Lysine Deprivation Suppresses Adipogenesis in 3T3-L1 Cells: A Transcriptome Analysis.

Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes' response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.

Hepatic Proteomic Analysis of Selenoprotein T Knockout Mice by TMT: Implications for the Role of Selenoprotein T in Glucose and Lipid Metabolism.

Selenoprotein T (SELENOT, SelT), a thioredoxin-like enzyme, exerts an essential oxidoreductase activity in the endoplasmic reticulum. However, its precise function remains unknown. To gain more understanding of SELENOT function, a conventional global Selenot knockout (KO) mouse model was constructed for the first time using the CRISPR/Cas9 technique. Deletion of SELENOT caused male sterility, reduced size/body weight, lower fed and/or fasting blood glucose levels and lower fasting serum insulin levels, and improved blood lipid profile. Tandem mass tag (TMT) proteomics analysis was conducted to explore the differentially expressed proteins (DEPs) in the liver of male mice, revealing 60 up-regulated and 94 down-regulated DEPs in KO mice. The proteomic results were validated by western blot of three selected DEPs. The elevated expression of Glycogen [starch] synthase, liver (Gys2) is consistent with the hypoglycemic phenotype in KO mice. Furthermore, the bioinformatics analysis showed that Selenot-KO-induced DEPs were mainly related to lipid metabolism, cancer, peroxisome proliferator-activated receptor (PPAR) signaling pathway, complement and coagulation cascades, and protein digestion and absorption. Overall, these findings provide a holistic perspective into SELENOT function and novel insights into the role of SELENOT in glucose and lipid metabolism, and thus, enhance our understanding of SELENOT function.

Universal Markers for hiPSCs Residue Detection.

BACKGROUND: Residual undifferentiated induced pluripotent stem cells (iPSCs) detection is essential for both Embryonic Stem Cells (ESCs) and iPSCs application in final cell therapy products. However, specific differentiated cells require specific genes for residual detection; identifying the suitable marker is costly and time-consuming. Thus, a universal marker for iPSCs residue detection for all three germline cells would greatly benefit PSC-derived cellular therapies. METHODS: Next-generation sequencing (NGS) was performed on total RNAs isolated from the iPSC cell lines and embryonic stem cells (H9), the top 30 expressed genes were selected as candidates. By analysis expression fold change comparing iPSC cells to the differentiated cells, seven genes were highly expressed in iPSCs but showed minimal background expression in differentiated cells. Tissue expression pattern of the candidate genes were explored in the Genotype-Tissue Expression (GTEx) project database, candidate genes were narrowed down to two genes. Spike-in experiments were performed to determine the detection limit and correlation with the number of iPSCs and gene expression by ddPCR. RESULTS: By next-generation sequencing (NGS), we identified two marker genes (ESRG and ZSCAN10) suitable for universal undifferentiated iPSC detection. Both ESRG and ZSCAN10 are highly expressed in iPSCs. ZSCAN10 is slightly expressed in the testis, pituitary, and cerebellum; ESRG is highly expressed in the vagina and scarcely expressed in the other tissues. Furthermore, the ddPCR method with a probe and primers for ESRG and ZSCAN10 detected a trace of undifferentiated hiPSCs to a spiked level of 0.0001%. CONCLUSIONS: These results suggest that targeting ESRG/ZSCAN10 transcripts is highly sensitive, quantitative, and could be broadly applied to quality control of almost all iPSC-derived cell therapy products.

Study on the bio-function of lipA gene in Aspergillus flavus.

Lipoic acid synthase (LipA) plays a role in lipoic acid synthesis and potentially affects the levels of acetyl-CoA, the critical precursor of tricarboxylic acid (TCA) cycle. Considering the potential effect of LipA on TCA cycle, whether the enzyme is involved in the growth and aflatoxin B1 (AFB1) biosynthesis, the significant events in Aspergillus flavus is yet known. The study was designed to explore the role of lipA gene in A. flavus, including growth rate, conidiation, sclerotia formation, and biosynthesis of AFB1. LipA coding lipoic acid synthetase was knocked out using homologous recombination. The role of lipA gene in A. flavus morphogenesis (including colony size, conidiation, and sclerotia formation) was explored on various media, and the bio-function of lipA gene in the biosynthesis of AFB1 was analyzed by thin layer chromatography analysis. The growth was suppressed in △lipA. The formation of conidia and sclerotia was also reduced when lipA gene was deleted. Moreover, AFB1 was down-regulated in ΔlipA compared with WT controls. LipA plays a role in the development of A. flavus and AFB1 biosynthesis, contributing to the full understanding of the lipA bio-function in A. flavus.