Pancreatic β-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in β-cell insulin receptor knockout (βIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, ∼7-mo-old βIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced β-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic βIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic βIRKOs and to a lesser degree in the nondiabetic βIRKO group. Pancreatic islets of nondiabetic βIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in β-cells leads primarily to profound defects in postnatal β-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.
- β-cell insulin receptor knockout
- insulin secretion
- insulin resistance
- glucose transporter 2
type 2 diabetes is characterized by the presence of insulin resistance and defects in pancreatic β-cell function (11, 27, 29). Although the molecular basis of the insulin resistance is not completely understood, defects in both the insulin receptor itself and the signaling pathways that are activated following binding of insulin to its receptor have been described (7, 11, 12, 32). The molecular basis of the defective insulin secretion is also complex and involves defects in the signal transduction mechanisms that are activated following exposure of pancreatic β-cells to increased concentrations of glucose and that are responsible for the production and secretion of insulin (8, 33, 35).
Recently, it has been demonstrated that insulin-dependent signaling pathways are present in the pancreatic β-cell that are activated following binding of insulin to its receptor (reviewed in Ref. 14). Disruption of these pathways in the β-cell is associated with defects in insulin secretion and/or growth (14). Although exposure of β-cells to increased concentrations of insulin has been shown to increase transcription of the insulin gene (25), other reports do not support this conclusion (26). Mice with a tissue-specific knockout of insulin receptors in β-cells (βIRKO) demonstrate defective insulin-secretory responses to glucose and progressive glucose intolerance (16); however, the role of the insulin receptor in pancreatic β-cell function is not fully understood. Freshly isolated islets from mice deficient in insulin receptor substrate-1 (IRS-1), a substrate of the insulin receptor, demonstrate reduced insulin-secretory responses to glucose and arginine and a decrease in insulin content of β-cells without a reduction in β-cell mass (18). Studies performed in single β-cells suggested that the autocrine-activated increase in intracellular Ca2+ concentration ([Ca2+]i) mediated by insulin was due to the release of intracellular Ca2+ stores especially from the endoplasmic reticulum and that these changes were mediated by IRS-1 and phosphatidylinositol (PI) 3-kinase (2), likely due to an interaction between IRS proteins and sarcoendoplasmic reticulum Ca2+ ATPase (5). Mice lacking IRS-2, another substrate of the insulin receptor, develop mild to severe diabetes, depending on the genetic background that is related, in part, to islet hypoplasia (13, 42, 44).
The present study was undertaken to further characterize the defects in β-cell growth and insulin secretion that result from disruption of the insulin-signaling pathways in pancreatic β-cells. We studied animals in which the insulin receptor gene had been specifically inactivated in β-cells by means of the Cre-loxP system (βIRKO mouse) and compared insulin-secretory function in these animals with controls (Lox/Lox) bearing a normal complement of insulin receptors on β-cells.
Animals. Experiments were performed using the previously described βIRKO mouse model, in which the animals lack insulin receptors specifically on pancreatic β-cells (16). Because the experimental animals were produced using the Cre-loxP system, both wild-type (WT) and Lox/Lox animals (6) were used as controls. All mice used in the present experiments were F1 female mice and their female littermates.
Assessment of glucose tolerance. Intraperitoneal glucose tolerance tests were performed following a 4-h fast. Blood was sampled from the tail vein before and 30, 60, and 120 min after injection of dextrose intraperitoneally (2 g/kg body wt).
Insulin secretion from in situ-perfused pancreas. The pancreas was perfused in situ in a humidified, temperature-controlled chamber by use of a modification of a previously described protocol (41). The perfusate, introduced through the aorta at the level of the celiac artery, consisted of oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 0.25% bovine serum albumin and a variable concentration of glucose. The perfusion system utilized three peristaltic pumps (Gilson Minipulse 2; Gilson, Middleton, WI), two of which were computer controlled, to allow insulin secretagogues to be administered while a constant total flow rate of 1 ml/min was maintained. In the perfusion experiments, before sample collection, the pancreas was perfused with KRB containing 5 mM glucose for a 30-min equilibration period. This was followed by a baseline sampling period during which insulin secretion in response to 5 mM glucose was measured for 20 min. The perfusate glucose was then increased to 20 mM for an additional 20 min. The perfusate glucose was then reduced to 5 mM between 40 and 60 min followed by 20 mM KCl in the continued presence of 5 mM glucose for a further 20 min. The experiment ended with a 20-min period during which the pancreas was again perfused with KRB containing 5 mM glucose. In the ramp experiments, before sample collection, the pancreas was perfused with KRB containing 2 mM glucose for a 40-min equilibration period. In additional experiments, after equilibration, the glucose concentration in the perfusate was increased gradually from 2 to 26 mM over a period of 100 min; i.e., the rate of glucose increase was 0.24 mM/min. At the end of this experiment, the pancreas was perfused with a solution containing 20 mM arginine in the continued presence of 26 mM glucose in KRB. Insulin concentrations (pmol/l) were measured in the effluent perfusate at 5-min intervals.
[Ca2+]i in pancreatic islets. Changes in [Ca2+]i were measured as previously described (40, 51) in the βIRKO, the Lox/Lox, and the WT mice in response to changes in the perifusate glucose concentration (a single-step increase from 2 to 14 mM glucose) or in response to exposure to 10 mM glyceraldehyde (GD) or 10 mM α-ketoisocaproic acid (KIC), respectively, for 10 min. After the administration of the secretagogues listed above (glucose, GD, and KIC), each islet was also exposed to 20 mM KCl for 10 min. Results are expressed as the ratio of the emitted light intensity (detected at 510 nm) following excitation at 340 and 380 nm (ratio 340/380). To compare the results in the different mouse groups, the area under curves (AUC) reflecting the ratio was measured after correction for the ratio of the baseline at 2 mM glucose.
To evaluate the adequacy of the intracellular stores of Ca2+, two protocols were used. In the first, the responses in [Ca2+]i to carbachol were evaluated, since it has been demonstrated that carbachol, a muscarinic antagonist, mobilizes Ca2+ from intracellular stores (9). The second protocol involved the administration of tolbutamide in the presence of 2 mM glucose followed by its administration in the presence of 20 mM glucose. When islets are exposed to tolbutamide in the presence of high glucose in this manner, there is a paradoxical decrease in [Ca2+]i if intracellular stores of Ca2+ fill normally (40).
Pancreatic insulin content. Pancreatic insulin content was measured after the whole pancreas was homogenized in 70% acid-ethanol (51). To allow the insulin content to be compared accurately in different groups, insulin levels were expressed as a function of pancreatic weight.
Measurement of NAD(P)H. Islet NAD(P)H was measured as previously described (37). To compare the results in the different mouse groups, the AUC reflecting the intensity of the NAD(P)H signal was measured after correction for the intensity of the baseline signal at 2 mM glucose.
Assay methods. Insulin concentrations were measured by a double-antibody radioimmunoassay using a rat insulin standard. The intra-assay coefficient of variation for this technique is 7%. All samples were assayed in duplicate.
Islet DNA content, islet insulin content, and quantitative morphometry. Islet DNA content was measured in aliquots of hand-picked islets from each mouse, as previously described (19). Islet insulin content was measured as described previously (51). β-Cell mass was calculated from sections obtained from paraffin-embedded pancreas and stained for non-β-cell hormones, as described previously (30).
Semiquantitative RT-PCR and Northern blotting. For measurement of steady-state mRNA levels, semiquantitative RT-PCR was performed with islet RNA (TRIzol, GIBCO-BRL) following the method of Wilson and Melton (43). Contaminating DNA was removed using 1 μl of RNase-free DNase-I (Boehringer) per 5 μg of RNA. cDNAs were synthesized and used as templates for PCRs by means of specific primers at annealing temperatures of 60-65°C in the presence of dNTPs and Taq polymerase. Typically, between 20 and 25 cycles were used for amplification in the linear range. Bands were quantitated using densitometry (Bio-Rad), and ratios of expression levels of knockout/control were calculated. Primer sequences are available on request.
Western blotting of islets. Islets were lysed in SDS buffer (2% SDS, 20 mM DTT, 1 mM EDTA, 50 mM Tris; pH 8.8), boiled, and separated by 14%-SDS PAGE, and proteins were transferred to nitrocellulose membranes. Insulin was detected using anti-insulin antibody (Linco), peroxidase-labeled secondary antibody (Sigma), and enhanced chemiluminescence. As a loading control, membranes were reprobed using anti-TATA box-binding protein antibody (gift of R. Roeder, New York, New York).
Statistical analysis. Results are expressed as means ± SE. In experiments involving multiple sampling times, appropriate summary measures were used for statistical comparison. Group means were compared using the Student's unpaired t-test. For data that were not normally distributed, a nonparametric test, the Mann-Whitney rank sum test, was used. Differences were considered to be significant at P < 0.05.
Mouse age, weight, and glucose tolerance. Mice were studied between the ages of 26 and 30 wk. On the basis of glucose tolerance tests the βIRKO mice were divided into two groups, diabetic and nondiabetic (based on blood glucose levels >11.1 mM 2 h after glucose injection). By analysis of variance, body weight was not different among the Lox/Lox (30.2 ± 1.2 g), the nondiabetic βIRKO (32.3 ± 1.4 g), and the diabetic βIRKO (29.4 ± 2.0 g) mice.
Glucose levels measured after the intraperitoneal administration of glucose (Fig. 1) demonstrated that the diabetic βIRKO mice had marked elevations in glucose levels in the fasting state (14.2 ± 2.5 mM), which further increased during the glucose tolerance test (27.4 ± 2.4 mM). In the nondiabetic βIRKO mice, fasting (6.7 ± 0.2 mM) and average glucose values (12.5 ± 0.6 mM) during the glucose tolerance test did not differ from those in the Lox/Lox mice (6.9 ± 0.2 and 12.4 ± 0.6 mM). Formal glucose tolerance testing was not performed in all diabetic βIRKO mice, but their random glucose values were 18.7 ± 2.2 mM, indicating the presence of diabetes. There was no significant difference in the WT mice compared with the Lox/Lox mice (data not shown).
Whole pancreatic insulin content, islet DNA content, islet insulin content, and β-cell mass. Because hyperglycemia is associated with degranulation of pancreatic β-cells, pancreatic mass and insulin content were measured. Pancreatic weight was similar in the Lox/Lox and the nondiabetic and diabetic βIRKO mice (358.6 ± 24.4, 344 ± 26.0, and 370.0 ± 35.6 mg). Pancreatic insulin content was reduced by ∼55% in the nondiabetic βIRKO compared with the Lox/Lox mice, although glucose tolerance was normal in these animals (P < 0.01; Table 1). Pancreatic insulin content was also reduced by ∼55% in the diabetic βIRKO mice compared with the Lox/Lox mice (P < 0.01; Table 1) but was not significantly different between the nondiabetic and the diabetic βIRKO mice. This was paralleled by a 40% decrease in islet DNA content in isolated islets. Islet DNA content was significantly greater in the Lox/Lox mice (0.97 ± 0.13 ng/islet) than in the diabetic βIRKO mice (0.58 ± 0.05 ng/islet, P < 0.05). Islet DNA content of the nondiabetic βIRKO mice (0.74 ± 0.03 ng/islet) did not differ from that of diabetic βIRKO mice. Because β-cells account for ∼80% of the cells in pancreatic islets, the decrease in islet DNA content suggests a decrease in β-cell number by ∼50%. Furthermore, islet insulin content, when expressed per nanogram of DNA, was lower in both the nondiabetic and diabetic βIRKO groups compared with the Lox/Lox controls (P < 0.05, Lox/Lox vs. diabetic or nondiabetic βIRKO, n = 325-350 islets in each group; Table 1). We also examined alterations in insulin at the protein level. Western blot analysis was performed using lysates of size-matched islets of control and nondiabetic and diabetic βIRKO mice. We observed no significant differences among the groups (Fig. 2). Together, this suggests that the decrease in insulin content is due to a reduction in β-cell mass and/or β-cell size. This was also confirmed by quantitative morphometric analysis of pancreata from 7- to 8-mo-old control (Lox/Lox), nondiabetic, and diabetic βIRKO mice that showed a reduced β-cell mass in the mutant animals (P < 0.05, Lox/Lox vs. diabetic or nondiabetic βIRKO, n = 3-6; Table 1) (30). A reduction in the number of islets appears to contribute to the reduction in β-cell mass in the βIRKO mice. The number of islets of size >50 μm was evaluated in at least eight serial sections (separated by 170 μm each) in control (n = 6), nondiabetic βIRKO (n = 5), and diabetic βIRKO (n = 3) mice. The total number of islets present in these sections was significantly reduced compared with control in both the nondiabetic (P < 0.02) and diabetic (P < 0.01) βIRKO mice (Table 1). The number of smaller islets (<50 μm) showed a trend toward a decrease in the mutant mice but did not reach statistical significance among the groups.
Insulin-secretory responses to glucose and KCl from in situ perfused pancreas. The effects of glucose and KCl on insulin secretion from the perfused pancreas are shown in Fig. 3A. Average pancreatic insulin secretion from the diabetic βIRKO mice was markedly reduced at 5 mM glucose (32.5 ± 9.7 pmol/l), and these animals demonstrated blunted insulin-secretory responses to 20 mM glucose (14.4 ± 4.5 pmol/l) by 99% reduction compared with the Lox/Lox mice (2,248.2 ± 618.8 pmol/l) and 20 mM KCl (256.6 ± 41.1 pmol/l) by 97% reduction compared with the Lox/Lox mice (9,385.7 ± 1,845.9 pmol/l). Average insulin secretion at 5 mM glucose in the nondiabetic βIRKO mice (604.0 ± 198.3 pmol/l) did not differ significantly from the values in the Lox/Lox mice (518.6 ± 75.7 pmol/l). After exposure to 20 mM glucose, there was a 58% reduction in the insulin-secretory response in the nondiabetic βIRKO animals (939.4 ± 567.1 pmol/l) compared with the Lox/Lox mice (P = 0.077 by the Mann-Whitney test) and a 50% decrease in insulin-secretory response to 20 mM KCl in the nondiabetic βIRKO mice (4,647.6 ± 841.7 pmol/l) compared with the Lox/Lox controls (P < 0.05). In the experiments in which the glucose concentrations in the perfusate were gradually increased from 2 to 26 mM glucose, average insulin secretion from the nondiabetic βIRKO mouse pancreata was significantly reduced compared with the amount of insulin secreted by pancreata from the Lox/Lox control mice during the 100-min duration of the experiment (P < 0.05; Fig. 3B). The response to 20 mM arginine in the presence of 26 mM glucose was also significantly reduced in the nondiabetic βIRKO mice (data not shown).
Secretion experiments using static incubation of islets. To evaluate the effects of exogenous GD (0, 1, and 10 mM) and KIC (0, 1, and 10 mM), we performed static incubation of size-matched islets isolated from control and diabetic and nondiabetic βIRKO mice by using protocols described earlier (18). We observed a dose-dependent increase (2- to 3-fold increase at 10 mM compared with unstimulated) in insulin secretion in response to both GD and KIC (Fig. 4, A and B). In contrast, a blunted secretion pattern was observed in the βIRKO mice, with a greater effect in the diabetic group.
Changes in islet [Ca2+]i. To determine whether the reduction in the insulin-secretory response to glucose was associated with reduced responses in [Ca2+]i, the 340/380 ratio AUC was measured in perifused islets from the nondiabetic βIRKO, the diabetic βIRKO, and the Lox/Lox mice during exposure to 14 mM glucose. We also used other secretagogues, including GD (10 mM), KIC (10 mM) and KCl (20 mM) to evaluate whether secretory pathways independent of glucose are altered. Representative [Ca2+]i responses following exposure to each secretagogue are shown in Fig. 5, A-I. The responses in [Ca2+]i tended to be lower in the islets from the nondiabetic βIRKO mice, but the differences were not statistically significant (14 mM glucose: P < 0.08, 10 mM GD: P < 0.09, 10 mM KIC: P < 0.10, 20 mM KCl: P < 0.37) from the Lox/Lox controls. On the other hand, the diabetic βIRKO mice exhibited significant reduction in the [Ca2+]i response to 14 mM glucose, 10 mM GD, 10 mM KIC, and 20 mM KCl. These results are summarized in Fig. 6.
Assessment of intracellular Ca2+ stores and NAD(P)H. The adequacy of the intracellular stores of Ca2+ was tested using the two protocols described in methods. The results did not demonstrate any evidence of a significant defect in intracellular Ca2+ stores in the nondiabetic βIRKO islets compared with the Lox/Lox controls. Changes in NAD(P)H autofluorescence were measured in response to an increase in glucose and the administration of the mitochondrial substrate KIC. NAD(P)H responses were similar in the WT mice and the nondiabetic βIRKO mice (data not shown) but were significantly reduced in the diabetic βIRKO mice compared with the Lox/Lox controls (P < 0.01 in 14 mM glucose, P < 0.05 in 10 mM KIC; Fig. 7, A-C).
Islet expression of GLUT2, glucokinase, and insulin. To evaluate whether the defect in insulin secretion is secondary to altered glucose-sensing proteins, we examined the expression of GLUT2 and glucokinase in islet RNA by semiquantitative RT-PCR (23). Significantly reduced levels of glucokinase and GLUT2 were evident in both the nondiabetic and diabetic βIRKO mice compared with the Lox/Lox controls (Fig. 8, B, C, and E). However, we did not observe significant differences in insulin mRNA between the nondiabetic or diabetic βIRKO and the control groups either by semiquantitative RT-PCR (Fig. 8, D and E) or by Northern blot analysis (Fig. 8F).
It has recently been demonstrated that mice that lack insulin receptors on pancreatic β-cells become diabetic due to defects in glucose-induced insulin secretion (16). Other data also suggest a role for insulin-mediated pathways in β-cell function (1, 10, 28, 39, 45-48, 50). The present studies were undertaken to further define the mechanisms underlying the potential defects in β-cell growth and insulin secretion in animals lacking β-cell insulin receptors. We demonstrate that mice lacking the insulin receptor in the pancreatic β-cells exhibit reduced pancreatic insulin content, islet insulin content per nanogram of DNA, and β-cell mass, reduced responses in insulin and [Ca2+]i to a variety of secretagogues, and reduced expression of GLUT2 and glucokinase. Although most defects are present in diabetic and nondiabetic βIRKO mice, alterations in [Ca2+]i were not statistically significant in the nondiabetic animals, and the reductions in insulin secretion were more severe in the diabetic animals. These results suggest that absence of the insulin receptor on the pancreatic β-cell is associated with a number of defects in pancreatic β-cell function. In contrast with nondiabetic βIRKO mice, diabetic mice also have severe defects in the [Ca2+]i-signaling pathways in the pancreatic β-cell, resulting in more marked reductions in insulin secretion in these animals. Interestingly, insulin mRNA and protein levels were not altered, indicating that changes in insulin content did not result from reduced expression of the insulin gene.
Hyperglycemia per se is associated with defects in the pancreatic β-cell due to exposure to high circulating levels of glucose (22, 33, 34). Our results in nondiabetic βIRKO mice allowed the effects of β-cell insulin receptor deficiency alone to be determined. Reduced insulin secretion in response to glucose and other secretagogues was observed in the nondiabetic βIRKO animals, although the defects were much less severe than in the diabetic group. Nondiabetic βIRKO mice also showed a significant reduction in pancreatic insulin content, islet insulin content per nanogram of DNA, and β-cell mass as well as reduced expression of GLUT2 and glucokinase. This indicates that defective β-cell function due to interruption of normal insulin signaling leads to alterations in mechanisms that maintain normal pancreatic insulin content and β-cell mass. The reduced expression of GLUT2 and glucokinase may also contribute to the abnormal glucose-stimulated insulin-secretory response. Furthermore, it is likely that genetic background (15) and diet modify these effects in the mice maintained on a C57Bl/6 × 129Sv × DBA/2 mixed background (Kulkarni RN, unpublished observations).
Insulin secretion from the isolated perfused pancreas was reduced by 58% to glucose and 50% to KCl in the nondiabetic βIRKO mice. Although significant, this defect in secretion was less severe than the secretory defect seen in the diabetic βIRKO animals, in which insulin secretion in response to glucose was reduced by 99% and by 97% in response to KCl. It is interesting that this 58% reduction in insulin secretion was still compatible with the maintenance of normal glucose tolerance in the nondiabetic βIRKO animals. Although pancreatic insulin content did not exhibit a significant difference between the nondiabetic and the diabetic βIRKO mice, insulin secretion in the diabetic group was much more reduced than in the nondiabetic cohort.
The reduction in pancreatic insulin content (55%) in the nondiabetic βIRKO animals was similar to the reduction in insulin secretion detected in the isolated perfused pancreas studies (58% to glucose). Similarly, we observed a decrease in the insulin content, when expressed per unit DNA, in islets isolated from both diabetic and nondiabetic βIRKOs. Because reduced insulin secretion could also be due to defects in the metabolic pathways in the pancreatic β-cell, we measured levels of [Ca2+]i and NAD(P)H after exposure to glucose and other secretagogues, since glucose metabolism in the β-cell leads to an increase in both [Ca2+]i and NAD(P)H. In the nondiabetic βIRKO mice, there was a tendency for reduced [Ca2+]i (14 mM glucose: 35%, P < 0.08; 10 mM GD: 40%, P < 0.09; 10 mM KIC: 40%, P < 0.10), whereas [Ca2+]i and NAD(P)H responses were significantly reduced in the diabetic βIRKO animals. These results suggest that insulin mediates its effects on the pancreatic β-cell via pathways that affect β-cell mass and consequently affect insulin content and secretion. Although the severe defects observed in the diabetic compared with the nondiabetic βIRKOs, particularly in responses in [Ca2+]i and insulin secretion, may have been exacerbated by the effects of hyperglycemia in the former group (22), these defects may also have played a primary pathogenic role in the development of diabetes in these animals.
Recent data from other groups have shown that exogenous addition of insulin affects transcription of the insulin gene via the insulin receptor/PI 3-kinase/p70 S6 kinase and calmodulin kinase pathways (25). Leibiger et al. (24) also reported that glucose-stimulated insulin biosynthesis depends on insulin-stimulated insulin gene transcription. In their report, treatment of β-cells with voltage-gated L-type Ca2+ channel blockers decreased glucose-stimulated proinsulin biosynthesis by ∼50% within 30 min. In the present study, although we did not examine the effects of addition of exogenous insulin on insulin gene expression in the βIRKO islets, we were unable to detect alterations in the basal levels of insulin gene expression or protein levels in the diabetic or nondiabetic knockouts. Previous studies have shown that insulin is a potent activator of general protein synthesis (21, 38) and can occur both in a c-Cbl-associating protein (CAP)-dependent and in a CAP-independent manner (3). Therefore, it is possible that the lower insulin content in βIRKO islets, without altered insulin gene expression, reflects a lack of insulin action due to absence of functional insulin receptors in the β-cells. Further detailed experiments are necessary to investigate the effects of insulin on phosphorylation of PHAS-1 (46) and other potential pathways as mechanisms of protein synthesis by translation in the βIRKO islets.
To evaluate whether the defect in glucose-stimulated insulin secretion in the βIRKO mice was secondary to defects in glucose sensing, we evaluated gene expression of glucose-sensing proteins. A modest but significant reduction in GLUT2 and glucokinase expression was evident in both the diabetic and nondiabetic βIRKO mice, suggesting that this may contribute to the reduced glucose-stimulated insulin secretion in the knockout mice. Indeed, a recent report indicates a role for insulin-mediated regulation of glucokinase activity in β-cells (39), pointing to one potential level of interaction between insulin-IGF-I and glucose-signaling pathways.
Aspinwall et al. (2) reported that, in dispersed single β-cells, the autocrine activation of insulin receptors by insulin increased [Ca2+]i due to release of intracellular Ca2+ stores, especially in the endoplasmic reticulum (ER), mediated by IRS-1 and PI 3-kinase. As we reported above in results, intracellular Ca2+ in isolated islets in the nondiabetic βIRKO mice tended to be reduced. One interpretation of these observations is that, in addition to the insulin receptor, IRS-1 can be activated by other receptors, including insulin-like growth factor I (IGF-I), growth hormone, and prolactin (4, 31, 36). In fact, recent data support a role for IGF-I signaling in modulating glucose-stimulated insulin secretion (17, 49). Therefore, it is possible that the tendency toward a reduction in [Ca2+]i, rather than an overt reduction, in the nondiabetic βIRKOs is due to potential alternative insulin receptor-signaling pathways that are functional and able to partially maintain [Ca2+]i in the mutant cells. Further studies in primary β-cells isolated from the βIRKO mice will be useful to define the potential downstream signaling pathways that may play a role in β-cell function.
In this study, we have shown that insulin-mediated signaling pathways in the pancreatic β-cell play a critical role in the maintenance of pancreatic insulin content and β-cell mass. In addition, gene expression defects, such as reduction of glucose-sensing genes, may contribute to the development of diabetes in mutant βIRKO mice. In nondiabetic βIRKO mice, insulin-secretory responses to glucose are reduced, but the decrease is sufficiently mild that blood glucose concentrations are normal. Intracellular Ca2+ responses to glucose and other secretagogues tended to be lower, but these differences were not statistically significant. In animals that develop diabetes, additional defects are present, including overtly abnormal responses in [Ca2+]i and NAD(P)H and a further reduction in the insulin-secretory response to glucose.
These novel observations add to the growing body of information indicating the importance of insulin-mediated signaling pathways in the maintenance of normal insulin secretion.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31842, DK-44840, DK-31036, a KO8 Clinical Scientist Award (DK-02885) to R. N. Kulkarni, and by the Diabetes Research and Training Center at Washington University School of Medicine (DK-20579).
We thank W. Pugh for outstanding technical assistance in the performance of the perfused pancreas experiments.
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.
↵* K. Otani and R. N. Kulkarni contributed equally to this study.
- Copyright © 2004 by American Physiological Society