Adenylyl cyclase P-site ligands accelerate differentiation in
Ob1771 preadipocytes
Azeddine
Ibrahimi,
Nada
Abumrad,
Hengameh
Maghareie,
Michael
Golia,
Ilana
Shoshani,
Laurent
Désaubry, and
Roger A.
Johnson
Department of Physiology and Biophysics, State University of New
York, Stony Brook, New York 11794-8661
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ABSTRACT |
Differentiation of Ob1771 preadipocytes to adipocytes was
characterized by morphological changes and elevated expression of the
specific marker enzyme, glycerol-3-phosphate dehydrogenase. A
differentiation response substantially more complete and rapid than
that obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases:
2',5'-dideoxyadenosine (2',5'-dd-Ado),
9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by
N6-(2-phenylisopropyl)adenosine
or 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively,
for adenosine A1 receptors. Also
ineffective were IBMX or forskolin, agents that can raise intracellular
cAMP levels. Potency of the differentiation response followed the order
2',5'-dd-Ado (1-20 µM) > 9-CP-Ade (10-100
µM) = 9-Ara-Ade (10-100 µM) >> inosine, consistent with
their potencies to inhibit adenylyl cyclases. The data suggest that
inhibition of adenylyl cyclase via the P-site and the consequent
reduction in cell cAMP levels facilitate the induction of
differentiation in Ob1771 cells. The findings raise the question
whether the known endogenous P-site ligands participate in the
differentiation response induced by hormones.
adipocytes; P-site inhibition; 2',5'-dideoxyadenosine; 9-(cyclopentyl)adenine; signal transduction; adenosine
3',5'-cyclic monophosphate
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INTRODUCTION |
DIFFERENTIATION OF THE Ob1771 clonal cell line is a
controlled multistep process characterized by emergence of early, late, and very late markers (2). Adipose precursor Ob1771 cells, defined as
preadipocytes, divide until confluence and then become committed to
differentiate by acquiring early markers but do not yet accumulate
triacylglycerol. These early markers include lipoprotein lipase and the
2-chain collagen VI (3). Further differentiation leads
to immature adipose cells (~6 days after confluence). These cells
acquire the protein machinery for triacylglycerol synthesis and
hydrolysis (~20 days after confluence) and begin accumulation of
triacylglycerol droplets. Terminal differentiation leads to the
formation of triacylglycerol-filled, mature adipose cells with the
emergence of specific genes, one of which is for glycerol-3-phosphate dehydrogenase (2, 3).
Transition to terminal differentiation is dependent on the sensitivity
of the preadipocyte to mitogenic and adipogenic stimuli (1). These
include growth hormone, glucocorticoid, prostacyclin, or
3,5,3'-triiodothyronine
(T3) and long-chain
fatty acids (1-3). Ob1771 cells undergo spontaneous
differentiation to adipocytes when cultured in medium supplemented with
8% FCS. However, the pattern of spontaneous differentiation seen with
FCS is usually incomplete and clustered. This process can
be promoted by insulin and T3 (12,
24). One of the main adipogenic factors in FCS is thought to be
arachidonic acid, which increases cellular cAMP levels and induces
polyphosphoinositide breakdown (21). It has been suggested that the
cAMP and IGF-I pathways are essential to terminal differentiation,
whereas diacylglycerol and insulin pathways play a modulating role (3).
Insulin is well known to lower cell cAMP levels (7) by activation of
type III phosphodiesterases (4, 9).
cAMP concentration can be regulated through changes in its formation,
catalyzed by adenylyl cyclases, or changes in its degradation, catalyzed by cyclic nucleotide phosphodiesterases. Numerous drugs have
been developed that act on cyclic nucleotide phosphodiesterases (4, 9),
whereas those that act directly on adenylyl cyclases have been less
well explored (36); the main class of such pharmacological agents
comprises forskolin and its analogs (15). Adenylyl cyclase is potently
and directly inhibited by analogs of adenosine via a configuration
distinct from that of the catalytic active site (27, 29, 37, 41). It is
referred to as the P-site from pharmacological characterizations of
inhibition that demonstrate a requirement for a purine moiety (26, 28,
30, 40). The most potent cell-permeable P-site ligands include
2',5'-dideoxyadenosine (2',5'-dd-Ado;
IC50 ~3 µM) (30),
9-(cyclopentyl)adenine (9-CP-Ade; IC50 ~20 µM) (40), and
9-(arabinofuranosyl)adenine (9-Ara-Ade; IC50 ~100 µM) (28, 39).
We utilized direct inhibitors of adenylyl cyclases to determine whether
and to what extent such agents may influence the differentiation process in Ob1771 cells. The cell-permeable P-site ligands used in this
study included 2',5'-dd-Ado, 9-CP-Ade, and 9-Ara-Ade. This
report compares differentiation responses in Ob1771 preadipocytes typically induced by insulin and
T3 with those observed in the presence of these nucleosides.
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EXPERIMENTAL PROCEDURES |
Materials. 9-CP-Ade was synthesized as
described by Montgomery and Temple (31), and 2',5'-dd-Ado
was synthesized by the procedure developed by Désaubry et al.
(14). The bicinchoninic acid (BCA) protein assay reagent kit was from
Pierce; culture cell media were from GIBCO; FCS was from Atlanta
Biologicals; protein kinase A was isolated from bovine muscle and
enriched by ammonium sulfate precipitation and chromatography on
DEAE-Sephadex by established procedures (34); kemptide was from Sigma
Chemical (K1127); the RIA kit for cAMP was from New England Nuclear;
and [
-32P]ATP was
from ICN. All the other chemicals were from Sigma.
Cell culture and treatment. Ob1771
preadipocyte cells were originally obtained after the subcloning of
Ob17 cells established from the periepididymal adipose tissue of
genetically obese B57BL6J mice (32). Cells were plated to a density of
~2,000/cm2 and grown in DMEM
supplemented with 8% fetal bovine serum, 200 units penicillin/ml, 50 µg streptomycin/ml, 33 µM biotin, and 17 nM pantothenate.
Confluence was reached within 5 days, after which differentiation was
promoted by the addition of the indicated concentrations of nucleoside
or of 17 nM insulin and 2 nM T3 to the medium. Nucleosides were added to prewarmed medium from
concentrated stocks in ethanol. Media were changed every other day, and
terminal differentiation of Ob1771 was followed for 2-4 wk,
typically being complete in 3-4 wk.
Stability of 9-CP-Ade and
2',5'-dd-Ado. Cells were exposed to either
30 µM 2',5'-dd-Ado or 30 µM 9-CP-Ade. At various times, a 200-µl portion of the medium above the cells was removed and frozen. The collected samples were thawed and passed through 0.22-µm syringe filters and then were subjected to HPLC by reverse-phase chromatography (Beckman Ultrasphere 5 mm C-18; 4.6 × 250 mm)
developed with a linear gradient from water to 100% methanol. Baseline
separations for both nucleosides were obtained. Nucleosides were
quantified as areas under peaks determined with a Waters 996 photo-diode array detector and the accompanying Millennium software
(version 2.10).
Glycerol-3-phosphate dehydrogenase.
Glycerol-3-phosphate dehydrogenase (GPDH) was assayed
spectrophotometrically from cell homogenates (12, 21) obtained at the
indicated day after confluence. Interassay variability as well as
variability among mean values from separate culture dishes maintained
under identical culture conditions never exceeded 7% within the same
series of cells. GPDH specific activity was expressed in milliunits per
milligram (nmol · min
1 · mg
protein
1).
Protein content was determined according to Smith et al. (35) with the
BCA protein assay.
Cell cAMP. Levels of cAMP were
determined on extracts of cells exposed to control medium or to medium
supplemented with adenine nucleosides. Samples were handled in either
of two ways. One involved extraction and a two-step chromatographic
purification before assay, and the other involved simply extraction and
dilution. For samples to be purified, medium was decanted from culture
dishes and cells were lysed by the successive addition and removal of two 0.6-ml portions of 0.3 M
HClO4, containing ~25,000
counts/min (cpm) of
[3H]cAMP, included to
allow the recovery of sample cAMP to be calculated. These extracts were
pooled and neutralized with
K2CO3.
The resulting precipitate of protein and
KClO4 was removed by
centrifugation. The supernatant fractions were then chromatographically
purified by sequential chromatography on
Al2O3
and Dowex-50 (H+ form). Samples,
eluted from a Dowex-50 column with water, were collected, lyophilized,
and then reconstituted with buffer. A portion of this solution was used
to determine sample recovery, which was typically >60%. For the
simpler extraction, medium was decanted from culture dishes and cells
were lysed by the addition of a solution containing 10 mM potassium
phosphate, pH 6.8, 10 mM EDTA, and 0.1 mM IBMX. cAMP was determined by
either of two techniques: by protein kinase A activation (10) or by
RIA. Protein kinase A was as per Corbin et al. (10) in a buffer
containing 20 mM magnesium acetate, 0.1 mM IBMX, 1 mg BSA/ml, 0.2 mM
ATP, ~200,000 cpm
[
-32P]ATP, 10 mM
potassium phosphate buffer, pH 6.8, and 130 µM kemptide as phosphate
acceptor. For assays utilizing the RIA kit from New England Nuclear,
the procedure followed that described by the manufacturer. For both
assays, samples were assayed in triplicate, with at least two dilutions
of sample, and also with added internal standard (usually 100 fmol)
with which to assess and adjust for any assay interference that may
have occurred.
RNA preparation. RNA was prepared with
the RNA STAT-60 kit. RNA was electrophoresed on denaturing agarose gel,
transferred to Hybond-N+
membranes, and hybridized at 42°C with randomly primed
32P-labeled DNA probes. After
washing, the membranes were exposed to Kodak Hyperfilm at
75°C. The mRNA for GPDH was used as an internal standard.
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RESULTS |
Effects of various P-site inhibitors on cell
differentiation. Several adenine nucleosides and
adenine 3'-nucleotides known to inhibit adenylyl cyclases
directly in vitro and in intact cells (Fig.
1) were tested for their effects on
differentiation of Ob1771 cells. Both 2',5'-dd-Ado (20 µM) and 9-Ara-Ade (30 µM) increased the number and the density of
clusters of differentiated cells well above levels seen in control
cells and in cells treated with insulin (17 nM) plus
T3 (2 nM; Fig.
2). By comparison, inosine, a nucleoside
structurally close to the class of inhibitory compounds designated as
P-site ligands but known not to affect adenylyl cyclase activity (28,
30, 40), was without effect, even at the relatively high concentration
of 100 µM (Fig. 2). This lack of response was consistent with its
lack of effect on the cyclase.

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Fig. 1.
Structures of several inhibitors of adenylyl cyclases.
A: 2',5'-dideoxyadenosine
(2',5'-dd-Ado). B:
9-(cyclopentyl)adenine (9-CP-Ade). C:
9-(arabinofuranosyl)adenine (9-Ara-Ade).
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Fig. 2.
Comparison of differentiation responses in Ob1771 cells elicited by
2',5'-dd-Ado, 9-Ara-Ade, inosine, and
insulin/triiodothyronine (T3).
At confluence, Ob1771 cells were exposed to medium + insulin/T3 or medium + nucleoside,
as indicated, with retreatment every other day. Concentrations were 30 µM 9-Ara-Ade; 20 µM 2',5'-dd-Ado; 100 µM inosine; and
17 nM insulin + 2 nM T3.
Photographs were taken after 22 days of treatment and are
representative of 3 experiments.
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The acceleration of differentiation was concentration dependent, as
evident in Fig. 3 for
2',5'-dd-Ado, with effects being noted with concentrations
as low as 1 µM. Although 2',5'-dd-Ado was more potent
than 9-Ara-Ade, the extent of the differentiation responses seen with
these nucleosides at their respectively optimal concentrations appeared
to be comparable. The effects of both nucleosides occurred at
concentrations within their ranges for inhibition of adenylyl cyclases.
This effect on differentiation was not related to the Ob1771 clone,
since similar effects were obtained in 3T3-F442A preadipocyte cells
after treatment with 2',5'-dd-Ado (not shown).

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Fig. 3.
Concentration dependence for induction of differentiation in Ob1771
cells by 2',5'-dd-Ado. Ob1771 cells were exposed to the
indicated concentrations of 2',5'-dd-Ado at confluence and
were maintained by retreatment every other day. Photographs were taken
after 16 days of treatment and are representative of 3 experiments.
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cAMP levels. Noteworthy was the
consistent observation that basal cAMP levels were also
reduced under conditions eliciting accelerated differentiation. This
occurred with 2',5'-dd-Ado in separate experiments (Table
1), assayed by different techniques, and
with 9-CP-Ade in a concentration-dependent manner (Fig.
4). For comparison, the effect of 30 µM
2',5'-dd-Ado in this experiment is also shown. In these
experiments cells were fixed after 3 wk of treatment with these
nucleosides and indicate that chronic treatment can reduce already low
cellular levels of cAMP in these unstimulated cells.

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Fig. 4.
Changes in cAMP levels in Ob1771 cells after chronic exposure to
9-CP-Ade. Ob1771 cells were exposed to the indicated concentrations of
9-CP-Ade or 2',5'-dd-Ado for 22 days. Cellular cAMP levels
were then determined on purified samples by the protein kinase
activation method described in EXPERIMENTAL
PROCEDURES. Individual values obtained
with 9-CP-Ade ( , ) or 2',5'-dd-Ado ( ) are shown; line shows
averages of values for 9-CP-Ade.
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Ligand stability. Because both
9-Ara-Ade and 2',5'-dd-Ado are ribose derivatives
containing the furanosyl oxygen, they are susceptible to enzymatic and
nonenzymatic depurination, which could affect the efficacy of these
compounds in experiments involving chronic treatments. Depurination
does not occur with 9-CP-Ade, which contains the chemically and
enzymatically much more stable adenine-cyclopentyl bond (Fig. 1).
9-CP-Ade is also a P-site ligand and exhibits
IC50 values for inhibition of
types I and VI adenylyl cyclases of ~20 to 100 µM, respectively
(25). 9-CP-Ade induced a more dramatic differentiation in Ob1771 cells
than that noted with either insulin plus
T3, 9-Ara-Ade, or
2',5'-dd-Ado (Fig. 5). In this
experiment, cells were exposed to either 10 or 100 µM 9-CP-Ade, and
the response was concentration dependent. Note also the very dense
concentration of lipid droplets in areas surrounding the several dark
spots. At higher magnification, the dark spots were actually floating
upwellings of cells that could be seen to be full of lipid droplets
(not shown). Although the concentrations of 9-CP-Ade necessary to
elicit this response were greater than those used to elicit responses
with 2',5'-dd-Ado, the extent of differentiation seen with
9-CP-Ade was greater than that seen with 2',5'-dd-Ado or
9-Ara-Ade.

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Fig. 5.
Accelerated differentiation in Ob1771 cells induced by 9-CP-Ade. Ob1771
cells were exposed at confluence to 9-CP-Ade at 10 or 100 µM, or
vehicle (control) for 16 days. Photographs represent 1 of 2 experiments.
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The response of these cells to 9-CP-Ade, when compared with the
response to 2',5'-dd-Ado, was consistent with both the
differences in potency of these adenine derivatives to inhibit adenylyl
cyclases and with their expected differences in chemical and metabolic stability. 9-CP-Ade is the less potent but chemically more stable ligand. To establish whether the stability of 9-CP-Ade or
2',5'-dd-Ado differed in this cell culture system as
expected, cells were exposed to either compound and samples were taken
over 2 days and analyzed by HPLC. The concentration of 9-CP-Ade was not
altered over several days, whereas 2',5'-dd-Ado exhibited a
half-life of ~24 h (Fig. 6). In these
experiments, no distinction was made whether disappearance of
2',5'-dd-Ado was due to medium, to cells, or both. Thus
9-CP-Ade at any given concentration would be effectively maintained
longer than would 2',5'-dd-Ado.

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Fig. 6.
Fate of 2',5'-dd-Ado and 9-CP-Ade on exposure to Ob1771
cells and medium. To cells plated in 10-ml standard medium on 100-mm
culture plates was added either 30 µM 2',5'-dd-Ado or 30 µM 9-CP-Ade. Thereafter, 200-µl portions were taken every hour for
the 1st day and then at additional times for 2 days. Nucleoside was
purified and quantified as described in EXPERIMENTAL
PROCEDURES. Values are relative concentrations of
nucleoside at sampling time (t) to
concentrations 1 min after addition of nucleoside
(t = 0).
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Effects of a single exposure to
nucleoside. To determine whether a single exposure of
Ob1771 cells to nucleoside was sufficient to promote differentiation,
cells were exposed for 48 h to 9-CP-Ade and 2',5'-dd-Ado.
The cells were then washed with fresh, nucleoside-free medium and were
maintained on the normal 2-day refeeding schedule but without
additional exposure to either compound. With either compound the
differentiation response of Ob1771 cells occurred at a rate
substantially faster than that which would have occurred had cells been
unexposed or exposed only to vehicle (not shown). This suggests that a
single, albeit 2-day, exposure to these P-site ligands was sufficient
to initiate the chain of events leading to the adipogenic response in
Ob1771 cells.
Induction of GPDH. Terminal
differentiation implies the capacity to accumulate triacylglycerol and
the enzymes necessary for this, one of which is GPDH. The activity of
GPDH correlates very well with the proportion of differentiated,
triacylglycerol-containing cells (21). To substantiate that the effects
of the nucleosides on cell morphology noted above were in fact
indicative of terminal differentiation to adipocytes, the activity of
GPDH was determined. The activity of expressed GPDH increased with
2',5'-dd-Ado and 9-CP-Ade at concentrations that elicited
the accelerated differentiation response, but not by inosine, even at
concentration up to 1 mM (Fig. 7). Thus the
morphological changes induced by these adenine derivatives were also
corroborated by elevated activity of GPDH.

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Fig. 7.
Increased expression of glycerol-3-phosphate dehydrogenase (GPDH) by
2',5'-dd-Ado or 9-CP-Ade. At confluence, Ob1771 cells were
exposed to 2',5'-dd-Ado, 9-CP-Ade, or the mixture of
insulin/T3, as indicated. For both
A and
B, nucleoside concentrations were as
indicated, and 0 (solid bar) indicates addition of the mixture of 17 nM
insulin + 2 nM T3.
A (Expt.
1): concentrations were millimolar and cells were
harvested after 22 days of treatment for determination of GPDH
activities. B (Expt.
2): concentrations were millimolar and cells were
harvested after 16 days of treatment for determining GPDH activities.
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Effect of ligands for adenosine
receptors. Given that the adenine derivatives eliciting
the accelerated differentiation response above also share structural
characteristics with ligands known to act on cell-surface receptors for
adenosine, such ligands were also tested for their effects on
differentiation. Neither
N6-(2-phenylisopropyl)adenosine,
an adenosine receptor agonist, nor 1,3-diethyl-8-phenylxanthine (DPX),
an adenosine receptor antagonist, induced changes in the
differentiation response of the Ob1771 cells (Fig.
8). This lack of response is similar to that reported by Borglum et al. (5) with
5'-(N-ethylcarboxamido)adenosine, an adenosine receptor agonist. These adenosine receptor ligands altered
neither the rate of spontaneous differentiation nor that induced by
2',5'-dd-Ado, suggesting no role of adenosine receptors in
the accelerated differentiation noted with the adenine derivatives we
have tested.

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Fig. 8.
Comparison of differentiation responses in Ob1771 cells elicited by
2',5'-dd-Ado, 1,3-diethyl-8-phenylxanthine (DPX), or
( )N6-(2-phenylisopropyl)adenosine
(PIA). At confluence, Ob1771 cells were exposed to medium + agents, as
indicated, with retreatment every other day. Concentrations were: 10 µM 2',5'-dd-Ado, 5 µM DPX, or 1 µM PIA. Photographs
were taken after 22 days of treatment and are representative of two
experiments.
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Effect of cAMP-elevating agents. The
acccelerated differentiation noted with the adenine derivatives above
was consistent with their effects on adenylyl cyclases via the
inhibitory P-site domain. These effects would suggest that elevations
in cellular cAMP levels by other agents would interfere with the
differentiation response to P-site ligands. Ob1771 cells were exposed
chronically to forskolin, an established activator of adenylyl cyclases
in vitro and in vivo, or IBMX, an established inhibitor of cyclic nucleotide phosphodiesterases (Fig. 9). In
this experiment, as with those with the adenine derivatives, exposure
was followed for several weeks, with refeeding every other day, and
morphological changes were monitored. Neither forskolin nor IBMX
promoted differentiation (Fig. 9), without or with
2',5'-dd-Ado (not shown).

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Fig. 9.
Comparison of differentiation responses in Ob1771 cells elicited by
2',5'-dd-Ado, forskolin, or IBMX. At confluence, Ob1771
cells were exposed to medium + agents, as indicated, with retreatment
every other day. Concentrations were 20 µM 2',5'-dd-Ado,
100 µM IBMX, and 50 µM forskolin. Photographs were taken after 22 days of treatment and are representative of 3 experiments.
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Changes in cell morphology were substantiated by analysis of the
expression of adipocyte markers under these conditions (Fig. 10). RNA analysis by Northern blotting
showed a high expression of genes for GPDH and fatty-acyl-CoA synthase
in cells treated with 2',5'-dd-Ado or insulin plus
T3. 2',5'-dd-Ado
appeared to upregulate the expression of these genes, in correlation
with the morphological changes of the cells. In contrast, the
expression of these genes was unaffected by treatment with either
forskolin or IBMX. These data are in line with the previously described argument that increasing cAMP concentration by these two agents did not
affect adipocyte differentiation (38). Moreover, the data are fully
consistent with both biochemical and morphological changes induced by
the adenine derivatives reported here.

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Fig. 10.
Expression of adipocyte marker genes for GPDH and acyl-CoA synthase
(ACS). At confluence, Ob1771 cells were exposed to medium + agents, as
indicated, with retreatment every other day for 14 days. Concentrations
were: 10 µM 2',5'-dd-Ado, 100 µM IBMX, 50 µM
forskolin, and 17 nM insulin + 2 nM
T3. Similar data were observed in
3 experiments. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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DISCUSSION |
The present studies demonstrate a concentration-dependent acceleration
in terminal differentiation of Ob1771 cells by several adenine
derivatives belonging to a class of noncompetitive inhibitors of
adenylyl cyclases. The differentiation response induced by 2',5'-dd-Ado was significantly more rapid and more complete
than that induced by insulin and
T3, agents typically used to
promote differentiation of preadipocytes. This was noted by changes in cell morphology, increased GPDH activity, and induced expression of
marker genes. In general, the efficacy and rank order of potency of the
adenine derivatives for inducing the differentiation response followed
that of P-site-mediated inhibition of adenylyl cyclases: 2',5'-dd-Ado > 9-CP-Ade > 9-Ara-Ade >> inosine.
Inosine does not inhibit adenylyl cyclases or induce adipocyte
differentiation. Consistent with its greater chemical and metabolic
stability, 9-CP-Ade caused a differentiation response that was more
dramatic than that seen with 2',5'-dd-Ado.
In 3T3-L1 cells, a fetal mouse fibroblast cell line, a dissociation
between cellular cAMP levels and the differentiation response was noted
(38); impaired expression or function of
Gs was associated with enhanced differentiation, while elevations in cell cAMP levels did
not have the opposite effect. In line with that, we also found that
neither forskolin nor IBMX influenced adipocyte differentiation. The
data we present, that P-site ligands accelerate differentiation, would
be consistent with reduced cellular cAMP levels promoting the process.
This was supported by the observations with 2',5'-dd-Ado and 9-CP-Ade noted in several experiments (Table 1 and Fig. 4). These
reductions in basal cAMP levels occurred following chronic exposure to
these nucleosides. These data are presented on the basis of cAMP levels
per dish and may represent an underestimation of actual
cellular changes, since both cell number and protein levels
approximately double when confluent cultures undergo differentiation (11, 19, 21). It is thus probable that the effects of this class of
compounds are mediated through reduction in cAMP and the cAMP-protein
kinase A signaling pathway subsequent to adenylyl cyclase inhibition.
We and others have used P-site ligands with a variety of mammalian
tissues and isolated cells (8, 16, 18, 20, 22, 23, 33) and have
demonstrated effects on different aspects of cell function. Rat
epididymal fat cells (18), isolated hepatocytes (8), primary cultures
of thyroid follicles (22), dorsal root ganglion neurons (23), bone
organ cultures (33), and cortical collecting tubules (16) have been
used, to name but a few. End points included changes in water
conductance (16), action potential afterhyperpolarization (23),
parathyroid hormone-stimulated bone resorption (33),
glycerol production (18), altered enzyme activities (8), and DNA
synthesis and cell growth (22). In this last example,
2',5'-dd-Ado suppressed thyroid-stimulating
hormone-stimulated cAMP levels while enhancing the stimulation of cell
growth and causing a two- to sevenfold increase in the
[3H]thymidine
incorporation into DNA caused by insulin, epidermal growth factor, and
FCS (22). In each of these instances the effects of
2',5'-dd-Ado on cell function and on cellular cAMP levels
were uniformly consistent with P-site inhibition of adenylyl cyclase.
It is possible that effects of P-site ligands on differentiation and
cell cAMP levels reflect cell-specific action(s) of the nucleoside
and/or the particular adenylyl cyclase isozyme(s) expressed. Sensitivity of adenylyl cyclases to P-site-mediated inhibition is
isozyme dependent (25), and the tissue-dependent level of expression of
different forms of adenylyl cyclase may influence the regulatory
importance of P-site ligands, whether intracellular or extracellular in
origin. Although the simplest interpretation of the data is that
nucleoside-induced differentiation was due to an inhibition of adenylyl
cyclase, P-site ligands may act selectively on other proteins. It has
been known for some time that some of these compounds can bind to DNA
polymerase (17) but are without effect on other enzymes (13). Potent
P-site ligands occur naturally within cells, e.g., 3'-AMP and
2'-d-3'AMP, and we have shown that levels of these
3'-nucleotides change in a chronic fashion (6). Therefore, while
2',5'-dd-Ado and 9-CP-Ade may affect cell differentiation by inhibiting adenylyl cyclase and lowering cAMP, it is not only their
effects per se that are important but also the putative natural
intracellular regulatory processes and ligands that they may mimic.
The ligands used in these studies represent a class of compounds
proving to be very useful as pharmacological tools for studies of cell
physiology and pathophysiology. The most effective compounds for work
with intact cells have been, in rank order of potency as inhibitors of
adenylyl cyclases, 2',5'-dd-Ado, 9-CP-Ade, 9-Ara-Ade, and
9-(2-tetrahydrofuryl)adenine (9-THF-Ade; SQ-22536). These compounds are
fairly potent (IC50
3-100
µM), soluble in water (e.g., to 10 mM), stable at neutral to alkaline
pH, and are readily taken up by cells. Because of the cyclic oxygen of
the ribose ring, 2',5'-dd-Ado, 9-Ara-Ade, and 9-THF-Ade are
subject to depurination at acidic pH, whereas 9-CP-Ade is stable.
Subsequent metabolism by cells is also less likely with 9-CP-Ade (cf.
Fig. 6), due to the lack of the ribosyl structure and its hydroxyl
groups. Thus, although 9-CP-Ade is less potent than
2',5'-dd-Ado, for example, its chemical and pharmacological
properties may make it the more useful compound for many applications.
It is likely, though, that if inhibition of adenylyl cyclase is
involved in the differentiation response we note here, previous work
indicates that 3-phosphorylated P-site ligands will prove to be much
more potent pharmacophores (6, 13, 14, 25, 27, 28). Once some of these
ligands become available with protected phosphate groups for use as
prodrugs, effects at substantially lower concentrations can be expected and these will become the agents of choice.
We believe that the differentiation response noted here, being a
derivative of the complex mechanisms regulating cell growth, division,
and development, will likely include effects of these ligands on
proteins other than adenylyl cyclase. Moreover, cells may produce
endogenous ligands to interact with adenylyl cyclase and specific other
proteins and thereby bring about changes in cell growth/differentiation
in response to stimuli or as part of a developmental event. The
identification of the naturally occurring ligands, the proteins with
which they interact, and the circumstances under which one or both
change, will allow an understanding of the mechanisms regulating cell
development to be delineated and then utilized to modify
pathophysiological conditions.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-38828 to R. A. Johnson and by a
grant from the Taher Fund to N. Abumrad.
 |
FOOTNOTES |
Address for reprint requests: R. A. Johnson, Dept. of Physiology and
Biophysics, State Univ. of New York, Stony Brook, NY 11794-8661.
Received 30 September 1997; accepted in final form 14 October
1998.
 |
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