Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 29:13:275.
doi: 10.3389/fnins.2019.00275. eCollection 2019.

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

Magdiel Salgado  1 , Patricio Ordenes  1 , Marcos Villagra  1 , Elena Uribe  2 , María de Los Angeles García-Robles  1 , Estefanía Tarifeño-Saldivia  2
Affiliations

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

Magdiel Salgado et al. Front Neurosci. .

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.

Keywords: GK regulatory protein; glucokinase; glucosensing; metabolic; tanycytes.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Expression and subcellular localization of glucose sensor machinery in insulinoma cells. (A) RT-PCR for GK (510 bp; first panel), GKRP (418 bp; second panel), GLUT2 (218 bp third panel), and β-actin (353 bp; fourth panel). PCR products were amplified from liver (lane 1), 832/13 culture (lane 2), negative control (RT -) of liver (line 3) and water (lane 4). (B) Western Blot from total protein extracted from liver (lane 1, liver, 50 μg of protein loaded) and 832/13 culture (lane 2, INS, 100 μg of protein loaded). Immunodetection was performed for GK (first panel, 52 kDa), GKRP (second panel, 69 kDa), GLUT2 (third panel, 60 Kda), and β-actin (fourth panel, 43 kDa). (C) Immunocytochemistry for GK. (D) Immunocytochemistry for GLUT2. (E–G) Immunocytochemistry for GK and GKRP in insulinoma cultured at 0.5 mM glucose and (H,I) 50 mM glucose. (E,H) Immunostaining for GKRP and nucleus, (F,I) GK, and (G,J) Merge. Nuclear staining performed by Hoechst. Scale bar: 50 μm. White arrows are indicating the cytoplasm of insulinoma cells.
FIGURE 2
FIGURE 2
Subcellular localization of GK and GKRP in insulinoma cells overexpressing GKRP. (A) Total hexokinase activity measured at 1, 10, and 30 mM glucose on Ad-GKRP (red bars) and Ad-Control (black bars). (B) Schema of glucose treatments for transduced cells to evaluate compartmentalization dynamics. (C,D) Immunocytochemistry for GK (C,F) and GKRP (D,G) of cells cultured at 0.5 mM (C–E) and 50 mM (F–H) glucose. (I,J) Quantification of nuclear and cytoplasmic fluorescence for GK (I) and GKRP (J) immunocytochemistry. (K) Immunodetection by western blot of GK (52 kDa, first panel) and GKRP (69 kDa, second panel) in nuclear and cytoplasmic protein extracts of transduced insulinoma cultured at 0.5 and 50 mM glucose. Lamin B1 (68 kDa, third panel) and β-actin (43 kDa, fourth panel) were used to confirm the purity of extracts and correct nuclear and cytoplasmic quantification. (L,M) Densitometry analysis of GK (L) and GKRP (M) at both glucose concentrations. Statistical T-test, ∗p-value < 0.05, ∗∗p-value < 0.01. White arrows are indicating the cytoplasm of insulinoma cells, while arrowheads indicate the nucleus.
FIGURE 3
FIGURE 3
Translocation dynamics for GK and GKRP in insulinoma cells overexpressing GKRP at increasing glucose concentrations. Transducted insulinoma cells were cultured for 3 h at 0.5 mM glucose with a posterior increase to 50 mM glucose for 30 min. (A) Dynamic subcellular localization of GKRP through fluorescence detection of RFP. (B) Dynamic subcellular localization of GK through immunocytochemistry. Scale Bar 20 μm. (C,D) Quantification of nuclear/cytoplasmic ratio measured by fluorescence intensity of both subcellular compartments for GKRP (C) and GK (D). Inserted box: Saccharose control treatments. Statistical ANOVA, ∗p-value < 0.05, ∗∗p-value < 0.01. Arrowheads show the nucleus of insulinoma cells, indicating nuclear exclusion.
FIGURE 4
FIGURE 4
Dynamic localization of GK in culture of tanycytes transduced with Ad-GKRP in response to high glucose. (A) Schematic representation of tanycytes transduction and glucose treatment. (B–M,B’–M’) Immunocytochemistry for GK in tanycytes transduced with Ad-control (B–M) and Ad-GKRP (B’–M’) incubated with 15 mM glucose for 0, 30, and 60 min. GK localization (green), Virus transduction (red), and nuclear staining with Hoechst. (N,O) Quantification of nuclear/cytoplasmatic ratio of GK fluorescence intensity for Ad-Control (N) and Ad-GKRP (O). Statistical T-test, ∗p-value < 0.05, ∗∗p-value < 0.01. White arrows are indicating the nucleus of tanycytes.
FIGURE 5
FIGURE 5
Relative expression of Gckr and Gck in different tissues. (A) Expression levels for Gckr and Gck were measured by qPCR and expression ratios were calculated for liver (L), pancreas (P), hypothalamus (H), and tanycytes (T). (B) Expression ratios for ad-GKRP transduced insulinoma (IG), and ad-GKRP tranduced tanycytes (TG). (C) Model to describe translocation dynamics determined by the ratio of expression between Gckr and Gck. When GKRP is in excess, in relation to GK, nuclear translocation is found at low glucose concentrations (as observed in hepatocytes). When GKRP is less expressed, in relation to GK, nuclear translocation is observed at high glucose concentrations (as observed in tanycytes). Statistical T-test, ∗∗p-value < 0.01, ∗∗∗p-value < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ainscow E. K., Mirshamsi S., Tang T., Ashford M. L. J., Rutter G. A. (2002). Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels. J. Physiol. 544 429–445. 10.1113/jphysiol.2002.022434 - DOI - PMC - PubMed
    1. Alvarez E., Roncero I., Chowen J. A., Vázquez P., Blázquez E. (2002). Evidence that glucokinase regulatory protein is expressed and interacts with glucokinase in rat brain. J. Neurochem. 80 45–53. 10.1046/j.0022-3042.2001.00677.x - DOI - PubMed
    1. Baltrusch S., Tiedge M. (2006). Glucokinase regulatory network in pancreatic -cells and liver. Diabetes 55 S55–S64. 10.2337/db06-S008 - DOI - PubMed
    1. Bosco D., Meda P., Iynedjian P. B. (2000). Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization. Biochem. J. 348 215–222. 10.1042/bj3480215 - DOI - PMC - PubMed
    1. Brown K. S., Kalinowski S. S., Megill J. R., Durham S. K., Mookhtiar K. A. (1997). Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus. Diabetes 46 179–186. 10.2337/diab.46.2.179 - DOI - PubMed

LinkOut - more resources