Expression Of Mitochondrial Enzymes Research Articles

Upregulation of ALOX12−12-HETE pathway impairs AMPK-dependent modulation of vascular metabolism in ApoE/LDLR−/− mice

Mitochondrial dysfunction and 12-lipoxygenase (ALOX12)-derived 12(S)-HETE production have been associated with vascular inflammation and the pathogenesis of atherosclerosis. However, the role of ALOX12 in regulating vascular energy metabolism in vascular inflammation has not been studied to date. Using mitochondrial and glycolysis functional profiling with the Seahorse extracellular flux analyzer, metabolipidomics, and proteomic analysis (LC-MS/MS), we characterized alterations in vascular energy metabolism in 2- and 6-month-old ApoE/LDLR−/− vs. control C57BL/6 mice. We identified that aorta of 6-month-old ApoE/LDLR−/− mice displayed compromised mitochondrial metabolism manifested by the reduced expression of mitochondrial enzymes, impaired mitochondrial respiration, and consequently diminished respiratory reserve capacity. An increased flux through the glycolysis/lactate shuttle, the hexosamine biosynthetic pathway (HBP), and the pentose phosphate pathway (PPP) was also recognized. Interestingly, ALOX12−12-HETE was the most upregulated axis in eicosanoid metabolism and histological examinations indicated that ApoE/LDLR−/− mice showed increased aortic expression of ALOX12, particularly in early atherosclerotic plaque areas. Remarkably, the joint blocking of ALOX12 and activation of AMPK, but not AMPK activation alone, resulted in the reprogramming of vascular metabolism, with improved mitochondrial respiration and suppressed auxiliary pathways (HBP, PPP, itaconate shunt). In conclusion, excessive activation of the ALOX12–12-HETE pathway in vascular inflammation in early atherosclerosis inhibits AMPK-dependent regulation of vascular metabolism. Consequently, ALOX12 may represent a novel target to boost impaired vascular mitochondrial function in pro-atherosclerotic vascular inflammation.

Abstract 4839: Chemotherapy alters mitochondrial metabolism in melanoma

Abstract Background: Advanced and metastatic melanoma carries significant mortality despite recent advances. Metabolic plasticity is a hallmark of cancer and an important resistance mechanism in the face of anti-neoplastic therapies. We sought to understand the effect of chemotherapy, Temozolomide (TMZ), on OXPHOS and TCA cycle metabolism in melanoma cell lines. By understanding these metabolic adaptations to chemotherapy, we hope to identify novel combination therapies. Methods: A375 and SK-MEL-28 cell lines were used for all experiments. Cell viability assays were performed with 1500 cells per well, treated with TMZ and oligomycin or phenformin, and viability was assessed using PicoGreen dsDNA assay. Western blot was used with primary antibodies against TOM20 and Β-actin. LCMS metabolites were assessed by QTRAP triple quadrupole mass spectrometry coupled to a Prominence UFLC HPLC system. This assessed approximately 299 metabolites. Seahorse XFp miniextracellular analyzer was used to obtain OCR with and without TMZ exposure, and after sequential injections of oligomycin, FCCP, and rotenone with antimycin A. Mitochondrial assays included TMRE staining (membrane potential) and Mitotracker Green (mitochondrial mass). Results: Metabolic profiles of melanoma cell lines change with exposure to chemotherapy. After melanoma cells are exposed to IC30 dose of TMZ, LCMS metabolomics revealed statistically significant increase in TCA cycle metabolites such as succinate, succinyl CoA, malate, oxaloacetate, isocitrate, alpha-ketoglutarate, and fumarate. Melanoma increases OXPHOS in the presence of chemotherapy. Mitochondrial mass, measured by Mitotracker, increased in response to TMZ exposure. We also found increased mitochondrial membrane potential and increased TOM20 expression after cells were treated with TMZ. Seahorse XF Cell Mito Stress Assay demonstrated increased oxygen consumption rate after exposure to TMZ. We then combined TMZ and two mitochondrial inhibitors, phenformin (complex I) and oligomycin (complex V). Combination of phenformin and TMZ was synergistic in cell viability assays as determined by Bliss equation. Oligomycin sensitized both cell lines to TMZ. Discussion: We have shown that melanoma cells rely on mitochondrial function when exposed to TMZ by increasing levels of TCA cycle intermediates, increasing mitochondrial tropism, and increased expression of mitochondrial enzymes. This demonstrates a metabolic resistance mechanism in response to chemotherapy to promote melanoma cell survival. Finally, we exploit this adaptation by pharmacologic inhibition of mitochondrial electron transport chain (ETC) with phenformin and oligomycin A. This treatment was synergistic with conventional chemotherapy. Our findings point out that the ETC compartment of mitochondria could be a novel and readily available combination treatment strategy for patients with advanced and refractory melanoma. Citation Format: Alexander W. Loftus, Mehrdad Zarei, Omid Hajihassani, Johnathan J. Hue, Hallie J. Graor, Ali Vaziri-Gohar, Jordan M. Winter, Luke D. Rothermel. Chemotherapy alters mitochondrial metabolism in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4839.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves.Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation.We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

PGC-1/ERR-Induced Regulator in Muscle (PERM1) Increases Mitochondrial Respiratory Capacity in Culture Muscle Cells

Peripheral arterial disease is the third leading cause of death of atherosclerotic cardiovascular mortality in the United states and the incidence of PAD increases age. Previous studies have shown that PAD displays impaired mitochondrial respiration, decreased expression of mitochondrial enzymes, increased oxidative stress, and mitochondrial DNA mutations within their ischemic limb muscles. We identified a potential transcriptional regulator of mitochondrial gene expression, PGC-1/ERR-induced regulator in muscle (PERM1) which is reduced 85% in patients with severe PAD. Interestingly, PERM1 regulates the expression of only a subset of genes induced by PGC-1a or ERRs expression in C2C12 myotubes, suggesting that PERM1 selectively functions in specific PGC-1/ERR-driven pathways. PURPOSE: The purpose of this study is to determine whether PERM1 is a potential gene target to aid in tissue recovery and regeneration from hypoxia in C2C12 myotubes. METHODS: We generated AAVs to overexpress the PERM1 gene or a green fluorescent protein (ZsGreen1). Following AAV infection, C2C12 myoblasts were differentiated into mature myotubes for assessments of mitochondrial biogenesis, mitochondrial respiration, and myogenesis were performed. To determine if PERM1 plays a role in recovering myotube respiration and myotube atrophy from hypoxia, C2C12 myotubes infected AAV-PERM1 were placed in hypoxia for 6-hours and mitochondrial respiratory function, content, and myosin heavy chain area were assessed during the recovery from hypoxic insult. RESULTS: AAV-PERM1 resulted in a ~16-fold increase in mRNA expression which drove a ~20% increase in complex I-supported respiration compared to the control cells (P<0.05, n=6). Increased Expression of PERM1 did not change the myotube fusion index (an indicator of myogenesis) (P=0.32, n=4). CONCLUSION: Our results indicate that PERM1 is a strong regulator of mitochondrial biogenesis in skeletal muscle cells, capable of increase both mitochondrial content and respiratory function. Based on these observations, future studies should be aimed at understanding the therapeutic potential of PERM1 for improving muscle energetics in conditions with impaired muscle metabolism, including peripheral arterial disease. Partially supported by AHA Grant 18CDA34110044 to TER.

Cytosolic lipid excess-induced mitochondrial dysfunction is the cause or effect of high fat diet-induced skeletal muscle insulin resistance: a molecular insight.

Mitochondria play a central role in the energy homeostasis in eukaryotic cells by generating ATP via oxidative metabolism of nutrients. Excess lipid accumulation and impairments in mitochondrial function have been considered as putative mechanisms for the pathogenesis of skeletal muscle insulin resistance. Accumulation of lipids in tissues occurs due to either excessive fatty acid uptake, decreased fatty acid utilization or both. Consequently, elevated levels cytosolic lipid metabolites, triglycerides, diacylglycerol and ceramides have been demonstrated to adversely affect glucose homeostasis. Several recent studies indicate that reduced insulin-stimulated ATP synthesis and reduced expression of mitochondrial enzymes and PPAR-γ coactivator, in high fat feeding (lipid overload) are associated with insulin resistance. Despite the fact, few notable studies suggest mitochondrial dysfunction is prevalent in type 2 diabetes mellitus; it is still not clear whether the defects in mitochondrial function are the cause of insulin resistance or the consequential effects of insulin resistance itself. Thus, there is a growing interest in understanding the intricacies of mitochondrial function and its association with cytosolic lipid excess. This review therefore critically examines the molecular cascades linking cytosolic lipid excess and mitochondrial dysfunction in the pathogenesis of high fat diet-induced insulin resistance in skeletal muscle. The sequential processes following the excess intake of high fat diet in skeletal muscle includes, accumulation of cytosolic fatty acids, increased production of reactive oxygen species, mutations and ageing, and decreased mitochondrial biogenesis. The consequent mitochondrial dysfunction is then leading to decreased β-oxidation, respiratory functions and glycolysis and increased glucolipotoxicity. These events collectively induce the insulin resistance in skeletal muscle.

Inter-individual variation in adaptive capacity at onset of lactation: Linking metabolic phenotype with mitochondrial DNA haplotype in Holstein dairy cows

Modern-day dairy cows express great variation in metabolic capacity to adapt to the onset of lactation. Although breeding programs increased the breeding value for longevity and robustness in the sires, a respective phenotype in female offspring has not been improving as predicted. Fundamental energy generating pathways such as mitochondrial fatty acid oxidation might have a crucial role for robustness and metabolic efficiency in dairy cows. Therefore, mitochondrial enzyme expression was examined in liver samples of one set of animals before and after calving. Furthermore, the mitochondrial DNA sequence was determined for each individual of a second set of animals using liver samples. Results from the first trial indicate that the expression and extent of phosphorylation of acetyl-CoA carboxylase (ACC) is the major key step for modulating fatty acid flux into the mitochondria at early onset of lactation in Holstein dairy cows. In the second trial, mitochondrial DNA sequencing and identification of mutation patterns yielded three major haplotypes. Haplotype H2 was closely associated with liver fat content, plasma glycerol and acyl-carnitine concentrations. The mitochondrial DNA haplotype, which is a feature of the maternal lines, might be related to the inter-individual variation in metabolic capacity of Holstein dairy cows.

Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis.

Based on previous findings that ozone can induce an inflammatory response in the ocular surface of an animal model and in cultured human conjunctival epithelial cells, we investigated whether exposure to ozone exacerbates symptoms of allergic conjunctivitis. We evaluated the effects of exposure to ozone on conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, production of inflammatory cytokines in tears, and aqueous tear production in a mouse model of allergic conjunctivitis. To validate our in vivo results, we used interleukin (IL)-1α-pretreated conjunctival epithelial cells as an in vitro substitute for the mouse model. We evaluated whether exposure to ozone increased the inflammatory response and altered oxidative status and mitochondrial function in IL-1α-pretreated conjunctival epithelial cells. In the in vivo study, ozone induced increases in conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, and production of inflammatory cytokines, accompanied by a decrease in tear volume. In the in vitro study, exposure to ozone led to additional increases in IL-6 and tumor necrosis factor-α mRNA levels, which were already induced by treatment with IL-1α. Ozone did not induce any changes in cell viability. Pretreatment with IL-1α increased the expression of manganese superoxide dismutase, and exposure to ozone led to additional increments in the expression of this antioxidant enzyme. Ozone did not induce any changes in mitochondrial activity or expression of mitochondrial enzymes and proteins related to mitochondrial function, with the exception of phosphor-mammalian target of rapamycin. Treatment with butylated hydroxyanisole, a free radical scavenger, attenuated the ozone-induced increases in IL-6 expression in IL-1α-pretreated conjunctival epithelial cells. Therefore, we conclude that exposure to ozone exacerbates the detrimental effects on the integrity of the ocular surface caused by conjunctival allergic reactions, and further increases the inflammatory response in IL-1α-pretreated conjunctival epithelial cells.

Adaptations of skeletal muscle mitochondria to exercise training.

Mitochondrial volume density (Mito(VD)) is composed of two distinct mitochondrial subpopulations--intermyofibrillar mitochondria (Mito(IMF)) and subsarcolemmal mitochondria (Mito(SS)). With exercise training, Mito(VD) may increase by up to 40% and is, for the most part, related to an increase in Mito(IMF). Exercise-induced adaptations in mitochondrial function depend on the intensity of training and appear to be explained predominately by an increased expression of mitochondrial enzymes that facilitate aerobic metabolism. Although mitochondrial content often increases with training, it seems that mitochondrial adaptations are not needed to facilitate maximal oxygen uptake, whereas such adaptations are of greater importance for endurance capacity.

Editorial on Cerebral endothelial and glial cells are more than bricks in the Great Wall of the brain: insights into the way the blood-brain barrier actually works (celebrating the centenary of Goldman's experiments).

EDITORIAL article Front. Cell. Neurosci., 01 April 2015Sec. Non-Neuronal Cells Volume 9 - 2015 | https://doi.org/10.3389/fncel.2015.00128

Exercise and mitochondrial function in adipose biology: all roads lead to NO.

Insulin resistance in rodents and humans is highly correlated with inflammation of white adipose tissue (WAT) and the presence of proinflammatory immune cells. Such chronic immune challenge leads to a variety of metabolic effects in the adipocyte, including endoplasmic reticulum stress, mitochondrial dysfunction, oxidative stress, and altered adipokine and cytokine secretion, which in sum play a major role in affecting whole-body insulin sensitivity (1–3). Of the factors regulating mitochondrial biogenesis, the regulation of the endothelial nitric oxide synthase (eNOS) has garnered considerable attention as a major metabolic control point. eNOS is a constitutively expressed nitric oxide (NO)–producing enzyme classically linked to smooth muscle contraction and platelet aggregation (4). However, eNOS and downstream NO signaling are now appreciated as a major metabolic determinant of the peroxisome proliferator–activated receptor γ coactivator 1α (PGC1α) (5). Although the molecular mechanism(s) is still not completely understood, NO signaling and activation of the soluble guanylyl cyclase–protein kinase G (PKG) system upregulate PGC1α expression and, moreover, are required for increased expression of SirT1, the major NAD+-dependent deacetylase controlling PGC1α activity (6–8). NO signaling via PKG stimulates the expression of the entire mitochondrial biogenesis program, including the activation of Nrf1 and Tfam, two major transcription factors regulating expression of mitochondrial enzymes. A second major theme controlling mitochondrial biogenesis linked to sympathetic drive and eNOS is G-protein–coupled receptor–dependent activation …

High-intensity exercise training induces morphological and biochemical changes in skeletal muscles.

In the present study we investigated the effect of two different exercise protocols on fibre composition and metabolism of two specific muscles of mice: the quadriceps and the gastrocnemius. Mice were run daily on a motorized treadmill, at a velocity corresponding to 60% or 90% of the maximal running velocity. Blood lactate and body weight were measured during exercise training. We found that at the end of training the body weight significantly increased in high-intensity exercise mice compared to the control group (P=0.0268), whereas it decreased in low-intensity exercise mice compared to controls (P=0.30). In contrast, the food intake was greater in both trained mice compared to controls (P < 0.0001 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively). These effects were accompanied by a progressive reduction in blood lactate levels at the end of training in both the exercised mice compared with controls (P=0.03 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively); in particular, blood lactate levels after high-intensity exercise were significantly lower than those measured in low-intensity exercise mice (P=0.0044). Immunoblotting analysis demonstrated that high-intensity exercise training produced a significant increase in the expression of mitochondrial enzymes contained within gastrocnemius and quadriceps muscles. These changes were associated with an increase in the amount of slow fibres in both these muscles of high-intensity exercise mice, as revealed by the counts of slow fibres stained with specific antibodies (P < 0.0001 for the gastrocnemius; P=0.0002 for the quadriceps). Our results demonstrate that high-intensity exercise, in addition to metabolic changes consisting of a decrease in blood lactate and body weight, induces an increase in the mitochondrial enzymes and slow fibres in different skeletal muscles of mice, which indicates an exercise-induced increase in the aerobic metabolism.

Aging-Associated Reductions in Lipolytic and Mitochondrial Proteins in Mouse Adipose Tissue Are Not Rescued by Metformin Treatment

Mitochondrial enzyme expression is reduced in adipose tissue from old mice, yet little is known regarding mechanisms that could be mediating, or interventions that could be used, to reverse these changes. The purpose of this study was to examine the relationship between lipolytic and fatty acid reesterification enzymes, 5' adenosine monophosphate-activated protein kinase and mitochondrial proteins in adipose tissue from young versus old mice. A second aim was to determine whether metformin treatment could rescue the age-associated decline in adipose tissue mitochondrial proteins. Approximately 22-month-old male C57BL/6 mice were fed a diet with or without 0.5% metformin for 8 weeks. Compared with young mice (~11 wk of age), the protein content/phosphorylation of hormone-sensitive lipase, adipose tissue triglyceride lipase, and phosphoenolpyruvate carboxykinase were reduced in old mice. This was paralleled by increases in the plasma nonesterified fatty acid:glycerol ratio and reductions in adipose tissue 5' adenosine monophosphate-activated protein kinase activity and select mitochondrial proteins in old mice. There were no differences in these variables when comparing adipose tissue from young and 6-month-old mice. While metformin improved glucose homeostasis, it did not increase 5' adenosine monophosphate-activated protein kinase phosphorylation or mitochondrial enzymes. Our findings demonstrate a co-ordinated down regulation of lipolytic, reesterification, and mitochondrial enzymes in adipose tissue with aging that is unresponsive to metformin treatment.

Aerobic training stimulates mitochondrial enzyme expression and initiates skeletal muscle fibre shifting in rat soleus muscle

Aerobic training stimulates mitochondrial enzyme expression and initiates skeletal muscle fibre shifting in rat soleus muscle

Abstract 1868: Metabolic characterization of prostate cancer cell lines.

Abstract Purpose: The tumor microenvironment has a major impact on tumor growth and progression; the composition and characteristics of the tumor microenvironment is influenced by the metabolism of both tumor and stromal cells. In preparation to determining the effects of cell metabolism on the anti-tumor T cell immune response, we studied the metabolism of two murine prostate cell lines (RM1 and Myc-Cap) under different conditions. The long-term goal of our study is to assess what features of an abnormal tumor microenvironment negatively impact T cell tumor targeting and the activation of T cells in orthotopic prostate cancer models. Methods: The metabolic profiles of the more aggressive rapidly growing RM1 and more moderate growing Myc-Cap cells were determined. A Seahorse XF96 Extracellular Flux Analyzer, which provides real-time measurements of the extracellular acidification rate (ECAR), was used to measure of glycolysis and lactate production, and the oxygen consumption rate (OCR), a measure of oxidative phosphorylation (OXPHOS). The effects of 24-hour glucose and glutamine deprivation were studied. Results: Myc-Cap cells utilized significantly less glucose, produced much less acid (lactate) and had a low proliferation rate compared to RM1 cells. RM1 cells exhibit a higher rate of glycolysis and showed higher glycolytic capacity, whereas OCR (a measure of mitochondrial respiration) was lower in RM1 compared to Myc-Cap cells. The expression of glycolytic enzymes was comparable in both cell lines; RM1 cells demonstrated higher LDHA and lower expression of mitochondrial enzymes, as measured by Western blot. For RM1, glucose withdrawal from the media resulted in reduced LDHA expression, a 77% decrease in lactate production (measured by ECAR), and a 50% decrease in OCR. Glutamine withdrawal resulted in a 35% decrease in ECAR, and an 82% decrease in OCR. Myc-Cap cells were slightly less affected by glucose withdrawal (slight change in LDHA expression, a 69% decrease in ECAR, and a 40% decrease in OCR), whereas glutamine withdrawal had a more profound effect on ECAR (75% decrease) and a similar effect on OCR (83% decrease). Glutamine withdrawal reduced the proliferation rate of both cell lines significantly more than glucose withdrawal. Conclusions: Our data demonstrate a major difference in acid production (lactate) between the two cell lines (RM1&amp;gt;Myc-Cap). RM1 cells are more glucose dependent, whereas Myc-Cap is more glutamine dependent. Understanding the metabolic differences between tumors and the impact of metabolism on the tumor microenvironment will provide specific targets that could enhance T cell therapy through a modification of the tumor microenvironment. Citation Format: Ekaterina S. Moroz, Ronald Blasberg, Inna Serganova. Metabolic characterization of prostate cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1868. doi:10.1158/1538-7445.AM2013-1868

Overexpression of the Mitochondrial Folate and Glycine–Serine Pathway: A New Determinant of Methotrexate Selectivity in Tumors

Previous studies have documented the roles of transport via the reduced folate carrier, retention via polyglutamylation, and increased levels of the target enzyme, dihydrofolate reductase in sensitivity to methotrexate. Recent studies have shown that the mitochondrial enzymes in the cellular metabolism of serine, folate, and glycine are overexpressed in a subset of human cancers and that their expression is required for tumor maintenance. In this Perspective article, we propose that the expression of mitochondrial enzymes in the metabolism of serine and glycine, in addition to those involved in folate metabolism, are determinants of the response to methotrexate. Furthermore, we show that myc activation in tumors is associated with upregulation of these enzymes. We propose that patients whose tumors show this phenotype will be sensitive to folate antagonists targeting thymidylate or purine biosynthesis.

Impaired myogenesis in estrogen‐related receptor γ (ERRγ)‐deficient skeletal myocytes due to oxidative stress

Specialized contractile function and increased mitochondrial number and oxidative capacity are hallmark features of myocyte differentiation. The estrogen-related receptors (ERRs) can regulate mitochondrial biogenesis or mitochondrial enzyme expression in skeletal muscle, suggesting that ERRs may have a role in promoting myogenesis. Therefore, we characterized myogenic programs in primary myocytes isolated from wild-type (M-ERRγWT) and muscle-specific ERRγ(-/-) (M-ERRγ(-/-)) mice. Myotube maturation and number were decreased throughout differentiation in M-ERRγ(-/-) primary myocytes, resulting in myotubes with reduced mitochondrial content and sarcomere assembly. Compared with M-ERRγWT myocytes at the same differentiation stage, the glucose oxidation rate was reduced by 30% in M-ERRγ(-/-) myotubes, while medium-chain fatty acid oxidation was increased by 34% in M-ERRγ(-/-) myoblasts and 36% in M-ERRγ(-/-) myotubes. Concomitant with increased reliance on mitochondrial β-oxidation, H(2)O(2) production was significantly increased by 40% in M-ERRγ(-/-) myoblasts and 70% in M-ERRγ(-/-) myotubes compared to M-ERRγWT myocytes. ROS activation of FoxO and NF-κB and their downstream targets, atrogin-1 and MuRF1, was observed in M-ERRγ(-/-) myocytes. The antioxidant N-acetyl cysteine rescued myotube formation and atrophy gene induction in M-ERRγ(-/-) myocytes. These results suggest that loss of ERRγ causes metabolic defects and oxidative stress that impair myotube formation through activation of skeletal muscle atrophy pathways.

Impaired expression of ketone metabolizing enzymes in malignant gliomas: Implication for ketogenic diet therapy.

2056 Background: Malignant glial cells may have altered expression of mitochondrial enzymes that are key for deriving energy from ketones. The novel strategy of treating malignant gliomas with a ketogenic diet has been tested in animal models with promising results. To evaluate the applicability of these findings to human patients we tested the hypothesis that high grade human gliomas may lack or have decreased expression of mitochondrial enzymes that are important for metabolizing ketones. Methods: We evaluated the expression of 2 key mitochondrial enzymes involved in ketone metabolism in 22 patients (17 males and 5 females, mean age 55,range 30-85) with either glioblastoma (GBM, WHO grade IV) or anaplastic glioma (WHO grade III) (Table). Immunohistochemistry reactions were performed on formalin-fixed paraffin-embedded sections from brain biopsies, using antibodies raised against succinyl CoA: 3-oxoacid CoA transferase (OXCT1) and 3-hydroxybutyrate dehydrogenase 1 (BDH1). Immunoreactivities were graded using a semi-quantitative scale based on the percentage of positive cells: negative (NEG) ≤ 5%; intermediate (INT) 5-20%; and positive (POS) more than 20%. Focal non-neoplastic “normal” brain tissue present within the specimens expressed both enzymes and served as an internal positive control. Results: Either absent or marked decreased expression of OXCT1 and BDH1 were observed in most GBM and anaplastic glioma. Concordance for absent or marked decrease expression of both OXCT1 and BDH1 was observed in 14 of the 17 (82%) GBM’s, and 1 of 5 (20%) anaplastic gliomas. Conclusions: These data support the rationale for a pilot clinical study investigating the therapeutic utility of a ketogenic diet in selected glioma patients. Malignant gliomas with diminished expression of key enzymes (e.g., OXCT1, BDH1) may benefit from the ketogenic diet therapy, whereas those with positive expression of these enzymes likely will not. [Table: see text]

Effects of in utero antiretroviral exposure on mitochondrial DNA levels, mitochondrial function and oxidative stress

HIV and antiretroviral (ART) exposure in utero may have deleterious effects on the infant, but uncertainty still exists. The objective of this study was to evaluate aspects of mitochondrial DNA (mtDNA) content, mitochondrial function and oxidative stress simultaneously in placenta, umbilical cord blood and infant blood in HIV/ART-exposed infants compared with uninfected controls. HIV-1-infected pregnant women and HIV-1-uninfected healthy pregnant controls were enrolled in the study prospectively. Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c-oxidase (COX IV)]- and mitochondrial (COX II)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative stress. Twenty HIV-positive/HIV-exposed and 26 control mother-infant pairs were enrolled in the study. All HIV-infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The cord blood COX II:IV ratio was lower in the HIV-positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV-exposed infants, but the infant peripheral blood COX II:IV ratio was similar. No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure. HIV-exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART-associated mitochondrial toxicity.

AMP Activated Protein Kinase-α2 Regulates Expression of Estrogen-Related Receptor-α, a Metabolic Transcription Factor Related to Heart Failure Development

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial estrogen-related receptor-α (ERRα), but not peroxisome proliferator-activated receptor-γ coactivator 1α, in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of left ventricular dysfunction, pulmonary congestion, and decreases of a group of myocardial energy metabolism-related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 knockout decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial peroxisome proliferator-activated receptor-γ coactivator 1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein, and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, overexpression of ERRα in AMPKα2 knockout neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism-related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes.