/. Embryol. exp. Morph. Vol. 33, 4, pp. 947-956, 1975
Printed in Great Britain
947
Nicotinamide adenine dinucleotide
levels in chick limb mesodermal cells in vitro:
effects of 3-acetylpyridine and nicotinamide
By MARTIN J. ROSENBERG 1 AND ARNOLD I. CAPLAN 1
From the Department of Biology,
Case Western Reserve University
SUMMARY
The studies reported here show that in cultures of developing mesodermal cells, chondrogenic expression is associated with a progressive decline in cellular NAD+ levels. Furthermore,
reduced cellular NAD + levels resulting from exposure to the nicotinamide analog 3-acetylpyridine are correlated with a 2- to 100-fold potentiation of chondrogenic expression. Conversely, elevated NAD + levels resulting from exposure to nicotinamide alone are correlated
with inhibition of chondrogenic expression. These data are consistent with the hypothesis
that pyridine nucleotides, or some derivative thereof, play a central role in the control of
muscle and cartilage development in embryonic chick limbs.
INTRODUCTION
Prior to stage 25, limb mesodermal cells have the capacity to develop into
either muscle or cartilage cells (Searls, 1965; Zwilling, 1968; Searls & Janners,
1969). Nicotinamide and/or pyridine nucleotide levels seem to be implicated in
the control processes governing whether myogenic or chondrogenic characteristics are expressed (Landauer, 1957; Caplan, Zwilling & Kaplan, 1968; Caplan,
1970, 1971, 1972 a, b, c, d; Rosenberg & Caplan, 1974). A previous report from
this laboratory (Rosenberg & Caplan, 1974) has shown that NAD+ levels in
chick limbs are quite low during the early, chondrogenic, phases of development,
while the NAD+ levels in the limb are quite high during the later, myogenic,
phases of development. We have also shown that limb mesodermal cells in
culture seem to mimic in vivo development, both morphologically and on the
basis of NAD+ levels. Furthermore, these mesodermal cells, in culture, can be
persuaded to express only the chondrogenic phenotype, with the elimination
of muscle cells, if they are exposed to the nicotinamide analog 3-acetylpyridine.
Under these conditions, 3-acetylpyridine has been shown to have no effect on
the rates of incorporation of exogenously added precursors of DNA, RNA or
1
Authors' address: Department of Biology, Case Western Reserve University, Cleveland,
Ohio 44106, U.S.A.
59
EMB
33
948
M. J. ROSENBERG AND A. I. CAPLAN
protein, while nicotinamide incorporation into pyridine nucleotides is lowered
by a factor of 5 to 10.
Quantitative evaluation of the NAD+ levels in cultured mesodermal cells,
in the presence and absence of 3-acetylpyridine or nicotinamide, is presented
here. The results of these experiments add support to, but do not prove, the
general hypothesis that pyridine nucleotide levels play a role in the differential
expression of chondrogenic and myogenic characteristics.
MATERIALS AND METHODS
Cells were obtained from stage-24 limb-buds as previously described (Caplan,
1970,19726). The cells were plated at a density of 107 cells/60 mm plastic culture
dish, except where otherwise indicated. Continuous exposure of the cultures to
3-acetylpyridine was initiated 24 h after plating; 4mg of 3-acetylpyridine, in
a volume of 0-1 ml of Tyrodes, was added to the culture dishes following the
daily change of medium. Control cultures were prepared from the same limb
mesodermal cell suspension and had 0-1 ml Tyrodes added to them. Analysis
for protein, DNA and NAD+ was exactly as described previously (Rosenberg
& Caplan, 1974). Appropriate controls were conducted with these analyses to
document that the presence of 3-acetylpyridine or nicotinamide did not affect
the recovery or accuracy of such analysis.
Total acid mucopolysaccharide in the cell cultures was estimated by a quantitative toluidine blue staining procedure described by Coleman, Coleman,
Kankel & Werner (1970), as modified from the method described by Schacter
(1970). Optical density at 625 nm, as read on a Zeiss PMQII spectrophotometer,
is linearly related to chondroitin sulfate levels.
RESULTS
Effects of 3-acetylpyridine
The morphological and biochemical parameters characteristic of limb mesodermal cultures, in both the presence and absence of 3-acetylpyridine, have been
described previously. In brief, untreated cultures exhibit areas of chondrogenic
activity by the third day of culture life. These areas have a distinctive morphology
in that they are bounded by a border of 'perichondrial-like' oblong cells, and
secrete copious amounts of acid mucopolysaccharide (Caplan, 1970, 19726).
When plated at a density of 107 cells/60 mm plate, these nodular areas cover
approximately 10 % of the area of the dish, beginning at the periphery of the
dish, and proceeding inward during the remaining period of culture life. Continuous exposure to 3-acetylpyridine, starting on day 2 of culture life, results
in a loss of about 20 to 30 % of the cells, without an impairment of the rates
of RNA, DNA or protein synthesis. The cells eliminated from culture are
presumably in part myogenic cells (Caplan & Stoolmiller, 1973).
949
NAD in developing chick limbs
25 -
20
Q
Control
'5
10
3AP
o
10
12
Days in culture
Fig. 1. Effect of continuous exposure of 3-acetylpyridine on total NAD+ in cultures
of limb mesodermal cells. Mesodermal cells were obtained from the limb-buds of
stage 23-24 chicks by a combination of trypsinization and mechanical agitation and
were plated at a density of 107 cells/60 mm plate. Culture medium was changed
daily (Caplan, 1972a). Four mg of 3-acetylpyridine, in a volume of 01 ml of sterile
Tyrode solution, was added to the experimental plates 24 h after plating, and was
added whenever the medium was changed. After the indicated periods of culture
growth, the cells from duplicate cultures were scraped in glass-distilled water,
homogenized, and analysed for protein, DNA and NAD+ as described in the text.
The standard deviation was as pictured in Rosenberg & Caplan (1974) and was
approximately equal to the diameter of the circles surrounding each data point.
As seen in Fig. 1, the NAD+ content of mesodermal cell cultures plated at an
initial density of 107 cells/60 mm culture dish increased almost linearly from
day 2 to day 12 of culture life; the control values (Figs. 1, 2, 3) have been
published previously (Rosenberg & Caplan, 1974) and are provided here for
comparison. In the first 24-h period following the addition of 3-acetylpyridine,
however, the NAD+ content of each plate was reduced by a factor of 100. At
no point during the entire culture period did the NAD+ content of 3-acetylpyridine-treated plates assume a value which was greater than 10 % of the
control NAD+ values. On the other hand, while the NAD+ content of untreated cultures increased by 50 % during the 12-day growth period, the NAD+
content of 3-acetylpyridine-treated cultures increased by a factor of eighteen.
A significant increase in the amount of NAD+ is observed, therefore, even in
the presence of 3-acetylpyridine. This suggests that treated cells retain the
capacity to synthesize and/or store NAD+.
When the NAD+ content of untreated mesodermal cultures is related to the
protein content of the cultures, and plotted as a function of days in culture
(Fig. 2), the NAD+ concentration is at its highest level, 11 nmols/mg protein,
59-2
950
M. J. ROSENBERG AND A. I. CAPLAN
12
10
I6
Control
i 4
3AP
-o
o - -
<?
10
12
Days in culture
Fig. 2. Effect of exposure of 3-acetylpyridine on NAD + concentration per mg
protein in cultures of limb mesodermal cells. See Fig. 1 for details.
on day 2 of culture life. During the next two days, there is a. 21 % decrease in
this level, which continues to decline during the next eight days, but at a slower
rate. By day 12, the NAD+ concentration falls to a level of 4 nmols/mg protein.
It is significant that the period between day 2 and day 4, when the NAD+ levels
are high, represents a period when myogenic cells are clearly discernible. The
following eight days, however, when the NAD+ levels are progressively decreasing, are characterized by cellular expression which is predominantly, if
not exclusively, chondrogenic in nature (Caplan, 1970; Schachter, 1970).
Cell cultures which have been treated with 3-acetylpyridine do not exhibit the
same trends of NAD+ concentration which have been seen with untreated cultures. In the untreated cultures, the NAD+ level falls almost 65 % during the
12-day period; in the 3-acetylpyridine-treated cultures, the NAD+ level increases
by a factor of eight during the same period. However, even after 12 days, the
NAD + level in 3-acetylpyridine-exposed cultures is only 25 % of that found in
control cultures (Fig. 2).
When plotted on the basis of DNA levels (Fig. 3), the NAD+ level in untreated
cultures falls from 374 pmols//tg DNA, on day 2, to 206 pmols//*g DNA, on
day 12. This decline is marked by three consecutive and overlapping phases:
from day 2 to day 4 there is a rapid decline, from 375 to 320 pmols//*g DNA.
From day 4 to day 8, the NAD+ concentration remains constant, at about
300pmols//*g DNA. During the next four days another decline is seen, the
NAD in developing chick limbs
951
400 -
Control
300
D 200
100
3AP
10
12
Days in culture
Fig. 3. Effect of exposure of 3-acetylpyridine on intracellular NAD+ concentration
(per /tg DNA) in cultures of limb mesodermal cells. See Fig. 1 for details.
final concentration on day 12 being 206 pmols//£g DNA. These data can be
related to the rate of cellular replication (Caplan & Stoolmiller, 1973): during
the initial period of rapid NAD+ decline, cell replication is occurring at a rapid
rate; during the middle, plateau period of NAD+ concentration, the number of
cells on the culture dish is increasing, but at a greatly reduced rate; and during
the last phase, when NAD+ levels once again begin to decline, the rate of cellular
replication increases at a steady rate which approaches that seen during the
first phase. Thus the curve generated by plotting NAD+ concentration in terms
of DNA levels is almost a mirror image of the 'DNA per plate' curve or 'cell
number per day in culture' curve.
When mesodermal cell cultures are exposed to 3-acetylpyridine, the intracellular concentration of NAD+ is greatly reduced (Fig. 3). On day 2, after 24 h of
exposure to this nicotinamide analog, the NAD + concentration is 4 pmols//tg
DNA, which is about 1 % of the control value. During the next 10 days the
NAD + concentration increases, but never attains a value which is greater than
15 % of the control level. In addition, the discrete changes in NAD+ levels, which
occur on day 4 and day 8 in untreated cultures, are lacking in cultures exposed
to 3-acetylpyridine.
Effects of nicotinamide
Previous reports from our laboratory have described nicotinamide's reversal
of 3-acetylpyridine-caused potentiation of chondrogenic expression, as well
as nicotinamide's individual capacity to inhibit chondrogenic expression
(Caplan et al. 1968; Caplan, 1970). Fig. 4 demonstrates that the inhibition of
952
M. J. ROSENBERG AND A. I. CAPLAN
2
Day 3
1 -
—i
Tr
T
T
2 -
Day 4
1 -
WW
T i
¥T
•i
T
Day 7
1-
ll T
m
T
0 —
1 -
II
II.
01
Day 10
- f10 mg
Fig. 4. Effects of exposure of 0,1,5 and 10 mg of nicotinamide on limb mesodermal
cell phenotypic expression as measured by a quantitative toluidine blue binding assay
described by Coleman et ah (1970). Optical density at 625 nm is linearly related to
acid mucopolysaccharide levels. On days 3, 4, 7 and 10, cultures in triplicate were
fixed, stained and destained and optical density readings of the destaining solvent
measured. The averaged value is plotted here. The standard deviation was maximally
±007 optical density unit.
chondrogenic expression, caused by continuous exposure to nicotinamide, is
concentration-dependent. Levels of 1 mg or less have no effect on cartilage
matrix synthesis or deposition, as seen by comparing the 0 and 1 mg levels of
nicotinamide. The optical density levels for exposure to 1 mg or below increase
as a function of days in culture, correlating well with visual estimates of increased chondrogenic expression. Levels of 5 or 10 mg of nicotinamide inhibit
chondrogenic expression by a factor of 5 to 12. Such inhibition is not the result
of chondrogenic cells producing less matrix, but is the result of many fewer cells
NAD in developing chick limbs
953
Table 1. Ratio ofNAD+ levels between cultured cells exposed to
5 and 0-1 mg of nicotinamide
Ratio of NAD+ Levels; 5 mg exposure: 0 1 mg exposure
Day
2
4
6
8
Calculated
Calculated
from per-plate from per-DNA
values
values
219
2-43
205
210
1-55
4-25
1-78
2-26
Mesodermal cells were plated on 60 mm Falcon plastic Petri dishes at a density of 107 cells
per dish. Medium was changed daily and just prior to pulsing with nicotinamide. Nicotinamide
was added to the plates on the days indicated for a 6 h period before the cells were collected.
Nicotinamide was added as a sterile solution of Tyrodes, 0 1 ml per plate, to a final concentration of 01 mg or 5 0 mg per 3 ml of medium. Ratios were calculated by taking the NAD+
values obtained from cells exposed to 5 mg of nicotinamide and dividing by the values
obtained for 0 1 mg exposure. For example, on day 8, 480 nmoles of NAD+ per plate
exposed to 5 mg of nicotinamide was divided by 22-9 nmoles of NAD + per plate exposed
to 01 mg nicotinamide, resulting in a ratio of 2-10. The NAD+ values obtained for the lower
nicotinamide concentration (01 mg) are comparable to those in Figs. 1 and 3, for untreated
cells.
exhibiting either morphological or biochemical characteristics of chondrocytes
(Caplan, 1970).
As might be expected from a consideration of the work of Kaplan et al.
(1954), Oide (1958) and others, high levels of nicotinamide cause a dramatic
increase in the cellular pool size of NAD + in cultured limb mesodermal cells.
Continuous exposure to nicotinamide can result in a 5- to 10-fold increase in
this pool size. Substantial increases can be observed even after a relatively
short exposure to nicotinamide, as seen in Table 1. Levels of 0-1 mg of nicotinamide have no morphological or biochemical effects on cultured cells; we have
therefore compared the NAD+ levels of cells exposed to 0-1 mg of nicotinamide
to those of cells exposed to 5-0 mg of nicotinamide, on various days during the
culture period. Table 1 shows that the NAD+ content of cultures exposed to
5-0 mg of nicotinamide for 6 h is more than double that of cultures exposed to
0-1 mg of nicotinamide for the same period. When calculated from cellular DNA
levels, nicotinamide-mediated stimulation varies from a 55 % increase to a 3- to 4fold increase. The variation in the NAD per DNA values reflects the fact that
some cells are dying or have been eliminated from the population. The great
variation reflects the fact that cells on day 4, for example, are more susceptible
to the effects of nicotinamide than those on day 8. The fact that NAD levels
per plate do not reflect such events is misleadingly fortuitous. However, one can
conclude that nicotinamide-caused inhibition of chondrogenic expression seems
to be correlated with increased NAD+ pool levels.
954
M. J. ROSENBERG AND A. I. CAPLAN
DISCUSSION
Data presented here show that the intracellular pool size of NAD + drops
when developing mesodermal cells in culture are exposed to the nicotinamide
analog 3-acetylpyridine. These low NAD+ levels are coincident with the elimination of about 30 % of the cells (Caplan & Stoolmiller, 1973) and seem to be
responsible for, or at least coincident with, the enhanced number of cells expressing chondrogenic properties as compared to untreated cultures. Previous
studies show that incorporation of exogenously added precursors of DNA,
RNA and protein, on a per-cell basis, does not seem to be affected by the
exposure to 3-acetylpyridine (Caplan, 19726). Also, high levels of exogenous
nicotinamide cause the intracellular pool size of NAD+ to increase. This increase
is correlated with the inhibition of chondrogenic expression. These observations
add support to the thesis that nicotinamide and/or pyridine nucleotides play
a controlling role in the differentiation of limb mesodermal cells into chondrogenic and myogenic expression while inhibiting chondrogenic development.
Low levels of NAD+ seem to favor chondrogenic expression while inhibiting
myogenic expression.
It should be stressed that 3-acetylypridine is unique in its ability to potentiate
chondrogenic expression. No other small molecule has been observed to mimic
or duplicate this potentiation of chondrogenic expression of limb mesodermal
cells, although hundreds have been tested (A. I. Caplan, unpublished observations). In addition, 3-acetylpyridine is capable of eliciting chondrogenic expression from the soft tissue, or myogenic areas of post stage-26 embryonic limb
(Caplan, 1970). In the mesodermal cells from these limbs from older embryos,
no chondrogenic expression is observed under normal conditions, presumably
since NAD+ levels are high in these soft tissue areas (Caplan & Koutroupas,
1973; Rosenberg & Caplan, 1974).
In cultures of limb mesodermal cells, exposure to 3-acetylpyridine results in
a potentiation of chondrogenic expression. This 'forced' differentiation of
phenotypically uncommitted cells into a chondrogenic phenotype could be
explained in one of four ways. (A) The lowering of the NAD+ pool size is toxic
to the cells. As these cells are now in the process of dying, their last act is to
express properties of a chondrogenic phenotype. There is little precedent for
this possibility since expression of chondrogenic properties is an active function
and would be an unlikely act for a dying cell to perform. (B) 3-Acetylpyridine
specifically affects genomic events and its action is totally unrelated to NAD+
pool size. There is no precedent for this possibility, and the data reported here
demonstrate the correlation between 3-acetylpyridine, nicotinamide and intracellular NAD+ pools. (C) 3-Acetylpyridine causes a reduction in NAD + levels,
enhancing the competition for NAD+ which serves as a coenzyme in both
glycolytic and mitochondrial energy transfer reactions. In the case of lowered
NAD + levels, one would expect glycolysis to prevail since so little NAD+ is
NAD in developing chick limbs .
955
necessary for the functioning of this pathway. If lactic acid is formed during
glycolytic activity, then this pathway assumes even more importance, since the
NAD + is recycled and is available for further use by the glycolytic enzymes.
NAD+ could be present in very small quantities yet would be catalytically
active relative to glycolysis. The favoring of glycolysis would support mucopolysaccharide synthesis, as opposed to protein synthesis, and would thus favor
chondrogenic over myogenic expression. (D) 3-Acetylpyridine causes a reduction in cellular NAD + levels. This reduction is sensed by the genome and the
genes coding for chondrogenic properties are selected. This possibility also
implies that high NAD+ levels select for the expression of myogenic genes.
Although none of the above-mentioned possibilities can be favored or eliminated by the experimental measurements in hand, we are sympathetic to the
last possibility, which correlates fluxes in NAD+ pool size with genomic events
and represents our current working hypothesis. The mechanism for sensing
changes in NAD+ levels and transmitting this information to the genome may
involve the synthesis and degradation of poly(ADPribose). This unique nucleic
acid is formed from NAD by the excision of nicotinamide and the formation of
a ribose-ribose linkage (Sugimura, 1973). Poly(ADPribose) is firmly associated
with chromatin and has been shown to be bound to histone (predominantly F J .
Its synthesis and degradation have been linked to the events of cell division, but
its possible role as a storage form of NAD or as a mediator of transcriptional
events has not been ruled out. High NAD+ levels would favor the formation of
poly(ADPribose) and myogenesis while low NAD+ levels would favor the
degradation of poly(ADPribose) and chondrogenesis. Also, since nicotinamide
is a product of the formation reaction, an inhibition of poly(ADPribose)
synthesis by nicotinamide would be expected. Experimentally, nicotinamide
causes the cellular level of NAD+ to increase, thus providing a situation of
high NAD+ with stable poly(ADPribose) levels (Caplan and Rosenberg, unpublished observations). Such a situation would not favor chondrogenic expression, and indeed we have reported that nicotinamide alone can inhibit chondrogenic expression. The above predictions, as well as others involving the relationships between myogenesis, chondrogenesis, NAD+ and poly(ADPribose) formation are the subjects of our current experimental efforts, and reflect our belief
that such an interrelationship is the most plausible mechanism to explain the
correlation between NAD+ levels and chondrogenic vs. myogenic expression.
The quantitative measurements presented here clearly show a correlation
between low intracellular levels of NAD and chondrogenesis and between high
intracellular levels of NAD and myogenesis. The present findings, taken together with our previous work, clearly suggest, but do not prove, that pyridine
nucleotides play a prominent if not controlling role in limb mesodermal cell
expression of chondrogenic and myogenic phenotypes.
956
M. J. ROSENBERG AND A. I. CAPLAN
Supported by grants from the National Science Foundation (GP-23030), American
Cancer Society (E-634), National Institutes of Health Training Grant (HD-00020) for M.J.R.,
and Research Career Development Award (HD-35,609) for A.I.C.
REFERENCES
CAPLAN, A.I. (1970). Effects of the nicotinamide-sensitive teratogen 3-acetylpyridine on
chick limb cells in culture. Expl Cell Res. 62, 341-355.
CAPLAN, A.I. (1971). The teratogenic action of the nicotinamide analogs 3-acetylpyridine
and 6-aminonicotinamide on developing chick embryos. /. exp. Zool. 178, 351-358.
CAPLAN, A. I. (1972a). Effects of a nicotinamide-sensitive teratogen 6-aminonicotinamide
on chick limb cells in culture. Expl Cell Res. 70, 185-195.
CAPLAN, A. I. (19726). The effects of the nicotinamide sensitive teratogen 3-acetylpyridine
on chick limb mesodermal cells in culture: biochemical parameters. /. exp. Zool. 180,
351-362.
CAPLAN, A. I. (1972c). The site and sequence of action of 6-aminonicotinamide in causing
bone malformations of embryonic chick limb and its relationship to normal development.
Devi Biol. 28, 71-83.
1
CAPLAN, A. I. (1972a ). Comparison of the capacity of nicotinamide and nicotinic acid to
relieve the effects of muscle and cartilage teratogens in developing chick embryos. Devi
Biol. 28, 344-351.
CAPLAN, A. I. & KOUTROUPAS, S. (1973). The control of muscle and cartilage development
in the chick limb: the role of differential vascularization. /. Embryol. exp. Morph. 29,
571-583.
CAPLAN, A. 1. & STOOLMILLER, A. C. (1973). Control of chondrogenic expression in mesodermal cells of embryonic chick limb. Proc. natn. Acad. Sci. U.S.A. 70, 1713-1717.
CAPLAN, A. I., ZWILLING, E. & KAPLAN, N. O. (1968). 3-Acetyl-pyridine: effects in vitro
related to teratogenic activity in chick embryos. Science, N. Y. 160, 1009-1010.
COLEMAN, A. W., COLEMAN, J. R., KANKEL, D. & WERNER, I. (1970). The reversible control
of animal cell differentiation by the thymidine analog 5-bromodeoxyruidine. Expl Cell
Res. 59, 319-328.
KAPLAN, N. O., GOLDIN, H., HUMPHREYS, S. R., CIOTTI, M. M. & VENDITTI, J. M. (1954).
Pyridine nucleotide synthesis in the mouse. /. biol. Chem. 219, 287-298.
LANDAUER, W. (1957). Niacin antagonists and chick development. /. exp. Zool. 136, 509-530.
OIDE, H. (1958). The influence of the intrasplenic implantation of nicotinamide on themitotic
activity and the phosphopyridine nucleotide content of regenerating liver. Gann 49, 49-56.
ROSENBERG, M. J. & CAPLAN, A. I. (1974). Nicotinamide adenine dinucleotide levels in cells
of developing chick limbs: possible control of muscle and cartilage development. Devi Biol.
38, 157-164.
SCHACHTER, L. P. (1970). Effect of conditioned media on differentiation in mass cultures of
chick limb bud cells. I. Morphological effects. Expl Cell Res. 63, 19-32.
35
SEARLS, R. L. (1965). An autoradiographic study of the uptake of S-sulfate during the
differentiation of limb-bud cartilage. Devi Biol. 11, 155-168.
SEARLS, R. L. & JANNERS, M. Y. (1969). The stabilization of cartilage properties in the
cartilage-forming mesenchyme of the embryonic chick limb. /. exp. Zool. 170, 365-376.
SUGIMURA, T. (1973). Poly(adenosine diphosphate ribose). In Progress in Nucleic Acid Research and Molecular Biology, vol. 13, pp. 127-151.
ZWILLING, E. (1968). Morphogenetic phases in development. Devi Biol. Suppl. 2, 184-207.
(Received 16 October 1974)