title: Characterization of glycerol uptake and glycerol kinase activity in rat hepatocytes cultured under different hormonal conditions.
abstract: 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.
authors.affiliation: Novo Nordisk, Bagsvaerd, Denmark. nwe@novo.dk
authors.forename: N
authors.initials: N
authors.lastname: Westergaard
authors.forename: P
authors.initials: P
authors.lastname: Madsen
authors.forename: K
authors.initials: K
authors.lastname: Lundgren
mesh_terms: 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
chemical_list: 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