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Endocrinology Vol. 142, No. 1 205-212
Copyright © 2001 by The Endocrine Society

ARTICLES

Phosphatidylinositol-3,4,5-Trisphosphate Is Required for Insulin-Like Growth Factor 1-Mediated Survival of 3T3-L1 Preadipocytes1

AnneMarie Gagnon2, Patti Dods, Nicolas Roustan-Delatour, Ching-Shih Chen and Alexander Sorisky

The Departments of Medicine and Biochemistry, Microbiology & Immunology (A.G., P.D., N.R.-D., A.S.), Loeb Health Research Institute, Ottawa Hospital, University of Ottawa, Ottawa, Canada; and the Division of Medicinal Chemistry and Pharmaceutics (C.-S.C.), College of Pharmacy, University of Kentucky, Lexington, Kentucky

Address all correspondence and requests for reprints to: Dr. Alexander Sorisky, Loeb Health Research Institute at the Ottawa Hospital, 725 Parkdale Avenue, Ottawa, Ontario, Canada, K1Y 4E9. E-mail: asorisky{at}Lri.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adipocyte number, a determinant of adipose tissue mass, reflects the balance between the rates of proliferation/differentiation vs. apoptosis of preadipocytes. The percentage of 3T3-L1 preadipocytes undergoing cell death following serum deprivation was reduced by 10 nM insulin-like growth factor (IGF)-1 (from 50.0 ± 0.7% for control starved cells to 27.5 ± 3.1%). TUNEL staining confirmed the apoptotic nature of the cell death. The protective effect of IGF-1 was blocked by phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin, and LY294002, but was unaffected by rapamycin, PD98059, or SB203580, which inhibit mammalian target of rapamycin (mTOR), ERK kinase (MEK1), and p38 MAPK respectively. Exogenous PI(3,4,5)P3 (10 µM), the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, had a modest survival effect on its own, reducing cell death from 47.9 ± 3.4% to 35.6 ± 3.5%. When added to the combination of IGF-1 and LY294002, PI(3,4,5)P3 reversed most of the inhibitory effect of LY294002 on IGF-1-dependent cell survival, protein kinase B/Akt phosphorylation, and caspase-3 activity. Taken together, these results implicate PI(3,4,5)P3 as a necessary signal for the anti-apoptotic action of IGF-1 on 3T3-L1 preadipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACCUMULATION of adipose tissue reflects an increase in both the number and the size of adipocytes (1). Storage of excess energy as triacylglycerol in existing adipocytes enlarges their volume to a finite limit, at which point it is postulated paracrine factors are released that influence preadipocyte proliferation and differentiation (2, 3). In addition, nutritional signals such as insulin and/or fatty acids may directly promote adipogenesis (4, 5). Newly formed adipocytes, derived from specialized fibroblasts committed to the adipose lineage (preadipocytes), increase the capacity for adipose tissue to store the required amount of energy as triacylglycerol (2). Adipocyte number may also be modulated by apoptosis, a coordinated series of proteolytic reactions that leads to cell shrinkage and internucleosomal DNA cleavage (6). This important process for the development and remodeling of various tissues has only recently been investigated in adipose cells (7, 8, 9). The precise identity of the cells within the adipose depot undergoing apoptosis is not entirely clear, but appears to include both preadipocytes as well as adipocytes (8).

The positive effect of insulin-like growth factor (IGF)-1 on cell survival has been described in several systems, using various apoptotic stimuli (10). IGF-1 action is mediated by its specific tyrosine kinase receptor, which phosphorylates insulin receptor substrates [IRS; (11)]. Activation of a variety of kinases ensues, including phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as Akt), and various mitogen-activated protein kinases [MAPKs; (11)]. In 3T3-L1 preadipocytes, IGF-1-stimulated PI3K leads to the production of PI(3, 4, 5)P3 (12). This lipid has a dual role in the activation of PKB/Akt. PI(3, 4, 5)P3 binding to PKB/Akt relocates the kinase to the plasma membrane, resulting in a modest activation. Full activation of PKB/Akt occurs upon phosphorylation of specific serine and threonine residues by 3-phosphoinositide-dependent kinases (PDKs) 1 and 2, which also seem to be regulated by PI(3, 4, 5)P3 (13, 14). Recent studies have identified several apoptosis-related proteins as substrates for PKB/Akt, implicating this kinase as an important regulator of the apoptotic process (15). Alternate IGF-1-dependent survival signaling pathways, including the Ras/mitogen-activated protein kinase (p42/44 MAPK) cascade and p38 MAPK, have also been proposed (16, 17, 18). However, current data suggest that IGF-1-mediated MAPK-dependent survival signals may predominate only when the PI3K pathway is disabled (19).

We have previously reported that differentiation of 3T3-L1 preadipocytes, a well-described model of adipose cell differentiation, is associated with resistance to apoptosis induced by serum deprivation (20, 21). The increased sensitivity of preadipocytes to apoptosis suggests that they may serve as a potential therapeutic target to stem the accumulation of adipose tissue. The current studies identify IGF-1 as a survival factor for 3T3-L1 preadipocytes and demonstrate that this effect is dependent on the activation of the PI3K-PKB/Akt pathway and the production of PI(3, 4, 5)P3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
3T3-L1 preadipocytes, maintained at low passage, were grown to confluence in DMEM supplemented with 10% calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from Life Technologies, Inc., Burlington, Ontario, Canada). At confluence, cells were either kept in growth medium, or serum-starved (i.e. growth factor deprivation) in DMEM, supplemented with penicillin and streptomycin (starving medium) for 3 to 18 h. IGF-1 (10 nM; Roche Molecular Biochemicals, Laval, Québec, Canada) was added to the starvation medium, as indicated. When specified, the following inhibitors or appropriate vehicle were added to the starvation medium, in the presence or absence of IGF-1: 100 nM wortmannin (Kamiya Biomedical, Thousand Oaks, CA), 10 µM LY294002 (Sigma, St. Louis, MO), 100 nM rapamycin (Sigma), 50 µM PD98059 (New England Biolabs, Inc., Mississauga, Ontario, Canada), or 10 µM SB203580 (Calbiochem, San Diego, CA). Alternatively, 3T3-L1 preadipocytes were incubated in starvation medium containing 10 µM each of dipalmitoyl-PI(3, 4, 5)P3, dipalmitoyl-PI(3, 4)P2, or dipalmitoyl-PI(4, 5)P2 (22), in the presence or absence of IGF-1 and/or LY294002 for 3 to 6 h. Lipids were stored in chloroform:methanol:water (1:1:0.3), dried down under a nitrogen stream, resuspended in the appropriate medium, and then dispersed by sonication. Following treatment, cells were assessed for apoptosis either by cell enumeration or by TUNEL staining, or lysed for immunoblot analysis.

Cell enumeration
Following the indicated treatments, floating cells were carefully washed away with PBS, and the remaining adherent viable cells from duplicate dishes of 3T3-L1 preadipocytes were trypsinized and counted in duplicate using a Neubauer hemacytometer. Cell viability was assessed by trypan blue dye exclusion and was shown to be >95% of adherent cells for all treatments. Data are expressed as % cell death according to the following equation: (number of adherent cells in serum-fed controls - number of remaining adherent cells after indicated treatment)/number of adherent cells in serum-fed controls. Results represent the mean ± SEM of at least three independent experiments, each done in duplicate.

TUNEL staining
3T3-L1 preadipocytes grown to confluence on coverslips were treated as indicated. Cells were processed for TUNEL staining using the In Situ Cell Death Detection Kit, AP (Roche Molecular Biochemicals), according to manufacturer’s directions. Briefly, cells were washed, and fixed in 4% paraformaldehyde for 30 min. Cells were washed, permeabilized with 0.1% sodium acetate/0.1% Triton X-100, and incubated with fluorescein-tagged nucleotides in the presence of terminal deoxynucleotidyl transferase (TdT) for 60 min. Cells were washed again and incubated with alkaline phosphatase conjugated anti-fluorescein antibodies. Following washes, cells were stained using the alkaline phosphatase substrate mix NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, Roche Molecular Biochemicals), and visualized with a Nikon TMS microscope, at 400x magnification. TUNEL staining was quantified for each sample by counting the number of positively stained cells in 10 random fields (200–300 cells/field). Photomicrographs were generated with an Olympus Corp. 1 x 70 microscope, connected to a dual-color CCD camera, using Northern Eclipse v5 software.

PKB/Akt analysis
Confluent 3T3-L1 preadipocytes were treated for 3 h as described, briefly washed with PBS, and then lysed in Laemmli buffer (23). Cellular proteins were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose. Membranes were then incubated with anti-phosphoPKB/Akt (ser473) antibody (New England Biolabs, Inc.; 1:1000) or anti-PKB/Akt antibody (a kind gift from P. Coffer; 1:9000), followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Baie d’Urfé; Québec, Canada). Immunoreactivity was detected by enhanced chemiluminescence (NEN Life Science Products, Boston, MA).

Caspase-3 analysis
Confluent 3T3-L1 preadipocytes were treated for 6 h with the indicated reagents. Floating and adherent cells from each dish were rinsed in PBS, lysed in Laemmli buffer (23), and pooled. Cellular proteins were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose. Membranes were then incubated with polyclonal anti-caspase-3 antibody (BDI; 1:1000), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Immunoreactivity was detected by enhanced chemiluminescence (NEN Life Science Products).

Statistical analysis
Statistical differences were determined by paired t test or by ANOVA, as indicated. A P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-1 reverses apoptosis in 3T3-L1 preadipocytes
We have previously demonstrated that, following serum deprivation, 3T3-L1 preadipocytes undergo apoptosis more readily than their differentiated counterparts (20, 21). The current studies investigate intracellular signaling pathways activated by IGF-1 and their role in 3T3-L1 preadipocyte survival. 3T3-L1 preadipocytes placed in starvation medium for 18 h underwent substantial cell death (50 ± 0.7%), as assessed by trypsinizing viable adherent cells and counting. Addition of 10 nM IGF-1 reduced cell death to 23.6 ± 3.4%, a 53% inhibition (P < 0.01; Fig. 1AGo). This dose of IGF-1 was shown to be optimal for the survival of 3T3-L1 preadipocytes (data not shown).



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  		Figure 1. IGF-1 promotes 3T3-L1 preadipocyte survival. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium, or placed in starvation medium in the absence (control) or presence of 10 nM IGF-1 for 3 (TUNEL staining) to 18 h (cell enumeration). A, Cells were trypsinized and counted in duplicate. Results are calculated as % cell death, as described in Materials and Methods, and represent the mean ± SEM of four independent experiments, each done in duplicate. B and C, Cells were fixed, permeabilized, and labeled using TdT. Positively staining cells in 10 random fields were counted. Results represent the mean ± SEM of three independent experiments, each done in duplicate (B). A representative photomicrograph (200x magnification) is shown for each treatment (C).

 
TUNEL analysis was performed to confirm that the observed reduction in cell death by IGF-1 was due to suppression of apoptosis. For these experiments, treatment time was reduced to 3 h to minimize detachment of apoptotic (floating) cells, and thereby permit the staining of adherent cells undergoing apoptosis. Serum deprivation of 3T3-L1 preadipocytes increased the number of TUNEL-positive stained cells compared with control cells maintained in serum-supplemented medium (Fig. 1Go, B and C). Addition of IGF-1 to the starvation medium reduced the number of apoptotic cells by 95% (P < 0.01), measured by TUNEL staining (Fig. 1Go, B and C). Taken together, these data indicate that IGF-1 is a survival factor for 3T3-L1 preadipocytes. The differences in the relative magnitude of the IGF-1 protective effect between the two techniques employed may reflect the duration of starvation. In addition, IGF-1 action may be more accentuated at the early stages of apoptosis, i.e. DNA fragmentation when the cells are still adherent (measured by TUNEL), compared with later stages, when apoptotic cells have detached from the substratum.

IGF-1 protects 3T3-L1 preadipocytes through activation of the PI3K pathway
To identify which signals are important for the protective effect of IGF-1, 3T3-L1 preadipocytes were incubated in starvation medium for 18 h, in the presence or absence of IGF-1 in combination with a variety of specific inhibitors. Each inhibitor was used at a concentration that effectively and specifically inhibits its respective target in this particular system (24, 25, 26, 27, 28). These inhibitors alone, in the absence of IGF-1, had no significant effect on the survival of 3T3-L1 preadipocytes (data not shown). Although both p42/44 MAPK and p38 MAPK have been implicated in IGF-1-mediated cell survival in other systems (16, 17, 18), neither 50 µM PD98059 nor 10 µM SB203580, which specifically inhibit MEK1 (the upstream regulator of p42/44 MAPK) and p38 MAPK respectively, modulated the survival effect of IGF-1 on 3T3-L1 preadipocytes deprived of serum (Fig. 2AGo). In contrast, the antiapoptotic properties of IGF-1 were completely blocked in the presence of 100 nM wortmannin or 10 µM LY294002, both inhibitors of PI3K (P < 0.05 compared with IGF-1 treatment alone; Fig. 2BGo). IGF-1 activates p70 S6 kinase, possibly via phosphorylation of the mammalian target of rapamycin (mTOR) by PKB/Akt (29, 30). However, 100 nM rapamycin, an inhibitor of mTOR/p70 S6 kinase, had no significant effect on IGF-1-dependent survival in 3T3-L1 preadipocytes (Fig. 2BGo), as observed in other models of cell survival (31).



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  		Figure 2. PI3K mediates IGF-1-dependent protection from apoptosis in 3T3-L1 preadipocytes. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium, or placed in starvation medium in the absence (control) or presence of 10 nM IGF-1, in combination with the following inhibitors: A, 50 µM PD98059, or 10 µM SB203580, and B. 100 nM wortmannin, 10 µM LY294002, or 100 nM rapamycin. After 18 h of treatment, cells were trypsinized and counted in duplicate. Results are calculated as % cell death, as described in Materials and Methods, and represent the mean ± SEM of three independent experiments, each done in duplicate.

 
PI(3, 4, 5)P3 protects 3T3-L1 preadipocytes from apoptosis
IGF-1-dependent activation of PI3K in 3T3-L1 preadipocytes produces PI(3, 4, 5)P3, but not PI(3, 4)P2 (12). We investigated whether PI(3, 4, 5)P3 was sufficient to replace IGF-1 and protect 3T3-L1 preadipocytes from apoptosis induced by serum deprivation. The addition of 10 µM PI(3, 4, 5)P3 to starvation medium modestly, but significantly, reduced 3T3-L1 preadipocyte death by 25% (P < 0.05), compared with control cells (Fig. 3Go). This effect of PI(3, 4, 5)P3 was unrelated to potential variations in membrane function due to interactions with charged phosphorylated phosphoinositides, because PI(3, 4)P2, or PI(4, 5)P2, had no effect on 3T3-L1 preadipocyte survival under these conditions (Fig. 3Go).



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  		Figure 3. PI(3 4 5 )P3 promotes 3T3-L1 preadipocyte survival. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium or placed for 6 h in starvation medium in the absence (control) or presence of 10 µM of the following lipids: PI(3 4 5 )P3, PI(3 4 )P2, or PI(4 5 )P2. Cells were trypsinized, and counted in duplicate. Results are calculated as % cell death, as described in Materials and Methods, and represent the mean ± SEM of four independent experiments, each done in duplicate.

 
As an alternate strategy to demonstrate the antiapoptotic effect of PI(3, 4, 5)P3, and to confirm the specificity of PI3K inhibitors, we tested the ability of exogenous PI(3, 4, 5)P3 to reverse the inhibitory effect of PI3K inhibitors on IGF-1-dependent survival signaling. As described above, IGF-1 reduced 3T3-L1 preadipocyte cell death from 54.2 ± 7.5% to 14.4 ± 2.3% (P < 0.05; Fig. 4AGo). This protective effect of IGF-1 was again blocked by LY294002 (P < 0.05), resulting in a level of cell death comparable to that observed in control serum-deprived cells (43.7 ± 4.7%). Addition of PI(3, 4, 5)P3 reversed much of the inhibition of the antiapoptotic effect of IGF-1 by LY294002, significantly reducing cell death from 43.7 ± 4.7% to 23.7 ± 6.6% (P < 0.05). PI(3, 4)P2 also decreased the inhibitory effect of LY294002 on IGF-1-dependent cell survival, but to a smaller extent than PI(3, 4, 5)P3 (Fig. 4AGo). PI(4, 5)P2 had no effect on death of serum-deprived cells treated with IGF-1 and LY294002. The inhibition of the IGF-1 protective effect by LY294002 (P < 0.01), and similar effects of adding the three inositol phospholipids (P < 0.05), were also observed by TUNEL staining (Fig. 4BGo). These results confirm that the reversal of the LY294002 inhibition by PI(3, 4, 5)P3 measured by cell enumeration (Fig. 4AGo) is due to suppression of apoptosis (Fig. 4BGo).



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  		Figure 4. PI(3 4 5 )P3 reverses the inhibitory effect of LY294002 on IGF-1-dependent 3T3-L1 preadipocyte survival. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium, or placed for 3 (TUNEL staining) to 6 h (cell enumeration) in starvation medium in the presence or absence of 10 nM IGF-1 ± 10 µM LY294002. PI(3 4 5 )P3, PI(3 4 )P2, or PI(4 5 )P2, were added (10 µM), as indicated. A, Cells were trypsinized and counted in duplicate. Results are calculated as % cell death, as described in Materials and Methods, and represent the mean ± SEM of four independent experiments, each done in duplicate. B, Cells were fixed, permeabilized, and labeled using TdT. Positively staining cells in 10 random fields were counted. An average of 73.5 positively stained cells/10 fields was observed in controls. Results represent the mean ± SEM of five independent experiments, each done in duplicate.

 
IGF-1 and PI(3, 4, 5)P3 stimulate PKB/Akt and suppress caspase-3 activation in 3T3-L1 preadipocytes
To investigate the role of PKB/Akt in IGF-1- and PI(3, 4, 5)P3-mediated survival signaling, we measured the phosphorylation state of PKB/Akt, which is indicative of its activity, in response to the various experimental conditions described in Fig. 4Go. IGF-1 strongly induced PKB/Akt phosphorylation, which was inhibited by LY294002 (Fig. 5AGo). Addition of PI(3, 4, 5)P3 to the IGF-1/LY294002 combination resulted in a small but significant increase in PKB/Akt phosphorylation, compared with the IGF-1/LY294002 condition (Fig. 5AGo). PKB/Akt expression was comparable in all lanes (Fig. 5BGo).



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  		Figure 5. Effect of PI(3 4 5 )P3 on the inhibitory effect of LY294002 on IGF-1-dependent phosphorylation of PKB/Akt. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium, or placed for 3 h in starvation medium in the presence or absence of 10 nM IGF-1 ± 10 µM LY294002. PI(3 4 5 )P3, or PI(4 5 )P2 was added (10 µM), as indicated. Cells were lysed and cellular proteins (35 58 g/lane) separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membrane, and immunoblotted with antibodies specific for phosphoPKB/Akt (A), or PKB/Akt (B). Results are representative of four independent experiments, each done in duplicate.

 
Intrinsic and extrinsic pathways of apoptosis converge at caspase-3 (32). Caspase-3 was activated when 3T3-L1 preadipocytes were deprived of serum, as measured by the appearance of the 17-kDa form of caspase-3 by immunoblot analysis (Fig. 6Go). Addition of IGF-1 to the starvation medium suppressed caspase-3 activation, and LY294002 inhibited this effect of IGF-1. PI(3, 4, 5)P3 reversed the LY294002-dependent inhibition of caspase-3 activation by IGF-1.



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  		Figure 6. Effect of PI(3 4 5 )P3 on the inhibitory effect of LY294002 on IGF-1-dependent suppression of active caspase-3. Confluent 3T3-L1 preadipocytes were either maintained in serum-supplemented medium, or placed for 6 h in starvation medium in the presence or absence of 10 nM IGF-1 ± 10 µM LY294002. PI(3 4 5 )P3 was added (10 µM), as indicated. Cells were lysed and cellular proteins (95 58 g/lane) separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membrane, and immunoblotted with antibodies specific for caspase-3. Results are representative of two independent experiments, each done in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified IGF-1 as a survival factor for 3T3-L1 preadipocytes and characterized its intracellular signaling targets. IGF-1 protects 3T3-L1 preadipocytes from apoptosis, induced by serum deprivation, by a mechanism dependent on the activation of PI3K. PI(3, 4, 5)P3, the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, is both required and sufficient, when added exogenously, to convey survival signals. Other potential targets of IGF-1, p42/44 and p38 MAPKs, are apparently not adequate for 3T3-L1 preadipocyte survival.

The dependence of IGF-1 on PI3K to promote 3T3-L1 preadipocyte survival was demonstrated using two structurally unrelated inhibitors of PI3K, wortmannin and LY294002 (33). Reports have challenged the specificity of PI3K inhibitors, by demonstrating that various other kinases can be directly affected upon exposure to wortmannin or LY294002 (34, 35). However, the concentrations of wortmannin and LY294002 used in our study are relatively low, and have been shown to be specific for the inhibition of PI3K (33). We have also addressed any remaining potential concerns about the specificity of the PI3K inhibitors by showing the reversal of their inhibition by the addition of the lipid product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, PI(3, 4, 5)P3. These observations further indicate that the PI3K-dependent survival signaling initiated by IGF-1 is solely mediated by PI(3, 4, 5)P3, and not other products of PI3K (36). Because PI(3, 4)P2 can be generated in 3T3-L1 preadipocytes by other growth factors (27), we tested whether it could also protect the cells from apoptosis. Addition of PI(3, 4)P2 to starvation medium did not protect 3T3-L1 preadipocytes from apoptosis on its own. However, it did enhance survival when added to IGF-1 and LY294002, albeit to lesser extent than PI(3, 4, 5)P3, suggesting PI(3, 4)P2, when added exogenously, can gain limited access to the survival signaling pathways used by PI(3, 4, 5)P3 in these cells.

When added on its own, PI(3, 4, 5)P3 only weakly reduced 3T3-L1 preadipocyte apoptosis, compared with the effect observed with IGF-1. Variations between the potency of IGF-1 and PI(3, 4, 5)P3, at the doses used, with respect to the activation of downstream antiapoptotic signals may explain such differences. More detailed dose-response and time course studies will be required for a thorough comparison of these two agents. Interestingly, PI(3, 4, 5)P3 was relatively more potent in promoting 3T3-L1 preadipocyte survival when added in the context of a PI3K-disabled IGF-1 signal (IGF-1 + LY294002), rather than when added alone. This intriguing finding suggests that alternate IGF-1-initiated pathways, other than those involving PI3K, may be permissive for 3T3-L1 preadipocyte survival. For example, IGF-1-mediated activation of c-Jun N-terminal kinase has recently been reported in a breast cancer cell line (37).

The use of synthetic phosphoinositides in cell cultures, while still relatively novel, has been successfully reported by us (27), and others (including 31, 38, 39). The PI(3, 4, 5)P3, and the weaker PI(3, 4)P2, response described here was unrelated to variations in membrane structure/integrity due to interactions with charged phosphoinositides, because PI(4, 5)P2 was without significant effect. The rates of incorporation into the cell membrane for PI(3, 4)P2 and PI(3, 4, 5)P3 are similar, and therefore cannot account for the distinct potency of each phosphoinositide (40). In recent years, it has become evident that each 3-phosphorylated phosphoinositide species activates a different array of cellular targets (41). Our data suggest PI(3, 4, 5)P3 may be more efficiently linked to cell survival pathways in preadipocytes.

In 3T3-L1 preadipocytes, IGF-1 activation of PI3K leads to the production of PI(3, 4, 5)P3, with no rise in PI(3, 4)P2 (12), and this is sufficient to activate PKB/Akt (27). PKB/Akt may use a variety of pathways to promote cell survival (15, 42). The identification of Bad, a proapoptotic member of the Bcl-2 family of proteins, as a direct substrate for PKB/Akt, provided a link between growth factor-generated signals and protection from apoptosis. Phosphorylation of Bad by PKB/Akt prevents Bad from binding to and inhibiting the activity of anti-apoptotic Bcl-2 family members, such as Bcl-2 and Bcl-xL (43). However, Bad has a restricted tissue expression profile (14) and, we, as well as others (M. Birnbaum, University of Pennsylvania, personal communication), have been unable to detect its expression in 3T3-L1 preadipocytes. Recent studies also implicate PKB/Akt upstream of caspase activation in the apoptosis cascade, regulating mitochondrial membrane integrity and cytochrome c release, independently of Bad phosphorylation (44). Growth factor-mediated induction of Bcl-2 expression has also been described as an important mechanism promoting cell survival in PC-12 cells (45). However, we observed no changes in Bcl-2 expression in our studies on 3T3-L1 preadipocytes (data not shown).

Caspase-9, which in turn activates caspase-3 (32), is another target of PKB/Akt. The importance of caspase-9 phosphorylation by PKB/Akt in promoting cell survival has been questioned recently (46). Variations in the consensus phosphorylation site indicate that caspase-9 phosphorylation by PKB/Akt may be restricted to human systems. Nevertheless, caspase-9 may be a relevant target of growth factor-initiated survival signaling in other mammalian species, because PKB/Akt activation inhibits caspase-9 activity, even in the absence of its phosphorylation (47, 48).

When added in combination with IGF-1 and LY294002, PI(3, 4, 5)P3 increased the levels of phosphoPKB/Akt, although not to the degree obtained with IGF-1 alone. The suppression of caspase-3 activation by PI(3, 4, 5)P3 in this context was comparable to IGF-1; possibly, PI(3, 4, 5)P3-stimulated PI3K-PKB/Akt meets a minimum threshold required for inhibition of caspase-3. The role of other PKB/Akt targets in preadipocyte survival remains to be investigated. These include glycogen synthase kinase 3ß, I{kappa}B kinases, and members of the forkhead family of transcription factors (15, 42, 49).

In summary, we have demonstrated that PI(3, 4, 5)P3 is required for IGF-1-regulated 3T3-L1 preadipocyte survival. Ongoing studies to delineate P(3, 4, 5)P3-dependent targets in preadipocyte should help to increase our understanding of adipose tissue development and remodeling.


   Acknowledgments
 
We thank Rania Rabie for technical assistance.


   Footnotes
 
1 This work was supported by a grant (to A.S.) from the Medical Research Council of Canada. Back

2 Recipient of a Canadian Diabetes Association Postdoctoral Research Fellowship. Back

Received May 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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