ARTICLE |
Correspondence to: Kevin D. Niswender, Box 359757, Harborview Medical Center, 300 9th Avenue, Seattle, WA 98104. E-mail: kniswend@u.washington.edu
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Summary |
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Intracellular signaling mediated by phosphatidylinositol 3-kinase (PI3K) is important for a number of cellular processes and is stimulated by a variety of hormones, including insulin and leptin. A histochemical method for assessment of PI3K signaling would be an important advance in identifying specific cells in histologically complex organs that are regulated by growth factors and peptide hormones. However, current methods for detecting PI3K activity require either homogenization of the tissue or cells or the ability to transfect probes that bind to phosphatidylinositol 3,4,5 trisphosphate (PIP3), the reaction product of PI3K catalysis. Here we report the validation of an immunocytochemical method to detect changes in PI3K activity, using a recently developed monoclonal antibody to PIP3, in paraformaldehyde-fixed bovine aortic endothelial cells (BAECs) in culture and in hepatocytes of intact rat liver. Treatment with either insulin or leptin increased BAEC PIP3 immunoreactivity, and these effects were blocked by pretreatment with PI3K inhibitors. Furthermore, infusion of insulin into the hepatic portal vein of fasted rats caused an increase of PIP3 immunostaining in hepatocytes that was associated with increased serine phosphorylation of the downstream signaling molecule protein kinase B/Akt (PKB/Akt). We conclude that immunocytochemical PIP3 staining can detect changes in PI3K activation induced by insulin and leptin in cell culture and intact liver.
(J Histochem Cytochem 51:275283, 2003)
Key Words: phosphatidylinositol 3-kinase, PIP3, insulin, leptin, endothelium, hepatocyte
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Introduction |
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PHOSPHATIDYLINOSITOL 3-KINASES (PI3K) are a family of ubiquitous signal transduction enzymes that participate in the intracellular signal transduction initiated by many growth factors and peptide hormones (
Methods currently available for assessing the activity of PI3K require generating cell or tissue homogenates from which biochemical measurements of enzyme activity are made by incubation with radiolabeled substrate (
PI3K catalyzes the phosphorylation of phosphatidylinositol (PI) at the 3'-OH position to generate PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 (abbreviated here as PIP3). Physiologically, PI(4,5)P2 appears to be the major substrate for PI3K, with PIP3 being the major reaction product (
Here we report the use of this PIP3 antibody to detect increased PIP3-like immunostaining of primary bovine aortic endothelial cells (BAECs) that were stimulated in vitro by insulin and leptin. Using a semiquantitative approach, immunological controls, and PI3K inhibitors, we demonstrate that the PIP3-like immunoreactivity detected by this antibody arises from activation of PI3K. Insulin and leptin stimulation resulted in a marked enhancement of immunoreactive PIP3 over a timecourse consistent with the well-characterized biochemical actions of these hormones (
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Materials and Methods |
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Cell Culture Studies
Cultured BAECs were established and maintained in DMEM supplemented with 10% fetal bovine serum, glutamine, and penicillin/streptomycin (
Studies In Vivo
All procedures involving the use of animals were approved by the Animal Research Committees at the University of Washington and the VA Puget Sound Health Care System. Mature (300340 g) male Wistar rats (Simonsen; Gilroy, CA) were housed under standard conditions. Animals were fasted for 48 hr (with water freely available), then anesthetized with a ketamine/xylazine mixture. A 5-mU injection of insulin in saline (n=3) (or saline alone, n=3) was administered via the hepatic portal vein, followed 15 min later by portal vein perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Liver samples were removed and placed in fresh fixative overnight, followed by the the buffer alone containing 25% sucrose (w/v) overnight at 4C. They were snap-frozen in freon cooled by dry ice. Cryostat sections (14 µm) were mounted on glass slides and stored at 70C.
Immunohistochemical Methods
Coverslips containing BAECs or slides with liver sections were equilibrated in PBS at room temperature. After blocking in 5% normal goat serum (NGS) and 2% bovine serum albumin (BSA), samples were incubated with mouse anti-PIP3 monoclonal antibody (Echelon; Salt Lake City, UT) at a 1:100 dilution overnight at 4C. Dilutions ranging from 1:50 to 1:500 were tested, with 1:100 giving the best signal-to-noise ratio. The negative immune control for this antibody was an equivalent concentration of non-immune mouse IgM (DakoCytomation; Carpinteria, CA) substituted for the primary antibody at an equivalent dilution. All antibodies were diluted in 0.1 M PBS containing 2% BSA and 5% NGS. All BAEC samples were counterstained with DAPI (Molecular Probes; Eugene OR) to count the number of nuclei per field of view. To detect PI3K-mediated serine phosphorylation of PKB/Akt- as a measure of activation of the PI3K pathway, we used a polyclonal antibody (1:100) to serine473-phosphorylated PKB/Akt (Cell Signaling Technology; Beverly, MA). The IRS-2 antibody [gift of M. Myers Jr, Joslin Diabetes Center, Harvard Medical School, Boston, MA (
Imaging Methods
Images of BAECs were captured via standard epifluorescence using a Nikon Eclipse E600 upright microscope equipped with a Diagnostic Instruments Spot RT Color digital camera. All images for a single experiment were obtained using identical acquisition parameters in one imaging session. PIP3 immunofluorescence was quantified using Scion Image freeware (Scion; Frederick, MD) as the simple average pixel intensity for the field of view. DAPI fluorescence was evaluated via particle analysis using Scion Image freeware, yielding the total number of cells present per field of view. Values are reported as the mean ± SEM of 610 fields of view per coverslip and at least two to four coverslips per measurement. The average pixel intensity was analyzed in this way from a set of coverslips (n=3) in which an equivalent concentration of non-immune mouse IgM was substituted for the PIP3 antibody. The average pixel intensity derived from these samples, representing cell autofluorescence and nonspecific immune interactions, was subtracted from the average pixel intensity derived from the experiments described in Fig 2 and Fig 3 to report a "corrected" cell fluorescence that more accurately reflects the true increase in immunodetectable PIP3 on hormone stimulation. Statistical significance was determined by ANOVA with Bonferroni's post test for multiple comparisions. Although separate experiments were not combined because we could not ensure identical staining and imaging parameters during different sessions on different days, the results are consistent among different experiments. Images of rat liver were acquired with a Zeiss Axiophot fluorescence microscope.
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Results |
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PIP3 Is an Immunodetectable Molecular Species in Cells
PIP3 immunoreactivity was readily visible in serum-fed BAECs stained with anti-PIP3 antibody (Fig 1A and Fig 1B) but was not detected in cells incubated with an equal concentration of non-immune mouse IgM (Fig 1C and Fig 1D). In comparison to serum-starved BAECs, PIP3 immunoreactivity was visibly increased in serum-fed cells and in cells stimulated by insulin or leptin (Fig 2).
Insulin and Leptin Stimulate PIP3-like Immunoreactivity in BAECs
Cultured BAECs are responsive to both leptin (
Immunoreactive PIP3 Arises from Activation of PI3K
To ensure that increased PIP3 immunofluoresence in BAECs treated with leptin was due specifically to activation of PI3K, rather than to a nonspecific effect, cells were pretreated with either of two PI3K inhibitors, LY294002 (Fig 3C and Fig 3E) or wortmannin (Fig 3D and Fig 3E) or with vehicle, followed by a 10-min incubation with leptin. Pretreatment with either PI3K inhibitor before hormone treatment blocked the increase of PIP3 immunoreactivity (Fig 3A and Fig 3E) observed when cells were pretreated with vehicle followed by leptin (Fig 3B and Fig 3E). Similar findings were observed with insulin treatment (data not shown). Because PI3K is the only enzyme known to be inhibited by both wortmannin and LY294002, we conclude that leptin-induced increases of PIP3 immunofluoresence in BAECs resulted specifically from activation of PI3K.
Immunoreactive PIP3 Co-localizes with IRS-2
To determine whether the PIP3-like immunoreactivity observed after insulin stimulation could represent PIP3 that participates in intracellular signaling, we sought to determine whether the insulin receptorPI3K coupling molecule IRS-2 co-localizes with PIP3 on stimulation by insulin. In the vehicle-treated state there is relatively weak immunocytochemical co-localization of IRS-2 (green fluorescence) with PIP3 (red fluorescence) (Fig 4A), indicated by the relative absence of yellow (red plus green) fluorescence. However, 5 min of insulin treatment resulted in a substantially increased immunological co-localization of IRS-2 and PIP3 (Fig 4B). The areas of greatest co-localization included the perinuclear area and the plasma membrane, where intracellular targets of PI3K activation are known to exist (
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Insulin Stimulation Generates Immunoreactive PIP3 and Phosphorylated PKB/Akt in Hepatocytes In Vivo
To determine whether the PIP3 immunoreactive species detected by this antibody could be used to detect changes in PI3K activity in situ, we examined PIP3 immunofluorescence in liver obtained from rats infused with either insulin or saline directly into the hepatic portal vein 15 min before sacrifice, a protocol that induces activation of insulin receptor signaling and phosphorylation of IRS proteins and PI3K in rat liver (
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Discussion |
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Our objective in developing this immunocytochemical method for detecting insulin- and leptin-stimulated PIP3 was to validate a direct assay of the activation of PI3K in histologically intact tissue. Current methods of quantifying PI3K activity require homogenization of the tissue or cells of interest, followed by biochemical analysis. Such methods work well for reasonably homogeneous tissues, such as liver and skeletal muscle, and for experiments in which information on cell variability, cell phenotype, or the response of individual cells is not required. They are obviously not appropriate for anatomically complex heterogeneous tissues, such as brain, in which the cell type of interest may be quite rare. The generation of an antibody that specifically recognizes PIP3 therefore promises to have great value in morphological studies of PI3K signaling.
The signaling cascade leading to activation of PI3K and generation of PIP3 is initiated by growth factor or hormone binding to its respective receptor, activating either an intrinsic tyrosine kinase (insulin) or an extrinsic tyrosine kinase (janus kinase in the case of leptin). Phosphorylation of the intracellular portion of the majority of these receptors by their respective kinase results in recruitment and binding of downstream signaling molecules to the phosphorylated tyrosine residues. In the case of insulin, receptor activation in this manner results in the tyrosine phosphorylation and activation of one of a family of insulin receptor substrate molecules (IRS 14) which, in turn, binds to the regulatory subunit of PI3K (
An emerging area of great interest in the fields of diabetes and obesity research is the concept of "crosstalk" or "convergence" between insulin and leptin intracellular signaling pathways. In several cell types, including muscle cells (
The experiments reported here demonstrate that immunocytochemistry with an antibody to PIP3 reveals increased immunofluorescence of BAECs treated with insulin and leptin and that this effect was blocked with PI3K inhibitors. These observations are consistent with PI3K catalysis stimulated by insulin and leptin. Although neither PI3K inhibitor is entirely specific, the only enzyme known to be inhibited by both LY294002 and wortmannin is PI3K (
Interestingly, the PIP3-like immunofluorescence levels in unstimulated cells were quite low, as expected, and there was very little co-localization with IRS-2. On insulin stimulation, marked enhancement of co-localization with IRS-2 was observed, particularly in the region occupied by perinuclear membranes and in the vicinity of the plasma membrane, both sites at which targets of PI3K signaling are known to exist (
Because PI3K is an evolutionarily conserved intracellular signaling system (
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Acknowledgments |
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Supported by NIH grants DK17047 to the Diabetes Endocrinology Research Center at the University of Washington, Department of Veterans Affairs Career Scientist and Merit Review Research Programs to DGB, and NIH grants NS32273, DK12829, and DK52989 to MWS, and HL30946 to MAC. KN is supported by NIH Training Grant T32 DK07247.
We acknowledge the skilled technical efforts of J. Murphy and T. Eakin.
Received for publication September 16, 2002; accepted October 30, 2002.
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