Lack of insulin stimulation on Percoll-prepared or high-speed-centrifuged rat liver hepatocytes.
Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.
Memon
R A
RA
Department of Pharmacology and Nutrition, University of Southern California, Los Angeles 90033.
Mohan
C
C
Geiger
P J
PJ
Bessman
S P
SP
Rogers
K S
KS
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D013386:Succinates / Q000378:metabolism
D007328:Insulin; D013386:Succinates; D002245:Carbon Dioxide