Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus

Abstract

Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254

“…osmoregulation, thyroid status, circadian rhythm, nutrient homeostasis, reproduction and growth (Canabal et al, 2007;Ding et al, 1994;Liu et al, 1998;Rettori et al, 1993), Aroclor 1254-induced disruption of NOS activity may represent one mechanism through which PCBs and similarly acting environmental toxicants may affect important regulatory physiological functions governed by the hypothalamus. These functions may include osmoregulatory capacity and blood pressure responses to stress in late adult rats which our lab has shown are significantly impaired by developmental exposure to similar organohalogens (Shah et al, 2011).…”

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).…”

“…Given previous results demonstrating that the effect of AICAR is inhibited when hypothalamic neurons are cultured in high glucose (5 mM) as opposed to physiologic glucose (2.5 mM) (Canabal, et al, 2007b) and our results that glucose levels present during neuronal culture maintenance altered C75-induced increases in ATP, we then analyzed the effect of ambient glucose on the ability of the AMP-mimetic, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), to increase AMPK phosphorylation. The response of AMPK to changes in neuronal energy status and metabolic stress makes the pAMPK/AMPK ratio a reasonable readout of neuronal responsiveness to changes in energy status.…”

“…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.…”