase, protein kinase B (Akt), Forkhead transcription factors, Bad, caspases, and microglial activation. Further knowledge acquired in regards to the ability of nicotinamide to foster cellular survival and regulate cellular lifespan should significantly promote the development of therapies against a host of disorders, such as aging, Alzheimer's disease, diabetes, cerebral ischemia, Parkinson's disease, and cancer.

Fig. 1

Nicotinamide (NIC) prevents the loss of mitochondrial membrane potential during oxygen glucose deprivation (OGD). (A) Representative pictures demonstrate that exposure to a 3 hour period of OGD produced a significant decrease in the red/green fluorescence intensity ratio in cultured rat hippocampal neurons using a cationic membrane potential indicator JC-1 within 3 hours when compared with untreated control cultures, suggesting that OGD results in mitochondrial membrane depolarization. Application of NIC (12.5 mM) 1 hour prior to OGD exposure significantly increased the red/green fluorescence intensity of neurons, indicating that mitochondrial membrane potential was restored. (B) The relative ratio of red/green fluorescent intensity of mitochondrial staining in both untreated (control) neurons and neurons exposed to OGD or NIC (12.5 mM) plus OGD 3 hour following the initial insult was measured in 4 independent experiments with analysis performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) (Control vs. OGD, *p<0.01; OGD vs. NIC/OGD, †p<0.01).

Fig. 2

Nicotinamide (NIC) modulates a variety of cellular mediators to oversee cellular metabolism, longevity, survival, and inflammatory microglial activation. Nicotinamide promotes cellular function and survival through a series of pathways that involve NAD⁺, cell senescence mechanisms, the serine-threonine kinase Akt and its downstream substrates of FOXO3a, Bad, and caspases. Closely to the cytoprotection by nicotinamide is the maintenance of mitochondrial membrane potential, mitochondrial energy reserves, cytochrome c (Cyto c) release, and PARP. Targeting by NIC of specific caspase pathways ultimately serves to preserve genomic integrity and prevent early apoptotic injury “tagging” for microglial disposal. NAD: β-nicotinamide adenine dinucleotide; Sir2: Silent information regulator 2; MG: microglia; Mito: mitochondria; PS: phosphatidylserine.

Fig. 3

Nicotinamide (NIC) increases phosphorylation of p-FOXO3a through activation of Akt during oxidative stress. (A) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted at 6 hours following NIC (12.5 mM) treatment with anti - phospho-FOXO3a (p- FOXO3a) antibody. NIC increased the expression of p-FOXO3a significantly over a 6 hour period. Co-application of NIC with phosphoinositol-3 kinase inhibitor wortmannin (W, 500 μM), which inhibits phosphorylation of Akt, decreased p-FOXO3a expression during NIC application. (B) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted at 6 hours following oxygen glucose deprivation (OGD), NIC (12.5 mM), or NIC (12.5 mM) following a 3 hour period of OGD with anti - phospho-FOXO3a (p- FOXO3a) antibody. OGD alone increased the expression of p-FOXO3a. The expression of p-FOXO3a was further increased in neurons with NIC applied 1 hour prior to OGD. Yet, application of wortmannin (W) significantly decreased the expression of p-FOXO3a during the administration of NIC during OGD.