Muscle weakness and mitochondrial stress occur before severe metastasis in a novel mouse model of ovarian cancer cachexia.

OBJECTIVES: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. METHODS: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ∼45, ∼75 and ∼90 days after EOC injection. RESULTS: Primary ovarian tumour sizes were progressively larger at each time point while severe metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-severe-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and severe metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. CONCLUSIONS: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes severe metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-severe-metastatic weakness during EOC in addition to therapies targeting cachexia.

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia.

Objectives: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. Methods: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ~45, ~75 and ~90 days after EOC injection. Results: Primary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. Conclusion: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime.

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime, prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and mH2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fiber, but reduced type I fibers in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Oral combined contraceptives induce liver mitochondrial reactive oxygen species and whole-body metabolic adaptations in female mice.

Compared to age-matched men, pre-menopausal women show greater resilience against cardiovascular disease (CVD), hepatic steatosis, diabetes and obesity - findings that are widely attributed to oestrogen. However, meta-analysis data suggest that current use of oral combined contraceptives (OC) is a risk factor for myocardial infarction, and OC use further compounds with metabolic disease risk factors to increase CVD susceptibility. While mitochondrial function in tissues such as the liver and skeletal muscle is an emerging mechanism by which oestrogen may confer its protection, effects of OC use on mitochondria and metabolism in the context of disease risk remain unexplored. To answer this question, female C57Bl/6J mice were fed a high fat diet and treated with vehicle or OCs for 3, 12 or 20 weeks (n = 6 to 12 per group) at a dose and ratio that mimic the human condition of cycle cessation in the low oestrogen, high progesterone stage. Liver and skeletal muscle mitochondrial function (respiratory capacity, H2 O2 , coupling) was measured along with clinical outcomes of cardiometabolic disease such as obesity, glucose tolerance, hepatic steatosis and aortic atherosclerosis. The main findings indicate that regardless of treatment duration, OCs robustly increase hepatic mitochondrial H2 O2 levels, likely due to diminished antioxidant capacity, but have no impact on muscle mitochondrial H2 O2 . Furthermore, OC-treated mice had lower adiposity and hepatic triglyceride content compared to control mice despite reduced wheel running, spontaneous physical activity and total energy expenditure. Together, these studies describe tissue-specific effects of OC use on mitochondria as well as variable impacts on markers of metabolic disease susceptibility. KEY POINTS: Oestrogen loss in women increases risk for cardiometabolic diseases, a link that has been partially attributed to negative impacts on mitochondria and energy metabolism. To study the effect of oral combined contraceptives (OCs) on hepatic and skeletal muscle mitochondria and whole-body energy metabolism, we used an animal model of OCs which mimics the human condition of cessation of hormonal cycling in the low oestrogen, high progesterone state. OC-treated mice have increased hepatic mitochondrial oxidative stress and decreased physical activity and energy expenditure, despite displaying lower adiposity and liver fat at this time point. These pre-clinical data reveal tissue-specific effects of OCs that likely underlie the clinical findings of increased cardiometabolic disease in women who use OCs compared to non-users, when matched for obesity.

A HIF-1 signature dominates the attenuation in the human skeletal muscle transcriptional response to high-intensity interval training.

High-intensity interval training (HIIT) generates profound metabolic adaptations in skeletal muscle. These responses mirror performance improvements but follow a nonlinear pattern comprised of an initial fast phase followed by a gradual plateau effect. The complete time-dependent molecular sequelae that regulates this plateau effect remains unknown. We hypothesize that the plateau effect during HIIT is restricted to specific pathways with communal upstream transcriptional regulation. To investigate this, 11 healthy men performed nine sessions of HIIT [10 × 4 min of cycling at 91% of maximal heart rate (HRmax)] over a 3-wk period. Before and 3 h after the 1st and 9th exercise bout, skeletal muscle biopsies were obtained, and RNA sequencing was performed. Almost 2,000 genes across 84 pathways were differentially expressed in response to a single HIIT session. The overall transcriptional response to acute exercise was strikingly similar at 3 wk, 83% (n = 1,650) of the genes regulated after the 1st bout of exercise were similarly regulated by the 9th bout, albeit with a smaller effect size, and the response attenuated to on average 70% of the 1st bout. The attenuation differed substantially between pathways and was especially pronounced for glycolysis and cellular adhesion compared to, e.g., MAPK and vascular endothelial growth factor (VEGF)-A signaling. The attenuation was driven by a combination of changes in steady-state expression and specific transcriptional regulation. Given that the exercise intensity was progressively increased, and the attenuation was pathway-specific, we suggest that moderation of muscular adaptation after a period of training stems from targeted regulation rather than a diminished exercise stimulus.NEW & NOTEWORTHY This is the first study to address the phenomena of attenuation of the acute exercise response on a global genomic scale with a focus on underlying regulatory machinery and it is, to the best of our knowledge, the first study conducted in humans was exercise-induced regulation of different canonical pathways and transcription factors are contrasted with regards to attenuation after a period with regular exercise training. These results provide evidence for a pathway-specific regulated augmentation of the response to acute exercise over time that tracks with the successive adaptation on the systemic level.

A Novel Multi-Ingredient Supplement Activates a Browning Program in White Adipose Tissue and Mitigates Weight Gain in High-Fat Diet-Fed Mice.

We investigated the effects of a novel multi-ingredient supplement comprised of polyphenol antioxidants and compounds known to facilitate mitochondrial function and metabolic enhancement (ME) in a mouse model of obesity. In this study, 6-week-old male C57/BL6J mice were placed on a high-fat diet (HFD; ~60% fat) for 6 weeks, with subsequent allocation into experimentalgroups for 4 weeks: HFD control, HFD + ME10 (10 components), HFD + ME7 (7 components), HFD + ME10 + EX, HFD + EX (where '+EX' animals exercised 3 days/week), and chow-fed control. After the intervention, HFD control animals had significantly greater body weight and fat mass. Despite the continuation of HFD, animals supplemented with multi-ingredient ME or who performed exercise training showed an attenuation of fat mass and preservation of lean body mass, which was further enhanced when combined (ME+EX). ME supplementation stimulated the upregulation of white and brown adipose tissue mRNA transcripts associated with mitochondrial biogenesis, browning, fatty acid transport, and fat metabolism. In WAT depots, this was mirrored by mitochodrial oxidative phosphorylation (OXPHOS) protein expression, and increased in vivo fat oxidation measured via CLAMS. ME supplementation also decreased systemic and local inflammation markers. Herein, we demonstrated that novel multi-ingredient nutritional supplements induced significant fat loss independent of physical activity while preserving muscle mass in obese mice. Mechanistically, these MEs appear to act by inducing a browning program in white adipose tissue and decreasing other pathophysiological impairments associated with obesity, including mitochondrial respiration alterations induced by HFD.

Promoting a pro-oxidant state in skeletal muscle: Potential dietary, environmental, and exercise interventions for enhancing endurance-training adaptations.

Accumulating evidence now shows that supplemental antioxidants including vitamin C, vitamin E and N-Acetylcysteine consumption can suppress adaptations to endurance-type exercise by attenuating reactive oxygen and nitrogen species (RONS) formation within skeletal muscle. This emerging evidence points to the importance of pro-oxidation as an important stimulus for endurance-training adaptations, including mitochondrial biogenesis, endogenous antioxidant production, insulin signalling, angiogenesis and growth factor signaling. Although sustained oxidative distress is associated with many chronic diseases, athletes have, on average, elevated levels of certain endogenous antioxidants to maintain redox homeostasis. As a result, trained athletes may have a better capacity to buffer oxidants during and after exercise, resulting in a reduced oxidative eustress stimulus for adaptations. Thus, higher levels of RONS input and exercise-induced oxidative stress may benefit athletes in the pursuit of continuous endurance training redox adaptations. This review addresses why athletes should be looking to enhance exercise-induced oxidative stress and how it can be accomplished. Methods covered include high-intensity interval training, hyperthermia and heat stress, dietary antioxidant restriction and modified antioxidant timing, dietary antioxidants and polyphenols as adjuncts to exercise, and vitamin C as a pro-oxidant.

Normal to enhanced intrinsic mitochondrial respiration in skeletal muscle of middle- to older-aged women and men with uncomplicated type 1 diabetes.

AIMS/HYPOTHESIS: This study interrogated mitochondrial respiratory function and content in skeletal muscle biopsies of healthy adults between 30 and 72 years old with and without uncomplicated type 1 diabetes. METHODS: Participants (12 women/nine men) with type 1 diabetes (48 ± 11 years of age), without overt complications, were matched for age, sex, BMI and level of physical activity to participants without diabetes (control participants) (49 ± 12 years of age). Participants underwent a Bergström biopsy of the vastus lateralis to assess mitochondrial respiratory function using high-resolution respirometry and citrate synthase activity. Electron microscopy was used to quantify mitochondrial content and cristae (pixel) density. RESULTS: Mean mitochondrial area density was 27% lower (p = 0.006) in participants with type 1 diabetes compared with control participants. This was largely due to smaller mitochondrial fragments in women with type 1 diabetes (-18%, p = 0.057), as opposed to a decrease in the total number of mitochondrial fragments in men with diabetes (-28%, p = 0.130). Mitochondrial respiratory measures, whether estimated per milligram of tissue (i.e. mass-specific) or normalised to area density (i.e. intrinsic mitochondrial function), differed between cohorts, and demonstrated sexual dimorphism. Mass-specific mitochondrial oxidative phosphorylation (OXPHOS) capacity with the substrates for complex I and complex II (CI + II) was significantly lower (-24%, p = 0.033) in women with type 1 diabetes compared with control participants, whereas mass-specific OXPHOS capacities with substrates for complex I only (pyruvate [CI pyr] or glutamate [CI glu]) or complex II only (succinate [CII succ]) were not different (p > 0.404). No statistical differences (p > 0.397) were found in mass-specific OXPHOS capacity in men with type 1 diabetes compared with control participants despite a 42% non-significant increase in CI glu OXPHOS capacity (p = 0.218). In contrast, intrinsic CI + II OXPHOS capacity was not different in women with type 1 diabetes (+5%, p = 0.378), whereas in men with type 1 diabetes it was 25% higher (p = 0.163) compared with control participants. Men with type 1 diabetes also demonstrated higher intrinsic OXPHOS capacity for CI pyr (+50%, p = 0.159), CI glu (+88%, p = 0.033) and CII succ (+28%, p = 0.123), as well as higher intrinsic respiratory rates with low (more physiological) concentrations of either ADP, pyruvate, glutamate or succinate (p < 0.012). Women with type 1 diabetes had higher (p < 0.003) intrinsic respiratory rates with low concentrations of succinate only. Calculated aerobic fitness (Physical Working Capacity Test [PWC130]) showed a strong relationship with mitochondrial respiratory function and content in the type 1 diabetes cohort. CONCLUSIONS/INTERPRETATION: In middle- to older-aged adults with uncomplicated type 1 diabetes, we conclude that skeletal muscle mitochondria differentially adapt to type 1 diabetes and demonstrate sexual dimorphism. Importantly, these cellular alterations were significantly associated with our metric of aerobic fitness (PWC130) and preceded notable impairments in skeletal mass and strength.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.

Muscle and serum myostatin expression in type 1 diabetes.

Type 1 diabetes (T1D) has been reported to negatively affect the health of skeletal muscle, though the underlying mechanisms are unknown. Myostatin, a myokine whose increased expression is associated with muscle-wasting diseases, has not been reported in humans with T1D but has been demonstrated to be elevated in preclinical diabetes models. Thus, the purpose of this study was to determine if there is an elevated expression of myostatin in the serum and skeletal muscle of persons with T1D compared to controls. Secondarily, we aimed to explore relationships between myostatin expression and clinically important metrics (e.g., HbA1c , strength, lean mass) in women and men with (N = 31)/without T1D (N = 24) between 18 and 72 years old. Body composition, baseline strength, blood sample and vastus lateralis muscle biopsy were evaluated. Serum, but not muscle, myostatin expression was significantly elevated in those with T1D versus controls, and to a greater degree in T1D women than T1D men. Serum myostatin levels were not significantly associated with HbA1c nor disease duration. A significant correlation between serum myostatin expression and maximal voluntary contraction (MVC) and body fat mass was demonstrated in control subjects, but these correlations did not reach significance in those with T1D (MVC: R = 0.64 controls vs. R = 0.37 T1D; Body fat: R = -0.52 controls/R = -0.02 T1D). Collectively, serum myostatin was correlated with lean mass (R = 0.45), and while this trend was noted in both groups separately, neither reached statistical significance (R = 0.47 controls/R = 0.33 T1D). Overall, while those with T1D exhibited elevated serum myostatin levels (particularly females) myostatin expression was not correlated with clinically relevant metrics despite some of these relationships existing in controls (e.g., lean/fat mass). Future studies will be needed to fully understand the mechanisms underlying increased myostatin in T1D, with relationships to insulin dosing being particularly important to elucidate.

A comparison of pain responses, hemodynamic reactivity and fibre type composition between Bergström and microbiopsy skeletal muscle biopsies.

This study tested the hypotheses that 1) skeletal muscle biopsies performed with the Bergström needle evoke larger perceptions of pain and greater hemodynamic reactivity compared to biopsies performed with the microbiopsy needle, and 2) both needles yield samples with similar fibre type compositions when samples are collected at similar skeletal muscle depths. Fourteen healthy (age: 21.6 ±â€¯3.2 years; VO2peak: 41.5 ±â€¯5.8 mL/kg/min) males (n = 7) and females (n = 7) provided two resting skeletal muscle biopsies, one with each needle type, following a randomized crossover design. Participants completed the short-form McGill Pain Questionnaire and the Brief Pain Inventory before, during, and after the skeletal muscle biopsies. Hemodynamic reactivity was assessed by measuring heart rate (HR) and mean arterial pressure (MAP) at rest and during the biopsy procedures. Immunofluorescence analysis was used to assess fibre type composition in vastus lateralis samples. Compared to the microbiopsy needle, the Bergström needle elicited a larger perception of pain but similar hemodynamic reactivity during the biopsy. Both needles yielded skeletal muscle samples with similar fibre type composition and resulted in similar perceptions of pain and pain-related interference during the post-biopsy recovery period. Collectively, these findings suggest that studies should consider using the microbiopsy needle rather than the Bergström needle unless large amounts of muscle tissue or certain muscle fibre lengths are required. However, future work should determine whether our findings are generalizable to biopsies performed with different procedures and/or types of Bergström/microbiopsy needles.

Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress.

Fatty acid stress can have divergent effects in various cancers. We explored how metabolic and redox flexibility in HepG2 hepatocarcinoma cells mediates protection from palmitoylcarnitine. HepG2 cells, along with HCT 116 and HT29 colorectal cancer cells were incubated with 100 µM palmitoylcarnitine for up to 48 h. Mitochondrial H2O2 emission, glutathione, and cell survival were assessed in HT29 and HepG2 cells. 100 µM palmitoylcarnitine promoted early growth in HepG2 cells by ~8% after 48 h versus decreased cell survival observed in HT29 and HCT 116 cells. Palmitoylcarnitine increased mitochondrial respiration at physiological and maximal concentrations of ADP, while lowering cellular lactate content in HepG2 cells, suggesting a switch to mitochondrial metabolism. HepG2 cell growth was associated with an early increase in H2O2 emission by 10 min, followed by a decrease in H2O2 at 24 h that corresponded with increased glutathione content, suggesting a redox-based compensatory mechanism. In contrast, abrogation of HT29 cell proliferation was related to decreased mitochondrial respiration (likely due to cell death) and decreased glutathione. Concurrent glutathione depletion with BSO prevented palmitoylcarnitine-induced growth in HepG2 cells, indicating that glutathione was critical for promoting growth following palmitoylcarnitine. Inhibiting UCP2 with genipin sensitized HepG2 cells to palmitoylcarnitine, suggesting that activation of UCP2 may be a 2nd redox-based mechanism conferring protection. These findings suggest that HepG2 cells possess inherent metabolic and redox flexibility relative to HT29 cells that confers protection from palmitoylcarnitine-induced stress via adaptive increases in mitochondrial respiratory control, glutathione buffering, and induction of UCP2.

The fatty acid derivative palmitoylcarnitine abrogates colorectal cancer cell survival by depleting glutathione.

Previous evidence suggests that palmitoylcarnitine incubations trigger mitochondrial-mediated apoptosis in HT29 colorectal adenocarcinoma cells, yet nontransformed cells appear insensitive. The mechanism by which palmitoylcarnitine induces cancer cell death is unclear. The purpose of this investigation was to examine the relationship between mitochondrial kinetics and glutathione buffering in determining the effect of palmitoylcarnitine on cell survival. HT29 and HCT 116 colorectal adenocarcinoma cells, CCD 841 nontransformed colon cells, and MCF7 breast adenocarcinoma cells were exposed to 0 µM, 50 µM, and 100 µM palmitoylcarnitine for 24-48 h. HCT 116 and HT29 cells showed decreased cell survival following palmitoylcarnitine compared with CCD 841 cells. Palmitoylcarnitine stimulated H2O2 emission in HT29 and CCD 841 cells but increased it to a greater level in HT29 cells due largely to a higher basal H2O2 emission. This greater H2O2 emission was associated with lower glutathione buffering capacity and caspase-3 activation in HT29 cells. The glutathione-depleting agent buthionine sulfoximine sensitized CCD 841 cells and further sensitized HT29 cells to palmitoylcarnitine-induced decreases in cell survival. MCF7 cells did not produce H2O2 when exposed to palmitoylcarnitine and were able to maintain glutathione levels. Furthermore, HT29 cells demonstrated the lowest mitochondrial oxidative kinetics vs. CCD 841 and MCF7 cells. The results demonstrate that colorectal cancer is sensitive to palmitoylcarnitine due in part to an inability to prevent oxidative stress through glutathione-redox coupling, thereby rendering the cells sensitive to elevations in H2O2. These findings suggest that the relationship between inherent metabolic capacities and redox regulation is altered early in response to palmitoylcarnitine.

Early myopathy in Duchenne muscular dystrophy is associated with elevated mitochondrial H2 O2 emission during impaired oxidative phosphorylation.

BACKGROUND: Muscle wasting and weakness in Duchenne muscular dystrophy (DMD) causes severe locomotor limitations and early death due in part to respiratory muscle failure. Given that current clinical practice focuses on treating secondary complications in this genetic disease, there is a clear need to identify additional contributions in the aetiology of this myopathy for knowledge-guided therapy development. Here, we address the unresolved question of whether the complex impairments observed in DMD are linked to elevated mitochondrial H2 O2 emission in conjunction with impaired oxidative phosphorylation. This study performed a systematic evaluation of the nature and degree of mitochondrial-derived H2 O2 emission and mitochondrial oxidative dysfunction in a mouse model of DMD by designing in vitro bioenergetic assessments that attempt to mimic in vivo conditions known to be critical for the regulation of mitochondrial bioenergetics. METHODS: Mitochondrial bioenergetics were compared with functional and histopathological indices of myopathy early in DMD (4 weeks) in D2.B10-DMDmdx /2J mice (D2.mdx)-a model that demonstrates severe muscle weakness. Adenosine diphosphate's (ADP's) central effect of attenuating H2 O2 emission while stimulating respiration was compared under two models of mitochondrial-cytoplasmic phosphate exchange (creatine independent and dependent) in muscles that stained positive for membrane damage (diaphragm, quadriceps, and white gastrocnemius). RESULTS: Pathway-specific analyses revealed that Complex I-supported maximal H2 O2 emission was elevated concurrent with a reduced ability of ADP to attenuate emission during respiration in all three muscles (mH2 O2 : +17 to +197% in D2.mdx vs. wild type). This was associated with an impaired ability of ADP to stimulate respiration at sub-maximal and maximal kinetics (-17 to -72% in D2.mdx vs. wild type), as well as a loss of creatine-dependent mitochondrial phosphate shuttling in diaphragm and quadriceps. These changes largely occurred independent of mitochondrial density or abundance of respiratory chain complexes, except for quadriceps. This muscle was also the only one exhibiting decreased calcium retention capacity, which indicates increased sensitivity to calcium-induced permeability transition pore opening. Increased H2 O2 emission was accompanied by a compensatory increase in total glutathione, while oxidative stress markers were unchanged. Mitochondrial bioenergetic dysfunctions were associated with induction of mitochondrial-linked caspase 9, necrosis, and markers of atrophy in some muscles as well as reduced hindlimb torque and reduced respiratory muscle function. CONCLUSIONS: These results provide evidence that Complex I dysfunction and loss of central respiratory control by ADP and creatine cause elevated oxidant generation during impaired oxidative phosphorylation. These dysfunctions may contribute to early stage disease pathophysiology and support the growing notion that mitochondria are a potential therapeutic target in this disease.

Holo-lipocalin-2-derived siderophores increase mitochondrial ROS and impair oxidative phosphorylation in rat cardiomyocytes.

Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

Decreased transcriptional corepressor p107 is associated with exercise-induced mitochondrial biogenesis in human skeletal muscle.

Increased mitochondrial content is a hallmark of exercise-induced skeletal muscle remodeling. For this process, considerable evidence underscores the involvement of transcriptional coactivators in mediating mitochondrial biogenesis. However, our knowledge regarding the role of transcriptional corepressors is lacking. In this study, we assessed the association of the transcriptional corepressor Rb family proteins, Rb and p107, with endurance exercise-induced mitochondrial adaptation in human skeletal muscle. We showed that p107, but not Rb, protein levels decrease by 3 weeks of high-intensity interval training. This is associated with significant inverse association between p107 and exercise-induced improved mitochondrial oxidative phosphorylation. Indeed, p107 showed significant reciprocal correlations with the protein contents of representative markers of mitochondrial electron transport chain complexes. These findings in human skeletal muscle suggest that attenuated transcriptional repression through p107 may be a novel mechanism by which exercise stimulates mitochondrial biogenesis following exercise.

p107 Determines a Metabolic Checkpoint Required for Adipocyte Lineage Fates.

We show that the transcriptional corepressor p107 orchestrates a metabolic checkpoint that determines adipocyte lineage fates for non-committed progenitors. p107 accomplishes this when stem cell commitment would normally occur in growth arrested cells. p107-deficient embryonic progenitors are characterized by a metabolic state resembling aerobic glycolysis that is necessary for their pro-thermogenic fate. Indeed, during growth arrest they have a reduced capacity for NADH partitioning between the cytoplasm and mitochondria. Intriguingly, this occurred despite an increase in the capacity for mitochondrial oxidation of non-glucose substrates. The significance of metabolic reprogramming is underscored by the disruption of glycolytic capacities in p107-depleted progenitors that reverted their fates from pro-thermogenic to white adipocytes. Moreover, the manipulation of glycolytic capacity on nonspecified embryonic and adult progenitors forced their beige fat commitment. These innovative findings introduce a new approach to increase pro-thermogenic adipocytes based on simply promoting aerobic glycolysis to manipulate nonspecified progenitor fate decisions. Stem Cells 2017;35:1378-1391.

Reduced ATGL-mediated lipolysis attenuates ß-adrenergic-induced AMPK signaling, but not the induction of PKA-targeted genes, in adipocytes and adipose tissue.

5'-AMP-activated protein kinase (AMPK) is activated as a consequence of lipolysis and has been shown to play a role in regulation of adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A (PKA)-targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, AMPK, and the ß-adrenergic-mediated regulation of gene expression. In 3T3-L1 adipocytes, the adipose tissue triglyceride lipase (ATGL) inhibitor ATGListatin attenuated the Thr(172) phosphorylation of AMPK by a ß3-adrenergic agonist (CL 316,243) independent of changes in PKA signaling. Similarly, CL 316,243-induced increases in the Thr(172) phosphorylation of AMPK were reduced in adipose tissue from whole body ATGL-deficient mice. Despite reductions in the activation of AMPK, the induction of PKA-targeted genes was intact or, in some cases, increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr(172) phosphorylation of AMPK. Taken together, our data provide evidence that AMPK is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release.

Modelling in vivo creatine/phosphocreatine in vitro reveals divergent adaptations in human muscle mitochondrial respiratory control by ADP after acute and chronic exercise.

KEY POINTS: Mitochondrial respiratory sensitivity to ADP is thought to influence muscle fitness and is partly regulated by cytosolic-mitochondrial diffusion of ADP or phosphate shuttling via creatine/phosphocreatine (Cr/PCr) through mitochondrial creatine kinase (mtCK). Previous measurements of respiration in vitro with Cr (saturate mtCK) or without (ADP/ATP diffusion) show mixed responses of ADP sensitivity following acute exercise vs. less sensitivity after chronic exercise. In human muscle, modelling in vivo 'exercising' [Cr:PCr] during in vitro assessments revealed novel responses to exercise that differ from detections with or without Cr (±Cr). Acute exercise increased ADP sensitivity when measured without Cr but had no effect ±Cr or with +Cr:PCr, whereas chronic exercise increased sensitivity ±Cr but lowered sensitivity with +Cr:PCr despite increased markers of mitochondrial oxidative capacity. Controlling in vivo conditions during in vitro respiratory assessments reveals responses to exercise that differ from typical ±Cr comparisons and challenges our understanding of how exercise improves metabolic control in human muscle. ABSTRACT: Mitochondrial respiratory control by ADP (Kmapp ) is viewed as a critical regulator of muscle energy homeostasis. However, acute exercise increases, decreases or has no effect on Kmapp in human muscle, whereas chronic exercise surprisingly decreases sensitivity despite greater mitochondrial content. We hypothesized that modelling in vivo mitochondrial creatine kinase (mtCK)-dependent phosphate-shuttling conditions in vitro would reveal increased sensitivity (lower Kmapp ) after acute and chronic exercise. The Kmapp was determined in vitro with 20 mm Cr (+Cr), 0 mm Cr (-Cr) or 'in vivo exercising' 20 mm Cr/2.4 mm PCr (Cr:PCr) on vastus lateralis biopsies sampled from 11 men before, immediately after and 3 h after exercise on the first, fifth and ninth sessions over 3 weeks. Dynamic responses to acute exercise occurred throughout training, whereby the first session did not change Kmapp with in vivo Cr:PCr despite increases in -Cr. The fifth session decreased sensitivity with Cr:PCr or +Cr despite no change in -Cr. Chronic exercise increased sensitivity ±Cr in association with increased electron transport chain content (+33-62% complexes I-V), supporting classic proposals that link increased sensitivity to oxidative capacity. However, in vivo Cr:PCr reveals a perplexing decreased sensitivity, contrasting the increases seen ±Cr. Functional responses occurred without changes in fibre type or proteins regulating mitochondrial-cytosolic energy exchange (mtCK, VDAC and ANT). Despite the dynamic responses seen with ±Cr, modelling in vivo phosphate-shuttling conditions in vitro reveals that ADP sensitivity is unchanged after high-intensity exercise and is decreased after training. These findings challenge our understanding of how exercise regulates skeletal muscle energy homeostasis.