| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Section for Molecular Signalling (J.W., T.R.L., P.B., E.D.), Department of Cell and Molecular Biology, Lund University, Sweden; and National Heart, Lung and Blood Institutes (V.M.), National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Eva Degerman, Section for Molecular Signalling, Department of Cell and Molecular Biology, University of Lund, P.O. Box 94, S-221 00 Lund, Sweden. E-mail: Eva.Degerman{at}medkem.lu.se
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
With the use of wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), it has been suggested that PI 3-kinase is involved in the antilipolytic action of insulin (7). Pretreatment of adipocytes with wortmannin inhibits phosphorylation/activation of PDE 3B, blocks the insulin-induced activation of a kinase that phosphorylates PDE 3B (8) and the antilipolytic action of insulin (7, 8). However, the components in the signaling pathway between PI 3-kinase and PDE 3B, including the kinase responsible for the phosphorylation of PDE 3B, have not been identified.
Mitogen-activated protein kinases (MAP kinases) (9), p70 S6 kinase (10) and protein kinase B (PKB), also known as RAC or Akt kinase (11), are activated in response to insulin stimulation of adipocytes (12, 13, 14, 15, 16) through wortmannin-sensitive mechanisms (15, 16, 17, 18, 19). Thus, these kinases could have a role in the signaling pathway between PI 3-kinase and PDE 3B. In this report, we show that insulin-induced activation of p70 S6 kinase and MAP kinases are not involved in the phosphorylation/activation of PDE 3B. However, our results strongly suggest that PKB is responsible for the phosphorylation of PDE 3B in vitro, and thereby constitutes a likely candidate involved in the insulin-induced antilipolytic signaling pathway in adipocytes. A minor part of these results has previously been reported in abstract form (20).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-32P]ATP was synthesized as
described (22). Protein A Sepharose was from Pharmacia (Uppsala,
Sweden).
Preparation and stimulation of adipocytes
Adipocytes were prepared from the epididymal adipose tissue of
36- to 38-day-old male Sprague-Dawley rats (B&K Universal, Stockholm,
Sweden) according to the method of Rodbell (23) with modifications
described by Honnor et al. (24). Cells were suspended in
Krebs-Ringer medium, pH 7.4, containing 25 mM HEPES, 200
nM adenosine, 2 mM glucose, and 1% BSA. PMA,
PD 98059, rapamycin and wortmannin were dissolved in dimethylsulfoxide
and added to cells resulting in a final concentration of
dimethylsulfoxide of less than 0.15%. Peroxovanadate was prepared
fresh by mixing 300 mM vanadate and 790 mM
H2O2 (final concentration; 12 mM of
each) in 40 mM HEPES, pH 7.4, and incubating the mixture at
20 C for 15 min. Adipocytes (1.5–2 ml 10% cell suspension) were
incubated at 37 C with various agents, centrifuged, and resuspended in
5–10 ml of homogenization buffer consisting of 50 mM
N-tris[Hydroxymetyl]metyl-2-aminoethanesulfonic acid
(TES), pH 7.5, 2 mM EGTA, 1 mM EDTA, 250
mM sucrose, 40 mM phenyl phosphate, 5
mM NaF, 1 mM phenylmethylsulfonylfluoride, 0.05
mM sodiumvanadate, 1 mM dithioerythritol (DTE),
antipain (10 µg/ml), leupeptin (10 µg/ml), and pepstatin A (1
µg/ml). Cells were centrifuged a second time, suspended in
homogenization buffer (1.0 ml final volume), homogenized (10 strokes)
at room temperature, and placed on ice. The homogenates were
centrifuged at 50,000 x g for 60 min at 4 C. Fat-free
supernatants (referred to as cytosol fractions) were withdrawn. To
determine PDE 3B activity, the membrane fractions were resuspended in
0.4 ml of 50 mM Tris, pH 7.5, 5 mM
MgCl2, 1 mM EDTA, 5% (wt/vol) glycerol,
antipain (10 µg/ml), leupeptin (10 µg/ml) and pepstatin A (1
µg/ml) and assayed as previously described (8). In experiments where
translocation of PKB and the kinase that phosphorylates PDE 3B was
investigated, the membrane fractions were resuspended in 1.0 ml of
homogenization buffer. Adipocytes (2 ml of 10% cell suspension) were
labeled with 32Pi (1 mCi/ml) for 1 h 15
min and after stimulation, PDE 3B was immunoisolated from solubilized
membrane fractions as previously described (8). The PDE 3B antibodies
quantitatively immunoprecipitated PDE 3B from both control and
insulin-stimulated cells.
Immunoprecipitation of p70 S6 kinase
Cytosol fractions (400 µl; equivalent to 80 µl packed cells)
from control and stimulated cells were immunoprecipitated with a
polyclonal antibody against p70 S6 kinase (1 µg IgG). After 16 h
incubation at 4 C, protein A Sepharose (35 µl 50% suspension) was
added, and the incubation continued for 2 h. After centrifugation,
the immunoprecipitates were washed twice with PBS, twice with
homogenization buffer, resuspended in 20 µl of homogenization buffer,
and assayed for kinase activity. One tenth of the immunoprecipitates
were mixed with Laemmli sample buffer (25) and subjected to SDS-PAGE,
followed by electrotransfer of proteins onto polyvinylidene difluoride
membrane (Millipore, Bedford, MA). After blocking with 0.5% gelatin in
PBS with 0.1% Tween 20, the membrane was incubated with a 1:1000
dilution of the p70 S6 kinase antibody followed by a horseradish
peroxidase-conjugated secondary antibody; protein was detected with
Amersham’s enhanced chemiluminescence system.
Protein kinase assays
The activity of p70 S6 kinase was determined in 15 µl
immunoprecipitates (equivalent to 60 µl packed cells, see above) by
incubation with 7.5 µl of a phosphorylation mixture containing 150
µM [-32P]ATP (10 µCi), 17
mM TES, pH 7.5, 40 mM MgSO4, 200
mM sucrose, 5 mM DTE, 15 µM
cAMP-dependent protein kinase inhibitor, and 13.3 µg of the peptide
KKRNRTLTK as substrate. After a 40-min incubation at 30 C, the
reactions were terminated by adding 15 µl 1% BSA and 1
mM ATP, pH 3.0, and 7.5 µl of 30% trichloroacetic acid.
Samples were centrifuged and supernatants applied onto phosphocellulose
papers (Whatman P81), washed three times with 75 mM
phosphoric acid, and once with acetone. The amount of 32P
incorporated into the substrate was determined by scintillation
counting.
PKB activity was determined in 10 µl of cytosol fractions or
chromatography fractions by incubation for 10 min at 30 C with 5 µl
of the phosphorylation mixture containing 13.3 µg of the peptide
KKRNRTLTK as substrate, as described above. Although these conditions
are essentially the same as for determining p70 S6 kinase activity, we
have found that the kinase activity detected in cytosol fractions from
insulin-stimulated adipocytes can, to a large extent (
95%), be
attributed to PKB with no significant contribution by p70 S6 kinase
(see Ref.16).
Activity of the MAP kinases were determined in an in-gel assay by
subjecting cytosol fractions (100 µl; equivalent to 20 µl packed
cells) to SDS-PAGE (13% polyacrylamide) (0.75-mm thick gels) with MBP
(0.5 mg/ml) in the separating gel. Subsequent to electrophoresis, gels
were treated as described by Kameshita and Fujisawa (26) and
modifications described by Cano et al. (27). The kinase
assay was initiated by addition of 10 ml buffer consisting of 40
mM HEPES, pH 8.0, 1 mM DTE, 9 mM
MgCl2, 0.2 mM EGTA, and 40 µM
[-32P]ATP (100 µCi) to the gels. After incubation at
room temperature for 1 h, the gels were washed five times with 5%
(wt/vol) trichloroacetic acid and 1% (wt/vol) sodium pyrophosphate,
dried, and 32P incorporated into MBP was visualized by
digital imaging (Fujix BAS 2000).
Kinase activity, with solubilized PDE 3B as substrate, was measured in cytosol fractions (70 µl, equivalent to 20 µl packed cells) or, in some cases, resuspended membrane fractions, as described previously (8).
Mono Q and Superdex chromatography
Cytosol fractions from control and insulin-stimulated adipocytes
(equivalent to 1.4 ml of packed cells) were chromatographed on a Mono Q
HR 5/5 FPLC column equilibrated in 50 mM TES, pH 7.5, 2
mM EGTA, 1 mM EDTA, 5% (wt/vol) glycerol, 40
mM phenyl phosphate, 5 mM NaF, 1 mM
DTE, 0.15 mM phenylmethylsulfonylfluoride, 0.05
mM sodiumvanadate (Buffer A). After a 15-ml wash, the
column was eluted with a 40-ml gradient of 0–700 mM NaCl
in buffer A at a flow rate of 1 ml/min and 1.5 ml fractions were
collected. Fractions were assayed for in vitro
phosphorylation of PDE 3B as well as subjected to SDS-PAGE and
immunoblot analysis with a 1:1000 dilution of antibodies against either
PKB, or p70 S6 kinase, or MAP kinases.
Selected fractions from the Mono Q chromatography were concentrated and subjected to gel filtration chromatography on a Superdex 75 HR 10/30 FPLC column equilibrated in buffer A containing 150 mM NaCl. The column was eluted at a flow rate of 0.5 ml/min and 0.5 ml fractions were collected. Fractions were assayed for in vitro phosphorylation of PDE 3B as well as subjected to SDS-PAGE and immunoblot analysis with antibodies against either PKB or MAP kinases.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Rapamycin was used to evaluate the role of p70 S6 kinase in the
phosphorylation/activation of PDE 3B. Pretreatment of adipocytes with
rapamycin had no effect on the insulin-induced phosphorylation (Fig. 1A
) or activation (Table 1) of PDE 3B. Furthermore, rapamycin did
not inhibit insulin-induced activation of the kinase that
phosphorylates PDE 3B (Fig. 1B). The 1.4-fold increase in activity of
PDE 3B seen in response to insulin stimulation (Table 1) is somewhat
low compared with the 1.5- to 3-fold activation previously reported for
adipocytes (8, 28, 29). An explanation for this difference could be
that the buffer we have used to homogenize the adipocytes was not
optimized to measure activation of PDE 3B and contains, e.
g. DTE, which has been shown to lower the insulin-induced
activation of PDE 3B without affecting the basal activity, thereby
lowering the fold activation (29). To confirm that rapamycin inhibited
the insulin-induced activation of p70 S6 kinase, experiments were
performed to measure kinase activity in immunoprecipitates of p70 S6
kinase. As seen in Fig. 2, insulin
induced activation of p70 S6 kinase (partial activation after 5 min and
maximal activation after 15-min stimulation), which was completely
inhibited by rapamycin. Pretreatment with wortmannin also inhibited the
insulin-induced activation of p70 S6 kinase. These results indicate
that p70 S6 kinase, although activated by insulin in a
wortmannin-sensitive manner, is not involved in the signaling pathway
that leads to phosphorylation/activation of PDE 3B.
|
|
|
). While the
insulin-induced activation of these MAP kinases was completely blocked
by wortmannin, the PMA-induced activation was unaffected by the
inhibitor. The activation of the MAP kinases in response to insulin
showed some variation between different experiments (2- to 5-fold);
however, it was always less pronounced than that induced by PMA
(8-fold).
|
), or the activation of
the kinase that phosphorylates PDE 3B (results not shown). Under the
same conditions, PD 98059 inhibited the insulin-induced activation of
MAP kinases by 71 ± 3% (mean ± SEM of three
separate experiments) (Fig. 3). The same extent of inhibition was also
seen when kinase activity was determined in immunoprecipitates of MAP
kinases (results not shown). Increasing the concentration of the
inhibitor (150 µM) or incubation time (60 min) reduced
the basal activity of MAP kinases but did not result in higher extent
of inhibition of the insulin-induced activation of MAP kinases.
Although PMA induced activation of MAP kinases to a greater extent than
insulin, it did not result in phosphorylation/activation of PDE 3B
(Fig. 1A and Table 1) or activation of the kinase that phosphorylates
PDE 3B (Fig. 1B). Using higher concentrations of PMA (450
nM) or extending the time of exposure to PMA (20 min) did
not result in activation of PDE 3B (not shown). Taken together, these
results suggest that MAP kinases are not involved in the
phosphorylation/activation of PDE 3B.
Mono Q and Superdex chromatography of the kinase that
phosphorylates PDE 3B: comparison with PKB, p70 S6 kinase and MAP
kinases
We and others (15, 16, 19) have recently shown that PKB is
activated in a wortmannin-sensitive manner in intact adipocytes in
response to insulin stimulation and could thus be involved in the
signaling pathway leading to phosphorylation/activation of PDE 3B.
Because inhibitors of PKB are not yet available, the role of PKB in the
insulin-induced antilipolytic pathway cannot easily be investigated in
the intact cell. To investigate the role of PKB as a PDE 3B kinase,
attempts were made to immunoprecipitate PKB from adipocytes to be used
in an immunocomplex kinase assay with PDE 3B as substrate. Despite the
use of several different antibodies against PKB, significant
immunoprecipitation of PKB from adipocytes was not successful although
these antibodies immunoprecipitated PKB from extracts of 3T3-L1 cells,
rat liver, and PKB expressed in yeast (16). However, it has not been
possible to obtain activated PKB from these sources. Therefore, to
evaluate the possibility that PKB is the kinase that phosphorylates PDE
3B in vitro, other approaches were used.
Mono Q and Superdex chromatrographies were performed and the elution
profiles of PKB and the kinase that phosphorylates PDE 3B were
compared. As shown in Fig. 4A, chromatography of cytosol fractions from control and insulin-stimulated
adipocytes on a Mono Q column revealed insulin-stimulated kinase
activity, assayed with PDE 3B as substrate, eluting as a single peak at
approximately 0.15 M NaCl (mainly fractions 19 and 20). No
kinase activity phosphorylating PDE 3B was detected in Mono Q fractions
from control cells. Immunoblot analysis of the Mono Q fractions with
antibodies against PKB (Fig. 4B) revealed elution of PKB from
insulin-stimulated cells in the same fractions as the kinase that
phosphorylates PDE 3B. These fractions also showed increased PKB
activity (see legend to Fig. 4). As noted previously, PKB protein from
insulin-stimulated cells eluted at a higher salt concentration from the
Mono Q column and showed reduced electrophoretic mobility on SDS-PAGE
compared with PKB from control cells (16). The reduced electrophoretic
mobility of stimulated PKB has been linked to phosphorylation of the
kinase (31). Reprobing the membranes with antibodies against MAP
kinases (Fig. 4D) revealed MAP kinases eluting in the same fractions as
PKB (fractions 19 and 20). p70 S6 kinase eluted later in the gradient
(fractions 24–26) (Fig. 4C), and no kinase activity phosphorylating
PDE 3B was detected in these Mono Q fractions (not shown). The
inability of p70 S6 kinase to phosphorylate PDE 3B in vitro
is consistent with the finding that rapamycin had no effect on the
insulin-induced phosphorylation/activation of PDE 3B in vivo
(Fig. 1A and Table 1).
|
) and the kinase that phosphorylates
PDE 3B (Fig. 5A) in fraction 21 (corresponding to a molecular mass of
about 60 kDa), whereas MAP kinases eluted in fractions 22 and 23 (Fig. 5C). Thus, from two consecutive chromatographies, the kinase that
phosphorylates PDE 3B co-eluted with PKB, suggesting that PKB is
responsible for the phosphorylation of PDE 3B.
|
, peroxovanadate
stimulation of adipocytes induced a wortmannin-sensitive translocation
to membranes of PKB (cf 16) as well as of the kinase that
phosphorylates PDE 3B. The concentration of peroxovanadate was chosen
to be in the range where wortmannin pretreatment did not efficiently
prevent peroxovanadate-induced activation of PKB and the kinase that
phosphorylates PDE3B (see Discussion). Insulin stimulation
also resulted in activation of PKB as well as the kinase that
phoshorylates PDE 3B; however, insulin stimulation did not induce
translocation to membranes (Fig. 6, lane 2). MAP kinases have been
shown to translocate to the nucleus in response to growth factor
stimulation (35). Immunoblotting of cytosol and membrane fractions from
adipocytes with antibodies against MAP kinases revealed MAP kinases
almost exclusively (more than 95%) in the cytosol fraction, with no
indication of translocation to membranes in response to insulin or
peroxovanadate stimulation (results not shown). Thus, data from the
chromatographies and the subcellular translocation experiments strongly
suggest that PKB is the kinase that phosphorylates PDE 3B.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
From the present work, we conclude that p70 S6 kinase is not involved in this signaling pathway because rapamycin did not prevent the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B, whereas it completely blocked the insulin-induced activation of p70 S6 kinase. We also conclude that MAP kinases are not involved in the insulin-induced signaling leading to activation of PDE 3B. The specific MAP kinase kinase inhibitor PD 98059 did not prevent insulin-induced activation of PDE 3B under conditions where the inhibitor blocked 70% of the insulin-induced activation of MAP kinases. However, we find it less likely that the residual MAP kinase activity is able to mediate maximal phosphorylation/activation of PDE 3B because extensive MAP kinase activation induced by PMA was not associated with phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B in vitro. In 3T3 cells (30) and 3T3-L1 adipocytes (37), the insulin-induced activation of MAP kinases has been shown to be completely inhibited by PD 98059. We do not know the reason for the incomplete inhibition of the activation of MAP kinases in primary adipocytes, but one possibility could be that these cells also contains other isoforms of MAP kinase kinase, such as MAP kinase kinase 2, which has been shown to be less sensitive to inhibition by PD 98059 (30).
Our data reported here strongly suggest that the insulin-stimulated kinase that phosphorylates PDE 3B in vitro is PKB. A rapid, reversible, and wortmannin-sensitive activation of PKB in response to insulin stimulation of primary adipocytes has recently been reported by us and others (15, 16, 19). Translocation has been suggested to be important in the activation mechanism of PKB that most likely is due to association between the pleckstrin homology domain of PKB and 3-phosphorylated phosphoinositides generated by PI 3-kinase (11). The 3-phosphorylated phosphoinositides have also been suggested to induce a conformational change of PKB leading to exposure of sites that are phosphorylated by upstream kinase(s) resulting in activation of PKB (38, 39). In a previous study we investigated the peroxovanadate-induced activation and membrane translocation of PKB under conditions where wortmannin pretreatment prevented both of these processes (16). However, we have also found that by increasing the concentration of peroxovanadate, eventually the activation of PKB cannot be prevented by wortmannin pretreatment of the adipocytes (results not shown). This is most likely due to the ability of high concentrations of peroxovanadate to induce dramatic translocation of PI 3-kinase to insulin receptor substrate 1, resulting in high activation of PI 3-kinase (Castan, I., unpublished results) and thereby presumably generation of large amounts of 3-phosphorylated phosphoinositides. The concentration of peroxovanadate that induces the apparent wortmannin-insensitive activation of PKB varies between preparations of peroxovanadate and is probably due to the formation of different amounts of peroxovanadate when mixing vanadate and H2O2 but is usually found in the range of 50 µM. In the present study, activation of PKB and the kinase that phosphorylates PDE 3B was induced by a high peroxovanadate concentration and pretreatment with wortmannin did not significantly prevent the activation of PKB (measured as reduction of electrophoretic mobility) or the activation of the kinase that phosphorylates PDE 3B but still prevented peroxovanadate-induced translocation. This suggests that the translocation process detected requires formation of more 3-phosphorylated phosphoinositides than the activation mechanism. Although insulin stimulation did not induce detectable translocation of PKB to membranes, it is possible that it occurs but is transient or reversed during homogenization and therefore difficult to detect.
Although information regarding the activation of PKB by insulin and growth factors has been obtained in a variety of cells, little is known about downstream targets for PKB. The first physiological substrate reported for PKB was glycogen synthase kinase-3 (40). Glycogen synthase kinase-3 is inactivated as a result of phosphorylation by PKB in vitro (40), and in L6 myotubes insulin-induced increase in glycogen synthesis has been suggested to be partially mediated by this mechanism (41). Most recently, it was reported that heart 6-phosphofructo-2-kinase was phosphorylated and activated by PKB in vitro (42). 6-phosphofructo-2-kinase catalyses the formation of fructose 2,6-bisphosphate, a key allosteric activator of 6-phosphofructo-1-kinase and the rate-limiting enzyme in glycolysis. A role for PKB in the translocation of Glut 4 to plasma membrane and increased glucose uptake has been suggested in 3T3-L1 adipocytes (43) and most recently also in rat adipocytes (44). The involvement of PKB is not restricted to metabolic actions because it has also been implicated in cell growth (45) and differentiation (43, 46) and data support that p70 S6 kinase is downstream of PKB (47), although not a direct substrate for PKB.
The conclusion that PDE 3B is a substrate for PKB in vitro is supported by results using a combination of methodologies. First, we found that during two consecutive chromatographies, PKB comigrated with the kinase that phosphorylates PDE 3B. Furthermore, upon peroxovanadate stimulation of adipocytes, PKB as well as the kinase that phosphorylates PDE 3B translocated from cytosol to membranes in a wortmannin-sensitive manner. So far we have not been able to detect activation of PDE 3B as a result of in vitro phosphorylation by PKB (Rahn Landström, T., unpublished results). One explanation could be that in addition to serine 302, which is the only site phosphorylated in response to insulin stimulation of intact cells and is associated with activation of the enzyme (5), also, other sites are phosphorylated in vitro by partially purified PKB (Rahn Landström, T., unpublished results), which could interfere with the activation of PDE 3B. It is also possible that activation of PDE 3B requires additional components such as lipids and proteins which are not present in the in vitro assay. Although our data support that PKB is the kinase that phosphorylates PDE 3B in vitro, the importance of this action of PKB in intact adipocytes remains to be established. This requires specific cell-permeable PKB inhibitors, dominant negative PKBs, or constitutively active PKB to be transfected into adipocytes or 3T3-L1 adipocytes. Such experiments have been initiated.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received June 3, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Curr Biol 7:261–269[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  W. L. Holland and S. A. Summers Sphingolipids, Insulin Resistance, and Metabolic Disease: New Insights from in Vivo Manipulation of Sphingolipid Metabolism Endocr. Rev., June 1, 2008; 29(4): 381 - 402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang and R. W. Colman Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A Blood, September 1, 2007; 110(5): 1475 - 1482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhao, J. Quilley, D. C. Montrose, S. Rajagopalan, Q. Guan, and C. J. Smith Differential effects of phosphodiesterase PDE-3/PDE-4-specific inhibitors on vasoconstriction and cAMP-dependent vasorelaxation following balloon angioplasty Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2973 - H2981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Towler and D. G. Hardie AMP-Activated Protein Kinase in Metabolic Control and Insulin Signaling Circ. Res., February 16, 2007; 100(3): 328 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, L. A. Reaves, and N. K. Edens Ginseng Extract Inhibits Lipolysis in Rat Adipocytes In Vitro by Activating Phosphodiesterase 4 J. Nutr., February 1, 2006; 136(2): 337 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Snyder, J. M. Esselstyn, K. Loughney, S. L. Wolda, and V. A. Florio The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis J. Lipid Res., March 1, 2005; 46(3): 494 - 503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Akesson, B. Ahren, V. C. Manganiello, L. S. Holst, G. Edgren, and E. Degerman Dual Effects of Pituitary Adenylate Cyclase-Activating Polypeptide and Isoproterenol on Lipid Metabolism and Signaling in Primary Rat Adipocytes Endocrinology, December 1, 2003; 144(12): 5293 - 5299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Yin, J. Mu, and M. J. Birnbaum Role of AMP-activated Protein Kinase in Cyclic AMP-dependent Lipolysis In 3T3-L1 Adipocytes J. Biol. Chem., October 31, 2003; 278(44): 43074 - 43080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Cohen, B. Razani, X. B. Wang, T. P. Combs, T. M. Williams, P. E. Scherer, and M. P. Lisanti Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue Am J Physiol Cell Physiol, July 1, 2003; 285(1): C222 - C235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Barry and J. M. Gibbins Protein Kinase B Is Regulated in Platelets by the Collagen Receptor Glycoprotein VI J. Biol. Chem., April 5, 2002; 277(15): 12874 - 12878. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. O’Rourke, S. J. Yeaman, and P. R. Shepherd Insulin and Leptin Acutely Regulate Cholesterol Ester Metabolism in Macrophages by Novel Signaling Pathways Diabetes, May 1, 2001; 50(5): 955 - 961. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. ZAULI, D. MILANI, P. MIRANDOLA, M. MAZZONI, P. SECCHIERO, S. MISCIA, and S. CAPITANI HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway FASEB J, February 1, 2001; 15(2): 483 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Conti Phosphodiesterases and Cyclic Nucleotide Signaling in Endocrine Cells Mol. Endocrinol., September 1, 2000; 14(9): 1317 - 1327. [Full Text]
|
|
|
|
 |
|
 F. Ahmad, L.-N. Cong, L. Stenson Holst, L.-M. Wang, T. Rahn Landstrom, J. H. Pierce, M. J. Quon, E. Degerman, and V. C. Manganiello Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells J. Immunol., May 1, 2000; 164(9): 4678 - 4688. [Abstract] [Full Text] [PDF]
|
|
