Dataset Information

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

ABSTRACT: The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.

SUBMITTER: Marshall SE

PROVIDER: S-EPMC1153330 | biostudies-other | 1984 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.

Project description:The administration in vivo of either adrenaline or glucagon alone resulted in increases of about 2-fold in the amounts of active, non-phosphorylated, pyruvate dehydrogenase in the livers of fed male or female rats, whereas when administered together increases of about 4-fold were obtained. Ca2+-dependent increases in the amount of active enzyme of up to about 5-fold could be achieved in isolated rat liver mitochondria by incubating them with increasing extramitochondrial [Ca2+]; from this, two conditions of Ca loading were chosen which caused increases in active enzyme similar to those with the hormone treatments given above. The increases in enzyme activity owing to these Ca loads persisted through the 're-isolation' of mitochondria and their incubation in Na+-free KCl-based media containing EGTA. Differences from values obtained with unloaded controls could be diminished by adding Na+ ions to cause the egress of Ca2+ from the mitochondria, or enough extramitochondrial Ca2+ to saturate the enzyme in its Ca2+-dependent activation; the effects of Na+ could be blocked by diltiazem, an inhibitor of mitochondrial Na+/Ca2+ exchange. The re-isolated, Ca-preloaded, mitochondria also exhibited enhanced activities of 2-oxoglutarate dehydrogenase when assayed at non-saturating [2-oxoglutarate] by two different methods; effects of Na+, Ca2+ or diltiazem on the persistent activations of this enzyme were similar to those for pyruvate dehydrogenase. Na+ caused a marked depletion, which could be blocked by diltiazem, of the 45Ca content of re-isolated mitochondria which had pre-loaded with Ca, containing 45Ca, to the same degrees as above. The activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in incubated liver mitochondria prepared from rats subjected to the hormone treatments given above were found to behave in a very similar manner to those exhibited in the re-isolated, Ca-preloaded, mitochondria. It is concluded that these hormones each bring about the activations of these rat liver enzymes by causing increases in intramitochondrial [Ca2+], and that their effects, as such, are additive.

Project description: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.

Project description:1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.

Dataset Information

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets