Evidence that hindbrain astrocytes in the rat detect low glucose with a glucose transporter 2-phospholipase C-calcium release mechanism

Abstract

Astrocytes generate robust cytoplasmic calcium signals in response to reductions in extracellular glucose. This calcium signal, in turn, drives purinergic gliotransmission, which controls the activity of catecholaminergic (CA) neurons in the hindbrain. These CA neurons are critical to triggering glucose counter-regulatory responses (CRRs) that, ultimately, restore glucose homeostasis via endocrine and behavioral means. Although the astrocyte low-glucose sensor involvement in CRR has been accepted, it is not clear how astrocytes produce an increase in intracellular calcium in response to a decrease in glucose. Our ex vivo calcium imaging studies of hindbrain astrocytes show that the glucose type 2 transporter (GLUT2) is an essential feature of the astrocyte glucosensor mechanism. Coimmunoprecipitation assays reveal that the recombinant GLUT2 binds directly with the recombinant Gq protein subunit that activates phospholipase C (PLC). Additional calcium imaging studies suggest that GLUT2 may be connected to a PLC-endoplasmic reticular-calcium release mechanism, which is amplified by calcium-induced calcium release (CICR). Collectively, these data help outline a potential mechanism used by astrocytes to convert information regarding low-glucose levels into intracellular changes that ultimately regulate the CRR.

Keywords: counter-regulation; ex vivo brain slice; live cell calcium imaging; low-glucose sensing; solitary nucleus.

Fig. 1.

Prelabeling with Cal-520 and SR101 allows for discrimination of astrocytes and neurons at the cellular level by comparing images captured using the 488-nm and 561-nm laser lines. A: at 561 nm, only astrocytes (prelabeled with SR101) will appear red. B: at 488 nm, both astrocytes and neurons (i.e., prelabeled with Cal-520) will appear green. C: these dual exposure images confirm the cell types being recorded. Dual labeled cells (astrocytes) are circled in white. The green cells are neurons. Scale bar = 30 μm.

Fig. 2.

Representative screenshots of identified astrocytes (color-coded encircled) and neurons (green arrows) adjacent to plots of the calcium-induced fluorescence signals over time evoked in these identified astrocytes by exposure to either ATP (viability test) and a glucoprivic [low glucose/2-deoxyglucose (LG/2DG)] challenge. Magnitudes of response to these challenges are reflected in the percent change in fluorescence of each individual astrocyte. Row 1 control conditions: A: screenshot of identified (double-labeled) astrocytes. B: adjacent trace shows the astrocytic responses to short exposure to ATP. C: the subsequent trace is representative of the robust astrocytic responses (color coded to the astrocytes encircled in A) to glucoprivic challenge under control conditions. Row 2 quercetin [glucose type 2 transporter (GLUT2) block] pretreatment: D: screenshot of identified astrocytes. E: adjacent trace are the astrocytic responses to short exposure to ATP. F: the subsequent trace is representative of diminished astrocytic responses (color coded to the astrocytes encircled in D) to glucoprivic challenge following pretreatment of slice with GLUT2 antagonist, quercetin. G: followed by another challenge with ATP to verify that the astrocytes were still viable after the GLUT2 blockade. Results following pretreatment with 2APB, U73122, and dantrolene are qualitatively similar (not shown here), and this is reflected in the summary data in Fig. 3. Row 3 fasentin (GLUT1/4 block) pretreatment: H: screenshot of identified astrocytes. I: adjacent trace showing astrocytic response to short exposure to ATP. J: astrocytes (color coded to cells encircled in H) response to LG/2DG challenge is not different from control. Row 4 U73122 (PLC block) pretreatment: K: screenshot of identified astrocytes. L: adjacent trace shows astrocytic response to short ATP exposure. M: U73122 blocks the effect of glucoprivic challenge on the identified astrocytes (color coded in K). Note that the ATP effect is mediated through a P2Y receptor that is also dependent on phospholipase C (PLC), so those effects are also blocked by U73122 (15) (null trace not shown).

Fig. 3.

Averaged magnitude of changes in fluorescence due to intracellular calcium fluxes in hindbrain astrocytes in response to glucoprivic challenge after specific pretreatment conditions (number of astrocytes studied per each group is noted in parentheses). Exposure of astrocytes in hindbrain slices to the various pretreatment conditions produced significant differences in response to subsequent glucoprivic challenge. The “control” low glucose/2-deoxyglucose (LG/2DG) challenge yielded robust responses in viable astrocytes. In contrast, pretreatment of hindbrain slices with the selective glucose type 2 transporter (GLUT2) transporter blocker (quercetin) produced a nearly complete block of the LG/2DG effect, whereas the other transport antagonists phlorizin (SGLT) and fasentin (GLUT1/4) were not effective. Pretreatment with the phospholipase C (PLC) antagonist (U73122) completely blocked the subsequent effects of the LG/2DG challenge, whereas the inactive enantiomer (U73343) was without effect. Additionally, 2APB (an IP3 antagonist) also blocked the LG/2DG effect as did the endoplasmic reticulum (ER) ryanodine receptor antagonist, dantrolene. Low calcium Krebs did not eliminate the astrocyte response to LG/2DG. An overall one-way ANOVA yielded F_8,441 = 33.11; P < 0.0001; Dunnett’s posttests: *P < 0.05.

Fig. 4.

Detection of a physical interaction between glucose type 2 transporter (GLUT2) and recombinant human Gαq protein (GNAQ). Recombinant GLUT2 and GNAQ were incubated at room temperature for 30 m followed by overnight immunoprecipitation (IP) with antibodies against either normal mouse serum (IgG) or GNAQ. Immunoprecipitated proteins were separated by SDS-PAGE, detected by immunoblotting (IB) with an antibody targeting GLUT2. The experiment was repeated on three separate occasions. The red arrow indicates detection of GLUT2 after immunoprecipitation using the GNAQ antibody.

Fig. 5.

Based on data from the current study, this cartoon representation proposes a hypothetical mechanism of astrocytic transduction of glucoprivic information into gliotransmission.