Mitochondrial Electron Transport Chain Function Research Articles

Stereotactically intracerebral transplantation of neural stem cells for ischemic stroke attenuated inflammatory responses and promoted neurogenesis: an experimental study with monkeys.

Ischemic stroke is a common neurovascular disorder with high morbidity and mortality. However, the underlying mechanism of stereotactically intracerebral transplantation of human neural stem cell (hNSC) is not well elucidated. Four days after ischemic stroke induced by Rose Bengal photo-thrombosis, 7 cynomolgus monkeys were transplanted with hNSCs or vehicles stereotactically and followed up for 84 days. Behavioral assessments, magnetic resonance imaging, blood tests, and pathological analysis were performed before and after treatment. The proteome profiles of the left and right precentral gyrus and hippocampus were evaluated. Extracellular vesicle micro-RNA (miRNA) from the peripheral blood was extracted and analyzed. hNSC transplantation reduced the remaining infarcted lesion volume of cynomolgus monkeys with ischemic stroke without remarkable side effects. Proteomic analyses indicated that hNSC transplantation promoted GABAergic and glutamatergic neurogenesis, and restored the mitochondrial electron transport chain function in the ischemic infarcted left precentral gyrus or hippocampus. Immunohistochemical staining and qRT-PCT confirmed the promoting effects on neurogenesis and revealed that hNSCs attenuated post-infarct inflammatory responses by suppressing resident glia activation and mediating peripheral immune cell infiltration. Consistently, miRNA-sequencing revealed the miRNAs which were related to these pathways were down-regulated after hNSC transplantation. This study indicates that hNSCs can be effectively and safely used to treat ischemic stroke by promoting neurogenesis, regulating post-infarct inflammatory responses, and restoring mitochondrial function in both the infarct region and hippocampus.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Tumour immunogenicity goes with the (mitochondrial electron) flow.

Mitochondrial metabolism and electron transport chain (ETC) function are essential for tumour proliferation and metastasis. However, the impact of ETC function on cancer immunogenicity is not well understood. In a recent study, Mangalhara etal. found that inhibition of complex II leads to enhanced tumour immunogenicity, T-cell-mediated cytotoxicity and inhibition of tumour growth. Surprisingly, this antitumour effect is mediated by succinate accumulation affecting histone methylation. Histone methylation promotes the transcriptional upregulation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes in a manner independent of interferon signalling. Modulating mitochondrial electron flow to enhance tumour immunogenicity provides an exciting new therapeutic avenue and may be particularly attractive for tumours with reduced expression of MHC-APP genes or dampened interferon signalling.

Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation.

Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox-sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

Diet-induced obesity augments ischemic myopathy and functional decline in a murine model of peripheral artery disease

Peripheral artery disease (PAD) causes an ischemic myopathy contributing to patient disability and mortality. Most preclinical models to date use young, healthy rodents with limited translatability to human disease. Although PAD incidence increases with age, and obesity is a common comorbidity, the pathophysiologic association between these risk factors and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of age, diet-induced obesity and chronic hindlimb ischemia (HLI) on (1) mobility, (2) muscle contractility, and markers of muscle (3) mitochondrial content and function, (4) oxidative stress and inflammation, (5) proteolysis, and (6) cytoskeletal damage and fibrosis. Following 16-weeks of high-fat, high-sucrose, or low-fat, low-sucrose feeding, HLI was induced in 18-month-old C57BL/6J mice via the surgical ligation of the left femoral artery at 2 locations. Animals were euthanized 4-weeks post-ligation. Results indicate mice with and without obesity shared certain myopathic changes in response to chronic HLI, including impaired muscle contractility, altered mitochondrial electron transport chain complex content and function, and compromised antioxidant defense mechanisms. However, the extent of mitochondrial dysfunction and oxidative stress was significantly greater in obese ischemic muscle compared to non-obese ischemic muscle. Moreover, functional impediments, such as delayed post-surgical recovery of limb function and reduced 6-minute walking distance, as well as accelerated intramuscular protein breakdown, inflammation, cytoskeletal damage, and fibrosis were only evident in mice with obesity. As these features are consistent with human PAD myopathy, our model could be a valuable tool to test new therapeutics.

Dysregulation of intracellular pH in the failing heart.

Despite significant advances in the therapeutic armamentarium, heart failure (HF) remains a deadly disease. Metabolic derangement occurs in HF, with a decrease in the overall ATP synthesis and alterations in substrate utilization. Our recent studies demonstrate beat-to-beat intracellular acidification, namely “intracellular pH (pHi) transient”, in cardiomyocytes, which is coupled to the contraction, myofilament ATP hydrolysis, and mitochondrial electron transport chain function, essential for ATP synthesis. However, the regulation of pHi in HF cardiomyocytes remains less understood. We hypothesize that pHi and pHi transients in HF cardiomyocytes may be subjected to dysregulation as a response to metabolic changes in HF.A HF mouse model was generated using transverse aortic constriction for eight weeks. We quantified pHi and sarcomere lengths simultaneously in single ventricular myocyte. We demonstrate that the baseline pHi and pHi transients are significantly reduced in HF cardiomyocytes compared to sham. Moreover, there are significant cell-to-cell variabilities in pHi and pHi transients in the HF mouse model. Similar alterations were observed in a rabbit model with pressure- and volume-overload induced HF. The phasic pHi during cardiac contraction dovetails with the prevailing dynamic electrical, Ca2+ and mechanical systems defining the heart as an efficient pump. Indeed, the dynamic acidification of pHi may serve as negative feedback to provide the needed relaxation during cardiac contraction. Therefore, the reduced amplitude in pHi transients may significantly contribute to the decrease in lusitropy in HF, well documented in HF. Our study demonstrates dysregulated cardiac pHi and pHi transients in HF ventricular myocytes with significantly increased variabilities. The abnormal pHi and pHi transients suggest significant dysregulation of acid-base balance in HF, which may contribute to cardiac dysfunction. Our study may help to unravel new therapeutic strategies targeting pHi and pHi transients to ameliorate cardiac dysfunction in the failing heart.

Mitochondrial DNA drives AD Phenotypes in Cell Models

AbstractBackgroundMitochondrial DNA (mtDNA) inheritance influences Alzheimer’s Disease (AD) risk. Adult children of mother’s afflicted with AD show decreased cognition and increased AD biomarkers (both peripheral and brain) at earlier ages than adult children with father’s afflicted with AD. These human derived data paired with prior in vitro work support a maternal inheritance association of AD risk. However, the exact mechanism linking mtDNA inheritance patterns and AD is not understood.MethodsCytoplasmic hybrid (cybrid) cells were derived by fusing platelet mitochondria with SH‐SY5Y cells devoid of mtDNA. Cybrid cells have the same nuclear DNA background with varying mtDNA sequences derived from human subjects. We examined mitochondrial function parameters in cybrids derived from 8 cognitively healthy (CH), 7 AD, and 5 mild cognitive impairment (MCI) subjects. Mitochondrial electron transport chain (ETC) function was measured using Seahorse XF technology. Mitochondrial membrane potential, mitochondrial superoxide, and mitochondrial calcium were measured using fluorescent assays (TMRE, MitoSox, and Rhod2AM). Mitophagy and mitochondrial biogenesis/mass were measured using adenoviral constructs (EGFP‐Cox8 and MitoTimer).ResultsCybrids derived from AD subjects had elevated mitochondrial superoxide production when compared to CH cybrid cells. MCI cybrids did not show changes to mitochondrial superoxide production. Mitochondrial membrane potential and mitochondrial calcium were not changed in MCI or AD cybrids compared to CH. AD cybrids, but not MCI cybrids, showed reduced flux through all ETC components, including complex I, II, III, and IV. Finally, AD cybrids showed increased mitophagy levels but no change in mitochondrial biogenesis. MCI cybrids did not reflect these changes. Data do indicate AD cybrids might have increased mitochondrial mass, but further assays are needed to verify these findings.ConclusionsTransfer of mtDNA from AD patients to cells harboring consistent nuclear DNA recapitulates mitochondrial phenotypes observed in AD. We are currently measuring AD biomarkers in these models and working to examine their correlation with clinical data from this cohort of research subjects.

Testosterone and long pulse width stimulation (TLPS) for denervated muscles after spinal cord injury: a study protocol of randomised clinical trial

IntroductionLong pulse width stimulation (LPWS; 120–150 ms) has the potential to stimulate denervated muscles and to restore muscle size in denervated people with spinal cord injury (SCI). We will determine...

Not-so-mighty mitochondria: Mitochondrial dysfunction in mismatch repair deficient endometrial cancer development (234)

<b>Objectives:</b> Thirty percent of endometrial cancers exhibit DNA mismatch repair deficiency (MMRd), and inherited or sporadic loss of mismatch repair proteins increases the risk of developing endometrial cancer (EC). Identifying the consequences of MMRd beyond hypermutability could provide novel targetable mechanisms during EC development. Mitochondrial dysfunction has been shown to be a therapeutic vulnerability in other advanced MMRd-related cancers but has yet to be described early in cancer development. The objective of our study was to define the mitochondrial function in MMRd-related EC development. <b>Methods:</b> Our novel mouse model for MMRd-related EC development uses the uterine-targeted loss of <i>Msh2</i> (a commonly mutated gene in MMRd EC), abbreviated Msh2KO. Transcriptomic profiling using the Mouse ClariomD Assay (Affymetrix) was used to compare Msh2KO normal endometrium (<i>n</i>=4) and Msh2KO EC tissue (<i>n</i>=7) versus <i>Msh2</i>-wildtype (WT) normal endometrium (<i>n</i>=5). Differentially expressed genes were further evaluated using Ingenuity Pathway Analysis. Mitochondrial function was evaluated using a panel of five tumor-derived cell lines from Msh2KO mice. Using the Msh2KO cell line panel and a mismatch repair proficient mouse EC line (<i>PTEN</i>-null and <i>KRAS</i>-mutant; abbreviated MecPK), we performed the Seahorse Mitochondrial Stress Test (Agilent) to measure basal and induced mitochondrial function. <b>Results:</b> A total of 1,295 genes were differentially expressed in Msh2KO normal endometrium and 2,938 in Msh2KO EC compared to <i>Msh2</i> WT normal endometrium (90% were downregulated). Fifty-two downregulated genes in Msh2KO EC and 32 in Msh2KO normal endometrium related to mitochondrial electron transport chain function (p<0.05). Pathway analysis showed that the top dysregulated pathway in Msh2KO EC was "Mitochondrial dysfunction" (p=1.5E-7), which was also significantly altered in Msh2KO normal endometrium. Within this pathway, Msh2KO EC and Msh2KO normal endometrium shared significant changes of <i>Casp8</i>, <i>Cpt1a</i>, <i>Gpd2</i>, <i>Ncstn</i>, <i>Ogdh</i>, <i>Ndufa8</i>, and <i>Ndufa9</i> with at least a 2-fold-change decrease in expression compared to Msh2 WT endometrium (p<0.001). Mitochondrial stress tests <i>in vitro</i> revealed that the Msh2KO cell line panel had significantly impaired mitochondrial function compared to MecPK cells. Basal oxygen consumption rate, a measure of baseline mitochondrial function, was decreased by 50-90% in four of five Msh2KO cell lines compared to MecPK (p<0.001). Spare respiratory capacity, a measure of mitochondrial response to induced stress, was decreased by 30-85% in all five Msh2KO cell lines compared to MecPK cells (p<0.001). Because there was no significant difference in extracellular acidification rate between Msh2KO cell lines and MecPK cells during the assay, diminished mitochondrial function was likely due to intrinsic mitochondrial dysfunction rather than an adaptive glycolytic shift. <b>Conclusions:</b> Endometrial cancers from Msh2KO mice exhibit mitochondrial dysfunction at a transcriptomic and functional level. Importantly, this study also revealed molecular changes consistent with mitochondrial dysfunction in histologically-normal endometrium from Msh2KO mice, suggesting intrinsic mitochondrial dysfunction early in MMRd EC development. Further studies are necessary to corroborate findings in human MMRd-related EC. Identifying a unique vulnerability of MMRd endometrium undergoing cancer development would provide a novel target for cancer interception efforts.

The MitoAging Project: Single nucleotide polymorphisms (SNPs) in mitochondrial genes and their association to longevity

Mitochondrial dysfunction is a major hallmark of aging. Mitochondrial DNA (mtDNA) mutations (inherited or acquired) may cause a malfunction of the respiratory chain (RC), and thus negatively affect cell metabolism and function. In contrast, certain mtDNA single nucleotide polymorphisms (SNPs) may be beneficial to mitochondrial electron transport chain function and the extension of cellular health as well as lifespan. The goal of the MitoAging project is to detect key physiological characteristics and mechanisms that improve mitochondrial function and use them to develop therapies to increase longevity and a healthy lifespan. We chose to perform a systematic literature review (SLR) as a tool to collect key mtDNA SNPs associated with an increase in lifespan. Then validated our results by comparing them to the MitoMap database. Next, we assessed the effect of relevant SNPs on protein stability. A total of 28 SNPs were found in protein coding regions. These SNPs were reported in Japan, China, Turkey, and India. Among the studied SNPs, the C5178A mutation in the ND2 gene of Complex I of the RC was detected in all the reviewed reports except in Uygur Chinese centenarians. Then, we found that G9055A (ATP6 gene) and A10398G (ND3 gene) polymorphisms have been associated with a protective effect against Parkinson’s disease (PD). Additionally, C8414T in ATP8 was significantly associated with longevity in three Japanese reports. Interestingly, using MitoMap we found that G9055A (ATP6 gene) was the only SNP promoting longevity not associated with any pathology. The identification of SNPs associated with an increase in lifespan opens the possibility to better understand individual differences regarding a decrease in illness susceptibility and find strategies that contribute to healthy aging.

Mitochondrial function and Aβ in Alzheimer's disease postmortem brain

IntroductionMitochondrial dysfunction is observed in Alzheimer's disease (AD). However, the relationship between functional mitochondrial deficits and AD pathologies is not well established in human subjects. MethodsPost-mortem human brain tissue from 11 non-demented (ND) and 12 AD subjects was used to examine mitochondrial electron transport chain (ETC) function. Data were analyzed by neuropathology diagnosis and Apolipoprotein E (APOE) genotype. Relationships between AD pathology and mitochondrial function were determined. ResultsAD subjects had reductions in brain cytochrome oxidase (COX) function and complex II Vmax. APOE ε4 carriers had COX, complex II and III deficits. AD subjects had reduced expression of Complex I-III ETC proteins, no changes were observed in APOE ε4 carriers. No correlation between p-Tau Thr 181 and mitochondrial outcomes was observed, although brains from non-demented subjects demonstrated positive correlations between Aβ concentration and COX Vmax. DiscussionThese data support a dysregulated relationship between brain mitochondrial function and Aβ pathology in AD.

Effects from disruption of mitochondrial electron transport chain function on bull sperm motility

Sperm mitochondrial function is essential for normal physiology and fertility, but the importance of mitochondrial activity to support specific sperm functions, such as motility, varies between species. It was previously believed that mitochondrial function was not necessary for bull sperm motility [1]; however, this theory is contradicted by recently reported findings that the upper fraction of bull sperm swim-up preparations had both high motility and elevated mitochondrial oxygen consumption rates [2]. The objective of this study was to investigate the relationship between mitochondrial function and motility in bull sperm. We hypothesized that sperm motility would be positively correlated with mitochondrial oxygen consumption (MITOX) but unaffected by pharmacological inhibition of electron transport chain (ETC) activity. This was accomplished by monitoring both mitochondrial oxygen consumption and motility parameters in the presence of mitochondrial effector drug treatments. Duplicate ejaculates were collected by electroejaculation from Black Angus bulls (n = 4). Oxygen consumption, as % air saturation (pO2; oxygen partial pressure), over time was monitored in the presence of 5 drug treatments: vehicle control, FCCP, Antimycin (ANTI), Oligomycin (Oligo), and FCCP + Oligomycin (FCCP + OLIGO). Duplicate aliquots were prepared for concurrent motility assessment by computer-assisted sperm analysis (CASA) at 6 and 30 min post-treatment (t6 and t30). The impact of treatments on pO2 (in % air saturation) over time were assessed by generalized linear mixed effects modeling (GLMM) which was also used to test for differences in average motility across treatment conditions and time points (t6 and t30). Pearson product-moment correlation was used to investigate relationships between oxygen consumption and motility parameters. Overall, pO2 differed over time between treatment conditions (p < 0.0001). When compared to the vehicle treatment, ANTI and OLIGO significantly inhibited oxygen consumption (p < 0.05, adjusted), and FCCP stimulated a marked increase in oxygen consumption. No significant differences in motility over time were observed between treatments, so comparison of motility parameters between treatment conditions was performed with pooled timepoints. Motility parameters were only observed to differ significantly from the vehicle with ANTI Treatment, for which significant decreases in numerous parameters, including total motility (p = 0.007), progressive motility (p = 0.01), DAP (p = 0.01), VAP (p = 0.01) and VSL (p = 0.02) were identified. For the vehicle treatment, correlational analysis did not reveal any significant correlations between pO2 and any motility parameters at t6; however, several significant correlations were identified at t30. Mean pO2 was negatively correlated with local motility (p < 0.01) and positively correlated with DCL, DAP, and VCL (p < 0.05). Results from this study suggest that bovine sperm motility is impacted by mitochondrial functionality, with ETC inhibition by ANTI causing significant reduction in motility parameters. This study also demonstrates the use of a new technology for the assessment of bovine sperm mitochondrial function. This modality for evaluation of bull sperm mitochondrial function will inform future efforts to understand bull sperm function and fertility and aid investigations into toxicological agents.

Abstract 2397: Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis, attributed to the limited understanding of its driver signaling pathways. Although TNBC exhibits unique characteristics, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are essential to reduce its severity. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a dominant metabolic pathway in TNBC. We have also reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC, via autophosphorylation of Src at Y419. However, single drug therapy using FAO inhibitors or Src inhibitors did not show a significant therapeutic advantage in TNBC. Biguanide such as metformin is one of the most widely administered anti-diabetic agents. Recently increasing evidence demonstrates that biguanide therapy can have an anticancer effect in various cancer types, including BC. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both biguanides can suppress mitochondrial electron transport chain (ETC) function because of its inhibitory effect on ETC complex I activity. However, biguanides also lead to the activation of AMPK, which plays a vital role in insulin signaling and energy-sensing. Notably, AMPK is an upstream regulator of the FAO pathway because it can phosphorylate ACC and activate FAO. Considering the dependency of TNBC on mitochondrial FAO and the role of AMPK in the activation of FAO, our computational modeling predicted that the combined targeting of both ‘arms' of the pathway could provide a significant advantage to the anticancer effect in TNBC. Thus, we have used the combination of biguanides and FAO inhibitors to target both ETC and FAO respectively. Pharmacological and genetic approaches with multiple in vitro and in vivo analysis using TNBC cell lines and PDX models suggest that the combination of biguanides and FAO inhibitors can provide significant advantage in TNBC therapy. Further studies also show that the combination of biguanides with Src inhibitors can provide a better therapeutic option to target metastatic TNBC. We are currently evaluating these findings' translational significance as a cost-effective approach to managing non-targetable TNBCs. Citation Format: Junhyoung Park, Kwang Hwa Jung, Dongya Jia, Sukjin Yang, Dharaniya Sakthivel, Abha Tiwari, José N. Onuchic, Benny Abraham Kaipparettu. Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2397.

Abstract P799: Nicotine Along With Oral Contraceptive Exposure Alters Brain Lipid Metabolism and Exacerbates Ischemic Injury in Female Rats

Background: Smoking-derived nicotine (N) and oral contraceptives (OC) synergistically exacerbate both global and focal ischemic brain damage in females. While the underlying mechanisms remain elusive, our published study showed that OC exacerbate N toxicity via altered mitochondrial electron transport chain function. Because mitochondria play a central role in cellular metabolism, we examined the metabolic fingerprint of adolescent and adult female rat brains exposed to N +/- OC. Methods: Adolescent (6 weeks old) and adult (12 weeks old) Sprague-Dawley female rats were randomly (n = 8/group) exposed to either saline, N (4.5 mg/kg) +/- OC for 16-21 days. Following treatment, brain tissue was harvested for unbias metabolomic analysis (performed by Metabolon Inc.). The metabolomic profile was complemented with western blot analysis and enzyme activity measurements. Results: Pathway enrichment analysis showed significant alterations in lipid metabolism. Adolescent but not adult females treated with N, OC and N+OC compared to saline showed significant increases in carnitine conjugated fatty acid metabolites such as arachidonoylcarnitine (C20:4), docosahexaenoylcarnitine (C22:6) and stearoylcarnitine (C18). These changes in fatty acyl carnitines were accompanied by an increase in a subset of free fatty acids, suggesting elevated fatty acid β-oxidation in the mitochondria to meet energy demand. In support, 3-hydroxybutyrate (BHBA) was significantly lower in OC and N+OC treatment group in adolescent animals, implying a complete shunting of acetyl CoA for energy production via TCA cycle. BHBA is a ketone body that increases in concentration as lipid oxidation rates increase with acetyl CoA accumulation. Reduced BHBA levels may also suggest mitochondrial dysfunction in response to OC and N+OC treatment. Conclusion: The observed changes in the metabolic fingerprint and fatty acid metabolism reflect a general alteration in energy metabolism with nicotine treatment exclusively in young animals and these changes are enhanced by N+OC treatment. Discerning the exact effects of N +/- OC on overall brain metabolism and the molecular mechanisms affecting mitochondrial function at different ages will open a new window for future therapeutic intervention.

Deficits in the Skeletal Muscle Transcriptome and Mitochondrial Coupling in Progressive Diabetes-Induced CKD Relate to Functional Decline.

Two-thirds of people with type 2 diabetes mellitus (T2DM) have or will develop chronic kidney disease (CKD), which is characterized by rapid renal decline that, together with superimposed T2DM-related metabolic sequelae, synergistically promotes early frailty and mobility deficits that increase the risk of mortality. Distinguishing the mechanisms linking renal decline to mobility deficits in CKD progression and/or increasing severity in T2DM is instrumental both in identifying those at high risk for functional decline and in formulating effective treatment strategies to prevent renal failure. While evidence suggests that skeletal muscle energetics may relate to the development of these comorbidities in advanced CKD, this has never been assessed across the spectrum of CKD progression, especially in T2DM-induced CKD. Here, using next-generation sequencing, we first report significant downregulation in transcriptional networks governing oxidative phosphorylation, coupled electron transport, electron transport chain (ETC) complex assembly, and mitochondrial organization in both middle- and late-stage CKD in T2DM. Furthermore, muscle mitochondrial coupling is impaired as early as stage 3 CKD, with additional deficits in ETC respiration, enzymatic activity, and increased redox leak. Moreover, mitochondrial ETC function and coupling strongly relate to muscle performance and physical function. Our results indicate that T2DM-induced CKD progression impairs physical function, with implications for altered metabolic transcriptional networks and mitochondrial functional deficits as primary mechanistic factors early in CKD progression in T2DM.

Molecular mechanisms for hypoxia development and adaptation to it. Part I

Hypoxia is a typical pathological process characterized by the occurrence of oxygen deficiency in tissues and cells and accompanied by the development of immediate and delayed compensatory and adaptive reactions. Reprogramming of the mitochondrial electron transport chain (ETC) function is one the most essential regulatory mechanisms that allow for immediate adaptation to hypoxia. Succinic acid, or succinate, is involved in this process not only as one of the intermediates of the tricarboxylic acid (TAC) cycle, but also as a signaling molecule. In this connection, the purpose of this review was to systematize the available data on the molecular mechanisms for the development of hypoxia and its adaptation at the ETC/TAC coupling site, as well as on the role of succinic acid in these processes.

Nicotine Exposure Along with Oral Contraceptive Treatment in Female Rats Exacerbates Post-cerebral Ischemic Hypoperfusion Potentially via Altered Histamine Metabolism.

Smoking-derived nicotine (N) and oral contraceptives (OCs) synergistically exacerbate ischemic brain damage in the female, and the underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial electron transport chain function. Because mitochondria play an important role in cellular metabolism, we investigated the global metabolomic profile of brains of adolescent and adult female Sprague-Dawley rats exposed to N with or without OC (N+/-OC). Rats were randomly exposed to saline or N+/-OC for 16-21days followed by random allocation into two cohorts. The first cohort was used to characterize the cortical metabolome. Pathway enrichment analysis showed a significant increase in several histamine metabolites including 1-methylhistamine, 1-methyl-4-imidazoleacetate, and 1-ribosyl-imidazleacetate, along with carnosine and homocarnosine in adolescent and adult animals treated with N and N+OC in relation to respective saline controls, which may be reflective of altered histamine metabolism with nicotine treatment. We also observed reduced levels of the neurotransmitters N-acetyl-aspartyl-glutamate (NAAG), gamma-aminobutyrate (GABA), and N-methyl-GABA in N+OC treatment in adolescent animals. The second cohort underwent bilateral carotid artery occlusion and hypotension followed by cerebral blood flow (CBF) assessment a day later. Autoradiographic images of the brain 24h after ischemic episodes showed severe reduction in cortical and hippocampal local CBF in N+/-OC-exposed rats compared with saline treated. Because GABA and histamine are critical for CBF maintenance, altered metabolism of these neurotransmitters may be responsible for observed severe post-ischemic hypoperfusion, which in turn exacerbates ischemic brain damage.

A Mitochondrial Stress-Specific Form of HSF1 Protects against Age-Related Proteostasis Collapse.

The loss of protein homeostasis (proteostasis) is a primary driver of age-related tissue dysfunction. Recent studies have revealed that the failure of proteostasis with age is triggered by developmental and reproductive cues that repress the activity of proteostasis-related pathways in early adulthood. In Caenorhabditis elegans, reduced mitochondrial electron transport chain (ETC) function during development can override signals that promote proteostasis collapse in aged tissues. However, it is unclear precisely how these beneficial effects are mediated. Here, we reveal that in response to ETC impairment, the PP2A complex generates a dephosphorylated, mitochondrial stress-specific variant of the transcription factor HSF-1. This results in the selective induction of small heat shock proteins in adulthood, thereby protecting against age-related proteostasis collapse. We propose that mitochondrial signals early in life can protect the aging cytosolic proteome by tailoring HSF-1 activity to preferentially drive the expression of non-ATP-dependent chaperones.

α-ketobutyrate links alterations in cystine metabolism to glucose oxidation in mtDNA mutant cells

Pathogenic mutations in the mitochondrial genome (mtDNA) impair organellar ATP production, requiring mutant cells to activate metabolic adaptations for survival. Understanding how metabolism adapts to clinically relevant mtDNA mutations may provide insight into cellular strategies for metabolic flexibility. In this study, we use 13C isotope tracing and metabolic flux analysis to investigate central carbon and amino acid metabolic reprogramming in isogenic cells containing mtDNA mutations. We identify alterations in glutamine and cystine transport which indirectly regulate mitochondrial metabolism and electron transport chain function. Metabolism of cystine can promote glucose oxidation through the transsulfuration pathway and the production of α-ketobutyrate. Intriguingly, activating or inhibiting α-ketobutyrate production is sufficient to modulate both glucose oxidation and mitochondrial respiration in mtDNA mutant cells. Thus, cystine-stimulated transsulfuration serves as an adaptive mechanism linking glucose oxidation and amino acid metabolism in the setting of mtDNA mutations.

Receptor for advanced glycation end products modulates oxidative stress and mitochondrial function in the soleus muscle of mice fed a high-fat diet.

Accumulation of advanced glycation end products (AGEs) and activation of the receptor for AGEs (RAGE) are implicated in the progression of pathologies associated with aging, chronic inflammation, diabetes, and cellular stress. RAGE activation is also implicated in cardiovascular complications of type 2 diabetes, such as nephropathy, retinopathy, accelerated vascular diseases, and cardiomyopathy. Studies investigating the effects of AGE/RAGE axis activation on skeletal muscle oxidative stress and metabolism are more limited. We tested whether a high-fat diet (HFD) would alter circulating AGE concentration, skeletal muscle AGE accumulation, and oxidative stress in wild-type and RAGE-deficient mice. The physiological significance of AGE/RAGE axis activation in HFD-fed mice was evaluated in terms of exercise tolerance and mitochondrial respiratory chain complex activity. HFD elicited adiposity, abnormal fat distribution, and oral glucose intolerance. HFD also induced accumulation of Nε-carboxymethyl-l-lysine, increased protein carbonyl levels, and impaired respiratory chain complex activity in soleus muscle. Ablation of RAGE had no effects on weight gain and oral glucose tolerance in HFD-fed mice. Peak aerobic capacity and mitochondrial cytochrome-c oxidase activity were restored in HFD-fed RAGE-/- mice. We concluded that RAGE signaling plays an important role in skeletal muscle homeostasis of mice under metabolic stress. Novelty HFD in mice induces accumulation of AGEs, oxidative stress, and mitochondrial dysfunction in the soleus muscle. RAGE, the multi-ligand receptor for AGEs, modulates oxidative stress and mitochondrial electron transport chain function in the soleus muscle of HFD-fed mice.