The purpose of this study was to assess the role of the hyperglucagonemia of total insulin deficiency in the lethal catabolic consequences of the disease and to determine if beneficial effects of glucagon blockade are sufficient and safe enough to qualify as a potential therapeutic target in human type 1 diabetes. This idea was proposed previously (9
) when somatostatin, the first glucagon-suppressing agent to be discovered (6
), was found to prevent the acute metabolic consequences of complete insulin lack (7
). The search for more practical suppressors or antagonists of glucagon has been reenergized by the recent demonstration that leptin can suppress diabetic hyperglucagonemia and reverse the catabolic effects of complete insulin deficiency for extended periods without apparent negative consequences (11
Although leptin is very effective in suppressing the hyperglucagonemia and in reversing the metabolic manifestations of type 1 diabetes (11
), the many other actions of the adipocyte hormone make it impossible to assume that suppression of glucagon by itself accounts for the remarkable remission of all clinical and laboratory manifestations of total insulin deficiency. The Gcgr−/−
mice of Gelling et al. (24
) provided an ideal model in which to assess the immediate and longer-term benefits and risks of permanent elimination of glucagon action without other actions of leptin unrelated to α-cell suppression, such as food restriction and lipopenia. We were able to overcome the previously reported resistance of Gcgr−/−
mice to STZ destruction of β-cells by using higher doses of STZ (24
). The β-cell destruction was as complete as in Gcgr+/+
controls, as evidenced by insulin assays of plasma, morphometric comparison of pancreata for insulin-positive cells, and qRT-PCR measurements of pancreatic preproinsulin mRNA. This enabled an assessment of the effects of complete elimination of glucagon action on the phenotype of mice with total insulin deficiency.
Whereas all STZ-treated Gcgr+/+
diabetic mice developed severe hyperglycemia, ketosis, and cachexia and had to be killed for humane reasons ~30 days after induction of diabetes, none of the insulin-deficient Gcgr−/−
mice developed any laboratory or clinical evidence of insulin deficiency. Without exception, all STZ-treated Gcgr−/−
mice appeared to be in a seemingly normal state of health after the high dose of β-cytotoxin for at least 6 weeks. This indicates that in the absence of glucagon action, insulin deficiency in mice is a silent disorder without overt metabolic or clinical manifestations, confirming an earlier preliminary observation in alloxan-treated mice (11
). It also suggests that glucagon suppression and/or antagonism may be an extremely useful strategy to treat type 1 diabetes.
Other than fasting hypoglycemia, no significant undesirable side effects of chronic blockade of glucagon action were observed in this brief study. The marked α-cell hyperplasia observed in Gcgr−/−
) has led to concerns that long-term glucagon receptor blockade could predispose to glucagonoma. However, thus far glucagonoma has not been observed in any of the Gcgr−/−
mice followed for up to 2 years (M.J.C., unpublished observation). Another theoretical hazard is the possibility of predisposition to lactic acidosis based on the high blood lactate levels in the Gcgr−/−
mice. This is attributed to reduced gluconeogenesis secondary to downregulation of gluconeogenic genes. A third potential issue relates to the role of glucagon in amino acid homeostasis. Amino acid deficiency has been described in glucagonoma patients (33
), raising the possibility that Gcgr−/−
mice might have high amino acid levels, as reported by Boden et al. (31
) in humans in whom glucagon was suppressed by somatostatin. For this reason, we measured amino acid levels in the livers of our normal and STZ-treated Gcgr+/+
mice. As shown in , eight of the nine hepatic amino acids measured were significantly higher in Gcgr−/−
mice with intact β-cells than in normal Gcgr+/+
mice. But no such differences occurred in STZ-treated mice, suggesting that insulin action, when unopposed by glucagon, was responsible for the increase.
By far the most surprising result of the study was the normal glucose tolerance of insulin-deficient Gcgr−/− mice. Currently, it is believed that it is the action of glucose-stimulated insulin secretion on hepatic glucose balance and glucose uptake in muscle and fat that is the essential determinant of glucose tolerance, with glucagon suppression playing a supporting role. Here we find that both oral and intraperitoneal glucose tolerance in Gcgr−/− mice with β-cell destruction is as normal as in Gcgr+/+mice with intact islets, even though no rise in insulin was detected in peripheral plasma of the former. Although we did not measure portal vein insulin levels, the lack of preproinsulin mRNA in the mice that received double-dose STZ argues against undetected insulin reaching the liver. The presence of residual granules in STZ-treated pancreata does not necessarily signify a residual insulin source because insulin granules may persist in apoptotic β-cells or in macrophages after all functioning β-cells have been destroyed. Certainly in wild-type mice, neither the “residual” insulin in the pancreas nor the low levels of “plasma insulin” were able to prevent a ketoacidotic death. The lack of preproinsulin mRNA detectable by qRT-PCR, which is far more sensitive and specific than either of the immunologic techniques, suggests that the STZ-treated mice were unable to produce insulin.
It seemed possible that the insulin-like responses were caused by an increase in a hormone with insulinometic activity. But neither leptin (not shown) (11
) nor IGF-1 rose significantly in response to the glucose challenge. Alternatively, the elevated glucagon-like peptide 1 levels might have increased muscle glucose uptake (32).
Recent studies by Meyer et al. (35
) indicate that in normal humans 90% of ingested glucose is taken up by liver, muscle, brain, and kidney, and hepatic glucose release declines 82%. Except for the brain, red cells, and possibly the kidney, glucose uptake is considered to be insulin-mediated. To account for the perfectly normal glucose tolerance in insulin-deficient mice with congenital lack of glucagon action, one can posit that insulin action during glucose absorption is largely directed toward overcoming the hepatic actions of glucagon during the preglucose fast. In this case, if glucagon action on the liver is absent, there is little, if anything, for insulin to do because the liver is already in a permanent storage mode. Therefore, when the liver has never experienced the action of glucagon, as in the Gcgr−/−
mice, glucose disposition without insulin is no different than when both hormones are normally active (36
). It should also be noted that fasting and fed FFA levels () were both lower in insulin-deficient Gcgr−/−
mice than in insulin-deficient Gcgr+/+
mice, suggesting that, at least when unopposed by insulin, glucagon may have lipolytic action in adipocytes, as it does in hepatocytes.
Finally, a persistent but fallacious argument against an essential role of glucagon in diabetes is the occurrence of diabetes in totally depancreatized humans and animals. It is not widely appreciated that when totally depancreatized animals or humans are deprived of insulin, there is an increased production of glucagon from extrapancreatic α-cells (37
) in the stomach and upper small bowel (38
). Suppression of the extrapancreatic hyperglucagonemia restores hyperglycemia to normal (44
Taken together these findings indicate in mice that type 1 diabetes can be converted into an asymptomatic, benign, noncatabolic, insulin-independent disorder by elimination of glucagon action. These studies support the clinical utility of the development of potent Gcgr antagonists and/or glucagon suppressors capable of eliminating the lethal glucagon-dependent component of type 1 diabetes (45