Increase In Mitochondrial Function Research Articles

Skeletal Muscle Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 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.

Adaptation of Oxidative Phosphorylation Machinery Compensates for Hepatic Lipotoxicity in Early Stages of MAFLD.

Alterations in mitochondrial function are an important control variable in the progression of metabolic dysfunction-associated fatty liver disease (MAFLD), while also noted by increased de novo lipogenesis (DNL) and hepatic insulin resistance. We hypothesized that the organization and function of a mitochondrial electron transport chain (ETC) in this pathologic condition is a consequence of shifted substrate availability. We addressed this question using a transgenic mouse model with increased hepatic insulin resistance and DNL due to constitutively active human SREBP-1c. The abundance of ETC complex subunits and components of key metabolic pathways are regulated in the liver of these animals. Further omics approaches combined with functional assays in isolated liver mitochondria and primary hepatocytes revealed that the SREBP-1c-forced fatty liver induced a substrate limitation for oxidative phosphorylation, inducing enhanced complex II activity. The observed increased expression of mitochondrial genes may have indicated a counteraction. In conclusion, a shift of available substrates directed toward activated DNL results in increased electron flows, mainly through complex II, to compensate for the increased energy demand of the cell. The reorganization of key compounds in energy metabolism observed in the SREBP-1c animal model might explain the initial increase in mitochondrial function observed in the early stages of human MAFLD.

An explanation for the decreased severity of liver malfunction in Niemann-Pick C1 disease with age.

Niemann-Pick C disease frequently presents as severe cholestatic disease in infants. However, it progressively becomes less of a problem as children age. We have found that, in an appropriate mouse model, liver cholesterol levels, which are initially very high, decrease while mitochondrial function, initially quite compromised, increases with age. The key mitochondrial regulator, MNRR1, increases in parallel with the increase in mitochondrial function. These changes appear to explain the amelioration of the liver disease that occurs with time in this disorder.

Repeated Aconitine Treatment Induced the Remodeling of Mitochondrial Function via AMPK-OPA1-ATP5A1 Pathway.

Aconitine is attracting increasing attention for its unique positive inotropic effect on the cardiovascular system, but underlying molecular mechanisms are still not fully understood. The cardiotonic effect always requires abundant energy supplement, which is mainly related to mitochondrial function. And OPA1 has been documented to play a critical role in mitochondrial morphology and energy metabolism in cardiomyocytes. Hence, this study was designed to investigate the potential role of OPA1-mediated regulation of energy metabolism in the positive inotropic effect caused by repeated aconitine treatment and the possible mechanism involved. Our results showed that repeated treatment with low-doses (0–10 μM) of aconitine for 7 days did not induce detectable cytotoxicity and enhanced myocardial contraction in Neonatal Rat Ventricular Myocytes (NRVMs). Also, we first identified that no more than 5 μM of aconitine triggered an obvious perturbation of mitochondrial homeostasis in cardiomyocytes by accelerating mitochondrial fusion, biogenesis, and Parkin-mediated mitophagy, followed by the increase in mitochondrial function and the cellular ATP content, both of which were identified to be related to the upregulation of ATP synthase α-subunit (ATP5A1). Besides, with compound C (CC), an inhibitor of AMPK, could reverse aconitine-increased the content of phosphor-AMPK, OPA1, and ATP5A1, and the following mitochondrial function. In conclusion, this study first demonstrated that repeated aconitine treatment could cause the remodeling of mitochondrial function via the AMPK–OPA1–ATP5A1 pathway and provide a possible explanation for the energy metabolism associated with cardiotonic effect induced by medicinal plants containing aconitine.

Effect of glucose concentration and cryopreservation on mitochondrial functions of bull spermatozoa and relationship with sire conception rate

Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P < 0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to “non-productive” oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.

General anesthesia activates the mitochondrial unfolded protein response and induces age-dependent, long-lasting changes in mitochondrial function in the developing brain

General anesthesia induces changes in dendritic spine number and synaptic transmission in developing mice. These changes are rather disturbing, as similar changes are seen in animal models of neurodevelopmental disorders. We previously suggested that mTor-dependent upregulation of mitochondrial function may be involved in such changes. To further understand the significance of mitochondrial changes after general anesthesia during neurodevelopment, we exposed young mice to 2.5 % sevoflurane for 2 h followed by injection of rotenone, a mitochondrial complex I inhibitor. In postnatal day 17 (PND17) mice, intraperitoneal injection of rotenone not only blocked sevoflurane-induced increases in mitochondrial function, it also prevented sevoflurane-induced changes in excitatory synaptic transmission. Interestingly, similar changes were not observed in younger, neonatal mice (PND7). We next assessed whether the mitochondrial unfolded protein response (UPRmt) acted as a link between anesthetic exposure and mitochondrial function. Expression of UPRmt proteins, which help maintain protein-folding homeostasis and increase mitochondrial function, was increased 6 h after sevoflurane exposure. Our results show that a single, brief sevoflurane exposure induces age-dependent changes in mitochondrial function that constitute an important mechanism for the increase in excitatory synaptic transmission in late postnatal mice, and also suggest mitochondria and UPRmt as potential targets for preventing anesthesia toxicity.

Abstract A69: Role of mitochondrial-STAT3 in promoting chemoresistance by modulating the energy metabolism in ovarian cancer

Abstract Signal Transducers and Activators of Transcription 3 (STAT3) is a transcription factor that is known to play a key role in cancer progression. In ovarian cancer, STAT3 overexpression leads to increased cancer cell proliferation and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. It is constitutively activated in patient-derived ovarian cancer cells and a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to translocate to mitochondria, facilitated by phosphorylation at S727 and modulate mitochondrial function to promote carcinogenesis. Our study aimed to investigate if mitochondrial-STAT3, rather than nuclear-STAT3, plays a major role in modulating cellular metabolism and promoting chemoresistance in ovarian cancer cells. We generated stable clones overexpressing STAT3 in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 resulted in increased proliferation, colony formation ability, and chemoresistance in vitro and led to large and aggressive ovarian tumors compared to parental and vector controls in the xenograft mouse model. STAT3 overexpressing clones exhibited higher mitochondrial respiration and glycolysis placing them in the “metabolically active” phenotype compared to parental and vector clones (metabolically less active phenotype). A STAT3 inhibitor, Stattic, inhibited both nuclear and mitochondrial STAT3 and also attenuated the STAT3-mediated growth of overexpressing clones both in vitro and in vivo. Stattic treatment also reversed the STAT3-mediated chemoresistance. In contrast, a selective inhibitor of STAT3-Y705, cryptotanshinone, was relatively less effective. Also, Stattic treatments reversed the “metabolically active” state of STAT3 overexpressing clones to a “lower metabolic state,” as the control cells. Stattic also inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200, and OVCAR3) that display a metabolically active phenotype, suggesting STAT3 plays a vital role in attaining a metabolically active phenotype by cancer cells. Further, silencing STAT3-Y705 (nuclear-STAT3) did not significantly affect the chemoresistance, while silencing STAT3-S727 (mitochondrial-STAT3) reversed chemoresistance. Although an increase in mitochondrial function was observed in overexpressing A2780 clones as evident from enhanced oxidative phosphorylation, there was no change in the mitochondrial mass or number, indicating the critical role of STAT3 in mitochondrial function rather than mitochondrial biogenesis in ovarian cancer cells. Thus, further evaluation of expression and function of mitochondrial STAT3 in ovarian cancers is warranted. Overall, mitochondrial-STAT3 can induce metabolic changes in ovarian cancer cells and enhance their cellular fitness by promoting chemoresistance. Citation Format: Vaishnavi Raja, Shailendra Giri, Suhail Hamid, Thomas Buekers, Adnan Munkarah, Ramandeep Rattan. Role of mitochondrial-STAT3 in promoting chemoresistance by modulating the energy metabolism in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A69.

Abstract GMM-049: STAT3 PROMOTES OVARIAN CANCER GROWTH AND DRUG RESISTANCE BY MODULATING THE ENERGY METABOLISM

Abstract Signal Transducers and Activators of Transcription 3 (STAT3) is a transcription factor that is known to play a key role in cancer progression. In ovarian cancer, STAT3 overexpression leads to increased cancer cell proliferation and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. It is constitutively activated in patient-derived ovarian cancer cells and a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to translocate to mitochondria facilitated by phosphorylation at S727 and modulate mitochondrial function to promote carcinogenesis. Our study aimed to investigate if STAT3 overexpression can modulate cellular metabolism and promote the growth of ovarian cancer cells. We generated stable clones overexpressing STAT3 in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 resulted in increased proliferation, colony formation ability and chemoresistance in vitro and led to large and aggressive ovarian tumors compared to parental and vector controls in xenograft mouse model. STAT3 overexpressing clones exhibited higher mitochondrial respiration and glycolysis placing them in the ‘metabolically active' phenotype compared to parental and vector clones (metabolically less active phenotype). A selective inhibitor of STAT3, Stattic, inhibited both nuclear and mitochondrial STAT3 and also attenuated the STAT3 mediated growth of over-expressing clones both in vitro and in vivo. Stattic treatment also reversed the STAT3-mediated chemoresistance. In contrast, a selective inhibitor of STAT3-Y705, Cryptotanshinone was relatively less effective. Also, Stattic treatments reversed the ‘metabolically active’ state of STAT3 overexpressing clones to a ‘lower metabolic state,’ as the control cells. Stattic also inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200, and OVCAR3) that display a metabolically active phenotype suggesting STAT3 plays a vital role in attaining a metabolically active phenotype by cancer cells. Although, an increase in mitochondrial function was observed in overexpressing A2780 clones as evident from enhanced oxidative phosphorylation, there is no change in the mitochondrial mass or number in overexpressing clones compared to parental A2780 and vector clones indicating the critical role of STAT3 in mitochondrial functions rather than mitochondrial biogenesis in ovarian cancer cells. Further, evaluation of expression and function of mitochondrial STAT3 in ovarian cancers is warranted. Overall, STAT3 can induce metabolic changes in ovarian cancer cells by facilitating mitochondrial function, maybe as a survival mechanism and enhances the cellular fitness of the ovarian cancer cell resulting in increased oncogenic abilities. Citation Format: Vaishnavi Raja, Shailendra Giri, Suhail Hamid, Adnan R Munkarah and Ramandeep Rattan. STAT3 PROMOTES OVARIAN CANCER GROWTH AND DRUG RESISTANCE BY MODULATING THE ENERGY METABOLISM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-049.

Reciprocal interactions between mtDNA and lifespan control in budding yeast.

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0 cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0 cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

Effect of TNFα on Mitochondrial Function and Mitochondrial Biogenesis in Human Airway Smooth Muscle

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity as well as airway remodeling. Proinflammatory cytokines, such as TNFα mediate this airway inflammatory response. Previously, we found that TNFα induces an increase airway smooth muscle (ASM) force and ATP hydrolysis. In response, TNFα may also induce an increase in ATP production in ASM. This could be achieved by either an increase in mitochondrial function and/or mitochondrial biogenesis. We previously found that TNFα decreases mitochondrial Ca2+ influx. Since dehydrogenase enzymes in the TCA cycle are Ca2+ dependent, this would tend to reduce mitochondrial function in response to agonist stimulation. We also found that TNFα increases mitochondrial fragmentation, an initial step in mitochondrial biogenesis. We hypothesized that TNFα increases mitochondrial biogenesis and decreases mitochondrial function in human ASM.Human ASM cells were isolated from lung specimens incidental to patient surgery. The patients were normal as evaluated by a clinical pathologist (no history of asthma or COPD). hASM cells were exposed to TNFα (20 ng/ml) for 24 h. To examine mitochondrial biogenesis, protein expression of porin (an indicator of mitochondrial density) and PGC1α (a regulator of mitochondria biogenesis) were determined by Western blot analysis. In addition, the relative number of copies of human mitochondrial DNA (mtDNA) was determined by PCR and normalized to nuclear DNA. Mitochondrial function was assessed by measuring O2 Consumption Rate (OCR – using a Seahorse Bioscience XF24 Analyzer), succinate dehydrogenase (SDH) activity (using a quantitative histochemical technique) and mitochondrial membrane potential – (DYm – using TMRM and confocal microscopy). OCR and SDH activity measurements were normalized to mitochondrial volume density. Results were analyzed by one‐way ANOVA with post hoc analysis when justified.We found that TNFα increases mitochondrial biogenesis as indicated by an increase in porin and PGC1α protein expression in hASM and an increase in mtDNA copy numbers. We also found that TNFα decreases mitochondrial membrane potential in hASM cells, and also decreases OCR and SDH activity when normalized for the increase in mitochondrial volume density induced by TNFα. In a normal homeostatic response, an increase in hASM force and ATP hydrolysis (energy demand) would be met by increased mitochondrial ATP production. Our results indicate that TNFα disrupts this normal homeostatic response by reducing mitochondrial Ca2+ influx and thereby reducing mitochondrial TCA activity. However, TNFα increase the number of mitochondria to compensate the decrease in mitochondrial TCA activity.Support or Funding InformationSupported by NIH grant HL126451This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition

BackgroundPlatinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. MethodsCisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). ResultsTwo out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α, and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles' heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. ConclusionThese results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.

Mitochondrial Uncoupling Protein 2 Knock-out Promotes Mitophagy to Decrease Retinal Ganglion Cell Death in a Mouse Model of Glaucoma.

Glaucoma is a neurodegenerative disorder characterized by mitochondrial dysfunction and an increase in oxidative damage, leading to retinal ganglion cell (RGC) death. The oxidative status of RGCs is regulated intrinsically and also extrinsically by retinal glia. The mitochondrial uncoupling protein 2 (UCP2) relieves oxidative and neuronal damage in a variety of neurodegenerative disease models. We hypothesized that deletion of Ucp2 in either RGCs or retinal glia would increase retinal damage and RGC death in a mouse model of glaucoma. Paradoxically, we found the reverse, and deletion of mitochondrial Ucp2 decreased oxidative protein modification and reduced RGC death in male and female mice. This paradox was resolved after finding that Ucp2 deletion also increased levels of mitophagy in cell culture and retinal tissue. Our data suggest that Ucp2 deletion facilitates increased mitochondrial function by improving quality control. An increase in mitochondrial function explains the resistance of Ucp2-deleted retinas to glaucoma and may provide a therapeutic avenue for other chronic neurodegenerative conditions.SIGNIFICANCE STATEMENT Many unsolved neurodegenerative conditions result from defects in mitochondrial function. Molecular tools that can manipulate mitochondria will therefore be central to developing neuroprotective therapies. Among the most potent regulators of mitochondrial function are the uncoupling proteins, particularly UCP2. In this manuscript, we show that, while loss of Ucp2 does increase mitochondrial membrane potential and the production of reactive oxygen species, it also initiates an increase in mitophagy that is ultimately neuroprotective. This novel protective consequence of uncoupling protein inhibition may lead to new therapeutic approaches to combat neurodegenerative disease, particularly because pharmacological compounds do exist that can selectively inhibit UCP2.

Parenteral L-Arginine Improves Mitochondrial Function in Children with Sickle Cell Disease Admitted for Vaso-Occlusive Pain Episodes

Introduction: Vaso-occlusive pain episodes (VOE) are the leading cause of emergency department visits and hospitalization in patients with sickle cell disease (SCD). There is currently no standard therapy for treating the underlying causes of VOE; supportive care & symptomatic pain relief with parenteral opioids is the mainstay of therapy. Prior work by Morris & colleagues has demonstrated that patients in VOE show depleted concentrations of plasma L-arginine (Arg), and that supplementation with Arg significantly decreases total parenteral opioid use and improves pain scores during hospitalization in these patients (Morris et al 2013, Haematologica). The mechanisms by which Arg mediates these positive effects remain unclear, but are potentially due to its ability to modulate metabolism through increased Arg transport or affect vascular signaling through the stimulation of nitric oxide production. The Shiva lab has previously demonstrated that platelet mitochondrial function is altered in patients with SCD compared to healthy controls (Cardenas et al 2014, Blood). This alteration is manifested as a decrease in mitochondrial electron transport Complex V (ATP Synthase) activity, which leads to decreased respiration and increased detrimental oxidant production. Here we hypothesized that treatment of SCD patients in VOE with Arg increases mitochondrial function.Methods: In a pharmacokinetic study, 10 subjects with Hb-SS or Sb0-thalassemia hospitalized for VOE (age 14±3 years, 70% male, 80% Hb-SS) were randomized to treatment utilizing one of 3 dosing schemes of L-arginine: 1) 100mg/kg IV three times/day (n=3); 2) loading dose (200mg/kg) then 100mg/kg three times/day (n=4); or 3) loading dose (200mg/kg) followed by continuous infusion (300mg/kg/day) until discharge (n=3). Platelet rich plasma was isolated & stored at baseline & discharge for each subject & mitochondrial electron transport complex activities, protein expression, as well as protein carbonyls were measured.Results: Compared to a cohort of SCD patients in steady state, all subjects in VOE had a significantly decreased complex V activity (Fig 1A). Notably, complex V activity was increased at discharge in subjects with VOE treated with Arg in all three dosing schemes. However, the increase in complex V activity was greatest when utilizing a loading dose. (Fig 1B, p<0.001). While complex IV & citrate synthase activities were not changed in platelets from subjects with VOE compared to SCD patients in steady state (data not shown), the activities of these enzymes were also significantly increased in VOE subjects after Arg treatment when utilizing a loading dose( Fig 1C-D, p<0.01). These changes with Arg do not appear to be due to increased mitochondrial number as quantification of mitochondrial DNA before and after Arg was not different (Fig 1E). Complex V protein expression was also unchanged after Arg treatment (data not shown). However, Arg treatment did significantly decrease levels of protein carbonyls in the platelet rich plasma across all treatment dosing schemes (Fig 1F, p<0.01 in all dosing groups), suggesting a decrease in oxidative stress.Conclusion: These data demonstrate for the first time that Arg supplementation increases mitochondrial activity and decreases oxidative stress in children with SCD and VOE. Our prior study showed that complex V inhibition leads to increased mitochondrial oxidant production in platelets from SCD patients. The improvement in complex V function & decreased oxidative markers observed with Arg treatment are consistent with Arg-induced decrease in mitochondrial oxidant generation. However, Arg treatment potentially regulates other oxidant sources and could decrease overall oxidative stress in SCD patients. Arg therapy has been reported to improve symptoms associated with MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) suggesting an impact of arginine on mitochondrial activity that may be beneficial for patients with SCD. With the recent FDA approval of L-glutamine, which is an Arg prodrug, this is also a potential consequence of glutamine that warrants further study. This increase in mitochondrial function may mechanistically contribute to decreased pain and ultimately less opioid requirement after IV Arg treatment in SCD, as well as have further implications for improved metabolism and oxidative signaling in these patients. DisclosuresDampier:Pfizer: Research Funding. Morris:Pfizer: Consultancy; MAST Therapeutics: Research Funding; UCSF-Benioff Children's Hospital Oakland: Patents & Royalties: For nutritional supplement licensed to Lifetrients; Calithera: Consultancy; FDA: Research Funding; NIH/NHLBI: Research Funding.

GPER and ERα mediate estradiol enhancement of mitochondrial function in inflamed adipocytes through a PKA dependent mechanism

Obesity is associated with inflammation, dysregulated adipokine secretion, and disrupted adipose tissue mitochondrial function. Estradiol (E2) has been previously reported to increase mitochondrial function and biogenesis in several cell lines, but neither the type of oestrogen receptor (ERα, ERβ and GPER) involved nor the mechanism whereby such effects are exerted have been fully described. Considering the anti-inflammatory activity of E2 as well as its effects in enhancing mitochondrial biogenesis, the aim of this study was to investigate the contribution of ERα, ERβ, and GPER signaling to the E2-mediated enhancement of adipocyte mitochondrial function in a pro-inflammatory situation.3T3-L1 cells were treated for 24 h with ER agonists (PPT, DPN, and G1) and antagonists (MPP, PHTPP, and G15) in the presence or absence of interleukin 6 (IL6), as a pro-inflammatory stimulus. Inflammation, mitochondrial function and biogenesis markers were analyzed. To confirm the involvement of the PKA pathway, cells were treated with a GPER agonist, a PKA inhibitor, and IL6. Mitochondrial function markers were analyzed.Our results showed that activation of ERα and GPER, but not ERβ, was able to counteract the proinflammatory effects of IL6 treatment, as well as mitochondrial biogenesis and function indicators. Inhibition of PKA prevented the E2- and G1-associated increase in mitochondrial function markers.In conclusion E2 prevents IL6 induced inflammation in adipocytes and promotes mitochondrial function through the combined activation of both GPER and ERα. These findings expand our understanding of ER interactions under inflammatory conditions in female rodent white adipose tissue.

Peripubertal stress increases play fighting at adolescence and modulates nucleus accumbens CB1 receptor expression and mitochondrial function in the amygdala

Play fighting is a highly rewarding behavior that helps individuals to develop social skills. Early-life stress has been shown to alter play fighting in rats and hamsters as well as to increase aggressive behaviors at adulthood. However, it is not known whether individual differences in stress-induced play fighting are related to differential developmental trajectories towards adult aggression. To address this question, we used a rat model of peripubertal stress (PPS)-induced psychopathology that involves increased aggression at adulthood. We report that, indeed, PPS leads to enhanced play fighting at adolescence. Using a stratification approach, we identify individuals with heightened levels of play fighting as the ones that show abnormal forms of aggression at adulthood. These animals showed as well a rapid habituation of their corticosterone responsiveness to repeated stressor exposure at peripuberty. They also showed a striking increase in mitochondrial function in the amygdala—but not nucleus accumbens—when tested ex vivo. Conversely, low, but not high players, displayed increased expression of the CB1 cannabinoid receptor in the nucleus accumbens shell. Our results highlight adolescence as a potential critical period in which aberrant play fighting is linked to the emergence of adult aggression. They also point at brain energy metabolism during adolescence as a possible target to prevent adult aggression.

Abstract 117: Regulation of Cardiac Mitochondrial Function by Chaperone Mediated Autophagy

Macroautophagy is required for normal cardiac function. Chaperone mediated autophagy (CMA) is distinct in that substrates are transported to lysosomes in complex with Hsc70, rather than within autophagosomes; restricted to cytoplasmic proteins; and selectively marked with a CMA-targeting motif. The role of CMA in the heart has not been assessed. We determined that CMA is induced in the heart by pressure overload with kinetics that differ markedly from those of macroautophagy in response to the same stimulus. In CMA, cargo is imported into the lysosome by LAMP2A, which is necessary, specific, and rate-limiting for this process. Accordingly, we created knockout mice lacking LAMP2A in cardiomyocytes to inactivate CMA specifically in those cells. Unexpectedly, these mice exhibited resistance to stress-induced cardiac dysfunction in both pressure overload and myocardial infarction models. Functional assessment of cardiac mitochondria from L2AKO mice and MEFs exhibited increased rates of O 2 consumption and ATP generation. Mitochondrial proteomics showed increased levels of ATP5L, a component of mitochondrial Complex V (ATP synthase). Overexpression of ATP5L phenocopied LAMP2A KO with respect to augmentation of mitochondrial function. Conversely, knockdown of ATP5L reversed increases in mitochondrial function resulting from L2AKO. We showed that ATP5L, which possesses a KFERQ motif, is a bona fide CMA substrate as it interacts with Hsc70 in a KFERQ-dependent manner; and localizes to lyososmes in a LAMP2A-dependent manner when CMA is induced. ATP5L is thought to mediate oligomerization of Complex V to enhance ATP production. In fact, we observed increases in complex V oligomerization in L2A KO mouse hearts in both basal and TAC-induced states. In summary, cardiomyocyte-specific inhibition of CMA, which renders mice resistant to stress-induced cardiac dysfunction, augments cardiac mitochondrial function through accumulation of ATP5L. Conversely, activation of CMA by cardiac stress may function as a new pathway to heart failure.

Mitochondrial adaptations in aged skeletal muscle: effect of exercise training.

The aging process is associated with a decline in mitochondrial functions. Mitochondria dysfunction is involved in initiation and progression of many health problems including neuromuscular, metabolic and cardiovascular diseases. It is well known that endurance exercise improves mitochondrial function, especially in the elderly. However, recent studies have demonstrated that resistance training lead also to substantial increases in mitochondrial function in skeletal muscle. A comprehensive understanding of the cellular mechanisms involved in the skeletal muscle mitochondrial adaptations to exercise training in healthy elderly subjects, can help practitioners to design and prescribe more effective exercise trainings.

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function

Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1α and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1α as cells deficient in PGC-1α (PGC-1α knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1α-dependent manner, P < 0.05. The increase in PGC-1α was associated with an increase in β-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1α is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1α signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation.

Phosphoinositide dependent protein kinase 1 is required for exercise-induced cardiac hypertrophy but not the associated mitochondrial adaptations

Phosphoinositide-dependent protein kinase-1 (PDPK1) is an important mediator of phosphatidylinositol 3-kinase (PI3K) signaling. We previously reported that PI3K but not Akt signaling mediates the increase in mitochondrial oxidative capacity following physiological cardiac hypertrophy. To determine if PDPK1 regulates these metabolic adaptations we examined mice with cardiomyocyte-specific heterozygous knockout of PDPK1 (cPDPK1+/−) after 5 wk. exercise swim training. Akt phosphorylation at Thr308 increased by 43% in wildtype (WT) mice but not in cPDPK1+/− mice following exercise training. Ventricular contractile function was not different between WT and cPDPK1+/− mice at baseline. In addition, exercise did not influence ventricular function in WT or cPDPK1+/− mice. Heart weight normalized to tibia length ratios increased by 13.8% in WT mice (6.2±0.2 vs. 7.1±0.2, P=0.001), but not in cPDPK1+/− (6.2±0.3 vs. 6.5±0.2, P=0.20) mice after swim training. Diastolic LV dimension increased in WT mice (3.7±0.1 vs. 4.0±0.1mm, P=0.01) but not in cPDPK1+/− (3.8±0.1 vs. 3.7±0.1mm, P=0.56) following swim training. Maximal mitochondrial oxygen consumption (VADP, nmol/min/mg) using palmitoyl carnitine as a substrate was significantly increased in mice of all genotypes following swim training (WT: 13.6±0.6 vs.16.1±0.9, P=0.04; cPDPK1+/−: 12.4±0.6 vs.15.9±1.2, P=0.04). These findings suggest that PDPK1 is required for exercise-induced cardiac hypertrophy but does not contribute to exercise-induced increases in mitochondrial function.

TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.

ObjectivsCytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.MethodsCultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.ResultsTreatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).ConclusionsTGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.