Reactivation of NAD(H) biosynthetic pathway by exogenous NAD⁺ in nil cells severely depleted of NAD(H)

Abstract

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm-MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150-200 pmoles/10(5) cells to less than 20 pmoles/10(5) cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3(0) NAm- cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One-hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)-biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.

Hexose transport derepressed and refractory to purine regulation in NAD(H)‐depleted Nil cells

“…Furthermore, growth of cells in the absence of nicotinamide or nicotinic acid results, first, in a dramatic reduction of of intracellular NAD(H) and NADP(H) and, second, in the cessation of cell division (Gardner et al, 1972;Jacobson et al, 1979). We have recently confirmed these findings using Nil hamster fibroblasts (Mandel et al, 1983).…”

“…Nil cells were grown in Eagle's (modified) MEM lacking nicotinamide, which we have termed NAm-MEM. The medium was prepared as described elsewhere (Mandel et al, 1983). NAm-MEM contained all vitamins (at their proper concentrations) normally present in MEM except for nicotinamide.…”

“…Tissue culture reagents, including antibiotics solution, essential and nonessential amino acids solutions, and calf serum, were purchased from Grand Island Biologicals. Prior to use calf serum was extensively dialyzed as described previously (Mandel et al, 1983). ADP-ribose, ATP, cytochalasin B, HEPES, 3-O-methylglucose, NADf, nicotinamide, and NMN were obtained from Sigma Chemical Company.…”

“…Growth of Nil cells in medium lacking nicotinamide results in the reported cessation of replication (Eagle, 1955;Swim and Parker, 1958;Ham, 1963;Sanford et al, 1963;Jacobson et al, 1979) after a number of cell divisions. In a n earlier publication (Mandel et al, 1983) the kinetics of growth of Nil cells through two subcultures in medium lacking pyridine were presented in detail. The extent of growth was found to be in part a function of the inoculum density, though the principal determinant of the time of cessation of cell division appears to be the level of pyridine nucleotide, estimated through determination of the NAD(H) content ( Fig.…”

Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells.

“…Alternatively, extracellular NAD+ could produce a transmembrane signal (11), causing stimulation of its biosynthesis, inhibition of its degradation, or both. Also, the increase could have resulted from extracellular NAD+ breakdown into membrane-permeable products reassembled into NAD+ after cellular uptake.…”