Hindbrain lactoprivic regulation of hypothalamic neuron transactivation and gluco-regulatory neurotransmitter expression: Impact of antecedent insulin-induced hypoglycemia
Introduction
The brain relies on a constant glucose supply to maintain vital high energy-demand nerve cell functions. Neuro-metabolic instability due to glucoprivation poses a substantial threat to brain function and well-being. Insulin-induced hypoglycemia (IIH) elicits hypothalamic activation of coordinated counter-regulatory autonomic, neuroendocrine, and behavioral functions that reverse glucose decrements. The hypothalamus is continuously apprised of cell energy imbalance by dedicated metabolic-sensory neurons in the brain that adjust synaptic firing in reaction to diminished substrate fuel supply. The hindbrain dorsal vagal complex (DVC) is a critical metabolic screening site in the brain as local deficits of the oxidizable glycolytic end-product L-lactate elevate blood glucose, whereas lactate repletion of the DVC intensifies hypoglycemia (Patil and Briski, 2005a). Caudal fourth ventricular (CV4) administration of the monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamic acid (4CIN) induces Fos protein expression, an indicator of change in cell functional status, in key hypothalamic metabolic loci. These findings verify operational connectivity of hindbrain lactoprivic-sensitive neurons with higher-order elements of the brain gluco-regulatory network (Briski and Patil, 2005). DVC A2 noradrenergic neurons are a plausible source of lactoprivic regulatory cues as these cells express Fos during monocarboxylate transporter blockade (Patil and Briski, 2005b), and exhibit activation of the metabolic sensor 5’-AMP-activated protein kinase (AMPK) in response to hypoglycemia-associated lactoprivation, coincident with L-lactate – reversible hypoglycemic augmentation of hypothalamic norepinephrine (NE) activity (Shrestha et al., 2014).
The role of A2 metabolic sensory signaling in counter-regulatory acclimation to recurring neuroglucopenia remains unclear. Iatrogenic hypoglycemia is an incessant complication of necessary strict glycemic management of type I diabetes mellitus (T1DM). In T1DM patients, antecedent IIH (AH) often leads to hypoglycemia-associated autonomic failure (HAAF), a pathophysiological mal-adaptation that manifests as diminished hypoglycemic awareness and defective glucose counter-regulatory outflow (Cryer et al., 2003; Cryer, 2010). Animal models for recurrent insulin-induced hypoglycemia that replicate insulin delivery route, frequency of administration, and duration of action in the clinical setting reveal blunted nerve cell transcriptional activation in hypothalamic metabolic loci and the DVC in male rat brain, inferring neural habituation to repeated hypoglycemia (Paranjape and Briski, 2005; Kale et al., 2006). A2 neurons harvested by laser-catapult microdissection from male rats after induction of a single bout of hypoglycemia show decreased monocarboxylate transporter-2 (MCT2) protein expression and elevated AMPK activity (Cherian and Briski, 2011). Evidence that hypoglycemic suppression of A2 MCT2 mRNA is reversed by exogenous lactate suggests that MCT2 expression and lactate uptake into these neurons may be proportionate to extracellular lactate concentrations (Briski et al., 2009). Between recurring hypoglycemia episodes, A2 cells maintain patterns of amplified AMPK activity and diminished MCT2 protein expression; moreover, neither sensor activity nor MCT2 content varies from these adaptive baseline profiles during ensuing re-exposure to hypoglycemia (Cherian and Briski, 2011). Reports that AH prevents IIH up-regulation of A2 catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) mRNA (Cherian and Briski, 2011) and protein (Mandal et al., 2017) support the notion of acquired A2 nerve cell desensitization to recurring hypoglycemia.
Current research addressed the hypothesis that hindbrain lactoprivic regulatory input to the hypothalamus may acclimate to recurring hypoglycemia as a consequence of diminished A2 neuron reactivity to hypoglycemia-related lactate decrements. Here, Fos immunostaining was used as a functional mapping tool to assess whether AH causes site-specific adjustments in hypothalamic neuron transcriptional sensitivity to hindbrain lactoprivation. Tissue sections through hypothalami of male rats pretreated by daily vehicle or insulin injections ahead of intra-CV4 4CIN administration were processed for visualization of Fos labeling patterns within distinctive hypothalamic metabolic loci, e.g. arcuate (ARH), ventromedial (VMN), and dorsomedial (DMN) hypothalamic nuclei and lateral hypothalamic area (LHA). A corroborative approach involving Western blot analysis of drug treatment on expression profiles of neuropeptide/biosynthetic enzyme protein markers of metabolic neurotransmitter signaling in these structures was also implemented. Micropunch samples were evaluated by NE ELISA to address the premise that hindbrain lactoprivic-driven activity of this catecholamine transmitter in the hypothalamus habituates to AH. Additionally, combinatory immunocytochemistry/laser-catapult microdissection techniques were utilized to procure pure hindbrain A2 nerve cell samples in order to characterize 4CIN effects on A2 neuron AMPK activation and MCT2, glucose transporter, and DβH protein expression, and to determine if these cellular responses to drug treatment may be modified by AH.
Section snippets
Experimental design
Adult male Sprague-Dawley rats (3–4 months of age) were maintained in groups (2–3 animals per cage) under a 14-h light/10-h dark lighting schedule (light on at 05:00 h), and allowed free access to standard laboratory rat chow (Harlan Teklad LM-485; Harlan Industries, Madison, WI, USA) and tap water. Animals were accustomed to daily handling for at least one week ahead of surgery. All protocols were conducted in accordance with NIH guidelines for care and use of laboratory animals, under ULM
Results
Laser-catapult microdissected A2 neuron samples were analyzed by Western blot to investigate whether AH modifies cellular nutrient transporter and AMPK responses to hindbrain lactate deficiency. As shown in Fig. 1, A2 DBH protein content was down-regulated by 4CIN (Panel A) [V/4CIN versus V/V; F(2,6) = 7.20; p = .02]; this inhibitory response to drug treatment was unaffected by AH. Data in Panels B-D indicate that MCT2 [F(2,6) = 10.78; p = .01] and GLUT3 [F(2,6) = 11.00; p = .004] expression
Discussion
Current research investigated whether AH alters hindbrain lactoprivic regulation of hypothalamic nerve cell transcriptional activity and metabolic neurotransmitter signaling in male rats. Results show that AH reduced intra-CV4 4CIN induction of hypothalamic Fos immunoreactivity, and may normalize (ARH NPY; VMN GABA) or modify (LHA ORX-A; DMN RFRP) treatment effects on select hypothalamic metabolic neurotransmitters. AH did not modify drug suppression of A2 nerve cell noradrenergic transmission
Acknowledgements
This research was funded by NIH DK 109382
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