Biliary Cholesterol Secretion Research Articles

Abstract 447: Cholesterol Excretion In Whole-body And Liver-specific ABCG5 ABCG8-deficient Mice

Background: The ABCG5 ABCG8 sterol transporter is the body’s primary defense against the accumulation of dietary sterols. Loss of function mutations in ABCG5 or ABCG8 result in Sitosterolemia, a rare disorder that presents as familial hypercholesterolemia, but is distinguished by recessive genetics and the accumulation of phytosterols in plasma and tissues. Biliary cholesterol is reduced by greater than 80% in ABCG5 ABCG8-deficient mice, but fecal neutral sterols are unaffected, suggesting a G5G8-independent pathway for cholesterol excretion. The objectives of the present study were to determine the ability of whole-body and liver-specific G5G8-deficient mice to maintain fecal neutral sterol output when challenged with cholesterol -enriched diet and determine maximal rates of biliary cholesterol secretion. Methods: Wild-type, whole-body and liver-specific ABCG5 ABCG8-deficient mice were maintained on cereal-based mixed diet, a phytosterol-free purified diet, or a purified diet supplemented with 0.2% cholesterol. Following acclimatization to each diet, fecal neutral sterol output was determined over a three-day period by gas chromatography/mass spectrometry. Maximal biliary cholesterol secretion rates were determined by biliary diversion with bile acid infusion over a range of 0 to 1000 nmol/minute. Biliary lipid secretion was determined by enzymatic colorimetric assays and molecular coupling of cholesterol to bile acid and phospholipid secretion calculated by linear regression. Results & Conclusions: Whole-body G5G8-deficient mice exhibited a 40% reduction in fecal neutral sterol output compared to wild-type mice when maintained on cereal-based or purified, low cholesterol containing diets. Cholesterol feeding resulted in a 10-fold increase in fecal neutral sterols in wild-type mice, an effect not observed in G5G8-deficient mice. These data indicate that ABCG5 ABCG8 is indispensable in opposing the accumulation of dietary cholesterol and that alternative pathways cannot compensate for G5G8 deficiency.

Two gallstone (Lith) genes, Lith9 and Lith18, produces a synergistic effect in enhancing cholelithogenesis by promoting hepatic secretion of biliary cholesterol in female mice

Estrogen is a critical risk factor for cholesterol gallstone disease because women are twice as likely as men to form gallstones. We have identified that the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, is a novel gallstone gene named Lith18 in mice. Supersaturated bile is mainly caused by persistent hepatic cholesterol hypersecretion, which is a prerequisite for gallstone formation. To explore the mechanisms underlying the critical role of GPR30 in promoting hepatic hypersecretion of biliary cholesterol and estrogen-induced gallstones in females, we tested the hypothesis that GPR30 may activate the ATP-binding cassette sterol efflux transporters G5 and G8 (ABCG5/G8), i.e., Lith9, on the canalicular membrane of hepatocytes, leading to a significant increase in hepatic cholesterol output and saturation of bile and enhancing cholelithogenesis. Methods: GPR30 and ABCG8 knockout (KO) mice on a gallstone-resistant AKR genetic background were created, respectively. The biliary and gallstone phenotypes, as well as cholesterol crystallization in gallbladder bile were studied in ovariectomized (OVX) GPR30 KO, ABCG8 KO, and wild-type (WT) mice injected intramuscularly with the potent GPR30-selective agonist G-1 at 0 or 1 μg/day and fed a lithogenic diet for 8 wk. Results: Compared to that in OVX WT mice receiving no G-1, hepatic expression of Abcg5/g8 is significantly increased by G-1 treatment in OVX WT, but not GPR30 or ABCG8 KO, mice fed the lithogenic diet. Moreover, no changes are found in hepatic expression of Abcb4 or Abcb11 for biliary phospholipid and bile acid secretion in these OVX mice treated with G-1 for 8 wk, suggesting that GPR30 activated by G-1 could promote biliary cholesterol hypersecretion by stimulating hepatic expression of Abcg5/G8 in mice. Feeding the lithogenic diet for 8 wk induces gallstone formation in 10% of OVX WT mice receiving no G-1. By contrast, G-1 treatment drastically increases gallstone prevalence to 80% in OVX WT mice. Many solid cholesterol monohydrate crystals embedded within mucin gels are observed in the remaining (20%) mice. However, no gallstones are found in OVX GPR30 KO or ABCG8 KO mice even treated with G-1 and fed the lithogenic diet for 8 wk. Moreover, gallbladder bile is unsaturated with cholesterol in two groups of KO mice. These results show that activation of GPR30 by G-1 enhances susceptibility to gallstone formation by stimulating hepatic Abcg5/g8 expression for promoting hepatic cholesterol hypersecretion in female mice. Conclusions: This study provides direct evidence for the first time that the interaction of GPR30 and ABCG5/G8, i.e., two Lith genes ( Lith9 and Lith18), exerts a synergistic lithogenic effect on promoting hepatic hypersecretion of biliary cholesterol, thereby leading to supersaturated bile and increased prevalence of cholesterol gallstone formation in female mice. NIH R01 DK126369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 314: Liver Specific Deletion Of Insulin Receptor Increases Biliary Cholesterol Secretion Independently Of Plasma Clearance Of HDL

Background: Insulin signaling regulates hepatic fatty acid, cholesterol, and bile acid metabolism. Liver-specific deletion of insulin receptor increases biliary cholesterol secretion and bile acid hydrophobicity, but the impact on hepatic clearance of HDL-cholesterol and transhepatic cholesterol excretion are unknown. The objectives of the present study were to understand the impact of hepatic insulin receptor on hepatic clearance of HDL-cholesterol. Methods: Mice harboring loxP sites within the insulin receptor gene were treated with either a control adeno-associated viral vector (AAV) or an AAV encoding Cre recombinase under the control of the liver-specific thyroxin binding globulin promoter to generate liver insulin receptor knockout (LIRKO) mice. The impact on hepatic clearance of HDL-cholesterol, biliary cholesterol secretion and the regulation of sterol transporting enzymes was determined. In a second experiment, the impact of FGF15/19 signaling and FGFR4 on both hepatic ABCG5 and ABCG8 and biliary lipid metabolism was assessed in control and LIRKO mice using recombinant FGF19 and an antisense oligonucleotide directed against FGFR4. Results & Conclusions: As previously reported, LIRKO mice exhibit decreased glucose tolerance and reduced hepatic insulin signaling. Hepatic SR-BI protein, plasma HDL clearance, hepatic HDL protein, and selective cholesteryl ester (CE) uptake did not differ between control and LIRKO mice. Hepatic ABCG5/ABCG8 protein and biliary cholesterol secretion were increased in LIRKO mice. However, biliary elimination of HDL-CE derived cholesterol was unaffected. These results indicate that the increase in biliary cholesterol secretion is independent of plasma clearance of HDL in the reverse cholesterol transport pathway. Loss of hepatic insulin receptor is associated with a more hydrophobic bile acid pool and an increase in ileal FGF15 mRNA in LIRKO mice. Administration of FGF19 to control mice increased hepatic ABCG5, ABCG8, and biliary cholesterol secretion. However, suppression of FGFR4 in LIRKO mice had no effect on hepatic ABCG5 or ABCG8. In addition, administration of FGF19 to ABCG5 ABCG8-deficient mice did not perturb hepatic cholesterol levels or alter expression of sterol sensing genes.

STUDY OF OCCURANCE OF GALL BLADDER STONES AMONG PATIENTS WITH TYPE 2 DIABETES MELLITUS IN A TERITIARY CARE HOSPITAL

Background: Diabetes could form cholesterol gallstones more frequently because of a reduced gallbladder contraction and increased biliary cholesterol secretion. A meta -analysis study suggested that there is a strong association between diabetes mellitus and the occurrence of gallstone disease. The incidence of gallstones is higher in Type 2 Diabetes both in males and females than the non-diabetics. To study the frequency of gallbladder stones among patients with Objective: Method: type 2 Diabetes mellitus. After institutional ethical committee approval and obtaining written informed consent from the patients, an observational cross-sectional study was conducted on patients admitted in Raja Rajeswari medical college and hospital over a period of one and half year on 100 known case of type II diabetes mellites for more than or equal to 5 years. In the present study, the Results: occurrence of gallbladder stones was 21% among Type 2 diabetes mellitus participants. Even though statistical association could not be proven, a higher HbA1c level was found to be among the study participants with gall stones when compared with the study participants without gall stones. 21% of the study participants were Conclusion: diagnosed with Gall stones. 79% of the study participants were not diagnosed with gall stones

Pectin Feeding Influences Fecal Bile Acid Excretion, Hepatic Bile Acid and Cholesterol Synthesis and Serum Cholesterol in Rats

This study was designed to investigate the effects of pectin on cholesterol and bile acid metabolism and to elucidate the mechanisms involved in its hypolipidemic effect in rats. The key regulatory enzymes in cholesterol and bile acid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cholesterol 7α-hydroxylase, were determined. Circulating, hepatic, and biliary lipid concentrations and fecal bile acid excretion were also measured. Male Wistar rats were fed a fiber-free or a pectin-supplemented (7 g/100 g) diet for 4 wk. Bile flow and the biliary secretion of both bile acids and cholesterol were not significantly different than controls in pectin-fed rats. The addition of pectin to the diet resulted in lower serum and liver cholesterol concentrations (-27 and -17%, respectively). Fecal bile acid excretion (+168%) and the hepatic activity of cholesterol 7α-hydroxylase (+70%) were significantly higher in pectin-fed animals. HMG-CoA reductase activity was also significantly greater (+11%) in the presence of dietary pectin. Results of our study indicate that pectin, by enhancing fecal bile acid excretion, may cause increased hepatic synthesis of bile acids and liver depletion of cholesterol in rats, which results in a higher rate of cholesterol synthesis and reduced serum cholesterol concentrations.

Abstract 439: Abcg5 Abcg8 Are Essential To Maintain Sterol Balance When Challenged With Dietary Cholesterol In Mice

Background: ABCG5/ABCG8 (G5/G8) is the primary biliary cholesterol transporter and accounts for >70% of biliary cholesterol secretion. Mice and humans maintain fecal cholesterol excretion under a variety of conditions that disrupt biliary cholesterol secretion, indicating the presence of a non-biliary route for cholesterol elimination. Cholesterol can be actively secreted from the intestine, a pathway termed transintestinal cholesterol excretion (TICE). The present study investigates rates of intestinal cholesterol secretion in mice lacking G5/G8 to determine if adaptive responses within the gut facilitate cholesterol excretion in the absence of the primary biliary cholesterol transporter. Methods: Wild-type (WT) and whole body Abcg5/Abcg8 deficient mice (G5/G8 KO) were maintained on a plant sterol free (PSF) diet to prevent the development of experimental sitosterolemia and potential confounding effects of biologically active phytosterols. Mice were anesthetized, the gall bladder cannulated, and the intestine perfused with model bile to simultaneously determine biliary and intestinal cholesterol secretion rates. Sterol balance studies were conducted in mice sequentially maintained on PSF diet, a PSF diet supplemented with 0.2% cholesterol, and standard rodent chow. Mice were allowed to acclimate to each diet for 14 days, then placed on wire-bottom cages for fecal collection. At termination of the experiment, basal bile, plasma, and tissues were collected. Fecal neutral sterols (FNS) were analyzed by GC-MS. Results: Despite a substantial reduction in biliary cholesterol secretion in G5/G8 KO mice, differences in intestinal cholesterol secretion rates were only observed in males, with KO mice excreting less cholesterol into the intestinal lumen. FNS did not differ between genotypes when maintained on the PSF diet. On chow diet, FNS increased 2x in both genotypes, but did not differ across genotypes. When PSF diet was supplemented with 0.2% cholesterol, FNS increased by 3x in WT mice, but were unchanged in G5/G8 KO mice, relative to PSF diet levels. Conclusions: Abcg5/Abcg8 is essential to maintain sterol balance when cholesterol is present in the diet. TICE is unable to compensate for Abcg5/Abcg8 deficiency in cholesterol fed mice.

Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion

Cholesterol gallstone disease is a worldwide common disease. Cholesterol supersaturation in gallbladder bile is the prerequisite for its pathogenesis, while the mechanism is not completely understood. In this study, we find enrichment of gut microbiota (especially Desulfovibrionales) in patients with gallstone disease. Fecal transplantation of gut microbiota from gallstone patients to gallstone-resistant strain of mice can induce gallstone formation. Carrying Desulfovibrionales is associated with enhanced cecal secondary bile acids production and increase of bile acid hydrophobicity facilitating intestinal cholesterol absorption. Meanwhile, the metabolic product of Desulfovibrionales, H2S increase and is shown to induce hepatic FXR and inhibit CYP7A1 expression. Mice carrying Desulfovibrionales present induction of hepatic expression of cholesterol transporters Abcg5/g8 to promote biliary secretion of cholesterol as well. Our study demonstrates the role of gut microbiota, Desulfovibrionales, as an environmental regulator contributing to gallstone formation through its influence on bile acid and cholesterol metabolism.

Cholesterol-to-Coprostanol Conversion by the Gut Microbiota: What We Know, Suspect, and Ignore.

Every day, up to 1 g of cholesterol, composed of the unabsorbed dietary cholesterol, the biliary cholesterol secretion, and cholesterol of cells sloughed from the intestinal epithelium, enters the colon. All cholesterol arriving in the large intestine can be metabolized by the colonic bacteria. Cholesterol is mainly converted into coprostanol, a non-absorbable sterol that is excreted in the feces. Interestingly, cholesterol-to-coprostanol conversion in human populations is variable, with a majority of high converters and a minority of low or inefficient converters. Two major pathways have been proposed, one involving the direct stereospecific reduction of the Δ5 double bond direct while the indirect pathway involves the intermediate formation of 4-cholelesten-3-one and coprostanone. Despite the fact that intestinal cholesterol conversion was discovered more than a century ago, only a few cholesterol-to-coprostanol-converting bacterial strains have been isolated and characterized. Moreover, the responsible genes were mainly unknown until recently. Interestingly, cholesterol-to-coprostanol conversion is highly regulated by the diet. Finally, this gut bacterial metabolism has been linked to health and disease, and recent evidence suggests it could contribute to lower blood cholesterol and cardiovascular risks.

MiRNA-223 Suppresses Mouse Gallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Depletion of hepatic forkhead box O1 does not affect cholelithiasis in male and female mice

Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.

Recent Advances in the Critical Role of the Sterol Efflux Transporters ABCG5/G8 in Health and Disease.

Cardiovascular disease is characterized by lipid accumulation, inflammatory response, cell death, and fibrosis in the arterial wall and is the leading cause of morbidity and mortality worldwide. Cholesterol gallstone disease is caused by complex genetic and environmental factors and is one of the most prevalent and costly digestive diseases in the USA and Europe. Although sitosterolemia is a rare inherited lipid storage disease, its genetic studies led to identification of the sterol efflux transporters ABCG5/G8 that are located on chromosome 2p21 in humans and chromosome 17 in mice. Human and animal studies have clearly demonstrated that ABCG5/G8 play a critical role in regulating hepatic secretion and intestinal absorption of cholesterol and plant sterols. Sitosterolemia is caused by a mutation in either the ABCG5 or the ABCG8 gene alone, but not in both simultaneously. Polymorphisms in the ABCG5/G8 genes are associated with abnormal plasma cholesterol metabolism and may play a key role in the genetic determination of plasma cholesterol concentrations. Moreover, ABCG5/G8 is a new gallstone gene, LITH9. Gallstone-associated variants in ABCG5/G8 are involved in the pathogenesis of cholesterol gallstones in European, Asian, and South American populations. In this chapter, we summarize the latest advances in the critical role of the sterol efflux transporters ABCG5/G8 in regulating hepatic secretion of biliary cholesterol, intestinal absorption of cholesterol and plant sterols, the classical reverse cholesterol transport, and the newly established transintestinal cholesterol excretion, as well as in the pathogenesis and pathophysiology of ABCG5/G8-related metabolic diseases such as sitosterolemia, cardiovascular disease, and cholesterol gallstone disease.

Bile acids in glucose metabolism and insulin signalling - mechanisms and research needs.

Of all the novel glucoregulatory molecules discovered in the past 20 years, bile acids (BAs) are notable for the fact that they were hiding in plain sight. BAs were well known for their requirement in dietary lipid absorption and biliary cholesterol secretion, due to their micelle-forming properties. However, it was not until 1999 that BAs were discovered to be endogenous ligands for the nuclear receptor FXR. Since that time, BAs have been shown to act through multiple receptors (PXR, VDR, TGR5 and S1PR2), as well as to have receptor-independent mechanisms (membrane dynamics, allosteric modulation of N-acyl phosphatidylethanolamine phospholipase D). We now also have an appreciation of the range of physiological, pathophysiological and therapeutic conditions in which endogenous BAs are altered, raising the possibility that BAs contribute to the effects of these conditions on glycaemia. In this Review, we highlight the mechanisms by which BAs regulate glucose homeostasis and the settings in which endogenous BAs are altered, and provide suggestions for future research.

Simultaneous Determination of Biliary and Intestinal Cholesterol Secretion Reveals That CETP (Cholesteryl Ester Transfer Protein) Alters Elimination Route in Mice.

Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol-fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway.

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium‐taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B–mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP‐binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 −/− mice. Stimulatory effects of NTCP inhibition were maintained in Sr‐b1 −/− mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high‐density lipoprotein–derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

Molecular Mechanisms for Protection of Hepatocytes against Bile Salt Cytotoxicity.

Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.

Biliary cholesterol secretion: more than a simple ABC.

Biliary cholesterol secretion is a process important for 2 major disease complexes, atherosclerotic cardiovascular disease and cholesterol gallstone disease. With respect to cardiovascular disease, biliary cholesterol secretion is regarded as the final step for the elimination of cholesterol originating from cholesterol-laden macrophage foam cells in the vessel wall in a pathway named reverse cholesterol transport. On the other hand, cholesterol hypersecretion into the bile is considered the main pathophysiological determinant of cholesterol gallstone formation. This review summarizes current knowledge on the origins of cholesterol secreted into the bile as well as the relevant processes and transporters involved. Next to the established ATP-binding cassette (ABC) transporters mediating the biliary secretion of bile acids (ABCB11), phospholipids (ABCB4) and cholesterol (ABCG5/G8), special attention is given to emerging proteins that modulate or mediate biliary cholesterol secretion. In this regard, the potential impact of the phosphatidylserine flippase ATPase class I type 8B member 1, the Niemann Pick C1-like protein 1 that mediates cholesterol absorption and the high density lipoprotein cholesterol uptake receptor, scavenger receptor class B type I, is discussed.

Abstract 597: Disruptions in Hepatic Insulin Signaling Reveal an Enterohepatic Signaling Pathway that Regulates the ABCG5 ABCG8 Sterol Transporter and Biliary Cholesterol Secretion

Insulin resistance is associated with increased risk for cholesterol gallstones as well as the development of diabetic dyslipidemia. HDL is the primary cholesterol carrier in the reverse cholesterol transport (RCT) pathway, the process by which cholesterol is delivered from peripheral organs to the liver for elimination in bile. Therefore, we hypothesized that insulin signaling regulates hepatobiliary cholesterol transport in the RCT pathway. To test the role of insulin signaling, we utilized mice harboring insulin receptor flanked by loxP sites (IR fl/fl ) in combination with adenoassociated viral vectors containing no transgene (empty) or Cre recombinase to generate control and liver insulin receptor knock out (LIRKO) mice, respectively. As with previous LIRKO models, our mice showed markedly reduced insulin receptor mRNA and protein in liver, but not skeletal muscle or adipose tissue, and impaired glucose tolerance. LIRKO mice had increased biliary cholesterol secretion as well as increased expression of the ABCG5 ABCG8 sterol transporter, the primary mediator of biliary cholesterol secretion. Levels of SR-BI, the primary HDL receptor, were unchanged as were rates of HDL clearance from plasma and selective delivery of HDL cholesterol to the liver. We also observed increased ileal fibroblast growth factor (FGF)15 mRNA. Wild-type mice treated with FGF19 exhibited increased ABCG5 ABCG8 protein expression in the liver. Immunofluorescence microscopy was used to examine the subcellular localization of ABCG5 in the liver following FGF19 administration. Under control conditions, G5 appeared in puncta, diffusely distributed within hepatocytes. Following treatment with FGF19, G5 signal intensity was substantially increased and juxtaposed to zonula occcludin-1 (ZO-1), a tight junction protein the delineates the canalicular channels within the liver. In conclusion, depletion of hepatic insulin receptors increases G5G8 abundance and promotes its localization to the canalicular surface. This effect is associated with an increase in ileal FGF15 expression which promotes its localization to the apical surface and drives biliary cholesterol secretion.

Cholesterol Absorption and Synthesis in Vegetarians and Omnivores

Vegetarian diets are considered health-promoting; however, a plasma cholesterol lowering effect is not always observed. We investigate the link between vegetarian-diet-induced alterations in cholesterol metabolism. We study male and female omnivores, lacto-ovo vegetarians, lacto vegetarians, and vegans. Cholesterol intake, absorption, and fecal sterol excretion are measured as well as plasma concentrations of cholesterol and noncholesterol sterols. These serve as markers for cholesterol absorption, synthesis, and catabolism. The biliary cholesterol secretion rate is estimated. Flux data are related to body weight. Individual vegetarian diet groups are statistically compared to the omnivore group. Lacto vegetarians absorb 44% less dietary cholesterol, synthesized 22% more cholesterol, and show no differences in plasma total and LDL cholesterol. Vegan subjects absorb 90% less dietary cholesterol, synthesized 35% more cholesterol, and have a similar plasma total cholesterol, but a 13% lower plasma LDL cholesterol. No diet-related differences in biliary cholesterol secretion and absorption are observed. Total cholesterol absorption is lower only in vegans. Total cholesterol input is similar under all vegetarian diets. Unaltered biliary cholesterol secretion and higher cholesterol synthesis blunt the lowered dietary cholesterol intake in vegetarians. LDL cholesterol is significantly lower only in vegans.

Bile Acid Metabolism in Liver Pathobiology.

Bile acids facilitate intestinal nutrient absorption and biliary cholesterol secretion to maintain bile acid homeostasis, which is essential for protecting liver and other tissues and cells from cholesterol and bile acid toxicity. Bile acid metabolism is tightly regulated by bile acid synthesis in the liver and bile acid biotransformation in the intestine. Bile acids are endogenous ligands that activate a complex network of nuclear receptor farnesoid X receptor and membrane G protein-coupled bile acid receptor-1 to regulate hepatic lipid and glucose metabolic homeostasis and energy metabolism. The gut-to-liver axis plays a critical role in the regulation of enterohepatic circulation of bile acids, bile acid pool size, and bile acid composition. Bile acids control gut bacteria overgrowth, and gut bacteria metabolize bile acids to regulate host metabolism. Alteration of bile acid metabolism by high-fat diets, sleep disruption, alcohol, and drugs reshapes gut microbiome and causes dysbiosis, obesity, and metabolic disorders. Gender differences in bile acid metabolism, FXR signaling, and gut microbiota have been linked to higher prevalence of fatty liver disease and hepatocellular carcinoma in males. Alteration of bile acid homeostasis contributes to cholestatic liver diseases, inflammatory diseases in the digestive system, obesity, and diabetes. Bile acid-activated receptors are potential therapeutic targets for developing drugs to treat metabolic disorders.