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.

Keywords: HAAF; catecholaminergic neurons; dorsomedial medulla; epinephrine; ventrolateral medulla.

Fig. 1.

Insulin-induced hypoglycemia protocols (inset) and blood glucose levels in all treatment groups: grouped data (n = 4 animals/group) indicating blood glucose (BG) levels in groups treated with insulin (I; 5 U/kg) or saline control (S). BG levels were measured before injections and 2 h after injections. Hypoglycemia was achieved if BG ≤ 3.9 mM. In the control group (A), animals were injected with volume-matched 0.9% saline once daily for 3 days. In the single-hypoglycemia (hypo) group (B), saline was injected on days 1 and 2, followed by insulin on day 3. In the repeated-hypo group (C), insulin was injected on all days. In the hypo prevention group (D), insulin was injected on days 1, 2, and 9. In the naloxone (N)-treated group (E), insulin was injected on days 1, 2, and 3, followed by intraperitoneal injection of naloxone (1 mg/kg) after the 2-h BG levels were recorded on days 1 and 2. Data are means ± SE. Statistical significance was determined by 1-way ANOVA and multiple-comparison analysis with a Holm-Šidák correction. ****P < 0.0001, significantly different from all other groups.

Fig. 2.

Antecedent insulin-induced hypoglycemia reduces plasma epinephrine levels after subsequent hypoglycemia: grouped data indicating plasma epinephrine levels measured from blood samples collected on the final day of injections from each animal. Plasma epinephrine levels were not affected in saline-treated control rats (n = 4). In the single-hypoglycemia (hypo) group, 1 episode of hypoglycemia significantly increased plasma epinephrine levels (n = 4). In the repeated-hypo group, plasma epinephrine levels after the last episode of hypoglycemia were significantly reduced compared with the single-hypo group (n = 4). Plasma epinephrine levels remained at levels similar to the repeated-hypo group after 1 wk of hypoglycemia prevention (n = 4) or naloxone treatment (n = 4). Data are means ± SE. Statistical significance was determined by 1-way ANOVA and multiple-comparison analysis with a Holm-Šidák correction. *P < 0.05, ***P < 0.001, significantly different from all other groups.

Fig. 3.

Representative images (×20) of treated C1 sections depicting Fos and phenylethanolamine-N-methyltransferase (PNMT) immunolabeling. A–E: representative photomicrographs (×20 magnification) of Fos (green) and PNMT (red) immunolabeling in the C1 medullary region of rat tissue (n = 4 animals/group). Cy3 fluorescence is pseudocolored red, and Alexa Fluor 488 is represented in green. Images represent staining in control (A), single-hypoglycemia (hypo) (B), repeated-hypo (C), hypo prevention (D), and naloxone-treated (E) animal tissue. Overlapping Fos+PNMT+ cells are indicated by arrows. F: bregma levels used for analysis. AmbC, ambiguus nucleus, compact part; Bo, Bötzinger complex; Sp5, spinal trigeminal nucleus; 4V, fourth ventricle.

Fig. 4.

Representative images (×40) of treated C1 sections depicting Fos and phenylethanolamine-N-methyltransferase (PNMT) immunolabeling: representative photomicrographs (×40 magnification) of Fos (green) and PNMT (red) immunolabeling in the C1 medullary region of rat tissue (n = 4 animals/group). Cy3 fluorescence is pseudocolored red, and Alexa Fluor 488 is represented in green. Images represent staining in control (A), single-hypoglycemia (hypo) (B), repeated-hypo (C), hypo prevention (D), and naloxone-treated (E) animal tissue. Overlapping Fos+PNMT+ cells are indicated by arrows.

Fig. 5.

Representative images (×20) of treated C3 sections depicting Fos and phenylethanolamine-N-methyltransferase (PNMT) immunolabeling. A–E: representative photomicrographs (×20 magnification) of Fos (green) and PNMT (red) immunolabeling in the C3 medullary region of rat tissue (n = 4 animals/group). Cy3 fluorescence is pseudocolored red, and Alexa Fluor 488 is represented in green. Images represent staining in control (A), single-hypoglycemia (hypo) (B), repeated-hypo (C), hypo prevention (D), and naloxone-treated (E) animal tissue. Overlapping Fos+PNMT+ cells are indicated by arrows. F: bregma levels used for analysis. AmbC, ambiguus nucleus, compact part; Bo, Bötzinger complex; Sp5, spinal trigeminal nucleus; 4V, fourth ventricle.

Fig. 6.

Representative images (×40) of treated C3 sections depicting Fos and phenylethanolamine-N-methyltransferase (PNMT) immunolabeling: representative photomicrographs (×40 magnification) of Fos and PNMT immunolabeling in the C3 medullary region of rat tissue (n = 4 animals/group). Cy3 fluorescence is pseudocolored red, and Alexa Fluor 488 is represented in green. Images represent staining in control (A), single-hypoglycemia (hypo) (B), repeated-hypo (C), hypo prevention (D), and naloxone-treated (E) animal tissue. Overlapping Fos+PNMT+ cells are indicated by arrows.

Fig. 7.

Antecedent insulin-induced hypoglycemia reduces activation of adrenergic neurons in medullary C1 and C3 nuclei after subsequent hypoglycemia: neuronal activation represented by double-stained Fos+ phenylethanolamine-N-methyltransferase (PNMT)+ neurons expressed as % of total PNMT+ neurons in the C1 (A) and C3 (B) regions (n = 4 animals/group). The ratio of activated catecholaminergic neurons in both regions was significantly higher in the single-hypoglycemia (hypo) group compared with the repeated-hypo group. There was no recovery in C1 or C3 neuronal activation after hypo prevention or naloxone treatment. Data are means ± SE. Statistical significance was determined by 1-way ANOVA and multiple-comparison analysis with a Holm-Šidák correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, significantly different from all other groups.

Fig. 8.

Correlation between plasma epinephrine levels and C1 and C3 neuronal activity: positive correlation between plasma epinephrine levels and the degree of C1 (A) and C3 (B) neuronal activation after hypoglycemia. Statistical significance was determined by a Pearson correlation test with GraphPad Prism software. PNMT, phenylethanolamine-N-methyltransferase.

Fig. 9.

Repeated hypoglycemia (hypo) does not alter neuronal phenylethanolamine-N-methyltransferase (PNMT) expression in the medullary C1 and C3 regions: neuronal cell counts depicting the total number of PNMT+ neurons in both the C1 and C3 regions (n = 4 animals/group). The total number of adrenergic neurons in the C1 and C3 regions did not vary between groups. Data are means ± SE. Statistical significance was determined by 2-way ANOVA. P > 0.05, not significantly different from all other groups.