Differential Effects of Biotin Deficiency and Replenishment on Rat Liver Pyruvate and Propionyl-CoA Carboxylases and on Their mRNAs

• Published in: Molecular genetics and metabolism 1999-01, Vol.66 (1), p.16-23
• Main Authors: Rodrı́guez-Meléndez, Rocı́o, Pérez-Andrade, Martha Elva, Dı́az, Alejandra, Deolarte, Alejandra, Camacho-Arroyo, Ignacio, Cicerón, Isabel, Ibarra, Isabel, Velázquez, Antonio
• Format: Article
• Language: English
• Subjects: 3-methylcrotonyl-CoA carboxylase; Animals; Biotin - deficiency; Biotin - pharmacology; Biotinylation; Carbon-Carbon Ligases - drug effects; Carbon-Carbon Ligases - metabolism; Carboxy-Lyases - drug effects; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; cultured hepatocytes; Electrophoresis, Polyacrylamide Gel; Liver - cytology; Liver - drug effects; Liver - enzymology; Male; Methylmalonyl-CoA Decarboxylase; propionyl-CoA carboxylase; pyruvate carboxylase; Pyruvate Carboxylase - drug effects; Pyruvate Carboxylase - genetics; Pyruvate Carboxylase - metabolism; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - drug effects; RNA, Messenger - genetics; Streptavidin; streptavidin blots
• ISSN: 1096-7192; 1096-7206
• DOI: 10.1006/mgme.1998.2777

Although the role of vitamins as prosthetic groups of enzymes is well known, their participation in the regulation of their genetic expression has been much less explored. We studied the effect of biotin on the genetic expression of rat liver mitochondrial carboxylases: pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC). Rats were made biotin-deficient and were sacrificed after 8 to 10 weeks, when deficiency manifestations began to appear. At this time, hepatic PCC activity was 20% of the control values or lower, and there was an abnormally high urinary excretion of 3-hydroxyisovaleric acid, a marker of biotin deficiency. Biotin was added to deficient primary cultured hepatocytes. It took at least 24 h after the addition of biotin for PCC to achieve control activity and biotinylation levels, whereas PC became active and fully biotinylated in the first hour. The enzyme's mass was assessed in liver homogenates from biotin-deficient rats and incubated with biotin to convert the apocarboxylases into holocarboylases, which were detected by streptavidin blots. The amount of PC was minimally affected by biotin deficiency, whereas that of the α subunits of PCC and of MCC decreased substantially in deficient livers, which likely explains the reactivation and rebiotinylation results. The expression of PC and αPCC was studied at the mRNA level by Northern blots and RT/PCR; no significant changes were observed in the deficient livers. These results suggest that biotin regulates the expression of the catabolic carboxylases (PCC and MCC), that this regulation occurs after the posttranscriptional level, and that pyruvate carboxylase, a key enzyme for gluconeogenesis, Krebs cycle anaplerosis, and fatty acid synthesis, is spared of this control.