S. Abraham

Factors involved in synthesis of fatty acids from acetate by a soluble fraction obtained from lactating rat mammary gland.

Abstract 1. 1. The conversion of acetate to fatty acids by a particle-free supernatant fraction obtained from the mammary glands of lactating rats was studied. 2. 2. Fatty acid synthesis by this particle-free supernatant shows an absolute requirement for (a) the cofactors concerned with acetate activation (ATP, Mg ++ , CoA, and glutathione), (b) triphosphopyridine nucleotide and (c) citrate. 3. 3. The requirement for triphosphopyridine nucleotide can be partially replaced by diphosphopyridine nucleotide, but the concentrations of diphosphopyridine nucleotide required are much higher than those of triphosphopyridine nucleotide. A conversion of diphosphopyridine nucleotide to triphosphopyridine nucleotide has been demonstrated in this system. 4. 4. Citrate can be completely replaced by cis -aconitate, but can be only partially replaced by d , l -isocitrate. 5. 5. Glucose 6-phosphate has only little effect on lipogenesis from acetate; in fact it strongly depresses the effect due to citrate in the presence of triphosphopyridine nucleotide. 6. 6. Fructose diphosphate has no effect on lipogenesis from acetate in the absence of citrate, whereas in the presence of this tricarboxylic acid its addition is inhibitory. 7. 7. α-ketoglutarate plus bicarbonate cannot replace citrate; however, in the presence of triphosphopyridine nucleotide and glucose 6-phosphate or l -malate, α-ketoglutarate plus bicarbonate shows a stimulatory effect which is about 25% of the citrate effect. 8. 8. Malonate stimulates fatty acid synthesis from acetate by the particle-free supernatant fraction. In the slice, malonate inhibits fatty acid synthesis in the absence of glucose, but it has little effect on fatty acid synthesis from acetate in the presence of glucose.

Fatty acid synthesis by complex systems The possibility of regulation by microsomes

Abstract 1. 1. Fatty acid synthesis from malonyl-CoA catalyzed by rat-liver microsomal protein does not seem to be due to contamination with enzymes from the particle-free supernatant or mitochondrial fractions. 2. 2. Optimum conditions for the conversion of malonyl-CoA to fatty acids by rat-liver microsomes with respect to substrate concentration, protein concentration, and incubation time were established. The enzyme systems concerned with fatty acid synthesis from malonyl-CoA in the particle-free supernatant fraction were 4–5 times more active than those in the microsomes. 3. 3. Preincubation of the microsomes caused a release of malonyl-CoA decarboxylase (EC 4.1.1.9) but not of fatty acid synthetase. This resulted in a greater conversion of the available malonyl-CoA to fatty acids. 4. 4. The products of synthesis in the supernatant system were free fatty acids bound to protein, whereas the products of the microsomal system were predominantly complex lipids (phospholipid). 5. 5. The predominant fatty acid synthesized from malonyl-CoA by the supernatant fraction was palmitate, and that synthesized by the microsomes was stearate. 6. 6. In the system composed of the supernatant fraction plus microsomes, the fatty acids synthesized were complex lipids (phospholipids), and the pattern of fatty acid synthesis resembled that found in the liver slice. 7. 7. The mechanism of the stimulatory effect of microsomes on fatty acid synthesis by the supernatant fraction is discussed and tentatively localized at the level of acetyl-CoA carboxylation. The stimulation may be due to a release of feedback inhibition.

Role of TPNH in fatty acid synthesis from acetate by normal and diabetic rat-liver homogenate fractions.

It is well known that fatty acid synthesis is depressed in liver slices (1, 2) and homogenates (3, 4) prepared from alloxan-diabetic rats. It has been suggested that this depression might result from a change in concentration or generation of cofactors, such as adenosine triphosphate (5)) coenzyme A (6)) and reduced triphosphopyridine nucleotide (7-10). It was the purpose of the study reported here to compare cofactor requirements for fatty acid synthesis in normal and diabetic rat liver homogenates.

Factors influencing the pattern of fatty acids synthesized by cell-free preparations of lactating rat mammary gland

Abstract 1. 1. The pattern of fatty acids synthesized from [1- 14 C]acetate, [1- 14 C]acetyl-CoA, [1,5- 14 C 2 ]citrate and [l,3- 14 C 2 ]malonyl-CoA by various homogenate preparations of mammary glands of lactating rats was studied with the aid of gas-liquid chromatography. 2. 2. Addition of microsomes to the 100000× g supernatant fraction significantly decreased the incorporation of labeled acetate into C 10 and C 12 fatty acids and increased that into C 16 and C 18 acids. 3. 3. When [ 14 C]acetate or acetyl-CoA were the substrates in experiments with the 100000× g supernatant fraction, the isotope was incorporated predominantly into C 10 fatty acid. The labeled malonyl-CoA was predominantly converted to C 16 fatty acid. The pattern of incorporation of the 14 C of [1,5- 14 C 2 ]citrate was intermediate between these two. 4. 4. When an ammonium sulfate fraction of the 100000× g supernatant fluid, precipitating between 0 and 40% saturation, was used as the source of acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase, the pattern of conversion of all labeled substrates to fatty acids was the same, i.e., predominautely to C 16 acids. Addition of fractions precipitating at higher satuiations of ammonium sulfate had no effect on the pattern of conversion. 5. 5. When the ammonium sulfate fraction (0–29% saturation) rich in acetyl-CoA carboxylase was added to incubation mixtures containing a constant amount of a fraction (29–40% saturation) rich in fatty acid synthetase, the average chain length of the fatty acids synthesized from acetyl-CoA was decreased as the concentration of acetyl-CoA carboxylase was decreased, but the chain length of the fatty acids formed from the labeled malonyl-CoA was unchanged. 6. 6. The results indicate that the concentration of malonyl-CoA is a factor in determining the pattern of fatty acids formed by mammary gland. The mechanism for expression of this influence is discussed, along with other factors that might affect the pattern of fatty acids synthesized.

Activity Patterns of Several Enzymes of Liver, Adipose Tissue, and Mammary Gland of Virgin, Pregnant, and Lactating Mice.

Summary Glucose-ATP phosphotransferases (glucokinase and hexokinase), glucose-6-PO4 dehydrogenase, malic enzyme, and citrate-cleavage enzyme were assayed in the 100,000 × g supernatant fraction (Fraction III) of liver, inguinal fat pads cleared of mammary gland tissue, abdominal adipose tissue, and mammary gland excised from virgin, pregnant, and lactating mice of the C3H strain. In addition, the activity of glucose-ATP phosphotransferases in the 350 × g supernatant fraction (Fraction I) and that associated with mitochondria of these same tissues was measured. Mitochondria isolated from adipose tissue contained only a small quantity of glucose-ATP phosphotransferase activity. No evidence for such activity was detected in mitochondria isolated from livers of lactating mice. Mitochondria from the mammary glands of lactating mice contributed more than 60% of the total glucose-ATP phosphotransferase activity of the cell, whereas those from virgin and pregnant mice contributed less than 17%. The specific activities of malic enzyme and citrate-cleavage enzyme were higher in mammary glands of virgin and lactating mice than in those of pregnant mice. Glucose-6-PO4 dehydrogenase activity was higher in liver and mammary gland of lactating mice than in virgin and pregnant mice. In adipose tissue, glucose-6-PO4 dehydrogenase was lower in lactating mice than in virgin and pregnant mice. The effect of lactation on the activity of the enzymes of adipose tissue and liver indicates that lactation involves more than just the mammary gland. The response cannot be brought about in all tissues by a generalized mechanism since the pattern of the enzymatic change was not the same in all tissues studied. The finding that the intracellular distribution of glucose-ATP phosphotransferase activity in liver and adipose tissue was unchanged during lactation adds strong support to the view that the increase in hexo-kinase activity associated with mitochondria of lactating mammary glands is due to an increase in the proportion of parenchymal tissue in the gland.

The role of microsomes in fatty acid synthesis from acetate by cell-free preparations of rat liver and mammary gland

Abstract 1. 1. The conversion of acetate to fatty acids by particle-free supernatant and microsomal fractions of rat livers and of lactating and non-lactating rat-mammary glands was studied. 2. 2. Stimulation of fatty acid synthesis by addition of microsomes to the supernatant fraction was related to the amounts of microsomal protein and ATP added to the incubation mixtures. Excessive amount of either one depressed the level of synthesis, which could be reversed by addition of larger amounts of the other. 3. 3. Microsomal stimulation cannot be explained solely by the presence of microsomal ATPase since the highest level of synthesis was observed at a critical ratio of microsomal protein to ATP concentration. This suggests that microsomal stimulation is related to an ATP-dependent process. 4. 4. The newly synthesized fatty acids were firmly bound to the incubated protein. Upon re-isolation of microsomes at the end of incubation, it was found that 80% of the newly synthesized fatty acids was bound to these particles. However, microsomal stimulation cannot be explained solely on the basis of a binding phenomenon. 5. 5. The possibility that microsomal stimulation of fatty acid synthesis from acetate or acetyl-CoA is due to an effect on acetyl-CoA carboxylase present in the supernatant fraction is discussed.

Stimulation of Microsomal Prostaglandin Synthesis by a Blood Plasma Constituent which Augments Autoregulation and Maintenance of Vascular Tone in Isolated Rabbit Hearts

Infusion of small amounts of plasma (1-2%) into isolated hearts perfused with Tyrode+-s solution causes vasoconstriction and augments regulation of coronary flow in response to changes in perfusion pressure (autoregulation). When plasma infusion is stopped, the vasoconstrictor effect dissipates within 5 minutes, whereas the autoregnlatory response remains for about 15 minutes. Thus the plasma-augmented autoregubtory response is not dependent on plasma-Induced vasoconstriction. Indomethadn (10 μml), an inhibitor of prostaglandin synthetase, causes coronary vasodflation and also abolishes the lingering autoregulatory response. These effects of indomethacin are counteracted by the addition of 1.5% plasma to the perfusate. Purification procedures led to the extraction of an active material from plasma which migrates as a single substance in thin layer chromatograms. This substance causes coronary vasoconstriction, augments autoregulation, and counteracts the effects of indometbacin in isolated rabbit hearts as effectively as plasma. The purified vasoactive substance stimulates a 2-fold increase in cardiac microsomal prostaglandin synthesis and a 5-fold increase using renal microsomal preparations. This substance counteracts indomethadn-induced inhibition of prostaglandin synthesis in microsomal preparations. These results provide convincing evidence that the effects of this pbtma constituent on the coronary vasculature are mediated by stimulation of prostaglandin synthesis. Thus it appears that prostaglandin synthesis plays an integral role In both the maintenance of coronary vascular tone and the autoregulatory response in isolated rabbit hearts.

Relative incorporation of the various propionate carbons into fatty acids by lactating rat mammary gland

Abstract 1. 1. Propionate labeled with 14 C in each of its carbons was incubated with slices prepared from lactating rat mammary glands. In the absence of added glucose, 14 C of [2- 14 C]propionate was more rapidly incorporated into fatty acids than was 14 C of either [1- 14 C]propionate or [3- 14 C]propionate. 2. 2. The addition of glucose to the medium increased fatty acid synthesis from all three carbons of propionate. The 14 C of [2- 14 C]propionate and [3- 14 C]propionate was incorporated at identical rates and exceeded that from [1- 14 C]propionate. 3. 3. Unlabeled acetate, at low concentrations in the presence of glucose, depressed conversion of 14 C of [1- 14 C]propionate into fatty acids. At higher acetate concentrations the conversion from all propionate carbons was depressed. 4. 4. The [ 14 C]fatty acids synthesized from [1- 14 C]-,[2- 14 C]- and [3- 14 C]propionate were subjected to gas-chromatographic analysis, and the presence of both-even-chain [ 14 C]fatty acids and odd-chain [ 14 C]fatty acids was demonstrated. 5. 5. The results suggest that there are at least three pathways for conversion of propionate carbon to fatty acids by lactating rat mammary gland. Direct condensation with malonyl-CoA to form odd-chain fatty acids is by far the most predominant pathway. Glucose stimulated incorporation of propionate carbon into fatty acids by all three pathways equally.

The relation of lipogenesis to reduced triphosphopyridine nucleotide generation and to certain enzyme activities in the liver of the "totally" depancreatized rat.

Abstract 1. 1. Evidence that the depressed lipogenesis observed in the liver of the diabetic animal is not the result of decreased TPNH generation via the hexose monophosphate oxidative pathway is provided by experiments carried out with rats 24 h after they were depancreatized by a procedure that ensured removal of more than 99.5% of the pancreas. For three days before pancreatectomy, these rats were fed a diet containing 60% glucose. The presence of diabetes at this early interval after pancreatectomy was indicated by the blood-sugar response, the presence of glucose and ketone bodies in the urine, and by increased amounts of fat in the liver. For controls we used normal rats and rats that had been subjected to laparotomy and manipulation of the abdominal viscera. 2. 2. Fatty acid synthesis from acetate was studied in a system consisting of a supernatant fraction plus microsomes prepared from homogenates of the livers. Lipogenesis from acetate was severely depressed at the 24-h interval after “total” pancreatectomy, but at that time the levels of activities of the two dehydrogenases concerned with glucose 6-phosphate and gluconic acid 6-phosphate were not depressed. 3. 3. Actual measurements of the TPNH produced from glucose 6-phosphate and citrate by the supernatant-microsomal system prepared from the livers of normal, sham-operated and “totally” depancreatized rats were made. TPNH generation by the diabetic system was well within the normal range. 4. 4. The levels of activities of isocitric dehydrogenase and aconitase, enzymes concerned with TPNH production from citrate, were about the same in the livers of normal controls and depancreatized rats. 5. 5. The existence of a powerful system for the reoxidation of TPNH, other than that concerned with fatty acid synthesis, and its effect upon TPN-dependent substrate oxidations are discussed.

Influence of enzymic activities on substrate oxidations in normal and diabetic rat liver and in mammary gland homogenate fractions

Abstract Activities of TPN-dependent enzymes that oxidize glucose 6-phosphate (glucose-6-phosphate and gluconic acid 6-phosphate dehydrogenases), isocitrate (isocitrate dehydrogenase) and malate (malic enzyme) were determined in particle-free supernatant fractions prepared from normal and diabetic rat liver homogenates and from lactating and nonlactating rat mammary-gland homogenates. These activities were compared with actual oxidations of their respective substrate under conditions where TPN availability was limited. 1. 1. The decrease in activities of glucose 6-phosphate-oxidizing enzymes in diabetic rat livers was not associated with a reduction in the rate of oxidation of glucose 6-phosphate. Thus, reoxidation of TPNH limits the oxidation of glucose 6-phosphate 2. 2. The inhibitory effect of citrate on oxidation of glucose 6-phosphate and malate in livers of normal and diabetic rats is principally due to high activities of isocitrate dehydrogenase. The inhibitory effect of glucose 6-phosphate on oxidation of citrate and malate by lactating gland fractions is due mainly to high activities of glucose 6-phosphate-oxidizing enzymes. 3. 3. Malic enzyme activity in diabetic liver was one-fourth that of normal liver. 4. 4. Weaning decreased activities of glucose 6-phosphate-oxidizing and malic enzymes in mammary gland preparations. 5. 5. Further evidence is presented for conversion of DPN to TPN in the presence of ATP in rat liver and lactating mammary gland fractions.