Richard M. Denton

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+‐sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin‐induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium‐dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+‐mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

Regulation of mitochondrial dehydrogenases by calcium ions.

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.

On the role of the calcium transport cycle in heart and other mammalian mitochondria

Much attention has been focussed recently on the transfer of Ca*+ across the inner membrane of mammalian mitochondria and within the last year or so this Journal has published two other review letters on the topic [ 1,2]. The elegant studies which formed the basis of these letters have shown that the calciumtransport system in mammalian mitochondria consists of separate influx and efflux components. These components taken together constitute a calcium-transport cycle which determines the distribution of Ca*+ across the inner mitochondrial membrane (fig.]). The main purpose of this letter is to suggest a role for this cycle. In the past, it appears to have been assumed rather generally amongst biochemists interested in Ca*+transport in mitochondria that the system is important in the regulation of cytoplasmic Ca*+ [3-61. We will argue in this article that this emphasis is probably misplaced. We will summarise evidence that Ca*+ enhances the activity of three key intramitochondrial dehydrogenases and that [Ca”] at 0.1-10 PM is potentially an important regulator of oxidative metabolism within mammalian mitochondria. If this view is correct then the function of the calcium-transport system in the inner mitochondrial membrane should be considered primarily as a means of determining the intramitochondrial (rather than extramitochondrial) [Ca”] concentration in the same sense that the system in the plasma membrane is usually viewed as a

Regulation of Protein Kinase B and Glycogen Synthase Kinase-3 by Insulin and β-Adrenergic Agonists in Rat Epididymal Fat Cells ACTIVATION OF PROTEIN KINASE B BY WORTMANNIN-SENSITIVE AND -INSENSITIVE MECHANISMS

Previous studies using L6 myotubes have suggested that glycogen synthase kinase-3 (GSK-3) is phosphorylated and inactivated in response to insulin by protein kinase B (PKB, also known as Akt or RAC) (Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and Hemmings, B. A. (1995) Nature 378, 785-789). In the present study, marked increases in the activity of PKB have been shown to occur in insulin-treated rat epididymal fat cells with a time course compatible with the observed decrease in GSK-3 activity. Isoproterenol, acting primarily through β3-adrenoreceptors, was found to decrease GSK-3 activity to a similar extent (approximately 50%) to insulin. However, unlike the effect of insulin, the inhibition of GSK by isoproterenol was not found to be sensitive to inhibition by the phosphatidylinositol 3′-kinase inhibitors, wortmannin or LY 294002. The change in GSK-3 activity brought about by isoproterenol could not be mimicked by the addition of permeant cyclic AMP analogues or forskolin to the cells, although at the concentrations used, these agents were able to stimulate lipolysis. Isoproterenol, but again not the cyclic AMP analogues, was found to increase the activity of PKB, although to a lesser extent than insulin. While wortmannin abolished the stimulation of PKB activity by insulin, it was without effect on the activation seen in response to isoproterenol. The activation of PKB by isoproterenol was not accompanied by any detectable change in the electrophoretic mobility of the protein on SDS-polyacrylamide gel electrophoresis. It would therefore appear that distinct mechanisms exist for the stimulation of PKB by insulin and isoproterenol in rat fat cells.

The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity

Here we report that the widely used protein kinase C inhibitors, bisindolylmaleimide I and IX, are potent inhibitors of glycogen synthase kinase‐3 (GSK‐3). Bisindolylmaleimide I and IX inhibited GSK‐3 in vitro, when assayed either in cell lysates (IC50 360 nM and 6.8 nM, respectively) or in GSK‐3β immunoprecipitates (IC50 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 μM) and IX (2 μM) reduced GSK‐3 activity in total cell lysates, to 25.1±4.3% and 12.9±3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 μM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK‐3, and that this may explain some of the previously reported insulin‐like effects on glycogen synthase activity.

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1)

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

Mitochondrial Ca2+ Transport and the Role of Intramitochondrial Ca2+ in the Regulation of Energy Metabolism

The mitochondrial inner membrane of all mammalian tissues, including brain tissues, has specific active transport systems for the uptake and egress of Ca2+. The primary role of this transport system is to relay changes in cytosolic [Ca2+], which stimulates energy-requiring processes in the cytosol (e.g. secretion), into the mitochondrial matrix where it stimulates several key steps in energy production. Thus using the same second-messenger molecule, the latter events allow energetic homeostasis to be maintained under conditions of cell stimulation. This appears to be brought about by a co-ordinated enhancement of steps throughout the pathways of oxidative phosphorylation, including substrate supply to the respiratory chain by dehydrogenase activation, activation of the respiratory chain itself by a mechanism which appears to involve changes in the matrix volume, and also possibly activation of the ATP synthetase where the release of a specific inhibitory subunit has been proposed.

The activation of p38 MAPK by the β-adrenergic agonist isoproterenol in rat epididymal fat cells

Here we report that the β‐adrenergic agonist isoproterenol increases the activity of the stress‐activated kinase p38 MAPK over 10‐fold in freshly isolated rat epididymal fat cells. Stimulation of the kinase was rapid, sustained for at least 60 min and sensitive to the specific p38 MAPK inhibitor, SB 203580. Half‐maximal stimulation of p38 MAPK by isoproterenol occurred at 13 nM isoproterenol. The cell permeable cyclic AMP analogue, chlorophenylthio‐cyclic AMP increased p38 MAPK activity to a similar extent to isoproterenol, suggesting that the effect of the β‐adrenergic agonist is mediated via increases in the activity of cyclic‐AMP dependent protein kinase. Although it had little or no effect on the activity of c‐Jun N‐terminal kinase, isoproterenol and a number of other treatments which activated p38 MAPK were found to stimulate AMP‐activated protein kinase in fat cells. Activation of AMPK and p38 MAPK were not, however, found to be directly linked.

Coupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism

Mitochondria are strategically localized at sites of Ca2+ release, such that increases in cytosolic free Ca2+ ([Ca2+]c) from either internal Ca2+ stores or Ca2+ influx across the plasma membrane can be rapidly transported into the mitochondrial matrix. The consequent elevation in mitochondrial Ca2+ ([Ca2+]m) stimulates the Ca2+-sensitive intramitochondrial dehydrogenases, resulting in elevation of NAD(P)H. The preferential coupling between increases in [Ca2+]c and [Ca2+]m is one proposed mechanism to coordinate mitochondrial ATP production with cellular energy demand. In liver cells, hormones that act through the second messenger inositol 1,4, 5-trisphosphate (IP3) generate oscillatory [Ca2+]c signals, which result from a periodic Ca2+- and IP3-mediated activation/deactivation of intracellular Ca2+ release channels. The [Ca2+]c spiking frequency increases with agonist dose, whereas the amplitude of each [Ca2+]c spike is constant. This frequency modulation of [Ca2+]c spiking encodes the signal from the extracellular agonist, which is then decoded by the internal Ca2+-sensitive proteins such as the Ca2+-sensitive intramitochondrial dehydrogenases. Our studies have investigated the relationship between IP3-dependent [Ca2+]c signals and [Ca2+]m in primary cultured hepatocytes. In addition, the changes in cellular [Ca2+] levels have been correlated with the regulation of intramitochondrial NAD(P)H levels, pyruvate dehydrogenase activity and the magnitude of the mitochondrial proton motive force.

The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues

The pyruvate, NAD(+)-isocitrate and 2-oxoglutarate dehydrogenases are key regulatory enzymes in intramitochondrial oxidative metabolism in mammalian tissues, and can all be activated by increases in Ca2+ in the micromolar range. There is now mounting evidence that hormones and other stimuli which act by increasing cytosolic Ca2+ also, as a result, cause increases in mitochondrial matrix Ca2+ and hence activation of these enzymes, suggesting that the primary physiological function of mitochondrial Ca2(+)-transport is to be involved in this relay mechanism. This may also explain how in such circumstances rates of ATP production may be increased to meet the greater demand, but without any decreases in ATP/ADP occurring.