Circadian tracking of nicotinamide cofactor levels in an immortalized suprachiasmatic nucleus cell line.

摘要

Nicotinamide adenine dinucleotides can exhibit a daily rhythm in plants and regulate the activity of mammalian clock-like transcription factors in vitro. Because one such redox-sensitive transcription factor is present in the master circadian clock of the brain (the suprachiasmatic nuclei, SCN) and the SCN exhibits a characteristic daily rhythm in glucose usage, nicotinamide cofactors might be expected to influence, exhibit, and/or reflect biological rhythms in SCN cells. Therefore, cofactors were extracted from a model SCN cell line at 3 h intervals over 1-2 day periods and samples were analyzed by capillary electrophoresis with multiphoton excitation of fluorescence. Natively fluorescent reduced cofactors (nicotinamide adenine dinucleotide, NADH, and its phosphorylated form, NADPH) were assayed directly, and nonfluorescent oxidized cofactors (nicotinamide adenine dinucleotide, NAD, and its phosphorylated form, NADP) were enzymatically reduced to their fluorescent counterparts before analysis. In the first day after a synchronizing pulse of fetal bovine serum, a dramatic upregulation in cellular NADH content was observed, consistent with a response to serum insulin; this was accompanied by a smaller decrease in NADPH redox state, which may indicate scavenging of reactive oxygen species generated by increased cellular metabolism. However, when cells were investigated after these early phenomena had recovered or stabilized, no circadian NAD(P)(H) rhythms were observed. During these studies, the NADH/NAD(H) concentration ratio in SCN2.2 cells (0.13+/-0.03) was not high enough to activate clock-like transcription factors. Although the NADPH/NADP(H) concentration ratio was more appropriate (0.8+/-0.1), the intracellular NADPH concentration was < or = 0.7 mM, far too low for half-maximal DNA binding of clock-like transcription factors in vitro. Moreover, these concentration and ratio values represent cellular averages, and free cofactors should be much lower in the cell nucleus. Our data show that SCN2.2 cells maintain nearly constant circadian NAD(P)(H) levels, and that the previously reported in vitro relationship between clock-like transcription factors and NAD(P)(H) does not appear to be biologically relevant.