Background: A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems. Results: We built a new lipid ontology (LION) that associates >50,000 lipid species to biophysical, chemical, and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including “plasma membrane", “headgroup with negative charge", "glycerophosphoserines", “above average bilayer thickness", and “below average lateral diffusion". A second validation was performed by analyzing the membrane fluidity of Chinese hamster ovary cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ("above average", "very high", and "high lateral diffusion" and "below average transition temperature"). Conclusions: The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research.

While heterogeneity is a key feature of cancer, understanding metabolic heterogeneity at the single-cell level remains a challenge. Here we present ¹³C-SpaceM, a method for spatial single-cell isotope tracing that extends the previously published SpaceM method with detection of ¹³C₆-glucose-derived carbons in esterified fatty acids. We validated ¹³C-SpaceM on spatially heterogeneous models using liver cancer cells subjected to either normoxia-hypoxia or ATP citrate lyase depletion. This revealed substantial single-cell heterogeneity in labelling of the lipogenic acetyl-CoA pool and in relative fatty acid uptake versus synthesis hidden in bulk analyses. Analysing tumour-bearing brain tissue from mice fed a ¹³C₆-glucose-containing diet, we found higher glucose-dependent synthesis of saturated fatty acids and increased elongation of essential fatty acids in tumours compared with healthy brains. Furthermore, our analysis uncovered spatial heterogeneity in lipogenic acetyl-CoA pool labelling in tumours. Our method enhances spatial probing of metabolic activities in single cells and tissues, providing insights into fatty acid metabolism in homoeostasis and disease.

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro. The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa^−/− mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). Dyslipidemia is associated with both excessive deposition of triacylglycerol (TAG) in lipid droplets (LDs) and lipotoxicity. Yet, it is unclear how these two effects correlate with each other in the kidney and how they are influenced by dietary patterns. By using a diabetes mouse model, we find here that high-fat diet enriched in the monounsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTCs) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). This effect was particularly evident in S2/S3 but not S1 segments of the proximal tubule. Combining transcriptomics, lipidomics, and functional studies, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. Mechanistically, ER stress is caused by elevated levels of saturated TAG precursors, reduced LD formation, and, consequently, higher membrane order in the ER. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and increasing both TAG and LD formation. Our study thus emphasizes the importance of monounsaturated fatty acids for the dietary management of DKD by preventing lipid bilayer stress in the ER and promoting TAG and LD formation in PTCs.

Lipid membranes are the border between living cells and their environments. The membrane's lipid composition defines fluidity, thickness, and protein activity and is controlled by the intricate actions of lipid gene-encoded enzymes. However, a comprehensive analysis of each protein's contribution to the lipidome is lacking. Here, we present such a comprehensive and functional overview of lipid genes in Escherichia coli by individual overexpression or deletion of these genes. We developed a high-throughput lipidomic platform, combining growth analysis, one-step lipid extraction, rapid LC-MS, and bioinformatic analysis into one streamlined procedure. This allowed the processing of more than 300 samples per day and revealed interesting functions of known enzymes and distinct effects of individual proteins on the phospholipidome. Our data demonstrate the plasticity of the phospholipidome and unexpected relations between lipid classes and cell growth. Modeling of lipidomic responses to short-chain alcohols provides a rationale for targeted membrane engineering.