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Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

ABSTRACT: The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.

SUBMITTER: Chisholm AB

PROVIDER: S-EPMC1152266 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

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Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

Project description:A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.

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The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats.

Project description:Treatment of rats for 3 h with dexamethasone was shown to stimulate both pyruvate carboxylation and decarboxylation in the subsequently isolated mitochondria. The effect of hormone treatment on pyruvate carboxylation was also apparent in liver homogenates assayed within minutes of killing the animal and was independent of the temperature at which the assay was performed, suggesting that it was not an artifact of the mitochondrial preparation procedure. The stimulation of both aspects of pyruvate metabolism in the intact organelle was independent of the induction of either pyruvate carboxylase or pyruvate dehydrogenase. Similarly, there was no change in the percentage of pyruvate dehydrogenase in the active form, indicating that the effect of steroid treatment on pyruvate oxidation was not via changes in the degree of phosphorylation of the enzyme. Adrenalectomizing the animals for a period of 14 days before the experiment had no effect on either parameter. Glucocorticoid treatment of the animals increased the rate of pyruvate uptake into the mitochondria, as measured by the titration of pyruvate metabolism with alpha-cyano-4-hydroxycinnamate, a specific inhibitor of the pyruvate translocator. It also increased the intramitochondrial concentrations of acetyl-CoA and ATP and led to an elevated [ATP]/[ADP] ratio within the mitochondria. It is suggested that both enzymes of pyruvate metabolism exist in the mitochondria under considerable restraint and that glucocorticoids act to relieve this restraint by alterations in substrate supply and the intramitochondrial concentrations of effector molecules.

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Role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion.

Project description:Pyruvate cycling has been implicated in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The operation of some pyruvate cycling pathways is proposed to necessitate malate export from the mitochondria and NADP(+)-dependent decarboxylation of malate to pyruvate by cytosolic malic enzyme (ME1). Evidence in favor of and against a role of ME1 in GSIS has been presented by others using small interfering RNA-mediated suppression of ME1. ME1 was also proposed to account for methyl succinate-stimulated insulin secretion (MSSIS), which has been hypothesized to occur via succinate entry into the mitochondria in exchange for malate and subsequent malate conversion to pyruvate. In contrast to rat, mouse beta-cells lack ME1 activity, which was suggested to explain their lack of MSSIS. However, this hypothesis was not tested. In this report, we demonstrate that although adenoviral-mediated overexpression of ME1 greatly augments GSIS in rat insulinoma INS-1 832/13 cells, it does not restore MSSIS, nor does it significantly affect GSIS in mouse islets. The increase in GSIS following ME1 overexpression in INS-1 832/13 cells did not alter the ATP-to-ADP ratio but was accompanied by increases in malate and citrate levels. Increased malate and citrate levels were also observed after INS-1 832/13 cells were treated with the malate-permeable analog dimethyl malate. These data suggest that although ME1 overexpression augments anaplerosis and GSIS in INS-1 832/13 cells, it is not likely involved in MSSIS and GSIS in pancreatic islets.

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Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes.

Project description:Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14 C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies.

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Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes.

Project description:At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPCFERM and MPCOX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPCOX has a higher transport activity than MPCFERM, which is dependent on the C-terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability.

| S-EPMC4388599 | biostudies-literature

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

Project description:1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.

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Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier.

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| S-EPMC7004878 | biostudies-literature

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Project description:Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied (13)C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.

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Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

Project description:It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

| S-EPMC4036342 | biostudies-literature

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