Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

Insulin-like effects of ATP on adipocyte pyruvate dehydrogenase and phosphorylase

Extracellular ATP stimulated adipocyte pyruvate dehydrogenase in a time- and dose-dependent manner with an EC50 of 0.1 mM. The maximal effect was observed at 0.5 mM ATP after a 15-min incubation with a lag period of about 5 min. Depletion of intracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid reduced the effect of ATP by 50% and completely abolished the stimulatory effect of vasopressin on adipocyte pyruvate dehydrogenase but had no effect on the stimulation induced by insulin or adenosine. The effects of insulin and ATP on pyruvate dehydrogenase were glucose-dependent whereas the effect of adenosine was glucose-independent. Furthermore, ATP, like insulin, partially blocked the stimulatory effect of isoproterenol on phosphorylase. Adenosine, at a concentration of 1 mM, did not affect either basal or isoproterenol-stimulated phosphorylase activities. It is concluded that ATP activates adipocyte pyruvate dehydrogenase by at least two separate mechanisms: one is Ca2(+)-dependent and the other is Ca2(+)-independent. However, neither is the result of the formation of adenosine from ATP through hydrolysis.     

Characterization of a pyruvate dehydrogenase modulator purified from insulin-treated rat brain plasma membranes

A factor able to stimulate pyruvate dehydrogenase when added to purified mitochondria was prepared from the supernatant of brain plasma membranes incubated with physiological concentrations of insulin (25 microU/ml). The factor completely reactivated pyruvate dehydrogenase previously inhibited with ATP and was active on pyruvate dehydrogenase from brain and liver mitochondria and from peripheral lymphocytes. The insulin-dependent stimulator of pyruvate dehydrogenase was heat and acid stable, was not absorbed on charcoal and displayed an isoelectric point of 5.5. The insulin mediator was purified by gel filtration, DEAE-cellulose and sulfonated polystyrene chromatography and, after dansylation, by high performance liquid chromatography. The purified mediator displayed a molecular weight of about 2800 and appeared as a peptide rich in glycine and serine and void of proline and sulfur containing aminoacids. It retained its stimulatory action on pyruvate dehydrogenase after dansylation and was completely inactivated by trypsin and chymotrypsin. Full reactivation of ATP-inhibited pyruvate dehydrogenase was attained when mitochondria were incubated with a mediator concentration of about 0.5 microM.     

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.     

Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase

A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 3–4 m, as well as from the hydroxylapatite column at a phosphate concentration of 2–3 m.     

Insulin-dependent production of low-molecular-weight compounds that modify key enzymes in metabolism

The mechanism by which the metabolic effects of insulin are transmitted is yet to be resolved. Second messengers mediating the action of insulin have been proposed and recently an insulin-dependent, peptide-like, heat- and acid-stable substance (Mr approx. 1000-3000) released from plasma membranes has been described which regulates the activity of key enzymes such as pyruvate dehydrogenase by altering its state of phosphorylation. It has been suggested that this material is the elusive second messenger of insulin and its discovery, generation, properties, isolation and mode of action are reviewed.     

Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose.

1. Adipocytes from rat epididymal fat-pads were incubated for 30 min with 5 mM-glucose and concentrations of lactate, pyruvate and amino acids typical of those found in rat plasma. 2. PDHa (active form of pyruvate dehydrogenase) activity was significantly increased after incubation of the cells with insulin (200 micro-i.u./ml), and decreased by incubation with palmitate (0.5--2 mM). 3. In the presence of insulin, palmitate did not decrease PDHa activity. 4. Dichloroacetate (1 mM) increased PDHa activity in the absence of palmitate to the same extent as did insulin. In the presence of dichloroacetate but the absence of insulin, palmitate decreased PDHa activity. In the presence of dichloroacetate and insulin, palmitate again did not decrease PDHa activity. 5. It is concluded that, in the presence of glucose, insulin has a strong protective action against inactivation of adipocyte PDHa by fatty acids.     

Insulin resistance in the liver in fasting and diabetes mellitus: the failure of insulin to stimulate the release of a chemical modulator of pyruvate dehydrogenase

To further define the mechanism(s) of insulin resistance in the liver associated with diabetes and fasting, we evaluated the ability of insulin to release an activator of pyruvate dehydrogenase activity from a liver particulate fraction. Insulin reproduceably and significantly enhanced the release of mediator from the liver particulate fraction of control animals. The particulate fractions from fasted and diabetic animals were resistant to this effect of insulin. Refeeding and insulin treatment, respectively, restored responsiveness to insulin. These data support the concept that alterations at or near the plasma membrane can be responsible for or accompany the insulin resistance observed in the liver in fasting and diabetes mellitus.     

Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N6-phenylisopropyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.     

Release of PAI-1 by human preadipocytes and adipocytes independent of insulin and IGF-1

The effect of insulin and insulin-like growth factor-1 (IGF-1) on plasminogen activator inhibitor-1 (PAI-1) release from primary cultures of human preadipocytes and adipocytes has been investigated. Initial experiments measuring basal PAI-1 release (ng/ml) indicated variability between individual cultures. Using a novel technique for adipocyte quantitation, additional experiments were performed to determine PAI-1 release per cell, indicating a significant reduction with differentiation. Insulin and IGF-1 over a range of concentrations had no effect on PAI-1 release, and RT-PCR of PAI-1 mRNA following treatment with insulin and IGF-1 also indicated similar expression between treatments. The cultures did exhibit insulin-stimulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and leptin synthesis following differentiation to the adipocyte phenotype. This is the first report of PAI-1 secretion by primary cultures of human preadipocytes and adipocytes, indicating PAI-1 release independent of insulin and IGF-1 and implicating other factors in the elevated plasma PAI-1 observed with insulin resistance.     

Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.     

Autophosphorylation of the insulin receptor in rat adipocytes is modulated by thyroid hormone status

The effect of thyroid status on the in vitro autophosphorylation of the insulin receptors was studied in triton-solubilized adipocyte plasma membranes obtained from normal and thyroidectomized rats. Thyroidectomy results in an increase (two to three times) of the in vitro insulin-dependent phosphorylation of the insulin beta-subunit receptor. Phosphorylation occurred on tyrosine residues. In vivo injection of triiodothyronine to thyroidectomized rats restored plasma membranes autophosphorylation of the beta-subunit to the values obtained for control euthyroid rats. This effect was independent of the number and affinity of the insulin receptors, which were not modified regardless of thyroid status.     

Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

Biochemical analysis of triggering signals induced by bridging of IgE receptors

Bridging of IgE receptors on rat mast cell plasma membranes induces phospholipid methylation and a monophasic increase in cyclic AMP. The stimulation of phospholipid methylation in the plasma membrane appears to be intrinsic to the processes leading to Ca2+ influx and histamine release. Evidence was obtained that IgE receptors are closely associated with methyltransferases and adenylate cyclase in the plasma membranes. The activation of one enzyme is regulated by the other. An increase in the cyclic AMP level before receptor bridging suppressed phospholipid methylation. On the other hand, inhibition of phospholipid methylation may affect the initial rise in cyclic AMP. Our experiments also indicated that bridging the receptor activates a membrane-associated proteolytic enzyme. Inasmuch as the inhibition of the enzyme activation results in the suppression of both phospholipid methylation and initial rise in cyclic AMP induced by receptor bridging, the proteolytic enzyme may be involved in the activation of methyltransferases and adenylate cyclase.     

Activation of pyruvate dehydrogenase by addition of insulin to a mixture of cell membranes and mitochondria from the brain of normal and alloxan treated rats. (Preliminary note)

The effect of insulin and ATP on pyruvate dehydrogenase activity has been studied in a mixture of plasma membranes/mitochondria from normal and diabetic rat brain. In both enzymatic preparations ATP inhibits almost completely pyruvate dehydrogenase activity; insulin on the contrary induces a conspicuous activation and removes the inhibiting action of ATP.     

The generation of inositolglycan mediators from rat liver plasma membranes: the role of guanine nucleotide binding proteins.

The guanine nucleotide dependence for the generation of inositolglycan second messengers from rat liver plasma membranes has been investigated. Plasma membranes, when treated with insulin release a soluble mediator substance which activates pyruvate dehydrogenase (PDH). Guanosine 5'-[3-thio]triphosphate (GTP gamma S) was found to be as potent as insulin in stimulating mediator release. The stimulatory effects of GTP gamma S required the presence of magnesium and following preincubation of membranes with guanosine 5'-[2-thio]diphosphate (GDP beta S) the stimulation of mediator release by either insulin or GTP gamma S was blocked. The activation of PDH by mediator fractions produced in response to either insulin or GTP gamma S was abolished following treatment of the fractions with anti-inositolglycan antibodies. The significance of these observations with respect to the possible involvement of a regulatory guanine-nucleotide binding protein (G-protein) in the generation of insulin mediators is discussed.     

Putative mediators of insulin action regulate hepatic acetyl CoA carboxylase activity

Incubation of rat liver plasma membranes with insulin enhances the production of small molecular weight substances which regulate the activity of liver acetyl CoA carboxylase. While low concentrations of insulin cause the release of a carboxylase stimulator from membranes, concentrations greater than 10^?9 M generate less stimulating activity. This biphasic concentration curve for insulin can be resolved by differential alcohol extraction into two fractions which have antagonistic activity. The production of both substances is enhanced by insulin. Chemical and chromatographic evidence suggest that these substances are identical to the previously described “mediators” which regulate both pyruvate dehydrogenase and adenylate cyclase activities.     

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.     

Fasting induced alterations in mitochondrial palmitoyl-CoA metabolism may inhibit adipocyte pyruvate dehydrogenase activity.

1. Adipocytes from fed and fasted (24 hr) groups of rats were fractionated into mitochondria, microsomes and plasma membranes. 2. Fasting significantly decreased the mitochondrial activity of palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, beta-oxidation and pyruvate dehydrogenase. 3. Fasting elevated intramitochondrial long-chain acyl-CoA. 4. Pyruvate dehydrogenase was inhibited 50% by addition of 30 microM palmitoyl-CoA. 5. Fasting-induced changes in palmitoyl-CoA metabolism may modulate pyruvate dehydrogenase activity in adipocyte mitochondria.     

Arachidonic acid and glucose uptake by freshly isolated human adipocytes

Fatty acid (FA) and glucose transport into insulin-dependent cells are impaired in insulin resistance (IR; type 2 diabetes mellitus). Studies done on the effects of FAs on glucose uptake, and the influence of insulin on FA uptake by adipocytes, have yielded contradictory results. In this study, isolated human adipocytes were exposed to arachidonic acid (AA) and to insulin, and FA uptake as well as glucose uptake was measured. AA uptake into adipocyte membranes and nuclei was also investigated. Glucose uptake was inhibited by 57 +/- 8% after 30 min of exposure to arachidonate. AA was significantly taken up into adipocyte membranes (49.6 +/- 29% and 123 +/- 74%) at 20 and 30 min of exposure, respectively, and into nuclei (147.6 +/- 19.2%) after 30 min. Insulin stimulated AA uptake (24.1 +/- 14.1%) at 30 min by adipocytes from a non-obese subject, while inhibiting it (16.6 +/- 12%) in adipocytes from an obese subject. These results suggest that: (1) AA inhibits glucose uptake by adipocytes exposed over a short period, probably by a membrane-associated mechanism, (2) insulin-dependent AA uptake is dependent on the body mass index (BMI) of the donor and the insulin sensitivity of their adipocytes.