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BackgroundNeurocognitive impairment (NCI) is associated with monocyte activation in people with HIV (PWH). Activated monocytes increase glycolysis, reduce oxidative phosphorylation, and accumulate citrate and succinate, tricarboxylic acid (TCA) cycle metabolites that promote inflammation-this metabolic shift may contribute to NCI and slowed gait speed in PWH.MethodsPlasma citrate and succinate were assayed by liquid chromatography-mass spectrometry from 957 participants upon entry to a multicenter, prospective cohort of older PWH. Logistic, linear, and mixed-effects linear regression models were used to examine associations between entry/baseline TCA cycle metabolites and cross-sectional and longitudinal NCI, neuropsychological test scores (NPZ-4), and gait speed.ResultsMedian age was 51 (range 40-78) years. Each 1 standard deviation (SD) citrate increment was associated with 1.18 higher odds of prevalent NCI at baseline (P = .03), 0.07 SD lower time-updated NPZ-4 score (P = .01), and 0.02 m/s slower time-updated gait speed (P < .0001). Age accentuated these effects. In the oldest age-quartile, higher citrate was associated with 1.64 higher odds of prevalent NCI, 0.17 SD lower NPZ-4, and 0.04 m/s slower gait speed (P ≤ .01 for each). Similar associations were apparent with succinate in the oldest age-quintile, but not with gait speed. In participants without NCI at entry, higher citrate predicted a faster rate of neurocognitive decline.ConclusionsHigher plasma citrate and succinate are associated with worse cross-sectional and longitudinal measures of neurocognitive function and gait speed that are age-dependent, supporting the importance of altered bioenergetic metabolism in the pathogenesis of NCI in older PWH.

Very little is known about how metabolic health status, insulin resistance or metabolic challenges modulate the endocannabinoid (eCB) or polyunsaturated fatty acid (PUFA)-derived oxylipin (OxL) lipid classes. To address these questions, plasma eCB and OxL concentrations were determined at rest, 10 and 20 min during an acute exercise bout (30 min total, ~45% of preintervention V̇O2peak , ~63 W), and following 20 min recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled diet conditions. We hypothesized that increased fitness and insulin sensitivity following a ~14-week training and weight loss intervention would lead to significant changes in lipid signatures using an identical acute exercise protocol to preintervention. In the first 10 min of exercise, concentrations of a suite of OxL diols and hydroxyeicosatetraenoic acid (HETE) metabolites dropped significantly. There was no increase in 12,13-DiHOME, previously reported to increase with exercise and proposed to activate muscle fatty acid uptake and tissue metabolism. Following weight loss intervention, exercise-associated reductions were more pronounced for several linoleate and alpha-linolenate metabolites including DiHOMEs, DiHODEs, KODEs, and EpODEs, and fasting concentrations of 9,10-DiHODE, 12,13-DiHODE, and 9,10-DiHOME were reduced. These findings suggest that improved metabolic health modifies soluble epoxide hydrolase, cytochrome P450 epoxygenase (CYP), and lipoxygenase (LOX) systems. Acute exercise led to reductions for most eCB metabolites, with no evidence for concentration increases even at recovery. It is proposed that during submaximal aerobic exercise, nonoxidative fates of long-chain saturated, monounsaturated, and PUFAs are attenuated in tissues that are important contributors to the blood OxL and eCB pools.

Insulin resistance has wide-ranging effects on metabolism, but there are knowledge gaps regarding the tissue origins of systemic metabolite patterns and how patterns are altered by fitness and metabolic health. To address these questions, plasma metabolite patterns were determined every 5 min during exercise (30 min, ∼45% of V̇o2peak, ∼63 W) and recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled conditions of diet and physical activity. We hypothesized that improved fitness and insulin sensitivity following a ∼14-wk training and weight loss intervention would lead to fixed workload plasma metabolomics signatures reflective of metabolic health and muscle metabolism. Pattern analysis over the first 15 min of exercise, regardless of pre- versus postintervention status, highlighted anticipated increases in fatty acid tissue uptake and oxidation (e.g., reduced long-chain fatty acids), diminution of nonoxidative fates of glucose [e.g., lowered sorbitol-pathway metabolites and glycerol-3-galactoside (possible glycerolipid synthesis metabolite)], and enhanced tissue amino acid use (e.g., drops in amino acids; modest increase in urea). A novel observation was that exercise significantly increased several xenometabolites ("non-self" molecules, from microbes or foods), including benzoic acid-salicylic acid-salicylaldehyde, hexadecanol-octadecanol-dodecanol, and chlorogenic acid. In addition, many nonannotated metabolites changed with exercise. Although exercise itself strongly impacted the global metabolome, there were surprisingly few intervention-associated differences despite marked improvements in insulin sensitivity, fitness, and adiposity. These results and previously reported plasma acylcarnitine profiles support the principle that most metabolic changes during submaximal aerobic exercise are closely tethered to absolute ATP turnover rate (workload), regardless of fitness or metabolic health status.

New findingsWhat is the central question of this study? Does improved metabolic health and insulin sensitivity following a weight-loss and fitness intervention in sedentary, obese women alter exercise-associated fuel metabolism and incomplete mitochondrial fatty acid oxidation (FAO), as tracked by blood acylcarnitine patterns? What is the main finding and its importance? Despite improved fitness and blood sugar control, indices of incomplete mitochondrial FAO increased in a similar manner in response to a fixed load acute exercise bout; this indicates that intramitochondrial muscle FAO is inherently inefficient and is tethered directly to ATP turnover. With insulin resistance or type 2 diabetes mellitus, mismatches between mitochondrial fatty acid fuel delivery and oxidative phosphorylation/tricarboxylic acid cycle activity may contribute to inordinate accumulation of short- or medium-chain acylcarnitine fatty acid derivatives [markers of incomplete long-chain fatty acid oxidation (FAO)]. We reasoned that incomplete FAO in muscle would be ameliorated concurrent with improved insulin sensitivity and fitness following a ∼14 week training and weight-loss intervention in obese, sedentary, insulin-resistant women. Contrary to this hypothesis, overnight-fasted and exercise-induced plasma C4-C14 acylcarnitines did not differ between pre- and postintervention phases. These metabolites all increased robustly with exercise (∼45% of pre-intervention peak oxygen consumption) and decreased during a 20 min cool-down. This supports the idea that, regardless of insulin sensitivity and fitness, intramitochondrial muscle β-oxidation and attendant incomplete FAO are closely tethered to absolute ATP turnover rate. Acute exercise also led to branched-chain amino acid acylcarnitine derivative patterns suggestive of rapid and transient diminution of branched-chain amino acid flux through the mitochondrial branched-chain ketoacid dehydrogenase complex. We confirmed our prior novel observation that a weight-loss/fitness intervention alters plasma xenometabolites [i.e. cis-3,4-methylene-heptanoylcarnitine and γ-butyrobetaine (a co-metabolite possibly derived in part from gut bacteria)], suggesting that host metabolic health regulated gut microbe metabolism. Finally, we considered whether acylcarnitine metabolites signal to muscle-innervating afferents; palmitoylcarnitine at concentrations as low as 1-10 μm activated a subset (∼2.5-5%) of these neurons ex vivo. This supports the hypothesis that in addition to tracking exercise-associated shifts in fuel metabolism, muscle acylcarnitines act as signals of exertion to short-loop somatosensory-motor circuits or to the brain.

Novel plasma metabolite patterns reflective of improved metabolic health (insulin sensitivity, fitness, reduced body weight) were identified before and after a 14-17 wk weight loss and exercise intervention in sedentary, obese insulin-resistant women. To control for potential confounding effects of diet- or microbiome-derived molecules on the systemic metabolome, sampling was during a tightly-controlled feeding test week paradigm. Pairwise and multivariate analysis revealed intervention- and insulin-sensitivity associated: (1) Changes in plasma xeno-metabolites ("non-self" metabolites of dietary or gut microbial origin) following an oral glucose tolerance test (e.g. higher post-OGTT propane-1,2,3-tricarboxylate [tricarballylic acid]) or in the overnight-fasted state (e.g., lower γ-tocopherol); (2) Increased indices of saturated very long chain fatty acid elongation capacity; (3) Increased post-OGTT α-ketoglutaric acid (α-KG), fasting α-KG inversely correlated with Matsuda index, and altered patterns of malate, pyruvate and glutamine hypothesized to stem from improved mitochondrial efficiency and more robust oxidation of glucose. The results support a working model in which improved metabolic health modifies host metabolism in parallel with altering systemic exposure to xeno-metabolites. This highlights that interpretations regarding the origins of peripheral blood or urinary "signatures" of insulin resistance and metabolic health must consider the potentially important contribution of gut-derived metabolites toward the host's metabolome.

Mitochondrial homeostasis is critical for tissue health, and mitochondrial dysfunction contributes to numerous diseases, including heart failure. Here, we have shown that the transcription factor Kruppel-like factor 4 (KLF4) governs mitochondrial biogenesis, metabolic function, dynamics, and autophagic clearance. Adult mice with cardiac-specific Klf4 deficiency developed cardiac dysfunction with aging or in response to pressure overload that was characterized by reduced myocardial ATP levels, elevated ROS, and marked alterations in mitochondrial shape, size, ultrastructure, and alignment. Evaluation of mitochondria isolated from KLF4-deficient hearts revealed a reduced respiration rate that is likely due to defects in electron transport chain complex I. Further, cardiac- specific, embryonic Klf4 deletion resulted in postnatal premature mortality, impaired mitochondrial biogenesis, and altered mitochondrial maturation. We determined that KLF4 binds to, cooperates with, and is requisite for optimal function of the estrogen-related receptor/PPARγ coactivator 1 (ERR/PGC-1) transcriptional regulatory module on metabolic and mitochondrial targets. Finally, we found that KLF4 regulates autophagy flux through transcriptional regulation of a broad array of autophagy genes in cardiomyocytes. Collectively, these findings identify KLF4 as a nodal transcriptional regulator of mitochondrial homeostasis., Introduction The heart has an unrelenting need for energy throughout life to sustain contractile function. Despite its high-energy demand, the heart contains relatively low ATP reserves, and thus, a continual [...]

We measured the loss of cardiac mitochondrial function related to aging in males of three rat strains presenting with different longevity and aging phenotypes: the Fischer 344 (F344), the Brown Norway (BN), and the hybrid F344xBN. The F344 rat has a short life span and a ~45% decrease in coupled mitochondrial oxidation in the cardiac permeabilized fibers from the old rats compared with the young rats. Citrate synthase activity in the permeabilized fibers (mitochondrial content) did not change significantly with aging. The BN live longer compared with the F344 and have a 15%-18% loss of mitochondrial respiration in the aged rats compared with the young rats. The differences are not significant. In hybrids, more resistant to aging than are the BN and the F344, mitochondrial function is preserved during aging. The difference in longevity of the different strains is correlated with mitochondrial dysfunction in the heart, suggesting the importance of mitochondria in cardiac aging. Key Words: Aging--Mitochondrial--Oxidative phosphorylation--Rat--Strains. doi: 10.1093/gerona/glq141

Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/ genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty acylcarnitines reflective of incomplete LCFA [beta]-oxidation in the latter (i.e. summed C 10- to C14-carnitine concentrations were ~300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin Alc (r = 0.544, P < 0.0001; and r = -0.308, P < 0.05, respectively) and with increasing total plasma acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 acylcarnitines significantly stimulated nuclear factor [kappa]-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA [beta]-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance.

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 rain global ischemia or following 40 min of perfusion alone as controls. [H.sub.2][O.sub.2] release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of [H.sub.2][O.sub.2] was increased by 178 [+ or -] 14% and 179 [+ or -] 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, [H.sub.2][O.sub.2] increased by 272 [+ or-] 22% and 171 [+ or -] 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased [H.sub.2][O.sub.2] production by 179 [+ or -] 24% and 155 [+ or -] 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of [H.sub.2][O.sub.2] from complex IH. Thus ischemic damage to the ETC increased both the capacity and the net production of [H.sub.2][O.sub.2] from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury. nicotinamide adenine dinucleotide:ubiquinone oxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III), cytochrome c; ischemia

Lipid accumulation in nonadipose tissue due to enhanced circulating fatty acids may play a role in the pathophysiology of heart failure, obesity, and diabetes. Accumulation of myocardial lipids and related intermediates, e.g., ceramide, is associated with decreased contractile function, mitochondrial oxidative phosphorylation, and electron transport chain (ETC) complex activities. We tested the hypothesis that the progression of heart failure would be exacerbated by elevated myocardial lipids and an associated ceramide-induced inhibition of mitochondrial oxidative phosphorylation and ETC complex activities. Heart failure (HF) was induced by coronary artery ligation. Rats were then randomly assigned to either a normal (10% kcal from fat; HF, n = 8) or high saturated fat diet (60% kcal from saturated fat; HF + Sat, n = 7). Sham-operated animals (sham; n = 8) were fed a normal diet. Eight weeks postligation, left ventricular (LV) function was assessed by echocardiography and catheterization. Subsarcolemmal and interfibrillar mitochondria were isolated from the LV. Heart failure resulted in impaired LV contractile function [decreased percent fractional shortening and peak rate of LV pressure rise and fall ([+ or -]dP/dt)] and remodeling (increased end-diastolic and end-systolic dimensions) in HF compared with sham. No further progression of LV dysfunction was evident in HF + Sat. Mitochondrial state 3 respiration was increased in HF + Sat compared with HF despite elevated myocardial ceramide. Activities of ETC complexes II and IV were elevated in HF + Sat compared with HF and sham. High saturated fat feeding following coronary artery ligation was associated with increased oxidative phosphorylation and ETC complex activities and did not adversely affect LV contractile function or remodeling, despite elevations in myocardial ceramide. oxidative phosphorylation; electron transport chain; ceramide; lipotoxicity

A study demonstrates that mutations in the BSC1L gene cause the Bjornstad syndrome by disrupting the assembly of mitochondrial respirasomes, which are the basic unit for respiration in human mitochondria. Mutations that cause the Bjornstad syndrome illustrate the acute sensitivity of ear and hair tissues to mitochondrial function, particularly to the production of reactive oxygen species.

Mitochondria are increasingly recognized as lynchpins in the evolution of cardiac injury during ischemia and reperfusion. This review addresses the emerging concept that modulation of mitochondrial respiration during and immediately following an episode of ischemia can attenuate the extent of myocardial injury. The blockade of electron transport and the partial uncoupling of respiration are two mechanisms whereby manipulation of mitochondrial metabolism during ischemia decreases cardiac injury. Although protection by inhibition of electron transport or uncoupling of respiration initially appears to be counterintuitive, the continuation of mitochondrial oxidative phosphorylation in the pathological milieu of ischemia generates reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c. The initial target of these deleterious mitochondrial-driven processes is the mitochondria themselves. Consequences to the cardiomyocyte, in turn, include oxidative damage, the onset of mitochondrial permeability transition, and activation of apoptotic cascades, all favoring cardiomyocyte death. Ischemia-induced mitochondrial damage carried forward into reperfusion further amplifies these mechanisms of mitochondrial-driven myocyte injury. Interruption of mitochondrial respiration during early reperfusion by pharmacologic blockade of electron transport or even recurrent hypoxia or brief ischemia paradoxically decreases cardiac injury. It increasingly appears that the cardioprotective paradigms of ischemic preconditioning and postconditioning utilize modulation of mitochondrial oxidative metabolism as a key effector mechanism. The initially counterintuitive approach to inhibit mitochondrial respiration provides a new cardio-protective paradigm to decrease cellular injury during both ischemia and reperfusion. cardiolipin; cytochrome c; complex I; cytochrome oxidase

A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cationexchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/ reversed-phase chromatography using a single non-endcapped C8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversedphase HPLC results in separation of reagent byproducts from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid-phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by MS and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.

Moderate severity heart failure does not involve a down-regulation of myocardial fatty acid oxidation. Am J Physiol Heart Circ Physiol 287: H1538-H1543. 2004. First published June 10, 2004; 10.1152/ajpheart.00281.2004.--Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of tree fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,[10-.sup.3]H]oteate, [[U-.sup.14]C]glucose, and [[1-.sup.13]C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity tot malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomemon. cardiac: metabolism; malonyl CoA

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrorne c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion. cytochrome c; oxygen radicals; phospholipids

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-[sup.13][C.sub.3]]propionate (0.25 mM) was infused in a coronary artery branch for I h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 [+ or -] 3.3% and 0.078 [+ or -] 0.009 [micro]mol * [min.sup.-1] * [g.sup.-1]. Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 [+ or -] 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 [+ or -] 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart. tricarboxylic acid cycle; cataplerosis; mass isotopomer analysis; mass spectrometry