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Liver fat represents a balance between input, secretion, and oxidation of fatty acids. As humans spend the majority of a 24-h period in a postprandial state, dietary fatty acids make an important contribution to liver fat metabolism. We compared hepatic fatty acid partitioning in healthy lean (n = 9) and abdominally obese (n = 10) males over 24 h. Volunteers received three mixed meals adjusted for basal metabolic rate. U-[sup.13]C-labeled fatty acids were incorporated into the meals, and [[sup.2][H.sub.2]]palmitate was infused intravenously to distinguish between sources of fatty acids incorporated into VLDL-TG. Immunoaffinity chromatography was used to isolate VLDL-TG of hepatic origin. Liver and whole body fatty acid oxidation was assessed by isotopic enrichment of 3-hydoxybutyrate and breath C[O.sub.2]. We found a similar contribution of dietary fatty acids to VLDL-TG in the two groups over 24 h The contribution of fatty acids from splanchnic sources was higher (P < 0.05) in the abdominally obese group. Ketogenesis occurred to a significantly greater extent in abdominally obese compared with lean males, largely due to lessened downregulation of postprandial ketogenesis (P < 0.001). The appearance of [sup.13]C in breath C[O.sub.2] was also greater (P < 0.001) in abdominally obese compared with lean men. Hepatic elongation and desaturation of palmitic acid were higher (P < 0.05) in abdominally obese than in lean males. Oxidation of dietary fatty acids and hepatic desaturation and elongation of palmitic acid occurred to a greater extent in abdominally obese men. These alterations may represent further pathways for redirection of fatty acids into export from the liver or oxidation to prevent liver fat accumulation. very low-density lipoprotein; stable isotopes; postprandial metabolism; oxidation; abdominal obesity; desaturation doi: 10.1152/ajpendo.00272.2010.

Aging presumably initiates shifts in substrate oxidation mediated in part by changes in insulin sensitivity. Similar shifts occur with cardiac hypertrophy and may contribute to contractile dysfunction. We tested the hypothesis that aging modifies substrate utilization and alters insulin sensitivity in mouse heart when provided multiple substrates. In vivo cardiac function was measured with microtipped pressure transducers in the left ventricle from control (4-6 mo) and aged (22-24 mo) mice. Cardiac function was also measured in isolated working hearts along with substrate and anaplerotic fractional contributions to the citric acid cycle (CAC) by using perfusate containing [sup.13]C-labeled free fatty acids (FFA), acetoacetate, lactate, and unlabeled glucose. Stroke volume and cardiac output were diminished in aged mice in vivo, but pressure development was preserved. Systolic and diastolic functions were maintained in aged isolated hearts. Insulin prompted an increase in systolic function in aged hearts, resulting in an increase in cardiac efficiency. FFA and ketone flux were present but were markedly impaired in aged hearts. These changes in myocardial substrate utilization corresponded to alterations in circulating lipids, thyroid hormone, and reductions in protein expression for peroxisome proliferator-activated receptor (PPAR)[alpha] and pyruvate dehydrogenase kinase (PDK)4. Insulin further suppressed FFA oxidation in the aged. Insulin stimulation of anaplerosis in control hearts was absent in the aged. The aged heart shows metabolic plasticity by accessing multiple substrates to maintain function. However, fatty acid oxidation capacity is limited. Impaired insulin-stimulated anaplerosis may contribute to elevated cardiac efficiency, but may also limit response to acute stress through depletion of CAC intermediates. diabetes; heart; efficiency; thyroid hormone; substrate oxidation doi: 10.1152/ajpheart.00931.2009.

Carbon 13 nuclear magnetic resonance (NMR) isotopomer analysis was used to measure the rates of oxidation of long-chain fatty acids, ketones, and pyruvate to determine the minimum pyruvate concentration ([pyruvate]) needed to suppress oxidation of these alternative substrates. Substrate mixtures were chosen to represent either the fed or fasted state. At physiological [pyruvate], fatty acids and ketones supplied the overwhelming majority of acetyl-CoA. Under conditions mimicking the fed state, 3 mM pyruvate provided ~80% of acetylCoA, but under fasting conditions 6 mM pyruvate contributed only 33% of acetyl-CoA. Higher [pyruvate], 10-25 mM, was associated with transient reduced cardiac output, but overall hemodynamic performance was unchanged after equilibration. These observations suggested that 3-6 mM pyruvate in the coronary arteries would be an appropriate target for studies with hyperpolarized [1-[sup.13]C]pyruvate. However, the metabolic products of 3 mM hyperpolarized [1-[sup.13]C]pyruvate could not be detected in the isolated heart during perfusion with a physiological mixture of substrates including 3% albumin. In the presence of albumin even at high concentrations of pyruvate, 20 mM, hyperpolarized [H.sup.13]C[O.sup.-.sub.3] could be detected only in the absence of competing substrates. Highly purified albumin (but not albumin from plasma) substantially reduced the longitudinal relaxation time of [1-[sup.13]C]pyruvate. In conclusion, studies of cardiac metabolism using hyperpolarized [1-[sup.13]C]pyruvate are sensitive to the effects of competing substrates on pyruvate oxidation. myocardium; pyruvate; dynamic nuclear polarization; substrate oxidation; isolated rat heart; carbon-13; nuclear magnetic resonance doi: 10.1152/ajpheart.00656.2009.

Sorensen LP, Gormsen LC, Nielsen S. VLDL-TG kinetics: a dual isotope study for quantifying VLDL-TG pool size, production rates, and fractional oxidation in humans. Am J Physiol Endocrinol Metab 297: E1324-E1330, 2009. First published September 22, 2009; doi: 10. 1152/ajpendo.00366.2009.--Very-low-density lipoproteins (VLDLs) are large, complex particles containing both surface proteins (e.g., ApoB100) and core lipids, e.g., cholesterol and triglycerides (TG). Whereas ApoB100 kinetics have been thoroughly studied, accurate measurement of VLDL-TG kinetics have proven difficult due to either complex mathematics or laborious procedures. The present study was therefore designed to measure VLDL-TG kinetics by dual isotope ex vivo labeled VLDL-TG tracers and well-established kinetics equations (bolus injection or the primed continuous infusion). Ten healthy Caucasian men [age, 23 [+ or -] 3 yr old (mean [+ or -] SD); body mass index, 24.7 [+ or -] 1.3 kg/[m.sup.2]] were included in the study. VLDL-TG rate of appearance (Ra) was measured using a dual-tracer technique ([9,[10.sup.-3]H]-labeled VLDL-TG and [1-[sup.14]C]- labeled VLDL-TG) to allow comparison of various bolus decay curve fits with the Ra obtained by the primed continuous infusion (PCI; considered the gold standard). In addition, VLDL-TG fatty acid oxidation was measured as [sup.14]C[O.sub.2] in exhaled breath, using the hyamine trapping technique. Following a bolus injection, tracer decay was better described by a biexponential than a monoexponential fit ([r.sup.2] = 0.99 [+ or -] 0.01 vs. 0.97 [+ or -] 0.04, respectively, P = 0.01). VLDL-TG Ra calculated using the PCI correlated significantly with the biexponential fit ([rho] = 0.62, P < 0.05), whereas this was not the case for the monoexponential fit ([rho] = -0.18, P = not significant). VLDL-TG Ra using the best fit of the bolus injection method (biexponential) was less than values obtained by the constant infusion technique [biexponential, 34.3 (range, 27.1-69.6) vs. PCI, 44.4 (range, 33.0-72.7), P < 0.05]. Fractional oxidation of VLDL-TG was 37.2 [+ or -] 8.8% at 240 min corresponding to 198.8 [+ or -] 55.9 kcal/day or 10.6 [+ or -] 3.3% of resting energy expenditure (REE). Our data demonstrate that VLDL-TG Ra measured by a biexponential fit to a bolus decay curve correlates well with VLDL-TG Ra measured by a primed continuous infusion, and therefore that a 'second' peripheral VLDL-TG compartment with rapid exchange of TG exists. VLDL-TG volume of distribution is therefore greater than previously anticipated. Finally our data supports that VLDL-TG contributes quantitatively to REE. lipoproteins; tracer kinetics; methods; margination doi: 10.1152/ajpendo.00366.2009

Type 1 diabetes is associated with a unique form of cardiomyopathy that is present without atherosclerosis. Redox imbalance and/or changes in vascular endothelial growth factor (VEGF) expression have been associated with diabetes-related cardiomyopathy. However, the mechanisms of these changes and their interrelationships remain unclear. Using a murine type 1 diabetes model, we tested the hypothesis that alterations in cardiac performance are associated with decreased cardiac microvascular prevalence, as well as downregulation of VEGF isoforms. We also investigated oxidative stress as a contributor to regulate individual VEGF isoforms and microvascular rarefaction. Significant and rapid hyperglycemia was observed at 1 wk post-streptozotocin (STZ) and persisted throughout the 5-wk study. Left ventricular (LV) fractional shortening was reduced at week 1 and 5 post-STZ insult relative to age-matched controls. We also observed the early reduction in E/A ratio at 1 wk. Immunostaining for CD31 and digital image analysis demonstrated a 35% reduction in microvessels/myocardial area, indicative of rarefaction, which was highly correlated with fractional shortening. Furthermore, a significant increase in the prevalence of protein 3-nitrotyrosine was observed in the diabetic cardiac tissue, which was inversely associated with microvascular rarefaction. The expressions of three VEGF isoforms were significantly reduced to different extents. The reduction of VEGF164 was associated with GSSG accumulation. These data demonstrate that the mouse model of STZ-induced diabetes has hallmark features observed in humans with respect to nonischemic systolic and diastolic performance and microvascular rarefaction, which are associated with changes in VEGF isoform expression and redox imbalance in the myocardium. microvascular density; vascular endothelial growth factor; cardiomyopathy; oxidants

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37[degrees]C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27[degrees]C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and ftavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27[degrees]C and 2 h of warm reperfusion. The [K.sub.ATP] channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27[degrees]C led to better recovery of function and less tissue damage on reperfusion than did 27[degrees]C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial [K.sub.ATP] channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion. nicotinamide adenine dinucleotide; flavin adenine dinucleotide; ATP-sensitive potassium channels; 5-hydroxydecanoic acid

The present study hypothesized that superoxide ([O.sup.-.sub.2]*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1[alpha] expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with [O.sup.-.sub.2]* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or [CoCl.sub.2]-induced increase in HIF-1[alpha] levels and completely blocked the increase in HIF-1[alpha] levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable [O.sup.-.sub.2]* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1[alpha] levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1[alpha] levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, [p22.sup.phox], into RMICs markedly increased HIF-1[alpha] levels. In contrast, the OH. scavenger tetramethylthiourea had no effect on the accumulation of HIF-1[alpha] in these renal cells. By Northern blot analysis, scavenging or dismutation of [O.sup.-.sub.2]* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1[alpha]-targeted gene, heme oxygenase-1. These results indicate that increased intracellular [O.sup.-.sub.2] levels induce HIF-1[alpha] degradation independently of [H.sub.2][O.sub.2] and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1[alpha]* in these cells under physiological conditions. reactive oxygen species; gene transcription; anoxia; renal interstitium; renal medulla

External surfaces of cells are normally protected by extracellular superoxide dismutase, SOD3, which binds to polyanions such as heparan sulfate. We constructed a fusion gene encoding a chimeric SOD consisting of the mature human mitochondrial SOD2 plus the COOH-terminal 26-amino acid heparin-binding 'tail' from SOD3. This tail is responsible for the enzyme's affinity for endothelial surfaces. The fusion gene was expressed in Escherichia coli, and the fully active enzyme SOD2/3 was purified. Although native SOD2 has no affinity for heparin, SOD2/3 binds to a heparin-agarose column. In a rat mode] of acute lung injury induced by intratracheal instillation of IL-1, SOD2/3, SOD2, and denatured SOD2/3 showed 92%, 13.8%, and 0% reduction of lung leak, respectively. Only SOD2/3 prevented neutrophil accumulation. In the carrageenan-induced foot edema model in the rat, SOD2/3 reduced edema by 62% (P < 0.003) at a dose in which native SOD2 produced no significant effect. Thus SOD2/3 appears to have properties as a therapeutic anti-inflammatory agent that are greatly superior to other available forms of the enzyme. inflammation; lung injury; interleukin-1; free radicals; oxidative injury

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on perlyentricular (PV) microvessels of fetal ([approximately equal to] 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 [+ or -] 2.6%) and newborn (22.14 [+ or -] 3.2%) pigs but was ineffective in adults ( newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TX[A.sub.2] formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects. peroxidation; age dependence; thromboxane; ischemia

Infusion of ANG II at a rate not sufficient to evoke an immediate vasoconstrictor response, produces a slow increase in blood pressure. Circulating levels of ANG II may be within ranges found in normotensive individuals, although inappropriately high with respect to sodium intake. When ANG II levels are dissociated from sodium levels, oxidative stress (OXST) occurs, which can increase blood pressure by several mechanisms. These include inadequate production or reduction of bioavailability of nitric oxide, alterations in metabolism of arachidonic acid, resulting in an increase in vasoconstrictors and decrease in vasodilators, and upregulation of endothelin. This cascade of events appears to be linked, because ANG II hypertension can be blocked by inhibition of any factor located distally, blockade of ANG II, OXST, or endothelin. Such characteristics are shared by other models of hypertension, such as essential hypertension, hypertension induced by reduction in renal mass, and renovascular hypertension. Thus these findings are clinically important because they reveal 1) uncoupling between ANG II and sodium, which can trigger pathological conditions; 2) the various OXST mechanisms that may be involved in hypertension; and 3) therapeutic interventions for hypertension developed with the knowledge of the cascade involving OXST. isoprostanes; endothelin; spontaneous hypertension; renovascular hypertension; sodium balance

Oxidative stress is one of the factors involved in age-related impairment of cardiac function. In the present study, we investigated the role of the catecholamine-degrading enzyme monoamine oxidase (MAO) in [H.sub.2][O.sub.2] production in the hearts of young, adult, and old rats. MAO-dependent [H.sub.2[O.sub.2] production, measured by a chemiluminescence-based assay, increased with age, reaching the maximum in 24-mo-old rats (7.5-fold increase vs. 1-mo-old rats). The following observations indicate that the age-dependent increase in [H.sub.2][O.sub.2] generation was fully related to the MAO-A isoform: 1) at all the ages tested, chemiluminescence production was inhibited by the MAO-A inhibitor clorgyline but not by the MAO-B inhibitor RO-19 6327; 2) enzyme assay, Western blot, and semiquantitative RT-PCR analysis showed an age-dependent increase in cardiac MAO-A activity, immunodetection, and mRNA expression, respectively; and 3) the MAO-B isoform was undetectable by enzyme assay and Western blot analysis. These results suggest that MAO-A could be a major source of [H.sub.2][O.sub.2] in the aging heart. monoamine oxidases; oxidative stress

Vascular oxidative stress contributes to endothelial dysfunction. Aerobic exercise training improves vascular function. The purpose of this study was to test the hypothesis that exercise training would improve the balance of antioxidant to prooxidant enzymes and reduce markers of oxidative stress in aortic endothelial cells (AEC). Female Yucatan miniature pigs either remained sedentary (SED) or were exercise trained (EX) for 16-19 wk. EX pigs had increased AEC SOD-1 protein levels and Cu/Zn SOD activity of the whole aorta compared with SED pigs. Protein levels of other antioxidant enzymes (SOD-2, catalase) were not affected by exercise training. Protein levels of [p67.sup.phox], a subunit of the prooxidant enzyme NAD(P)H oxidase, were reduced in EX vs. SED AEC. These EX adaptations were associated with lower AEC malondialdehyde levels and decreased phosphorylation of ERK-1/2. Endothelial nitric oxide synthase protein, protein nitrotyrosine content, and heine oxygenase-1 protein were not different in EX vs. SED pigs. We conclude that chronic aerobic exercise training influenced both antioxidant and prooxidant enzymes and decreased indexes of oxidative stress in AEC. These adaptations may contribute to improved endothelial function with exercise training. superoxide dismutase; nitric oxide; NAD(P)H oxidase; endothelial function; antioxidant enzymes; ERK

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and [H.sub.2][O.sub.2] generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular [H.sub.2][O.sub.2] release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study. mitochondria; superoxide; hydrogen peroxide; aconitase

Antiretroviral therapy in human immunodeficiency virus (HIV)-positive patients can induce a lipodystrophy syndrome of peripheral fat wasting and central adiposity, dyslipidemia, and insulin resistance. To test whether in this syndrome insulin resistance is associated with abnormal muscle handling of fatty acids, 12 HIV-1 patients (8 females/4 males, age = 26 [+ or -] 2 yr, HIV duration = 8 [+ or -] yr, body mass index = 22.0 [+ or -] 1.0 kg/[m.sup.2], on protease inhibitors and nucleoside analog RT inhibitors) and 12 healthy subjects were studied. HIV-1 patients had a total body fat content (assessed by dual-energy X-ray absorptiometry) similar to that of controls (22 [+ or -] 1 vs. 23 [+ or -] 2%; P = 0.56), with a topographic fat redistribution characterized by reduced fat content in the legs (18 [+ or -] 2 vs. 32 [+ or -] 3%; P < 0.01) and increased fat content in the trunk (25 [+ or -] 2 vs. 19 [+ or -] 2%; P = 0.03). In HIV-positive patients, insulin sensitivity (assessed by QUICKI) was markedly impaired (0.341 [+ or -] 0.011 vs. 0.376 [+ or -] 0.007; P = 0.012). HIV-positive patients also had increased total plasma cholesterol (216 [+ or -] 20 vs. 174 [+ or -] 9 mg/dl; P = 0.05) and triglyceride (298 [+ or -] 96 vs. 87 [+ or -] 11 mg/dl; P = 0.03) concentrations. Muscular triglyceride content assessed by means of [sup.1]H NMR spectroscopy was higher in HIV patients in soleus [92 [+ or -] 12 vs. 42 [+ or -] 5 arbitrary units (AU); P < 0.01] and tibialis anterior (26 [+ or -] 6 vs. 11 [+ or -] 3 AU; P = 0.04) muscles; in a stepwise regression analysis, it was strongly associated with QUICKI ([R.sup.2] = 0.27; P < 0.0093). Even if the basal metabolic rate (assessed by indirect calorimetry) was comparable to that of normal subjects, postabsorptive lipid oxidation was significantly impaired (0.30 [+ or -] 0.07 vs. 0.88 [+ or -] 0.09 mg * [kg.sup.-1] * [min.sup.-1]; P < 0.01). In conclusion, lipodystrophy in HIV-1 patients in antiretroviral treatment is associated with intramuscular fat accumulation, which may mediate the development of the insulin resistance syndrome. human immunodeficiency virus

After birth, a dramatic increase in fatty acid oxidation occurs in the heart, which has been attributed to an increase in L-carnitine levels and a switch from the liver (L) to muscle (M) isoform of carnitine palmitoyltransferase (CPT)-1. However, because M-CPT-1 is more sensitive to inhibition by malonyl CoA, a potent endogenous regulator of fatty acid oxidation, a switch to the M-CPT-1 isoform should theoretically decrease fatty acid oxidation. Because of this discrepancy, we assessed the contributions of myocardial L-carnitine content and CPT-1 isoform expression and kinetics to the maturation of fatty acid oxidation in newborn rabbit hearts. Although fatty acid oxidation rates increased between 1 and 14 days after birth, myocardial L-carnitine concentrations did not increase. Changes in the expression of L-CPT-1 or M-CPT-1 mRNA after birth also did not parallel the increase in fatty acid oxidation. The [K.sub.m] of CPT-1 for carnitine and the I[C.sub.50] for malonyl CoA remained unchanged between 1 and 10 days after birth. However, malonyl CoA levels dramatically decreased, due in part to an increase in malonyl CoA decarboxylase activity. Our data suggest that a decrease in malonyl CoA control of CPT-1 is primarily responsible for the increase in fatty acid oxidation seen in the newborn heart. carnitine palmitoyltransferase-1; pyruvate dehydrogenase; acetyl CoA carboxylase; glucose oxidation

The present study focused on the molecular signaling pathways of monocyte chemoattractant protein-1 (MCP-1) induction by interleukin-4 (IL-4) in human umbilical vein endothelial cells (HUVEC). RT-PCR showed that MCP-1 mRNA accumulation was markedly increased in IL-4-treated HUVEC in a time-and dose-dependent manner. Antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), significantly inhibited IL-4-induced MCP-1 mRNA expression. These effects correlated well with the PDTC-mediated inhibition of MCP-1 promoter transcriptional activity observed in IL-4-treated HUVEC. IL-4-induced MCP-1 gene expression was paralleled by a concomitant production of MCP-1 protein. In agreement with MCP-1 gene expression, PDTC attenuated IL-4-mediated induction of MCP-1 protein expression. In addition, IL-4 dramatically increased the transcription factor signal transducers and activators of transcription 1 (STAT1) DNA binding activity, an effect that was attenuated by PDTC. The role of STAT1 in the regulation of the IL-4-induced MCP-1 gene expression was further confirmed in HUVEC transfected with a reporter construct of the MCP-1 promoter with a mutated STAT1 binding site. These results demonstrate that IL-4-dependent MCP-1 induction in HUVEC is mediated by redox-regulated STAT1 activation. inflammatory cytokine; atherosclerosis; transcriptional regulation; antioxidants; signal transducers and activator of transcription 1; interleukin-4; monocyte chemoattractant protein-1

We investigated the hypothesis that birth-related pulmonary vasodilation is mediated in part by an increase in oxidative phosphorylation and ATP release in response to oxygen exposure at birth. Studies were done in fetal lambs to evaluate the independent effects of oxygen, lung distension alone, or lung distension accompanied by oxygenation and shear stress on fetal pulmonary blood flow and resistance and plasma ATP levels in the pulmonary artery. The effect of each intervention was evaluated in lambs assigned to one of three groups: control or pretreatment with 2,4-dinitrophenol or antimycin-A, inhibitors of oxidative phosphorylation. Exposure to oxygen alone or with lung distension was associated with increases in plasma ATP levels and pulmonary blood flow and a decrease in pulmonary vascular resistance. Plasma ATP levels did not change during lung distension alone. 2,4-Dinitrophenol and antimycin-A attenuated the pulmonary vasodilator response to oxygen but did not attenuate the response to lung distension alone. An increase in oxidative phosphorylation and ATP release during oxygen exposure may contribute to birth-related pulmonary vasodilation in fetal lambs. pulmonary circulation; purine nucleotides; oxygen; newborn

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial effiux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846-E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-[sup.13]C](lactate + pyruvate) and [1-[sup.13]C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This [sup.13]C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-[sup.13]C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis. gas chromatography-mass spectrometry; adenosine 5'-triphosphate-citrate lyase; hydroxycitrate; acetyl-coenzyme A; citric acid cycle; [sup.13]C-substrate; isotopomer analysis

Two 'blood substitutes,' a diaspirin cross-linked human hemoglobin [bis(3,5 dibromosalicyl)fumarate, DBBF-Hb] and a bovine polymerized hemoglobin (PolyHbBv), advanced to clinical trials, are used in this study. Previously, we have shown that injection of DBBF-Hb into the rat circulation produces venular leakage and intestinal epithelial disruption. The purpose of this study was to determine whether PolyHbBv, currently approved for veterinary use in the United States, shows similar effects. In anesthetized Sprague-Dawley rats, the mesenteric microvasculature was perfused with DBBF-Hb (n = 6), PolyHbBv (n = 5), cyanomet Hb (CNmet-DBBF-Hb), or HEPES-buffered saline with 0.5% bovine serum albumin (HBS-BSA) (controls, n = 7) for 10 min, followed by FITC-albumin for 3 min, and then fixed for microscopy. For DBBF-Hb, the mean leak number per micrometer venule length [2.41 [+ or -] 0.33 ([+ or -]SE) x [10.sup.-3]] was significantly greater than for PolyHbBv (0.53 [+ or -] 0.14 x [10.sup.-3]), CNmet-DBBF-Hb (0.36 [+ or -] 0.14 x [10.sup.-3]), and HBS-BSA (0.12 [+ or -] 0.08 x [10.sup.-3]) (P < 0.01). Corresponding quantities for leak area were 0.10 [+ or -] 0.03, 0.010 [+ or -] 0.003, 0.005 [+ or -] 0.003, and 0.02 [+ or -] 0.02 [micro][m.sup.2]/[micro]m. In rats injected with DBBF-Hb (n = 8), intestinal epithelial integrity was significantly compromised compared with those injected with PolyHbBv (n = 5) or saline (n = 6). These results indicate that intravascular PolyHbBv produces significantly less disruption of the intestinal exchange barrier than does DBBF-Hb, probably because the heme is not so easily oxidized. blood substitutes; fluorescence microscopy; venular leakage; electron microscopy

Hearts from diabetic db/db mice, a model of Type 2 diabetes, exhibit left ventricular failure and altered metabolism of exogenous substrates. Peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha] ligands reduce plasma lipid and glucose concentrations and improve insulin sensitivity in db/db mice. Consequently, the effect of 4- to 5-wk treatment of db/db mice with a novel PPAR-[alpha] ligand (BM 17.0744; 25-38 mg*[kg.sup.-1]*[day.sup.-1]), commencing at 8 wk of age, on ex vivo cardiac function and metabolism was determined. Elevated plasma concentrations of glucose, fatty acids, and triacylglycerol (34.0 [+ or -] 3.6, 2.0 [+ or -] 0.4, and 0.9 [+ or -] 0.1 mM, respectively) were reduced to normal after treatment with BM 17.0744 (10.8 [+ or -] 0.6, 1.1 [+ or -] 0.1, and 0.6 + 0.1 mM). Plasma insulin was also reduced significantly in treated compared with untreated db/db mice. Chronic treatment of db/db mice with the PPAR-[alpha] agonist resulted in a 50% reduction in rates of fatty acid oxidation, with a concomitant increase in glycolysis (1.7-fold) and glucose oxidation (2.3-fold). Correction of the diabetes-induced abnormalities in systemic and cardiac metabolism after BM 17.0744 treatment did not, however, improve left ventricular contractile function. substrate oxidation; isolated working mouse hearts

The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal. glutathione; redox; Bid; BCL-[x.sub.1]; caspase-8; apoptosis

The effects of intracranial transforming growth factor (TGF)-[beta]3 on spontaneous motor activity and energy metabolism were examined in rats. After injection of TGF-[beta]3 into the cisterna magna of the rat, spontaneous motor activity decreased significantly for 1 h. The intracranial injection of TGF-[beta]3 produced an immediate decrease in respiratory exchange ratio (RER). No significant changes were observed in energy expenditure. TGF-[beta]3 induced a significant increase in total fat oxidation and a decrease in total carbohydrate oxidation. Furthermore, the serum substrates associated with fat metabolism were significantly altered in rats injected with TGF-[beta]3. Both lipoprotein lipase activity in skeletal muscle and the concentration of serum ketone bodies increased, suggesting that the increase in fat oxidation caused by TGF-[beta]3 may have occurred in the liver and muscle. Intracranial injection of TGF-[beta]3 appeared to evoke a switch in the energy substrates accessed in energy expenditure. These results suggest that the release of TGF-[beta]3 in the brain by exercise is a signal for regulating energy consumption. spontaneous motor activity; respiratory exchange ratio; energy metabolism

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-[alpha] (TNF-[alpha]) inhibits myogenic differentiation via the activation of nuclear factor-[kappa]B (NF-[kappa]B). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-[alpha] causes a decrease in reduced glutathione (GSH) during myogenic differentiation of [C.sub.2][C.sub.12] cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-[alpha] on troponin I promoter activation and only partially restored creatine kinase activity in TNF-[alpha]-treated cells. In contrast, the administration of NAC before treatment with TNF-[alpha] almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-[alpha]-induced activation of NF-[kappa]B only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-[alpha] occurred independently, or downstream of NF-[kappa]B. Our observations suggest that the inhibitory effects of TNF-[alpha] on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression. inflammation; glutathione; nuclear factor-[kappa]B; myotube; myogenic differentiation

Behavioral tests, tightrope success, and exploratory activity in a T maze were conducted with male and female mice for 65 wk. Four groups were defined: the lower performance slow males and slow females and the higher performance fast males and fast females. Fast females showed the longest life span and the highest performance, and slow males showed the lowest performance and the shortest life span. Oxidative stress and mitochondrial electron transfer activities were determined in brain of young (28 wk), adult (52 wk), and old (72 wk) mice in a cross-sectional study. Brain thiobarbituric acid reactive substances (TBARS) were increased by 50% in old mice and were ~15% higher in males than in females and in slow than in fast mice. Brain Cu,Zn-superoxide dismutase (SOD) activity was increased by 52% and Mn-SOD by 108% in old mice. The activities of mitochondrial enzymes NADH-cytochrome c reductase, cytochrome oxidase, and citrate synthase were decreased by 14-58% in old animals. The cumulative toxic effects of oxyradicals are considered the molecular mechanism of the behavioral deficits observed on aging. neuromuscular impairment; NADH-cytochrome c reductase

The type 1 ryanodine receptor (RyR1) from rabbit skeletal muscle displayed two distinct degrees of response to cytoplasmic [Ca.sup.2+] [high- and low-open probability ([P.sub.o]) channels]. Here, we examined the effects of adenine nucleotides and caffeine on these channels and their modulations by sulfhydryl reagents. High-[P.sub.o] channels showed biphasic [Ca.sup.2+] dependence and were activated by adenine nucleotides and caffeine. Unexpectedly, low-[P.sub.o] channels did not respond to either modulator. The addition of a reducing reagent, dithiothreitol, to the cis side converted the high-[P.sub.o] channel to a state similar to that of the low-[P.sub.o] channel. Treatment with p-chloromercuriphenylsulfonic acid (pCMPS) transformed low-[P.sub.o] channels to a high-[P.sub.o] channel-like state with stimulation by [beta],[Gamma]-methylene-ATP and caffeine. In experiments under redox control using glutathione buffers, shift of the cis potential toward the oxidative state activated the low-[P.sub.o] channel, similar to that of the high-[P.sub.o] or the pCMPS-treated channel, whereas reductive changes inactivated the high-[P.sub.o] channel. Changes in trans redox potential, in contrast, did not affect channel activity of either channel. In all experiments, channels with higher [P.sub.o] were stimulated to a great extent by modulators, but ones with lower [P.sub.o] were unresponsive. These results suggest that redox states of critical sulfhydryls located on the cytoplasmic side of the RyR1 may alter both gating properties of the channel and responsiveness to channel modulators. calcium-induced calcium release; adenine nucleotide; caffeine; sulfhydryl reagents; redox potential

Birds have high metabolic rates, body temperatures, and plasma glucose concentrations yet physiologically age at a rate slower than comparably sized mammals. These studies were designed to test the hypothesis that the antioxidant uric acid protects birds against oxidative stress. Mixed sex broiler chicks (3 wk old) were fed diets supplemented or not with purines (0.6 mol hypoxanthine or inosine). Study 1 consisted of 18 female Cobb x Cobb broilers that were fed purines for 7 days, whereas study 2 consisted of 12 males in a 21-day trial. Study 3 involved 30 mixed sex broilers that were fed 40 or 50 mg allopurinol/kg body mass (BM) for 21 days, a drug that lowers plasma uric acid (PUA). PUA and leukocyte oxidative activity (LOA) were determined weekly for all studies. For study 2, pectoralis major shear force, relative kidney and liver sizes (RKS and RLS), and plasma glucose concentrations were also determined. In study 1, PUA concentration was increased three- and twofold (P < 0.001) in birds fed inosine or hypoxanthine, respectively, compared with control birds. LOA of birds supplemented with inosine was lower (P < 0.05) than that of control or hypoxanthine birds. In study 2, PUA concentrations were increased fivefold (P < 0.001) in birds fed inosine and twofold (P < 0.001) in birds fed hypoxanthine compared with control birds at day 21. RKS (g/kg BM) was greater (P < 0.001) for chicks fed purine diets compared with control chicks. Muscle shear value was lower (P < 0.05) in chicks fed purine diets. PUA concentration was decreased (P < 0.001) in birds consuming allopurinol diets, whereas LOA was increased (P < 0.01) in study 3. These studies show that PUA concentrations can be related to oxidative stress in birds, which can be linked to tissue aging. purines; allopurinol

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 [micro]M Cu[Cl.sub.2] had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors. ferrireductase; divalent metal transporter-1; hephaestin; ferroportin-1; duodenal cytochrome b

High carbohydrate availability increases LCFA uptake and decreases LCFA oxidation in perfused muscle. Am J Physiol Endocrinol Metab 282: E177-183, 2002; 10.1152/ajpendo.00284.2001.--To determine whether changes in long-chain fatty acid (LCFA) oxidative metabolism induced by elevated intracellular carbohydrate availability are due to changes in LCFA uptake or in mitochondrial transport capacity, rat hindquarters were perfused with 500 [micro]M palmitate and [1-[sup.]14]C]palmitate or [1-[sup.14]C]octanoate as well as with either low (LG) or high (HG) carbohydrate availability. Glucose uptake was higher in the HG vs. LG group (23.6 [+ or -] 1.5 vs 4.7 [+ or -] 0.9 [micro]mol * [g.sup.-1] * [h.sup.-1], P < 0.05). Palmitate delivery was not significantly different between groups and averaged 97.1 [+ or -] 4.6 nmol * [min.sup.-1] * [g.sup.-1] (P > 0.05). Fractional and total palmitate uptake values were 60% higher (P < 0.05) in the HG (0.125 [+ or -] 0.012 and 7.4 [+ or -] 1.2 nmol * [min.sup.-1] * [g.sup.-1]) vs. LG (0.079 [+ or -] 0.009 and 11.8 [+ or -] 1.5 nmol * [min.sup.-1] * [g.sup.-1]) group. Values of percent and total palmitate oxidized were significantly lower (P < 0.05) in the HG (9.1 [+ or -] 1.1% and 1.31 [+ or -] 0.16 nmol * [min.sup.-1] * [g.sup.-1]) vs. LG (23.4 [+ or -] 5.2% and 0.76 [+ or -] 0.08 nmol * [min.sup.-1] * [g.sup.-1]) group. Conversely, values of fractional uptake and percent oxidation of octanoate were not significantly different between groups (P > 0.05). Malonyl-CoA levels were inversely correlated with LCFA oxidation (P < 0.05). These results demonstrate that high carbohydrate availability in muscle is associated with a decrease in LCFA oxidation that is not due to a parallel decrease in LCFA uptake; rather, the decrease in LCFA oxidation could be due to malonyl-CoA inhibition of mitochondrial LCFA transport. long-chain fatty acid; medium-chain fatty acid; malonyl-co-enzyme A; carbohydrate; free fatty acid metabolism; free fatty acid oxidation; free fatty acid uptake

Lipid oxidation fuels recovery from exhaustive exercise in white muscle of rainbow trout. Am J Physiol Regulatory Integrative Comp Physiol 282: R89-R99, 2002; 10.1152/ajpregu.00238.2001.--The oxidative utilization of lipid and carbohydrate was examined in white muscle of rainbow trout (Oncorhynchus mykiss) at rest, immediately after exhaustive exercise, and for 32-h recovery. In addition to creatine phosphate and glycolysis fueling exhaustive exercise, near maximal activation of pyruvate dehydrogenase (PDH) at the end of exercise points to oxidative phosphorylation of carbohydrate as an additional source of ATP during exercise. Within 15 min postexercise, PDH activation returned to resting values, thus sparing accumulated lactate from oxidation. Glycogen synthase activity matched the rate of glycogen resynthesis and represented near maximal activation. Decreases in white muscle free carnitine, increases in long-chain fatty acyl carnitine, and sustained elevations of acetyl-CoA and acetyl carnitine indicate a rapid utilization of lipid to supply ATP for recovery. Increases in malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palmitoyltransferase-1 in trout muscle during recovery, but instead it may act to elongate short-chain fatty acids for mitochondrial oxidation. In addition, decreases in intramuscular triacylglycerol and in plasma nonesterified fatty acids indicate that both endogenous and exogenous lipid fuels may be oxidized during recovery. pyruvate dehydrogenase; glycogen synthase; carbohydrate; lactate; metabolism; malonyl-coenzyme A; nonesterified fatty acids

Reduced synthesis of NO causes marked alterations in myocardial substrate metabolism in conscious dogs. Am J Physiol Endocrinol Metab 282: E197-E206, 2002.--To test whether the acute reduction of nitric: oxide (NO) synthesis causes changes in cardiac substrate metabolism and in the activity of key enzymes of fatty acid and glucose oxidation, we blocked NOS by giving [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME; 35 mg/kg iv two times) to nine chronically instrumented dogs. [[sup.3]H]oleate, [[sup.14]C]glucose, and [[sup.13]C]lactate were infused to measure the rate of cardiac substrate uptake and oxidation. Glyceraldehyde-3-phosphate dehydrogenase, acetyl-CoA carboxylase, and malonyl-CoA decarboxylase activities were measured in myocardial biopsies. In eight control dogs, ANG II was infused (20-40 ng * [kg.sup.-1] * [min.sup.-1]) to mimic the hemodynamic effects of L-NAME. After L-NAME, significant changes occurred for fatty acid oxidation (from 9.8 [+ or -] 0.8 to 7.1 [+ or -] 1.2 [micro][mol/min), glucose uptake (from 12.9 [+ or -] 5.5 to 45.0 [+ or -] 14.2 [micro]mol/min), and oxidation (from 4.4 [+ or -] 1.2 to 19.9 [+ or -] 2.3 [micro]mol/min). ANG caused only a significantly lower increase in glucose oxidation. Lactate uptake increased by more than twofold in both groups. The enzyme activities did not differ significantly between the two groups. In conclusion, the acute inhibition of NO synthesis causes marked metabolic alterations that do not involve key rate-controlling enzymes of fatty acid oxidation nor glyceraldehyde-3-phosphate dehydrogenase. fatty acids; glucose; lactate; oxidation; heart

Hemodialysis stimulates muscle and whole body protein loss and alters substrate oxidation. Am J Physiol Endocrinol Metab 282: E107-E116, 2002.--The hemodialysis (HD) procedure has been implicated as a potential catabolic factor predisposing the chronic HD (CHD) patients to protein calorie malnutrition. To assess the potential effects of HD on protein and energy metabolism, we studied 11 CHD patients 2 h before, during, and 2 h after HD by use of primed constant infusion of L-[1-[sup.13]C]leucine and L-[ring-[sup.2][H.sub.5]]phenylalanine. Our results showed that HD led to increased whole body (10%) and muscle protein (133%) proteolysis. Simultaneously, whole body protein synthesis did not change, and forearm synthesis increased (120%). The net result was increased net whole body protein loss (96%) and net forearm protein loss (164%). During the 2-h post-HD period, the muscle protein breakdown trended toward baseline, whereas whole body protein breakdown increased further. Substrate oxidation during the post-HD was significantly altered, with diminished carbohydrate and accelerated lipid and amino acid oxidation. These data demonstrate that hemodialysis is an overall catabolic event, decreasing the circulating amino acids, accelerating rates of whole body and muscle proteolysis, stimulating muscle release of amino acids, and elevating net whole body and muscle protein loss. catabolism; malnutrition; inflammation; energy expenditure; metabolism

Effect of acute exercise on uncoupling protein 3 is a fat metabolism-mediated effect. Am J Physiol Endocrinol Metab 282: E11-E17, 2002.--Human and rodent uncoupling protein (UCP)3 mRNA is upregulated after acute exercise. Moreover, exercise increases plasma levels of free fatty acid (FFA), which are also known to upregulate UCP3. We investigated whether the upregulation of UCP3 after exercise is an effect of exercise per se or an effect of FFA levels or substrate oxidation. Seven healthy untrained men [age: 22.7 [+ or -] 0.6 yr; body mass index: 23.8 [+ or -] 1.0 kg/[m.sup.2]; maximal [O.sub.2] uptake (V[O.sub.2 max]): 3,852 [+ or -] 211 ml/min] exercised at 50% V[O.sub.2 max] for 2 h and then rested for 4 h. Muscle biopsies and blood samples were taken before and immediately after 2 h of exercise and 1 and 4 h in the postexercise period. To modulate plasma FFA levels and fat/glucose oxidation, the experiment was performed two times, one time with glucose ingestion and one time while fasting. UCP3 mRNA and UCP3 protein were determined by RT-competitive PCR and Western blot. In the fasted state, plasma FFA levels significantly increased (P < 0.0001) during exercise (293 [+ or -] 25 vs. 1,050 [+ or -] 127 [micro]mol/l), whereas they were unchanged after glucose ingestion (335 [+ or -] 54 vs. 392 [+ or -] 74 [micro]mol/l). Also, fat oxidation was higher after fasting (P < 0.05), whereas glucose oxidation was higher after glucose ingestion (P < 0.05). In the fasted state, UCP3L mRNA expression was increased significantly (P < 0.05) 4 h after exercise (4.6 [+ or -] 1.2 vs. 9.6 [+ or -] 3.3 amol/[micro]g RNA). This increase in UCP3L mRNA expression was prevented by glucose ingestion. Acute exercise had no effect on UCP3 protein levels. In conclusion, we found that acute exercise had no direct effect on UCP3 mRNA expression. Abolishing the commonly observed increase in plasma FFA levels and/or fatty acid oxidation during and after exercise prevents the upregulation of UCP3 after acute exercise. Therefore, the previously observed increase in UCP3 expression appears to be an effect of prolonged elevation of plasma FFA levels and/or increased fatty acid oxidation. fat oxidation; uncoupling protein; fatty acids; mitochondria

A study was conducted to examine the redox changes in heme a + a3 and copper in cerebral tissue as related to the mitochondrial energy state. Near-infrared spectroscopy was performed to measure the redox behavior of copper. The findings illustrate a similarity between the redox behavior of copper and the reduction of heme a3 and heme a. The results indicate that rapid reduction of heme a can precede decreases in adenosine triphosphate levels which can aid in monitoring tissue oxygenation and energy states.

Analysis of the influences of glucose-to-lipid ratio and lipid type on substrate oxidation rate in post-operative patients reveals that large quantities of lipid, a mixture of long and medium-chain triglycerides (MCT/LCT) or long chain triglycerides LCT, do not affect oxidation rate in post-operative patients. Enhancing the level of lipids in parenteral nutrition for 24-h periods causes a minimal increase in lipid oxidation rate and does not reduce the minute ventilation when total caloric uptake is fixed at the estimated energy expenditure.

The effects of exercise on glycerol kinetics were studied in running African goats. Fatty acid (FA) mobilization and oxidation were monitored and compared with low aerobic controls and published data on aerobic canines and humans. High and low aerobic subjects demonstrated identical FA mobilization at identical maximal oxygen consumption (VO2max) levels suggesting that role of FA in body energy production is independent of the VO2max.

A dual role of cysteine as a generator of hydrogen peroxide and as modulator of its effects is demonstrated using fluorescence cytometry. Exposure of cysteine to catalytically active metal sucah as copper promotes autoxidation which leads to the generation of active oxygen species. Pyruvate was demonstrated to cause a diminution of this response. Further studies revealthat cysteine can reduce active oxygen-induced toxicity by binding metals that active oxygen affect.

A surface fluorescence technique was employed in monitoring the reduction status of NAD(P)H in isolated rabbit hearts. The conditions tested are substrate limitation, alteredafterload and faster heart rate. Only oxygen debt resulting from elevated heartrate without concommitant increase in oxygen supply caused increased fluorescence of NAD(P)H. This suggests that the level reducing equivalent in the electron transport chain parallels that of oxygen supply.

15-[F.sub.2t]-isoprostane (15-[F.sub.2t]-IsoP), also termed 8-isoprostaglandin [F.sub.2[Alpha]], is one of a series of prostanoids formed by free radical-mediated peroxidation of arachidonic acid and exerts potent biological actions such as vasoconstriction. We recently demonstrated that 15-[F.sub.2t]-IsoP is metabolized in humans to a major metabolite, 2,3-dinor-5,6-dihydro-15-[F.sub.2t]-IsoP (15-[F.sub.2t]-IsoP-M). 15-[F.sub.2t]-IsoP-M can also potentially be formed as a product of free radical-induced oxidation of the low abundance fatty acid [Gamma]-linolenic acid. We confirmed that 15-[F.sub.2t]-IsoP-M is generated during oxidation of [Gamma]-linolenic acid and explored whether it may exhibit biological activity. 15-[F.sub.2t]-IsoP-M caused marked constriction of porcine surface retinal and intraparenchymal brain microvessels, comparable to that observed with 15-[F.sub.2t]-IsoP. These effects were associated with increased thromboxane [A.sub.2] ([TXA.sub.2]) formation and were virtually abolished by [TXA.sub.2]-synthase and -receptor inhibitors (CGS-12970 and L-670596). Vasoconstriction induced by either 15-[F.sub.2t]-IsoP or 15-[F.sub.2t]-IsoP-M on perfused ocular choroid was also abrogated by [TXA.sub.2]-synthase inhibition as well as by removal of endothelium. Similar to 15-[F.sub.2t]-IsoP, 15-[F.sub.2t]-IsoP-M evoked vasoconstriction and [TXA.sub.2] generation by activating [Ca.sub.2+] influx from nonvoltage-gated channels (SK&F96365 sensitive) in the retina and from both nonvoltage-and N-type voltage-gated [Ca.sub.2+] channels ([Omega]-conotoxin MVIIA sensitive), respectively, in brain endothelial and astroglial cells; smooth muscle cells were unresponsive to both agents. Cross-desensitization experiments further suggest that 15-[F.sub.2t]-IsoP and 15-[F.sub.2t]-IsoP-M act on the same receptor mechanism. Findings reveal a novel concept by which a [Beta]-oxidation metabolite of 15-[F.sub.2t]-IsoP that can also be formed by nonenzymatic oxidation of [Gamma]-linolenic acid is equivalently bioactive to 15-[F.sub.2t]-IsoP and may prolong the vascular actions of [F.sub.2]-IsoPs. calcium; thromboxane; peroxidation

Bassenge, Eberhard, Olaf Sommer, Michael Schwemmer, and Rolf Bunger. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state. Am J Physiol Heart Circ Physiol 279: H2431-H2438, 2000.--Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], implying the NADH concentration-to-[NAD.sup.+] concentration ratio ([NADH]/[[NAD.sup.+]]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and post-ischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[[NAD.sup.+]]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 [+ or -] 2%, 58 [+ or -] 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate. reduced nicotinamide adenine dinucleotide oxidase; heart; ischemia-reperfusion

Springett, R., J. Newman, M. Cope, and D. T. Delpy. Oxygen dependency and precision of cytochrome oxidase signal from full spectral NIRS of the piglet brain. Am J Physiol Heart Circ Physiol 279: H2202-H2209, 2000.--Oxidation changes of the copper A (CUA) center of cytochrome oxidase in the brain were measured during brief anoxic swings at both normocapnia and hypercapnia (arterial [Pco.sub.2] =55 mmHg). Hypercapnia increased total hemoglobin from 37.5 [+ or -] 9.1 to 50.8 [+ or -] 12.9 [micro]mol/l (means [+ or -] SD; n = 7), increased mean cerebral saturation [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from 65 [+ or -] 4 to 77 [+ or -] 3%, and oxidized [Cu.sub.A] by 0.43 + 0.23 [micro]mol/l. During the onset of anoxia, there were no significant changes in the [Cu.sub.A] oxidation state until [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] had fallen to 43 [+ or -] 5 and 21 [+ or -] 6% at normocapnia and hypercapnia, respectively, and the maximum reduction during anoxia was not significantly different at hypercapnia (1.49 [+ or -] 0.40 [micro]mol/l) compared with normocapnia (1.53 [+ or -] 0.44 [micro]mol/l). Residuals of the least squares fitting algorithm used to convert near-infrared spectra to concentrations are presented and shown to be small compared with the component of attenuation attributed to the [Cu.sub.A] signal. From these observations, we conclude that there is minimal interference between the hemoglobin and [Cu.sub.A] signals in this model, the [Cu.sub.A] oxidation state is independent of cerebral oxygenation at normoxia, and the oxidation after hypercapnia is not the result of increased cerebral oxygenation. near-infrared spectroscopy; cytochrome oxidase; hypercapnia

Sakamoto, Jun, Rick L. Barr, Katherine M. Kavanagh, and Gary D. Lopaschuk. Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart. Am J Physiol Heart Circ Physiol 278: H1196-H1204, 2000.--Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [[sup.14]C]glucose and 0.4 mM [[sup.3]H]palmitate (low-fat/low-glucose buffer) or 20 mM [[sup.14]C] glucose and 1.2 mM [[sup.3]H] palmitate (high-fat/high-glucose buffer) [+ or -] 100 [micro]U/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 [+ or -] 85 to 1,150 [+ or -] 147 nmol [multiplied by] g dry [wt.sup.-1] [multiplied by] [min.sup.-1] (P [is less than] 0.05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P [is less than] 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P [is less than] 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low-fat/low-glucose buffer (9 [+ or -] 1 nmol [multiplied by] g dry [wt.sup.-1] [min.sup.-1]) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl-CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts. adenosine 5'-monophosphate-activated protein kinase; acetylcoenzyme A carboxylase; glucose oxidation; malonyl-coenzyme A; carnitine palmitoyltransferase 1