Ghrelin does not impact the blunted counterregulatory response to recurrent hypoglycemia in mice

Abstract

Introduction: Recurrent episodes of insulin-induced hypoglycemia in patients with diabetes mellitus can result in hypoglycemia-associated autonomic failure (HAAF), which is characterized by a compromised response to hypoglycemia by counterregulatory hormones (counterregulatory response; CRR) and hypoglycemia unawareness. HAAF is a leading cause of morbidity in diabetes and often hinders optimal regulation of blood glucose levels. Yet, the molecular pathways underlying HAAF remain incompletely described. We previously reported that in mice, ghrelin is permissive for the usual CRR to insulin-induced hypoglycemia. Here, we tested the hypothesis that attenuated release of ghrelin both results from HAAF and contributes to HAAF.

Methods: C57BL/6N mice, ghrelin-knockout (KO) + control mice, and GhIRKO (ghrelin cell-selective insulin receptor knockout) + control mice were randomized to one of three treatment groups: a "Euglycemia" group was injected with saline and remained euglycemic; a 1X hypoglycemia ("1X Hypo") group underwent a single episode of insulin-induced hypoglycemia; a recurrent hypoglycemia ("Recurrent Hypo") group underwent repeated episodes of insulin-induced hypoglycemia over five successive days.

Results: Recurrent hypoglycemia exaggerated the reduction in blood glucose (by ~30%) and attenuated the elevations in plasma levels of the CRR hormones glucagon (by 64.5%) and epinephrine (by 52.9%) in C57BL/6N mice compared to a single hypoglycemic episode. Yet, plasma ghrelin was equivalently reduced in "1X Hypo" and "Recurrent Hypo" C57BL/6N mice. Ghrelin-KO mice exhibited neither exaggerated hypoglycemia in response to recurrent hypoglycemia, nor any additional attenuation in CRR hormone levels compared to wild-type littermates. Also, in response to recurrent hypoglycemia, GhIRKO mice exhibited nearly identical blood glucose and plasma CRR hormone levels as littermates with intact insulin receptor expression (floxed-IR mice), despite higher plasma ghrelin in GhIRKO mice.

Conclusions: These data suggest that the usual reduction of plasma ghrelin due to insulin-induced hypoglycemia is unaltered by recurrent hypoglycemia and that ghrelin does not impact blood glucose or the blunted CRR hormone responses during recurrent hypoglycemia.

Keywords: Blood glucose; counterregulation; ghrelin; hypoglycemia unawareness; hypoglycemia-associated autonomic failure; mouse models.

Figure 1

Validation of a recurrent hypoglycemia model of hypoglycemia-associated autonomic failure in C57BL/6N mice. (A) Table outlining the days on which mice in the “Euglycemia”, “1X Hypo”, and “Recurrent Hypo” groups were acclimated to i.p. injections (via handling and saline injections; highlighted in gold) and were administered saline on the final Day (“Euglycemia”), insulin on the final day (“1X Hypo”), or insulin for 5 days in a row (“Recurrent Hypo”). (B) Schematic diagram depicting the schedule on the days of antecedent hypoglycemia and the final Days. (C) Blood glucose levels, (D) plasma glucagon, (E) plasma epinephrine, (F) plasma norepinephrine, and (G) plasma ghrelin measured on the final day of the “Euglycemia”, “1X Hypo” and “Recurrent Hypo” protocols at 30 min post i.p. injection of saline or insulin. Data are represented as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. n=9-12. Statistically significant differences are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns represents no statistical significance.

Figure 2

Effect of ghrelin deletion on blood glucose and the CRR following recurrent hypoglycemia. (A) Blood glucose levels, (B) plasma glucagon, (C) plasma epinephrine, (D) plasma norepinephrine, and (E) plasma ghrelin at 30 min post i.p injection of saline or insulin on the final day of the “Euglycemia”, “1X Hypo”, and “Recurrent Hypo” protocols. Data are represented as mean ± SEM and were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test. n=8-13. Effects of genotype, treatment, and the interaction between the two are listed above the panels whereas post-hoc analysis results appear as connecting lines and asterisks above the bars. Statistically significant differences are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns represents no statistical significance.

Figure 3

Effect of raising plasma ghrelin as a result of ghrelin cell-selective IR deletion on blood glucose and the CRR to recurrent hypoglycemia. (A) Blood glucose levels, (B) plasma glucagon, (C) plasma epinephrine, (D) plasma norepinephrine, and (E) plasma ghrelin at 30 min post i.p injection of saline or insulin on the final day of the “Euglycemia”, “1X Hypo”, and “Recurrent Hypo” protocols. Data are represented as mean ± SEM and were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test. n=9-12. Effects of genotype, treatment, and the interaction between the two are listed above the panels whereas post-hoc analysis results appear as connecting lines and asterisks above the bars. Statistically significant differences are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns represents no statistical significance.