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SUMMARY Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferasemediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

Protein N-glycosylation stands out for its intrinsic and functionally related heterogeneity. Despite its biomedical interest, Glycoprofile analysis still remains a major scientific challenge. Here, we present an NMR-based strategy to delineate the N-glycan composition in intact glycoproteins and under physiological conditions. The employed methodology allowed dissecting the glycan pattern of the IgE high-affinity receptor (FcεRIα) expressed in human HEK 293 cells, identifying the presence and relative abundance of specific glycan epitopes. Chemical shifts and differences in the signal line-broadening between the native and the unfolded states were integrated to build a structural model of FcεRIα that was able to identify intramolecular interactions between high-mannose N-glycans and the protein surface. In turn, complex type N-glycans reflect a large solvent accessibility, suggesting a functional role as interaction sites for receptors. The interaction between intact FcεRIα and the lectin hGal3, also studied here, confirms this hypothesis and opens new avenues for the detection of specific N-glycan epitopes and for the studies of glycoprotein-receptor interactions mediated by N-glycans.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program