Characterization of glycerol uptake and glycerol kinase activity in rat hepatocytes cultured under different hormonal conditions.
Glycerol uptake and glycerol kinase activity were studied in primary cultures of rat hepatocytes in the presence of either 1 nM insulin, 1 nM glucagon, or 100 nM dexamethasone, alone or in combination in the culture medium. Glycerol uptake exhibited saturation kinetic with K(m) values (microM) and Vmax (nmol/min x mg protein) ranging from 250-402, and 7.9-10.1, respectively. The corresponding K(m) and Vmax values for glycerol kinase activity were 36-46 and 8.7-12.7. Using the metabolic uncoupler 2,4-dinitrophenol, glycerol uptake and the cellular content of glycerol phosphorylated metabolites were reduced 33% and 43%, respectively, whereas no decrease in the cellular content of glycerol was seen. The glycerol analogues monoacetin, monobutyrin and dihydroxypropyl dichloroacetate were able in a concentration-dependent manner to inhibit glycerol uptake into hepatocytes with the two latter having IC50 values of approximately 1 mM. Moreover, it was demonstrated that the three glycerol analogues were substrates for glycerol kinase, which indicates a competitive mode of inhibition. The kinetic parameters for these substrates were calculated by using glycerol kinase from Candida Mycoderma. Monobutyrin was found to be 4 times lees efficient as substrate compared to the other substrates. Overall, these results indicate that independently of the culture conditions, glycerol uptake is the rate-limiting step in glycerol metabolism, and that the investigated glycerol analogues are metabolized via the same route as glycerol.
Westergaard
N
N
Novo Nordisk, Bagsvaerd, Denmark. nwe@novo.dk
Madsen
P
P
Lundgren
K
K
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000085:Acetates / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005934:Glucagon / Q000494:pharmacology; D005989:Glycerides / Q000494:pharmacology; D005990:Glycerol / Q000378:metabolism*; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006728:Hormones / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism*; D008297:Male; D011409:Propylene Glycols / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D000085:Acetates; D005989:Glycerides; D006728:Hormones; D007328:Insulin; D011409:Propylene Glycols; C063895:1-butyrylglycerol; D003907:Dexamethasone; C038923:monoacetin; D005934:Glucagon; C043658:2,3-dihydroxypropyl dichloroacetate; D005991:Glycerol Kinase; D005990:Glycerol; D019297:2,4-Dinitrophenol