A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo*

(Received for publication, November 22, 1996, and in revised form, December 30, 1996)

Jeroen van der Kaay Dagger §, Ian H. Batty Dagger , Darren A. E. Cross Dagger , Pete W. Watt and C. Peter Downes Dagger

From the Dagger  Department of Biochemistry, Medical Sciences Institute, and the  Department of Anatomy and Physiology, University of Dundee, DD1 4HN Dundee, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). Cellular levels of PtdIns(3,4,5)P3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32PO4, extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P3, are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P4. The latter is measured by its ability to displace [32P]Ins(1,3,4,5)P4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.


INTRODUCTION

PI 3-kinases1 represent a family of enzymes which phosphorylate phosphoinositides on the 3-position of the inositol ring (1). The role of PI 3-kinases in mitogenic signaling, membrane ruffling, trafficking, cell motility, inflammatory and immune cell responses, activation of neutrophils, and the metabolic effects of insulin (2-8) has been well established using biochemical, pharmacological, and genetic approaches. At least two major classes of PI 3-kinases have been identified which differ in their substrate specificity. Agonist-stimulated forms of PI 3-kinase can utilize PtdIns, PtdIns 4-phosphate, and PtdIns(4,5)P2 as substrates in vitro (9, 10), but several lines of evidence suggest that PtdIns(4,5)P2 is the preferred substrate in intact cells (11, 12). The resulting PtdIns(3,4,5)P3 has been shown to increase rapidly and transiently in many cells in response to a wide variety of stimuli and since there are no known phospholipases that can metabolize this lipid (13), it has been suggested that PtdIns(3,4,5)P3 itself is a second messenger. At present there are reports that PtdIns(3,4,5)P3 can activate atypical forms of protein kinase C (14) and can bind to protein kinase B (also named Akt or Rac) (15-17) and the recently cloned centaurin (18), encoding for a novel gene, which is highly expressed in brain and which shows homology to yeast and mammalian Arf GTPase-activating proteins.

Measuring PtdIns(3,4,5)P3 levels has so far been done by labeling cells with [3H]inositol or [32P]orthophosphate. Cell labeling with inositol is often not very efficient, and labeling to isotopic equilibrium can take days. Labeling cells with [32P]orthophosphate is inconvenient because of the precautions needed for the large amounts of radioactivity required. Both labeling procedures suffer from the drawback of elaborate, expensive and time-consuming HPLC analysis of deacylated lipid extracts which are required to resolve the glycerol derivative, GroPtdIns(3,4,5)P3, from 32P-labeled contaminants. We therefore developed a straightforward, highly specific and sensitive method that allows the detection of picomole amounts of PtdIns(3,4,5)P3. The assay is based on cleavage of the polar head group of PtdIns(3,4,5)P3 to yield Ins(1,3,4,5)P4, the mass of which can be measured using an isotope dilution assay with a sensitivity limit of approximately 0.3 pmol. In general, lipid extracts from samples containing 1 mg of cellular protein are sufficient for determination of the level of PtdIns(3,4,5)P3. The preparation of a highly specific Ins(1,3,4,5)P4-binding protein (obtained from sheep cerebellum) and high specific activity [3-32P]Ins(1,3,4,5)P4 (prepared from Ins(1,4,5)P3 and [gamma -32P]ATP using a recombinant Ins(1,4,5)P3 3-kinase from rat brain) are described. We show that measurements of PtdIns(3,4,5)P3 mass determined by isotope dilution match well with measurements from isotopic equilibrium cell labeling experiments. Furthermore, the assay can be applied in situations where radioactive labeling is impractical. Insulin administration to anesthetized rats resulted in a rapid increase of PtdIns(3,4,5)P3 levels, which was paralleled by an increase in PKB activity, in extracts from freeze-clamped hind limb skeletal muscle.


EXPERIMENTAL PROCEDURES

Materials

[gamma -32P]ATP (3000 Ci/mmol) and [3H]Ins(1,4,5)P3 (20-60 Ci/mmol) were from Amersham. Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were from CellSignals. Folch fraction I from bovine brain and calmodulin immobilized on agarose beads were from Sigma. Calmodulin was from Calbiochem. [32P]PtdIns(3,4,5)P3 was prepared from PtdIns(4,5)P2 and [gamma -32P]ATP using immunoprecipitated PI 3-kinase from U937 cells. Anti-PKB antibodies were raised in sheep. GroPtdIns(3,4,5)P3 was prepared from PtdIns(3,4,5)P3 by deacylation for 20 min with methylamine (25%)/methanol/butanol (42.8:45.7:11.5 (v/v/v)) at 53 °C (19). A parallel reaction with [32P]PtdIns(3,4,5)P3 internal standard showed a 95% conversion to GroPtdIns(3,4,5)P3.

Partial Purification of Recombinant Rat Brain Ins(1,4,5)P3 3-Kinase

Expression and purification of recombinant rat brain Ins(1,4,5)P3 3-kinase was performed according to Ref. 20 with minor modifications. A single Escherichia coli colony, containing the Bluescript plasmid with the cloned DNA insert (C5), was used to inoculate LB medium supplemented with 50 µg/ml ampicillin at 37 °C to an A600 of 1.5. The culture was then diluted to an A600 of 0.5 with fresh prewarmed medium (30 °C), and expression was induced by the addition of isopropyl-beta -D-thiogalactopyranoside (1 mM) after which the cells were grown for an additional 2 h at 30 °C. Bacteria were harvested (1200 × g, 15 min) and resuspended in cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, 0.4 mM benzamidine, 5 µM leupeptin, 5 µM pepstatin, and calpain inhibitors I and II at 5 µg/ml). After sonication in ice, Triton X-100 was added to a final concentration of 1% (v/v) and the lysate was shaken for 1 h at 4 °C. After a 30-min centrifugation at 15,000 × g, the supernatant was applied to a CaM-affinity column which was eluted as described in Ref. 20. The active fraction was concentrated using an Amicon Centriprep 30 filter.

Preparation of [3-32P]Ins(1,3,4,5)P4

[3-32P]Ins(1,3,4,5)P4 was prepared from Ins(1,4,5)P3 and [gamma -32P]ATP using recombinant Ins(1,4,5)P3 3-kinase partially purified as described above. A reaction mixture of 200 µl, containing 100 µM Ins(1,4,5)P3, 20 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 1.018 mM CaCl2, 1 mM EGTA, 10 µM CaM, 1 mg/ml bovine serum albumin, 1 mCi of [gamma -32P]ATP (3000 Ci/mmol), and 20 µl of enzyme, was incubated for 1 h at 37 °C, and the reaction was terminated by the addition of 0.8 ml of 10 mM EDTA followed by 2 min of boiling. The sample was applied to a HPLC Partisphere-SAX column, eluted with a nonlinear gradient made of water and 1.0 M NH4H2PO4 (pH 3.8). Fractions (2 ml) containing [3-32P]Ins(1,3,4,5)P4 were identified by Cerenkov counting, pooled, and dialyzed 3 times against 1000 ml of water for 45 min (the desalting of inositol polyphosphates by dialysis is described in more detail in Ref. 21). This effectively removed the inorganic phosphate which would otherwise interfere in the Ins(1,3,4,5)P4 binding assay. The recovery of [32P]Ins(1,3,4,5)P4 following dialysis was approximately 50%. Starting with 100 µM Ins(1,4,5)P3 and 1 mCi of [gamma -32P]ATP (~3000 Ci/mmol), 0.5 mCi of [32P]Ins(1,3,4,5)P4 with a specific radioactivity of ~3000 Ci/mmol was routinely produced.

Preparation of Lipid Extracts

1321N1 Astrocytoma cells were grown to confluence on 6-well plates as described previously (22). Prior to treatment, cells were washed twice with modified Krebs-Henseleit buffer and incubated in this buffer for 30 min at 37 °C (22). Cells were treated for 7.5 min with dimethyl sulfoxide carrier or wortmannin (100 nM) and then stimulated for 10 min with insulin (10 µg/ml). The medium was aspirated and the cells were quenched with 1 ml of 10% trichloroacetic acid. After 15 min on ice, dishes were scraped and washed once with 0.5 ml of 10% trichloroacetic acid. The cell lysates were centrifuged for 5 min at 13,000 × g, and the resulting pellet was washed twice with 0.5 ml of 5% trichloroacetic acid, 1 mM EDTA. Lipids were extracted in 0.75 ml of CHCl3/MeOH/HCl (40:80:1, by volume) for 20 min, and phases were then split by the addition of 0.25 ml of CHCl3 and 0.45 ml of 0.1 M HCl. The lower phase, obtained after a 1-min centrifugation at 13,000 × g, was transferred to a screw cap tube, and the upper phase was re-extracted once with 0.45 ml of the synthetic lower phase. The lower phases were pooled and dried down. Alkaline hydrolysis of dried lipids was carried out by vortexing in 50 µl of 1.0 M KOH and boiling for 30 min. Following neutralization with 50 µl of 1.0 M acetic acid, the fatty acids were removed by 2 extractions with 0.5 ml of water-saturated butan-1-ol/petroleum ether (40-60 °C)/ethyl acetate (20:4:1, by volume). Finally, the resulting water-soluble fractions were dried down and resuspended in 100 µl of 0.2 M acetic acid, resulting in a 0.5 M potassium acetate solution with a final pH of 5.0, which could be used directly in the isotope dilution assay. The volumes could be adjusted to take into account the amount of cellular lipid present in each sample. As a general rule, samples which originally contained 1 mg of cell protein (about 0.3 mg of total lipid) could be efficiently hydrolyzed using the volumes of reagents noted above (see "Results and Discussion").

Ins(1,3,4,5)P4 Isotope Dilution Assay

The Ins(1,3,4,5)P4-binding protein was obtained from sheep cerebellum and prepared as described in Ref. 23. Briefly, cerebella were homogenized in ice cold buffer (20 mM NaHCO3, pH 8.0, 1 mM dithiothreitol, 2 mM EDTA) and centrifuged for 10 min at 5000 × g. The pellet was re-extracted once, and the pooled supernatants were centrifuged for 20 min at 38,000 × g. The pellet was washed twice and resuspended in homogenization buffer at a final protein concentration of 10-20 mg/ml.

Ins(1,3,4,5)P4 concentration was determined as in Ref. 23 which is an adaptation of Ref. 24. Assays of 320 µl comprised 80 µl of assay buffer (0.1 M NaAc, 0.1 M KH2PO4, pH 5.0, 4 mM EDTA, 80 µl of 3 × 105 dpm of [32P]Ins(1,3,4,5)P4), 80 µl of sample, and 80 µl of binding protein. Both standard Ins(1,3,4,5)P4 and samples were in 0.5 M KOH/acetic acid (pH 5.0) and were assayed directly. Samples were diluted in 0.5 M KOH/acetic acid (pH 5.0) to allow measurements in the most sensitive range of the displacement curve. After addition of binding protein, samples were incubated on ice for 30 min and subsequently subjected to rapid filtration using GF/C filters. Filters were washed twice with 5 ml of ice cold buffer (25 mM NaAc, 25 mM KH2PO4, pH 5.0, 1 mM EDTA, and 5 mM NaHCO3). Radioactivity was determined after the filters were extracted for 12 h in 4 ml of scintillant.

In Vivo Stimulation of Rats with Insulin

Male Wistar rats (200 g) were starved overnight and anesthetized with sodium pentobarbital, and one hind limb muscle was isolated prior to treatment. Rats were injected via the hind limb saphenous vein with 0.5 ml of 150 mM NaCl, containing propranolol (3.0 mg/kg) or propranolol (3.0 mg/kg) plus insulin (1.0 unit/kg). After various times, one hind limb skeletal muscle was freeze-clamped (with aluminum tongs cooled in liquid nitrogen), excised, and then powdered under liquid nitrogen using a precooled pestle and mortar. One gram of powdered muscle was used for the PtdIns(3,4,5)P3 mass determination, and three rats were used for each time point. To each gram of powdered muscle, 10 ml of 10% trichloroacetic acid was added, and after 10 min the samples were centrifuged at 3000 rpm for 10 min. The pellets were washed and extracted as described above, but using 10 volumes of extraction solution. The extracted lipids were hydrolyzed in 0.8 ml of 1 M KOH, to account for the greater mass of starting material. Efficient hydrolysis was checked by HPLC analysis of spiked [32P]PtdIns(3,4,5)P3. For measurements of PKB activity, 1 g of powdered muscle was homogenized in 3 ml of ice-cold homogenization buffer (4 mM EDTA (pH 8.0), 50 mM NaF, 1.0 mM orthovanadate (pH 10.0), 1 µM microcystin-LR, 0.1% beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine) using a Polytron homogenizer at 4 °C. The homogenate was spun at 13,000 × g for 10 min in a refrigerated centrifuge. Immunoprecipitation of PKB from 500 µg of muscle extract and subsequent measurement of enzymatic activity was done as in Ref. 25.


RESULTS AND DISCUSSION

Principles of the Assay

The assay procedure is based upon alkaline cleavage of a total cell phospholipid extract which generates Ins(1,3,4,5)P4 from PtdIns(3,4,5)P3. The water-soluble head group can then be measured using a highly specific radioligand displacement assay. This approach, which avoids tedious chromatographic procedures, is based on a previously reported assay for PtdIns(4,5)P2, which utilized alkaline hydrolysis coupled to the radioligand displacement-based measurement of Ins(1,4,5)P3 (26).

In order to assess the utility of such an approach for analysis of PtdIns(3,4,5)P3, it was first necessary to determine the recovery of Ins(1,3,4,5)P4 through the lipid extraction and hydrolysis procedures. The recovery of PtdIns(3,4,5)P3 was analyzed by spiking tracer [32P]PtdIns(3,4,5)P3 into total cell lipid extracts or lipids from Folch fraction I from bovine brain.

A single back-extraction of the upper phase of such extracts with synthetic lower phase (see "Experimental Procedures") was sufficient to recover 98% of the spiked radioactivity (not shown). The recovery of the radiolabeled Ins(1,3,4,5)P4 was then followed by anion exchange HPLC analysis of the products. For these experiments, it was important to ensure an excess of KOH for hydrolysis of the ester bonds in the sample. Cell samples containing 1 mg of protein contain approximately 0.3 mg of lipid. Assuming an average molecular weight of 1000 and 3 susceptible bonds per mol of lipid, 50 µl of 1 M KOH gives a 50-fold excess of hydroxyl ions. When this approach was applied to astrocytoma cell lipid extracts, HPLC analysis revealed 4 major peaks of radioactivity which accounted for >95% of the radioactivity applied to the column and which corresponded with the mobilities of Ins(3,4,5)P3 (10.9 ± 0.59%), GroPtdIns(3,4,5)P3 (3.9 ± 0.66%), Ins(1,3,4,5)P4 (62 ± 0.41%), and Ins(2,3,4,5)P4 (24 ± 1.2%). These are the expected products of alkaline hydrolysis of PtdIns(3,4,5)P3 based on the original studies of PtdIns(4,5)P2 hydrolysis by Brockerhoff and Ballou (27). As expected, the recovery of Ins(1,3,4,5)P4 through this procedure depended on the relative amounts of lipid and KOH. Thus, hydrolysis of 0.3 mg of astrocytoma cell lipids with 0.1 M and 0.5 M KOH yielded 17.0% and 52.4% Ins(1,3,4,5)P4, respectively. All subsequent experiments used 50 µl of 1 M KOH to hydrolyze samples containing not more than 0.6 mg of lipid; the volume of M KOH was increased correspondingly for samples containing greater amounts of lipid.

Selectivity and Sensitivity

Previous reports established the presence of highly specific Ins(1,3,4,5)P4 binding sites in membranes prepared from mammalian cerebella and platelets (23, 24, 28), which formed the basis of a radioreceptor assay for Ins(1,3,4,5)P4 (24). This assay requires a radioligand with a higher specific radioactivity than the commercially available [3H]Ins(1,3,4,5)P4, and hence it was necessary to synthesize [32P]Ins(1,3,4,5)P4 as detailed under "Experimental Procedures." Sheep cerebellum proved in our hands to be a convenient source of membranes with a sufficiently high density of high affinity Ins(1,3,4,5)P4 binding sites, although pig and rat cerebellum could also be used (23, 24). At least 5 different preparations of Ins(1,3,4,5)P4-binding protein, isolated from sheep cerebellum, showed consistent characteristics with regard to the Kd for Ins(1,3,4,5)P4 (3.97 ± 1.23; n = 5) and Ins(1,4,5)P3, maximal binding, nonspecific binding, and the presence of a low affinity binding site.

Fig. 1 shows a typical calibration curve in which [32P]Ins(1,3,4,5)P4 binding to cerebellar membranes from sheep was displaced by increasing concentrations of unlabeled ligand. The binding assays contained 125 mM potassium acetate (pH 5.0) to mimic the conditions for samples which had undergone alkaline hydrolysis and were analyzed using a computer-assisted curve-fitting program. Ins(1,3,4,5)P4 bound with a Kd of 4.1 ± 0.96 nM and nonspecific binding, defined in the presence of 2.5 µM unlabeled ligand, was approximately 20% of total binding. Scatchard analysis indicated the presence of a second, low affinity site which never amounted to more than 10% of total specific binding. The sensitivity range for accurate measurements of Ins(1,3,4,5)P4 was approximately 0.3 to 5 pmol (25-75% displacement of specific binding). Since the assay volume was 0.32 ml, this could be improved slightly by reducing the assay volume. These results are essentially similar to those reported in Ref. 23.


Fig. 1. Displacement of [32P]Ins(1,3,4,5)P4 by authentic Ins(1,3,4,5)P4 and Ins(1,4,5)P3 using Ins(1,3,4,5)P4-binding protein isolated from sheep cerebellum. The displacement of [32P]Ins(1,3,4,5)P4 (3 × 105 dpm) by Ins(1,3,4,5)P4 (black-diamond ) and Ins(1,4,5)P3 (black-square) at indicated concentrations was measured in a final assay volume of 0.32 ml after a 30-min incubation on ice. Each data point represents a determination in triplicate ± S.D. obtained from seven independent experiments.
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When the above assay was applied to synthetic PtdIns(3,4,5)P3 that had been subjected to alkaline hydrolysis, an apparent Kd of 8.1 ± 2.9 nM was obtained, consistent with the expected yield of Ins(1,3,4,5)P4 of 62%. This suggests that none of the side-products of PtdIns(3,4,5)P3 hydrolysis have a significant impact on displacement of the radioligand in the assay. This was established directly in the case of GroPtdIns(3,4,5)P3, which gave a Kd of 7.6 ± 1.72 nM, but amounts to less than 5% of the products of alkaline hydrolysis (see above).

Another issue to be dealt with was the affinity of the receptor for Ins(1,4,5)P3 since this is the polar head group derived from alkaline hydrolysis of PtdIns(4,5)P2, which in unstimulated cells is usually present at a 1000-fold excess over PtdIns(3,4,5)P3 (29). As shown in Fig. 1, Ins(1,4,5)P3 did not displace any [32P]Ins(1,3,4,5)P4 at concentrations less than 1 µM (confirming previous reports (23, 24, 28)) and therefore is unlikely to contribute to the measured values obtained by the radioligand displacement assay.

Application of the Assay to Measurements of PtdIns(3,4,5)P3 in Tissue Extracts

A calibration curve, as shown in Fig. 1, was constructed for each experiment, and the assay values obtained for each sample were corrected for recovery of PtdIns(3,4,5)P3 during extraction (98%) and yield of Ins(1,3,4,5)P4 through alkaline hydrolysis (62%). To establish that displacement in the assay was due to authentic, cell-derived PtdIns(3,4,5)P3, we made use of a highly specific PtdIns(3,4,5)P3/Ins(1,3,4,5)P4-5-phosphatase that was partially purified from bovine brain.2 A large scale lipid extract from astrocytoma cells was hydrolyzed as described above. The resulting sample was dissolved in water (giving a final solution of 0.5 M potassium acetate, pH 7.5) and split into 2 portions, which were incubated with the 5-phosphatase under conditions in which hydrolysis of the available Ins(1,3,4,5)P4 was between 50 and 90%. One sample was spiked with [32P]Ins(1,3,4,5)P4, and the products were analyzed by anion exchange to determine the degradation of authentic Ins(1,3,4,5)P4. The other sample was analyzed by the radioligand displacement assay. The 2 assays gave similar results with 73% hydrolysis of the internal radiolabeled standard versus 85% loss of radioligand displacing activity (no significant loss was detected by either approach using boiled enzyme as control). These results confirm that the radioligand displacement assay detects only material present in alkali-hydrolyzed total cell lipid extracts that is degraded by a specific 5-phosphatase preparation at a similar rate to authentic Ins(1,3,4,5)P4, strongly suggesting that the assay is specifically measuring the latter compound.

We next applied the assay to measurements of PtdIns(3,4,5)P3 in control and insulin-stimulated 1321N1 astrocytoma cells in parallel with analysis of cells which had been labeled to isotopic equilibrium with [3H]inositol as described previously (22). The latter procedure allowed an independent assessment of the mass of PtdIns(3,4,5)P3 which can be determined from the radioactivity recovered in this lipid and measurement of the specific radioactivity of the inositol lipid pool. The results from these experiments are shown in Table I. The basal levels of PtdIns(3,4,5)P3 (2.16 ± 1.0 pmol/well) increased approximately 5-fold upon stimulation with insulin (11.3 ± 1.85 pmol/well). These values are remarkably close to those determined from the specific radioactivity measurements.

Table I.

PI 3-kinase stimulation in 1321N1 astrocytoma cells

The data, showing insulin-stimulated PtdIns(3,4,5)P3 production in 1321N1 astrocytoma cells, are expressed as dpm of [3H]PtdIns(3,4,5)P3 or pmol of PtdIns(3,4,5)P3 per one well of a six-well plate. The determined radioactivity and subsequent calculation of specific activity represent duplicates from at least two independent experiments (ND is not determined). The mass determinations were triplicates from 7 independent experiments, except the data for the inhibition with wortmannin which were from 2 independent experiments.
1321N1 astrocytoma cells PtdIns(3,4,5)P3 (HPLC analysis) PtdIns(3,4,5)P3 (specific activity) PtdIns(3,4,5)P3 (mass assay)

dpm/well pmol/well pmol/well
Control 2467  ± 154 0.82  ± 0.051 2.16  ± 1.00
Insulin 23862  ± 775 7.95  ± 0.258 11.32  ± 1.85
Wortmannin ND ND 1.41  ± 0.13

The use of wortmannin, a potent inhibitor of PI 3-kinases, gave further confirmation of the assay's specificity. In 1321N1 astrocytoma cells preincubated with wortmannin at a concentration of 100 nM, a dose which is considered to be selective for PI 3-kinases, the insulin-stimulated increase in PtdIns(3,4,5)P3 was completely abolished. The radioligand displacement assay was also used for measurements on Rat-1 fibroblasts, rat adipocytes, and HEK 293 cells, which all showed wortmannin-sensitive increases in PtdIns(3,4,5)P3 upon stimulation with platelet-derived growth factor or insulin (not shown).

Regulation of PtdIns(3,4,5)P3 Levels and PKB Activity in Skeletal Muscle after in Vivo Stimulation of Rats with Insulin

PKB is the cellular homologue of the v-Akt protein encoded in the genome of the Akt-8 retrovirus, isolated from a rodent T-cell lymphoma (30). Some isoforms of PKB are significantly overexpressed in several types of cancer (17, 31, 32). PKB is activated rapidly in response to a wide range of stimuli which also activate PI 3-kinase. A direct role for PI 3-kinase in the activation of PKB has been suggested by a combination of pharmacological and molecular genetic approaches (33-35). Moreover, PtdIns(3,4,5)P3 can bind directly to PKB although this in itself is not sufficient for activation (36) which requires phosphorylation of specific serine and threonine residues by an unidentified protein kinase.3

Using the new assay it is possible to monitor PtdIns(3,4,5)P3 levels in tissues and/or cells which are not suitable for labeling to high specific radioactivity. Thus we now report the first measurements of this lipid second messenger in an animal tissue, rat hind limb skeletal muscle, and compare the effects of intravenous injection of insulin on PtdIns(3,4,5)P3 levels and PKB activity in the same tissue samples. The results are shown in Fig. 2. PtdIns(3,4,5)P3 levels increased approximately 3-fold, 5 min after insulin administration, remained elevated at 10 min, and returned to basal levels within 15 min. PKB activity was elevated up to 4-fold and followed a similar time course which persisted at 15 min, but also returned to basal levels within 30 min. The larger and more prolonged PKB response is compatible with the hypothesis that PI 3-kinase lies upstream of PKB in a pathway which serves to amplify this initial signal.


Fig. 2. PtdIns(3,4,5)P3 production and PKB activation in response to insulin in rat skeletal muscle. PtdIns(3,4,5)P3 was extracted from 1 g of powdered muscle, and PKB was immunoprecipitated from 500 µg of muscle extract. The mass of PtdIns(3,4,5)P3 and the activity of PKB were measured using three rats for each time point. Data were standardized for protein content using the method of Bradford and are expressed as fold stimulation over control levels. Similar results were obtained in 3 independent experiments. square , PI 3-kinase; black-square, PKB.
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In summary, the present report establishes the feasibility of measuring PtdIns(3,4,5)P3 in cell and tissue extracts by a method which avoids time-consuming chromatographic procedures and hazardous amounts of radioactivity. The specificity and precision of the assay was demonstrated enzymologically, using an enzyme which specifically degrades PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4; pharmacologically, by showing that the signal detected by the assay was enhanced by agonists which stimulate PI 3-kinase in a wortmannin-sensitive manner; and by comparison with PtdIns(3,4,5)P3 levels determined by an independent method. In addition to the ease of analysis, an important advance is the ability to monitor PtdIns(3,4,5)P3 in animal tissues which are not suited to metabolic labeling procedures. As PtdIns(3,4,5)P3 has been implicated as a critical signal in both normal and pathological growth control, an important application of the assay will be the measurement of this lipid in clinical samples such as tumor biopsies.


FOOTNOTES

*   This work was supported by Medical Research Council Program Grant G9403619 (to C. P. D.) and by a CASE studentship from the Biochemical Biophysical Sciences Research Council and SmithKline Beecham Pharmaceuticals (to D. A. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 13-82-344729; Fax: 13-82-201063; E-mail: jvdkaay{at}bad.dundee.ac.uk.
1    The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Gro, glycero; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; CaM, calmodulin; HPLC, high performance liquid chromatography.
2    D. M. Hickinson and C. P. Downes, unpublished results.
3    D. Alessi, personal communication.

Acknowledgments

The E. coli clone expressing the recombinant Ins(1,4,5)P3 3-kinase was a gift from C. Erneux (Free University of Brussels). PtdIns(3,4,5)P3 was provided by R. Gigg, and [3-32P]PtdIns(3,4,5)P3 was prepared by D. M. Hickinson.


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