Sterile inflammation, resulting from hepatocyte death and subsequent release of damage-associated molecular patterns (DAMPs), significantly contributes to liver disease pathogenesis. Neutrophils, as primary responders to liver injury, undergo NETosis - an immune response generating neutrophil extracellular traps (NETs), further amplifying inflammatory damage. Extracellular DNA (ecDNA), a major constituent of NETs and released cell fragments, potentiates inflammation through pattern recognition receptor activation. Mitochondrial DNA, released during hepatocyte damage, especially provokes robust immune responses due to its bacterial DNA-like structure and unmethylated CpG motifs. Concurrently, purinergic signaling - particularly via ATP release and its conversion into adenosine by ectonucleotidases CD39 and CD73 - critically modulates immune homeostasis and inflammatory responses. Dysregulated expression of CD39/CD73, driven by altered aryl hydrocarbon receptor (AhR) signaling, exacerbates inflammatory states through disturbed regulatory T (Treg) and T helper (Th) 17 cell balance. Recent insights highlight that neutrophils and NETs not only drive innate inflammatory responses but significantly influence adaptive immunity by modulating T cell differentiation. NET components, such as cathelicidin and histones, actively promote Th17 differentiation while simultaneously impairing Treg functions, thereby sustaining inflammatory conditions. Additionally, T cells reciprocally influence neutrophil activation and recruitment, predominantly through IL-17A production. Detailed mechanisms underlying neutrophil-T cell crosstalk in autoimmune hepatitis, acute liver failure, ischemia/reperfusion injury, alcoholic liver disease, and metabolic dysfunction-associated steatotic liver disease underscore potential therapeutic targets. Future strategies targeting NET formation, ecDNA clearance via DNase therapy, purinergic receptor modulation, and restoring AhR signaling hold promise for effectively attenuating sterile inflammation and immune dysregulation in liver diseases.

Serotonin (5-HT) is a multifunctional signaling molecule in the gastrointestinal (GI) tract. 5-HT synthesis is regulated by the gut microbiota. Microbial dysbiosis has been implicated in visceral pain and persistent alterations in gut function that occur following inflammation. Here we tested the hypothesis that alterations in gut microbiota in a post-inflammatory model of visceral pain contribute to dysregulated 5-HT signaling. We used mice treated with dextran sodium sulfate (DSS) 42 days earlier (post-colitis) or untreated mice as donors for fecal microbiota transplants (FMTs) into germ-free mice to explore changes in enterochromaffin (EC) cell populations, expression of 5-HT synthesis, transport, and degradation genes, levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT release. Significant differences were observed in EC cells, , , and gene expression, 5-HT and 5-HIAA levels and 5-HT release between germ-free mice and mice receiving an FMT from either control or post-colitis donor mice. We observed no differences in the total number of EC cells, , or expression of mice after FMT from post-colitis or control mice. However, there was a significant increase in gene expression in the terminal ileum, an increased 5-HIAA/5-HT ratio in the proximal colon and reduced 5-HT release to mechanical and chemical stimulation in the proximal and distal colon after FMT from post-colitis mice. Collectively, these findings provide additional evidence that the gut microbiota regulates 5-HT signaling. Moreover, they reveal functional changes in EC cell sensitivity in the presence of an altered microbiota after recovery from inflammation.

Excessive intake of fructose and fats disrupts hepatocyte function by overwhelming endoplasmic reticulum (ER) capacity, leading to unresolved protein stress and progression to metabolic dysfunction-associated steatohepatitis (Shepherd EL, Saborano R, Northall E, Matsuda K, Ogino H, Yashiro H, Pickens J, Feaver RE, Cole BK, Hoang SA, Lawson MJ, Olson M, Figler RA, Reardon JE, Nishigaki N, Wamhoff BR, Günther UL, Hirschfield G, Erion DM, Lalor PF. 3: 100217, 2021). Ketohexokinase (KHK), the primary enzyme for fructose metabolism, is increasingly recognized for nonmetabolic roles (Peng C, Yang P, Zhang D, Jin C, Peng W, Wang T, Sun Q, Chen Z, Feng Y, Sun Y. 14: 2959-2976, 2024; Li X, Qian X, Peng LX, Jiang Y, Hawke DH, Zheng Y, Xia Y, Lee JH, Cote G, Wang H, Wang L, Qian CN, Lu Z. 18: 561-571, 2016), but its function in regulating ER proteostasis under nutrient stress remains unclear. We show that steatogenic conditions synergistically induce lipid accumulation and robust KHK expression, accompanied by activation of the IRE1α-XBP1 branch of the unfolded protein response. This adaptive axis was observed in HepG2 cells, primary hepatocytes from Gubra Amylin NASH, (GAN) diet-fed mice, and liver biopsies from MASLD patients, establishing a conserved KHK-IRE1α axis across species. Khk knockdown disrupted this balance, causing accumulation of misfolded and ubiquitinated proteins, proteotoxic stress, and a shift toward PERK-CHOP-driven apoptosis. Similar signatures in Khk-deficient mouse livers further underscore KHK's role in sustaining ER homeostasis. Our findings identify KHK as a dual-function enzyme: a metabolic gatekeeper of fructose flux and a proteostatic regulator that safeguards hepatocyte survival. Although KHK contributes to steatosis, its complete loss destabilizes ER proteostasis, suggesting that selective inhibition of KHK enzymatic activity may offer therapeutic benefit without compromising ER function. This study uncovers a noncanonical role for ketohexokinase (KHK) in maintaining ER proteostasis during nutrient overload. In hepatocytes exposed to fructose and saturated fat, KHK promotes adaptive IRE1α-XBP1 signaling and prevents proteotoxic stress and apoptosis. These findings position KHK as a metabolic checkpoint linking fructose metabolism to ER stress resolution and offer new insight into liver survival pathways relevant to MASLD and MASH.

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. 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.

Fructose consumption contributes to metabolic dysfunction-associated steatohepatitis (MASH). Retatrutide is a novel triple receptor agonist that improves obesity and hepatic steatosis in humans. The aims of this study were to develop a shortened and clinically relevant dietary mouse model of diet-induced steatohepatitis, and to evaluate the effects of a retatrutide intervention in this model. C57BL/6N mice were subjected to a single fructose binge (10 mg/g body wt), or a new 31-day mouse model of diet-induced steatohepatitis using a Western diet, fructose, and sucrose in the drinking water, and a final fructose binge with or without retatrutide. A single fructose binge resulted in significantly elevated alanine aminotransferase (ALT) and hepatic triglyceride levels in female mice after 6 h; male mice showed less hepatotoxicity. The novel 31-day feeding model significantly increased body weight, ALT levels, hepatic triglycerides and cholesterol, and hepatic inflammatory markers in female and male mice compared with their chow-fed controls. The overall hepatic gene expression profile per RNA sequencing of treated mice correlated with that of human MASH in children and adults. Retatrutide intervention over the final 2 weeks of the 31-day mouse model significantly reduced body weight, ALT levels, hepatic triglycerides and cholesterol, and hepatic inflammatory markers in female mice compared with their vehicle-treated counterparts. Our findings indicate that female mice develop more severe liver injury due to a single fructose binge than males. The novel 31-day mouse model induces robust steatohepatitis and correlates with human disease. An intervention with retatrutide improves steatohepatitis in this shortened mouse model. Female mice are more prone to liver injury due to a single fructose binge compared with male mice. The new 31-day mouse model induces robust steatohepatitis in mice and correlates with MASLD in children and adults. An intervention with retatrutide improves steatohepatitis in this novel mouse model, indicating despite its short duration, the model can be used to trial pharmacological interventions.

Lithogenic diet exposure disrupts biliary lipid homeostasis to promote precipitation of excess biliary cholesterol; however, the underlying pathogenic signaling mechanism remains unclear. Protein kinase Cbeta (PKCβ) is involved in regulating hepatic cholesterol and bile acid metabolism. In this study, we aimed to identify the initiating signaling and biological changes in the liver upon loss of hepatic PKCβ function under lithogenic stress. Transcriptome analysis of the liver revealed that hepatic deletion of PKCβ altered the expression of 183 liver genes, 118 of which were upregulated and 65 were downregulated. We identified marked increases in the expression of genes involved in bile acid biosynthesis ( and ) and a decrease in retinol metabolism () as the most relevant changes, with blunted expression of genes involved in bile acid and phosphatidylcholine transporters. Mechanistic studies revealed that the hepatic PKCβ deficiency was associated with reduced ERK1/2 phosphorylation in concert with increased p38 phosphorylation in the liver. Overexpression of PKCβ in the liver blocked p38 activation as well as resulted in increased ERK1/2 phosphorylation and was accompanied by suppression of both and expression, demonstrating that hepatic PKCβ functions as a positive regulator of ERK1/2 to suppress the expression of both genes by antagonizing p38. Furthermore, depletion of liver p38 in PKCβ mice resulted in enhanced ERK1/2 phosphorylation and suppression of and expression. The findings yielded by this study support our understanding of the intricate interplay among PKCβ, p38, and ERK1/2 signaling in vivo and provide valuable insights into potential therapeutic targets for the development of novel strategies to combat cholelithiasis. This study underscores the pivotal role of hepatic PKCβ in controlling biliary lipid composition under lithogenic stress. Our findings on the distinct and combined effects of downstream p38 and ERK1/2 offer key insights into the mechanisms driving lithogenic diet-induced dysregulation of biliary lipid composition. This research reveals that the potential of PKCβ/p38/ERK1/2 signaling axis offers the possibility for the integration of different inputs to modulate the signaling output balancing in a way most appropriate for context.

Mitochondrial bioenergetics and hydrogen peroxide (HO) 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 (Jo), membrane potential (ΔΨ), and HO 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). Jo and ΔΨ were measured simultaneously using an Oroboros Oxygraph-2k respirometer with the cationic rhodamine dye tetramethylrhodamine methyl ester. HO 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 Jo, whereas PM yielded the lowest Jo. Notably, female mitochondria exhibited higher Jo than males across all substrates. Both ΔΨ and HO 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 HO production. 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 HO. Female mitochondria exhibited higher respiration and HO 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.

Radiation exposure impairs rapidly renewing tissues like the intestinal epithelium, yet translational insights from murine models have been limited by species-specific responses. Here, we use human intestinal organoids (HIOs) derived from jejunal epithelium to evaluate human epithelial responses to low-dose proton and photon (γ) radiation. γ irradiation induces a unique developmental and metabolic shift in crypt-like organoids, including enrichment of amino acid metabolism pathways and activation of fetal-associated transcription factors and morphology. Integrated multiomic profiling reveals serotonin biosynthesis as a central regenerative node. HIOs can complement animal models and are emerging as a powerful tool in modeling human radiation responses and identifying candidate biomarkers for intestinal injury. Radiation damages the intestinal epithelium, but murine models often fail to capture human-specific responses. Using human intestinal organoids, we show that γ irradiation triggers a distinct developmental and metabolic reprogramming, including enrichment of amino acid metabolism and induction of fetal-associated transcription factors and morphology. These findings highlight human organoids as a translational platform to model radiation injury and uncover candidate biomarkers for intestinal damage.

Gastroesophageal reflux disease (GERD) is common and often medically refractory. Abnormal gastric myoelectrical function may contribute to pathogenesis. This prospective observational study with matched controls assessed if myoelectrical abnormalities measured using body surface gastric mapping were correlated with reflux measured by 24 hr pH testing, and symptom severity. Gastric Alimetry® was performed simultaneously on patients undergoing 24 hr pH testing for investigation of reflux symptoms, with a standardised 4.5 hr test and validated symptom logging. Data were segmented into 15-minute epochs. Forty subjects were recruited (mean age 46.5 years, 60% female): 20 undergoing pH testing (12 with GERD and 8 symptomatic patients without), and 20 controls. GERD patients displayed lower gastric rhythm stability measured by the Gastric Alimetry® Rhythm-Index (GA-RI) when compared with controls (p=0.011), but not with non-GERD patients (p=0.605). Lower gastric rhythm stability measured by GA-RI was associated with increased esophageal acid exposure (DeMeester score; r=-0.46, p=0.042). Periods of decreased gastric rhythm stability measured by GA-RI were not temporally correlated with reflux (r=0.08, p=0.182), or heartburn severity (r=0.04, p=0.309), but were correlated with nausea (r=-0.22, p<0.001) and excessive fullness (r=-0.28, p<0.001). We demonstrated that gastric rhythm instability is associated with increased symptom severity and overall acid exposure in GERD patients. While there was no temporal link between rhythm instability and heartburn found, rhythm instability was temporally associated with increased nausea and fullness. GA-RI offers an emerging biomarker of concurrent gastric neuromuscular dysfunction in patients with GERD.

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 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. 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 commensals in an ex vivo peritonitis model compared with fluid from conventional LPS-treated animals.

Liver fibrosis is driven by the accumulation of scar tissue in response to injury. Activated hepatic stellate cells (HSCs) secrete fibrogenic proteins that deposit into the extracellular matrix, leading to fibrosis. Increased production of fibrogenic proteins by HSCs leads to ER stress, triggering the Unfolded Protein Response (UPR). The UPR is important in regulating HSC activation and fibrogenesis, but mechanisms driving this regulation are unclear. A key process regulated by the UPR is degradation of misfolded proteins through various pathways, including ER-to-Lysosome-Associated Degradation (ERLAD). ERLAD targets proteins for lysosomal degradation and can involve autophagosomes engulfing portions of the ER, termed ER-phagy. ER-phagy is implicated in degradation of misfolded fibrillar collagen, but its role in fibrogenesis is unknown. We show that collagen I levels are post-translationally regulated by autophagy, and this correlates with ER-phagy receptor expression. Furthermore, activation of HSCs induces ER-phagy flux and expression of ER-phagy receptors, including FAM134B, in a process dependent on UPR transducer ATF6α. Loss of FAM134B decreases intracellular collagen I without affecting COL1A1 mRNA. Moreover, FAM134B deletion blocks TGFβ-induced collagen I deposition despite increased secretion. Together, we show that ER-phagy receptor FAM134B is pivotal for collagen I deposition during fibrogenesis.

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 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 . Consistent with this observation, 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. 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 . These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.

The role of -methyladenosine (mA) RNA methylation in liver regeneration is unclear. This study aimed to determine the role of mA 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 , , or (HEPA1, 3, or 7) in control (Flox) mice. In Flox mice, cyclin D1 protein expression was highest on postoperative (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 mA 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 mA-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 mA RNA methylation being significant in decorating genes involved in fibrinogen synthesis in liver regeneration. This study uncovers a previously unrecognized mechanism for regulation of the fibrinogen pathway in -methyladenosine (mA) RNA methylation-mediated liver regeneration.

DRA (Downregulated in adenoma, SLC26A3) is a major apical intestinal Cl/HCO exchanger, which is expressed in complex and hybrid -glycosylated forms. Although the importance of -glycosylation is evident from the significantly reduced transport activity of non--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 -glycosylation does not affect transport activity through trafficking and localization in these cell compartments. To gain insight into potential intramolecular effects of -glycosylation on DRA, its three-dimensional structure was predicted using AlphaFold3 with complex -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. Deficient -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.

Some forms of Gastroesophageal Reflux Disease (GERD) are associated with crural diaphragm (CD) dysfunction, suggesting that GERD may be influenced by skeletal muscle deficiencies. Skeletal muscle atrophy has been strongly linked to alterations in the ubiquitin-proteasome system, the primary pathway for protein degradation. This study aimed to assess the expression of muscle atrophy-related proteins in the CD of patients with reflux esophagitis compared to those without esophagitis. Additionally, we examined the correlation between these proteins, esophagitis severity, and esophageal acid exposure. CD biopsies were obtained from 15 volunteers (8 males, 7 females; mean age 43 years) during anti-reflux laparoscopic Nissen fundoplication (GERD group) or gallbladder surgery (control group). The GERD group was further classified based on the Los Angeles classification into grades A (n=5), B (n=7), and C (n=3). We analyzed key signaling pathways involved in muscle atrophy, including AKT, pAKT, MuRF-1, and MAFbx/Atrogin-1, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). No significant differences were observed in MuRF-1, pAKT/AKT ratio, or MAFbx/Atrogin-1 expression between the control and GERD groups. However, MuRF-1 expression was significantly elevated in the GERD C group compared to GERD B. The control group showed no differences from GERD A or B. Notably, MuRF-1 expression correlated with esophageal total reflux time in the supine position. These findings suggest that increased MuRF-1 expression may contribute to CD fiber atrophy and weakness in GERD patients, potentially impairing gastroesophageal junction function and influencing 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. 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.

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 knockout mice () 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 deletion did not affect body weight, hepatic lipid accumulation, glucose tolerance, or insulin sensitivity. However, mice exhibited significantly increased hepatic inflammation compared to wild-type controls. These changes were associated with higher hepatic expression of tumor necrosis factor () and interleukin-1b (), 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 () expression, suggesting activation of hepatic stellate cells. In addition, 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 signaling promotes hepatic inflammation, highlighting a neuroimmune mechanism that may be targeted to prevent disease progression. 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 exacerbates hepatic inflammatory responses without affecting steatosis or systemic metabolism. These findings reveal a previously unknown immune mechanism in liver disease progression.

The global population is aging, with one in six people projected to be 65 years or older by 2050. Since people aged 65 and older experience higher rates of morbidity and mortality after burn injury, there is an increased need to develop effective burn treatments in this age group. Heightened morbidity and risk of mortality may stem from increased gut leakiness and death of intestinal epithelial cells of aged individuals. Herein, we used our clinically relevant model of scald burn injury in young and aged mice to ascertain whether the colon, isolated colonic epithelium, and organoids grown from the colon, have deficiencies in cell growth, senescence, and apoptosis pathways. Aged, burn-injured mice displayed increased senescence marker in the colon and isolated epithelium, and displayed a reduction in proliferation marker in the colon when compared to young mice. Changes in senescence and proliferation coincided with a reduction in stem cell marker in the colon and colonic epithelium, suggesting a burn-related reduction in the stemness of the epithelial crypt. While we failed to see histological changes in the colonic epithelium, we observed an increase in pro-apoptotic cleaved caspase 3 and 9 within the colons of aged, burn-injured mice. Finally, there was a decrease in the expression antimicrobial peptide , and not in the colons of aged, burn-injured mice. Taken together, these data suggest that in the colon, disruption of proliferation and apoptosis in aged burn-injured mice occurs primarily in the non-epithelial compartment.

Germline mutations in the gene encoding carboxypeptidase A1 were found in association with chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC). The mutations increase pancreatic disease risk, presumably, by causing proenzyme misfolding and endoplasmic reticulum stress. Previously, we showed that mice that carry the p.N256K misfolding human mutation in the mouse gene develop spontaneous chronic pancreatitis. Here, our aim was to investigate whether mice have increased susceptibility to PDAC induced by a mutation. We generated × () and × × () mice and compared the development of pancreas pathology in the two strains at 1, 3, 6, and 12 mo of age. We observed progressive parenchymal remodeling in both strains, with more rapid changes in mice. Thus, histological analysis revealed loss of normal pancreas parenchyma, extensive fibrosis, and aberrant ductal structures such as acinar-to-ductal metaplasia and precancerous pancreatic intraepithelial neoplasia. At 3 mo, these microscopic changes were significantly more abundant in versus mice. Owing to the massive fibrosis, the pancreas weight of and mice was significantly increased relative to C57BL/6N and controls, with the largest increase observed in 3-mo-old animals. The observations indicate that a misfolding mutation accelerated the development of precancerous lesions and fibro-inflammatory remodeling in the pancreas of mice, providing support for the notion that mutations might be risk factors for human PDAC. Inborn mutations in the gene encoding carboxypeptidase A1 have been proposed to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by causing enzyme misfolding and endoplasmic reticulum stress in the pancreas. Here, we demonstrated in a novel mouse model that a misfolding mutation accelerated the development of precancerous lesions driven by mutant in the pancreas. The observations offer experimental support for the notion that mutations are risk factors for human PDAC.