104 research outputs found

    Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin.

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    The extracellular matrix is a master regulator of tissue homeostasis in health and disease. Here we examined how the small, leucine-rich, extracellular matrix proteoglycan decorin regulates cardiomyocyte metabolism during fasting in vivo. First, we validated in Dcn-/- mice that decorin plays an essential role in autophagy induced by fasting. High-Throughput metabolomics analyses of cardiac tissue in Dcn-/- mice subjected to fasting revealed striking differences in the hexosamine biosynthetic pathway resulting in aberrant cardiac O-β-N-Acetylglycosylation as compared with WT mice. Functionally, Dcn-/- mice maintained cardiac function at a level comparable with nonfasted animals whereas fasted WT mice showed reduced ejection fraction. Collectively, our results suggest that reduced sensing of nutrient deprivation in the absence of decorin preempts functional adjustments of cardiac output associated with metabolic reprogramming. © 2018 Gubbiotti et al

    Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION.

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    The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (\u3e85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high

    Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice

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    AbstractCalorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated ‘energy-wasting’ process during CR

    Natural and induced mitochondrial phosphate carrier loss: differential dependence of mitochondrial metabolism and dynamics, and cell survival, on the extent of depletion

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    The relevance of PiC, encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3. First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the 5th matrix loop is incompatible with survival on non-fermentable substrate whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants, and HeLa cells with varying degrees of PiC depletion, PiC loss of ~60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos) whereas lower maximal oxphos was evident at ~85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete media, however proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1, MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss-of-function may be independent of phosphate limitation when ATP requirements are not high

    The Abandoned Radical Hysterectomy for Cervical Cancer: Clinical Predictors and Outcomes

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    Objective. Cervical cancer patients who had an abandoned radical hysterectomy were evaluated for preoperative clinical predictors, complication rates, and outcomes. Study Design. IRB approval was obtained for this retrospective analysis and chart review was performed. Results. From 268 women with early-stage (IA2 to IIA) cervical cancer, 19 (7%) had an abandoned hysterectomy for finding grossly positive lymph nodes (84%) or pelvic spread of tumor (16%). No clinical characteristics clearly identified women preoperatively at risk of having an abandoned hysterectomy. In the abandoned group, 26% suffered major morbidities, compared to 34% in the completed group (OR 0.69, [CI 0.16–2.57], P = .789). Thirty-seven percent recurred in the abandoned group, compared to 18% in the completed group (P = .168). Overall survival in the abandoned group was 73% versus 80% in the completed group (P = .772). Conclusion. The practice of abandoning a planned radical hysterectomy for unexpected metastatic disease may not worsen the outcome

    TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

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    A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer

    Long-Chain Fatty Acid Combustion Rate Is Associated with Unique Metabolite Profiles in Skeletal Muscle Mitochondria

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    Incomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA beta-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate.Large-scale unbiased metabolomics analysis was performed using GC/TOF-MS on buffer and mitochondrial matrix fractions obtained prior to and after 20 min of palmitate catabolism (n = 7 mice/condition). Three palmitate concentrations (2, 9 and 19 microM; corresponding to low, intermediate and high oxidation rates) and 9 microM palmitate plus tricarboxylic acid (TCA) cycle and electron transport chain inhibitors were each tested and compared to zero palmitate control incubations. Paired comparisons of the 0 and 20 min samples were made by Student's t-test. False discovery rate were estimated and Type I error rates assigned. Major metabolite groups were organic acids, amines and amino acids, free fatty acids and sugar phosphates. Palmitate oxidation was associated with unique profiles of metabolites, a subset of which correlated to palmitate oxidation rate. In particular, palmitate oxidation rate was associated with distinct changes in the levels of TCA cycle intermediates within and effluxed from mitochondria.This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle

    Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis.

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    Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1-deficient model, we observed a cyclin D1-mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis

    Addressing disparities in pharmacogenomics through rural and underserved workforce education

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    Introduction: While pharmacogenomic (PGx) testing is routine in urban healthcare institutions or academic health centers with access to existing expertise, uptake in medically-underserved areas is lagging. The primary objective of this workforce education program is to extend access to didactic, case-based and clinical PGx training for pharmacists serving rural Minnesota and populations experiencing health disparities in Minnesota.Methods: A PGx workforce training program funded through the Minnesota Department of Health was offered through the University of Minnesota College of Pharmacy (COP) to pharmacists working in rural and/or underserved areas in the state of Minnesota. Learning activities included a 16-week, asynchronous PGx didactic course covering PGx topics, a 15-min recorded presentation, an in-person PGx case-based workshop, and a live international PGx Conference hosted by the University of Minnesota COP and attendance at our PGx Extension of Community Health Outcomes (ECHO).Results: Twenty-nine pharmacists applied for the initial year of the program, with 12 (41%) being accepted. Four (33%) practiced in a hospital setting, four (33%) in retail pharmacy, two (17%) in managed care, and two (17%) in other areas. The majority had not implemented a PGx program as part of their practice, although nearly all responded definitely or probably yes when asked if they expected their organization to increase its use of PGx testing services over the next three years. All participants either strongly or somewhat agreed that this program helped them identify how and where to access clinical PGx guidelines and literature and improved their ability to read and interpret PGx test results. Eight participants (67%) strongly or somewhat agreed that they expected to increase the number of PGx consultations in their practice, while ten (83%) strongly or somewhat agreed they would be able to apply what they learned in this program to their practice in the next six months to a year.Discussion: This novel PGx training program focused exclusively on pharmacists in rural and/or underserved areas with a delivery method that could be accomplished conveniently and remotely. Although most participants’ organizations had yet to implement PGx testing routinely, most anticipated this to change in the next few years

    Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells.

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    Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C \u3e 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR–IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer
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