Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice

OBJECTIVE

To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency.

RESEARCH DESIGN AND METHODS

We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr^−/−) mice and wild-type (Gcgr^+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction.

RESULTS

Gcgr^+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr^−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr^+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower.

CONCLUSIONS

We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.The development of a radioimmunoassay for glucagon (1) established the hormonal status of this peptide (2), originally considered to be a contaminant of the insulin extraction process. Glucagon was immunocytochemically localized to pancreatic α-cells (3) and shown to be secreted in response to increased glucose need, as in starvation and exercise (4). By 1973, it was recognized that α-cell function was grossly abnormal in diabetes, particularly in type 1 diabetes (5). Here, β-cells are largely replaced by α-cells, and, without the inhibitory action of insulin, their secretion of glucagon is unrestrained, and glucagon action on the liver is unopposed. The result is a lethal hypercatabolic state. In 1973, the discovery of somatostatin (6), a potent inhibitor of glucagon secretion, made it possible to demonstrate the essential role of glucagon in the metabolic phenotype of type 1 diabetes (7–10).Those studies led to a search for a therapeutic suppressor of diabetic hyperglucagonemia or blocker of its action on the liver. In the 37 years since the discovery of somatostatin, only one other potent glucagon-suppressing substance, leptin, has been identified (11,12). By contrast, inactivators of glucagon have been less elusive. High affinity antiglucagon antibodies have benefited diabetic animals (13), as have a variety of molecules that block binding of glucagon to the glucagon receptor and/or block its signaling (14–18). Diabetic mice with glucagon receptor knockout (19) or mice treated with Gcgr antisense oligonucleotide similarly benefit from the elimination of glucagon action (20,21). Although all of the foregoing reports demonstrate that abrogation of glucagon action reduces hepatic overproduction of glucose, a potential therapeutic asset in diabetes management, none of the foregoing diabetic models studied thus far have been totally insulin-deficient, as in type 1 diabetes. In type 1 diabetes, islets are virtually devoid of β-cells, and are largely made up of hyperplastic α-cells. In contrast to normal α-cells, which are restrained by the high local concentrations of intraislet insulin (22), type 1 diabetic α-cells are unregulated, which results in inappropriate hyperglucagonemia (5). Moreover, with no insulin to oppose the hepatic actions of the hyperglucagonemia, unrestrained glycogenolysis, gluconeogenesis, ketogenesis, and hypercatabolism lead rapidly to ketoacidosis, cachexia, coma, and death.An essential role of hyperglucagonemia in the pathogenesis of this lethal syndrome has long been suspected but never fully proven. Recent studies of adenovirally induced hyperleptinemia in type 1 diabetic mice (12) indicate that glucagon suppression normalizes all metabolic parameters for more than a month despite a total absence of insulin. More recently, the same antidiabetic efficacy has been demonstrated with recombinant leptin (11,23). However, leptin-induced actions other than suppression of glucagon, such as increased insulin-like growth factor-1 (IGF-1) (12) and insulin-like growth factor-binding protein-2 (IGFBP-2) (23), may have contributed. To obtain unassailable proof that glucagon action by itself causes the lethal consequences of insulin deficiency, we induced insulin deficiency in glucagon receptor-null (Gcgr^−/−) mice. Gcgr^−/− mice were treated with streptozotocin (STZ), the most commonly used β-cytotoxins in rodents, in an effort to achieve complete insulin deficiency in the complete absence of glucagon activity. We compared the metabolic phenotype of complete β-cell destruction in mice in which glucagon action had been transgenically abrogated by knockout of the glucagon receptor (24). Because Gcgr^−/− mice are resistant to STZ-induced β-cell destruction (24), it was necessary to use a double dose of the β-cell poison STZ. Despite β-cell destruction equivalent to the wild-type (Gcgr^+/+) mice, Gcgr^−/− remained free of all manifestations of insulin deficiency.