Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus

Abstract

Canabal DD, Song Z, Potian JG, Beuve A, McArdle JJ, Routh VH. Glucose, insulin and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus. Am J Physiol Regul Integr Comp Physiol 292: R1418 -R1428, 2007. First published December 24, 2006; doi:10.1152/ajpregu.00216.2006.-Glucose-sensing neurons in the ventromedial hypothalamus (VMH) are involved in the regulation of glucose homeostasis. Glucosesensing neurons alter their action potential frequency in response to physiological changes in extracellular glucose, insulin, and leptin. Glucose-excited neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency when extracellular glucose is reduced. Central nitric oxide (NO) synthesis is regulated by changes in local fuel availability, as well as insulin and leptin. NO is involved in the regulation of food intake and is altered in obesity and diabetes. Thus this study tests the hypothesis that NO synthesis is a site of convergence for glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. With the use of the NO-sensitive dye 4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein in conjunction with the membrane potential-sensitive dye fluorometric imaging plate reader, we found that glucose and leptin suppress, whereas insulin stimulates neuronal nitric oxide synthase (nNOS)-dependent NO production in cultured VMH GI neurons. The effects of glucose and leptin were mediated by suppression of AMP-activated protein kinase (AMPK). The AMPK activator 5-aminoimidazole-4-carboxamide-1-␤-4-ribofuranoside (AICAR) increased both NO production and neuronal activity in GI neurons. In contrast, the effects of insulin on NO production were blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Furthermore, decreased glucose, insulin, and AICAR increase the phosphorylation of VMH nNOS, whereas leptin decreases it. Finally, VMH neurons express soluble guanylyl cyclase, a downstream mediator of NO signaling. Thus NO may mediate, in part, glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. ventromedial hypothalamus; glucose-sensing neurons; leptin; insulin; nitric oxide; adenosine 5Ј-monophosphate-activated protein kinase THE VENTROMEDIAL HYPOTHALAMUS (VMH), which contains the ventromedial (VMN) and arcuate (ARC) nuclei, is critical for the regulation of glucose and energy homeostasis (34). This region contains specialized neurons whose activity is regulated by physiologically relevant changes in extracellular glucose (36,37,42). Glucose-excited (GE) neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency (APF) when extracellular glucose is reduced (36). GE neurons activate ATP-sensitive K ϩ (K ATP ) channels in response to decreased glucose. GI neurons appear to close an ATP-activated Cl Ϫ channel, although the identity of this channel is unknown (36). Glucose-sensing neurons also possess insulin and leptin receptors (22,39,40,42). Thus gluc...

Leptin responsiveness restored by amylin agonism in diet-induced obesity: Evidence from nonclinical and clinical studies

“…Amylin augmented leptin-stimulated signaling by Ϸ43% in the VMH without improving leptin signaling in the ARC. Within the VMH, enhanced leptin signaling would be expected to positively impact energy regulation because leptin receptors are abundantly expressed on glucose-sensitive VMH neurons, and potent anorexigenic and weight-reducing effects are observed after VMH leptin microinjection (27)(28)(29). Leptin-responsive neurons in the VMH also contain SF-1 (22), a transcription factor whose deficiency induces DIO in mice (30).…”

Physiological glucose is critical for optimized neuronal viability and AMPK responsiveness in vitro

“…For these studies, a previously established primary dissociated cortical neuronal culture (Dawson, et al, 1993;Landree, et al, 2004) was maintained in a glucose-free media supplemented with glutamine and B27 and either a physiological concentration of 3 mM glucose (NB-A 3 ), or a non-physiological high concentration of 25 mM glucose (NB-A 25 ). The present study was designed to further develop previous studies that considered the role of media glucose concentrations present during short-term culture (used by DIV4) and/or experimental medias (Wang, et al, 2004;Song, et al, 2005;Morgenthaler, et al, 2006;Abe, et al, 2006;Bak, et al, 2006;Kang, et al, 2006;Canabal, et al, 2007a;Canabal, et al, 2007b).…”

“…There are a small number of studies involving neuronal cultures and AMPK modulation with AICAR Chau-Van, et al, 2007;Dasgupta, et al, 2007;Canabal, et al, 2007a;Canabal, et al, 2007b;Mountjoy, et al, 2007), but few which utilize physiological glucose culture conditions Canabal, et al, 2007a;Canabal, et al, 2007b). A previous study using neuroblastoma cell lines cultured in 25 mM glucose demonstrated that basal levels of pAMPK are increased following incubation (16 hour) in media containing glucose below physiological concentrations (0 and 1 mM) relative to those switched to 2.5 mM glucose, and decreased in media containing glucose above physiological levels (5, 10, and 25 mM) .…”

“…In these situations, brain glucose can rise to 4.5 mM or drop as low as 0.16 mM (Silver, et al, 1994). Select studies acknowledge these relationships, and emphasize the importance of in vitro culture conditions for CNS research (Wang, et al, 2004;Song, et al, 2005;Morgenthaler, et al, 2006;Abe, et al, 2006;Bak, et al, 2006;Kang, et al, 2006;Canabal, et al, 2007a;Canabal, et al, 2007b); however, most studies do not. It is not unusual for culture media to contain 25 mM glucose, a level perhaps seen in the plasma of obese ob/ob mice (Schwartz, et al, 1996), but never seen in the brain.…”

“…The experiments reported here were designed as an expansion of previous studies that utilized short-term (used by DIV4) dissociated hypothalamic neurons, cultured in media containing 2.5 mM glucose (Wang, et al, 2004;Song, et al, 2005;Canabal, et al, 2007a;Canabal, et al, 2007b). We have expanded this concept to the preparation and maintenance of long-term dissociated cortical neuronal cultures (DIV10-13) in both physiologic and non-physiologic concentrations of glucose.…”