American Journal of Physiology-Regulatory, Integrative and Comparative Physiology

Hypoxia (low oxygen availability) is a common environmental stressor in estuarine ecosystems that negatively affects fish survival as well as physiological and behavioral responses. However, the effects of hypoxia on the brain remains poorly understood, particularly in non-model species. Here, we investigated how prolonged hypoxia influences neural, vascular, and molecular responses in the brain of the speckled sanddab (Citharichthys stigmaeus), an ecologically relevant estuarine flatfish. Fish were exposed to normoxic or hypoxic conditions for seven days, and responses were assessed using histological analyses of neural proliferation and vascular structure, alongside transcriptomic and proteomic profiling. Hypoxia increased neural cell proliferation and progenitor activation in the hypothalamic nucleus recessus lateralis (NRL) and optic tectum, while reducing survival of newly generated cells. At the tissue level, hypoxia induced region-specific vascular remodeling, characterized by increased vessel area and vessel number without evidence of widespread endothelial proliferation. At the molecular level, transcriptomic and proteomic analyses revealed consistent enrichment of biological processes related to stress responses, development, metabolism, and cellular homeostasis, despite limited overlap between individual genes and proteins. Gene- and protein-level analyses further indicated activation of hypoxia-responsive pathways, including HIF signaling and oxidative stress protection, alongside selective metabolic reprogramming. Together, these findings demonstrate that hypoxia induces multi-level changes in the brain, linking neural plasticity, vascular remodeling, and molecular responses. This integrated response likely supports brain function under reduced oxygen availability in dynamic estuarine environments and highlights the role of the brain in regulating responses to environmental stress.

BackgroundHydrogen sulfide (H2S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H2S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H2S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H2S-mediated vasodilation requires HO-2-derived CO. MethodsPressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H2S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. ResultsH2S Selicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H2S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H2S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. ConclusionsThese findings support a model in which H2S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H2S-mediated vascular reactivity.

The expression and secretion of sulfated colonic-type mucins is a feature of high-risk metaplasias of the gastrointestinal foregut (Barretts esophagus, type III intestinal metaplasia of the stomach, and pancreatic intraepithelial neoplasia). Galectin-3 is a lectin that preferentially associates with galactose modified by a 3-O-sulfate relative to its unmodified counterparts and is upregulated as the tissue transitions to high-risk metaplasia, dysplasia, and cancer. Since both galectin-3 and sulfated glycotopes are aberrantly and concurrently overexpressed in high-risk premalignant and malignant tissue transformations, we sought to investigate the role of galectin-3 in the metaplastic reaction. We found that injury induces the expression of Lgals3 at the RNA and protein levels. Unlike cancer cell lines, we show that in vivo galectin-3 colocalized with sulfomucins in zymogenic granules of the gastric chief cell. Utilizing a synchronous, chemically-induced murine model that produces spasmolytic polypeptide expressing metaplasia, we found that galectin-3 facilitates cathartocytosis of the vesicles it resides in, but not organelles lacking LGALS3. Inhibition of cellular downscaling resulted in delayed expression of the metaplastic transcription factor Sox9 as well as proliferation. Here, we present a new role for galectin-3 in promoting the transition from normal, homeostatic tissue to metaplasia and our data suggest that cathartocytosis represents an unconventional secretory pathway for galectin-3, which has been a matter of controversy as galectins are not secreted via canonical pathways.

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

Heightened sympathetic outflow is a major contributor to the development of hypertension. The hypothalamic paraventricular nucleus (PVN) and the rostral ventrolateral medulla (RVLM) are critical regions for generating and regulating sympathetic activity associated with hypertension. Although presympathetic neural circuitry in the healthy brain is well characterized, it remains unclear whether these pathways undergo alterations in hypertension. Here, we determined presympathetic neural circuits by injecting pseudorabies virus (PRV), a transsynaptic retrograde tracer, into the adrenal gland of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Adult SHR exhibited a significantly greater number of PRV-labeled neurons in the PVN and RVLM, but not in the spinal intermediolateral column, compared with WKY. In contrast, the numbers of PRV-labeled neurons in the PVN and RVLM were comparable between young, prehypertensive SHR and age-matched WKY. Remarkably, long-term treatment with losartan--a brain-penetrant angiotensin II AT1 receptor antagonist-- initiated in young, prehypertensive SHR blunted the age-dependent hypertension development and reversed the increase in neuronal labeling in both the PVN and RVLM. However, losartan treatment had no effects in WKY. Additionally, electrophysiological recordings showed an elevated frequency of miniature excitatory postsynaptic currents in PVN presympathetic neurons of SHR, which was also normalized by long-term losartan treatment. These findings reveal an age-dependent expansion of presympathetic neuronal connectivity from the hypothalamus and brainstem to the adrenal gland during hypertension development in SHR. Enhanced AT1 receptor activity contributes to hypertension by increasing active glutamatergic synaptic input and promoting the recruitment of additional presympathetic neurons in the hypothalamus and brainstem. Key Points1. The numbers of neurons labeled by PRV injected into the adrenal gland are increased in the PVN and RVLM, but not in the spinal cord IML, in adult SHR compared to normotensive WKY. 2. The numbers of neurons in the PVN, RVLM, and spinal cord labeled by PRV injected into the adrenal gland are comparable in young, prehypertensive SHR and age-matched WKY. 3. Losartan treatment, initiated at a young age, blunts the hypertension development and reverses the increased numbers of PRV-labeled neurons in the PVN and RVLM of adult SHR but has no such effects in WKY. 4. The active glutamatergic synapses in PVN presympathetic neurons are elevated in adult SHR, and this elevation is reversed by long-term losartan treatment.

ABSTACTGalectin-8 is a lectin that binds N-acetyllactosamine moieties with preference towards those with acidic, terminal modifications (-O-sialyated, 3-O-sulfated). As CD44-variants (CD44v) are biomarkers of metaplasia and cancer, a hyaluronic acid receptor that modulates STAT3 signaling, and specifically expresses 3-Sialyl-LeA/X glycotopes, we asked whether galectin-8 might play a role in gastric metaplasia. Using a synchronous, chemically induced murine model that produces gastric spasmolytic polypeptide expressing metaplasia (SPEM), we compared Lgals8-/- mice to congenic wild-type C57BL/6J mice. We found that galectin-8 was necessary for membrane localization of CD44v on SPEM cells at the base of the glands, suggesting a physical interaction between galectin-8 and CD44v. Metaplastic glands from Lgals8-/- mice had an increase in nuclear pSTAT3 compared to C57BL/6J mice, suggesting that galectin-8 restrains CD44 -> STAT3 signaling. This effect was more prominent in the neck compared to the base, which has greater abundance of CD44v after injury in Lgals8-/- mice. Derepression of STAT3 signaling may explain why low galectin-8 levels is associated with a worse prognosis in gastric cancer.

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

The formation of inert gas bubbles during decompression can lead to decompression sickness (DCS), a major operational risk for divers, compressed-gas workers, astronauts, and high-altitude aviators. In diving, DCS risk is typically inferred from post-dive ultrasound detection of venous gas emboli (VGE), precluding modification of decompression schedules based on real-time physiological feedback. Two-dimensional ultrasound imaging could provide additional insight into decompression-related physiological changes; however, its use in hyperbaric environments has been largely precluded by fire risk associated with elevated oxygen partial pressures (ppO2) in enclosed spaces. Here, we developed a workflow for operating a programmable ultrasound system under hyperbaric conditions and acquiring ultrasound data from the subclavian vein and calf muscle during decompression. A total of 42 dives were conducted by 26 individuals using a previously characterized dive profile to 132 feet seawater (FSW) for 20 min with 9 min of decompression. Three exposure conditions were evaluated: non-exercising, exercising, and a brief pause at 20 FSW during compression. Twelve dives included programmable ultrasound imaging during decompression. Post-dive VGE responses were consistent with prior reports while demonstrating substantial inter-individual variability and sensitivity to modest profile modifications. VGE were detected in the subclavian vein during decompression in two participants and subsequently confirmed by post-dive echocardiography. Calf muscle ultrasound brightness typically increased from pre-dive to decompression measurements, before decreasing below baseline in the 120 min post dive measurement period. These findings demonstrate the feasibility of programmable ultrasound imaging during human decompression and establish a practical framework for ultrasound operation under hyperbaric conditions. This approach may support future physiological studies and development of automated decompression monitoring technologies. New and NoteworthyThis study demonstrates the first use of a programmable ultrasound system to acquire and quantitatively analyze ultrasound data during human decompression. The approach enabled direct visualization of venous gas emboli during decompression and revealed calf muscle ultrasound signal changes, providing a new tool for investigating physiological responses during decompression that are not accessible through conventional post-dive monitoring.

Aberrant changes in circulating cell-free mitochondrial DNA (ccf-mtDNA) across gestation are associated with adverse pregnancy outcomes. Given the inflammatory properties of ccf-mtDNA via pattern recognition receptors such as Toll-like receptor 9 (TLR9), we hypothesized that extracellular mtDNA induces placental inflammation via TLR9 signaling and that this response differs by fetal sex. Pregnant Sprague-Dawley rats were treated intravenously with purified mtDNA (300 g/kg), nuclear DNA (nDNA), saline, and/or the TLR9 antagonist ODN2088 across five studies. Placental responses were evaluated 4 h (Studies 1-3) and 24 h (Study 4) post-treatment; pregnancy and neonatal outcomes were assessed at delivery (Study 5). Exposure to mtDNA, but not nDNA, increased placental il1{beta}, tnf, and il10 mRNA (p < 0.05), establishing response specificity. mtDNA-induced placental inflammation was fetal sex-dependent: mtDNA increased il6 and il1{beta} mRNA in male placentas (p [&le;] 0.0004) but not female placentas, whereas ifn{gamma} was selectively induced in female placentas (p = 0.0004). TLR9 and MyD88 abundance increased in female but not male placentas, and TLR9 antagonism modified selected inflammatory responses with sex-specific patterns. The 4 h inflammatory transcriptional signature resolved by 24 h, whereas mtDNA exposure was associated with a sex-specific shift in antioxidant enzyme expression persisting to 24 h. Despite no effects on gestational length or neonatal biometrics, mtDNA exposure was associated with a higher estimated stillbirth count per litter (IRR = 4.23, 95% CI [0.89, 20.1], p = 0.069). These findings establish extracellular mtDNA as an acute, sex-differentiated placental inflammatory stimulus with partial TLR9 dependence and a potential impact on fetal viability. New & NoteworthyThis study demonstrates that acute exposure to extracellular mtDNA induces placental inflammatory responses in vivo. This response is specific to mtDNA, fetal-sex dependent, and partially mediated by TLR9, with male and female placentas engaging distinct inflammatory signals within hours of exposure. The biological effects extend beyond the initial inflammatory window, with mtDNA exposure producing lasting, sex-specific changes in antioxidant enzyme expression. mtDNA-exposed dams had higher expected stillbirth counts, suggesting extracellular mtDNA may affect fetal viability.

Objective: Circadian rhythms are frequently disrupted in patients in the intensive care unit (ICU), potentially worsening clinical outcomes. Continuous enteral nutrition throughout the day and night is common in the ICU, but eliminates feeding-fasting cycles that serve as important timing cues for the circadian system. The objective of this study was to determine the effect of providing enteral nutrition in a cyclic daytime pattern, compared with continuous administration, on circadian rhythmicity in critically ill patients in the ICU. Design: Single-center randomized controlled trial Setting: Mixed medical-surgical tertiary intensive care unit in the Netherlands Patients: Adult ICU patients ([&ge;]18 yr) receiving enteral nutrition. Intervention: Patients were randomized to receive either continuous, or cyclic daytime enteral feeding (08:00-20:00), initiated from the start of nutritional support. Measurements and Main Results: Sixty-two ICU patients were enrolled, of whom 51 were included in the per-protocol analysis. While the amplitude of the 24-hour rhythm in core body temperature did not differ significantly between the cyclic daytime and continuous feeding groups (0.17 [interquartile range: 0.09-0.24] vs. 0.20 [0.13-0.30], p=0.182), the 24-hour rhythm in heart rate was enhanced in patients receiving cyclic daytime feeding, as reflected by significantly higher amplitudes and more synchronized peak times. No significant differences in 24-hour rhythmicity were observed between groups for the other vital signs or melatonin. Conclusions: Our findings suggest that cyclic daytime feeding may strengthen circadian rhythms in critically ill patients. Further studies are warranted to evaluate its impact on clinical outcomes.

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by [~]50{+/-}12% and calculated fluid volume displacement by each contraction by [~]35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7{micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9nL versus BODIPY cholesterol: 3.3{+/-}1.2nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11{micro}m, volume displacement: 5.5{+/-}2.4nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

Obesity alters protein metabolism in skeletal muscle, and although exercise and amino acids act synergistically to regulate muscle anabolism in healthy humans, this interaction may be impaired in obesity. We examined whether acute aerobic exercise alters amino acid-stimulated muscle protein synthesis during the immediate postexercise period in subjects with obesity. Sixteen sedentary adults with a body mass index >30 kg/m2 underwent stable-isotope tracer infusion studies to determine mixed-muscle fractional synthesis rate (FSR) in the basal (fasted) state and under two experimental conditions: eight subjects received an amino acid infusion (AA), while another eight performed 45 min of cycling at [~]65% heart rate reserve immediately prior to the amino acid infusion (EX+AA). Amino acid infusion significantly increased muscle protein FSR in AA (P < 0.0001). In contrast, no significant increase was observed in EX+AA (P > 0.05), and the amino acid-stimulated increase in muscle protein FSR in EX+AA was 78% lower than that in the AA (P < 0.01). Amino acid infusion increased plasma amino acid concentrations in both conditions (P < 0.05); however, plasma concentrations of essential and branched-chain amino acids, including leucine, were lower in the EX+AA condition (P < 0.05). Changes in muscle protein FSR were positively associated with plasma leucine concentrations during the amino acid infusion (P < 0.05). These findings suggest that, in humans with obesity, aerobic exercise may abolish amino acid-stimulated muscle protein synthesis during the immediate postexercise period, with implications when considering nutritional strategies designed to optimize muscle anabolism in this population. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/732200v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1c9d4e3org.highwire.dtl.DTLVardef@1b7c399org.highwire.dtl.DTLVardef@18a99aborg.highwire.dtl.DTLVardef@6ed880_HPS_FORMAT_FIGEXP M_FIG C_FIG

Systemic inflammatory diseases can be influenced by dietary intake, with gastrointestinal dysfunction driving both metabolic and behavioural changes mirroring the altered inflammatory profile. Additionally, the use of live biotherapeutic products (LBPs) shows promise for treating metabolic and inflammatory diseases, but their efficacy is limited by poor persistence in inflamed gut environments. Designed to utilize inflammatory byproducts, the LBP EcN::ttr has proven efficacy in the treatment of acute and chronic colitis, however its effects on the metabolic and behavioural patterns remain uncharacterized. We evaluated the effects of EcN::ttr on mice fed a proinflammatory omega-6 PUFA-rich diet. EcN::ttr-treated mice exhibited notable changes in the gut, including an improved expression of tight junction protein occludin, accompanied by reduced serum lipopolysaccharide (LPS) - binding protein, indicating protection against endotoxemia. EcN::ttr improved insulin sensitivity compared to the parental strain, associated with increased hepatic insulin receptor expression and reduced GSK3{beta} activation and endoplasmic reticulum stress. Secondary bile acids in mice treated with EcN::ttr were more abundant, with increases in those associated with resolving diarrhea and bile acid detoxification. Behavioural assessment highlighted a normalization of long-term memory along with a reduction of stress management behaviours. Altogether, EcN::ttr restores gut-liver-brain axis function through coordinated modulation of inflammation, barrier integrity, and bile acid metabolism. HighlightsO_LILive Biotherapeutic Product EcN::ttr, designed with a fitness advantage to survive inflammation, and provides protection against a proinflammatory omega 6-rich diet C_LIO_LIAdministration of EcN::ttr improved metabolic outcomes including increasing insulin sensitivity C_LIO_LIEcN::ttr increased the abundance of secondary bile acids including those that modulate bile acid detoxification C_LIO_LIBehavioural parameters were normalized in mice given EcN::ttr C_LIO_LIEcN::ttr partially normalizes gut-liver-brain axis through restoring barrier integrity, modulating inflammation and improving secondary bile acid metabolism C_LI

BackgroundDietary sodium restriction is a common nutritional and physiological challenge that activates electrolyte-conserving endocrine pathways, but its impact on systemic lipid metabolism remains incompletely defined. We examined whether short-term dietary sodium deprivation alters the circulating lipidome and identifies lipid signatures of metabolic adaptation. MethodsMale Sprague-Dawley rats were maintained on sodium-sufficient (NaS) or sodium-deprived (NaD) diets for 7 days (n=3 per group). Serum lipids were profiled by untargeted LC-MS/MS in positive and negative ion modes. Lipidomic differences were evaluated using class-level and species-level analyses, principal component analysis, volcano plots, heatmaps, and pathway-oriented interpretation. ResultsNaD rats exhibited a distinct serum lipidomic profile compared with NaS controls, indicating global remodeling of circulating lipid composition. Sodium deprivation produced class-specific and species-resolved changes, including selective depletion of subsets of neutral lipid species, prominent wax ester remodeling, increased phosphatidylcholine and lysophosphatidylcholine abundance, and altered acylcarnitine profiles. These signatures are consistent with coordinated changes in lipid storage, membrane phospholipid turnover, and mitochondrial fatty-acid handling. ConclusionsDietary sodium deprivation induces coordinated serum lipidome remodeling in rats, supporting the concept that nutritional electrolyte status can influence systemic lipid metabolism. These exploratory findings identify sodium deprivation as a metabolic stressor linked to neutral lipid mobilization, phospholipid remodeling, and altered mitochondrial substrate handling, and provide a foundation for future mechanistic studies.

BackgroundHypertension, the persistent elevation of blood pressure (BP), is characterized by chronic low-grade inflammation and systemic cytokine release. Circulating cytokines contribute to the development of hypertension and end-organ damage. However, the specific immune profile associated with the progression of hypertension remains unclear. We hypothesize that a plasma cytokine signature reflects early BP changes in older adults. MethodsSeventy participants aged 57-81 years were categorized as normotensive (n = 17), elevated BP (n = 10), or hypertensive (n = 43) based on 24-hour ambulatory BP monitoring and antihypertensive treatment status. Plasma IL-1{beta}, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, and TNF- were quantified using immunoassays. Partial Pearson correlations adjusted for demographic and biochemical covariates were used to assess associations between cytokines, BP, and cytokine-cytokine networks. ResultsIn untreated hypertensive individuals, plasma IL-23 was positively correlated with 24-hour diastolic BP. Antihypertensive treatment was associated with reduced IL-17A concentrations, which are negatively associated with 24-hour systolic BP. In the elevated BP group, IL-21 concentrations were higher than in normotensive individuals. To further characterize the cytokine signature, cytokine-cytokine correlations were examined. IL-23 and IL-17A were positively correlated with most interleukins, whereas TNF- showed few associations. IL-1{beta} exhibited strong correlations with both IL-23 and IL-17A, particularly in untreated participants. ConclusionIL-23 and IL-17A are associated with BP status and are broadly interconnected with other inflammatory cytokines, highlighting the potential importance of the IL-23/IL-17A axis in the hypertension of development. Early alterations in IL-21 in elevated BP may reflect immune changes that precede the onset of hypertension.

The coexistence of obesity and insulin resistance is associated with elevated plasma amino acid concentrations. However, it remains unclear whether adiposity or insulin resistance is the stronger determinant of plasma amino acid dysregulation in this setting. Twenty-two adults (10 women, 12 men) spanning a broad range of body mass index (BMI) and insulin resistance underwent a 75-g oral glucose tolerance test (OGTT) after an overnight fast. Plasma glucose, insulin, and amino acid concentrations were measured serially, and insulin resistance/sensitivity was estimated from OGTT-derived glucose and insulin responses, using the homeostasis model assessment of insulin resistance (HOMA-IR) and the Matsuda insulin sensitivity index (Matsuda-ISI). Principal component analysis (PCA) of fasting plasma amino acid concentrations showed no clear separation by obesity or insulin resistance classifications. In contrast, PCA of OGTT-stimulated plasma amino acid concentrations revealed clearer clustering by BMI, fat mass, and waist circumference, whereas separation by HOMA-IR and Matsuda-ISI was less distinct. Importantly, regression analyses showed that BMI, fat mass, and waist circumference were significant predictors of OGTT-stimulated, but not fasting, amino acid responses, with waist circumference accounting for the greatest proportion of the variance in branched-chain amino acid responses during the OGTT (R2 = 0.54). In conclusion, measures of adiposity, particularly total fat mass and waist circumference, accounted for a greater proportion of the variance in plasma amino acid responses under physiologically stimulated conditions than indices of insulin resistance. These findings support the view that plasma amino acid concentrations reflect adiposity-related metabolic alterations more strongly than insulin resistance.

Tear fluid is amenable to non-invasive and repeated collection, making it a practical specimen for evaluating exercise-related physiological responses. However, the immediate proteome-wide alterations in tear fluid following acute exercise have not been characterised. In this study, we performed quantitative proteomic profiling of paired tear samples from healthy female participants before and immediately after a single exercise session using data-independent acquisition liquid chromatography-tandem mass spectrometry (DIA-LC-MS/MS). Among the 3,173 identified proteins, 744 were significantly altered post-exercise, of which 484 were up-regulated and 260 down-regulated. Functional enrichment analysis revealed that up-regulated proteins were predominantly associated with translation and ribosome biogenesis, whereas down-regulated proteins were involved in glycan metabolism, lysosomal processing, and extracellular matrix organisation. Collectively, these findings indicate that acute exercise elicits a rapid and coordinated reconfiguration of the tear proteome. This investigation provides a molecular basis for understanding exercise-mediated modulation of tear composition and ocular surface homeostasis.