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1.
Mol Pain ; 17: 17448069211041853, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34505814

RESUMO

Provoked vestibulodynia (PVD) is a chronic vulvar pain disorder characterized by hypersensitivity and severe pain with pressure localized to the vulvar vestibule. Knowledge regarding pathophysiological mechanisms contributing to the etiology and production of symptoms in PVD remains incomplete but is considered multifactorial. Using a cross-sectional observational study design, data from untargeted metabolomic profiling of vaginal fluid and plasma in women with PVD and healthy women was combined with pain testing and brain imaging in women with PVD to test the hypotheses that women with PVD compared to healthy women show differences in vaginal and plasma metabolites involved in steroid hormone biosynthesis. Steroid hormone metabolites showing group differences were correlated with vulvar vestibular pain and vaginal muscle tenderness and functional connectivity of brain regions involved in pain processing in women with PVD to provide insight into the functional mechanisms linked to the identified alterations. Sensitivity analyses were also performed to determine the impact of hormonal contraceptive use on the study findings. Women with PVD compared to healthy controls had significant reductions primarily in vaginal fluid concentrations of androgenic, pregnenolone and progestin metabolites involved in steroidogenesis, suggesting localized rather than systemic effects in vagina and vulvar vestibule. The observed reductions in androgenic metabolite levels showed large effect size associations with increased vulvar vestibular pain and vulvar muscle tenderness and decreases in androgenic and progestin metabolites were associated with decreased connectivity strength in primary sensorimotor cortices. Women with PVD showed symptom-associated reductions in vaginal fluid concentrations of metabolites involved in the biosynthesis of steroid hormones previously shown to affect the integrity of vulvar and vaginal tissue and nociceptive processing. Deficiency of certain steroids may be an important mechanism contributing to the pathophysiology of symptoms in PVD may provide potential diagnostic markers that could lead to new targets for therapeutic intervention.


Assuntos
Mialgia/fisiopatologia , Córtex Sensório-Motor/fisiopatologia , Vagina/fisiopatologia , Vulvodinia/fisiopatologia , Adulto , Estudos Transversais , Feminino , Humanos , Metabolômica/métodos , Pessoa de Meia-Idade , Mialgia/metabolismo , Medição da Dor/métodos , Córtex Sensório-Motor/metabolismo , Vagina/metabolismo , Vulvodinia/metabolismo , Adulto Jovem
2.
J Pain ; 22(12): 1586-1605, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34029688

RESUMO

Provoked vestibulodynia (PVD) is a chronic pain disorder characterized by local hypersensitivity and severe pain with pressure localized to the vulvar vestibule. Despite decades of study, the lack of identified biomarkers has slowed the development of effective therapies. The primary aim of this study was to use metabolomics to identify novel biochemical mechanisms in vagina and blood underlying brain biomarkers and symptoms in PVD, thereby closing this knowledge gap. Using a cross-sectional case-control observational study design, untargeted and unbiased metabolomic profiling of vaginal fluid and plasma was performed in women with PVD compared to healthy controls. In women with PVD, we also obtained assessments of vulvar pain, vestibular and vaginal muscle tenderness, and 24-hour symptom intensity alongside resting-state brain functional connectivity of brain regions involved in pain processing and modulation. Compared to healthy controls, women with PVD demonstrated differences primarily in vaginal (but not plasma) concentrations of metabolites of the sphingolipid signaling pathways, suggesting localized effects in vagina and vulvar vestibule rather than systemic effects. Our findings reveal that dysregulation of sphingolipid metabolism in PVD is associated with increased vulvar pain and muscle tenderness, sexual dysfunction, and decreased functional connectivity strength in pain processing/modulatory brain regions. This data collectively suggests that alterations in sphingolipid signaling pathways are likely an important molecular biomarker in PVD that could lead to new targets for therapeutic intervention. PERSPECTIVE: This manuscript presents the results of a robust, unbiased molecular assessment of plasma and vaginal fluid samples in women with provoked vestibulodynia compared to healthy controls. The findings suggest that alterations in sphingolipid signaling pathways are associated with symptoms and brain biomarkers and may be an important molecular marker that could provide new targets for therapeutic intervention.


Assuntos
Encéfalo/fisiopatologia , Conectoma , Esfingolipídeos/metabolismo , Vulvodinia , Adulto , Biomarcadores , Encéfalo/diagnóstico por imagem , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Imageamento por Ressonância Magnética , Metaboloma/fisiologia , Transdução de Sinais/fisiologia , Vulvodinia/diagnóstico , Vulvodinia/metabolismo , Vulvodinia/fisiopatologia
3.
J Clin Invest ; 131(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33878036

RESUMO

The ability to adapt to low-nutrient microenvironments is essential for tumor cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription factor pathway associates with advanced disease stages and shorter survival in patients with CRC. NF-κB has been shown to drive tumor-promoting inflammation, cancer cell survival, and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in patients with CRC is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB-regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC cell survival via cell-autonomous mechanisms that fuel fatty acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight patients with CRC. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype 4 (CMS4), which is associated with obesity, stemness, and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator HNF4A in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavorable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Neoplasias Colorretais/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Triglicerídeos/metabolismo , Hidrolases de Éster Carboxílico/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Triglicerídeos/genética
4.
Sci Rep ; 10(1): 16353, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004997

RESUMO

The influence of maternal high-fat diet (HFD) on metabolic response to ozone was examined in Long-Evans rat offspring. F0 females were fed control diet (CD; 10%kcal from fat) or HFD (60%kcal from fat) starting at post-natal day (PND) 30. Rats were bred on PND 72. Dietary regimen was maintained until PND 30 when all offspring were switched to CD. On PND 40, F1 offspring (n = 10/group/sex) were exposed to air or 0.8 ppm ozone for 5 h. Serum samples were collected for global metabolomic analysis (n = 8/group/sex). Offspring from HFD dams had increased body fat and weight relative to CD. Metabolomic analysis revealed significant sex-, diet-, and exposure-related changes. Maternal HFD increased free fatty acids and decreased phospholipids (male > female) in air-exposed rats. Microbiome-associated histidine and tyrosine metabolites were increased in both sexes, while 1,5-anhydroglucitol levels decreased in males indicating susceptibility to insulin resistance. Ozone decreased monohydroxy fatty acids and acyl carnitines and increased pyruvate along with TCA cycle intermediates in females (HFD > CD). Ozone increased various amino acids, polyamines, and metabolites of gut microbiota in HFD female offspring indicating gut microbiome alterations. Collectively, these data suggest that maternal HFD increases offspring susceptibility to metabolic alterations in a sex-specific manner when challenged with environmental stressors.


Assuntos
Dieta Hiperlipídica , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Ozônio/administração & dosagem , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Estresse Fisiológico/fisiologia , Animais , Ácidos Graxos não Esterificados/sangue , Feminino , Microbioma Gastrointestinal , Masculino , Metabolômica , Fosfolipídeos/sangue , Gravidez , Ratos , Ratos Long-Evans , Fatores Sexuais
5.
Am J Med Genet A ; 182(11): 2781-2787, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32909658

RESUMO

Riboflavin transporter deficiency (RTD) (MIM #614707) is a neurogenetic disorder with its most common manifestations including sensorineural hearing loss, peripheral neuropathy, respiratory insufficiency, and bulbar palsy. Here, we present a 2-year-old boy whose initial presentation was severe macrocytic anemia necessitating multiple blood transfusions and intermittent neutropenia; he subsequently developed ataxia and dysarthria. Trio-exome sequencing detected compound heterozygous variants in SLC52A2 that were classified as pathogenic and a variant of uncertain significance. Bone marrow evaluation demonstrated megaloblastic changes. Notably, his anemia and neutropenia resolved after treatment with oral riboflavin, thus expanding the clinical phenotype of this disorder. We reiterate the importance of starting riboflavin supplementation in a young child who presents with macrocytic anemia and neurological features while awaiting biochemical and genetic work up. We detected multiple biochemical abnormalities with the help of untargeted metabolomics analysis associated with abnormal flavin adenine nucleotide function which normalized after treatment, emphasizing the reversible pathomechanisms involved in this disorder. The utility of untargeted metabolomics analysis to monitor the effects of riboflavin supplementation in RTD has not been previously reported.


Assuntos
Anemia Macrocítica/patologia , Paralisia Bulbar Progressiva/patologia , Perda Auditiva Neurossensorial/patologia , Metaboloma , Deficiência de Riboflavina/patologia , Riboflavina/metabolismo , Adulto , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Paralisia Bulbar Progressiva/genética , Paralisia Bulbar Progressiva/metabolismo , Feminino , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Humanos , Lactente , Masculino , Mutação , Receptores Acoplados a Proteínas G/genética , Deficiência de Riboflavina/genética , Deficiência de Riboflavina/metabolismo
6.
Sci Rep ; 10(1): 10250, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581232

RESUMO

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.


Assuntos
Metabolômica/métodos , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Mieloma Múltiplo/diagnóstico , Plasmócitos/metabolismo , Aminoácidos de Cadeia Ramificada/análise , Diagnóstico Diferencial , Humanos , Cinurenina/análise , Lactosilceramidas/análise , Lipidômica/métodos , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Mieloma Múltiplo/sangue , Mieloma Múltiplo/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidilinositóis/análise , Estudos Prospectivos
7.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R1004-R1013, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32292063

RESUMO

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.


Assuntos
Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoproteção , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Masculino , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismo
8.
Sci Rep ; 9(1): 10070, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296900

RESUMO

Muscular dystrophy-dystroglycanopathies comprise a heterogeneous and complex group of disorders caused by loss-of-function mutations in a multitude of genes that disrupt the glycobiology of α-dystroglycan, thereby affecting its ability to function as a receptor for extracellular matrix proteins. Of the various genes involved, FKRP codes for a protein that plays a critical role in the maturation of a novel glycan found only on α-dystroglycan. Yet despite knowing the genetic cause of FKRP-related dystroglycanopathies, the molecular pathogenesis of disease and metabolic response to therapeutic intervention has not been fully elucidated. To address these challenges, we utilized mass spectrometry-based metabolomics to generate comprehensive metabolite profiles of skeletal muscle across diseased, treated, and normal states. Notably, FKRP-deficient mice elicit diverse metabolic abnormalities in biomarkers of extracellular matrix remodeling and/or aging, pentoses/pentitols, glycolytic intermediates, and lipid metabolism. More importantly, the restoration of FKRP protein activity following AAV-mediated gene therapy induced a substantial correction of these metabolic impairments. While interconnections of the affected molecular mechanisms remain unclear, our datasets support the notion that global metabolic profiling can be valuable for determining the involvement of previously unsuspected regulatory or pathological pathways as well as identifying potential targets for drug discovery and diagnostics.


Assuntos
Distroglicanas/metabolismo , Metabolômica/métodos , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular Animal/metabolismo , Pentosiltransferases/metabolismo , Animais , Terapia Genética , Glicosilação , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distrofias Musculares/genética , Distrofias Musculares/terapia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Mutação/genética , Pentosiltransferases/genética
9.
Mol Cell Biol ; 39(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31235477

RESUMO

In this report, we show that nitric oxide suppresses DNA damage response (DDR) signaling in the pancreatic ß-cell line INS 832/13 and rat islets by inhibiting intermediary metabolism. Nitric oxide is known to inhibit complex IV of the electron transport chain and aconitase of the Krebs cycle. Non-ß cells compensate by increasing glycolytic metabolism to maintain ATP levels; however, ß cells lack this metabolic flexibility, resulting in a nitric oxide-dependent decrease in ATP and NAD+ Like nitric oxide, mitochondrial toxins inhibit DDR signaling in ß cells by a mechanism that is associated with a decrease in ATP. Non-ß cells compensate for the effects of mitochondrial toxins with an adaptive shift to glycolytic ATP generation that allows for DDR signaling. Forcing non-ß cells to derive ATP via mitochondrial respiration (replacing glucose with galactose in the medium) and glucose deprivation sensitizes these cells to nitric oxide-mediated inhibition of DDR signaling. These findings indicate that metabolic flexibility is necessary to maintain DDR signaling under conditions in which mitochondrial oxidative metabolism is inhibited and support the inhibition of oxidative metabolism (decreased ATP) as one protective mechanism by which nitric oxide attenuates DDR-dependent ß-cell apoptosis.


Assuntos
Reparo do DNA/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Óxido Nítrico/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Hep G2 , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NAD/metabolismo , Ratos , Ratos Sprague-Dawley
10.
J Biol Chem ; 294(13): 4843-4853, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30659092

RESUMO

Oxidative stress is thought to promote pancreatic ß-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that ß-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which ß-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of ß-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that ß-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized ß-cells to continuously generated H2O2 In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased ß-cell viability independently of thioredoxin reductase inhibition. These findings show that ß-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase-dependent mechanism and are not as sensitive to oxidative damage as previously thought.


Assuntos
Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animais , Sobrevivência Celular , Dano ao DNA , Secreção de Insulina , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Tiorredoxina Dissulfeto Redutase/metabolismo
11.
J Nutr ; 148(2): 194-201, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490096

RESUMO

Background: Individuals with phenylketonuria (PKU) have a risk of cognitive impairment and inflammation. Many follow a low-phenylalanine (low-Phe) diet devoid of animal protein in combination with medical foods (MFs). Objective: To assess lipid metabolism in participants with PKU consuming amino acid MFs (AA-MFs) or glycomacropeptide MFs (GMP-MFs), we conducted fatty acid and metabolomics analyses. Methods: We used subsets of fasting plasma and urine samples from our randomized crossover trial in which participants with early-treated classical and variant (milder) PKU consumed a low-Phe diet combined with AA-MFs or GMP-MFs for 3 wk each. Fatty acid profiles of red blood cell (RBC) membranes were determined for 25 adults (aged 18-49 y) with PKU and 143 control participants. Metabolomics analyses of plasma and urine samples were conducted by Metabolon for 9-10 adolescent and adult participants with PKU and for 15 control participants. Results: RBC fatty acid profiles were not significantly different with AA-MFs or GMP-MFs. PKU participants showed higher total n-6:n-3 (ω-6:ω-3) fatty acids (mean ± SD percentages of total fatty acids: AA-MF = 5.45% ± 1.07%; controls = 4.33%; P < 0.001) and lower docosahexaenoic acid (DHA; AA-MF = 3.21% ± 0.98%; controls = 3.70% ± 1.01%; P = 0.02) and eicosapentaenoic acid (AA-MF = 0.33% ± 0.12%; controls = 0.60% ± 0.43%; P < 0.001) in RBCs than did control participants. Despite higher carnitine intake from AA-MFs than GMP-MFs (mean ± SE intake: AA-MFs = 58.6 ± 5.3 mg/d; GMP-MFs = 0.3 ± 0.01 mg/d; P < 0.001), plasma concentrations of carnitine were similar and not different from those in the control group (AA-MF compared with GMP-MF, P = 0.73). AA-MFs resulted in higher urinary excretion of trimethylamine N-oxide (TMAO), which is synthesized by bacteria from carnitine, compared with GMP-MFs (mean ± SE scaled intensity-TMAO: AA-MFs = 1.2 ± 0.1, GMP-MFs = 0.9 ± 0.1; P = 0.005). Plasma deoxycarnitine was lower in PKU participants than in control participants, suggesting reduced carnitine biosynthesis in PKU (AA-MF = 0.9 ± 0.1; GMP-MF = 1.0 ± 0.1; controls = 1.3 ± 0.1; AA-MF compared with controls, P = 0.01; GMP-MF compared with controls, P = 0.04). Conclusions: Supplementation with DHA is needed in PKU. Carnitine supplementation of AA-MFs shows reduced bioavailability due, in part, to bacterial degradation to TMAO, whereas the bioavailability of carnitine is greater with prebiotic GMP-MFs. This trial was registered at www.clinicaltrials.gov as NCT01428258.


Assuntos
Biomarcadores/análise , Carnitina/metabolismo , Colesterol/metabolismo , Ácidos Graxos Essenciais/metabolismo , Metabolômica , Fenilcetonúrias/metabolismo , Adolescente , Adulto , Aminoácidos/administração & dosagem , Betaína/análogos & derivados , Betaína/sangue , Biomarcadores/sangue , Biomarcadores/urina , Carnitina/administração & dosagem , Carnitina/sangue , Caseínas/administração & dosagem , Estudos Cross-Over , Suplementos Nutricionais , Eritrócitos/química , Jejum , Ácidos Graxos/administração & dosagem , Ácidos Graxos/sangue , Feminino , Microbioma Gastrointestinal/fisiologia , Humanos , Metabolismo dos Lipídeos , Masculino , Metilaminas/urina , Pessoa de Meia-Idade , Fragmentos de Peptídeos/administração & dosagem , Fenilcetonúrias/dietoterapia
12.
Int J Mol Sci ; 19(2)2018 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-29439449

RESUMO

The FecB gene has been discovered as an important gene in sheep for its high relationship with the ovulation rate, but its regulatory mechanism remains unknown. In the present study, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were adopted to detect the metabolic effects of FecB gene in follicular fluid (FF) and ovarian vein serum (OVS) in Small Tail Han (STH) sheep. ANOVA and random forest statistical methods were employed for the identification of important metabolic pathways and biomarkers. Changes in amino acid metabolism, redox environment, and energy metabolism were observed in FF from the three FecB genotype STH ewes. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) showed that metabolic effects of FecB gene are more pronounced in FF than in OVS. Therefore, the difference of the metabolic profile in FF is also affected by the FecB genotypes. In Spearman correlation analysis, key metabolites (e.g., glucose 6-phosphate, glucose 1-phosphate, aspartate, asparagine, glutathione oxidized (GSSG), cysteine-glutathione disulfide, γ-glutamylglutamine, and 2-hydrosybutyrate) in ovine FF samples showed a significant correlation with the ovulation rate. Our findings will help to explain the metabolic mechanism of high prolificacy ewes and benefit fertility identification.


Assuntos
Fertilidade , Líquido Folicular/metabolismo , Genótipo , Ovulação/sangue , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Ovulação/metabolismo , Ovinos
13.
Stem Cell Reports ; 9(6): 1839-1852, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29129681

RESUMO

Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitocôndrias/patologia , Doença de Parkinson/genética , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Histona Desacetilases do Grupo III/genética , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/genética , Mutação , Neuritos/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia
14.
Stem Cells Transl Med ; 6(4): 1191-1201, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28224719

RESUMO

To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201.


Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/metabolismo , Acrilamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Miócitos Cardíacos/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo
15.
Mol Cell Biol ; 36(15): 2067-77, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185882

RESUMO

Nitric oxide, produced in pancreatic ß cells in response to proinflammatory cytokines, plays a dual role in the regulation of ß-cell fate. While nitric oxide induces cellular damage and impairs ß-cell function, it also promotes ß-cell survival through activation of protective pathways that promote ß-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents ß-cell apoptosis by attenuating the DNA damage response (DDR). Nitric oxide suppresses activation of the DDR (as measured by γH2AX formation and the phosphorylation of KAP1 and p53) in response to multiple genotoxic agents, including camptothecin, H2O2, and nitric oxide itself, despite the presence of DNA damage. While camptothecin and H2O2 both induce DDR activation, nitric oxide suppresses only camptothecin-induced apoptosis and not H2O2-induced necrosis. The ability of nitric oxide to suppress the DDR appears to be selective for pancreatic ß cells, as nitric oxide fails to inhibit DDR signaling in macrophages, hepatocytes, and fibroblasts, three additional cell types examined. While originally described as the damaging agent responsible for cytokine-induced ß-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes ß-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis.


Assuntos
Camptotecina/farmacologia , Reparo do DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Óxido Nítrico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Dano ao DNA/efeitos dos fármacos , Células Hep G2 , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
17.
Am J Physiol Regul Integr Comp Physiol ; 309(5): R525-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26084699

RESUMO

While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling ß-cell function and viability, translating findings to human ß-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human ß-cell line, EndoC-ßH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-ßH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-ßH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-ßH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-ßH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-ßH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-ßH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-ßH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-ßH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.


Assuntos
Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Metabolismo Energético/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Insulinoma/patologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias Pancreáticas/patologia , Fenótipo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/farmacologia
18.
Virology ; 483: 264-74, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26001649

RESUMO

Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.


Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Células Mieloides/virologia , Ativação Viral , Adulto , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doença Crônica , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout
19.
Stem Cells Transl Med ; 4(5): 483-93, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25834119

RESUMO

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF-31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF-31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF-31 as a specific NAD⁺ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD⁺ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC-derived cell-based therapies.


Assuntos
Diferenciação Celular/efeitos dos fármacos , NAD/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Piridinas/farmacologia , Técnicas de Cultura de Células , Citocinas/antagonistas & inibidores , Humanos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
20.
J Biol Chem ; 290(12): 7952-60, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25648890

RESUMO

Cytokines impair the function and decrease the viability of insulin-producing ß-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause ß-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that ß-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When ß-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and ß-cell viability. In this study, we show that the ß-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of ß-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within ß-cells, superoxide protects ß-cells by scavenging nitric oxide.


Assuntos
Ilhotas Pancreáticas/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Ilhotas Pancreáticas/citologia , Camundongos , Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
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