Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes
- Univ. of Rochester School of Medicine and Dentistry, NY (USA)
Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using (35S)methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorporation of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBH with an intra-cellular retention half-time of 50 min compared with 85 min in control (t1/2 = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin-treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively. When hepatocyte monolayers were continuously labeled with (35S)methionine and then incubated in chase medium with and without insulin, labeled apoBH was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase.
- OSTI ID:
- 7045426
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 265:15; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
APOLIPOPROTEINS
BIOSYNTHESIS
INSULIN
BIOCHEMICAL REACTION KINETICS
ANTIBODIES
CELL CULTURES
ELECTROPHORESIS
IMMUNOASSAY
LIVER CELLS
METHIONINE
RATS
SECRETION
SULFUR 35
TRACER TECHNIQUES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
EVEN-ODD NUCLEI
HORMONES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPIDS
LIPOPROTEINS
LIPOTROPIC FACTORS
MAMMALS
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HORMONES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SOMATIC CELLS
SULFUR ISOTOPES
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques