ABSTRACT
We report the preparation of specific polyclonal antibodies raised against two synthetic peptides deduced from the cDNA sequence for the rat liver mitochondrial 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) synthase gene. Immunoelectron microscopy using these antibodies on hepatic cryoultrathin sections confirms the mitochondrial localization of this protein in hepatocytes. Immunofluorescence microscopy on frozen sections of adult rat liver revealed fluorescence inside all hepatocytes, with no evidence of zonation, indicating that ketogenesis may not be limited to specific regions of rat liver but is extended to all hepatocytes.
Subject(s)
Hydroxymethylglutaryl-CoA Synthase/analysis , Liver/enzymology , Mitochondria, Liver/enzymology , Amino Acid Sequence , Animals , Antibodies/analysis , Antibodies/immunology , DNA, Complementary/analysis , DNA, Complementary/genetics , Fluorescent Antibody Technique , Hydroxymethylglutaryl-CoA Synthase/genetics , Hydroxymethylglutaryl-CoA Synthase/immunology , Immunohistochemistry , Liver/cytology , Liver/ultrastructure , Male , Microscopy, Immunoelectron , Mitochondria, Liver/ultrastructure , Molecular Sequence Data , Precipitin Tests , Rats , Rats, WistarABSTRACT
Ketogenesis has been thought to occur exclusively in the mitochondrial compartment of liver cells. After analysis of five different rat tissues, it was shown that the gene for mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, one of the major control points in the pathway (1992. Casals et al. Biochem. J. 283: 261-264) was expressed only in liver (1990. Ayté et al. Proc. Natl. Acad. Sci. USA. 87: 3874-3878). However, exhaustive analysis of organs and tissues has shown that, in addition to liver cells, testis and ovary express this committed gene in levels similar to those of liver, not only as mRNAs but also as immunodetectable mitochondrial HMG-CoA synthase protein. Immunocytochemical studies locate the mitochondrial HMG-CoA synthase protein in Leydig cells, theca interna cells of ovarian follicle, corpus luteum cells of ruptured ovarian follicle, and epidermal cells of the oviduct. The development of gonadal function appears to be accompanied by mitochondrial HMG-CoA synthase gene expression, as hypophysectomy reduces the expression pattern in gonads. Changes induced in mitochondrial HMG-CoA synthase levels after the depletion of lipoprotein levels in blood closely mimic those of the cholesterogenic cytosolic HMG-CoA synthase and HMG-CoA reductase. These results suggest that mitochondrial HMG-CoA synthase could perform a function similar to that of cytosolic HMG-CoA synthase in de novo cholesterogenesis in gonads, at variance with its ketogenic role in liver.
