sb-334867-a and Hypoglycemia

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Topics: Animals; Arousal; Behavior, Animal; Benzoxazoles; Blood Glucose; Brain; Conditioning, Psychological; Disease Models, Animal; Feeding Behavior; Hypoglycemia; Male; Naphthyridines; Orexin Receptor Antagonists; Orexin Receptors; Orexins; Rats, Sprague-Dawley; Reward; Sertraline; Signal Transduction; Time Factors; Urea

Iatrogenic hypoglycemia in response to insulin treatment is commonly experienced by patients with type 1 diabetes and can be life threatening. The body releases epinephrine in an attempt to counterregulate hypoglycemia, but the neural mechanisms underlying this phenomenon remain to be elucidated. Orexin neurons in the perifornical hypothalamus (PeH) project to the rostral ventrolateral medulla (RVLM) and are likely to be involved in epinephrine secretion during hypoglycemia. In anesthetized rats, we report that hypoglycemia increases the sympathetic preganglionic discharge to the adrenal gland by activating PeH orexin neurons that project to the RVLM (PeH-RVLM). Electrophysiological characterization shows that the majority of identified PeH-RVLM neurons, including a subpopulation of orexin neurons, are activated in response to hypoglycemia or glucoprivation. Furthermore, the excitatory input from the PeH is mediated by orexin type 2 receptors in the RVLM. These results suggest that activation of orexin PeH-RVLM neurons and orexin type 2 receptors in the RVLM facilitates epinephrine release by increasing sympathetic drive to adrenal chromaffin cells during hypoglycemia.

Topics: Adrenal Glands; Animals; Benzoxazoles; Brain; Epinephrine; Fornix, Brain; Hypoglycemia; Hypoglycemic Agents; Hypothalamus; Insulin; Isoquinolines; Medulla Oblongata; Naphthyridines; Neural Pathways; Neurons; Orexin Receptor Antagonists; Orexin Receptors; Pyridines; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; Urea

Previous reports suggested an important role for serotonin (5-hydroxytryptamine [5-HT]) in enhancing the counterregulatory response (CRR) to hypoglycemia. To elucidate the sites of action mediating this effect, we initially found that insulin-induced hypoglycemia stimulates 5-HT release in widespread forebrain regions, including the perifornical hypothalamus (PFH; 30%), ventromedial hypothalamus (34%), paraventricular hypothalamus (34%), paraventricular thalamic nucleus (64%), and cerebral cortex (63%). Of these, we focused on the PFH because of its known modulation of diverse neurohumoral and behavioral responses. In awake, behaving rats, bilateral PFH glucoprivation with 5-thioglucose stimulated adrenal medullary epinephrine (Epi) release (3,153%) and feeding (400%), while clamping PFH glucose at postprandial brain levels blunted the Epi response to hypoglycemia by 30%. The PFH contained both glucose-excited (GE) and glucose-inhibited (GI) neurons; GE neurons were primarily excited, while GI neurons were equally excited or inhibited by 5-HT at hypoglycemic glucose levels in vitro. Also, 5-HT stimulated lactate production by cultured hypothalamic astrocytes. Depleting PFH 5-HT blunted the Epi (but not feeding) response to focal PFH (69%) and systemic glucoprivation (39%), while increasing PFH 5-HT levels amplified the Epi response to hypoglycemia by 32%. Finally, the orexin 1 receptor antagonist SB334867A attenuated both the Epi (65%) and feeding (47%) responses to focal PFH glucoprivation. Thus we have identified the PFH as a glucoregulatory region where both 5-HT and orexin modulate the CRR and feeding responses to glucoprivation.

Topics: Animals; Astrocytes; Benzoxazoles; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Fornix, Brain; Hypoglycemia; Hypothalamus; Intracellular Signaling Peptides and Proteins; Male; Midline Thalamic Nuclei; Naphthyridines; Neurons; Neuropeptides; Orexins; Paraventricular Hypothalamic Nucleus; Rats, Sprague-Dawley; Receptors, Neuropeptide; Serotonin; Urea; Ventromedial Hypothalamic Nucleus

The potent orexigenic neuropeptide, orexin-A (ORX-A), acts at multiple sites within the central neuroaxis to control autonomic responses to energy imbalance, including the dorsal vagal motor nucleus (DMV), where it regulates pancreatic efferent nerve firing. Recent evidence that recurrent insulin-induced hypoglycemia (RIIH) attenuates lateral hypothalamic ORX-A-ergic neuronal transcriptional activation and prepro-orexin gene expression suggests that this phenotype undergoes functional adaptation to repeated glucoprivation. We examined the hypothesis that RIIH-associated patterns of ORX-A neurotransmission and/or orexin-receptor-1 (OR-1) expression within the DMV may be correlated with exacerbated hypoglycemic and impaired pancreatic counterregulatory responses to repeated insulin administration. Male rats were pretreated by bilateral intra-DMV infusion of the OR-1 antagonist, SB-334867, or vehicle prior to s.c. injection of Humulin NPH (NPH), or diluent alone. Other animals were injected with one or four doses of NPH, on as many days, or diluent alone, and pretreated by bilateral intra-DMV administration of graded doses of ORX-A or vehicle on the final day of the study. Effects of acute versus repeated insulin administration on ORX-A and OR-1 protein levels in the microdissected dorsal vagal complex (DVC) were evaluated by radioimmunoassay and Western blot analyses, respectively. SB-334867 treatment prior to acute NPH administration decreased plasma glucose and suppressed peak glucagon secretion, whereas exogenous ORX-A administration prior to RIIH did not reverse amplified patterns of hypoglycemia. RIIH did not alter intra-DVC ORX-A tissue concentrations, but diminished OR-1 levels in that site. These results show that DMV OR-1 function is critical for optimal glucagon secretory responsiveness to acute hypoglycemia, and that RIIH-associated downregulation of receptor expression in that brain site may contribute to impaired restoration of euglycemia. The current data provide unique evidence that ORX-A acts via OR-1-dependent mechanisms within DMV to regulate glucagon counterregulatory function during hypoglycemia, and that decreased receptor-mediated signaling during RIIH may underlie characteristic intensification of hypoglycemia.

Topics: Animals; Benzoxazoles; Blood Glucose; Glucagon; Hypoglycemia; Hypoglycemic Agents; Insulin; Intracellular Signaling Peptides and Proteins; Male; Motor Neurons; Naphthyridines; Neuropeptides; Neurotransmitter Agents; Orexin Receptors; Orexins; Rats; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Signal Transduction; Urea; Vagus Nerve

Circulating glucose levels significantly affect vagal neural activity, which is important in the regulation of pancreatic functions. Little is known about the mechanisms involved. This study investigates the neural pathways responsible for hypoglycemia-induced vagal efferent signaling to the pancreas and identifies the neurotransmitters involved. Vagal pancreatic efferent nerve activities were recorded in anesthetized rats. Insulin-induced hypoglycemia, a decrease of blood glucose levels from 114 +/- 5 to 74 +/- 6 mg dl(-1), stimulated an increase in pancreatic efferent nerve firing from a basal rate of 1.1 +/- 0.3 to 19 +/- 3 impulses 30 s(-1). In contrast, vagal primary afferent neuronal discharges recorded in the nodose ganglia were unaltered by systemic hypoglycemia. Vagal afferent rootlet section plus splanchnicotomy had no effect on hypoglycemia-induced vagal efferent firing, suggesting a central site of action. Decerebration reduced the increase in nerve firing stimulated by hypoglycemia from 21 +/- 4 to 9.6 +/- 2 impulses 30 s(-1). Chemical ablation of the lateral hypothalamic area, but not the arcuate nucleus, inhibited pancreatic nerve firing evoked by hypoglycemia. Microinjection of the orexin-A receptor antagonist SB-334867 into the dorsal motor nucleus of the vagus (DMV) inhibited pancreatic nerve firing evoked by insulin-induced hypoglycemia by 56%. In contrast, injection of orexin-A (20 pmol) into the DMV elicited a 30-fold increase in pancreatic nerve firing. We concluded that systemic hypoglycemia stimulates pancreatic efferent nerve firing through a central mechanism. Full expression of pancreatic nerve activities during hypoglycemia requires both the forebrain and the brain stem. In addition to activating neurons in the brain stem, central neuroglucopenia activates subpopulations of neurons in the lateral hypothalamic area that contain orexin. The released orexin acts on DMV neurons to stimulate pancreatic efferent nerve activities and thus regulate pancreatic functions.

Topics: Action Potentials; Animals; Benzoxazoles; Blood Glucose; Brain Stem; Carrier Proteins; Dose-Response Relationship, Drug; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Agonists; Glucose; Hypoglycemia; Hypothalamus; Immunohistochemistry; Insulin; Intracellular Signaling Peptides and Proteins; Kainic Acid; Male; Microinjections; Naphthyridines; Neural Pathways; Neurons, Efferent; Neuropeptide Y; Neuropeptides; Nodose Ganglion; Orexins; Pancreas; Rats; Rats, Sprague-Dawley; Serotonin; Splanchnic Nerves; Urea; Vagus Nerve