Glial glucokinase expression in adult and post-natal development of the hypothalamic region

Abstract

It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic beta-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT-PCR [quantitative RT-PCR (reverse transcription-PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immuno-ultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of beta1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.

Keywords: CSF, cerebrospinal fluid; DIG, digoxigenin; DTT, dithiothreitol; GFAP, glial fibrillary acidic protein; GK, glucokinase; GKRP, GK regulatory protein; GLUT2, glucose transporter 2; RT–PCR, reverse transcription–PCR; TFIIB, transcription factor IIB; arcuate nucleus; brain; glia; glucokinase; glucosensing; tanycyte.

Figure 1. RT–PCR, Western-blot and immunohistochemical analyses of GK expression

(A) RT–PCR for GK mRNA using mRNAs isolated from pancreas, liver and hypothalamus. Left-hand lane, DNA 100 bp standard; lane 1, amplified sequence of 510 bp using mRNA from rat adult hypothalamus; lane 2, RT–PCR product obtained using mRNA isolated from rat liver; lane 3, RT–PCR product obtained using mRNA isolated from rat pancreas; lane 4, RT–PCR product obtained using mRNA isolated from rat spleen (negative control); lane 5, RT(−) of hypothalamus. (B) Western-blot analysis using two different antibodies for human GK. Lane 1, sheep anti-GK and rat liver extract; lanes 2–4, rabbit anti-GK and rat liver (2), pancreas (3) and hypothalamus (4) extracts; lane 5, control using hypothalamic extract. (C–G) Frontal sections of rat brain immunostained with sheep anti-GK and rabbit anti-GLUT2 antibodies followed by secondary antibodies labelled with Cy3 (red) or Cy2 (green). Topro-3 was used for nuclear staining. GK immunoreactivity is detected in the cytosol of ependymal cells and GLUT2 is detected in the ciliar membranes. III V, third ventricle; Ep, ependymal cells. Scale bars in (C–H), 30 μm.

Figure 2. Immunohistochemical and ultrastructural analysis of the hypothalamic tanycytes

(A–C) Frontal hypothalamic sections immunostained with anti-vimentin antibody and using a secondary antibody labelled with Cy2. Topro-3 was used for nuclear staining. Z-stack sections were obtained using confocal analysis and are shown in X–Y and X–Z projection (C). (D–F) Transmission electron microscopy of the ventricular wall. The area analysed is framed in (C). The association between tanycytes and neuron is shown (N). (E) The β1-tanycytes present blebs (B) in the apical region of the cells. (F) Contact region between tanycytes and neurons (arrows). (G–I) Scanning electron microscopy of the ventricular wall associated with the hypothalamic basal area. (H) Transition between ependymal cells and α-tanycytes. (I) Scanning microscopy of blebs present in the apical region of β-tanycytes. III V, third ventricle; Ep, ependymal cells; ME, median eminence; M, mitochondrion. Scale bar in (A–C), 150 μm; scale bars in (D–F), 1 μm; scale bar and in (G), 100 μm; scale bars in (H and I), 25 μm.

Figure 3. Glial GK mRNA detection by in situ hybridization

Frontal section of rat hypothalamus probed with a GK antisense riboprobe. (A) A high hybridization signal was observed in glial cells of the periventricular area. (B) The sense riboprobe for GK was used as a control. (C–F) The α- and β1-tanycytes present positive hybridization with the antisense GK riboprobe. In α- and β1-tanycytes, the positive reaction is observed in the proximal area of the cells. Also some neurons show a positive reaction for the GK riboprobe (F). III V, third ventricle; ME, median eminence. Scale bars in (A and B), 100 μm; in (C–F), 20 μm.

Figure 4. GK and vimentin are co-localized in the ventricular tanycytes

(A and B) Frontal hypothalamic sections immunostained with anti-vimentin and anti-GK antibodies and using a secondary antibody labelled with Cy3 (red) and Cy2 (green). (C) A frontal hypothalamic section used as a control. (D) Quantitative analysis of GK-positive nuclei in tanycytes and neurons. Statistical analysis was performed using the Student’s t test with Welch correction; P values<0.05 were considered significant. Results represent the average GK-positive nuclei in a total of 16 areas within the ventricular and arcuate nuclei. (E–H) Brain sections co-immunostained with anti-GFAP (astrocytes marker) and sheep anti-GK. (I–L) Brain sections co-immunostained with anti-vimentin (blood vessels marker) and rabbit anti-GK. (M–O) Frontal hypothalamic sections immunostained with rabbit anti-GK using a secondary antibody labelled with Cy3 (red) and Topro-3 (blue) for nuclear staining. GK is mainly detected in the nucleus of tanycytes (the inset and merged image in O). (P) Western-blot study using nuclear protein extracts isolated from the periventricular hypothalamus and analysed with rabbit anti-GK and anti-TFIIb antibodies. III V, third ventricle. Scale bars in (A and B), 100 μm; scale bars in (C and M–O), 50 μm; scale bars in (E–L), 150 μm.

Figure 5. Ultrastructural immunocytochemistry of hypothalamic ventricular wall

Frontal section through the rat medial basal hypothalamus. Immunohistochemical analysis using rabbit anti-GK antibody and anti-IgG labelled with 10-nm gold particles. (A, B) Apical region of β1-tanycytes. The immunoreaction is mainly observed in the apical region of the cells, depicted over the segment line. (C, D) Nuclear region of β1-tanycytes. Stronger immunostaining was detected that is associated with the chromatin. III V, third ventricle; N, nucleus. Scale bars in (A, C), 1 μm; scale bars in (B, D), 0.5 μm.

Figure 6. GK and GLUT2 expressions in the ventricular tanycytes

(A–F) Frontal hypothalamic sections immunostained with anti-GLUT2 and sheep anti-GK antibodies and using a secondary antibody labelled with Cy2 (green) or Cy3 (red). Topro-3 (blue) was used for nuclear staining. Confocal analysis and Z-stack projection confirmed GK and GLUT2 co-localization in the apical region of the tanycytes. Nuclear staining was also detected. Insets: negative controls. III V, third ventricle. Scale bars in (A–C), 50 μm; scale bars in (D–F), 30 μm.

Figure 7. GK levels during post-natal tanycyte development

Frontal hypothalamic sections through the rostral hypothalamic region immunostained with rabbit anti-GK (green) or and anti-vimentin (red). (A–D) Post-natal 10-day-old (PN10). (E–H) Post-natal 15-day-old (PN15). (I–L) Post-natal 20-day-old (PN20). (M) RT–PCR analysis. Lane 1, 100 bp standard; lane 2, RT–PCR product obtained using mRNA isolated from 1-day-old rat hypothalamus; lane 3, mRNA isolated from 5-day-old rat hypothalamus; lane 4, mRNA isolated from 15-day-old rat hypothalamus. (N) QRT–PCR analysis of the mRNA GK levels in samples isolated from 1-day-old rat hypothalamus (pn1), 5-day-old rat hypothalamus (pn5), 10-day-old rat hypothalamus (pn10) and 15-day-old rat hypothalamus (pn15). *P values <0.05; **P values <0.01. Results are the ratio of GK mRNA to cyclophilin mRNA and represent the means±S.D. for three independent experiments. III V, third ventricle. Scale bars in (A–L), 100 μm.