Differential effects of biotin deficiency and replenishment on rat liver pyruvate and propionyl-CoA carboxylases and on their mRNAs

Although the role of Vitamins as prosthetic groups of enzymes is well known , their participation in the regulation of their genetic expression has bee n much less explored. We studied the effect of biotin on the genetic expres sion of rat liver mitochondrial carboxylases: pyruvate carboxylase (PC), pr opionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC). Rats were made biotin-deficient and were sacrificed after 8 to 10 weeks, wh en deficiency manifestations began to appear. At this time, hepatic PCC act ivity was 20% of the control values or lower, and there was an abnormally h igh urinary excretion of 3-hydroxyisovaleric acid, a marker of biotin defic iency. Biotin was added to deficient primary cultured hepatocytes. It took at least 24 h after the addition of biotin for PCC to achieve control activ ity and biotinylation levels, whereas PC became active and fully biotinylat ed in the first hour. The enzyme's mass was assessed in liver homogenates f rom biotin-deficient rats and incubated with biotin to convert the apocarbo xylases into holocarboylases, which were detected by streptavidin blots. Th e amount of PC was minimally affected by biotin deficiency, whereas that of the cu subunits of PCC and of MCC decreased substantially in deficient liv ers, which likely explains the reactivation and rebiotinylation results. Th e expression of PC and alpha PCC was studied at the mRNA level by Northern blots and RT/PCR; no significant changes were observed in the deficient liv ers. These results suggest that biotin regulates the expression of the cata bolic carboxylases (PCC and MCC), that this regulation occurs after the pos ttranscriptional level, and that pyruvate carboxylase, a key enzyme for glu coneogenesis, Krebs cycle anaplerosis, and fatty acid synthesis, is spared of this control. (C) 1999 Academic Press.