Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels

Abstract

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured β-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

Figure 1. Immunocytochemistry characterization of cultured tanycytes

Tanycytes obtained from rat hypothalamus at 1 day postnatal were cultured for 3 weeks. (A–E) Representative confocal images depicting vimentin (A, green), GFAP (B, green), MAP2 (C, green), βIII-tubulin (D, green), Kir6.1 (E, green) in rat tanycytes under control conditions. (F) MAP2 (red) and vimentin (green) staining in mixed hypothalamic cultures of tanycytes and neurons. In blue are shown nuclei stained with TOPRO-3. (G) Quantification of immunopositive expression normalized to total cells of vimentin, Kir6.1, GLUT2, GK, MCT1, MCT4, GFAP, βIII-tubulin, MAP2 and Von Willebrand factor (VWF) in tanycytes under control conditions. Scale bar = 80 µm.

Figure 2. Glucose-induced increase in intracellular free Ca²⁺ signal occurs by a glucokinase- and K_ATP channel-dependent pathway in rat tancytes

(A–C) Representative plots of relative changes in Ca²⁺ signal (340/380 nm) over time in cultured rat tanycytes subjected to changes in glucose concentrations (horizontal bars, from 2 to 10 mM glucose) under control conditions (A), in presence of 500 µM alloxan, a glucokinase inhibitor (B) or in presence of 1 µM diazoxide (C), a K_ATP channel activator. In each panel, three photomicrographs of time-lapse images show changes in Fura-2 ratio (pseudo-colored scale). The delay between the addition of glucose and the increase in Fura-2 ratio is represented by the vertical gray line. (D) Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to increasing glucose concentration. (E) Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 10 mM glucose alone or in combination with the following blockers: 100 µM Cyto-B, 100 mM 4,6,-O-ethylidene-D-glucose (ETDG), 500 µM alloxan, 1 mM iodoacetate (IA), 200 nM AA (AA) and 1 µM diazoxide (Diazox). ^*** p < 0.001, effect of 10 mM glucose compared to control; ^# p < 0.05, ^## p < 0.005, effect of blockers compared to glucose treatment. Averaged data were obtained from at least five independent experiments.

Figure 3. Rat tanycytes exhibit functional Cx43 hemichannels that are involved in the glucose-induced increase in intracellular Ca²⁺ signal

(A–B) Fluorescence micrographs of Etd uptake (10 min exposure) in tanycytes under control conditions (A) and then exposed for 10 min to a divalent cation (Ca²⁺/Mg²⁺)-free solution (DCFS) (B). (C) Time-lapse measurement of Etd uptake in rat tanycytes under control conditions (basal) or exposed to divalent cation free solution (DCFS). La³⁺ (200 µM), a connexin hemichannel blocker, applied at ~7 min of Etd uptake measurements reduced the Etd uptake to a value even lower than that recorded under basal condition. (D) Averaged data normalized to control (dashed line) of Etd uptake rate by tanycytes treated with the following Cx43 hemichannel blockers co-applied during dye uptake recording: 200 µM La³⁺, 200 µM Gap26 and 1:500 Cx43^E2; or with the following Panx1 hemichannel blockers: 200 µM ¹⁰panx1 or 500 µM probenecid (Prob). Also, it is shown the Etd uptake rate induced by DCFS conditions alone or plus the anti-Cx43 hemichannel blocker, Cx43^E2. ^*** p < 0.001, ^** p < 0.005, treatments compared to control; ^# p < 0.05, effect of DCFS conditions compared to blockers. Averaged data were obtained from at least 5 independent experiments. Scale bar = 30 µm. (E) Representative confocal image depicting vimentin (red) and Cx43 (green) immunolabeling of cultured rat tanycytes under control conditions. In blue are shown nuclei stained with TOPRO-3. Scale bar = 15 µm. (F) Representative plot of relative changes in Ca²⁺ signal over time in cultured tanycytes subjected to changes in glucose concentrations (horizontal bars, from 2 to 10 mM glucose) in absence of extracellular Ca²⁺ (Ca²⁺-free) (G) Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 10 mM glucose alone or in combination with the following blockers: 200 µM ¹⁰panx1, 500 µM probenecid (Prob), 200 µM Gap26, 1:500 Cx43^E2 antibody and in absence of extracellular Ca²⁺. All blockers were applied 10 min before treatment with glucose. ^# p < 0.005, effect of blockers compared to glucose treatment. Averaged data were obtained from at least five independent experiments.

Figure 4. Increased Etd uptake induced by glucose is mediated by a glucokinase/K_ATP channel-dependent pathway in cultured rat tanycytes

(A–C) Representative immunofluorescence images depicting vimentin (white) labeling and Edt (red) nuclei-staining from dye uptake experiments (10 min exposure to dye) in cultures of tanycytes under control conditions (A) or treated with 10 mM glucose for 5 min alone (B) or glucose plus 1:500 Cx43^E2, an anti-Cx43 antibody that blocks Cx43 hemichannel (C). (D) Time-lapse measurement of Etd uptake in rat tanycytes treated with 2, 10 or 20 mM glucose. La³⁺ (200 µM) applied at ~10 min of Etd uptake measurement inhibited dye uptake. (E) Average of Etd uptake rate normalized to control (dashed line) in tanycytes exposed to increasing concentrations of glucose. * p<0.001, treatments compared to control. (F) Averaged Etd uptake rate normalized to control (dashed line) by tanycytes treated with 10 mM glucose alone or in combination with the following blockers: 100 µM cytochalasin B (Cyto-B), 100 mM ETDG, 500 µM alloxan, 1 mM iodocetic acid (IA), 200 nM antimycin A (AA), 1 µM diazoxide (Diazox), 200 µM La³⁺, 200 µM Gap26, 1:500 Cx43^E2, 200 µM ¹⁰panx1, or 500 µM Prob. ^** p < 0.005, 10 mM glucose treatment compared to control; ^# p < 0.05, ^## p < 0.005, effect of 10 mM glucose treatment compared to blockers. Averaged data were obtained from at least four independent experiments. Scale bar = 15 µm.

Figure 5. Glucose increases Cx43 hemichannel currents, but does not affect the surface Cx43 levels in cultured rat tanycytes

(A) Voltage ramps from −80 to +80 mV, 5 s in duration, were applied. Each ramp was initiated and finished by a transition from 0 to −80 and +80 to 0 mV, respectively. Membrane currents were measured in a whole-cell voltage-clamp configuration using low-density cultures of rat tanycytes. (B) Voltage ramp from −80 to +80 mV, 5 s duration in tanycytes under control conditions (black line) or treated for 3 min with 10 mM glucose alone (magenta line) or 10 mM glucose plus 1:500 Cx43^E2 (green line). A record section at aboy +60 mV the current trace was point-by-point converted in conductance values showing unitary events of about 220 pS. (C) Cultured tanycytes were treated for 5 min with 10 mM glucose. Total (left panel) and surface (right panel) levels of Cx43 in tanycytes under control conditions (lane 1) or treated for 5 min with 10 mM glucose alone (lane 2), 10 mM glucose plus 500 µM alloxan (lane 3) or plus 1 µM diazoxide (Diazox) (lane 4). The Cx43 phosphorylated (P1-P2) and nonphosphorylated (NP) forms are indicated in the left. Total levels of each analyzed protein were normalized according to the levels of α-tubulin detected in each lane. Surface levels of each analyzed protein were normalized according to the total protein loaded as revealed by staining with Ponceau red (PR) in each lane. Similar observations were made in two other independent experiments. (D) Averaged data on current events at +60 (white bars) or −60 mV (black bars) in tanycytes under control conditions, exposed to 10 mM glucose alone or 10 mM glucose plus Cx43^E2. ^* p < 0.001, glucose treatment compared to control; ^# p < 0.001, inhibitory effect of blocker on the glucose-induced response. Averaged data were obtained from three independent experiments in which at least 8 cells were analyzed per treatment. (E) Quantification of total (white bars) and surface (black bars) levels of Cx43 normalized to control (dashed line) in tanycytes treated for 5 min with 10 mM glucose alone, plus 500 µM alloxan or 1 µM Diazox. Averaged data were obtained from three independent experiments.

Figure 6. Glucose uptake occurs via GLUTs and Cx43 hemichannels in rat tanycytes

Averaged data at 1 min of 2-DOG (A) and 2-NBDG (B) uptake in tanycytes under control conditions or treated with 100 mM ETDG, 100 µM cytochalasin B (Cyto-B), 1:500 Cx43^E2 or 1:500 Cx43^E2 plus 100 µM Cyto-B. In addition, 2-DOG and 2-NBDG uptake by tanycytes exposed to DCFS conditions for 1 min alone or plus 100 mM (ETDG), 100 µM Cyto-B, 1:500 Cx43^E2 or 1:500 Cx43^E2 plus 100 µM Cyto-B was analyzed. ^*** p < 0.001, ^** p < 0.005, ^* p < 0.05; treatments compared to control; ^# p < 0.05, treatments compared to DCFS conditions. Averaged data were obtained from at least four independent experiments.

Figure 7. Glucose increases the intracellular free Ca²⁺ signal in tanycytes by ATP released via Cx43 hemichannels

(A–H) Representative fluorescence micrographs of simultaneous time-lapse imaging showing changes in Fura-2 ratio (A–D, pseudo-colored scale) and Etd uptake (E–H, red) in tanycytes treated with 10 mM glucose for 0 (A and E), 48 (B and F), 50 (C and G) and 200 s (D and H). (I) Simultaneous plots of relative changes in [Ca²⁺]_i and Etd uptake over time of cells 1 (red), 2 (green), 3 (yellow) and 4 (cyan) depicted in panel A. The delay between the increase in Etd uptake and Fura-2 ratio is indicated by the vertical dashed line. (J) Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 10 mM glucose alone or in combination with the followed blockers: 10 U/mL apyrase (Apyr), 200 µM oxidized ATP (oATP), 10 µM (BBG), 200 µM suramin (Sur), 10 µM MRS2179, 2 µM thapsigargin (TG), 5 µM xestospongin C (XeC) and 5 µM xestospongin (XeB). ^** p < 0.005, 10 mM glucose treatment compared to blockers. (K) Averaged values of ATP released by tanycytes under control conditions or treated with 10 mM glucose alone or in combination with the following blockers: 100 µM cytochalisin B (Cyto-B), 500 µM alloxan, 1 mM IA, 1 µM diazoxide (Diazox), 200 µM ¹⁰panx1, 500 µM probenecid (Prob), 200 µM La³⁺, 200 µM Gap26 or 1:500 Cx43^E2. ^*** p < 0.001, 10 mM glucose treatment compared to control; ^## p < 0.005, effect of 10 mM glucose treatment compared to blockers. Averaged data were obtained from at least four independent experiments. Scale bar = 35 µm.

Figure 8. ATP increases the intracellular free Ca²⁺ signal in tanycytes via P2Y receptors

(A) Representative plots of relative changes in Ca²⁺ signal over time in cultured rat tanycytes subjected to 100 µM ATP under control conditions or pretreated with MRS21. (B) Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 100 µM ATP alone or in combination with the followed blockers: 200 µM oxidized ATP (oATP), 10 µM brilliant blue G (BBG), 200 µM suramine (Sur), 10 µM MRS2179, 2 µM thapsigargin (TG) 5 µM xestospongin (XeC) and 5 µM xestospongin (XeB). ^* p < 0.001, 100 µM ATP treatment compared to control; ^# p < 0.001, 100 µM ATP treatment compared to blockers.

Figure 9. Model of glucosensing mechanism in tanycytes

Upon a rise in extracellular glucose concentration, glucose enters (1) mainly through GLUTs and to a minor extent via Cx43 hemichannels, leading to glucokinase-dependent phosphorylation of glucose (2). The ATP generated during glycolysis (3) via processing of glucose-derived substrates causes closure of K_ATP channels (4). This event promotes by an unknown mechanism the opening of Cx43 hemichannels (5). The ATP released via Cx43 hemichannels (6) activates P2Y receptors (7) and induces formation of cytoplasmic inositol (1,4,5)-trisphosphate (IP₃), which promotes the release of Ca²⁺ stored in the endoplasmic reticulum (8), raising the [Ca²⁺]_i (9).