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.     

Deficiency in hepatic ATP-citrate lyase affects VLDL-triglyceride mobilization and liver fatty acid composition in mice

ATP-citrate lyase (ACL) is a key lipogenic enzyme that converts citrate in the cytoplasm to acetyl-CoA, the initial precursor that yields malonyl-CoA for fatty acid biosynthesis. As cytosolic citrate is derived from the tricarboxylic acid cycle in the mitochondrion, ACL catalyzes a critical reaction linking cellular glucose catabolism and lipid synthesis. To investigate the metabolic action of ACL in lipid homeostasis, we specifically knocked down hepatic ACL expression by adenovirus-mediated RNA interference in mice maintained on a low-fat or high-fat diet. Hepatic ACL abrogation markedly reduced the liver abundance of both acetyl-CoA and malonyl-CoA regardless of dietary fat intake, which was paralleled with decreases in circulating levels of triglycerides and free fatty acids. Moreover, hepatic ACL knockdown resulted in diet-dependent changes in the expression of other lipogenic enzymes, accompanied by altered fatty acid compositions in the liver. Interestingly, ACL deficiency led to reduced serum VLDL-triglyceride levels but increased hepatic triglyceride content, resulting at least partially from decreased hepatic secretion of VLDL-containing apolipoprotein B-48. Together, these results demonstrate that hepatic ACL suppression exerts profound effects on triglyceride mobilization as well as fatty acid compositions in the liver, suggesting an important role for ACL in lipid metabolism.     

The influence of diet on the lipogenic response to thyroxine in rat liver.

ATP citrate lyase, acetyl-CoA synthetase, malic enzyme and hexose monophosphate dehydrogenase activities and rates of $\text{denovo}$ synthesis of long chain fatty acids from labeled acetate and citrate were measured in cell-free fractions of liver from rats fed various diets, with and without D- or L- thyroxine. Diets containign sucrose (vs. isocaloric glucose) or lard (vs. isocaloric corn oil) stimulated hepatic lipogenesis both in control and in thyroxine-treated rats. The lipogenic response to thyroxine was greatly modified by diet, except for an invariable rise in malic enzyme activity. With diets providing less than 6% of calories as linoleic acid, thyroxine increased fatty acid synthesis, depleted liver glycogen and retarded growth; when linoleic acid was increased to 16% of calories, thyroxine had no effect on fatty acid synthesis or growth and liver glycogen depletion was significantly attenuated. This response to dietary linoleic acid suggests that these phenomena may be largely secondary to the increased requirement for essential fatty acid in thyrotoxicosis. Further study should reveal the extent to which observed effects of excess thyroid hormone are amenable to control by dietary polyunsaturated fat.     

Dependence of ATP citrate lyase activity on cell density of isolated rat hepatocytes

The activities of two enzymes involved in the lipogenic process, ATP citrate lyase and NADP-linked malic enzyme were evaluated as a function of cell density in isolated rat hepatocytes. The activity of ATP citrate lyase was profoundly affected by cell density with the activity/cell being higher at low cell densities than at high cell densities. Malic enzyme was not similarly affected, nor was cellular ATP content. The effect was observed regardless of dietary state but was most dramatic with hepatocytes from fasted-refed rats. Both an activator and an inhibitor of ATP citrate lyase have been isolated from conditioned medium from cells at low density and at high density, respectively. The activator fraction was heat stable while the inhibitor fraction was heat labile, and both factors had molecular weights in excess of 10,000 daltons.     

Biochemical and ultrastructural hepatic changes during vitamin B12 deficiency in animals and man.

Vitamin B12 deficiency has been shown to result in an increase in content and activity of the hepatic cytosolic enzymes of fatty acid synthesis. The present study demonstrated that ATP citrate lyase, an enzyme whose activity has been positively correlated with rates of fatty acid biosynthesis, also increased in the livers of B12-deficient animals. Total and specific activity of hepatic citrate synthase, an enzyme whose activity is unaffected by a variety of dietary and hormonal changes, also was found to be increased in the B12-deprived state. By contrast, the activity of hepatic succinate-cytochrome c reductase, a portion of a multicomponent enzyme complex synthesized in part within the mitochondria, was unchanged in B12 deficiency. Vitamin B12 deprivation resulted in an increase in hepatic mitochondrial cristae membranes in both animals and man. Histochemical and chemical analysis demonstrated increased glycogen in the liver cells from B12-deficient animals and man. Thus, in the livers from vitamin B12-deficient animals there is an increased activity of the otherwise highly constant Krebs cycle enzyme citrate synthase, and in both animals and man there are increased mitochondrial cristae membranes.     

Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats

Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.     

Citrate and the conversion of carbohydrate into fat. Citrate cleavage in obesity and lactation

1. The specific activity of ATP citrate lyase is two to four times as great in livers of mice with hereditary obesity as in livers of their non-obese siblings. The enzyme activity in both types of mice can be reduced by starvation, and can be increased by refeeding starved animals. The specific activity of acetyl-CoA synthetase is approximately the same in both types of mice. 2. ATP citrate lyase of mammary gland of the rat undergoes large increases in activity after the onset of lactation. It declines rapidly upon weaning. 3. The changes in activity of ATP citrate lyase in obesity and lactation are consistent with the hypothesis that the enzyme supplies extramitochondrial acetyl-CoA for fatty acid synthesis.     

Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes

To elucidate the physiological role of sterol regulatory element-binding protein-1 (SREBP-1), the hepatic mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all lipogenic enzymes were consistently low in both wild-type and SREBP-1(-/-) mice. However, the absence of SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and S14 mRNAs, were completely abolished in SREBP-1(-/-) mice. In contrast, mRNA levels for cholesterol biosynthetic genes were elevated in the refed SREBP-1(-/-) livers accompanied by an increase in nuclear SREBP-2 protein. When fed a high carbohydrate diet for 14 days, the mRNA levels for these lipogenic enzymes were also strikingly lower in SREBP-1(-/-) mice than those in wild-type mice. These data demonstrate that SREBP-1 plays a crucial role in the induction of lipogenesis but not cholesterol biosynthesis in liver when excess energy by carbohydrates is consumed.     

Coordinate control of rat liver lipogenic enzymes by insulin

Recent evidence has established that insulin is required for the dietary induction of rat liver fatty acid synthetase [Proc. Nat. Acad. Sci. USA69, 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 prolonged ethanol ingestion on hepatic lipogenesis and related enzyme activities

1. Hepatic lipogenesis in vivo and the activities of enzymes associated with fatty acid synthesis in the liver were studied in rats fed for 21 days on liquid diets containing ethanol. 2. The ethanol-fed rats developed a moderate hepatic triacylglycerol accumulation during this period. When carbohydrate was replaced by ethanol in the diet, the rate of fatty acid synthesis was slower in the ethanol-fed rats on low-, medium- and high-fat diets than in the appropriate controls. However, when the fat/carbohydrate ratio was kept the same in the ethanol-fed and control rats, ethanol had no influence on the rate of fatty acid synthesis. 3. Glucose 6-phosphate dehydrogenase activity was lower in the ethanol-fed group. ;Malic' enzyme activity did not change during the ethanol treatment when the fat/carbohydrate ratio was kept unchanged. 4. The ATP citrate lyase activity was lower in the ethanol-fed rats on all diets, whereas acetyl-CoA synthetase activity was independent of the composition of the control diet, but was lower in the ethanol-fed rats, in which the concentration of the active form of pyruvate dehydrogenase was also lower. 5. It is concluded that hepatic fatty acid synthesis does not play any major role in ethanol-induced triacylglycerol accumulation. Careful design of the diets is necessary to reveal the specific effects of ethanol on the enzymes associated with lipogenesis.     

Calcitonin and hepatic fatty acid synthesis: effect of thyroparathyroidectomy on the elevation of ATP citrate lyase activity by refeeding of starved rats

The effect of thyroparathyroidectomy (TPTX) on ATP citrate lyase regulation, a rate-limiting enzyme of fatty acid synthesis in hepatic cytosol, was investigated in rats refed after a 24 h fast. ATP citrate lyase activity in the hepatic cytosol was increased 2-fold by refeeding. This increase was suppressed about 50% by TPTX. The suppression of the enzyme activity by TPTX was completely restored by administration of calcitonin (CT; 80 MRC mU/100 g body weight). This hormonal effect was also observed at 20 MRC mU/100 g dose of CT. CT administration to refeeding-TPTX rats produced a significant increase in the calcium content of the liver tissue and the cytosol. The cytosolic ATP citrate lyase activity increase with CT administration was completely blocked by treatment of cytosol with EGTA (10 microM). This inhibition was clearly reversed by addition of calcium ion (1.25-5.0 microM). In addition, CT-induced rise in enzyme activity was markedly reduced by the presence of W-7 (5 and 50 microM), a calmodulin inhibitor, in the enzyme assay system. The present results suggest that CT plays a role in the elevation of hepatic ATP citrate lyase activity brought about by refeeding of fasted rats, and that this hormonal regulation might depend on Ca2+-calmodulin.     

Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells

The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.     

Role of pyruvate carboxylase in fatty acid synthesis: Alterations during preadipocyte differentiation

Transport of mitochondrial acetyl units to the cytoplasm for fatty acid synthesis via the citrate cleavage pathway requires replenishment of mitochondrial oxaloacetate. Pyruvate carboxylase is though to fulfill this role although compelling evidence has been lacking. During lipogenic differentiation of 3T3-L1 preadipocytes, pyruvate carboxylase activity rises 18-fold in close coordination with fat accumulation and the activity of ATP-citrate lyase, an established lipogenic enzyme. The activities of enzymes less directly related to lipogenesis rise only 3–5-fold while other unrelated enzymes do not increase significantly. These results indicate that pyruvate carboxylase is in fact a lipogenic enzyme.     

On the significance of the prosthetic group composition of citrate lyase.

1. Klebsiella aerogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase. 2. The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes. 3. As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes. 4. Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes. 5. The prosthetic group of citrate lyase, phosphoribosyl dephospho-CoA, apparently represents no incidental, phosphopantetheine-replacing aberration. The requirement of citrate lyase for the CoA-like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.     

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.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

The effects of estradiol administration of the hepatic activities of some enzymes of carbohydrate, amino acid and lipid metabolism in the immature pullet.

Two experiments were performed to examine the effects of intramuscular estradiol administration on the hepatic specific activities of some enzymes of lipid, carbohydrate and amino acid metabolism in the immature fowl. Estradiol increased the specific activities of the hepatic lipogenic enzymes, ATP citrate lyase and malate dehydrogenase (decarboxylating) (NADP), but had no effects on the activities of the glycolytic, gluconeogenic and amino acid metabolising enzymes except for pyruvate kinase and glutamate dehydrogenase which were reduced in activity in both experiments. The results indicate that the estrogen-induced increase in hepatic lipid biosynthesis is due to a specific effect on lipid metabolism and not to a general increase in liver metabolism.     

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.     

The lipogenic enzyme acetoacetyl-CoA synthetase and ketone body utilization for denovo lipid synthesis,a review

Acetoacetyl-CoA synthetase (AACS) is the key enzyme in the anabolic utilization of ketone bodies (KBs) for denovo lipid synthesis, a process that bypasses citrate and ATP citrate lyase. This review shows that AACS is a highly regulated, cytosolic, and lipogenic enzyme and that many tissues can readily use KBs for denovo lipid synthesis. AACS has a low micromolar K_m for acetoacetate, and supply of acetoacetate should not limit its activity in the fed state. In many tissues, AACS appears to be regulated in conjunction with the need for cholesterol, but in adipose tissue, it seems tied to fatty acid synthesis. KBs are readily utilized as substrates for lipid synthesis in lipogenic tissues, including liver, adipose tissue, lactating mammary gland, skin, intestinal mucosa, adrenals, and developing brain. In numerous studied cases, KBs served several-fold better than glucose as substrates for lipid synthesis, and when present, KBs suppressed the utilization of glucose for lipid synthesis. Here, it is hypothesized that a physiological role for the utilization of KBs for lipid synthesis is a metabolic process of lipid interconversion. Fatty acids are converted to KBs in liver, and then, the KBs are utilized to synthesize cholesterol and other long-chain fatty acids in liver and nonhepatic tissues. The conversion of fatty acids to cholesterol via the KBs may be a particularly important example of lipid interconversion. Utilizing KBs for lipid synthesis is glucose sparing and probably is important with low carbohydrate diets. Metabolic situations and tissues where this pathway may be important are discussed.     

Levels of mRNAs coding for lipogenic enzymes in rat lung upon fasting and refeeding and during perinatal development

The relative amounts of mRNAs coding for fatty-acid synthase (EC 2.3.1.85), acetyl-CoA carboxylase (EC 6.4.1.2), ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) were determined in lungs and livers of adult rats that were normally fed, starved for 48 h or starved for 48 h and subsequently refed for 72 h with a carbohydrate-rich, fat-free diet. In the liver, starvation caused a small decrease in the relative abundance of the mRNAs which was not statistically significant. Subsequent refeeding caused a statistically significant increase in mRNAs for all of the enzymes studied. In the lung, no significant changes were found, indicating that the regulation of the abundance of mRNAs encoding the lipogenic enzymes in the lung differs from that in the liver. In the developing rat lung, mRNA for fatty-acid synthase increased 3-fold in abundance between fetal days 18 and 20 and decreased directly after birth (at day 22 of gestation). A similar pattern was observed for ATP citrate lyase mRNA. The level of acetyl-CoA carboxylase mRNA decreased significantly after birth. These observations indicate that in perinatal rat lungs, pretranslational regulation is involved in the control of the synthesis of these enzymes. The abundance of acetyl-CoA carboxylase mRNA did not change in the prenatal period, a time during which the specific activity of this enzyme increases. This lack of correlation between the specific activity of acetyl-CoA carboxylase and the abundance of its mRNA may indicate that translational regulation of the synthesis of the enzyme or post-synthetic regulatory effects on enzyme molecules are involved in the control of this enzyme in the prenatal period. No changes in the abundance of lung malic enzyme mRNAs were observed throughout the perinatal period.