Polycystic ovary syndrome (recently renamed to polyendocrine metabolic ovarian syndrome [PMOS]) is the most common endocrine disorder in women of reproductive age. Women with PMOS not only suffer from reproductive symptoms but are also more prone to develop metabolic dysfunction and aberrant eating behavior. The gut hormone ghrelin affects both metabolism and eating behavior in a sex-dependent fashion. Therefore, this study explored the effects of ghrelin deficiency on the phenotype of female mice with prepubertal, chronic dihydrotestosterone (DHT)-exposure, a widely used mouse model of PMOS. DHT-exposure markedly suppressed estrus cycling and corpora luteal number, increased lean mass (but not fat mass), and elevated inactive-phase food intake, but ghrelin deficiency had no marked effect on these outcomes. Interestingly, in wild-type mice, eating time and meal number of DHT-treated mice responded more strongly to acute ghrelin administration than those of controls. In ghrelin-deficient mice, responses to acute ghrelin administration were like DHT-treated wild-type mice, regardless of treatment. Beside effects on eating behavior, chronic DHT-exposure also reduced locomotor activity in both wild-type and ghrelin-deficient mice, which is in line with reports on physical activity in women with PMOS. Locomotor activity was also decreased upon acute ghrelin administration, especially in ghrelin-deficient mice. Overall, chronic DHT exposure modified the behavioral responses to acute ghrelin administration in females, without evidence for additive effects of ghrelin deficiency, suggesting overlapping mechanisms of action. This work emphasizes the added value of detailed behavioral data in translational models of human disease to help elucidate potential mechanisms underlying aberrant eating behavior.
This study investigates the individual and combined effects of Roux-en-Y gastric bypass (RYGB), sleeve gastrectomy (SG), and metformin on glucose regulation in a non-obese, insulin-deficient model of type 2 diabetes. Female Goto-Kakizaki (GK) rats underwent RYGB, SG, or sham surgery. Three weeks postoperatively, animals received metformin (50 mg/kg/day, 5 days/week) or vehicle for three additional weeks. Glucose tolerance was assessed using a standardized meal test, and insulin sensitivity was evaluated by insulin tolerance test. Plasma levels of GLP-1, GIP, insulin, and leptin were measured. RYGB and SG reduced body weight, food intake, and leptin levels, and improved fasting glucose, glucose tolerance, insulin sensitivity, and postprandial incretin and insulin secretion. Metformin alone improved glucose tolerance and insulin sensitivity independently of incretin or insulin changes. When combined with surgery, metformin further reduced postprandial glycemic excursions but did not enhance insulin sensitivity or hormone secretion beyond surgery alone. In conclusion, metabolic-bariatric surgery and metformin independently improve glucose regulation in non-obese diabetic GK rats. Their combination provides additional benefits on postprandial glucose control, despite no additional effects on insulin sensitivity or hormone levels. These findings support the use of metformin as an adjunct to metabolic-bariatric surgery in insulin-deficient diabetes and highlight the need for longer-term, sex-inclusive studies to enhance translational relevance.
Leptin, secreted by adipocytes, conveys the status of peripheral energy stores to the brain to regulate appetite and metabolism. Although sympathetic activation via β3-adrenergic receptors (β3-ARs) has been shown to suppress leptin expression, it remains unclear whether this regulation arises directly within adipocytes or through other populations. Additionally, it is unclear whether β3-AR signaling contributes to the fasting-induced reduction in leptin. To address this, we generated a novel Adrb3 transcriptional block mouse (Adrb3TB/TB) enabling global β3-AR inactivation and adipocyte-specific re- expression (Adrb3TB/TB; Adipoq-Cre). Global loss of Adrb3 increased plasma leptin levels, while re-expression in adipocytes normalized leptin and restored β3-AR agonist-induced leptin suppression. Analysis of Lep mRNA revealed depot specific regulation, with β3-AR signaling predominantly affecting gonadal and subcutaneous white adipose tissue, while brown adipose tissue responses were modest and variable. Despite these effects, fasting for 48 hours comparably reduced plasma leptin and Lep mRNA in both wild-type (WT) and Adrb3TB/TB mice, indicating that β3-AR signaling is not required for the fasting-induced decline in leptin. Collectively, these findings establish adipocyte β3-AR signaling as a key regulator that constrains leptin synthesis under basal and stimulated conditions in male mice, but not during energy deprivation. Together, these findings clarify the role of adipocyte β3-adrenergic signaling in leptin regulation and provide new insight into the sympathetic control of adipose tissue function.
Glucagon-like peptide-1 receptor agonists (GLP-1RAs), such as semaglutide, are highly effective treatments for obesity and type 2 diabetes but are often associated with gastrointestinal side effects including delayed transit times through the gastrointestinal tract. We present a non-invasive breath test to measure oral-cecal transit time (OCTT) in mice that could be readily translated to clinical studies. Current methods for measuring OCTT are limited by invasiveness, terminal procedures, anesthesia, radioactive tracers, or stress-induced influence on the outcomes. Here we demonstrate that 13 C-labeled mannitol is metabolized by murine cecal microbiota, producing measurable 13 CO₂ in exhaled breath. Using indirect calorimetry combined with real-time measurement of the 13 C/ 12 C ratio, this approach provides a highly granular, non-invasive method to measure OCTT in mice. This technique is performed in unrestrained freely moving animals while enabling high-throughput measurements performed longitudinally. The method improves experimental rigor and reproducibility and provides a valuable tool for studying gastrointestinal physiology and the effects of pharmacologic therapies.
Population aging is accelerating worldwide, and is accompanied by a growing burden of chronic metabolic diseases. Adipose tissue dysfunction represents a central mechanism linking aging and obesity to metabolic decline, contributing to chronic low-grade inflammation, impaired adipokine signaling, ectopic lipid deposition, and the whitening of thermogenic fat depots. Brown adipose tissue (BAT) plays a key role in adaptive thermogenesis through UCP1 mediated mitochondrial uncoupling, however, its activity progressively declines with advancing age and excess adiposity. This narrative review synthesizes emerging biomolecular mechanisms that underlie impaired thermogenic function in the context of aging and obesity. We integrate evidence across three major regulatory domains: (1) intracellular thermogenic signaling, highlighting salt-inducible kinases (SIK2/3), as transcriptional repressors that limit UCP1 expression when ß3-adrenergic/PKA signaling is impaired, (2) autophagy-dependent secretion of acyl-CoA binding protein (ACBP), which suppresses lipolysis, ß-oxidation, and thermogenic signaling and may contribute to BAT whitening, and (3) immune mediated regulation of thermogenic adipose tissue, with macrophage modulating sympathetic signaling, inflammatory tone, and extracellular matrix remodeling through pathways involving NLRP3-MAOA, SLIT3/ETS1, prolidase (PEPD) and immunoglobulin G (IgG). Collectively, these convergent mechanisms illustrate how aging and obesity impose coordinated constraints on brown and beige adipose tissue activation. By framing thermogenic decline as a regulated process, this review provides a conceptual basis for future studies aimed at preserving metabolic flexibility and promoting healthier aging.
The ISS (Insulin Secretion and Sensitivity) differential equation model has been previously shown to provide simultaneous estimates of SI and BCF during an oral glucose tolerance test. Using the Federal Women’s Study, we adapted the ISS model to fit glucose and insulin dynamics and estimate SI and BCF during mixed meal tests. Our goal was to develop an effective, straightforward, and reliable tool that would be simpler to use than currently available approaches that require technically challenging deconvolution methods to assess BCF. The ISS model for mixed meal tests strongly correlated with oral glucose tolerance tests and gold-standard estimates from intravenous glucose tolerance tests and were reliable when using a shortened 2-hour MMT protocol. The ISS model is versatile, publicly available, and expands the toolbox for assessing post-prandial glucose-insulin homeostasis.
The oral glucose tolerance test (OGTT) is widely used to assess glucose tolerance, but the conventional 2-h postprandial glucose value faces limited reliability. Alternative OGTT-derived indices, such as the shape of the glucose curve and the time to glucose peak, may offer additional insights into metabolic health. This study examined whether these indices could serve as biomarkers of metabolic risk. Normoglycemic adults [n = 85 (males: 40; females: 45); age: 26.9 ± 4.6 yr; body mass index: 23.1 ± 2.4 kg/m2] underwent a 3-h OGTT, with repeated measurements of blood glucose, insulin, and free fatty acids (FFAs). Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp. Energy expenditure (EE) and sleep respiratory quotient (RQ) were measured by metabolic chamber, and body composition by the EchoMRI-AH. Participants were classified by glucose curve morphology (monophasic vs. biphasic) and glucose peak time (early: ≤30 min vs. late: >30 min). Compared with the monophasic group, the biphasic group showed lower postprandial glucose (P < 0.05), with no differences in 3-h glucose area under the curve (AUC), insulin AUC, insulin sensitivity, FFA, EE, RQ, or body fat. In contrast, the early peak exhibited lower postprandial glucose, 3-h insulin AUC, 2-h FFA AUC, RQ, and body fat, with higher EE (P < 0.05) and a trend toward higher insulin sensitivity (P = 0.0592), compared with the late-peak group. Biphasic glucose curves and early glucose peak time indicate better glucose tolerance and insulin sensitivity. Compared with glucose curve morphology, glucose peak time demonstrated a stronger discriminative index of metabolic health and could serve as an additional biomarker of the OGTT for identifying potential diabetes risks.NEW & NOTEWORTHY Participants in biphasic groups showed better glucose tolerance than monophasic participants. Participants in early-peak groups showed better glycemic control and energy metabolism than late-peak participants. Glucose peak time demonstrates a stronger discriminative index of metabolic health than glucose curve morphology. Glucose peak time could serve as a useful biomarker of the OGTT for identifying potential diabetes risks.
We examined liver and muscle glycogen utilization during high-intensity interval cycling, and the impact of carbohydrate (CHO) feeding, using noninvasive 13C magnetic resonance spectroscopy (MRS). Following 24 h of standardized dietary intake, nine male cyclists completed 8 × 5-min intervals (1-min recovery), ingesting either placebo (PLA), 60 g maltodextrin (CHO), or 60 g maltodextrin plus caffeine, taurine, l-theanine, l-citrulline, and citicoline (CHO+) in a randomized crossover design. 13C MRS and 1H imaging were performed pre- and postexercise to determine liver and muscle glycogen and liver volume, respectively. Liver glycogen utilization was not significantly different between trials (P = 0.101) despite lower postexercise plasma glucagon concentrations in CHO and CHO+ (P = 0.001). In contrast, muscle glycogen utilization was significantly lower (∼40%) with CHO feeding compared with PLA (P = 0.006), yet this sparing effect was not evident with CHO+ (P = 0.073) in accordance with a higher mean power output during the late intervals (+2.8%, P = 0.046). Plasma glucose was comparable between trials (P = 0.175), whereas plasma lactate was higher in CHO+ versus CHO (P = 0.003), alongside lower blood bicarbonate (P = 0.005), base excess (P < 0.001), and total CO2 (P = 0.004). These findings demonstrate preferential use of skeletal muscle glycogen during high-intensity interval training (HIIT), which is attenuated under conditions of CHO feeding. This sparing effect is, however, not evident with the coingestion of a caffeine-containing multi-ingredient blend, potentially due to an increased capacity to sustain higher power outputs resulting in greater glycogen utilization.NEW & NOTEWORTHY Using 13C MRS, we provide data demonstrating preferential use of skeletal muscle glycogen during HIIT. Furthermore, data show muscle glycogen utilization is attenuated with CHO feeding, yet sparing is not evident when coingesting a caffeine-containing formulation, potentially reflecting increased capacity to perform more total work rather than a direct metabolic effect of caffeine. In contrast, liver glycogen utilization was not significantly different with CHO feeding despite a modest reduction of ∼5 g versus placebo.
Autoimmune diseases, including Type 1 diabetes (T1D), are often characterized by overactive inflammatory signaling pathways. The proinflammatory cytokine interleukin-1β (IL-1β) elicits global gene expression changes in islet β-cells which overlap with islets obtained from human donors with T1D. The direct transcriptional link between NF-κB subunit p65 and target genes involved with autoimmune events was investigated. We used a multiomics approach including bulk RNA-sequencing (RNA-Seq), single-cell RNA-sequencing (scRNA-Seq), and chromatin immunoprecipitation coupled to deep sequencing (ChIP-Seq), alongside molecular docking simulations, and transcriptional assays. Through the various experimental modalities, we identified early response genes driven by IL-1β that were differentially expressed in pancreatic islets from human T1D donors and also conserved across mouse, rat, and human tissues. ChIP-Seq revealed genes that are direct genomic targets of the NF-κB p65 transcription factor. Moreover, regions that gained RNA polymerase II binding following cellular exposure to IL-1β were identified, complementing the early response gene profile induced by β-cell exposure to IL-1β. Molecular docking simulations predicted that mutations reducing p65 transcriptional capacity do not alter DNA binding ability. These findings clearly show that IL-1β signaling in pancreatic β-cells directs p65 to specific genomic regions congruent with increased gene expression relevant to T1D in β-cell lines as well as mouse and human islets exposed to cytokines. Islets from human donors with T1D express genes identified as direct p65 targets using unbiased approaches, implicating heightened NF-κB activity as a critical component of autoimmune disease etiology.NEW & NOTEWORTHY Using multiple Seq-based approaches, this study identified genes expressed in human pancreatic tissue from donors with Type 1 diabetes that are regulated acutely by exposure to the cytokine interleukin-1beta. The NF-kB transcription factor p65 (RelA) was determined via ChIP-Seq to be a major control node regulating this immediate early response. These collective datasets are consistent with a paradigm of overactive NF-kB signaling as a critical component of autoimmunity in both rodents and humans.
Obesity is strongly associated with elevated blood glucose levels, glucose intolerance, insulin resistance, and type 2 diabetes. The Nr4a family of orphan nuclear receptors is essential for proliferation, cell survival, mitochondrial function, and fuel utilization in a tissue-dependent manner. Nr4a3 overexpression has been shown to decrease blood glucose levels and improve glucose tolerance. Here, we present the effects of full body Nr4a3 deletion in mice fed a standard chow diet. We demonstrate that male and female Nr4a3 knockout mice fed a standard chow diet have elevated nonfasting blood glucose and impaired glucose tolerance. Male Nr4a3 knockout mice have increased body weight, without changes in body length, food intake, movement, or energy expenditure. Interestingly, male, but not female, Nr4a3 knockout mice have increased weight of all adipose depots with increased adipocyte cell size. Furthermore, male Nr4a3 mice have impaired adipose mitochondrial respiration, with normal liver and soleus respiration. Finally, we show a significant decrease in Drp1 mRNA, Drp1 protein, and phosphorylated DRP1 levels. These data suggest that Nr4a3 loss impairs expression of the key mitochondrial fission gene Drp1, resulting in impaired adipose mitochondrial respiration and ultimately increasing adipocyte size, adipose depot mass, and body mass. These data demonstrate that Nr4a3 is critical for proper adipocyte function.NEW & NOTEWORTHY This study reveals that full-body Nr4a3 deletion in mice leads to elevated blood glucose, impaired glucose tolerance, and increased adiposity, particularly in males. Notably, male Nr4a3 knockout mice exhibited enlarged adipocytes and impaired mitochondrial respiration in adipose tissue, which is linked to reduced expression of the mitochondrial fission gene DRP1. These findings highlight Nr4a3's critical role in regulating glucose metabolism and adipose tissue function, with implications for understanding obesity and type 2 diabetes.
Glucagon signaling through the glucagon receptor (GCGR) plays a central role in systemic metabolic regulation. However, its role in skeletal muscle protein homeostasis remains poorly understood. In this study, we demonstrated that skeletal muscle from male GCGR-deficient mice exhibited preferential gastrocnemius atrophy accompanied by elevated intramuscular free amino acid levels and hyperaminoacidemia. Transcriptomic and biochemical analyses revealed activation of glucocorticoid receptor (GR) signaling together with induction of proteolytic pathways, as reflected by increased tripartite motif containing 63 (TRIM63) and F-box protein 32 (FBXO32) levels, elevated light chain 3B-II/I (LC3B-II/I) ratio, and reduced p62 levels. Notably, GCGR deficiency was associated with increased corticosterone and subsequent GR activation. In cultured myotubes, serum from male Gcgr-/- mice induced GR activation, enhanced proteolysis, and increased intracellular free amino acid levels, all of which were attenuated by GR antagonism. Consistently, dexamethasone stimulation increased net amino acid accumulation in the culture medium. Moreover, the GR activation-resulted gastrocnemius atrophy was alleviated by glucagon-like peptide-1 receptor (GLP-1R) antagonism. These findings link GCGR deficiency to GR activation, gastrocnemius proteolysis, hyperaminoacidemia, and elevated GLP-1 signaling, suggesting a role for skeletal muscle in systemic amino acid homeostasis under impaired GCGR signaling.NEW & NOTEWORTHY Glucagon receptor signaling is classically viewed as acting primarily in the liver to regulate amino acid homeostasis. Here, we show that loss of GCGR signaling in male mice is associated with preferential gastrocnemius atrophy, accompanied by enhanced glucocorticoid receptor activation and increased proteolytic markers in gastrocnemius, which can be rescued by GLP-1R antagonism. These alterations occur together with intramuscular free amino acid accumulation and hyperaminoacidemia, suggesting that skeletal muscle is affected by disrupted GCGR signaling.
The ability to tightly regulate and maintain a warm core body temperature (Tb) is a defining characteristic of homeotherms. Moving to a thermally preferred place is an energetically efficient first-line thermoregulatory mechanism. We optimized a mouse binary thermal preference assay, selecting floor plate temperatures of 38°C versus 25°C for males and 39°C versus 30°C for females as having no baseline preference. Warm preference was increased in females, by fasting, and with age. Genetic manipulations that increased warm preference included ablation of uncoupling protein 1 (Ucp1) or of all four adenosine receptors (Adora1, Adora2a, Adora2b, Adora3). Ablation of estrogen receptor α (Esr1) reduced the preference for the warmer side in females but had no effect on preference in males. Mice treated with the β3 adrenergic receptor agonist CL-316243 or the A3 adenosine receptor agonist MRS5698 sought the cooler surface. Thermal preference assays complement the measurement of Tb, providing information about whether a change in Tb reflects a new target or set point Tb caused by the intervention, or if the mouse is trying to maintain an unchanged Tb. Thermal preference can be more sensitive than baseline Tb for detecting changes in thermal physiology.NEW & NOTEWORTHY A thermal preference assay optimized for each sex demonstrated preference differences depending on physiologic state, genetic manipulation, and drug treatment. Thermal preference assays provide information that is complementary to measurement of core body temperature. Thermal preference can be more sensitive to perturbations than baseline body temperature and informs the understanding of how genetic, pharmacologic, and physiologic interventions alter thermoregulatory drive.
Norepinephrine (NE)-induced Ca 2+ signaling in PVAT is mediated by adrenergic receptors with depot-specific roles. All three receptor subtypes contribute in mPVAT and aPVAT, whereas α1a predominates in white adipose tissue. Internal Ca 2+ stores are the primary Ca 2+ source for NE-induced signaling across depots. Ca 2+ imaging with selective agonists supports α1a’s role, and NO imaging highlights depot diversity. Immunofluorescence data confirm receptor expression on adipocytes and vasculature, indicating complex signaling pathways in adipose tissues studied here across depots.
Using a [U- 13 C 6 ]glucose breath test during a 3-h OGTT, we show that normal-weight females oxidize a greater proportion of ingested glucose than males despite similar blood glucose and insulin responses. This sex difference is attenuated in obesity, which reduces overall glucose oxidation. These findings reveal sex-specific regulation of postprandial glucose metabolism not captured by conventional OGTT metrics and highlight breath testing as a sensitive, noninvasive approach for assessing glucose metabolism outside the laboratory.
This study was aimed at systematically characterizing cell-type-specific cholesterol metabolism in human blood samples and investigating the impact of hypercholesterolemia on the composition and gene expression of immune and stromal populations. Single-cell RNA sequencing (scRNA-seq) data from 31 human blood samples, encompassing 472,204 high-quality circulating cells, were analyzed. Pathway activities were assessed via gene set variation analysis (GSVA), and cell-type-specific markers were identified using differential expression analysis. Clinical cholesterol levels were used to stratify samples for comparative analysis of cell abundance and transcriptional changes. Monocytes and macrophages were found to exhibit heightened activity in cholesterol transport and dynamic response pathways. Five monocyte-enriched cholesterol metabolism genes (colony-stimulating factor 3 receptor, formyl peptide receptor 1, myeloid cell nuclear differentiation antigen, macrophage-expressed gene 1, and versican) were identified. Hypercholesterolemia was associated with increased abundance of macrophages, T cells, and natural killer cells, as well as decreased B cells and monocytes. Significant upregulation of proinflammatory genes such as VCAM1 in high-cholesterol conditions was revealed by differential gene expression analysis. Our study provided a comprehensive cell-type-specific atlas of cholesterol metabolism in human blood cells, highlighting metabolic heterogeneity and hypercholesterolemia-induced immune remodeling in the circulation. This resource offered novel insights into cholesterol-mediated immunomodulation.NEW & NOTEWORTHY Monocytes and macrophages display elevated activity in cholesterol transport and dynamic response pathways. Five monocyte-enriched genes (CSF3R, FPR1, MNDA, MPEG1, VCAN) are implicated in cholesterol metabolism regulation. Hypercholesterolemia reshapes circulating immune cell composition and upregulates proinflammatory genes (e.g., VCAM1), mediating immunomodulatory effects.
Children born after preeclampsia, the leading hypertensive disorder of pregnancy, are predisposed to long-term cardiometabolic and reproductive disorders that are likely associated with prenatal exposure to a dysregulated maternal endocrine milieu. We have previously described abnormal pubertal development and hyperandrogenism in female offspring from the preeclamptic-like blood pressure high subline 5 (BPH/5) mouse, recapitulating patterns observed in adolescents born after preeclampsia. Herein, our objective was to elucidate BPH/5 offspring hypothalamic-pituitary-gonadal (HPG) axis programming and the associated hormonal profile of late-gestation BPH/5 dams. Reproductive and metabolic phenotypes of BPH/5 and blood pressure normal subline 3 (BPN/3) offspring were assessed from birth to adulthood, including anogenital distance, pubertal onset, ovarian function, and adiposity. Maternal late-gestation circulating hormones and placental steroidogenic enzymes were also investigated. Anogenital distance, indicative of prenatal androgen exposure, was longer in BPH/5 male and female offspring. BPH/5 female offspring exhibited precocious pubertal onset and, during adulthood, abnormal estrous cycles, increased visceral adiposity, increased serum anti-Mullerian hormone (AMH), and ovarian morphology consistent with a polycystic ovary syndrome (PCOS)-like phenotype. Serum testosterone did not differ between late-gestation BPH/5 and BPN/3 dams. Conversely, serum AMH concentrations were threefold higher in late-gestation BPH/5 dams, a maternal endocrine disruption previously linked to abnormal female fetus HPG axis programming and PCOS-like offspring phenotype. In conclusion, BPH/5 offspring recapitulate the aberrant reproductive phenotype seen in children born after preeclampsia and major PCOS-like characteristics. Furthermore, maternal AMH excess was identified in late-gestation BPH/5 females, highlighting this as a valuable model of the interplay between PCOS and preeclampsia.NEW & NOTEWORTHY This study explored hypothalamic-pituitary-gonadal (HPG) axis development in offspring from the preeclamptic-like BPH/5 mouse model. Female BPH/5 offspring exhibited precocious pubertal development, elevated AMH, abnormal estrous cycles, abnormal ovarian histomorphology, and increased visceral adiposity. Interestingly, male and female offspring had longer anogenital distance, indicative of prenatal androgen exposure. Late-gestation BPH/5 dams exhibited a threefold higher AMH concentration, elucidating a novel developmental link between preeclampsia, offspring HPG axis programming, and postnatal ovarian and endocrine dysfunction.
Cancer cachexia is a wasting condition characterized by muscle loss and reduced quality of life in cancer patients. Although biological sex differences in the progression of cancer cachexia have been increasingly recognized, their role during chemotherapy-treated cancer cachexia remains largely unexplored. We evaluated such potential differences in male and female mice implanted subcutaneously with Colon-26 (C26) allografts. Our novel approach to disentangle the effects of chemotherapy consisted of administering two cycles of 75% of the maximum tolerated dose of 5-fluorouracil, cisplatin, or paclitaxel in the presence and absence of cancer (n = 6-11/condition). Muscles and organs were collected 25 days after tumor implant, and protein turnover markers, gene expression through RNA sequencing, and mitochondrial function were evaluated in gastrocnemius. A two-way factorial analysis was conducted to assess main effects and interactions across groups (P < 0.05). We demonstrate that chemotherapy exhibited preserved fat mass in males and maintained body weight in females. Chemotherapy elicited negative impacts on muscle in both sexes, even with a reduced or absent tumor burden. In males, protein synthesis was lower in the presence of cancer and chemotherapy without corresponding differences in atrogenes. Cluster analysis revealed largest differences in muscle transcriptome between cancer control and C26-paclitaxel in male mice, highlighting altered regulation of ubiquitin-mediated proteolysis. These findings point to divergent mechanisms during protein processing in endoplasmic reticulum regulation in muscle atrophy associated with cancer in the presence of chemotherapy. Our results highlight distinct mechanisms underlying cancer cachexia alone versus the addition of chemotherapy and further indicate that responses to chemotherapy differ between biological sexes.NEW & NOTEWORTHY Our study highlights critical differences in the mechanisms mediating muscle loss between cancer and chemotherapy-treated cancer, particularly those by which proteins are degraded and processed. Our data point to biological sex differences in the responsiveness to chemotherapy agents as well as the wasting response. Major differences were observed between sexes, specifically fat mass preservation in male mice and the preservation of body weight in female mice, both despite significant reduction in muscle mass.
Liver function is impaired in metabolic dysfunction-associated fatty liver disease. Previous studies have demonstrated that oxygen availability in the tissue microenvironment affects adipose tissue and skeletal muscle function, but its hepatic effects remain unclear. This study aimed to investigate the impact of oxygen levels on metabolic pathways in HepG2 cells. Nonlipid-loaded and lipid-loaded HepG2 cells were exposed to different physiological O2 levels (5% and 10%) or standard laboratory conditions (21% O2) for 24 h. Thereafter, we determined lipid content, gene expression of metabolic markers, glycogen content, and glucose release. Furthermore, mitochondrial respiration and glycolytic activity were assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Exposure to 5% O2 increased the expression of the gluconeogenic gene glucose-6-phosphatase catalytic subunit 1 (G6PC1) in both nonlipid-loaded and steatotic HepG2 cells compared with 21% O2 (P < 0.001). Furthermore, 5% O2 decreased the expression of lipogenic genes [sterol regulatory element binding transcription factor 1 (SREBF1), acetyl-CoA carboxylase beta (ACACB), and fatty acid synthase (FASN)] in nonlipid-loaded and/or steatotic cells (all P < 0.05), whereas genes involved in fatty acid oxidation [peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), P < 0.001 and peroxisome proliferator-activated receptor alpha (PPARA), P = 0.038] were downregulated in steatotic cells. Low oxygen exposure increased glycogen content in nonlipid-loaded and steatotic cells (both P < 0.001) and reduced glucose release (P < 0.05). Finally, low oxygen exposure reduced OCR (P < 0.05) and increased glycolysis (P < 0.001) in both nonlipid-loaded and steatotic cells compared with 21%. In conclusion, our findings demonstrate that reduced oxygen availability in the microenvironment has marked effects on metabolic pathways in nonlipid-loaded and steatotic hepatocytes, inducing a metabolic shift to enhanced reliance on glucose as an energy source.NEW & NOTEWORTHY Oxygen availability in the tissue microenvironment affects adipose tissue and skeletal muscle function, but the effects of oxygen levels on hepatic metabolism are unclear. Low oxygen exposure altered expression of genes involved in glucose and lipid metabolism, increased glycogen content, decreased glucose release and oxygen consumption, and increased glycolytic rate compared with exposure to 21% O2 in both nonlipid-loaded and steatotic HepG2 cells, indicative of a shift to enhanced reliance on glucose as an energy source.
The aim of this study was to determine, in vitro, the effect of endothelial cell-derived extracellular vesicles (EEVs) from adults with obesity on brain microvascular endothelial cell nitric oxide (NO) and endothelin (ET)-1 production as well as tissue-type plasminogen activator (t-PA) release. Circulating EEVs (CD144+ extracellular vesicles) were identified, enumerated, and isolated (flow cytometry) from 24 midlife and older sedentary adults (45-71 yr): 12 normal weight [6 M/6 F; body mass index (BMI) ≥18.5 and ≤25 kg/m2] adults and 12 adults with obesity (6 M/6 F; BMI ≥ 30.0 kg/m2). Human cerebral microvascular endothelial cells (hCMECs) were cultured and treated with EEVs from either normal-weight adults or adults with obesity. Expression of phosphorylated (p)-eNOS (Ser1177) was ∼20% lower (31.5 ± 5.6 vs. 39.1 ± 7.9 AU) and p-eNOS (Thr495) expression ∼40% higher (47.3 ± 13.2 vs. 33.4 ± 10.5 AU) in hCMECs treated with EEVs from obese compared with normal weight adults. As a result, NO production was significantly lower (∼20%) in hCMECs treated with EEVs from adults with obesity (4.9 ± 0.4 vs. 5.9 ± 0.5 µmol/L). Cell expression of Big ET-1 (317.8 ± 51.8 vs. 241.6 ± 65.0 AU) and endothelin-converting enzyme (838.3 ± 160.8 vs. 631.8 ± 126.3 AU) as well as ET-1 production (21.9 ± 1.6 vs. 18.0 ± 3.9 pg/mL) were significantly higher in hCMECs treated with EEVs from adults with obesity. t-PA release in response to thrombin was significantly lower in hCMECs treated with EEVs from obese (from 49.3 ± 6.6 to 52.3 ± 8.2 pg/mL) compared with normal weight (from 52.3 ± 8.8 to 62.0 ± 8.1 pg/mL) adults. Circulating EEVs are a potential mediator of obesity-related cerebrovascular dysfunction and stroke risk.NEW & NOTEWORTHY Despite an improved understanding of many of the pathological consequences associated with human obesity, factors that initiate, promote, and accelerate cerebrovascular events are not completely understood. This study provides novel data demonstrating that circulating EEVs from adults with obesity adversely affect eNOS activity, reducing nitric oxide bioavailability, enhancing ET-1 production, and impairing t-PA release in brain microvascular endothelial cells in vitro. These changes in endothelial cell phenotype have been linked with cerebrovascular dysfunction and ischemic stroke risk.
Aerobic exercise training (AET) and numerous dietary interventions, including nitrate and resveratrol supplementation, display overlapping mechanisms affecting mitochondrial bioenergetics and metabolism in diverse tissues. However, it remains unclear if a combination of these interventions results in additive benefits for the prevention of obesity-related comorbidities. To investigate this, C57Bl/6N mice consumed a high-fat diet and remained sedentary (HFD) or performed AET for 6 wk in the absence (HFD + AET) or presence of nitrate + resveratrol supplementation (HFD + AET + NR). As expected, AET attenuated body weight gain, reduced adipocyte cross-sectional area and markers of cellular stress/inflammation within white adipose tissue, and increased mitochondrial respiratory capacity and decreased lipid content within skeletal muscle independent of supplementation. Although AET alone was sufficient to improve glucose tolerance, the addition of +NR provided modest liver-specific enhancements, including increased mitochondrial respiratory capacity, reduced reactive lipid accumulation, and a unique proteomic signature associated with altered amino acid metabolism, corresponding to further reductions in systemic fasting blood glucose levels. These data suggest that although AET remains a primary lifestyle intervention to drive metabolic improvements during high-fat feeding, targeted dietary supplementation may provide tissue-specific enhancements, particularly within the liver, that complement exercise adaptations.NEW & NOTEWORTHY Dietary supplementation may alter the adaptive response to aerobic exercise training (AET). We investigated whether combined dietary nitrate + resveratrol supplementation could complement adaptations to AET in a high-lipid environment. AET attenuates various metabolic impairments associated with high-fat feeding, whereas nitrate + resveratrol supplementation provides modest, liver-specific enhancements. Although AET serves as the primary therapeutic intervention for high-fat diet-induced cardiometabolic disease, targeted dietary supplementation may provide complementary tissue-specific benefits.
