Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver

The activities of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and acetyl-CoA carboxylase (extracted with or without phosphatase inhibitor) in rat liver did not vary significantly during 24 h. The hepatic levels of glucose 6-phosphate and malate increased coordinately 3-6 h after the beginning (1900 h) of food intake and were high until morning, whereas the levels of acetyl-CoA and citrate peaked at 1900 h and then decreased. However, it is remarkable that the in vivo incorporation of 3H from tritiated water into fatty acids in liver increased with the level of malonyl-CoA after food intake. Comparing the substrate and effector levels with the Km and Ka values for the enzymes, the levels of acetyl-CoA, malonyl-CoA and citrate appear to limit the enzyme activities. It is suggested that, after food intake, the physiological activity of acetyl-CoA carboxylase was increased with the substrate increase and/or with the catalytic activation with citrate, and consequently, the fatty acid synthetase activity was also increased, whereas the enzyme activities measured under optimum conditions were not.     

Regulation of hepatic fatty acid-synthesizing enzymes of diabetic animals by thyroid-hormone

Subcutaneous administration of l-triiodothyronine (T₃) to diabetic rats restored hepatic acetyl-CoA carboxylase and fatty acid synthetase enzymes to normal levels. T₃ stimulated the fatty acid-synthesizing enzymes of diabetic animals by two different mechanisms. Between 4 and 12 h after T₃ administration, carboxylase and synthetase increased slowly, after which both the enzyme activities increased at faster rate. Carboxylase and synthetase induction could be inhibited by cycloheximide or actinomycin D during the first 12 h. The incorporation of [¹⁴C]pantothenate into the fatty acid synthetase during 4–12 h followed the same pattern as the development of the enzyme activity. Moreover, liver supernatants from T₃-treated diabetic rats were able to compete with pure fatty acid synthetase for antibody binding sites, the degree of competition increased with increasing period of T₃ treatment. The results suggest that enzymatically inactive precursors of synthetase in the diabetic livers are converted to enzymatically active enzyme as a result of T₃ treatment. The second part of T₃-mediated stimulation (24 to 72 h following T₃ treatment) was inhibited by cycloheximide and actinomycin D. Antibody-antigen titration and measurement of rate of protein synthesis suggest that the increased activity of hepatic synthetase is due to enhanced synthesis of the enzyme for that period. These results indicate that T₃ might play a significant regulatory role in hepatic fatty acid synthesis.     

Prolonged feeding of mice with conjugated linoleic acid increases hepatic fatty acid synthesis relative to oxidation

Feeding mice conjugated linoleic acid (9 cis,11 trans/9 trans,11 cis-and 10 trans,12 cis-CLA in equal amounts) resulted in triacylglycerol accumulation in the liver. The objective of this study was to examine whether this steatosis is associated with changes in hepatic fatty acid synthesis and oxidation. Therefore, we measured the activities of key enzymes of fatty acid synthesis, i.e., acetyl-CoA carboxylase and fatty acid synthase and of fatty acid oxidation, i.e., 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase in livers of mice fed a diet with 0.5% (w/w) CLA. CLA (a 1:1 mixture of the 10 trans, 12 cis and 9 cis, 11 trans isomers of octadecadenoic acid) was administered for 3 and 12 weeks with high-oleic sunflower oil fed as control. The proportion of body fat was significantly lower on the CLA than on the control diet and this effect was already significant after 3 weeks. The specific activites of 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase were unaffected by CLA both after 3 and 12 weeks. The specific activity of fatty acid synthase was nonsignificantly raised (by 12%) after 3 weeks on the CLA diet but had increased significantly (by 34%) after 12 weeks of feeding. The specific activity of acetyl-CoA carboxylase had also increased both after 3 weeks (by 53%) and 12 weeks (by 23%) on the CLA diet, but this effect did not reach statistical significance. Due to CLA-induced hepatomegaly, the overall capacity for both fatty acid oxidation and synthesis-as evidenced by the total hepatic activities of 3-hydroxy-acyl-CoA dehydrogenase, citrate synthase, acetyl-CoA carboxylase, and fatty acid synthase-was significantly greater in the CLA-fed group after 12 weeks, although the overall capacity for fatty acid synthesis had increased more than that for fatty acid oxidation. Thus, this study indicates that prolonged, but not short-term, feeding mice with CLA increased hepatic fatty acid synthesis relative to oxidation, despite the decrease in body fat and the increase in liver weight seen earlier. It is concluded that the observed CLA-induced changes in hepatic fatty acid synthesis and oxidation are the result, rather than the cause, of the lowering of body fat.     

The effect of starvation and alloxan-diabetes on the contents of citrate and other metabolic intermediates in rat liver

1. The content of citrate in ;freeze-clamped' livers from starved and alloxan-diabetic rats was measured by using the specific citrate assay method of Gruber & Moellering (1966). 2. The content of citrate fell progressively during a period of 48hr. starvation to reach a plateau value that is 50% of the value for livers from fed rats. Some possible explanations for the conflicting reports of changes in hepatic citrate content during starvation are discussed. 3. The hepatic contents of ATP, pyruvate, lactate, glycogen and the hexose phosphates were decreased during starvation, whereas those of acetyl-CoA and AMP were increased. 4. Acute alloxan-diabetes produced similar changes in the contents of these metabolic intermediates. 5. The effects of starvation and diabetes on the citrate and acetyl-CoA contents are discussed in relation to control of gluconeogenesis, fatty acid synthesis and the activity of citrate synthase.     

Stimulation of the synthesis of hepatic fatty acid synthesizing enzymes of hypophysectomized rats by 3,5,3'-l-triiodothyronine.

The levels of hepatic fatty acid synthesizing enzymes, acetyl-CoA carboxylase and fatty acid synthetase, are lowered to about one-tenth of the controls in hypophysectomized animals, whereas the lung enzymes decrease by only 25–30%. Administration of 3,5,3′-l-triiodothyronine to the hypophysectomized animals returns the hepatic and lung enzyme activities to the control values. Optimum levels are achieved at a dose of about 150 μg/100 g body wt and 3–4 days after triiodothyronine administration. The triiodothyronine response can be reduced by 80% with actinomycin-D or cycloheximide but not with hydrocortisone hemisuccinate. Antibody-antigen titrations and measurements of the rate of synthesis of fatty acid synthetase are indicative of increased synthesis of fatty acid synthetase and not of activation of the preexisting inactive species. These measurements provide evidence for the involvement of hormones other than insulin in the control of synthesis of fatty acid synthesizing enzymes.     

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.     

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

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-¹⁴C]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. USA69, 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.     

Polymer-protomer transition of acetyl-CoA carboxylase as a regulator of lipogenesis in rat liver

Data are presented which indicate that the transition of acetyl-CoA carboxylase between the active polymeric and inactive protomeric conformations defined for the purified enzyme also occurs with the enzyme in vivo, depends upon the nutritional state of the animal, and is an important physiological phenomenon in the acute regulation of liver fatty acid synthesis. This conclusion utilized the observation that the protomeric form of purified acetyl-CoA carboxylase is inactivated by the binding of avidin to the biotinyl prosthetic group; the catalytically active filamentous form of the enzyme is resistant to avidin. Acetyl-CoA carboxylase activity was 75% avidin-resistant (polymeric) in the liver of meal-fed rats that had completed the consumption of a high glucose meal. This avidin resistance gradually decreased to 20% during the 21-h interval between meals. Peak resistance to avidin of liver carboxylase was attained within 30 min of initiating meal ingestion. The rise in carboxylase resistance to avidin could not be mimicked by insulin injection alone, but could be greatly attenuated by the addition of fat to the glucose meal. The amount of avidin-resistant acetyl-CoA carboxylase was closely associated with the concentration of hepatic malonyl-CoA and the subsequent rate of fatty acid synthesis.     

Time course of hormonal effects on acetyl-CoA carboxylase as measured in digitonin-permeabilized rat hepatocytes

Acetyl-CoA carboxylase activity was measured in digitonin-permeabilized rat hepatocytes by coupling the carboxylase reaction to the fatty acid synthase reaction. Using this assay the activity of acetyl-CoA carboxylase was covariant with the rate of fatty acid synthesis. Insulin and the tumor promotor phorbol myristate acetate were found to stimulate, and glucagon and noradrenaline to inhibit both cellular parameters. The stimulation of acetyl-CoA carboxylase by insulin developed slowly (15 to 30 min) whereas the phorbol myristate acetate effect developed faster (within 15 min). The inhibition of the enzyme caused by glucagon was already apparent within 1 min after hormone addition. Inhibition by noradrenaline, in the presence of propranolol, was also quite rapid and occurred within 2 min after addition of the agonist.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Temporal changes in plasma and liver lipids and in the hepatic activities of acetyl-coenzyme a carboxylase and fatty acid synthetase after oestrogen treatment of the male chicken (Gallus domesticus)

1. Male chickens (Gallus domesticus) were treated with a single intramuscular injection of oestradiol-17 beta, then changes in the liver and plasma levels of triacylglycerol, phospholipid, nonesterified fatty acids and in the hepatic activities of acetyl-CoA carboxylase and fatty acid synthetase were measured at various times after injection. 2. The results suggest that the initial phase (less than 20 hr) of oestrogen-induced hyperlipidaemia occurs in the absence of changes in the hepatic activities of the major enzymes of fatty acid biosynthesis, but a subsequent increase in these enzyme activities may contribute to the later phase (greater than 20 hr) of oestrogen-induced lipogenesis in avian liver.     

Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells.

The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect.     

Regulation of palmitic acid synthesis in cultured glial cells: effects of glucocorticoid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C-6 glial cells in culture were utilized to define the role of glucocorticoid in the regulation of palmitic acid synthesis and the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase. Particular emphasis was given to fatty acid synthetase which exhibited more than a 50% reduction in specific activity when cells were exposed to hydrocortisone (10 μg/ml) for 1 week. Coordinate changes in acetyl-CoA carboxylase activity and in palmitic acid (and sterol) synthesis from acetate accompanied the alterations in fatty acid synthetase. Immunochemical techniques were utilized to show that the decrease in synthetase activity involved an alteration in enzyme content, not in catalytic efficiency. The changes in content of fatty acid synthetase were caused by alterations in enzyme synthesis. Glucocorticoids may regulate fatty acid synthetase in C-6 glial cells by a mechanism similar to that suggested for adipose tissue. The inhibition of palmitic acid synthesis may be relevant to other effects of glucocorticoids on developing brain.     

Regulation of fatty acid synthesis and malonyl-CoA content in mouse brown adipose tissue in response to cold-exposure, starvation or re-feeding.

1. The effect of nutritional status on fatty acid synthesis in brown adipose tissue was compared with the effect of cold-exposure. Fatty acid synthesis was measured in vivo by 3H2O incorporation into tissue lipids. The activities of acetyl-CoA carboxylase and fatty acid synthetase and the tissue concentrations of malonyl-CoA and citrate were assayed. 2. In brown adipose tissue of control mice, the tissue content of malonyl-CoA was 13 nmol/g wet wt., higher than values reported in other tissues. From the total tissue water content, the minimum possible concentration was estimated to be 30 microM 3. There were parallel changes in fatty acid synthesis, malonyl-CoA content and acetyl-CoA carboxylase activity in response to starvation and re-feeding. 4. There was no correlation between measured rates of fatty acid synthesis and malonyl-CoA content and acetyl-CoA carboxylase activity in acute cold-exposure. The results suggest there is simultaneous fatty acid synthesis and oxidation in brown adipose tissue of cold-exposed mice. This is probably effected not by decreases in the malonyl-CoA content, but by increases in the concentration of free long-chain fatty acyl-CoA or enhanced peroxisomal oxidation, allowing shorter-chain fatty acids to enter the mitochondria independent of carnitine acyltransferase (overt form) activity.     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

Evidence of a biotin dependent acetyl-coenzyme A carboxylase in rat muscle.

Rat hindlimb muscle tissue was extracted from male Sprague-Dawley rats exsanguinated under light ether anesthesia. Muscle homogenates (50,000 x g supernatant) were incubated with ATP, bicarbonate, acetyl-CoA, and citrate. The quantity of malonyl-CoA synthesized was determined by malonyl-CoA incorporation into long acyl chains using tritiated acetyl-CoA and fatty acid synthetase. Malonyl-CoA synthesis was found to be dependent on the presence of ATP, bicarbonate, citrate, and acetyl-CoA in the incubation medium. Incubation with avidin showed near complete inhibition of carboxylation that was restored with the addition of biotin. These results represent strong evidence of a biotin containing acetyl-CoA carboxylase in skeletal muscle.     

Effect of tumor necrosis factor on acetyl-coenzyme A carboxylase gene expression and preadipocyte differentiation

Tumor necrosis factor (TNF) is secreted by macrophages in response to various stimuli and blocks lipid accumulation during the conversion of preadipocytes to adipocytes in culture. In the present report, we investigate the effect of recombinant TNF on the expression of acetyl-coenzyme-A (CoA) carboxylase, the rate-limiting enzyme for long-chain fatty acid biosynthesis. We used a preadipocyte cell line, 30A-5, derived from 10T1/2 mouse fibroblasts after treatment with 5-azacytidine. Treatment of the preadipocyte cell line with dexamethasone and insulin triggers the conversion of these cells to mature adipocytes as evidenced by the accumulation of lipid. The mRNA and enzyme levels of acetyl-CoA carboxylase as well as the enzyme activity increase markedly during the conversion process. TNF prevents the conversion of preadipocytes to adipocytes with a concomitant inhibition in the accumulation of acetyl-CoA carboxylase mRNA and decrease in enzyme activity. This observed reduction in acetyl-CoA carboxylase mRNA levels is reversible upon removal of TNF. Acetyl-CoA carboxylase mRNA levels and enzyme activity also decrease when fully differentiated adipocytes are exposed to TNF but to a much lesser extent. These results suggest that TNF affects de novo lipid synthesis in part by altering the mRNA levels of acetyl-CoA carboxylase.     

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.