GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

Abstract

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Figure 1

Localization of the expression of glut2 in zebrafish. Whole-mount in situ hybridization showing expression of zebrafish glut2 mRNA at 24 hours post fertilization (hpf; A and B), 48 hpf (C), 72 hpf (D–F), and 120 hpf (G–I). Left (A–C, E, and H), right lateral views (F and I), and dorsal views of the hindbrain region (D and G) are shown. To confirm glut2 expression in endocrine pancreas, double in situ hybridization showing pre-proinsulin expression was performed (I, inset). (D'–I') Schematic representations of D–I highlighting with different colors the regions where glut2 is expressed. Black dotted contours outline the hindbrain region (D' and G') and the pancreas (I'). Anterior intestine (ai), corpus cerebelli (cce), endocrine pancreas (ep), hindbrain (hb), liver (li), medulla oblongata (mo), pronephric duct (pd), pronephric tubule (pt), telencephalon (t).

Figure 2

glut2 abrogation disrupts brain development. Phenotype of embryos injected with a control morpholino (Con MO) at 24 hours post fertilization (hpf; A and B) and at 48 hpf (C), a morpholino targeting the translational start site (ATG MO) at 24 hpf (D and E), and at 48 hpf (F) and a morpholino targeting the splice acceptor between exons 5 and 6 (Splice MO) at 24 hpf (G and H) and at 48 hpf (I). Embryos injected with both morpholinos displayed a defective formation of the hindbrain that could be observed from 24 hpf onwards. Hindbrain ventricle (hbv), midbrain/hinbrain boundary (mbh), tectal ventricle (tctv), telencephalon (t).

Figure 3

Morphant phenotype caused by abrogation of glut2 can be rescued with full-length zebrafish glut2 and rat GLUT2 mRNA. Phenotype of embryos injected with a control morpholino (Con MO) at 48 hpf (A), ATG morpholino (ATG MO) at 48 hpf (B) and splice morpholino (Splice MO) at 48 hpf (C). Rescue of overall morphant phenotype at 48 hpf by co-injection of ATG MO with rat GLUT2 mRNA (rGLUT2; D) and of Splice MO with zebrafish glut2 (zfGLUT2; E). (F–J) Shows higher magnification images of the cephalic region of Con MO (F), ATG MO (G), Splice MO (H), rescued ATG morphants (I), and rescued splice morphants (J). The hindbrain region (outlined by black dotted line) is affected in the morphants (G and H), while brain development in rescued embryos (I and J) is similar to that observed in control embryos (F).

Figure 4

Knockdown of glut2 results in inhibition of glucose uptake in vivo. (A) (a–i) Bright field (upper line, a–c), fluorescent (middle line, d–f), and overlay (bottom line, g–i) pictures of control, ATG morphants, and rescued embryos at 24 hpf. (B) Measurement of fluorescent signal in embryos injected with 2-NBDG. Control injected embryos (a, d, and g) displayed significant amounts of fluorescent glucose throughout the embryo; in contrast, ATG morphants (b, e, and h) showed very minimal fluorescent glucose visible at 60 minutes after injection. Embryos injected with ATG MO+rat GLUT2 mRNA (ATG MO Rescued) recovered glucose uptake to levels similar to Con MO (c, f, and i). * indicates significant differences compared with the Con MO injected embryos (*P<0.05; **P<0.01; ***P<0.001). ^# indicates significant differences compared with ATG MO injected embryos (^##P<0.01; ^###P<0.001).

Figure 5

glut2 abrogation causes hindbrain disorganization and affects the expression of cerebellar proneural genes. To study the consequences of glut2 knockdown in the hindbrain structure we performed immunostaining using an antibody against acetylated tubulin in con MO, ATG MO, and ATG MO+rat GLUT2 mRNA rescued embryos at 24 hpf (i, ii, iii). At this stage, morphant embryos showed disorganized axon tracts. Rescued embryos showed a hindbrain structure similar to control injected embryos. Lateral longitudinal fascicles (llf); medial longitudinal fascicles (mlf). To further study the consequences of glut2 abrogation in the neural progenitor cells we performed ISH for the proneural genes ptf1a (A–D), atoh1b (G–J), neurod (M–P) in control injected embryos at 24 hpf (A, G, and M), and 48 hpf (C, I, and O), and in ATG morphants at 24 hpf (B, H, and N) and 48 hpf (D, J, and P). To better illustrate the effects caused by the abrogation of glut2, immunostained medial longitudinal fascicles have been outlined (i', ii', iii'). The expression patterns observed by ISH of the proneural genes are represented with diagrams overlapping the expression patterns in control and ATG morphants at 24 hpf and 48 hpf of ptf1a (E and F), atoh1b (K and L), and neurod (Q and R). A, anterior; L, left; R, right.

Figure 6

Abrogation of glut2 enhances cell death mainly in the head region. To assay for cell death, embryos injected with control morpholino (Con MO; A), ATG morpholino (ATG MO; B and C), and ATG MO+rat GLUT2 mRNA (ATG MO Rescued; D) were stained with the vital dye acridine orange. To assay for apoptosis, embryos injected with Con MO (E) and ATG MO (F) were analyzed by TUNEL assay. At 24 hpf, there was an overall increase in cell death and apoptosis primarily localized in the hindbrain region (B, C, and F). Fluorescent signal analysis (G) and counting of TUNEL-positive cells (H) confirmed a significant increase in cell death and apoptosis, respectively, in ATG morphants which appears reverted in rescued embryos (G). * indicates significant differences compared with the Con MO injected embryos (**P<0.01; ***P<0.001). Hindbrain ventricle (hbv), midbrain/hindbrain boundary (mbh), telencephalon (t). In the images (A–F), the position of the embryos is indicated by a representation of a zebrafish embryo.