Frans Stellaard

Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

Improved Glycemic Control with Colesevelam Treatment in Patients with Type 2 Diabetes Is Not Directly Associated with Changes in Bile Acid Metabolism

Bile acids (BAs) are essential for fat absorption and appear to modulate glucose and energy metabolism. Colesevelam, a BA sequestrant, improves glycemic control in type 2 diabetes mellitus (T2DM). We aimed to characterize the alterations in BA metabolism associated with T2DM and colesevelam treatment and to establish whether metabolic consequences of T2DM and colesevelam are related to changes in BA metabolism. Male subjects with T2DM (n = 16) and controls (n = 12) were matched for age and body mass index. BA pool sizes and synthesis/input rates were determined before and after 2 and 8 weeks of colesevelam treatment. T2DM subjects had higher cholic acid (CA) synthesis rate, higher deoxycholic acid (DCA) input rate, and enlarged DCA pool size. Colesevelam resulted in a preferential increase in CA synthesis in both groups. CA pool size was increased whereas chenodeoxycholic acid and DCA pool sizes were decreased upon treatment. Fasting and postprandial fibroblast growth factor 19 (FGF19) levels did not differ between controls and diabetics, but were decreased by treatment in both groups. Colesevelam treatment reduced hemoglobin A1C by 0.7% (P < 0.01) in diabetics. Yet, no relationships between BA kinetic parameters and changes in glucose metabolism were found in T2DM or with colesevelam treatment. Conclusion: Our results reveal significant changes in BA metabolism in T2DM, particularly affecting CA and DCA. Colesevelam treatment reduced FGF19 signaling associated with increased BA synthesis, particularly of CA, and resulted in a more hydrophilic BA pool without altering total BA pool size. However, these changes could not be related to the improved glycemic control in T2DM. (HEPATOLOGY 2010;)

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated. Methodology/Principal Findings To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-13C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized. Conclusions/Significance High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.

Restoration of impaired intestinal barrier function by the hydrolysed casein diet contributes to the prevention of type 1 diabetes in the diabetes-prone BioBreeding rat.

Aims/hypothesisImpaired intestinal barrier function is observed in type 1 diabetes patients and animal models of the disease. Exposure to diabetogenic antigens from the intestinal milieu due to a compromised intestinal barrier is considered essential for induction of the autoimmune process leading to type 1 diabetes. Since a hydrolysed casein (HC) diet prevents autoimmune diabetes onset in diabetes-prone (DP)-BioBreeding (BB) rats, we studied the role of the HC diet on intestinal barrier function and, therefore, prevention of autoimmune diabetes onset in this animal model.MethodsDP-BB rats were fed the HC diet from weaning onwards and monitored for autoimmune diabetes development. Intestinal permeability was assessed in vivo by lactulose–mannitol test and ex vivo by measuring transepithelial electrical resistance (TEER). Levels of serum zonulin, a physiological tight junction modulator, were measured by ELISA. Ileal mRNA expression of Myo9b, Cldn1, Cldn2 and Ocln (which encode the tight junction-related proteins myosin IXb, claudin-1, claudin-2 and occludin) and Il-10, Tgf-ß (also known as Il10 and Tgfb, respectively, which encode regulatory cytokines) was analysed by quantitative PCR.ResultsThe HC diet reduced autoimmune diabetes by 50% in DP-BB rats. In DP-BB rats, prediabetic gut permeability negatively correlated with the moment of autoimmune diabetes onset. The improved intestinal barrier function that was induced by HC diet in DP-BB rats was visualised by decreasing lactulose:mannitol ratio, decreasing serum zonulin levels and increasing ileal TEER. The HC diet modified ileal mRNA expression of Myo9b, and Cldn1 and Cldn2, but left Ocln expression unaltered.Conclusions/interpretationImproved intestinal barrier function might be an important intermediate in the prevention of autoimmune diabetes by the HC diet in DP-BB rats. Effects on tight junctions, ileal cytokines and zonulin production might be important mechanisms for this effect.

Treatment of refractory cholestatic pruritus after liver transplantation with albumin dialysis

Albumin dialysis has been shown to improve the outcome in patients with cholestatic liver failure caused by chronic liver disease. This study reports 7 liver transplant recipients who were treated with albumin dialysis for intractable pruritus of different origin (ductopenic graft rejection, non‐anastomotic strictures, and recurrence of hepatitis C). Treatment with histamine (H1) blockers, opioid antagonists, and cholestyramine had not been effective. The Molecular Adsorbent Recirculating System (MARS; Teraklin, Rostock, Germany) was used for albumin dialysis. All patients presented with numerous scratch marks, 6 of whom had a pronounced icterus. Six patients (86%) responded to 3 consecutive treatments with significant reduction of pruritus. The mean pruritus score, which was quantified by a visual analog scale (VAS), decreased from 9.7 ± 0.5 to 3.7 ± 0.8 (SD). The mean duration of 1 treatment was 15.6 hours. The procedure was well tolerated by all patients. The mean total serum bilirubin in patients who responded to therapy declined from 19.11 ± 16.96 mg/dL (SD) before MARS therapy to 9.24 ± 3.52 mg/dL after treatment. The mean serum concentration of 3α‐hydroxy bile acids decreased from 192.67 ± 58.12 μmol/L (SD) to 42.33 ± 31.58 μmol/L (SD). Follow‐up in 3 cases showed sustained improvement of pruritus lasting for more than 3 months. In 3 patients, however, pruritus relapsed. One patient, who showed severe pruritus, without relevant elevation of serum bile acids before treatment, did not respond to albumin dialysis. Our data indicate that MARS is an effective therapeutic option for patients with intractable cholestatic pruritus. (Liver Transpl 2004;10:107–114.)

Bile salt sequestration induces hepatic de novo lipogenesis through farnesoid X receptor– and liver X receptorα–controlled metabolic pathways in mice

Diabetes is characterized by high blood glucose levels and dyslipidemia. Bile salt sequestration has been found to improve both plasma glycemic control and cholesterol profiles in diabetic patients. Yet bile salt sequestration is also known to affect triglyceride (TG) metabolism, possibly through signaling pathways involving farnesoid X receptor (FXR) and liver X receptor α (LXRα). We quantitatively assessed kinetic parameters of bile salt metabolism in lean C57Bl/6J and in obese, diabetic db/db mice upon bile salt sequestration using colesevelam HCl (2% wt/wt in diet) and related these to quantitative changes in hepatic lipid metabolism. As expected, bile salt sequestration reduced intestinal bile salt reabsorption. Importantly, bile salt pool size and biliary bile salt secretion remained unchanged upon sequestrant treatment due to compensation by de novo bile salt synthesis in both models. Nevertheless, lean and db/db mice showed increased, mainly periportally confined, hepatic TG contents, increased expression of lipogenic genes, and increased fractional contributions of newly synthesized fatty acids. Lipogenic gene expression was not induced in sequestrant‐treated Fxr−/− and Lxrα−/− mice compared with wild‐type littermates, in line with reports indicating a regulatory role of FXR and LXRα in bile salt–mediated regulation of hepatic lipid metabolism. Conclusion: Bile salt sequestration by colesevelam induces the lipogenic pathway in an FXR‐ and LXRα‐dependent manner without affecting the total pool size of bile salts in mice. We speculate that a shift from intestinal reabsorption to de novo synthesis as source of bile salts upon bile salt sequestration affects zonation of metabolic processes within the liver acinus. (HEPATOLOGY 2010.)

Pulsatile drug delivery to ileo-colonic segments by structured incorporation of disintegrants in pH-responsive polymer coatings.

Conventional pH-responsive coatings used for oral drug delivery to the lower parts of the gastro-intestinal tract often show a poor performance. A new system for site-specific pulsatile delivery in the ileo-colonic regions is described. The system is based on the non-percolating incorporation of disintegrants in a coating which consists further of a continuous matrix of pH-responsive polymer (Eudragit S). Extensive in vitro release studies were performed in which coatings with different concentrations and disintegrants were studied and compared to non-disintegrant containing coatings. In vitro data show that the incorporation of swelling agents in an Eudragit S-coating still allows delayed release in the simulated terminal ileum. The pulse time and the robustness could be improved compared to conventional Eudragit S-coatings. The augmented pH-responsiveness of the new coating was related to the swelling index of the applied disintegrant. Based on the in vitro data comparing different swelling agents, Ac-di-sol appears to be the best performing swelling agent. A proof-of-concept study in human subjects was performed to investigate the performance of the new system in vivo. Coated capsules containing the stable isotope (13)C(6)-glucose as the test compound were administered and the occurrence of (13)CO(2) in the breath of the subjects was measured. It could be shown that the coating is able to resist the environmental conditions in the stomach and duodenum and delay release until deeper parts of the intestines are reached. Furthermore, the capsule is able to maintain a pulsatile release profile. It is concluded that the structured incorporation of swelling agents in pH-responsive polymers improves the delayed, pulsatile release kinetics of coated capsules. In a proof-of-concept in vivo study it was shown that the newly developed coating enables pulsatile delivery of the content to the lower parts of the intestines.

Lactose intolerance: analysis of underlying factors

Background We studied the degree of lactose digestion and orocecal transit time (OCTT) as possible causes for the variability of symptoms of lactose intolerance (LI) in a sample of a population with genetically determined low lactase activity.

Citrulline as a Marker for Chemotherapy Induced Mucosal Barrier Injury in Pediatric Patients

The currently used National Cancer Institute (NCI) adverse events criteria for mucosal barrier injury (MBI) are insufficient for use in children. We searched for objective, easily measurable indicators for MBI in children with cancer.

Plasma bile acids are not associated with energy metabolism in humans.

Bile acids (BA) have recently been shown to increase energy expenditure in mice, but this concept has not been tested in humans. Therefore, we investigated the relationship between plasma BA levels and energy expenditure in humans. Type 2 diabetic (T2DM) patients (n = 12) and gender, age and BMI-matched healthy controls (n = 12) were studied before and after 8 weeks of treatment with a BA sequestrant. In addition, patients with liver cirrhosis (n = 46) were investigated, since these display elevated plasma BA together with increased energy expenditure. This group was compared to gender-, age- and BMI-matched healthy controls (n = 20). Fasting plasma levels of total BA and individual BA species as well as resting energy expenditure were determined. In response to treatment with the BA sequestrant, plasma deoxycholic acid (DCA) levels decreased in controls (-60%, p < 0.05) and T2DM (-32%, p < 0.05), while chenodeoxycholic acid (CDCA) decreased in controls only (-33%, p < 0.05). Energy expenditure did not differ between T2DM and controls at baseline and, in contrast to plasma BA levels, was unaffected by treatment with the BA sequestrant. Total BA as well as individual BA species did not correlate with energy expenditure at any time throughout the study. Patients with cirrhosis displayed on average an increase in energy expenditure of 18% compared to values predicted by the Harris-Benedict equation, and plasma levels of total BA (up to 12-fold) and individual BA (up to 20-fold) were increased over a wide range. However, neither total nor individual plasma BA levels correlated with energy expenditure. In addition, energy expenditure was identical in patients with a cholestatic versus a non-cholestatic origin of liver disease while plasma total BA levels differed four-fold between the groups. In conclusion, in the various (patho)physiological conditions studied, plasma BA levels were not associated with changes in energy expenditure. Therefore, our data do not support an important role of circulating BA in the control of human energy metabolism.