Rethinking Microbiome Metabolites 

Isotope tracing shows that mammalian cells directly produce several circulating indole and phenol metabolites often attributed to the gut microbiome.

Many circulating indole and phenol metabolites commonly attributed to the gut microbiome can also be produced directly by mammalian cells, according to an isotope-tracing study that separated host and microbial contributions to their synthesis.

Using liquid chromatography-mass spectrometry, the researchers followed ^13C-labeled tryptophan, phenylalanine, and tyrosine through mice and rats, cultured human cells, and tissue lysates. Short intravenous infusions exposed host tissues to the tracers without delivering detectable labeled amino acids to the gut lumen, distinguishing rapidly formed host products from metabolites requiring microbial processing.

Longer infusions, germ-free and antibiotic-treated mice, and a digestion-resistant ^13C-labeled protein diet provided complementary tests of origin. Mammalian metabolism generated aryl-pyruvates, aryl-lactates, aryl-acetates, and aryl-carboxylic acids in physiologically relevant quantities, whereas aryl-propionates and the carbon skeletons of free indole, phenol, and p-cresol remained microbiome-dependent.

The experiments also began to define the host pathway. Cultured cells converted aromatic amino acids into pyruvate and lactate derivatives, while liver lysates generated additional downstream products. Proteomic analysis of fractionated lysates showed that GOT1 abundance closely tracked transaminase activity, and purified GOT1 converted tryptophan and phenylalanine into their corresponding aryl-pyruvates.

Human data supported the same division: host-derived compounds remained comparatively stable during antibiotic-associated disruption of the gut microbiota, while microbiome-dependent metabolites declined.

The authors argue that resolving metabolite origin is a necessary step toward understanding what controls circulating levels and how they might be manipulated therapeutically, while cautioning that microbiome studies must account for host production when interpreting indole and phenol measurements.

The Cell Types Behind the Secretome

An endoplasmic-reticulum TurboID platform traces secreted proteins back to specific cell types and follows how they change across metabolic states. 

Researchers at Rockefeller University have developed an in vivo proximity-labeling platform that traces secreted proteins back to specific cell types and follows how those signals change during fasting, inflammation, and obesity.

The team engineered mice to express TurboID, a promiscuous biotin ligase, within the endoplasmic reticulum. As secreted and membrane proteins passed through the ER, they were biotinylated, enriched with streptavidin, and quantified using tandem mass tag-based LC–MS/MS/MS.

Recovering low-abundance factors from a plasma proteome spanning roughly ten orders of magnitude presented a major analytical challenge. Refinements to sample processing, enrichment, mass spectrometry, and data analysis reduced missing values and pushed detection into the sub-nanomolar range.

“Without optimization, the platform identified only the most abundant proteins, which generally are the things people already know about,” said co-senior author Paul Cohen in RU’s press release. “It’s only by refining the technique that we were able to get deeper into the unknown areas of biology.”

Applied across metabolic states, the workflow revealed distinct remodeling of adipocyte- and hepatocyte-derived secretory proteomes during fasting and inflammation. Advanced obesity altered hundreds of proteins in subcutaneous and visceral fat, including established signals such as leptin and less studied candidates including γ-synuclein and the orphan receptor MTR1L.

Comparison with UK Biobank data linked 65 stress-responsive proteins to conditions including type 2 diabetes, cardiovascular disease, and sepsis. Although these associations do not establish causal roles, they highlight candidates for further investigation across metabolic and inflammatory disease.

“We’re hoping that it opens the door to building an organ interactome across the entire spectrum of health and disease,” said co-senior author Ekaterina Vinogradova.

A Proteomic Route to Cardiac Regeneration 

The study identifies a proteomic shift away from fatty acid oxidation and toward glycolysis in cardiomyocytes with regenerative potential. 

Single-cell proteomic analysis of nearly 650 adult mouse cardiomyocytes has linked Myc, a transcription factor known to promote cardiomyocyte proliferation, to metabolic reprogramming and the emergence of a pro-regenerative cell population.

Researchers at the Centro Nacional de Investigaciones Cardiovasculares combined optimized cardiomyocyte isolation with TMT-based LC–MS and the iSanXoT statistical framework. The workflow was designed to correct two persistent sources of distortion in single-cell proteomics: differences in cell size and batch effects introduced during multiplexed analysis.

The choice of isolation method proved critical. Fluorescence-activated sorting reduced signal intensity and obscured established Myc-related changes in fatty acid oxidation, whereas manually isolated cardiomyocytes produced more reproducible and biologically interpretable profiles. Using this approach, the team analyzed 323 control and 324 Myc-overexpressing cells, quantifying 1,624 proteins overall and approximately 440 proteins per cell.

The researchers then integrated protein abundance with information about subcellular localization, improving separation between control and Myc-expressing cardiomyocytes. Myc shifted metabolic protein expression away from fatty acid β-oxidation and toward glycolysis, consistent with a less mature cellular state.

Clustering identified five cardiomyocyte populations, including one strongly enriched in Myc-overexpressing cells. This group combined increased glycolysis and lactate production with reduced fatty acid oxidation, fetal myosin expression, and lower DNA content, collectively defining a pro-regenerative signature.

“Our results show that Myc expression alters the levels of metabolic enzymes differently in each individual cell, generating distinct states of cellular immaturity and giving rise to a subpopulation of cardiomyocytes with regenerative potential,” said first author Consuelo Marín-Vicente in a recent press release.

The findings provide a basis for testing whether this proteomic state directly supports cardiac repair and for refining Myc-based regenerative strategies.

Lipids at the Macrophage Front Line 

Imaging at cell-resolved scale separates macrophage phenotypes and identifies phospholipid metabolism as a major axis of variation. 

A cell-resolved MALDI imaging workflow has shown that antitumor M1 macrophages synthesize more phospholipids than tumor-promoting M2 cells, with inhibition experiments supporting a functional role for that metabolism.

The researchers used MALDI-MSI to map metabolites across resting, M1, and M2 macrophages cultured on conductive slides. Imaging at 20 μm spatial resolution, followed by principal component and segmentation analysis, separated the three phenotypes and highlighted lipid metabolism as a major source of variation.

Stable isotope tracing provided a dynamic view of those differences. MALDI imaging followed ^13C-labeled linoleic acid into newly synthesized phospholipids, while tandem MS confirmed its incorporation into representative lipid species. Linoleic acid uptake changed little between macrophage states, but M1 cells produced substantially more labeled phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, and phosphatidic acids than either resting or M2 cells.

That phospholipid signature changed upon exposure to MDA-MB-231 breast cancer cells. Across 24 hours of coculture, most newly labeled phospholipids declined progressively in M1 macrophages, whereas M2 cells showed more variable, lipid-specific responses.

Functional experiments supported a link between phospholipid metabolism and antitumor activity. Inhibiting cytosolic phospholipase A2 with pyrrophenone reduced expression of the M1 marker CD86 and weakened the ability of M1 macrophages to suppress tumor-cell viability in both two-dimensional cultures and three-dimensional spheroids.

The authors caution that the 20 μm laser spot provides cell-resolved rather than true single-cell measurements, while ion suppression may affect quantitative accuracy.

Newsletters

Receive the latest analytical science news, personalities, education, and career development – weekly to your inbox.

I have read and understand the Privacy Notice*

Subscribe