American journal of physiology. Gastrointestinal and liver physiology最新文献

Peritonitis is a well-known complication of bowel perforation and abdominal surgery, leading to sepsis and high mortality. Despite its prevalence and severity, the pathogenesis of peritonitis remains incompletely understood, limiting our ability to develop targeted medical therapies. Specifically, little is known about the determinants of the peritoneal nutrient environment for pathogens. The gut microbiome is a well-established source of infectious bacteria in peritonitis, but whether it also modulates levels of nutrients that enable and sustain these infections remains unknown. Using multiple murine models of peritonitis (lipopolysaccharide and cecal slurry), multiple methods of microbiome modulation (germ-free mice and antibiotic-treated mice), novel ex vivo modeling of peritonitis, and nuclear magnetic resonance (NMR) metabolomics of the peritoneal microenvironment, we performed a series of experiments to determine how the gut microbiome influences peritoneal metabolite concentration during peritonitis. We found that both lipopolysaccharide and cecal slurry peritonitis caused consistent changes in high-abundance peritoneal metabolites and that many of these changes were blunted or completely abrogated in antibiotic-treated and germ-free mice. Moreover, we found that peritoneal washings from septic, microbiome-depleted animals supported less bacterial growth of common intra-abdominal pathogens compared with washings from septic conventional animals. We identified the peritoneal nutrients consumed by two common pathogens from the Enterobacteriaceae family and found that supplementation of gut microbiome-mediated nutrients was sufficient to alter bacterial growth in an ex vivo model. Taken together, we identify the gut microbiome as a key driver of the peritoneal nutrient environment, mediating pathogen growth. These findings suggest that microbiome-targeted therapies could mitigate peritonitis risk.NEW & NOTEWORTHY Peritonitis induced via cecal slurry or LPS induced similar changes in prevalent metabolites in the murine peritoneal microenvironment, increasing glutamine and pyruvate and decreasing glucose and butyrate. Gut microbiome depletion using germ-free mice or antibiotic pretreatment blunted many of these peritoneal metabolite changes. Peritoneal fluid from microbiome-depleted LPS-treated mice exhibited reduced bacterial growth of two Enterobacteriaceae commensals in an ex vivo peritonitis model compared with fluid from conventional LPS-treated animals.

Early-life adversity, including abrupt weaning, imposes significant psychosocial and environmental stress during a critical window of gastrointestinal (GI) development, leading to long-term consequences for gut function and disease susceptibility. In piglets, early weaning profoundly disrupts GI development, altering the intestinal epithelial barrier, reshaping immune function, and inducing lasting changes in the enteric nervous system. Despite these adverse outcomes, the early molecular mechanisms that initiate these alterations and set the gut on a divergent developmental trajectory remain poorly understood. Here, we used RNA sequencing and bioinformatic analyses to delineate early transcriptional changes in the jejunal mucosa of early-weaned male castrates compared with unweaned littermates. Ex vivo Ussing chamber experiments validated functional changes associated with these transcriptional alterations. Weaning triggered rapid transcriptional shifts observable within 3 h, including suppressed mitochondrial energy production and increased glucose transporter expression. Pathway analysis revealed upregulation of ion channel transport genes (KCN, SCN, TRP, SLC) and neurotransmitter receptors (cholinergic, dopaminergic, GABAergic, glutamatergic), indicating early neuronal adaptations. Functional assays confirmed enhanced SGLT-mediated glucose transport and neural-evoked secretory responses 24 h postweaning, supporting transcriptomic findings. These findings reveal previously unexamined early transcriptional and functional changes that may serve as inciting mechanisms altering gut trajectory during this critical developmental window, providing new insight into how psychosocial stress and early weaning contribute to long-term gut dysfunction, with broader implications for preterm birth, neonatal GI injury, and other early-life stressors that impact lifelong GI health.NEW & NOTEWORTHY Early-life stress is linked to long-term gut dysfunction, but the initiating events remain unclear. This study shows that early weaning triggers a rapid, integrated jejunal response involving metabolic suppression, altered glucose transport, and heightened enteric nervous system activity. These findings identify a critical developmental inflection point in gut maturation and offer new mechanistic insight into how early adversity shapes lifelong gastrointestinal health, informing strategies to prevent chronic disease in animals and humans.

Mitochondrial bioenergetics and hydrogen peroxide (H₂O₂) production play a central role in maintaining liver metabolic function and redox balance. Understanding sex dimorphism and substrate dependency in these mitochondrial processes is crucial for elucidating the regulatory mechanisms that govern male versus female differences in liver physiology in health and disease. This study aimed at investigating sex-specific and substrate-dependent alterations in liver mitochondrial respiratory rates (Jo₂), membrane potential (ΔΨ), and H₂O₂ production and their metabolic regulation. Liver mitochondria were isolated from adult male and female Sprague-Dawley rats. Four substrate combinations-pyruvate + malate (PM), glutamate + malate (GM), succinate, and succinate with complex I inhibitor rotenone-were used to determine their impact on the activities of the electron transport chain (ETC) and TCA cycle complexes. Adenosine diphosphate (ADP) was added to determine the influence of substrates on oxidative phosphorylation (OxPhos). Jo₂ and ΔΨ were measured simultaneously using an Oroboros Oxygraph-2k respirometer with the cationic rhodamine dye tetramethylrhodamine methyl ester. H₂O₂ production was measured spectrofluorometrically using the Amplex Red and horseradish peroxidase assay. Our results show that male and female liver mitochondria displayed distinct respiratory patterns for different substrates. GM and succinate yielded higher Jo₂, whereas PM yielded the lowest Jo₂. Notably, female mitochondria exhibited higher Jo₂ than males across all substrates. Both ΔΨ and H₂O₂ production showed substrate-dependent patterns, with females exhibiting higher values than males across all substrates. These findings reveal sex-specific differences in liver mitochondrial function, driven by substrate-dependent engagement of the ETC and TCA cycle complexes toward OxPhos, with females showing higher respiratory capacity and H₂O₂ production.NEW & NOTEWORTHY We examined sex-specific and substrate-dependent differences in liver mitochondrial function of adult SD rats. Liver mitochondria preferentially use GM over PM for respiration, whereas PM produces more H₂O₂. Female mitochondria exhibited higher respiration and H₂O₂ production than males across all substrates, likely driven by hormonal factors and sex-specific regulatory pathways. These findings highlight the importance of both substrate and sex in shaping liver mitochondrial bioenergetics and redox function, offering insight into intrinsic metabolic differences.

DRA (Downregulated in adenoma, SLC26A3) is a major apical intestinal Cl^-/HCO₃^- exchanger, which is expressed in complex and hybrid N-glycosylated forms. Although the importance of N-glycosylation is evident from the significantly reduced transport activity of non-N-glycosylated DRA constructs (DRA-N0), the underlying molecular mechanisms are controversial. Therefore, plasma membrane expression and lipid raft localization of glycosylation-deficient DRA-N0 were analyzed in HEK cells. The activity of DRA-N0 was reduced by 70% compared with the wild-type construct. Absolute expression of DRA-N0 was significantly reduced by ∼57% in the cell lysate and by 34 and 45% in the plasma membrane and in plasma membrane-derived lipid rafts, respectively. These amounts are insufficient to account for the reduction in activity. Furthermore, the statistical analysis did not support a difference in the relative expression of DRA and DRA-N0 in the plasma membrane and in plasma membrane-derived lipid rafts, indicating that N-glycosylation does not affect transport activity through trafficking and localization in these cell compartments. To gain insight into potential intramolecular effects of N-glycosylation on DRA, its three-dimensional structure was predicted using AlphaFold3 with complex N-glycans covalently attached to N153, N161, and N164 in the transport domain. This revealed multiple inward- and outward-facing conformations of the protein. The number of interdomain contacts of the transport domain-bound glycans with the scaffold domain was higher in the inward-facing state. Because substrate release to the cytoplasm represents the rate-limiting step in many transport proteins, this suggests that in DRA, glycans stabilize the inward-facing state facilitating anion transport.NEW & NOTEWORTHY Deficient N-glycosylation decreases DRA transport activity but does not significantly affect trafficking to the plasma membrane or to lipid rafts. Meanwhile, molecular modeling predicts stabilizing interdomain contacts of the glycans, covalently attached to the transport domain, with the scaffold domain having more contacts in the inward-facing state. Favoring the inward-facing state may facilitate more efficacious anion transport, as substrate release from this state into the cytoplasm is a rate limiting step for numerous transport proteins.

The brain regulates liver metabolism through neuroendocrine and autonomic pathways, which can be disrupted in metabolic dysfunction-associated steatotic liver disease (MASLD). Although autonomic dysfunction, including liver neuropathy, has been reported in MASLD, the role of hepatic sympathetic signaling in disease progression remains unclear. Recent studies show that liver innervation is predominantly of a sympathetic nature, suggesting that adrenergic receptors in hepatocytes may influence the pathogenesis of MASLD. We previously identified adrenoceptor alpha-1b (ADRA1B) as the dominant hepatic adrenergic receptor. Here, we hypothesized that ADRA1B plays a protective role in MASLD progression. To test this, we generated hepatocyte-specific Adra1b knockout mice (Adra1b^LKO) and induced MASLD with the Gubra Amylin NASH diet for up to 32 wk. Liver pathology was quantified by automated image analysis (MorphoQuant), and metabolic phenotyping included glucose tolerance, insulin sensitivity, and bile acid composition. Hepatocyte-specific Adra1b deletion did not affect body weight, hepatic lipid accumulation, glucose tolerance, or insulin sensitivity. However, Adra1b^LKO mice exhibited significantly increased hepatic inflammation compared to wild-type controls. These changes were associated with higher hepatic expression of tumor necrosis factor (Tnf) and interleukin-1b (Il1b), as well as an increase in monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). We also observed elevated transforming growth factor beta (TGF-β) and α-smooth muscle actin (Acta2) expression, suggesting activation of hepatic stellate cells. In addition, Adra1b^LKO mice displayed higher circulating bilirubin levels, with no significant alterations in albumin and bile acid pool composition. These findings reveal a previously unrecognized role for hepatic ADRA1B in restraining inflammatory responses in MASLD. Loss of Adra1b signaling promotes hepatic inflammation, highlighting a neuroimmune mechanism that may be targeted to prevent disease progression.NEW & NOTEWORTHY This study identifies the hepatic α1b adrenoceptor (ADRA1B) as a regulator of inflammation in metabolic dysfunction-associated steatotic liver disease (MASLD). Using a hepatocyte-specific knockout model, we show that loss of Adra1b exacerbates hepatic inflammatory responses without affecting steatosis or systemic metabolism. These findings reveal a previously unknown immune mechanism in liver disease progression.

α-1 Antitrypsin deficiency (AATD) is a genetic disorder characterized by accumulation of misfolded Z α-1 antitrypsin (ZAAT) in hepatocytes, leading to liver injury and metabolic dysfunction. There is no therapy to reduce ZAAT accumulation and restore proteostasis. Pioglitazone activates AMP-activated protein kinase (AMPK), enhance autophagy, and modulate endoplasmic reticulum stress responses, suggesting a potential effect on ZAAT clearance. Our objective is to examine whether pioglitazone can protect against AATD-mediated liver disease. Huh7.5 cells expressing ZAAT (HuhZ) and Pi*Z transgenic mice were used to investigate pioglitazone treatment on hepatic ZAAT accumulation, autophagy activation, and AMPK signaling. Histological, molecular, and metabolic analyses were conducted to assess changes in ZAAT content, autophagy markers, AMPK phosphorylation, and proteostasis. Pioglitazone significantly reduced intracellular ZAAT and decreased lipid droplet accumulation in HuhZ cells. Pioglitazone markedly lowered hepatic ZAAT content in Pi*Z mice, suggesting enhanced degradation. This reduction was mediated through the AMPK pathway, indicated by increased phosphorylation of AMPK and ULK1. Pioglitazone induced autophagy, shown by decreased p62 and increased ATG5 and LC3B-II. This is indicative of enhanced autophagy. Although total hepatic AAT levels were reduced, periodic acid-Schiff with diastase-positive ZAAT aggregates exhibited only a downward trend, suggesting these may be more resistant to clearance. These findings demonstrate pioglitazone reduces hepatic ZAAT accumulation by activating AMPK and inducing autophagy in AATD-associated liver disease, supporting its potential for therapeutic repurposing. As pioglitazone is FDA-approved with benefits for metabolic liver health, further studies are warranted to evaluate efficacy in restoring proteostasis and reducing hepatic ZAAT.NEW & NOTEWORTHY α-1 Antitrypsin deficiency (AATD)-mediated liver disease lacks therapies that reduce hepatic ZAAT accumulation and liver manifestations. We demonstrate that pioglitazone activates AMPK and induces autophagy, leading to decreased ZAAT and improved proteostasis in Pi*Z mouse livers and human hepatocyte models. As an FDA-approved drug with metabolic benefits, pioglitazone holds promise for repurposing in AATD-related liver disease. These findings offer a mechanistic rationale for targeting autophagy to alleviate hepatic injury in protein misfolding disorders.

During cholestasis, cholangiocytes become activated, promoting macrophage-associated periductal infiltration and fibrosis. The cholangiocyte-specific mechanisms responsible for these processes are unclear. To gain insight into the cholangiocyte signaling mechanisms contributing to these pathophysiologic processes, mice were fed a 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet for 10 days to induce liver injury and then switched to a chow diet to permit recovery, designated as R days. Profiling of isolated intrahepatic leukocytes by mass spectrometry revealed an abundant CX3CR1⁺ macrophage population on the DDC diet that declined during the recovery period. This observation was confirmed using Cx3cr1^GFP mice. Next, cholangiocytes were isolated from control, DDC, and R15 mice, and RNA sequencing (RNAseq) was performed. Cholangiocyte CX3CL1 expression, the cognate ligand for CX3CR1, increased in DDC-fed mice and returned to basal values by R15, implicating cholangiocytes in CX3CR1⁺ macrophage recruitment. Ingenuity pathway analysis (IPA) of the RNAseq data revealed upregulation of the pathogen-induced cytokine storm pathway in cholangiocytes activated from DDC fed mice, and resolution of this pathway in R15 isolated cholangiocytes. SCENIC regulon analysis identified that NF-Y, a transcription factor complex, was activated only on the DDC diet, but not in control or R15 mice. Finally, siRNA targeted suppression of NF-YA in normal human cholangiocytes (NHC) reduced cholangiocyte expression of the profibrogenic ligand TGFβ1. Consistent with this observation, Tgfβ1 was increased in cholangiocytes from DDC-fed animals that returned to control values at day R15. Collectively, these observations provide mechanistic insights into cholangiocyte pathobiology during cholestasis.NEW & NOTEWORTHY Cholangiocyte pathophysiological activation was examined in a model of murine cholestasis. CX3CR1⁺ macrophages are recruited to the periportal region, likely mediated by cholangiocyte expression of CX3CL1. Cholangiocyte transcriptomics from cholestatic mice display activation of a "pathogen-induced cytokine storm" pathway, and exhibit activation of the transcription factor NF-Y. In human cholangiocytes, NF-Y promotes expression of the profibrogenic ligand TGFβ1. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.

The role of N⁶-methyladenosine (m⁶A) RNA methylation in liver regeneration is unclear. This study aimed to determine the role of m⁶A methylation in liver regeneration after a 70% hepatectomy (HEPA) using liver-specific methyltransferase-like 14 (Mettl14) knockout (KO) male mice. Analysis was conducted on postoperative days 1, 3, or 7 (HEPA1, 3, or 7) in control (Flox) mice. In Flox mice, cyclin D1 protein expression was highest on postoperative day 3 (HEPA3) consistent with a dynamic increase in hepatocyte replication. The abundance of Mettl14 protein presented a similar pattern on HEPA3. Then, we performed hepatectomy in Mettl14 KOs (M14KO) and Flox controls and observed significantly higher postsurgical mortality in mutants. In Flox mice, cyclin D1 protein levels and Ki-67 were markedly increased on HEPA3 compared to sham operation, while being downregulated in M14KO. Characterizing the m⁶A epitranscriptomic changes in Flox mice after hepatectomy and contrasting them to hepatectomy in M14KO in HEPA3 revealed enrichment for gene ontology terms associated with endoplasmic reticulum, inflammation, and apoptosis. Differentially methylated genes in M14KO compared to Flox on HEPA3 were also enriched for peroxisome proliferator-activated receptor (PPAR) and AMPK signaling. Finally, we identified hypomethylated transcripts involved in fibrinogen synthesis, such as Fga, Fgb, and Fgg, by comparing differentially m⁶A-decorated genes in M14KO vs. Flox on HEPA3. Knockdown of fibrinogen leads to suppression of proliferation via activation of p21 protein in AML12 cells. Together, these data point to m⁶A RNA methylation being significant in decorating genes involved in fibrinogen synthesis in liver regeneration.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism for regulation of the fibrinogen pathway in N⁶-methyladenosine (m⁶A) RNA methylation-mediated liver regeneration.