When a Little Bit More Makes the Difference: Expression Levels of GKRP Determines the Subcellular Localization of GK in Tanycytes

Abstract

Glucose homeostasis is performed by specialized cells types that detect and respond to changes in systemic glucose concentration. Hepatocytes, β-cells and hypothalamic tanycytes are part of the glucosensor cell types, which express several proteins involved in the glucose sensing mechanism such as GLUT2, Glucokinase (GK) and Glucokinase regulatory protein (GKRP). GK catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6P), and its activity and subcellular localization are regulated by GKRP. In liver, when glucose concentration is low, GKRP binds to GK holding it in the nucleus, while the rise in glucose concentration induces a rapid export of GK from the nucleus to the cytoplasm. In contrast, hypothalamic tanycytes display inverse compartmentalization dynamic in response to glucose: a rise in the glucose concentration drives nuclear compartmentalization of GK. The underlying mechanism responsible for differential GK subcellular localization in tanycytes has not been described yet. However, it has been suggested that relative expression between GK and GKRP might play a role. To study the effects of GKRP expression levels in the subcellular localization of GK, we used insulinoma 832/13 cells and hypothalamic tanycytes to overexpress the tanycytic sequences of Gckr . By immunocytochemistry and Western blot analysis, we observed that overexpression of GKRP, independently of the cellular context, turns GK localization to a liver-like fashion, as GK is mainly localized in the nucleus in response to low glucose. Evaluating the expression levels of GKRP in relation to GK through RT-qPCR, suggest that excess of GKRP might influence the pattern of GK subcellular localization. In this sense, we propose that the low expression of GKRP (in relation to GK) observed in tanycytes is responsible, at least in part, for the compartmentalization pattern observed in this cell type. Since GKRP behaves as a GK inhibitor, the regulation of GKRP expression levels or activity in tanycytes could be used as a therapeutic target to regulate the glucosensing activity of these cells and consequently to regulate feeding behavior.

Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells

“…In homeostatic state, GCKR is usually present in molar excess of HK1 in the cell. However, the glycolytic rate is affected by the level of GCKR and the binding status of GCKR/HK1, which is dynamically and intricately modulated and can be rapidly changed by metabolic conditions (22). Thus, the expression of GCKR and its posttranslational modifications are essential regulatory mechanisms in glycolysis (23).…”

GKRP-dependent modulation of feeding behavior by tanycyte-released monocarboxylates

“…To evaluate the effect of GKRP overexpression on GK activity, 832/13 cells were transduced with 5 x 10 7 IFU/mL each adenovirus and incubated for 72 h. After that, cells were lysed by ultrasound, and HK activity was promptly determined as previously described with slight modifications 50 . Briefly, a glucose 6-phosphatase (G-6P) dehydrogenase-coupled reaction was used, and the activity was followed by measuring the increase in absorbance at 340 nm after 5 min incubation at 37°C.…”

“…Highly enriched primary tanycyte cultures from 1-day postnatal brains (10-12 rats) were isolated and immunocharacterized following the method described previously 13 , 16 , 50 , 51 . Briefly, the hypothalamus was removed and further dissected to obtain a region close to the ependymal layer.…”