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Pharmacology & Physiology Program
M.S. 2006, University Denis Diderot, Paris VII, France
Thesis Advisor: Andrew P. Thomas, Ph.D.
Professor and Chair
Department of Pharmacology and Physiology
Friday, March 25, 2011
MSB H-609, 1:00 P.M.
In the brain, the ventromedial hypothalamus (VMH) plays a critical role in the regulation of energy metabolism, and in particular glucose homeostasis. Thus, changes in VMH glucose level participate in the control of food intake and the control of the counter-regulatory response (CRR) to hypoglycemia. Specialized VMH glucose sensors known as glucose-sensitive neurons (GSNs) have been characterized and suggested to participate in this regulation of feeding behavior and peripheral metabolism. Two main populations of GSNs have been described, the glucose-excited (GE) neurons and the glucose-inhibited (GI) neurons. More recently, fructose has been shown to increase food intake and potentiate the CRR to hypoglycemia. Studies have suggested that fructose might act centrally. Nevertheless, the cellular and molecular mechanisms involved are unknown. Interestingly, the fuel sensor AMP-activated kinase (AMPK) in VMH GI neurons has been suggested to be involved in the effects of fructose. In this study, we hypothesize that fructose modifies the control of glucose homeostasis by activating VMH GI neurons, leading to two important consequences: (1) fructose causes a hyperglycemic increase in blood glucose under euglycemic conditions; (2) fructose potentiates the CRR to increase blood glucose under hypoglycemic conditions. Although we did not directly address the effects of fructose on food intake, we also hypothesize that the activation of GI neurons could contribute to the hyperphagic effects of fructose. By using calcium imaging on VMH dissociated neurons, we showed that fructose (100 ÁM) directly stimulates 7.8% (called Fructose(F)-activated neurons) and inhibits 3.6% (called Fructose(F)-inhibited neurons) of VMH neurons. The direct effect of fructose on VMH neurons was confirmed in vivo by c-fos immunohistochemistry. Injection of fructose (5 mM in the injected solution) into the carotid artery toward the brain significantly increased the number of c-fos immunoreactive cells 2 h post-injection in euglycemic rats. Characterization of the F-activated neurons in vitro demonstrated that the majority (60%) were also activated in response to decreased glucose from 2.5 to 0.1 mM, and were thus identified as GI neurons. Interestingly, fructose caused a substantial increase (76%) in the magnitude of the calcium response of GI neurons to a decrease in glucose from 2.5 to 0.1 mM, suggesting that fructose potentiates the activity of GI neurons to decreased glucose. We found that fructose increases VMH AMPK phosphorylation ex vivo by western-blot. From calcium imaging studies using the AMPK inhibitor compound C (30 ÁM) and the AMPK activator AICAR (0.5 mM), we showed that activation of AMPK is necessary for the fructose response of VMH F-activated neurons. We also obtained evidence for a role of neuronal nitric oxide synthase (nNOS) and the cystic fibrosis transmembrane conductance regulator (CFTR) channel using inhibitors. Finally, we found in vivo that fructose injected toward the brain causes transient increases in blood glucose concentration, and stimulates the release of glucagon in euglycemic rats. However, intra-carotid fructose injection failed to potentiate the CRR to hypoglycemia in the presence of a bolus intravenous injection of insulin. In conclusion, this study showed for the first time the central role of fructose in the regulation of glucose homeostasis. The characterization of fructose action on VMH neurons and identification of the signaling pathways involved may improve knowledge of Type 2 Diabetes etiology, which would offer the potential for development of new therapeutic targets to improve the treatment of Type 2 Diabetes patients.