Hypoglycemic mechanism of intestinal bypass surgery in type 2 diabetic rats.

To investigate the effect of duodenal-jejunal bypass (DJB) surgery on postoperative blood glucose in type 2 diabetic rats, and further explore possible mechanisms for the effect of surgical treatment of type 2 diabetes. Forty rats with type 2 diabetes were randomly assigned to 4 groups (n = 10 rats per group), which subsequently underwent DJB, new biliopancreatic diversion (NBPD) or duodenal-jejunal exclusion (DJE) surgery or a sham operation (SHAM). Fasting glucose, 2-h postprandial glucose and blood lipids were measured, and the mRNA in liver and intestinal tissue for bile acid receptor (FXR), as well as the FXR protein expression in the liver tissues were determined. Postprandial blood glucose and fasting TG and FFA in the DJB and NBPD groups were significantly lower than those in the SHAM group and preoperative (p < 0.05) at 8 weeks postoperation. Liver FXR protein was expressed at significantly higher in the DJB and NBPD groups than in the other two (p < 0.05), and the intestinal FXR mRNA in the DJE group were highest. DJB up-regulates the expression of bile acid receptors in the liver and down-regulates those receptors in the intestinal tract via biliopancreatic diversion. This process reduces TG levels, and subsequently any lipotoxicity to islet cells to produce a hypoglycemic effect.

Colesevelam Reduces Ethanol-Induced Liver Steatosis in Humanized Gnotobiotic Mice.

Alcohol-related liver disease is associated with intestinal dysbiosis. Functional changes in the microbiota affect bile acid metabolism and result in elevated serum bile acids in patients with alcohol-related liver disease. The aim of this study was to identify the potential role of the bile acid sequestrant colesevelam in a humanized mouse model of ethanol-induced liver disease. We colonized germ-free (GF) C57BL/6 mice with feces from patients with alcoholic hepatitis and subjected humanized mice to the chronic-binge ethanol feeding model. Ethanol-fed gnotobiotic mice treated with colesevelam showed reduced hepatic levels of triglycerides and cholesterol, but liver injury and inflammation were not decreased as compared with non-treated mice. Colesevelam reduced hepatic cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) protein expression, although serum bile acids were not lowered. In conclusion, our findings indicate that colesevelam treatment mitigates ethanol-induced liver steatosis in mice.

Glucagon-like peptide 1 analogue prevents cholesterol gallstone formation by modulating intestinal farnesoid X receptor activity.

BACKGROUND & AIMS: Cholesterol gallstone disease (CGD) is a common gastrointestinal disease. Liraglutide, an analogue of glucagon-like peptide 1, has been approved to treat type 2 diabetes. Clinical studies have suggested a potential role of liraglutide in CGD. METHODS: Mice were subcutaneously injected with liraglutide, then fed a lithogenic diet. Bile duct cannulation was performed to collect bile output in mice. Intestinal-specific ablation or pharmacological inhibition of farnesoid X receptor (FXR) was used to study its functions in CGD. RESULTS: Liraglutide could protect mice against CGD. Liraglutide treatment increased the biliary concentration of cholesterol, phospholipids and bile acids and thereby decreased the cholesterol saturation index. The resistance to CGD conferred by liraglutide is likely a result of increased bile acid synthesis and efficient bile acid transport. The expression of a key bile acid synthetic enzyme, Cyp7a1, was significantly increased in liraglutide-treated mice. The increased expression of Cyp7a1 resulted from a relieved suppression signal of Fgf15 from the ileum. Mechanistically, liraglutide treatment altered bile acid composition and suppressed FXR activity in the ileum. Genetic ablation or pharmacological inhibition of FXR in the intestine protected mice against CGD. More importantly, intestinal FXR was required for liraglutide-mediated regulation of hepatic expression of Cyp7a1. CONCLUSION: Liraglutide improved CGD by increasing bile acid secretion and decreasing cholesterol saturation index. Liraglutide attenuates the negative feedback inhibition of bile acids through inhibiting intestinal FXR activity. Our results suggest that liraglutide may represent a novel way for treating or preventing cholesterol gallstones in individuals with high risk of CGD.

The effects, underlying mechanism and interactions of dexamethasone exposure during pregnancy on maternal bile acid metabolism.

As important members in steroids related signal pathways, bile acids are very important in regulating substance metabolism and immune homeostasis. However, bile acids are highly cytotoxic, and the excessive accumulation can induce several abnormalities such as cholestatic liver injury. It is known that the bile acid metabolism alters during pregnancy and mostly will not result in pathologies. However, the effect of dexamethasone exposure during pregnancy on bile acid metabolism is still unknown. In this study, pregnant Wistar rats were subcutaneously administered dexamethasone (0.2 mg/kg.d) or saline from gestation day 9-21, while virgin rats were given the same treatment for 13 days. We found that, physiological pregnancy or dexamethasone exposure during non-pregnancy did not affect maternal serum TBA level and liver function. Nevertheless, dexamethasone exposure during pregnancy increased serum TBA level and accompanied with liver injury. Furthermore, we discovered that the conservation of bile acid homeostasis under pregnancy or dexamethasone exposure was maintained through compensatory pathways. However, dexamethasone exposure during pregnancy tipped the balance of liver bile acid homeostasis by increasing classical synthesis and decreasing efflux and uptake. In addition, dexamethasone exposure during pregnancy also increased serum estrogen level and nuclear receptors mRNA expression levels. Finally, two-way ANOVA analysis showed that dexamethasone exposure during pregnancy could induce or facilitate maternal cholestasis and liver injury by up-regulating ERα and CYP7A1 expression. This study confirmed that dexamethasone exposure during pregnancy was related to maternal intrahepatic cholestasis of pregnancy and should be carefully monitored in clinical settings.

Interactions Between Regulatory Variants in CYP7A1 (Cholesterol 7α-Hydroxylase) Promoter and Enhancer Regions Regulate CYP7A1 Expression.

BACKGROUND: CYP7A1 (cholesterol 7α-hydroxylase) catalyzes the rate-limiting step in bile acid biosynthesis from cholesterol-a main pathway for cholesterol removal from the body. CYP7A1 single-nucleotide polymorphisms (SNPs) are associated with total cholesterol and LDL (low-density lipoprotein) levels, risk of cardiovascular diseases, and other phenotypes; however, results are inconsistent, and causative variants remain uncertain, except for a frequent promoter SNP (rs3808607). METHODS: We used chromatin conformation capture (4C assay), chromatin immunoprecipitation qPCR assay in hepatocytes, and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing in hepatocellular carcinoma cell line cells to identify regulatory regions for CYP7A1. We then screened for SNPs located in regulatory regions, testing effects on reporter gene assays and on hepatic CYP7A1 expression by measuring allelic mRNA expression imbalance. RESULTS: 4C assays showed several regions interacting with CYP7A1 promoter. CRISPR-mediated genome editing in hepatocellular carcinoma cell line cells revealed a novel CYP7A1 enhancer and a repressor region, located >10 kb downstream of the CYP7A1 promoter. SNP screening with an allelic mRNA expression imbalance in human livers and reporter gene assays identified a frequent functional SNP (rs9297994) located in the downstream CYP7A1 enhancer region. SNP rs9297994 is in high linkage disequilibrium with promoter SNP rs3808607 but has opposite effects on CYP7A1 mRNA expression. Their combined effects using a 2-SNP model robustly associate with hepatic CYP7A1 mRNA expression, ranging >2 orders of magnitude. Moreover, only the 2-SNP model, but not each SNP alone, is significantly associated with LDL levels, risk of coronary artery disease, statin response, and diabetes mellitus in several clinical cohorts, including CATHGEN (Catheterization Genetics) and Framingham. CONCLUSIONS: Two interacting regulatory SNPs modulate CYP7A1 expression and are associated with risk of coronary artery disease and diabetes mellitus.

Alterations in gene expression in vitamin D-deficiency: Down-regulation of liver Cyp7a1 and renal Oat3 in mice.

The vitamin D-deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D-deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α-hydroxylase (Cyp7a1, rate-limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up-regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D-deficiency. When vitamin D-sufficient or D-deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D-deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D-sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)2 D3 /cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D-deficient mice, while 1,25(OH)2 D3 treatment led to hypercalcemia. In conclusion, vitamin D-deficiency resulted in down-regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.

On the Role of Illness Duration and Nutrient Restriction in Cholestatic Alterations that Occur During Critical Illness.

BACKGROUND AND AIMS: Elevated markers of cholestasis are common in response to critical illness, and associated with adverse outcome. The role of illness duration and of nutrient restriction on underlying molecular pathways of such cholestatic responses have not been thoroughly investigated. METHODS: In a mouse model of surgery- and sepsis-induced critical illness, molecular pathways of cholestasis were investigated up to 7 days. To assess which changes are explained by illness-induced lack of feeding, nutrient-restricted healthy mice were studied and compared with ad libitum fed healthy mice. Furthermore, serum bile acid (BA) concentrations were quantified in 1,114 human patients with either short or long intensive care unit (ICU) stay, matched for type and severity of illness, up to ICU-day-7. RESULTS: In critically ill mice, either evoked by surgery or sepsis, circulating and hepatic BA-levels progressively increased with time from day-3 onward, preceded by unsuppressed or upregulated CYP7A1 and CYP27A1 protein expression. From 30 h onward, nuclear farnesoid-X-receptor-retinoid-X-receptor staining was significantly suppressed in both critically ill groups, followed from day-3 onward by decreased gene expression of the apical exporter BA-specific export pump and increased expression of basolateral exporters multidrug resistance-associated protein 3 (MRP3) and MRP4. Nutrient restriction in healthy mice only partly mirrored illness-induced alterations in circulating BA and BA-transporters, without changing nuclear receptors or synthesis markers expression. Also in human critically ill patients, serum BA increased with time in long-stay patients only, similarly for patients with or without sepsis. CONCLUSIONS: Circulating BA concentrations rose days after onset of sepsis- and surgery-induced, critical illness, only partially explained by lack of feeding, preceded by suppressed nuclear feedback-sensors and ongoing BA synthesis. Expression of transporters suggested ongoing reversed BA-flow toward the blood.

Improvement of hepatogenic differentiation of iPS cells on an aligned polyethersulfone compared to random nanofibers.

The application of stem cells holds great promises in cell and tissue transplants. This study was designed to compare the hepatogenic differentiation of iPSCs on aligned PES/COL versus random. Aligned and random PES/COL nanofibrus scaffolds were fabricated by electrospining and their surface modified through plasma treatment and collagen coating. The scaffolds were characterized using scanning electron microscopy (SEM) and ATR-FTIR. Morphology and biochemical activities of the differentiated hepatocyte-like cells (HLCs) were examined after 5 and 20 days of differentiation. Real-Time RT-PCR and ICC showed no significant difference in the mRNA and protein levels of two important definitive endoderm specific markers, including Sox17 and Foxa2 between two scaffolds. However, Real-Time RT-PCR analysis indicated an increase in the expression of Cyp7A1 gene over the period of the differentiation procedure on the aligned nanofibers but there was no difference in other genes such as Albumin and CK19. Moreover, comparison of hepatogenic differentiation evaluated by Albumin production in conditioned media of HLCs differentiated on aligned PES/COL, showed increase expression of these markers after 20 days compared to that of the random nanofibers. Taken together, the results of this study may indicate that aligned PES/COL nanofibrous scaffolds can improve terminal differentiation of HLCs from iPSCs.

p38α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure.

The p38α mitogen-activated protein kinase (MAPK) has been related to gluconeogenesis and lipid metabolism. However, the roles and related mechanisms of p38α MAPK in intestinal failure (IF)-associated liver steatosis remained poor understood. Here, our experimental evidence suggested that p38α MAPK significantly suppressed the fat accumulation in livers of IF patients mainly through two mechanisms. On the one hand, p38α MAPK increased hepatic bile acid (BA) synthesis by upregulating the expression of the rate-limiting enzyme cholesterol 7-α-hydroxylase (CYP7A1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which in turn activated the transcription of the CYP7A1. On the other hand, p38α MAPK promoted fatty acid (FA) ß-oxidation via upregulating peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes carnitine palmitoyltransferase 1A (CPT1A) and peroxisomal acyl-coenzyme aoxidase 1 (ACOX1). Dual luciferase assays indicated that p38α MAPK increased the transcription of PPARα, PGC-1α and CYP7A1 by upregulating their promoters' activities. In addition, in vitro and in vivo assays indicated p38α MAPK negatively regulates the hepatic steatosis by controlling JNK activation. In conculsion, our findings demonstrate that hepatic p38α MAPK functions as a negative regulator of liver steatosis in maintaining BA synthesis and FAO by antagonizing the c-Jun N-terminal kinase (JNK).

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats.

Osteopontin Deficiency Alters Biliary Homeostasis and Protects against Gallstone Formation.

The precipitation of excess biliary cholesterol as solid crystals is a prerequisite for cholesterol gallstone formation, which occurs due to disturbed biliary homeostasis. Biliary homeostasis is regulated by an elaborate network of genes in hepatocytes. If unmanaged, the cholesterol crystals will aggregate, fuse and form gallstones. We have previously observed that the levels of osteopontin (OPN) in bile and gallbladder were reduced in gallstone patients. However, the role and mechanism for hepatic OPN in cholesterol gallstone formation is undetermined. In this study, we found that the expression of hepatic OPN was increased in gallstone patients compared with gallstone-free counterparts. Then, we observed that OPN-deficient mice were less vulnerable to cholesterol gallstone formation than wild type mice. Further mechanistic studies revealed that this protective effect was associated with alterations of bile composition and was caused by the increased hepatic CYP7A1 expression and the reduced expression of hepatic SHP, ATP8B1, SR-B1 and SREBP-2. Finally, the correlations between the expression of hepatic OPN and the expression of these hepatic genes were validated in gallstone patients. Taken together, our findings reveal that hepatic OPN contributes to cholesterol gallstone formation by regulating biliary metabolism and might be developed as a therapeutic target for gallstone treatments.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

BACKGROUND: Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. RESULTS: We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. CONCLUSIONS: HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression.

Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism.

Anti-obesity effect of total phenylpropanoid glycosides from Ligustrum robustum Blume in fatty diet-fed mice via up-regulating leptin.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese folk medicine, the leaves of Ligustrum robustum Blume (LR) were commonly used in the treatment of obesity and hyperlipidemia. This study aimed to evaluate the anti-obesity effect and mechanisms of total phenylpropanoid glycosides from Ligustrum robustum Blume (LRTPG) in fatty diet-fed C57BL/6J mice. MATERIALS AND METHODS: C57BL/6J mice were divided randomly into 6 groups, i.e., control, model, positive (Orlistat 0.12g/kg), and LRTPG at three dosages (0.3, 0.6 or 1.2g/kg), respectively. Control mice were fed with standard diet; the others were fed with fatty diet. After 4 weeks׳ modeling, therapy mice were intragastrically administrated with positive drug or LRTPG for 5 weeks, respectively. Pharmacodynamic effects including body weight, fat weight, Lee׳s index, serum lipid levels, morphological changes and adipocyte area ratio were evaluated. The mechanisms were explored as the factors related to lipids metabolism in gene expressions by real-time PCR, and assured as the protein level of differential gene by Western blotting. RESULTS: The anti-obesity effects of LRTPG in all treated mice were shown as decreased body weight, fat mass, Lee׳s index, total cholesterol (TC) level, and adipocyte area. The mechanisms were demonstrated as elevated mRNA and protein levels of adipose leptin, and consequently decreasing mRNA of adipose acyl coenzyme A: diacylglycerol acyltransferase (DGAT) with increasing mRNA of hepatic cholesterol 7α-hydroxylase (CYP7A1), which led to inhibition of triglyceride (TG) synthesis and promotion of cholesterol catabolism. CONCLUSIONS: The anti-obesity effect of LRTPG in fatty diet-fed mice was related to the up-regulation of leptin, which may provide scientific evidence supporting the traditional usage of LR on obesity in China.

Influence of dietary tender cluster beans (Cyamopsis tetragonoloba) on biliary proteins, bile acid synthesis and cholesterol crystal growth in rat bile.

Tender cluster beans (CBs; Cyamopsis tetragonoloba) are observed to possess anti-lithogenic potential in experimental mice. Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile in addition to supersaturation of cholesterol. This study aimed at evaluating the influence of CB on biliary glycoproteins, low molecular weight (LMW) and high molecular weight (HMW) proteins, cholesterol nucleation time, and cholesterol crystal growth in rat hepatic bile. Groups of rats were fed for 10 weeks with 0.5% cholesterol to render the bile lithogenic. Experimental dietary interventions were: 10% freeze-dried CB, 1% garlic powder or their combination. Incorporation of CB into HCD decreased the cholesterol saturation index in bile, increased bile flow and biliary glycoproteins. Dietary CB prolonged cholesterol nucleation time in bile. Electrophoresis of biliary proteins showed the presence of high concentration of 27 kDa protein which might be responsible for the prolongation of cholesterol nucleation time in the CB fed group. Proteins of 20 kDa and 18 kDa were higher in CB treated animals, while the same were less expressed in HCD group. Biliary proteins from CB fed animals reduced cholesterol crystal growth index which was elevated in the presence of proteins from HCD group. Cholesterol-7α-hydroxylase and cholesterol-27-hydroxylase mRNA expression was increased in CB treated animals contributing to the bile acid synthesis. Thus, the beneficial anti-lithogenic effect of dietary CB which primarily is due to reduced cholesterol saturation index was additionally affected through a modulation of the nucleating and anti-nucleating proteins that affect cholesterol crystallization.

Bile acid flux through portal but not peripheral veins inhibits CYP7A1 expression without involvement of ileal FGF19 in rabbits.

It was proposed that CYP7A1 expression is suppressed through the gut-hepatic signaling pathway fibroblast growth factor (FGF) 15/19-fibroblast growth factor receptor 4, which is initiated by activation of farnesoid X receptor in the intestine rather than in the liver. The present study tested whether portal bile acid flux alone without ileal FGF19 could downregulate CYP7A1 expression in rabbits. A rabbit model was developed by infusing glycodeoxycholic acid (GDCA) through the splenic vein to bypass ileal FGF19. Study was conducted in four groups of rabbits: control; bile fistula + bovine serum albumin solution perfusion (BF); BF + GDCA (by portal perfusion); and BF + GDCA-f (by femoral perfusion). Compared with only BF, BF + GDCA (6 h portal perfusion) suppressed CYP7A1 mRNA, whereas BF + GDCA-f (via femoral vein) with the same perfusion rate of GDCA did not show inhibitory effects. Meanwhile, there was a decrease in ileal FGF19 expression and portal FGF19 protein levels, but an equivalent increase in biliary bile acid outputs in both GDCA perfusion groups. This study demonstrated that portal bile acid flux alone downregulated CYP7A1 expression with diminished FGF19 expression and protein levels, whereas the same bile acid flux reaching the liver through the hepatic artery via femoral vein had no inhibitory effect on CYP7A1. We propose that bile acid flux through the portal venous system may be a kind of "intestinal factor" that suppresses CYP7A1 expression.

Involvement of hepatic IL-1 in the strain-dependent sex differences in serum total cholesterol levels in rats.

The strain and sex differences in serum total cholesterol (TC) levels were examined in F344 and Sprague-Dawley (SD) rats. A sex difference (male

On the formation of 7-ketocholesterol from 7-dehydrocholesterol in patients with CTX and SLO.

A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX.

Loss of small heterodimer partner protects against atherosclerosis in apolipoprotein E-deficient mice.

Small heterodimer partner (SHP) is involved in bile, lipid, and glucose metabolism. The aim of this study was to investigate the effect of SHP on the development of atherosclerosis. Apolipoprotein E knockout (ApoE-/-) mice were crossed with SHP knockout (SHP-/-) mice to generate double knockout (ApoE-/-SHP-/-) mice. ApoE-/- and ApoE-/-SHP-/- male mice were fed a western diet for 20 weeks. Body weight in ApoE-/-SHP-/) mice was significantly lower than that in ApoE-/- mice (37±1 g vs. 42±1 g, p<0.01). Loss of SHP in ApoE-/- mice decreased the size of adipocytes in white adipose tissue and reduced lipid accumulation in the liver. Glucose intolerance was improved in ApoE-/-SHP-/- mice as compared with ApoE-/- mice (p<0.01). There was no statistical difference in non-high density lipoprotein cholesterol levels between ApoE-/-SHP-/- mice and ApoE-/- mice despite an increase of cholesterol 7α-hydroxylase expression in the liver. The proportion of atherosclerotic lesions in the aorta was significantly lower in ApoE-/-SHP-/- mice than in ApoE-/- mice (2.8±2.0% vs. 9.1±1.9%, p<0.01). In conclusion, loss of SHP function can prevent atherosclerosis, and resistance to diet-induced obesity is the primary factor contributing to this protective effect.