p110β Is Up-regulated during Differentiation of 3T3-L1 Cells and Contributes to the Highly Insulin-responsive Glucose Transport Activity*

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85α) and one of two widely distributed isoforms of the p110 catalytic subunit: p110α or p110β. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110β was up-regulated ∼10-fold, whereas expression of p110α was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110β and p110α in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110β was lower than that of p110α. When p110α and p110β were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85α and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110β, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110α were observed. Interestingly, overexpression of p110β increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110α increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110β neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110α neutralizing antibody did only slightly. Together, these findings suggest that p110β plays a crucial role in cellular activities evoked acutely by insulin.

Activation of phosphatidylinositol (PI) 1 3-kinase has been implicated in the regulation of various cellular activities, including proliferation (1,2), differentiation (3), membrane ruffling (4,5), and prevention of apoptosis (6 -12). It is now clear that at least four types of PI 3-kinases exist, including mammalian homologs of Saccharomyces cerevisiae VPS34 (13), a G-protein-activated form termed p110␥ (14), and p170 having C-terminal sequences similar to the phosphoinositide-binding C2 domain (15). The best known form of PI 3-kinase is p85/ p110, which exists as a heterodimer consisting of a regulatory and a catalytic subunit. The regulatory subunit contains two Src homology 2 domains and functions as an adaptor protein transmitting the signal from a tyrosine-phosphorylated protein to the catalytic p110 subunit (16). To date, five isoforms of the regulatory subunit and three isoforms of the catalytic subunit (p110␣, p110␤, and p110␦) have been identified. The regulatory subunits include two 85-kDa proteins (p85␣ and p85␤) (17), two 55-kDa proteins (p55␣ and p55␥) (18 -20), and one 50-kDa protein (p50␣) (21,22). The respective tissue distributions of the five isoforms differ, as do the levels of their insulin-induced activation of the associated catalytic subunits (21)(22)(23). This suggests that the different regulatory subunit isoforms function within specific signal transduction pathways induced by various growth factors and hormones. On the other hand, although some reports suggest different roles for p110␣ and p110␤, far less is known about functional differences of the catalytic subunits.
Insulin-induced activation of PI 3-kinase in 3T3-L1 adipocytes is itself sufficient to elicit translocation of the GLUT4 glucose transporter to the cell surface with a resultant increase in glucose uptake (24 -26). In addition, we have observed that expression of 110␤ is markedly increased in 3T3-L1 cells during their differentiation into adipocytes. The aim of the present study was to better understand the respective roles of the p110␣ and p110␤ subunits in the regulation of phosphoinositide metabolism by insulin. This was accomplished by examining the effects of p110 catalytic subunits overexpressed in 3T3-L1 adipocytes using an adenovirus transduction system and by microinjecting neutralizing anti-p110␣ and anti-p110␤ antibodies into the cells.
Antibodies-Anti-phosphotyrosine antibody (Ab) 4G10 and anti-p85 antiserum were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal anti-GLUT4 Ab was from Genzyme (Cambridge, MA). The anti-p110␣ and anti-p110␤ Abs were raised in rabbits against synthetic peptides corresponding to residues 1048 -1068 of the p110␣ protein and to residues 1039 -1070 of the p110␤ protein, respectively. These Abs were affinity-purified on Affi-Gel-10 (Bio-Rad) columns to which the corresponding peptides had been coupled. They were then extensively dialyzed against phosphate-buffered saline (PBS). Abs raised against IRS-1 and the C-terminal GLUT2 tag were prepared as described previously (24,27).
Cell Culture-3T3-L1 fibroblasts to be transduced using recombinant adenovirus were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% donor calf serum (Life Technologies, Inc.) under an atmosphere of 10% CO 2 /90% air at 37°C. Two days after the fibroblasts reached confluence, differentiation was induced by incubating them for 48 h in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 4 mg/ml dexamethasone, and 10% fetal bovine serum. Thereafter, the cells were maintained in DMEM supplemented with 10% fetal bovine serum, which was renewed every other day. The cells were infected with the indicated adenoviruses on day 4 after inducing differentiation, and the experiments were conducted on day 6 when Ͼ90% of cells expressed the adipocyte phenotype and GLUT4 was almost fully expressed.
Cloning of Human p110␤ cDNA-Cloning and construct reverse transcription-polymerase chain reaction was carried out on human embryonic heart cDNA; the cDNA encoding p110␤ was amplified based on its reported sequence (28), yielding its entire coding region. The coding region of p110␣ cDNA was obtained as described in our previous paper (24). A portion of human GLUT2 cDNA corresponding to residues 510 -524 was then ligated to p110␣ and to p110␤, generating PI kinase catalytic subunits that were tagged at their C termini (24).
Gene Transduction-The entire coding regions of p110␣ and p110␤, along with the GLUT2 epitope tag, were cloned into the expression cosmid cassette pAdexCAwt. Recombinant adenoviruses were obtained by homologous recombination of those cosmids and the parental virus genome as described previously (24).
Immunoprecipitation and Western Blotting-Cells were lysed in PBS containing 1% Triton, 0.35 mg/ml phenylmethylsulfonyl fluoride, and 100 M sodium vanadate, after which the lysates were centrifuged for 10 min at 15,000 ϫ g and 4°C to remove insoluble materials. For immunoprecipitation, the supernatants were incubated with the indicated antibodies, after which protein A-Sepharose was added. The immune complexes were then collected by centrifugation, washed with PBS containing 1% Triton, boiled in Laemmli sample buffer containing 100 mM dithiothreitol, and subjected to SDS-PAGE. Immunoblotting was performed with an ECL system according to the manufacturer's instructions. In some experiments, band intensities were quantitated using a Molecular Imager GS-525 (Bio-Rad).
PI 3-Kinase Assay-PI 3-kinase assays were carried out using transduced 3T3-L1 adipocytes. 3T3-L1 adipocytes plated in 24-well culture dishes were serum-starved for 3 h in DMEM containing 0.2% bovine serum albumin and then incubated with or without selected concentrations of insulin for 5 min, washed with ice-cold PBS, and lysed with PBS containing 1% Nonidet P-40, 0.35 mg/ml phenylmethylsulfonyl fluoride, and 100 M sodium vanadate. Cell lysates were cleared of insoluble materials by centrifugation (15,000 ϫ g, 4°C, 10 min) and immunoprecipitated with the indicated Ab and protein A-Sepharose. PI 3-kinase activities in the immunoprecipitates were measured as described previously (24) using PI as a substrates. The results were quantitated with Fuji BAS2000 (Tokyo, Japan).
Glucose Transport Assay-3T3-L1 adipocytes plated in 24-well culture dishes were serum-starved as indicated above, after which they were incubated in Krebs-Ringer phosphate buffer for an additional 45 min prior to incubation with or without 10 Ϫ7 M insulin for 15 min. The assay was initiated by adding 2-deoxy-D-[ 3 H]glucose (0.5 mCi/sample, 0.1 mmol) and was terminated 4 min later by washing the cells once with ice-cold Krebs-Ringer phosphate buffer containing 0.3 mM phloretin and then twice with ice-cold Krebs-Ringer phosphate buffer. The cells were then solubilized in 0.1% SDS, and the incorporated radioac-tivity was determined by scintillation counting.
Microinjection of Anti-p110 Abs into 3T3-L1 Adipocytes-3T3-L1 adipocytes were trypsinized, reseeded onto acid-washed glass coverslips, and then incubated in serum-free DMEM for 3 h. Microinjection of the affinity-purified anti-p110 Abs or control rabbit IgG at a final concentration of 2 mg/ml in PBS was carried out using a semiautomated Eppendorf microinjection system with an injection pressure of 100 hectopascals and an injection time of 0.8 s. The injection volume is approximately 10% of the cell volume under these conditions. In each experiment, 200 -250 cells were microinjected with each anti-p110␣, anti-p110␤ Ab, or control rabbit IgG.
Membrane Sheet Assay-After microinjection, 3T3-L1 adipocytes were incubated with/without insulin for 15 min. Plasma membrane sheets were prepared by sonication as described previously (29). Adherent plasma membrane were fixed in 2% paraformaldehyde and processed for indirect immunofluorescence using a monoclonal anti-GLUT4 Ab followed by fluorescein-conjugated, anti-mouse IgG secondary Ab. The intensity of fluorescence from GLUT4 on the plasma membrane of each cell was analyzed using Molecular Analyst (Bio-Rad). The quantitation was carried out based on the statistical analysis from approximately 100 cells for each injected Ab.

RESULTS
Altered Expression of p110␣ and p110␤ during Differentiation of 3T3-L1-Differentiation of 3T3-L1 cells into adipocytes was induced as described under "Materials and Methods," and expression of p110␣ and p110␤, along with that of C/EBP␣ and GLUT4 as differentiation markers, was measured by immunoblotting using their specific Abs, respectively. To avoid influences from cell division after induction, each sample was normalized by protein contents. As shown in Fig. 1, C/EBP␣ was detected from Day 2, showed maximum expression on Day 4 and continued to express at a high level (Fig. 1A). GLUT4 could be detected from Day 2, and its expression level continued to increase (Fig. 1B). Expression level of p110␣ was unchanged during the differentiation (Fig. 1C). On the other hand, although the expression of p110␤ was under detectable level before induction, it could be detected from Day 2 and continued to increase significantly during differentiation, of which the time course was very similar to that of GLUT4 expression (Fig. 1D).
p110␣ and p110␤ Overexpressed in 3T3-L1 Adipocytes-p110␣ or p110␤ was overexpressed in 3T3-L1 adipocytes by using recombinant adenovirus such that similar levels of p110␣ and p110␤ proteins were expressed ( Fig. 2A, top panel). From the optical density of the blots, the degree to which p110␣ and p110␤ were overexpressed was calculated to be approximately 8-fold over their endogenous expression levels in 3T3-L1 cells ( Fig. 2A, middle and bottom panels). Overexpressed p110␣ and p110␤ were not recognized by the anti-p110␤ and anti-p110␣ Abs, respectively ( Fig. 2A, middle and bottom panels), indicating that these antibodies are highly specific for their corresponding isoforms.
The association of the overexpressed p110␣ and p110␤ isoforms with the p85␣ regulatory subunit was shown by the presence of p85␣ in the respective immunoprecipitates (Fig.  2B). p110␣ and p110␤ exhibited a very similar binding ability with the regulatory subunit, p85␣, in 3T3-L1 cells. In addition, it was also revealed that IRS-1, which was tyrosine-phosphorylated and bound to p85␣ in the presence of insulin, complexed with p110␣ or p110␤ with equal efficiency (Fig. 2C). Thus, it appears that p110␣ and p110␤ are not different in regard to their molecular association with the regulatory subunit or with IRS-1.
PI 3-Kinase Activities of p110␣ and p110␤ at the Basal Level and Their Responses to Insulin Stimulation-To compare PI 3-kinase activities of p110␣ and p110␤, either catalytic subunit was overexpressed in 3T3-L1 cells and immunoprecipitated with anti-GLUT2 tag Ab (Fig. 3A). Without insulin stimulation, overexpressed p110␣ exhibited 34-fold higher PI 3-kinase activity as compared with overexpressed p110␤, despite these catalytic subunits having similar affinity for p85␣, as shown in Fig. 2B.
The concentration response curves for endogenous p110␣ and p110␤ were next investigated. As shown in Fig. 3B, insulin-stimulated lipid kinase activity in the p110␤ immunoprecipitate was increased as much as 3.4-fold over basal activity in the absence of insulin. In contrast, insulin stimulation increased the kinase activity in the p110␣ immunoprecipitate by only about 60%. When the two catalytic subunits were then overexpressed using the adenovirus expression system, the kinase activity in the p110␤ immunoprecipitates from the cells overexpressing p110␤ were insulin-dependently increased as much as ϳ19-fold (Fig. 3C), whereas the kinase activity in the p110␣ immunoprecipitate from the cells overexpressing p110␣ was unaffected. To avoid any effect caused by using different Abs, anti-p110␣ and anti-p110␤, for immunoprecipitation, overexpressed p110␣ or p110␤ were immunoprecipitated by anti-GLUT2 tag Ab (Fig. 3D). Essentially the same results were obtained, clearly indicating that PI 3-kinase activity catalyzed by p110␤ is highly insulin-sensitive, whereas the activity catalyzed by p110␣ is not. or with anti-GLUT2 tag Ab (D); thereafter, the immunoprecipitates were assayed for PI 3-kinase activity. Squares represent PI 3-kinase activities in p110␣ immunoprecipitates, whereas circles represent those in p110␤ immunoprecipitates. Data are expressed as fold increases over basal PI 3-kinase activity, which is arbitrarily set to a value of 1. Two other independent experiments yielded similar results.

Effect of Overexpressing p110␣ or p110␤ on Basal and Insulin-induced Glucose
Uptake-3T3-L1 adipocytes were subjected to various multiplicities of infection (M.O.I.) with p110␣, p110␤, or control Lac-Z adenovirus, and the resulting effects on glucose transport were assessed. Infection with less than 250 M.O.I. of Lac-Z adenovirus had no affect on glucose transport into 3T3-L1 cells in either the absence or presence of insulin (Fig. 4, upper panel). Overexpression of p110␣ increased basal as well as insulin-stimulated glucose uptake in a M.O.I-dependent manner, which confirms the findings in our previous report (24). In contrast, despite the fact that overexpression of p110␤ did not significantly affect basal uptake, it increased the insulin-stimulated glucose transport as much as 2-fold over control. The lower panel in Fig. 4 shows the relationship between the degree of overexpression of each catalytic subunit and the basal and insulin-stimulated glucose uptake into the cells. Note that overexpression of p110␣ increased both basal and insulin-stimulated uptake, whereas overexpression of p110␤ increased only the stimulated uptake.
Inhibition of p110␣ and p110␤ Lipid Kinase Activity by Corresponding Specific Abs-After confirming the specificity of the anti-p110␣ and anti-p110␤ Abs ( Fig. 2A), we found that these Abs exert inhibitory effects on their corresponding isoforms. The anti-p110␣ Ab drastically reduced the lipid kinase activity of p110␣ by 96 -99% (30), whereas the Ab raised against residues 1039 -1070 of p110␤ protein reduced the lipid kinase activity by approximately 98% based on the same method as that used for anti-p110␣ Ab (data not shown).
Effect of Microinjecting p110 Abs on Insulin-induced Translocation of GLUT4 to the Cell Surface-To analyze the isoformspecific functions of endogenously expressed p110␣ and p110␤, inhibitory anti-p110␣ or anti-p110␤ Ab was microinjected into 3T3-L1 adipocytes, followed by membrane sheet assay. Microinjection of control rabbit IgG had no effect on insulin-induced translocation of the GLUT4 transporter to the cell surface (Fig.  5, A and B). Microinjection of either anti-p110␣ or anti-p110␤ Ab into cells had no effect on GLUT4 localization in the basal state (data not shown). Microinjection of anti-p110␣ Ab attenuated insulin-evoked GLUT4 translocation by approximately 25% (Fig. 5C; also quantified in Fig. 5E). In contrast, microinjection of anti-p110␤ Ab inhibited insulin-evoked GLUT4 translocation to the cell surface by approximately 94% (Fig. 5D; also quantified in Fig. 5E). DISCUSSION p85/p110 type PI kinase has been implicated in a wide range of cellular activities, including control of proliferation (1, 2), cytoskeletal organization (4, 5), prevention of apoptosis (6 -12), neurite outgrowth (3), vesicular trafficking (31), and insulininduced translocation of the glucose transporter to the cell surface (1,32). Most of these findings have been derived from experiments in which lipid kinase activity was diminished by antagonists such as wortmannin and LY294002 or by microinjection or overexpression of a dominant negative p85␣ mutant that binds to tyrosine phosphorylated proteins but lacks a p110 binding site. More recent studies entailing overexpression of constitutively active p110␣ or GLUT2-tagged p110␣ have shown that elevation of this lipid kinase activity is itself sufficient to induce some of these cellular activities, including translocation of GLUT4 glucose transporter and activation of an intracellular signaling pathway known to mediate proliferation (24 -26).
The aim of the present study was to better understand regulation of the PI kinase activities associated with two isoforms of its catalytic subunit, p110␣ and p110␤, and to assess how regulatory differences might influence the cellular effects of insulin. In fact, during differentiation of 3T3-L1 cells into adipocytes, in which they acquire insulin-induced glucose uptake, expression of p110␣ is unchanged, whereas that of p110␤ is markedly up-regulated (Fig. 1). On the basis of in vitro PI 3-kinase assays of p110␣ and p110␤ expressed in 3T3-L1 adipocytes, basal p110␤ activity is substantially lower than that of p110␣ (Fig. 3A). However, p110␤ appears to be highly insulinsensitive, whereas p110␣ was activated only slightly by insulin (Fig. 3, B-D). Although the two isoforms bind to p85␣ and to IRS-1 with similar efficiency (Fig. 2, B and C), by these bindings, p110␤ may change its conformation more dramatically than does p110␣. Such a difference may be attributable to isoform specific binding of p110 to unknown molecules, although further investigations are required. It should be noted, however, that each isoform-specific anti-p110 Abs markedly inhibited PI kinase activity of p110␣ or p110␤ (30). Nonetheless, because the lipid kinase activity of p110 incubated with each concentration of insulin should be similarly inhibited, it still seems reasonable to conclude that the p110␤ isoform is a highly insulin-sensitive form of the catalytic subunit, whereas the p110␣ isoform is a rather insulin-insensitive form.
In addition, we can speculate the reason why the insulininduced response of endogenous p110␤ was lower than that obtained from the experiment using overexpressed p110␤. In the plain or Lac-Z expressing 3T3-L1 cells, p85␣ is present as a heterodimer with endogeneous p110␣ or p110␤. Because IRS-1 possesses four potential binding sites for the Src homology 2 domain of p85␣ and thus can bind to two molecules of p85␣, a detectable amount of p110␣ was co-immunoprecipitated by the anti-p110␤ antibody in the insulin-stimulated condition, although this amount was small (data not shown). This co-immunoprecipitated "low-insulin responsive" p110␣ is likely to reduce the insulin responsiveness of the anti-p110␤ immunoprecipitate as compared with the control 3T3-L1 cells. In case of p110␤-overexpressing 3T3-L1 cells, most p85␣ is occupied by overexpressed p110␤, and the amount of p110␣ bound with p85␣ is much smaller than in control cells, and thus the effect of co-immunoprecipitated p110␣ becomes much smaller.
Earlier reports have suggested that different p110 catalytic subunit isoforms have distinct functions. For example, the p110␣ subunit is involved in platelet-derived growth factorand epidermal growth factor-mediated mitogenic responses, but not in responses mediated by bombesin or lysophosphatidic acid (33). In contrast, p110␤ is apparently necessary for insulin-and lysophosphatidic acid-mediated mitogenic responses but not platelet-derived growth factor-mediated responses (34). Moreover, in response to insulin stimulation, p110␤ but not p110␣ associates with the GLUT4 glucose transporter compartment (35). It should be noted that the previous studies showing that overexpressed p110␣ induces GLUT4 translocation may not directly indicate the importance of p110␣ itself, because these membrane-targeted or constitutively active forms of p110␣ are considered to function in all of the membrane fractions but are not limited to the membrane of a specific compartment. Such an ectopic expression of p110␣, which is always active even without insulin stimulation, might have brought insulin-independent phosphoinositide production at a place where phosphoinositide is critical to cause GLUT4 translocation. Because p110␤ seems to require insulin stimulation to be an active form, overexpression of p110␤ without insulin treatment might not cause translocation of GLUT4. Thus, it seems very likely that both subcellular localization of the enzyme and kinetics of the kinase activity are key factors in determining the specific functions of the various PI kinase isoforms.
Taking into consideration that both the subcellular localization and kinetics of p110 may influence cellular function, we tried to determine which endogenous catalytic subunit, p110␣ or p110␤, is involved in insulin-induced GLUT4 translocation, by microinjecting isoform-specific anti-p110 to neutralize activ-ities of each p110 (Fig. 5). The neutralizing antibodies used in this study were highly isoform-specific and very strongly inhibited the lipid kinase activity of the corresponding isoform. The obtained data clearly suggested that p110␤ plays a major role in transmitting the signals to translocate GLUT4. On the other hand, the contribution of p110␣ to insulin-induced GLUT4 translocation is likely to be relatively small.
To date, some evidence has been obtained suggesting that high levels of basal PI 3-kinase activity associated with p110 may be important for prevention of apoptosis. On the other hand, acute cellular activities rapidly evoked by growth factors or hormones may require a large increase in PI kinase activity against a background of low basal activity. In this way, maintenance of the appropriate levels of basal and stimulated PI kinase activities may be governed by the ratio of the expressions of the p110␣ and p110␤ isoforms. We therefore speculate that PI kinase activity derived from p110␣ functions mainly in carrying out such "housekeeping" activities as prevention of apoptosis and sustaining basal glucose uptake, whereas p110␤ plays a crucial role in carrying out cellular responses to insulin and possibly other hormones as well.
This is the first report differentiating the two isoforms of the PI kinase catalytic subunit as regards regulation of basal and insulin-stimulated lipid kinase activity and glucose transport. The respective roles played by p110␣ and p110␤ may be applicable to the signal transduction cascades activated not only by insulin but also by other growth factors. In addition, we have recently reported that p85/p110 type PI kinase phosphorylates both the D-3 and the D-4 position of the inositol ring in vivo (36). In this report, it was shown that p110␤ exhibits a higher ratio of PI 4-kinase activity/PI 3-kinase activity than p110␣. Additional studies will be required before a full understanding of the mechanisms underlying the localization and regulation of p110 activities under basal and stimulated conditions can be obtained.