Decline in Glucokinase Activity in the Arcuate Nucleus of Streptozotocin-Induced Diabetic Rats

Abstract

Glucokinase (GK) is known to be the critical glucose sensor of pancreatic B-cells. However, the localization and functional role of GK in the brain remains to be elucidated. In this study, we measured both the activity and mRNA level of GK in the hypothalamic nuclei and the cortex of rats injected intraperitoneally with streptozotocin or vehicle. GK activity was measured by a fluorometric assay; and the GK mRNA level, by use of the realtime reverse transcription polymerase chain reaction. GK activity in vehicle-treated rats was high in the arcuate nucleus, moderate or low in the ventromedial nucleus, lateral hypothalamic area, and paraventricular nucleus, and very low in the cortex. The order of GK mRNA level was almost the same as that of GK activity. GK activity and GK mRNA level only in the arcuate nucleus of streptozotocin-treated rats at 7 d, but not at 2 d, after treatment were lower than those of vehicle-treated rats. The results suggest that prolonged hyperglycemia induced by diabetes decreased the activity of GK in the arcuate nucleus.Key words glucokinase activity; glucokinase mRNA; hyperglycemia; hypothalamus; arcuate nucleus © 2006 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: q0391504@ccmailg.meijo-u.ac.jp the reaction was started by the addition of glucose. The rate of increase in fluorescence of NADH at excitation and emission wavelengths of 340 and 450 nm, respectively, was recorded for about 5 min. HK activity was measured at a glucose concentration of 0.5 mM, and GK activity was estimated as the difference between activities at 0.5 and 50 mM glucose.Fluorescence Real-Time RT-PCR for GK mRNA Brain samples (about 1 mg) were transferred to 100 ml of TRIzol (Invitrogen, Carlsbad, CA, U.S.A.) immediately after their collection and homogenized with a Teflon pestle. Total RNA was extracted according to the protocol of the manufacturer. Single-stranded cDNA was synthesized from 1 mg of total RNA with a 1st strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Mannheim, Germany). GK mRNA was quantified with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster, CA, U.S.A.). For detection of GK mRNA (GenBank accession no. M25807), PCR primer sets for GK (forward: 5Ј-CAAGCTGCACCC-GAGCTT-3Ј, reverse: 5Ј-TGATTCGATGAAGGTGATTTC-G-3Ј) and carboxyfluorescein (FAM)-labeled and carboxytetramethylrhodamine (TAMRA)-labeled TaqMan probes for GK (5Ј-TCAGCCTGCGCACACTGGCG-3Ј) were purchased from Nihon Gene Research Laboratories (Sendai, Japan). In each well of the plate, 12.5 ml of TaqMan Universal PCR Master mix (4304437; Applied Biosystems, Foster, CA, U.S.A.), 10 pmol of primers, 1.5 ml of cDNA samples, and 5.0 pmol of GK probe were applied to give a total volume of 25 ml. PCR settings were as follows: 40 cycles for 15 s at 95°C and for 1 min at 60°C. The amount of each mRNA was standardized by the graded dilution of highly concentrated cDNA samples from the hypothalamus. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level, an endogenous reference...

Effects of Caudal Fourth Ventricular Lactate Infusion on Hypoglycemia-Associated MCT2, GLUT3, GLUT4, GCK, and Sulfonylurea Receptor-1 Gene Expression in the Ovariectomized Female Rat LHA and VMH: Impact of Estradiol

“…We surmise that these measures may reflect local patterns of GCK enzyme activity, as recent studies on the LHA and VMH show that hyperglycemia elicits parallel modifications in GCK gene and protein expression in both sites (Nishio et al 2006). While our observations of unvaried LHA GCK gene expression is consistent with apparent maintenance of GLUT3-and GLUT4-mediated glucose uptake, the data showing diminished GCK transcripts in the VMH were surprising, as local GLUT3 and GLUT4 mRNA profiles were either unaltered or increased.…”

Effects of glucose and insulin on glucokinase activity in rat hypothalamus

“…5 mM for low Km hexokinase activities, a concentration at which GK is essentially inactive (Bedoya et al 1984, Nishio et al 2006, and 25 mM glucose, a concentration at which all phosphotransferase activities were measured. Glucose phosphorylation rates were measured using an isotopic method (Bedoya et al 1984).…”

Molecular and functional changes in glucokinase expression in the brainstem dorsal vagal complex in a murine model of type 1 diabetes

“…A similar decrease in GCK expression and activity has been reported in the arcuate nucleus, but not in other glucose-responsive hypothalamic nuclei, of STZ-treated rats (Nishio et al, 2006). The present findings suggest that responses of NTS neurons to increased glucose concentration may be altered as a consequence of chronic hyperglycemia or hypoinsulinemia in a GCK-dependent manner, similar to glucose-sensitive neurons of the arcuate nucleus.…”

“…Neurons in the NTS are glucose sensors, and this sensitivity may be especially prominent in GABAergic NTS neurons (Boychuk et al 2015a;Lamy et al 2014). Since GCK expression was reduced in the NTS of STZtreated mice after several days of hyperglycemia, we examined action potential frequency of A similar decrease in GCK expression and activity was reported in the arcuate nucleus, but not in other glucose-responsive hypothalamic nuclei, of STZ-treated rats (Nishio et al 2006). The present findings suggest that responses of NTS neurons to increased glucose concentration may be altered as a consequence of chronic hyperglycemia or hypoinsulinemia in a GCK-dependent manner, similar to glucose-sensitive neurons of the arcuate nucleus.…”