Integration of bile proteomics and metabolomics analyses reveals novel insights into different types of gallstones in a high-altitude area.

BACKGROUND: To explore the pathogenesis of different subtypes of gallstones in high-altitude populations from a molecular perspective. METHODS: We collected bile samples from 20 cholesterol gallstone disease (CGD) patients and 20 pigment gallstone disease (PGD) patients. Proteomics analysis was performed by LC/MS DIA, while metabolomics analysis was performed by UPLC- Q-TOF/MS. RESULTS: We identified 154 up-regulated and 196 down-regulated differentially expressed proteins, which were significantly enriched in neurodegenerative diseases, energy metabolism, amino acid metabolism etc. In metabolomics analysis, 20 up-regulated and 63 down-regulated differentially expressed metabolites were identified, and they were significantly enriched in vitamin B6 metabolism. Three pathways of integrated proteomics and metabolomics were significantly enriched: porphyrin and chlorophyll metabolism, riboflavin metabolism and aminoacyl-tRNA biosynthesis. Remarkably, 7 differentially expressed proteins and metabolites showed excellent predictive performance and were selected as potential biomarkers. CONCLUSION: The findings of our metabolomics and proteomics analyses help to elucidate the underlying mechanisms of gallstone formation in high-altitude populations.

Accumulation of rare earth elements in human gallstones: a perspective from dietary and human health.

BACKGROUND: Gallstone disease poses a global threat to human health and is strongly linked to environmental factors. However, there is currently no data on the presence of rare earth elements (REEs) in human gallstones. This paper investigates the concentration and distribution of REEs in gallstones for the first time, aiming to explore the environmental implications on human health. METHODS: A total of 25 gallstone samples were collected in Shanghai and the content of REEs was measured by Inductively coupled plasma-Mass Spectrometry (ICP-MS) to explore the distribution of REEs in gallstones. RESULTS: The concentration of REEs in gallstones ranged from 4.89 to 190.8 ng/g (mean 39.21). In most of the gallstone analyses, REEs have been detected and generally attributed to environmental exposure or food contamination. The Y/Ho ratio of gallstones was lower than that of continental rocks, similar to that in the blood, indicating limited fractionation during fluid transport processes in the gallbladder. CONCLUSIONS: The upper continental crust (UCC)-normalized REEs pattern in gallstones showed depletion of light REEs, while most showed enrichment of heavy REEs. Positive Gd anomalies were found in most samples, while few samples suggested anthropogenic influence. Whether exogenous inputs or in vivo biofractionation lead to changes in REEs fractionated patterns require further analyses.

Association between triglyceride-glucose index and gallstones: a cross-sectional study.

This study used data from the National Health and Nutrition Examination Survey (NHANES) to investigate the relationship between the triglyceride-glucose (TyG) index and gallstones. We evaluated the data collected between 2017 to 2020. To evaluate the relationship between TyG index and gallstones, logistic regression analysis, basic characteristics of participants, subgroup analysis, and smooth curve fitting were utilized. The study included 3870 participants over the age of 20 years, 403 of whom reported gallstones, with a prevalence rate of 10.4%. After adjusting for all confounding factors, the risk of gallstones increased by 41% for each unit increase in the TyG index (OR 1.41, 95% CI 1.07, 1.86). The smooth curve fitting also showed a positive correlation between the TyG index and gallstones. Subgroup analysis revealed a significant positive relationship between the TyG index and the risk of gallstones in those aged < 50 years, women, individuals with total cholesterol levels > 200 mg/dL, individuals with body mass index (BMI) > 25, and individuals without diabetes. The risk of gallstones is positively correlated with a higher TyG index. Thus, the TyG index can be used as a predictor of the risk of gallstones.

Proteomics and metabolomics analyses of urine for investigation of gallstone disease in a high-altitude area.

BACKGROUND: The incidence of gallstones is high in Qinghai Province. However, the molecular mechanisms underlying the development of gallstones remain unclear. METHODS: In this study, we collected urine samples from 30 patients with gallstones and 30 healthy controls. The urine samples were analysed using multi-omics platforms. Proteomics analysis was conducted using data-independent acquisition, whereas metabolomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Among the patients with gallstones, we identified 49 down-regulated and 185 up-regulated differentially expressed proteins as well as 195 up-regulated and 189 down-regulated differentially expressed metabolites. Six pathways were significantly enriched: glycosaminoglycan degradation, arginine and proline metabolism, histidine metabolism, pantothenate and coenzyme A biosynthesis, drug metabolism-other enzymes, and the pentose phosphate pathway. Notably, 10 differentially expressed proteins and metabolites showed excellent predictive performance and were selected as potential biomarkers. CONCLUSION: The findings of our metabolomics and proteomics analyses provide new insights into novel biomarkers for patients with cholelithiasis in high-altitude areas.

Predictive value of bile acids as metabolite biomarkers for gallstone disease: A systematic review and meta-analysis.

BACKGROUND: The profiles of bile acids (BAs) in patients with gallstone disease (GSD) have been found to be altered markedly though in an inconsistent pattern. This study aims to characterize the variation of the BA profiles in GSD patients, thereby to discover the potential metabolite biomarkers for earlier detection of GSD. METHODS: Literature search of eight electronic database in both English and Chinese was completed on May 11, 2023. The qualitative and quantitative reviews were performed to summarize the changes of BA profiles in GSD patients compared with healthy subjects. The concentrations of BAs were adopted as the primary outcomes and the weighted mean differences (WMDs) and 95% confidence interval (CI) were generated by random-effects meta-analysis models. RESULTS: A total of 30 studies were enrolled which included 2313 participants and reported the 39 BAs or their ratios. Qualitative review demonstrated serum Taurocholic Acid (TCA), Glycochenodeoxycholic acid (GCDCA), Glycocholic acid (GCA), Taurochenodeoxycholic acid (TCDCA), Glycodeoxycholic acid (GDCA) and Deoxycholic acid (DCA) were significantly increased in GSD patients compared with healthy subjects. Meta analysis was performed in 16 studies and showed that serum Total BAs (TBA) (WMD = 1.36µmol/L, 95%CI = 0.33; 2.4) was elevated however bile TBA (WMD = -36.96mmol/L, 95%CI = -52.32; -21.6) was declined in GSD patients. GCA (WMD = 0.83µmol/L, 95%CI = 0.06; 1.6) and TCA (WMD = 0.51µmol/L; 95%CI = 0.18; 0.85) were both increased in serum sample; TCDCA (WMD = 2.64mmol/L, 95%CI = 0.16; 5.12) was rising, however GCDCA (WMD = -13.82mmol/L, 95%CI = -21.86; -5.78) was falling in bile sample of GSD patients. The level of serum DCA in the GSD patients was found to be increased by using chromatography, yet decreased by chromatography mass spectrometry. CONCLUSION: The profiles of BAs demonstrated distinctive changes in GSD patients compared with healthy control subjects. Serum GCA, TCA and GCDCA, as the typically variant BAs, presented as a potential marker for earlier diagnosis of GSD, which could facilitate early prophylactic intervention. Yet, further validation of these biomarkers by longitudinal studies is still warranted in the future. PROSPERO registration number CRD42022339649.

Antimicrobial glycoprotein 2 (GP2) in gallstones, bile fluid and peribiliary glands of patients with primary sclerosing cholangitis.

BACKGROUND: Glycoprotein-2 (GP2) IgA is a predictor of disease severity in primary sclerosing cholangitis (PSC). We examined GP2's occurrence in the biliary tract, the site of inflammation. METHODS: GP2 was analyzed using ELISA, immunoblotting, mass spectrometry, and immunohistochemistry. The samples included: 20 bile and 30 serum samples from PSC patients, 23 bile and 11 serum samples from patients with gallstone disease (GD), 15 bile samples from healthy individuals undergoing liver-donation surgery (HILD), 20 extracts of gallstones (GE) obtained during cholecystectomy, and 101 blood-donor sera. RESULTS: Biliary GP2 concentrations were significantly higher in patients with PSC and GD than in HILD (p < 0.0001). Serum GP2 levels were similar in PSC and GD patients, and controls, but lower than in bile (p < 0.0001). GP2 was detected in all 20 GEs. Mass spectrometry identified GP2 in the bile of 2 randomly selected GD and 2 PSC patients, and in none of 2 HILD samples. GP2 was found in peribiliary glands in 8 out of 12 PSC patients, showing morphological changes in acinar cells, but not in GD-gallbladders. CONCLUSIONS: GP2 is present in bile of PSC and GD patients. It is synthesized in the peribiliary glands of PSC patients, supporting a pathogenic role for biliary GP2 in PSC.

Role of Secretory Mucins in the Occurrence and Development of Cholelithiasis.

Cholelithiasis is a common biliary tract disease. However, the exact mechanism underlying gallstone formation remains unclear. Mucin plays a vital role in the nuclear formation and growth of cholesterol and pigment stones. Excessive mucin secretion can result in cholestasis and decreased gallbladder activity, further facilitating stone formation and growth. Moreover, gallstones may result in inflammation and the secretion of inflammatory factors, which can further increase mucin expression and secretion to promote the growth of gallstones. This review systematically summarises and analyses the role of mucins in gallstone occurrence and development and its related mechanisms to explore new ideas for interventions in stone formation or recurrence.

Immunohistochemical inflammation in histologically normal gallbladders containing gallstones.

BACKGROUND: The aim of this study was to establish features of inflammation in histologically normal gallbladders with gallstones and compare the expression of inflammatory markers in acutely and chronically inflamed gallbladders. METHODS: Immunohistochemistry was performed on formalin-fixed paraffin-embedded gallbladders for tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-2R, and substance p in three groups: Group I (n = 60) chronic cholecystitis, Group II (n = 57) acute cholecystitis and Group III (n = 45) histologically normal gallbladders with gallstones. Expression was quantified using the H-scoring system. RESULTS: Median, interquartile range expression of mucosal IL-2R in Groups I (2.65, 0.87-7.97) and II (12.30, 6.15-25.55) was significantly increased compared with group III (0.40, 0.10-1.35, p < 0.05). Submucosal IL-2R expression in Groups I (2.0, 1.12-4.95) and II (10.0, 5.95-14.30) was also significantly increased compared with Group III (0.50, 0.15-1.05, p < 0.05). There was no difference in the lymphoid cell IL-6 expression between Groups I (5.95, 1.60-18.15), II (6.10, 1.1-36.15) and III (8.30, 2.60-26.35, p > 0.05). Epithelial IL-6 expression of Group III (8.3, 2.6-26.3) was significantly increased compared with group I (0.5, 0-10.2, p < 0.05) as was epithelial TNF-α expression in Group III (85.0, 70.50-92.0) compared with Groups I (72.50, 45.25.0-85.50, p < 0.05) and II (61.0, 30.0-92.0, p < 0.05). Lymphoid cell Substance P expression in Groups I (1.90, 1.32-2.65) and II (5.62, 2.50-20.8) was significantly increased compared with Group III (1.0,1.0-1.30, p < 0.05). Epithelial cell expression of Substance P in Group III (121.7, 94.6-167.8) was significantly increased compared with Groups I (75.7, 50.6-105.3, p < 0.05) and II (78.9, 43.5-118.5, p < 0.05). CONCLUSION: Histologically normal gallbladders with gallstones exhibited features of inflammation on immunohistochemistry.

Hif-1α expression targets the TMA/Fmo3/TMAO axis to participate in gallbladder cholesterol stone formation in individuals living in plateau regions.

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.

Gallbladder microbial species and host bile acids biosynthesis linked to cholesterol gallstone comparing to pigment individuals.

Gallstones are crystalline deposits in the gallbladder that are traditionally classified as cholesterol, pigment, or mixed stones based on their composition. Microbiota and host metabolism variances among the different types of gallstones remain largely unclear. Here, the bile and gallstone microbial species spectra of 29 subjects with gallstone disease (GSD, 24 cholesterol and 5 pigment) were revealed by type IIB restriction site-associated DNA microbiome sequencing (2bRAD-M). Among them (21 subjects: 18 cholesterol and 3 pigment), plasma samples were subjected to liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. The microbiome yielded 896 species comprising 882 bacteria, 13 fungi, and 1 archaeon. Microbial profiling revealed significant enrichment of Cutibacterium acnes and Microbacterium sp005774735 in gallstone and Agrobacterium pusense and Enterovirga sp013044135 in the bile of cholesterol GSD subjects. The metabolome revealed 2296 metabolites, in which malvidin 3-(6''-malonylglucoside), 2-Methylpropyl glucosinolate, and ergothioneine were markedly enriched in cholesterol GSD subjects. Metabolite set enrichment analysis (MSEA) demonstrated enriched bile acids biosynthesis in individuals with cholesterol GSD. Overall, the multi-omics analysis revealed that microbiota and host metabolism interaction perturbations differ depending on the disease type. Perturbed gallstone type-related microbiota may contribute to unbalanced bile acids metabolism in the gallbladder and host, representing a potential early diagnostic marker and therapeutic target for GSD.

Improved detection of cholesterol gallstones using quasi-material decomposition images generated from single-energy computed tomography images via deep learning.

In this study, we developed a method for generating quasi-material decomposition (quasi-MD) images from single-energy computed tomography (SECT) images using a deep convolutional neural network (DCNN). Our aim was to improve the detection of cholesterol gallstones and to determine the clinical utility of quasi-MD images. Four thousand pairs of virtual monochromatic images (70 keV) and MD images (fat/water) of the same section, obtained via dual-energy computed tomography (DECT), were used to train the DCNN. The trained DCNN can automatically generate quasi-MD images from the SECT images. Additional SECT images were obtained from 70 patients (40 with and 30 without cholesterol gallstones) to generate quasi-MD images for testing. The presence of gallstones in this dataset was confirmed by ultrasonography. We conducted a receiver operating characteristic (ROC) observer study with three radiologists to validate the clinical utility of the quasi-MD images for detecting cholesterol gallstones. The mean area under the ROC curve for the detection of cholesterol gallstones improved from 0.867 to 0.921 (p = 0.001) when quasi-MD images were added to SECT images. The clinical utility of quasi-MD imaging for detecting cholesterol gallstones was showed. This study demonstrated that the lesion detection capability of images obtained from SECT can be improved using a DCNN trained with DECT images obtained using high-end computed tomography systems.

Plasma campesterol and ABCG5/ABCG8 gene loci on the risk of cholelithiasis and cholecystitis: evidence from Mendelian randomization and colocalization analyses.

The causal relationships between plasma metabolites and cholelithiasis/cholecystitis risks remain elusive. Using two-sample Mendelian randomization, we found that genetic proxied plasma campesterol level showed negative correlation with the risk of both cholelithiasis and cholecystitis. Furthermore, the increased risk of cholelithiasis is correlating with the increased level of plasma campesterol. Lastly, genetic colocalization study showed that the leading SNP, rs4299376, which residing at the ABCG5/ABCG8 gene loci, was shared by plasma campesterol level and cholelithiasis, indicating that the aberrant transportation of plant sterol/cholesterol from the blood stream to the bile duct/gut lumen might be the key in preventing cholesterol gallstone formation.

Identification of changes in bile composition in pancreaticobiliary reflux based on liquid chromatography/mass spectrometry metabolomics.

INTRODUCTION: Pancreaticobiliary reflux (PBR) can induce gallstone formation; however, its pathogenic mechanism remains unclear. In this study, we explored the mechanism of PBR by the non-targeted metabolomic analysis of bile in patients with PBR. OBJECTIVE: The aim of this study was to investigate the pathogenic mechanism in PBR by the non-targeted metabolomic analysis of bile collected during surgery. METHODS: Sixty patients who underwent gallstone surgery at our center from December 2020 to May 2021 were enrolled in the study. According to the level of bile amylase, 30 patients with increased bile amylase ( > 110 U/L) were classified into the PBR group, and the remaining 30 patients were classified into the control group (≤ 110 U/L). The metabolomic analysis of bile was performed. RESULTS: The orthogonal projections to latent structure-discriminant analysis of liquid chromatography mass spectrometry showed significant differences in bile components between the PBR and control groups, and 40 metabolites were screened by variable importance for the projection value (VIP > 1). The levels of phosphatidylcholine (PC) and PC (20:3(8Z,11Z,14Z)/14:0) decreased significantly, whereas the levels of lysoPC (16:1(9z)/0:0), lysoPC (15:0), lysoPC (16:0), palmitic acid, arachidonic acid, leucine, methionine, L-tyrosine, and phenylalanine increased. CONCLUSIONS: Significant differences in bile metabolites were observed between the PBR and control groups. Changes in amino acids and lipid metabolites may be related to stone formation and mucosal inflammation.

Sphincter of Oddi Dysfunction Induces Gallstone by Inhibiting the Expression of ABCB11 via PKC-α.

The abnormal increase of Oddi sphincter pressure and total bile duct pressure may play an important role in the formation of cholesterol stones, but the specific molecular mechanism is still unclear. This study aims to investigate it through in vitro and in vivo experiments. A mouse model of Oddi sphincter dysfunction was constructed by stone-inducing diet. We compared the two groups with PKC-α inhibitor GÖ6976 and PKC-α agonist thymeleatoxin. Oddi sphincter pressure and total bile duct pressure were measured. Biochemical analysis of total cholesterol, bile acid and bilirubin was then conducted. The histopathologic changes of bile duct were observed by HE staining and the ultrastructure of liver cells and surrounding tissues was observed by transmission electron microscopy. Through the above experiments, we found that the change of PKC-α expression may affect the formation process of gallstones. The relationship between PKC-α and ABCB11 was further verified by in vitro and in vivo experiments. Our results suggest that ABCB11 and PKC-α are co-expressed in the tubule membrane of hepatocytes and interact with each other in hepatocytes. The high cholesterol diet further enhances the activation of PKC-α and thus reduces the expression of ABCB11. The formation of cholesterol stones is associated with the down-regulation of ABCB11 expression in the tubule membrane of hepatocytes due to kinase signaling. This is the first study to demonstrate that sphincter of Oddi dysfunction induces gallstones through PKC-α inhibition of ABCB11 expression.

Hyodeoxycholic acid attenuates cholesterol gallstone formation via modulation of bile acid metabolism and gut microbiota.

BACKGROUND & AIMS: Hyodeoxycholic acid (HDCA), a hydrophilic bile acid (BA), may prevent and suppress the formation of cholesterol gallstones (CGs). However, the mechanism by which HDCA prevents CGs formation remains unclear. This study aimed to investigate the underlying mechanism of HDCA in preventing CG formation. METHODS: C57BL/6J mice were fed either a lithogenic diet (LD), a chow diet, or LD combined with HDCA. The concentration of BAs in the liver and ileum were determined using liquid chromatography-mass spectrometry (LC-MS/MS). Genes involved in cholesterol and BAs metabolism were detected using polymerase chain reaction (PCR). The gut microbiota in the faeces was determined using 16S rRNA. RESULTS: HDCA supplementation effectively prevented LD-induced CG formation. HDCA increased the gene expression of BA synthesis enzymes, including Cyp7a1, Cyp7b1, and Cyp8b1, and decreased the expression of the cholesterol transporter Abcg5/g8 gene in the liver. HDCA inhibited LD-induced Nuclear farnesoid X receptor (Fxr) activation and reduced the gene expression of Fgf15 and Shp in the ileum. These data indicate that HDCA could prevent CGs formation partly by promoting BA synthesis in the liver and reduced the cholesterol efflux. In addition, HDCA administration reversed the LD-induced decrease in the abundance of norank_f_Muribaculaceae, which was inversely proportional to cholesterol levels. CONCLUSIONS: HDCA attenuated CG formation by modulating BA synthesis and gut microbiota. This study provides new insights into the mechanism by which HDCA prevents CG formation. LAY SUMMARY: In this study, we found that HDCA supplementation suppressed LD-induced CGs in mice by inhibiting Fxr in the ileum, enhancing BA synthesis, and increasing the abundance of norank_f_Muribaculaceae in the gut microbiota. HDCA can also downregulate the level of total cholesterol in the serum, liver, and bile.

Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice.

BACKGROUND: Cholesterol gallstone disease is a common disease. Reducing cholesterol burden is important to prevent/treat gallstone. In this study, we investigated the application of diosgenin (DG) to prevent the formation of gallstone in mice. METHODS: Adult male C57BL/6J mice were fed with the lithogenic diet (LD) only or LD supplemented with DG or ezetimibe for 8 weeks. Incidences of gallstone formation were documented. Intestine and liver tissues were collected to measure the lipid contents and expression of genes in cholesterol metabolism. Caco2 cells were treated with DG to monitor the regulation on cholesterol absorption and the transcriptional regulation of Npc1l1 gene. Changes of gut microbiota by DG was analyzed. Intraperitoneal injection of LPS on mice was performed to verify its effects on STAT3 activation and Npc1l1 expression in the small intestine. RESULTS: LD led to 100% formation of gallstones in mice. In comparison, dietary DG or ezetimibe supplementary completely prevents gallstones formation. DG inhibited intestinal cholesterol absorption in mice as well as in Caco2 cells by down-regulation of Npc1l1 expression. DG could directly inhibit phosphorylation of STAT3 and its transcriptional regulation of Npc1l1 expression. Furthermore, DG could modulate gut microbiota profiles and LPS mediated STAT3 activation and Npc1l1 expression. CONCLUSION: Our results demonstrated that dietary DG could inhibit intestinal cholesterol absorption through decreasing NPC1L1 expression to prevent cholesterol gallstone formation.

Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation.

OBJECTIVE: Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. METHODS: We first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1-/-) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. RESULTS: We found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1-/- mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. CONCLUSION: Our findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.

The role of hypoxia-inducible factor 1α in hepatic lipid metabolism.

Chronic liver disease is a major public health problem with a high and increasing prevalence worldwide. In the progression of chronic liver disease, steatosis drives the progression of the disease to cirrhosis or even liver cancer. Hypoxia-inducible factor 1α (HIF-1α) is central to the regulation of hepatic lipid metabolism. HIF-1α upregulates the expression of genes related to lipid uptake and synthesis in the liver and downregulates the expression of lipid oxidation genes. Thus, it promotes intrahepatic lipid deposition. In addition, HIF-1α is expressed in white adipose tissue, where lipolysis releases free fatty acids (FFAs) into the blood. These circulating FFAs are taken up by the liver and accumulate in the liver. The expression of HIF-1α in the liver condenses bile and makes it easier to form gallstones. Contrary to the role of hepatic HIF-1α, intestinal HIF-1α expression can maintain a healthy microbiota and intestinal barrier. Thus, it plays a protective role against hepatic steatosis. This article aims to provide an overview of the current understanding of the role of HIF-1α in hepatic steatosis and to encourage the development of therapeutic agents associated with HIF-1α pathways. KEY MESSAGES: • Hepatic HIF-1α expression promotes lipid uptake and synthesis and reduces lipid oxidation leading to hepatic steatosis. • The expression of HIF-1α in the liver condenses bile and makes it easier to form gallstones. • Intestinal HIF-1α expression can maintain a healthy microbiota and intestinal barrier.

Effects of Biliary Phospholipids on Cholesterol Crystallization and Growth in Gallstone Formation.

The prevalence of cholesterol gallstone disease is increasing, primarily due to the global epidemic of obesity associated with insulin resistance, and this trend leads to a considerable healthcare, financial, and social burden worldwide. Although phospholipids play an essential role in maintaining cholesterol solubility in bile through both mixed micelles and vesicles, little attention has been paid to the impact of biliary phospholipids on the pathogenesis of cholesterol gallstone formation. A reduction or deficiency of biliary phospholipids results in a distinctly abnormal metastable physical-chemical state of bile predisposing to supersaturation with cholesterol. Changes in biliary phospholipid concentrations influence cholesterol crystallization by yielding both liquid crystalline and "anhydrous" crystalline metastable intermediates, evolving into classical parallelogram-shaped cholesterol monohydrate crystals in supersaturated bile. As a result, five distinct crystallization pathways, A-E, have been defined, mainly based on the prime habits of liquid and solid crystals in the physiological or pathophysiological cholesterol saturation of gallbladder and hepatic bile. This review concisely summarizes the chemical structures and physical-chemical properties of biliary phospholipids and their physiological functions in bile formation and cholesterol solubility in bile, as well as comprehensively discusses the latest advances in the role of biliary phospholipids in cholesterol crystallization and growth in gallstone formation, largely based on the findings from clinical and animal studies and in vitro experiments. The insights gleaned from uncovering the cholelithogenic mechanisms are expected to form a fundamental framework for investigating the hitherto elusive events in the earliest stage of cholesterol nucleation and crystallization. This may help to identify better measures for early diagnosis and prevention in susceptible subjects and effective treatment of patients with gallstones.

Hepatobiliary Thyroid Hormone Deficiency Impacts Bile Acid Hydrophilicity and Aquaporins in Cholestatic C57BL/6J Mice.

Women are more prone to develop either hypothyroidism or cholesterol gallstones than men. However, a male predominance in cholesterol gallstones under hypothyroidism was reported. Recently, a novel pathogenic link between thyroid hormone (TH) deficiency and cholesterol gallstones has been described in male mice. Here, we investigate if TH deficiency impacts cholesterol gallstone formation in females by the same mechanism. Three-month-old C57BL/6J mice were randomly divided into a control, a TH deficient, a lithogenic, and a lithogenic + TH deficient group and diet-treated for two, four, and six weeks. Gallstone prevalence, liver function tests, bile composition, hepatic gene expression, and gallbladder aquaporin expression and localization were investigated. Cholesterol gallstones were observed in lithogenic + TH deficient but not lithogenic only female mice. Diminished hydrophilicity of primary bile acids due to decreased gene expression of hepatic detoxification phase II enzymes was observed. A sex-specific expression and localization of hepatobiliary aquaporins involved in transcellular water and glycerol permeability was observed under TH deficient and lithogenic conditions. TH deficiency promotes cholesterol gallstone formation in female C57BL/6J mice by the same mechanism as observed in males. However, cholesterol gallstone prevalence was lower in female than male C57BL/6J mice. Interestingly, the sex-specific expression and localization of hepatobiliary aquaporins could protect female C57BL/6J mice to cholestasis and could reduce biliary water transport in male C57BL/6J mice possibly contributing to the sex-dependent cholesterol gallstone prevalence under TH deficiency.