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Am J Physiol Endocrinol Metab 292: E187-E195, 2007. First published August 15, 2006; doi:10.1152/ajpendo.00283.2006
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Increased IRS-2 content and activation of IGF-I pathway contribute to enhance beta-cell mass in fetuses from undernourished pregnant rats

Elisa Fernández,* M. Angeles Martín,* Susana Fajardo, Fernando Escrivá, and Carmen Álvarez

Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, Madrid, Spain

Submitted 15 June 2006 ; accepted in final form 10 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that fetuses from undernourished (U) pregnant rats exhibited an increased beta-cell mass probably related to an enhanced IGF-I replicative response. Because IGF-I signaling pathways have been implicated in regulating beta-cell growth, we investigated in this study the IGF-I transduction system in U fetuses. To this end, an in vitro model of primary fetal islets was developed to characterize glucose/IGF-I-mediated signaling that specially influences beta-cell proliferation. We found that U fetal islets showed a greater replicative response to glucose and IGF-I than controls. Furthermore, insulin receptor substrate (IRS)-2 protein and its association with p85 were also increased. In the complete absence of IGF-I or stimulatory glucose, U islets presented an increased basal phosphorylation of downstream signals of the phosphatidylinositol 3-kinase (PI3K) pathway such as PKB, glycogen synthase kinase (GSK)3{alpha}/beta, PKC{zeta}, and mammalian target of rapamycin (mTOR). Similarly, phosphorylation of these proteins (except GSK3{alpha}/beta) by glucose and IGF-I was augmented even though total protein content remained unchanged. Downstream of PKB, direct glucose activation of mTOR was increased as well. In contrast, ERK1/2 phosphorylation was unaffected by undernutrition, but ERK activation seemed to be required to induce a higher proliferative response in U islets. In conclusion, we have demonstrated that fetal U islets show increased IRS-2 content and an enhancement in both basal and glucose/IGF-I activations of the IRS-2/PI3K/PKB pathway. These molecular changes may be responsible for the greater glucose/IGF-I islet replication and contribute to the increased beta-cell mass found in these fetuses.

undernutrition; fetal islet; insulin-like growth factor I pathway; insulin receptor substrate-2

AN ADVERSE FETAL OR NEONATAL ENVIRONMENT can lead to changes in the development of a key endocrine axis with deleterious consequences for function in later life (reviewed in Ref. 16). In this regard, the endocrine pancreas seems to be particularly affected by the metabolic conditions of the mother. Perinatal period is considered to be critical for beta-cell growth, and maternally derived changes in the fetal plasma concentration of nutrients clearly influence the development and function of the fetal endocrine pancreas (24). For example, maternal diabetes and experimentally induced hyperglycemia result in increased insulin secretion and hyperplasia of beta-cells in the fetuses, while maternal malnutrition is generally associated with low insulin secretion and decreased development of beta-cell (reviewed in Ref. 24). We have also shown that a 65% food restriction during the last week of gestation provoked glucose intolerance in pregnant rats, leading to an increase in beta-cell mass and hyperinsulinemia in their fetuses at term (3). Consequently, the adaptation of the endocrine pancreas to an adverse intrauterine environment can lead to an inappropriate pancreatic beta-cell mass, which is a well-known causative factor for subsequent type 2 diabetes (18). Identifying the factors and the mechanisms that control endocrine pancreas growth in physiological and pathological conditions has thus become a priority in the prevention of diabetes and later diseases.

The number of islet beta-cells present at birth is mainly determined by the proliferation and differentiation of pancreatic progenitor cells in a process called neogenesis (28). The growth of the endocrine pancreas is controlled by many factors including nutrients, like glucose and certain amino acids, and a variety of peptide growth factors (22). Among these, insulin-like growth factors (IGFs) have been specially implicated in fetal pancreas development, since they potentiate beta-cell growth, maturation, and function and are expressed by beta-cells in early life (reviewed in Ref. 46). The potential of IGFs as beta-cell mitogenic factors has been demonstrated in vitro on primary islets (23) and in beta-cell lines (25) and in vivo using transgenic mice (17). Furthermore, the mitogenic action of IGF-I is glucose dependent, and glucose itself is able to promote beta-cell proliferation in a manner that is dependent on glucose metabolism (25).

Recent investigations have focused on the importance of the IGF signaling transduction pathway to endocrine pancreas development. Growth-promoting effects of IGFs on beta-cells are mediated by their binding to the IGF-I receptor (IGF-IR) and subsequent tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) (47). Different studies have widely shown that IRS-2 plays a critical role in the regulation of beta-cell growth. IRS-2 signaling has been implicated in beta-cell mitogenesis and survival (33) and has also been associated with beta-cell neogenesis in pancreatectomized rats (26). Downstream of IRS-2, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) pathway seems to be highly related to cell cycle regulation. Expression of a constitutively active PI3K increases cell size by affecting mTOR and the ribosomal S6 kinase (p70S6K) activity (2). On the other hand, IGF-I can also activate the mitogen-activated protein kinase (MAPK) pathway, leading to activation of the extracellular signal-related kinases-1 and -2 (ERK1/2), which are required for beta-cell proliferation (35). In addition, glucose can activate these pathways in beta-cells independently of growth factors (35). Taken together, these studies point out the relevance of these signalings as regulators of beta-cell growth and show the importance of the study of the molecular mechanism that controls this process.

The aim of the present study was to investigate the intracellular glucose- and IGF-I-mediated signal transduction pathway(s) in islets of fetuses from undernourished (U) pregnant rats (65% daily reduction of food intake during the last week of gestation). These fetuses exhibited an increased beta-cell mass that has been recently related to a higher stimulation of beta-cell replication induced by an increase in both local IGF-I expression (36) and glucose oxidation (37).


   MATERIAL AND METHODS
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 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and diets. Wistar rats bred in our laboratory under controlled temperature and an artificial dark-light cycle (from 0700 to 1900; 24-h clock) were used throughout the study. Females were caged with males, and mating was confirmed by the presence of spermatozoa in a vaginal smear. Each dam was housed individually from the 14th day, and maternal food restriction was established. All animals were fed a standard laboratory diet (19 g protein, 56 g carbohydrate, 3.5 g lipid, 4.5 g cellulose/100 g, plus salt and vitamin mixtures) and were divided into two groups. Control (C) pregnant dams were fed ad libitum, and the U group received 35% of the food intake of a C pregnant dam during the third part of pregnancy. Water was given ad libitum. Food intake of C and U rats has been previously reported (15).

All studies were conducted in strict conformance with the National Institutes of Health guidelines for animal care, and the studies were submitted to and approved by the Bioethics Committee of the Universidad Complutense, Ciudad Universitaria, Madrid.

Islet isolation and culture. Islets from fetal rats (21.5 days of gestation) were obtained according to the method of Hellerstrom et al. (19). Briefly, five to seven pancreases were minced in sterile Hanks’ solution. The fragments were transferred to a sterile vial containing Hanks’ solution supplemented with 5–7 mg of collagenase (Boehringer Mannheim). The vial was shaken for 10 min at 37°C, and the tissue digested was washed three times with Hanks’ solution. The pellet was resuspended in culture medium and transferred to six plastic dishes containing the same medium. The culture medium consisted of RPMI 1640 (ICN, Nuclear Iberica) supplemented with 2 mmol/l glutamine, penicillin, and streptomycin and 10% fetal bovine serum (ICN, Nuclear Iberica). The islets were maintained at 37°C in an atmosphere of 5% CO2 during 2 days to allow for recovery and eliminate acinar cells. After that, the islets were gently detached from the plates, handpicked under a dissecting microscope, washed with PBS, and used for the diverse experiments. As it has been shown previously (1), the structural integrity of the islets after 2 days of culture was well preserved, and islets were ready to use for different functional studies.

Cell proliferation assay. A colorimetric immunoassay [Cell Proliferation ELISA, 5-bromo-2'-deoxyuridine (BrdU); Roche Diagnostic] was used for the quantification of cell proliferation. This method is based on the measurement of BrdU incorporation into genomic DNA during DNA synthesis of proliferating cells. Islets were cultured for 2 days with 11 mM glucose and maintained in a serum-free medium with 3 mM glucose for 20 h. After that, groups of 20 islets were seeded in 96-well multiwells and grown for 48 h at different glucose concentrations (3, 9, and 17 mM glucose) with or without IGF-I (1–1,000 ng/ml). During the last 24 h, they were labeled by the addition of BrdU. Then the anti-BrdU antibody was added, and the immune complexes were detected by the subsequent substrate (tetramethylbenzidine) reaction and quantified by measuring the absorbance at 620 nm, using a scanning multiwell spectrophotometer (ELISA reader).

Islet protein determination. Batches of 300–500 islets cultured for 2 days with 11 mM glucose were maintained in a serum-free medium with 3 mM glucose for 20 h. After that, islets were incubated at 37°C with basal (3 mM) or stimulatory (17 mM) concentrations of glucose with or without 100 ng/ml IGF-I (Recombinant Human IGF-I, R&D Systems) at 5, 10, or 15 min. After the incubations, the islets were sonicated in a homogenization buffer (10 µM leupeptin, 2 mM o-vanadate, 2 mM benzamidine, 10 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride in 12.5 mM EGTA, 1.25 mM EDTA, and 0.25% Triton X, pH 7.6). The tissue extracts were centrifuged at 12,000 rpm at 4°C for 15 min, and the supernatant was used for the determination of protein. The concentration of protein was determined by the Bradford method (4) using a protein assay (Bio-Rad Laboratories), with {gamma}-globulin as standard.

Immunoprecipitation. Islet extracts containing 150–300 µg of protein were immunoprecipitated overnight at 4°C with gentle rotation in the presence of 2–5 µg of the corresponding primary antibody, followed by the addition of protein A-agarose (Roche Diagnostics, Indianapolis, IN) or anti-mouse IgG-agarose (Sigma, St. Louis, MO) for the rabbit polyclonal and mouse monoclonal antibodies, respectively. After mixing for 2 h, the pellets were collected by centrifugation, and the supernatants were discarded. Then the pellets were washed and saved for Western blot analyses.

Western blot analyses. Equal amounts of protein were submitted to SDS-PAGE on 10% polyacrylamide gels at 125 V. Proteins were then electrophoretically transferred to polyvinylidene difluoride (PVDF) filters (PVDF Protein Sequencing Membrane, Bio-Rad Laboratories) at 100 V for 2 h. After the transfer, the filters were blocked with 5% (wt/vol) nonfat dry milk (for general antibodies) or 3% BSA (for anti-phosphotyrosine antibodies) in PBS followed by incubation with primary antibodies overnight. The PVDF filters were then washed four times for 10 min at 37°C with PBS and 0.1% Tween 20, followed by 1-h incubation with appropriate secondary antibody conjugated to horseradish peroxidase (Sigma BioSciences, St. Louis, MO). The PVDF membranes were then washed as indicated above and subsequently exposed to an enhanced chemiluminescence reagent (Amersham Life Sciences, Little Chalfont, Buckinghamshire, UK). The bands were quantified by laser scanning densitometry (Molecular Dynamics, Sunnyvale, CA). Immunoblots were performed under conditions of linearity according to the amount of protein loaded on the gel. The PVDF filters were finally stained with Coomassie blue to confirm that, in the same Western assay, equal amounts of protein were analyzed as well as to ensure the heterogeneity of the protein composition pattern in the different samples.

Antibodies used. Antibodies included the following: anti-IGF-IR beta-subunit sc713 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-IRS-2 (Upstate Biotechnology, Lake Placid, NY); anti-p85, which recognizes all variants of p85{alpha} and p85beta (Upstate Biotechnology); anti-phosphotyrosine (Upstate Biotechnology); anti-PKB and anti-phospho-PKB, recognizing phosphorylated Ser473 of PKB (Cell Signaling Technology, Beverly, MA); anti PKC{zeta} sc216 (Santa Cruz Biotechnology) and anti-phospho-PKC{zeta}, recognizing phosphorylated Thr410 (Cell Signaling Technology); anti-phospho-GSK3{alpha}/beta, recognizing phosphorylated Ser21 of glycogen synthase kinase-3{alpha} (GSK3{alpha}) or Ser9 of GSK3beta (Cell Signaling Technology); anti-mTOR (Upstate Biotechnology); anti-phospho-mTOR, recognizing phosphorylated Ser2448 of mTOR (Biosource International); anti-p70S6K and anti-phospho-p70S6K, recognizing phosphorylated Thr421/Ser424 of p70S6K (Cell Signaling Technology); anti-4E-BP1 sc6936 (Santa Cruz Biotechnology) and anti-phospho-4E-BP1, recognizing phosphorylated Thr70 of the initiation factor 4E-binding protein1 (4E-BP1) (Cell Signaling Technology); anti-ERK1/2 and anti-phospho-ERK1/2, recognizing phosphorylated Thr202/Tyr204 of ERK1/2 (Cell Signaling Technology).

Statistics. Values are given as means ± SE. The two-tailed t-test for independent observations was used for comparisons of the two groups. For multiple comparisons, significance was evaluated by two-way analysis of variance (ANOVA), followed by the protected least significant difference test.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of the U animals. The present animal model matches all physiological characteristics of those reported in previously published studies (3, 36, 37, 38). At the end of gestation, body weight of U mothers was significantly lower than that of C mothers (394.1 ± 12.3 vs. 312.2 ± 11.5 g, P < 0.05). Accordingly, maternal food restriction provoked a clear decrease in body weight of fetuses at term compared with C (5.1 ± 0.3 vs. 4.3 ± 0.2 g, P < 0.05). Nevertheless, no significant differences were found in the size of C and U fetal islets.

Proliferation response induced by glucose and IGF-I in pancreatic fetal islets. Because glucose and IGF-I are able to stimulate beta-cell growth in the fetal period in a synergistic way, we addressed the effect of increasing IGF-I concentrations on islet proliferation in C and U fetal islets at different glucose concentrations. After 2 days of culture, isolated fetal islets were cultured with 3 mM glucose without serum for 20 h. Then groups of 20 islets were treated for 2 more days with 3, 9, or 17 mM glucose with or without IGF-I at different concentrations (1–1,000 ng/ml). As an index of islet proliferation, DNA synthesis was determined by BrdU incorporation. To normalize the data, the value of mitogenesis at 3 mM glucose without IGF-I in C islets was set as the value 1. For the rest of the conditions, islet proliferation was expressed as its relative increase over this value. In the absence of IGF-I, increasing glucose concentration up to 9 and 17 mM provoked an increase in DNA synthesis in C and U islets (Fig. 1). Besides, addition of IGF-I (10–1,000 ng/ml) was able to further increase islet proliferation above values of glucose alone when glucose was at 9 and 17 mM in C and at 3, 9, and 17 mM in U islets. However, islet mitogenic response to glucose (9 and 17 mM) and IGF-I was significantly higher in U islets than in C. In both C and U groups, maximal mitogenic response was found at 17 mM glucose with 100 ng/ml IGF-I. Consequently, these glucose and IGF-I concentrations were considered as optimal for further experiments.


Figure 1
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  		Fig. 1. Effect of increasing glucose and IGF-I concentrations on control (C) and undernourished (U) islet proliferation. Islet proliferation was measured by incorporation of 5-bromo-2'-deoxyuridine (BrdU). Groups of 20 islets cultured for 2 days were further cultured with 3 mM glucose and without serum for 20 h. After that, islets were grown 48 h in 3, 9, or 17 mM glucose, with or without IGF-I, at different concentrations (1–1,000 ng/ml). Four different wells (samples) for each condition and 4 different experiments (n = 16) were used for statistical purposes. Results are expressed as the relative increase over the value of C group at 3 mM glucose without IGF-I and are means ± SE. aP < 0.05 when U group is compared with the respective C group within the same treatment. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) at 3 mM glucose without IGF-I. cP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I within the same glucose concentration.

 
IGF-IR protein levels and its tyrosine phosphorylation in pancreatic fetal islets. The mitogenic effect of IGF-I is mediated mainly through interactions with the IGF-IR. For this reason, we then determined the total protein levels of IGF-IR and the IGF-I ability to induce its tyrosine phosphorylation in C and U fetal islets. After 2 days of culture at 11 mM glucose, C and U fetal islets were cultured with 3 mM glucose and without serum for 20 h. Islets were lysed, and their total protein content was evaluated. Because no difference was found between C and U islets in total protein content (0.72 ± 0.02 and 0.68 ± 0.04 ng/islets, respectively), this parameter was used for normalization of samples in the immunoblot analysis. Equal protein content (100 µg) was analyzed by Western blotting with the anti-IGF-IR antibody. As shown in Fig. 2A, IGF-IR protein levels were increased 30% (P < 0.05) in U fetal islets compared with C. To analyze the tyrosine phosphorylation of the IGF-IR, islets were stimulated for 5 min with IGF-I. At the end of the culture period, islets were lysed, and equal amounts of total protein (150 µg) were immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted with antibodies against IGF-IR. The increase in the concentration of glucose did not affect binding of the IGF-I to its receptor in any of the considered populations (data not shown). In both groups, the stimulation induced a significant increase in tyrosine phosphorylation of IGF-IR (Fig. 2B), but the ratio P-IGF-IR/IGF-I was significantly higher in U fetal islets compared with C (Fig. 2C).


Figure 2
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  		Fig. 2. Islet content and phosphorylation of IGF-I receptor (IGF-IR). Two-day-cultured U and C fetal islets were further cultured with 3 mM glucose and without serum for 20 h. A: islets were lysed, and total protein (100 µg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-IGF-IR antibody. A representative experiment of 3 is shown. B: C and U islets stimulated 5 min with 100 ng/ml IGF-I were lysed, and 150 µg of protein were subjected to immunoprecipitation (IP) with the anti-Tyr(P) (pY) antibody. The resulting immune complexes were analyzed by immunoblotting (IB) with the anti-IGF-I antibody. The results shown are representative of 3 experiments. C: densitometric quantification of 6–9 independent determinations. Results are expressed as IGF-IR phosphorylated relative to IGF-IR protein level and are means ± SE. aP < 0.05 when U group is compared with the respective C within the same treatment. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I.

 
IRS-2 protein levels and its association with p85 in pancreatic fetal islets. The autophosphorylation of the IGF-IR and activation of its tyrosine kinase activity lead to tyrosine phosphorylation of various intracellular substrates, including IRS-2. To assay the protein content of IRS-2, C and U islets were cultured for 20 h in a serum-free medium containing 3 mM glucose. After the culture period, islets were lysed, and equal protein content (100 µg) was analyzed by Western blotting with anti-IRS-2 antibody. As shown in Fig. 3A, the protein levels of IRS-2 were twofold increased in islets from U fetuses compared with those in C islets. However, we could not detect the tyrosine phosphorylation of IRS-2 because it was below the sensitivity of the immunoblotting techniques employed.


Figure 3
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  		Fig. 3. Insulin receptor substrate (IRS)-2 protein content and its association with p85. Two-day-cultured U and C fetal islets were further cultured with 3 mM glucose and without serum for 20 h. A: islets were lysed, and total protein (100 µg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-IRS-2 antibody. A representative experiment of 3 is shown. B: C and U islets were cultured for 5 min with 3 or 17 mM glucose, with or without 100 ng/ml of IGF-I, and 300 µg of protein were subjected to IP with anti-p85 antibody. The resulting immune complexes were analyzed by IB with anti-IRS-2 and anti-p85 antibodies. Shown are representative blots and bars corresponding to densitometric quantification of 6–8 independent determinations. Results are expressed as the relative increase over C value at 3 mM glucose without IGF-I and are means ± SE. aP < 0.05 when U group is compared with the respective C group within the same treatment. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) at 3 mM glucose without IGF-I. cP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I within the same glucose concentration.

 
Once phosphorylated, IRS-2 binds proteins that contain Src homology 2 domains, such as the p85 regulatory subunit of PI3K. To evaluate the association of IRS-2 with the p85 subunit of PI3K, fetal islets were cultured for 20 h in a serum-free medium containing 3 mM glucose. Then islets were stimulated for 5 min with 3 and 17 mM glucose with or without IGF-I. Next, equal amounts of protein (300 µg) were immunoprecipitated with the 85-kDa regulatory subunit of PI3K antibody and immunoblotted with anti-IRS-2 and anti-p85 antibodies. As shown in Fig. 3B, total p85 levels were equivalent between C and U fetal islets. IRS-2-p85 complexes were ninefold higher in U islets than in C at the basal glucose concentration. When glucose was augmented in the culture medium to 17 mM, the association of IRS-2 with PI3K increased in C islets but did not change in U. Similarly, the presence of IGF-I in C islets led to marked increases of this association, both at 3 and 17 mM, but in U islets the IRS-2-p85 complexes were further increased only in presence of 17 mM glucose and IGF-I. Levels of IRS-2 binding the p85-kDa regulatory subunit of PI3K were significantly higher in the U group throughout the study.

Protein expression and phosphorylation of PKB, PKC{zeta}, and GSK3{alpha}/beta. To investigate the participation of downstream proteins of the PI3K pathway, C and U fetal islets were cultured for 20 h in a serum-free medium containing 3 mM glucose and treated for different time periods with basal (3 mM) or stimulating (17 mM) concentrations of glucose, with or without IGF-I. Then islets were lysed and separated by SDS-PAGE, and equal amounts of protein (70 µg) were blotted with anti-PKB, anti-phospho-PKB, anti-GSK3{alpha}/beta, anti-PKC{zeta}, and anti-phospho-PKC{zeta} antibodies. The maximum activation time was established at 10 min for PKB and 15 min for PKC{zeta} and GSK3{alpha}/beta (data not shown).

As shown in Fig. 4A, the amount of total PKB was not altered in the U group. The presence of 17 mM glucose significantly increased PKB phosphorylation in C islets (1.8-fold). However, PKB was found significantly increased at 3 mM glucose (8-fold) and remained constitutively activated in the presence of 17 mM glucose in U rats compared with C. In both groups, the magnitude of the phosphorylation was further increased by the addition of IGF-I. In all cases, the amount of phosphorylated PKB in the U group was markedly higher than in C.


Figure 4
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  		Fig. 4. Protein content and phosphorylation of PKB, PKC{zeta}, and glycogen synthase kinase (GSK)3{alpha}/beta on glucose/IGF-I stimulation. Two-day-cultured U and C fetal islets were cultured with 3 mM glucose without serum for 20 h. After this period, islets were cultured for 10–15 min with 3 or 17 mM glucose with or without 100 ng/ml IGF-I. C and U islets were lysed, and total protein (70 µg) was submitted to SDS-PAGE and analyzed by Western blotting with anti-phospho-PKB and anti-PKB antibodies (A), anti-PKC{zeta} and anti-phospho-PKC{zeta} antibodies (B), and anti-phospho-GSK3 antibodies (C). Blots are representative of 6–8 independent determinations. Bars represent the relative increase over C value at 3 mM glucose without IGF-I and are means ± SE. aP < 0.05 when U group is compared with respective C group within the same treatment. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) at 3 mM glucose without IGF-I. cP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I within the same glucose concentration.

 
Once phosphorylated, PKB can evoke PKC{zeta} activation. Total PKC{zeta} levels were equivalent between samples in both C and U fetal islets (Fig. 4B). PKC{zeta} phosphorylation was increased in C islets with 17 mM glucose (2.1-fold) and further by the addition of IGF-I. Compared with C, U islets showed a significantly higher basal phosphorylation of PKC{zeta} (5.2-fold) that was only slightly increased in the presence of both glucose (17 mM) and IGF-I.

Downstream of PKB, phosphorylation of GSK3{alpha}/beta takes place. Compared with C, in U islets GSK3{alpha}/beta phosphorylation was significantly increased at basal (3 mM) glucose (1.7-fold). In the C group, increasing glucose concentration and the additional presence of IGF-I at 3 mM enhanced the amount of GSK3{alpha}/beta phosphorylated. Unlike PKB and PKC{zeta}, there were no differences in GSK3{alpha}/beta phosphorylation between C and U islets under these conditions (Fig. 4C).

Protein expression and phosphorylation of mTOR, p70S6K, and 4E-BP1. PKB phosphorylation of mTOR leads to mTOR activation, which in turn phosphorylates at least two proteins involved in translational control of protein synthesis, 4E-BP1 and p70S6K. Besides, glucose itself can activate mTOR independently of PKB. To evaluate the effect of glucose and IGF-I in these proteins, C and U fetal islets were cultured for 20 h in a serum-free medium containing 3 mM glucose and stimulated for different time periods with basal (3 mM) and stimulating (17 mM) concentrations of glucose, with or without IGF-I. Then islets were lysed and separated by SDS-PAGE, and equal amounts of protein (100 µg) were blotted with anti-mTOR, anti-phospho-mTOR, anti-p70S6K, anti-phospho-p70S6K, anti-4E-BP1, and anti-phospho-4E-BP1 antibodies. The maximum activation time was established at 15 min (data not shown).

Figure 5A shows that there were no differences in mTOR total content between U and C fetal islets. The presence of IGF-I at 3 mM glucose and the increase in glucose concentration were able to induce mTOR phosphorylation in C islets. Compared with C, in U islets, the mTOR phosphorylation was significantly increased at basal (3 mM) glucose (2.4-fold). In U islets, increasing glucose concentration enhanced the mTOR phosphorylation, which slightly increased further in the presence of IGF-I. Thus mTOR phosphorylation was ever higher in the U group compared with C.


Figure 5
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  		Fig. 5. Protein content and phosphorylation of mammalian target of rapamycin (mTOR), p70S6K, and the initiation factor 4E-binding protein1 (4E-BP1) on glucose/IGF-I stimulation. Two-day-cultured U and C fetal islets were cultured in 3 mM glucose without serum for 20 h. After this period, islets were cultured for 15 min with 3 or 17 mM glucose with or without 100 ng/ml IGF-I. C and U islets were lysed, and total protein (100 µg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-phospho-mTOR and anti-mTOR antibodies (A), the anti-phospho-p70S6K and phospho-p70S6K antibodies (B), and anti-phospho-4E-BP1 and anti-4E-BP1 antibodies (C). Blots representative of 6 independent determinations are shown. Bars represent the relative increase over C value at 3 mM glucose without IGF-I and are means ± SE. aP < 0.05 when U group is compared with respective C group within the same treatment. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) at 3 mM glucose without IGF-I. cP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I within the same glucose concentration.

 
As shown in Fig. 5B, glucose and IGF-I stimulation evoked an increase in the phosphorylation of p70S6K in C islets. In the U group, p70S6K phosphorylation was found elevated at basal (3 mM) glucose (6.5-fold over C value), and it underwent a slight increase in the presence of 17 mM with IGF-I. Therefore, p70S6K phosphorylation was significantly higher in the U group than in the C group, even though total p70S6K levels were similar in both populations.

No differences were found in 4E-BP1 protein content between C and U islets (Fig. 5C). In C and U islets, the phosphorylation of 4E-BP1 was increased in the presence of 17 mM glucose and further enhanced by the addition of IGF-I. However, levels of phosphorylated 4E-BP1 were significantly higher in the U group, even at basal (3 mM) glucose (2.1-fold over C value).

Protein expression and phosphorylation of ERK1/2. Activation of ERK1/2 has been shown to be an essential requirement for glucose- and IGF-I-induced beta-cell mitogenic responses. Consequently, to evaluate potential effects of undernutrition on the ERK1/2 pathway, we determined the glucose- and IGF-I-induced ERK phosphorylation in C and U fetal islets. Therefore, fetal islets were cultured for 20 h in a serum-free medium containing 3 mM glucose and treated for different time periods with basal (3 mM) and stimulating (17 mM) concentrations of glucose with or without IGF-I. Then, islets were lysed and separated by SDS-PAGE, and equal amounts of protein (40 µg) were blotted with anti-ERK1/2 and anti-phospho-ERK1/2 antibodies. The maximum activation time was established at 15 min (data not shown).

As shown in Fig. 6, undernutrition did not alter the total amount of ERK1/2. At basal (3 mM) glucose, IGF-I promoted the activation of ERK1/2 in C and U islets. Increasing the glucose concentration to 17 mM further enhanced phosphorylation in both groups of islets. There was no difference in phosphorylated ERK1/2 between C and U islets.


Figure 6
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  		Fig. 6. Protein content and phosphorylation of ERK1/2 on glucose/IGF-I stimulation. Two-day-cultured U and C fetal islets were cultured at 3 mM glucose and without serum for 20 h. After this period, islets were cultured for 15 min with 3 or 17 mM glucose with or without 100 ng/ml IGF-I. C and U islets were lysed, and total protein (40 µg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. Blots representative of 6–7 independent determinations are shown. Bars represent the relative increase over C value at 3 mM glucose without IGF-I and are means ± SE. aP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) at 3 mM glucose without IGF-I. bP < 0.05 when a group (C or U) is compared with the value of the identical group (C or U) without IGF-I within the same glucose concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this work, we have investigated the IGF-I signal transduction system in islets from fetuses with increased beta-cell mass proceeding from U pregnant rats (36). To this end, we have developed an in vitro model of primary fetal islets in which elements of the above-mentioned signal transduction pathway are present and can be stimulated by glucose and IGF-I. Through this in vitro system, we have established that, compared with C, U fetal islets show a higher protein content of IRS-2 and an increased basal and glucose/IGF-I-induced phosphorylation of several proteins implicated in the mitogenic and survival pathway.

In the present study, islet mitogenesis was evaluated in freshly isolated islets that were cultured with 11 mM glucose for 48 h. After that, islets were maintained for 20 h in a serum-free medium with 3 mM glucose. This experimental procedure allowed us to obtain better physiological conditions and alleviate the effects on the IGF-I signaling of exposing the islets to glucose and serum for a prolonged period of time (5). By using this in vitro system, we found that glucose in the physiological range (3–17 mM) was able to stimulate islet cell replication in fetal islets, especially in the U group. Thus, the additional presence of IGF-I (10–1,000 ng/ml) induced a greater islet replication in both groups. As previously shown in pancreatic beta-cells (25), the effect of IGF-I on increased DNA synthesis in C islets was apparent only at 9 and 17 mM glucose; however, in the U group, this increase was also evident at 3 mM glucose. These results suggest that U fetal islets show increased sensitivity to glucose and to IGF-I compared with C.

To identify the mechanism underlying the islet replication response observed in the U population, we evaluated in fetal islets the levels of different proteins implicated in the IGF-I mitogenic pathway and their phosphorylation on stimulation by IGF-I and glucose. It is widely known that the growth effect of IGF-I is mediated by its interaction with IGF-IR and the subsequent activation of IRS-2 within the beta-cells (47). Here we show that IGF-IR content and its phosphorylation on IGF-I stimulation were increased in U fetal islets compared with C. Although IGF-IR is not crucial for beta-cell development (29), it seems to be instrumental in activating IRS-2-mediated signaling, strongly implicated in beta-cell growth. Therefore, the increased IGF-IR content and phosphorylation shown by U islets may be favoring the mitogenic action of IGF-I, which is also enhanced in U fetal pancreas (36).

Unlike IGF-IR, the importance of IRS-2 to pancreatic beta-cell growth has become apparent from several in vivo and in vitro studies. Increased IRS-2 in vitro can promote beta-cell replication, neogenesis, and survival, whereas decreased IRS-2 causes beta-cell apoptosis (reviewed in Ref. 12). In addition, transgenic expression of IRS-2 in vivo promotes beta-cell survival (20), while IRS-2 knockout mice show a marked decrease in beta-cell mass (48). Interestingly, we found that IRS-2 protein levels were markedly increased in U islets. As previously shown in INS-1 cells (34) and pancreatic islets (41), the endogenous expression levels of IRS-2 can be specifically increased by glucose. This effect seems to be dependent on glucose metabolism and requires a downstream increase in cytosolic Ca2+ concentration (32). In addition, enhanced glucose oxidation is also able to increase IRS-2 levels via cAMP response element-binding protein (CREB) activation (27). Accordingly, we have previously demonstrated that U islets show a greater glucose oxidation (37). Therefore, we suggest that the enhanced content of IRS-2 found in these islets can be related to the higher glucose oxidative metabolism shown by the U group.

Increased IRS-2 in U islets was accompanied by a rise in both basal and glucose/IGF-I-induced association of IRS-2 with p85, the regulatory subunit of PI3K. Surprisingly, the magnitude of the increase found in the basal association of p85 with IRS-2 in U islets was greater than that expected just from the increase of IRS-2 content. Thus it seems fair to assume that tyrosine phosphorylation of IRS-2 could be augmented in U islets under basal conditions too. In this regard, it has been shown recently (32) that glucose not only regulates IRS-2 expression levels but also IRS-2 intracellular localization. At stimulatory glucose concentrations (32) or in the presence of IGF-I (33), IRS-2 is translocated to the beta-cell plasma membrane where it can be tyrosine phosphorylated, leading to an increase in IRS-2/p85 association. Accordingly, it is possible that, in U islets at 3 mM glucose, IRS-2 was mostly localized in the plasma membrane, favoring the tyrosine phosphorylation of IRS-2. Although there was no activation of the IGF-IR at 3 mM glucose in fetal islets, IRS-2 could then be tyrosine phosphorylated by basal tyrosine kinase activities localized in the basal membrane (40, 43).

The increase in IRS-2/p85 association in both populations leads to a consequential increase in PKB phosphorylation. PKB has been involved in the regulation of beta-cell mass and function (14). The phosphorylation activation of this protein plays a pivotal role mediating beta-cell mitogenesis (12) and beta-cell survival (45, 49). Besides, it has been shown that glucose itself can promote pancreatic beta-cell survival through activation of PI3K/PKB signaling pathway (44). It can be assumed then that the greater PKB phosphorylation activation found in U islets, even at basal conditions, could lead to increased beta-cell replication and promote survival under restricted conditions.

To determine the role of PKB in mediating mitogenic response in fetal islets, we examined downstream PKB targets that could be involved in promoting this process. PKB can activate an atypical isoform of PKC (PKC{zeta}) that has been implicated in increasing beta-cell mitogenesis in response to glucagon-like peptide-1 (GLP-1) (8) and to IGF-I (21). In this regard, basal and glucose/IGF-I-induced PKC{zeta} phosphorylation was increased in U islets compared with C, suggesting that PKC{zeta} could be implicated in the increased replication response found in U islets. Unlike PKC{zeta}, the increased PKB phosphorylation in U islets did not evoke higher levels of GSK3{alpha}/beta in response to IGF-I and/or glucose, and it was increased only under basal conditions. It can be assumed then that GSK3{alpha}/beta is not implicated in the increased replication response found in U islets. Nevertheless, inactivation by phosphorylation of GSK3{alpha}/beta decreases phosphorylation of beta-catenin, which has been previously associated with increased beta-cell survival (9). Therefore, the increase phosphorylation found in the basal situation in U islets could be playing an important role in promoting beta-cell survival at low levels of glucose.

Downstream of PKB, mTOR regulates cell growth through at least two proteins, 4E-BP1 and p70S6K, whose activation results in a general increase in protein synthesis in beta-cells (39). Our results showed that the phosphorylation levels of these proteins were again significantly enhanced in U fetal islets. These findings suggest that the greater activation of these proteins is related to the higher mitogenic response observed in U islets. In addition, it has been shown that glucose can induce mTOR (11) and ERK1/2 (7) phosphorylation in beta-cells independently of IRS signaling. It is reasonable to suppose then that the altered glucose oxidation reported in U islets could also be contributing to increased DNA synthesis in these islets by activation of mTOR independently of IRS-2 stimulation. As recently suggested (30), the appropriate regulation of mTOR by nutrients may be an effective strategy to enhance growth and proliferating capacity of beta-cells. However, glucose activation in U islets did not evoke an increased stimulation of ERKs compared with C, supporting the idea that glucose-induced ERK activation is mediated by a distinct signaling pathway from mTOR (7).

The increased glucose/IGF-I activation of the PI3K pathway found in U islets correlated with an enhanced glucose- and IGF-I-induced mitogenesis except at basal (3 mM) glucose. Interestingly, in this basal condition, U islets showed increased phosphorylation activation of several proteins implicated in DNA synthesis such as PKB, PKC{zeta}, mTOR, 4E-BP1, and p70S6K but not ERK1/2. Thus these results suggest that a minimum ERK activation needs to be reached to elicit the greater mitogenic response in U islets. As previously shown in the pancreatic beta-cell line INS-1 (13) and in the rat clonal beta-cell line RIN 1046-38 (21), there is a requirement for the activation of both ERK1/2 and PI3K signal transduction pathways for a full commitment to induce pancreatic beta-cell mitogenesis.

Finally, it is worth remarking that the effects of undernutrition on beta-cell in our model are different from those described in other models of maternal low-protein diets (42) and intrauterine growth restriction (31), in which beta-cell mass and beta-cell replication were decreased. However, it has been shown recently that several processes leading to beta-cell failure and death seem to be preceded by periods of beta-cell hyperfunction (10). It is possible that our U islets are in such a previous hyperfunction stage and that this overstimulation leads to a decrease in beta-cell mass later, as it has previously been shown at 4 days of life and at adult age (38). In support of this idea, a recent report (6) has demonstrated that chronic activation of mTOR, as in our U islets, could lead to a decreased IRS-2 expression and an increased beta-cell apoptosis, a mechanism that may be contributing to reduce beta-cell mass.

In conclusion, we have demonstrated that U islets are characterized by multiple alterations in the IGF-I signal transduction system, leading to an improved activation of the IRS-2/PI3K/PKB/mTOR pathway and to a better mitogenic response to glucose and IGF-I. Thus, all these molecular changes may be contributing to the increased beta-cell mass found in U fetuses. Since an increase in IRS-2 has a role in promoting beta-cell survival (34) and neogenesis (26), the finding of a raised content of this factor suggests that both processes might also participate in the improved beta-cell mass exhibited by U fetuses. Interestingly, the in vitro system shown herein represents a valuable tool for testing the effect that different factors may have on important aspects of beta-cell life such as proliferation, survival, or neogenesis. Understanding the intracellular signaling molecules implicated in beta-cell growth under physiological and pathological conditions will provide future strategies aimed at the prevention and the treatment of diabetes and other related metabolic disorders.


   GRANTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grant Ref. No. BFU2005-02849 from the Ministerio de Ciencia y Tecnología of Spain.


   ACKNOWLEDGMENTS
 
We thank Dr. Luis Goya for critical review of this manuscript.

Parts of this work were presented at the 41st European Association for the Study of Diabetes (EASD), Athens, Greece (Diabetologia 48, Suppl 1: 160, 2005).

Present addresses: M. A. Martín, Departamento de Metabolismo y Nutrición, Instituto del Frío, Consejo Superior de Investigaciones Científicas, Madrid, Spain, and S. Fajardo, Instituto de Recursos Materiales, Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Científicas, Madrid, Spain.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Álvarez, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain (e-mail: calvarez{at}farm.ucm.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

*E. Fernández and M. A. Martín contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Al-Mashat F, Sibyani AM, El-Naggar MM, Badawoud MH, Alayat AA, Ardawi MS. Structural study on cultured isolated fetal rat pancreatic islets. Saudi Med J 25: 1352–1355, 2004.[Web of Science][Medline]
  2. Alvarez B, Garrido E, García-Sanz JA, Carrera AC. Phosphoinositide 3-kinase activation regulates cell division time by coordinated control of cell mass and cell cycle progression rate. J Biol Chem 278: 26466–26473, 2003.[Abstract/Free Full Text]
  3. Alvarez C, Martín MA, Goya L, Bertin E, Portha B, Pascual-Leone AM. Contrasted impact of maternal rat food restriction on the fetal endocrine pancreas. Endocrinology 138: 2267–2273, 1997.[Abstract/Free Full Text]
  4. Bradford M. A rapid and sensitive method for the quantification of microgram quanties of protein, utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
  5. Briaud I, Dickson LM, Lingohr MK, McCuaig JF, Lawrence JC, Rhodes CJ. Insulin receptor substrate-2 proteosomal degradation mediated by a mammaliam target of rapamicin (m-TOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta cells. J Biol Chem 280: 2282–2293, 2005.[Abstract/Free Full Text]
  6. Briaud I, Dickson LM, Lingohr MK, McCuaig JF, Lawrence JC, Rhodes CJ. Insulin receptor substrate-2 proteosomal degradation mediated by a mammalian target of rapamycin (m-TOR)-induced negative feedback down-regulates protein kinase B-mediated signalling pathway in beta cells. J Biol Chem 280: 2282–2293, 2005.[Abstract/Free Full Text]
  7. Briaud I, Lingohr MK, Dickson LM, Wrede CE, Rhodes CJ. Differential activation of Erk-1/2 and p70S6K by glucose in pancreatic beta cells. Diabetes 52: 974–983, 2003.[Abstract/Free Full Text]
  8. Buteau J, Foisy S, Rhodes CJ, Carpenter L, Biden TJ, Prentki M. Protein kinase Czeta activation mediates glucagon-like peptide-1-induced pancreatic beta cell proliferation. Diabetes 50: 2237–2243, 2001.[Abstract/Free Full Text]
  9. Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, Franklyn RA, McCubrey JA. Involvement of PI3K/AKT pathway in cell cycle progression, apoptosis and neoplastic transformation: a target for cancer chemotherapy. Leukemia 17: 590–603, 2003.[CrossRef][Web of Science][Medline]
  10. Dahlquist G. Can we slow the rising incidence of childhood-onset autoimmune diabetes? The overload hypothesis. Diabetologia 49: 20–24, 2006.[CrossRef][Web of Science][Medline]
  11. Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G. Mammalian TOR: a homeostatic ATP sensor. Science 294: 1102–1105, 2001.[Abstract/Free Full Text]
  12. Dickson LM, Rhodes CJ. Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt? Am J Physiol Endocrinol Metab 287: E192–E198, 2004.[Abstract/Free Full Text]
  13. Dickson LM, Lingohr MK, McCuaig J, Hugl SR, Snow L, Kahn BB, Myers MG, Rhodes CJ. Differential activation of protein kinase B and p70S6K by glucose and insulin-like growth factor 1 in pancreatic beta cells (INS-1). J Biol Chem 276: 21110–21120, 2001.[Abstract/Free Full Text]
  14. Elghazi L, Balcazar N, and Bernal-Mizrachi E. Emerging role of protein kinase B/AKT signaling in pancreatic beta cell mass and function. Int J Biochem Cell Biol 38: 157–163, 2006.[Web of Science][Medline]
  15. Escrivá F, Rodriguez C, Cacho J, Alvarez C, Portha B, Pascual-Leone AM. Glucose utilization and insulin action in adult rats submitted to prolonged food restriction. Am J Physiol Endocrinol Metab 263: E1–E7, 1992.[Abstract/Free Full Text]
  16. Fowden AL, Giussani DA, Forhead AJ. Endocrine and metabolic programming during intrauterine development. Early Hum Dev 81: 723–734, 2005.[CrossRef][Web of Science][Medline]
  17. George M, Ayuso E, Casellas A, Costa C, Devedjian JC, Bosch F. Beta cell expression of IGF-1 leads to recovery from type I diabetes. J Clin Invest 109: 1153–1163, 2002.[CrossRef][Web of Science][Medline]
  18. Hales CN, Barker DJP. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty hypothesis. Diabetologia 35: 595–601, 1992.[CrossRef][Web of Science][Medline]
  19. Hellerstrom C, Lewis NJ, Borg H, Johnson R, Freinkel N. Methods for large scale isolation of pancreatic islets by tissue culture of fetal rat pancreas. Diabetes 28: 769–776, 1979.[Web of Science][Medline]
  20. Hennige AM, Burks DJ, Ozcan U, Kulkarni RN, Ye J, Park S, Schubert M, Fisher TL, Dow MA, Leshan R, Zakaria M, Mossa-Basha M, White MF. Upregulation of insulin receptor substrate-2 in pancreatic beta cells prevents diabetes. J Clin Invest 112: 1521–1532, 2003.[CrossRef][Web of Science][Medline]
  21. Hennige AM, Fritsche A, Strack V, Weigert C, Mischak H, Borboni P, Renn W, Haring HU, Kellerer M. PKCzeta enhances insulin-like growth factor 1-dependent mitogenic activity in the rat clonal beta cell line RIN 1046-38. Biochem Biophys Res Commun 290: 85–90, 2002.[CrossRef][Web of Science][Medline]
  22. Hill DJ. Development of the endocrine pancreas. Rev Endocr Metab Disord 6: 229–238, 2005.[CrossRef][Web of Science][Medline]
  23. Hogg J, Han VK, Clemmons DR, Hill DJ. Interactions of nutrients, insulin-like growth factors (IGFs) and IGF-binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. J Endocrinol 138: 401–412, 1993.[Abstract/Free Full Text]
  24. Holemans K, Aerts L, Van Assche FA. Lifetime consequences of abnormal fetal pancreatic development. J Physiol 547: 11–20, 2003.[Abstract/Free Full Text]
  25. Hugl SR, White MF, Rhodes CJ. Insulin-like growth factor (IGF-1) stimulated pancreatic beta cell growth is glucose-dependent: synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-1 in INS-1 cells. J Biol Chem 273: 17771–17779, 1998.[Abstract/Free Full Text]
  26. Jetton TL, Liu YQ, Trotman WE, Nevin PW, Sun XJ, Leahy JL. Enhanced expression of insulin receptor substrate-2 and activation of protein kinase B/Akt in regenerating pancreatic duct epithelium of 60% partial pancreatectomy rats. Diabetologia 44: 2056–2065, 2001.[CrossRef][Web of Science][Medline]
  27. Jhala US, Canettieri G, Screaton RA, Kulkarni RN, Krajewski S, Reed J, Walker J, Lin X, White M, Montminy M. cAMP promotes pancreatic beta cell survival via CREB-mediated induction of IRS-2. Genes Dev 17: 1575–1580, 2003.[Abstract/Free Full Text]
  28. Kaung HL. Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 200: 163–175, 1994.[Web of Science][Medline]
  29. Kido Y, Nakae J, Hribal ML, Xuan S, Efstratiadis A, Accili D. Effects of mutations in the insulin-like growth factor signaling system on embryonic pancreas development and beta cell compensation to insulin resistance. J Biol Chem 277: 36740–36747, 2002.[Abstract/Free Full Text]
  30. Kwon G, Marshall CA, Liu H, Pappan KL, Remedi MS, McDaniel ML. Glucose-stimulated DNA synthesis through mTOR is regulated by K ATP channels: effects on cell cycle progression in rodent islets. J Biol Chem 281: 3261–3267, 2006.[Abstract/Free Full Text]
  31. Limesand SW, Jensen J, Hutton JC, Hay WW Jr. Diminished beta-cell replication contributes to reduced beta-cell mass in fetal sheep with intrauterine growth restriction. Am J Physiol Regul Integr Comp Physiol 288: R1297–R1305, 2005.[Abstract/Free Full Text]
  32. Lingohr MK, Briaud I, Dickson LM, McCuaig JF, Alarcon C, Wicksteed BL, Rhodes CJ. Specific Regulation of IRS-2 expression by glucose in rat primary pancreatic islet beta cell. J Biol Chem 281: 15884–15892, 2006.[Abstract/Free Full Text]
  33. Lingohr MK, Dickson LM, Wrede CE, McCuaig JF, Myers MG, Rhodes CJ. IRS-3 inhibits IRS-2-mediated signaling in pancreatic beta cells. Mol Cell Endocrinol 204: 85–99, 2003.[CrossRef][Web of Science][Medline]
  34. Lingohr MK, Dickson LM, Wrede CE, Briaud I, McCuaig JF, Myers MG, Rhodes CJ. Decreasing IRS-2 expression in pancreatic beta cells (INS-1) promotes apoptosis, which can be compensated for by introduction of IRS-4 expression. Mol Cell Endocrinol 209: 17–31, 2003.[CrossRef][Web of Science][Medline]
  35. Lingohr MK, Dickson LM, McCuaig JF, Hugl SR, Twardzik DR, Rhodes CJ. Activation of IRS-2-mediated signal transduction by IGF-1, but not TGFalpha or EGF, augments pancreatic beta cell proliferation. Diabetes 51: 966–976, 2002.[Abstract/Free Full Text]
  36. Martín MA, Serradas P, Ramos S, Fernández E, Goya L, Gangnerau MN, Lacorne M, Pascual-Leone AM, Escrivá F, Portha B, Alvarez C. Protein-caloric food restriction affects insulin-like growth factor system in fetal Wistar rat. Endocrinology 146: 1364–1371, 2005.[Abstract/Free Full Text]
  37. Martín MA, Fernández E, Pascual-Leone AM, Escrivá F, Alvarez C. Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas. Am J Physiol Endocrinol Metab 286: E542–E550, 2004.[Abstract/Free Full Text]
  38. Martín MA, Alvarez C, Goya L, Portha B, Pascual-Leone AM. Insulin secretion in adult rats that had experienced different underfeeding patterns during their development. Am J Physiol Endocrinol Metab 272: E634–E640, 1997.[Abstract/Free Full Text]
  39. McDaniel ML, Marshall CA, Pappan KL, Know G. Metabolic and autocrine regulation of the mammalian target of rapamicin by pancreatic beta cells. Diabetes 51: 2877–2885, 2002.[Abstract/Free Full Text]
  40. Myers MG, White MF. Insulin signal transduction and the IRS proteins. Annu Rev Pharmacol Toxicol 36: 615–658, 1996.
  41. Paris M, Bernard-Kargar C, Vilar J, Kassis N, Ktorza A. Role of glucose in IRS signaling in rat pancreatic islets: specific effects and interplay with insulin. Exp Diabesity Res 5: 257–263, 2004.[CrossRef][Web of Science][Medline]
  42. Petrik J, Reusens B, Arany E, Remacle C, Hoet JJ, Hill DJ. A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat, and is associated with a reduced pancreatic expression of insulin-like growth factor-II. Endocrinology 140: 4861–4873, 1999.[Abstract/Free Full Text]
  43. Rhodes CJ, White MF. Molecular insights into insulin action and secretion. Mol Cell Biochem 182: 3–11, 1998.[CrossRef][Web of Science][Medline]
  44. Srinivasan S, Bernal-Mizrachi E, Ohsugi M, Permutt MA. Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway. Am J Physiol Endocrinol Metab 283: E784–E793, 2002.[Abstract/Free Full Text]
  45. Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A, Birnbaum MJ. Regulation of pancreatic beta cell growth and survival by the serine/threonine protein kinase Akt/PKBalpha. Nat Med 7: 1133–1137, 2001.[CrossRef][Web of Science][Medline]
  46. Van Haeften TW, Twickler ThB. Insulin-like growth factors and pancreas beta cells. Eur J Clin Invest 34: 249–255, 2004.[CrossRef][Web of Science][Medline]
  47. Withers DJ, Burst DJ, Towery HH, Altamuro SL, Flint CL, White MF. Irs-2 coordinates IGF-1 receptor mediated beta cell development and peripheral insulin signaling. Nat Genet 23: 32–40, 1999.[Web of Science][Medline]
  48. Withers DJ, Gutierrez JS, Towery H, Burks DJ, Rem JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900–904, 1998.[CrossRef][Medline]
  49. Wrede CE, Dickson LM, Lingohr MK, Briaud I, McCuaig JF, Rhodes CJ. Protein kinase B/Akt prevents fatty acid induced apoptosis in pancreatic beta cells (INS-1). J Biol Chem 277: 49676–49684, 2002.[Abstract/Free Full Text]




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