Acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, exists as an oligodendrocyte-associated enzyme in brain and plays an important role in supplying fatty acid for myelination. Rat brain acetyl-CoA carboxylase (ACC) has been identified as a single isozyme of M(r) = 265,000 daltons, indistinguishable immunologically from the isozyme in rat adipose tissue and liver. Total activity of brain ACC declines from birth to 4 weeks of age in the newborn rat. This change in activity can entirely be accounted for by changes in enzyme content, not enzyme specific activity, and is paralleled by decreases in ACC mRNA. In contrast, cardiac, skeletal muscle and liver ACC does not change in content over this developmental period. Unlike ACC in liver and adipose tissue, the enzyme content and specific activity of brain ACC is invariant during various states of nutrition. These data indicate that the brain ACC is subject to unique regulation, as compared to non-neural enzyme. The mechanisms underlying the control of neural ACC activity may be important to understanding the process of myelination during development and to a more general understanding of the factors regulating ACC expression/activity in other tissues.

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

Acetyl-CoA carboxylase (ACC) is a major rate-limiting enzyme of fatty acid biosynthesis; its product, malonyl-CoA, also contributes to the regulation of fatty acid oxidation and elongation. Using monospecific antibodies directed against rat liver ACC and N- and C-terminal antipeptide antibodies raised against predicted sequences of the cloned ACC of Mr 265,000, we have identified a unique biotin-containing cytosolic protein of molecular mass 280,000 daltons that is distinct from this 265,000-dalton protein. This protein is uniquely expressed in rat cardiac and skeletal muscle but is co-expressed with the 265,000-dalton protein in rat liver, mammary gland, and brown adipose tissue. In the fed rat, white adipose tissue contains only the 265,000-dalton protein. Like the 265,000-dalton protein, the 280,000-dalton protein is present predominantly in the cytosolic fraction of liver. In the liver, the content of both proteins is diminished on fasting and increases on fasting/refeeding with a high carbohydrate diet. In contrast, the cardiac and skeletal muscle 280,000-dalton protein content is unaltered by nutritional manipulation. Avidin-Sepharose isolates of citrate-dependent ACC from the heart reveal only the 280,000-dalton protein, while white adipose tissue isolates show only the 265,000 form. These species differ in the sensitivity to citrate activation and in the Km for acetyl-CoA. Antibodies reactive with the 280,000-dalton protein on immunoblotting precipitate ACC activity in heart isolates, while white adipose ACC is precipitated only by antibodies specific for the 265,000-dalton species. However, in ACC isolates where both proteins are present, a heteroisozyme complex can be detected both by immunoprecipitation and by a sandwich enzyme-linked immunosorbent assay. We conclude that the 280,000-dalton protein is an isozyme of ACC, distinct from the previously cloned 265,000-dalton species. Its presence in cardiac and skeletal muscle, where fatty acid synthesis rates are low, suggest that it might play alternative roles in these tissues such as regulation of fatty acid oxidation or microsomal fatty acid elongation.

Changes in the mass and subunit structure of liver acetyl-CoA carboxylase (ACC) accompany altered nutrition in vivo. Enzyme activity in different tissues and cell lines is also, in part, determined by variations in both total mass and ACC isoenzyme composition. ACC isoenzyme mass and hetero/homo-isoenzyme association were quantified by three sandwich e.l.i.s.a. assays, i.e. an avidin-based assay that measured total isoenzyme mass and two antibody-sandwich assays which measure polypeptide association. Results from the avidin-based assay reveal that the two major isoenzymes, of molecular mass 265 kDa (ACC 265) and 280 kDa (ACC 280), are present in markedly variable concentration in several rat and mouse tissues and in cell lines of rat and mouse origin. Hepatic ACC mass has been reported to be distributed between mitochondrial and cytosolic fractions and to undergo only a change in subcellular distribution without alteration in total mass on induction/repression of activity in vivo [Roman-Lopez, Shriver, Joseph & Alfred (1989) Biochem. J. 260, 927-930]. However, in the present study, immunoblotting and e.l.i.s.a. analysis reveals that, in rat liver, the mass of both isoenzymes is predominantly cytosolic in distribution, is markedly diminished on fasting and rises 6-8-fold on refeeding of a high-carbohydrate diet. These data support the results of several other investigations of hepatic ACC mass, and are consistent with known nutritionally altered changes in ACC mRNA content. By the two antibody-sandwich e.l.i.s.a. assays, isoenzyme complexes either composed of both ACC 280 and 265 or with multiple copies of ACC 265 are detectable in rat liver enzyme; their concentration varies independently of total ACC mass with the nutritional state of the rat, being lowest in fasting and highest on fasting/refeeding. E.l.i.s.a. analysis, applicable to crude tissue/cell extracts, provides a simple, sensitive and quantitative measurement of ACC mass and subunit composition. Its use may permit needed quantitative insight into the role of variable total ACC and isoenzyme mass and of alterations in ACC subunit composition that occur in vivo or in isolated cells in response to a variety of hormonal and nutritional influences.

Zonal distribution of insulin stimulation of hepatic protein tyrosine phosphorylation, detected by immunoblotting with an anti-phosphotyrosine antibody, has been studied in the in situ perfused rat liver by dual-digitonin-pulse perfusion. Insulin promotes the rapid and sustained tyrosine phosphorylation of two proteins (pp150 and pp69) that are present only in the perivenous hepatocytes, while three others (pp46, pp48 and pp96) are stimulated identically in the periportal and perivenous cells. The ability of insulin to rapidly activate acetyl-CoA carboxylase is indistinguishable between the hepatic zones. Hepatic zonation of insulin-stimulated tyrosine phosphorylation could underly differential hepatic insulin responses and might provide clues to the identification of tyrosine phosphorylated proteins linked to insulin regulation of intracellular events.