Previous attempts to minimize the contribution of the compen

Previous attempts to minimize the contribution of the compensatory changes due to chronic loss of Gcgr, have used temporally-controlled reduction of glucagon levels by alpha-cell ablation, glucagon immunoneutralization, and the use of small molecule or antibody glucagon receptor antagonists [10], [35]. Similar to our results, most of these acute disruptions of glucagon signaling did not mitigate the hyperglycemia of STZ diabetes [35]. In Cy3.5 NHS ester (non-sulfonated) to these previously described methods, we used the “cre-loxP” approach to temporally control the disruption of Gcgr in adulthood upon treatment with tamoxifen. In contrast to what occurs with alpha-cell deletion and pharmacologic approaches, mice with TMX-induced Gcgr deletion completely lack a hyperglycemic response to a Gcgr agonist, ensuring near total time-controlled loss of GCGR function in the liver. The literature points to the liver as the organ mediating most of the contribution of GCGR signaling to whole-body glucose control in mice. For example, liver-specific CGGR KO mice faithfully recapitulate the glucose metabolism and other phenotypical characteristics, including alpha cell hyperplasia, typical of mice with congenital global GCGR deletion [12]. Furthermore, re-expression of GCGR in the liver of congenital, global GCGR KO mice prevents the protection from hyperglycemia following STZ injection [4]. Importantly, our model recapitulates hallmarks of congenital deletion of Gcgr, including reduced baseline blood glucose, improved glucose tolerance and loss of a hyperglycemic response following a challenge with a Gcgr agonist, as soon as one week after disruption of Gcgr expression. The comparison of short versus long-term Gcgr deletion, specifically in STZ-treated mice, reveals a clear influence of the duration of Gcgr disruption in the protection from hyperglycemia. Thus, while the deletion of the Gcgr for 6 weeks provides near complete protection from hyperglycemia after STZ, short term Gcgr deletion in adult mice previously treated with STZ provides only comparatively modest protection. While the compensations to loss of GCGR were qualitatively similar in the 2 and 6 week knockout models based on our survey of relevant gene expression, there were quantitative difference as exemplified by the circulating levels of GLP-1 and FGF21. Moreover, the relative correction of hyperglycemia with acute and chronic Gcgr deletion did correspond with the marked reductions in Pck1 and G6pc and increase in Gck expression in the 6 week knockout animals. These results suggest a gradual change in physiology after loss of GCGR signaling with a critical accumulation necessary for the full protection from insulinopenic diabetes. Previous studies suggest that some of the compensatory mechanisms in response to Gcgr deletion act by increasing insulin action. Hence, Gcgr mice exhibit increased insulin sensitivity [36], and treatment with a monoclonal antibody against Gcgr reduces insulin requirements in T1DM humans [37]. Our data suggest that factors other than different levels of basal plasma c-peptide (insulin) or insulin signaling measured as pAKT levels must contribute to the increased insulin action due to loss of GCGR signaling. Since suppression of mTOR signaling is one of the mechanisms whereby glucagon opposes the action of insulin in the liver ( [28], we postulated that chronic Gcgr loss of function could result in insulin-like activity by promoting increased mTOR signaling. Indeed, some of the changes in gene expression seen following chronic loss of Gcgr are consistent with increased mTORC activity, including the reduction in Igfbp1 and the increase in Gck [30], as well as the increase in lipogenic genes [38] and the increase in Fgf21 [39]. In addition, a potential contribution of extrahepatic mTOR signaling was supported by the increase in plasma amino acid levels and the suppression of the hepatic expression of transporters and enzymes involved in amino acid metabolism exhibited by mice lacking glucagon [40] or Gcgr expression [18]. These were confirmed by our gene expression analysis. Amino acids are one of the principal activators of the mTOR pathway, and recent evidence strongly suggests that hyperaminoacidemia contributes to the alpha-cell hyperplasia exhibited by rodent models of reduced glucagon levels [11], [41] or Gcgr signaling [2], [12], as well as those exhibited by human subjects with Gcgr mutations [16], [17], [42]. Thus, alpha cell proliferation can be greatly blunted by chronic administration of the mTOR inhibitor rapamycin [16], [17], [18]. We adopted the rapamycin administration protocol used by those groups to determine the impact of chronic reduction of mTORC signaling on the development of compensatory mechanisms leading to improved glucose control in mice with long-term Gcgr deletion in conditions of acute reduction of insulin action achieved by repeated injections with the insulin antagonist S961. In contrast to the expected hyperglycemia exhibited by the S961-treated control counterparts [43], chronic treatment with rapamycin did not change blood glucose very much in Gcgr deficient mice. In fact, mice lacking GCGR had a minimal glycemic response to S961 alone, suggesting the contribution of an insulin-independent mechanism to glycemic control in mice with long-term Gcgr deletion. These results also suggest that additional mechanisms contributing to the improved glucose control exhibited by long-term deletion of Gcgr develop and function under conditions of reduced mTORC signaling and independently of differences in circulating insulin. This notion is consistent with the maintenance of normoglycemia and survival under insulinopenic conditions to a degree that cannot be explained solely by the action the residual insulin [5], [7], [10]. On the other hand, the fact that loss of Gcgr does not prevent hyperglycemia and death under conditions of complete loss of insulin [8], [9] demonstrates that such additional mechanisms are impotent preventing hyperglycemia in conditions of nearly absent insulin.