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Ras-GRF1 signaling is required for normal -cell development and glucose homeostasis

2003; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês

10.1093/emboj/cdg280

1460-2075

Jaime Font de Mora,

FOXO transcription factor regulation

Article16 June 2003free access Ras-GRF1 signaling is required for normal β-cell development and glucose homeostasis Jaime Font de Mora Jaime Font de Mora Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Luis Miguel Esteban Luis Miguel Esteban Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Deborah J. Burks Deborah J. Burks Instituto de Neurociencias de Castilla y León, Spain Search for more papers by this author Alejandro Núñez Alejandro Núñez Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Carmen Garcés Carmen Garcés Unidad de Lípidos, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Spain Search for more papers by this author María José García-Barrado María José García-Barrado Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author María Carmen Iglesias-Osma María Carmen Iglesias-Osma Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author Julio Moratinos Julio Moratinos Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author Jerrold M. Ward Jerrold M. Ward Veterinary Pathology, NCI, Frederick, MD, 21702 USA Search for more papers by this author Eugenio Santos Corresponding Author Eugenio Santos Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Jaime Font de Mora Jaime Font de Mora Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Luis Miguel Esteban Luis Miguel Esteban Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Deborah J. Burks Deborah J. Burks Instituto de Neurociencias de Castilla y León, Spain Search for more papers by this author Alejandro Núñez Alejandro Núñez Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Carmen Garcés Carmen Garcés Unidad de Lípidos, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Spain Search for more papers by this author María José García-Barrado María José García-Barrado Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author María Carmen Iglesias-Osma María Carmen Iglesias-Osma Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author Julio Moratinos Julio Moratinos Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain Search for more papers by this author Jerrold M. Ward Jerrold M. Ward Veterinary Pathology, NCI, Frederick, MD, 21702 USA Search for more papers by this author Eugenio Santos Corresponding Author Eugenio Santos Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain Search for more papers by this author Author Information Jaime Font de Mora1, Luis Miguel Esteban1, Deborah J. Burks2, Alejandro Núñez1, Carmen Garcés3, María José García-Barrado4, María Carmen Iglesias-Osma4, Julio Moratinos4, Jerrold M. Ward5 and Eugenio Santos 1 1Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, Spain 2Instituto de Neurociencias de Castilla y León, Spain 3Unidad de Lípidos, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Spain 4Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain 5Veterinary Pathology, NCI, Frederick, MD, 21702 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3039-3049https://doi.org/10.1093/emboj/cdg280 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Development of diabetes generally reflects an inadequate mass of insulin-producing β-cells. β-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interest ingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of β-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired β-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal β-cell number and function. Introduction Ras proteins are able to regulate various signaling pathways controlling cell growth, differentiation or survival. They do so through their ability to integrate multiple, different upstream signals and to elicit a variety of different cellular responses. The broad array of signals emanating from Ras reflects the binding and activation of different downstream effectors. Known effectors of Ras signaling pathways include the Raf–MEK–Erk kinase cascade, the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, some protein kinase C isoforms, RalGDS family members, and AF-6, Nore1, Mekk1 and Rin1 (reviewed in Campbell et al., 1998; Boettner and Van Aelst, 2002). Insulin controls glucose homeostasis by directing peripheral tissues to transport and metabolize glucose. Insulin production depends on the proper development and maintenance of the endocrine pancreas. Pancreatic β-cells are developed by a series of differentiation and maturation processes and by cell proliferation. The precise molecular mechanisms governing β-cell development are largely unknown, but insights into these events are being discovered by physiological studies of genetically modified animals and isolated islets. Many signals have been implicated in pancreatic β-cell proliferation and function, including insulin-like growth factor-I (IGF-I), prolactin, growth hormone (GH), glucagon-like peptide-1 (GLP1) and others (Nielsen et al., 2001). However, the specific pathways that mediate development of a normal β-cell mass are poorly understood. Several molecules that are upstream and downstream modulators of Ras have been implicated in β-cell proliferation. Both insulin receptor substrate-2 (IRS-2) (Withers et al., 1998) and cyclin-dependent kinase 4 (Cdk4) (Rane et al., 1999) play critical roles in β-cell physiology, since deletion of either of these intracellular signaling molecules produces severe diabetes in mice. The IGF-I signaling pathway is an important contributor to β-cell mass, since transgenic animals overexpressing IGF-I specifically in β-cells develop islet hyperplasia (George et al., 2002). Conversely, heterozygous animals (+/−) for the IGF-I receptor show a reduction in β-cell mass, and this effect is diminished even more potently by the lack of IRS-2 (Withers et al., 1999). In addition, activation of IRS-2-mediated signaling pathways triggered by IGF-I, but not transfrorming growth factor-α (TGF-α) or epidermal growth factor (EGF), augments pancreatic β-cell proliferation in vitro (Lingohr et al., 2002), further demonstrating a link between IGF-I and IRS-2. More recently, transgenic animals overexpressing the PI3K effector Akt/PKB in β-cells revealed its important contribution to the mitogenic signal (Tuttle et al., 2001). Also, knockout mice for S6K1 (p70s6k), another kinase downstream of PI3K, exhibit a reduction in pancreatic β-cell mass (Pende et al., 2000). Interestingly, transgenic mice expressing v-ras specifically in pancreatic islets develop β-cell apoptosis and diabetes (Efrat et al., 1990). Similarly, c-Myc activation in β-cells induces apoptosis and β-cell mass reduction, whereas suppression of the apoptotic effects of c-Myc reveals a potent proliferative activity that results in neoplasia (Pelengaris et al., 2002). Thus, these various observations demonstrate that a balance among the different signaling pathways activated by Ras is essential for normal β-cell development and function. Ras cycling between an inactive GDP-bound conformation and an active GTP-bound conformation is controlled by guanine nucleotide-releasing factors (GRFs) and GTPase-activating proteins (GAPs), respectively. Thus, GRFs play a critical role in the cell by allowing signals from the extracellular environment to reach the internal response machinery. Different GRF homologs have been cloned and characterized (Bowtell et al., 1992; Martegani et al., 1992; Fam et al., 1997; Ebinu et al., 1998) and their distinct roles are determined by distinct mechanisms of activation and cell-specific expression. Hence, Sos1 and Sos2 are activated preferentially by binding to docking proteins containing SH2 and SH3 domains, as well as the interaction with the PI3K products. On the other hand, GRF1 and GRF2 can be activated by binding to βγ-subunits of heterotrimeric G proteins coupled to receptors (Mattingly and Macara, 1996) as well as by calmodulin-mediated Ca2+ influxes (Farnsworth et al., 1995; Cullen and Lockyer, 2002). Whereas Sos1 and Sos2 are ubiquitously expressed, expression of GRF1 and GRF2 is limited to brain and a few other tissues (Guerrero et al., 1996). Interestingly, GRF1 and GRF2 exhibit different patterns of expression within the brain (Fernandez-Medarde et al., 2002), suggesting different physiological roles despite the high homology between these factors. GRF1 is an imprinted gene which is expressed only after birth (Ferrari et al., 1994; Itier et al., 1998). Previous reports have demonstrated that GRF1-deficient mice exhibit defects in learning and memory, although some controversy exists regarding the molecular nature of these impairments (Brambilla et al., 1997; Giese et al., 2001; Tonini et al., 2001). Similar to two other GRF1 knockout strains (Itier et al., 1998; Giese et al., 2001), our mice display a reduced body size and low IGF-I levels. Surprisingly, we have observed that GRF1 knockout mice have low levels of circulating insulin and are glucose intolerant due to a reduction of β-cell mass. Here we report that β-cells express GRF1, and deletion of this Ras-GTP exchange factor causes an impairment of β-cell proliferation and neogenesis. Glucose sensing and insulin production are not affected by the absence of Ras-GRF1 signaling since β-cells from knockout animals secrete and synthesize insulin comparably with control mice. One explanation for the impaired β-cell proliferation observed in GRF1-deficient mice is the failure to activate PI3K–Akt and Erk signaling pathways within these cells. Thus, our data reveal an important role for Ras-GRF1-mediated signaling in the regulation of β-cell proliferation and glucose homeostasis. Results To study the physiological role of GRF1, we used gene targeting technology in embryonic stem (ES) cells to generate mutant mice deficient for GRF1 (Figure 1A). A deletion in two exons coding for the catalytic domain inactivated all of the potential splicing isoforms of GRF1 (Cen et al., 1992). As expected, our mutant mice did not express GRF1 (Figure 1B and C) based on Southern analysis and RT–PCR. These results were confirmed further by immunoblotting with GRF1-specific antibodies (Figure 1C). GRF1 knockout animals characterized by others have been reported to display alterations in learning and long-term memory (Brambilla et al., 1997; Giese et al., 2001; Tonini et al., 2001) and we did not study such phenotypes in our animals. On the other hand, in agreement with a previous report (Itier et al., 1998), our GRF1 knockout mice displayed a marked reduction in body size and weight (∼70% of wild-type counterparts; Fernandez-Medarde et al., 2002) without any other observable somatic phenotype. GRF1 is expressed preferentially, but not exclusively, in brain, suggesting its importance in the central nervous system (CNS). However, we have also detected the expression of GRF1 in wild-type testis and faintly in whole pancreas (Figure 1C) as well as in other tissues (Guerrero et al., 1996). Figure 1.Generation and verification of the targeted deletion of the mouse GRF1 gene. (A) Protein structure of GRF1 and genomic targeting strategy. Relevant structures including two pleckstrin homology (PH) domains, the DH domain and CDC25-like domain are indicated in the schematic of p140 GRF1. The seven exons encoding amino acid sequences through the CDC25-like catalytic domain were mapped and sequenced in the murine GRF1 gene (vertical bars, arbitrarily assigned I–VII). A 1.8 kb region of GRF1 containing two exons of the catalytic region was replaced by the PGK promoter-driven neomycin cassette. Restriction sites: B, BamHI; H, HindIII; S′, SacI; Sp, SpeI; Sw, SwaI. (B) This modification produced a diagnostic reduction of 6 kb in a SpeI fragment which was detected by Southern blot analysis. (C) Tissue distribution of GRF1 expression in wild-type and knockout mice. RNA from the indicated tissues was isolated and used in an RT–PCR with specific primers from two consecutive exons (V and VI) for the catalytic region of GRF1 gene: 5′-CTGTGTCCCTTACCTGGGGATGTATCTCAC-3′ and 5′-GGCTGGGGGTCGATTTTGTAGGTAGTCTGC-3′. Primers for the housekeeping gene G3PDH were 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′. Tissues were also collected for western blot analysis using immunopurified anti-GRF1 antibodies. Download figure Download PowerPoint Surprisingly, a detailed hormonal study of GRF1−/− mice revealed low serum insulin levels, without significant alterations of glucagon levels (Figure 2A). Other hormones, including prolactin, thyroid-stimulating hormone, testosterone, estradiol and corticosterone did not change. In both male and female GRF1 knockouts, insulin levels are diminished by ∼30%. This reduction in circulating insulin was detected as early as 6 weeks of age and persisted throughout adulthood. Based on this observation, we examined whether GRF1 is expressed in pancreatic β-cells. RT–PCR analysis revealed the presence of full-length GRF1 as well as Sos1 and Sos2 in isolated murine islets (Figure 2B). However, expression of GRF2 was not detected in islets. Furthermore, GRF2 knockout mice have normal insulin levels (unpublished results). Thus, despite the similarities in the structure and mechanisms of activation shared between GRF1 and GRF2, these results indicate that GRF2 is not involved in β-cell development and function. Our findings suggest a potential direct and specific role for GRF1 in β-cell function (see below). Therefore, we focused on elucidating the role of Ras exchange factor in islet physiology. Figure 2.Deletion of GRF1 results in low circulating levels of insulin. (A) Analysis of serum insulin and glucagon levels from adult mice under fed conditions. Data presented correspond to the average ± SEM of seven wild-type and seven GRF1−/− animals. Statistical analysis: *P = 0.05; **P = 0.3. (B) Expression of different GEFs as detected by RT–PCR analysis of isolated wild-type islets, liver, brain and gonadal fat. Download figure Download PowerPoint To assess whether low insulin circulating levels altered glucose metabolism, we first analyzed glucose tolerance in GRF1−/− animals. GRF1-deficient mice displayed a notable intolerance to glucose (Figure 3A), although by the end point of the test (120 min), glucose concentrations were restored to the normal levels of control animals. We also evaluated glucose-stimulated insulin release under similar in vivo conditions. Interestingly, GRF1 knockouts displayed a dampened release of insulin at the early time point (15 min), but circulating insulin levels were comparable with control values at 120 min. These results parallel the behavior of the GRF1 knockouts during the glucose tolerance test, providing an explanation for both the hyperglycemia and the return to normal glucose levels at the end of the test (Figure 3B). Thus, these assays indicate that GRF1-deficient mice are glucose intolerant due to diminished circulating insulin. Figure 3.Analysis of glucose tolerance and insulin release. (A) For evaluation of glucose tolerance, male animals (12 weeks of age) were fasted for 14–16 h. Following intraperitoneal injection of glucose (2 mg/g), serum glucose levels were measured by tail bleed at the indicated time points. Data represent the average ± SEM of six wild-type and six GRF1−/− animals. The experiment shown is representative of at least four independent glucose tolerance tests. (B) Glucose-stimulated insulin release was analyzed in vivo using fasted male animals. Animals were injected intraperitoneally with glucose (2 mg/g body weigth) and serum samples were collected by tail bleed at the indicated times. Insulin levels were determined by ELISA. Results are the average ± SEM of four wild-type and four GRF1−/− animals. *P = 0.04; **P = 0.08; ***P = 0.3. Download figure Download PowerPoint To determine whether the observed insulin deficiency was accompanied by insulin resistance in peripheral tissues, we performed a set of experiments to test insulin sensitivity in fat, liver and skeletal muscle. First, we evaluated the response of GRF1−/− mice during an insulin tolerance test (ITT). GRF1-deficient animals displayed the same sensitivity to insulin as controls; the reduction of blood glucose levels in response to the insulin injection paralleled that of wild-type animals (Figure 4A). Thus, based on this classic test, peripheral tissues of GRF1 knockouts are not resistant to the physiological effects of insulin and, therefore, insulin resistance is produced by a failure in the insulin signaling pathways (Pessin and Saltiel, 2000). The PI3K–Akt signaling pathway is activated by insulin in a broad variety of tissues and participates in the regulation of glucose homeostasis. To evaluate insulin action in GRF1 knockouts at the molecular level, we analyzed insulin signaling in skeletal muscle and liver. Fasted animals were stimulated with a bolus of insulin and tissues were lysed and studied for activation of insulin signaling pathways. Expression and activation of the insulin receptor in GRF1−/− skeletal muscle and liver (Figure 4B) were comparable with levels detected in control animals. Insulin produced a potent activation of Akt/PKB in muscle and liver in wild-type and knockout mice; Erk was potently activated in muscle, but weakly or not activated in liver from both wild-type and knockout mice. However, no differences in this activation were noted between knockouts and controls (Figure 4B). Therefore, the results of these signaling experiments further confirm that peripheral tissues of GRF1-deficient mice respond properly to insulin; these animals simply have less insulin, but this reduction is not accompanied by insulin resistance. Figure 4.Assessment of insulin responsiveness. (A) An insulin tolerance test was performed on fed animals at 8–12 weeks of age. Insulin (1 U/kg) was injected intraperitoneally and glucose levels were sampled at the indicated time. The graph represents the average ± SEM of six wild-type and six GRF1−/− animals. Similar results were obtained in two additional insulin tolerance tests. (B) Insulin-stimulated signal transduction was analyzed in tissues from wild-type and knockout animals. Animals were fasted overnight and then treated with a bolus insulin injection (5 U). Non-stimulated controls were subjected to a saline injection. Tissues were lysed and evaluated by western analysis as shown. Download figure Download PowerPoint During the study of peripheral insulin signaling, we observed that GRF1 knockouts appeared to have less abdominal fat than control animals. Indeed, a careful analysis revealed that the weight of gonadal fat in GRF1−/− mice is reduced ∼30% compared with wild-type males (Figure 5A). The reduction in fat accumulation was not due to a dysregulation of appetite, as food intake was not reduced in these mice (Figure 5B). Adipose tissue modulates metabolic events by secreting bioactive substances that regulate appetite and insulin resistance. Hence, we measured the levels of leptin and resistin, two hormones produced by fat that have been implicated in these processes (Steppan et al., 2001; Friedman, 2002). However, the expression levels of these hormones were similar in the fat of wild-type and GRF1−/− animals (Figure 5C and D). Figure 5.Analysis of adipose storage and metabolism. (A) Gonadal fat pads were collected from male animals at 12–16 weeks of age. Weight of fat (wild type, 1.50 ± 0.07 g; knockout, 0.78 ± 0.16 g) was compared with overall body weight (wild type, 40.2 ± 0.7 g; knockout, 29.5 ± 1.5 g) and used to calculate the given ratios. Data represent the average fat:body comparisons ± SEM of four wild-type and six GRF1−/− mice. *P = 0.04. (B) Food intake was monitored daily in groups of wild-type and knockout animals during a period of 4 weeks. Prior to onset of the study, animals were given 1 week to adjust to feeding jars and ground chow. Results represent the average chow consumed per animal per day ± SEM from five wild-type and eight knockout animals. (C) Serum leptin was assessed from fed animals by ELISA. Results represent the average ± SEM circulating leptin levels of five wild-type and eight knockout animals. (D) For analysis of resistin expression, adipose tissue was collected from fasted animals and used for preparation of RNA. RT–PCR was performed to detect resistin expression, and densitometric analysis was used to assess relative levels of resistin from ethidium bromide-stained gels. RNA was prepared and evaluated independently from the fat pads of five wild-type and eight knockout animals. (E) Lipolysis was analyzed in isolated adipocytes of fed male mice (12 weeks of age). The rate of basal, insulin-stimulated (10−8 M), and isoproterenol-stimulated (ISO; 10−6 M) glycerol formation was measured. Data represent the average ± SEM rates of lipolysis of three wild-type and three knockout animals. Download figure Download PowerPoint One classic action of insulin is to suppress lipolysis in adipose tissue. Thus, given that serum insulin levels are reduced in GRF1−/− animals, we postulated that the reduced fat storage probably reflects failed inhibition of lipolysis in this animal model. Isolated abdominal fat preparations from wild-type and knockout animals were used to determine the lipolytic activity in response to insulin. Interestingly, basal lipolysis levels were almost doubled in GRF1-deficient mice (Figure 5E). However, both groups of animals responded equally well to insulin (Figure 5E) in a dose-dependent manner (data not shown) by blocking lipolysis activity. Additionally, the β-adrenergic agonist isoproterenol induced the lipolytic activity of fat proportionally from both wild-type and GRF1-deficient animals. Thus, the results of the lipolysis study confirm (i) that basal lipolytic activity is higher in adipose tissue of GRF1 knockouts, possibly owing, at least in part, to lower circulating insulin levels; and (ii) that based on the normal response of GRF1−/− fat to insulin, this tissue displays no insulin resistance. Finally, no significant elevations in serum-free fatty acids were detected in GRF1 knockouts (3–5 months of age: 0.9 ± 0.1 mEq/l, wild type; and 0.8 ± 0.2 mEq/l, GRF1−/−), suggesting that free fatty acids liberated by the increased lipolysis in these animals serve as a source of fuel. Taken together, these data indicate that GRF1 knockout mice display abnormalities in fat storage due to enhanced basal lipolysis but not due to differences in food consumption. The results thus far indicate that GRF1 knockout mice are glucose intolerant due to insulin deficiency. Hence we focused on β-cell physiology to elucidate the nature of the defects underlying the reduction in serum insulin. We reasoned that three different possibilities might account for the hypoinsulinemia observed in the GRF1 knockouts: a reduction in insulin production; a defect in glucose sensing or insulin secretion; and/or an overall reduction in the mass of β-cells. Strikingly, we observed a reduction in β-cell area and islet size (Figure 6A). Based on insulin immunostaining of pancreatic sections taken from animals at 3 months of age, quantification of the β-cell area (percentage of the total pancreas) revealed a 30% reduction in GRF1−/− mice as compared with controls (Figure 6B). Moreover, the average size of GRF1−/− islets was noticeably smaller (Figure 6C). The reduction in size and overall area occupied by β-cells might reflect fewer and/or smaller cells. To distinguish between these possibilities, we analyzed β-cell density within a constant area of islets. 4′,6-diamidino-2-phenylindole (DAPI)-staining nuclei revealed that the number of insulin-positive cells per area was equivalent between GRF1 knockouts and wild-type controls (Figure 6D), demonstrating that deletion of this Ras exchange factor does not alter β-cell size but rather reduces the overall number of cells. As described above, isolated islets from wild-type pancreas contain high levels of GRF1 mRNA, as well as Sos1 and low levels of Sos2. However, expression of GRF2 is not detected in normal islets, and GRF2−/− mice have normal insulin levels (Figure 2B). These observations suggest specifically that the lack of Ras-GRF1 activity may account for the reduction in β-cell area, which underlies the hypoinsulinemia and glucose intolerance in the knockout animals. Figure 6.Morphological analysis of pancreatic β-cells. (A) Representative insulin immunostaining of pancreatic sections from wild-type and knockout male mice. Upper panels: low magnification immunohistochemical analysis with anti-insulin antibodies. Lower panels: higher magnification of immunofluorescence studies. (B) The percentage of total pancreatic area occupied by β-cells was calculated using insulin-stained pancreas sections. For this analysis, three sections from each animal were immunostained and data were collected from eight independent fields of each section using Openlabs software. Results represent examination of sections from four wild-type and four knockout animals (16 weeks of age; P = 0.06). (C) Average islet size was assessed using pancreatic sections double-stained with glucagon and insulin. Measurements were performed using Openlabs software. Results represent examination of sections from four wild-type and four knockout animals (16 weeks of age; P = 0.03). (D) To approximate the density of β-cells/islet, paraffin-embedded pancreas sections were immunostained with insulin. To reveal the nuclei, sections were also exposed to DAPI. The area represented by insulin-containing cells was tabulated as described above, and nuclei were counted manually from these cells stained for insulin. Download figure Download PowerPoint To investigate further the mechanisms by which Ras-GRF1 contributes to β-cell function, we considered the various developmental processes that ultimately determine β-cell number: neogenesis, growth and apoptosis. In mice, formation of the pancreatic bud occurs approximately at embryonic day 9.5 and although islets do not form properly until the end of gestation, insulin-producing cells can be detected in the developing pancreas as early as E10 (Sander and German, 1997). However, GRF1 is not expressed until after birth (Ferrari et al., 1994; Itier et al., 1998), excluding the possibility that this molecule exerts its role during embryonic development of the endocrine pancreas. Thus, we focused our study on postnatal development of β-cell number. The rodent pancreas undergoes major remodeling during the postnatal phase, and apoptosis is a critical mechanism in this process. In the rat, a major wave of apoptosis occurs in the neonatal pancreas between days 2 and 18, but is followed by increased apoptosis just prior to weaning (Scaglia et al., 1997). A positive outcome, i.e. development of a normal β-cell mass, requires a fine balance between growth and apoptosis; a dysregulation of remodeling events could have important effects on β-cell mass. As GRF1 is not fully expressed until after postnatal day 5, we performed our analysis of proliferation and apoptosis at weaning (postnatal day 21). Apoptotic events within β-cells were investigated at postnatal day 21 by TUNEL assay and activated caspase-3 on pancreatic histological sections, but no differences in rates of apoptosis were observed between knockouts and controls (data not shown). However, quantification of bromodeoxyuridine (BrdU) incorporation from these same samples revealed a significant reduction in the proliferation of GRF1−/− β-cells (Figure 7A), suggesting that Ras-GRF1 functions in the β-cells by mediating proliferative signals. Moreover, fewer neogenic islets were noted in GRF1−/− pancreas sections (Figure 7B). Substantial evidence indicates that β-cell proliferation and/or survival is dependent on IGF-I (George et al., 2002). Thus, we reasoned that IGF-I signaling might be altered in GRF1−/− β-cells. To test this possibility, we stimulated isolated islets from both knockouts and controls with IGF-I and anal