Implications of glucose transporter protein type 1 (GLUT1)-haplodeficiency in embryonic stem cells for their survival in response to hypoxic stress.
Glucose transporter protein type 1 (GLUT1) is a major glucose transporter of the fertilized egg and preimplantation embryo. Haploinsufficiency for GLUT1 causes the GLUT1 deficiency syndrome in humans, however the embryo appears unaffected. Therefore, here we produced heterozygous GLUT1 knockout murine embryonic stem cells (GT1+/-) to study the role of GLUT1 deficiency in their growth, glucose metabolism, and survival in response to hypoxic stress. GT1(-/-) cells were determined to be nonviable. Both the GLUT1 and GLUT3 high-affinity, facilitative glucose transporters were expressed in GT1(+/+) and GT1(+/-) embryonic stem cells. GT1(+/-) demonstrated 49 +/- 4% reduction of GLUT1 mRNA. This induced a posttranscriptional, GLUT1 compensatory response resulting in 24 +/- 4% reduction of GLUT1 protein. GLUT3 was unchanged. GLUT8 and GLUT12 were also expressed and unchanged in GT1(+/-). Stimulation of glycolysis by azide inhibition of oxidative phosphorylation was impaired by 44% in GT1(+/-), with impaired up-regulation of GLUT1 protein. Hypoxia for up to 4 hours led to 201% more apoptosis in GT1(+/-) than in GT1(+/+) controls. Caspase-3 activity was 76% higher in GT1(+/-) versus GT1(+/+) at 2 hours. Heterozygous knockout of GLUT1 led to a partial GLUT1 compensatory response protecting nonstressed cells. However, inhibition of oxidative phosphorylation and hypoxia both exposed their increased susceptibility to these stresses.
Heilig
Charles
C
Division of Nephrology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 947, Baltimore, MD 21205, USA. cheilig@jhmi.edu
Brosius
Frank
F
Siu
Brian
B
Concepcion
Luis
L
Mortensen
Richard
R
Heilig
Kathleen
K
Zhu
Min
M
Weldon
Richard
R
Wu
Guimei
G
Conner
David
D
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015152:Blotting, Northern; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051272:Glucose Transporter Type 1; D006239:Haplotypes; D006579:Heterozygote; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D009004:Monosaccharide Transport Proteins / Q000172:deficiency* / Q000235:genetics; D010085:Oxidative Phosphorylation / Q000187:drug effects; D019810:Sodium Azide / Q000494:pharmacology; D013234:Stem Cells / Q000473:pathology* / Q000502:physiology*
D004791:Enzyme Inhibitors; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495678:Slc2a1 protein, mouse; D019810:Sodium Azide; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases