Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.     

Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase.

1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.     

Glutamine metabolism in isolated incubated adipocytes of the rat.

1. Phosphate-dependent glutaminase activity in the epididymal fat-pad was 15.1 nmol/min per mg of protein. Glutaminase activity demonstrated differences with respect to adipose-tissue sites. Considerable variation was found in different sites of adipose tissue from lean control and Zucker obese animals. 2. Adipocytes incubated in the presence of 2 mM-glutamine utilized glutamine at a rate of 1.8 mumol/h per g dry wt., and glutamate, ammonia, lactate and alanine were produced. Addition of glucose plus insulin increased the rates of glutamine utilization and glutamate, ammonia, lactate and alanine production. Isoprenaline alone or plus glucose further stimulated the rate of glutamine utilization and formation of end products. 3. The rate of incorporation of 14C from glutamine into CO2 was similar to that of glucose, but the rate of incorporation into triacylglycerol was much less. Addition of unlabelled glucose or glucose plus insulin stimulated the rate of incorporation of [14C]glutamine into triacylglycerol, but had no effect on that of 14CO2 formation. Isoprenaline plus glucose increased the rate of incorporation of [14C]glutamine into CO2, but decreased the rate of incorporation into triacylglycerol. 4. In the absence of insulin, the rate of [14C]glutamine incorporation into triacylglycerol was related to the glucose concentration (0-10 mM). However, in the presence of insulin, the rate of incorporation of [14C]glutamine was maximal at 1 mM-glucose.     

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.     

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.     

Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

Altered lipolytic response to glucagon and adenosine deaminase in adipocytes from starved rats.

1. Adipocytes isolated from epididymal adipose tissue of fed or 24 h-starved rats were incubated with a range of glucagon concentrations in the presence and absence of adenosine deaminase (4 munits/ml). 2. With adenosine deaminase present, the lipolytic response to low concentrations of glucagon (1-6 ng/ml) was considerably enhanced in cells from starved rats. 3. The effect of adenosine deaminase on basal lipolysis was altered after starvation. 4. D-3-Hydroxybutyrate (5 mM) decreased the sensitivity of lipolysis to glucagon. 5. The possible involvement of glucagon-stimulated lipolysis in the regulation of ketogenesis is briefly discussed.     

Regulation of pyruvate dehydrogenase activity in rat epididymal fat-pads and isolated adipocytes by adrenaline.

1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.     

Stimulation by a tumor-promoting phorbol ester of acetyl-CoA carboxylase activity in isolated rat hepatocytes

Acetyl-CoA carboxylase (EC 6.4.1.2) in hepatocytes from meal-fed rats was activated by phorbol myristate acetate (PMA) in a time- and concentration-dependent fashion. This activation can account for the PMA-induced stimulation of de novo fatty acid synthesis. Purified rat-liver acetyl-CoA carboxylase was found to be phosphorylated and activated by protein kinase C, thus providing a possible mechanism for the metabolic action of PMA in intact hepatocytes.     

Modulation by insulin and glucagon of noradrenaline-induced activation of isolated brown adipocytes from the rat

1. The effects of insulin (2 nM and 4 nM) upon oxygen consumption (VO2), lipolysis rates and indirectly derived rates of fatty acid utilization, by isolated brown adipocytes from warm-acclimated (W cells) and cold-acclimated (C cells) animals, induced by noradrenaline and glucagon separately and conjointly, are reported. 2. Changes in interrelationships (coupling) between the parameters under different treatment regimes were assessed using bivariate regression analyses. 3. Administration of glucagon with noradrenaline increased lipolysis/fatty acid utilization coupling without concomitant increase of VO2 suggesting that glucagon may increase re-esterification through glycogenolytic generation of glycerol 3-phosphate, trapping intracellular fatty acid in excess of the capacity of disposal mechanisms, thus conserving respiratory substrate. 4. W cells were unresponsive to glucagon in terms of lipolysis and VO2, C cells responded to glucagon with parallel increases in lipolysis rate and VO2. Both cell types responded to noradrenaline alone and conjointly with glucagon; C cells were more sensitive to these agonists than W cells. 5. Lipolysis/VO2 coupling was reduced in C cells suggesting that in cold acclimation, noradrenaline-induced lipolysis rates are in excess of the capacity of cellular oxidation/re-esterification mechanisms. 6. Insulin inhibited noradrenaline and glucagon-induced lipolysis, simultaneously increasing VO2, supporting the hypothesis that glucose may be a thermogenic substrate in brown adipase tissue, permitting concurrent thermogenesis and lipogenesis. C cells were more insulin-sensitive than W cells. 7. The data indicate that insulin may mediate its effects (additively with noradrenaline) by activation of pyruvate dehydrogenase, generating glycolytic flux and, in the presence of noradrenaline-inhibited lipogenesis, generate additional oxaloacetate, permitting increased beta-oxidation.     

Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity

The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon.     

Phosphatase activity in rat adipocytes: effects of insulin and insulin resistance

Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.     

Adrenalectomy-induced alterations in glucagon binding and lipolysis in isolated rat adipocytes.

Adipocytes from adrenalectomized rats nearly lost their lipolytic response to glucagon concomitant with a 90% decrease in the number of glucagon receptors per cell. Quantitative analysis of the relation between amount of cell-bound glucagon and hormone-stimulated lipolysis revealed that the ability of the remaining 10% of glucagon receptors to induce lipolysis was not impaired. Binding of the beta-adrenergic antagonist [3H]dihydroalprenolol and maximal lipolysis induced by (-)-isoproterenol, (Bu)2cAMP, 3-isobutyl-1-methylxanthine, and adenosine deaminase were reduced only 10 to 20% after adrenalectomy. Furthermore, glucagon-stimulated cAMP production was greatly decreased in adrenalectomized animals, but isoproterenol-stimulated cAMP production was not. Hydrocortisone replacement in adrenalectomized rats only partially prevented the loss of glucagon receptors and glucagon effects on both cAMP production and lipolysis. These findings suggest that lipolytic cascade distal to hormone receptors was not greatly impaired in adipocytes after adrenalectomy and that the unresponsiveness of these cells to glucagon was mostly due to a marked reduction in the number of glucagon receptors.     

Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes.

1. In isolated rat adipocytes, acetyl-CoA carboxylase is inactivated by treatment of the cells with adrenaline or the beta-agonist isoproterenol, but not by the alpha-agonist phenylephrine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30-40% increase in the labelling of enzyme isolated from 32P-labelled cells. 2. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser-Ser77-Met-Ser79-Gly-Leu-His-Leu-Val-Lys, containing Ser-77 (phosphorylated by cyclic-AMP-dependent protein kinase) and Ser-79 (phosphorylated by the AMP-activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser-79 in both basal and hormone- or agonist-stimulated cells. Treatment of adipocytes with various agents which activate cyclic-AMP-dependent protein kinase by receptor-independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl-CoA carboxylase and increased phosphorylation at Ser-79. 3. The (Rp)-[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic-AMP-dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl-CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic-AMP-dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl-CoA carboxylase. 4. As shown by okadaic acid inhibition, greater than 95% of the acetyl-CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase-2A, with less than 5% attributable to protein phosphatase-1. Inhibition of protein phosphatase-1 via phosphorylation of inhibitor-1 is therefore unlikely to be the mechanism by which cyclic-AMP-dependent protein kinase indirectly increases phosphorylation of acetyl-CoA carboxylase. Various other potential mechanisms are discussed.     

The effects of clofibrate on acetyl-CoA carboxylase activity in hepatocytes

Summary Cytochemical localization techniques and electron microscopy were used to evaluate the effects of clofibrate on acetyl-CoA carboxylase activity. It was demonstrated that the drug inhibited the activity of acetyl-CoA carboxylase in rat hepatocytes. Although the results in one of these experiments were somewhat variable, it is suggested that the inhibition of acetyl-CoA carboxylase may be the mechanism by which clofibrate exerts its hypolipidemic effects.Research supported by USPHS Grants HE 12751, NS 05665 and 00690.Recipient of Career Research Development Award 1K3 GM 28064. The authors would like to express their appreciation to Richard L. Hogg of the Ayerst Laboratories for supplying the drug.     

Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.