Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression.

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.

D-glucose stimulates mesangial cell GLUT1 expression and basal and IGF-I-sensitive glucose uptake in rat mesangial cells: implications for diabetic nephropathy.

The complications of diabetes arise in part from abnormally high cellular glucose uptake and metabolism. To determine whether altered glucose transporter expression may be involved in the pathogenesis of diabetic nephropathy, we investigated the effects of elevated extracellular glucose concentrations on facilitative glucose transporter (GLUT) expression in rat mesangial cells. GLUT1 was the only transporter isoform detected. Cells exposed to 20 mmol/l glucose medium for 3 days demonstrated increases in GLUT1 mRNA (134%, P < 0.002), GLUT1 protein (68%, P < 0.02), and V(max) (50%, P < 0.05) for uptake of the glucose analog [3H]2-deoxyglucose (3H2-DOG), when compared to cells chronically adapted to physiologic glucose concentrations (8 mmol/l). The increase in GLUT1 protein was sustained at 3 months, the latest time point tested (77% above control, P < 0.01). In contrast, hypertonic mannitol had no effect on GLUT1 protein levels. Insulin-like growth factor I (IGF-I; 30 ng/ml) increased the uptake of 3H2-DOG by 28% in 8 mmol/l glucose-treated cells (P < 0.05) and by 75% in cells switched to 20 mmol/l glucose for 3 days (P < 0.005). These increases in 3H2-DOG uptake occurred despite a lack of effect of IGF-I on GLUT1 protein levels (P > 0.5 vs. control). Therefore, hyperglycemia and IGF-I treatment both lead to increases in mesangial cell glucose uptake, and hyperglycemia induces increased GLUT1 expression, which can directly lead to the pathological changes of diabetic nephropathy. The effects of high glucose and of IGF-I to stimulate 3H2-DOG uptake also appear to be additive.

Variable denaturation of ovalbumin by incorporation of amino acid analogs.

Denaturation of hen ovalbumin synthesized in a cell-free system was assayed by examining its sensitivity to trypsin. The native ovalbumin resisted digestion by trypsin, and it remained resistant to digestion when some amino acid analogs, including azetidine-2-carboxylic acid and meta-flourotyrosine, were incorporated into its peptide chain. However, when other amino acid analogs such as beta-hydroxyleucine and 4-thiaisoleucine were incorporated during protein systhesis, ovalbumin became very lablie to trypsin. These experiments demonstrate a sensitive system for detecting protein denaturation and suggest a variable effect of different amino acid analogs on the native conformation of a protein.