Past-A, a novel proton-associated sugar transporter, regulates glucose homeostasis in the brain

Abstract

The ventral medullary surface (VMS) of the medulla oblongata is known to be the site of the central chemosensitive neurons in mammals. These neurons sense excess H+/CO2 dissolved in the CSF and induce hyperventilation. To elucidate the mechanism of neuronal cell adaptation to changes of H+/CO2, we screened for hypercapnia-induced genes in the VMS. Here, we report cloning and characterization of a novel gene called proton-associated sugar transporter-A (Past-A), which is induced in the brain after hypercapnia and mediates glucose uptake along the pH gradient. Past-A comprises 751 amino acid residues containing 12 membrane-spanning helices, several conserved sugar transport motifs, three proline-rich regions, and leucine repeats. Past-A transcript was expressed predominantly in the brain. Moreover, the Past-A-immunoreactive neural cells were found in the VMS of the medulla oblongata, and the number of immunoreactive cells was increased by hypercapnic stimulation. Transient transfection of Past-A in COS-7 cells leads to the expression of a membrane-associated 82 kDa protein that possesses a glucose transport activity. The acidification of extracellular medium facilitated glucose uptake, whereas the addition of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, inhibited glucose import. Together, our results indicate that Past-A is a brain-specific glucose transporter that may represent an adaptation mechanism regulating sugar homeostasis in neuronal cells after hypercapnia.

Fig. 1.

Structural analysis of Past-A.A, Amino acid sequences of rat Past-A. The approximate positions of the transmembrane domains are underlined. Three proline-rich regions are boxed. The leucine repeats are marked with filled circles. The sucrose–H⁺ transport motif is indicated withasterisks. Numbers to theright show amino acid positions. B, Putative membrane topology of Past-A. Three proline-rich regions areboxed. The leucine repeats are marked with dark background. The sucrose–H⁺ transport motif is indicated with asterisks. Numbers show amino acid positions. C, Phylogenic tree of a multiple alignment the Past-A with other transporters. c, Carrot;h, human; m, mouse; o,Oryzias latipes (medaka); r, rat;z, zea mays (maize).

Fig. 2.

Tissue distribution and ontogenic changes of Past-A mRNA. Total RNAs (20 μg) extracted from various tissues (A) or from brains at different developmental stages (E20 to P300) (B) were analyzed by Northern blot. Arrowheads mark hybridizing bands corresponding to mRNA encoding rat Past-A. The positions of standard molecular masses (in kilobases) are indicated at left. Ribosomal RNA (28S) stained with ethidium bromide is shown as a control.

Fig. 3.

Induction of Past-A by hypercapnic stimulation in the medulla oblongata. A, Analysis of differential expression of Past-A by hypercapnic stimulation by Northern blotting. Representative example of autoradiogram sampled from four experiments. Arrowhead shows hybridizing band corresponding to mRNA encoding rat Past-A. Positions of standard molecular masses (in kilobases) are indicated at left. Ribosomal RNA (28S) stained with ethidium bromide is shown as a control. B, Histograms of density units of the expression of Past-A mRNA after air or CO₂ inhalation. The content of Past-A mRNA was measured densitometrically on the autoradiogram, and the results are expressed as means ± SEM of arbitrary units of density (n = 4). The significance of differences in the density of Past-A mRNA was evaluated by the Mann–Whitney U test, with *p< 0.05 considered indicative of a statistically significant difference. C, Photomicrographs of Past-A-immunoreactive neural cells in the VMS after inhalation of air. Right, Magnified subregion indicated by arrow atleft. D, Photomicrographs of Past-A-immunoreactive neuronal cells in the VMS after inhalation of air containing 7% CO₂ for 5 min. Bottom, Magnified subregions indicated by arrows attop. py, Pyramidal tract;RPA, nucleus raphe pallidus. Scale bars: panoramic picture, 200 μm; magnified picture, 20 μm.

Fig. 4.

Characterization of sugar transport activity of Past-A. A, COS-7 cells were transfected with blank vector (Vec), Past-A (Past), or rat GLUT4 cDNA. Sixty-four hours after transfection, transfected cells were incubated with one of radiolabeled sugars and solubilized as described in Materials and Methods. Results are averages of four individual experiments, with SEM indicated by error bars. B, Western blot of the membrane fractions. Transporter proteins Past-A and GLUT4 were detected with each antiserum. The positions of standard molecular masses (in kilodaltons) are indicated at left.C, Inhibition of glucose uptake of COS-7 transfected with Past-A cDNA by the protonophore CCCP. Glucose uptake data are expressed as fold stimulation relative to the extent of uptake observed with COS-7 transfected with blank vector not exposed to CCCP. Results are averages of four individual experiments, with SEM indicated by error bars. D, COS-7 transfected cells with Past-A cDNA were incubated at different pH values for glucose uptake. Glucose uptake data are expressed as fold stimulation relative to the extent of uptake observed with COS-7 transfected with Past-A cDNA at a pH of 7.5. Results are averages of four individual experiments, with SEM indicated by error bars. (▴), pH 6.5; (■), pH 7.0; (●), pH 7.5.