Fig 1.
Procedure for determining the rates of NAD+ synthesis and breakdown with the deuterium-labeled Nam.
(A) Cellular synthesis of labeled NAD+ and Nam from d4-Nam. The structures of d4-Nam, formed NAD+, and its breakdown product are shown together with their m/z values. Key enzymes in the metabolism of NAD+ are also shown. NMNAT, Nam mononucleotide adenylyltransferase; ADPR, ADP-ribose. (B) Experimental protocol for determination of the rates of NAD+ synthesis and breakdown using LC/MS/MS with d4-Nam. Cells were pre-incubated over night in the medium containing unlabeled Nam (d0-Nam) and then the medium was changed to that containing the same concentration of d4-Nam for the indicated times. After the incubation, NAD+ and Nam were extracted from cells and their incubation medium and were quantified by LC/MS/MS. (C) Definition of parameters representing the rates of NAD+ synthesis (RS) and breakdown (RB). RS and RB were calculated from the amounts of d3-NAD+ appeared (a) and d0-NAD+ decreased (b) during a given incubation time (t).
Fig 2.
Determination of the rates of NAD+ synthesis and breakdown with d4-Nam.
HepG2 cells (4.04 x 105 cells) were cultured in d0-Nam-free MEM supplemented with 2 μM d4-Nam for the indicated times. After the incubation, isotopomers of NAD+ in the cells and Nam in the culture medium were extracted, separated by HPLC, and quantified. Amounts of d3- (filled circles) and d0-NAD+ (open circles), and d3- (filled triangles) and d0-Nam (open triangles) are plotted, as a function of incubation times. The total amounts of NAD+ (crosses) consisting of d3- and d0-NAD+ are also shown.
Fig 3.
The effect of concentrations of d4-Nam in medium on RS.
Fao (A), HepG2 (B), and HeLa (C) cells were incubated with d0-Nam-free MEM supplemented with indicated concentrations of d4-Nam for 3 h. After the incubation, d3-NAD+ was quantified to determine RS (circles).
Fig 4.
Absolute values of NAD+ level, RS, RB, total Nampt activity in mammalian cells.
Fao, 293T, 293, HeLa, Caco-2, HepG2, C2C12, H9c2, and the primary cultured cardiomyocytes were incubated in d0-Nam-free MEM supplemented with 2 μM d4-Nam for 3 h. After the incubation, d3- and d0-NAD+ were quantified to determine RS (B) and RB (C) as well as total NAD+ content (D). Total Nampt activity was determined in cells incubated with 2 μM d0-Nam for 3 h (A). The suspended cells were counted using an automated cell counter, which also provides a readout of the approximate volume of the counted cells (E). Total Nampt activity, RS, RB, and total NAD+ content were normalized to cell volume to obtain absolute values (F-I). Half-life of cellular NAD+ (J) was calculated from RB and cellular NAD+ concentration. Data shown are results from 3, 3, 4, 3, 5, 3, 4, 5, and 3 independent experiments for Fao, 293T, 293, HeLa, Caco-2, HepG2, C2C12, H9c2, and the primary cultured cells, respectively. The smallest values within those obtained from Fao, 293T, 293, HeLa, Caco-2, and HepG2 cells in each panel were set to 1.0 and relative values are shown in parentheses.
Fig 5.
Relationship among total Nampt activity, RS, RB, cellular NAD+ concentration, and calculated NAD+ half-life.
Data in Fig 4 were re-plotted. (A) Relation of RS, RB, and the NAD+ concentration to total Nampt activity. (B) Relation between RS and RB. (C) Relation of the NAD+ concentration to RS or RB. (D) Relation of NAD+ half-life to RB or the NAD+ concentration. Determination coefficience (R2) is also indicated.
Fig 6.
The effects of induced expression of Nampt on RS, RB, cellular NAD+ concentration, and half-life of NAD+.
Nampt expression was induced in HeLa cells by incubating with 0.1, 0.2, 0.3, or 1.0 μg/mL of Dox. (A) Relation of RS, RB, and cellular NAD+ concentration to total Nampt activity. (B) Relation between RS and RB. (C) Relation of cellular NAD+ concentration to RS or RB. (D) Relation of NAD+ half-life to RB or the NAD+ concentration. Determination coefficience (R2) is also indicated.