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Endocrinology, doi:10.1210/en.2005-0831
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Endocrinology Vol. 147, No. 4 1959-1968
Copyright © 2006 by The Endocrine Society

Differential Mitogenic Signaling in Insulin Receptor-Deficient Fetal Pancreatic ß-Cells

C. Guillen1, P. Navarro1, M. Robledo, A. M. Valverde and M. Benito

Institute of Biochemistry/Department of Biochemistry and Molecular Biology, Joint Center Consejo Superior Investigacion Cientifica/Universidad Complutense, School of Pharmacy, Complutense University, 28040 Madrid, Spain

Address all correspondence and requests to: M. Benito, Instituto de Bioquímica/Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: benito{at}farm.ucm.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin receptor (IR) may play an essential role in the development of ß-cell mass in the mouse pancreas. To further define the function of this signaling system in ß-cell development, we generated IR-deficient ß-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing ß-cell lines (ß-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (ß-IR–/–). ß-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In ß-IRLoxP ß-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S6K phosphorylation and ß-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S6K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S6K signaling and mitogenesis. Interestingly, in ß-IR–/– ß-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S6K phosphorylation and ß-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S6K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of ß-IR–/– cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic ß-cell lines. However, in the absence of the insulin receptor, glucose induces ß-cell mitogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES mellitus is a complex metabolic disease with environmental and genetic origins that affects more than 5% of the population in Western societies. The pathogenesis of type 2 diabetes involves abnormalities in both peripheral insulin action and insulin secretion by pancreatic ß-cells (1). Transgenic technology has yielded various animal models of type 2 diabetes, and these have advanced significantly the analysis of insulin signaling molecules implicated in the regulation of glucose homeostasis (2). Recently conditional knockout mice have been generated to define the specific roles of the insulin receptor (IR) and the IGF-I in pancreatic ß-cell development (3, 4). ß-Cell-specific IR deficiency (BIRKO mice) causes defective insulin secretion, which leads to progressive glucose intolerance and hyperglycemia. This diabetic phenotype reflects an impairment of acute insulin secretion in response to glucose in fed mice, which is characteristic of patients with type 2 diabetes (3). Total pancreas insulin content is severely reduced in adult BIRKO mice as compared with controls. Moreover, gene expression analysis has revealed a significant reduction of GLUT 2 and glucokinase in both diabetic and nondiabetic BIRKO mice. However, reductions of ß-cell mass, islet number, and insulin secretion are more pronounced in diabetic than in nondiabetic mutant mice (5). Taken together, these data implicate a direct role for the IR in the ß-cell growth and suggest that this receptor may also modulate the glucose-sensing machinery required for normal insulin secretion.

Interestingly, the ß-cell-specific deletion of the IGF-IR creates a phenotype similar to BIRKO mice. These transgenic animals display a defect in glucose-stimulated insulin secretion due to reduced expression of GLUT 2 and glucokinase, resulting in impaired glucose tolerance. However, no effect on ß-cell mass and islet number was noted in mutant mice as compared with controls (4). These data suggest that IGF-IR is not essential for ß-cell growth and development but participates in the mechanisms of glucose-stimulated insulin secretion of pancreatic ß-cells.

Although still controversial, it appears that pancreatic ß-cells arise from two main sources. New islets can be formed via budding from the pancreatic ductal epithelium (neogenesis) or through the proliferation of existing islets (6, 7). Whereas neogenesis occurs during fetal development (8) and the regeneration of adult pancreas (9), pancreatic ß-cell proliferation has been observed in the late fetal stages and also in normal adult pancreatic islet cells (6, 8). Of the signals involved in triggering ß-cell mitogenesis, IGF-I is one of the best characterized. However, IGF-I-induced ß-cell proliferation appears to depend on glucose, which alone is classic ß-cell mitogen (10, 11, 12). Thus, glucose at low concentrations (5 mM) activates p42/p44 MAPK and p70S6K by differential mechanisms in insulinoma cells; whereas glucose stimulates p42/p44 MAPK in a Ca2+-dependent manner, p70S6K activation was achieved in an ATP-dependent manner (13). At high concentrations (16.5 mM), glucose induced insulin secretion in islets and lines of insulinomas, but this effect required the expression of the transcriptional factor insulin promoter factor-1/pancreatic duodenal homeobox gene (PDX)-1. In fact, mutation of the Pdx-1 transactivation domain impaired insulin secretion in response to 16.7 mM glucose in insulinoma-1 ß-cells. Interestingly, the expression of PDX-1 in insulinoma-1 ß-cells is 4-fold higher than in isolated islets; consequently, insulin secretion by these insulinoma-derived ß-cells is highly responsive to glucose. However, it is uncertain whether the effect of glucose on insulinoma proliferation might reflect an underlying defect in insulin secretion. To address this important issue, we generated fetal pancreatic ß-cells without the insulin receptor. These cell lines have allowed us to examine the role of insulin in ß-cell proliferation and, conversely, to analyze the glucose signaling component of ß-cell mitogenesis in the absence of IR signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fetal calf serum and culture media were obtained from Life Technologies, Inc. (Gaithersburg, MD). Insulin, crystal violet (no. 0775), and antimouse IgG-agarose were from Sigma Chemical Co. (St. Louis, MO). Protein A-agarose was from Roche Molecular Biochemicals (Mannheim, Germany). The anti-phosphotyrosine (Py20) (sc-508), proliferating cell nuclear antigen (PCNA; no. sc-56) and antiinsulin (sc-9168) antibodies were purchased from Santa Cruz Biotechnology (Palo Alto, CA). The antiphospho Akt (Ser 473, no. 9271), antiphospho p70S6K (Ser424/Thr421, no. 9204), anti p70S6K (Thr 389, no. 9205), anti-Akt (no. 9272), anti-p70S6K (no. 9202), antiphospho p44/p42 MAPK (Thr 202/Tyr 204, no. 9101S), anti-p44/p42 MAPK (no. 9102), antiphospho mammalian target of rapamycin (mTOR) (Ser 2448, no. 2971), antiphospho Foxo1 (Ser 256, no. 9461), and anti-Foxo1 (no. 9462) antibodies were purchased from Cell Signaling (Beverly, MA). The anti-IR ß-subunit antibodies for immunoprecipitation and Western blot (Ab3 and Ab4, respectively) were from Oncogene (Cambridge, MA). For immunofluorescence fluorescein isothiocyanate-conjugated sheep antimouse immunoglobulins and monoclonal antivimentin (cloneV9) antibody were from Roche Molecular Biochemicals. Immunofluorescence mounting medium was from Vector (Burlingame, CA). Insulin and IGF-I were purchased from Sigma and Preprotech (Rocky Hill, NY), respectively. Wortmannin (no. W1628), monoclonal anti-ß-actin clone AC-15 (no. A5441), and atractyloside (no. 6882) were from Sigma. PD98059 (no. 13000) and rapamycin (no. 53210) were from Calbiochem (La Jolla, CA).

Generation of immortalized ß-floxed allele of the IR (IRLoxP) and ß-IR–/– ß-cell lines
Fetal pancreata were harvested from IRLoxP mice at late gestation and digested with collagenase. Animals were handled in accordance with approved institutional procedures. Subsequently, 50–100 ß-islets per pancreas were hand picked under a dissection microscope and plated as previously described (14). At this stage of development, ß-islets showed a differentiated phenotype yet maintained their proliferative capacity. Viral Bosc-23 packaging cells were transfected by the calcium phosphate method with 3 µg per 6-cm dish of the puromycin-resistance retroviral vector pBabe containing attenuated simian virus 40 large T antigen. Then the ß-islets growing on a fibroblasts monolayer were infected with the retroviral particles. After 72 h, islets were carefully hand picked from the monolayer using a dissection microscope and replated. ß-Cells obtained from these islets were expanded for 1 wk in DMEM with 20% fetal bovine serum (FBS) to eliminate contaminating fibroblasts and subsequently subjected to puromycin selection (1 µg/ml) for 1 wk. In addition, IRLoxP ß-cells were subcloned twice to select them from other islet-derived cell types. Immortalized ß-cells were established and grown in 10% FBS-DMEM. These cells maintained their cell-cell contact inhibition. For in vitro deletion of the insulin receptor, ß-cells harboring a IRLoxP were first placed at a subconfluent density. After 24 h, cells were infected with adenovirus encoding cre recombinase at a titer of 109 plaque-forming units. After 1 h, the viral supernatant was replaced with culture medium. Individual colonies were selected for IR recombination by PCR. Then, although these IR-deficient cells grew more slowly, they were cloned twice and resubjected to viral infection to assure complete deletion of the IR.

Cell signaling
For cell signaling experiments, both lines of ß-cells, ß-IRLoxP and ß-IR–/–, were serum-starved for 16–20 h and subsequently stimulated with insulin or glucose for 5 and 15 min, respectively. Afterward, cells were washed twice with PBS and lysed for total protein extraction according to standard procedure (14). This protocol was used in Fig. 1Go.


Figure 1
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  		FIG. 1. Characterization of immortalized ß-IRLoxP and ß-IR–/– ß-cells. A, ß-IRLoxP and ß-IR–/– ß cells and also NIH3T3 were cultured to confluence in the presence of 10% FBS-DMEM. Representative immunofluorescence detection of insulin and vimentin of growing immortalized fetal ß-IRLoxP ß-cells and also vimentin in NIH3T3 cells is shown. B, ß-IRLoxP ß cells were cultured for 16–20 h in glucose-free DMEM and further stimulated with glucose at 3 or 25 mM for 24 h. Representative RT-PCR for insulin mRNA expression of ß-cells, mouse islets as a positive control, and NIH3T3 cells as negative is shown. C, ß-IRLoxP and insulinoma MIN-6 ß-cells were cultured for 16–20 h in glucose-free DMEM and further stimulated with glucose 5 or 16 mM for 24 h. Total RNA (10 µg) was isolated, submitted to Northern blot analysis, and hybridized with Pdx-1 and 18S cDNAs. Densitometric study corresponding to three independent experiments is shown. D, ß-IRLoxP and ß-IR–/– ß-cells were lysed, and 600 µg total protein were immunoprecipitated with the anti-IR ß-chain antibody, the resulting immune complexes were analyzed by Western blotting (WB) with the anti-IR ß-chain antibody, respectively. Alternatively, ß-IRLoxP and ß-IR–/– ß-cells were cultured for 16–20 h in glucose-free DMEM and further stimulated with insulin 1–10 nM for 5 min, 600 µg total protein immunoprecipitated with anti-IR antibodies, and the resulting immune complexes analyzed by Western blotting with the anti-P-Tyr antibody, respectively. Representative autoradiograms of three are shown.

 
Immunofluorescence and confocal imaging
Cells were grown on glass coverslips until 80% confluence was reached. Then cells were washed twice with PBS, fixed in methanol (–20 C) for 2 min, and processed for immunofluorescence. Primary antibodies (antiinsulin and antivimentin) were applied for 1 h at 37 C in PBS 1% BSA, followed by 4 x 5 min washes in PBS, a 45-min incubation with fluorescence conjugated antibodies (fluorescein isothiocyanate-conjugated sheep antimouse and Cy3-conjugated goat antirabbit), and four final washes of 5 min each in PBS. Immunofluorescence was examined in an MRC-1024 (Bio-Rad Laboratories, Hempstead, UK) confocal microscope adapted to an inverted Eclipse TE 300 microscope (Nikon, Tokyo, Japan).

RT-PCR
Immortalized ß-cells, primary islets, and NIH3T3 fibroblasts (80–90% confluence) were serum-starved for 16–20 h and stimulated with glucose at 3 or 25 mM for 24 h. Afterward, cells were washed twice with PBS and lysed for total RNA extraction according to standard procedure (15). Total RNA was reverse transcribed into cDNA, as previously published (16).

Immunoprecipitations and Western blot analysis
Quiescent cells (20 h serum starved) were treated with several doses of insulin and glucose and lysed as previously described (17). After protein content determination, Western blot analysis was performed (17).

Phosphatidylinositol (PI) 3-kinase activity
PI 3-kinase activity was measured in the anti-phosphotyrosine immunoprecipitates by in vitro phosphorylation of phosphatidylinositol as previously described (17).

Cell proliferation
Cells were plated in 6-cm-multiwell plates and cultured in 10% FBS-DMEM until 40–50% of confluence was reached. Then cells were serum starved for a minimum of 4 h in medium A (DMEM supplemented with 0.2 mM glucose, 0.5% BSA) to assure cell survival. ß-IRLoxP cells were preincubated with different inhibitors for 30 min and subsequently stimulated with 10 nM insulin for 2 h. The 2-h time point was chosen as compromise between maximal proliferation response and cell survival. A shorter exposure did not reach maximal mitogenic response. A longer treatment with insulin, however, elicited increasingly cell death apoptotic rates. After 2 h, culture medium containing insulin was removed and replaced by medium A for up to 24 h. In parallel, ß-IR–/– cell were preincubated with inhibitors for 30 min and subsequently stimulated with 5 mM glucose for 24 h. Then both lines of ß-cells were washed with PBS and stained with crystal violet (0.2% in 2% ethanol) for 10 min. Finally, plates were rinsed with water, dried, and 1% sodium dodceyl sulfate was added. Absorbance of each plate was read at 560 nm. This protocol was used in Figs. 2–5GoGoGoGo.


Figure 2
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  		FIG. 2. PI 3 kinase-dependent and -independent signaling in ß-IRLoxP and ß-IR–/– ß-cells. ß-IRLoxP and ß-IR–/– ß-cells were cultured to confluence in the presence of 10% FBS-DMEM. Then cells were serum starved in medium A (DMEM supplemented with 0.2 mM glucose, 0.5% BSA). A, ß-IRLoxP and ß-IR–/– ß-cells, serum deprived for 4 h, were cultured and stimulated with 10 nM insulin (Ins) vs. IGF-I for 2 h or with several concentrations of glucose for 24 h vs. 10 nM IGF-I for 2 h, respectively. At the end of the culture time, cells were lysed and 600 µg total protein immunoprecipitated with antityrosine phosphate antibodies, and the resulting immune complexes were washed and immediately used for an in vitro PI 3 kinase assay. The conversion of PI to phosphatidylinositol 4,5-bisphosphate (PIP) in the presence of [{gamma}32-P]ATP was analyzed by thin-layer chromatography. Representative autoradiograms of three are shown. B, ß-IRLoxP and ß-IR–/– ß-cells, serum deprived for 4 h, were cultured and stimulated with 10 nM insulin vs. IGF-I for 2 h or with several concentrations of glucose for 24 h vs. 10 nM IGF-I for 2 h, respectively. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-Akt (Ser 473), total Akt, phospho-Foxo1 (Ser 256), total Foxo, phospho-p44/p42 MAPKs, and total p44/p42 MAPKs. Representative autoradiogram of three is shown.

 

Figure 3
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  		FIG. 3. Differential regulation of mTOR signaling pathway in ß-IRLoxP and ß-IR–/– ß-cells. ß-IRLoxP and ßIR–/– ß-cells were grown to confluence in the presence of 10% FBS-DMEM. Then cells were serum starved in medium A (DMEM supplemented with 0.2 mM glucose, 0.5% BSA). A, ß-IRLoxP ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059 (PD), 20 nM wortmannin (Wort), PD98059 plus wortmannin, or 20 nM rapamycin (Rapa), for 30 min and then were stimulated with 10 nM insulin (Ins) for 2 h. Total protein (50–120 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-mTOR (Ser 2448) and ß-actin. B, ß-IR–/– ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059, 20 nM rapamycin, or PD98059 plus rapamycin for 30 min (upper left panel). Then cells were stimulated with 5 mM glucose (Gluc) for 24 h. Total protein (50–120 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-mTOR (Ser 2448) and ß-actin. A representative experiment of four is shown. Densitometric analysis of four independent experiments is also shown. Results are means ± SEM. #, P < 0.05 PD + Wort + Insulin or rapamycin + insulin vs. insulin (A) and PD + glucose, rapamycin + glucose, and PD + rapamycin + glucose vs. glucose (B). Alternatively, cells were preincubated with 20 nM wortmannin and further stimulated with or without 5 mM glucose for 24 h (upper right panel). A representative experiment of three is shown. Fold increase values referred to their corresponding basal values from densitometric analysis of three independent experiments are represented above lanes. Inset on wortmannin or PD98059 control effect on PI 3 kinase activity or p42/p44 MAPKs phosphorylation, respectively, is also shown. phosphatidylinositol 4,5-bisphosphate (PIP).

 

Figure 4
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  		FIG. 4. Differential regulation of p70S6K signaling pathway in ß-IRLoxP and ß-IR–/– ß-cells. ß-IRLoxP and ßIR–/– ß-cells were grown to confluence in the presence of 10% FBS-DMEM. Then ß-IRLoxP and ßIR–/– ß-cells were serum starved in medium A (DMEM supplemented with 0.2 mM glucose, 0.5% BSA). A, ß-IRLoxP ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059 (PD), 20 nM wortmannin (Wort), PD98059 plus wortmannin, or 20 nM rapamycin (Rapa) for 30 min and then were stimulated with 10 nM insulin for 2 h. Total protein (50–120 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-p70S6K (Thr 421/Ser 424), phospho-p70S6K (Thr 389), and ß-actin. B, ß-IR–/– ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059, 20 nM rapamycin, or PD98059 plus rapamycin for 30 min. Then cells were stimulated with 5 mM glucose for 24 h. Total protein (50–120 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-p70S6K (Thr 421/Ser 424), phospho-p70S6K (Thr 389), and ß-actin. A representative experiment of four is shown. Densitometric analysis of four independent experiments is also shown. Results are means ± SEM. #, P < 0,05 PD + insulin, Wort + insulin, PD + Wort + insulin, or rapamycin + insulin vs. insulin 5 min (A) and PD + glucose, rapamycin + glucose, and PD + rapamycin + glucose vs. glucose 5 mM (B).

 

Figure 5
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  		FIG. 5. ß-Cell proliferation in ß-IRLoxP and ß-IR–/– ß-cells ß-IRLoxP and ßIR–/– ß-cells were grown to 50% confluence in the presence of 10% FBS-DMEM. Then ß-IRLoxP and ßIR–/– ß-cells were serum starved in medium A (free-glucose DMEM supplemented with 0.2 mM glucose, 0.5% BSA). ß-IRLoxP ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059 (PD), 20 nM wortmannin (Wort), PD98059 plus wortmannin, or 20 nM rapamycin (Rapa) for 30 min and further stimulated with 10 nM insulin for 2 h. Also, cells were stimulated with 10 nM IGF-I for 2 h or 5 mM glucose for 24 h as controls. Then culture medium was withdrawn and ß-IRLoxP ß-cells were stained with violet crystal after 24 h (A). ß-IRLoxP ß-cells, serum starved for 4 h, were stimulated with 10 nM insulin for 2 h, in a time-dependent manner. Then PCNA protein expression was measured by Western blot (C). ß-IR–/– ß-cells, serum starved for 4 h, were preincubated with 20 µM PD98059, 20 nM rapamycin, PD98059 plus rapamycin, or 20 nM wortmannin for 30 min and further stimulated with 5 mM glucose for 24 h. Also, they were stimulated with 10 nM insulin or IGF-I for 2 h as controls. Then culture medium was withdrawn, and ß-IR–/– ß-cells were stained with violet crystal (B). ß-IRLoxP ß-cells, serum starved for 4 h, were stimulated with 5 mM glucose for 24 h in a time-dependent manner. Then PCNA protein expression was measured by Western blot (D). Results are means ± SEM of six independent experiments. *, P < 0.05 insulin, glucose, or IGF-I vs. control (A), glucose, or IGF-I vs. control (B). #, P < 0.05 PD + Wort or rapamycin vs. insulin or PD, rapamycin, or PD + rapamycin vs. glucose (A and B, respectively).

 
Protein determination
Protein determination was by the Bradford dye method (18), using the Bio-Rad reagent and BSA as the standard.

RNA extraction and Northern blot analysis
Primary ß-cells and MIN-6 cells were grown until confluence. Then cells were serum starved for 20 h and further incubated for 24 h in the presence of glucose (5 and 25 mM). Then total RNA was isolated as described (15) and submitted to Northern blot analysis (16).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of immortalized ß-cells
Our first goal was to characterize the phenotype of these new cell lines. Thus, we performed immunofluorescence with antibodies against insulin (a ß-cell-specific marker) and vimentin (a marker for cells of mesenchymal origin). ß-IRLoxP ß-cells stained positive for insulin, indicating that these cells maintained ß-cell phenotypic features. Conversely, the ß-cell lines were negative for vimentin (Fig. 1AGo) in contrast to NIH3T3 cells, which served as a positive control for this antibody. The expression of insulin in ß-IRLoxP ß cells and isolated mouse islets was assessed by RT-PCR analysis. Clearly these immortalized cells express insulin, which could be induced by glucose in a concentration-dependent manner (Fig. 1BGo). Previous data have demonstrated that glucose-stimulated insulin production is regulated by PDX-1 expression. Insulinoma MIN-6 ß-cells and ß-IRLoxP ß-cells were stimulated with glucose and PDX-1 expression was evaluated by Northern blot. An increase in PDX-1 expression was noted in both types of ß-cells in response to different glucose concentrations (Fig. 1CGo).

To assess the distinct contributions of glucose vs. insulin signaling, we generated ß-cells lacking the IR (Fig. 1DGo). The deletion of the IR was further confirmed by analysis of IR phosphorylation. We observed an increase in IR ß-chain tyrosine phosphorylation in a dose-dependent manner in ß-IRLoxP ß-cells. In contrast, insulin stimulation produced no detectable IR phosphorylation in Iß-IR–/– ß-cells, as expected (Fig. 1DGo).

Downstream PI 3-kinase-dependent and -independent signaling in ß-IRLoxP and ß-IR–/– ß-cells
The insulin signaling cascade begins when the activated IR phosphorylates tyrosine residues of insulin receptor substrate (IRS) docking proteins (19, 20). Then phosphorylated IRSs bind proteins such as the p85 regulatory subunit of PI 3-kinase. Treatment of the ß-IRLoxP ß-cell line with insulin or IGF-I for 2 h stimulated phosphotyrosine-associated PI 3-kinase activity. However, exposure to various concentrations of glucose for 24 h had no effect on phosphotyrosine-associated PI 3 kinase activity (Fig. 2AGo). These results exclude the possibility that glucose might activate insulin signaling pathways indirectly by triggering insulin secretion in fetal ß-cells. In ß-IR–/– ß-cells, phosphotyrosine-associated PI 3-kinase was not activated on glucose stimulation. However, IGF-I stimulated phosphotyrosine-associated PI 3 kinase activity in a dose-dependent manner in this cell line (Fig. 2AGo).

ß-Cells were serum deprived for 4 h, and then the ß-IRLoxP line was stimulated with 10 nM insulin or IGF-I, whereas ß-IR–/– ß-cells received 5 mM glucose or 10 nM IGF-I for 2 h (Fig. 2BGo). Downstream of PI 3 kinase, insulin and IGF-I stimulated Akt and also Foxo-1 phosphorylation in ß-IRLoxP ß-cells. Neither Akt nor Foxo-1 was phosphorylated on glucose stimulation in the IR-deficient cells. However, IGF-I induced both Akt and Foxo-1 phosphorylation, the level of phosphorylation reached was lower in IR-deficient cells, compared with ß-IRLoxP ß-cells. Another substrate recruited by activated IR is SHC (21). On insulin stimulation, Src-homology collagen protein (SHC) proteins associate with Grb-2, resulting in the activation of the ras/MAPKs pathway. In ßIR-LoxP ß-cells, there was a dose-dependent increase in p44/p42 MAPK phosphorylation on insulin or IGF-I stimulation.

Similarly, both glucose and IGF-I stimulated p44/p42 MAPKs phosphorylation in ß-IR–/– ß-cells; the level of phosphorylation induced by IGF-I was lower in IR-deficient cells, compared with ß-IRLoxP ß-cells. In fact, IR-deficient cells grew more slowly, compared with ß-IRLoxP ß-cells.

Differential regulation of mTOR /p70S6K signaling pathways in ß-IRLoxP and ß-IR–/– ß-cells
A step further, stimulation with 10 nM insulin for 2 h induced mTOR and p70S6K phosphorylation in ß-IRLoxP ß cells. Similarly, stimulation with 5 mM glucose for 24 h induced mTOR and p70S6K phosphorylation in ß-IR–/– ß-cells (Figs. 3Go and 4Go). ß-Cell lines with or without insulin receptors were preincubated in the presence of 20 nM wortmannin (PI 3 kinase inhibitor), or 20 µM PD98059 [MAPK kinase (MEK)1 inhibitor]. Neither of these inhibitors produced a significant effect on insulin-induced mTOR phosphorylation in ß-IRLoxP ß-cells. However, 20 nM wortmannin or 20 µM PD98059 inhibited phosphotyrosine-associated PI 3 kinase activity and p42/p44 MAPKs phosphorylation, respectively, as expected (Fig. 3Go, inset). The combined treatment with wortmannin and PD98059 totally abolished the mTOR activation in response to 2 h stimulation with 10 nM insulin, as statistically quantified by densitometry (Fig. 3AGo). Wortmannin had only a statistically significant partial effect on insulin-induced p70S6K Thr421/Ser424 and Thr389 phosphorylation sites. PD98059 had also a statistically significant partial effect on insulin-induced p70S6K Thr421/Ser424 and Thr389 phosphorylation sites. The combined presence of wortmannin and PD98059 totally prevented the p70S6K full phosphorylation stimulated by insulin, as statistically quantified by densitometry (Fig. 4AGo). Finally, 20 nM rapamycin, mTOR kinase inhibitor, totally eliminated phosphorylation of Ser 2448 of mTOR as well as p70S6K full phosphorylation induced by insulin in ß-IRLoxP ß-cells; its inhibitory effect was even below their corresponding basal levels (Figs. 3AGo and 4AGo, respectively). These results suggest a convergence of PI 3 kinase and MEK-1 signaling on the mTOR/p70S6K pathway on insulin stimulation in ß-IRLoxP fetal pancreatic ß-cells.

PD98059 inhibited phosphorylation of mTOR Ser 2448, which was induced by stimulation with 5 mM glucose for 24 h in ß-IR–/– ß-cells, as statically quantified (Fig. 3BGo, left panel). In addition, wortmannin did not produce any significant direct effect on mTOR phosphorylation as densitometrically quantified (Fig. 3BGo, right panel). Moreover, we noted a differential effect of PD98059 on glucose-induced phosphorylation of p70S6K sites; whereas this inhibitor exerted no effect on Thr421/Ser424 phosphorylation sites, phosphorylation of Thr389 was completely blocked, as statistically quantified by densitometry (Fig. 4BGo). Finally, treatment with rapamycin totally abolished the glucose-mediated phosphorylation of mTOR Ser 2448 and p70S6K Thr421/Ser424 and Thr389 in ß-IR–/– ß-cells (Figs. 3BGo and 4BGo, respectively). In the presence of rapamycin plus PD98059, the effect on p70S6K Thr421/Ser424 phosphorylation site was even below its basal level (Fig. 4BGo). These results suggest that glucose may activate a MEK-1/mTOR/p70S6K signaling pathway in insulin-receptor deficient ß-cell lines.

Mitogenic effects of insulin or glucose in immortalized ß-cell lines
Next, we studied the effects of insulin and glucose on ß-cell proliferation as determined by PCNA protein expression and ß-cell number. We observed that, during a 24-h time course, 10 nM insulin or 5 mM glucose increased PCNA protein expression in ß-IRLoxP and ßIR–/– cells, respectively (Fig. 5Go, C and D, respectively). In addition, there was a 20% increase in ß-cell number on stimulation with 10 nM insulin, 10 nM IGF-I, or 5 mM glucose stimulation in ß-IRLoxP ß-cells (Fig. 5AGo). This is a remarkable effect, considering that none of the cell lines was synchronized. Glucose stimulated proliferation by 25% in ß-IR–/– ß-cells; however, insulin had no mitogenic effect on this IR-deficient cell line, as would be expected. Interestingly, IGF-I increased proliferation by 17% in ß-cell line lacking insulin receptors (Fig. 5BGo). To assess which signaling pathways participate in the mitogenic effects of insulin and glucose, we tested the different inhibitors used in the experiments described above. Treatment with either 20 nM wortmannin or 20 µM PD98059 did not effect insulin-induced proliferation of the ß-IRLoxP ß-cell line. However, the combined addition of PD98059 and wortmannin completely blocked insulin-induced ß-cell mitogenesis (Fig. 5AGo). These results suggest that activation of both IRS-dependent PI 3 kinase and MEK-1 pathways is required for ß-cell mitogenesis in response to insulin. Finally, 20 nM rapamycin abolished insulin-mediated mitogenesis in ß-IRLoxP ß-cells (Fig. 5AGo). These results are consistent with those seen above in Figs. 3AGo and 4AGo, suggesting that signals from the PI 3 kinase and MEK-1 pathways converge on the mTOR/p70S6K pathway to mediate fetal ß-cell proliferation in response to insulin.

By contrast, in IR-deficient ß-cells, the sole addition of PD98059 totally blunted glucose-stimulated mitogenesis. However, wortmannin had no effect on proliferation in this cell line (Fig. 5BGo), which was expected, given that glucose did not activate PI 3 kinase signaling in IR-deficient cells. As observed in the IR-expressing ß-cell line, rapamycin produced a total block of ß-cell mitogenesis in response to glucose, paralleling its inhibitory effect on mTOR/p70S6K signaling. Finally, combined addition of PD98059 and rapamycin also inhibited mitogenesis of ß-IR–/– ß-cells (Fig. 5BGo). These data are also consistent with data of Figs. 3BGo and 4BGo, suggesting that the MEK-1/mTOR/p70S6K pathway mediates fetal ß-cell proliferation in response to glucose.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGF-I is an important mitogen in many types of eukaryotic cells (22). In the developing and regenerating pancreas, IGF-I plays a major role in increasing the population of islet-cells (6, 9). However, the IGF-I-induced mitogenic response appears to be glucose dependent, at least in vitro (10, 11, 12). Glucose-dependent activation of MAPK is required to maintain the balance between MAPKs/PI-3 kinase/Akt in IGF-I-mediated ß-cell proliferation (23). Additionally, glucose-dependent activation of p70S6K might also be crucial due to the mTOR-mediated degradation of IRS-2 during the mitogenic response to glucose and/or IGF-I (24). Evidence from knockout mice has challenged classic notions about the roles of insulin and IGF-I in ß-cell physiology. Deletion of the IGF-IR specifically in ß-cells produces defective glucose-stimulated insulin secretion but does not effect ß-cell mass (4). ß-cell specific IR deficiency also results in an insulin secretion defect (3). However, a decrease in ß-cell mass and islet number occurs in most adult BIRKO mice as compared with controls (5). These results strongly suggest that IR signaling is essential for pancreatic ß-cell growth throughout development.

To address whether insulin indeed induces ß-cell proliferation, we studied the insulin-induced mitogenic signaling in immortalized lines of mouse ß-cells that do or do not express the insulin receptor. Upon insulin stimulation, the IR recruited docking proteins such as IRSs and also SHC in the ß-IRLoxP cells. As expected, insulin activated the PI 3 kinase/Akt signaling pathway. However, 5 mM glucose failed to stimulate phosphotyrosine-associated PI 3 kinase in our ßIR-LoxP line of ß-cells. These data rule out the possibility that low glucose might indirectly activate insulin signaling by stimulating insulin secretion in fetal ß-cells. The MAPK pathway was also stimulated by insulin in these cells. Neither wortmannin nor PD98059 blocked the mitogenic response to insulin. Consequently, neither IRS/PI 3 kinase signaling nor the IRS/ras/MAPK pathway is sufficient to mediate the mitogenic activity of insulin in these ß-cell lines. However, combined treatment with wortmannin and PD98059 completely abolished the insulin-mediated mitogenic response. Thus, activation of both IRS-dependent signaling pathways is required for insulin-induced mitogenesis in these ß-cells.

Furthermore, combined treatment with PD98059 and wortmannin inhibited the insulin-stimulated phosphorylation of mTOR. Wortmannin alone partially blocked the insulin-induced phosphorylation of p70S6K at Thr421/Ser424 and Thr389 sites. In the same way, the MEK-1-inhibitor PD98059 had only a partial effect on insulin-induced phosphorylation of p70S6K at Thr421/Ser424 and Thr389 sites. Treatment with wortmannin plus PD98059 totally blocked p70S6K phosphorylation sites and mitogenesis as stimulated by insulin. Our results suggest that complete phosphorylation of p70S6K is required to achieve activation of p70S6K activity and the resulting mitogenesis, as recently reported (25). Moreover, these data suggest that the PI 3 kinase and MEK-1 pathways converge on mTOR/p70S6K signaling in response to insulin in our fetal pancreatic ß-cell lines. In fact, this convergent signaling has been previously suggested to function in the EGF/insulin pathway, in which active tuberin/hamartin heterocomplex playing a central role in the negative regulation of mTOR (26). Rapamycin totally abolished p70S6K phosphorylation sites and the phosphorylation of mTOR on serine 2448, thereby impairing insulin-induced mitogenesis in ß-IRLoxP ß-cells. These results are entirely consistent with recent evidence suggesting that p70S6K, but not Akt, directly leads to phosphorylation of mTOR on serine 2448 in cells (27, 28). Thus, IRS-dependent complete activation of mTOR/p70S6K is necessary and sufficient to induce mitogenesis in response to insulin in fetal pancreatic ß-cells. Moreover, these results demonstrate that insulin, acting independently of glucose signaling, induces mitogenesis in fetal ß-cells. Therefore, our data provide further evidence that the IR is essential for the development of ß-cell mass in BIRKO mice (5).

Glucose itself stimulates mitogenesis in primary pancreatic ß-cells via a glucose-metabolism-dependent mechanism (6, 8). Glucose metabolism increases intracellular cAMP and subsequently protein kinase A activity, protein kinase C, activity and cytosolic Ca2+ in ß-cells (29, 30, 31). Additionally, glucose induces IRS-1/IRS-2 phosphorylation and PI 3 kinase activity with downstream activation of p70S6K (10, 11). Thus, the glucose-mediated increase of intracellular Ca2+ leads activation of protein kinase A that acts downstream of Ras and upstream of MEK-1 to mediate p44/p42 MAPKs activation. Glucose also stimulates p70S6K via the mTOR kinase, which functions as an ATP sensor (32). However, these studies were performed in ß-cell lines derived from insulinomas in which insulin secretion is highly sensitive to glucose. Thus, it is possible that these reported effects of glucose on signaling and mitogenesis in insulinoma cell lines are the indirect result of abnormal insulin secretion.

To address whether glucose itself directly triggers ß-cell mitogenesis, we generated immortalized lines of fetal pancreatic ß-cells lacking the insulin receptor. In these ß-IR–/– ß-cells, glucose failed to stimulate phosphotyrosine-associated PI 3 kinase activity and its downstream effectors, Akt and Foxo-1. These observations contradict previous data regarding IRS-mediated PI 3 kinase activation in response to glucose in insulinoma-derived ß-cells, suggesting that altered insulin secretion or other mechanisms must account for PI 3 kinase activity in these cells. However, treatment with IGF-I activated phosphotyrosine-associated PI 3 kinase and its downstream effectors in ß-IR–/– ß-cells. Thus, despite the lack of the insulin receptor, these cells maintain postreceptor signaling machinery.

Exposure to glucose alone increased p44/p42MAPKs phosphorylation in our IR-deficient ß-cell line. These data are consistent with those previously reported in INS-1 ß-cells, in which glucose activates phosphorylation of p44/p42 MAPKs in the presence of a dominant-negative form of ras (13). In addition, glucose evoked complete phosphorylation of mTOR and p70S6K via a PI 3 kinase-independent pathway in our studies. PD98059 inhibited glucose-mediated mTOR phosphorylation but exerted a site-specific effect on glucose-induced phosphorylation of p70S6K. Whereas this inhibitor had no effect on Thr421/Ser424 phosphorylation sites, phosphorylation of Thr389 was completely eliminated. In parallel, PD98059 blocked glucose-induced mitogenesis in ß-cells lacking insulin receptors. These results suggest the MEK-1/mTOR/p70S6K signaling pathway mediates mitogenic signaling in these cells, with the phosphorylation on Thr389 of p70S6K being required for glucose-induced mitogenesis. Furthermore, rapamycin inhibited phosphorylation of p70S6K as well as the ser2448 site of mTOR in ß-IR–/– ß-cells. These results also agree with recent evidence, suggesting that p70S6K, but not Akt, directly leads to phosphorylation of mTOR on serine 2448 in cells (27, 28). No rapamycin-insensitive glucose-induced p70S6K phosphorylation sites were found in ß-cells as previously described (25). Thus, rapamycin impaired ß-cell mitogenesis in response to glucose. Taken together, these data indicate that glucose-induced activation of MEK-1/mTOR/p70S6K Thr389 signaling pathway is necessary and sufficient to induce mitogenesis in ß cells lacking insulin receptors.

In conclusion, our results demonstrate that the insulin receptor, independent of glucose signaling, plays an essential role in the regulation of mitogenesis in fetal pancreatic ß-cell lines, requiring both the PI 3 kinase and MEK-1 pathways to fully activate mTOR/p70S6K. Moreover, our cell lines reveal that glucose induces ß-cell mitogenesis in the absence of the IR by activating signals, which are not dependent on PI 3 kinase, revealing that phosphorylation at Thr389 of p70S6K is necessary and sufficient to mediate mitogenesis in insulin-receptor deficient fetal ß-cell lines (Fig. 6Go).


Figure 6
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  		FIG. 6. Converging mTOR/p706K signaling pathway in ß-cell mitogenesis. Insulin independently of glucose signals through IRS-dependent PI 3 kinase/Akt and MEK-1 pathways in ß-IRLoxP ß-cells. However, glucose, in a PI 3 kinase-independent manner, induces MEK-1 in ß-IR–/– ß-cells. Both signalings converge on mTOR/p70S6K pathway to mediate fetal ß-cell proliferation. TSC, Tuberous sclerosis complex; AMPK, AMP-activated protein kinase.

 


   Footnotes
 
This work was supported by Grants SAF 2001/1302 and SAF 2002/00863 from Ministerio de Educación y Ciencia, Spain, and "Red de Grupos de Diabetes Mellitus" (G03/212), Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.

First Published Online January 5, 2006

1 C.G. and P.N. were equal contributors to this manuscript. Back

Abbreviations: BIRKO, ß-Cell-specific IR deficiency; FBS, fetal bovine serum; IR, insulin receptor; IRLoxP, floxed allele of the insulin receptor; IRS, insulin receptor substrate; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; PCNA, proliferating cell nuclear antigen; PDX, pancreatic duodenal homeobox gene; PI, phosphatidylinositol; SHC, Src-homology collagen protein.

Received July 7, 2005.

Accepted for publication December 23, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
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