0013.7227/92/1311-0441$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 131, No. 1 Printed in U.S.A.
Society
Stimulatory Effect of Vanadate on 3’,5’-Cyclic Guanosine Monophosphate-Inhibited Low MichaelisMenten Constant 3’,5’-Cyclic Adenosine Monophosphate Phosphodiesterase Activity in Isolated Rat Fat Pads HIROSHI NOBUAKI
UEKI, RYUJI TOMINAGA,
OKUHAMA, MISAKI SERA, TOMOHIRO AND TETSUO MORITA
Department Fukuyama,
of Biochemistry, Faculty of Pharmacy Hiroshima 729-02, Japan
and Pharmaceutical
INOUE, Sciences, Fukuyama
University,
ABSTRACT When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (K,) CAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dosedependent manner with vanadate. 3’,5’-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulinstimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K’, Na+, or Ca” in the medium, while preincubation of the fat pads with a chelator of intracellular Ca*+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results
suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca*+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3’,5’cyclic GMP-inhibited low K, CAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes. (Endocrinology 131: 441-446, 1992)
V
cytes, and the insulin-stimulated PDE activity is inhibited by 3’,5’-cyclic GMP (cGMP) at a low concentration (14-17). Mechanisms of the stimulatory effect of vanadate on the PDE activity in fat pads or adipocytes are unknown. In the present paper, we show that vanadate, as well as
ANADIUM is an essential trace element for animals owing to its physiological and biochemical activities (l-3). Vanadium compounds mimic many in vitro and in viva effects of insulin including the stimulation of glucose transport, the suppression of the hormone-dependent lipolysis, the stimulatory phosphorylation of insulin receptor in isolated rat adipocytes, and the normalization of blood glucoselevels of streptozotocin-induced diabetic rats (4-9). We previously reported that sodium orthovanadate (vanadate) increased the lipoprotein lipase (LPL) activity in isolated rat fat pads and stimulated the release of LPL activity from the fat pads into medium (10, 11). The increasing effect of vanadate on the LPL activity was almost completely inhibited by adrenaline, (Bu)~cAMP, or 3-isobutyl-1-methylxanthine which was a potent inhibitor of the cyclic nucleotide phosphodiesterase (PDE) activity in adipocytes (11, 12). These results suggest that the action of vanadate is inhibited with an increase in the intracellular level of CAMP. It is known that many different PDEs selectively catalyze the hydrolysis of cyclic nucleotide but it still is not known precisely how many different PDE isozymes are present in most mammalian tissues(13). Of these many PDEs, insulin stimulates the low Michaelis-Menten constant (K,) CAMP PDE activity in the microsomal fraction of isolated rat fat pads and adipo-
insulin,
stimulates
the
cGMP-inhibited
low
K,
CAMP
PDE
activity in the microsomal fraction of rat fat pads, and that the stimulatory effect of vanadate is dependent on the intracellular level of Ca*+ and, in part, associated with tyrosine kinase- and protein kinase C (PKC)-mediated processes.In addition, we discuss that the increasing effect of vanadate on the LPL activity is not due to the stimulation of PDE activity by vanadate. Materials
and Methods
Materials The sources of chemicals used in this work were as follows: Vanadate (Na3V04), collagenase (Clostridium histolyticum, 300 U/m&, and tetrakis (acetoxymethyl)ester of quin 2 (quin 2-AM) from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan); insulin (bovine pancreas, 24.4 IU/mg), amiloride, biochanin A, cGMP, and Crotalus atrox snake venom from Sigma Chemical Co. (St. Louis, MO); CAMP, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan); BSA (Fraction V) from Boehringer-Mannheim Biochemicals (Mannheim, Germany); genistein from K & K Laboratories (Cleveland, OH); [2,8-3H]cAMP (1.158 TBq/mmol) from New England Nuclear (Boston, MA); ACS II and Protein kinase C enzyme assay kit from Amersham Japan Co. (Tokyo, Japan). Staurosporine was a gift from Dr.
Received January 13, 1992. Address all correspondence and requests for reprints to: Dr. H. Ueki, Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-02, Japan. 441
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442
STIMULATION
OF PHOSPHODIESTERASE
Y. Matsuda of Institute of Tokyo, Kyowa Hakko Co. Ltd. (Tokyo, All other chemicals used were of analytical grade.
Japan).
Animals Male Wistar rats weighing ZOO-220 g were obtained from Shimizu Laboratory Supplies Co. (Kyoto, Japan), fed on a commercial laboratory chew ad libitum, and given free access to water for 1 week. All animals were starved for 24 h before use.
Preparation of fat pads and adipocytes vanadate or insulin
and incubation
with
Epididymal adipose tissue was quickly removed from rats killed under ether anesthesia and cut into small pieces (30-40 mg) with scissors, Stock solutions of vanadate (100 mM, pH 7.4) and insulin (174 mM, pH 3) were diluted with deionized water to the desired concentration immediately before use. The fat pads (1 g) were incubated with 2 rnM vanadate or 3 nM insulin in 10 ml Krebs-Ringer bicarbonate buffer containing 5 mM glucose and 2% BSA, pH 7.4 (KRBGA) at 37 C for 60 min. Adipocytes were prepared as described by Rodbell (18). Briefly, the fat pads (0.7 g) were digested with collagenase (2.5 mg) in 2 ml KRBGA at 37 C for 60 min. The dispersed cells were filtered through nylon mesh, washed three times with fresh buffer. The freshly prepared adipocytes (0.2 g) were incubated with 2 rnM vanadate in 2 ml KRBGA at 37 C for 60 min. The fat pads and adipocytes incubated were washed three times with physiological saline at 4 C and used as materials of next step.
Preparation
of subcellular
fractions
Subcellular fractions were prepared by a slight modification of the method of Kono et al. (17). Briefly, the fat pads incubated were homogenized with a Potter-Elvejehm homogenizer at three strokes and 500 rpm in 2 ml 10 mM Tris-HCl buffer containing 1 rnM EDTA, 0.1 M KCl, and 0.25 M sucrose, pH 7.4. After centrifugation at 1,000 x g for 10 min, the infranatant was further centrifuged at 12,000 x g and 17,500 x g for 4.5 min each to remove a mitochondrial fraction and at 105,000 X 8 for 60 min to separate a microsomal fraction, The adipocytes incubated were homogenized with a Dounce tissue grinder at eight strokes in 5 ml 10 rnM Tris-HCI buffer, pH 7.4, containing 0.25 M sucrose. The homogenate defatted by centrifugation at 1,000 x g for 10 min was further centrifuged at 20,000 X g for 2 min and 30,000 X g for 30 min to separate a crude microsomal fraction.
of low K, CAMP PDE activity
The PDE activity was determined by a slight modification of procedure reported by Kono et al. (17). Briefly, microsomal fractions (25 pg protein) were incubated with 250 nM [3H]cAMP (0.9 KBq) in 0.25 ml 33 mM Tris-HCl buffer containing 4 rnM MgClz, pH 7.4, at 30 C for 5 min. The incubation was terminated by addition of 0.1 ml 0.1 N HCI. A mixture (0.05 ml) of 5 mM AMP and CAMP was added to the incubated solution, allowed to stand at 70 C for 4 min, cooled, and neutralized with 0.1 ml 0.1 N NaOH. After the addition of 50 PLg snake venom in 0.05 ml 0.1 M Tris-HCI buffer, pH 8.0, to the neutralized solution, the incubation was carried out at 37 C for 20 min and terminated by the addition of a mixed solution (0.05 ml, pH 7.0) of 200 rnM sodium EDTA and 5 mM adenosine. The reaction mixture (0.5 ml) was applied to a 5.5 X 30 mm column of Dowex l-X8 (200-400 mesh in chloride form from Bio-Rad, Richmond, CA), which had been washed with water and eluted with water. The first 1.4 ml of the effluent was discarded, and the next 2.5 ml was collected. The radioactivity of adenosine contained in the 2.5 ml fraction was determined in ACS II with an Aloka liquid scintillation counter (LSC-700). Protein was determined by the method of Lowry et al. (19). The PDE activity was expressed as picomoles of CAMP hydrolyzed per minute per milligram protein.
Determination
of PKC activity
The fat pads (0.5 g) were incubated with vanadate (O-10 mM) in 2 ml KRBGA at 37 C for O-150 min and homogenized in 1 ml 10 mM Tris-
1992 No 1
HCl buffer, pH 7.4, containing 1 rnM EDTA, 0.1 M KCl, and 0.25 M sucrose. After centrifugation, a microsomal fraction obtained was dissolved in 100 ~150 mM Tris-HCI buffer, pH 7.5, containing 5 rnM EDTA, 10 mM EGTA and benzamidine, 0.3% fl-mercaptoethanol, and 0.005% phenylmethylsulfonyl fluoride. The PKC activity was determined with a Protein kinase C enzyme assay system kit, essentially according to the method supplied by the manufacturer (Amersham International plc, Arlington Heights, IL), except that contents of L-a-phosphatidyl-L-serine and phorbol 12.myristate 13.acetate were reduced by one-tenth. The activity was expressed in terms of picomole substrate phosphorylated per minute per milligram protein.
Statistical
analysis
Results are shown separated experiments. unpaired observations.
as mean t SE of four The data were analyzed
observations by Student’s
for three t test for
Results First, kinetic constants of the vanadate-stimulated PDE activities were compared between both microsomal fractions of the fat pads and adipocytes (Table 1). Increases in maximum velocity (V,,,) by vanadate were 1.6- and 2.0-fold on the fat pads and adipocytes, respectively. No appreciable change in K,, values by vanadate was observed with both microsomal fractions. cGMP (0.01-10 PM) inhibited the vanadate-stimulated PDE activity of fat pads with similar profile to the insulin-stimulated one (data not shown). Thus, the stimulatory effect of vanadate appears not to be different between the fat pads and adipocytes. Therefore, the microsomal fraction of fat pads was used as the source of PDE through this study. Figure 1 shows changes in the PDE activity in the fat pads TABLE fractions
1. Kinetic of adipocytes
constants of PDE activity in microsomal and fat pads treated with or without vanadate
Km (PM) Control
Determination
Endo. Voll31.
BY VANADATE
Adipocytes Fat pads
V,,.
Control
Vanadate
0.55 0.59
162 248
326 406
The adipocytes and fat pads were incubated vanadate in KRBGA for 60 min. K, and V,,, from double reciprocal plots of the PDE activity concentrations of 0.17-2 PM.
30
mg)
Vanadate
0.59 0.57
0
(pmol/min.
60
Incubation
90 time
FIG. 1. Time course of stimulatory effect PDE activity. The fat pads were incubated nM insulin or without either one for O-150
with or without 2 mM values were calculated in a range of substrate
120
150
(min) of vanadate with 2 mM min.
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or insulin on vanadate or 3
STIMULATION
OF PHOSPHODIESTERASE
incubated with 2 mM vanadate or 3 nM insulin up to 150 min. The PDE activity was increased 1.5- and 1.8-fold by vanadate with 5- and 30-min incubation periods, respectively, then declined with 120- and 150-min incubation ones. Insulin almost linearly increased the PDE activity up to 150 min. Time course of the vanadate action showed a profile differing from that of the insulin action. When the fat pads were incubated for 60 min with vanadate over the concentration range of 0.5-10 mM, the PDE activity was increased in a dose-dependent manner up to 10 mM (Fig. 2). To determine whether activations of the insulin receptor and/or Na+/H+ exchange system are involved in the stimulatory effect of vanadate on the PDE activity, the fat pads pretreated with amiloride, biochanin A, or genistein were incubated with vanadate or insulin (Table 2). A significant inhibition of both actions of vanadate and insulin was observed with three inhibitors. Furthermore, the requirement for potassium, sodium, or calcium in the medium in which the fat pads were incubated with vanadate or insulin was investigated. No change in both actions of vanadate and insulin was found by replacement of Na’ with choline ion, addition of Na’ instead of K+, or omission of Ca2+ in the medium (data not shown). These results suggest that vana-
BY VANADATE
443
date increases the PDE activity in the fat pads through mechanismsinvolving activation of the insulin receptor but not the Na’/H+ exchange system. Next, the fat pads were loaded with quin 2-AM to remove intracellular Ca*+. The stimulatory effect of vanadate was progressively decreasedwith an increasein the concentration of quin 2-AM (Fig. 3). That of insulin was not decreasedat tested concentrations of quin 2-AM. The incubation with 100 FM quin 2-AM had no effect on the basal activity. Thus, the stimulatory effect of vanadate, in contrast to that of insulin, was dependent on the intracellular concentration of Ca’+. When the fat pads were preincubated with H-7, the stimulatory effect of vanadate was progressively decreasedwith an increasein the concentration of H-7 (Fig. 4). A significant inhibition of the vanadate action was observed with staurosporine at concentrations of 3 and 10 nM (data not shown). The stimulatory effect of insulin was not inhibited by inhibitors of PKC. When the fat pads were incubated with vanadate in the presence of insulin, the stimulatory effect of vanadate was additive to that of insulin (Table 3). These results suggest that vanadate exerts its action through mechanismsdiffering
0 Control0 Vanadatc A Insulin (3nM)
1
PmW
I 0
I
I
I
I
2
4
6
8
vanadatc
TABLE
2. Effects of inhibitors of tyrosine activity by vanadate or insulin
effect of vanadate on PDE vanadate at the indicated
kinases
PDE activity Inhibitors
Control
Biochanin
Genistein
on stimulation
(pmol/min
A
0 bM) 1 2 0 (he/ml) 100 250 0 b.dml) 5 25
63 62 61 66 67 64 60 59 59
f 0.8 f 0.7 + 1.0 xi 0.9 + 2.1 + 1.3 k 1.3 AZ 1.1 + 1.7
Vanadate
171 129 112 133 137 62 127 92 59
k + + + f + + + +
80
100
(PM)
3. Effect of quin 2-AM loading on vanadateor insulin-stimulated PDE activity. ” Quin - 2-AM was dissolved in dimethvl sulfoxide and 50 ~1 of it was added to the incubation mixture. The fat pads were preincubated with quin 2-AM at the indicated concentrations in 10 ml KRBGA containing 10 mM sodium pyruvate for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min.
FIG.
0
Insulin
8.6 3.2” 7.1b 3.8 4.9 0.9* 1.6 2.3” 1.8’
60 2-AM
mg)
Concentration
Amiloride
40 Quin
(mM)
2. Dose-response curve for stimulatory activity. The fat pads were incubated with concentrations for 60 min. FIG.
of PDE
20
0
10
93 69 61 104 98 62 95 76 62
+ + f + + f + -c +
Control
l
Vanadatc
10
20
30
2.0 0.7” 1.7* 3.2 0.6 1.4’ 1.7 2.4 l.Ob
Inhibitors were dissolved in dimethyl sulfoxide and their 50-111 was added to the incubation mixture. The fat pads (1 g) were preincubated with inhibitors at the indicated concentrations in 10 ml KRBGA for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min. n P < 0.05. * P < 0.01 compared to vanadateor insulin-stimulated group without inhibitors.
H-7
40
50
(M)
FIG. 4. Effect of H-7 on vanadateor insulin-stimulated PDE activity. H-7 was dissolved in dimethyl sulfoxide and 20 ~1 of it was added to the incubation mixture. The fat pads were preincubated with H-7 at the indicated concentrations in 10 ml KRBGA for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min.
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444 TABLE presence
STIMULATION 3. PDE activity in fat pads or absence of insulin
incubated
PDE activity Without Control Vanadate (0.5 mM) (2 n-1
OF PHOSPHODIESTERASE
with
vanadate
(pmol/min~
insulin
Endo. Vol 131.
With
in the
insulin
69 + 0.7
95 f 0.8
76 + 0.6 132 k 1.8
108 + 0.5” 147 + 0.8”
I
in the presence
I
20 -
F----t
I
J
0 Control 0 Vanad;rtc (2mM) A Insulin (3nM)
\
0
I 2
I
4 Vanadatc
6 (mM)
8
FIG. 6. Dose-response curve for stimulation date. The fat pads were incubated with concentrations for 60 min.
of PKC vanadate
TABLE
on stimulation
4. Effects by vanadate
activity
Inhibitors
of H-7
and amiloride
30
60 Incubation
90 time
FIG. 5. Time course of stimulation of PKC fat pads were incubated with 2 mM vanadate either one for O-150 min.
120 (min)
150
activity by vanadate. The or 3 nM insulin or without
from the action of insulin. Next, we examined whether or not changes in PKC activity are involved in mechanisms of the action of vanadate. The standard activity of PKC was determined to be 49.3 pmol/ min.mg in the nontreated fat pads with a complete assay system kit. A small increase in the activity (7 pmol/min. mg) was observed with vanadate-treated fat pads. When the kit omitted phorbol 12-myristate 13-acetate, an activator of PKC, used for the assay also, vanadate showed a slight increase in the activity from 1 to 2.3 pmol/min. mg in the fat pads. Therefore, the appropriate concentration of the activator (4 mM) for the assay was used through all determinations of the PKC activity. Incubation of the fat pads with 2 mM vanadate increased 2.5-fold the PKC activity with 60and 120-min incubation periods (Fig. 5). In contrast, insulin never increased the PKC activity. When the fat pads were incubated for 60 min with vanadate over the concentration range of 0.5-10 mM, the PKC activity was increased in a dose-dependent manner up to 2 mM (Fig. 6). Preincubation of the fat pads with H-7 or amiloride showed a significant inhibition of stimulation of the PKC activity by vanadate (Table 4). These results suggest that vanadate stimulates the PKC activity in fat pads via an amiloride-sensitive process. Discussion The insulin-stimulated PDE is reported to be a membranebound enzyme which is recovered in a crude microsomal
H-7
Amiloride
0 (FM) 5 20 0 (mM) 1 2
10 activity at the
by vanaindicated
of PKC
PKC activity (pmol/min. mg)
Concentration
Control 0
1992 No 1
mg)
The fat pads were incubated with vanadate for 60 min or absence of 3 nM insulin. ’ P < 0.05 compared to groups without insulin.
G \E .e
BY VANADATE
8.8 7.5 8.0 11.8 11.6 11.2
f + + ++ +
Vanadate 1.2 0.7 0.3 0.2 1.4 1.2
17.7 14.4 11.8 22.4 19.8 13.6
f + f + + +
1.8 1.0” 0.8’ 1.2 0.2” 0.2*
The fat pads (0.8 g) were preincubated with inhibitors at the indicated concentrations in 5 ml KRBGA for 15 min and further incubated with 2 mM vanadate for 60 min. o P < 0.05. b P < 0.01 compared to vanadate-stimulated group without inhibitors.
fraction, probably endoplasmic reticulum fraction, of rat adipocytes (17, 20, 21). It also is designated as the type IV PDE which is calmodulin-insensitive and inhibited by cGMP at a low concentration (22-24). The incubation of fat pads with vanadate stimulated the PDE activity in the microsomal fraction but neither mitochondrial nor cytosolic one (data not shown). The vanadate-stimulated PDE in microsomal fraction of fat pads, as well as that of adipocytes, had a low K, value toward CAMP and its activity was inhibited by cGMP at a low concentration. These results suggest that vanadate, as well as insulin, stimulates the cGMP-inhibited low K, CAMP PDE activity in the microsomal fraction. However, whether vanadate stimulates the insulin-stimulated PDE itself or its isoforms remains to be elucidated. Amiloride, an inhibitor of activation of the Na+/H+ exchange system and the tyrosine kinase activities of insulin and growth factor receptors (25-28), and biochanin A and genistein, inhibitors of tyrosine kinase activity of epidermal growth factor receptor (29), all inhibited the stimulation of PDE activity by vanadate or insulin to various extents. Both actions of vanadate and insulin were well preserved with incubation of the fat pads in the absence of either K’, Na+, or Ca2+ in the
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STIMULATION
OF PHOSPHODIESTERASE
medium. It is reported that vanadate, as well as insulin, stimulates selective phosphorylation of tyrosine residues in a 95-kilodalton insulin receptor subunit of rat adipocytes to produce an intracellular event such as the activation of glycogen synthase (4). Our results, therefore, suggest that the action of vanadate is associated with stimulation of tyrosine kinase activity of insulin receptor and is independent of activation of the Na+/H+ exchange system. Vanadate is a potent inhibitor of Naf, K+-adenosine triphosphatase (3). However, inhibition of the enzyme by vanadate is not involved in mechanisms of the insulinmimetic actions of vanadate such as the stimulation of hexose transport and the activation of glycogen synthase (5). Our result, obtained with the treatment of fat pads in K+-free medium, shows that the action of vanadate is not due to inhibition of the activity of Naf, K’-adenosine triphosphatase. When the fat pads were loaded with quin 2-AM, a chelator of intracellular Ca*+ (30), the stimulation of PDE activity by vanadate was decreased in a dose-dependent manner. The stimulatory effect of insulin, on the contrary, was not decreased at tested concentrations of quin 2-AM. These results show that the action of vanadate is dependent on the intracellular concentration of Ca”. PKC is a Ca2+- and phospholipid-dependent enzyme and plays an important role in the transduction of extracellular signalsinto intracellular events and of cellular proliferation in many tissues(31-34). The enzyme is activated by diacylglycerol which is transiently produced from inositol phospholipids in responseto extracellular signals. Several workers failed to demonstrate the effect of insulin on the PKC activity and the subcellular distribution of the enzyme in isolated and cultured adipocytes (35-37), although insulin increased the PKC activity in rat diaphragms (38). Phorbol ester is a tumor-promoting agent known to act through PKC (39) and activates the low K, CAMP PDE in fat pads (40). Our results were that the stimulation of PDE activity by vanadate was in part inhibited by H-7 and staurosporine, potent inhibitors of PKC (41-43), but the action of insulin was not inhibited. Furthermore, vanadate increased PKC activity in the fat pads and showed the maximum effect with incubations at concentrations of 2 and 5 mM for 60-120 min. Insulin never showed any stimulation of PKC activity in fat pads. Preincubation of the fat pads with H-7 or amiloride showed a significant inhibition of the stimulation of PKC activity by vanadate. Theseresults suggestthat a PKC-mediated processis, in part, involved in the stimulation of PDE activity by vanadate. Recent reports show that addition of the purified catalytic subunit of CAMP-dependent protein kinase to the crude microsomal fractions, containing the low K, CAMP PDE, of rat adipocytes produces a 2-fold activation of the enzyme activity (44), that incubation of rat adipocytes with insulin induces serine phosphorylation of the low K, CAMP PDE via the activation of an unidentified serine protein kinase (45), and that a protein kinase, which is more H-7-resistance than CAMP-dependent protein kinase, is involved in the stimulation of PDE activity by insulin (46). Thus, a protein kinase associated with the insulin action on stimulation of the low K, CAMP PDE activity appears to be different from
BY VANADATE
445
that involved in the vanadate action. In contrast to the stimulatory effect of vanadate on the LPL activity, the action of vanadate on the PDE activity was markedly inhibited by quin 2-AM loading and, in part, inhibited by amiloride. The requirement for Na+ or Ca*+ in the medium differed between actions of vanadate on the LPL and PDE activities (11). These results suggest that the increasing effect of vanadate on the LPL activity is not due to the stimulation of the PDE activity by vanadate. In conclusion, vanadate appears to stimulate in part the cGMP-inhibited low K, CAMP PDE activity in the microsoma1 fraction through the activation of tyrosine kinase- and protein kinase C-mediated processes. References
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15.
16.
17. 18.
Nechay BR 1984 Mechanisms of action of vanadium. Annu Rev Pharmacol Toxicol24:501-524 Chasteen ND 1983 The biochemistry of vanadium. Struct Bonding 53:105-138 Ramasarma T, Crane FL 1981 Does Vanadium play a role in cellular regulation? Curr Top Cell Regul 20:247-301 Tamura S, Brown TA, Dubler RE, Larner J 1983 Insulin-like effect of vanadate on adipocyte glycogen synthase and on phosphorylation of 95,000 dalton subunit of insulin receptor. Biochem Biophys Res Commun 113:80-86 Tamura S, Brown TA, Whipple JH, Fujita-Yamaguchi Y, Dubler RE, Cheng K, Larner J 1984 A novel mechanism for the insulinlike effect of vanadate on glycogen synthase in rat adipocytes. J Biol Chem 259:6650-6658 Duckworth WC, Solomon SS, Liepnieks J, Hamel FG, Hand S, Peavy DE 1988 Insulin-like effects of vanadate in isolated rat adipocytes. Endocrinology 122:2285-2289 Heyliger CE, Tahiliani AG, McNeil1 JH 1985 Effect of vanadate on-elevated blood glucose and depressed cardiac performance of diabetic rats. Science 227:1474-1477 Meyerovitch J, Farfel Z, Sack J, Shechter Y 1987 Oral Administration of vanadate normalizes blood glucose levels in streptozotocintreated rats. J Biol Chem 262:6658-6662 Bollen M, Miralpeix M, Ventura F, Toth B, Bartrons R, Stalmans W 1990 Oral administration of vanadate to streptozotocin-diabetic rats restores the glucose-induced activation of liver glycogen synthase. Biochem J 267:269-271 Ueki H, Sera M, Tanaka K 1989 Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate. Arch Biochem Biophys 272:18-24 Sera M, Tanaka K, Morita T, Ueki H 1990 Increasing effect of vanadate on lipoprotein lipase activity in isolated rat fat pads. Arch Biochem Biophys 279:291-297 Beavo JA, Rogers NL, Crofford OB, Hardman JG, Sutherland EW, Newman EV 1970 Effects of xanthine derivatives on lipolysis and on adenosine 3’,5’-monophosphate phosphodiesterase activity. Mol Pharmacol 6:597-603 Beavo J 1990 Multiple phosphodiesterase isoenzymes: Background, nomenclature and implications. Mol Pharmacol Cell Regul 2:3-15 Loten EG, Sneyd JGT 1970 An effect of insulin on adipose-tissue adenosine 3’:5’-cyclic monophosphate phosphodiesterase. Biochem J 120:187-193 Manganiello V, Vaughan M 1973 An effect of insulin on cyclic adenosine 3’:5’-monophosphate phosphodiesterase activity in fat cells. J Biol Chem 248:7164-7170 Zinman B, Hollenberg CH 1974 Effect of insulin and lipolytic agents on rat adipocyte low Km cyclic adenosine 3’5.monophosphate phosphodiesterase. J Biol Chem 249:2182-2187 Kono T, Robinson FW, Sarver JA 1975 Insulin-sensitive 1phosphoI diesterase. J Biol Chem 250:7826-7835 Rodbell M 1964 Metabolism of isolated fat cells I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem
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PJ, Witters LA, Girard PR, Kuo JF, Quamo SN 1985
Glynn BP, Colliton
phosphorylation
JW, McDermott
in 3T3-Ll
cells.
JM, Witters LA 1986 Phorbol
Walaas SI, Horn RS, Adler A, Albert protein
protein kinase 135:1119-l 125 KA, Walaas 0 1987 Insulin C activity in rat diaphragm.
K, Sano K, Kikkawa
U, Nishizuka
Y 1982 Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257:7847-7851 Solomon SS, Palazzolo M 1986 Rapid communication: Activation of cyclic AMP phosphodiesterase by phorbol and protein kinase C pathway. Am J Med Sci 292:182-184 Hidaka H, Inagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamide, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:50365041
Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F 1986 Staurosporine, a potent inhibitor of phospholipid/Ca” protein
kinase.
Biochem
Biophys
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135:397-
Fujita-Yamaguchi
Y, Kathuria S 1988 Characterization of receptor tyrosine-specific protein kinases by the use of inhibitors. Staurosporine is a loo-times more potent inhibitor of insulin receptor than IGF-I receptor. Biochem Biophys Res Commun 157:955-962 Gettys TW, Vine AJ, Simonds MF, Corbin JD 1988 Activation of the particulate low Km phosphodiesterase of adipocytes by addition of CAMP-deoendent urotein kinase. I Biol Chem 263:10359-10363 Degerman 6, Smith’CJ, Tornqvist H, Vasta V, Belfrage I’, Manganiello VC 1990 Evidence that insulin and isoprenaline activate che cCMP-inhibited cells bv uhosohorvlation.
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Castagna M, Takai Y, Kaibuchi
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lymphocytes: cytoplasmic free calcium cellularly trapped fluorescent indicator.
Takai Y, Kishimoto
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binding and J Biol Chem
Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y 1987 Genistein, a specific inhibitor of
R, Takai Y, Yu B, Nishizuka Y 1981 Widespread occurrence of calcium-activated, phospholipid dependent protein kinase in mammalian tissue. I Biochem 89:1651-1654 Nishizuka Y 1984 The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693-698 Tabarini D. Heinrich I. Rosen OM 1985 Activation of S6 kinase activity in 3T3-Ll cells by insulin and phorbol ester. Proc Nat1 Acad Sci USA 82:4369-4373
increases membrane FEBS Lett 220:311-318
Kadota S, Fantus IG, Deragon
G, Guyda
77:7039-7143
Minakuchi
esters, but not insulin, promote depletion of cytosolic C in rat adipocytes. Biochem Biophys Res Commun
39.
growth factor II receptor activity in rat adipocytes.
Endo. 1992 Voll31. No 1
Growth factor-stimulated protein J Biol Chem 260:13304-13315
of the putative hormonesensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity hgand. J Biol Chem 262:5797-5807 Rothenberg P, Glaser L, Schlesinger I’, Cassel D 1983 Epidermal growth factor stimulates amiloride-sensitive “Na+ uptake in A431 cells. J Biol Chem 258:4883-4889 Frelin C, Vigne P, Lazdunski M 1983 The amiloride-sensitive Na+/ Ht antiport in 3T3 fibroblasts. J Biol Chem 258:6272-6276 Davis RJ, Czech MP 1985 Amiloride directly inhibits growth factor receptor tyrosine kinase activity. J Biol Chem 260:2543-2551
29.
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36.
Phosphorylation
Degerman E, Belfrage P 1987 Purification
Stimulation of insulin-like insulin receptor kinase 262:8252-8256
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low Km cyclic AMP phosphodiesterase J Biol Chem 257:5339-5341
phosphodiesterase. Res 16:149-158 24.
cyclic
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FW, Smith CJ, Flanagan JE, Shibata H, Kono T 1989
Weber HW, Appleman independent pose tissue.
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sensitivity of adipocyte J 262:867-872
Cyclic GMP-dependent stimulation lin-sensitive CAMP phosphodiesterase Chem 264:16458-16464 22.
RJ 1951 Protein
BY VANADATE
low
Km CAMP phosphodiesterase in rat fat Proc Nat1 Acad Sci USA 87:533-537 Shibata’H, Robinson FW, Soderling TR, Kono T 1991 Effects of okadaic acid on insulin-sensitive CAMP phosphodiesterase in rat adipocytes. J Biol Chem 266:17948-17953
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Vol. 131, No. 1 Printed in U.S.A.
Society
Stimulatory Effect of Vanadate on 3’,5’-Cyclic Guanosine Monophosphate-Inhibited Low MichaelisMenten Constant 3’,5’-Cyclic Adenosine Monophosphate Phosphodiesterase Activity in Isolated Rat Fat Pads HIROSHI NOBUAKI
UEKI, RYUJI TOMINAGA,
OKUHAMA, MISAKI SERA, TOMOHIRO AND TETSUO MORITA
Department Fukuyama,
of Biochemistry, Faculty of Pharmacy Hiroshima 729-02, Japan
and Pharmaceutical
INOUE, Sciences, Fukuyama
University,
ABSTRACT When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (K,) CAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dosedependent manner with vanadate. 3’,5’-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulinstimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K’, Na+, or Ca” in the medium, while preincubation of the fat pads with a chelator of intracellular Ca*+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results
suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca*+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3’,5’cyclic GMP-inhibited low K, CAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes. (Endocrinology 131: 441-446, 1992)
V
cytes, and the insulin-stimulated PDE activity is inhibited by 3’,5’-cyclic GMP (cGMP) at a low concentration (14-17). Mechanisms of the stimulatory effect of vanadate on the PDE activity in fat pads or adipocytes are unknown. In the present paper, we show that vanadate, as well as
ANADIUM is an essential trace element for animals owing to its physiological and biochemical activities (l-3). Vanadium compounds mimic many in vitro and in viva effects of insulin including the stimulation of glucose transport, the suppression of the hormone-dependent lipolysis, the stimulatory phosphorylation of insulin receptor in isolated rat adipocytes, and the normalization of blood glucoselevels of streptozotocin-induced diabetic rats (4-9). We previously reported that sodium orthovanadate (vanadate) increased the lipoprotein lipase (LPL) activity in isolated rat fat pads and stimulated the release of LPL activity from the fat pads into medium (10, 11). The increasing effect of vanadate on the LPL activity was almost completely inhibited by adrenaline, (Bu)~cAMP, or 3-isobutyl-1-methylxanthine which was a potent inhibitor of the cyclic nucleotide phosphodiesterase (PDE) activity in adipocytes (11, 12). These results suggest that the action of vanadate is inhibited with an increase in the intracellular level of CAMP. It is known that many different PDEs selectively catalyze the hydrolysis of cyclic nucleotide but it still is not known precisely how many different PDE isozymes are present in most mammalian tissues(13). Of these many PDEs, insulin stimulates the low Michaelis-Menten constant (K,) CAMP PDE activity in the microsomal fraction of isolated rat fat pads and adipo-
insulin,
stimulates
the
cGMP-inhibited
low
K,
CAMP
PDE
activity in the microsomal fraction of rat fat pads, and that the stimulatory effect of vanadate is dependent on the intracellular level of Ca*+ and, in part, associated with tyrosine kinase- and protein kinase C (PKC)-mediated processes.In addition, we discuss that the increasing effect of vanadate on the LPL activity is not due to the stimulation of PDE activity by vanadate. Materials
and Methods
Materials The sources of chemicals used in this work were as follows: Vanadate (Na3V04), collagenase (Clostridium histolyticum, 300 U/m&, and tetrakis (acetoxymethyl)ester of quin 2 (quin 2-AM) from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan); insulin (bovine pancreas, 24.4 IU/mg), amiloride, biochanin A, cGMP, and Crotalus atrox snake venom from Sigma Chemical Co. (St. Louis, MO); CAMP, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan); BSA (Fraction V) from Boehringer-Mannheim Biochemicals (Mannheim, Germany); genistein from K & K Laboratories (Cleveland, OH); [2,8-3H]cAMP (1.158 TBq/mmol) from New England Nuclear (Boston, MA); ACS II and Protein kinase C enzyme assay kit from Amersham Japan Co. (Tokyo, Japan). Staurosporine was a gift from Dr.
Received January 13, 1992. Address all correspondence and requests for reprints to: Dr. H. Ueki, Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-02, Japan. 441
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442
STIMULATION
OF PHOSPHODIESTERASE
Y. Matsuda of Institute of Tokyo, Kyowa Hakko Co. Ltd. (Tokyo, All other chemicals used were of analytical grade.
Japan).
Animals Male Wistar rats weighing ZOO-220 g were obtained from Shimizu Laboratory Supplies Co. (Kyoto, Japan), fed on a commercial laboratory chew ad libitum, and given free access to water for 1 week. All animals were starved for 24 h before use.
Preparation of fat pads and adipocytes vanadate or insulin
and incubation
with
Epididymal adipose tissue was quickly removed from rats killed under ether anesthesia and cut into small pieces (30-40 mg) with scissors, Stock solutions of vanadate (100 mM, pH 7.4) and insulin (174 mM, pH 3) were diluted with deionized water to the desired concentration immediately before use. The fat pads (1 g) were incubated with 2 rnM vanadate or 3 nM insulin in 10 ml Krebs-Ringer bicarbonate buffer containing 5 mM glucose and 2% BSA, pH 7.4 (KRBGA) at 37 C for 60 min. Adipocytes were prepared as described by Rodbell (18). Briefly, the fat pads (0.7 g) were digested with collagenase (2.5 mg) in 2 ml KRBGA at 37 C for 60 min. The dispersed cells were filtered through nylon mesh, washed three times with fresh buffer. The freshly prepared adipocytes (0.2 g) were incubated with 2 rnM vanadate in 2 ml KRBGA at 37 C for 60 min. The fat pads and adipocytes incubated were washed three times with physiological saline at 4 C and used as materials of next step.
Preparation
of subcellular
fractions
Subcellular fractions were prepared by a slight modification of the method of Kono et al. (17). Briefly, the fat pads incubated were homogenized with a Potter-Elvejehm homogenizer at three strokes and 500 rpm in 2 ml 10 mM Tris-HCl buffer containing 1 rnM EDTA, 0.1 M KCl, and 0.25 M sucrose, pH 7.4. After centrifugation at 1,000 x g for 10 min, the infranatant was further centrifuged at 12,000 x g and 17,500 x g for 4.5 min each to remove a mitochondrial fraction and at 105,000 X 8 for 60 min to separate a microsomal fraction, The adipocytes incubated were homogenized with a Dounce tissue grinder at eight strokes in 5 ml 10 rnM Tris-HCI buffer, pH 7.4, containing 0.25 M sucrose. The homogenate defatted by centrifugation at 1,000 x g for 10 min was further centrifuged at 20,000 X g for 2 min and 30,000 X g for 30 min to separate a crude microsomal fraction.
of low K, CAMP PDE activity
The PDE activity was determined by a slight modification of procedure reported by Kono et al. (17). Briefly, microsomal fractions (25 pg protein) were incubated with 250 nM [3H]cAMP (0.9 KBq) in 0.25 ml 33 mM Tris-HCl buffer containing 4 rnM MgClz, pH 7.4, at 30 C for 5 min. The incubation was terminated by addition of 0.1 ml 0.1 N HCI. A mixture (0.05 ml) of 5 mM AMP and CAMP was added to the incubated solution, allowed to stand at 70 C for 4 min, cooled, and neutralized with 0.1 ml 0.1 N NaOH. After the addition of 50 PLg snake venom in 0.05 ml 0.1 M Tris-HCI buffer, pH 8.0, to the neutralized solution, the incubation was carried out at 37 C for 20 min and terminated by the addition of a mixed solution (0.05 ml, pH 7.0) of 200 rnM sodium EDTA and 5 mM adenosine. The reaction mixture (0.5 ml) was applied to a 5.5 X 30 mm column of Dowex l-X8 (200-400 mesh in chloride form from Bio-Rad, Richmond, CA), which had been washed with water and eluted with water. The first 1.4 ml of the effluent was discarded, and the next 2.5 ml was collected. The radioactivity of adenosine contained in the 2.5 ml fraction was determined in ACS II with an Aloka liquid scintillation counter (LSC-700). Protein was determined by the method of Lowry et al. (19). The PDE activity was expressed as picomoles of CAMP hydrolyzed per minute per milligram protein.
Determination
of PKC activity
The fat pads (0.5 g) were incubated with vanadate (O-10 mM) in 2 ml KRBGA at 37 C for O-150 min and homogenized in 1 ml 10 mM Tris-
1992 No 1
HCl buffer, pH 7.4, containing 1 rnM EDTA, 0.1 M KCl, and 0.25 M sucrose. After centrifugation, a microsomal fraction obtained was dissolved in 100 ~150 mM Tris-HCI buffer, pH 7.5, containing 5 rnM EDTA, 10 mM EGTA and benzamidine, 0.3% fl-mercaptoethanol, and 0.005% phenylmethylsulfonyl fluoride. The PKC activity was determined with a Protein kinase C enzyme assay system kit, essentially according to the method supplied by the manufacturer (Amersham International plc, Arlington Heights, IL), except that contents of L-a-phosphatidyl-L-serine and phorbol 12.myristate 13.acetate were reduced by one-tenth. The activity was expressed in terms of picomole substrate phosphorylated per minute per milligram protein.
Statistical
analysis
Results are shown separated experiments. unpaired observations.
as mean t SE of four The data were analyzed
observations by Student’s
for three t test for
Results First, kinetic constants of the vanadate-stimulated PDE activities were compared between both microsomal fractions of the fat pads and adipocytes (Table 1). Increases in maximum velocity (V,,,) by vanadate were 1.6- and 2.0-fold on the fat pads and adipocytes, respectively. No appreciable change in K,, values by vanadate was observed with both microsomal fractions. cGMP (0.01-10 PM) inhibited the vanadate-stimulated PDE activity of fat pads with similar profile to the insulin-stimulated one (data not shown). Thus, the stimulatory effect of vanadate appears not to be different between the fat pads and adipocytes. Therefore, the microsomal fraction of fat pads was used as the source of PDE through this study. Figure 1 shows changes in the PDE activity in the fat pads TABLE fractions
1. Kinetic of adipocytes
constants of PDE activity in microsomal and fat pads treated with or without vanadate
Km (PM) Control
Determination
Endo. Voll31.
BY VANADATE
Adipocytes Fat pads
V,,.
Control
Vanadate
0.55 0.59
162 248
326 406
The adipocytes and fat pads were incubated vanadate in KRBGA for 60 min. K, and V,,, from double reciprocal plots of the PDE activity concentrations of 0.17-2 PM.
30
mg)
Vanadate
0.59 0.57
0
(pmol/min.
60
Incubation
90 time
FIG. 1. Time course of stimulatory effect PDE activity. The fat pads were incubated nM insulin or without either one for O-150
with or without 2 mM values were calculated in a range of substrate
120
150
(min) of vanadate with 2 mM min.
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or insulin on vanadate or 3
STIMULATION
OF PHOSPHODIESTERASE
incubated with 2 mM vanadate or 3 nM insulin up to 150 min. The PDE activity was increased 1.5- and 1.8-fold by vanadate with 5- and 30-min incubation periods, respectively, then declined with 120- and 150-min incubation ones. Insulin almost linearly increased the PDE activity up to 150 min. Time course of the vanadate action showed a profile differing from that of the insulin action. When the fat pads were incubated for 60 min with vanadate over the concentration range of 0.5-10 mM, the PDE activity was increased in a dose-dependent manner up to 10 mM (Fig. 2). To determine whether activations of the insulin receptor and/or Na+/H+ exchange system are involved in the stimulatory effect of vanadate on the PDE activity, the fat pads pretreated with amiloride, biochanin A, or genistein were incubated with vanadate or insulin (Table 2). A significant inhibition of both actions of vanadate and insulin was observed with three inhibitors. Furthermore, the requirement for potassium, sodium, or calcium in the medium in which the fat pads were incubated with vanadate or insulin was investigated. No change in both actions of vanadate and insulin was found by replacement of Na’ with choline ion, addition of Na’ instead of K+, or omission of Ca2+ in the medium (data not shown). These results suggest that vana-
BY VANADATE
443
date increases the PDE activity in the fat pads through mechanismsinvolving activation of the insulin receptor but not the Na’/H+ exchange system. Next, the fat pads were loaded with quin 2-AM to remove intracellular Ca*+. The stimulatory effect of vanadate was progressively decreasedwith an increasein the concentration of quin 2-AM (Fig. 3). That of insulin was not decreasedat tested concentrations of quin 2-AM. The incubation with 100 FM quin 2-AM had no effect on the basal activity. Thus, the stimulatory effect of vanadate, in contrast to that of insulin, was dependent on the intracellular concentration of Ca’+. When the fat pads were preincubated with H-7, the stimulatory effect of vanadate was progressively decreasedwith an increasein the concentration of H-7 (Fig. 4). A significant inhibition of the vanadate action was observed with staurosporine at concentrations of 3 and 10 nM (data not shown). The stimulatory effect of insulin was not inhibited by inhibitors of PKC. When the fat pads were incubated with vanadate in the presence of insulin, the stimulatory effect of vanadate was additive to that of insulin (Table 3). These results suggest that vanadate exerts its action through mechanismsdiffering
0 Control0 Vanadatc A Insulin (3nM)
1
PmW
I 0
I
I
I
I
2
4
6
8
vanadatc
TABLE
2. Effects of inhibitors of tyrosine activity by vanadate or insulin
effect of vanadate on PDE vanadate at the indicated
kinases
PDE activity Inhibitors
Control
Biochanin
Genistein
on stimulation
(pmol/min
A
0 bM) 1 2 0 (he/ml) 100 250 0 b.dml) 5 25
63 62 61 66 67 64 60 59 59
f 0.8 f 0.7 + 1.0 xi 0.9 + 2.1 + 1.3 k 1.3 AZ 1.1 + 1.7
Vanadate
171 129 112 133 137 62 127 92 59
k + + + f + + + +
80
100
(PM)
3. Effect of quin 2-AM loading on vanadateor insulin-stimulated PDE activity. ” Quin - 2-AM was dissolved in dimethvl sulfoxide and 50 ~1 of it was added to the incubation mixture. The fat pads were preincubated with quin 2-AM at the indicated concentrations in 10 ml KRBGA containing 10 mM sodium pyruvate for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min.
FIG.
0
Insulin
8.6 3.2” 7.1b 3.8 4.9 0.9* 1.6 2.3” 1.8’
60 2-AM
mg)
Concentration
Amiloride
40 Quin
(mM)
2. Dose-response curve for stimulatory activity. The fat pads were incubated with concentrations for 60 min. FIG.
of PDE
20
0
10
93 69 61 104 98 62 95 76 62
+ + f + + f + -c +
Control
l
Vanadatc
10
20
30
2.0 0.7” 1.7* 3.2 0.6 1.4’ 1.7 2.4 l.Ob
Inhibitors were dissolved in dimethyl sulfoxide and their 50-111 was added to the incubation mixture. The fat pads (1 g) were preincubated with inhibitors at the indicated concentrations in 10 ml KRBGA for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min. n P < 0.05. * P < 0.01 compared to vanadateor insulin-stimulated group without inhibitors.
H-7
40
50
(M)
FIG. 4. Effect of H-7 on vanadateor insulin-stimulated PDE activity. H-7 was dissolved in dimethyl sulfoxide and 20 ~1 of it was added to the incubation mixture. The fat pads were preincubated with H-7 at the indicated concentrations in 10 ml KRBGA for 15 min and further incubated with 2 mM vanadate or 3 nM insulin for 60 min.
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444 TABLE presence
STIMULATION 3. PDE activity in fat pads or absence of insulin
incubated
PDE activity Without Control Vanadate (0.5 mM) (2 n-1
OF PHOSPHODIESTERASE
with
vanadate
(pmol/min~
insulin
Endo. Vol 131.
With
in the
insulin
69 + 0.7
95 f 0.8
76 + 0.6 132 k 1.8
108 + 0.5” 147 + 0.8”
I
in the presence
I
20 -
F----t
I
J
0 Control 0 Vanad;rtc (2mM) A Insulin (3nM)
\
0
I 2
I
4 Vanadatc
6 (mM)
8
FIG. 6. Dose-response curve for stimulation date. The fat pads were incubated with concentrations for 60 min.
of PKC vanadate
TABLE
on stimulation
4. Effects by vanadate
activity
Inhibitors
of H-7
and amiloride
30
60 Incubation
90 time
FIG. 5. Time course of stimulation of PKC fat pads were incubated with 2 mM vanadate either one for O-150 min.
120 (min)
150
activity by vanadate. The or 3 nM insulin or without
from the action of insulin. Next, we examined whether or not changes in PKC activity are involved in mechanisms of the action of vanadate. The standard activity of PKC was determined to be 49.3 pmol/ min.mg in the nontreated fat pads with a complete assay system kit. A small increase in the activity (7 pmol/min. mg) was observed with vanadate-treated fat pads. When the kit omitted phorbol 12-myristate 13-acetate, an activator of PKC, used for the assay also, vanadate showed a slight increase in the activity from 1 to 2.3 pmol/min. mg in the fat pads. Therefore, the appropriate concentration of the activator (4 mM) for the assay was used through all determinations of the PKC activity. Incubation of the fat pads with 2 mM vanadate increased 2.5-fold the PKC activity with 60and 120-min incubation periods (Fig. 5). In contrast, insulin never increased the PKC activity. When the fat pads were incubated for 60 min with vanadate over the concentration range of 0.5-10 mM, the PKC activity was increased in a dose-dependent manner up to 2 mM (Fig. 6). Preincubation of the fat pads with H-7 or amiloride showed a significant inhibition of stimulation of the PKC activity by vanadate (Table 4). These results suggest that vanadate stimulates the PKC activity in fat pads via an amiloride-sensitive process. Discussion The insulin-stimulated PDE is reported to be a membranebound enzyme which is recovered in a crude microsomal
H-7
Amiloride
0 (FM) 5 20 0 (mM) 1 2
10 activity at the
by vanaindicated
of PKC
PKC activity (pmol/min. mg)
Concentration
Control 0
1992 No 1
mg)
The fat pads were incubated with vanadate for 60 min or absence of 3 nM insulin. ’ P < 0.05 compared to groups without insulin.
G \E .e
BY VANADATE
8.8 7.5 8.0 11.8 11.6 11.2
f + + ++ +
Vanadate 1.2 0.7 0.3 0.2 1.4 1.2
17.7 14.4 11.8 22.4 19.8 13.6
f + f + + +
1.8 1.0” 0.8’ 1.2 0.2” 0.2*
The fat pads (0.8 g) were preincubated with inhibitors at the indicated concentrations in 5 ml KRBGA for 15 min and further incubated with 2 mM vanadate for 60 min. o P < 0.05. b P < 0.01 compared to vanadate-stimulated group without inhibitors.
fraction, probably endoplasmic reticulum fraction, of rat adipocytes (17, 20, 21). It also is designated as the type IV PDE which is calmodulin-insensitive and inhibited by cGMP at a low concentration (22-24). The incubation of fat pads with vanadate stimulated the PDE activity in the microsomal fraction but neither mitochondrial nor cytosolic one (data not shown). The vanadate-stimulated PDE in microsomal fraction of fat pads, as well as that of adipocytes, had a low K, value toward CAMP and its activity was inhibited by cGMP at a low concentration. These results suggest that vanadate, as well as insulin, stimulates the cGMP-inhibited low K, CAMP PDE activity in the microsomal fraction. However, whether vanadate stimulates the insulin-stimulated PDE itself or its isoforms remains to be elucidated. Amiloride, an inhibitor of activation of the Na+/H+ exchange system and the tyrosine kinase activities of insulin and growth factor receptors (25-28), and biochanin A and genistein, inhibitors of tyrosine kinase activity of epidermal growth factor receptor (29), all inhibited the stimulation of PDE activity by vanadate or insulin to various extents. Both actions of vanadate and insulin were well preserved with incubation of the fat pads in the absence of either K’, Na+, or Ca2+ in the
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STIMULATION
OF PHOSPHODIESTERASE
medium. It is reported that vanadate, as well as insulin, stimulates selective phosphorylation of tyrosine residues in a 95-kilodalton insulin receptor subunit of rat adipocytes to produce an intracellular event such as the activation of glycogen synthase (4). Our results, therefore, suggest that the action of vanadate is associated with stimulation of tyrosine kinase activity of insulin receptor and is independent of activation of the Na+/H+ exchange system. Vanadate is a potent inhibitor of Naf, K+-adenosine triphosphatase (3). However, inhibition of the enzyme by vanadate is not involved in mechanisms of the insulinmimetic actions of vanadate such as the stimulation of hexose transport and the activation of glycogen synthase (5). Our result, obtained with the treatment of fat pads in K+-free medium, shows that the action of vanadate is not due to inhibition of the activity of Naf, K’-adenosine triphosphatase. When the fat pads were loaded with quin 2-AM, a chelator of intracellular Ca*+ (30), the stimulation of PDE activity by vanadate was decreased in a dose-dependent manner. The stimulatory effect of insulin, on the contrary, was not decreased at tested concentrations of quin 2-AM. These results show that the action of vanadate is dependent on the intracellular concentration of Ca”. PKC is a Ca2+- and phospholipid-dependent enzyme and plays an important role in the transduction of extracellular signalsinto intracellular events and of cellular proliferation in many tissues(31-34). The enzyme is activated by diacylglycerol which is transiently produced from inositol phospholipids in responseto extracellular signals. Several workers failed to demonstrate the effect of insulin on the PKC activity and the subcellular distribution of the enzyme in isolated and cultured adipocytes (35-37), although insulin increased the PKC activity in rat diaphragms (38). Phorbol ester is a tumor-promoting agent known to act through PKC (39) and activates the low K, CAMP PDE in fat pads (40). Our results were that the stimulation of PDE activity by vanadate was in part inhibited by H-7 and staurosporine, potent inhibitors of PKC (41-43), but the action of insulin was not inhibited. Furthermore, vanadate increased PKC activity in the fat pads and showed the maximum effect with incubations at concentrations of 2 and 5 mM for 60-120 min. Insulin never showed any stimulation of PKC activity in fat pads. Preincubation of the fat pads with H-7 or amiloride showed a significant inhibition of the stimulation of PKC activity by vanadate. Theseresults suggestthat a PKC-mediated processis, in part, involved in the stimulation of PDE activity by vanadate. Recent reports show that addition of the purified catalytic subunit of CAMP-dependent protein kinase to the crude microsomal fractions, containing the low K, CAMP PDE, of rat adipocytes produces a 2-fold activation of the enzyme activity (44), that incubation of rat adipocytes with insulin induces serine phosphorylation of the low K, CAMP PDE via the activation of an unidentified serine protein kinase (45), and that a protein kinase, which is more H-7-resistance than CAMP-dependent protein kinase, is involved in the stimulation of PDE activity by insulin (46). Thus, a protein kinase associated with the insulin action on stimulation of the low K, CAMP PDE activity appears to be different from
BY VANADATE
445
that involved in the vanadate action. In contrast to the stimulatory effect of vanadate on the LPL activity, the action of vanadate on the PDE activity was markedly inhibited by quin 2-AM loading and, in part, inhibited by amiloride. The requirement for Na+ or Ca*+ in the medium differed between actions of vanadate on the LPL and PDE activities (11). These results suggest that the increasing effect of vanadate on the LPL activity is not due to the stimulation of the PDE activity by vanadate. In conclusion, vanadate appears to stimulate in part the cGMP-inhibited low K, CAMP PDE activity in the microsoma1 fraction through the activation of tyrosine kinase- and protein kinase C-mediated processes. References
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15.
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