Dual elimination of the glucagon and GLP-1 receptors in mice reveals plasticity in the incretin axis

Disordered glucagon secretion contributes to the symptoms of diabetes, and reduced glucagon action is known to improve glucose homeostasis. In mice, genetic deletion of the glucagon receptor (Gcgr) results in increased levels of the insulinotropic hormone glucagon-like peptide 1 (GLP-1), which may contribute to the alterations in glucose homeostasis observed in Gcgr^–/– mice. Here, we assessed the contribution of GLP-1 receptor (GLP-1R) signaling to the phenotype of Gcgr^–/– mice by generating Gcgr^–/–Glp1r^–/– mice. Although insulin sensitivity was similar in all genotypes, fasting glucose was increased in Gcgr^–/–Glp1r^–/– mice. Elimination of the Glp1r normalized gastric emptying and impaired intraperitoneal glucose tolerance in Gcgr^–/– mice. Unexpectedly, deletion of Glp1r in Gcgr^–/– mice did not alter the improved oral glucose tolerance and increased insulin secretion characteristic of that genotype. Although Gcgr^–/–Glp1r^–/– islets exhibited increased sensitivity to the incretin glucose-dependent insulinotropic polypeptide (GIP), mice lacking both Glp1r and the GIP receptor (Gipr) maintained preservation of the enteroinsular axis following reduction of Gcgr signaling. Moreover, Gcgr^–/–Glp1r^–/– islets expressed increased levels of the cholecystokinin A receptor (Cckar) and G protein–coupled receptor 119 (Gpr119) mRNA transcripts, and Gcgr^–/–Glp1r^–/– mice exhibited increased sensitivity to exogenous CCK and the GPR119 agonist AR231453. Our data reveal extensive functional plasticity in the enteroinsular axis via induction of compensatory mechanisms that control nutrient-dependent regulation of insulin secretion.

Glucagon, a 29-amino-acid peptide hormone, is produced in pancreatic α cells and, together with insulin, plays a key role in regulating hepatic glucose production, serving as the first line of defense against hypoglycemia (1). Disordered control of glucagon secretion is observed in type 2 diabetes mellitus (T2DM) (2–4) and together with insulin deficiency or resistance results in metabolic derangements characteristic of T2DM (5). Glucagon exerts its actions through a class B family G protein–coupled receptor (GPCR), the glucagon receptor (Gcgr), that is expressed not only in the liver, but in a broad range of tissues, including the heart, kidney, endocrine pancreas, and the central nervous system (6–10).

The observations that inappropriately increased levels of glucagon promote increased hepatic glucose production and hyperglycemia (3, 11–14) have fostered efforts targeting suppression of glucagon action for the treatment of T2DM (15). Reduction of glucagon activity using glucagon antagonists, immunoneutralizing antisera, or antisense oligonucleotides (ASOs) directed against Gcgr attenuates hyperglycemia in experimental models of diabetes (16–23). The importance of glucagon action has also been examined via generation and characterization of the Gcgr^–/– mouse. Surprisingly, mice with complete germline disruption of Gcgr are viable and grow normally (24, 25). Gcgr^–/– mice exhibit normal body weight, food intake, and energy expenditure, yet display improved glucose tolerance, enhanced β cell function and insulin sensitivity, resistance to streptozotocin-induced (STZ-induced) diabetes, and delayed gastric emptying (25–27). These favorable phenotypes support reduction of glucagon action for the therapy of T2DM.

Interpretation of metabolic derangements arising from loss of Gcgr signaling is complicated by the concomitant finding of markedly increased circulating levels of glucagon-like peptide 1 (GLP-1) in Gcgr^–/– mice (25). GLP-1, a related proglucagon-derived peptide normally secreted from enteroendocrine L cells, produces many of the actions described in Gcgr^–/– mice. The increased circulating levels of GLP-1 in Gcgr^–/– mice likely reflect increased production of bioactive GLP-1 in the Gcgr^–/– pancreas (25). As GLP-1 improves β cell function and glucose tolerance, reduces gastric emptying, and promotes islet cell proliferation and resistance to STZ-induced diabetes, attribution of precise mechanisms responsible for the improved metabolic phenotype in Gcgr^–/– mice is challenging. We now demonstrate that a functional GLP-1 receptor (GLP-1R) is essential for control of fasting and ambient glycemia, enhancement of glucose-stimulated insulin secretion following i.p. glucose challenge, and inhibition of gastric emptying in Gcgr^–/– mice. Unexpectedly, Gcgr^–/–Glp1r^–/– mice continued to exhibit improved oral glucose tolerance and enhanced β cell function despite loss of two key insulinotropic β cell receptors. Although loss of GLP-1 action is classically compensated for by upregulation of the other major incretin, glucose-dependent insulinotropic polypeptide (GIP) (28), we demonstrate that reduction of Gcgr expression in mice with combined inactivation of both Glp1r and the GIP receptor (Gipr) genes continues to be associated with preservation of oral glucose tolerance and enhanced insulin secretion. Disruption of the classical (GLP-1R and GIPr) enteroinsular axis was associated with enhanced expression and activity of the cholecystokinin A receptor (Cckar) and G protein–coupled receptor 119 (Gpr119), functionally related insulinotropic receptors that respond to nutrient-sensitive signals. These findings reveal considerable plasticity in the incretin-related mechanisms regulating β cell function and glucose homeostasis.

Glp1r is not required for pancreas enlargement or α cell hyperplasia in Gcgr^–/– mice. Body weight, food intake, physical activity, and energy expenditure were comparable in WT, single incretin receptor knockout, and Gcgr^–/–Glp1r^–/– mice (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI43615DS1). As Gcgr^–/– mice exhibit increased pancreatic mass and marked islet and α cell hyperplasia (25) and GLP-1R activation promotes expansion of islet and pancreatic mass (29–31), we assessed the contribution of Glp1r to the development of these abnormalities in Gcgr^–/– mice. Consistent with previous findings, Gcgr^–/– mice exhibited very high circulating levels of GLP-1 (Supplemental Figure 2A), significantly increased pancreas weight, and an approximately 4-fold increase in islet area (Figure 1); however, islet area and pancreatic mass remained significantly increased to a similar extent in Gcgr^–/–Glp1r^–/– mice (Figure 1, A and B). Immunohistochemical analysis revealed that the increased islet area was predominantly due to α cell hyperplasia, with most Gcgr^–/– and Gcgr^–/–Glp1r^–/– islets containing a core of β cells surrounded by an expanded mantle of hyperplastic α cells (Figure 1C). Hence, Glp1r is not required for development of increased pancreatic mass and islet hyperplasia following loss of Gcgr action.

Figure 1

Glp1r is not required for development of increased pancreas weight or α cell hyperplasia in Gcgr^–/– mice. (A) Pancreas weight of 20- to 24-week-old mice shown as percentage of the final body weight (n = 7–20 mice per group). (B) Islet area shown as a percentage of total pancreas area (n = 4–12 mice per group) (C) Representative histological sections of pancreas stained for insulin or glucagon alone. Final magnification, ×80. Values are expressed as mean ± SEM. *P < 0.05, Gcgr^–/– mice versus WT littermate controls; ^#P < 0.05, Gcgr^–/–Glp1r^–/– mice versus WT littermate control mice; ^†P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– mice.

Disruption of Glp1r leads to increased fasting glycemia in Gcgr^–/– mice. Both fasting and random glucose were reduced in Gcgr^–/– mice (Figure 2, A and B), consistent with the central role of glucagon in the maintenance of euglycemia (25). Basal GLP-1R signaling also regulates fasting glycemia (32), classically through suppression of glucagon secretion (33). Surprisingly, a significant increase in fasting glucose was observed in Gcgr^–/–Glp1r^–/– compared with Gcgr^–/– mice (Figure 2A) and elimination of the Glp1r normalized random-fed glycemia in Gcgr^–/–Glp1r^–/– mice (Figure 2B). Hence, loss of Glp1r substantially attenuates improvements in both ambient and fasting glycemia in Gcgr^–/– mice (25).

Figure 2

Glp1r controls fasting and fed glycemia in Gcgr^–/– mice. (A) Blood glucose following 5 or 16 hours of fasting in 8- to 12-week-old WT, Gcgr^–/–, Glp1r^–/–, and Gcgr^–/–Glp1r^–/– mice (n = 5–30 mice per genotype). (B) Weekly random-fed blood glucose levels in 8- to 20-week-old Gcgr^–/–Glp1r^–/–, Gcgr^–/–, Glp1r^–/–, and littermate control WT mice (n = 3–20 mice per group). Values are expressed as mean ± SEM. *P < 0.05, Gcgr^–/– versus Gcgr^–/–Glp1r^–/– mice; ^#P < 0.05, Gcgr^–/– versus WT mice; ^‡P < 0.05, Glp1r^–/– versus WT mice; ^†P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– mice.

Elimination of Glp1r reverses improvements in i.p. glucose tolerance in Gcgr^–/– mice. To elucidate the contribution of enhanced GLP-1 receptor signaling to improved β cell function and glucose tolerance in Gcgr^–/– mice (25), we first assessed clearance of i.p. glucose in mice of different genotypes. Intraperitoneal glucose tolerance was significantly enhanced and plasma insulin levels increased in Gcgr^–/– mice; conversely, i.p. glucose tolerance was impaired in Glp1r^–/– mice (Figure 3A), consistent with previous studies (25, 32). Furthermore, disruption of Glp1r in Gcgr^–/– mice reversed the improvements in i.p. glucose tolerance and normalized plasma insulin levels in Gcgr^–/–Glp1r^–/– mice (Figure 3, A and B), whereas insulin sensitivity, approximated by insulin tolerance, was comparable among all genotypes (Figure 3C). Hence, the elevated levels of GLP-1 leading to increased GLP-1R signaling is primarily responsible for enhanced β cell function and improved glucose clearance after i.p. glucose challenge in Gcgr^–/– mice.

Figure 3

Loss of Glp1r reverses improvements in i.p. glucose tolerance without altering insulin sensitivity in Gcgr^–/– mice. (A) IPGTT in 8- to 10-week-old WT, Gcgr^–/–, Glp1r^–/–, and Gcgr^–/–Glp1r^–/– mice (n = 9–24 mice per group). (B) Area under the curve and plasma insulin levels at 0 and 15 minutes following i.p. glucose challenge (n = 4–8 mice per group). (C) Insulin tolerance test in 12- to 14-week-old mice; values are normalized to basal glucose, with right graph showing area under the glucose curve (n = 5–20 mice per group). Values are expressed as mean ± SEM. *P < 0.05, Gcgr^–/– versus Gcgr^–/–Glp1r^–/– mice; ^#P < 0.05, Gcgr^–/– versus WT mice; ^‡P < 0.05, Glp1r^–/– versus WT mice; ^†P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– mice.

The GLP-1 receptor mediates reduced gastric emptying; however, oral glucose tolerance remains improved independent of Glp1r in Gcgr^–/– mice. As GLP-1–mediated reduction in gastric emptying may substantially account for the improved oral glucose tolerance in Gcgr^–/– mice (25, 26), we quantified gastric emptying with two complementary methods. Both liquid-phase gastric emptying, assessed via measurement of plasma acetaminophen levels, and solid-phase gastric emptying were significantly reduced in Gcgr^–/– mice and normalized in Gcgr^–/–Glp1r^–/– mice (Figure 4, A and B). We hypothesized that normalization of gastric emptying would be associated with deterioration of oral glucose tolerance in Gcgr^–/–Glp1r^–/– mice (25). Unexpectedly, oral glucose tolerance remained significantly improved in Gcgr^–/–Glp1r^–/– mice to an extent comparable to that in Gcgr^–/– mice alone (Figure 4, C and D). Furthermore, in contrast to the normalization of plasma insulin levels seen following i.p. glucose challenge in Gcgr^–/–Glp1r^–/– versus Gcgr^–/– mice (Figure 3B), Gcgr^–/–Glp1r^–/– mice continued to exhibit significantly increased levels of plasma insulin following oral glucose challenge (Figure 4D).

Figure 4

Glp1r mediates reduced gastric emptying but not improved oral glucose tolerance in Gcgr^–/– mice. (A) Liquid-phase gastric emptying (as determined by the appearance of acetaminophen in the circulation after 15 minutes) in 10- to 11-week-old mice (n = 4–14 mice per group). (B) Solid-phase gastric emptying in 20-week-old mice (n = 4–10 mice per group). Values are expressed as mean ± SEM. *P < 0.05, Gcgr^–/– versus Gcgr^–/–Glp1r^–/– mice; ^#P < 0.05, Gcgr^–/– versus WT mice. (C) Blood glucose levels during an OGTT in 10- to 11-week-old mice (n = 11–22 mice per group). (D) Area under the glucose curve and plasma insulin levels 0 and 15 minutes following oral glucose challenge (n = 4–9 mice per group). Values are expressed as mean ± SEM. In C and D: *P < 0.05, Gcgr^–/– versus WT littermate control mice; ^#P < 0.05, Gcgr^–/–Glp1r^–/– versus WT mice; ^†P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– littermate control mice; ^‡P < 0.05, Glp1r^–/– versus WT littermate control mice.

Islets from Gcgr^–/–Glp1r^–/– mice display increased sensitivity to GIP. As Glp1r^–/– mice exhibit enhanced GIP secretion and increased sensitivity to GIP (28), we explored whether GIP-related mechanisms underlie the enhanced enteral glucose-stimulated insulin secretion in Gcgr^–/–Glp1r^–/– mice. Although levels of GIP were modestly elevated in Glp1r^–/– mice, GIP levels were not significantly increased in Gcgr^–/–Glp1r^–/– mice (Supplemental Figure 2B). We next examined control of insulin secretion from WT, Glp1r^–/–, Gcgr^–/–, and Gcgr^–/–Glp1r^–/– islets. No significant differences across genotypes were detected in response to 16.7 mM glucose with a modest but nonsignificant reduction in insulin secretion observed with Gcgr^–/–Glp1r^–/– islets (Figure 5A). Consistent with the loss of the Glp1r, the insulinotropic response to exendin-4 was absent in Glp1r^–/– and Gcgr^–/–Glp1r^–/– islets (Figure 5A). Although GIP sensitivity was not enhanced in Glp1r^–/– or Gcgr^–/– islets, the insulinotropic response to GIP was significantly increased in Gcgr^–/–Glp1r^–/– islets (Figure 5A).

Figure 5

Function of GPCRs in isolated islets. Islet insulin secretion was assessed by preincubation of islets in KRB for 60 minutes at 2.8 mM glucose at 37°C before distribution in batches of 10 islets per condition into wells containing 16.7 mM glucose with or without (A) exendin-4 (Ex-4, 10 nM), [d-Ala²]GIP (GIP, 10 nM), (B) PACAP (10 nM), tolbutamide (Tol, 100 μM), or l-arginine (L-arg, 10 mM) for 1 hour at 37°C. Levels of insulin in the secretion medium were normalized to levels of islet insulin content and are expressed as a fold change in insulin secretion relative to WT high-glucose treatment. Insulin content values averaged approximately 30–40 ng/islet for Glp1r^–/– and WT mice and 15–25 ng/islet for Gcgr^–/– and Gcgr^–/–Glp1r^–/– mice (n = 3 mice per group). Data shown are representative of 2–3 independent experiments, each with 3 replicates per condition. (C) Total cellular and media cAMP in islets from WT, Glp1r^–/–, Gcgr^–/–, and Gcgr^–/–Glp1r^–/– mice was quantified following treatment of the islets with 0, 1, 3, or 10 nM [d-Ala²]GIP. Levels of cAMP in the secretion medium were normalized to levels of islet insulin content and are expressed as a fold change in islet cAMP levels relative to WT high-glucose treatment (n = 3 mice per group). Values are expressed as mean ± SEM. ^§P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– mice; ^#P < 0.05, Gcgr^–/– versus Gcgr^–/–Glp1r^–/– mice; ^‡P < 0.05, Gcgr^–/–Glp1r^–/– versus WT mice; ^†P < 0.05, Glp1r^–/– versus WT mice; *P < 0.05, Gcgr^–/– versus WT mice.

To explore the selectivity of the enhanced response to GIP, we tested a range of other insulin secretagogues. In contrast to the enhanced response to GIP, Gcgr^–/–Glp1r^–/– islets exhibited a normal response to pituitary adenylate cyclase–activating peptide (PACAP) but significantly reduced insulin secretory responses to tolbutamide and l-arginine (Figure 5B). Consistent with the increased GIP sensitivity demonstrated for insulin secretion (Figure 5A), Gcgr^–/–Glp1r^–/– islets also exhibited enhanced cAMP accumulation in response to GIP (Figure 5C). To evaluate GIP sensitivity in vivo, we administered i.p. glucose in the presence or absence of submaximal doses of exogenous GIP to WT, Glp1r^–/–, Gcgr^–/–, and Gcgr^–/–Glp1r^–/– mice. Both Glp1r^–/– and to a greater extent Gcgr^–/–Glp1r^–/– mice exhibited enhanced sensitivity to GIP, as revealed by reduced glycemic excursions and increased circulating levels of plasma insulin in response to exogenous GIP administration (Figure 6, A–D, and Supplemental Figure 3, A–D).

Figure 6

Gcgr^–/–Glp1r^–/– mice exhibit enhanced sensitivity to [d-Ala²]GIP. An IPGTT was performed in 20- to 22-week-old (A) WT, (B) Gcgr^–/–, (C) Glp1r^–/–, and (D) Gcgr^–/–Glp1r^–/– mice following treatment with 1 nmol/kg [d-Ala²]GIP or saline (vehicle [Veh]). Insets depict the area under the glucose excursion curve (AUC) in mM×min and plasma insulin levels at 0 and 15 minutes following glucose challenge (n = 5–8). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, [d-Ala²]GIP–treated versus saline-treated group.

Plasticity of the incretin axis revealed through reduction of Gcgr action in Glp1r^–/–Gipr^–/– mice. The available data strongly suggested that preservation of improved glucose tolerance and enhanced insulin secretion despite loss of GLP-1 action reflects increased GIP sensitivity in Gcgr^–/–Glp1r^–/– islets. To more rigorously test this hypothesis, we reduced Gcgr expression using ASOs in mice lacking both functional incretin receptors, i.e., Glp1r^–/–Gipr^–/– (double incretin receptor knockout [DIRKO]) mice (34). Hepatic Gcgr mRNA transcripts were markedly decreased and plasma levels of GLP-1 progressively increased in WT and DIRKO mice following ASO treatment (Figure 7, A and B, respectively). WT mice treated with Gcgr ASOs showed improved i.p. glucose tolerance and increased insulin levels following i.p. glucose challenge (Figure 7C). In contrast, DIRKO mice treated with Gcgr ASOs showed no improvement in glucose tolerance or insulin levels following i.p. glucose challenge (Figure 7D), consistent with the importance of the GLP-1R for improved β cell function following reduction of Gcgr expression (21, 22, 32). WT mice treated with Gcgr ASOs also showed improved oral glucose tolerance and significantly higher plasma insulin levels than vehicle-treated controls (Figure 7E). Remarkably, despite loss of both classical incretin receptors, DIRKO mice treated with Gcgr ASOs also exhibited improved oral glucose tolerance and significantly increased plasma insulin levels (Figure 7F). Hence, preferential improvement of glucose tolerance and enhanced β cell function following glucose administration in the gut can be achieved despite loss of both incretin receptors.

Figure 7

Enteroinsular axis is maintained in DIRKO mice treated with Gcgr ASOs. (A) mRNA expression of Gcgr in the liver (n = 3 per group) following treatment with 6 injections of 25 mg/kg Gcgr ASOs. (B) Total plasma GLP-1 levels following 2, 4, or 6 injections of 25 mg/kg saline or Gcgr ASOs (n = 5 per group). (C and D) An i.p. glucose challenge was performed on 13- to 14-week-old male (C) WT mice and (D) DIRKO mice that had been treated with 3 injections of vehicle or 25 mg/kg Gcgr ASOs (n = 5 per group). (E and F) An OGTT was performed on 15- to 16-week-old (E) WT mice and (F) DIRKO mice that had been treated with 4 injections of vehicle or 25 mg/kg Gcgr ASOs (n = 5 per group). Insets depict plasma insulin levels at 0 and 15 minutes following glucose challenge for saline- or Gcgr ASO–treated mice (n = 5 per treatment group). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vehicle- versus Gcgr ASO–treated WT or DIRKO mice.

To identify mechanisms responsible for improvement of oral glucose tolerance and β cell function despite absence of both GLP-1 and GIP receptors, we assessed the expression of insulinotropic receptors in islets from (a) DIRKO mice treated with Gcgr ASOs and (b) Gcgr^–/–Glp1r^–/– mice. Remarkably, levels of mRNA transcripts for the insulinotropic receptors gastrin-releasing peptide receptor (Grpr), Cckar, and Gpr119 were significantly increased in islet RNA from Gcgr^–/–Glp1r^–/– mice (Figure 8A). Similarly Gpr119 and Cckar mRNA transcripts were also significantly increased in islet RNA from DIRKO mice following Gcgr ASO administration (Figure 8B). These findings raised the possibility that increased activity of related insulinotropic receptors may compensate for the loss of GLP-1 and GIP action on islet β cells.

Figure 8

Expression of insulinotropic GPCRs in islets. (A) Islets were isolated from WT, Gcgr^–/–, Glp1r^–/–, and Gcgr^–/–Glp1r^–/– mice, followed by isolation of mRNA for real-time PCR of basal levels of transcripts encoding Gipr, Pacapr, Gpr40, Grpr, Cckar, and Gpr119. (B) Islets were isolated from WT or DIRKO mice following 6 injections of vehicle or 25 mg/kg Gcgr ASOs, and mRNA levels of Grpr, Gpr119, and Cckar were determined. Levels of transcripts were normalized to levels of cyclophilin for each RNA sample. n = 4 mice per genotype. Values are expressed as mean ± SEM. ^§P < 0.05, Glp1r^–/– versus Gcgr^–/–Glp1r^–/– mice; ^#P < 0.05, Gcgr^–/– versus Gcgr^–/–Glp1r^–/– mice; ^‡P < 0.05, Gcgr^–/–Glp1r^–/– versus WT mice; ^¶P < 0.01, WT Gcgr ASO– vs DIRKO Gcgr ASO–treated mice; *P < 0.01, WT saline- versus DIRKO Gcgr ASO–treated mice; ^†P < 0.01, DIRKO saline- versus DIRKO Gcgr ASO–treated mice.

Gcgr^–/–Glp1r^–/– mice display increased sensitivity to Gpr119 and Cckar agonists. To assess the functional significance of increased islet receptor expression, we carried out glucose tolerance tests in the presence or absence of exogenous GRP, CCK, PACAP, and the GPR119 agonist AR231453 (35) in WT, Gcgr^–/–, Glp1r^–/–, and Gcgr^–/–Glp1r^–/– mice. We did not detect enhanced sensitivity to exogenous GRP or PACAP (Supplemental Figure 4 and Supplemental Figure 5, A–D). However, although AR231453 failed to improve i.p. glucose tolerance in WT, Gcgr^–/–, or Glp1r^–/– mice, a robust improvement in glucose tolerance and marked stimulation of plasma insulin levels were observed following AR231453 administration in Gcgr^–/–Glp1r^–/– mice (Figure 9, A–D, and Supplemental Figure 6, A–D). Similarly, doses of CCK that failed to improve glucose tolerance in WT or single Gcgr^–/– or Glp1r^–/– mice produced a significant reduction in glycemic excursion and significantly elevated plasma insulin levels in Gcgr^–/–Glp1r^–/– mice, consistent with increased sensitivity to CCK (Figure 10, A–D, and Supplemental Figure 7, A–D).

Figure 9

Gcgr^–/–Glp1r^–/– mice exhibit enhanced sensitivity to the GPR119 agonist AR231453. An IPGTT was performed in 22- to 24-week-old (A) WT (B), Gcgr^–/– (C), Glp1r^–/–, and (D) Gcgr^–/–Glp1r^–/– mice 30 minutes following treatment with 5 mg/kg AR231453 or vehicle. Insets depict the area under the glucose excursion curve in mM.min and plasma insulin levels at 0 and 15 minutes following glucose challenge (n = 5–8). Values are expressed as mean ± SEM. **P < 0.01, AR231453- versus vehicle-treated mice.

Figure 10

Gcgr^–/–Glp1r^–/– mice exhibit increased sensitivity to CCK. An IPGTT was performed in 22- to 24-week-old (A) WT, (B) Gcgr^–/–, (C) Glp1r^–/–, and (D) Gcgr^–/–Glp1r^–/– mice following treatment with 9 μg/kg of CCK-8 or vehicle. Insets depict the area under the glucose excursion curve (AUC) in mM.min and plasma insulin levels at 0 and 15 minutes following glucose challenge (n = 5–8). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P <0.001, CCK-8– versus saline-treated mice.

The central importance of glucagon action for the maintenance of euglycemia, together with observations that glucagon secretion may be inappropriately increased in many subjects with T2DM, has fostered great interest in reduction of glucagon action for the treatment of T2DM. Indeed, multiple therapeutic modalities, including insulin, amylin analogs, GLP-1R agonists, and dipeptidyl peptidase–4 inhibitors, exert their anti-diabetic actions in part through suppression of glucagon secretion (36). The Gcgr^–/– phenotype — healthy mice with reduced fasting and postprandial glycemia (25), resistance to diet-induced obesity, and enhanced β cell function and survival (26) — further supports the concept of reducing glucagon receptor signaling for treatment of T2DM. Our studies clearly show that a substantial component of the improved metabolic phenotype of Gcgr^–/– mice, including control of fasting and fed glycemia, reduced gastric emptying, improved i.p. glucose tolerance, and enhanced β cell function, reflects concomitant upregulation of GLP-1 action in Gcgr^–/– mice (Figure 11).

Figure 11

Schematic of proposed model. (A) Loss of Gcgr in the liver markedly elevates plasma GLP-1 levels, which promotes glucose-stimulated insulin secretion during i.p. glucose challenge and inhibits gastric emptying in Gcgr^–/– mice. (B) Loss of Glp1r in the Gcgr^–/– mice results in preservation of the enteroinsular axis via compensatory mechanisms such as upregulation of Gpr119 and Cckar action in β cells, independent of the increase in GIP sensitivity.

Although incretin action classically controls postprandial glucose excursion, considerable evidence supports a role for GLP-1 in the regulation of fasting glucose. GLP-1 receptor agonists reduce fasting glucose in human subjects, in association with reduced levels of circulating glucagon (37). Moreover, the GLP-1R antagonist exendin_9–39 increases fasting glucose and glucagon levels in baboons (38), and genetic disruption of the Glp1r is associated with fasting hyperglycemia in Glp1r^–/– mice, without detectable changes in levels of fasting glucagon (32). Surprisingly, elimination of GLP-1 action significantly increased fasting glycemia in Gcgr^–/–Glp1r^–/– mice. These observations further demonstrate that GLP-1R–dependent pathways may regulate fasting glucose independent of glucagon action, perhaps through incompletely understood neural mechanisms (39).

Reduction or loss of Gcgr signaling (22, 25) or resistance to glucagon action (40) leads to hyperplasia of the exocrine and endocrine pancreas. Selective liver-specific deletion of the gene encoding the G protein G_sα generates a similar phenotype characterized by markedly increased levels of glucagon and GLP-1, increased pancreatic mass, and islet hyperplasia (40). Moreover, this constellation of pancreatic abnormalities is reversible, as glucagon replacement in glucagon-deficient PC2-knockout mice normalizes a substantial proportion of these pancreatic phenotypes (41). Although GLP-1R activation promotes expansion of islet and pancreatic mass (29, 31), α cell hyperplasia is not generally observed following administration of GLP-1R agonists. Consistent with these findings, our data demonstrate that the Glp1r does not mediate pancreatic or islet/α cell proliferation in Gcgr^–/– mice. As Gcgr^–/– mice are born with normal pancreatic mass and only modest α cell hyperplasia (25, 42), the precise mechanisms linking loss of the Gcgr to marked postnatal expansion of the exocrine and endocrine islet compartments remain unclear.

The finding of reduced gastric emptying in Gcgr^–/– mice (26) might be explained by enhanced action of multiple proglucagon-derived peptides. GLP-1, oxyntomodulin, and to a lesser extent GLP-2 inhibit gastric emptying (43, 44), and GLP-1 and oxyntomodulin exert their actions on gastric emptying through the GLP-1 receptor. Unexpectedly, however, despite normalization of gastric emptying in Gcgr^–/–Glp1r^–/– mice, oral glucose tolerance remained substantially improved in Gcgr^–/–Glp1r^–/– mice, despite elimination of GLP-1R action. Moreover, levels of circulating insulin remained significantly increased following oral glucose challenge despite loss of GLP-1R action in Gcgr^–/–Glp1r^–/– mice. These findings implicate sustained enhancement of β cell function as a mechanism underlying persistent improvement in oral glucose tolerance despite loss of GLP-1R action in Gcgr^–/–Glp1r^–/– mice.

The demonstration that the Gcgr^–/–Glp1r^–/– β cell maintains enhanced insulin secretion in response to oral glucose was unexpected given the previous demonstration of selectively impaired β cell function in Gcgr^–/– mice compared with age- and sex-matched WT C57BL/6 mice (27) and the multiple lines of evidence supporting important roles for both GLP-1 and glucagon in the control of glucose-stimulated insulin secretion. Glucagon directly stimulates insulin secretion in the rat pancreas independent of GLP-1 receptor action (45), and transgenic Gcgr expression in murine β cells significantly augments glucose-stimulated insulin secretion and reduces random glycemia in RIP-Gcgr transgenic mice (46). Moreover, the glucagon antagonist des-His1-[Glu9]-glucagon-amide significantly reduced glucose-stimulated insulin release in human islets (47). Similarly, GLP-1 induces β cell glucose competence, upregulates insulin biosynthesis and secretion, and markedly enhances glucose sensitivity and glucose-stimulated insulin secretion even in poorly responsive diabetic β cells (48). Hence, given the results of previous observations reporting impaired β cell function in Gcgr^–/– islets (27), we initially predicted that a further deterioration in β cell function would ensue following removal of the GLP-1R signaling system in Gcgr^–/– mice.

Surprisingly, however, elimination of Glp1r in Gcgr^–/– mice did not lead to deterioration in glycemic excursion after oral glucose loading in Gcgr^–/–Glp1r^–/– mice. The preferential preservation of improved oral glucose tolerance and enhanced insulin secretion following glucose administration via the gastrointestinal tract, despite functional elimination of both the Gcgr and Glp1r, strongly implicates the existence of one or more gut-derived compensatory factors that augment β cell function in an “incretin-like” manner in Gcgr^–/–Glp1r^–/– mice. As compensatory upregulation of GIP action has been described in Glp1r^–/– mice, we postulated that upregulation of the GIP-Gipr axis might similarly explain preservation of improved oral glucose tolerance in mice following loss of glucagon and GLP-1 action. Although circulating levels of GIP and levels of GIP RNA transcripts were not increased in islets from Gcgr^–/–Glp1r^–/– mice, we detected increased insulin secretion and enhanced cAMP accumulation following treatment of Gcgr^–/–Glp1r^–/– islets with GIP, consistent with increased GIP sensitivity. Furthermore, glucose tolerance was significantly improved and plasma insulin levels were markedly increased following administration of GIP in vivo, further supporting enhanced sensitivity to GIP as a compensatory mechanism augmenting β cell function in Gcgr^–/–Glp1r^–/– mice.

Unexpectedly, however, we continued to observe preferential improvement of oral glucose tolerance and β cell function in DIRKO mice treated with ASOs to reduce Gcgr expression, implying the existence of additional incretin-like mechanisms compensating for the lack of insulinotropic activity normally subserved by the glucagon, GLP-1, and GIP receptors. Our data demonstrating increased islet expression and functional activity of Cckar and Gpr119 mRNA transcripts from Gcgr^–/–Glp1r^–/– mice, and in islets from DIRKO mice treated with Gcgr ASOs, reveal a mechanism compensating for the loss of insulinotropic incretin receptors (Figure 11B). The finding of greatly enhanced sensitivity in vivo (improved glucose tolerance and plasma insulin levels) to exogenous ligands for both the Cckar and Gpr119 receptors is consistent with an important role for these receptors in maintaining an incretin response to enteral glucose administration despite loss of GLP-1 and GIP action.

It is notable that the putative ligands for both Cckar, namely CCK, and Gpr119, principally lipid-derived amides such as oleoylethanolamide and N-oleoyldopamine, would be expected to increase significantly following oral, but not i.p., glucose administration, consistent with our findings of enhanced oral, but not i.p., glucose clearance in Gcgr^–/–Glp1r^–/– mice. Intriguingly, β cells from DIRKO mice appeared to compensate for loss of incretin receptor and Gcgr expression by similar induction of Cckar and Gpr119 expression, extending the findings of islet incretin receptor plasticity to a second related, yet genetically distinct model.

Studies of islet adaptation classically carried out in the context of β cell injury and regeneration, pregnancy, or high-fat feeding have revealed numerous changes in islet gene expression thought to be linked to the need to expand β cell mass and/or enhance β cell function in response to insulin resistance (49–51). Hence, the concept of β cell plasticity is reasonably well established under physiological conditions requiring enhanced functional β cell mass. There is considerably less information concerning the potential plasticity of the gut-islet axis in circumstances associated with impairment or disruption of classic incretin receptor signals normally emanating from the GLP-1 and GIP receptors. Our observations made using two distinct genetic models, the Gcgr^–/–Glp1r^–/– mouse and the DIRKO mouse treated with Gcgr ASOs, reveal a heretofore unrecognized capacity for β cell adaptation to loss of insulinotropic receptor signaling (Figure 11). Intriguingly, upregulation of Cckar and Gpr119 expression was also observed in murine islets from pregnant mice, although the functional importance of these findings was not ascertained (51). Our findings may explain why the metabolic phenotype arising from loss of one or both incretin receptors is comparatively mild (32, 34, 52) and identify new models for exploring the expanding importance of incretin-related mechanisms that potentiate β cell function following oral nutrient ingestion.

View Supplemental data

We would like to thank Xiemin Cao for technical assistance with islet experiments and collection of blood samples during glucose tolerance tests. This work was supported in part by operating grants from the Canadian Institutes for Health Research (CIHR) MOP 93749 and MOP 82700. D.J. Drucker is supported by a Canada Research Chair in Regulatory Peptides and a Banting and Best Diabetes Centre–Novo Nordisk Chair in Incretin Biology, and S. Ali is supported by a Banting and Best Diabetes Centre Studentship Award.

Address correspondence to: Daniel J. Drucker, Mt. Sinai Hospital SLRI, 600 University Ave TCP5-1004, Toronto, Ontario, Canada M5G 1X5. Phone: 416.361.2661; Fax: 416.361.2669; E-mail: d.drucker@utoronto.ca.

Conflict of interest: D.J. Drucker has been an advisor or consultant for Amylin Pharmaceuticals, Arena Pharmaceuticals Inc., Arisaph Pharmaceuticals Inc., Eli Lilly Inc., GlaxoSmithKline, Hoffman LaRoche Inc., Merck Research Laboratories, Metabolex Inc., Novartis Pharmaceuticals, Novo Nordisk Inc., and Transition Pharmaceuticals Inc.

Reference information: J Clin Invest. 2011;121(5):1917–1929. doi:10.1172/JCI43615.

1. Cryer PE. Hypoglycaemia: the limiting factor in the glycaemic management of Type I and Type II diabetes. Diabetologia. 2002;45(7):937–948.
   View this article via: PubMed CrossRef Google Scholar
2. Jaspan JB, Rubinstein AH. Circulating glucagon: plasma profiles and metabolism in health and disease. Diabetes. 1977;26(9):887–902.
   View this article via: PubMed Google Scholar
3. Unger RH, Aguilar-Parada E, Muller WA, Eisentraut AM. Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest. 1970;49(4):837–848.
   View this article via: JCI PubMed CrossRef Google Scholar
4. Sherwin RS, Fisher M, Hendler R, Felig P. Hyperglucagonemia and blood glucose regulation in normal, obese and diabetic subjects. N Engl J Med. 1976;294(9):455–461.
   View this article via: PubMed Google Scholar
5. Unger RH. Glucagon physiology and pathophysiology in the light of new advances. Diabetologia. 1985;28(8):574–578.
   View this article via: PubMed Google Scholar
6. Campos RV, Lee YC, Drucker DJ. Divergent tissue-specific and developmental expression of receptors for glucagon and glucagon-like peptide-1 in the mouse. Endocrinology. 1994;134(5):2156–2164.
   View this article via: PubMed CrossRef Google Scholar
7. Marks J, Debnam ES, Dashwood MR, Srai SK, Unwin RJ. Detection of glucagon receptor mRNA in the rat proximal tubule: potential role for glucagon in the control of renal glucose transport. Clin Sci (Lond). 2003;104(3):253–258. .
   View this article via: PubMed CrossRef Google Scholar
8. Dunphy JL, Taylor RG, Fuller PJ. Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression. Mol Cell Endocrinol. 1998;141(1–2):179–186.
   View this article via: PubMed Google Scholar
9. Hansen LH, Abrahamsen N, Nishimura E. Glucagon receptor mRNA distribution in rat tissues. Peptides. 1995;16(6):1163–1166.
   View this article via: PubMed CrossRef Google Scholar
10. Svoboda M, Tastenoy M, Vertongen P, Robberecht P. Relative quantitative analysis of glucagon receptor mRNA in rat tissues. Mol Cell Endocrinol. 1994;105(2):131–137.
    View this article via: PubMed Google Scholar
11. Shah P, Basu A, Basu R, Rizza R. Impact of lack of suppression of glucagon on glucose tolerance in humans. Am J Physiol. 1999;277(2 pt 1):E283–E290.
    View this article via: PubMed Google Scholar
12. Consoli A. Role of liver in pathophysiology of NIDDM. Diabetes Care. 1992;15(3):430–441.
    View this article via: PubMed CrossRef Google Scholar
13. Muller WA, Faloona GR, Aguilar-Parada E, Unger RH. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion. N Engl J Med. 1970;283(3):109–115.
    View this article via: PubMed CrossRef Google Scholar
14. Dinneen S, Alzaid A, Turk D, Rizza R. Failure of glucagon suppression contributes to postprandial hyperglycaemia in IDDM. Diabetologia. 1995;38(3):337–343.
    View this article via: PubMed CrossRef Google Scholar
15. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab. 2003;284(4):E671–E678.
    View this article via: PubMed Google Scholar
16. Johnson DG, Goebel CU, Hruby VJ, Bregman MD, Trivedi D. Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science. 1982;215(4536):1115–1116.
    View this article via: PubMed CrossRef Google Scholar
17. Brand CL, Rolin B, Jorgensen PN, Svendsen I, Kristensen JS, Holst JJ. Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats. Diabetologia. 1994;37(10):985–993.
    View this article via: PubMed Google Scholar
18. Unson CG, Cypess AM, Wu CR, Goldsmith PK, Merrifield RB, Sakmar TP. Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding. Proc Natl Acad Sci U S A. 1996;93(1):310–315.
    View this article via: PubMed Google Scholar
19. Dallas-Yang Q, et al. Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists. Eur J Pharmacol. 2004;501(1–3):225–234.
    View this article via: PubMed CrossRef Google Scholar
20. Qureshi SA, et al. A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects. Diabetes. 2004;53(12):3267–3273.
    View this article via: PubMed CrossRef Google Scholar
21. Liang Y, et al. Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice. Diabetes. 2004;53(2):410–417.
    View this article via: PubMed Google Scholar
22. Sloop KW, et al. Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors. J Clin Invest. 2004;113(11):1571–1581.
    View this article via: JCI PubMed Google Scholar
23. Winzell MS, et al. Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet. Diabetologia. 2007;50(7):1453–1462.
    View this article via: PubMed CrossRef Google Scholar
24. Parker JC, Andrews KM, Allen MR, Stock JL, McNeish JD. Glycemic control in mice with targeted disruption of the glucagon receptor gene. Biochem Biophys Res Commun. 2002;290(2):839–843.
    View this article via: PubMed CrossRef Google Scholar
25. Gelling RW, et al. Lower blood glucose, hyperglucagonemia, and pancreatic {alpha} cell hyperplasia in glucagon receptor knockout mice. Proc Natl Acad Sci U S A. 2003;100(3):1438–1443.
    View this article via: PubMed CrossRef Google Scholar
26. Conarello SL, et al. Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia. Diabetologia. 2007;50(1):142–150.
    View this article via: PubMed CrossRef Google Scholar
27. Sorensen H, et al. Glucagon receptor knockout mice display increased insulin sensitivity and impaired {beta}-cell function. Diabetes. 2006;55(12):3463–3469.
    View this article via: PubMed Google Scholar
28. Pederson RA, et al. Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor^–/– mice. Diabetes. 1998;47(7):1046–1052.
    View this article via: PubMed CrossRef Google Scholar
29. Baggio LL, Huang Q, Cao X, Drucker DJ. The long-acting albumin-exendin-4 GLP-1R agonist CJC-1134 engages central and peripheral mechanisms regulating glucose homeostasis. Gastroenterology. 2008;134(4):1137–1147.
    View this article via: PubMed CrossRef Google Scholar
30. Brubaker PL, Drucker DJ. Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut and central nervous system. Endocrinology. 2004;145(6):2653–2659.
    View this article via: PubMed CrossRef Google Scholar
31. Koehler JA, Baggio LL, Lamont BJ, Ali S, Drucker DJ. GLP-1 receptor activation modulates pancreatitis-associated gene expression but does not modify the susceptibility to experimental pancreatitis in mice. Diabetes. 2009;58(9):2148–2161.
    View this article via: PubMed CrossRef Google Scholar
32. Scrocchi LA, et al. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide receptor gene. Nature Med. 1996;2(11):1254–1258.
    View this article via: PubMed Google Scholar
33. Creutzfeld WO, Kleine N, Willms B, Orskov C, Holst JJ, Nauck MA. Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I(7-36) amide in type I diabetic patients. Diabetes Care. 1996;19(6):580–586.
    View this article via: PubMed CrossRef Google Scholar
34. Hansotia T, et al. Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure. J Clin Invest. 2007;117(1):143–152.
    View this article via: JCI PubMed CrossRef Google Scholar
35. Flock G, Holland D, Seino Y, Drucker DJ. GPR119 regulates murine glucose homeostasis through incretin receptor-dependent and independent mechanisms. Endocrinology. 2011;152(2):374–383.
    View this article via: PubMed CrossRef Google Scholar
36. Ali S, Drucker DJ. Benefits and limitations of reducing glucagon action for the treatment of type 2 diabetes. Am J Physiol Endocrinol Metab. 2009;296(3):E415–E421.
    View this article via: PubMed CrossRef Google Scholar
37. Drucker DJ, Nauck MA. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet. 2006;368(9548):1696–1705.
    View this article via: PubMed Google Scholar
38. D’Alessio DA, et al. Elimination of the action of glucagon-like peptide 1 causes an impairment of glucose tolerance after nutrient ingestion by healthy baboons. J Clin Invest. 1996;97(1):133–138.
    View this article via: JCI PubMed CrossRef Google Scholar
39. Knauf C, et al. Brain glucagon-like peptide 1 signaling controls the onset of high-fat diet-induced insulin resistance and reduces energy expenditure. Endocrinology. 2008;149(10):4768–4777.
    View this article via: PubMed CrossRef Google Scholar
40. Chen M, et al. Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific G(s)alpha deficiency. J Clin Invest. 2005;115(11):3217–3227.
    View this article via: JCI PubMed CrossRef Google Scholar
41. Webb GC, Akbar MS, Zhao C, Swift HH, Steiner DF. Glucagon replacement via micro-osmotic pump corrects hypoglycemia and alpha-cell hyperplasia in prohormone convertase 2 knockout mice. Diabetes. 2002;51(2):398–405.
    View this article via: PubMed CrossRef Google Scholar
42. Vuguin PM, et al. Ablation of the glucagon receptor gene increases fetal lethality and produces alterations in islet development and maturation. Endocrinology. 2006;147(9):3995–4006.
    View this article via: PubMed CrossRef Google Scholar
43. Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci. 1993;38(4):665–673.
    View this article via: PubMed CrossRef Google Scholar
44. Schjoldager B, Mortensen PE, Myhre J, Christiansen J, Holst JJ. Oxyntomodulin from distal gut. Role in regulation of gastric and pancreatic functions. Dig Dis Sci. 1989;34(9):1411–1419.
    View this article via: PubMed CrossRef Google Scholar
45. Kawai K, Yokota C, Ohashi S, Watanabe Y, Yamashita K. Evidence that glucagon stimulates insulin secretion through its own receptor in rats. Diabetologia. 1995;38(3):274–276.
    View this article via: PubMed CrossRef Google Scholar
46. Gelling RW, et al. Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass. Am J Physiol Endocrinol Metab. 2009;297(3):E695–E707.
    View this article via: PubMed CrossRef Google Scholar
47. Huypens P, Ling Z, Pipeleers D, Schuit F. Glucagon receptors on human islet cells contribute to glucose competence of insulin release. Diabetologia. 2000;43(8):1012–1019.
    View this article via: PubMed Google Scholar
48. Drucker DJ. The biology of incretin hormones. Cell Metab. 2006;3(3):153–165.
    View this article via: PubMed CrossRef Google Scholar
49. Rieck S, et al. The transcriptional response of the islet to pregnancy in mice. Mol Endocrinol. 2009;23(10):1702–1712.
    View this article via: PubMed Google Scholar
50. Kim H, et al. Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med. 2010;16(7):804–808.
    View this article via: PubMed Google Scholar
51. Layden BT, et al. Regulation of pancreatic islet gene expression in mouse islets by pregnancy. J Endocrinol. 2010;207(3):265–279.
    View this article via: PubMed CrossRef Google Scholar
52. Miyawaki K, et al. Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice. Proc Natl Acad Sci U S A. 1999;96(26):14843–14847.
    View this article via: PubMed CrossRef Google Scholar
53. Maida A, Hansotia T, Longuet C, Seino Y, Drucker DJ. Differential importance of GIP versus GLP-1 receptor signaling for beta cell survival in mice. Gastroenterology. 2009;137(6):2146–2157.
    View this article via: PubMed CrossRef Google Scholar
54. Maida A, Lovshin JA, Baggio LL, Drucker DJ. The glucagon-like peptide-1 receptor agonist oxyntomodulin enhances {beta}-cell function but does not inhibit gastric emptying in mice. Endocrinology. 2008;149(11):5670–5678.
    View this article via: PubMed CrossRef Google Scholar