Carl M. Nepokroeff

[6] Fatty acid synthase from rat liver

Publisher Summary The fatty acid synthase complex exists in mammalian and avian liver in the soluble portion of the cell. Negligible activity for the de novo synthesis of fatty acids is associated with the microsomal and mitochondrial fractions. The fatty acid synthase complex can be assayed by either radiochemical or spectrophotometric methods. In the radioisotopic method, incorporation of 1- 14 C-labeled acetyl-CoA into fatty acids is measured in the presence of malonyl-CoA and reduced nicotinamide adenine dinucleotide phosphate (NADPH). This method is reliable for either crude or purified enzyme preparations. The spectrophotometric method measures the malonyl-CoA- and acetyl-CoA-dependent rate of oxidation of NADPH at 340 nm and is best suited for purified enzyme preparations. However, it can be used with crude preparations providing appropriate corrections are made. A unit of enzymatic activity is defined as the amount of enzyme protein required to synthesize 1 nmole of palmitic acid (equivalent to the oxidation of 14 nmoles of NADPH) per minute under the conditions of the assay. The specific activity is defined as the number of activity units per milligram of protein. Protein is determined by the biuret method.

Regulation of the diurnal rhythm of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity by insulin, glucagon, cyclic AMP and hydrocortisone☆

Abstract Rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and the amplitude of the diurnal variation of this enzyme are progressively reduced to very low levels within 1 week after the onset of diabetes induced by streptozotocin. Daily insulin therapy to 7-day diabetic rats restores the activity and the amplitude of this diurnal variation in enzyme activity to near-normal levels within 4 days. Insulin also produces a rapid 2-hr stimulation of the reductase activity in diabetic rats to the level found in normal animals at that time of day regardless of the duration of diabetes. Hence, insulin is required for the diurnal rise of reductase activity in rat liver. Glucagon, dibutyryl cyclic AMP, and hydrocortisone, in contrast, markedly inhibit the diurnal rise of reductase activity in normal rats. Therefore, the relative concentrations of insulin, glucagon, and glucocorticoids are important in the regulation of the diurnal variation of hepatic reductase activity.

Coordinate control of rat liver lipogenic enzymes by insulin

Abstract Recent evidence has established that insulin is required for the dietary induction of rat liver fatty acid synthetase [ Proc. Nat. Acad. Sci. USA 69 , 3516 (1972)]. Since other hepatic lipogenic enzymes as well as fatty acid synthetase exhibit coordinate adaptation to nutritional changes [ Advan. Enzyme Regul. 10 , 187(1972)], the role of insulin in the dietary induction of these enzymes has been investigated. When a high-carbohydrate, fat-free diet was fed to diabetic rats previously fasted for 48 hr, insulin was shown to be required for the dietary induction of acetyl-CoA carboxylase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fatty acid synthetase, and glucokinase. Activity of serine dehydrase, selected as a model gluconeogenic enzyme, was increased in diabetic rats, whereas insulin treatment reduced the activity of this enzyme during the course of refeeding. The behavior of serine dehydrase was consistent with its gluconeogenic role. The activity of the cytosol isocitrate dehydrogenase did not change during refeeding in the diabetic or insulin-treated diabetic rat. Glucagon, the physiological antagonist of insulin, inhibited the increase in activity of each of the lipogenic enzymes requiring insulin for induction. Our results indicate that insulin is required for the coordinate regulation of the lipogenic enzymes of mammalian liver.

Effect of dietary change on the rates of synthesis and degradation of rat liver fatty acid synthetase

Abstract Investigations were carried out to determine whether diet-induced changes in the level of rat liver fatty acid synthetase are due to changes in the rate of synthesis or degradation of this enzyme. Assays for radioactivity in fatty acid synthetase were carried out through isolation of the enzyme by a standard purification procedure or by immunoprecipitation with antiserum prepared specifically against rat liver fatty acid synthetase. Studies on the incorporation of radioactive leucine into the fatty acid synthetase during steady-state conditions showed that the relative rate of synthesis of this enzyme was decreased to one-fifth the normal value in the fasted state and increased to 14.3 times the normal value in rats refed a fat-free diet. Studies on the degradation of the fatty acid synthetase, reported as half-life of the enzyme, yielded identical values, 69 hr, for normal animals, as well as for animals refed a fat-free diet. The rates of synthesis of enzyme ( k s ) calculated from these half-life values and the enzyme content present in the liver in a given nutritional state agreed well with the relative rates of synthesis. On the basis of these results it is concluded that changes in the level of liver fatty acid synthetase which are diet induced are due primarily to changes in the rate of synthesis rather than degradation of this enzyme. However, transient changes in the rate of degradation may also be important during the transition period, particularly in the case of fasting.

Regulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase by the interplay of hormones☆

We have previously established that insulin causes a marked and rapid stimulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity in normal and diabetic rats [Biochem. Biophys. Res. Commun.50, 504 (1973)], whereas l-triiodothyronine stimulates the reductase activity to supranormal levels in hypophysectomized rats two days after administration [Proc. Nat. Acad. Sci. (1974) In press]. In the present investigation it is demonstrated that the stimulation of the reductase activity in hypophysectomized-diabetic rats requires the mediation of both insulin and l-triiodothyronine. Neither hormone alone is effective. The rapid stimulation of reductase activity by insulin and the delayed stimulation elicited by l-triiodothyronine are both inhibited by either glucagon or hydrocortisone. Thus, an interplay of hormones regulates reductase activity and consequently cholesterol biosynthesis.

Adaptive synthesis of fatty acid synthetase and acetyl-CoA carboxylase by isolated rat liver cells

Abstract Hepatocytes were isolated at specified times from livers of diabetic and insulin-treated diabetic rats during the course of a 48-h refeeding of a fat-free diet to previously fasted rats. The rates of synthesis of fatty acid synthetase and acetyl-CoA carboxylase in the isolated cells were determined as a function of time of refeeding by a 2-h incubation with l -[U- 14 C]leucine. Immunochemical methods were employed to determine the amount of radioactivity in the fatty acid synthetase and acetyl-CoA carboxylase proteins. The amount of radioactivity in the fatty acid synthetase synthesized by the isolated cells was also determined following enzyme purification of the enzyme to homogeneity. Enzyme activities of the fatty acid synthetase and acetyl-CoA carboxylase in the cells were measured by standard procedures. The results show that isolated liver cells obtained from insulintreated diabetic rats retain the capacity to synthesize fatty acid synthetase and acetyl-CoA carboxylase. The rate of synthesis of the fatty acid synthetase in the isolated cells was similar to the rate found in normal refed animals in in vivo experiments [Craig et al. (1972) Arch. Biochem. Biophys . 152 , 619–630; Lakshmanan et al. (1972) Proc. Nat. Acad. Sci. USA 69 , 3516–3519]. In addition the relative rate of synthesis of fatty acid synthetase was stimulated greater than 20-fold in the diabetic animals treated with insulin. Immunochemical assays, when compared with enzyme activities, indicated the presence of an immunologically reactive, but enzymatically inactive, form or “apoenzyme” for both the fatty acid synthetase and acetyl-CoA carboxylase. The synthesis of these immunoreactive and enzymatically inactive species of protein, as well as the synthesis of the “holoenzyme” forms of both enzymes, requires insulin.

Identification and quantitation of polysomes synthesizing rat liver fatty acid synthetase during nutritional or hormonal regulation

Abstract 1. 1. Fatty acid synthetase synthesizing polysomes from rat liver have been identified and quantitated by sucrose density gradient centrifugation with [125I]labeled anti-fatty acid synthetase antibody. 2. 2. The [125I]labeled antibody binding to nascent fatty acid synthetase peptides demonstrated that the relative content of the fatty acid synthetase mRNA-containing polysomes in rat liver increases dramatically during dietary induction. 3. 3. This increase correlates well with the relative rate of in vivo synthesis of the fatty acid synthetase under the same conditions. 4. 4. The maximum quantity of the synthetase mRNA-containing polysomes was observed at the 12-hr period of dietary induction. Thereafter the quantity of fatty acid synthesizing polysomes declined. 5. 5. Insulin also markedly increased the quantity of synthetase polysomes in diabetic animals, which suggests that this hormone affects regulation of the synthetase at the transcriptional level. 6. 6. Most of the fatty acid synthetase synthesizing polysomes were “free”, rather than membrane-bound.

Cell-free translation and partial purification of rat liver fatty acid synthetase mRNA

Abstract The cell-free synthesis of rat liver fatty acid synthetase has been demonstrated in a modified reticulocyte lysate translation system. The mRNA was partially purified from membrane-free polysomes by oligo (dT)-cellulose chromatography and subsequent sucrose density gradient centrifugation.

Cell-free synthesis of pigeon liver fatty acid synthetase☆

Abstract Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the in vitro synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K+ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5.10(6) Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (Mr = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.