Defective insulin secretion in pancreatic β cells lacking type 1 IGF receptor

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of β cell mass and impaired glucose-dependent insulin release. β cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in β cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect β cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

The ability of pancreatic β cells to synthesize, store, and release insulin in response to variations in circulating metabolite levels and intracellular glucose metabolism is regulated by changes in ATP/ADP ratios resulting in Ca²⁺ mobilization (1). Alterations of this sensing loop occur early in the pathogenesis of type 2 diabetes, but are initially compensated by an increase of β cell mass (2). In this respect, pancreatic β cells appear to differ from other terminally differentiated cell types by retaining their ability to proliferate, as demonstrated in both physiological conditions (growth, gestation) and disease states (obesity, insulin resistance) (3). In addition to presumptive proliferation of existing β cells, there is evidence for β cell neogenesis from undifferentiated progenitors, apparently arising from the epithelial lining of pancreatic ducts (4–8).

The factors inducing β cell proliferation under normal or pathological conditions are largely unknown, although some evidence exists about the involvement of FGFs (9), HGFs (10), and placental lactogen (11). Moreover, signaling by receptor tyrosine kinases has been implicated as a regulatory mechanism in both β cell proliferation (12–14) and insulin release (9, 15–19). In particular, insulin/IGF signaling through insulin receptor substrate (IRS) and phosphoinositide 3-kinase (PI 3-kinase) appears to regulate several aspects of β cell function. Thus, ablation of the insulin/IGF receptor substrate IRS-2 impairs β cell proliferation (20, 21), whereas ablation of p70^s6k1, an Akt substrate, is associated with a decrease in β cell size (22). Conversely, overexpression of a constitutively active mutant Akt1 increases β cell mass and protects from streptozotocin-induced diabetes (23, 24). However, mutations affecting the components of the IRS-2→Akt→p70^s6k1 axis do not perturb glucose sensing or insulin production. In contrast, mutations of Insulin receptor (Ir) (25) or Irs1 (26) impair insulin synthesis and secretion mediated by PI 3-kinase–dependent pathways (17, 19, 26). These observations suggest that signals regulating β cell proliferation and insulin secretion diverge downstream of PI 3-kinase; i.e., Akt is not the sole effector of PI 3-kinase. Nevertheless, the role of growth factor signaling through PI 3-kinase as related to insulin secretion remains poorly understood. Additional difficulties arise because, against expectation, knockout of the gene encoding the p85α subunit of PI 3-kinase results in increased insulin secretion (27), consistent with studies using PI 3-kinase inhibitors (28). Because of the known relationship between IGF and PI 3-kinase signaling and results indicating that IGFs inhibit insulin secretion (29–31), we sought to examine in mutant mice the contribution of type 1 IGF receptor (IGF1R) function to insulin secretion and β cell proliferation, which could differ from that of the IR pathway.

Although the roles of IGFs in β cells have not been completely elucidated, these ligands and their cognate receptors are both expressed in this tissue (32–35). Interestingly, the developmental wave of β cell apoptosis observed in neonatal rats (36) is associated with a decrease in IGF-2 expression (32, 37), leading to the suggestion that the IGFs have an antiapoptotic function in β cells. On the other hand, overexpression of IGF-2 in β cells (38), but not in liver (39, 40), results in diabetes, presumably through increased insulin production with concomitant downregulation of insulin sensitivity.

A potential role for IGF1R acting as a mediator of β cell proliferation through IRS-2 has been implied by observations indicating that the β cell failure observed in Irs2^–/– mice is exacerbated by Igf1r haploinsufficiency (13). However, generalized lack of IGF1R is lethal in the immediate postnatal period (41), thus precluding an extensive analysis of the role of IGF signaling in β cell physiology. To circumvent this limitation and analyze the role of IGF1R in β cells, we have used conditional mutagenesis with the cre/loxP site-specific recombination system to generate mice lacking IGF1R specifically in pancreatic β cells. Here we show that these mutant mice are glucose intolerant and exhibit an insulin secretion defect.

Generation of mice lacking IGF1R in β cells. Tissue-specific conditional mutagenesis requires crosses between Cre-producing and Cre-responding strains of mice. For Cre expression in pancreatic β cells of producer mice we used an InsPr-Cre transgenic line, in which Cre transcription is driven by the rat insulin-2 gene promoter (43). These mice express Cre in β cells as early as embryonic day 11.5 (E11.5), and show very limited extrapancreatic expression in the amygdala (P. Herrera, unpublished observations). As responder mice we used previously described compound heterozygotes (42) derived from matings between heterozygotes carrying an Igf1r null allele (Igf1r+/–) and Igf1rlox/+ conditional mutants carrying a modified Igf1r locus in which exon 3 of the gene is flanked by loxP sites (“floxed”). PCR analysis of DNA extracted from various tissues of progeny carrying the InsPr-Cre transgene and possessing an Igf1rlox/+ genotype showed that DNA excision leaving behind a single loxP site had occurred specifically in pancreatic islets (Figure 1a). Additional Southern blot analysis of DNA from several other tissues from which adequate amounts of DNA could be extracted indicated the presence of an exclusively unrecombined allele (data not shown). The extent of recombination detected in islets was approximately 80%, consistent with the ratio of β/non-β (α, δ, PP, vascular, and connective) cells in normal islets (Figure 1b). Western blot analysis of pancreatic islets with anti-IGF1R antiserum confirmed that the levels of proreceptor and mature β subunit were reduced by 85% in Igf1rΔlox/– mice, consistent with ablation of the protein product exclusively in β cells (Figure 1c). Parallel immunohistochemical analysis yielded consistent results (data not shown). Igf1rΔlox/– mice were born at the expected Mendelian frequency and did not exhibit abnormalities.

Phenotypic characterization of mice lacking IGF1R in β cells. To assess the consequences of IGF1R ablation in β cells on glucose metabolism, we measured glucose and insulin levels in neonates and adult mice in the fasting and fed states at various ages. No differences were detected between mice with Igf1r+/–, Igf1rlox/+, Igf1rlox/–, Igf1rΔlox/+, and Igf1rΔlox/– genotypes (Table 1). Glucose tolerance tests were then performed in adult (6-month-old) mice to assess potential metabolic defects not resulting in overt diabetes. In Igf1r+/– and Igf1rΔlox/+ mice, glucose values rose to approximately 250 mg/dl 30 minutes after intraperitoneal glucose administration and returned to nearly normal values by 2 hours. In contrast, in Igf1rΔlox/– mice glucose values rose to approximately 400 mg/dl and did not decrease appreciably thereafter, consistent with impaired glucose disposal. Igf1rlox/– mice had similar 30-minute glucose values as Igf1rΔlox/– mice, but gradually returned to nearly normal values (Figure 2). These data could be interpreted as indicating that the combination of a single null allele and a floxed allele results in haploinsufficiency for Igf1r and causes a mild impairment of glucose tolerance. The impairment of glucose tolerance in Igf1rlox/– and Igf1rΔlox/– mice is apparently not due to insulin resistance, since insulin tolerance tests failed to demonstrate differences between the two genotypes and wild-type mice (data not shown).We next examined the consequences of ablation of IGF1R function on pancreatic morphology. Immunohistochemical analysis of pancreata from Igf1rΔlox/– mice showed that the islet architecture was normal (Figure 3). Thus, β cell mass was similar in all genotypes examined at various time points, as was mean β cell diameter (Table 2). In addition, the pattern of expression of the β cell–restricted transcription factor Pdx1, as assessed by immunohistochemistry, was similar (data not shown). In contrast, real-time PCR analysis of the expression levels of mRNA encoding the β cell glucose transporter GLUT-2 demonstrated a significant decrease in the islets of Igf1rΔlox/– mice (by approximately 40%) when compared with Igf1rlox/+ and Igf1rlox/– mice (Figure 4a). Immunostaining of pancreatic sections with anti–GLUT-2 antiserum showed decreased immunoreactivity in Igf1rΔlox/– mice (Figure 4b).To determine whether the lack of IGF1R affects β cell proliferation, we measured the number of Ki67-positive β cells in Igf1rlox/–, Igf1rΔlox/+, and Igf1rΔlox/– mice at various ages as an index of mitotic activity. The use of Ki67 for this purpose was validated by staining adjacent sections with two additional proliferation markers, phosphohistone H3 and BrdU, and then analyzing the correlation between the values obtained with different methods. As shown in Figure 5, phosphohistone H3– and BrdU-positive cells showed a highly significant positive correlation with Ki67-positive cells (r² > 0.8, P < 0.0001). Thus, Ki67 was employed for all subsequent analyses. A developmental analysis showed that the number of β cells undergoing replication decreased sharply with age. However, no differences were detected between the various genotypes under study (Figure 6). These data indicate that lack of IGF1R does not impair β cell replication. Similarly, we observed no differences in the levels of apoptotis ascertained at P10, P20, and P30 using TUNEL staining, although the small number of detectable apoptotic cells did not permit accurate quantitation (data not shown).

Figure 2

Intraperitoneal glucose tolerance tests in mice lacking IGF1R in β cells. Tests were performed in overnight-fasted mice as indicated in Methods. The difference between Igf1r^Δlox/– and Igf1r^Δlox/+ is significant at all time points except 0. The difference between Igf1r^Δlox/– and Igf1r^lox/– is statistically significant at times 90 and 120. The difference between Igf1r^Δlox/+ and Igf1r^lox/– is statistically significant at times 30, 60, and 90 (P < 0.05 by ANOVA). Bars represent SEM.

Figure 3

Islet morphology in mice lacking IGF1R in β cells. Examples of pancreatic sections from P30 mice of the indicated genotypes that were stained with anti-insulin and anti-glucagon Ab’s are shown.

Figure 4

Analysis of Glut-2 expression. (a) Real-time PCR amplification. mRNA was isolated from Igf1r^Δlox/–, Igf1r^lox/–, and Igf1r^Δlox/+ mice (n = 3 each; equal amounts were pooled). Amplification was performed using a one-step RT-PCR reaction. The amount of RNA in the reaction was normalized using β-actin RNA as a control. *P < 0.05 by ANOVA. (b) Immunohistochemistry with anti-GLUT-2 antiserum. Representative sections of Igf1r^Δlox/+ and wild-type mice are shown.

Figure 5

Ki67, histone H3, and BrdU labeling of β cells. Adjacent 4-μm-thick pancreatic section from mice at E18.5, P10, P30, and P120 were costained with anti-insulin Ab’s (to identify β cells) and anti-Ki67, or anti-histone H3 or anti-BrdU Ab’s (to provide an index of proliferation). Positive cells were visually scored, and the number of Ki67-positive cells was plotted as a function of the number of histone H3–positive (upper panel) or BrdU-positive cells (lower panel). Regression analysis was performed using the Statsview software. r² = 0.9 for the H3/Ki67 and 0.9 for the BrdU/Ki67 correlation (P < 0.0001).

Figure 6

Developmental analysis of Ki67 labeling. A Ki67 labeling index was determined in mice of different ages by multiplying the mean number of Ki67/insulin double-positive cells per section by β cell mass (μg). The values at E18.5 were normalized to 100%. None of the differences observed between the different genotypes are statistically significant. Two mice for each genotype were used at each time point, except for Igf1r^lox/+ and Igf1r^Δlox/+ at P10, where one mouse was used. Three to five sections per pancreas were scored as described in Methods.

Table 1

Metabolic parameters

Table 2

β cell mass and size

Measurements of insulin secretion in purified islets. To examine whether the conditional mutation under study affects insulin secretion, we purified islets from Igf1rlox/+, Igf1rΔlox/+, and Igf1rΔlox/– mice, which were incubated in the presence of glucose or arginine. As shown in Figure 7a, glucose induced a dose-dependent increase in insulin secretion up to a maximum of 15- to 20-fold over the basal level in islets from Igf1rlox/+ and Igf1rΔlox/+ controls. In Igf1rΔlox/– mutants, glucose-induced insulin secretion was also normal at low glucose concentrations, but decreased by approximately 70% at high concentrations (P < 0.05 by ANOVA). A similar pattern of response was observed in arginine-induced insulin secretion in Igf1rΔlox/– mutant mice (Figure 7b). On the other hand, the islet insulin content was similar in the Igf1rlox/+, Igf1rΔlox/+, and Igf1rΔlox/– genotypes (Figure 7c), indicating that the defective insulin secretion in Igf1rΔlox/– mutants, which apparently leads to impairment of glucose tolerance, is not due to paucity of insulin. To assess whether the observed defect in insulin secretion could be correlated with β cell structure abnormalities, we analyzed β cells from Igf1rΔlox/– mice and wild-type controls by electron microscopy. The results showed that there were no identifiable morphologic abnormalities in β cells lacking IGF1R, except for a hypertrophic Golgi system, indicating that abnormal insulin secretion is not due to overt disruption of cellular architecture and failure to assemble insulin secretory granules (Figure 8).

Figure 7

Insulin secretion and content in purified islets. Insulin release was determined in response to varying glucose (a) and arginine (b) concentrations. For each glucose concentration, ten islets derived from two or three mice of each genotype were used. Three separate experiments were performed and the results were averaged. (c) Insulin content in islets of the indicated genotypes was measured by high-salt buffer extraction followed by radioimmunoassay.

Figure 8

Electron microscopy of β cells. Representative images of β cells in wild-type and mutant mice are shown at ×11,300 magnification.

Analysis of transcripts encoding proteins involved in exocytosis. Considering that the secretion data in isolated islets are consistent with a defect in insulin release, we analyzed the expression of several genes involved in insulin exocytosis using expression profiling with an oligonucleotide microarray. This analysis confirmed that Igf1r mRNA was decreased by approximately 80% in Igf1rΔlox/– mice. However, the assay did not reveal differences in expression levels of several candidate genes, including those encoding the β-granule components synaptotagmin II and III, synaptobrevin 2 (VAMP-2), VAMP-3, and Rab-3a; the cytosolic proteins NSF, 14-3-3, Rab-3 GDI, Mss-4, Munc-18, and annexin II; the plasma membrane proteins SNAP-23, SNAP-25, syntaxin-1A, -3, -4A, -5A, -6; and the synaptic-like vesicles synaptophysin, synapsin I and II. Expression of the sulfonylurea receptor and calmodulin kinase II was also unaffected. Thus, the observed defect in insulin secretion is apparently not due to alterations in members of the insulin exocytotic pathway, at least at the transcriptional level.

Type 2 diabetes results from combined abnormalities of β cell function and insulin action. Although the exact nature of the β cell defect remains elusive, one of the earliest demonstrable abnormalities in individuals at high risk of developing type 2 diabetes is an exaggerated insulin response to a glucose challenge, suggestive of an intrinsic secretory defect (48, 49). Moreover, unaffected first-degree relatives of type 2 diabetics exhibit abnormalities of the first-phase insulin response to glucose, similar to the defect observed in patients with active disease (50). Notably, β cell secretory defects are exemplified by maturity onset diabetes of the young (51, 52), a genetically heterogeneous, highly penetrant, monogenic form of diabetes caused by mutations in genes that couple glucose metabolism to insulin release (53).

In most type 2 diabetics, defective insulin secretion is compounded with peripheral insulin resistance in various tissues (54). Initially, in response to this resistance, there is a compensatory increase in β cell mass to preserve adequate insulin production. Over time, however, this compensation cannot be sustained. The mechanism of this response and the pathophysiology of its failure in type 2 diabetes remain unclear, but there is some evidence for circulating factors affecting islet proliferation (55). Alternatively, transient oscillations in glucose levels, occurring before the onset of overt hyperglycemia, may be sufficient to promote β cell growth (56), or glucose may have a permissive effect in stimulating β cell proliferation acting in concert with growth factors (12).

The observations that mice lacking IRS-1 develop defective insulin secretion, whereas mice lacking IRS-2 develop impaired β cell proliferation, have raised the question of whether insulin signaling on one hand and IGF signaling on the other serve overlapping or distinct functions related to these phenotypic manifestations (57). In this regard, conditional ablation of Ir in β cells results in a secretory defect similar to that observed in type 2 diabetes (25), but less severe than that observed in IRS-1–deficient β cells (26). Insulin acting through IR appears to be required for regulation of its own synthesis and secretion (17), while the IGFs have been proposed to be involved in the promotion of β cell proliferation, based on the observation that Igf1r haploinsufficiency increases the severity of diabetes in mice lacking IRS-2 (13). While our data do not support this view, there are potential explanations for the discrepancy between our observations and those in Irs2–/–Igf1r+/– mice. First, the tissue-specific phenotype that we describe here is not necessarily directly comparable with the state of ubiquitous haploinsufficiency in Igf1r+/– heterozygotes carrying a null allele. Second, it is possible that IRS-2, in addition to mediating IGF signaling, mediates signaling by other growth factors, potentially acting in a strain-dependent manner. Finally, it remains to be seen whether IR could play a compensatory role in β cell proliferation in the absence of IGF1R, as suggested by the observation that not only Igf1r haploinsufficiency, but also Ir haploinsufficiency results in accelerated β cell failure in Irs2–/– mice (58). Combined ablation of both the Ir and Igf1r functions in β cells is required to address this question.

In contrast to the absence of a proliferative defect, β cells lacking IGF1R exhibit a profound decrease of insulin secretion in response to both glucose and arginine. This impairment is reminiscent of the secretory defect observed in Irs1–/– β cells (26). Although the decrease in glucose-induced insulin secretion could be accounted for partly by the observed decrease in GLUT-2 expression, the inability of IGF1R-deficient β cells to respond to arginine suggests that the defect lies in the exocytotic pathway. Since arginine promotes insulin release through direct membrane depolarization and Ca²⁺ influx (59, 60), our data indicate that IGF1R signaling could be involved in regulating insulin secretion at a step distal to that of ATP production and closure of ATP-sensitive K channels (61). These findings are consistent with the observation that insulin stimulates exocytosis through changes in intracellular Ca²⁺ stores in a PI 3-kinase–dependent fashion (19). In this regard, it is important to note that while some steps of the exocytotic process are stimulated by insulin signaling, others may be inhibited. It was shown, for example, that intracellular translocation of storage granules into the early releasable pool is inhibited by PI 3-kinase activation (27). By analogy, it can be hypothesized that the early stimulatory effect of IGF-1 on insulin secretion in cultured β cells, which is followed by a late phase of inhibition (62), is due to activation of different steps in the exocytotic process.

Expression profiling using microarray analysis showed that there are no detectable differences in the transcription of known genes involved in insulin exocytosis. However, IGF1R signaling could promote this process by several additional mechanisms. For example, IGF1 may directly stimulate Ca²⁺ entry into β cells, occurring through translocation of a recently described growth factor–regulated calcium-permeable channel belonging to the TRP family (63). Consistent with this hypothesis, there are reports suggesting that IGF-2 promotes exocytosis in β cells (64), while tyrosine kinase inhibitors apparently prevent insulin secretion in response to several secretagogues (18). Alternatively, IGF signaling may activate ATP-sensitive K channels in a PI 3-kinase–dependent manner, as already shown for insulin (15), or could impinge upon G protein–mediated exocytotic steps through one of the insulin/IGF-sensitive G protein subunits (65). Finally, an intriguing possibility is that IGFs regulate insulin secretion through an autocrine mechanism. This hypothesis has emerged from studies of mice lacking HNF-1α (the MODY3 gene homologue) or overexpressing dominant negative mutant forms of HNF-1α in β cells (66–68), which develop a defect similar to that described here for β cell–specific Igf1r knockouts. Interestingly, IGF-1 appears to be an HNF-1α target gene in β cells that is downregulated in mice expressing a dominant negative HNF-1α (69). Furthermore, the hypothesis mentioned above could explain why overexpression of another IGF1R ligand, IGF-2, in β cells of transgenic mice results in increased insulin secretion (38).

While this manuscript was under review, Kulkarni et al., using different strains of Cre-producing and Cre-responding mice, reported the generation of mice lacking IGF1R in β cells (70). Their findings are in agreement with ours, except that they did not study the response of purified islets to arginine, but reported normal arginine-induced insulin secretion in vivo. Interestingly, both we and Kulkarni report an increase in basal insulin values, although in our study the values did not reach statistical significance.

The similarities between the phenotype described here and those of Ir and Irs1 knockouts indicate that IGF1R could have overlapping functions with those of Ir and could be signaling through IRS-1 in β cells. The identification of IGF-1–dependent mechanisms of exocytosis potentially could provide novel targets for therapeutic intervention in β cell dysfunction.

This work was supported by Juvenile Diabetes Research Foundation grant 2000-893 to D. Accili and A. Efstratiadis. We thank members of the Accili and Efstratiadis laboratories for helpful discussions.

Conflict of interest: No conflict of interest has been declared.

Nonstandard abbreviations used: phosphoinositide 3-kinase (PI 3-kinase); insulin receptor substrate (IRS); type 1 IGF receptor (IGF1R); insulin receptor (IR); 5-bromo-2-deoxyuridine (BrdU); embryonic day (E).

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