Targeted quantitative metabolic profiling of brain-derived cell cultures by semi-automated MEPS and LC-MS/MS

The accurate characterisation of metabolic profiles is an important prerequisite to determine the rate and the efficiency of the metabolic pathways taking place in the cells. Changes in the balance of metabolites involved in vital processes such as glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), as well as in the biochemical pathways related to amino acids, lipids, nucleotides, and their precursors reflect the physiological condition of the cells and may contribute to the development of various human diseases. The feasible and reliable measurement of a wide array of metabolites and biomarkers possesses great potential to elucidate physiological and pathological mechanisms, aid preclinical drug development and highlight potential therapeutic targets. An effective, straightforward, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was developed for the simultaneous quali-quantitative analysis of 41 compounds in both cell pellet and cell growth medium obtained from brain-derived cell cultures. Sample pretreatment miniaturisation was achieved thanks to the development and optimisation of an original extraction/purification approach based on digitally programmed microextraction by packed sorbent (eVol®-MEPS). MEPS allows satisfactory and reproducible clean-up and preconcentration of both low-volume homogenate cell pellet lysate and cell growth medium with advantages including, but not limited to, minimal sample handling and method sustainability in terms of sample, solvents, and energy consumption. The MEPS-LC-MS/MS method showed good sensitivity, selectivity, linearity, and precision. As a proof of concept, the developed method was successfully applied to the analysis of both cell pellet and cell growth medium obtained from a line of mouse immortalised oligodendrocyte precursor cells (OPCs; Oli-neu cell line), leading to the unambiguous determination of all the considered target analytes. This method is thus expected to be suitable for targeted, quantitative metabolic profiling in most brain cell models, thus allowing accurate investigations on the biochemical pathways that can be altered in central nervous system (CNS) neuropathologies, including e.g., mitochondrial respiration and glycolysis, or use of specific nutrients for growth and proliferation, or lipid, amino acid and nucleotide metabolism

Statins reduce intratumor cholesterol affecting adrenocortical cancer growth

Mitotane causes hypercholesterolemia in ACC patients. We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to ER\u3b1 action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production and ER\u3b1 activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ER\u3b1 function. A mitochondrial target of ER\u3b1, the respiratory complex IV (COX IV) was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. In vivo experiments confirmed the ability of simvastatin to reduce tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ER\u3b1, COX IV expression and activity and increase TUNEL positive cells. Collectively these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production, inhibits mitochondrial respiratory chain inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth

In Vitro Effects of Low-energy Ultrasound Treatment on Healthy CD3/CD8+ Lymphocytes, Red blood cells, Acute Myeloid leukemia cells, and Jurkat cell line

: The study of the biological effects of low-energy ultrasound and its applications is a rapidly expanding research area. Low-energy ultrasound could be used as anti-tumoral therapy with or without the pharmacological combination even if the second situation has been scarcely investigated up to now. Very little information is available about the ultrasound effects on healthy red blood cells, CD3, and mainly CD8 subset lymphocytes which is the main subset cell having cytotoxic function towards cancer cells. In this study, we investigated in vitro the bioeffects of low energy ultrasound on red blood cells and PBMCs isolated from healthy donors as well as on two myeloid leukemia cell lines (OCI- AML-3 MOLM-13) and lymphoblastic Jurkat cell line. Using low-energy ultrasound (US), a study was conducted to determine how it affects CD3/CD8 lymphocytes and leukemia cells, as well as its potential role in treating blood cancers, by analyzing changes in mitochondrial membrane potential, phosphatidylserine asymmetry, morphological changes for myeloid AML cell lines, proliferation and cytotoxic activation of healthy lymphocytes, and apoptosis for RBCs after US exposure. Overall, we demonstrated that CD3/CD8 lymphocytes proliferation/activation and cytotoxic functions are fully preserved after ultrasound treatments, whereas leukemia cell lines undergo apoptosis and stop proliferating suggesting a potential method of treating blood cancer

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production

BackgroundMitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries.ResultsIn this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica.ConclusionsThe Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis

Transcriptional and metabolic effects of aspartate-glutamate carrier isoform 1 (AGC1) downregulation in mouse oligodendrocyte precursor cells (OPCs)

: Aspartate-glutamate carrier isoform 1 (AGC1) is a carrier responsible for the export of mitochondrial aspartate in exchange for cytosolic glutamate and is part of the malate-aspartate shuttle, essential for the balance of reducing equivalents in the cells. In the brain, mutations in SLC25A12 gene, encoding for AGC1, cause an ultra-rare genetic disease, reported as a neurodevelopmental encephalopathy, whose symptoms include global hypomyelination, arrested psychomotor development, hypotonia and seizures. Among the biological components most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination processes, and their precursors [oligodendrocyte progenitor cells (OPCs)]. The AGC1 silencing in an in vitro model of OPCs was documented to cause defects of proliferation and differentiation, mediated by alterations of histone acetylation/deacetylation. Disrupting AGC1 activity could possibly reduce the availability of acetyl groups, leading to perturbation of many biological pathways, such as histone modifications and fatty acids formation for myelin production. Here, we explore the transcriptome of mouse OPCs partially silenced for AGC1, reporting results of canonical analyses (differential expression) and pathway enrichment analyses, which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further investigate the cellular effects of AGC1 deficiency through the identification of most affected transcriptional networks and altered alternative splicing. Transcriptional data were integrated with differential metabolite abundance analysis, showing downregulation of several amino acids, including glutamine and aspartate. Taken together, our results provide a molecular foundation for the effects of AGC1 deficiency in OPCs, highlighting the molecular mechanisms affected and providing a list of actionable targets to mitigate the effects of this pathology