Obesity is a risk factor for estrogen receptor (ER) positive breast cancer. Beyond body mass index, adult weight gain increases breast cancer risk. During weight gain, hypertrophic adipocytes produce fibroblast growth factor 1 (FGF1), which drives estrogen-independent growth of ER-positive tumors. Effects of FGF1 on breast cancer cells include elevated proliferation and enhanced glycolytic activity. We identified the Ets transcription factor ETV4 as a target of FGF1 treatment across multiple breast cancer cell lines. Our objective was to define the role of ETV4 in mediating the tumor-promotional effects of FGF1, to better understand how weight gain and obesity drive breast cancer risk and progression. Here, we determined that ETV4 directly associates with a poor prognosis for patients with ER-positive tumors and positively correlates with FGF1 levels in the context of obesity. We demonstrate that ETV4 is required to mediate the pro-tumorigenic effects of FGF1 on cell proliferation, glycolytic reprogramming, and tamoxifen sensitivity in vitro, and on tumor growth in the presence of estrogen in obese mice. In vitro, ETV4 overexpression enhances proliferation and metabolic activity, mimicking effects of FGF1 on breast cancer cells, but it is not sufficient to promote ER-positive tumor growth before or after estrogen deprivation in vivo in lean females. This study reveals a potentially novel mechanism through which weight gain, characterized by excess FGF1 production, drives the development of aggressive features in the prevalent ER-positive breast cancer subtype. ### Competing Interest Statement The authors have declared no competing interest. National Cancer Institute, https://ror.org/040gcmg81, CA241156

Midlife obesity is a major risk factor for vascular cognitive impairment (VCI) and dementia, but the cellular mechanisms linking obesity to brain microvascular dysfunction remain unclear. Here, we show that high-fat diet (HFD)-induced obesity accelerates cellular senescence within the neurovascular unit (NVU), resulting in structural and functional microcirculatory deficits. Combining multimodal in vivo longitudinal imaging with single-cell RNA sequencing, we identify a senescence-associated transcriptional program in endothelial cells and neurons, coinciding with reduced brain microvascular density, impaired neurovascular coupling (NVC), and disruption of blood-brain barrier (BBB) integrity. These vascular abnormalities associate with cognitive decline in behavioral assays. Transcriptomic profiling further revealed cell-type-specific senescence signatures, including dysregulation of angiogenic, mitochondrial, and inflammatory pathways, which were alleviated by senescent-cell clearance. Notably, clearing p16+ senescent cells partially restored BBB integrity, improved NVC responses, and reduced neuroinflammation. Together, these findings identify cellular senescence as a mechanistic driver of midlife obesity-induced cerebrovascular and cognitive dysfunction and provide proof-of-concept that senescence-targeted therapies may preserve brain health in individuals with midlife obesity who are at risk for dementia.

High-fat diet (HFD) intake has been linked to an increased risk of pancreatic ductal adenocarcinoma (PDAC), a lethal and therapy-resistant cancer. However, whether and how specific dietary fats drive cancer development remains unresolved. Leveraging an oncogenic Kras -driven mouse model that closely mimics human PDAC progression, we screened a dozen isocaloric HFDs differing solely in fat source and representing the diversity of human fat consumption. Unexpectedly, diets rich in oleic acid - a monounsaturated fatty acid (MUFA) typically associated with good health - markedly enhanced tumorigenesis. Conversely, diets high in polyunsaturated fatty acids (PUFAs) suppressed tumor progression. Relative dietary fatty acid saturation levels (PUFA/MUFA) governed pancreatic membrane phospholipid composition, lipid peroxidation, and ferroptosis sensitivity in mice, concordant with circulating PUFA/MUFA levels being linked to altered PDAC risk in humans. These findings directly implicate dietary unsaturated fatty acids in controlling ferroptosis susceptibility and tumorigenesis, supporting potential "precision nutrition" strategies for PDAC prevention.

The role of dietary fat in human health remains debated, particularly in aging. The diet-heart hypothesis of the 1950s linked saturated fat to cardiovascular disease (CVD), shaping nutrition guidelines for decades. However, later trials yielded inconsistent results, and recent reviews have questioned the strength of this association. At the same time, studies of ketogenic and other high-fat diets suggest that, in specific metabolic contexts, higher fat intake may offer metabolic and vascular benefits. These findings highlight the need to move beyond simplified views of dietary fat by considering fat type, metabolic state, and eating patterns. Beyond cardiovascular outcomes, interest has grown in how diet influences brain aging and cerebrovascular health. Cerebrovascular dysfunction is central to vascular cognitive impairment and dementia (VCID), yet dietary effects on these mechanisms remain incompletely understood. This review examines evidence on how dietary fat composition and eating strategies affect endothelial function, cerebral blood flow, blood-brain barrier integrity, inflammation, and mitochondrial function within the aging neurovascular unit, identifying pathways that may support cerebrovascular resilience and cognitive health.

Obesity remains a major global health challenge with limited durable pharmacotherapies. Disulfiram (DSF), an FDA-approved drug reported to inhibit gasdermin D (GSDMD), has been proposed to improve metabolic outcomes through suppression of inflammasome signaling. Here, we demonstrate that GSDMD is dispensable for high-fat diet-induced obesity and insulin resistance, as neither genetic deletion nor antisense-mediated inhibition of GSDMD confers metabolic protection. In contrast, DSF robustly protects against obesity and IR through a GSDMD-independent mechanism. These effects are not attributable to reduced caloric intake but instead reflect a coordinated reprogramming of systemic lipid handling. Under steady-state conditions, DSF suppresses basal lipid oxidation while promoting fecal fatty acid excretion. In striking contrast, during acute lipid challenge, DSF enhances tissue lipid utilization and accelerates systemic clearance. Together, these findings overturn the prevailing inflammasome-centric model and establish context-dependent regulation of lipid partitioning-rather than inflammasome inhibition-as the primary mechanism underlying DSF's anti-obesity effects.

Adipose dysfunction contributes to age-related systemic decline primarily through its adverse effects on energy metabolism, insulin sensitivity, circulating adipokines, and inflammation. Time-restricted feeding (TRF) has emerged as a promising approach to correct adipose and metabolic dysfunction. However, most of these studies were carried out in young animals. Whether TRF could exert similar beneficial effects in the adipose tissue during aging remains unknown. To address this, 18-month-old C57BL/6 mice were placed on either a TRF diet (food intake restricted to a 6-h time window every day in the dark phase) or an unrestricted diet for 6 months. Young animals on an unrestricted diet acted as additional controls to compare the effects of aging. Here, we demonstrate that a 6-month TRF regimen induces a biphasic pattern in whole-body energy metabolism characterized by a selective increase in energy expenditure and oxygen consumption during the active dark phase, aligning with the feeding schedule. TRF increased uncoupling protein 1 (UCP1) expression in the white adipose tissue (WAT) and reverses age-associated whitening of brown adipose tissue (BAT) in aged mice. In addition, TRF selectively enhances mitochondrial metabolism in WAT depots. Furthermore, TRF reduces macrophage infiltration, induces a favorable shift in macrophage polarization (lower M1/M2 ratio), and decreases fibrosis in adipose tissue. Overall, our findings indicate that TRF promotes a metabolically beneficial adipose phenotype characterized by beiging and reduced fibro-inflammation during aging. These results underscore the potential of TRF as a dietary intervention to mitigate adipose dysfunction and promote metabolic health in the aging population.

Maternal obesity alters breast milk composition in ways that may predispose infants to excess adiposity. Although maternal exercise during lactation has been associated with favorable shifts in milk metabolites in humans, the mechanisms by which exercise remodels the mammary gland and milk lipid profile to influence offspring metabolism remain unclear. We developed a mouse model incorporating daily moderate treadmill exercise only during lactation, using lean (LN) and diet-induced obese (OB) dams, and leveraged indirect calorimetry, stable isotope tracer respirometry, and mammary epithelial cell (MEC) proteomics assays. Maternal obesity broadly remodeled the MEC proteome, decreasing enzymes of de novo fatty acid synthesis and altering lipid transport and oxidative pathways. These molecular adaptations in OB dams corresponded to higher milk triglyceride content and shifts in fatty acid composition, including suppressed medium-chain fatty acids (MCFAs). The exercise (EX) intervention during lactation reset MEC protein networks, enhancing protein translation and vesicle transport pathways, whereas decreasing fatty acid desaturation, relative to the sedentary (SED) group. In OB dams, the exercise intervention increased milk MCFA levels and partially corrected the proinflammatory omega-6 fatty acid bias. Offspring suckling OB-EX dams exhibited enhanced in vivo fatty acid oxidation, partially rescuing obesity-associated impairments in metabolic fuel preference. Together, maternal exercise during lactation remodels mammary metabolism and milk fatty acid composition in obese dams, which in turn, enhances postnatal lipid oxidation. These findings highlight lactation as a modifiable window, wherein maternal activity influences milk composition and early life metabolism. NEW & NOTEWORTHY Maternal obesity alters milk fatty acid composition, with consequences for postnatal metabolism. Maternal exercise during lactation in obese dams remodeled the mammary epithelial cell proteome, increasing medium-chain fatty acids in milk and enhancing offspring lipid oxidation.

Acquired lipodystrophy in the dermal white adipose tissue (DWAT) is a salient feature of skin fibrosis, and is followed by accumulation of extracellular matrix (ECM). Lipodystrophy syndromes, often associated with metabolic co-morbidities, are estimated to affect 1 in 20,000 people. We recently showed that fibrosis-associated lipodystrophy is dependent on sustained Wnt signaling, but the mechanism is unclear. Transcriptomic profiling of mature dermal adipocytes in vivo reveal that Wnt activation downregulates the de novo -lipogenesis (DNL) axis enzymes within 48 hours. We found that protein expression of Fatty Acid Synthase (FASN), a key DNL enzyme, is dependent on sustained Wnt activation in vitro and in vivo . In human systemic sclerosis, FASN mRNA is significantly downregulated during two years of disease. Remarkably, pharmacological inhibition of FASN enzyme during reversal from Wnt induced fibrosis impedes recovery of DWAT lipid content as well as ECM accumulation and topography. All together, we demonstrate that acquired lipodystrophy in the context of skin fibrosis is mediated by a new role of the Wnt-DNL axis. These findings underscore the importance of this pathway in lipodystrophy and fibrosis, opening new avenues for therapeutic targets in skin fibrosis.

Primary adipocytes exhibit striking variability in size, yet the functional consequences of adipocyte hypertrophy remain unclear due to insufficient experimental approaches to control for cell size. Here, we establish methods to culture large and small primary adipocytes isolated from the same adipose depot, enabling size-resolved analyses independent of systemic obesity. Using transcriptomic, lipidomic, and functional profiling across two mouse models of obesity, as well as human clinical samples, we show that adipocyte size—rather than body weight—drives distinct phenotypic cell states. Notably, large adipocytes increase extracellular vesicle-mediated lipid release. In coculture assays, this shift enhances lipid uptake, migration, and proliferation of breast cancer cells through fatty acid oxidation. Consistent with these findings, individuals with larger mammary adipocytes exhibit elevated fasting triglycerides independent of body mass index. Together, our results identify adipocyte size as a key determinant of adipose tissue function with implications for both metabolic disease and cancer progression.

ABSTRACT Broadly protective immunity to the Lyme disease spirochete, Borreliella burgdorferi, is constrained by antibodies against type-specific epitopes on outer surface protein C (OspC), a homodimeric helix-rich lipoprotein essential for early stages of spirochete dissemination in vertebrate hosts. However, the molecular basis for type-specific immunity has not been fully elucidated. In this report, we produced and characterized an OspC mouse monoclonal antibody, 8C1, that recognizes native and recombinant OspC type A (OspCA) but not OspC type B or K. Epitope mapping by hydrogen–deuterium exchange mass spectrometry (HDX-MS) localized 8C1’s epitope to a protruding ridge on the apex of OspCAα-helix 3 (residues 130–150) previously known to be an immunodominant region of the molecule. Alanine scanning pinpointed 8C1’s core binding motif to a solvent exposed patch consisting of residues K141, H142, T143, and D144. Analysis of 26 Lyme disease-positive serum samples confirmed human antibody reactivity with this region of OspCA, with residues E140 and D144 as being the most consequential. Our results underscore the importance of α-helix 3 as a target of type-specific epitopes on OspCA that should be taken into consideration in Lyme disease vaccine design.IMPORTANCEA central challenge in the development of vaccines against Lyme disease, the most common vector-borne infection in the United States, is the antigenically variable nature of the lipoproteins displayed on the surface of the disease-causing spirochete, Borreliella burgdorferi. For example, antibodies against one type of outer surface protein C (OspC), a lipoprotein involved in B. burgdorferi transmission and early stages of infection, may have little or no cross reactivity with another seemingly closely related variant of OspC, thereby hampering the use of a single OspC type as a vaccine antigen. For the sake of vaccine design, it is critical to identify the specific epitopes on OspC that both restrict and enable cross-reactivity.

Human milk oligosaccharides (HMOs) are integral to infant health. Yet, their complex biosynthesis pathways in the mammary gland during lactation remain under-characterized. To address this knowledge gap, we performed integrated analyses of single-cell RNA-sequencing (scRNA-seq) datasets combined with select HMO concentration measures. We identify differential expression patterns of known HMO synthesis genes in epithelial subsets and nominate several candidate genes that vary with HMO concentration. Additionally, we identify novel gene patterns and transcription factors that may regulate the expression of HMO biosynthesis genes and the cellular pathways supporting HMO production. Finally, we demonstrate that co-expression of HMO synthesis genes and milk fat synthesis genes is limited, suggesting that distinct epithelial cell subtypes may be responsible for the production of different milk components. Our study suggests that HMO synthesis may be achieved through cell-type specialization within the lactocyte compartment.

Dietary fat composition has changed substantially during the obesity epidemic. As adipocyte hyperplasia is a major mechanism of adipose expansion, we aim to ascertain how dietary fats affect adipogenesis during obesity. We employ an unbiased dietary screen to identify oleic acid (OA) as the only dietary fatty acid that induces obesogenic hyperplasia at physiologic levels and show that plasma monounsaturated fatty acids (MUFAs), which are mostly OA, are associated with human obesity. OA stimulates adipogenesis in mouse and human adipocyte precursor cells (APCs) by increasing AKT2 signaling, a hallmark of obesogenic hyperplasia, and reducing LXR activity. High OA consumption decreases LXRα Ser196 phosphorylation in APCs, while blocking LXRα phosphorylation results in APC hyperproliferation. As OA is increasingly being incorporated into dietary fats due to purported health benefits, our finding that OA is a unique physiologic regulator of adipose biology underscores the importance of understanding how high OA consumption affects metabolic health.

Introduction and Objective: The Developmental Origins of Health and Disease (DOHaD) hypothesis posits that early-life nutritional exposures shape long-term metabolic health. Here, we show that perinatal excess n6-FA exposure programs neonatal Adipocyte Stem-like Cells (ASCs) toward a lipogenic, metabolically inflexible phenotype, increasing susceptibility to obesity later in life. We hypothesize that n6-FA suppression of NR2F2 underlies these effects. Methods: C57BL/6J dams were provided an n6-FA rich or a balanced n6/n3 (control) diet at pairing. Offspring were evaluated at postnatal day 12 for whole-body metabolic responses, adipose morphology, cellular metabolism, and molecular signatures. ASCs were isolated, and NR2F2 expression was manipulated to assess its role in adipocyte metabolic programming. Metabolic, proteomic, and transcriptomic analyses were conducted. Results: Perinatal high n6-FA exposure reduced fatty acid oxidation (FAO) in both ASC-derived adipocytes and whole-body metabolism, accompanied by mitochondrial dysfunction, increased lipogenesis, and suppressed thermogenic adipocyte markers. Morphological changes included adipocyte hypertrophy, increased triglyceride storage, and greater body fat in exposed pups. Mechanistically, n6-FA exposure suppressed NR2F2 expression in ASCs. ASC-specific deletion of NR2F2 recapitulated the metabolic impairments observed with n6-FA exposure, including reduced fatty acid oxidation and thermogenic gene expression. Conversely, transient activation of NR2F2 in n6-FA-exposed ASCs restored FAO by activating the PPARγ-PGC1α axis and upregulating mitochondrial respiratory chain components. Conclusion: These findings establish NR2F2 as a central regulator of ASC metabolic programming, revealing how early-life n6-FA exposure promotes a pro-adipogenic, metabolically inflexible phenotype. Our study highlights NR2F2 as a potential therapeutic target for mitigating obesity and associated metabolic disorders. S. Das: None. R. Varshney: None. G. Kyere-Davies: None. A.E. Martinez: None. K.B. Hill: None. M. Kinter: None. G.P. Mullen: None. M. Rudolph: None. The Oklahoma Center for Adult Stem Cell Research, Presbyterian Health Foundation Bridge Fund, College of Medicine Alumni Association Support, IDeA National Resource for Quantitative Proteomics (R24GM137786); Oklahoma INBRE (P20GM103447)

Primary adipocytes possess a dramatic capacity to expand and retract in volume, leading to high variability in cell size within and between individuals. Yet, how adipocyte size impacts cell function remains unclear as adipocyte size is not tunable with traditional experimental approaches, forcing previous work to rely on correlative studies. Here, we develop protocols to separate primary adipocytes from the same donor into large and small populations and maintain these size-sorted cells in culture. Using these methods, we perform transcriptomic, lipidomic, and functional analyses on large and small adipocytes across two orthogonal mouse models of obesity and validate our results with human clinical samples. Our findings indicate that changes to cell size, rather than global differences mediated by weight gain, drive the transcriptional response of primary adipocytes to obesity. Moreover, large adipocytes shift from a traditional, lipase-mediated mode of lipid release to a non-canonical, extracellular vesicle-mediated mechanism. In functional coculture studies, this change promotes lipid accumulation in neighboring breast cancer cells, increasing their migration and proliferation via enhanced tumor cell fatty acid oxidation. Consistent with our experimental data, human patients with large adipocytes present with greater rates of dyslipidemia and higher concentrations of fasting triglycerides, even when accounting for differences in body mass index. Collectively, our results provide direct evidence that large and small adipocytes from the same donor differ in gene expression, lipid composition, and function with implications for the management of adipose tissue-related pathologies such as breast cancer.

Context: Exercise is recommended for postpartum health, but its effects on breast milk composition and offspring are understudied. Objective: This work aimed to test whether the breast milk metabolome is altered with (i) acute exercise and/or (ii) habitual physical activity, and (iii) whether exercise-altered metabolites are associated with infant adiposity. Methods: Milk metabolites were assessed before and after acute exercise and in association with habitual activity score in 2 independent cohorts at 2 academic medical centers. The acute exercise cohort had 15 mother-infant dyads. The habitual activity nested case-control analysis had 84 physically active "cases" and 35 inactive "controls," and was conducted in a subset of the Mothers and Infants Linked for Healthy Growth (MILk)/4M study (N = 348). The acute exercise exposure was a 30-minute moderate-intensity treadmill session. The habitual activity exposure was based on Physical Activity Recall questionnaire scores. Main outcome measures included milk metabolite relative abundance at 1-month post partum by liquid chromatography-gas chromatography mass spectrometry, and infant anthropometric and body composition measures at 1, 3, and 6 months. Results: An acute exercise bout altered milk concentrations in 28 of 511 detectable metabolites (false discovery rate [FDR] < .05). In the habitual activity analysis, 4 of 454 detectable metabolites differed between active cases vs inactive controls (FDR < .05). Ten metabolites were altered (P < .05) by both exercise exposures. Of these, 4 were positively associated with fat mass index at 1 month, and 2 were associated with greater increase in body mass index z score between 1 and 3 months. Conclusion: Maternal exercise was associated with differences in the breast milk metabolome. Metabolites that were associated both with acute exercise and habitual activity correlated with infant adiposity measures.

Due to the growing obesity epidemic in the United States, it is now estimated that approximately one third of all children are born to obese moms. These data, coupled with data indicating that obesity is associated with accelerated cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD), led us to hypothesize that maternal obesity may influence the rate of disease progression in offspring. To test this hypothesis, we induced maternal obesity by high-fat diet (HFD) feeding in the orthologous Pkd1RC/RC mouse model of ADPKD and followed polycystic kidney disease (PKD) progression in offspring for up to 1 year. Surprisingly, and in contrast to our initial hypothesis, exposure to maternal obesity during pregnancy and lactation did not significantly impact PKD severity in offspring at 3 mo or 1 yr of age. In contrast, reexposure to HFD for ∼3 m beginning at 12 wk of age worsened PKD severity in female, but not male, offspring born to obese dams as measured by cystic index, cyst number, and cyst area. Despite worsened cystic parameters, fibrosis and blood urea nitrogen were not altered in these animals. Collectively, these findings indicate that maternal obesity may accelerate PKD severity in female offspring exposed to an obesogenic diet.NEW & NOTEWORTHY Due to the growing obesity pandemic, almost one third of all children are born to mothers with obesity; however, the impact of maternal obesity on polycystic kidney disease (PKD) is unknown. In this manuscript, we found that maternal obesity did not worsen PKD severity in Pkd1RC/RC mice at 3 mo or 1 yr of age when weaned onto normal chow diet. However, rechallenging pups born to obese mothers worsened PKD severity in female but not male mice.

Introduction and Objective: High levels of omega-6 (n6) fatty acids (FA) relative to omega-3 (n3) FA correlate with increased adipose (AT) accumulation during development and enhanced obesity risk in later life. Intriguingly, AT macrophages (ATMs), vital for tissue development, lipid metabolism, and immune regulation, are dysregulated during adult obesity. In vitro, n3 FA promote anti-inflammatory cytokine production while n6 FA promote inflammatory cytokine production; however, how perinatal FA exposures shape ATM phenotypes is unclear. We hypothesize that elevated maternal dietary n6:n3 FA predisposes offspring AT to enhanced inflammation by shifting ATM away from pro-resolving phenotypes. Methods: C57BL/6J mice were provided n6-rich or balanced control diets at pairing through lactation. On post-natal day 12 (PND12), inguinal AT was dissected from pups for flow cytometry and GC-MS lipidomics, and a separate cohort was used for whole AT global gene expression at PND14. Results: Flow cytometry revealed a 5.2-fold reduction in Tim4+/CD206+ ATMs (p<0.0001), a nearly 1.8-fold increase in CD9+/MHC2+/CD11c+ ATMs (p=0.0386), and a strong trend toward lower levels of patrolling monocytes (p=0.0551) relative to controls. Lipidomics showed an elevated n6:n3 FA, driven by reduced n3-FA bioactive precursors. Global gene expression of subcutaneous adipose from high n6 pups indicated significant enrichment in macrophage activation, cytokine signaling, and fibrosis pathways compared to controls. Conclusion: Elevated maternal dietary n6:n3 FA in the perinatal window shifts offspring ATM away from resolving phenotypes with a loss of critical resolving lipid precursors, potentially predisposing AT for impaired responses to metabolic stress and exaggerated inflammation. Further studies will assess the mRNA signatures and role of ATMs in AT programming and later life metabolic dysfunction. Our data confirm that ATM populations are sensitive to developmental n6-FA. K.B. Hill: None. K.A. Zimmerman: None. M. Rudolph: None. G.P. Mullen: None. R. Varshney: None. G. Kyere-Davies: None. OCASCR, Research Grant 2024-2025PHF-HHDC, Team Science, Bridge Fund, and Equipment Grants 2022-2024R01 HD117197, NIH NICHD (4th Percentile); COMAA, College of Medicine Alumni Association Award 2024

Cannulae are structurally rigid tubular protein filaments that accumulate on the extracellular surface of archaea within the family Pyrodictiaceae during cell growth. These obligate anaerobes propagate under hyperthermophilic conditions in which cannulae form a biomatrix that interconnects and sustains cells. The persistence of cannulae in this environment suggests that these filaments display significant thermostability, which has attracted technological interest in their development as synthetic protein-based biomaterials. Here, we report cryoEM structural analyses of ex vivo and in vitro assembled recombinant cannulae. We demonstrate that the interactions between protomers in native and recombinant cannulae is based on donor strand complementation (DSC), a form of non-covalent polymerization previously observed for bacterial chaperone-usher pili. Unexpectedly, calcium ion coordination at the subunit interfaces reinforces the network of donor strand interactions in the cannulae. This study provides insight into the mechanism of assembly of cannulae and the structural origin of their high stability and rigidity.

Ricin is a category B agent for bioterrorism, and Shiga toxins are the primary virulence factors of Shiga toxin (Stx) producing Escherichia coli. Ricin and Stxs bind the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop on the eukaryotic ribosome and inhibit translation. Both toxins are prime targets for therapeutic intervention because no effective therapy exists for ricin intoxication or Shiga toxin producing Escherichia coli infection. Binding of ricin toxin A subunit (RTA) to the ribosomal P-stalk stimulates depurination of the sarcin/ricin loop by an unknown mechanism. We previously identified compounds that bind the P-stalk pocket of RTA and inhibit catalytic activity. Here we characterize a second-generation lead compound, which binds the P-stalk pocket of RTA with over 30-fold improved affinity relative to the original compound and inhibits the cytotoxicity of ricin holotoxin in Vero cells with no apparent cellular toxicity by itself. This compound also shows protection against Stx2A1. X-ray crystal structure of RTAinhibitor complexes suggests that the orientation of the carboxylic acid influences the inhibitor contacts at the P-stalk site of RTA and contributes to inhibitor potency. The structural changes triggered at the P-stalk site of RTA were validated by solution NMR-based chemical shift perturbation analysis. A key finding by NMR is that binding-induced conformational changes extend beyond the P-stalk site to residues in the active site cleft of RTA. Collectively, these results provide valuable new insight into the conformational flexibility in the C-terminal domain of RTA and its potential role in mediating the remarkable catalytic activity of ricin.

Breast cancer endocrine therapy, which systemically disrupts estrogen receptor signaling, increases type 2 diabetes (T2D) risk in some women. Sustained treatment with low-dose tamoxifen depletes subcutaneous adipocyte progenitors and promotes glucose intolerance and hepatic lipid deposition in obese female mice. Hyperplastic adipose tissue expansion, especially in subcutaneous depots, preserves metabolic health during a chronic positive energy balance by facilitating nutrient storage and attenuating inflammation. Adipocyte progenitors are renewed in part through Wnt signaling pathway activation, which is altered in women with obesity or T2D. Estrogen receptors are expressed in several adipose cell types, but the distinct actions of tamoxifen in adipocyte progenitors and the mechanisms that explain their depletion during endocrine therapy are not defined. The direct impact of tamoxifen was evaluated in subcutaneous adipose stromal cells from humans and adult mice. Self-renewal, proliferation, and differentiation were measured, and analyses of gene expression and progenitor or preadipocyte populations were performed. Mechanistic insight was gained from primary adipose stromal cells of obese female mice, in which the Wnt1 inducible signaling pathway protein 2 (Wisp2) was lost following endocrine therapy. Wisp2 gain and loss of function studies were carried out in adipose stromal cells to define the link between estrogen signaling and adipocyte progenitor maintenance. We found that tamoxifen treatment disrupts the protection of adipocyte progenitors by estrogen, mediated through suppression of Wisp2. These studies reveal potential metabolic effects of tamoxifen therapy that precede and could drive T2D development in breast cancer survivors. ### Competing Interest Statement The authors have declared no competing interest.