Hindbrain dorsal vagal complex AMPK controls hypothalamic gluco-regulatory transmitter and counter-regulatory hormone responses to hypoglycemia

Abstract

Pharmacologic activation of the hindbrain dorsal vagal complex energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK) causes site-specific adjustments in hypothalamic AMPK activity. DVC A2 noradrenergic neurons are a likely source of metabolo-sensory cues to downstream network components as they express substrate fuel-sensitive AMPK. This study investigated the hypothesis that DVC AMPK controls hypothalamic sensor, metabolic effector transmitter, and counter-regulatory hormone responses to insulin-induced hypoglycemia. Male rats were injected into the caudal fourth ventricle with the AMPK inhibitor compound C (Ccor vehicle before hypoglycemia. Arcuate (ARH), ventromedial (VMN), and dorsomedial (DMN) nuclei and lateral hypothalamic area (LHA) were micropunch-dissected for norepinephrine ELISA and Western blot analyses. Hypoglycemic stimulation of norepinephrine activity in each site was impeded by compound C. Hypoglycemia caused drug-revocable (ARH) or -refractory (VMN, DMN) reductions in AMPK, alongside hindbrain AMPK-dependent augmentation of phospho-AMPK expression in each location. Compound C prevented hypoglycemic augmentation of gluco-stimulatory ARH neuropeptide Y, VMN neuronal nitric oxide synthase, and LHA orexin-A expression, while hypoglycemic suppression of the catabolic neuron protein markers ARH pro-opiomelanocortin and VMN glutamate decarboxylase_65/67 was respectively averted or unaffected by drug treatment. DMN RFamide-related peptide-1 and -3 profiles were correspondingly amplified or suppressed hindbrain AMPK-reliant mechanisms during hypoglycemia. Results show that DVC AMPK is required for hypoglycemic intensification of norepinephrine activity in characterized hypothalamic gluco-regulatory structures, and that this sensor regulates AMPK activation and metabolic effector transmission in those sites.

Keywords: AMPK; Compound C; Dopamine-beta hydroxylase; Laser-catapult microdissection; Norepinephrine.

Figure 1.

Effects of Caudal Fourth Ventricular (CV4) Administration of the 5’-adenosine monophosphate-activated protein kinase (AMPK) Inhibitor Compound C (Cc) on Hypoglycemic Patterns of Arcuate Hypothalamic Nucleus (ARH) AMPK Activation and Neuropeptide Y (NPY), and Proopiomelanocortin (POMC) Protein Expression. Groups of adult male rats were pretreated by vehicle (V) or Cc injection into the CV4 prior to sc insulin (INS) administration. Data depict mean normalized protein optical density (O.D) measures of ARH AMPK (Panel A), phosphoAMPK (pAMPK; Panel B), NPY (Panel C), and POMC (Panel D) + S.E.M. for the following treatment groups: V/V (solid white bars; n=5); V/INS (solid gray bars; n=5); Cc/INS (diagonal-striped gray bars; n=5). *p<0.05; **p<0.01; ***p<0.001.

Figure 2.

Impact of Cc Pretreatment on Ventromedial Hypothalamus Nucleus (VMN) AMPK and, Glutamate Decarboxylate_65/67 (GAD_65/67), and Neuronal Nitric Oxide Synthase (nNOS) Protein Expression during Insulin-Induced Hypoglycemia (IIH). Results show mean normalized VMN AMPK (Panel A), pAMPK (Panel B), GAD_65/67 (Panel C), and nNOS (Panel D) O.D. values + S.E.M. for V/V, V/INS, and Cc/INS treatment groups (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Figure 3.

Role of Dorsal Vagal Complex (DVC) AMPK in IIH-Associated Patterns of Dorsomedial Hypothalamus Nucleus (DMN) AMPK Activation and RFamide-Related Peptide-1 (RFRP-1) and −3 (RFRP-3) Protein Expression. Data illustrate indicate mean normalized DMN AMPK (Panel A), pAMPK (Panel B), RFRP-1 (Panel C), and RFRP-3 (Panel D) O.D. values + S.E.M. for groups of V-pretreated, V-injected (solid white bars; n=5) controls and INS-injected animals pretreated with V (solid gray bars; n=5) or Cc (diagonal-striped gray bars; n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Figure 4.

Effects of Intra-CV4 Cc Administration on Lateral Hypothalamic Area (LHA) AMPK Activity and Orexin-A (ORX-A) Activity and RFamide-Related Peptide-1 (RFRP-1) and −3 (RFRP-3) Protein Expression in Hypoglycemic Male Rats. Data indicate mean normalized LHA AMPK (Panel A), pAMPK (Panel B), and ORX-A (Panel C) O.D. values + S.E.M. for V/V, V/INS, and Cc/INS treatment groups (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Figure 5.

Role of DVC AMPK in Hypoglycemia-Associated Patterns of Hypothalamic Norepinephrine (NE) Activity. Results depict mean ARH (Panel A), VMN (Panel B), DMN (Panel C), LHA (Panel D), and paraventricular hypothalamic nucleus (PVN) NE tissue content + S.E.M. for groups of V/V-, V/INS-, and Cc/INS-treated animals (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Figure 6.

A2 Noradrenergic Neuron AMPK, pAMPK, Dopamine-Beta-Hydroxylase (DβH), and Monocarboxylate Transporter-2 (MCT) Protein Responses to IIH; Impact of Cc Pretreatment. Data illustrate mean A2 AMPK (Panel A), pAMPK (Panel B), DβH (Panel C), and MCT (Panel D) protein O.D. measures + S.E.M. for V/V, V/INS, and Cc/INS treatment groups (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Figure 7.

Effects of Intra-CV4 Cc Administration on Glycemic, Glucagon, and Corticosterone Responses to INS Injection in Male Rats. Results depict circulating glucose (Panel A), glucagon (Panel B), and corticosterone (Panel C) levels + S.E.M. for groups of V/V-, V/INS-, and Cc/INS-treated animals (n=5/group). *p<0.05; **p<0.01; ***p<0.001.