William M. Pandak

Bile acids as regulatory molecules

In the past, bile acids were considered to be just detergent molecules derived from cholesterol in the liver. They were known to be important for the solubilization of cholesterol in the gallbladder and for stimulating the absorption of cholesterol, fat-soluble vitamins, and lipids from the intestines. However, during the last two decades, it has been discovered that bile acids are regulatory molecules. Bile acids have been discovered to activate specific nuclear receptors (farnesoid X receptor, preganane X receptor, and vitamin D receptor), G protein coupled receptor TGR5 (TGR5), and cell signaling pathways (c-jun N-terminal kinase 1/2, AKT, and ERK 1/2) in cells in the liver and gastrointestinal tract. Activation of nuclear receptors and cell signaling pathways alter the expression of numerous genes encoding enzyme/proteins involved in the regulation of bile acid, glucose, fatty acid, lipoprotein synthesis, metabolism, transport, and energy metabolism. They also play a role in the regulation of serum triglyceride levels in humans and rodents. Bile acids appear to function as nutrient signaling molecules primarily during the feed/fast cycle as there is a flux of these molecules returning from the intestines to the liver following a meal. In this review, we will summarize the current knowledge of how bile acids regulate hepatic lipid and glucose metabolism through the activation of specific nuclear receptors and cell signaling pathways.—Hylemon, P. B., H. Zhou, W. M. Pandak, S. Ren, G. Gil, and P. Dent. Bile acids as regulatory molecules.

Modulation of the fecal bile acid profile by gut microbiota in cirrhosis.

BACKGROUND & AIMS The 7α-dehydroxylation of primary bile acids (BAs), chenodeoxycholic (CDCA) and cholic acid (CA) into the secondary BAs, lithocholic (LCA) and deoxycholic acid (DCA), is a key function of the gut microbiota. We aimed at studying the linkage between fecal BAs and gut microbiota in cirrhosis since this could help understand cirrhosis progression. METHODS Fecal microbiota were analyzed by culture-independent multitagged-pyrosequencing, fecal BAs using HPLC and serum BAs using LC-MS in controls, early (Child A) and advanced cirrhotics (Child B/C). A subgroup of early cirrhotics underwent BA and microbiota analysis before/after eight weeks of rifaximin. RESULTS Cross-sectional: 47 cirrhotics (24 advanced) and 14 controls were included. In feces, advanced cirrhotics had the lowest total, secondary, secondary/primary BA ratios, and the highest primary BAs compared to early cirrhotics and controls. Secondary fecal BAs were detectable in all controls but in a significantly lower proportion of cirrhotics (p<0.002). Serum primary BAs were higher in advanced cirrhotics compared to the rest. Cirrhotics, compared to controls, had a higher Enterobacteriaceae (potentially pathogenic) but lower Lachonospiraceae, Ruminococcaceae and Blautia (7α-dehydroxylating bacteria) abundance. CDCA was positively correlated with Enterobacteriaceae (r=0.57, p<0.008) while Ruminococcaceae were positively correlated with DCA (r=0.4, p<0.05). A positive correlation between Ruminococcaceae and DCA/CA (r=0.82, p<0.012) and Blautia with LCA/CDCA (r=0.61, p<0.03) was also seen. Prospective study: post-rifaximin, six early cirrhotics had reduction in Veillonellaceae and in secondary/primary BA ratios. CONCLUSIONS Cirrhosis, especially advanced disease, is associated with a decreased conversion of primary to secondary fecal BAs, which is linked to abundance of key gut microbiome taxa.

Prevention of free fatty acid–induced hepatic lipotoxicity by 18β‐glycyrrhetinic acid through lysosomal and mitochondrial pathways

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and affects millions of people worldwide. Despite the increasing prevalence of NAFLD, the exact molecular/cellular mechanisms remain obscure and effective therapeutic strategies are still limited. It is well‐accepted that free fatty acid (FFA)‐induced lipotoxicity plays a pivotal role in the pathogenesis of NAFLD. Inhibition of FFA‐associated hepatic toxicity represents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive component of licorice root extract, has a variety of pharmacological properties including anti‐inflammatory, antioxidant, and immune‐modulating activities. GL has been used to treat hepatitis to reduce liver inflammation and hepatic injury; however, the mechanism underlying the antihepatic injury property of GL is still poorly understood. In this report, we provide evidence that 18 β‐glycyrrhetinic acid (GA), the biologically active metabolite of GL, prevented FFA‐induced lipid accumulation and cell apoptosis in in vitro HepG2 (human liver cell line) NAFLD models. GA also prevented high fat diet (HFD)‐induced hepatic lipotoxicity and liver injury in in vivo rat NAFLD models. GA was found to stabilize lysosomal membranes, inhibit cathepsin B expression and enzyme activity, inhibit mitochondrial cytochrome c release, and reduce FFA‐induced oxidative stress. These characteristics may represent major cellular mechanisms, which account for its protective effects on FFA/HFD‐induced hepatic lipotoxicity. Conclusion: GA significantly reduced FFA/HFD‐induced hepatic lipotoxicity by stabilizing the integrity of lysosomes and mitochondria and inhibiting cathepsin B expression and enzyme activity. (HEPATOLOGY 2008.)

LXRα is the dominant regulator of CYP7A1 transcription

Abstract Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of bile acid biosynthesis. Dietary cholesterol stimulates CYP7A1 transcription via activation of oxysterol receptor, LXRα, whereas bile acids repress transcription through FXR-mediated induction of SHP protein. The aim of this study was to determine the quantitative role that LXR- and FXR-regulated pathways play in regulating CYP7A1 and SHP in both rat and hamster models. In rats fed a 2% cholesterol diet, both SHP and CYP7A1 mRNA levels were elevated. The inability to induce CYP7A1 mRNA levels by cholesterol feeding in hamsters led to a decline in SHP mRNA levels. Elimination of hepatic bile acid flux by cholestyramine or bile fistula resulted in a marked repression of rat SHP mRNA levels. These results suggest that under conditions of both SHP and LXRα activation, stimulatory effect of LXRα overrides the inhibitory effect of FXR and results in an induction of rat CYP7A1 mRNA levels.

Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G‐protein–coupled receptor (GPCR) membrane‐type bile acid receptor (TGR5/M‐BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant‐negative Gαi in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid‐activated phylogenetic family (expressed in HEK293 cells) identified sphingosine‐1‐phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P‐induced activation of ERK1/2 and AKT were significantly inhibited by JTE‐013, a S1P2 antagonist, in primary rat hepatocytes. JTE‐013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P2 in primary rodent hepatocytes. (HEPATOLOGY 2012)

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes.

Bile acid synthesis occurs mainly via two pathways: the “classic” pathway, initiated by microsomal cholesterol 7α-hydroxylase (CYP7A1), and an “alternative” (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the “alternative” pathway. Overexpression of the gene encoding steroidogenic acuteregulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of 14C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.

Randomised clinical trial: Lactobacillus GG modulates gut microbiome, metabolome and endotoxemia in patients with cirrhosis.

Safety of individual probiotic strains approved under Investigational New Drug (IND) policies in cirrhosis with minimal hepatic encephalopathy (MHE) is not clear.

Short course of omeprazole: a better first diagnostic approach to noncardiac chest pain than endoscopy, manometry, or 24-hour esophageal pH monitoring.

Noncardiac chest pain (NCCP) presents as a frequent diagnostic challenge, with patients tending to use a disproportionate level of health care resources. Gastroesophageal reflux disease (GERD) is the most frequent cause of NCCP. Goals To test the efficacy of a potent acid-suppressing agent as a diagnostic test in the evaluation of NCCP and to compare it with three commonly used tests. Study Eighteen men and 24 women, aged 22 to 77 years, who presented with recurrent chest pain complaints of a noncardiac etiology, as determined by rest/stress perfusion imaging with technetium Tc99m sestamibi (MIBI), were enrolled in a prospective, double-blinded, placebo-controlled, crossover trial using high-dose omeprazole. Thirty-seven patients completed both arms of the trial. Findings were compared with those of endoscopy, manometry, and ambulatory 24-hour two-channel esophageal pH monitoring. All patients underwent initial diagnostic upper endoscopy, esophageal manometry, and 24-hour pH monitoring. Patients were then randomly assigned to either placebo or omeprazole (40 mg/d orally twice daily) for 14 days, washed out for 21 days, and then crossed over. Patients symptoms were determined using a Visual Analogue Scale to measure the severity of chest pain before and after each period. Results Seventy-one percent of patients in the omeprazole arm reported improved chest pain, whereas only 18% in the placebo arm did. Abnormal results on manometry (20%), 24-hour pH monitoring (42%), or endoscopy with visual evidence of esophagitis (26%) were found less frequently. Combination of the three tests did not significantly increase their usefulness. In NCCP patients with GERD, as defined by positive results on a 24-hour pH test or presence of esophagitis on endoscopy, omeprazole treatment led to a response in 95% of patients, whereas 90% of GERD-positive patients treated with placebo did not respond. Of NCCP patients determined to be GERD negative, 39% responded to omeprazole. Conclusions Omeprazole as a first diagnostic tool in the evaluation of MIBI-negative NCCP is sensitive and specific for determining the cause of NCCP. Endoscopy, manometry, and 24-hour pH monitoring were not only less sensitive in diagnosing NCCP, but they were significantly more expensive.

Regulation of sterol 12α-hydroxylase and cholic acid biosynthesis in the rat

Abstract Background & Aims: Sterol 12α-hydroxylase (CYP8b1) is required for the biosynthesis of cholic acid (CA) and hence helps determine the ratio of CA to chenodeoxycholic acid (CDCA) in bile. This study examined the in vivo regulation of CYP8b1 in the rat by bile acids, cholesterol, and thyroxine. Methods: The specific activities (SAs), messenger RNA (mRNA) levels, and transcriptional activities of CYP8b1 were determined in intact rats and rats with biliary diversion. Results: CA, CDCA, and deoxycholic acid (DCA), fed as a supplement to the diet, down-regulated CYP8b1 SAs by 99% ± 0%, 72% ± 10%, and 98% ± 1%, respectively. Under these same conditions, mRNA levels decreased by 93% ± 7%, 60% ± 11%, and 93% ± 4%, respectively. Intraduodenal infusion of taurocholate (36 μmol/h · 100 g rat −,1 ) decreased SAs and mRNA levels by 63% ± 8% and 74% ± 8%, respectively. Ursodeoxycholic acid (UDC) and hyocholic acid (HC) feeding increased CYP8b1 SAs by 119% ± 21% and 65% ± 18%, respectively. CA feeding decreased CYP8b1 transcriptional activity by 72%. Complete biliary diversion increased CYP8b1 SAs and mRNA levels by 150% ± 30% and 287% ± 51%, respectively. Cholesterol feeding decreased CYP8b1 mRNA by 39% ± 8%. In intact rats, a single injection of thyroid hormone eliminated CYP8b1 activity. Conclusions: CYP8b1 is transcriptionally down-regulated by hydrophobic but not hydrophilic bile acids. Cholesterol feeding and a single thyroid hormone injection repressed CYP8b1 in the face of induction of cholesterol 7α-hydroxylase (CYP7a1 by the new nomenclature) SAs. These results suggest that cholesterol, thyroid hormone, and hydrophobic bile acids are important regulators of CYP8b1 and consequently of the bile acid pool composition. GASTROENTEROLOGY 2000;118:599-607

Simultaneous measurement of cholesterol 7α-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.