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. 2016 May 30;5(8):731-736.
doi: 10.1016/j.molmet.2016.05.014. eCollection 2016 Aug.

Glucagon receptor gene deletion in insulin knockout mice modestly reduces blood glucose and ketones but does not promote survival

Ursula H Neumann  1 , Jessica S S Ho  2 , Majid Mojibian  3 , Scott D Covey  4 , Maureen J Charron  5 , Timothy J Kieffer  6
Affiliations

Glucagon receptor gene deletion in insulin knockout mice modestly reduces blood glucose and ketones but does not promote survival

Ursula H Neumann et al. Mol Metab. .

Abstract

Objective: It has been thought that the depletion of insulin is responsible for the catabolic consequences of diabetes; however, evidence suggests that glucagon also plays a role in diabetes pathogenesis. Glucagon suppression by glucagon receptor (Gcgr) gene deletion, glucagon immunoneutralization, or Gcgr antagonist can reverse or prevent type 1 diabetes in rodents suggesting that dysregulated glucagon is also required for development of diabetic symptoms. However, the models used in these studies were rendered diabetic by chemical- or immune-mediated β-cell destruction, in which insulin depletion is incomplete. Therefore, it is unclear whether glucagon suppression could overcome the consequence of the complete lack of insulin.

Methods: To directly test this we characterized mice that lack the Gcgr and both insulin genes (GcgrKO/InsKO).

Results: In both P1 pups and mice that were kept alive to young adulthood using insulin therapy, blood glucose and plasma ketones were modestly normalized; however, mice survived for only up to 6 days, similar to GcgrHet/InsKO controls. In addition, Gcgr gene deletion was unable to normalize plasma leptin levels, triglycerides, fatty acids, or hepatic cholesterol accumulation compared to GcgrHet/InsKO controls.

Conclusion: Therefore, the metabolic manifestations associated with a complete lack of insulin cannot be overcome by glucagon receptor gene inactivation.

Keywords: Gcgr, glucagon receptor; Glucagon; Glucose metabolism; Het, heterozygous; Ins1, insulin 1; Ins2, insulin 2; InsKO, insulin knockout; Insulin; KO, knockout; Lipid metabolism; Mice; P, post-natal day; STZ, streptozotocin; Type 1 diabetes; WT, wildtype.

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Figures

Figure 1
Figure 1
Gcgr gene deletion does not promote survival in InsKO pups. Ins1KO pups with 0–1 alleles of Gcgr and 0–2 alleles of Ins2 were tracked twice daily for body weight (A) and survival (B) for 8 days after birth. For A statistical analysis was not performed, for B no statistical differences were observed between GcgrHet/InsKO and GcgrKO/InsKO pups. Data are graphed as individual pups (A) and % survival (B), n = 6–17.
Figure 2
Figure 2
Gcgr gene deletion modestly improves body weight, blood glucose and plasma ketones but does not normalize plasma leptin, triglycerides, fatty acids or hepatic cholesterol accumulation in InsKO pups. On P1, Ins1KO pups with 0–1 alleles of Gcgr and 0–2 alleles of Ins2 were harvested to measure body weight (A), trunk blood was collected to measure blood glucose (B) and plasma β-hydroxybutyrate (C), glucagon (D), leptin (E), triglycerides (F), fatty acids (G), glycerol (H), and cholesterol (I), and liver was collected to quantify hepatic cholesterol (J) and triglycerides (K). Different superscripts (a, b, c) are significantly different from each other within each graph. Statistical analysis was not performed on leptin measurements (D) as some samples were below the limit of detection. Data are mean ± SEM, n = 19–59 for A & B, n = 6 for C–K.
Figure 3
Figure 3
Gcgr gene deletion modestly lowers blood glucose but does not promote survival in adult InsKO mice. InsKO mice with and without Gcgr gene deletion were kept alive to 4 weeks of age using insulin injections and an islet transplant into the eye. On day 0 of the study, the eye containing islets was enucleated rendering the mice completely insulin deficient. Day 0 absolute body weight (A) and blood glucose levels (B) prior to enucleation. Following enucleation, survival (C) and day 0.75 (D), 1.25 (E) and 1.75 (F) body weight loss were measured. Blood glucose at day 0.75 (G), 1.25 (H), 1.75 (I) and throughout the study (H) are graphed. Different superscripts (a, b, c) are significantly different from each other within each graph. Statistical analysis was not performed on G and H as one blood glucose value fell above the limit of detection and was assigned a value of 33.3 mM, or in K because the n value decreased over time. Data are graphed as mean ± SEM (A, B, D–I), % survival (C), or individual mice (K), n = 4–7, throughout study InsKO mice reached humane endpoint therefore n is reduced in E, F, H, and I.
Figure 4
Figure 4
Gcgr gene deletion increases plasma glucagon, normalizes plasma ketones, and does not affect plasma cholesterol or fatty acids in adult InsKO mice. At humane endpoint (InsKO mice) or the end of the study (controls), cardiac blood was collected to measure plasma glucagon (A), β-hydroxybutyrate (B), cholesterol (C), and fatty acids (D). Different superscripts (a, b, c) are significantly different from each other within each graph. Data are mean ± SEM, n = 6–8.
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