Glucose-induced toxicity in insulin-producing pituitary cells that coexpress GLUT2 and glucokinase. Implications for metabolic engineering.
We have shown that intermediate lobe (IL) pituitary cells can be engineered to produce sufficient amounts of insulin (ins) to cure diabetes in nonobese diabetic mice but, unlike transplanted islets, ILins cells evade immune attack. To confer glucose-sensing capabilities into these cells, they were further modified with recombinant adenoviruses to express high levels of GLUT2 and the beta-cell isoform of glucokinase (GK). Although expression of GLUT2 alone had negligible effects on glucose usage and lactate production, expression of GK alone resulted in approximately 2-fold increase in glycolytic flux within the physiological (3-20 mm) glucose range. GLUT2/GK coexpression further increased glycolytic flux at 20 mm glucose but disproportionately increased flux at 3 mm glucose. Despite enhanced glycolytic fluxes, GLUT2/GK-coexpressing cells showed glucose dose-dependent accumulation of hexose phosphates, depletion of intracellular ATP, and severe apoptotic cell death. These studies demonstrate that glucose-sensing properties can be introduced into non-islet cells by the single expression of GK and that glucose responsiveness can be augmented by the coexpression of GLUT2. However, in the metabolic engineering of surrogate beta cells, it is critical that the levels of the components be closely optimized to ensure their physiological function and to avoid the deleterious consequences of glucose-induced toxicity.
Faradji
R N
RN
Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Havari
E
E
Chen
Q
Q
Gray
J
J
Tornheim
K
K
Corkey
B E
BE
Mulligan
R C
RC
Lipes
M A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D016923:Cell Death; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005941:Glucokinase / Q000096:biosynthesis*; D005947:Glucose / Q000378:metabolism / Q000633:toxicity*; D051273:Glucose Transporter Type 2; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000096:biosynthesis*; D051379:Mice; D016688:Mice, Inbred NOD; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis*; D010766:Phosphorylation; D010902:Pituitary Gland / Q000378:metabolism*; D020033:Protein Isoforms; D014018:Tissue Distribution
D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005947:Glucose