Role of Endogenous Glucagon-Like Peptide-1 in Islet Regeneration After Partial Pancreatectomy

For the antagonist experiment, 8- to 9-week-old male BALB/c mice were obtained from Charles River Laboratories, housed under standard conditions, and allowed free access to standard mouse food and water except for the times specified. At 1 day before surgery, Alzet mini-osmotic pumps (model 2002; Alza, Palo Alto, CA) were implanted subcutaneously to deliver Ex9-39 (Bachem Bioscience, King of Prussia, PA) at a rate of 50 pmol · kg⁻¹ · min⁻¹. The pumps were removed at the end of 2 weeks to clearly demarcate the termination of treatment. GLP-1R^−/− mice were also housed and fed under standard conditions. These mice have been previously described in detail (12). These studies were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Mice were anesthetized by intraperitoneal administration of a mixture of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The abdomen was opened through an upper midline incision. The spleen and the entire splenic portion of the pancreas were surgically removed, but the mesenteric pancreas between the portal vein and duodenum was left intact. This resulted in a ∼70% pancreatectomy, confirmed by weighing the removed and remnant portions during a pilot study. Sham operation was performed by removing the spleen while leaving the pancreas intact. The incision was closed using 5-0 silk sutures.

Random blood glucose was measured weekly using a handheld glucometer (FreeStyle; TheraSense, Alameda, CA). Intraperitoneal glucose tolerance testing was carried out at 2 and 4 weeks after surgery. Mice were injected intraperitoneally with 2 g/kg of glucose after a 12- to 16-h fast. Blood was sequentially sampled from the tail vein and analyzed using a GL5 glucose analyzer (Analox Instruments, Lunenburg, MA).

β-Cell mass was determined 5 weeks after Ppx as previously described (4). A section through the maximal footprint region was stained with either guinea pig anti-human insulin (1:5,000; Linco Research, St. Charles, MO) or a non–β-cell hormone cocktail (rabbit anti-somatostatin 1:5,000 from Peninsula Laboratories, San Carlos, CA; rabbit anti-glucagon 1:5,000 from Biodesign International, Kennebunk, ME; and rabbit anti-pancreatic polypeptide 1:15,000 from Linco Research). Secondary antisera were biotinylated anti–guinea pig and anti-rabbit (1:200; Vector Laboratories, Burlingame, CA). Color development was with diamino benzidine (DAB; Vector Laboratories). Sections were evaluated using a Nikon E600 microscope attached to a Nikon Coolpix 995 digital camera with direct video output to a Sony Trinitron video monitor. Using a 9×10 grid of points, the percentage of points overlying β-cells was determined and then multiplied by the weight of the pancreas to determine the mass of β-cells. At least 200 random fields were assessed for each pancreas.

Values of blood glucose and β-cell mass represent the means ± SE. Fasting blood glucose levels and β-cell mass values were compared using standard t tests. Glucose tolerance test results were compared using repeated-measures ANOVA. If ANOVA detected a significant effect, post hoc analysis was carried out by Fisher’s protected least-significant difference (PLSD) (StatView, 3rd edition; SAS Institute, Cary, NC). Differences were accepted as significant at P < 0.05.

In our first approach to establish the role of endogenous GLP-1 in pancreatic regeneration after Ppx, we administered the GLP-1 receptor antagonist Ex9-39 for 2 weeks after surgery. Once-daily injections of Ex9-39 at 1 nmol · kg body wt⁻¹ · day⁻¹ i.p. had no effect on glucose tolerance after Ppx in mice (data not shown). Therefore, Ex9-39 was administered continuously via a mini-osmotic pump. The optimal dose of Ex9-39 was determined in a pilot study. Based on acute infusion studies in humans and primates (20–22), we investigated the ability of two doses of Ex9-39 to antagonize the incretin effect of endogenous GLP-1. Pumps delivering a low dose (4 pmol · kg⁻¹ · min⁻¹, n = 2) or high dose (30 pmol · kg⁻¹ · min⁻¹, n = 2) of Ex9-39 were implanted subcutaneously in 7- to 8-week-old male BALB/c mice. Oral glucose tolerance was evaluated 24 h after pump implantation. Controls were untreated (n = 3). Mice were fasted for 12 h and given 2 g/kg glucose orally by gavage using a feeding needle. Mice receiving 30 pmol · kg⁻¹ · min⁻¹ had significantly impaired glucose tolerance when compared with control mice (P < 0.05) (data not shown).

Based on the results of the pilot dose-finder study, we used a slightly higher dose of 50 pmol · kg⁻¹ · min⁻¹ delivered continuously for 2 weeks for the pancreatic regeneration studies. Twenty 8- to 9-week-old male BALB/c mice were divided into four groups: Sham (n = 5), Sham + Ex9-39 (n = 5), Ppx (n = 5), and Ppx + Ex9-39 (n = 5). Surgeries were performed 24 h after pump implantation (Fig. 1). At 2 weeks after surgery and just before pump removal, fasting blood glucose levels were significantly higher in Ppx mice compared with Sham mice (5.2 ± 0.4 vs. 3.4 ± 0.3 mmol/l [93.9 ± 8.1 vs. 61.8 ± 4.7 mg/dl], P = 0.01). The efficacy of Ex9-39 administration was evident in the Sham + Ex9-39 group, which had significantly higher fasting blood glucose compared with the untreated Sham group (4.8 ± 0.1 vs. 3.4 ± 0.3 mmol/l [87.2 ± 1.8 vs. 61.8 ± 4.7 mg/dl], P = 0.004) (Fig. 2A). This group had also impaired glucose tolerance when compared with untreated Sham (repeated-measures ANOVA: P = 0.05; Fisher’s PLSD: P = 0.001 [0 min] and P = 0.04 [30 min]). Intraperitoneal glucose tolerance was significantly impaired in Ppx mice compared with the Sham mice, (repeated measures ANOVA: P = 0.004; Fisher’s PLSD: P = 0.009 [0 min, 30 min], P = 0.004 [60 min], P = 0.02 [90 min], and P = 0.002 [120 min]); however, Ex9-39 did not further exacerbate fasting hyperglycemia or glucose tolerance in the Ppx animals (Fig. 2B).

At 1 week after the removal of the pumps, the effect on fasting glucose levels in Sham mice treated with Ex9-39 persisted. Fasting blood glucose levels in these mice were significantly higher than in those of untreated Sham mice (6.3 ± 0.2 vs. 4.7 ± 0.1 mmol/l [113.9 ± 2.9 vs. 84.6 ± 1.7 mg/dl], P = 0.0001) (Table 1). Similarly, Ppx mice continued to exhibit significantly higher fasting blood glucose levels compared with the Sham group (7.1 ± 0.3 vs. 4.7 ± 0.1 mmol/l [127.9 ± 5.8 vs. 84.6 ± 1.7 mg/dl], P = 0.001) (Table 1). At 5 weeks after surgery (3 weeks after pump removal), fasting blood glucose levels were not different in Ppx compared with Sham animals (4.7 ± 0.3 vs. 4.2 ± 0.2 mmol/l [84.7 ± 4.7 vs. 75.2 ± 3.5 mg/dl]), but Sham + Ex9-39 mice continued to exhibit higher fasting blood glucose levels than those of untreated Sham mice (4.9 ± 0.2 vs. 4.2 ± 0.2 mmol/l [88.2 ± 4.0 vs. 75.2 ± 3.5 mg/dl], P = 0.04) (Fig. 3A). At this time, intraperitoneal glucose tolerance was impaired in Ppx mice when compared with Sham controls (repeated measures ANOVA: P = 0.05; Fisher’s PLSD: P = 0.02 [15 min], P = 0.01 [30 min], and P = 0.007 [60 min]), but treatment with Ex9-39 during the first 2 weeks after surgery did not further exacerbate this glucose intolerance (Fig. 3B).

These results correlate with the results of β-cell mass determination. At 5 weeks after 70% Ppx, β-cell mass in Ppx mice was 50% of the β-cell mass in the Sham controls (0.9 ± 0.4 vs. 1.8 ± 0.2 mg, P = 0.01). Similarly, the weight of the remnant pancreas was 50% of the Sham pancreas weight (138 ± 13.6 vs. 272 ± 13.2 mg, P = 0.0001). Ex9-39 therapy during the first 2 weeks after surgery did not affect β-cell mass in the Ppx (0.7 ± 0.1 vs. 0.9 ± 0.4 mg, Ppx + Ex9-39 vs. Ppx, respectively) or Sham (1.8 ± 0.2 vs. 1.8 ± 0.2 mg, Sham + Ex9-39 vs. Sham, respectively) groups (Fig. 4). Overall, these data indicate that doses of Ex9-39 sufficient to antagonize the incretin effect of endogenous GLP-1 do not antagonize islet mass regeneration in BALB/c mice. Furthermore, the prolonged action of Ex9-39 in Sham mice indicates a long-lasting effect on glucose homeostasis.

The second model we used to address the hypothesis was the GLP-1R^−/− mouse (12). These mice are bred on a CD1 genetic background. Controls were age-matched wild-type CD1 mice. Thirty-six 7- to 9-week-old male mice were divided into four groups: wild-type CD1 Sham (WT Sham; n = 9), wild-type CD1 Ppx (WT Ppx; n = 8), GLP-1R^−/− Sham (KO Sham; n = 9), and GLP-1R^−/− Ppx (KO Ppx; n = 10). At 2 weeks after 70% Ppx, glucose tolerance was impaired to a greater degree in the KO Ppx group as compared with the WT CD1 Ppx group (repeated measures ANOVA: P = 0.007; Fisher’s PLSD: P = 0.0009 [30 min, 60 min] and P = 0.02 [120 min]). WT Ppx mice had a tendency toward higher blood glucose levels after an intraperitoneal glucose tolerance test (IPGTT) when compared with WT Sham mice, but this was not statistically significant. Similarly, KO Ppx mice had a nonsignificant tendency to higher blood glucose levels at 2 weeks compared with KO Sham mice (Fig. 5A).

At 4 weeks after surgery, WT Ppx mice again showed a nonsignificant tendency to higher blood glucose levels compared with WT Sham animals. In contrast, the KO Ppx mice showed a significant worsening in glucose tolerance compared with KO Sham mice (repeated measures ANOVA: P = 0.0008; Fisher’s PLSD: P = 0.002 [15 min], P = 0.007 [30 min], P = 0.0003 [60 min], and P = 0.002 [120 min]) (Fig. 5B). These observations were also reflected in the results of β-cell mass determination. At 5 weeks after 70% Ppx, β-cell mass in the WT Ppx mice was not different from the WT Sham controls (0.4 ± 0.1 vs. 0.4 ± 0.1 mg, P = NS), indicating a full recovery of β-cell mass (Fig. 6). In contrast, β-cell mass in the KO Ppx mice was 40% of the KO Sham β-cell mass (0.3 ± 0.1 vs. 0.7 ± 0.2 mg, P = 0.05) (Fig. 6). Despite this marked difference in β-cell mass recovery, the pancreas remnant weight was similar for WT Ppx (50% of Sham) and KO Ppx (45% of Sham) mice. These data indicate that, in contrast to BALB/c mice, CD1 mice have a robust regeneration of β-cell mass after Ppx and that GLP-1 does play a role in adaptive islet regeneration after Ppx in this background strain.

Contrasting results were obtained in the two models used to address the role of endogenous GLP-1 in islet mass regeneration after Ppx. The GLP-1 receptor antagonist Ex9-39 effectively altered glucose homeostasis in Sham BALB/c mice, whereas in Ppx mice glucose tolerance was not exacerbated beyond the effect of surgery. The continuous infusion of Ex9-39 after Ppx did not impair β-cell mass regeneration, nor did it impact β-cell mass in Sham mice, despite a long-lasting functional effect in Sham mice. In contrast, β-cell mass regeneration in GLP1R^−/− mice was markedly impaired.

The effectiveness of Ex9-39 antagonism of endogenous GLP-1 in our experiment was proven by the hyperglycemic effect in the sham-operated animals. GLP-1 levels during therapy were not measured, but previous studies in baboons have shown that plasma GLP-1 levels during treatment with Ex9-39 do not change, despite the effect on glucose levels (20). Interestingly, the elevation of fasting glucose levels in these mice persisted well beyond the termination of treatment, in the absence of any alteration in β-cell mass. A similar “memory” effect has been observed during a 48-h GLP-1 infusion that resulted in a maximal effect on insulin secretion at 1 week. This was attributed to neogenesis, and therefore an increase in β-cell mass, although indexes of β-cell neogenesis and mass were not quantitated at the 1-week time point in this study (23). We hypothesize that the long-term functional effect of GLP-1 receptor antagonism in our study could result from underlying changes in islet gene expression. Additional studies will be required to demonstrate that islet function remains impaired after chronic Ex9-39 treatment. Furthermore, the prolonged action of Ex9-39 to impair glucose homeostasis even in the presence of normal β-cell mass suggests that Ex9-39 may have clinical application in the treatment of hypoglycemic disorders.

The pharmacology of Ex9-39 appears to vary in pancreatic and peripheral tissues. Ex9-39 was created by the deletion of eight NH₂-terminal amino acids from Ex, a potent GLP-1 agonist isolated from the venom of Heloderma suspectum (24,25). Ex9-39 maintains high-affinity binding to the GLP-1 receptor (26). In β-cells, Ex9-39 dose-dependently decreases basal intracellular levels of cAMP. On this basis, Ex9-39 is considered to be an inverse agonist of the GLP-1 receptor (26). In contrast, in some studies Ex9-39 does not antagonize GLP-1 action or may even behave as an agonist (27–30). These observations raise the possibility that although Ex9-39 effectively antagonized the incretin effect of endogenous GLP-1, the growth-promoting effect was not antagonized. It is also possible that the processes of proliferation and neogenesis involved in the regeneration of β-cell mass after Ppx were reactivated in the mice after the termination of Ex9-39 treatment, allowing for a normal recovery of β-cell mass by the end of 5 weeks. We do not favor this explanation because of the previously reported kinetics of β-cell mass regeneration after Ppx (31).

In contrast to the results of the Ex9-39 experiment, in the GLP1R^−/− model, Ppx resulted in a progressive deterioration in glucose metabolism, and β-cell mass regeneration was severely affected. When compared with wild-type CD1 mice, in which the β-cell mass of Ppx mice fully recovered by 5 weeks, GLP-1R^−/− Ppx mice had only 40% of sham mass at 5 weeks after Ppx. The significant impairment in β-cell mass regeneration in GLP-1R^−/− mice suggests a role for the continuous action of endogenous GLP-1 during the process of regeneration after pancreatectomy.

Several considerations in the interpretation of these results include: 1) the role of background strain, 2) the potential role of hyperglycemia in the regenerative response, and 3) the potential adaptive compensations in genetically modified mice. The background strain of the GLP-1R^−/− mice is CD1, an outbred strain that develops mild age-related alterations in glucose metabolism (D.D.D.L. and D.A.S., unpublished observations). To avoid this complication, we used younger mice that maintain normal glucose homeostasis. Nevertheless, strain-dependent variations in β-cell function and compensation in the face of excess demand (e.g., insulin resistance) are well recognized (32). Further evidence for a role of background strain derives from the marked variation in β-cell mass recovery in BALB/c versus CD1 control mice in this study. The response to a disruption in GLP-1 receptor signaling could also be strain dependent.

Fasting glycemia and glucose tolerance are mildly impaired in GLP1R^−/− mice (12). Glucose appears to be a potent stimulus of pancreatic β-cell growth in vivo and in vitro (32). High glucose levels may also lead to a reduction in β-cell proliferation and an increase in β-cell death (33,34). These studies have led to the concept that glucose toxicity may affect β-cell mass homeostasis when hyperglycemia is of high level and long duration. In the animal models examined in this study, however, the level of hyperglycemia was mild, suggesting that glucose toxicity is not the explanation for the defect in β-cell mass regeneration in GLP1R^−/− mice.

Adaptive changes in genetically modified mice can also influence metabolic phenotypes. In GLP-1R^−/− mice, enhanced glucose-dependent insulinotropic polypeptide (GIP) secretion and action has been reported, in part explaining the mild phenotype of these mice (35). Like GLP-1, GIP also appears to be a growth factor for insulin-producing β-cells (36). The upregulation of GIP (as well as the mild hyperglycemia) may explain the tendency toward increased β-cell mass in sham-operated GLP-1R^−/− mice. After Ppx, however, enhanced GIP action did not ameliorate the impairment in β-cell regeneration.

In summary, we found discordant results in the two models used in this study. Although a limited period of GLP-1 receptor antagonism after Ppx did not affect β-cell mass regeneration in BALB/c mice, CD1 mice harboring a null mutation of the GLP-1 receptor had a significant defect in β-cell mass regeneration after Ppx. The data point to a significant influence of genetic background in β-cell growth. Taken together, these studies demonstrate that endogenous GLP-1 plays a significant role in the adaptive regeneration of β-cell mass after Ppx.

This study was supported by an American Diabetes Association Career Development Award, National Institutes of Health Grant DK49210 (to D.A.S.), National Institutes of Health Training Grant 5T32DK07314, an Eli Lilly-Lawson Wilkins Pediatric Endocrinology Society Training Grant (to D.D.D.L.), and a JDRF grant (to D.J.D.). This study also received support from the Morphology Core of the University of Pennsylvania Center for Molecular Studies in Digestive and Liver Disease (P30 DK50306).

The authors acknowledge Susan Bonner-Weir for guidance with the determination of β-cell mass and Karen Teff for help with the statistical analysis.

This study was presented in part at the 62nd Annual Meeting of the American Diabetes Association, San Francisco, California, 14–18 June 2002, and at the 2002 Pediatric Academic Societies’ Meeting, Baltimore, Maryland, 3–6 May 2002.