Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is comprised of tandem enzymatic thioesterase domains and a C-terminal lipid-binding (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and enhances catalytic activity, whereas lysophosphatidylcholine (LPC) binding has the opposite effect. Them1 forms biomolecular condensates (puncta) in brown adipocytes that diffuse in response to β-adrenergic stimulation with norepinephrine (NE), or by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). Diffuse Them1 enters the nucleus to regulate gene expression related to energy substrate production. This study aimed to determine the mechanism by which diffuse-state Them1 traffics to the nucleus. METHODS: cDNA constructs encoding full-length Them1, a phosphomimetic mutant (S15, S18, and S25 substituted with D; DDD) where Them1 is constitutively diffuse, a phospho-deficient mutant (S15, S18, and S25 substituted with A; AAA) where Them1 is constitutively in puncta, and DDD with additional deletions or loss-of-function mutations in the nuclear localization signal (NLS; aa 353-362) were cloned and fused to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal microscopy via EGFP and nuclei with NucSpot, and signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated with or without NE, PMA, aristolochic acid (phospholipase A2 inhibitor), leptomycin B (nuclear exit blocker), LPC, or edelfosine (non-metabolized LPC analog). Molecular dynamics (MD) simulations of Them1 with or without bound LPC were compared, and 95D, 96D and 268E were identified as potential key residues mediating LPC-dependent regulation. Homologous residues in Them1-DDD were mutated, cloned, and evaluated by live cell microscopy. RESULTS: Them 1 lacking the NLS or harboring mutations in the NLS did not enter the nucleus in iBAs. Although diffuse Them1 (DDD) showed some nuclear localization, treatment with LPC or edelfosine, but not other lipid species, significantly increased Them1 nuclear translocation. In contrast, Them1-AAA (in puncta), with or without LPC or edelfosine, showed no nuclear translocation. Stimulation with NE, but not PMA, led to Them1 nuclear trafficking; adding LPC to PMA-treated cells restored Them1 nuclear trafficking whereas inhibition of LPC synthesis prior to NE stimulation prevented Them1 nuclear trafficking. Because the NLS lies buried in the compact hinge region between the thioesterase and START domains, it is normally hidden. MD simulations identified key interactions between residues in the thioesterase and hinge region domains that were disrupted with LPC binding. Mutating these residues mimicked LPC binding, unmasked the NLS, and led to robust nuclear translocation of Them1. This event correlated with the down-regulation of mRNA expression of genes involved in lipid biosynthesis. CONCLUSIONS: Our data suggest that LPC, which is generated during thermogenesis, binds to the START domain of diffuse-state Them1, inducing a conformational change that disrupts interdomain interactions that normally sequester the NLS. LPC binding thus exposes the NLS, and enables the nuclear trafficking of diffuse Them1 with subsequent regulation of thermogenic genes. Supported by NIH DK103046 to DEC, SJH, and EAO. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Tropospheric ozone (O3) harms vegetation by reducing tree biomass and crop yield. Accurate risk assessment based on O3 uptake depends on quantifying the plant's capacity to cope with O3-generated reactive oxygen species (ROS) at the cellular level. Young leaves were shown to have better ability than mature leaves to deal with O3 exposure but the mechanisms behind remain unclear. The aim of this study was to assess the crosstalk between O3 response and leaf development. Time-course response to O3 concentrations (0, 80 and 100 ppb) was studied at transcriptome and cellular level in a growing leaf (GL) and an expanded leaf (EL) of young poplar trees. The emergence of hypersensitive response-like lesions and the loss of chlorophyll content induced by O3 was less pronounced in GL than EL showing a higher tolerance of GL to O3. The leaf transcriptome response to O3 involved many genes (15,784) mainly involved in leaf development resulting in a stress-induced acceleration of leaf aging and senescence. In addition to detoxification mechanisms, O3 tolerance at early stages of leaf development might result from an insensitivity to senescence induction though further validation is needed. Understanding this process could offer new perspectives for O3 tolerance improvement or assessment.

Dysregulated blood lipids are a major predictor of cardiovascular events. A recent genome-wide association study (GWAS) with five clinically relevant lipid traits in 1.65 million individuals implicated over 770 genomic regions in regulating blood lipid metabolism. To translate these associations into clinical applications, a functional understanding of their roles in lipoprotein metabolism, transport and remodeling (LPmtr) is required. Here, we report the deep molecular fine-mapping of 554 of these lipid risk loci using 168 lipoprotein-related traits and all possible ratios between them in over 273,000 participants of the UK Biobank. We identified new ratio-based markers of pathways shared by multiple LPmtr genes, such as the linoleic acid fraction of the polyunsaturated fatty acid pool to reveal potential causal genes at poorly characterized lipid risk loci, the percentage of esterified cholesterol moieties in LDL particles as a proxy for soluble LDL receptor levels, and the HDL fraction of total lipoprotein particle number as a predictor of incident myocardial infarction. We demonstrate how lipoprotein fine-mapping can generate new hypotheses for drug target development while uncovering new mechanisms relevant to hyperlipidemia. Ratio-driven clustering further implicated miR-148 in TG secretion, linking ER-stress responses at postprandial state to VLDL metabolism via mTORC1, shown through series of integrated cellular assays and mouse studies. Moreover, consistent with its regulatory influence on lipid flux we identify miR-148a a previously unrecognized determinat of Lp(a) levels. Our study implements a novel approach of using metabolomic data to follow-up on genetic evidence from GWAS with clinical traits and generates new insights into the biology of lipoprotein particles, supporting the emerging view that assessing lipoprotein size and composition is essential for the understanding, prevention, and treatment of lipid-related disorders.

Though incidence and mortality of colorectal cancer (CRC) in individuals older than 50 (late-onset, LO-CRC) have been declining over the last 30 years, they have been increasing among adults less than 50 (early onset, EO-CRC), constituting a significant public health concern. Detailed molecular comparison of EO-CRC to LO-CRC tumors has thus far been limited to whole-exome sequencing and confounded by the complex tumor immune microenvironment (TME), with significant presence of non-tumor cells. Here, we present integrated comparison of EO- and LO-CRC by single-cell RNA-Sequencing (scRNA-Seq) and multi-plex immuno-fluorescence (mIF). We combine public data with in-house clinical samples to comprise a rich resource of scRNA-Seq on 43, 000 cells from 11 EO-CRC tumors and 19 LO-CRC tumors and perform mIF on a tissue microarray of 50 EO- and 50 LO-CRC samples, focusing on major immune cell subsets. Our ScRNA-Seq analysis utilizes an algorithm that leverages gene regulatory networks for Virtual Inference of Protein Activity by Enriched Regulon (VIPER) to cluster cell populations in the TME, identify cell types over-represented in EO-CRC (by Fisher’s Exact Test), and characterize regulatory proteins aberrantly activated in those cell types. By this approach, we have found small, but statistically significant elevation in fibroblasts (p=0.042), endothelial cells (p=0.025), and B-cells (p=0.042) in EO-CRC compared with LO-CRC, though no difference in T-cells. Further, scRNA-Seq VIPER analysis of tumor cells demonstrates a sub-phenotype conserved across patients and significantly over-represented in EO-CRC (p=0.028), marked by up-regulation of TLR4, C5AR2, LILRB4, PTPRE, and CCR5 (proteins known to play a role in immune signaling and inflammation) – and with specific activity of druggable proteins EPHB4, FGFR4, and IKBKB. Overall, we find that EO-CRC is transcriptionally similar to LO-CRC and is composed of a similar cellular milieu. mIF validates no differences in T-cell subsets but is unable to confirm increase in endothelial cells in EO-CRC found in our scRNA-Seq data set. Our finding of similar T-cell density is of particular interest and helps dispel the notion that EO-CRC is more immune “cold” than sporadic CRC in older cohorts. Most strikingly, we find that EO-CRC is marked by a particular cluster of TLR4-high tumor cells. It has previously been found that high fat diets can lead to increased CRC growth in a TLR4-dependent manner in murine models – thus suggesting a link between a known environmental risk factor and EO-CRC. Work is ongoing to validate TLR4-high tumor cells in human tissues and patient-derived organoids (PDOs). Further studies will utilize PDO as an in vitro model for drug screens targeting aberrantly active druggable protein targets in EO- CRC tumors and to further explore the mechanisms that contribute to development of EO-CRC. Multi-omics comparison of early vs late-onset colorectal cancer reveals distinct tumor phenotype of early-onset patients. Multi-omics comparison of early vs late-onset colorectal cancer reveals distinct tumor phenotype of early-onset patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3442.

Early onset colorectal cancer (EO-CRC) is growing steadily, despite increased efforts in screening and early detection. Although genomically similar to average-onset colorectal cancer (AO-CRC), younger patients face distinct clinical, psychosocial and economic challenges. While the general guidelines for detection and treatment are the same across age groups, we aim here to describe some of the unique challenges faced by younger patients. These include diagnostic delays, fertility concerns, and unique emotional and financial burdens. In this review, we explore the multidimensional needs of EO-CRC patients, including barriers to early detection, the impact of treatment on reproductive health and relationships, and the long-term financial and psychological toll. Addressing these challenges requires a tailored, multidisciplinary approach that goes beyond standard oncologic care.

Activation of thermogenic brown adipose tissue (BAT) and inducible beige adipose tissue (BeAT) is triggered by environmental or metabolic stimuli, including cold ambient temperatures and nutrient stress. Thioesterase superfamily member 1 (Them1), a long-chain fatty acyl-CoA thioesterase that is enriched in BAT, suppresses acute cold-induced thermogenesis. Here, we demonstrate that Them1 expression was induced in BAT and BeAT by the carbohydrate response element binding protein (ChREBP) in response to chronic cold exposure or to the activation of the integrated stress response (ISR) by nutrient excess. Under either condition, Them1 suppressed energy expenditure. Consequently, mice lacking Them1 in BAT and BeAT exhibited resistance to obesity and glucose intolerance induced by feeding with a high-fat diet. During chronic cold exposure or ISR activation, Them1 accumulated in the nucleus, where it interacted with ChREBP and reduced the expression of its target genes, including those encoding enzymes that mediate glycolysis and de novo lipogenesis. These findings demonstrate that in response to chronic cold- or nutrient-induced stress, the induction of Them1 by ChREBP limits thermogenesis while coordinately reducing glucose utilization and lipid biosynthesis through its distinct cytoplasmic and nuclear activities. Targeted inhibition of Them1 could be a potential therapeutic approach to increase the activity of BAT and BeAT to enhance energy expenditure in the management of obesity-associated metabolic disorders.

Diastolic dysfunction is increasingly common in preterm infants exposed to supplemental oxygen (hyperoxia). Previous studies in neonatal mice showed hyperoxia suppresses fatty acid synthesis genes required for proliferation and survival of atrial cardiomyocytes. The loss of atrial cardiomyocytes creates a hypoplastic left atrium that inappropriately fills the left ventricle during diastole. Here, we show that hyperoxia stimulates adenosine monophosphate-activated kinase (AMPK) and peroxisome proliferator activated receptor-gamma (PPARγ) signaling in atrial cardiomyocytes. While both pathways can regulate lipid homeostasis, PPARγ was the primary pathway by which hyperoxia inhibits fatty acid gene expression and inhibits proliferation of mouse atrial HL-1 cells. It also enhanced the toxicity of hyperoxia by increasing expression of activating transcription factor (ATF) 5 and other mitochondrial stress response genes. Silencing PPARγ signaling restored proliferation and survival of HL-1 cells as well as atrial cardiomyocytes in neonatal mice exposed to hyperoxia. Our findings reveal PPARγ enhances the toxicity of hyperoxia on atrial cardiomyocytes, thus suggesting inhibitors of PPARγ signaling may prevent diastolic dysfunction in preterm infants.

Thioesterase superfamily member 2 (Them2), a long-chain fatty acyl-CoA thioesterase that is highly expressed in oxidative tissues, interacts with phosphatidylcholine transfer protein (PC-TP) to regulate hepatic lipid and glucose metabolism and to suppress insulin signaling. High-fat diet-fed mice lacking Them2 globally or specifically in skeletal muscle, but not liver, exhibit reduced hepatic steatosis and insulin resistance. Here, we report that the capacity of Them2 in skeletal muscle to promote hepatic steatosis and insulin resistance depends on both its catalytic activity and interaction with PC-TP. Two residues of Them2 catalytic site were mutated (N50A/D65A) to produce the inactive enzyme while maintaining its homotetrameric structure and interaction with PC-TP. Restoration of skeletal muscle expression in Them2-/- mice using recombinant adeno-associated virus revealed that WT, but not N50A/ D65A Them2, promoted high-fat diet-induced weight gain and hepatic steatosis. This was accompanied by greater impairment of insulin sensitivity in WT than N50A/D65A Them2. Pharmacological inhibition or genetic ablation of PCTP attenuated these effects. In reductionist experiments, conditioned medium collected from WT primary cultured myotubes promoted excess lipid accumulation in oleic acid- treated primary cultured hepatocytes relative to Them2-/- myotubes, which was attributable to secreted extracellular myotubes affirmed the requirements for catalytic activity and PC-TP interactions for extracellular vesicles to promote lipid accumulation in hepatocytes. These studies provide valuable as attractive targets for managing metabolic dysfunctionassociated steatotic liver disease.

Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non-ectomycorrhizal P. trichocarpa cuttings to short-term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short-term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi-omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa-L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non-mycorrhizal roots to abiotic challenges.

ABSTRACTThioesterase superfamily member 1 (Them1; synonyms Acyl-CoA thioesterase 11 (Acot11) and steroidogenic acute regulatory protein-related lipid transfer (START) domain 14 (StarD14) is a long chain acyl-CoA thioesterase comprising two N-terminal hot-dog fold enzymatic domains linked to a C-terminal lipid-sensing START domain, which allosterically modulates enzymatic activity. Them1 is highly expressed in thermogenic adipose tissue, where it functions to suppress energy expenditure by limiting rates of fatty acid oxidation. Its expression is also induced markedly in liver in response to high fat feedings, where it suppresses fatty acid oxidation and promotes hepatic glucose production. Mice lacking the gene (Them1-/-) are protected against diet-induced non-alcoholic fatty liver disease (NAFLD), suggesting Them1 as a therapeutic target. The current study was designed to develop small molecule inhibitors of Them1 and to establish their activitiesin vitroand in cell culture. High-throughput screening combined with counter screening assays were leveraged to identify two lead allosteric inhibitors that selectively inhibited Them1 by binding the START domain. In primary mouse brown adipocytes, these inhibitors promoted fatty acid oxidation, as evidence by increased rates of oxygen consumption. In primary mouse hepatocytes, they similarly promoted fatty acid oxidation, but also reduced glucose production. Optimized Them1 inhibitors could provide an attractive modality for the pharmacologic management of NAFLD and obesity-associated metabolic disorders.