* This work was supported by National Institutes of Health Grants DK58282 and DK57539 (to D. A.) and Juvenile Diabetes Research Foundation Grant 893 (to D. A. and A. E.).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. § Present address: Second Dept. of Internal Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan.
Insulin and insulin-like growth factors (IGF) play overlapping and complementary roles in pancreatic β-cell function and peripheral metabolism. In this study, we have analyzed mice bearing loss-of-function mutations of the insulin/IGF signaling systems. Combined inactivation of insulin receptor (Insr) and Igf1 receptor (Igf1r), but not of either receptor alone, resulted in a 90% decrease in the size of the exocrine pancreas, because of decreased cellular proliferation. In contrast to the findings in the exocrine compartment, endocrine α- and β-cell development was unperturbed. Combined ablation of Igf1 and Igf2, the ligands for these two receptors, resulted in an identical phenotype. We also examined the effect of heterozygous null Igf1r mutations on glucose homeostasis in adult mice. Igf1r haploinsufficiency did not affect insulin action and compensatory β-cell growth in insulin-resistant mice with combined Insr andIgf1r heterozygous null mutations, resulting in a considerably milder phenotype than combined haploinsufficiency forInsr and its main signaling substrates, Irs1and Irs2. We conclude that Igf1r andInsr are required for embryonic development of the exocrine but not of the endocrine pancreas and that defects of Igf1rdo not alter glucose homeostasis as long as the insulin receptor system remains intact.
insulin-like growth factor
analysis of variance
Type 2 diabetes is a complex metabolic disease characterized by impaired insulin action in peripheral tissues and perturbed pancreatic β-cell function (
). Although these two abnormalities are interdependent, and lead in concert to the onset of hyperglycemia, it is unclear whether they share a common pathogenesis.
Increasing evidence indicates that insulin and IGF1 signalings play overlapping roles in metabolism and pancreatic β-cell function. For example, ablation of Insr function in skeletal muscle, a key target tissue in glucose disposal, does not cause significant metabolic abnormalities (
). At present, however, the details of the involvement of INSR and IGF1R in β-cell proliferation and insulin secretion and also in compensatory functions in response to insulin resistance remain unclear (
To investigate further the functional overlap between INSR and IGF1R signaling in development, especially as it pertains to the pancreas, and also in metabolic control, we examined different mouse models. First, we analyzed pancreatic development in embryos homozygous for null mutations of the genes encoding Igf1r andInsr, alone or in combination. We also studied double mutant embryos lacking both IGF ligands, Igf1 andIgf2. Perinatal lethality precludes a metabolic analysis of nullizygotes for Insr or Igf1rmutations in adulthood. Thus, usingInsr+/−Igf1r+/− double heterozygotes, we asked whether the insulin resistance caused by theInsr mutation (
) is exacerbated by Igf1rhaploinsufficiency. Because we have previously shown thatInsr haploinsufficiency has a strong diabetogenic effect in appropriate predisposing backgrounds (Irs1+/−or Irs2+/−), it was important to examine whether a similar predisposing effect of Igf1rhaploinsufficiency would be detected by this experimental approach.
Generation and genotyping of mice with null alleles of Igf1, Igf2, Insr, andIgf1r have been reported in previous publications (
). Timed pregnancies were set up to retrieveInsr−/− and Igf1r−/−embryos at gestational stages E12.5 and E18.5. Mice were housed in a barrier facility in ventilated cages with filtered air in a 12-h light-dark cycle.
Guinea pig anti-insulin, mouse anti-glucagon (Sigma), and rabbit polyclonal anti-pancreatic α-amylase antibodies (Calbiochem) were used at a 1:1,000 dilution for immunohistochemistry. Immunoprecipitation and immunoblotting of IGF1R was performed with rabbit polyclonal antibody C-20 (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-phosphotyrosine antibody (Transduction Laboratories, Lexington, KY) was used at a 1:1,000 dilution and detected using enhanced chemiluminescence (ECL, Amersham Biosciences). Anti- BrdUrd antibody (Sigma) was used at a 1:1,000 dilution followed by detection with alkaline phosphatase-conjugated secondary antibody at a 1:100 dilution (
Mice were fed regular chow ad libitum or fasted for 16 h. Blood was drawn from the retroorbital sinus of anesthetized animals between 0900 and 1100 h. For glucose tolerance test, mice were fasted starting at 1800 for 16 h. Thereafter, mice were injected intraperitoneally with glucose (2 g/kg body weight). Blood samples were obtained immediately before and at the indicated time points after injection. Glucose levels were measured using a glucometer (Accucheck; Roche Diagnostics). Insulin was measured in plasma samples by radioimmunoassay using a rat insulin standard (Linco, St. Charles, MO).
Immunoprecipitation, Immunoblotting, and PI 3-Kinase Assays
Experiments were carried out in overnight fasted, 4–6-month-old male mice. Animals were anesthetized by the intraperitoneal administration of sodium pentobarbital (65 mg/kg), and human insulin (5 units) was injected through the inferior vena cava. Extracts of liver and hindlimb muscles (gluteus and soleus) were prepared as described (
Pancreatic Immunohistochemistry and Morphometry: Adult Mice
Pancreata were removed from adult mice, cleared of fat and spleen, weighed, and fixed overnight in Bouin's solution. Tissues were embedded in paraffin, and consecutive 5-μm sections were mounted on slides (
). Sections at 200-μm intervals were immunostained with various antibodies used at the dilutions indicated above. The cross-sectional area of the pancreas was covered systematically by accumulating images from a series of non-overlapping fields at a ×40 magnification. Images were recorded on a digital camera Nikon Coolpix 950 (Nikon) and analyzed using the “image analysis” macro of the NIH Image software, version 1.60. Areas corresponding to the whole pancreas, α- and β-cells were measured by manual tracing of the acquired images. α- and β-cell areas were calculated by dividing the area of glucagon (α-cells) and insulin-positive (β-cells) immunoreactivity by whole pancreas area (
To measure pancreatic size, E18.5 embryos were sacrificed; the abdominal cavity was cut open to facilitate tissue fixation, and serial transverse abdominal sections covering the entire pancreatic length were obtained. Total pancreatic size was estimated from the number of pancreas-containing sections, because pancreatic weights could not be reliably ascertained due to the small size of some mutant embryos. However, total embryo weight could be used to normalize the various comparisons, as we have shown that in normal embryos the total number of pancreatic sections obtained correlated with whole embryo weight (r2 = 0.8, p < 0.001). Three sections, spaced at least 50 μm apart, were chosen for morphometric measurements. To closely match the position of these sections among different genotypes, we used the duodenum, liver, and spine as reference points. Three non-overlapping microscopic fields at low magnification (×10) were scored on each section, and four to six embryos were analyzed per genotype. Islet area was defined as the sum of α- and β-cells areas after staining with insulin and glucagon antisera, respectively, because the number of somatostatin- and pancreatic polypeptide-positive cells was too small to be quantitated reliably.
Pregnant females were injected intraperitoneally with BrdUrd (100 mg/kg body weight) and 5-fluoro-2′-deoxyuridine (6.7 mg/kg body weight) at E18.5 and sacrificed 1 h later. Embryos were dissected, fixed, and analyzed by immunohistochemistry using anti-BrdUrd antibody as described (
). The number of BrdUrd-positive and total nuclei within a microscopic field was determined using the “cell counting” macro of the NIH Image 1.60 software. Ten sections from three different WT animals and eight sections from three differentIgf1−/−Igf2+/p−mice were analyzed.
Descriptive statistics and analysis of variance (ANOVA) were calculated using the Statsview software (Abacus Concepts, Inc., Berkeley, CA).
Effect of Null Insr and Igf1r Mutations on Fetal Pancreatic Development
To determine the consequences of ablation of INSR or IGF1R signaling on pancreatic development, we analyzed the morphology, size, and composition of pancreata in embryos lacking INSR, IGF1R, or both. Embryos were examined at embryonic day E18.5, corresponding to the developmental stage in which aggregation of endocrine cells into distinct islet structures was first recognizable (
). A representative immunohistochemical analysis for markers of endocrine (insulin) and exocrine (amylase) differentiation is shown in Fig.1, a andb. As we have shown in the past, mutations of the IGF signaling system result in embryonic growth retardation. Thus, the approximate weights of mutant embryos relative to normal wereInsr−/− 90% (
). The weight values were used to normalize pancreatic sizes, determined as described under “Experimental Procedures” and expressed in arbitrary units (Fig. 1c). This morphometric analysis indicated that the normalized total pancreatic size inInsr−/− embryos was similar to WT controls, whereas it was increased by ∼2-fold inIgf1r−/− and was decreased by ∼50% inInsr−/−Igf1r−/−embryos (Fig. 1c). Immunostaining with anti-amylase antiserum indicated that the decrease in pancreatic size observed in the double nullizygous embryos could be accounted for by a decrease of exocrine pancreatic size (Fig. 1b) (p < 0.05 by ANOVA). In contrast, α- and β-cell areas, as assessed by immunohistochemistry with anti-insulin and anti-glucagon antisera, were not significantly different from WT in Insr−/−and Igf1r−/− embryos (Fig. 1d). In double mutant embryos, however, relative α- and β-cell areas were increased ∼3-fold, as a result of the decreased area of exocrine pancreas (Fig. 1d). The α/β-cell area ratio within islets was unchanged in all embryos exceptIgf1r−/−, in which a 60% increase in the relative area of α-cells was detected (p < 0.05 by ANOVA). Notably, islet architecture appeared to be disrupted in doubleInsr−/−Igf1r−/−mutants, apparently as result of the overall impairment of exocrine pancreas development.
These analyses indicated that, in Insr−/−embryos, pancreatic acinar size is reduced proportionately to body weight (isometric change). However, inIgf1r−/− and double nullizygous embryos the relationship of pancreatic size to body size was altered in opposite directions (i.e. increased and decreased, respectively; allometric changes). To obtain an absolute measure of exocrine pancreatic size (i.e. independently of body size), we measured islet and acinar diameters in pancreata of each genotype. Mean acinar diameter was reduced by ∼40% inInsr−/−Igf1r−/−embryos, as compared with wild-type mice, with no changes in the two types of single mutants (Fig. 1e). In contrast, islet diameter was similar in all four genotypes (Fig. 1f). These data indicate that exocrine pancreas growth is selectively blunted inInsr−/−Igf1r−/−embryos.
Mice Lacking Both Igf1 and Igf2 Exhibit the Same Phenotype of Reduced Exocrine Pancreatic Growth
Because of the known interaction of IGF2 with both IGF1R and INSR, the decrease of pancreatic exocrine tissue in double mutantsInsr−/−Igf1r−/−, but not in single Insr−/− orIgf1r−/− mutant embryos, is consistent with the hypothesis that acinar tissue growth is mediated by the IGF2 ligand (
), we examined pancreatic development in E12.5 embryos. Interestingly, the change in pancreatic size was already apparent at this early stage, consistent with an IGF2-dependent effect (Fig.2).
For further verification, we examined embryos lacking both IGF1 and IGF2 ligands, and we showed that they are phenotypically indistinguishable in their histopathology fromInsr−/−Igf1r−/−mice. Thus, they display normal endocrine pancreas architecture but a decrease of exocrine pancreatic size (Fig.3a).
Because loss of IGF function results in reduced cell proliferation (
), we examined the proliferation potential ofIgf1−/−Igf2+/p−mice using BrdUrd labeling. The ratio of BrdUrd-positive cells to total number of cells scored inIgf1−/−Igf2+/p−mice was reduced by 35% (p < 0.04), consistent with previous observations (
Effects of IGF1R Haploinsufficiency on Mouse Growth
We next examined adult mice with heterozygous Igf1r mutations, either alone or in combination with Insr heterozygous mutations. Determination of body weights at different postnatal ages indicated that there was no statistically significant difference between WT and Insr+/− mutant mice or betweenIgf1r+/− and double heterozygousInsr+/−Igf1r+/− mutant mice. In agreement with the results of a detailed analysis of post-natal growth of Igf1r+/−mice,
E. Chiao and A. Efstratiadis, unpublished results.
we observed that the relative weights of mice carrying the heterozygous Igf1rmutation, alone or in combination with Insr heterozygosity, was ∼85% of WT (Fig. 4a). Accordingly, we conclude that there is growth haploinsufficiency of IGF1R function, which is not aggravated by concomitant heterozygosity for Insr. Haploinsufficiency results from a gene dosage effect, as evidenced by the decrease in the expression of IGF1R, at least as assayed in muscle extracts (Fig. 4b).
Effect of a Null Igf1r Mutation on Metabolic Control in Insulin-resistant Insr+/− Mice
To determine the metabolic effects of the combined Insr/Igf1r mutations, various parameters were analyzed in 6-month-old mice. Insulin levels were measured in the fasted and fed states (Fig.5, a andb). Igf1r+/− mice had normal insulin levels. In contrast, insulin levels inInsr+/− mice were 1.8- and 2.9-fold higher than in fasted and fed WT mice, respectively. CombinedInsr+/−Igf1r+/−mutations caused a further increase to 2.2- and 4.5-fold higher than WT (p < 0.05 by ANOVA versus WT andIg1r+/−).
To examine the effect of these mutations on pancreatic islets, we performed immunohistochemistry of pancreatic sections with anti-insulin and anti-glucagon antisera to analyze islet morphology and morphometry. Islets were moderately enlarged in Insr+/− andInsr+/−Igf1r+/− mice compared with WT mice. Insulin (Fig. 5h) and glucagon (not shown) immunoreactivities were unevenly distributed within islets, consistent with altered islet architecture, as typically seen in insulin-resistant states (
). Mean β-cell area in 4-week-oldIgf1r+/− mice was similar to WT mice. In contrast, Insr+/− andInsr+/−Igf1r+/−mice showed a 1.7- and 1.9-fold increase in β-cell area, respectively (p < 0.05 by ANOVA) (Fig. 5c). In addition, we observed an altered distribution of islets size, with an increase in the ratio of “large” islets to total islet number from 14% (WT) to 23 and 30% in Insr+/− andInsr+/−Igf1r+/− mice, respectively (p < 0.05 by ANOVA) (Fig. 5d). These results indicate that Igf1r haploinsufficiency is associated with normal β-cell area and that combined haploinsufficiency in Insr+/−Igf1r+/− mice does not result in significant alterations of β-cell area compared with single heterozygous Insr+/− mice.
Fasting glucose levels were maintained within a normal range in all mice (Fig. 5e). Measurements of glucose levels in the fed state indicated that ∼10% ofInsr+/−Igf1r+/− mice had developed diabetes (defined as an elevation of glucose values more than the mean + 2 S.D. on at least two separate measurements), as had 1 of 10 Insr+/− mice analyzed in this cross (Fig. 5f). These data are consistent with larger data sets reported in previous analyses (
). An intraperitoneal glucose tolerance test showed thatInsr+/− and Igf1r+/−mice had normal glucose tolerance. In contrast, double heterozygousInsr+/−Igf1r+/− mice had abnormal glucose tolerance, with a 2-h glucose value of 250 mg/ml (p < 0.05 by ANOVA versus WT andIgf1r+/− mice) (Fig. 5). However, intraperitoneal insulin tolerance tests failed to demonstrate a difference in insulin sensitivity among the four genotypes studied (data not shown), consistent with previous observations inInsr+/− mice (
). These results suggest that Igf1r haploinsufficiency does not cause a major deterioration of metabolic homeostasis inInsr+/− mice. These data stand in sharp contrast to those we reported previously (
) in mice with combined haploinsufficiency of Insr/Irs1 orInsr/Irs2.
We next examined the effect of the combinedInsr+/−Igf1r+/−mutations on insulin signaling in muscle, because muscle is the main site of insulin-dependent glucose disposal, and combined ablation of INSR and IGF1R by a dominant negative Igf1rtransgene has been shown to cause diabetes (
). To this end, we analyzed patterns of Igf1r expression and phosphorylation, as well as insulin-induced activation of PI 3-kinase in skeletal muscle extracts. Western blotting analysis revealed that Igf1r was expressed in skeletal muscle and that expression was reduced inIgf1r+/− mice compared with WT, as expected based on the presence of a null allele (Fig. 5b). Expression of Igf1r was not detected in liver extracts (not shown). In skeletal muscle extracts, both insulin and IGF1 induced tyrosine phosphorylation of Igf1r (data not shown). Insulin-stimulated PI 3-kinase activity was assessed in vivoin skeletal muscle from mice with various combinations of mutations.Igf1r+/− mice showed normal insulin-stimulated PI 3-kinase activity. In contrast, Insr+/− mice showed a ∼30% decrease andInsr+/−Igf1r+/− mice a 37% decrease (Fig. 6). However, the difference between Insr+/− mice andInsr+/−Igf1r+/− mice was not statistically significant.
The IGF System in Endocrine Pancreatic Development
The current consensus regarding development of endocrine islet cells is that they derive from Ngn3-positive precursors (
). Because islet cells are still present in embryos lacking IGF signaling, our data indicate that INSR and IGF1R are not required for embryonic islet development and proliferation. It remains to be seen whether they participate in postnatal β-cell growth (
), a heterotrimeric protein that directs exocrine pancreas-specific gene expression. Mice lacking the p48 subunit of PTF-1 die shortly after birth due to the complete absence of exocrine pancreatic tissue and display abnormal localization of endocrine cells to the spleen (
In contrast to the wealth of information available on the transcription factors required to initiate pancreatic differentiation, little is known about growth factors that mediate exocrine pancreatic proliferation (
). Several studies have shown that exocrine pancreatic development is crucially dependent upon mesenchymal/epithelial interactions, suggesting that locally produced growth factors play an essential role in this process (
Our developmental analysis indicates that exocrine pancreas growth is blunted in embryos with combinedInsr−/−Igf1r−/−mutations. Because this phenotype is not observed in singleInsr−/− or Igf1r−/−mutant embryos, it is unlikely to result from impaired insulin or IGF1 signaling, which activates only the cognate receptor. Moreover, the phenotype is already apparent in E12.5 embryos, a stage at which absence of IGF2, but not of IGF1 or insulin, has manifestations (
). Further studies will be required to examine mechanistic aspects of the role of IGF2 in exocrine pancreatic growth and to determine whether this factor is the long-sought and elusive promoter of acinar development (
The generation of mice with combined mutations of Insr and Igf1rhas enabled us to begin to address the role of IGF1R in metabolic control. Although conclusive evidence indicates that INSR plays a role in promoting embryonic growth (
) that IGF1 administration to Insr−/− mice decreases glucose levels, without affecting overall survival. We have suggested that this results from the ability of IGF1R to compensate for INSR in skeletal muscle but not in liver (
) in which a dominant negative igf1rtransgene, overexpressed in skeletal muscle, caused diabetes as a result of combined inhibition of INSR and IGF1R function, probably mediated by heterotetrameric receptors composed of an INSR monomer and an IGF1R monomer (
From our characterization, there does not appear to be further deterioration of insulin sensitivity inInsr+/−Igf1r+/− mice compared with Insr+/− mice. The lack of a marked synergism between the Insr and Igf1rmutations stands in sharp contrast to similar experiments in whichInsr+/− mice were crossed with mice bearingIrs1 and Irs2 mutations (
), who showed that simultaneous disruption of INSR and IGF1R in muscle is required to cause insulin-resistant diabetes. We suggest that the two receptors have the ability to compensate for each other, but neither is necessary for the metabolic response in muscle. These data should not be interpreted to suggest that muscle metabolism is unimportant for overall fuel homeostasis, because the phenotype of mice with a conditional Glut4 knockout in muscle shows that blocking glucose uptake causes severe insulin resistance (
In summary, we present an extensive analysis of the interactions between INSR and IGF1R in mice. The data reveal a novel role for IGF signaling in exocrine pancreatic development and indicate that IGF1R signaling plays an ancillary role to insulin signaling in adult mice.
We thank Youping Liu for skillful assistance with immunohistochemistry and members of the Accili and Efstratiadis laboratories for helpful discussions.