Stem cells, poised to revolutionize current medicine, stand as major workhorses for monitoring changes in cell fate. Characterizing metabolic phenotypes is key to monitor in differentiating cells transcriptional and epigenetic shifts at a functional level and provides a non-genetic means to control cell specification. Expanding the arsenal of analytical tools for metabolic profiling of cell differentiation is therefore of importance. Here, we describe the metabolome of whole pluripotent stem cells (PSCs) using highresolution magic angle spinning (HR-MAS), a non-destructive approach for Nuclear Magnetic Resonance (NMR) analysis. The integrated 1 H NMR analysis results in detection of metabolites of various groups, including energy metabolites, amino acids, choline derivatives and short chain fatty acids. It unveils new metabolites that discriminate PSCs from differentiated counterparts and directly measures substrates and co-factors of histone modifying enzymes, suggesting that NMR stands as a strategic technique for deciphering metabolic regulations of histone post-translational modifications. HR-MAS NMR analysis of whole PSCs complements the much used solution NMR of cell extracts. Altogether, our multi-platform nMR investigation provides a consolidated picture of pSc metabolic signatures and of metabolic pathways involved in differentiation. open Scientific RepoRtS | (2020) 10:1622 | https://doi.org/10.1038/s41598-020-58377-w www.nature.com/scientificreports www.nature.com/scientificreports/ NMR-based analysis, suggesting that NMR is an important tool for monitoring histone modifying metabolic processes in general, and PSC metabolic state in particular.NMR offers critical insights into cell metabolism, and has previously been used for profiling many cell systems, including adult stem cells, with important clinical implications [25][26][27][28][29] . Several studies have notably described the metabolism of PSCs using solution NMR 12,28,30,31 . A comprehensive evaluation of the metabolic fingerprints provided by complementary NMR approaches relevant to the challenges of deciphering metabolic regulations of gene expression is yet to be described.Traditionally, NMR metabolomics has relied on the analysis of solution samples obtained by extraction of biological materials. However, the analysis of cell extracts suffers from several disadvantages: i) the resulting metabolic profiles are partial and biased towards the extraction system used; ii) sample preparation may cause the loss or chemical modification of cellular components, limiting the information about in vivo metabolic events; iii) it may also introduce technical variability to the studied replicates. High-resolution magic angle spinning (HR-MAS) NMR spectroscopy enables the direct characterization of whole cells or tissues, allowing the simultaneous detection of polar and nonpolar metabolites, in a more global insight into their metabolic profiles. Rapid spinning of a sample at an angle of 54.7° ("magic angle") relative to the applied magnetic field reduces ...

Follicular fluid (FF) is a liquid that surrounds the ovum. Its metabolite and, specifically, its lipid content have been associated with oocyte development. To characterize possible association between the lipid composition of FF and the outcome of pregnancy, we carried out a lipidomics study and compared the abundance of lipids from FF of patients with positive and negative outcomes. We found a differential lipid network wiring in positive‐outcome FF, with a significant decrease (∼2 fold; P < 0.001) in triacylglycerol levels and higher accumulation (10–50%; P < 0.001) of membrane lipids groups (phospholipids and sphingolipids). In addition to this major metabolic alteration, other lipid groups such as cholesteryl esters showed lower levels in positive‐outcome patients, whereas derivatives of vitamin D were highly accumulated in positive‐outcome FF, supporting previous studies that associate vitamin D levels in FF to pregnancy outcome. Our data also point to specific lipid species with a differential accumulation pattern in positive‐outcome FF that predicted pregnancy in a receiver operating characteristic analysis. Altogether, our results suggest that FF lipid network is associated with the oocyte development, with possible implications in diagnostics and treatment.—Shehadeh, A., Bruck‐Haimson, R., Saidemberg, D., Zacharia. A., Herzberg, S., Ben‐Meir, A., Moussaieff, A. A shift in follicular fluid from triacylglycerols to membrane lipids is associated with positive pregnancy outcome. FASEB J. 33, 10291–10299 (2019). http://www.fasebj.org

Follicular fluid (FF) constitutes the microenvironment of the developing oocyte. We recently characterized its lipid composition and found lipid signatures of positive pregnancy outcome after in vitro fertilization (IVF). In the current study, we aimed to test the hypothesis that unexplained female infertility is related to lipid metabolism, given the lipid signature of positive-outcome IVF patients we previously found. Assuming that FF samples from IVF patients with male factor infertility can represent a non-hindered metabolic microenvironment, we compared them to FF taken from women with unexplained infertility. FF from patients undergoing IVF was examined for its lipid composition. We found highly increased triacylglycerol levels, with a lower abundance of monoacylglycerols, phospholipids and sphingolipids in the FF of patients with unexplained infertility. The alterations in the lipid class accumulation were independent of the body mass index (BMI) and were altogether kept across the age groups. Potential lipid biomarkers for pregnancy outcomes showed a highly discriminative abundance in the FF of unexplained infertility patients. Lipid abundance distinguished IVF patients with unrecognized infertility and provided a potential means for the evaluation of female fertility.

Background Adipose tissue plays important roles in health and disease. Given the unique association of visceral adipose tissue with obesity-related metabolic diseases, the distribution of lipids between the major fat depots located in subcutaneous and visceral regions may shed new light on adipose tissue–specific roles in systemic metabolic perturbations. Objective We sought to characterize the lipid networks and unveil differences in the metabolic infrastructure of the 2 adipose tissues that may have functional and nutritional implications. Methods Paired visceral and subcutaneous adipose tissue samples were obtained from 17 overweight patients undergoing elective abdominal surgery. Ultra-performance LC-MS was used to measure 18,640 adipose-derived features; 520 were putatively identified. A stem cell model for adipogenesis was used to study the functional implications of the differences found. Results Our analyses resulted in detailed lipid metabolic maps of the 2 major adipose tissues. They point to a higher accumulation of phosphatidylcholines, triacylglycerols, and diacylglycerols, although lower ceramide concentrations, in subcutaneous tissue. The degree of unsaturation was lower in visceral adipose tissue (VAT) phospholipids, indicating lower unsaturated fatty acid incorporation into adipose tissue. The differential abundance of phosphatidylcholines we found can be attributed at least partially to higher expression of phosphatidylethanolamine methyl transferase (PEMT). PEMT-deficient embryonic stem cells showed a dramatic decrease in adipogenesis, and the resulting adipocytes exhibited lower accumulation of lipid droplets, in line with the lower concentrations of glycerolipids in VAT. Ceramides may inhibit the expression of PEMT by increased insulin resistance, thus potentially suggesting a functional pathway that integrates ceramide, PEMT, and glycerolipid biosynthetic pathways. Conclusions Our work unveils differential infrastructure of the lipid networks in visceral and subcutaneous adipose tissues and suggests an integrative pathway, with a discriminative flux between adipose tissues.

Stem cells are defined by their ability to self-renew and differentiate, both shown in multiple studies to be regulated by metabolic processes. To decipher metabolic signatures of self-renewal in blastocyst-derived stem cells, we compared early differentiating embryonic stem cells (ESCs) and their extra-embryonic counterparts, trophoblast (T)SCs to their self-renewing counterparts. A metabolomics analysis pointed to the desaturation of fatty acyl chains as a metabolic signature of differentiating blastocyst-derived SCs via the upregulation of delta-6 desaturase (D6D; FADS2) and delta-5 desaturase (D5D; FADS1), key enzymes in the biosynthesis of polyunsaturated fatty acids (PUFAs). The inhibition of D6D or D5D by specific inhibitors or SiRNA retained stemness in ESCs and TSCs, and attenuated endoplasmic reticulum (ER) stress-related apoptosis. D6D inhibition in ESCs upregulated stearoyl-CoA desaturase-1 (Scd1), essential to maintain ER homeostasis. In TSCs, however, D6D inhibition downregulated Scd1. TSCs show higher Scd1 mRNA expression and high levels of monounsaturated fatty acyl chain products in comparison to ESCs. The addition of oleic acid, the product of Scd1 (essential for ESCs), to culture medium, was detrimental to TSCs. Interestingly, TSCs express a high molecular mass variant of Scd1 protein, hardly expressed by ESCs. Taken together, our data suggest that lipid desaturation is a metabolic regulator of the balance between differentiation and self-renewal of ESCs and TSCs. They point to lipid polydesaturation as a driver of differentiation in both cell types. Monounsaturated fatty acids (MUFAs), essential for ESCs are detrimental to TSCs.