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Endocrinology, doi:10.1210/en.2003-1191
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Endocrinology Vol. 145, No. 5 2507-2515
Copyright © 2004 by The Endocrine Society

In Vitro Increase in Intracellular Calcium Concentrations Induced by Low or High Extracellular Glucose Levels in Ependymocytes and Serotonergic Neurons of the Rat Lower Brainstem

Ryutaro Moriyama, Hiroko Tsukamura, Mika Kinoshita, Hirokatsu Okazaki, Yukio Kato and Kei-ichiro Maeda

Laboratory of Animal Reproduction, Graduate School of Bioagricultural Sciences, Nagoya University (R.M., H.T., M.K., K.M.), Nagoya 464-860, Japan; and Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, Meiji University School of Agriculture (H.O., Y.K.), Kawasaki, Kanagawa 214-8571, Japan

Address all correspondence and requests for reprints to: Dr. Kei-ichiro Maeda, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. E-mail: keimaeda{at}agr.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pancreatic glucokinase (GK)-like immunoreactivities are located in ependymocytes and serotonergic neurons of the rat brain. The present study investigated in vitro changes in intracellular calcium concentrations ([Ca2+]i) in response to low (2 mM) or high (20 mM) extracellular glucose concentrations in isolated cells from the wall of the central canal (CC), raphe obscurus nucleus (ROb), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LHA) in male rats. An increase in [Ca2+]i was found in cells from the CC (21.1% or 9.8% of ependymocytes), ROb (10.9% or 14.5% of serotonergic neurons), VMH (7.8% and 25.2% of neurons), and LHA (20% or 15.7% of neurons), when extracellular glucose levels were changed from 10 to either 2 or 20 mM, respectively. Most of the ependymocytes and serotonergic neurons responding to the glucose changes were immunoreactive to the anti-GK in the CC (96.8% for low glucose and 100% for high glucose) and ROb (100% for low and high glucose). The [Ca2+]i increase was blocked with calcium-free medium or L-type calcium channel blocker. Cells with an increase in [Ca2+]i in response to low glucose did not respond to high glucose and vice versa. Inhibition of GK activity with acute alloxan treatment blocked low or high glucose-induced [Ca2+]i increases in most GK-immunoreactive cells from the CC or ROb. The glucose-sensitive [Ca2+]i increase in neurons of the VMH and LHA was also alloxan-sensitive, but no cells taken from the VMH and LHA were immunoreactive to the antibody used. The present study further indicates that ependymocytes of the CC and serotonergic neurons in the ROb are also sensitive to the changes in extracellular glucose in a GK-dependent manner, but that the subtype of GK in these cells could be different from that in the VMH and LHA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BRAIN GLUCOSE SENSING has been thought to be involved in regulating glucose homeostasis, food intake (1, 2), and reproductive functions (3). Neurons in several hypothalamic regions, such as the hypothalamic ventromedial nucleus (VMH), lateral hypothalamic area (LHA), and arcuate nucleus, have been reported to respond to hyper- or hypoglycemic stimulation (4, 5, 6, 7, 8, 9, 10, 11). In particular, the neurons located in the VMH and LHA, the satiety and feeding centers, are considered to play important roles in controlling satiety and appetite (4, 6, 8).

Blood glucose levels have also been reportedly sensed by the lower brainstem. Lateral ventricular injection of 2-deoxy-D-glucose (2DG), an antimetabolic glucose analog, increases food intake, but local injection of this drug into the VMH, LHA, or other hypothalamic regions had no effect on food intake (12), whereas local injection of 5-thio-D-glucose (5TG), another glucose antagonist, into the lower brainstem induced food intake and a hyperglycemic response (2). Ritter et al. (13) reported that the feeding response induced by lateral ventricular administration of 5TG was blocked by obstruction of the cerebral aqueduct with silicon glue, but that a fourth ventricular administration of 5TH induced food intake in animals with the same obstruction. In addition, the administration of 2DG limited to the fourth ventricle suppressed pulsatile LH secretion (14). These results support the idea that a glucose-sensing mechanism is located in the lower brainstem to control feeding and reproductive functions.

Pancreatic glucokinase (GK), an enzyme that plays a principal role in sensing blood glucose levels in pancreatic ß-cells (15), has been found in the hypothalamus and lower brainstem in rats (16, 17, 18, 19, 20). Our previous results have shown that the ependymocytes of the wall of the central canal (CC) and fourth ventricle and serotonergic neurons in the raphe nuclei, such as the raphe obscurus nucleus (ROb) and raphe pallidus, showed pancreatic GK-like immunoreactivities in rats (21).

The present study was conducted to determine whether the pancreatic GK-like immunoreactive cells in the lower brainstem respond to hyper- or hypoglycemic stimuli. For this purpose, in vitro changes in the intracellular calcium concentration ([Ca2+]i) in response to low or high extracellular glucose levels were determined in isolated cells taken from various brain areas, such as the wall of the CC, ROb, VMH, and LHA in adult male rats. In addition, the effects of alloxan, a GK inhibitor, on the changes in [Ca2+]i in response to hypo- or hyperglycemic conditions were examined to determine whether the response of [Ca2+]i is mediated by the GK activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Intact male Wistar-Imamichi strain rats (390 ± 30 g), kept in a controlled environment (14 h of light and 10 h of darkness; lights on at 0500 h; temperature, 24 ± 2 C), were used. The present study was approved by the committee on animal experiments of Graduate School of Bioagricultural Sciences, Nagoya University.

Preparation of isolated brain cells
After decapitation, the whole brain was removed and placed in cold HEPES-buffered Krebs-Ringer bicarbonate buffer (HKRB; 129 mM NaCl, 5.0 mM NaHCO3, 3.7 mM KCl, 1.2 mM KH2PO4, 1.8 mM CaCl2, 1.2 mM MgSO4, and 10 mM HEPES, pH 7.3) containing 10 mM glucose. Two coronal sections, 0.5 and 1 mm thick, respectively, were taken from the hindbrain (approximately –14.0 to –13.5 mm bregma) and forebrain (approximately –2.0 to –3.0 mm bregma), respectively (22), with a hand-made brain blocker placed in ice-cold HKRB. The CC, ROb, VMH, and LHA were punched out using a 18-gauge stainless-steel tubing. The tissues were washed with ice-cold HKRB and then dissociated with 6.8 µg/ml papain in HKRB containing 0.2 mg/ml BSA, 0.2 mg/ml L-cystein, and 1.8 mg/ml glucose for 15 min at 31 C. The tissues were gently and mechanically dispersed in the cold HKRB, then filtered through a 70-µm pore size nylon mesh (BD Biosciences, Franklin Lakes, NJ). A cell pellet was obtained by centrifugation at 1000 rpm for 5 min. Cells were resuspended in HKRB and plated onto CELLocate coverslips (square size, 55 µm; Eppendorf, Hamburg, Germany). The CELLocate coverslips had grids with numbers and letters to indicate the individual cell locations. The cells were kept at 20 C for at least 1.5 h for adhesion to the coverslip. The O2/CO2 condition was not controlled throughout the experiments.

[Ca2+]i measurement
The [Ca2+]i was measured 3–10 h after cell preparation. Fura-2 was loaded onto the cells by incubating with 5 µM fura-2/acetoxymethylester (Molecular Probes, Eugene, OR) in HKRB for 40 min at 20 C. The cells were washed three times with HKRB and kept at 35 C throughout the Ca2+ measurement, then superfused with 10 mM glucose for 3 min, with 2 or 20 mM for 20 min, with 10 mM for 15 min, and then with 2 or 20 mM glucose for 20 min. The flow rate was 1 ml/min throughout the superfusion, and the glucose was dissolved in HKRB at various concentrations. Fluorescence images were taken every 6 sec with a fluorescent microscope (Olympus, Tokyo, Japan) and an ICCD camera (Hamamatsu Photonics, Hamamatsu, Japan) with excitation wavelengths at both 340 and 380 nm. Ratio images were obtained by the Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan). The [Ca2+]i was calculated from the ratio values according to a calibration curve obtained with a solution mimicking cytosol using Ca2+-EGTA buffer and Fura-2 free acid (Molecular Probes) (23).

The functional condition of the cells was determined at the end of each experiment by a calcium increase in response to 100 mM KCl. Data were taken only from cells that responded to the KCl stimulation. The cells that showed a [Ca2+]i increase of more than 50 nM within 15 min after changing the glucose concentration to 2 or 20 mM from 10 mM were considered as having a positive response. The same criteria were used in previous studies with pancreatic ß-cells and hypothalamic neurons (9, 24, 25). The osmolarity of each medium was kept constant by replacing glucose with xylose.

In some incubations, extracellular calcium was chelated by 0.1 mM EGTA or the L-type calcium channel was blocked by 1 µM nifedipine (Sigma-Aldrich Corp., St. Louis, MO) to determine whether the [Ca2+]i increase was triggered by an influx of extracellular Ca2+. In other incubations, alloxan, a GK inhibitor, was added to the medium to determine the role of GK in mediating the [Ca2+]i response to the changes in glucose concentration. Alloxan monohydrate (Sigma-Aldrich Corp.) was dissolved in ice-cold 0.05 M citric acid buffer (pH 4.2) at 50 mM, and the solution was diluted to 0.5 mM with incubation medium immediately before use.

The remaining tissues were fixed with 4% paraformaldehyde after various regions were punched out. Coronal sections (50 µm) were made with a cryostat and stained with thionine, and the dissected areas were verified under a microscope. Data were taken only from the experiments in which correct areas were punched out in these sections.

Immunocytochemistry
After [Ca2+]i measurement, cells were fixed with 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.5) and immunohistochemically stained with antipancreatic GK antiserum (1:25; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as in an earlier study (21). The immunoreactivity was visualized with Alexa Fluor 488-conjugated donkey antigoat IgG (1:200; Molecular Probes) and observed by confocal laser microscopy (MRC-1024, Bio-Rad Laboratories, Hercules, CA).

To determine cell types, cells taken from each group were double immunostained with antipancreatic GK and one of the following antibodies: a mouse monoclonal antibody to vimentin, a marker protein for ependymocytes (1:200; Roche, Mannheim, Germany), a mouse monoclonal antibody to MAP2, a marker for neuron (1:500; Sigma-Aldrich Corp.), or a rabbit polyclonal antibody to serotonin (1:1000; DiaSorin, Stillwater, MN). Cy3-conjugated donkey antimouse IgG (1:800; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or antirabbit IgG (1:800; Jackson ImmunoResearch Laboratories, Inc.) was used to visualize the immunoreactivities of the above-mentioned cell markers.

Preparation of RNA and RT-PCR
Total RNAs were extracted from the rat liver and wall of the fourth ventricle, consisting of the ependymocytes, and their cDNAs were synthesized using the Ready-to-Go kit (Amersham Pharmacia Biotech, Piscataway, NJ), followed by PCR. Two specific primers were synthesized to distinguish the pancreas- and liver-type GK mRNAs, pairing with a universal primer, 5'-CACGTAGGTGGGTAACATCTTTACAC-3', a complimentary sequence for the both mRNA types. The primers was 5'-AATCTTGCGGAACACTGAG-3' for pancreas type and 5'-AGGAGTCAGGAACATCTCT-3' for liver type, respectively. The amplification was performed in a reaction mixture (10 µl) containing a set of primers (10 pmol each) and 0.5 U AmpliTaq Gold polymerase (PerkinElmer Cetus, Norwalk, CT) with 36 cycles of denaturation (95 C, 30 sec), annealing (55 C, 30 sec), and extension reaction (72 C, 3 min) steps. The resulting PCR products (240 bp for both products) were separated on 2% agarose gel. The nucleotide sequences of the PCR products, which were extracted from the agarose gel, were determined by the fluorescence-labeled dye terminator reaction using the BigDye terminator version 2 system (PerkinElmer Cetus) according to the manufacturer’s instructions.

Data analysis
Statistical differences in [Ca2+]i between the periods with low and high glucose were determined by one-way ANOVA, followed by Fisher’s protected least significant difference test. Statistical comparison was made by binomial test between the percentages of cells that responded to low and high glucose and between the percentages of cells in which alloxan was effective and ineffective. Differences at P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histology
Figure 1Go shows a photomicrograph of a representative coronal section of the rat lower brainstem (A) and hypothalamus (B). The entire CC and most of the ROb, VMH, and LHA were punched out.



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  		FIG. 1. Coronal sections of the lower brainstem (A) and hypothalamus (B) from which the wall of the CC, ROb, VMH, and LHA were punched out. Scale bars, 1 mm. 3V, Third ventricle.

 
Effects of low or high glucose concentrations on [Ca2+]i in selected brain areas
Figure 2Go shows a representative pattern of changes in and staining of the cells taken from the CC, ROb, VMH, and LHA and differential interference contrast images of the representative cells indicating the [Ca2+]i responses (Fig. 2Go, upper photos in each panel) and the dual immunostaining with GK and respective cell markers (Fig. 2Go, lower photos in each panel). [Ca2+]i increased beyond the resting level when the glucose concentration in the medium was reduced from 10 to 2 mM (Fig. 2Go, left panels). The mean [Ca2+]i values in the CC cells were significantly (P < 0.05) higher with 2 mM glucose (239 ± 22 and 201 ± 13 nM for the first and second 20 min; mean ± SEM[SCAP]; n = 39) than with 10 mM (138 ± 8 nM for the first 3 min and 125 ± 7 nM for the second 20 min; by Fisher’s protected least significant difference test). Similar increases were found when the glucose concentration was increased from 10 to 20 mM (Fig. 2Go, right panels). In both cases, [Ca2+]i was kept at a high level at 2 or 20 mM and then decreased to the resting level or below after changing the glucose concentration to 10 mM. In the cells satisfying the criteria, the mean [Ca2+]i values with 2 or 20 mM glucose were significantly higher than those with 10 mM glucose in each brain area.



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  		FIG. 2. The [Ca2+]i increase evoked by low (left panels) and high (right panels) glucose concentrations in cells from the CC, ROb, VMH, or LHA. Horizontal bars at the top indicate changes in glucose concentrations in the medium and the addition of 100 mM KCl to the medium. Each panel is accompanied by two photomicrographs of the cell showing the [Ca2+]i increase: upper photographs present differential interference images of the cell, and lower photographs provide confocal microscopic images of the dual immunostaining of cells. Immunoreactivity to the pancreatic GK antibody is shown in green. Cell markers in red indicate vimentin for ependymocytes in the CC, serotonin for serotonergic neurons in the ROb, and MAP2 for neurons in the VMH and LHA. Scale bars, 10 µm.

 
The percentages of cells responding to the changes in glucose concentrations to the cells immunoreactive to respective cell markers were as follows: 21.1% (31 of 147) or 9.8% (12 of 123) of the vimentin-immunoreactive cells in the CC responded to low or high glucose, respectively; 10.9% (20 of 184) or 14.5% (19 of 131) of the serotonin-immunoreactive cells in the ROb responded to low or high glucose, respectively; 7.8% (8 of 102) and 25.2% (36 of 143) of the MAP2-immunoreactive cells in the VMH responded to low or high glucose, respectively; and 20% (13 of 65) and 15.7% (31 of 198) of the MAP2-immunoreactive cells in the LHA responded to low or high glucose, respectively.

Most of the vimentin- or serotonin-immunoreactive glucose-responsive cells were also stained with pancreatic GK in the CC [30 of 31 cells (96.8%) for low glucose; 12 of 12 cells (100%) for high glucose] or ROb [20 of 20 cells (100%) for low glucose; 19 of 19 (100%) cells for high glucose], although glucose-responsive cells immunonegative to vimentin or serotonin were not stained with pancreatic GK in the CC and ROb. Furthermore, cells responding to either low or high glucose in the VMH and LHA were not stained with the pancreatic GK antibody used.

Effects of calcium-free medium and nifedipine on either low or high glucose-induced [Ca2+]i increases
The [Ca2+]i increases in response to either 2 or 20 mM glucose in the cells taken from the CC, ROb, VMH, or LHA were blocked by chelating extracellular Ca2+ (Fig. 3Go). Likewise, either low or high glucose-induced [Ca2+]i increases were blocked by 1 µM nifedipine in cells taken from the CC and ROb.



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  		FIG. 3. Effects of Ca2+-free medium on either low or high glucose-induced [Ca2+]i increases in representative cells from the CC (n = 7 or 3 for low or high glucose, respectively), ROb (n = 8 or 7), VMH (n = 6 or 15), or LHA (n = 4 or 19). Effects of 1 µM nifedipine were examined only in cells from the CC (n = 5 or 3 for low or high glucose, respectively) and ROb (n = 9 or 3). Horizontal bars at the top indicate changes in glucose concentrations and the duration of Ca2+-free medium or 1 µM nifedipine.

 
Effects of low and high glucose concentrations on [Ca2+]i in a single cell
Figure 4Go shows the [Ca2+]i increases in representative CC cells exposed to low and high glucose concentrations in an incubation (Fig. 4Go, upper panels). Cells with a [Ca2+]i increase in response to low glucose did not respond to high glucose and vice versa. Likewise, when the cells taken from ROb, VMH, and LHA were exposed to both low and high glucose levels, the cells responded to either level (data not shown).



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  		FIG. 4. Effects of low and high glucose concentrations on the [Ca2+]i in cells from the CC (upper panels) and the percentages of cells responding to either low or high glucose taken from the CC, ROb, VMH, or LHA (lower panel). Horizontal bars at the top of the upper panels indicate the changes in glucose concentrations. *, P < 0.05, differences in the percentage between the cells responding to low and high glucose (by binomial test).

 
The percentages of CC cells (75%) and LHA cells (90.9%) responding to low glucose were significantly (P < 0.05) higher than those in the corresponding groups responding to high glucose (by binomial test, 25% in the CC and 9.1% in the LHA; Fig. 4Go, lower panel). The percentage of VMH cells (83.3%) responding to high glucose was significantly (P < 0.05) higher than that in the corresponding group responding to low glucose (16.7%, by binomial test). In ROb, the population of cells responding to high glucose was higher than that responding to low glucose, but there was no significant (P > 0.05) difference.

Effects of alloxan on either low or high glucose-induced [Ca2+]i increases and immunoreactivity to pancreatic GK antibody in selected brain areas
Figure 5Go, A and B, shows representative [Ca2+]i profiles of the GK-immunopositive CC cells in which 0.5 mM alloxan inhibited the [Ca2+]i increase. In Fig. 5Go, C and D, alloxan had no effect on the high or low glucose-induced [Ca2+]i increase in GK-immunonegative CC cells.



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  		FIG. 5. Effects of 0.5 mM alloxan on low or high glucose-induced [Ca2+]i increases in cells from the CC (A–D), and percentages of cells from the CC, ROb, VMH, or LHA in which alloxan inhibited such increases and GK immunoreactivities (E). Horizontal bars in A–D indicate the changes in glucose concentrations; black lines show the duration of the addition of 0.5 mM alloxan or 100 mM KCl to the medium. A–D are accompanied by a differential interference image and confocal microscopic images of GK immunostaining of the cell showing an increase in [Ca2+]i. E, The statistical significance (P < 0.05) between the percentages of cells in which alloxan was effective and ineffective is indicated (*; by binomial test). Scale bars, 10 µm.

 
The [Ca2+]i increase was blocked by alloxan in most CC cells responding to either low (14 of 16 cells, 87.5%) or high (eight of 11 cells, 72.7%) glucose (Fig. 5Go, A and B). In the ROb, alloxan blocked [Ca2+]i increases in 11 of 12 (91.7%) cells responding to low glucose and four of four cells (100%) responding to high glucose. All of the cells in the CC and ROb, in which the alloxan blocked low or high glucose-induced [Ca2+]i increases, had pancreatic GK immunoreactivities (Fig. 5EGo). On the other hand, alloxan had no effect on the glucose-induced [Ca2+]i increases in cells from the CC, which is not immunoreactive to the present anti-GK antibody (Fig. 5Go, C and D). Alloxan had similar effects on cells in the VMH and LHA. Alloxan blocked the [Ca2+]i increase in four of five (80%) or eight of 12 (66.7%) cells responding to low glucose and in four of eight (50%) or eight of 10 (80%) cells responding to high glucose in the VMH or LHA, respectively (Fig. 5EGo). None of the cells was stained with pancreatic GK antibody in those areas.

Statistical analysis revealed that the percentage of the cells in which alloxan inhibited low or high glucose-induced [Ca2+]i increase was significantly higher than that cells in which alloxan did not inhibit the increase in the CC, ROb,VMH, and LHA, except for those cells responding to 20 mM glucose in the VMH (Fig. 5EGo).

Pancreatic GK mRNA expression in ependymocytes
RT-PCR analysis of pancreas- or liver-type GK transcripts prepared from RNAs of ependymocytes and liver is shown in Fig. 6Go. The pancreas-type primer set gave a product only for ependymocyte cDNAs, and the liver-type primer set gave a similar product only for liver cDNAs. A subsequent direct sequence analysis of the PCR products confirmed that the amplified DNA from ependymocytes and liver encodes pancreas-type exon 1 and liver-type exon 1 (data not shown), respectively.



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  		FIG. 6. RT-PCR analysis of GK transcripts for rat ependymocytes and liver total RNAs. A, Positions of the pancreas (P)- and liver (L)-type 5'-primers and the common (P/L) 3'-primers with arrows at P- and L-type exon 1 (Ex1) and common exon 2 (Ex2). The translation initiation site (+1) is indicated ({blacktriangledown}+1). The product size for both primer sets is assumed to be 240 bp. B, Agarose gel electrophoresis is shown with size markers (M). PGK, P-type primer set for the cDNAs from ependymocytes or liver; LGK, L-type primer set for cDNAs from ependymocytes or liver.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that a population of cells taken from the lower brainstem, such as the wall of the CC or ROb, shows an apparent increase in [Ca2+]i in response to either a low or high concentration of extracellular glucose. Those cells showing positive responses are identified as ependymocytes for the cells taken from ventricular walls or as serotonergic neurons for those from ROb, because they were immunoreactive to respective specific cell markers, such as vimentin and serotonin. In addition, the responses of isolated cells to the changes in extracellular glucose levels were blocked by calcium-free medium or nifedipine, an L-type calcium blocker, suggesting that those responses imply a physiological mechanism mediated by an influx of extracellular calcium through the L-type calcium channel. Interestingly, individual cells responded to either low or high glucose levels, but none responded to both low and high glucose stimulations in the present study. Thus, the glucose-sensitive cells in the lower brainstem consist of two distinct populations: high and low glucose responders.

It has been well established that some populations of neurons in the hypothalamus, such as VMH and LHA, are electrophysiologically sensitive (4, 5, 6, 18) and show [Ca2+]i increases (7, 11) in response to the changes in extracellular glucose levels. Neurons in the VMH and LHA have been found to respond to hyperglycemia or hypoglycemia (8). In addition, a previous study using intracellular calcium measurements revealed that a population of neurons in the arcuate nucleus is sensitive to changes in extracellular glucose levels (9). The present results are consistent with the above-mentioned reports, as the neurons in the VMH or LHA showed an increase in [Ca2+]i when the extracellular glucose level was manipulated. The percentages of neurons responding to high glucose in the VMH and to low glucose in the LHA of the total neurons were approximately 25% and 20%, respectively, in the present study and were similar to those in the previous studies (5, 7, 11, 18).

In the present study, most of the ependymocytes in the CC and neurons in the LHA responded only to low glucose, but neurons responding to high glucose were predominant in the VMH. This supports the findings of previous studies, which reported that most of the glucose-sensitive neurons in the VMH and LHA respond to hyperglycemia and hypoglycemia, respectively (4, 8). Thus, the present findings support the presence of glucose-sensitive cells in the hypothalamus and further suggest that the activity of groups of cells, such as ependymocytes and serotonergic neurons in the lower brainstem, is regulated by either a low or a high extracellular glucose level.

The present results suggest that in GK-immunoreactive ependymocytes and serotonergic neurons, GK mediates the [Ca2+]i increase induced by high or low glucose stimulation, because the addition of alloxan, a GK inhibitor (26, 27), blocked the increases in [Ca2+]i induced by high or low glucose stimulation in most of the GK-immunoreactive ependymocytes and serotonergic neurons taken from the CC and ROb, respectively. The present study also showed pancreas-type GK exon 1 mRNA expression in ependymocytes from the fourth ventricular wall. Those facts may indicate that the ependymocytes and serotonergic neurons have a type of GK that is immunoreactive to the present anti-GK and also mediates the [Ca2+]i response to extracellular glucose. It should be noted that alloxan blocked low or high glucose-induced [Ca2+]i increases in 50–80% of the glucose-sensitive neurons taken from the VMH and LHA that were immunonegative to the anti-GK serum used in the present study. Some reports have indicated that a population of neurons in those hypothalamic nuclei in rats have pancreatic GK immunoreactivities and mRNA expression (11, 16, 18, 19, 20). Furthermore, GK inhibitors (alloxan, mannoheptulose, glucosamine, and N-acetylglucosamine) blocked high glucose-induced [Ca2+]i responses in 29–100% of the glucose-sensitive neurons in the VMH (11). The reason for the absence of immunoreactivity to the present anti-GK in the hypothalamic neurons is unknown, but it could be due to the different isoforms of GK in these brain regions. We have recently cloned cDNAs for several isoforms of GK from the hindbrain ependymocytes and hypothalamus (unpublished data). Detailed analysis on the GK isoforms could provide a molecular basis for the discrepancy in immunohistochemical results.

In contrast, alloxan did not block the increase in some cells taken from the CC and ROb, which were immunonegative to the anti-GK used in the present study. One possibility is that those cells have a different type of GK that is not immunoreactive to the present anti-GK. The other possibility is that the [Ca2+]i increase in those cells is not mediated by GK. Mobbs et al. (28) previously suggested that some neurons respond to the change in extracellular glucose levels from 0 to 20 mM via a global and ubiquitous mechanism. The above-mentioned GK-independent [Ca2+]i increase found in the present study may involve such a mechanism.

The mechanism mediating the high glucose-induced [Ca2+]i increase via the voltage-gated L-type Ca2+ channel in the ependymocytes and serotonergic neurons could be similar to those in neurons in the VMH and pancreatic ß-cell. In those cells, an increase in the ATP:ADP ratio inhibits the specific ATP-sensitive K+ (KATP) channel to cause membrane depolarization (29, 30, 31) and then an increase in [Ca2+]i (11). Interestingly, the mRNA of the Kir6.2, a pore-forming unit of the KATP channel, is expressed in the dorsal and medial raphe, raphe magnus, and raphe pontis nuclei, where the serotonergic neurons are abundantly localized (32). In contrast, it is also widely acknowledged that a decreased cytosolic ATP:ADP ratio activates the membrane Na+-K+ pump (5) or KATP channel (11) leading to cell firing and an increase in [Ca2+]i through the voltage-gated, L-type Ca2+ channel. In accordance with the above-mentioned mechanism, Dunn-Meynell et al. (11) reported that alloxan induced an [Ca2+]i increase in VMH neurons that showed a [Ca2+]i increase in response to low glucose. In the present study alloxan blocked the low glucose-induced [Ca2+]i increase in a population of cells taken from the CC, ROb, VMH, and LHA. Thus, the present results could indicate a mechanism that is not explained by the change in the ATP:ADP ratio. Further studies are needed to clarify the mechanism mediating the low glucose-induced [Ca2+]i increase.

The physiological role of those glucose-responsive ependymocytes and serotonergic neurons remains to be determined. Previous reports suggest that cells of the rat hindbrain are involved in sensing glucose levels to control feeding, reproduction, or energy homeostasis (2, 13, 14, 33, 34, 35). Ritter et al. (13) first suggested that a glucose-sensing mechanism that controls feeding behavior in rats is located around the fourth ventricle. Local implants of 5TG in the lower brainstem in rats induce feeding behavior and increase blood glucose (2). Administration of a glucose antagonist to the fourth ventricle suppresses pulsatile LH release and food intake in male rats (14). These results are consistent with the present results suggesting the presence of a glucose-sensing mechanism in ependymocytes on the wall of the ventricle of the hindbrain. In addition, serotonergic neurons have reportedly been involved in the satiety response (33) and cessation of feeding after food intake (34). Serotonin release increases in the hypothalamic paraventricular nucleus after food intake (35). These results together suggest that GK-immunoreactive serotonergic neurons may also sense changes in the blood glucose level to regulate feeding behavior, gonadal activity, and energy homeostasis. It is speculated that ependymocytes or serotonergic neurons monitor the glucose level in cerebrospinal fluid (CSF) rather than the blood glucose level. Ependymocytes in the hindbrain exhibit GLUT2 immunoreactivities on their cilia (21), and serotonergic neurons have been known to contact the CSF in rats (36). In addition, CSF glucose levels have been reported to be well correlated with blood glucose levels (37). It is thus plausible that ependymocytes and serotonergic neurons sense the changes in CSF glucose levels to regulate reproduction and feeding.

In conclusion, the present study suggests that ependymocytes and serotonergic neurons derived from the lower brainstem directly respond to the changes in extracellular glucose levels. Those glucose-sensitive cells seem to consist of two populations: one responding to low and the other to high glucose levels. Such responses may be mediated by either GK-dependent or -independent mechanisms of the cells in these areas. These cells most likely play a key role in the glucose-sensing mechanism in the brain to regulate reproductive function and feeding behavior in response to changes in nutritional conditions.


   Acknowledgments
 
We are grateful to Drs. H. I’Anson and Y. Uenoyama for valuable suggestions, and to Y. Niwa for technical assistance.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research 14360177 (to K.M.) and 15380193 and 15658082 (to H.T.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Abbreviations: [Ca2+]i, Intracellular calcium concentration; CC, central canal; CSF, cerebrospinal fluid; 2DG, 2-deoxy-D-glucose; GK, glucokinase; HKRB, HEPES-buffered Krebs-Ringer bicarbonate buffer; KATP, ATP-sensitive K+; LHA, lateral hypothalamic area; ROb, raphe obscurus nucleus; 5TG, 5-thio-D-glucose; VMH, ventromedial hypothalamus.

Received September 9, 2003.

Accepted for publication February 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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