Effects of wortmannin on glucose uptake and protein kinase C activity in rat adipocytes

ELSEVIER

Diabetes Research and Clinical Practice 29 (1995) 143-152

Effects of wortmannin on glucose uptake and protein kinase C activity in rat adipocytes Tatsuo Ishizuka* a, Toshihiko Nagashimaa, Mayumi Yamamoto”, Kazuo Kajita”, Kouji Yamadaa, Hiroaki Wada”, Satomi Itayaa, Keigo Yasudaa, Yoshinori Nozawab aThe Third Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasamachi, Gifu 500,Japan bDepartment of Biochemistry, G$i University School of Medicine, 40 Tsukasamachi, Gifu 500, Japan

Received 1I January 1995; revision received 27 June 1995;accepted 17 July 1995

Abstract

Wortmannin is known to be an inhibitor of myosin light chain kinase and phosphatidylinositol3-kinase (PI 3-kinase) (J. Biol. Chem. 268, 25846, 1993). We studied the effects of wortmannin on insulin- and 12-O-tetradecanoylphorbol 13-acetate(TPA)-induced glucose uptake, purified PKC activity and in vitro 80 kDa protein phosphorylation to elucidate the relationship between insulin-induced PI 3-kinase and PKC activations. Pretreatment with 10-12-10m6M wortmannin for 60 min resulted in a dose-responsive reduction of 10 nM insulin-stimulated glucose uptake in rat adipocytes. Pretreatment with 10m6M wortmannin resulted in 80% and 20% decreasesof glucose uptake stimulated by insulin and TPA, respectively. Partially purified rat brain PKC activity and 80 kDa protein in vifro phosphorylation of rat adipocyte cytosol by addition of Ca2+ and phospholipid were dose-dependently decreasedby lO-8-lO-6 M wortmannin; 20% decreaseof PKC activity and 50% decreaseof 80 kDa protein phosphorylation by lO-6 M wortmannin were observed.These results suggestthat wortmannin has a potent inhibitory effect on PI 3-kinase and a weak inhibitory effect on PKC activity, and both effects cause a significant inhibition of insulin-stimulated glucose uptake in rat adipocytes.

Keywords: Wortmannin; Insulin; Glucose uptake; Protein kinase C; Adipocytes

1. Introduction Activation of protein kinase C (PKC) is an important role for transduction of signals generated upon external stimulation of cells by hormones [ 11. In particular, insulin and phorbol esters provoke * Corresponding author, Tel.: +81 058 2672328; Fax: +81 058 2672956.

increases in membrane-associated PKC in rat adipocytes [2,3]. Further, it has previously been reported that PKC 0 is translocated from cytosol to membrane in response to treatment of rat adipocytes [2] and soleus muscle [4,5] with insulin and/or 12-0-tetradecanoyl phorbol- 13-acetate (TPA). However, it is known that many cell types contain a variety of PKC isoforms 161. On the other hand, phosphatidylinositol 3-

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kinase (PI 3-kinase) is associatedwith and activated by a number of proteins containing tyrosine kinase activities, including the receptor for platelet-derived growth factor [7], insulin [8,9], and the products of oncogenes [lo]. PI 3-kinase, which consists of an 85kDa regulatory subunit and a 1IO-kDa catalytic subunit [ 1l- 131,is not in the formal phosphatidylinositol (PI) turnover pathway, but catalyzes the formation of a family of phosphoinositides with phosphate at the D-3 position of the inositol ring. Its activation and the association of this activity with tyrosine kinase to induce mitogenesisor cellular transformation, suggesting it plays important roles in the transduction of mitogenic signals [14]. Although receptormediated activation of PI 3-kinase was also found in fMLP-stimulated human neutrophils and thrombin stimulated human platelets [ 15,161,the physiological role of PI3-kinase in these cells is still somewhat uncertain. Recently, activation of the PKC[ isoform by phosphatidylinositol 3,4,5trisphosphate (PIP,) which is produced by PI 3-kinase has been reported [17]. Therefore, PKC activation by PIP3 would produce a novel link between tyrosine kinase activity of insulin receptor and serine/ threonine phosphorylation cascades. Here, we have examined that the effect of wortmannin, a specific inhibitor of PI 3-kinase on insulin- or phorbol ester-induced glucose uptake, PKC activation, and phosphorylation of 80 kDa protein to clarify the interrelationship between diacylglycerol-PKC pathway and PI 3-kinase activating pathway in rat adipocytes. 2. Materials and methods

MO). Silicon oil was obtained from Aldrich Chemical Co. (Milwaukee, Wis). H7 (l-(5isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) was purchased from Seikagaku Kogyo (Tokyo, Japan). Wortmannin was kindly supplied by Dr. Nonomura (Department of Pharmacology, Faculty of Medicine, University of Tokyo). All other chemicals were of reagent grade. 2.2. Adipocyte experiments

Male Wistar rats weighing 150-200 g were fed ad libitum and killed by decapitation. Isolated adipocytes were obtained by collagenasedigestion of rat epididymal fat pads [ 181 in Krebs Ringer phosphate buffer (pH 7.4) containing 127 mM NaCl, 12.3 mM NaH2P04, 5.1 mM KCl, 1.3 mM MgSO,, 1.4 mM CaC12,3% BSA, and 2.5 mM glucose. Adipocytes were washed and preincubated at 37°C in glucose-free Krebs-Ringerphosphate buffer containing 1 % BSA for 30 min, and then incubated with or without 10-12- 10e6M wortmannin, followed by incubation with insulin for 30 min. [3H]2-DOG (0.08 &i) and unlabelled 2-DOG (0.05 mM) were then added to 300 ~1 of a 10% (v/v) adipocyte suspension, and uptake of [ 3H]2-DOG was measured over 1 min [ 191. In PKC experiments, reactions were terminated by addition of 10 ml ice-cold buffer I (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1.2 mM EGTA, 0.1 mM PMSF, 20 &ml leupeptin, 20 mM 20-mercaptoethanol). The adipocytes were washed twice and homogenized in buffer I. Homogenates were centrifuged at 1000 x g for 2 min, and floating fatty materials were removed. Resultant homogenates were centrifuged at 105 000 x g for 60 min to obtain cytosol and membrane fractions as described below.

2.1. Materials

Pork insulin was obtained from Novo (Copenhagen, Denmark). [T-~~P]ATP (3000 Ci/mmol) and [ 1,2-3H]2-deoxyglucose ([ 3H]2DOG), and L-[ l- “C]glucose (47 mCi/mmol) were purchased from New England Nuclear (Boston, MA). Phosphatidylserine (PS), diolein, histone (type III-S), phenylmethylsulfonyl fluoride (PMSF), leupeptin, 12-O-tetradecanoyl phorbol13-acetate(TPA), BSA, D-glucose and ATP were purchased from Sigma Chemical Co. (St. Louis,

2.3. PKC studies

Rat adipocytes or brain was homogenized with a polytron homogenizer in 20 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1.2 mM EGTA, 0.1 mM phenylsulfonyl fluoride, 20 &ml leupeptin, and 20 mM 2-mercaptoethanol (buffer I). The homogenate of adipocytes was centrifuged for 60 min at 105 000 x g to obtain cytosol and membrane fractions. After membrane fractions were resuspended in buffer I containing 5 mM

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EGTA, 2 mM EDTA, and 1%Triton X-100 for 30 min at 4”C, they were sonicated, and centrifuged at 105 000 x g to obtain solubilized membrane fractions. To measure PKC enzyme activity, adipocytes or brain cytosol or solubilized membrane fractions were diluted with 20 mM Tris/HCl buffer (pH 7.5) containing 0.5 mM EGTA, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol (buffer II). The sampleswere then applied to a Mono Q column (0.5 x 5 cm, Pharmacia HR 5/5) that had beenequilibrated with buffer II and connected to a high performance liquid-chromatography system, as described previously [2,4]. PKC was eluted by application of a 20 ml linear gradient of NaCl (O-O.7 M) in buffer II at a flow rate of 0.65 ml/min. Fractions of 1 ml were collected, and PKC activity of each fraction was assayedby measuring the phosphorylation of histone III-S, as described previously [2]. Activation of PKC in rat adipocytes was also assayedby changesin the subcellular distribution of immunoreactive PKC using methods described previously [2,4]. Partially purified rat brain PKC, which was obtained from the peak of cytosolic Mono Q column puritiedPKC activity, was concentrated by Centricon tube (Amicon, Rikaken Co., Tokyo), kept in 20 mM Tris/HCl (pH 7.5) containing 10% glycerol at -8O”C, and used for experiments of wortmannin effects on PKC activity in vitro. Equal amounts of cytosol or membrane fractions were prepared as described above and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Gels were transferred to nitrocellulose and incubated first with rabbit polyclonal antiserum raised to synthetic peptide specific to PKC OLand /3 (GIBCO, NY) and second with goat antirabbit y-globulin complexed to alkaline phosphatase. As reported [2], this immunoblotting method detected a single major immunoreactive band that comigrated on SDS-PAGE and blotted identically with purified rat brain 80 OOO-MrPKC. The intensity of immunoreactivity was scanned with a laser densitometer (Pharmacia LKB Biotechnology, Tokyo) to determine the relative value. 2.4. Phosphorylation studies

Cytosolic and membrane-associatedproteins of rat brain and adipocytes (30 pg) with [y-32P]ATP

were incubated with or without 10-‘2-10-6 M wortmannin or 1O-7-1O-6 M H7 in the presence of 0.5 mM Ca*+ alone, 0.5 mM Ca*+/40 &ml PS10.4pg./ml diolein, 0.5 mM EGTA/40 &ml PS10.4&ml diolein or 0.5 mM EGTA alone for 5 min at 37°C. The reaction was terminated by the addition of Laemli sample buffer [20]. All samples were subjected to SDS-PAGE. After electrophoresis, the gels were stained with Coomasie Blue, dried, and autoradiographed with Kodak XOmat film for 48-96 h. Phosphorylated 80 kDa and 50 kDa proteins were apparently identified as Ca*+-dependentPKC and Ca*+-independent PKC substrates, respectively. Effect

of wortmannin on insulin-stimulated glucose uptake in rat adipocytes so0 I ,

0

0.01

10

0.1

wortmanAin

Effect

1001000

(nM)

of wortmannin on TPA-stimulated glucose uptake in rat adipocytes

-0 0.01 wortmannin

0.1

1 10 concentration

1001000 fnMl

Fig. 1. Effects of various concentrations of wortmannin on insulin- or TPA-stimulated [3H]2-deoxyglucose (DOG) uptake in rat adipocytes. Isolated adipocytes were incubated with or without 1O-“-1O-6 M wortmannin for 60 min, followed by incubation with insulin for 30 min. [3H]2-DOG (0.08 PCi) and unlabelled 2-DOG (0.05 mM) were then added to 300 pl of a 10% adipocyte suspension, and uptake of [3H]2-DOG was measured over 1 min. Data are shown as mean * SE of five separate experiments. l P < 0.05, l *P < 0.01 by standard t test vs. without wortmannin pretreatment.

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Statistical comparisons were performed by standard Student’s t-test for planned paired and unpaired comparisons where appropriate. Unless otherwise stated, all data are expressed as the mean f SE.

for 60 min, followed by incubation with 10 nM insulin for 30 min, dose-responsive inhibition of insulin-stimulated [3H]2-DOG uptake by wortmannin where occuring up to 80% was observed (insulin-stimulated [ 3H]2-DOG uptake without wortmannin pretreatment vs. with pretreatment by lo-*, lo-’ and lO-‘j M wortmannin: 657 f 55% vs. 493 f 39%, 329 f 36% and 269 f 25%, respectively, P < 0.05 or 0.01 by standard t test). Half-maximal inhibitory effect of wortmannin on insulin-stimulated [ ‘H]ZDOG uptake was observed at lo-’ M. On the other hand, TPAstimulated [3H]2-DOG uptake was also inhibited

3. Results

3.1. Effect of wortmannin on insulin- or TPAstimulated [3H]2-deoxyglucose (DOG) uptake in rat adipocytes

When isolated adipocytes were incubated with or without (control) 10-“-10-6 M wortmannin

A

lmmunoblot adipocytes

0

analysis

5 1020

0

of PKC B in rat

5

10 20

min of treatment with insulin

10-6 M CONT 0 5 1020

Wortmannin 0

5 10

20

60 min

min of treatment with insulin

Membrane Fig. 2. Effects of wortmannin on insulin-induced translocation of PKC B (A, B) and PKC (Y (C) in rat adipocytes. After treatment with or without (control) 10-‘“-10-6 M wortmannin (B) for 60 min, isolated adipocytes were stimulated with 10 nM insulin for 0, 5, 10 and 20 min (A, C). Reactions were terminated by the addition of ice cold buffer. The adipocytes were washed, homogenized, and centrifuged to obtain cytosol and membrane fractions. Equal amounts of cytosol or membrane fractions were subjected to SDSPAGE, transferred to nitrocellulose, and incubated first with rabbit polyclonal antiserum raised to synthetic peptide specific to PKC f3 and a (GIBCO, NY) and second with goat anti-rabbit -r-globulin complexed to alkaline phosphatase. Similar results were observed in at least five other experiments.

T. lshizuka et al. /Diabetes

Research and Clinical Practice 29 (1995) 143-152

147

B

Immunoblot

analysis

of PKC R in rat adipocytes

80 kDa

CONT 1 o-6 10-8 10-j 6 Wortmannin

60

min

+

10 nM insulin

10 min

Fig. 2(B).

by 1O-7-1O-6 M wortmannin only up to 20% (TPA-stimulated [3H]2-DOG uptake without wortmannin pretreatment vs. with pretreatment by lo-’ and 10M6M wortmannin: 472 f 46% vs. 415 f 28% and 392 f 35%, respectively, P < 0.05 by standard t test) (Fig. 1). 3.2. Effect of wortmannin on insulin-induced translocation of PKC@, and (Y immunoreactivity in rat adipocytes

Wortmannin (lo-“- 10s6 M) pretreatment of adipocytes for 60 min resulted in a dose-responsive inhibition of insulin-induced decreasesof cytosolic and increases of membrane-associated PKCp immunoreactivity (Fig. 2-B). At 10e6M wortmannin suppressed 10 nM insulin-mediated, timedependent translocation of PKC p (Fig. 2-A) and (Y(Fig. 2-C) immunoreactivity.

3.3. Effect of wortmannin on insulin- or TPAinduced translocation of PKC activity in rat adipocytes

After pretreatment with 10m6M wortmannin for 60 min, 10nM insulin-induced translocation of Mono Q column-purified PKC activity from cytosol to membrane was suppressed(PKC activity without wortmannin pretreatment vs. with wortmannin treatment). Cytosol (mean f S.D.): before insulin treatment, 100%vs. 90 f 12%,after insulin treatment for 10 min, 51 f 10% vs. 65 f 11%. Membrane (mean f S.D.): before insulin treatment, 100%vs. 89 f 1l%, after insulin treatment for 10 min 210 f 12% vs. 140 l 12%, *P < 0.01) (Fig. 3-upper panel). But pretreatment with less than lo-* M wortmannin for 60 min resulted in formal insulin-induced PKC translocation (data not shown).

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’

lmmunoblot adipocytes

analysis

of PKC a in rat

80 kDa

10 -6 M Wortmannin

0

10 20

0 10 20

min of treatment with 10 nM insulin

80 kDa

Membrane

Fig; 2(C).

The wortmannin pretreatment resulted in an inhibition of TPA-induced PKC translocation at 10m6M but not at lo-’ M (PKC activity without wortmannin pretreatment vs. with wortmannin pretreatment for 60 min). Cytosol (mean f S.D.): before TPA treatment, 100% vs. 90 f 12%, after TPA treatment for 10 min, 58 f 12% vs. 75 f lo%, *P < 0.05. Membrane (mean f SD): before TPA treatment, 100%vs. 89 f 1l%, after TPA treatment for 10 min, 155 f 14% vs. 130 i 13%, *P < 0.05) (Fig. 3-lower panel). 3.4. Effect of wortmannin or H7 on calciumlphospholipid-dependent phosphorylation of 80 kDa protein in adipocyte cytosol

Fig. 4 showed calcium/phospholipid-dependent phosphorylation of 80 kDa protein, PKC substrate. In the presenceof wortmannin ( 10-12- 10m6

M) for 5 min, 80 kDa protein phosphorylation was dose-responsivelydecreasedcompared with that in the absenceof wortmannin. Calcium-independent, phospholipid-dependent phosphorylation of 80 kDa protein, a novel PKC substrate was also decreasedby wortmannin (10-‘2-10-6M) (Fig. 4). The inhibitory profile of wortmannin-induced 80 kDa protein phosphorylation was similar to that of H7-induced 80 kDa phosphorylation, as indicated in Fig. 5. Wortmannin at 1 FM has a similar potent inhibitory effect on 80 kDa protein phosphorylation to H7 at 1 PM, a selective PKC inhibitor. 3.5. Effect of wortmannin on Mono Q-column purified histone III-S phosphorylation in vitro

Mono Q column-purified PKC activity was dose-dependently inhibited by measuring histone

T. Ishizuka et al. /Diabetes Research and Clinical Practice 29 (1995) 143-152

Effect of wortmannin on 80 kDa protein phosphorylation in cytosol of rat adipocytes

Effect of wortmannin on insulin-induced translocation of PKC in rat adipocytes

0

200

uz

n q

Z ;E c.m SO .-CL al z t

B. C .g

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CytOSOl Membrane

100 SO kDa s 0 COlll

INS

10 min

WM

WM+INS

Effect of wortmannin on TPA-induced translocation of PKC in rat adipocytes 200

T

Cant

TPA

10 min

WY

WM+TPA

Fig. 3. Effects of I gM wortmannin on insulin- or TPA-induced translocation of Mono Q column-purified PKC activity in rat adipocytes. Equal amounts of cytosol and membrane fractions were prepared as indicated in ‘materials and methods’. To measure PKC activity of adipocytes, cytosol or solubilized membrane fractions were applied to a Mono Q column. PKC was eluted by application of a 20 ml linear gradient of NaCl (O-O.7 M) at a flow rate of 0.65 mhmin. Fractions of 1 ml were collected, and PKC activity of each fraction was assayed by measuring the phosphorylation of histone III-S. Total elutable PKC activity was calculated by the sum of PKC activity of each fraction. Data are plotted as mean f SE of 5 separate experiments (*P < 0.05-0.01 by standard t- test vs. without wortmannin pretreatment).

III-S phosphorylation for 5 min in vitro (P < 0.01 vs. control) (Fig. 6). 4. Discussion Wortmannin, which was first isolated as an artificial antibiotic from the culture of Penicillium wortmunnii [2 I], has immunosuppressive activity [22], strong anti-inflammatory activities [23] and acute toxicity [24] in animals. Wortmannin has also been shown to suppress cellular responses such as respiratory burst and

Fig. 4. Effects of various concentrations of wortmannin on calcium/phospholipid-dependent 80 kDa protein phosphorylation in adipocyte cytosol. Cytosolic fraction of adipocyte (30 pg) was incubated with [y-32P]ATP in the presence of EGTA, Ca*+/phosphatidylserine (PS)/diolein Ca*+, (DL) or EGTAiPS/DL in addition to IO-‘*-IO-* M wortmannin for 5 min. The reaction was terminated by addition of Laemli sample buffer. All samples were subjected to SDS-PAGE, and the gels were stained, dried, and autoradiographed. Abbreviations were used as follows; E, 0.5 mM EGTA; Ca*+, 0.5 mM Ca*+; PS, 40 &ml phosphatidylserine; DL; 0.4 p&ml diolein. Similar results were observed in three other experiments.

exocytosis in neutrophils [25,26]. Aggregation and serotonin release in platelets [27,28], and catecholamine release in adrenal chromaffin cells [29]. Moreover, this compound inhibited the activation of phospholipase D in response to fMLP without direct inhibition of the enzyme [30]. Nakanishi et al. have reported that wortmannin inactivated purified MLCK without affecting other protein kinases such as protein kinase C, CAMP-dependent protein kinase, calmodulindependent protein kinase II, and cGMPdependentprotein kinase [31]. Recently, wortmannin was found to be a PI 3-kinase inhibitor in RBL-2H3 cells [32], rat adipocytes [33], and neu-

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Effect of wortmannin on 80 kDa protein phosphorylation in cytosol of rat adipocytes

80 kDa

Fig. 5. Differential effect of low6 M wortmannin and IO-‘10W6M H7 on calcium/phospholipid-dependent 80 kDa protein phosphorylation in adipocytes cytosol. Cytosolic proteins were incubated with [T-~*P]ATP in the various condition as indicated in Fig. 4 legend. Similar results were obtained in three other experiments. Effect

of

wottmannin

on

tat

brain

I-

,-

I-

,-

PKC

activity

trophils [34]. Insulin-induced [3H]2-deoxyglucose uptake was dramatically inhibited by 0.1 PM wortmannin in which concentration PI 3-kinase activity was completely blocked without affecting the insulin receptor tyrosine kinase, and accordingly insulin-induced activation of PI 3-kinase has a pivotal role in the intracellular signaling pathway 1331. In our experiment, half-maximal inhibitory effect of wortmannin on insulin-induced glucose uptake was 0.1 PM, where PI 3-kinase activity was completely blocked [32-341. Therefore, we have examined the effect of wortmannin on insulininduced PKC activation through diacylglycerol production. As indicated in Fig. 1, 1 PM wortmannin suppressedinsulin- and TPA-induced glucose uptake up to 80% and 20%, respectively, indicating that differential effect of wortmannin on insulininduced and TPA-induced glucose uptake in rat adipocytes. Although lower concentration (0.1 FM) of wortmannin blocks PI 3-kinase activity [33], 50% of insulin-induced glucose uptake still occurs. Insulin- and TPA-induced translocation of PKC from cytosol to membrane were also suppressedby 1 PM wortmannin as indicated in Figs. 2 and 3. Theseresults suggestthat PI 3-kinase activating pathways exist neither downstream nor upstream of DG-PKC signaling pathways and furthermore wortmannin also inhibits insulininduced PKC activation, in addition to PI 3-kinase and myosin light chain kinase activities [31], and that some cross talking between PI 3-kinase and DG-PKC signaling (i.e. phorbol ester provoking increasesof tyrosine kinase activity and/or PI 3kinase activity, and PKCC being activated by PIP3 [ 171and subsequently stimulating glucose uptake) may exist in insulin sensitive tissue. When the adipocyte cytosol was used as PKC substrate, wortmannin suppressed 80 kDa protein phos-

I-

I-

wottmannin

concentration

(nM)

Fig. 6. Effect of various concentrations of wortmannin on Mono Q column-purified PKC activity from rat brain cytosol. Cytosolic fraction of rat brain was used for PKC assay. PKC was assayedby measuring the phosphorylation of histone III-S. Data are plotted as mean f SE of five separate experiments (**P c 0.01 by standard r-test vs. without wortmannin treatment).

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phorylation by Ca*+/phospholipid, which was similar to H7, a PKC selective inhibitor in vitro. These results indicated that inhibition of PKC activity using 80 kDa protein by wortmannin was more distinct compared to that using histone HI. In summary, first, wortmannin, an inhibitor of PI 3-kinase, inhibited insulin-stimulated [3H]2deoxyglucose (DOG) uptake up to 70% with an EDso value of 0.1 PM. On the other hand, 1 PM wortmannin inhibited TPA-stimulated [ 3H]2DOG uptake only by 20%. Second, 1 FM wortmannin, however, apparently suppressedinsulinor TPA-induced translocation of PKC from cytosol to membrane. Moreover, PKC-mediated phosphorylation of 80 kDa protein in adipocyte cytosol was inhibited up to 40% by 1 PM wortmannin which was similar to the effect of 1 PM H7 in vitro. Third, Mono Q column-purified PKC activity was reduced to 20% for 5 min by the addition of 1 PM wortmannin in in vitro histone phosphorylation. Finally, wortmannin should be used in less than 100 nM for insulin signal transduction experiments. References [I]

[2]

[3]

[4]

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[7]

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