Expression and subcellular distribution of phosphoenolpyruvate carboxykinase in primary cultures of rabbit kidney proximal tubule cells: comparative study with renal and hepatic PEPCK in vivo.
The behaviour of the phosphoenolpyruvate carboxykinase (PEPCK) in rabbit proximal tubule cells in primary culture was investigated and compared with renal and hepatic PEPCK in vivo. The enzyme activity decreased rapidly in rabbit proximal tubule cells developed in hormonally defined medium supplemented with glucose and insulin. In this condition, the cytosolic form disappears with time. Without glucose and insulin, the subcellular location of PEPCK is similar to the location observed in proximal tubule freshly isolated and in renal cortex, with approx. 50% of mitochondrial form and approx. 50% of cytosolic form. However, the levels of mRNA that encode the cytosolic PEPCK are not detectable in cell cultures, whatever the medium composition. Treatment with dibutyryl cAMP caused a 14-fold induction of PEPCK mRNA in 6 h. This result indicates that the transcription of cytosolic PEPCK can be induced in cell cultures. Lactate or pyruvate additions did not modify the levels of PEPCK mRNA whereas specific activity increased rapidly, suggesting an activation of an inactive form in cell cultures. Moreover, lactate induced increased specific activity of the sole mitochondrial form while pyruvate induced increased specific activities of both mitochondrial and cytosolic form. Thus, subcellular location of PEPCK in rabbit proximal tubule cells appears to be modulated by the available substrate in culture medium. This observation parallels the changes observed in vivo since a modification of subcellular location of this enzyme was seen between fed and fasted rabbit, when subcellular distribution remains similar between fed and starved rats. Moreover, in the fasted liver of rabbit, a decrease of the mitochondrial PEPCK specific activity is seen concomitant with an increase in cytosolic PEPCK activity. These results point out the relative contributions of the cytosolic and mitochondrial PEPCK to rabbit gluconeogenesis.
Monteil
C
C
INSERM U-295-Université de Rouen, Saint Etienne du Rouvray, France.
Fillastre
J P
JP
Morin
J P
JP
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media; D003600:Cytosol / Q000201:enzymology; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D008099:Liver / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000201:enzymology*
D003470:Culture Media; D007328:Insulin; D007773:Lactates; D011773:Pyruvates; D012333:RNA, Messenger; D019344:Lactic Acid; D003994:Bucladesine; D019289:Pyruvic Acid; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose