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The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-13C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of 13C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of 13C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-13C] pyruvate. [ABSTRACT FROM AUTHOR]

Hormones such as glucagon are secreted by Ca2+-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca2+-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca2+ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca2+ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca2+-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca2+-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca2+-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking synaptotagmin-7 exhibited little Ca2+-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca2+ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca2+ sensors in neurotransmitter, neuropeptide, and hormone secretion. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Many patients with heart failure have whole-body insulin resistance and reduced cardiac fluorodeoxyglucose uptake, but whether these metabolic changes have detrimental effects on the heart is unknown. Here, we tested whether there is a link between insulin resistance and ischemic damage in the chronically infarcted Wistar rat heart, postulating that the heart would have decreased insulin sensitivity, with lower GLUT4 glucose transporter protein levels due to high circulating free fatty acid (FFA) concentrations. A decreased capacity for glucose uptake would lower glycolytic adenosine triphosphate (ATP) production and thereby increase ischemic injury in the infarcted heart. Methods and results: In vivo left ventricular ejection fractions, measured using echocardiography, were 40% lower in rats 10weeks after coronary artery ligation than in sham-operated control rats. Insulin-stimulated d[2-3H]glucose uptake was 42% lower in isolated, perfused, infarcted hearts. Myocardial GLUT4 glucose transporter protein levels were 28% lower in the infarcted hearts and correlated negatively with ejection fractions and with fasting plasma FFA concentrations. Compared with controls, chronically infarcted hearts had 46% lower total glucose uptake and three-fold faster ATP hydrolysis rates, measured using phosphorus-31 nuclear magnetic resonance spectroscopy, during 32-min ischemia at 0.4ml/min/gww. During reperfusion, recovery of left ventricular developed pressure in infarcted hearts was 42% lower than in control hearts. Conclusions: Glucose uptake, in response to insulin or ischemia, was lower in the chronically infarcted rat heart and associated with increased circulating FFA concentrations and decreased GLUT4 levels. Thus, infarcted hearts had greater ATP depletion, and consequently incurred greater damage, during ischemia. [Copyright &y& Elsevier]

Objective: Whole body insulin resistance and diabetes are risk factors for cardiovascular diseases, yet little is known about insulin resistance in the diabetic heart. The aim of this work was to define the insulin response in hearts of the Goto–Kakizaki (GK) rat, a polygenic model of spontaneous type 2 diabetes. Methods: We measured d[2-3H]glucose uptake before and after insulin stimulation, plus initial steps of the insulin signaling pathway after insulin infusion via the caudal vena cava in hearts from the male Wistar and spontaneously diabetic GK rats. Results: Despite normal basal d[2-3H]glucose uptake, insulin-stimulated glucose uptake was 50% (p<0.03) lower in GK rat hearts compared with their Wistar controls. Total GLUT4 protein was depleted by 28% (p<0.01) in GK rat hearts. We found 31% (p<0.0001) and 38% (p<0.001) decreased protein levels of insulin receptor β (IRβ)-subunit and insulin receptor substrate-1 (IRS-1), respectively, in GK rat hearts with 37% (p<0.02) and 45% (p<0.01) lower insulin-stimulated tyrosine phosphorylation of these proteins. Owing to the decreased IRS-1 protein levels, GK rat hearts had a 41% (p<0.0001) decrease in insulin-stimulated IRS-1 protein association with the p85 subunit of phosphatidylinositol 3-kinase, despite normal phosphatidylinositol 3-kinase protein expression. Insulin-stimulated serine phosphorylation of protein kinase B was the same in all hearts, as was protein kinase B expression. Conclusion: We conclude that decreased insulin receptor β, IRS-1 and GLUT4 proteins are associated with insulin resistance in type 2 diabetic rat hearts. [Copyright &y& Elsevier]

The accumulation of 2-deoxy-d-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38–60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a Km of 17.5 mM and Vmax of 1.4 μmol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution. [Copyright &y& Elsevier]

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the ieucine methyl and γ-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two β turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity. [ABSTRACT FROM AUTHOR]

The structure of the chromaffin granule membrane has been probed using a number of different win labels. Both the effect of temperature and high levels of calcium have been studied 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which under- goes a structural transition at 32- 36 C. characterized h an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 C, This transition corresponds to an increased degree of motional freedom of tile spin label above the transition temperature. 3. The lipid-soluble spin label 2.2.6.6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in tile membrane lipid, again possibly involving a redistribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzmic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al. Eur, J. Biochem. 67. 105-114 (1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion. [ABSTRACT FROM AUTHOR]

The interaction of phosphofructokinase from rabbit skeletal muscle with 2-(N-methylanilino)-naphthalene-6-sulphonate was studied. The fluorescence maximum of the probe was shifted to 430 nm and was enhanced by the enzyme. The intensity of emitted light was reduced by the presence of either one of the substrates, fructose 6-phosphate or ATP. The reduction of fluorescence by fructose 6-phosphate was accompanied by a decrease in the number of binding sites for the probe on the protein. The number of binding sites for the probe was also dependent on the concentration of the enzyme. Decrease of enzyme concentration resulted in relatively higher fluorescence and in rise of the number of binding sites. Phosphofructokinase is inactivated after dilution as well in the absence as in the presence of the probe as a consequence of dissociation into inactive subunits. In the presence of the probe, the loss in activity followed the same kinetics as the increase in fluorescence. The rise in the number of probe binding sites, therefore, is most probably due to the dissociation of the enzyme. The effect of substrates on fluorescence intensity was used to follow inter. actions of the enzyme with its substrates. At low substrate concentrations the fluorescence changes were dependent on the concentration of the enzyme. At higher enzyme levels, higher ATP but lower fructose 6-phosphate concentrations were required to obtain half-maximal effects, thus suggesting that the binding of the substrates is dependent on the association state of the enzyme. [ABSTRACT FROM AUTHOR]