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.     

Effect of adenosine on insulin activation of rat adipocyte pyruvate dehydrogenase

Adenosine and its analogue N6-phenylisopropyladenosine stimulated pyruvate dehydrogenase activity of isolated rat adipocytes. Maximal stimulation was obtained with concentrations between 50 and 100 mu M, with the effect decreasing at higher concentrations. The effects of insulin on this enzyme was modified by adenosine. The concentration of insulin (10 mu units/ml) that produced almost half-maximal stimulation, had little or no effect, when adenosine deaminase was present. Adenosine also enhanced the effect of suboptimal but not optimal concentrations of insulin. Thus, the mechanism of adenosine action on adipocyte pyruvate dehydrogenase could in some way be similar or related to that of insulin.     

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+.     

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. Relationship to the metabolic effects of insulin and insulin-like agents.

Exposure to phospholipase C increased the incorporation of [32P]Pi into phosphatidate, CMP-phosphatidate and phosphatidylinositol in rat adipose tissue and isolated adipocytes. A similar effect was observed in response to insulin and oxytocin. Theophylline, 3-isobutyl-1-methylxanthine and adenosine deaminase decreased [32P]Pi incorporation, and adenosine and N6-phenylisopropyladenosine reversed these effects. As with insulin, exposure of adipose tissue to phospholipase C stimulated oxidation of glucose, pyruvate and leucine and activated pyruvate dehydrogenase. Oxytocin and adenosine also mimicked the effects of insulin on leucine oxidation and pyruvate dehydrogenase. However, only insulin stimulated glycogen synthase activity, indicating that the regulation of synthase may be achieved by intracellular events distinct from those regulating changes in phospholipid metabolism, sugar transport and mitochondrial enzyme activities. It is postulated that exposure to phospholipase C forms diacylglycerol, which is phosphorylated to yield phosphatidate. The increased labelling of CMP-phosphatidate and phosphatidylinositol results from the conversion of phosphatidate into these lipids. The correlation between the effects of phospholipase C on phosphatidate synthesis and changes in adipose-tissue metabolism suggests the possibility that increased phosphatidate may directly or indirectly produce changes in membrane transport and enzyme activities. The pattern of phospholipid labelling produced by insulin, adenosine and oxytocin suggests that these stimuli may also increase phosphatidate synthesis, and, if so, changes in phospholipid metabolism could account for some of the metabolic actions of these stimuli.     

Mechanisms regulating adipose tissue pyruvate dehydrogenase

1. Isolated rat epididymal fat-cell mitochondria showed an inverse relationship between ATP content and pyruvate dehydrogenase activity consistent with competitive inhibition of pyruvate dehydrogenase kinase by ADP. At constant ATP concentration pyruvate rapidly activated pyruvate dehydrogenase in fat-cell mitochondria, an observation consistent with inhibition of fat-cell pyruvate dehydrogenase kinase by pyruvate. Pyruvate dehydrogenase in fat-cell mitochondria was also activated by nicotinate (100mum) and by extramitochondrial Na(+) (replacing K(+)) but not by ouabain or insulin. 2. In rat epididymal fat-pads incubated in vitro pyruvate dehydrogenase was activated by addition of insulin in the absence of substrate or in the presence of glucose (10mm) or fructose (10mm). Glucose and fructose activated the dehydrogenase in the absence or in the presence of insulin, and pyruvate also activated in the absence of insulin. It is concluded that extracellular glucose, fructose and pyruvate may activate the dehydrogenase by raising intracellular pyruvate and that insulin may activate the dehydrogenase by some other mechanism. 3. Ouabain (300mum) and medium in which K(+) was replaced by Na(+), activated pyruvate dehydrogenase in epididymal fat-pads. Prostaglandin E(1) (1mug/ml), 5-methylpyrazole-3-carboxylate (10mum) and nicotinate (10mum), which are as effective as insulin as inhibitors of lipolysis and which like insulin lower tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate), did not activate pyruvate dehydrogenase. Higher concentrations of prostaglandin E(1) (10mug/ml) and nicotinate (100mum) produced some activation of the dehydrogenase. 4. It is concluded that the activation of pyruvate dehydrogenase by insulin is not due to the antilipolytic effect of the hormone and that the action of insulin in lowering adipose-cell concentrations of cyclic AMP does not afford an obvious explanation for the effect of the hormone on pyruvate dehydrogenase. The possibility that the effects of insulin, ouabain and K(+)-free medium may be mediated by Ca(2+) is discussed.     

Effect of S-adenosylhomocysteine on insulin-independent release of pyruvate dehydrogenase activator from rat adipocyte plasma membranes

The addition of insulin to adipocyte plasma membranes has been shown to release a low molecular weight, acid stable mediator which activates mitochondrial pyruvate dehydrogenase.The insulin-dependent release of this activator is dependent on the method used to prepare the plasma membranes. Adipocyte plasma membranes prepared in 0.25 M sucrose, 10 mM MOPS, pH 7.4 released an activator of pyruvate dehydrogenase in an insulin-independent manner. Insulin is required to stimulate phospholipid methylation in these membranes. The inhibition of insulin-stimulated phospholipid methylation with 1 mM S-adenosylhomocysteine resulted in a significant increase in amount and/or activity of the pyruvate dehydrogenase activator. The insulin-dependent dependent release of mediators of insulin action from adipocyte plasma may be regulated by phospholipid methylation.     

Insulin stimulates the release from liver plasma membranes of a chemical modulator of pyruvate dehydrogenase

Incubation of a rat liver particulate fraction with physiological concentrations of insulin enhances the production of a small molecular weight substance which modulates adipocyte as well as liver mitochondrial pyruvate dehydrogenase. While low concentrations of insulin enhance production of this activity, levels of greater than 10^?9M produce significantly less. Similarly, while increasing concentrations of mediator cause increased stimulation of pyruvate dehydrogenase activity, higher concentrations no longer exhibit this effect. The putative insulin mediator was partially purified on HPLC and Sephadex G-25 columns. Its molecular weight was about 1000–2000. These results indicate the presence of a chemical mediator of insulin action in liver similar to that observed in other insulin target tissues.     

Regulation of adipose-tissue pyruvate dehydrogenase by insulin and other hormones

1. In epididymal adipose tissue synthesizing fatty acids from fructose in vitro, addition of insulin led to a moderate increase in fructose uptake, to a considerable increase in the flow of fructose carbon atoms to fatty acid, to a decrease in the steady-state concentration of lactate and pyruvate in the medium, and to net uptake of lactate and pyruvate from the medium. It is concluded that insulin accelerates a step in the span pyruvate-->fatty acid. 2. Mitochondria prepared from fat-cells exposed to insulin put out more citrate than non-insulin-treated controls under conditions where the oxaloacetate moiety of citrate was formed from pyruvate by pyruvate carboxylase and under conditions where it was formed from malate. This suggested that insulin treatment of fat-cells led to persistent activation of pyruvate dehydrogenase. 3. Insulin treatment of epididymal fat-pads in vitro increased the activity of pyruvate dehydrogenase measured in extracts of the tissue even in the absence of added substrate; the activities of pyruvate carboxylase, citrate synthase, glutamate dehydrogenase, acetyl-CoA carboxylase, NADP-malate dehydrogenase and NAD-malate dehydrogenase were not changed by insulin. 4. The effect of insulin on pyruvate dehydrogenase activity was inhibited by adrenaline, adrenocorticotrophic hormone and dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate). The effect of insulin was not reproduced by prostaglandin E(1), which like insulin may lower the tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and inhibit lipolysis. 5. Adipose tissue pyruvate dehydrogenase in extracts of mitochondria is almost totally inactivated by incubation with ATP and can then be reactivated by incubation with 10mm-Mg(2+). In this respect its properties are similar to that of pyruvate dehydrogenase from heart and kidney where evidence has been given that inactivation and activation are catalysed by an ATP-dependent kinase and a Mg(2+)-dependent phosphatase. Evidence is given that insulin may act by increasing the proportion of active (dephosphorylated) pyruvate dehydrogenase. 6. Cyclic AMP could not be shown to influence the activity of pyruvate dehydrogenase in mitochondria under various conditions of incubation. 7. These results are discussed in relation to the control of fatty acid synthesis in adipose tissue and the role of cyclic AMP in mediating the effects of insulin on pyruvate dehydrogenase.     

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.     

Stimulation of pyruvate dehydrogenase activity in adipocytes by oxytocin: Evidence for mediation of the insulin-like effect by endogenous hydrogen peroxide independent of glucose transport

Oxytocin, a nonapeptide posterior pituitary hormone, which is known to increase glucose oxidation in fat cells like insulin, is shown here to stimulate pyruvate dehydrogenase activity in these cells. The process appears to involve the activation of preexisting molecules since there was no change in the total enzyme content after full activation. The effect of oxytocin, as well as of insulin, appears to be mediated by endogenous H₂O₂ formation, as evident from (i) the enhanced [¹⁴C]formate oxidation and its greater inhibition by 3-amino-1,2,4-triazole in the hormone-treated cells than in the control. This is a measure of the catalase:H₂O₂ complex, and the dose dependence of this response is found to be identical with that of glucose oxidation via the hexose monophosphate shunt pathway and of pyruvate dehydrogenase activity; and (ii) treatment of the cells with low concentration of exogenous H₂O₂ causes the activation of pyruvate dehydrogenase to the extent which is comparable with the effect of the hormones. The ED₅₀ of oxytocin was 7 × 10^?9m, whereas the ED₅₀ of insulin was 5 × 10^?11m. The reduced, inactive (SH) derivatives of the hormones had the same dose-response relationship, but considerably lower effect (10 to 20% of the native molecules of the hormones), indicating the significant role of the disulfide bridge(s) in eliciting these metabolic responses. The stimulation of PDH by oxytocin or insulin is found to be essentially independent of medium glucose which, however, can sustain the response apparently by recycling the intracellular oxidation-reduction state. However, unlike insulin, oxytocin fails to stimulate the rapid uptake of 3-O-[³H]methyl-d-glucose in these cells. The data illustrate that the major metabolic actions of insulin, viz., glucose utilization and lipogenesis, are shared by another heterologous polypeptide hormone, e.g., oxytocin, through a common effector, H₂O₂. It is suggested that (i) oxytocin may play a limited surrogate role for insulin in these cells; and (ii) H₂O₂ production may be the general basis of oxytocin's action.     

Adenosine modulation of fat cell responsiveness to insulin and oxytocin

We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 microM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its ED50. A change in ED50, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (ED50 approximately equal to 3 microM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes.     

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.     

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.     

Effects of oxytocin and vasopressin on the preadipocyte 3T3-F442A cell line

The 3T3-F442A mouse fibroblast cell line, triggered by factors present in fetal calf serum (FCS), converts either spontaneously or, in the simultaneous presence of FCS and insulin, at an accelerated rate into cells exhibiting the adipocyte phenotype. The effects of the neurohypophysial hormones in differentiated cells on glucose metabolism (glucose oxidation and lipogenesis) were compared with the stimulatory actions of insulin, which had its most pronounced effects in cells differentiated spontaneously with FCS in the absence of insulin. The differentiated 3T3-F442A cells were sensitive to physiological levels of insulin and exhibited manyfold increases in glucose metabolism in response to it. This result demonstrated that these cultured cells respond to insulin, in a manner analogous to freshly isolated adipocytes. In contrast to its insulin-like effects in isolated epididymal adipocytes, oxytocin was not reproducibly able to stimulate glucose metabolism in differentiated 3T3-F442A cells. Vasopressin was similarly inactive. In contrast, both oxytocin and vasopressin blocked adipocyte conversion triggered by FCS, either in the presence or absence of insulin; vasopressin was more potent than oxytocin, indicating that a vasopressin receptor was responsible for the observed inhibition of differentiation. Our work suggests that vasopressin could potentially play a role in the regulation of the adipocyte differentiation process.     

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.     

Pyruvate accumulation may contribute to acceleration-induced impairment of physical and cognitive abilities: an experimental study

Background: Fatigue can be induced after acceleration exposure, however its mechanism is still unclear. The aim of the present study was to examine whether metabolites’ changes can decrease cognitive and physical function after acceleration. Methods: Graybiel scale and Fatigue Self-rating scale were used to assess the seasickness and fatigue degrees of 87 male seafarers respectively after sailing. To test the effect of pyruvate on cognitive and physical functions, five different doses of pyruvate were administrated into rats. Insulin can reduce the accumulation of pyruvate. To observe the insulin effect on pyruvate, cognitive and physical functions after acceleration, insulin administration or treatment of promoting insulin secretion was used. Physical and cognitive functions were assessed using open field test (OFT), morris water maze (MWM) and loaded swimming test (LST) in animals. Results: Physical and cognitive abilities were decreased obviously, and serum pyruvate increased mostly in human and rats after acceleration. Compared with vehicle group, physical and cognitive abilities were significantly decreased after pyruvate administration. Besides, we found a significant decline in adenosine triphosphate (ATP) concentration and pyruvate dehydrogenase (PDH) activity in the hippocampus, prefrontal cortex, liver, and muscle of rats treated with acceleration or pyruvate injection, while insulin administration or treatment of promoting insulin secretion markedly alleviated this decline and the impairment of physical and cognitive abilities, compared with the control group. Conclusion: Our results indicate that pyruvate has a negative effect on physical and cognitive abilities after acceleration. Insulin can inhibit pyruvate accumulation and cognitive and physical function after acceleration exposure.     

Impact of perturbed pyruvate metabolism on adipocyte triglyceride accumulation

This study aimed to test the hypothesis that adipocyte TG accumulation could be altered by specifically perturbing pyruvate metabolism. We treated cultured 3T3-L1 adipocytes with chemical inhibitors of lactate dehydrogenase (LDH) and pyruvate carboxylase (PC), and characterized their global effects on intermediary metabolism using metabolic flux and isotopomer analysis. Inhibiting the enzymes over several days did not alter the adipocyte differentiation program as assessed by the expression levels of peroxisome proliferator-activated receptor-γ and glycerol-3-phosphate dehydrogenase. The main metabolic effects were to up-regulate intracellular lipolysis and decrease TG accumulation. Inhibiting PC also up-regulated glycolysis. Flux estimates indicated that the reduction in TG was due to decreased de novo fatty acid synthesis. Exogenous addition of free fatty acids dose-dependently increased the cellular TG level in the inhibitor-treated adipocytes, but not in untreated control cells. The results of this study support our hypothesis regarding the critical role of pyruvate reactions in TG synthesis.     

‘Insulin-like’ effects of lithium ion on isolated rat adipocytes II. Specific activation of glycogen synthase

Lithium ion, like insulin, activated adipocyte glycogen synthase with or without glucose in the medium. However, the effect of lithium ion was much greater than that of insulin under both conditions. The lithium-activated glycogen synthase was stable to both Sephadex chromatography and ethanol precipitation of the enzyme, indicating that the effect of lithium ion on glycogen synthase was through covalent modification of the enzyme. Glycogen synthase was significantly activated by lithium ion under conditions where concentrations of cellular ATP were unaffected. The effect of lithium ion on glycogen synthase was rapid and observed at concentrations as low as 1 to 3 mM, reaching a maximum at the concentration of 40 mM. It was thus the most sensitive of all the effects studied (see previous paper). Insulin further stimulated glycogen synthase at low concentrations but not at maximal concentration of lithium ion. Lithium-activated glycogen synthase was inhibited by both epinephrine and dibutyryl cyclic AMP, but was not affected by the removal of extracellular Ca++. Interestingly, lithium ion had no detectable effect on basal pyruvate dehydrogenase as well as on epinephrine-stimulated phosphorylase. The failure of lithium ion to thus mimic insulin actions on pyruvate dehydrogenase and on phosphorylase suggests that the action of lithium ion on glycogen synthase is quite specific and may be mediated by stimulating a phosphatase or by inhibiting a protein kinase acting specifically on glycogen synthase.     

Enhanced glucose homeostasis in BHE/cdb rats with mutated ATP synthase

The BHE/cdb rat has a mutation in adenosine triphosphate (ATP) synthase that impairs insulin secretion. However, male BHE/cdb rats have normal circulating glucose and enhanced glucose tolerance. The aim of the current study was to identify mechanisms of enhanced glucose tolerance. The respiratory exchange ratio was increased, indicating increased oxidation of carbohydrate in BHE/cdb rats, consistent with increases in liver pyruvate dehydrogenase activity and muscle citrate synthase activity. Liver also exhibited diminished phosphoenol pyruvate carboxykinase content, which correlated with a decreased counter-regulatory response in the insulin tolerance test. Signaling via Akt or AMP-dependent kinase pathways in the liver could not account for lower blood glucose. We conclude that chronically low insulin secretion leads to adaption in glucose metabolism primarily in liver to maintain euglycemia.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.