title: Biotin deficiency affects both synthesis and degradation of pyruvate carboxylase in rat primary hepatocyte cultures.
abstract: Pyruvate carboxylase (PC) is a biotin-dependent enzyme that plays a crucial role in gluconeogenesis, lipogenesis, Krebs cycle anaplerosis and amino acid catabolism. Biotin deficiency reduces its mass besides its activity. Enzyme mass is the result of its cellular turnover, i.e., its rates of synthesis and degradation. We have now investigated, by a pulse and chase approach in cultured primary hepatocytes, the effects of biotin deficiency on these rates. Wistar rats were fed a biotin-deficient diet and the controls were fed the same diet supplemented with biotin; their biotin status was monitored measuring lymphocytes propionyl-CoA carboxylase activity and urinary 3-hydroxyisovaleric acid. After 6-7 weeks primary hepatocytes were cultured in biotin-deficient or complete DMEM. PC activity was determined by measuring the incorporation of (14)C-bicarbonate into acid-non-volatile products, and its mass by streptavidin Western blots. Its synthesis rate was estimated from [(35)S] methionine incorporation into anti-PC antibody immunoprecipitate. Its degradation rate was calculated from the loss of radioactivity from previously labeled hepatocytes, in a medium containing an excess of non-radioactive methionine. PC synthesis rate in biotin-deficient hepatocytes was approximately 4.5-fold lower than in the controls, and its degradation rate was 5.1-fold higher. Therefore, the decrement of PC mass during biotin deficiency results both from a decrease in its synthesis and an increase in its degradation rates. To our knowledge, this is the first instance where a mammalian enzyme cofactor is necessary to sustain both processes.
authors.affiliation: Instituto de Investigaciones Biomédicas de la Universidad Nacional Autónoma de Mexico, Instituto Nacional de Pediatría, Department of Nutritional Genetics, Torre de Investigacion, Av. del IMAN No. 1, 4o. piso, D.F. 04530 México City, Mexico.
authors.forename: Nayeli
authors.initials: N
authors.lastname: Rodríguez-Fuentes
authors.forename: Itzel
authors.initials: I
authors.lastname: López-Rosas
authors.forename: Gabriela
authors.initials: G
authors.lastname: Román-Cisneros
authors.forename: Antonio
authors.initials: A
authors.lastname: Velázquez-Arellano
mesh_terms: D000818:Animals; D001710:Biotin / Q000172:deficiency*; D019921:Biotinylation; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology*; D008297:Male; D043586:Methylmalonyl-CoA Decarboxylase / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014631:Valerates / Q000652:urine
chemical_list: D014631:Valerates; C004961:beta-hydroxyisovaleric acid; D001710:Biotin; D011766:Pyruvate Carboxylase; D043586:Methylmalonyl-CoA Decarboxylase