Heightened sterile inflammation and mitochondrial metabolic dysfunction drives the pathophysiology of heart failure in ischemic cardiomyopathy. Yet, the transcriptional regulators within cardiomyocytes driving crosstalk between inflammation and energy metabolism remain ill-defined. Here we identify elevated Ser396/Ser398 phosphorylation of the type I interferon (IFN) response regulating transcription factor IRF3 in the myocardium of patients and male mice with ischemic cardiomyopathy. Cardiomyocyte-specific IRF3 deficiency attenuates ischemia induced contractile dysfunction. Conversely, IRF3 activation in cardiomyocytes through a phosphomimetic IRF3 mutant represses Ppargc1α expression leading to dysfunctional mitochondrial oxidative phosphorylation, altered metabolic flux in the pentose phosphate pathway/TCA cycle, impaired NAD metabolism and an excessive type I IFN activation, collectively detrimental for cardiac function. Restoring cardiomyocyte-specific Ppargc1α expression in IRF3-overexpressor male mice attenuates contractile dysfunction by augmenting a metabolic shift towards fatty acid oxidation and decreasing inflammatory fibrotic responses. These findings identify IRF3 activation in cardiomyocytes as a transcriptional nexus between cardiac inflammation and metabolic fuel switch contributing to heart failure progression.

Bile acids (BAs) play an important role in systemic metabolic improvements following bariatric surgery. In this study, we found that orally administered norursodeoxycholic acid (norUDCA), a conjugation-resistant C23 derivative of naturally occurring UDCA, accumulated in peripheral organs including heart and brown adipose tissue (BAT). Moreover, norUDCA decreased systemic levels of endogenous conjugated BAs, while increasing unconjugated BAs. Notably, in addition to beneficial effects in a cholestatic liver disease model, norUDCA also lowered plasma glucose and fat mass in mice, suggesting that this BA derivative could be repurposed for treating obesity-associated cardiometabolic diseases. Metabolic energy expenditure studies, however, revealed that norUDCA-treated mice have impaired BAT capacity and developed intolerance to cold stress, a phenotype exacerbated in mice lacking adipose ATGL-dependent lipolysis. Transcriptomic and metabolic analyses demonstrated tissue remodeling in heart and BAT that involved pronounced changes in energy substrate utilization, including enhanced cardiac glucose uptake and higher ketone body utilization in BAT. Importantly, co-administration of a low-carb diet prevented cold stress-induced metabolic deficits. Mechanistic studies in human engineered heart tissue indicated that norUDCA compromised contractile function. In conclusion, these data suggest that conjugation- resistant BA derivatives like norUDCA impair myocardial and BAT energetics by altering glucose, lipid, and energy metabolism, particularly during catabolic cold stress conditions.

Accurate quantification of bile acids is vital as they are potential biomarkers for several diseases. To solubilize bile acids from tissues, homogenization is required, typically performed using mechanical methods such as the TissueLyser method. Drawbacks like available tissue amount and low abundance of bile acids interfere with the analysis. We aim to establish a nanosecond infrared laser (NIRL) as a possible tissue homogenization tool to circumvent problems associated with limited sample amounts for research and to target low abundance bile acids. We quantify bile acids from mouse and human liver tissues comparing both the classic TissueLyser and the NIRL methods for homogenization prior to LC-MS/MS analysis. The applicability of our approach is tested using mice lacking Cyp2c70, a well-established mouse model with an altered bile acid composition compared to wild type mice. We found the NIRL method to require an almost 14 times smaller starting tissue amount than the TissueLyser method. The NIRL is also comparable to the TissueLyser for high abundance bile acids. While the TissueLyser provides efficient mechanical homogenization, the NIRL potentially enables quantification of low abundance bile acids. The well-established biological differences in bile acid profiles from the Cyp2c70 knockout mouse model were also observed with NIRL homogenization. These results show that the NIRL is potentially useful as smaller tissue amounts are required for analysis and low abundance bile acids are quantifiable.

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.

Loss of brown adipose tissue (BAT) activity observed during ageing, obesity and living at thermoneutrality is associated with lipid accumulation, fibrosis and tissue inflammation in BAT. The mechanisms that promote this degenerative process of BAT remain largely enigmatic. Here, we show that an imbalance between sympathetic activation and mitochondrial energy handling causes BAT degeneration, which leads to impaired energy expenditure and systemic metabolic disturbances. Mechanistically, we demonstrate that brown adipocytes secrete ATP in response to imbalanced thermogenic activation, which activates P2X4 and P2X7 of BAT-resident macrophages. Notably, mice lacking activity of these purinergic receptors in myeloid cells are protected against BAT inflammation, thermogenic dysfunction and systemic metabolic disturbances under conditions of imbalanced BAT activation, thermoneutrality or overnutrition. These results highlight the relevance of extracellular ATP released by brown adipocytes as a paracrine signal for myeloid cells to initiate BAT degeneration.

Objective and methods: Brown adipose tissue (BAT) comprises a heterogeneous population of adipocytes and non-adipocyte cell types. To characterize these cellular subpopulations and their adaptation to cold, we performed single-nucleus mRNA-sequencing (snRNA-seq) on interscapular BAT from mice maintained at room temperature or exposed to acute (24h) or chronic (10 days) cold (6 °C). To investigate the role of the de novo lipogenesis (DNL)-regulating transcription factor carbohydrate response element-binding protein (ChREBP), we analyzed control and brown adipocyte-specific ChREBP knockout mice. Results: We identified different cell populations, including seven brown adipocyte subtypes with distinct metabolic profiles. One of them highly expressed ChREBP and DNL enzymes. Notably, these lipogenic adipocytes were highly sensitive to acute cold exposure, showing a marked depletion in BAT of control mice that was compensated by other brown adipocyte subtypes maintaining DNL. Chronic cold exposure resulted in an expansion of basal brown adipocytes and adipocytes putatively derived from stromal and endothelial precursors. In ChREBP-deficient mice, lipogenic adipocytes were almost absent under all conditions, identifying the transcription factor as a key determinant of this adipocyte subtype. Detailed expression analyses revealed Ttc25 as a specific marker of lipogenic brown adipocytes and as a downstream target of ChREBP. Furthermore, pathway and cell–cell interaction analyses implicated a Wnt–ChREBP axis in the maintenance of lipogenic adipocytes, with Wnt ligands from stromal and muscle cells providing instructive cues. Conclusions: Our findings provide a comprehensive atlas of BAT cellular heterogeneity and reveal a critical role for ChREBP in lipogenic adipocyte identity, with implications for BAT plasticity and metabolic function.

Brown adipose tissue (BAT) is a heat-generating organ burning significant amounts of calories from fatty acids and glucose. The importance of glucose metabolism in the context of thermogenic function has been underlined by several studies. However, fructose metabolism and consequences of fructose overfeeding are poorly studied in BAT. Here we provide evidence that brown adipocytes use fructose as a substrate, however to a lesser extent than glucose. Furthermore, our data suggest that carbohydrate response element binding protein (ChREBP) and its target glucose transporter 5 (GLUT5) are not essential for fructose uptake and metabolism in BAT. Notably, we report that high fructose feeding has no effect on ChREBP activity and thus de novo fatty acid synthesis in BAT as opposed to liver and intestine. Instead, excessive carbohydrate loading of brown adipocytes induced by both, high-fructose feeding and impairment of ChREBP-dependent glucose metabolism, causes a massive accumulation of hexosylceramide species, as revealed by mass spectrometry-based lipidomics. Based on our data we hypothesize a reprogramming of fructose utilization upon impaired carbohydrate metabolism from canonical glycolysis and pentose phosphate pathway towards glycosphingolipid synthesis.

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.

Brown adipose tissue (BAT) comprises a heterogeneous population of adipocytes and non-adipocyte cell types. To characterize these cellular subpopulations and their adaptation to cold, we performed single-nucleus mRNA-sequencing (snRNA-seq) on interscapular BAT from mice maintained at room temperature or exposed to acute (24h) or chronic (10 days) cold (6°C). To investigate the role of the de novo lipogenesis (DNL)-regulating transcription factor carbohydrate response element-binding protein (ChREBP), we analyzed control and brown adipocyte-specific ChREBP knockout mice. We identified different cell populations, including seven brown adipocyte subtypes with distinct metabolic profiles. One of them highly expressed ChREBP and DNL enzymes. Notably, these lipogenic adipocytes were highly sensitive to acute cold exposure, showing a marked depletion in BAT of control mice that was compensated by other brown adipocyte subtypes maintaining DNL. Chronic cold exposure resulted in an expansion of basal brown adipocytes and adipocytes putatively derived from stromal and endothelial precursors. In ChREBP-deficient mice, lipogenic adipocytes were almost absent under all conditions, identifying the transcription factor as a key determinant of this adipocyte subtype. Pathway and cell-cell interaction analyses implicated a Wnt-ChREBP axis in the maintenance of lipogenic adipocytes, with Wnt ligands from stromal and muscle cells providing instructive cues. Our findings provide a comprehensive atlas of BAT cellular heterogeneity and reveal a critical role for ChREBP in lipogenic adipocyte identity, with implications for BAT plasticity and metabolic function.

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

Objective Recombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. Methods To achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. Results We demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. Conclusion Taken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids – a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

During chronic liver disease, hepatocytes may undergo proliferative arrest, leading to the activation, expansion and differentiation of hepatic progenitor cells (HPCs). Here we observe that expression of A Disintegrin And Metalloprotease (ADAM) 10 is increased in human and murine chronic liver disease correlating with HPC expansion. We report that proteolytic processing of ADAM10 by ADAM9 and generation of an ADAM10 intracellular domain that translocates to the nucleus, rather than ADAM10 enzymatic activity is essential for the regulation of HPC gene expression and differentiation. Genetic loss of ADAM10 in vitro and in vivo enhances stemness gene expression, increases the accumulation of undifferentiated HPCs and promotes the formation of liver fibrosis. Taken together, we demonstrate that a non-catalytic function of ADAM10 is an essential regulator of HPC fate and HPC-driven regeneration. Our data ascribe a non-proteolytic function to ADAM proteases which may be a general concept in adult tissue stem cells. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVE:In brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive. METHODS:Using an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed. RESULTS:Deletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs. CONCLUSIONS:We identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor.

Disturbed cholesterol homeostasis is associated with multiple diseases, such as atherosclerotic cardiovascular disease, lysosomal storage disorders, and neurodegenerative disorders. The endo-lysosomal network plays a central role in the distribution of cholesterol between subcellular membranes, but the processes controlling this transport are still not well-defined. Here, we investigate the impact of hepatic Retromer, an endosomal sorting complex consisting of VPS35, VPS26, and VPS29, on cholesterol homeostasis by using a liver-specific VPS35-deficient ( Vps35 HepKO ) mouse model. Hepatic VPS35 deficiency strongly reduces the function of the lysosomal proteins lysosomal acid lipase (LAL), scavenger receptor class B member 2 (SCARB2), and Niemann-Pick type C1 (NPC1), concomitant with hepatic cholesterol accumulation in lysosomal compartments, delayed transport of endocytosed cholesterol through the endo-lysosomal network, and increased cholesterol biosynthesis. In addition, the levels and intracellular distribution of low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) are affected, likely explaining increased plasma LDL-cholesterol in Vps35 HepKO mice. High-fat/high-cholesterol diet feeding exacerbates hepatic and plasma cholesterol content in Vps35 HepKO mice and leads to mild liver injury, hepatic inflammation, and increased hepatocellular proliferation. Altogether, we here uncover that, in addition to its established function in the endosomal sorting of integral membrane proteins, Retromer plays a key role in lysosomal cholesterol egress.

Background and Aims: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co−expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine−rich protein 1 ( SMLR1 ), encoding for small leucine−rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. Approach and Results: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR−associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 −LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis−Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet−induced hyperlipidemia and atherosclerosis but causes NASH. On a high−fat, high−cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 −LKO mice versus controls. Conclusions: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis−Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.