Glucokinase inhibitor glucosamine stimulates feeding and activates hypothalamic neuropeptide Y and orexin neurons

Abstract

Maintaining glucose levels within the appropriate physiological range is necessary for survival. The identification of specific neuronal populations, within discreet brain regions, sensitive to changes in glucose concentration has led to the hypothesis of a central glucose-sensing system capable of directly modulating feeding behaviour. Glucokinase (GK) has been identified as a glucose-sensor responsible for detecting such changes both within the brain and the periphery. We previously reported that antagonism of centrally expressed GK by administration of glucosamine (GSN) was sufficient to induce protective glucoprivic feeding in rats. Here we examine a neurochemical mechanism underlying this effect and report that GSN stimulated food intake is highly correlated with the induction of the neuronal activation marker cFOS within two nuclei with a demonstrated role in central glucose sensing and appetite, the arcuate nucleus of the hypothalamus (ARC) and lateral hypothalamic area (LHA). Furthermore, GSN stimulated cFOS within the ARC was observed in orexigenic neurons expressing the endogenous melanocortin receptor antagonist agouti-related peptide (AgRP) and neuropeptide Y (NPY), but not those expressing the anorectic endogenous melanocortin receptor agonist alpha-melanocyte stimulating hormone (α-MSH). In the LHA, GSN stimulated cFOS was found within arousal and feeding associated orexin/hypocretin (ORX), but not orexigenic melanin-concentrating hormone (MCH) expressing neurons. Our data suggest that GK within these specific feeding and arousal related populations of AgRP/NPY and ORX neurons may play a modulatory role in the sensing of and appetitive response to hypoglycaemia.

Supplementary Fig. S1

A series of low-power photomicrographs summarizing FOS-IR brain expression in a representative rat treated with glucosamine (150 nmol/min, ICV). Brain sections are an arranged in a rostral-to-caudal manner (A–L). One red dot indicates two FOS-IR cells. Scale bar in L, 1 mm. Abbreviations: 3V, third ventricle; 4V, fourth ventricle; 7n, facial nerve; 12, hypoglossal nucleus; ac, anterior commissure; AcbSh, shell portion of the accumbens nucleus; AOP, posterior part of the anterior olfactory nucleus; AP, area postrema; Aq, aqueduct; ARC, arcuate nucleus of the hypothalamus; cp, cerebral peduncle, basal part; CPu, caudate putamen; D3V, dorsal third ventricle; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; f, fornix; fmi, forceps minor of the corpus callosum; fr, fasciculus retroflexus; ic, internal capsule; IC, inferior colliculus; IL, infralimbic cortex; LHA, lateral hypothalamic area; LS, lateral septal nucleus; LSV, lateral septal nucleus, ventral part; LV, lateral ventricle; MeA, medial amygdaloid nucleus; ml, medial lemniscus; MPA, medial preoptic area; mt, mammillothalamic tract; PAG, pariaqueductal gray; PH, posterior hypothalamic area; Pir, piriform cortex; pm, principal mammillary tract; PMV, premammillary nucleus, ventral part; PVN, paraventricular nucleus of the hypothalamus; py, pyramidal tract; SO, supraoptic nucleus; sol, solitary tract; STh, subthalamic nucleus; str, superior thalamic radiation; Tu, olfactory tubercle; VMH, ventromedial nucleus of the hypothalamus; xscp, decussation of the superior cerebellar peduncle; ZI, zona incerta.

Supplementary Fig. S2

A series of low-power photomicrographs summarizing FOS-IR brain expression in a representative rat treated with ICV aECF. Brain sections are arranged in a rostral-to-caudal manner (A–L). One red dot indicates two FOS-IR cells. Scale bar in L, 1 mm. For abbreviations see Supplemental Fig. S1.

Fig. 1

GSN significantly increased food intake and FOS-IR in the ARC and LHA. (a) GSN (150 nmol/min, i.c.v.) significantly increased 2 h food intake and FOS-IR in the (b) ARC and (c) LHA in rats compared to GSN (15 nmol/min, i.c.v.) and aECF. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 compared to aECF.

Fig. 2

GSN significantly activates ARC NPY and LHA ORX neurons, but not ARC α-MSH or LHA MCH neurons. (a) In the ARC, GSN (150 nmol/min, i.c.v.) induced FOS-IR in less than 1% of α-MSH-IR neurons, but induced FOS-IR in approximately one-third of NPY neurons. (b) In the LHA, GSN did not increase FOS-IR in MCH-IR neurons, but produced a significant increase in FOS-IR in ORX neurons. **p ≤ 0.01, ***p ≤ 0.001.

Fig. 3

Colocalization of FOS-IR with ARC NPY mRNA and α-MSH-IR neurons and with LHA ORX-IR and MCH-IR neurons in rats treated with GSN (150 nmol/min, i.c.v.). (a–d) are merged micrographs showing representative regions of FOS-IR and neuropeptide co-expression. (a¹–a³, b¹–b³, c¹–c³, and d¹–d³) are higher-power magnification of boxes area in a–d, respectively, with a¹, b¹, c¹, and d¹ illustrating FOS-IR positive cells (green); a² illustrating NPY mRNA, b² illustrating α-MSH-IR, c² illustrating ORX-IR and d² illustrating MCH-IR (red); and a³, b³, c³, and d³ illustrating merged photographs. Arrows indicate colocolization. Scalebar in a, 75 μm, also applies to b; scalebar in c, 100 μm, also applies to d; scalebar in d³, 25 μm, applies to all other images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)