Mitochondrial Oxidative Capacity Research Articles

Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3−/−, Grx2−/−), and double-knockout mice (Grx2−/−/Sirt3−/−) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2−/−/Sirt3−/− mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2−/− and the Sirt3−/− we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3−/−. Similar changes were observed but not worsened in hearts from Grx2−/−/Sirt3−/− mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity

Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

Metabolic Characterization and Consequences of Mitochondrial Pyruvate Carrier Deficiency in Drosophila melanogaster.

In insect, pyruvate is generally the predominant oxidative substrate for mitochondria. This metabolite is transported inside mitochondria via the mitochondrial pyruvate carrier (MPC), but whether and how this transporter controls mitochondrial oxidative capacities in insects is still relatively unknown. Here, we characterize the importance of pyruvate transport as a metabolic control point for mitochondrial substrate oxidation in two genotypes of an insect model, Drosophila melanogaster, differently expressing MPC1, an essential protein for the MPC function. We evaluated the kinetics of pyruvate oxidation, mitochondrial oxygen consumption, metabolic profile, activities of metabolic enzymes, and climbing abilities of wild-type (WT) flies and flies harboring a deficiency in MPC1 (MPC1def). We hypothesized that MPC1 deficiency would cause a metabolic reprogramming that would favor the oxidation of alternative substrates. Our results show that the MPC1def flies display significantly reduced climbing capacity, pyruvate-induced oxygen consumption, and enzymatic activities of pyruvate kinase, alanine aminotransferase, and citrate synthase. Moreover, increased proline oxidation capacity was detected in MPC1def flies, which was associated with generally lower levels of several metabolites, and particularly those involved in amino acid catabolism such as ornithine, citrulline, and arginosuccinate. This study therefore reveals the flexibility of mitochondrial substrate oxidation allowing Drosophila to maintain cellular homeostasis.

Progress Update and Challenges on O2max Testing and Interpretation.

The maximal oxygen uptake (O2max) is the primary determinant of endurance performance in heterogeneous populations and has predictive value for clinical outcomes and all-cause mortality. Accurate and precise measurement of O2max requires the adherence to quality control procedures, including combustion testing and the use of standardized incremental exercise protocols with a verification phase preceded by an adequate familiarization. The data averaging strategy employed to calculate the O2max from the breath-by-breath data can change the O2max value by 4–10%. The lower the number of breaths or smaller the number of seconds included in the averaging block, the higher the calculated O2max value with this effect being more prominent in untrained subjects. Smaller averaging strategies in number of breaths or seconds (less than 30 breaths or seconds) facilitate the identification of the plateau phenomenon without reducing the reliability of the measurements. When employing metabolic carts, averaging intervals including 15–20 breaths or seconds are preferable as a compromise between capturing the true O2max and identifying the plateau. In training studies, clinical interventions and meta-analysis, reporting of O2max in absolute values and inclusion of protocols and the averaging strategies arise as imperative to permit adequate comparisons. Newly developed correction equations can be used to normalize O2max to similar averaging strategies. A lack of improvement of O2max with training does not mean that the training program has elicited no adaptations, since peak cardiac output and mitochondrial oxidative capacity may be increased without changes in O2max.

Temperament influences mitochondrial capacity in skeletal muscle from 8 through 18 mo of age in Brahman heifers.

Temperamental cattle tend to yield carcasses of poorer quality, and Brahman cattle are reportedly more temperamental than non-indicus cattle breeds. A potential link between temperament and product quality may be mitochondrial activity. We hypothesized that mitochondrial measures would be greater in temperamental compared with calm heifers and that the relationships between temperament and mitochondria would persist as heifers age. Serum cortisol and skeletal muscle (longissimus thoracis [LT] and trapezius [TRAP]) mitochondrial profiles and antioxidant activities were quantified from the same calm (n = 6) and temperamental (n = 6) Brahman heifers at 8, 12, and 18 mo of age. Data were analyzed using a mixed model ANOVA in SAS (9.4) with repeated measures. Serum cortisol was greater in temperamental compared with calm heifers throughout the study (P = 0.02). Mitochondrial volume density (citrate synthase [CS] activity) increased over time (P < 0.0001) but was similar between temperament and muscle groups. Mitochondrial function (cytochrome c oxidase activity) was greatest in the temperamental LT at 8 mo of age (P ≤ 0.0006), greatest in the temperamental TRAP at 18 mo of age (P ≤ 0.003), and did not differ by temperament at 12 mo of age. Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI+II), noncoupled electron transfer system capacity (ECI+II), and E with functional complex II only (ECII) were greater in the TRAP than LT for calm heifers at all ages (P ≤ 0.002), but were similar between muscle groups in temperamental heifers. Overall, calm heifers tended to have greater intrinsic (relative to CS activity) PCI and flux control of PCI+II (P ≤ 0.1) than temperamental heifers, indicating greater utilization of complex I paired with greater coupling efficiency in calm heifers. Within the LT, integrative PCI+II was greater (P = 0.05) and ECI+II tended to be greater (P = 0.06) in temperamental compared with calm heifers. From 8- to 18-mo old, glutathione peroxidase (GPx) activity decreased (P < 0.0001) and superoxide dismutase activity increased (P = 0.02), and both were similar between muscle groups. The activity of GPx was greater in temperamental compared with calm heifers at 8 (P = 0.004) but not at 12 or 18 mo of age. These results detail divergent skeletal muscle mitochondrial characteristics of live Brahman heifers according to temperament, which should be further investigated as a potential link between temperament and product quality.

Cancer-induced Cardiac Atrophy Adversely Affects Myocardial Redox State and Mitochondrial Oxidative Characteristics.

Determine how cancer-induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery and in-vitro oxidative characteristics. Lewis lung carcinoma (LLC) tumors were implanted in C57BL6/J mice and grown for 28days to induce cardiac atrophy. Endogenous metabolic species, and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC-conditioned media with(out) the antioxidant MitoTempo. Cells were analyzed for ROS, oxidative capacity, and hypoxic resistance. LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio (FAD/FAD+NADH) compared to PBS controls. When compared to PBS, LLC hearts showed ~50% greater COX-IV and VDAC, attributed to ~50% lower mitophagy markers. mt-mRNA translation machinery was elevated similarly to markers of mitochondrial content. mitochondrial DNA-encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx-3 and GPx-7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC-conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal, 75% at maximal), and greater susceptibility to hypoxia (~25%) -- which was reversed by MitoTempo. These results substantiate metabolic cardiotoxic effects attributable to tumor-associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity and hypoxia resistance.

Heat acclimation during low-intensity exercise increases and Hsp72, but not markers of mitochondrial biogenesis and oxidative phosphorylation, in skeletal tissue.

What is the central question of this study? Heat acclimation increases tolerance to exercise performed in the heat and may improve maximal oxygen uptake (VO2 max) and performance in temperate environments. However, it is unknown if HA affects the expression of proteins related to mitochondrial biogenesis and oxidative capacity in skeletal muscle. What is the main finding and its importance? We showed that heat acclimation increased VO2 max in a temperate environment but did not change markers of mitochondrial biogenesis and oxidative phosphorylation in the skeletal muscle. Heat acclimation (HA) increases tolerance to exercise performed in the heat and may improve maximal oxygen uptake ( ) in temperate environments. However, it is unknown if HA affects the expression of proteins related to mitochondrial biogenesis and oxidative capacity in skeletal muscle. The purpose of this study was to investigate the effect of HA on skeletal muscle markers of mitochondrial biogenesis and oxidative phosphorylation in recreationally trained adults. Thirteen (7 males and 6 females) individuals underwent 10days of HA. Participants performed two 45min bouts of exercise (walking at 30-40% maximal velocity at 3% grade) with 10min rest per session in a hot environment (∼42°C and 30-50% relative humidity). , ventilatory thresholds (VT), and protein expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (TFAM), calcium/calmodulin-dependent protein kinase (CaMK), electron transport chain (ETC) complexes I-IV, and heat shock protein 72 (Hsp72) in skeletal muscle were measured pre- and post-HA. Comparing day1 to day10, HA was confirmed by lower resting core temperature (Tcore ) (P=0.026), final Tcore (P<0.0001), mean heart rate (HR) (P=0.002), final HR (P=0.003), mean ratings of perceived exertion (RPE) (P=0.026) and final RPE (P=0.028). Pre- to post-HA (P=0.045) increased but VT1 (P=0.263) and VT2 (P=0.239) were unchanged. Hsp72 (P=0.007) increased, but skeletal muscle protein expression (PGC-1α, P=0.119; TFAM, P=0.763; CaMK, P=0.19; ETC I, P=0.629; ETC II, P=0.724; ETC III, P=0.206; ETC IV, P=0.496) were not affected with HA. HA during low-intensity exercise increased in a temperate environment and Hsp72 but it did not affect markers of mitochondrial biogenesis and oxidative phosphorylation in the skeletal muscle.

Cbl Negatively Regulates NLRP3 Inflammasome Activation through GLUT1-Dependent Glycolysis Inhibition.

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis.

Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals

ObjectiveSkeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. Twenty-four-hour rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans remains unknown. MethodsTwelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at 5 time points (8 AM, 1 PM, 6 PM, 11 PM, 4 AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry. ResultsMitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBα showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level, resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease during the night, indicating metabolic inflexibility. ConclusionsMitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed. ClinicalTrials.gov IDNCT03733743.

Mitochondrial division inhibitor 1 (mdivi-1) increases oxidative capacity and contractile stress generated by engineered skeletal muscle.

In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.

Skeletal Muscle Mitochondrial Capacity Is Similar In Ambulatory And Non-ambulatory Children With Cerebral Palsy

PURPOSE: Cerebral palsy (CP) is the most common childhood movement dysfunction secondary to a brain injury around birth. These children can be classified as ambulatory or non-ambulatory based on their functional abilities. Importantly, they expend significantly increased energy expenditure during movement. Muscle mitochondria, specifically the electron transport system are responsible for oxidative capacity, energy production and are associated with functional capacity. Unfortunately, mitochondrial oxidative capacity in children with CP and its association with different functional levels is not known. We measured maximum mitochondrial respiration rates directly from biopsies in children with CP, compared across ambulatory levels. METHODS: Twenty children (6-16 years, 10 M/6F, Ambulatory-12), undergoing surgery participated in this study. Twenty-nine biopsies were obtained from adductors, vastus lateralis, gastrocnemius. Carbohydrate and fatty acid respirometry substrate-uncoupler-inhibitor titration (SUIT) protocols were performed on permeabilized muscle fiber bundles. In addition, muscle homogenate was used to measure citrate synthase activity as a marker of mitochondrial content. Ambulatory capacity was measured using 6-minute walk tests (n=7), muscle strength, and gait velocity (n=15) during routine therapy visits. RESULTS: Surprisingly, maximal mitochondrial phosphorylation capacity was similar across between functionally ambulatory and non-ambulatory children (77.1± 23.9 vs. 84.9± 24.0 pmol O2/s/mg). This was uncorrelated with mitochondrial content (p>0.1), as measured by citrate synthase. Mitochondria in children with CP still preferentially used carbohydrates over fatty acids based on state-3 respiration. Functionally ambulatory children showed positive associations between mitochondrial function and ambulatory capacity measures (r2 values for gait velocity=0.50, 6-meter walk test=0.33, p< 0.05). CONCLUSIONS: Mitochondrial function are typically associated with activity level. Surprisingly, functionally ambulatory children with CP did not have greater mitochondrial function compared to non-ambulatory children and might even be lower. Importantly, within ambulatory children walking capacity was related to maximal mitochondrial function.

Does Heat Acclimation Upregulate Skeletal Muscle Markers Of Oxidative Metabolism And Mitochondrial Biogenesis?

Heat acclimation (HA) increases tolerance to exercise performed in the heat and may improve aerobic fitness and performance in highly trained individuals. However, it is unknown if HA effects the expression of proteins related to mitochondrial biogenesis and oxidative capacity in skeletal muscle in active young individuals. PURPOSE: To investigate the effect of HA on the expression of oxidative and mitochondrial proteins in the skeletal muscle of healthy, active young-adult males and females. METHODS: In a quasi-experimental design, thirteen (7 males, 6 females) aerobically fit (VO2max > 75th percentile) individuals underwent 10-days of HA over 14-days. Participants performed two 45-minute bouts of exercise (walking at 30-40% maximal velocity) with 10 minutes rest per session in a hot environment (dry temperature ~42°C, relative humidity 30-50%). Pre- and post-HA VO2max (room temperature), second ventilatory threshold (VT), protein expression of PGC-1α, TFAM, CaMK, Cytochromes I-IV, and HSP72 were measured pre-HA and post-HA. Heat acclimation was determined comparing heart rate (HR), core temperature (CT), sweat rate, and ratings of perceived exertion (RPE) on days 1 and 10. RESULTS: Participants were acclimated as indicated by pre- and post-HA resting-CT (37.3 ± 0.38 vs. 37.1 ± 0.28°C; p = 0.04), maximal-CT (38.8 ± 0.35 vs. 38.2 ± 0.38°C; p<0.0001), mean-HR (138.5 ± 17.7 vs. 125.9 ± 14.0 bpm; p=0.03), maximal-HR (170.2 ± 26.4 vs. 149.4 ± 24.5 bpm; p=0.003), mean-RPE (12.4 ± 1.2 vs. 9.8 ± 1.1; p< 0.0001), and maximal-RPE (15.0 ± 1.6 vs. 12.2 ± 1.9; p=0.002). Pre- to Post-HA VO2max (50.2 ± 8.6 vs. 51.7 ± 11.2 mL/kg/min; p=0.08) and VT (31.2 ± 7.0 vs. 33.9 ± 8.7; p=0.19) were unchanged. Protein expression was unchanged pre- to post-HA; (PGC-1α p=0.21, TFAM p=0.34, CaMK 0-0.19, Cytochromes I p=0.73; II p=0.85, III p=0.62, IV p=0.70, Hsp72 p=0.27). CONCLUSIONS: 10-days of low intensity exercise in the heat resulted in HA, but did not increase aerobic fitness or markers of mitochondrial biogenesis and oxidative metabolism in physically active individuals. Supported by a research grant from the College of Education, University of New Mexico.

Skeletal Muscle Mitochondrial Plasticity Is Reduced In Liver Transplant Recipients

PURPOSE Sarcopenic obesity is a hallmark of liver transplant recipients, of which the mechanisms are not yet fully understood. Decline in mitochondrial plasticity could play a mechanistic role in muscle wasting, loss of strength, and increased adiposity via impaired energy substrate utilization. The purpose of this study is to determine if skeletal muscle mitochondrial plasticity is reduced in liver transplant (LT) recipients. METHODS In this prospective cohort study, 15 LT patients (Mean±SD; Age: 58±10 years; months since transplant: 55±81 months) and 10 age matched healthy controls (Age: 55±4 years), underwent assessment of mitochondrial oxidative capacity of the wrist flexor muscle group. Near infrared spectroscopy coupled with repeated, transient arterial occlusions, measured the recovery kinetics of oxygen consumption following a 10 second bout of hand grip exercise. Post exercise metabolic recovery rate constant was calculated and reported as an index of mitochondrial oxidative capacity. RESULTS Post exercise metabolic recovery rates were significantly greater in LT recipients (Mean±SEM; Recovery Time Constant: 72±7 s) compared to HC (51±4 s; p=0.03) indicative of diminished mitochondrial plasticity in this patient cohort (Figure 1). CONCLUSION Skeletal muscle mitochondrial plasticity is reduced in LT patients compared to matched healthy controls. Further investigations are warranted to determine the role of mitochondrial dysfunction in the development of sarcopenic obesity and frailty following LT.

Association between muscle aerobic capacity and whole-body peak oxygen uptake.

Decline in skeletal muscle mitochondrial oxidative capacity (MOC) is associated with reduced aerobic capacity and increased risk ofcardiovascular and metabolicdisease. Measuring skeletal muscle MOC may be an alternative method to assess aerobic capacity, especiallyfor individuals unable to perform a whole-body maximum oxygen uptake protocol. In this study, linear regression analysis in two leg muscles was performed to determine whether MOC values could be used to predict whole-body peak oxygen uptake. MOC was measured with near infrared spectroscopy (NIRS) inthemedial gastrocnemius (MG) and vastus lateralis (VL) musclesof 26 participants (age, 27.1 ± 5.8years old). Whole-body peak oxygen uptake (VO2 peak) was determinedby indirect calorimetry during a continuous ramp protocol on a cycle ergometer. VO2 peak values were significantly correlated with the muscle recovery rate constant (k)ofthe MG (kMG,r = 0.59;p < 0.01) and VL (kVL,r = 0.63; p < 0.01) muscles. Summingrecovery rate constantsof both muscles together (kMG + kVL)improved thestrength of thecorrelation with VO2 peak (r = 0.78;p < 0.0001)andcouldexplain amajorityof the variance(R2 = 0.61)between the two measurements. Data suggest that NIRS can provide reliable MOC measurements on two leg muscles that correlate well with whole-body peak oxygen uptake.

Poor mitochondrial health and systemic inflammation? Test of a classic hypothesis in the Baltimore Longitudinal Study of Aging.

Although a persistent inflammatory state has long been associated with aging and negative health outcomes, the underlying mechanisms remain unclear. Mitochondrial dysfunction has been proposed as a cause of inflammaging, but evidence of an association in humans is lacking. In this study, we analyzed the cross-sectional association between inflammatory biomarkers and mitochondrial oxidative capacity in skeletal muscle, assessed as post-exercise phosphocreatine recovery time constant by phosphorus magnetic resonance spectroscopy, in a population of 669 adults (mean age 67years) from the Baltimore Longitudinal Study of Aging. We observed that participants with lower mitochondrial oxidative capacity exhibited hallmarks of inflammation, specifically markedly higher levels of interleukin-6 and C-reactive protein, as well as increased erythrocyte sedimentation rate when compared with participants with better oxidative capacity, independent of age and sex. We speculate that this association reflects the observation that products of damaged mitochondria, such as mitochondrial DNA, activate multiple pathways that lead to inflammation. Furthermore, excess production of oxidative species (ROS) by dysfunctional mitochondria could trigger inflammation either directly via NF-κB or through oxidative damage to proteins, lipids, and nucleic acids. Longitudinal studies are necessary to ascertain whether and through which mechanisms mitochondrial dysfunction activate inflammation or whether both these phenomena derive from a common root.

Acute insulin deprivation results in altered mitochondrial substrate sensitivity conducive to greater fatty acid transport.

Type 1 and type 2 diabetes are both tightly associated with impaired glucose control. Although both pathologies stem from different mechanisms, a reduction in insulin action coincides with drastic metabolic dysfunction in skeletal muscle and metabolic inflexibility. However, the underlying explanation for this response remains poorly understood, particularly since it is difficult to distinguish the role of attenuated insulin action from the detrimental effects of reactive lipid accumulation, which impairs mitochondrial function and promotes reactive oxygen species (ROS) emission. We therefore utilized streptozotocin to examine the effects of acute insulin deprivation, in the absence of a high-lipid/nutrient excess environment, on the regulation of mitochondrial substrate sensitivity and ROS emission. The ablation of insulin resulted in reductions in absolute mitochondrial oxidative capacity and ADP-supported respiration and reduced the ability for malonyl-CoA to inhibit carnitine palmitoyltransferase I (CPT-I) and suppress fatty acid-supported respiration. These bioenergetic responses coincided with increased mitochondrial-derived H2O2 emission and lipid transporter content, independent of major mitochondrial substrate transporter proteins and enzymes involved in fatty acid oxidation. Together, these data suggest that attenuated/ablated insulin signaling does not affect mitochondrial ADP sensitivity, whereas the increased reliance on fatty acid oxidation in situations where insulin action is reduced may occur as a result of altered regulation of mitochondrial fatty acid transport through CPT-I.

An improvement in skeletal muscle mitochondrial capacity with short-term aerobic training is associated with changes in Tribbles 1 expression.

Exercise training and physical activity are known to be associated with high mitochondrial content and oxidative capacity in skeletal muscle. Metabolic diseases including obesity and insulin resistance are associated with low mitochondrial capacity in skeletal muscle. Certain transcriptional factors such as PGC‐1α are known to mediate the exercise response; however, the precise molecular mechanisms involved in the adaptation to exercise are not completely understood. We performed multiple measurements of mitochondrial capacity both in vivo and ex vivo in lean or overweight individuals before and after an 18‐day aerobic exercise training regimen. These results were compared to lean, active individuals. Aerobic training in these individuals resulted in a marked increase in mitochondrial oxidative respiratory capacity without an appreciable increase in mitochondrial content. These adaptations were associated with robust transcriptome changes. This work also identifies the Tribbles pseudokinase 1, TRIB1, as a potential mediator of the exercise response in human skeletal muscle.

Low-intensity exercise stimulates bioenergetics and increases fat oxidation in mitochondria of blood mononuclear cells from sedentary adults.

AimExercise training induces adaptations in muscle and other tissue mitochondrial metabolism, dynamics, and oxidative phosphorylation capacity. Mitochondrial fatty acid oxidation was shown to be pivotal for the anti‐inflammatory status of immune cells. We hypothesize that exercise training can exert effects influence mitochondrial fatty acid metabolism in peripheral blood mononuclear cells (PBMCs). The aim was to investigate the effect of exercise on the fatty acid oxidation‐dependent respiration in PBMCs.DesignTwelve fasted or fed volunteers first performed incremental‐load exercise tests to exhaustion on a cycle ergometer to determine the optimal workload ensuring maximal health benefits in volunteers with a sedentary lifestyle. In addition, the same volunteers performed 60 min of low‐intensity constant‐load exercise.ResultsIn the incremental‐load exercise, the maximal whole‐body fat oxidation rate measured by indirect calorimetry was reached at the fasted state already at a 50 W workload. At the 75–175 W workloads, the contribution of fat oxidation significantly decreased to only 11%, the heart rate increased to 185 BPM, and the study participants reached exhaustion. These results show that low‐intensity exercise (50W) is optimal for maximal whole‐body fat utilization. After low‐intensity exercise, the ROUTINE mitochondrial respiration, as well as fatty acid oxidation‐dependent respiration in PBMCs at LEAK and OXPHOS states, were significantly increased by 31%, 65%, and 76%, respectively. In addition, during 60 min of low‐intensity (50W) exercise, a 2‐fold higher lipolysis rate was observed and 13.5 ± 0.9 g of fat was metabolized, which was 57% more than the amount of fat that was metabolized during the incremental‐load exercise.ConclusionsIn individuals with a sedentary lifestyle participating in a bicycle ergometry exercise program, maximal lipolysis and whole‐body fat oxidation rate is reached in a fasted state during low‐intensity exercise. For the first time, it was demonstrated that low‐intensity exercise improves bioenergetics and increases fatty acid oxidation in PBMCs and may contribute to the anti‐inflammatory phenotype.

1891-P: Palmitate-Enriched Lipid Ingestion Acutely Induces Insulin Resistance Associated with PKCγ Activation and Impaired Mitochondrial Function in Human Skeletal Muscle

Hypercaloric diets cause dyslipidemia, insulin resistance and contribute to the development of type 2 diabetes. Nevertheless, the degree of fatty acid (FA) saturation may differently affect metabolism and risk of cardiovascular diseases, with saturated FA (SAFA) considered to be harmful and polyunsaturated FA (PUFA) considered to be beneficial. Thus, we aimed at characterizing the acute effects of palm oil (POL, SAFA-rich) and safflower oil (SOL, PUFA-rich) on tissue-specific insulin sensitivity and skeletal muscle mitochondrial function in glucose-tolerant volunteers (10 m/6 f; age 24±2 years; BMI 23.1±0.9 kg/m2), who ingested 1.2 g/kg BW of POL, SOL or control (CON, water) on three occasions. Whole-body glucose disposal (Rd) was assessed with [2H2]glucose before and during hyperinsulinemic-euglycemic clamps. Skeletal muscle biopsies were performed to assess protein kinase C (PKC) ԑ and θ translocation and oxygen fluxes by high-resolution respirometry at 1 h before and 2, 4 and 7 h after the interventions. Before the clamp, POL but not SOL ingestion raised plasma palmitic acid levels by 102% (p&amp;lt;0.01) compared to CON. Only POL decreased Rd by 28% and muscle mitochondrial oxidative capacity by 38% from succinate and by 39% from octanoyl-carnitine, but increased PKCɛ activity by 57% (all p&amp;lt;0.05 vs. CON). During insulin stimulation, Rd decreased by 38% after POL and by 26% after SOL (all p&amp;lt;0.05 to CON). Non-oxidative glucose disposal was lower after POL than after SOL (p&amp;lt;0.05). In conclusion, oral ingestion of palmitate-enriched lipids rapidly induces whole-body insulin resistance, which is likely mediated through skeletal muscle lipid-mediated PKCԑ activation and possibly accelerated by impaired mitochondrial function. Thus, the amount of dietary lipids determines acute insulin resistance, but its composition may differentially regulate skeletal muscle oxidative capacity. Disclosure T. Sarabhai: None. C. Koliaki: None. S. Kahl: None. D. Pesta: None. L. Mastrototaro: None. M. Apostolopoulou: None. D.F. Markgraf: None. C. Herder: Research Support; Self; Sanofi-Aventis. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S. Funding German Federal Ministry of Health and Ministry of Culture and Science of the State North Rhine-Westphalia; German Federal Ministry of Education and Research by the European Regional Development Fund (KomIT, EFRE-0400191); German Research Foundation (DFG; SFB 1116/2)

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.

Using an unbiased high‐throughput microRNA (miRNA)‐silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA‐204‐5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA‐204‐5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA‐204‐5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC‐1α (3.71‐fold; p < .01), predicted as an miR‐204‐5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA‐204‐5p silencing in C2C12 myotubes. Silencing of miRNA‐204‐5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA‐204‐5p silencing. Finally, miRNA‐204‐5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA‐204‐5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA‐204‐5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA‐204‐5p as an interesting modulator of skeletal muscle mitochondrial metabolism.