Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Control analysis of systems with reaction blocks that 'cross-talk'.

Practical application of metabolic control analysis has been facilitated by use of the top-down approach, which divides a metabolic system into a small number of reaction blocks, linked by a few key intermediates. Previous papers have stressed that communication between blocks should be only through the explicit intermediates, 'cross-talk' between reaction blocks invalidated the approach. Here we show how the restriction is a result of the use of inhibitors of the blocks, and can be overcome if other system modulations are used. We also show a way to treat the related problem of enzymes that appear in more than one block such as the analysis of glycolytic substrate cycles into ATP consuming and net flux activities.

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria.

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

The causes and functions of mitochondrial proton leak.

The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion.

Islet beta-cells express low levels of lactate dehydrogenase and have high glycerol phosphate dehydrogenase activity. To determine whether this configuration favors oxidative glucose metabolism via mitochondria in the beta-cell and is important for beta-cell metabolic signal transduction, we have determined the effects on glucose metabolism and insulin secretion of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH)-A. Monitored in single MIN6 beta-cells, LDH hyperexpression (achieved by intranuclear cDNA microinjection or adenoviral infection) diminished the response to glucose of both phases of increases in mitochondrial NAD(P)H, as well as increases in mitochondrial membrane potential, cytosolic free ATP, and cystolic free Ca2+. These effects were observed at all glucose concentrations, but were most pronounced at submaximal glucose levels. Correspondingly, adenoviral vector-mediated LDH-A overexpression reduced insulin secretion stimulated by 11 mmol/l glucose and the subsequent response to stimulation with 30 mmol/l glucose, but it was without significant effect when the concentration of glucose was raised acutely from 3 to 30 mmol/l. Thus, overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in the islet beta-cell type, and it may therefore be directly responsible for insulin secretory defects in some forms of type 2 diabetes. The results also reinforce the view that glucose-derived pyruvate metabolism in the mitochondrion is critical for glucose-stimulated insulin secretion in the beta-cell.

Quantifying elasticity analysis: how external effectors cause changes to metabolic systems.

The sites of action of external effectors, such as inhibitors or hormones, on metabolic systems can be described qualitatively by elasticity analysis, or quantitatively by regulation analysis. The use of the latter approach has been limited, due to its practical complexity. In this study, we report mathematical relationships that relate the finite changes in system variables (fluxes and metabolite concentrations) to changes in activity of metabolic processes brought about by a single step addition of an effector. The activation or inhibition of a process by an effector is measured from changes in flux and intermediate levels. The changes in activity of each process can be used to describe, semi-quantitatively, which activations or inhibitions of the system processes are important in bringing about the observed levels of system variables.

Top-down control analysis of systems with more than one common intermediate.

The analysis of the control of complex metabolic systems can be greatly simplified by application of the top-down approach of metabolic control analysis, in which the reactions of the system are grouped together into a small number of blocks connected by a common intermediate. The experimental application of the top-down approach has so far been limited to systems that have only a single intermediate. In this study, we demonstrate that the connectivity and summation theorems of metabolic control analysis hold with any number of intermediates between the metabolic blocks, and in doing so show that top-down analysis is valid for systems with multiple intermediates and so can be applied to most metabolic systems regardless of their complexity; an example of such an application is provided. Top-down control analysis has successfully described the control of mitochondrial respiration by dividing the system into three blocks, the respiratory chain, phosphorylation system and proton leak, all linked by a single intermediate, proton motive force. Here, we subdivide the respiratory chain into succinate consumers and cytochrome oxidase so that a second intermediate, cytochrome c redox state, is generated. Despite the fact that the redox state of cytochrome c is not measured, we solve the control over the system fluxes. In common with previous studies, we find that under conditions where there is no ATP turnover (state 4), respiration is largely controlled by proton leak, while at maximal ATP turnover (state 3) respiration is controlled by the respiratory chain and the phosphorylating system. In state 4,85% of the control by the respiratory chain resides with cytochrome oxidase. As ATP turnover increases, the respiration rate increases, and the control by the respiratory chain shifts from cytochrome oxidase to the succinate consumers, so that in state 3 83% of the control by the respiratory chain lies in the reactions between succinate and cytochrome c and only 17% resides with cytochrome oxidase.

The responses of rat hepatocytes to glucagon and adrenaline. Application of quantified elasticity analysis.

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6-phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.

Errors associated with metabolic control analysis. Application Of Monte-Carlo simulation of experimental data.

The errors associated with experimental application of metabolic control analysis are difficult to assess. In this paper, we give examples where Monte-Carlo simulations of published experimental data are used in error analysis. Data was simulated according to the mean and error obtained from experimental measurements and the simulated data was used to calculate control coefficients. Repeating the simulation 500 times allowed an estimate to be made of the error implicit in the calculated control coefficients. In the first example, state 4 respiration of isolated mitochondria, Monte-Carlo simulations based on the system elasticities were performed. The simulations gave error estimates similar to the values reported within the original paper and those derived from a sensitivity analysis of the elasticities. This demonstrated the validity of the method. In the second example, state 3 respiration of isolated mitochondria, Monte-Carlo simulations were based on measurements of intermediates and fluxes. A key feature of this simulation was that the distribution of the simulated control coefficients did not follow a normal distribution, despite simulation of the original data being based on normal distributions. Consequently, the error calculated using simulation was greater and more realistic than the error calculated directly by averaging the original results. The Monte-Carlo simulations are also demonstrated to be useful in experimental design. The individual data points that should be repeated in order to reduce the error in the control coefficients can be highlighted.

Top-down control analysis of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

Control analysis was used to analyse the internal control of rat hepatocyte metabolism. The reactions of the cell were grouped into nine metabolic blocks linked by five key intermediates. The blocks were glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption. The linking intermediates were intracellular glucose-6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential. The steady-state fluxes through the blocks and the levels of the intermediates were measured in the absence and presence of specific effectors of hepatocyte metabolism. Application of the multiple modulation approach gave the kinetic responses of each block to each intermediate (the elasticities). These were then used to calculate all of the control coefficients, which describe the degree of control each block had over the level of each intermediate, and over the rate of each process. Within this full description of control, many different interactions could be identified. One key finding was that the processes that consumed ATP had only 35% of the control over the rate of ATP consumption. Instead, the reactions that produced ATP exerted the most control over ATP consumption rate; particularly important were mitochondrial phosphorylation (30% of control) and glycolysis (19%). The rate of glycolysis was positively controlled by the glycolytic enzymes themselves (66% of control) and by ATP consumption (47%). Mitochondrial production of ATP, including oxidative, proton leak and phosphorylation processes, had negative control over glycolysis (-26%; the Pasteur effect). In contrast, glycolysis had little control over the rate of ATP production by the mitochondria (-10%; the Crabtree effect). Control over the flux through the mitochondrial phosphorylation block was shared between pyruvate oxidation (23%), ATP consumption (28%) and the mitochondrial phosphorylation block itself (64%).

Internal regulation of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

Previously [Ainscow, E.K. & Brand, M.D. (1999) Eur. J. Biochem. 263, 671-685], top-down control analysis was used to describe the control pattern of energy metabolism in rat hepatocytes. The system was divided into nine reaction blocks (glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption) linked by five intermediates (intracellular glucose 6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential). The kinetic responses (elasticities) of reaction blocks to intermediates were determined and used to calculate control coefficients. In the present paper, these elasticities and control coefficients are used to quantify the internal regulatory pathways within the cell. Flux control coefficients were partitioned to give partial flux control coefficients. These describe how strongly one block of reactions controls the flux through another via its effects on the concentration of a particular intermediate. Most flux control coefficients were the sum of positive and negative partial effects acting through different intermediates; these partial effects could be large compared to the final control strength. An important result was the breakdown of the way ATP consumption controlled respiration: changes in ATP level were more important than changes in mitochondrial membrane potential in stimulating oxygen consumption when ATP consumption increased. The partial internal response coefficients to changes in each intermediate were also calculated; they describe how steady state concentrations of intermediates are maintained. Increases in mitochondrial membrane potential were opposed mostly by decreased supply, whereas increases in glucose-6-phosphate, NADH/NAD and pyruvate were opposed mostly by increased consumption. Increases in ATP were opposed significantly by both decreased supply and increased consumption.

Mitochondrial priming modifies Ca2+ oscillations and insulin secretion in pancreatic islets.

Increases in mitochondrial [Ca(2+)] ([Ca(2+)](m)) have recently been reported to cause long-term alterations in cellular ATP production [Jouaville, Bastianutto, Rutter and Rizzuto (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 13807-13812]. We have determined the importance of this phenomenon for nutrient sensing in pancreatic islets and beta-cells by imaging adenovirally expressed Ca(2+) and ATP sensors (aequorin and firefly luciferase). [Ca(2+)](m) increases provoked by KCl or tolbutamide evoked an immediate increase in cytosolic and mitochondrial free ATP concentration ([ATP](c) and [ATP](m) respectively) at 3 mM glucose. Subsequent increases in [glucose] (to 16 or 30 mM) then caused a substantially larger increase in [ATP](c) and [ATP](m) than in naïve cells, and pre-stimulation with tolbutamide led to a larger secretory response in response to glucose. Whereas pre-challenge of islets with KCl altered the response to high [glucose] of [Ca(2+)](m) from periodic oscillations to a sustained elevation, oscillations in [ATP](c) were observed neither in naïve nor in stimulated islets. Hence, long-term potentiation of mitochondrial ATP synthesis is a central element in nutrient recognition by pancreatic islets.

Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase.

Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta-cells, stimulation of insulin secretion is important for the activation by glucose of L-PK as well as the PPI promoter, but does not cause increases in glucokinase gene expression or glucose metabolism.

Molecular mechanisms involved in secretory vesicle recruitment to the plasma membrane in beta-cells.

Glucose stimulates the release of insulin in part by activating the recruitment of secretory vesicles to the cell surface. While this movement is known to be microtubule-dependent, the molecular motors involved are undefined. Active kinesin was found to be essential for vesicle translocation in live beta-cells, since microinjection of cDNA encoding dominant-negative KHC(mut) (motor domain of kinesin heavy chain containing a Thr(93)-->Asn point mutation) blocked vesicular movements. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Thus, vesicles released during the first phase of insulin secretion exist largely within a translocation-independent pool. Kinesin-driven anterograde movement of vesicles is then necessary for the sustained (second phase) of insulin release. Kinesin may, therefore, represent a novel target for increases in intracellular ATP concentrations in response to elevated extracellular glucose and may be involved in the ATP-sensitive K+channel-independent stimulation of secretion by the sugar.

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.