CONTEXT:Short-chain fatty acids (SCFAs) produced from dietary fiber fermentation can regulate adipose tissue metabolism through signaling pathways involving G protein-coupled receptors and histone deacetylase inhibition. While preclinical studies suggest they enhance thermogenesis, their role in human brown adipose tissue (BAT) under different thermal conditions remains unclear. OBJECTIVE:This study explores the associations between circulating SCFAs and human BAT metabolism at room temperature and after cold exposure. METHODS:This cross-sectional study included data from 71 adults (aged 20-55 years, body mass index 19-44 kg/m2). Dynamic [15O]O2, [15O]H2O, [¹⁸F]FDG, and [¹⁸F]FTHA positron emission tomography/computed tomography scans were used to assess BAT metabolism. Serum SCFAs were quantified using liquid chromatography-mass spectrometry, and gene expression in biopsy-excised BAT samples (n = 14) was analyzed. Participants were stratified into low- and high-BAT groups based on [¹⁸F]FDG or [¹⁸F]FTHA uptakes. RESULTS:Cold-induced acetate and propionate were positively associated with key in vivo BAT metabolism indicators, namely nonesterified fatty acid (NEFA) uptake and oxygen consumption. Only in the high-BAT group were circulating SCFAs maintained after cold exposure. BAT transcriptome revealed that genes involved in SCFA metabolism (such as conversion to acetyl-CoA) correlated with thermogenic and lipid metabolism genes exclusively in the high-BAT group, suggesting a distinct molecular link between SCFA pathways and BAT function. CONCLUSION:Circulating SCFAs are linked with BAT oxidative metabolism and NEFA uptake during cold exposure. The observed correlations between SCFA catabolic genes and thermogenic markers suggest that metabolically active BAT may selectively engage SCFA-related pathways, pointing to a potential mechanistic role of SCFAs in supporting BAT function in humans.

Resting metabolic rate (RMR) is modulated by a variety of factors. Accurate prediction of RMR is essential for planning energy requirements but remains challenging due to interindividual variability. This study aimed to develop and evaluate machine learning models for predicting RMR using comprehensive data from the cross-sectional enable study and to identify the most predictive and stable features across different study populations. RMR was predicted using data from 454 participants of the enable phenotyping platform (Freising and Nuremberg cohort). We systematically compared linear and nonlinear machine learning models trained on either the full set of 94 predictors or a reduced set of routinely accessible variables, including sex, age, body weight, fat mass, and fat-free mass. Model performance was assessed by cross-validation. The best-performing model (Lasso) was further evaluated on independent test datasets from other cohorts. Feature importance and stability were assessed using repeated cross-validation and marginal variance decomposition. Lasso regression consistently outperformed other models, particularly when trained on the enable cohort feature set. The final model explained 76.8% of RMR variance in the Freising cohort. Key predictive features included fat-free mass, body weight, and mean outdoor temperature. Blood-based features contributed marginally, whereas microbiota and fecal short-chain fatty acids variables did not contribute to explaining RMR. This novel prediction model for RMR shows improved accuracy in comparison with traditional models. Although microbiota composition did not contribute to explain the residual variation in RMR, the inclusion of clinical blood parameters and outdoor temperature improved predictive performance. Clinical Trial Registry Number: DRKS00009797.NEW & NOTEWORTHY We introduce a novel machine learning framework for predicting resting metabolic rate (RMR), emphasizing the superior performance of Lasso regression. Our analysis incorporates both standard clinical variables and previously underexplored factors such as gut microbiota, fecal short-chain fatty acids (SCFAs), and mean outdoor temperature.

Bats are the only mammals capable of powered flight, allowing them to cover relatively long distances in a short time. However, the general inability of mammals to fuel endurance exercise solely by oxidizing fatty acids may prevent bats from undertaking long-distance intercontinental migrations-like birds do. Here, we conducted untargeted metabolomics to reveal the oxidative fuels used by wild caught Nathusius' pipistrelles. We investigated polar metabolites and lipids in whole blood from bats flying under controlled wind tunnel or field conditions and how metabolites respond to the physiological challenge. Around 70% of detected acyl carnitines were significantly elevated after flight in the wind tunnel compared to resting bats. The phospholipid levels varied; some increased while others decreased significantly after flight, and most did not return to resting levels within 1 h of recovery. During migration season, we observed a significant increase of phosphatidylethanolamines with unsaturated fatty acids and a bulk increase of several phosphatidylcholines and their lyso-derivatives. While migration had a clear effect on phospholipids, recovering after flight in both seasons was less pronounced and only 24% of acyl carnitines were increased after 1 h of rest. We conclude that endurance exercise such as migration has a greater influence on lipid composition and their abundance than short flights, which indicates a relatively high relevance of fatty acid oxidation to fuel migration in bats.

Circulating branched-chain amino acids (BCAAs) are linked with insulin resistance, but the human tissues contributing to systemic BCAA homeostasis remain incompletely defined. Brown adipose tissue (BAT) is a metabolically active adipose depot associated with favourable insulin sensitivity, yet its role in BCAA metabolism in humans remains unclear. We tested whether human BAT metabolism is associated with circulating BCAA levels, BAT-resident BCAA-catabolic signatures, and longitudinal changes in systemic BCAA homeostasis. We studied 83 adults who underwent metabolic phenotyping, PET-CT assessment of cold-stimulated BAT metabolism, and serum metabolomic profiling at room temperature and during acute mild cold exposure. Supraclavicular BAT biopsies from 25 participants were analysed by transcriptomics and metabolomics, and 40 participants were re-examined for circulating BCAA profiles after approximately five years. Participants with high BAT metabolism had lower circulating BCAA levels than those with low BAT metabolism. Within BAT, metabolically active individuals exhibited lower relative BCAA abundance together with higher expression of genes involved in BCAA catabolism. These BAT BCAA-catabolic signatures aligned with thermogenic capacity and indices of systemic insulin sensitivity. In contrast, individuals with low BAT metabolism showed increases in circulating BCAAs over five years. Integrative analyses further linked circulating lipopolysaccharide, a marker of metabolic endotoxemia, with higher BAT BCAA and aminomalonate abundance, together with transcriptional patterns involving inflammatory and mitochondrial pathways. Together, these findings identify human BAT metabolism as a tissue phenotype linked to systemic BCAA homeostasis and extend the role of human BAT beyond thermogenesis, suggesting that BAT-associated BCAA handling may contribute to systemic metabolic health.

The gut microbiota influences host metabolism, but the mechanisms of lipid uptake from food remain mysterious. Here we used stable isotope-labelled tracers in gnotobiotic mouse models, which revealed that host uptake of dietary lipids depends on microbial colonization. Systemic lipid metabolism modelling predicted that the gut microbiota restricts intestinal lipid absorption, and labelled lipid administration verified that the gut contents of microbiota-colonized mice contained up to 12-fold more lipids than those of germ-free animals. A combination of lipidomics and proteomics showed that gut microbes trigger Myd88 signalling, leading to a downregulation of hepatic Cyp7b1 activity and increased taurocholate production. Taurocholate stimulates phospholipase A1 activity in bile, causing the degradation of phosphatidylcholine that is essential for luminal micelle formation and lipid uptake. A diverse microbiome was associated with lower phosphatidylcholine content. This previously unrecognized host-gut microbiota interplay via enzymes in bile could provide future targets to modulate dietary lipid absorption.

OBJECTIVES:Brown adipose tissue (BAT) dissipates energy via non-shivering thermogenesis, but durable thermogenic benefit requires sustained cold remodeling that stabilizes a cold-adapted tissue state. While most studies have focused on adipocyte-intrinsic pathways that drive acute activation, how stromal niche cells-particularly the vasculature-sense and coordinate long-term adaptation remains poorly defined. Because GPCRs are key sensors of extracellular and neurohumoral cues, we mapped GPCR expression across mouse and human BAT at single-nucleus resolution and identified adhesion GPCRs as a prominent family enriched in vascular cells, with endothelial ADGRF5(GPR116) emerging as a leading candidate regulator. METHODS:Single-nucleus RNA sequencing of mouse and human BAT was used to map GPCR expression across cell types. Global, inducible endothelial-specific, and adipocyte-specific ADGRF5(GPR116) knockout mouse models were each challenged with acute and prolonged cold exposure. Endothelial and adipocyte states were analyzed using single-nucleus RNA sequencing transcriptional profiling, functional vascular assays, and cell-cell communication modeling. RESULTS:Endothelial deletion of ADGRF5(GPR116) impaired the ability of mice to sustain thermogenesis during prolonged cold exposure, whereas adipocyte-specific deletion did not affect thermogenic capacity in vivo. Loss of endothelial ADGRF5(GPR116) did not alter endothelial cell abundance, but induced endothelial transcriptional reprogramming characterized by disrupted quiescent remodeling programs, shifts in endothelial state with EndMT-like features, and context-dependent alterations in barrier-associated pathways, occurring in the absence of immune cell infiltration or overt fibrosis. Adipocyte reclustering revealed a failure to acquire a fully cold-adapted thermogenic state, with thermogenically inefficient programs and adrenergic hyporesponsiveness, despite preserved sympathetic input. CellChat and NicheNet analyses predicted altered endothelial-derived paracrine signaling capable of reshaping adipocyte identity. CONCLUSIONS:Endothelial ADGRF5(GPR116) is a critical regulator of vascular adaptation during sustained cold exposure and supports full acquisition of the thermogenic adipocyte state through endothelial identity and paracrine signaling.

Adipose tissue plasticity and functional heterogeneity play a central role in maintaining energy homeostasis, and their malfunction leads to metabolic disorders such as obesity, diabetes, and cardiometabolic disease. Rapid, single-cell metabolic imaging of intact fat tissue not only extends our understanding of metabolic dynamics and heterogeneity but also holds great potential as a tool for clinical diagnosis. However, the use of exogenous labels and dyes in conventional optical microscopy results in tissue deformation and requires time-consuming tissue preparation. Here, we demonstrated single-cell imaging of metabolic changes and heterogeneity in freshly excised adipose tissues that can distinguish tissue types without the need for exogenous labels using bond-specific, non-destructive, mid-infrared optoacoustic microscopy (MiROM) that allows preserving the native tissue architecture with minimal sample preparation time. Further leveraging MiROM, we monitored intracellular molecular and morphological changes during postnatal remodeling of adipose tissue when metabolic characteristics of adipocytes undergo a transient drastic change. Additionally, we developed a quantitative spatial tissue analysis tool (Q-SAT) to predict the spatial distribution of white fat- and brown fat-like features, providing a robust digital scoring method for adipose tissue phenotypic assessment. Collectively, we implemented MiROM as an enabling technology to provide fast, label-free metabolic imaging of unprocessed adipose tissue, opening a new perspective for understanding and characterizing the morpho-functional dynamics of adipose tissue remodeling.

Indirect calorimetry is a cornerstone technique for metabolic phenotyping of animal models in preclinical research, with well-established experimental protocols and platforms. However, a flexible, extensible, and user-friendly software suite that enables standardized integration of data and metadata from diverse metabolic phenotyping platforms—followed by unified statistical analysis and visualization—remains absent. We present Shiny-Calorie, an open-source interactive web application for transparent data and metadata integration, comprehensive statistical data analysis, and visualization of indirect calorimetry datasets. Shiny-Calorie is compatible with data formats from widely used commercial metabolic phenotyping platforms, such as TSE and Sable Systems, and includes functionality for exporting processed data in these formats. Built using GNU R and a Shiny-based reactive interface, Shiny-Calorie enables intuitive exploration of complex, multi-modal longitudinal datasets comprising categorical, continuous, ordinal, and count variables. The platform incorporates state-of-the-art statistical methods for robust hypothesis testing, thereby facilitating biologically meaningful interpretation of energy metabolism phenotypes, including resting metabolic rate and energy expenditure. Overall, Shiny-Calorie streamlines routine analysis workflows and enhances reproducibility and transparency in metabolic phenotyping studies. ### Competing Interest Statement The authors have declared no competing interest.

Cold exposure stimulates the sympathetic nervous system (SNS) to activate brown fat thermogenesis and maintain optimal body temperature, while simultaneously triggering compensatory hyperphagia to restore energy balance. The mechanisms coordinating energy expenditure and intake, however, remain unclear. Here, we reveal that the hypothalamic-pituitary-adrenal (HPA) axis plays a dual role in this process: endogenous adrenocorticotropic hormone (ACTH) directly stimulates the melanocortin-2 receptor (MC2R) in brown adipocytes to promote thermogenesis, whereas glucocorticoids drive cold-induced hyperphagia and act permissively to enhance ACTH-mediated energy expenditure. These findings uncover previously unrecognized functions of the HPA axis and a delicate hormonal interplay that orchestrates energy homeostasis during cold stress. Targeting these pathways may offer novel strategies to mitigate hyperphagic responses associated with increased energy expenditure, with potential implications for obesity treatment. ![Figure][1] Highlights ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, DFG-TRR 333/1-450149205, DFG-Emmy Noether program-441904031, KL 973/20-1, #532683878, SCHN 1696/1-1, #532683878, FI 1546/8-1, #532683878 European Research Council, ERC starting grant: 101078516 National Natural Science Foundation of China (NSFC), 32330012 [1]: pending:yes

Increasing energy expenditure in brown adipose (BAT) tissue by cold-induced lipolysis is discussed as a potential strategy to counteract imbalanced lipid homeostasis caused through unhealthy lifestyle and cardiometabolic disease. Yet, it is largely unclear how liberated fatty acids (FA) are metabolized. We investigated the liver and BAT lipidome of mice housed for 1 week at thermoneutrality, 23 degrees C and 4 degrees C using quantitative mass spectrometry-based lipidomics. Housing at temperatures below thermoneutrality triggered the generation of phosphatidylethanolamine (PE) in both tissues. Particularly, the concentrations of PE containing polyunsaturated fatty acids (PUFA) in their acyl chains like PE 18:0_20:4 were increased at cold. Investigation of the plasma's FA profile using gas chromatography coupled to mass spectrometry revealed a negative correlation of PUFA with unsaturated PE in liver and BAT indicating a flux of FA from the circulation into these tissues. Betaadrenergic stimulation elevated intracellular levels of PE 38:4 and PE 40:6 in beige wildtype adipocytes, but not in adipose triglyceride lipase (ATGL)-deficient cells. These results imply an induction of PE synthesis in liver, BAT and thermogenic adipocytes after activation of the beta-adrenergic signaling cascade.

Mitochondrial abnormalities drive subcutaneous white adipose tissue dysfunction in obesity, yet in vitro models to study adipocyte mitochondria remain limited. Here, we establish a human subcutaneous adipocyte spheroid model to characterize mitochondrial metabolism under obesity-relevant conditions and drug exposure. Human preadipocyte spheroids were differentiated in ultra-low attachment plates for 3 weeks using thiazolidinedione-free medium. Matrigel embedding was incorporated into the protocol as it promoted mitochondrial network and respiration compared to scaffold-free conditions. Differentiated spheroids showed increased lipid accumulation, adipogenic gene expression, mitochondrial respiration, adiponectin secretion, and hormonal responsiveness. Lipid mixture administration during differentiation induced metabolic disturbances, including mitochondrial respiration failure alongside increased mitochondrial biogenesis. Post-differentiation treatment with rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, improved mitochondrial bioenergetics and adiponectin secretion in lipid mixture-administered adipocyte spheroids. Our model enables precise measurement of adipocyte mitochondria metabolism, providing a platform for mitochondria-focused research and drug discovery in obesity.

Motivation:The availability of bulk-omic data is steadily increasing, necessitating collaborative efforts between experimental and computational researchers. While software tools with graphical user interfaces (GUIs) enable rapid and interactive data assessment, they are limited to pre-implemented methods, often requiring transitions to custom code for further adjustments. However, the most available tools lack GUI-independent reproducibility such as direct integration with R, resulting in very limited support for transition. Results:We introduce the customizable Omics Analysis and reporting tool-cOmicsArt. cOmicsArt aims to enhance collaboration through integration of GUI-based analysis with R. The GUI allows researchers to perform user-friendly exploratory and statistical analyses with interactive visualizations and automatic documentation. Downloadable R scripts and results ensure reproducibility and seamless integration with R, supporting both novice and experienced programmers by enabling easy customizations and serving as a foundation for more advanced analyses. This versatility also allows for usage in educational settings guiding students from GUI-based analysis to R Code. Availability and implementation:cOmicsArt is freely available at https://shiny.iaas.uni-bonn.de/cOmicsArt/. User documentation is available at https://icb-dcm.github.io/cOmicsArt/. Source code is available at https://github.com/ICB-DCM/cOmicsArt. A docker available from https://hub.docker.com/r/pauljonasjost/comicsart/tags. A snapshot upon publication available from https://zenodo.org/records/14907620. A screen recording of cOmicsArt is available at: https://www.youtube.com/watch?v=pTGjtIYQOakp.

Motivation:Indirect calorimetry is the standard method for metabolic phenotyping of animal models in pre-clinical research, supported by mature experimental protocols and widely used commercial platforms. However, a flexible, extensible, and user-friendly software suite that enables standardized integration of data and metadata from diverse metabolic phenotyping platforms-followed by unified statistical analysis and visualization-remains absent. Results:We present Shiny-Calorie, an open-source interactive application for transparent data and metadata integration, comprehensive statistical data analysis, and visualization of indirect calorimetry datasets. Shiny-Calorie supports the majority of standard data formats across commercial metabolic phenotyping platforms, such as TSE and Sable Systems, COSMED platform and CLAMS/Columbus instruments, and provides export functionality of processed data into standardized formats. Built using GNU R with a reactive interface, Shiny-Calorie enables intuitive exploration of complex, multi-modal longitudinal datasets comprising categorical, continuous, ordinal, and count variables. The platform incorporates state-of-the-art statistical methods for robust hypothesis testing, thereby facilitating biologically meaningful interpretation of energy metabolism phenotypes, including resting metabolic rate and energy expenditure. Together, these features, streamline routine analysis workflows and enhances reproducibility and transparency in metabolic phenotyping studies. Availability and implementation:Shiny-Calorie is freely available at https://shiny.iaas.uni-bonn.de/Shiny-Calorie/. User documentation and source code are available at https://github.com/ICB-DCM/Shiny-Calorie. A docker image is available from https://hub.docker.com/r/stephanmg/Shiny-Calorie. Instructional screen recordings are available on https://www.youtube.com/@shiny-calorie.

Understanding the complex factors influencing mammalian metabolism and body weight homeostasis is a long-standing challenge requiring knowledge of energy intake, absorption and expenditure. Using measurements of respiratory gas exchange, indirect calorimetry can provide non-invasive estimates of whole-body energy expenditure. However, inconsistent measurement units and flawed data normalization methods have slowed progress in this field. This guide aims to establish consensus standards to unify indirect calorimetry experiments and their analysis for more consistent, meaningful and reproducible results. By establishing community-driven standards, we hope to facilitate data comparison across research datasets. This advance will allow the creation of an in-depth, machine-readable data repository built on shared standards. This overdue initiative stands to markedly improve the accuracy and depth of efforts to interrogate mammalian metabolism. Data sharing according to established best practices will also accelerate the translation of basic findings into clinical applications for metabolic diseases afflicting global populations. The authors highlight inconsistencies and divergencies in the literature reporting data on indirect calorimetry for studies on whole-body energy homeostasis, and propose harmonization of standards to facilitate data comparison and interpretation across different datasets.

Objective Brown adipose tissue (BAT) dissipates energy via non-shivering thermogenesis, and it is a promising therapeutic target for metabolic disease. While most research focuses on thermogenic adipocytes, emerging data point to critical contributions from the surrounding stromal niche. Here, we investigated the role of adhesion G protein–coupled receptors (aGPCRs) in BAT function, focusing on Adgrf5 (Gpr116), a receptor enriched in endothelial cells. Methods We used single-nuclei RNA sequencing to map aGPCRs expression across mouse and human BAT. We then examined the consequences of Adgrf5(Gpr116) loss using global, brown adipocyte, and endothelial-specific knockout mouse models under acute and prolonged cold exposure. Results Inducible endothelial deletion of Adgrf5(Gpr116) impaired the maintenance of thermogenic capacity during prolonged—but not acute—cold exposure. This was not associated with defective angiogenesis, but rather with endothelial fibro-inflammatory reprogramming. Single-nuclei RNA sequencing analysis revealed endothelial-to- mesenchymal transition (EndMT) features, including induction of mesenchymal markers, collagens, and metalloproteinases, and loss of barrier genes. Adgrf5(Gpr116)-deficient endothelial cells also exhibited cytoskeletal remodeling and activation of stress fiber pathways, implicating Adgrf5(Gpr116) as a mechanosensory safeguard of endothelial identity. Conclusion Endothelial Adgrf5(Gpr116) preserves thermogenic competence in BAT by suppressing EndMT and maladaptive matrix remodeling. Our findings establish vascular mechanosensing as a critical determinant of thermogenic tissue homeostasis. Highlights ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 450149205-TRR333/1 to S.H and A.G (Project P03)

Despite the multifaceted role of leptin for energy homeostasis and its broad therapeutic potential, the FDA/EMA-approved metreleptin constitutes the only leptin drug to date. To translate the promising results from previous studies on murine PASylated leptin with improved solubility and extended plasma half-life using PASylation technology─a biological alternative to PEGylation─we have developed a second-generation human leptin drug candidate and tested it rigorously in vitro and in vivo. To this end, the exposed hydrophobic Trp residue at position 100 in human leptin was replaced by Gln, which, together with the genetic fusion with a 600-residue PAS polypeptide, yielded a protein with high solubility, folding stability and receptor-stimulatory activity. In a pharmacokinetic (PK) study with wild-type mice, this modified human leptin showed an extended plasma half-life of 18.8 ± 3.6 h after subcutaneous (s.c.) injection. Furthermore, leptin-deficient mice were dosed s.c. with the modified human leptin carrying two different PAS fusion tags, PAS#1 or P/A#1, each comprising 600 residues. After only four doses, the disease phenotype, including morbid adiposity, hyperphagia, and hepatic steatosis, was completely reversed by both PASylated leptin versions, but not by the non-PASylated leptin if administered at the same dose. To assess its tissue distribution, P/A(200)-huLeptinW100Q was doubly labeled with two fluorescent dyes, which were specifically attached to the leptin and the PAS moiety, respectively. Analysis of relevant mouse organs by light sheet fluorescence microscopy after clearance revealed colocalized signals in the kidney and liver, thus indicating general stability of the PAS-leptin fusion protein in vivo. However, discrete signals were observed in the hypothalamic region, only with leptin detectable in the choroid plexus, which implies cleavage of the PAS tag during transcytosis across the physiological barriers. This study should pave the way toward a second-generation leptin drug enabling prolonged dosing intervals.