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Glycemic Control in Mice with Targeted Disruption of the Glucagon Receptor Gene
Biochemical and Biophysical Research Communications 290, 839 – 843 (2002) doi:10.1006/bbrc.2001.6265, available online at http://www.idealibrary.com on
Glycemic Control in Mice with Targeted Disruption of the Glucagon Receptor Gene Janice C. Parker, 1 Kim M. Andrews, Melanie R. Allen, Jeffrey L. Stock, and John D. McNeish Pfizer Global Research & Development, Groton Laboratories, Groton, Connecticut 06340
Received November 20, 2001
The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR ⴚ/ⴚ mice). Blood glucose levels were somewhat reduced in GR ⴚ/ⴚ mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude. © 2002 Elsevier Science
Key Words: glucagon; glycemia; glucagon receptor; glucose tolerance; pancreas; ␣-cell.
Glucagon is a 29-amino-acid hormone secreted by the ␣-cells of the pancreas. It has an important physiological role in the regulation of blood glucose levels and in other aspects of metabolism. Glucagon binding to specific receptors, principally on liver membranes, activates the glycogenolytic and gluconeogenic pathways, thereby increasing hepatic glucose production. The glucagon receptor is a 7-membrane spanning G-coupled receptor (1) that is known to be coupled to adenylate cyclase (2) and may also cause hydrolysis of inositol phospholipids (3, 4) and increases in intracellular Ca 2⫹ concentrations (5). There is evidence to indicate that the hyperglycemia that characterizes diabetes mellitus is a consequence of a bihormonal disorder, in which relatively insufficient 1 To whom correspondence and reprint requests should be addressed at Pfizer Global Research & Development, Groton Laboratories 8220-0375, Eastern Point Road, Groton, CT 06340. Fax: (860) 441-0548. E-mail: [email protected].
levels of insulin and excessive levels of glucagon together contribute to a net increase in hepatic glucose production (6, 7). Hyperglucagonemia has been shown to be present in Type 2 diabetic subjects (8, 9) and antagonists that interfere with the binding of glucagon to its receptor (10) or with its signal transduction (11) can reduce glucose output from liver cells (11) and reduce hyperglycemia in diabetic animals (10). To further examine the role of glucagon and of glucagon receptor function in glucose homeostasis we have generated mice in which the glucagon receptor gene was inactivated. This study describes the effects of this genetic intervention on pancreatic hormone levels and on glycemic control. MATERIALS AND METHODS Construction of the GR-KO targeting vector. A 1.4-kb murine glucagon receptor genomic fragment (corresponding to exons 1–3) was used as a probe to identify genomic clones from a DBA/1LacJ genomic phage library (Stratagene). The GR-KO targeting vector (Fig. 1) was constructed by cloning 8.4 kb of 5⬘ homology and 2.9 kb of 3⬘ homology into a pJNS2 (PGK-NEO/PGK-TK) backbone vector. The targeting vector replaced ⬃1.8 kb of the GR locus, corresponding to amino acids 56 –303 of GenBank Accession No. L38613, with PGK-NEO. Generation of GR-KO targeted embryonic stem cells. DBA/252 embryonic stem cells (12) were cultured on primary embryonic fibroblasts (PEFs) in DMEM containing high glucose, 15% heatinactivated fetal bovine serum, 0.2 mM L-glutamine, 0.1 mM 2-ME, 0.1 mM MEM nonessential amino acids, penicillin-streptomycin, and 400 U/ml recombinant murine leukemia inhibitory factor (SCML). The GR targeting vector (25 g) was electroporated at 1.0 ⫻ 10 8 embryonic stem cells/ml (0.4 ml total volume), and the electroporation was achieved with a BTX Electrocell Manipulator 600 (200 V, 50 F, 360 ⍀). Cells were then subjected to positive/negative selection with 200 g/ml G418 (geneticin; Life Technologies) and 2 M gancyclovir (Syntex Laboratories) by a published method (13). Gene targeting by homologous recombination was determined in surviving embryonic stem cell clones by Southern analysis using an 800-bp probe from a region 3⬘ of the 2.9-kb homology arm in conjunction with NsiI–XbaI digested embryonic stem cell DNA. The endogenous allele yielded a band hybridizing at 5.3 kb, whereas the targeted allele revealed a predicted RFLP at 3.7 kb due to the introduction of a novel XbaI restriction site from the GR targeting vector Homologously targeted embryonic stem cell clones were also screened for the incidence of multiple insertions by Southern analysis using a NEO
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FIG. 1. Schema employed to achieve homologous recombination of the murine glucagon receptor locus. The targeting vector was constructed by cloning 8.4 kb of 5⬘ homology and 2.9 kb of 3⬘ homology into a pJNS 2 (PGK-NEO/PGK-TK) backbone vector. Approximately 1.8 kb of endogenous genomic sequence was replaced by the neomycin gene. This created a deletion from glutamate 56 to glycine 303 within the native protein.
probe. Karyotyping analysis was completed on several targeted embryonic stem cell clones to rule out any chromosome abnormalities (Coriel Inst.). Generation of F2 ⫺/⫺ mice. GR ⫹/⫺ embryonic stem cells were microinjected into day 3.5 C57BL/6J blastocysts and resulting chimeric mice were backcrossed to DBA/1LacJ mates to identify germline transmission. GR ⫺/⫺ mice were derived from crosses of GR ⫹/⫺ males and females. F2 generation genotypes were determined by PCR using primer sets specific for either the endogenous or the targeted alleles as indicated in Figs. 2A and 2B.
Glucose assay, hormone assays, and triglyceride and cholesterol assays. Blood was collected from the retroorbital sinus. Fed animals had free access to chow, fasted animals had food withheld 14 –16 h prior to blood collection. Plasma glucose, triglyceride and cholesterol concentrations were measured enzymatically. Insulin and glucagon levels were measured by radioimmunoassay. Oral glucose tolerance test (OGTT). Mice were fasted overnight (⬃16 h) and then administered a 1 g/kg oral bolus of glucose. Blood samples were obtained from the retroorbital sinus of each animal at 0, 30, 60, and 90 min postdosing for glucose and insulin measurements.
FIG. 2. (A) Embryonic stem cell clones surviving selection were screened for homologous recombination by Southern blot analysis with a unique 3⬘-external probe to distinguish a wild-type 5.3-kb NsiI fragment from a 3.7-kb NsiI/XbaI fragment generated from the targeted allele. (B) Following microinjection of targeted embryonic stem cells into blastocyst stage DBA/1LacJ embryos, resulting offspring were subsequently genotyped by a PCR scheme as indicated on the diagram, employing specific primers to distinguish between the endogenous and targeted alleles. 840
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TABLE 2
Effect of Glucagon Gene Disruption on Plasma Glucose and Hormone Levels
Effect of Glucagon Gene Disruption on Pancreatic Hormone Content
WT
GR ⫹/⫹
GR ⫺/⫺
Plasma glucose (mg/dl) Fasting 159.0 ⫾ 6.5 139.3 ⫾ 5.0 99.5 ⫾ 5.2* ,• Fed 184.3 ⫾ 11.6 152.7 ⫾ 12.7 121.6 ⫾ 4.2* ,• Plasma insulin (ng/ml) Fasting 1.06 ⫾ 0.13 0.67 ⫾ 0.12 0.60 ⫾ 0.10 Fed 1.42 ⫾ 0.18 2.14 ⫾ 0.67 0.88 ⫾ 0.17 Plasma glucagon (pg/ml) Fasting 81.0 ⫾ 17.5 497.6 ⫾ 156.7* 5623.9 ⫾ 400.7* ,• Fed 82.9 ⫾ 11.5 905.1 ⫾ 149.7* 6417.2 ⫾ 420.6* ,•
Pancreatic weight (g) Glucagon content (g/g) Insulin content (g/g)
WT
GR ⫺/⫺
0.22 ⫾ 0.01 3.00 ⫾ 0.93 165.1 ⫾ 5.6
0.35 ⫾ 0.03* 131.56 ⫾ 29.62* 100.6 ⫾ 18.0
Note. Food was withheld for 14 –16 h prior to blood collection from the fasting animals. Insulin and glucagon were extracted from each whole pancreas acid-ethanol extraction and the hormone contents measured by radioimmunoassay as described under Materials and Methods. Data are means ⫾ SEM for measurements from three to five animals. * P ⬍ 0.05 vs WT.
Note. Food was withheld for 14 –16 h prior to blood collection from the fasting animals. Glucose insulin and glucagon concentrations were measured as described under Materials and Methods. Data are means ⫾ SEM for measurements from 4 to 10 animals. * P ⬍ 0.05 vs WT; • P ⬍ 0.05 vs GR ⫹/⫹ control.
Data analysis. All data represent means ⫾ SEM for measurements from 3 to 10 animals. Statistical significance was assessed by Student’s t test. Age-matched wild type DBA/1LacJ males were purchased from The Jackson Labs, Maine, and used as controls. Data presented is for male GR ⫺/⫺ and GR ⫹/⫹ animals, the latter group included as an additional control. Qualitatively similar results were obtained with the corresponding female animals (data not shown). All procedures involving animals were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.
RESULTS AND DISCUSSION Mice genetically modified to lack glucagon receptors (GR ⫺/⫺ mice) did not demonstrate any gross abnormalities of glucose homeostasis. Their plasma glucose levels, both under fed and fasting conditions, were somewhat lower than in either GR ⫹/⫹ mice or wild type mice of the same strain that had not been subject to genetic intervention (Table 1), but still fell within the range of normality. Plasma insulin levels appeared slightly lower in GR ⫺/⫺ mice, but this difference did not achieve statistical significance (Table 1). GR ⫺/⫺ mice had elevated levels of circulating glucagon, 2 orders of magnitude greater than the plasma concentrations present in wild-type mice (Table 1). Plasma glucagon levels were somewhat higher in GR ⫹/⫹ mice than in wild-type animals, suggesting that the manipulations to which the GR ⫹/⫹ mice were subjected may have resulted in a reduced level of glucagon receptor expression, with a compensatory increase in circulating glucagon. The large increase in the circulating concentration of glucagon in the GR ⫺/⫺ was associated with a corresponding increase in the pancreatic content of glucagon, which was 20-fold greater than in the correspond-
ing WT animals (Table 2). The overall size of the pancreata in the GR ⫺/⫺ mice was also significantly increased. The increase in wet weight and in glucagon content presumably reflects ␣-cell hyperplasia, necessary to sustain the extraordinarily high levels of glucagon observed in the circulation. The pancreatic insulin levels appeared somewhat lower in GR ⫺/⫺ than in WT mice, but this difference did not achieve statistical significance (Table 2). The phenotype observed in these mice is strikingly similar to that reported for a human subject with a splice site mutation of the glucagon receptor gene that resulted in non-functional glucagon receptors (14). As with the GR ⫺/⫺ mice, this individual had hyperglucagonemia and pancreatic hyperplasia together with normal plasma insulin levels and no impairment of glucose tolerance. The very large increase in plasma glucagon concentrations and in pancreatic glucagon content presumably represents a compensatory response that enables GR ⫺/⫺ to maintain normal or near-normal glycemia and lipidemia and would be an expected finding in animals with reduced levels of receptor. However, it is not clear what could be the mechanism for this adaptive response in the total absence of glucagon receptors (con-
TABLE 3
Effect of Glucagon Gene Disruption on Plasma Lipid Levels in Fasting Mice
Plasma cholesterol (mg/dl) Plasma triglycerides (mg/dl)
WT
GR ⫹/⫹
GR ⫺/⫺
54.8 ⫾ 3.2 101.3 ⫾ 11.1
58.9 ⫾ 8.6 92.8 ⫾ 17.0
66.0 ⫾ 3.8* 91.7 ⫾ 7.1
Note. Food was withheld for 14 –16 h prior to blood collection. Cholesterol and triglyceride concentrations were measured as described under Materials and Methods. Data are means ⫾ SEM for measurements from three to seven animals. * P ⬍ 0.05 vs WT.
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The results of an oral glucose tolerance test administered to GR ⫺/⫺ mice were consistent with improved insulin sensitivity (Fig. 3). It has been proposed that a glucagon receptor antagonist might be an effective anti-diabetic agent and a number of studies with glucagon analogs (4, 10) and with small molecular weight glucagon receptor antagonists (11, 17) have produced data consistent with this suggestion. The results of the present study support the proposal that reducing glucagon signaling through its receptor would serve to improve insulin sensitivity and reduce blood glucose levels. REFERENCES
FIG. 3. Mice were fasted overnight (⬃16 h) and then administered a 1 g/kg bolus of glucose by oral gavage. Blood samples were obtained from the retroorbital sinus of each animal at 0, 30, 60, and 90 min postdosing. Plasma glucose concentrations were measured as described under Materials and Methods.
firmed by RT-PCR and by [ 125I]glucagon binding studies using isolated liver membranes, data not shown.) Wakelam et al. (3) have proposed that in hepatocytes there are two signal transduction pathways that are activated by glucagon, one involving adenylate cyclase and cAMP kinase, the other utilizing phospholipase C and protein kinase C. They hypothesize that these independent pathways are activated by different receptors that they designate GR-2 and GR-1 respectively (by analogy with the vasopressin receptor system.) If the observations of the present study were interpreted according to this hypothesis, one might speculate that though the GR ⫺/⫺ mice lack the GR-2 receptor, the GR-1 receptor would still be present in these animals and capable of sensing, and attempting to compensate for, the drastic reduction in total glucagon receptor numbers. This hypothesis does not, however, explain the failure to detect measurable levels of [ 125I]glucagon binding on liver membranes prepared from GR ⫺/⫺ mice, unless the receptor density of GR-1 is itself very low. Glucagon administration to normal animals has been shown to have hypolipidemic effects, lowering plasma cholesterol levels (15, 16). In GR ⫺/⫺ mice fasting plasma cholesterol levels were slightly elevated relative to WT controls, though not significantly different from GR ⫹/⫹ animals (Table 3). This may imply that normal circulating levels of glucagon have a tonic hypocholesterolemic effect, which is mediated via glucagon receptors and so is lost in GR ⫺/⫺ mice.
1. Jelinek, L. J., Lok, S., Rosenberg, G. B., Smith, R. A., Grant, F. J., Biggs, S., Bensch, P. A., Kuijper, J. L., Sheppard, P. O., Sprecher, C. A., O’Hara, P. J., Foster, D., Walker, K. M., Chen, L. H. J., McKernan, P. A., and Kindsvogel, W. (1993) Expression cloning and signaling properties of the rat glucagon receptor. Science 259, 1614 –1616. 2. Rodbell, M., Krans, M. J., Pohl, S. L., and Birnbaumer, L. (1971) The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. J. Biol. Chem. 246, 1861–1871. 3. Wakelam, M. J. O., Murphy, G. J., Hruby, V. J., and Houslay, M. D. (1986) Activation of two signal transduction systems in hepatocytes by glucagon. Nature 328, 68 –71. 4. Unson, C. G., Gurzenda, E. M., and Merrifield, R. B. (1989) Biological activities of des-His 1[Glu 9]glucagon amide, a glucagon antagonist. Peptides 10, 1171–1177. 5. Sistare, F. D., Picking, R. A., and Haynes, J., R. C. (1985) Sensitivity of the response of cytosolic calcium in Quin-2-loaded rat hepatocytes to glucagon, adenine nucleosides, and adenine nucleotides. J. Biol. Chem. 260, 12744 –12747. 6. Dobbs, R. E., Sakurai, H., Sasaki, H., Faloona, G., Valverde, I., Baetens, D., Orci, L., and R. H., U. (1975) Glucagon: Role in the hyperglycemia of diabetes mellitus. Science 187, 544 –547. 7. Unger, R. H., and Orci, L. (1975) The essential role of glucagon in the pathogenesis of diabetes mellitus. Lancet 1, 14 –16. 8. Baron, A. D., Schaeffer, L., Shragg, P., and Kolterman, O. G. (1987) Role of hyperglucagonemia in the maintenance of increased rates of hepatic glucose output in Type 2 diabetics. Diabetes 36, 274 –283. 9. Reaven, G. M., Chen, Y.-D. I., Golay, I., Swislocki, A. L. M., and Jaspan, J. B. (1987) Documentation of hyperglucagonemia throughout the day in nonobese and obese patients with noninsulin-dependent diabetes mellitus. J. Clin. Endocrinol. Metab. 64, 106 –110. 10. Johnson, D. G., Goebel, C. U., Hruby, V. J., Bregman, M. D., and Trivedi, D. (1982) Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science 215, 1115–1116. 11. Parker, J. C., Mcpherson, R. K., Andrews, K. M., Levy, C. B., Dubins, J. S., Chin, J. E., Perry, P. V., Hulin, B., Perry, D. A., Inagaki, T., Dekker, K. A., Tachikawa, K., Sugie, Y., and Treadway, J. L. (2000) Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes. Diabetes 49, 2079 – 2086. 12. Roach, M. L., Stock, J. L., Byrum, R., Koller, B. H., and McNeish, J. D. (1995) A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human inflammation. Exp. Cell. Res. 221, 520 –525. 13. Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived
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stem cells: A general strategy for targeting mutations to nonselectable genes. Nature 336, 348 –352. 14. Hansen, L. H., Larger, E., Chaput, J.-C., Vissuzaine, C., Holst, J. J., Madsen, O. D., Yakishiji, F., De Meyts, P., Assan, R., and Nishimura, E. (1999) Identification of a glucagon receptor gene deletion mutation in a patient with hyperglucagonemia and pseudoadenomatous hyperplasia of pancreatic alpha-cells. In 81st Annual Meeting of the Endocrine Society, San Diego, CA, P3– 441.
15. Friedman, M., Byers, S. O., and Rosenman, R. H. (1971) Effect of glucagon on blood cholesterol levels in rats. Lancet 2, 464 – 466. 16. Byers, S. O., Friedman, M., and Elek, S. R. (1975) Further studies concerning glucagon-induced hypocholesterolemia. Proc. Soc. Exp. Biol. Med. 149, 151–157. 17. Petersen, K. F., Sullivan, J. T., Amatruda, J. M., Livingston, J. N., and Shulman, G. I. (1998) The effects of a specific glucagon antagonist on glucagon stimulated glucose production. Diabetologia 42(Suppl. 1), A42.
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Glycemic Control in Mice with Targeted Disruption of the Glucagon Receptor Gene Janice C. Parker, 1 Kim M. Andrews, Melanie R. Allen, Jeffrey L. Stock, and John D. McNeish Pfizer Global Research & Development, Groton Laboratories, Groton, Connecticut 06340
Received November 20, 2001
The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR ⴚ/ⴚ mice). Blood glucose levels were somewhat reduced in GR ⴚ/ⴚ mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude. © 2002 Elsevier Science
Key Words: glucagon; glycemia; glucagon receptor; glucose tolerance; pancreas; ␣-cell.
Glucagon is a 29-amino-acid hormone secreted by the ␣-cells of the pancreas. It has an important physiological role in the regulation of blood glucose levels and in other aspects of metabolism. Glucagon binding to specific receptors, principally on liver membranes, activates the glycogenolytic and gluconeogenic pathways, thereby increasing hepatic glucose production. The glucagon receptor is a 7-membrane spanning G-coupled receptor (1) that is known to be coupled to adenylate cyclase (2) and may also cause hydrolysis of inositol phospholipids (3, 4) and increases in intracellular Ca 2⫹ concentrations (5). There is evidence to indicate that the hyperglycemia that characterizes diabetes mellitus is a consequence of a bihormonal disorder, in which relatively insufficient 1 To whom correspondence and reprint requests should be addressed at Pfizer Global Research & Development, Groton Laboratories 8220-0375, Eastern Point Road, Groton, CT 06340. Fax: (860) 441-0548. E-mail: [email protected].
levels of insulin and excessive levels of glucagon together contribute to a net increase in hepatic glucose production (6, 7). Hyperglucagonemia has been shown to be present in Type 2 diabetic subjects (8, 9) and antagonists that interfere with the binding of glucagon to its receptor (10) or with its signal transduction (11) can reduce glucose output from liver cells (11) and reduce hyperglycemia in diabetic animals (10). To further examine the role of glucagon and of glucagon receptor function in glucose homeostasis we have generated mice in which the glucagon receptor gene was inactivated. This study describes the effects of this genetic intervention on pancreatic hormone levels and on glycemic control. MATERIALS AND METHODS Construction of the GR-KO targeting vector. A 1.4-kb murine glucagon receptor genomic fragment (corresponding to exons 1–3) was used as a probe to identify genomic clones from a DBA/1LacJ genomic phage library (Stratagene). The GR-KO targeting vector (Fig. 1) was constructed by cloning 8.4 kb of 5⬘ homology and 2.9 kb of 3⬘ homology into a pJNS2 (PGK-NEO/PGK-TK) backbone vector. The targeting vector replaced ⬃1.8 kb of the GR locus, corresponding to amino acids 56 –303 of GenBank Accession No. L38613, with PGK-NEO. Generation of GR-KO targeted embryonic stem cells. DBA/252 embryonic stem cells (12) were cultured on primary embryonic fibroblasts (PEFs) in DMEM containing high glucose, 15% heatinactivated fetal bovine serum, 0.2 mM L-glutamine, 0.1 mM 2-ME, 0.1 mM MEM nonessential amino acids, penicillin-streptomycin, and 400 U/ml recombinant murine leukemia inhibitory factor (SCML). The GR targeting vector (25 g) was electroporated at 1.0 ⫻ 10 8 embryonic stem cells/ml (0.4 ml total volume), and the electroporation was achieved with a BTX Electrocell Manipulator 600 (200 V, 50 F, 360 ⍀). Cells were then subjected to positive/negative selection with 200 g/ml G418 (geneticin; Life Technologies) and 2 M gancyclovir (Syntex Laboratories) by a published method (13). Gene targeting by homologous recombination was determined in surviving embryonic stem cell clones by Southern analysis using an 800-bp probe from a region 3⬘ of the 2.9-kb homology arm in conjunction with NsiI–XbaI digested embryonic stem cell DNA. The endogenous allele yielded a band hybridizing at 5.3 kb, whereas the targeted allele revealed a predicted RFLP at 3.7 kb due to the introduction of a novel XbaI restriction site from the GR targeting vector Homologously targeted embryonic stem cell clones were also screened for the incidence of multiple insertions by Southern analysis using a NEO
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FIG. 1. Schema employed to achieve homologous recombination of the murine glucagon receptor locus. The targeting vector was constructed by cloning 8.4 kb of 5⬘ homology and 2.9 kb of 3⬘ homology into a pJNS 2 (PGK-NEO/PGK-TK) backbone vector. Approximately 1.8 kb of endogenous genomic sequence was replaced by the neomycin gene. This created a deletion from glutamate 56 to glycine 303 within the native protein.
probe. Karyotyping analysis was completed on several targeted embryonic stem cell clones to rule out any chromosome abnormalities (Coriel Inst.). Generation of F2 ⫺/⫺ mice. GR ⫹/⫺ embryonic stem cells were microinjected into day 3.5 C57BL/6J blastocysts and resulting chimeric mice were backcrossed to DBA/1LacJ mates to identify germline transmission. GR ⫺/⫺ mice were derived from crosses of GR ⫹/⫺ males and females. F2 generation genotypes were determined by PCR using primer sets specific for either the endogenous or the targeted alleles as indicated in Figs. 2A and 2B.
Glucose assay, hormone assays, and triglyceride and cholesterol assays. Blood was collected from the retroorbital sinus. Fed animals had free access to chow, fasted animals had food withheld 14 –16 h prior to blood collection. Plasma glucose, triglyceride and cholesterol concentrations were measured enzymatically. Insulin and glucagon levels were measured by radioimmunoassay. Oral glucose tolerance test (OGTT). Mice were fasted overnight (⬃16 h) and then administered a 1 g/kg oral bolus of glucose. Blood samples were obtained from the retroorbital sinus of each animal at 0, 30, 60, and 90 min postdosing for glucose and insulin measurements.
FIG. 2. (A) Embryonic stem cell clones surviving selection were screened for homologous recombination by Southern blot analysis with a unique 3⬘-external probe to distinguish a wild-type 5.3-kb NsiI fragment from a 3.7-kb NsiI/XbaI fragment generated from the targeted allele. (B) Following microinjection of targeted embryonic stem cells into blastocyst stage DBA/1LacJ embryos, resulting offspring were subsequently genotyped by a PCR scheme as indicated on the diagram, employing specific primers to distinguish between the endogenous and targeted alleles. 840
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TABLE 2
Effect of Glucagon Gene Disruption on Plasma Glucose and Hormone Levels
Effect of Glucagon Gene Disruption on Pancreatic Hormone Content
WT
GR ⫹/⫹
GR ⫺/⫺
Plasma glucose (mg/dl) Fasting 159.0 ⫾ 6.5 139.3 ⫾ 5.0 99.5 ⫾ 5.2* ,• Fed 184.3 ⫾ 11.6 152.7 ⫾ 12.7 121.6 ⫾ 4.2* ,• Plasma insulin (ng/ml) Fasting 1.06 ⫾ 0.13 0.67 ⫾ 0.12 0.60 ⫾ 0.10 Fed 1.42 ⫾ 0.18 2.14 ⫾ 0.67 0.88 ⫾ 0.17 Plasma glucagon (pg/ml) Fasting 81.0 ⫾ 17.5 497.6 ⫾ 156.7* 5623.9 ⫾ 400.7* ,• Fed 82.9 ⫾ 11.5 905.1 ⫾ 149.7* 6417.2 ⫾ 420.6* ,•
Pancreatic weight (g) Glucagon content (g/g) Insulin content (g/g)
WT
GR ⫺/⫺
0.22 ⫾ 0.01 3.00 ⫾ 0.93 165.1 ⫾ 5.6
0.35 ⫾ 0.03* 131.56 ⫾ 29.62* 100.6 ⫾ 18.0
Note. Food was withheld for 14 –16 h prior to blood collection from the fasting animals. Insulin and glucagon were extracted from each whole pancreas acid-ethanol extraction and the hormone contents measured by radioimmunoassay as described under Materials and Methods. Data are means ⫾ SEM for measurements from three to five animals. * P ⬍ 0.05 vs WT.
Note. Food was withheld for 14 –16 h prior to blood collection from the fasting animals. Glucose insulin and glucagon concentrations were measured as described under Materials and Methods. Data are means ⫾ SEM for measurements from 4 to 10 animals. * P ⬍ 0.05 vs WT; • P ⬍ 0.05 vs GR ⫹/⫹ control.
Data analysis. All data represent means ⫾ SEM for measurements from 3 to 10 animals. Statistical significance was assessed by Student’s t test. Age-matched wild type DBA/1LacJ males were purchased from The Jackson Labs, Maine, and used as controls. Data presented is for male GR ⫺/⫺ and GR ⫹/⫹ animals, the latter group included as an additional control. Qualitatively similar results were obtained with the corresponding female animals (data not shown). All procedures involving animals were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.
RESULTS AND DISCUSSION Mice genetically modified to lack glucagon receptors (GR ⫺/⫺ mice) did not demonstrate any gross abnormalities of glucose homeostasis. Their plasma glucose levels, both under fed and fasting conditions, were somewhat lower than in either GR ⫹/⫹ mice or wild type mice of the same strain that had not been subject to genetic intervention (Table 1), but still fell within the range of normality. Plasma insulin levels appeared slightly lower in GR ⫺/⫺ mice, but this difference did not achieve statistical significance (Table 1). GR ⫺/⫺ mice had elevated levels of circulating glucagon, 2 orders of magnitude greater than the plasma concentrations present in wild-type mice (Table 1). Plasma glucagon levels were somewhat higher in GR ⫹/⫹ mice than in wild-type animals, suggesting that the manipulations to which the GR ⫹/⫹ mice were subjected may have resulted in a reduced level of glucagon receptor expression, with a compensatory increase in circulating glucagon. The large increase in the circulating concentration of glucagon in the GR ⫺/⫺ was associated with a corresponding increase in the pancreatic content of glucagon, which was 20-fold greater than in the correspond-
ing WT animals (Table 2). The overall size of the pancreata in the GR ⫺/⫺ mice was also significantly increased. The increase in wet weight and in glucagon content presumably reflects ␣-cell hyperplasia, necessary to sustain the extraordinarily high levels of glucagon observed in the circulation. The pancreatic insulin levels appeared somewhat lower in GR ⫺/⫺ than in WT mice, but this difference did not achieve statistical significance (Table 2). The phenotype observed in these mice is strikingly similar to that reported for a human subject with a splice site mutation of the glucagon receptor gene that resulted in non-functional glucagon receptors (14). As with the GR ⫺/⫺ mice, this individual had hyperglucagonemia and pancreatic hyperplasia together with normal plasma insulin levels and no impairment of glucose tolerance. The very large increase in plasma glucagon concentrations and in pancreatic glucagon content presumably represents a compensatory response that enables GR ⫺/⫺ to maintain normal or near-normal glycemia and lipidemia and would be an expected finding in animals with reduced levels of receptor. However, it is not clear what could be the mechanism for this adaptive response in the total absence of glucagon receptors (con-
TABLE 3
Effect of Glucagon Gene Disruption on Plasma Lipid Levels in Fasting Mice
Plasma cholesterol (mg/dl) Plasma triglycerides (mg/dl)
WT
GR ⫹/⫹
GR ⫺/⫺
54.8 ⫾ 3.2 101.3 ⫾ 11.1
58.9 ⫾ 8.6 92.8 ⫾ 17.0
66.0 ⫾ 3.8* 91.7 ⫾ 7.1
Note. Food was withheld for 14 –16 h prior to blood collection. Cholesterol and triglyceride concentrations were measured as described under Materials and Methods. Data are means ⫾ SEM for measurements from three to seven animals. * P ⬍ 0.05 vs WT.
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The results of an oral glucose tolerance test administered to GR ⫺/⫺ mice were consistent with improved insulin sensitivity (Fig. 3). It has been proposed that a glucagon receptor antagonist might be an effective anti-diabetic agent and a number of studies with glucagon analogs (4, 10) and with small molecular weight glucagon receptor antagonists (11, 17) have produced data consistent with this suggestion. The results of the present study support the proposal that reducing glucagon signaling through its receptor would serve to improve insulin sensitivity and reduce blood glucose levels. REFERENCES
FIG. 3. Mice were fasted overnight (⬃16 h) and then administered a 1 g/kg bolus of glucose by oral gavage. Blood samples were obtained from the retroorbital sinus of each animal at 0, 30, 60, and 90 min postdosing. Plasma glucose concentrations were measured as described under Materials and Methods.
firmed by RT-PCR and by [ 125I]glucagon binding studies using isolated liver membranes, data not shown.) Wakelam et al. (3) have proposed that in hepatocytes there are two signal transduction pathways that are activated by glucagon, one involving adenylate cyclase and cAMP kinase, the other utilizing phospholipase C and protein kinase C. They hypothesize that these independent pathways are activated by different receptors that they designate GR-2 and GR-1 respectively (by analogy with the vasopressin receptor system.) If the observations of the present study were interpreted according to this hypothesis, one might speculate that though the GR ⫺/⫺ mice lack the GR-2 receptor, the GR-1 receptor would still be present in these animals and capable of sensing, and attempting to compensate for, the drastic reduction in total glucagon receptor numbers. This hypothesis does not, however, explain the failure to detect measurable levels of [ 125I]glucagon binding on liver membranes prepared from GR ⫺/⫺ mice, unless the receptor density of GR-1 is itself very low. Glucagon administration to normal animals has been shown to have hypolipidemic effects, lowering plasma cholesterol levels (15, 16). In GR ⫺/⫺ mice fasting plasma cholesterol levels were slightly elevated relative to WT controls, though not significantly different from GR ⫹/⫹ animals (Table 3). This may imply that normal circulating levels of glucagon have a tonic hypocholesterolemic effect, which is mediated via glucagon receptors and so is lost in GR ⫺/⫺ mice.
1. Jelinek, L. J., Lok, S., Rosenberg, G. B., Smith, R. A., Grant, F. J., Biggs, S., Bensch, P. A., Kuijper, J. L., Sheppard, P. O., Sprecher, C. A., O’Hara, P. J., Foster, D., Walker, K. M., Chen, L. H. J., McKernan, P. A., and Kindsvogel, W. (1993) Expression cloning and signaling properties of the rat glucagon receptor. Science 259, 1614 –1616. 2. Rodbell, M., Krans, M. J., Pohl, S. L., and Birnbaumer, L. (1971) The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. J. Biol. Chem. 246, 1861–1871. 3. Wakelam, M. J. O., Murphy, G. J., Hruby, V. J., and Houslay, M. D. (1986) Activation of two signal transduction systems in hepatocytes by glucagon. Nature 328, 68 –71. 4. Unson, C. G., Gurzenda, E. M., and Merrifield, R. B. (1989) Biological activities of des-His 1[Glu 9]glucagon amide, a glucagon antagonist. Peptides 10, 1171–1177. 5. Sistare, F. D., Picking, R. A., and Haynes, J., R. C. (1985) Sensitivity of the response of cytosolic calcium in Quin-2-loaded rat hepatocytes to glucagon, adenine nucleosides, and adenine nucleotides. J. Biol. Chem. 260, 12744 –12747. 6. Dobbs, R. E., Sakurai, H., Sasaki, H., Faloona, G., Valverde, I., Baetens, D., Orci, L., and R. H., U. (1975) Glucagon: Role in the hyperglycemia of diabetes mellitus. Science 187, 544 –547. 7. Unger, R. H., and Orci, L. (1975) The essential role of glucagon in the pathogenesis of diabetes mellitus. Lancet 1, 14 –16. 8. Baron, A. D., Schaeffer, L., Shragg, P., and Kolterman, O. G. (1987) Role of hyperglucagonemia in the maintenance of increased rates of hepatic glucose output in Type 2 diabetics. Diabetes 36, 274 –283. 9. Reaven, G. M., Chen, Y.-D. I., Golay, I., Swislocki, A. L. M., and Jaspan, J. B. (1987) Documentation of hyperglucagonemia throughout the day in nonobese and obese patients with noninsulin-dependent diabetes mellitus. J. Clin. Endocrinol. Metab. 64, 106 –110. 10. Johnson, D. G., Goebel, C. U., Hruby, V. J., Bregman, M. D., and Trivedi, D. (1982) Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science 215, 1115–1116. 11. Parker, J. C., Mcpherson, R. K., Andrews, K. M., Levy, C. B., Dubins, J. S., Chin, J. E., Perry, P. V., Hulin, B., Perry, D. A., Inagaki, T., Dekker, K. A., Tachikawa, K., Sugie, Y., and Treadway, J. L. (2000) Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes. Diabetes 49, 2079 – 2086. 12. Roach, M. L., Stock, J. L., Byrum, R., Koller, B. H., and McNeish, J. D. (1995) A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human inflammation. Exp. Cell. Res. 221, 520 –525. 13. Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived
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stem cells: A general strategy for targeting mutations to nonselectable genes. Nature 336, 348 –352. 14. Hansen, L. H., Larger, E., Chaput, J.-C., Vissuzaine, C., Holst, J. J., Madsen, O. D., Yakishiji, F., De Meyts, P., Assan, R., and Nishimura, E. (1999) Identification of a glucagon receptor gene deletion mutation in a patient with hyperglucagonemia and pseudoadenomatous hyperplasia of pancreatic alpha-cells. In 81st Annual Meeting of the Endocrine Society, San Diego, CA, P3– 441.
15. Friedman, M., Byers, S. O., and Rosenman, R. H. (1971) Effect of glucagon on blood cholesterol levels in rats. Lancet 2, 464 – 466. 16. Byers, S. O., Friedman, M., and Elek, S. R. (1975) Further studies concerning glucagon-induced hypocholesterolemia. Proc. Soc. Exp. Biol. Med. 149, 151–157. 17. Petersen, K. F., Sullivan, J. T., Amatruda, J. M., Livingston, J. N., and Shulman, G. I. (1998) The effects of a specific glucagon antagonist on glucagon stimulated glucose production. Diabetologia 42(Suppl. 1), A42.
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