Repetitive hypoglycemia reduces activation of glucose-responsive neurons in C1 and C3 medullary brain regions to subsequent hypoglycemia

Abstract

The impaired ability of the autonomic nervous system to respond to hypoglycemia is termed “hypoglycemia-associated autonomic failure” (HAAF). This life-threatening phenomenon results from at least two recent episodes of hypoglycemia, but the pathology underpinning HAAF remains largely unknown. Although naloxone appears to improve hypoglycemia counterregulation under controlled conditions, hypoglycemia prevention remains the current mainstay therapy for HAAF. Epinephrine-synthesizing neurons in the rostroventrolateral (C1) and dorsomedial (C3) medulla project to the subset of sympathetic preganglionic neurons that regulate peripheral epinephrine release. Here we determined whether or not C1 and C3 neuronal activation is impaired in HAAF and whether or not 1 wk of hypoglycemia prevention or treatment with naloxone could restore C1 and C3 neuronal activation and improve HAAF. Twenty male Sprague-Dawley rats (250–300 g) were used. Plasma epinephrine levels were significantly increased after a single episode of hypoglycemia ( n = 4; 5,438 ± 783 pg/ml vs. control 193 ± 27 pg/ml, P < 0.05). Repeated hypoglycemia significantly reduced the plasma epinephrine response to subsequent hypoglycemia ( n = 4; 2,179 ± 220 pg/ml vs. 5,438 ± 783 pg/ml, P < 0.05). Activation of medullary C1 ( n = 4; 50 ± 5% vs. control 3 ± 1%, P < 0.05) and C3 ( n = 4; 45 ± 5% vs. control 4 ± 1%, P < 0.05) neurons was significantly increased after a single episode of hypoglycemia. Activation of C1 ( n = 4; 12 ± 3%, P < 0.05) and C3 ( n = 4; 19 ± 5%, P < 0.05) neurons was significantly reduced in the HAAF groups. Hypoglycemia prevention or treatment with naloxone did not restore the plasma epinephrine response or C1 and C3 neuronal activation. Thus repeated hypoglycemia reduced the activation of C1 and C3 neurons mediating adrenal medullary responses to subsequent bouts of hypoglycemia.

Slow but Steady—The Responsiveness of Sympathoadrenal System to a Hypoglycemic Challenge in Ketogenic Diet-Fed Rats

“…While we did not directly measure brain tissue consumption of BHB, we can speculate on the basis of BHB measurements in the cerebral venous blood that central glucose-inhibited neurons (i.e., neurons that increase their firing rate in the presence of low glucose) were not sufficiently activated until a substantial amount of BHB disappeared from the circulation, which manifested as a delayed increase in ASNA and epinephrine release. One of the important brain nuclei necessary for the CRR and specifically for the sympathetically mediated epinephrine secretion is the C1 area in the rostral ventrolateral medulla [ 24 , 41 ]. In our study, a smaller proportion of activated C1 neurons in KD-fed rats may indicate a delayed response to hypoglycemia due to the time-course of Fos expression, which is reported to peak at 2 h post stimulus in the medulla [ 42 ]; however, in the absence of a longer time-course (e.g., 3 h post insulin), lower (but not delayed) activation of C1 neurons cannot be excluded.…”

“…Brainstem ( n = 3 per treatment group) was sectioned coronally at 40 µm in 1:5 series. Free-floating sections underwent routine immunohistochemistry [ 24 ]. Primary antibodies for adrenal gland immunohistochemistry were anti-PNMT (phenylethanolamine- N -methyltransferase, a marker for epinephrine-producing cells [ 30 ]; 1:1000, rabbit polyclonal, generated and characterized by P.R.…”

“…For that reason, and the fact that glucagon injections are less effective in patients who follow a low-carbohydrate diet [ 22 ], our study focused specifically on the role of the sympathoadrenal system and epinephrine, which are expected to be the major contributors to the CRR in the setting of type 1 diabetes. Therefore, the aim of this study was to test the effects of dietary ketosis, induced by a 3 week KD, on components of the sympathoadrenal CRR to severe insulin-induced hypoglycemia in healthy, nondiabetic rats, by measuring adrenal sympathetic nerve activity (ASNA), adrenal gland activity, and epinephrine release, as well as the activation state of catecholaminergic C1 neurons in the brainstem, which are glucose-sensitive and involved in the hypoglycemic CRR [ 23 , 24 ]. The adrenal sympathetic nerve, unlike renal, lumbar, or muscle sympathetic nerves, is activated by hypoglycemia independent of elevated insulin levels [ 25 ] and can be acutely recorded in anesthetized rats [ 26 ].…”

Pentobarbital Anesthesia Suppresses the Glucose Response to Acute Intermittent Hypoxia in Rat

“…It is well established that acute IHx in rodents under anesthesia produces long lasting sympathoexcitation ( Dick et al, 2007 ; Xing and Pilowsky, 2010 ; Roy et al, 2018 ; Farnham et al, 2019 ), which can be blocked at the level of carotid bodies, the spinal cord, and brainstem ( Kakall et al, 2018b ; Kim et al, 2018 ; Farnham et al, 2019 ). The involvement of the sympathetic system in glucose regulation is similarly well established and involves regions within the hypothalamus ( Frohman and Bernardis, 1971 ; Grayson et al, 2013 ) including the ventromedial hypothalamus ( Meek et al, 2016 ; Shimazu and Minokoshi, 2017 ) and paraventricular nucleus ( Sharpe et al, 2013 ; Menuet et al, 2014 ; Zhao et al, 2017 ), the brainstem ( Verberne and Sartor, 2010 ; Kakall et al, 2019 ), the adrenal gland ( Jun et al, 2014 ), and carotid bodies ( López-Barneo, 2003 ). Increases in blood glucose following acute, conscious IHx can be blocked by adrenergic blockade or adrenal medullectomy ( Rafacho et al, 2013 ; Jun et al, 2014 ) indicating sympathetic involvement via catecholamine release.…”

Brain-Body Control of Glucose Homeostasis—Insights From Model Organisms

“…The rat is a particularly useful model to study the neuroendocrine regulation of the CRR to acute and recurrent hypoglycemia, particularly the Sprague-Dawley and Wistar rats, which have been the workhorse of the hypoglycemia field for the last 30 years. The hyperinsulinemic-hypoglycemic clamp, together with insulininduced hypoglycemia and 2-DG induced glucoprivation, have been extensively used in Sprague-Dawley rats to investigate CRR, glucoprivic feeding and impaired awareness of hypoglycemia (49)(50)(51)(52)(53)(54)(55)(56). This latter aspect of hypoglycemia "awareness" can be studied using a conditioned place preference test, which has so far been validated in rats but not in mice (57), largely because of the more rapid induction of defective counter-regulation in rats and their tractability for behavioral tests.…”