Effect of glucose and insulin deprivation on differentiation and carbohydrate metabolism of rabbit proximal tubular cells in primary culture.
Rabbit proximal tubule cells in primary culture revert from gluconeogenesis to glycolysis. To determine whether glucose and insulin deprivation of the culture medium could prevent this metabolic conversion without a loss of differentiation, rabbit proximal tubule cells were cultured in hormonally defined medium free of glucose and insulin and compared to rabbit proximal tubule cells cultured in medium supplemented with 17.5 mM glucose and 5 micrograms/ml insulin. In the two culture conditions, RPT cells grew at a similar rate and reached confluency within 4-5 days. Patterns of enzyme activity, including brush-border hydrolases, N-acetyl-beta-D-glucosaminidase and glutathione-S-transferases as a function of culture time were comparable in the two media. During the growth phase in glucose- and insulin-free medium, cells showed higher sodium-dependent glucose uptake. Scanning electron microscopy revealed a high density of microvilli at confluency regardless of the culture conditions. In both the presence and absence of glucose and insulin, the activities of gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase, as well as basal and pyruvate-stimulated glucose production fell markedly as a function of time. By contrast, glucose and insulin deprivation greatly reduced both the lactate production rate and the activities of glycolytic enzymes, pyruvate kinase, hexokinase and lactate dehydrogenase.
Département Sécurité du Médicament, Rhône-Poulenc Rorer S.A., Vitry sur Seine, France.
F
F
Courjault
J
J
Chevalier
D
D
Leroy
H
H
Toutain
D000118:Acetylglucosaminidase / Q000032:analysis; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000378:metabolism; D005260:Female; D006597:Fructose-Bisphosphatase / Q000032:analysis; D005943:Gluconeogenesis; D005947:Glucose / Q000096:biosynthesis / Q000172:deficiency* / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis; D006019:Glycolysis; D006593:Hexokinase / Q000032:analysis; D007328:Insulin / Q000172:deficiency*; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000648:ultrastructure; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D007773:Lactates / Q000096:biosynthesis; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis; D011770:Pyruvate Kinase / Q000032:analysis; D011817:Rabbits; D013997:Time Factors
D007328:Insulin; D007773:Lactates; D007770:L-Lactate Dehydrogenase; D005982:Glutathione Transferase; D006593:Hexokinase; D011770:Pyruvate Kinase; D006597:Fructose-Bisphosphatase; D000118:Acetylglucosaminidase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose