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1.
Nat Commun ; 15(1): 4711, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830841

RESUMO

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.


Assuntos
Feto , Lipopolissacarídeos , Fígado , Pulmão , Placenta , Feminino , Gravidez , Placenta/metabolismo , Placenta/imunologia , Animais , Feto/imunologia , Feto/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Fígado/metabolismo , Fígado/imunologia , Ácidos Docosa-Hexaenoicos/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Camundongos , Inflamação/imunologia , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Adaptação Fisiológica/imunologia , Desenvolvimento Fetal/imunologia , Troca Materno-Fetal/imunologia , Interleucina-6/metabolismo , Interleucina-6/imunologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-38820114

RESUMO

CONTEXT: Given the promising effects of prolonged treatment with beta2-agonist on insulin sensitivity in animals and non-diabetic individuals, the beta2-adrenergic receptor has been proposed as a target to counter peripheral insulin resistance. On the other hand, rodent studies also reveal that beta2-agonists acutely impair insulin action, posing a potential caveat for their use in treating insulin resistance. OBJECTIVE: To assess the impact of beta2-agonist on muscle insulin action and glucose metabolism and identify the underlying mechanism(s) in 10 insulin-resistant subjects. METHODS AND PARTICIPANTS: In a cross-over design, we assessed the effect of beta2-agonist on insulin-stimulated muscle glucose uptake during a 3-h hyperinsulinemic isoglycemic clamp with and without intralipid infusion in 10 insulin-resistant overweight subjects. Two hours into the clamp, we infused beta2-agonist. We collected muscle biopsies before, two hours into and by the end of the clamp and analyzed them using metabolomic and lipidomic techniques. RESULTS: We establish that beta2-agonist, independently from and additively to intralipid, impairs insulin-stimulated muscle glucose uptake via different mechanisms. In combination, beta2-agonist and intralipid nearly eliminates insulin-dependent muscle glucose uptake. While both beta2-agonist and intralipid elevated muscle glucose-6-phosphate, only intralipid caused accumulation of downstream muscle glycolytic intermediates, whereas beta2-agonist attenuated incorporation of glucose into glycogen. CONCLUSIONS: Our findings suggest that beta2-agonist inhibits glycogenesis while intralipid inhibits glycolysis in skeletal muscle of insulin-resistant individuals. These results should be addressed in future treatment of insulin resistance with beta2-agonist.

3.
Comp Med ; 74(2): 55-69, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38508697

RESUMO

Disturbances in gut microbiota are prevalent in inflammatory bowel disease (IBD), which includes ulcerative colitis (UC). However, whether these disturbances contribute to development of the disease or are a result of the disease is unclear. In pairs of human twins discordant for IBD, the healthy twin has a higher risk of developing IBD and a gut microbiota that is more similar to that of IBD patients as compared with healthy individuals. Furthermore, appropriate medical treatment may mitigate these disturbances. To study the correlation between microbiota and IBD, we transferred stool samples from a discordant human twin pair: one twin being healthy and the other receiving treatment for UC. The stool samples were transferred from the disease-discordant twins to germ-free pregnant dams. Colitis was induced in the offspring using dextran sodium sulfate. As compared with offspring born to mice dams inoculated with stool from the healthy cotwin, offspring born to dams inoculated with stool from the UC-afflicted twin had a lower disease activity index, less gut inflammation, and a microbiota characterized by higher α diversity and a more antiinflammatory profile that included the presence and higher abundance of antiinflammatory species such as Akkermansia spp., Bacteroides spp., and Parabacteroides spp. These findings suggest that the microbiota from the healthy twin may have had greater inflammatory properties than did that of the twin undergoing UC treatment.


Assuntos
Colite Ulcerativa , Microbioma Gastrointestinal , Animais , Colite Ulcerativa/microbiologia , Humanos , Camundongos , Feminino , Vida Livre de Germes , Sulfato de Dextrana/toxicidade , Fezes/microbiologia , Gravidez , Masculino , Modelos Animais de Doenças , Transplante de Microbiota Fecal
4.
Mol Ther Nucleic Acids ; 35(1): 102101, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38204914

RESUMO

Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder propionic acidemia by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. Interestingly, the PCCA pseudoexon offers an unexploited potential to upregulate gene expression because healthy tissues show relatively high inclusion levels. By blocking inclusion of the nonactivated wild-type pseudoexon, we can increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels in not only patient fibroblasts harboring PCCA missense variants but also those harboring PCCB missense variants. This is a potential treatment strategy for propionic acidemia.

5.
Cell Rep ; 42(11): 113381, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37930887

RESUMO

Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.


Assuntos
Ribossomos , Ubiquitina , Ribossomos/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Autofagia/fisiologia , Oncogenes , Senescência Celular
6.
EMBO Mol Med ; 15(12): e17836, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37766669

RESUMO

The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Propionatos/farmacologia , Propionatos/uso terapêutico , Camundongos Nus , Linhagem Celular Tumoral , Pulmão/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Movimento Celular
7.
Endocrinology ; 164(10)2023 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-37610219

RESUMO

Hepatic lipid metabolism is highly dynamic, and disruption of several circadian transcriptional regulators results in hepatic steatosis. This includes genetic disruption of the glucocorticoid receptor (GR) as the liver develops. To address the functional role of GR in the adult liver, we used an acute hepatocyte-specific GR knockout model to study temporal hepatic lipid metabolism governed by GR at several preprandial and postprandial circadian timepoints. Lipidomics analysis revealed significant temporal lipid metabolism, where GR disruption results in impaired regulation of specific triglycerides, nonesterified fatty acids, and sphingolipids. This correlates with increased number and size of lipid droplets and mildly reduced mitochondrial respiration, most noticeably in the postprandial phase. Proteomics and transcriptomics analyses suggest that dysregulated lipid metabolism originates from pronounced induced expression of enzymes involved in fatty acid synthesis, ß-oxidation, and sphingolipid metabolism. Integration of GR cistromic data suggests that induced gene expression is a result of regulatory actions secondary to direct GR effects on gene transcription.


Assuntos
Metabolismo dos Lipídeos , Receptores de Glucocorticoides , Masculino , Animais , Camundongos , Metabolismo dos Lipídeos/genética , Receptores de Glucocorticoides/genética , Hepatócitos , Fígado , Adipogenia
8.
Int J Obes (Lond) ; 47(7): 529-537, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37029207

RESUMO

BACKGROUND/OBJECTIVES: Obesity in pregnancy associates with changes in the glucose-insulin axis. We hypothesized that these changes affect the maternal metabolome already in the first trimester of human pregnancy and, thus, aimed to identify these metabolites. PATIENTS/METHODS: We performed untargeted metabolomics (HPLC-MS/MS) on maternal serum (n = 181, gestational weeks 4+0-11+6). For further analysis, we included only non-smoking women as assessed by serum cotinine levels (ELISA) (n = 111). In addition to body mass index (BMI) and leptin as measures of obesity and adiposity, we metabolically phenotyped women by their fasting glucose, C-peptide and insulin sensitivity (ISHOMA index). To identify metabolites (outcome) associated with BMI, leptin, glucose, C-peptide and/or ISHOMA (exposures), we used a combination of univariable and multivariable regression analyses with multiple confounders and machine learning methods (Partial Least Squares Discriminant Analysis, Random Forest and Support Vector Machine). Additional statistical tests confirmed robustness of results. Furthermore, we performed network analyses (MoDentify package) to identify sets of correlating metabolites that are coordinately regulated by the exposures. RESULTS: We detected 2449 serum features of which 277 were annotated. After stringent analysis, 15 metabolites associated with at least one exposure (BMI, leptin, glucose, C-peptide, ISHOMA). Among these, palmitoleoyl ethanolamine (POEA), an endocannabinoid-like lipid endogenously synthesized from palmitoleic acid, and N-acetyl-L-alanine were consistently associated with C-peptide in all the analyses (95% CI: 0.10-0.34; effect size: 21%; p < 0.001; 95% CI: 0.04-0.10; effect size: 7%; p < 0.001). In network analysis, most features correlating with palmitoleoyl ethanolamide and N-acetyl-L-alanine and associated with C-peptide, were amino acids or dipeptides (n = 9, 35%), followed by lipids (n = 7, 27%). CONCLUSIONS: We conclude that the metabolome of pregnant women with overweight/obesity is already altered early in pregnancy because of associated changes of C-peptide. Changes of palmitoleoyl ethanolamide concentration in pregnant women with obesity-associated hyperinsulinemia may reflect dysfunctional endocannabinoid-like signalling.


Assuntos
Endocanabinoides , Leptina , Feminino , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Peptídeo C , Espectrometria de Massas em Tandem , Peso ao Nascer , Obesidade , Índice de Massa Corporal , Glucose
9.
Proc Natl Acad Sci U S A ; 120(14): e2220102120, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-36996103

RESUMO

Molecular clocks in the periphery coordinate tissue-specific daily biorhythms by integrating input from the hypothalamic master clock and intracellular metabolic signals. One such key metabolic signal is the cellular concentration of NAD+, which oscillates along with its biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT). NAD+ levels feed back into the clock to influence rhythmicity of biological functions, yet whether this metabolic fine-tuning occurs ubiquitously across cell types and is a core clock feature is unknown. Here, we show that NAMPT-dependent control over the molecular clock varies substantially between tissues. Brown adipose tissue (BAT) requires NAMPT to sustain the amplitude of the core clock, whereas rhythmicity in white adipose tissue (WAT) is only moderately dependent on NAD+ biosynthesis, and the skeletal muscle clock is completely refractory to loss of NAMPT. In BAT and WAT, NAMPT differentially orchestrates oscillation of clock-controlled gene networks and the diurnality of metabolite levels. NAMPT coordinates the rhythmicity of TCA cycle intermediates in BAT, but not in WAT, and loss of NAD+ abolishes these oscillations similarly to high-fat diet-induced circadian disruption. Moreover, adipose NAMPT depletion improved the ability of animals to defend body temperature during cold stress but in a time-of-day-independent manner. Thus, our findings reveal that peripheral molecular clocks and metabolic biorhythms are shaped in a highly tissue-specific manner by NAMPT-dependent NAD+ synthesis.


Assuntos
NAD , Nicotinamida Fosforribosiltransferase , Animais , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Ritmo Circadiano/fisiologia , Tecido Adiposo Marrom/metabolismo , Obesidade/metabolismo , Citocinas/metabolismo
10.
Aging Cell ; 21(12): e13726, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36217558

RESUMO

Several epidemiological studies have suggested that obesity complicated with insulin resistance and type 2 diabetes exerts deleterious effects on the skeleton. While obesity coexists with estrogen deficiency in postmenopausal women, their combined effects on the skeleton are poorly studied. Thus, we investigated the impact of high-fat diet (HFD) on bone and metabolism of ovariectomized (OVX) female mice (C57BL/6J). OVX or sham operated mice were fed either HFD (60%fat) or normal diet (10%fat) for 12 weeks. HFD-OVX group exhibited pronounced increase in body weight (~86% in HFD and ~122% in HFD-OVX, p < 0.0005) and impaired glucose tolerance. Bone microCT-scanning revealed a pronounced decrease in trabecular bone volume/total volume (BV/TV) (-15.6 ± 0.48% in HFD and -37.5 ± 0.235% in HFD-OVX, p < 0.005) and expansion of bone marrow adipose tissue (BMAT; +60.7 ± 9.9% in HFD vs. +79.5 ± 5.86% in HFD-OVX, p < 0.005). Mechanistically, HFD-OVX treatment led to upregulation of genes markers of senescence, bone resorption, adipogenesis, inflammation, downregulation of gene markers of bone formation and bone development. Similarly, HFD-OVX treatment resulted in significant changes in bone tissue levels of purine/pyrimidine and Glutamate metabolisms, known to play a regulatory role in bone metabolism. Obesity and estrogen deficiency exert combined deleterious effects on bone resulting in accelerated cellular senescence, expansion of BMAT and impaired bone formation leading to decreased bone mass. Our results suggest that obesity may increase bone fragility in postmenopausal women.


Assuntos
Diabetes Mellitus Tipo 2 , Dieta Hiperlipídica , Feminino , Camundongos , Animais , Humanos , Dieta Hiperlipídica/efeitos adversos , Diabetes Mellitus Tipo 2/complicações , Camundongos Endogâmicos C57BL , Obesidade/complicações , Obesidade/metabolismo , Osso e Ossos/metabolismo , Estrogênios , Ovariectomia/efeitos adversos
11.
J Clin Endocrinol Metab ; 107(12): 3261-3274, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-36111559

RESUMO

CONTEXT: Glucose-dependent insulinotropic polypeptide (GIP) has been proposed to exert insulin-independent effects on lipid and bone metabolism. OBJECTIVE: We investigated the effects of a 6-day subcutaneous GIP infusion on circulating lipids, white adipose tissue (WAT), brown adipose tissue (BAT), hepatic fat content, inflammatory markers, respiratory exchange ratio (RER), and bone homeostasis in patients with type 1 diabetes. METHODS: In a randomized, placebo-controlled, double-blind, crossover study, 20 men with type 1 diabetes underwent a 6-day continuous subcutaneous infusion with GIP (6 pmol/kg/min) and placebo (saline), with an interposed 7-day washout period. RESULTS: During GIP infusion, participants (26 ± 8 years [mean ± SD]; BMI 23.8 ± 1.8 kg/m2; glycated hemoglobin A1c 51 ± 10 mmol/mol [6.8 ± 3.1%]) experienced transiently increased circulating concentrations of nonesterified fatty acid (NEFA) (P = 0.0005), decreased RER (P = 0.009), indication of increased fatty acid ß-oxidation, and decreased levels of the bone resorption marker C-terminal telopeptide (P = 0.000072) compared with placebo. After 6 days of GIP infusion, hepatic fat content was increased by 12.6% (P = 0.007) and supraclavicular skin temperature, a surrogate indicator of BAT activity, was increased by 0.29 °C (P < 0.000001) compared with placebo infusion. WAT transcriptomic profile as well as circulating lipid species, proteome, markers of inflammation, and bone homeostasis were unaffected. CONCLUSION: Six days of subcutaneous GIP infusion in men with type 1 diabetes transiently decreased bone resorption and increased NEFA and ß-oxidation. Further, hepatic fat content, and supraclavicular skin temperature were increased without affecting WAT transcriptomics, the circulating proteome, lipids, or inflammatory markers.


Assuntos
Reabsorção Óssea , Diabetes Mellitus Tipo 1 , Masculino , Humanos , Transcriptoma , Tecido Adiposo Marrom/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Estudos Cross-Over , Proteoma/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Tecido Adiposo Branco , Termogênese , Reabsorção Óssea/metabolismo
12.
Cell Metab ; 34(3): 473-486.e9, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35120589

RESUMO

Fasting metabolism and immunity are tightly linked; however, it is largely unknown how immune cells contribute to metabolic homeostasis during fasting in healthy subjects. Here, we combined cell-type-resolved genomics and computational approaches to map crosstalk between hepatocytes and liver macrophages during fasting. We identified the glucocorticoid receptor (GR) as a key driver of fasting-induced reprogramming of the macrophage secretome including fasting-suppressed cytokines and showed that lack of macrophage GR impaired induction of ketogenesis during fasting as well as endotoxemia. Mechanistically, macrophage GR suppressed the expression of tumor necrosis factor (TNF) and promoted nuclear translocation of hepatocyte GR to activate a fat oxidation/ketogenesis-related gene program, cooperatively induced by GR and peroxisome proliferator-activated receptor alpha (PPARα) in hepatocytes. Together, our results demonstrate how resident liver macrophages directly influence ketogenesis in hepatocytes, thereby also outlining a strategy by which the immune system can set the metabolic tone during inflammatory disease and infection.


Assuntos
Jejum , Receptores de Glucocorticoides , Animais , Jejum/metabolismo , Hepatócitos/metabolismo , Humanos , Corpos Cetônicos/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , PPAR alfa/metabolismo , Receptores de Glucocorticoides/metabolismo
13.
Cell Rep ; 37(5): 109938, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34731602

RESUMO

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.


Assuntos
Ingestão de Alimentos , Glucoquinase/metabolismo , Glucose/metabolismo , Glicogênio Hepático/metabolismo , Fígado/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Sítios de Ligação , Glicemia/metabolismo , Dexametasona/farmacologia , Regulação Enzimológica da Expressão Gênica , Glucocorticoides/farmacologia , Glucoquinase/genética , Células HEK293 , Humanos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Ratos Sprague-Dawley , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica
14.
Metabol Open ; 12: 100125, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34622190

RESUMO

BACKGROUND: Alzheimer's Disease (AD) is a complex and multifactorial disease and novel approaches are needed to illuminate the underlying pathology. Metabolites comprise the end-product of genes, transcripts, and protein regulations and might reflect disease pathogenesis. Blood is a common biofluid used in metabolomics; however, since extracellular vesicles (EVs) hold cell-specific biological material and can cross the blood-brain barrier, their utilization as biological material warrants further investigation. We aimed to investigate blood- and EV-derived metabolites to add insigts to the pathological mechanisms of AD. METHODS: Blood samples were collected from 10 AD and 10 Mild Cognitive Impairment (MCI) patients, and 10 healthy controls. EVs were enriched from plasma using 100,000×g, 1 h, 4 °C with a wash. Metabolites from serum and EVs were measured using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Multivariate and univariate analyses were employed to identify altered metabolites in cognitively impaired individuals. RESULTS: While no significant EV-derived metabolites were found differentiating patients from healthy individuals, six serum metabolites were found important; valine (p = 0.001, fold change, FC = 0.8), histidine (p = 0.001, FC = 0.9), allopurinol riboside (p = 0.002, FC = 0.2), inosine (p = 0.002, FC = 0.3), 4-pyridoxic acid (p = 0.006, FC = 1.6), and guanosine (p = 0.004, FC = 0.3). Pathway analysis revealed branched-chain amino acids, purine and histidine metabolisms to be downregulated, and vitamin B6 metabolism upregulated in patients compared to controls. CONCLUSION: Using a combination of LC-MS and NMR methodologies we identified several altered mechanisms possibly related to AD pathology. EVs require additional optimization prior to their possible utilization as a biological material for AD-related metabolomics studies.

15.
Stem Cell Reports ; 16(6): 1510-1526, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34048689

RESUMO

PARK2 (parkin) mutations cause early-onset Parkinson's disease (PD). Parkin is an ubiquitin E3 ligase that participates in several cellular functions, including mitochondrial homeostasis. However, the specific metabolomic changes caused by parkin depletion remain unknown. Here, we used isogenic human induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) to investigate the effect of parkin loss of function by comparative metabolomics supplemented with ultrastructural and functional analyses. PARK2 KO neurons displayed increased tricarboxylic acid (TCA) cycle activity, perturbed mitochondrial ultrastructure, ATP depletion, and dysregulation of glycolysis and carnitine metabolism. These perturbations were combined with increased oxidative stress and a decreased anti-oxidative response. Key findings for PARK2 KO cells were confirmed using patient-specific iPSC-derived neurons. Overall, our data describe a unique metabolomic profile associated with parkin dysfunction and show that combining metabolomics with an iPSC-derived dopaminergic neuronal model of PD is a valuable approach to obtain novel insight into the disease pathogenesis.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Metabolismo Energético , Células-Tronco Pluripotentes Induzidas/metabolismo , Metaboloma , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Trifosfato de Adenosina/metabolismo , Ciclo do Ácido Cítrico , Técnicas de Inativação de Genes/métodos , Glicólise , Humanos , Redes e Vias Metabólicas , Mitocôndrias/ultraestrutura , Mutação , Estresse Oxidativo , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética
16.
Aging Cell ; 20(4): e13342, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33724708

RESUMO

One of the most fundamental challenges for all living organisms is to sense and respond to alternating nutritional conditions in order to adapt their metabolism and physiology to promote survival and achieve balanced growth. Here, we applied metabolomics and lipidomics to examine temporal regulation of metabolism during starvation in wild-type Caenorhabditis elegans and in animals lacking the transcription factor HLH-30. Our findings show for the first time that starvation alters the abundance of hundreds of metabolites and lipid species in a temporal- and HLH-30-dependent manner. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by supplementation of exogenous fatty acids, and that HLH-30 is required for complete oxidation of long-chain fatty acids. We further show that RNAi-mediated knockdown of the gene encoding carnitine palmitoyl transferase I (cpt-1) only impairs survival of wild-type animals and not of hlh-30 animals. Strikingly, we also find that compromised generation of peroxisomes by prx-5 knockdown renders hlh-30 animals hypersensitive to starvation, which cannot be rescued by supplementation of exogenous fatty acids. Collectively, our observations show that mitochondrial functions are compromised in hlh-30 animals and that hlh-30 animals rewire their metabolism to largely depend on functional peroxisomes during starvation, underlining the importance of metabolic plasticity to maintain survival.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais/genética , Inanição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Suplementos Nutricionais , Ácidos Graxos/administração & dosagem , Ácidos Graxos/metabolismo , Técnicas de Silenciamento de Genes , Longevidade/genética , Mutação , Oxirredução , Peroxissomos/metabolismo , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Inanição/genética
17.
Nucleic Acids Res ; 48(16): 9204-9217, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32766806

RESUMO

The type III CRISPR-Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR-Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.


Assuntos
Sistemas CRISPR-Cas/genética , Imunidade/genética , Streptococcus thermophilus/genética , Transcrição Gênica/genética , Cromatografia Líquida de Alta Pressão , Endonucleases/genética , Escherichia coli/genética , Escherichia coli/imunologia , Domínios Proteicos/genética , Estabilidade de RNA/genética , Estabilidade de RNA/imunologia , Ribonucleases/genética , Transdução de Sinais/genética , Streptococcus thermophilus/imunologia , Transcrição Gênica/imunologia
18.
Metabolites ; 10(7)2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32668656

RESUMO

Mitochondrial dysfunction after transient cerebral ischemia can be monitored by cerebral microdialysis (CMD) using changes in the lactate and pyruvate concentrations and ratio. Other metabolites associated with mitochondrial (dys)function are, e.g., tricyclic acid (TCA) and purine metabolites. Ethyl pyruvate (EP) is a putative neuroprotectant, supposedly targeting mitochondrial energy metabolism, but its effect on cerebral energy metabolism has never been described using microdialysis. In this study we monitored the metabolic effects of EP in the endothelin-1 (ET-1) rat model using perfusion with 13C-succinate and analysis of endogenous and 13C-labeled metabolites in the dialysates by liquid chromatography-mass spectrometry (LC-MS). Adult Sprague Dawley rats (n = 27 of which n = 11 were included in the study) were subjected to the microdialysis experiments. Microdialysis probes were perfused with 13C-labeled succinate (1 mM), and striatal dialysates were collected at 30 min intervals before induction of the insult, during intracerebral application of ET-1, and during intravenous treatment with either EP (40 mg/kg) or placebo, which was administered immediately after the insult. The rats were subjected to transient cerebral ischemia by unilateral microinjection of ET-1 in the piriform cortex, causing vasoconstriction of the medial cerebral artery. Monitoring was continued for 5 h after reperfusion, and levels of endogenous and 13C-labeled energy metabolites before and after ischemia-reperfusion were compared in EP-treated and control groups. Infarct volumes were assessed after 24 h. In both the EP-treated and placebo groups, ET-1-induced vasoconstriction resulted in a transient depression of interstitial glucose and elevation of lactate in the ipsilateral striatum. In the reperfusion phase, the concentrations of labeled malate, isocitrate, and lactate as well as endogenous xanthine were significantly higher in the EP-group than in the placebo-group: (mean ± SEM) labeled malate: 39.5% ± 14.9, p = 0.008; labeled isocitrate: 134.8% ± 67.9, p = 0.047; labeled lactate: 61% ± 22.0, p = 0.007; and endogenous xanthine: 93.9% ± 28.3, p = 0.0009. In the placebo group, significantly elevated levels of uridine were observed (mean ± SEM) 32.5% ± 12.7, p = 0.01. Infarct volumes were not significantly different between EP-treated and placebo groups, p = 0.4679. CMD labeled with 13C-succinate enabled detection of ischemic induction and EP treatment effects in the ET-1 rat model of transient focal cerebral ischemia. EP administered as a single intravenous bolus in the reperfusion-phase after transient cerebral ischemia increased de novo synthesis of several key intermediate energy metabolites (13C-malate, 13C-isocitrate, and endogenous xanthine). In summary, mitochondria process 13C-succinate more effectively after EP treatment.

19.
Nucleic Acids Res ; 48(12): 6906-6918, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32459340

RESUMO

The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.


Assuntos
Desulfurococcaceae/genética , Nanoarchaeota/genética , Conformação de Ácido Nucleico , RNA de Transferência/ultraestrutura , Archaea/genética , Archaea/ultraestrutura , Escherichia coli/genética , Metilação , Filogenia , RNA de Transferência/genética , tRNA Metiltransferases/genética , tRNA Metiltransferases/ultraestrutura
20.
Int J Mol Sci ; 21(4)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102473

RESUMO

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O2 in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca2+-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.


Assuntos
Hemoglobinas/metabolismo , Hordeum/metabolismo , Metaboloma , Oxigênio/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Anaerobiose , Clorofila/metabolismo , Clorofila A/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Hemoglobinas/genética , Hordeum/genética , Metabolômica/métodos , Óxido Nítrico/metabolismo , Proteínas de Plantas/genética , Proteoma/genética , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/metabolismo
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