Partial blockade of nicotinic acetylcholine receptors improves the counterregulatory response to hypoglycemia in recurrently hypoglycemic rats

Abstract

Recurrent exposure to hypoglycemia can impair the normal counterregulatory hormonal responses that guard against hypoglycemia, leading to hypoglycemia unawareness. This pathological condition known as hypoglycemia-associated autonomic failure (HAAF) is the main adverse consequence that prevents individuals with type 1 diabetes mellitus from attaining the long-term health benefits of tight glycemic control. The underlying molecular mechanisms responsible for the progressive loss of the epinephrine response to subsequent bouts of hypoglycemia, a hallmark sign of HAAF, are largely unknown. Normally, hypoglycemia triggers both the release and biosynthesis of epinephrine through activation of nicotinic acetylcholine receptors (nAChR) on the adrenal glands. We hypothesize that excessive cholinergic stimulation may contribute to impaired counterregulation. Here, we tested whether administration of the nAChR partial agonist cytisine to reduce postganglionic synaptic activity can preserve the counterregulatory hormone responses in an animal model of HAAF. Compared with nicotine, cytisine has limited efficacy to activate nAChRs and stimulate epinephrine release and synthesis. We evaluated adrenal catecholamine production and secretion in nondiabetic rats subjected to two daily episodes of hypoglycemia for 3 days, followed by a hyperinsulinemic hypoglycemic clamp on day 4. Recurrent hypoglycemia decreased epinephrine responses, and this was associated with suppressed TH mRNA induction (a measure of adrenal catecholamine synthetic capacity). Treatment with cytisine improved glucagon responses as well as epinephrine release and production in recurrently hypoglycemic animals. These data suggest that pharmacological manipulation of ganglionic nAChRs may be promising as a translational adjunctive therapy to avoid HAAF in type 1 diabetes mellitus.

Keywords: cytisine; epinephrine release; hypoglycemia-associated autonomic failure; partial nicotinic acetylcholine receptor agonists; tyrosine hydroxylase.

Fig. 1.

Effect of cytisine on epinephrine release and adrenal tyrosine hydroxylase (TH) mRNA levels: dose response. Experimental groups are as follows: Sal (saline-injected animals), 0.3 Cyt (animals injected ip with 0.3 mg/kg cytisine), 1 Cyt (1 mg/kg cytisine), 3 Cyt (3 mg/kg cytisine), and Nic (1 mg/kg nicotine). Each dose/drug was given twice daily, for a total of 7 episodes, to mimic the HAAF protocol. On day 4 the catheters were extended out of the cages, and arterial blood samples were collected stress free before and every 30 min after the last drug application (iv) for a total of 2 h. A: the results (plasma epinephrine values) are from 2 independent experiments (n = 6) and are presented as means ± SE. *P ≤ 0.05, Cyt vs. Sal; **P ≤ 0.002, Nic vs. Cyt. B: for RNA analyses, animals were euthanized 5 h after treatment and changes in adrenal medullary TH gene expression analyzed by Northern blots.

Fig. 2.

Schematic diagram depicting the main protocol used in these experiments, designed to determine the effect of cytisine given before each insulin treatment. Experimental groups are as follows: RH, recurrent hypoglycemic animals; RS, recurrent saline controls; RC, recurrent cytisine controls; CRH, animals who received cytisine before each insulin treatment. ON, overnight.

Fig. 3.

Plasma glucose levels during each antecedent treatment period. Results are presented as means ± SE; n = 9.

Fig. 4.

A: plasma glucose concentrations during the hyperinsulinemic hypoglycemic glucose clamp. B: glucose infusion rates during the clamp. Results are presented as means ± SE; n ≤ 9.

Fig. 5.

Effect of cytisine pretreatment on counterregulatory hormone release during insulin-induced recurrent hypoglycemia. Plasma epinephrine (A), norepinephrine (B), glucagon (C), and corticosterone (D) responses during hypoglycemic clamp in RS (maximal response group), RH (hypoglycemia-associated autonomic failure group), RC, and CRH. The results are summarized from 3 independent experiments (n = 9) and are presented as means ± SE. *P ≤ 0.05; **P ≤ 0.002 RH vs. RS; ^xP ≤ 0.05 and ^xxP ≤ 0.002, RH vs. CRH.

Fig. 6.

Relative adrenal TH mRNA levels correlate with the magnitude of the epinephrine response. A: after the hypoglycemic clamp the animals were provided food and euthanized 3.5 h later. The results (Northern blot) are summarized from 2 independent experiments (n = 6) and are calculated as fold induction from CON (absolute control; nontreated animals) and given as means ± SE. B: animals (n = 3 for each group) were maintained in hypoglycemic state until euthanization (5 h after the initiation of hypoglycemic clamp). *P ≤ 0.05.