| Hypothalamic glucose-sensing neurons have been implicated in the regulation of several neuroendocrine functions such as energy balance and glucose homeostasis, and thus contribute to the pathogenesis of metabolic disorders such as diabetes and obesity. However, the molecular mechanisms of glucose-sensing are poorly understood. In this thesis, we employed a hypothalamic neuronal cell line N-38 in which gene expression of an orexigenic neuropeptide agouti-related peptide (AgRP) was regulated by glucose, to examine hypothalamic glucose-sensing mechanisms. Initially using DNA microarrays, we discovered eight glucose-induced genes including the highly-induced thioredoxin interacting protein (Txnip), which not only proved valuable in analyzing the "gene memory" effect in glucose-regulated gene expression, but also suggest plausible roles in mediating neuroendocrine and neurotoxic effects of glucose. We then examined in detail molecular mechanisms mediating effects of glucose on AgRP gene expression. Our results demonstrated that a product of glycolysis, but not oxidative phosphorylation, the pentose pathway, or the hexosamine pathway, mediates effects of glucose on AgRP gene expression in the hypothalamic cell line N-38, whereas in contrast the pentose and the hexosamine pathways mediate effects of glucose on Txnip gene expression. We hypothesize that the production of NADH by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), through the regulation of the transcription factor C-terminal binding protein (CtBP), mediates effects of glucose on AgRP. Since CtBP antagonizes transactivational effects of CBP through an NADH-sensitive mechanism, our further studies strongly support the hypothesis that cAMP-response-element-binding protein (CREB), together with its co-activator CREB-binding protein (CBP), mediates at least in part the effects of glucose on the gene transcription of AgRP and possibly other hypothalamic genes. In support of this hypothesis, we observed in an unbiased survey of the activity of transcription factors that glucose most prominently reduced the activity of CREB. Since estradiol reduces hypothalamic CREB phosphorylation in vivo, we tested the prediction that estradiol would enhance hypothalamic responses to glucose. Our results demonstrated that estradiol enhanced glucose tolerance and attenuated hypoglycemia-induced gene expression in hypothalamus, implying enhanced sensitivity to glucose and suggesting that at least some metabolic effects of glucose are mediated by enhancing hypothalamic sensitivity to glucose. |
