Pharmacological doses of triiodothyronine upregulate lipogenic enzyme gene expression in rat white adipose tissue.

Triiodothyronine (T (3)) is known to increase liver lipogenic enzyme gene expression both in vivo and in tissue culture. Conflicting results have been reported on the effect of T (3) on lipogenic enzyme gene expression in white adipose tissue. The results presented in this paper indicate that administration of pharmacological doses of T (3) in rats leads to increased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL) and malic enzyme (ME) activity in white adipose tissue. The increase in lipogenic enzyme activity was associated with increased FAS, ACC, ACL and ME mRNA levels. The response was dose-dependent. Activity of lipogenic enzyme and the lipogenic enzyme mRNA levels were positively correlated to serum T (3) concentration. The in vivo effect of T (3) on lipogenic enzyme gene expression could be reproduced in primary white rat adipocyte culture. In conclusion, the results presented in this paper indicate that T (3) exerts a stimulatory effect on lipogenic enzyme gene expression in white adipose tissue both in vivo and in tissue culture. Significant effects of T (3) on lipogenic enzyme gene expression were only observed in the presence of relatively high (pharmacological) concentrations of the hormone.     

Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet

We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.     

Lipogenesis at the suckling-weaning transition in liver and brown adipose tissue of the rat

The responses of rat hepatic and brown adipose tissue in vivo lipogenesis to premature (15 days) and normal (21 days) weaning have been correlated to changes in the activities of acetyl-CoA carboxylase and two NADPH-producing enzymes, malic enzyme and glucose-6-phosphate dehydrogenase. Both tissues show an induction of lipogenesis in response to weaning. In the liver, lipogenic flux is closely linked to the activity of acetyl-CoA carboxylase, but not necessarily that of malic enzyme or glucose-6-phosphate dehydrogenase, whereas no such dissociation between enzyme activity and flux rate occurs in brown adipose tissue. Thyroid hormones, implicated in many physiological changes around weaning, do not seem to play a primary role in the adaptation of lipogenesis to the dietary change at this time, although a permissive role in both tissues is possible.     

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.     

Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning

1. The enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, UDP-glucose pyrophosphorylase, phosphofructokinase, ATP-citrate lyase and acetyl-CoA carboxylase have been assayed in rat mammary glands in various stages of involution after hypophysectomy and weaning. 2. After hypophysectomy all seven enzymes decline in activity over a 12–16hr. period but the extent of the decline varies, with acetyl-CoA carboxylase becoming almost totally inactive, ATP-citrate lyase and phosphofructokinase showing a large decrease, and the remaining enzymes a less marked decline. 3. Within 24hr. of removing the litter a change in the pattern of enzyme activity is found very similar to that after hypophysectomy. 4. The significance of these results is discussed in relation to the endocrine control of mammary gland metabolism and the mechanisms of involution.     

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 mm lactate with or without 2 mm glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 mm glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.     

Glucose stimulation of lipogenic enzyme gene expression in cultured white adipose tissue. A role for glucose 6-phosphate.

The expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) is low in the adipose tissue of suckling rats and increases markedly at weaning to a high carbohydrate diet. We have studied in vitro the factors regulating this phenomenon. Inguinal adipose tissue pieces from 19-day-old suckling rats were cultured for 6 or 24 h in minimal essential medium. Insulin (100 nM) added in the presence of lactate and pyruvate did not stimulate the expression of FAS and ACC. Glucose (20 mM) alone resulted in a 5-7-fold increase of FAS and ACC mRNA. Insulin potentiated the effect of glucose. 3-O-Methylglucose, a glucose analog that is transported into the cell but not metabolized, had no effect on FAS and ACC mRNA accumulation. However, 2-deoxyglucose (1 mM), a glucose analog which is phosphorylated to 2-deoxyglucose 6-phosphate, stimulated the expression of FAS and ACC to the same extent as 20 mM glucose. Glucose 6-phosphate concentrations in adipose tissue pieces cultured in various conditions changed in parallel with the FAS and ACC mRNA levels. We conclude that glucose 6-phosphate could be the metabolite involved in the stimulation of lipogenic enzyme gene expression in response to glucose.     

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.     

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.     

Effect of clofibrate on malic enzyme and leptin mRNAs level in rat brown and white adipose tissue.

Two previous studies have reported contradictory results regarding the effect of fibrates treatment on obese (ob) gene expression in rodents. The purpose of the present study was to reinvestigate this issue. We examined the effect of clofibrate (fibrate derivative) administration for 14 days to rats on malic enzyme (as an adequate control of fibrates action) and leptin mRNAs level in the white and brown adipose tissues (WAT and BAT, respectively). The malic enzyme activity and malic enzyme mRNA level in white adipose tissue increased significantly after clofibrate feeding. In brown adipose tissue, the drug treatment resulted in depression of malic enzyme activity and malic enzyme mRNA level. Under the same conditions, leptin mRNA level did not change in these tissues. The results presented in this paper provide further evidence that the clofibrate (activator of peroxisome proliferator activated receptor alpha), feeding is without effect on ob gene expression in rat white and brown adipose tissue. Furthermore, the present study demonstrates that clofibrate causes opposite effects on malic enzyme gene expression in WAT (up-regulation) and BAT (down-regulation).     

Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism

Using primary cultures of adult rat hepatocytes, the regulation of the following lipogenic enzymes was studied: glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. The addition to the culture medium of either insulin or triiodothyronine produced a 2-3-fold increase in each of the individual enzyme activities whereas glucagon slightly decreased enzyme activities. The addition to the medium of 8-bromoguanosine 3,'5'-monophosphate had no effect on any of the enzyme activities unless glucose was also added to the culture medium. Glucose addition alone to the culture medium was without any effect; however, glucose enhanced the stimulation of enzyme activity due to insulin. The addition of fructose or glycerol, even in the absence of insulin, increased the activities of each of the enzymes studied 2-3-fold. The increases in enzyme activity brought about by insulin or fructose were apparently the result of de novo enzyme synthesis, as indicated by the observation that the increases were not noted in the presence of cordycepin or cycloheximide. Immunoprecipitation of ATP-citrate lyase from hepatocytes pulse-labeled with [3H]leucine indicated that the induction of this enzyme in response to the addition of fructose or glycerol to the culture medium was the result of an increase in the rate of synthesis of the enzyme. These results indicate that the activity and synthesis of individual enzymes involved in lipogenesis are increased in response to the metabolism of carbohydrate independently in part from hormonal effects.     

Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat

The effects of vanadate administration on the plasma lipids and hepatic lipogenic enzymes were investigated in Zucker (fa/fa) rat, a model for obesity and non insulin-dependent diabetes. These animals were administered sodium orthovanadate through drinking water for a period of four months. The plasma levels of insulin, triacylglycerols and total cholesterol were significantly (p<0.001) elevated in untreated obese control rats as compared to the lean animals. In the livers of obese rats, the number of insulin receptors decreased by 60% and the activities of lipogenic enzymes acetyl-CoA carboxylase and ATP-citrate lyase increased by 4.7- and 5.6-folds, respectively. The messenger RNA for ATP-citrate lyase as measured by Northern blot analysis showed a parallel increase in obese control rats. Treatment of these rats with vanadate caused 56–77% decreases in the plasma levels of insulin, triacylglycerols and total cholesterol. The insulin receptor numbers in vanadate-treated obese rats increased (119%) compared to levels in untreated obese animals. The elevated activities of acetyl-CoA carboxylase and ATP-citrate lyase observed in livers of obese rats were significantly reduced by vanadate. The messenger RNA for ATP-citrate lyase also decreased in vanadate-treated obese rats back to the lean control levels. This study demonstrates that vanadate exerts potent actions on lipid metabolism in diabetic animals in addition to the recognized effects on glucose homeostasis.     

mRNA levels of SREBP-1c do not coincide with the changes in adipose lipogenic gene expression

Physiological differences in lipid metabolism exist according to adipose sites. To delineate at which step such gene regulation could occur, mRNA levels of various proteins involved in the overall lipogenic process were determined in subcutaneous (SC) and retroperitoneal (RP) adipose tissues. Fatty acid synthase, malic enzyme, ATP citrate lyase, insulin-sensitive glucose transporter, and glucose-6-phosphate dehydrogenase mRNA levels were coordinately reduced (by up to 50-fold) during fasting in RP and in SC relative to fed rats, and restored or overexpressed (by up to 5- to 6-fold) during refeeding. The response was most often delayed and lower in SC compared to RP. This could contribute to site-specific differences. Interestingly, SREBP-1c mRNA levels were markedly decreased by fasting in SC but remained unchanged in RP. Refeeding tended to restore levels close to fed group values. We conclude that mRNA levels of SREBP-1c do not coincide with the expected changes in adipose lipogenic gene expression of fasted/refed rats.     

Modulation of the activity of insulin-dependent enzymes of lipogenesis by glucocorticoids.

Administration of triamcinolone or dexamethasone to rats led to a prompt, marked and persistent rise in liver acetyl-CoA carboxylase activity. The activity of fatty acid synthetase increased to a lesser extent and after a more prolonged glucocorticoid treatment, whereas the changes in that of NADP-malate dehydrogenase and ATP-citrate lyase were not appreciable. The overall channeling of [1-14-C]acetyl-CoA to fatty acids was enhanced. The triamcinolone effect on acetyl-CoA carboxylase activity appeared to be dependent on the coincident hyperinsulinemia since it was not obtained in alloxan-diabetic rats, whereas the alanine-aminotransferase-inducing effect of this hormone was additive to that of insulin deficiency. In adipose tissue triamcinolone treatment caused a reduction in the activity of all lipogenesis enzymes and blunted their response to insulin administration. The antagonism of glucocorticoids toward insulin, selectively modulating the responses of the insulin-sensitive enzymes in liver and adipose tissue is discussed. The rise in hepatic lipogenic capacity, through the retention of the ability of insulin to induce acetyl-CoA carboxylase, may be physiologically important in restraining the ketogenesis from acetyl-CoA despite the increased fat utilization during glucocorticoid excess.     

Up-regulation of stearoyl-CoA desaturase 1 and elongase 6 genes expression in rat lipogenic tissues by chronic food restriction and chronic food restriction/refeeding

Successful treatment of obesity and related diseases by chronic food restriction requires the understanding of the effect of such nutritional therapy on the expression of genes which have been implicated to be involved in some diseases associated with obesity. The purpose of this study was to examine the effect of chronic food restriction and chronic food restriction/refeeding on lipogenic enzymes, especially the expression of genes encoding the stearoyl-CoA desaturase 1 (Scd1) and elongase6 (Elovl6) in rat liver and adipose tissue. We found that both chronic food restriction and chronic food restriction/refeeding caused increased expression of the Scd1 and Elovl6 genes in both the liver and adipose tissue. The increase was more pronounced in case of chronic food restriction/refeeding (several-fold increase) than that in chronic food restriction alone (two to threefold increase). Essentially, similar results were obtained when the expression of fatty acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme genes was studied. Moreover, we found that chronic food restriction and short-term fasting exert opposite effects on the expression of lipogenic enzymes genes. The increased expression of the genes encoding Scd1, Elovl6, and other key lipogenic enzymes may favor fat storage after chronic food restriction/refeeding and may be part of the molecular mechanism by which food restriction/refeeding increases body weight and enhances susceptibility to insulin resistance.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Regulation of glucose-6-phosphate dehydrogenase activity during caloric restriction in human adipose tissue

Glucose-6-phosphate dehydrogenase activity was significantly lower in adipose tissue of human subjects after 7 days of severe caloric restriction on low-carbohydrate diets and had returned to normal values 4 days after the subjects resumed normal diets. Three other enzyme activities (malate-NADP dehydrogenase, oxaloacetate decarboxylating; ATP-citrate lyase and 6-phosphofructokinase) were not significantly affected by these dietary changes. These results are consistent with separate control of glucose-6-phosphate dehydrogenase activity versus other 'lipogenic' enzyme activities in human adipose tissue.     

Long-term consumption of a diet with moderate medium chain triglyceride content does not inhibit the activity of enzymes involved in hepatic lipogenesis in the rat

The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.     

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.