Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V_max. of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30–40% in starvation. There was no change in apparent K_m for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3–0.4 and 0.5–0.65μmol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3μmol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44μmol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.     

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.     

Lipid metabolism by rat lung in vitro. Utilization of citrate by normal and starved rats

1. The utilization of [1,5-(14)C(2)]citrate by lung slices and cell cytosol preparations, and the activities of liver and lung cytosol citrate-cleavage enzyme (EC 4.1.3.8), l-malate-NAD oxidoreductase (malate dehydrogenase, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (EC 4.1.1.32) were examined in normal and starved rats. 2. Lipogenesis from citrate was decreased by approx. 70% in both the phospholipid and neutral lipid fractions of lung slices from starved rats as compared with fed controls. 3. Incorporation of citrate by lung cytosol preparations into fatty acids was decreased by approx. 35% in the starved rats. The apparent inhibition by avidin of fatty acid synthesis was overcome partially by preincubation of lung cytosol preparations with biotin. These results are consistent with the presence in lung tissue of the malonyl-CoA pathway for fatty acid synthesis. 4. Lung citrate-cleavage enzyme activity decreased in rats that had been starved for 72h whereas malate dehydrogenase and phosphoenolpyruvate carboxylase activities remained unchanged. The results suggest that the pattern of utilization of lipid precursors by rat lung may be altered during various nutritional states.     

Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes

Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or perfluorosuccinate, but were overcome by α-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of α-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglycerol synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglycerol synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.     

Relationship between the stimulation of citric acid cycle oxidation and the stimulation of fatty acid esterification and inhibition of ketogenesis by lactate in isolated rat hepatocytes.

Isolated hepatocytes from fasted rats were used to study the effects of lactate on palmitate metabolism. Lactate was found to stimulate fatty acid esterification and citric acid cycle oxidation and to inhibit ketone body synthesis. These effects of lactate were largely maintained when gluconeogenesis was inhibited with either quinolinate or perfluorosuccinate, but were overcome by alpha-cyano-4-hydroxycinnamate. However, the responses of hepatocytes to lactate could be restored in the presence of alpha-cyano-4-hydroxycinnamate by the further addition of propionate. The stimulation of triacylglycerol synthesis by lactate was not associated with an increase in the concentration of glycerol 3-phosphate. Rather, there was a correlation between flux through the citric acid cycle and the rate of triacylglycerol synthesis. In all instances reduction of ketone body formation in the presence of lactate was accompanied by a stimulation of citric acid cycle oxidation.     

The role of malate in hormone-induced enhancement of mitochondrial respiration

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

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.     

Dietary protein and the control of fatty acid synthesis in rat adipose tissue

Fatty acid synthesis in adipose tissue normally proceeds at a high rate when fasted animals are refed a diet containing carbohydrate, protein, and low levels of fat. This study investigated the effect of omitting protein from the refeeding diet. Rats were fasted for 48 hr and refed either a protein-free diet or a balanced diet, and the rate of fatty acid synthesis from glucose, pyruvate, lactate, and aspartate was measured. Refeeding the animals a diet devoid of protein resulted in a low rate of fatty acid synthesis from each of these substrates as well as a reduction in carbon flow over the citrate cleavage pathway. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-malate dehydrogenase, and ATP-citrate lyase were also reduced in epididymal fat pads from these rats. On the other hand, adipose tissue phosphoenolpyruvate carboxykinase activity was five times as great as that in tissue from animals refed a balanced diet. This difference could be eliminated if actinomycin D was injected coincident with refeeding. Refeeding rats diets high in carbohydrate is not, therefore, capable of inducing high rates of fatty acid synthesis in adipose tissue in the absence of dietary proteins. Thus, liver and adipose tissue respond differently to dietary protein.     

Modulation by glycerol of hepatic glycero-3-phosphate concentration, ketogenesis, and output of triglyceride and glucose in perfused livers from hyperthyroid and euthyroid rats

Decreased glycero-3-phosphate (glycero-3-P) concentration, decreased output of triglyceride and glucose, increased output of apolipoprotein A-I, and increased ketogenesis were observed with isolated perfused livers from triiodothyronine-treated rats in comparison to livers from euthyroid animals. Infusion of glycerol produced a concentration-dependent accumulation of glycero-3-P in perfused livers from hyperthyroid and euthyroid rats, which was considerably enhanced in the euthyroid group. The antiketogenic effect of glycerol in livers from triiodothyronine-treated rats was accompanied by increased output of glucose and triglyceride, while no change in the output of apolipoprotein A-I was observed. The reduction of ketogenesis (49%) in euthyroid livers by glycerol was not accompanied by increased triglyceride output, while with the largest amount of glycerol infused, decreased output of apolipoprotein A-I was seen. Output of triglycerides by livers from hyperthyroid rats correlated with hepatic concentration of glycero-3-P and was maximal at a glycero-3-P concentration (0.5 mumol/g), similar to that observed in livers from euthyroid rats in the absence of glycerol. Availability of glycero-3-P appears to be rate-limiting for synthesis and secretion of triglyceride by livers from hyperthyroid animals, whereas the glycero-3-P concentrations in euthyroid livers were sufficient to support maximal production of triglyceride limited only by the supply of free fatty acid.     

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.     

Increased activity of glycerol 3-phosphate dehydrogenase and other lipogenic enzymes in human bladder cancer.

Common molecular changes in cancer cells are high carbon flux through the glycolytic pathway and overexpression of fatty acid synthase, a key lipogenic enzyme. Since glycerol 3-phosphate dehydrogenase creates a link between carbohydrates and the lipid metabolism, we have investigated the activity of glycerol 3-phosphate dehydrogenase and various lipogenic enzymes in human bladder cancer. The data presented in this paper indicate that glycerol 3-phosphate dehydrogenase activity in human bladder cancer is significantly higher compared to adjacent non-neoplastic tissue, serving as normal control bladder tissue. Increased glycerol 3-phosphate dehydrogenase activity is accompanied by increased enzyme activity, either directly (fatty acid synthase) or indirectly (through ATP-citrate lyase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and citrate synthase) involved in fatty acid synthesis. Coordinated upregulation of glycerol 3-phosphate dehydrogenase and lipogenic enzymes activities in human bladder cancer suggests that glycerol 3-phosphate dehydrogenase supplies glycerol 3-phosphate for lipid biosynthesis.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.     

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.     

Gluconeogenesis from L-serine in rat liver.

Livers of fed and fasted rats were perfused $\text{in situ}$ in the presence and absence of 4.8 mM quinolinate, an $\text{in vivo}$ inhibitor of phosphoenolpyruvate carboxykinase. An assay of the hepatic activities of serine dehydratase and serine pyruvate transaminase and a comparison of the $\text{in vivo}$ incorporation of radioactivity from serine 3-¹⁴C and serine U-¹⁴C into blood glucose were also carried out in the above nutritional states. Our results demonstrate that gluconeogenesis from L-serine proceeds through two pathways. One, involving the reversal of the biosynthetic route of serine, bypasses conversion to pyruvate phosphoenolpyruvate and oxaloacetate and is not inhibited by quinolinate. This pathway appears to be the only one active in the fed state but produces a very insignificant amount of glucose. The other involves serine dehydratase mediated conversion of serine to pyruvate, is inhibited by quinolinate and becomes predominant during starvation.     

Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO(2) production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77-86% as C(8:0)+C(10:0) acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO(2) production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP(+)-isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP(+)-malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).     

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.     

Effects of thyroid hormones on enzymes involved in fatty acid and glycerolipid synthesis.

The influence of thyroid hormones on lipid biosynthesis was studied after administration of L-thyroxine to rats for 5 days. Their weights remained the same as those of control animals, despite an approximately 3-fold increment in plasma L-thyroxine and L-triiodothyronine concentrations. The activity of acetyl-CoA carboxylase and fatty acid synthetase as well as incorporation of tritium into fatty acids were depressed significantly in epididymal adipose tissue and enhanced significantly in livers of thyroxine-treated rats. Using antibodies specific against rat liver fatty acid synthetase, it was determined that the changes in activity of this multienzymic complex were due to alterations in amount of enzyme protein. In the presence of optimal concentrations of fatty acids, radioactive sn-glycero-3-phosphate, and co-substrates, total glycerolipid synthesis (defined in this study as the sum of newly formed radioactive mono- and diacyl-sn-glycero-3-phosphate, diglyceride, and triglyceride) was decreased significantly in adipose tissue and increased in liver and heart. Thus, administration of thyroid hormone results in tissue-specific alterations in lipid biosynthesis which, at least in the case of fatty acid synthetase, are due to changes in enzyme protein content.