High-resolution Respirometry Research Articles

Two Weeks of Nrf2 Activation Improves Maximal Mitochondrial Respiration in Older Adults with Lower Starting Values

Background: The impact of aging on skeletal muscle mitochondrial function, as assessed by maximal mitochondrial respiration (OXPHOS), is a developing field of research with no clear consensus. Older individuals often display a greater range of OXPHOS, compared to younger individuals, possibly reflecting heterogeneity in the aging process. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a role in the transcription of genes involved in many pathways including the regulation of metabolism, inflammation, oxidative stress, and mitochondrial physiology. Since Nrf2 is downregulated with aging and significantly inactivated with sedentary behaviors, we hypothesized that activating this pathway with the nutraceutical PB125 would improve OXPHOS in older individuals with lower OXPHOS starting values. Methods: OXPHOS (CI&II, State 3) was assessed in permeabilized muscle fibers via high-resolution respirometry (Oroboros, O2k) on samples biopsied from the vastus lateralis in 19 young (26 ± 5 years; 13 female) and 18 older (67 ± 7 years; 11 female) volunteers. Sixteen of the older individuals were then randomized to receive a two-week supplementation of either PB125 (n = 10) or Placebo (n = 6) and OXPHOS was reassessed post-supplementation. Results: Baseline OXPHOS was not different between the young (32 ± 8 pmol/s/mg; Range: 18-44) and old (35 ± 12 pmol/s/mg; Range: 14-60; P = 0.49). The supplement-induced change in OXPHOS was not different between the older groups (PB125: 3 ± 9 vs Placebo: -0 ± 10 pmol/s/mg; P = 0.56) and neither was different from baseline (both, P > 0.05). However, a “threshold-like” relationship between the baseline OXPHOS and the PB125-induced change was identified within the PB125-treated group, whereby older individuals with a relatively lower baseline OXPHOS (<25 pmol/s/mg) had a significant improvement (11 ± 7 pmol/s/mg, n = 4; P = 0.03) compared to baseline, while individuals with a normal baseline OXPHOS had no significant change (-2 ± 2 pmol/s/mg, n = 6; P > 0.05). Conclusion: These findings support previous reports that mitochondrial function, as measured by OXPHOS, is not impaired in older individuals compared to their younger counterparts. However, older individuals with low mitochondrial function exhibited improved OXPHOS following Nrf2 activation via PB125 supplementation. These findings highlight the importance of heterogeneity in aging and provide novel evidence that activating Nrf2 may prove beneficial to improve maximal mitochondrial respiration. NIH: T32HL007576, T32HL139451, R01HL142603; VA: I01CX001999 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle microcirculatory and mitochondrial function in patients with claudication and critical limb ischemia

Introduction: Peripheral artery disease (PAD) is an atherosclerotic progressive disorder that impairs blood flow to the lower extremities. Early-stage patients often present with intermittent claudication (leg pain during walking, CLD), but as the disease progresses, patients can experience pain at rest and tissue death (critical limb ischemia, CLI). We recently reported that skeletal muscle microvascular endothelial dysfunction presented in claudicating patients with PAD. However, it is unknown if this microvascular dysfunction continues to progress with disease severity. Objective: To compare a direct investigation of the skeletal muscle microcirculation and the mitochondrial respiratory environment in patients with claudication and patients with CLI. Hypothesis: We hypothesize that (1) CLI will have greater attenuation in microcirculatory endothelial-dependent and endothelial-independent vasodilatory function (smooth muscle function) than CLD and (2) impairments in skeletal muscle microcirculatory vasodilatory function will contribute to attenuated mitochondrial respiratory function in CLD and CLI. Methods: Medial gastrocnemius muscles were harvested from PAD patients with CLD ( n = 5), CLI ( n = 6), and age-matched controls ( n = 6) by both muscle biopsies and open incisions. Arterioles were isolated to assess endothelium-dependent and independent vasodilatory responses to flow (30 mL·min -1 ), acetylcholine (ACh), and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiratory function was determined by high-resolution respirometry. Results: Endothelium-dependent vasodilation was attenuated ( P < 0.05) in response to flow (CON: 53.6 ± 23%, PAD: 26.7 ± 10.4%, CLI: 20.1 ± 14.5%) and ACh (10 -3 M, CON: 64 ± 6.9%, PAD: 39.1 ± 6.7%, CLI: 20.1 ± 14.5%) in both PAD and CLI compared to CON. However, endothelium-independent vasodilation with SNP was not different in any condition ( P > 0.05). Complex I+II state 3 mitochondrial respiration was blunted in both CLD and CLI compared to CON ( P < 0.05). However, state 4 respiration in CON was greater than CLI ( P < 0.05) but lower than CLD. Additionally, complex I+II state 3 respiration was positively associated with both flow ( r = 0.5, P = 0.01) and ACh mediated vasodilation ( r = 0.6, P = 0.009). No differences were found between CLD and CLI conditions. Conclusions: This was the first the first study to directly examine and compare skeletal muscle microvascular function in PAD patients with CLD and CLI. Our initial data indicate that functional characteristics of the skeletal muscle microvasculature and mitochondria are altered in PAD and deteriorate with disease progression. There is a correlation between alterations in the endothelium-dependent microvessel function and the dysfunction of mitochondria in the ischemic muscle raising the possibility that the microvascular dysfunction in PAD is produced by defects in the mitochondria of the microvascular endothelium. National Institutes of Health (R01 AG034995, R01 AG049868) NIH National Institute of General Medical Sciences, which funds the Great Plains IdeA-CTR Network (U54 GM115458) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

1 Repeated muscle collections induce few physiological changes in mature sedentary horses

Repeated skeletal muscle collections are often used to determine tissue-specific responses to interventions or stressors but little is known about the physiological impacts of the collections themselves. Twenty-six mature sedentary horses (8 geldings, 18 mares; mean ± SD 9.5 ± 3.5y) were used to test the hypothesis that repeated muscle collections would not impact mitochondrial function or markers of inflammation and muscle damage. Horses were stratified by age and sex and randomly assigned to have gluteus medius samples collected at: 1) 0 and 24 h (n = 7); 2) 0 and 6 h (n = 6); 3) 0, 6, and 12 h (n = 7); or 4) 0, 6, 12, and 24 h (n = 6). Blood was collected from all horses every 6 h for 72 h total, starting 24 h before the first muscle collection. Muscle mitochondrial oxidative phosphorylation and electron transfer (E) capacities were determined via high-resolution respirometry, and mitochondrial volume density and function by citrate synthase (CS) and cytochrome c oxidase activities. Muscle and plasma concentrations of interleukin (IL)- 4, IL-8, IL-10, and tumor necrosis factor (TNF)α were quantified, as well as serum amyloid A (SAA) and cortisol concentrations and creatine kinase (CK) activity. Data were analyzed using linear models in SAS with time as a repeated measure, horse as the subject, time as a fixed effect, and sex when significant. For blood parameters, additional fixed effects includedcollection group and the time × group interaction. Integrative (per mg tissue) E supported by complex II decreased from 0 to 6 h (P = 0.004) but returned to baseline by 12 h post-initial muscle collection. Intrinsic (per unit CS) Leak was greater at 6 than at 0 and 12 h (P < 0.03) but did not differ from 24 h. Activity of CS was greater at 0 than 12 and 24 h (P ≤ 0.02). Regardless of group, serum CK activity was similar from −24 through 0 h but increased in all horses at 6 h and remained elevated through 48 h, peaking at 24 and 30 h post-initial muscle collection (P < 0.05). Geldings had greater serum CK activity and plasma IL-8 concentrations than mares (P ≤ 0.006). Serum cortisol concentrations displayed a circadian pattern throughout the 72-h collection period (P < 0.0001) but were not impacted by tissue collection group. Overall, horses in group 2 (0 and 6 h) had greater SAA concentrations than all other groups (P < 0.006). No other variable was impacted by repeated tissue collections. In unstressed, mature horses, repeated gluteal muscle collections did not profoundly impact local and systemic markers of mitochondrial function, select cytokines, and stress indicators; however, serum CK should be interpreted with caution in studies employing repeated tissue collections.

The acute removal of metformin induces mitochondrial remodeling and increases mitochondrial respiration

Metformin is a candidate to slow biological aging and extend healthspan, as it reduces the risk of morbidity and mortality in people with type 2 diabetes. However, it is not clear how metformin impacts individuals absent of chronic disease. This unknown is important to answer before prescribing metformin to healthy individuals to prolong the healthspan. It has been shown that metformin creates an inefficiency in energy transfer by accumulating within the mitochondria. Therefore, we hypothesized that the effects of metformin on healthspan are context specific; beneficial in the context of low energy demand/mitochondrial capacity but detrimental in the context of high energy demand/mitochondrial capacity. To test this hypothesis, we used 18-month, male rats selectively bred for low and high treadmill running capacity (LCR/HCR) as models of low and high intrinsic mitochondrial function. For both HCR and LCR, we used high-resolution respirometry (Oroboros O2K) on skeletal muscles from controls (CON), 4 weeks of metformin-treatment (MET), 4 weeks of metformin-treatment with a 48-hour metformin washout (MET-WO), and 48-hour washout with metformin added back to the respirometry chamber (MET-WO-SPIKE). A week prior to sacrifice, we labeled the rats with deuterium oxide to assess protein turnover as an indicator of mitochondrial remodeling. In gastrocnemius muscle, the HCR MET-WO had greater Complex I (CI), CI+Complex II (CII), and maximal respiration (MaxR) (115 ± 23, 164 ± 38, 157 ± 35 pmol/(s✕mg), respectively) compared to the CON (70 ± 24, p = 0.02, 114 ± 32, p = 0.01, 115 ± 40 pmol/(s✕mg), p = 0.04, respectively), and MET (63 ± 54, p = 0.007, 90 ± 50, p &lt; 0.001, 91 ± 48 pmol/(s✕mg), p = 0.002, respectively). The HCR MET-SPIKE group had greater CII (83 ± 33 pmol/(s✕mg)) and MaxR (86 ± 35 pmol/(s✕mg)) compared to the MET (p = 0.007 and p = 0.002, respectively). In the soleus, MET-WO (4.15 ± 0.33%/day) had greater mitochondrial fractional synthesis rates (FSRs) compared to MET (3.12 ± 0.62%/day, p = 0.001) in the HCRs. In the LCRs, MET-WO (4.61 ± 0.42%/day) had greater FSRs compared to CON (p &lt; 0.001, 3.07 ± 0.67%/day) and MET (3.12 ± 0.61%/day, p = 0.001). In the tibialis anterior, MET-WO (3.47 ± 0.80%/day) had greater FSRs compared to the MET (2.41 ± 0.47%/day, p = 0.04) within the HCRs with no differences in the LCR. These data show that metformin reduces mitochondrial respiration in HCR rats but not in LCR rats. The presence of metformin produced a greater subsequent response in HCR compared to LCR as shown by its acute removal revealing metformin-induced mitochondrial remodeling. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

O008 The impact of home-based exercise prehabilitation on skeletal muscle mitochondrial function in patients awaiting curative surgery for cancer

Abstract Introduction Cancer cachexia is associated with poor surgical outcomes, including increased length of stay, morbidity and mortality. Cachexia is a complex multi-faceted pathological process consistently associated with energy imbalance via hypermetabolism. Beyond this, pre-clinical animal models suggest mitochondrial dysfunction to be an important component of cachexia, although this is unconfirmed in humans. This study aimed to investigate the mitochondrial function of pre-surgical cancer patients, and the impact of exercise prehabilitation on this. Methods This study was approved by an NHS research ethics committee (IRAS: 275264). Male patients awaiting surgery with curative intent for colorectal and prostate cancer were eligible, with healthy volunteers recruited for comparison. Muscle biopsies of vastus lateralis were taken before and after 4-weeks home-based exercise (3x/wk). Mitochondrial oxidative phosphorylation (OXPHOS) was analysed using high-resolution respirometry (Oroboros Instruments). Results Eleven cancer patients and 6 healthy volunteers were recruited. Cancer patients had significantly lower maximal coupled (44.49pmol/(s*mg) vs. 74.81pmol/(s*mg), p=0.004) and uncoupled (55.82pmol/(s*mg) vs. 100.00pmol/(s*mg), p&amp;lt;0.001) OXPHOS capacity compared to healthy controls. Exercise training significantly increased maximal coupled (44.10pmol/(s*mg) vs. 54.64pmol/(s*mg), p=0.045) and uncoupled (56.20pmol/(s*mg) vs. 71.96pmol/(s*mg), p=0.001) OXPHOS capacity in this cancer cohort. Conclusion Declines in mitochondrial function appear to play a role in cancer cachexia. Short-term exercise training, in keeping with target timeframes for surgery (i.e., &amp;lt;31-days from decision-to-treat), appears to attenuate these declines. Further work is needed to determine if declines in mitochondrial function are causative or reactive, and to identify optimal exercise regimes to mitigate these declines.

The effect of chronic unpredictable stress on renal mitochondrial function in female mice

Background: Chronic stress can induce high blood pressure and lead to decreased renal function by mechanisms that remain unclear. The exposure of rodents to chronic unpredictable stress (CUS) is a translational model used for investigating the physiological impact of stress. The CUS protocol consists of unpredictable and random stressors that mimic real life exposure to stress. While stress is known to impact mitochondrial function, little is understood about the direct impact of chronic stress on renal mitochondrial respiration. Moreover, while studies have shown a link between estrogen and mitochondria through estrogen receptors, the relation between stress induced renal mitochondrial dysfunction and estrous cycle is unknown. Hypothesis: Exposing female mice to CUS will increase blood pressure and cause decreased renal mitochondrial respiration. Methods: We exposed adult female C57BL/6 mice to CUS for 28 days. Before and after stress, all control (CON, n=8) and CUS (n=10) mice were placed in metabolic cages for overnight urine collection. Urine albumin was measured by dipstick assay. On day 28, mice were assessed for blood pressure measurement via tail cuff. 24 hours later, mice were euthanized, and vaginal lavage was performed. The left kidney was removed, transected into thirds, and the poles were placed in relaxing buffer. Kidney mitochondrial oxygen consumption was assessed using high-resolution respirometry. Results: Data were stratified by phases of the estrus cycle. Neither blood pressure nor urinary albumin was significantly altered by CUS in stressed females. Females in estrus and proestrus exposed to CUS (n=5) exhibited a decrease of 20.8% ± 5.1 (n=5, p=0.03) in renal mitochondrial maximal uncoupled respiration compared to non-stressed CON. Renal mitochondrial respiration in diestrus was similar between CUS and CON. Conclusion: Results from this study reveal a link between the female estrus cycle, stress, and mitochondrial function. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. &amp; F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

30 Intrinsic skeletal muscle mitochondrial function decreases while mitochondrial volume density increases from 6 months to 3-years-old in Thoroughbreds

As the primary source of cellular energy, mitochondria are critical for athletic performance, and the intricacies of mitochondrial electron transfer may be related to Thoroughbred race performance. However, little is known about specific mitochondrial adaptations to growth in the equine athlete. To test the hypothesis that mitochondrial metrics would increase with age, gluteus medius samples were collected from the same Thoroughbred horses as weanlings (4 colts, 9 fillies; mean ± SD, 6 ± 1mo), yearlings (4 colts, 10 fillies; 20 ± 1mo), 2-year-olds (2yo; 3 geldings, 10 mares; 32 ± 1mo), and 3yo (2 geldings, 3 mares; 44 ± 1mo) from the same Kentucky farm over 4 years. Thoroughbreds were broke to ride in August of their yearling year then began race training. Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were determined via high-resolution respirometry. Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were quantified as measures of mitochondrial volume density and function, respectively. Data were analyzed using PROC MIXED in SASv9.4 with age and sex as fixed effects, sire and dam as random effects, and age as a repeated effect with horse as the subject. Intrinsic (relative to CS activity) CCO activity, P supported by complex I (PCI), maximal P (PCI+II), and maximal E (ECI+II) weregreatest in weanlings compared with all other ages (P ≤ 0.003) and greater in yearlings than 2yo (P ≤ 0.05). Intrinsic E supported by complex II only (ECII) was greater in weanlings than 2 or 3yo (P ≤ 0.01) and greater in yearlings than 2yo (P ≤ 0.04). Weanlings also had a greater contribution of PCI+II to ECI+II than yearlings, 2yo, or 3yo (P ≤ 0.05). However, CS activity was greatest in 3yo (P ≤ 0.03) and lowest in weanlings (P ≤ 0.0001) with yearlings and 2yo intermediate, indicating expansion of the mitochondrial network with age and training. Additionally, 3yo had the greatest integrative (per mg tissue) PCI of all ages (P ≤ 0.005). Two and 3yo had greater integrative PCI+II, ECI+II, and ECII capacities than weanlings (P ≤ 0.05). Overall, weanlings had the greatest intrinsic mitochondrial capacities and function but 2 and 3yo Thoroughbreds had greater mitochondrial volume density resulting in greater integrative capacities. Skeletal muscle mitochondria of young Thoroughbreds appear to adapt to meet energetic demands of growth and race training by increasing volume density rather than intrinsic capacity, but further research is warranted to characterize the impacts of various training programs on mitochondrial metrics.

High fat diet feeding induces brain mitochondrial dysfunction in female Ossabaw swine

Study objective: Emerging evidence now suggest reduced mitochondrial function as a key initiator in Alzheimer’s disease (AD)-like pathology increasing the need for models of AD that display mitochondrial dysfunction. Dyslipidemia is a common aliment from overconsumption of a high fat diet (HFD) that increases the risk of AD and may contribute to mitochondrial dysfunction. Rodent studies utilize HFDs to induce brain pathology resembling that of AD. Unsurprisingly, rodent studies are rarely recapitulated in humans, which leads to a very low clinical success rate and highlights the need for more translatable models to study AD-like pathology. Swine seem to share many characteristics of humans and might offer a more translatable model to study HFD induced pathology. Thus, the objective of this study was to determine if HFD feeding in swine could offer a more translatable model to study mitochondrial dysfunction during neurodegeneration. Hypothesis: 6-months of HFD feeding will decrease indices of mitochondrial function and biogenesis in female Ossabaw swine. Methods: To characterize the effects of a HFD on brain mitochondrial health in a highly translatable swine model, high resolution respirometry measurements were taken from isolated brain mitochondria from in female Ossabaw swine fed a HFD for 6-months. Additionally, indices of mitochondrial biogenesis in whole tissue was measured (transcriptional markers of biogenesis and citrate synthase activity). Results: HFD feeding significantly increased body weight (p&lt;0.05) with trending increases in blood glucose (p&lt;0.10). Maximal uncoupled mitochondrial respiration was significantly decreased (p&lt;0.05) with state 3-complex I and state 3 complex I &amp; II trending down in the dorsal hippocampus (DH) after HFD diet feeding (p&lt;0.10). Additionally, state 2 respiration was significantly decreased in the prefrontal cortex (PFC) after HFD feeding (p&lt;0.05). HFD feeding increased transcriptional markers of mitochondrial biogenesis in the DH and PFC (p&lt;0.05), without altering whole homogenate citrate synthase activity in either brain region. Conclusions: These data indicate HFD feeding in Ossabaw pigs could offer a potential model to study pathologies relating to mitochondrial dysfunction during neurodegenerative process. Additionally, the DH was found to be more vulnerable to HFD-induced perturbations, evident by the exacerbated deficits in mitochondrial respiration in this brain region. Finally, these data suggest HFD-induced increase in transcriptional markers of mitochondrial biogenesis may be a compensatory response to HFD-induced mitochondrial respiration deficits. However, citrate synthase activity was not different between groups indicating this upregulation of transcriptional markers was unable to translate to an increase in mitochondrial content in the DH and PFC. The project was supported by the MU Office of Postdoctoral Education Research Grant Program. Additional funding was also generously provided Scott Rector. Images in figure 2 adapted and modified from “Krebs cycle template”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates . This work was also supported with resources and the use of facilities at the University of Missouri and Harry S. Truman Memorial Veterans Hospital in Columbia, Missouri. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations

PurposeTreatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.MethodsWe treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.ResultsThe HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.ConclusionHAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells’ respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.

Impairment of substrate-mediated mitochondrial respiration in cardiac cells by chloroquine.

Chloroquine (CQ) has a long clinical history as an anti-malarial agent and also being used for the treatment of other infections and autoimmune diseases. Recently, this lysosomotropic agent and its derivatives are also been tested as adjuncts alongside conventional anti-cancer treatments in combinatorial therapies. However, their reported cardiotoxicity tends to raise concern over their indiscriminate use. Even though the influence of CQ and its derivatives on cardiac mitochondria is extensively studied in disease models, their impact on cardiac mitochondrial respiration under physiological conditions remains inconclusive. In this study, we aimed to evaluate the impact of CQ on cardiac mitochondrial respiration using both in-vitro and in-vivo model systems. Using high-resolution respirometry in isolated cardiac mitochondria from male C57BL/6 mice treated with intraperitoneal injection of 10mg/kg/day of CQ for 14days, CQ was found to impair substrate-mediated mitochondrial respiration in cardiac tissue. In an in-vitro model of H9C2 cardiomyoblasts, incubation with 50µM of CQ for 24h disrupted mitochondrial membrane potential, produced mitochondrial fragmentation, decreased mitochondrial respiration and induced superoxide generation. Altogether, our study results indicate that CQ has a deleterious impact on cardiac mitochondrial bioenergetics which in turn suggests that CQ treatment could be an added burden, especially in patients affected with diseases with underlying cardiac complications. As CQ is an inhibitor of the lysosomal pathway, the observed effect could be an outcome of the accumulation of dysfunctional mitochondria due to autophagy inhibition.

Astrocytic aerobic glycolysis provides lactate to support neuronal oxidative metabolism in the hippocampus.

Under physiological conditions, the energetic demand of the brain is met by glucose oxidation. However, ample evidence suggests that lactate produced by astrocytes through aerobic glycolysis may also be an oxidative fuel, highlighting the metabolic compartmentalization between neural cells. Herein, we investigate the roles of glucose and lactate in oxidative metabolism in hippocampal slices, a model that preserves neuron-glia interactions. To this purpose, we used high-resolution respirometry to measure oxygen consumption (O2 flux) at the whole tissue level and amperometric lactate microbiosensors to evaluate the concentration dynamics of extracellular lactate. We found that lactate is produced from glucose and transported to the extracellular space by neural cells in hippocampal tissue. Under resting conditions, endogenous lactate was used by neurons to support oxidative metabolism, which was boosted by exogenously added lactate even in the presence of excess glucose. Depolarization of hippocampal tissue with high K+ significantly increased the rate of oxidative phosphorylation, which was accompanied by a transient decrease in extracellular lactate concentration. Both effects were reverted by inhibition of the neuronal lactate transporter, monocarboxylate transporters 2 (MCT2), supporting the concept of an inward flux of lactate to neurons to fuel oxidative metabolism. We conclude that astrocytes are the main source of extracellular lactate which is used by neurons to fuel oxidative metabolism, both under resting and stimulated conditions.

Cryopreserved placental biopsies maintain mitochondrial activity for high-resolution respirometry

BackgroundHigh-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need.MethodsWe optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and − 80 °C storage using a specific DMSO-based buffer.ResultsPlacenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions.ConclusionsBy this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity.Graphical

Weight loss increases skeletal muscle mitochondrial energy efficiency in obese mice.

Weight loss from an overweight state is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype (WT) male C57BL6/N mice were fed with high fat diet for 10 weeks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 weeks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2 consumed, or P/O) in skeletal muscle. However, weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

PGC 1α-Mediates Mitochondrial Damage in the Liver by Inhibiting the Mitochondrial Respiratory Chain as a Non-cholinergic Mechanism of Repeated Low-Level Soman Exposure.

This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague-Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I-IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I + II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I-IV, I-III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.

Mixed Berry Juice and Cellulose Fiber Have Differential Effects on Peripheral Blood Mononuclear Cell Respiration in Overweight Adults.

Berries and other anthocyanin-rich foods have demonstrated anti-obesity effects in rodents and humans. However, the bioactive components of these foods and their mechanisms of action are unclear. We conducted an intervention study with overweight and obese adults to isolate the effects of different berry components on bioenergetics. Subjects consumed whole mixed berries (high anthocyanin, high fiber), pressed berry juice (high anthocyanin, low fiber), berry-flavored gelatin (low anthocyanin, low fiber), or fiber-enriched gelatin (low anthocyanin, high fiber) for one week prior to a meal challenge with the same treatment food as the pre-feed period. Peripheral blood mononuclear cells were collected 2 h after the meal challenge, and cellular respiration was assessed via high-resolution respirometry. The high-anthocyanin, low-fiber treatment (berry juice) and the low-anthocyanin, high-fiber treatment (fiber-enriched gelatin) had opposite effects on cellular respiration. In the fasted state, berry juice resulted in the highest oxygen-consumption rate (OCR), while fiber-enriched gelatin resulted in the highest OCR in the fed state. Differences were observed in multiple respiration states (basal, state 3, state 4, uncoupled), with the greatest differences being between the pressed berry juice and the fiber-enriched gelatin. Different components of berries, specifically anthocyanins/flavonoids and fiber, appear to have differential effects on cellular respiration.

Sepsis-like Energy Deficit Is Not Sufficient to Induce Early Muscle Fiber Atrophy and Mitochondrial Dysfunction in a Murine Sepsis Model.

Sepsis-induced myopathy is characterized by muscle fiber atrophy, mitochondrial dysfunction, and worsened outcomes. Whether whole-body energy deficit participates in the early alteration of skeletal muscle metabolism has never been investigated. Three groups were studied: "Sepsis" mice, fed ad libitum with a spontaneous decrease in caloric intake (n = 17), and "Sham" mice fed ad libitum (Sham fed (SF), n = 13) or subjected to pair-feeding (Sham pair fed (SPF), n = 12). Sepsis was induced by the intraperitoneal injection of cecal slurry in resuscitated C57BL6/J mice. The feeding of the SPF mice was restricted according to the food intake of the Sepsis mice. Energy balance was evaluated by indirect calorimetry over 24 h. The tibialis anterior cross-sectional area (TA CSA), mitochondrial function (high-resolution respirometry), and mitochondrial quality control pathways (RTqPCR and Western blot) were assessed 24 h after sepsis induction. The energy balance was positive in the SF group and negative in both the SPF and Sepsis groups. The TA CSA did not differ between the SF and SPF groups, but was reduced by 17% in the Sepsis group compared with the SPF group (p < 0.05). The complex-I-linked respiration in permeabilized soleus fibers was higher in the SPF group than the SF group (p < 0.05) and lower in the Sepsis group than the SPF group (p < 0.01). Pgc1α protein expression increased 3.9-fold in the SPF mice compared with the SF mice (p < 0.05) and remained unchanged in the Sepsis mice compared with the SPF mice; the Pgc1α mRNA expression decreased in the Sepsis compared with the SPF mice (p < 0.05). Thus, the sepsis-like energy deficit did not explain the early sepsis-induced muscle fiber atrophy and mitochondrial dysfunction, but led to specific metabolic adaptations not observed in sepsis.

Enhanced mitochondrial oxidative metabolism in peripheral blood mononuclear cells is associated with fatty liver in obese young adults

Systemic inflammation underlies the association between obesity and nonalcoholic fatty liver disease (NAFLD). Here, we investigated functional changes in leukocytes’ mitochondria in obese individuals and their associations with NAFLD. We analyzed 14 obese male Japanese university students whose body mass index was > 30 kg/m2 and 15 healthy age- and sex-matched lean university students as controls. We observed that the mitochondrial oxidative phosphorylation (OXPHOS) capacity with complex I + II-linked substrates in peripheral blood mononuclear cells (PBMCs), which was measured using a high-resolution respirometry, was significantly higher in the obese group versus the controls. The PBMCs’ mitochondrial complex IV capacity was also higher in the obese subjects. All of the obese subjects had hepatic steatosis defined by a fatty liver index (FLI) score ≥ 60, and there was a positive correlation between their FLI scores and their PBMCs’ mitochondrial OXPHOS capacity. The increased PBMCs’ mitochondrial OXPHOS capacity was associated with insulin resistance, systemic inflammation, and higher serum levels of interleukin-6 in the entire series of subjects. Our results suggest that the mitochondrial respiratory capacity is increased in the PBMCs at the early stage of obesity, and the enhanced PBMCs’ mitochondrial oxidative metabolism is associated with hepatic steatosis in obese young adults.

Cognitive heterogeneity reveals molecular signatures of age-related impairment.

The greatest risk factor for cognitive decline is aging. The biological mechanisms for this decline remain enigmatic due, in part, to the confounding of normal aging mechanisms and those that contribute to cognitive impairment. Importantly, many individuals exhibit impaired cognition in age, while some retain functionality despite their age. Here, we establish a behavioral testing paradigm to characterize age-related cognitive heterogeneity in inbred aged C57BL/6 mice and reliably separate animals into cognitively "intact" (resilient) and "impaired" subgroups using a high-resolution home-cage testing paradigm for spatial discrimination. RNA sequencing and subsequent pathway analyses of cognitively stratified mice revealed molecular signatures unique to cognitively impaired animals, including transcriptional down-regulation of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) and sirtuin (Sirt1 and Sirt3) expression in the hippocampus. Mitochondrial function assessed using high-resolution respirometry indicated a reduced OXPHOS coupling efficiency in cognitively impaired animals with subsequent hippocampal analyses revealing an increase in the oxidative damage marker (3-nitrotyrosine) and an up-regulation of antioxidant enzymes (Sod2, Sod1, Prdx6, etc.). Aged-impaired animals also showed increased levels of IL-6 and TNF-α gene expression in the hippocampus and increased serum levels of proinflammatory cytokines, including IL-6. These results provide critical insight into the diversity of brain aging in inbred animals and reveal the unique mechanisms that separate cognitive resilience from cognitive impairment. Our data indicate the importance of cognitive stratification of aging animals to delineate the mechanisms underlying cognitive impairment and test the efficacy of therapeutic interventions.

Effects of acute cigarette smoke concentrate exposure on mitochondrial energy transfer in fast- and slow-twitch skeletal muscle

The mechanisms underlying cigarette smoke-induced mitochondrial dysfunction in skeletal muscle are still poorly understood. Accordingly, this study aimed to examine the effects of cigarette smoke on mitochondrial energy transfer in permeabilized muscle fibers from skeletal muscles with differing metabolic characteristics. The electron transport chain (ETC) capacity, ADP transport, and respiratory control by ADP were assessed in fast- and slow-twitch muscle fibers from C57BL/6 mice (n = 11) acutely exposed to cigarette smoke concentrate (CSC) using high-resolution respirometry. CSC decreased complex I-driven respiration in the white gastrocnemius (CONTROL:45.4 ± 11.2 pmolO2.s−1.mg−1 and CSC:27.5 ± 12.0 pmolO2.s−1.mg−1; p = 0.01) and soleus (CONTROL:63.0 ± 23.8 pmolO2.s−1.mg−1 and CSC:44.6 ± 11.1 pmolO2.s−1.mg−1; p = 0.04). In contrast, the effect of CSC on Complex II-linked respiration increased its relative contribution to muscle respiratory capacity in the white gastrocnemius muscle. The maximal respiratory activity of the ETC was significantly inhibited by CSC in both muscles. Furthermore, the respiration rate dependent on the ADP/ATP transport across the mitochondrial membrane was significantly impaired by CSC in the white gastrocnemius (CONTROL:-70 ± 18 %; CSC:-28 ± 10 %; p < 0.001), but not the soleus (CONTROL:47 ± 16 %; CSC:31 ± 7 %; p = 0.08). CSC also significantly impaired mitochondrial thermodynamic coupling in both muscles. Our findings underscore that acute CSC exposure directly inhibits oxidative phosphorylation in permeabilized muscle fibers. This effect was mediated by significant perturbations of the electron transfer in the respiratory complexes, especially at complex I, in both fast and slow twitch muscles. In contrast, CSC-induced inhibition of the exchange of ADP/ATP across the mitochondrial membrane was fiber-type specific, with a large effect on fast-twitch muscles.

Impact of GLP-1 receptor agonist versus omega-3 fatty acids supplement on obesity-induced alterations of mitochondrial respiration.

To compare administration of the glucagon-like peptide-1 (GLP-1) analogue, exenatide, versus dietary supplementation with the omega-3 fatty acid-rich Calanus oil on obesity-induced alterations in mitochondrial respiration. Six-week-old female C57BL/6JOlaHSD mice were given high fat diet (HFD, 45% energy from fat) for 12 weeks to induce obesity. Thereafter, they were divided in three groups where one received exenatide (10 μg/kg/day) via subcutaneously implanted mini-osmotic pumps, a second group received 2% Calanus oil as dietary supplement, while the third group received HFD without any treatment. Animals were sacrificed after 8 weeks of treatment and tissues (skeletal muscle, liver, and white adipose tissue) were collected for measurement of mitochondrial respiratory activity by high-resolution respirometry, using an Oroboros Oxygraph-2k (Oroboros instruments, Innsbruck, Austria). It was found that high-fat feeding led to a marked reduction of mitochondrial respiration in adipose tissue during all three states investigated - LEAK, OXPHOS and ETS. This response was to some extent attenuated by exenatide treatment, but not with Calanus oil treatment. High-fat feeding had no major effect on hepatic mitochondrial respiration, but exenatide treatment resulted in a significant increase in the various respiratory states in liver. Mitochondrial respiration in skeletal muscle was not significantly influenced by high-fat diet or any of the treatments. The precise evaluation of mitochondrial respiration considering absolute oxygen flux and ratios to assess flux control efficiency avoided misinterpretation of the results. Exenatide increased hepatic mitochondrial respiration in high-fat fed mice, but no clear beneficial effect was observed in skeletal muscle or fat tissue. Calanus oil did not negatively affect respiratory activity in these tissues, which maintains its potential as a dietary supplement, due to its previously reported benefits on cardiac function.