Glucagon receptor antibody completely suppresses type 1 diabetes phenotype without insulin by disrupting a novel diabetogenic pathway

Insulin monotherapy can neither maintain normoglycemia in type 1 diabetes (T1D) nor prevent the long-term damage indicated by elevated glycation products in blood, such as glycated hemoglobin (HbA1c). Here we find that hyperglycemia, when unaccompanied by an acute increase in insulin, enhances itself by paradoxically stimulating hyperglucagonemia. Raising glucose from 5 to 25 mM without insulin enhanced glucagon secretion ∼two- to fivefold in InR1-G9 α cells and ∼18-fold in perfused pancreata from insulin-deficient rats with T1D. Mice with T1D receiving insulin treatment paradoxically exhibited threefold higher plasma glucagon during hyperglycemic surges than during normoglycemic intervals. Blockade of glucagon action with mAb Ac, a glucagon receptor (GCGR) antagonizing antibody, maintained glucose below 100 mg/dL and HbA1c levels below 4% in insulin-deficient mice with T1D. In rodents with T1D, hyperglycemia stimulates glucagon secretion, up-regulating phosphoenolpyruvate carboxykinase and enhancing hyperglycemia. GCGR antagonism in mice with T1D normalizes glucose and HbA1c, even without insulin.Ninety years of insulin treatment in patients with type 1 diabetes (T1D) have made it clear that insulin alone cannot normalize glucose homeostasis or glycated hemoglobin (HbA1c) levels. Even optimally controlled patients may exhibit postprandial surges of glucose levels to three or four times normal (1, 2), which may explain why HbA1c levels below 6% are so rare in patients with T1D. Current thinking attributes these spikes in peripheral plasma glucose to insufficient uptake of incoming dietary glucose by peripheral target tissues as a result of a lack of insulin. As a consequence, they are often managed by a preprandial bolus of insulin and restriction of dietary carbohydrate. This strategy results in chronic iatrogenic hyperinsulinemia (3) in patients with “well-controlled” T1D and is responsible for a high incidence of hypoglycemic events, which can be life-threatening.In nondiabetic subjects, a glucose load suppresses glucagon levels by stimulating an acute transient rise in paracrine insulin from β-cells juxtaposed to the glucagon-producing α cells (4–6). This glucagon suppression converts the liver from an organ of glucose production to an organ of glucose storage (7). In T1D, paracrine insulin is lacking and is replaced by peripherally injected insulin. The resulting intraislet insulin concentrations are but a small fraction of the paracrine concentrations of undiluted insulin that suppress glucagon in nondiabetic subjects (8, 9). In 1974, it was reported that hyperglycemia paradoxically stimulates glucagon secretion in dogs with chemically induced diabetes (10). More recently, plasma glucagon concentrations were reported to rise, with a tripling of hepatic glucose production, in normal rats continuously infused with glucose at a constant rate (11). Thus, there is evidence that in the absence of adequate insulin, elevated glucose might stimulate glucagon production, which in turn aggravates hyperglycemia. In this setting, the liver would not be reprogrammed to store incoming glucose but, rather, would continue to produce glucose as if it were still in the unfed state (12). This may play a major role in postprandial hyperglycemia (10).Here we find that in T1D, hyperglycemia stimulates, rather than suppresses, glucagon secretion. This suggests that in T1D, a positive hormonal feedback loop enhances hyperglycemia by adding endogenously produced glucose to diet-derived glucose. If this is an important factor in the hyperglycemic surges that plague patients with T1D, then suppressing glucagon secretion or blocking glucagon action should eliminate the surges of hyperglycemia observed in T1D in mice.To measure the normal response of pancreatic islets to elevated glucose, pancreata were isolated from normal mice and perfused with 5 or 25 mM glucose. Glucagon concentrations were measured in the perfusate. Raising the glucose concentration fivefold decreased glucagon concentration in the perfusate approximately sixfold (Fig. 1A). To determine the effect of increased glucose concentration on glucagon secretion without paracrine insulin, we measured glucagon levels in the medium of cultured InR1-G9 α cells in 5, 10, and 25 mM glucose. The rise from 5 to 10 mM glucose caused an approximately threefold increase in glucagon secretion, and the rise from 10 to 25 mM caused another twofold rise (Fig. 1B). Because cultured cells may not reflect the behavior of native α cells in situ in T1D, we isolated pancreata from streptozotocin-induced insulin-deficient T1D rats and perfused them with 5, 10, and 25 mM glucose concentrations. When the perfusate glucose was increased from 5 to 10 mM, glucagon secretion increased fourfold (Fig. 1C). An increase in glucose from 10 to 25 mM increased glucagon secretion another fourfold. Insulin concentrations in all of these perfusates were below the detection limit of a radioimmune assay (RIA) (EMD Millipore). Previously, we reported that the streptozotocin treatment protocol we used resulted in the destruction of 93.4% of β cells (13).Fig. 1.In the absence of paracrine insulin, glucagon secretion increases in response to increases in glucose. (A) An increase from 5 to 25 mM in the glucose perfused into the isolated pancreata of nondiabetic mice causes profound suppression of glucagon secretion. ...The fact that elevations of glucose stimulated glucagon secretion in the absence of an acute paracrine insulin release suggested that in animals with T1D, any rise in glucose would stimulate glucagon secretion and give rise to a cycle of self-enhancing hyperglycemia (3, 14). To investigate the possibility of such a diabetogenic pathway, we compared plasma glucagon levels in insulin-treated NOD/ShiLtJ T1D mice during and between hyperglycemic surges (Fig. 1D). The mice were treated with 0.1 U Levemir twice daily, and blood glucose was measured in the morning 17 h after an insulin injection (high blood glucose) and in the afternoon 7 h after an insulin injection (low blood glucose) (Fig. 1E). In mice receiving this insulin regimen, glucagon averaged 138 ± 41 pg/mL and insulin 3.9 ± 1.1 ng/mL in samples in which glucose averaged 500 ± 37 mg/dL This glucagon concentration was significantly higher (P < 0.05) than the mean glucagon level of 55 ± 35 pg/mL, measured in samples from the same mice when their glucose levels averaged 130 ± 71 mg/dL and insulin averaged 14.3 ± 4.5 ng/mL These findings are consistent with a glucagon-mediated contribution to the surges of hyperglycemia.To assess directly the effect on the liver of the hyperglucagonemia accompanying hyperglycemia in the absence of endogenous insulin, we compared activation of key markers of glucagon action in liver. The phosphorylation of cAMP response element binding protein (CREB), a transducer of the glucagon signal, and the expression of a gluconeogenic glucagon target, phosphoenolpyruvate carboxykinase (PEPCK), were measured in T1D and nondiabetic mice. Compared with nondiabetic liver, there was a 3.5-fold elevation in phosphorylated CREB and a 2.5-fold increase in PEPCK expression in T1D livers (Fig. 2 A–C). To demonstrate that these differences were glucagon-mediated, we treated T1D mice with a single injection of 5 mg/kg mAb B (15), a fully human, antiglucagon receptor (GCGR) antibody drug candidate under development by REMD Biotherapeutics, Inc. (15–17). In mice treated with the monoclonal antibody mAb B, daily 10:00 AM blood glucose measurements averaged 85 ± 5 mg/dL and remained normoglycemic for 8 d (Fig. 2D), at which time livers were harvested. In the mAb B-treated T1D livers, phosphorylated CREB protein was reduced to nondiabetic levels and PEPCK protein expression was reduced below that of nondiabetic mice (Fig. 2 A–C). Thus, the activation of hepatic gluconeogenesis in T1D mice was a result of their hyperglucagonemia and disappeared when glucagon actions were blocked.Fig. 2.Glucagon action is chronically high in the livers of diabetic animals. (A) Hepatic markers of glucagon signaling (P-CREB) and of glucagon action (PEPCK) were measured by immunoblotting liver samples from nondiabetic and diabetic mice. (B) The ratio of ...If self-enhancing action of hyperglycemia is mediated by glucose-stimulated increase in glucagon in T1D, it follows that suppressing glucagon secretion should eliminate or reduce the problem. To test this, we placed T1D mice on a low dose of insulin (0.01 U twice daily) and then began continuous s.c. infusions of four peptides known to suppress glucagon directly or indirectly (18–22). A fifth, nonpeptidic suppressor (23), GABA, was given mixed in the chow. Each of the five reagents lowered glucagon levels, and in each case, this was accompanied by reduction of hyperglycemia from >600 mg/dL to 160 ± 75 mg/dL (P < 0.001; Fig. 3A). The average insulin concentrations in these samples were not correlated with either glucagon or glucose concentrations. With the exception of leptin, which reduced food intake by 50% compared with diabetic animals receiving insulin monotherapy, none of these glucagon suppressors caused a significant reduction in food intake.Fig. 3.Agents that suppress glucagon secretion or antagonize GCGR normalize glucagon action in liver and plasma glucose in diabetic mice. (A) Plasma glucose and glucagon levels in diabetic NOD mice (n= 3–10) treated with the agents shown. (B) Blood glucose ...If the glucose-lowering effects of glucagon suppressors resulted entirely from reduced glucagon secretion, rather than from off-target actions, therapy with a GCGR antibody should cause as dramatic an improvement as the glucagon-suppressing agents. Mice with chemically (streptozotocin)-induced T1D and a starting hyperglycemia of ∼325 ± 72 mg/dL were injected i.p. once each week with 7.5 mg/kg anti-GCGR antibody mAb Ac (15) (Fig. 3B), and blood glucose was measured weekly for 12 wk. Blood glucose concentrations returned to normal (∼90 mg/dL) in the mice treated with the antibody 1 wk after a single dose (first time point), and this normalization continued for the duration of treatment. Body weight did not change significantly between the control and antibody-treated groups of mice from the start to finish of the experiment. In the vehicle-treated control mice, blood glucose levels rose to 540 ± 70 mg/dL during the 12-wk study. At the end of this treatment, HbA1c was measured as an indication of chronic hyperglycemia. In the mice treated with mAb Ac, HbA1c levels were normal (4 ± 1%), whereas in the control mice, HbA1c averaged 11 ± 1% (Fig. 3C). GLP-1 averaged 3.64 ± 0.9 pmol/L in the control mice and 3.63 ± 0.52 pmol/L in the mice treated with antibody. By immunohistochemistry, the ratio of insulin-positive cells to glucagon-positive cells in islets observed in sections of pancreas taken from five control mice at the end of the study was 0.15, and using cells from five antibody treated mice, the ratio was 0.16. Because the ratio of β cells to α cells in wild-type mice is ∼6.0 (24), the ratios observed are those expected for severe ablation of β cells by streptozotocin. The fact that the ratio did not change between the two groups of animals indicates that the antibody treatment did not induce α-cell hyperplasia in this experiment.