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Pharmacology and Physiology Program
B.S. 2007, Beijing Normal University, China
Thesis Advisor: Andrew P. Thomas, Ph.D.
Department of Pharmacology, Physiology and Neuroscience
Wednesday, April 27, 2016
2:00 P.M., MSB Room H609
Fructose consumption has been increasing during recent decades, and both short-term and long-term studies have shown that fructose consumption is related to various metabolic problems. Nevertheless, the cellular and molecular mechanisms involved are unclear. One of the proposed ideas for fructose-induced metabolic syndromes involves its central effects. Using calcium imaging as an indicator of neuron depolarization and activation, previous studies from our lab showed that in rats, fructose can directly stimulate (termed F-activated or FA neurons) or inhibit (termed F-inhibited or FI neurons) ventromedial hypothalamus neurons. In ventromedial hypothalamus, there are two classes of neurons that regulate food intake and energy homeostasis. One is suppressed by hypoglycemia, called glucose-excited (GE) neurons, and the other is activated by hypoglycemia, called glucose-inhibited (GI) neurons. Interestingly in rats, the majority (77%) of rat GI neurons are also FA neurons, and the fructose-induced neuron activation is dependent on stimulation of AMPK, a key nutrition sensor in hypothalamus that is suggested to be activated by hypoglycemia.
It has been shown that fructose, if metabolized by fructokinase (KHK), will cause dramatic drop of ATP, leading to activation of AMPK. Since previously RT-PCR showed mRNA of KHK and GLUT5 (fructose specific transporter) exist in ventromedial hypothalamus neurons of rats, in this project, we hypothesize that fructose can act centrally in the ventromedial hypothalamus as a result of transport by GLUT5 and metabolism via fructokinase to elicit responses normally elicited by hypoglycemia at the euglycemic level.
Calcium imaging showed similar effects of fructose in ventromedial hypothalamus neurons dissociated from mice as that from rats. That is, fructose directly modulated neuron activities, and the fructose activation effect significantly overlapped with GI neurons, though the overlap in mice (41%) was not as large as that in rats (77%). The low fructose concentration applied during calcium imaging (0.1mM fructose on a base of 2.5mM glucose) implied that high Km KHK isoform C might be responsible for rapid changes rather than the low Km KHK isoform A. RT-PCR suggested that both KHK-A and KHK-C isoform are expressed in ventromedial hypothalamus. Single cell (sc) PCR showed that both KHK-C and total KHK (includes both isoforms) are more likely to be present in FA neurons than in non-fructose-sensitive neurons, reflected by percentage of positive cells and CT values. However, GLUT5 cannot be identified in either FA neurons or non-fructose sensitive neurons, indicating fructose uptake by neurons might be dependent on other fructose transporters. Dissociated neurons of KHK KO mice showed no activation by fructose, and hypothalamic neurons from GLUT5 KO mice showed a significantly decreased fructose activation. Taken together, KHK is indispensable for fructose-induced neuron activation and neuronal fructose uptake is probably via other fructose transporters such as sodium-dependent glucose cotransporters (or sodium-glucose liked transporter, SGLT). GLUT5 may still contribute to fructose activation mechanism rather than the direct transporter for neurons to take up fructose.
scPCR also indicated a much higher potential of co-expression of KHK and/or AMPK and/or nNOS in the population of FA neurons compared to non-fructose sensitive neurons. This again supported the idea that KHK, AMPK activation and nNOS were likely to be involved in neuronal fructose activation mechanism.
Finally, measured by quantative PCR, incubation of neurons with fructose resulted in an upregualtion of KHK and GLUT5, a down regulation of brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating peptide (PACAP) and a potential increase of somatostatin (SST) and nNOS compared to control. These were important factors that impact energy balance.
In conclusion, under euglycemic condition, fructose inappropriately activates an important subset of neurons that are supposed to be stimulated by hypoglycemia, and this mechanism is dependent on fructose metabolism via KHK. Extracellular fructose induced changes of expression levels of KHK, GLUT5, nNOS, BDNF, PACAP and SST, indicating that fructose consumption might potentiate the improper activation of GI neurons by fructose. And fructose disruption on expression levels of various other genes that regulate energy balance suggests fructose is likely to stimulate food intake under euglycemic conditions, which might involve complicated combination of pathways.