Generation of Gipr–/– and Glp-1R–/–Gipr–/– mice.
We generated mice with inactivation of the Gipr gene by replacing exons 1–6 with a neomycin cassette (Figure a). Transmission of the mutant allele was confirmed by Southern blot analysis using a 3′ probe, external to the targeting vector (Figure b). We then intercrossed Gipr–/– mice with Glp-1R–/– mice to obtain Glp-1R–/–Gipr–/– (double-KO) mice. Controls were derived from littermates of double-KO mice and single-KO mice.
To confirm the absence of the hormone receptor from pancreatic islets of each set of mutants, we measured the insulin secretion from isolated islets incubated in the presence of either hormone. Figure c demonstrates that inactivation of each receptor led to suppression of the insulinotropic action of the respective hormone, and inactivation of both receptor genes abolished the effects of both hormones.
Double-KO mice were fertile and exhibited no gross behavioral abnormalities. Their body weight was similar to that of control mice, and their fed and fasted blood glucose concentrations were normal (Table ). The plasma insulin levels of fasted females tended to be lower in the KO mice as compared to controls, although the difference sometimes did not reach statistical significance (see Figures b and d). In males no statistically significant differences were observed in plasma insulin levels in the fasted state (Figure d).
Body weight, blood glucose, and plasma insulin levels in experimental mice 3–4 months old
Figure 2 Oral glucose tolerance tests (3 mg/g) in 3- to 4-month-old mice. (a and c) Glycemic excursions for (a) female WT (n = 11), Glp-1R–/– (n = 6), Gipr–/– (n = 9), and double-KO mice (n = 11) (more ...)
Figure 3 IPGTTs (1 mg/g). (a) Glycemic excursions in 3- to 4-month-old male WT (n = 9), Glp-1R–/– (n = 12), Gipr–/– (n = 19), and double-KO (n = 17) mice. Insets: quantification as AUC of the glycemic (more ...) Double-KO mice are intolerant to oral glucose.
To evaluate the impact of gluco-incretin receptor gene inactivation on disposal of a glucose load, we performed oral glucose tolerance tests. Female mice with a single-receptor gene inactivation showed glucose intolerance as revealed by a significant increase in the AUC of the glucose tolerance test (AUC glucose) (Figure a). Female mice with double-receptor KO displayed greater intolerance, with an AUC glucose approximately twice that of WT and 40–50% higher than in single-KO mice (Figure a). Glucose intolerance in single-KO mice was associated with a 34–43% lower plasma insulin level measured at 15 minutes after glucose injection compared to WT control mice (Figure b). Double-KO mice exhibited an even lower insulin response (–57% vs. WT; –24 to –34% vs. single-KO mice) (Figure b).
In male mice, glucose intolerance was less marked, with AUC glucose for double-KO mice increased by 17–40% as compared to single-KO mice, and by 50% as compared to WT mice (Figure c). The corresponding insulin responses were significantly blunted, with a 60% reduction as compared to WT mice and a 40% reduction as compared to the GLP-1 receptor KO mice (Figure d). These data indicate an additive insulinotropic action of GIP and GLP-1 during an oral glucose load. The incretin effect of each hormone seemed to be of similar magnitude in females when comparing the AUC glucose and AUC insulin in single-receptor KO mice. In light of these criteria, the incretin effect of GIP was more important than that of GLP-1 in male mice.
Intraperitoneal glucose intolerance and impaired first-phase insulin secretion in double-KO mice.
Oral, but not intraperitoneal, glucose administration induces the secretion of gluco-incretin hormones. To evaluate whether single or combined suppression of the GIP and GLP-1 receptors also led to altered glucose tolerance and insulin secretion when secretion of these hormones was not stimulated, we performed intraperitoneal glucose tolerance tests (IPGTTs). Male mice with single- and double-receptor gene KO showed normal basal glycemia as well as normal glucose tolerance after intraperitoneal glucose injection, at both 14–16 and 40 weeks of age (Figure , a and b). Thus glucose tolerance was not impaired in male KO mice.
In 14- to 16-week-old single- and double-KO female mice, basal glycemia was normal (Figure c). However, single-KO mice had greater glycemic excursions after intraperitoneal glucose injection (AUC glucose 40–45% higher than in WT mice), and double-KO mice had still higher AUC glucose, approximately twice that of WT and 40% higher than single-KO mice (Figure c).
Glucose intolerance in single- and double-KO female mice was associated with changes in plasma insulin concentration (Figure d). Remarkably, the 2-minute plasma insulin peak, which represents first-phase insulin secretion, was totally absent in the double-KO mice as well as in mice lacking the GLP-1 receptor, but was present in the Gipr–/– mice (Figure d). The 30-minute peak insulin levels were significantly reduced in the single-KO mice, but the reduction was not statistically significant in the double-KO mice (Figure d).
To evaluate whether glucose intolerance could be associated with peripheral insulin resistance, we performed hyperinsulinemic euglycemic clamp experiments in double-KO and WT female mice. Insulin was infused at either high (18 U/kg/min) or intermediate (6 U/kg/min) rates. In both conditions, double-KO mice had normal insulin sensitivity compared to WT mice, as revealed by identical glucose infusion rates to maintain euglycemia (84.1 ± 6.4 vs. 75.5 ± 5.4 mg/kg/min in double-KO and control mice, respectively, at high insulin infusion rate, and 60.5 ± 3.4 vs. 60.1 ± 6.0 mg/kg/min, in double-KO and control mice, respectively, at intermediate insulin infusion rate; n = 5–6).
Analysis of insulin secretion in the perfused pancreas.
To determine the insulin-secretory capacity of the endocrine pancreas of the mutant mice, we first performed pancreas perfusion experiments. In our setting, the pancreas is still connected to the duodenum and has normal connections to the local autonomic nervous system. Moreover, the hepatoportal glucose sensor should be exposed to the perfusate, which is introduced via the aorta (30
). Glucose-induced insulin secretion in perfused pancreata from control female mice showed a strong first-phase secretion (Figure , a and d). Perfusion of pancreata from GIP-receptor KO mice showed a first phase of secretion that was not significantly different from that of control pancreas (Figure , a and d). In contrast, as compared to WT pancreas, Glp-1R–/–
and double-KO pancreas perfusions revealed a markedly blunted first phase of insulin secretion (50 ± 7 vs. 17 ± 7 and 11 ± 3 in control vs. Glp-1R–/–
and double-KO pancreata, respectively, Figure , b–d). The second phase of insulin secretion was significantly decreased in single-KO but not in double-KO pancreata, similar to the observation on second-phase secretion in IPGTTs. Notably, those defects occurred despite normal pancreatic insulin contents in single- and double-KO mice (Table ). The excellent correlation between these results and those from the IPGTT indicate that the factors responsible for the loss of first-phase insulin secretion in the double-KO and GLP-1 receptor-KO mice remain in the perfused-pancreas setup.
Figure 4 Glucose-induced insulin secretion in perfused pancreata from (a) Gipr–/– (n = 4), (b) Glp-1R–/– (n = 4), and (c) double-KO (n = 4) mice compared to WT mice (n = 6). Pancreata were perfused (more ...) Double-KO pancreatic islets exhibit impaired insulin secretion.
We next evaluated the kinetics of insulin secretion in islet-perifusion experiments. Figure a shows insulin secretion from control and single-KO islets when glucose concentration was raised to 11.1 or 16.7 mM. The kinetics of secretion in single-KO islets was indistinguishable from that of control islets, with a very rapid onset of secretion at high glucose concentration and suppression of secretion when glucose levels returned to basal values. Although there was no clear separation of first- and second-phase insulin secretion in the present experiments, as is usually the case in mouse islets (33
), the initial rapid secretory response over the first 5 minutes of high-glucose perfusion is considered to represent the first phase of insulin secretion. The total amount of insulin released from single-KO islets was indistinguishable from those of control islets at 11.1 or 16.7 mM glucose (Figure c). Moreover, as shown in Figure d, the ratio of insulin to DNA content of the different types of islets was identical.
Figure 5 Insulin secretion in perifused islets from (a) Glp-1R–/–, Gipr–/–, and (b) double-KO mice compared with WT mice. (c) Quantification as AUC of insulin responses shown in a and b. n = 4. Data are expressed as means (more ...)
Perifusion of double-KO islets (Figure b) revealed a pattern of insulin secretion characterized by a rapid initiation of secretion over the first 5 minutes of high-glucose perifusion, suggesting a normal first phase of insulin release. However, this secretion was of reduced amplitude, and the total amount of secreted insulin from the double-KO islets was markedly reduced (Figure c) despite a normal islet insulin/DNA ratio (Figure d).
To evaluate whether the secretory defect in double-KO islets occurred upstream or downstream of the depolarization event, we stimulated insulin secretion by K+-induced depolarization. Figure e shows a markedly reduced secretory response to K+ in double-KO mice, strongly suggesting that there is a major defect distal to plasma membrane depolarization.
The above data thus demonstrate that in the absence of a single hormone receptor, first-phase and total insulin secretion were normal. In the absence of both receptors, onset of secretion was kinetically normal but total secretion was markedly reduced and associated with a defect in glucose signaling at a step distal to plasma membrane depolarization. This defect could result from reduced intracellular cAMP levels, to a defect in the insulin-secretory pathway as a consequence of alterations in the expression of genes controlling exocytosis of insulin granules, or both.
To distinguish between these alternatives, we first tested whether the secretory defect was observed when the perifusion experiments were performed in the presence or absence of IBMX combined or not combined with forskolin. As shown in Figure a, the secretory response to 16.7 mM glucose was similarly reduced in double-KO islets whether IBMX was included in the perifusate or not. Moreover, the addition of forskolin in the presence of IBMX strongly stimulated insulin secretion in response to 16.7 mM glucose, and the insulin secretion rate was similar in double-KO and control islets (Figure b). In Figure , c and d, in static incubations, we measured insulin secretion from control and double-KO islets and their intracellular cAMP levels. The results show that secretion from double-KO islets incubated with or without IBMX was markedly reduced as compared to secretion from control islets. Furthermore, addition of forskolin to the incubation medium strongly increased secretion in both control and double-KO islets, and secretion was quantitatively identical from both types of islets. These results are similar to those obtained in perifusion experiments (see Figure , a and b). The intracellular cAMP levels from islets incubated in the presence of 16.7 mM glucose and IBMX were identical in control and double-KO islets (Figure d), suggesting that the difference in cAMP levels cannot explain the reduced secretory activity in the mutant islets. In the presence of forskolin and IBMX, cAMP levels were strongly elevated in both types of islets (Figure d), conditions in which insulin secretion was normalized in double-KO islets (Figure c). Together, these data suggest that impaired insulin secretion in double-KO islets was not due to lower cAMP levels but rather to a reduced sensitivity to this second messenger.
Figure 6 cAMP, carbachol, and insulin secretion. (a) IBMX and insulin secretion. Islets were perifused successively with 2.8 mM glucose for 10 minutes, 16.7 mM glucose for 10 minutes, 2.8 mM glucose for 20 minutes, first in the absence and then in the presence (more ...)
To evaluate whether the secretory defect was restricted to cAMP-controlled mechanisms, we performed islet-perifusion experiments in the presence or absence of carbachol. As shown in Figure e, the secretory defect observed in double-KO islets perifused with high glucose concentrations was suppressed when the islets were perifused in the presence of carbachol, an activator of the G-coupled muscarinic receptors.
Despite their secretory defect, double-KO islets exhibited no obvious abnormality in insulin content (Table ), size range, gross morphology, and distribution of insulin- and glucagon-producing cells (data not shown). Hence, double-KO islets have a β cell autonomous defect in stimulated insulin secretion that is probably due to the absence of both GLP-1- and GIP-receptor signaling. However, strikingly, first-phase insulin secretion was not suppressed in GLP-1 receptor KO islets and only moderately in double-KO islets. This suggests that the blunted first-phase insulin secretion obtained via IPGTT and in the perfused pancreas may reflect an integrative response, including glucose sensors located outside the endocrine pancreas.
The first phase of insulin secretion is inhibited when the hepatoportal sensor is inactivated.
We and others previously demonstrated that a glucose sensor was located in the hepatoportal vein region. This sensor controls insulin secretion, through autonomic nervous connections (34
). Using different mouse models, we demonstrated that this sensor required GLUT2 gene expression and the presence of an activated GLP-1 receptor (20
) for normal function. Our present data suggest that this sensor could be involved in the control of first-phase insulin secretion, since GLP-1 receptor KO mice lack first-phase secretion during an IPGTT and in perfused pancreata but not in perifused isolated islets.
Thus, to test the involvement of the hepatoportal sensor in the control of first-phase insulin secretion, we performed two sets of experiments. First, we measured insulin secretion in RipGlut1Glut2–/–
mice. These mice have a nonfunctional hepatoportal sensor due to GLUT2 gene inactivation; however, they have normal biphasic glucose-stimulated insulin secretion, as assessed in islet-perifusion experiments, due to the transgenic expression of a GLUT1 transporter in their β cells (33
). Figure a shows that the 2-minute peak of plasma insulin normally induced by an intraperitoneal glucose injection was absent from RipGlut1Glut2–/–
mice. Similarly, when evaluated in the perfused pancreas, this first peak of insulin secretion was almost completely blunted (Figure b).
Figure 7 Glucose-induced insulin secretion in RipGlut1Glut2–/– female mice. (a) IPGTTs (1 mg/g) in RipGlut1Glut2–/– compared to C57BL/6J WT mice (n = 7). §P < 0.005. (b) Glucose-induced insulin secretion (more ...)
In a second set of experiments, we inactivated the hepatoportal glucose sensor in control mice by portal vein infusion of the GLP-1 receptor antagonist exendin-(9–39). The perfusion rate was chosen so as to block the function of the hepatoportal sensor without interfering with blood glucose levels (0.5 pmol/kg/min), as previously described (21
). Thirty minutes after initiation of the perfusion experiments, mice were injected intraperitoneally with glucose. Figure c shows the glycemic profiles over the time course of the experiments, and Figure d shows the plasma insulin levels measured at time 0, at the initiation of peptide or saline infusion (time 60 minutes), and 2 and 15 minutes (time 92 and 105 minutes) after the injection of glucose. The basal insulinemic levels (0 and 60 minutes) were similar in the saline- and antagonist-perfused mice. The first peak of insulin secretion (2 minutes after glucose injection, time 92 minutes) reached 75 ± 11 μU/ml for the saline-infused mice and 30 ± 2 μU/ml for the exendin-(9–39)–infused mice. Fifteen minutes after injection (time 105 minutes), the plasma insulin concentrations were reduced to the same level in both groups. Thus, the above data demonstrate that inhibition of the hepatoportal glucose sensor by GLUT2 gene inactivation or exendin-(9–39) portal infusion dramatically reduced first peak insulin secretion after intraperitoneal glucose injection.