Potential of therapeutic bile acids in the treatment of neonatal Hyperbilirubinemia.

Neonatal hyperbilirubinemia or jaundice is associated with kernicterus, resulting in permanent neurological damage or even death. Conventional phototherapy does not prevent hyperbilirubinemia or eliminate the need for exchange transfusion. Here we investigated the potential of therapeutic bile acids ursodeoxycholic acid (UDCA) and obeticholic acid (OCA, 6-α-ethyl-CDCA), a farnesoid-X-receptor (FXR) agonist, as preventive treatment options for neonatal hyperbilirubinemia using the hUGT1*1 humanized mice and Ugt1a-deficient Gunn rats. Treatment of hUGT1*1 mice with UDCA or OCA at postnatal days 10-14 effectively decreased bilirubin in plasma (by 82% and 62%) and brain (by 72% and 69%), respectively. Mechanistically, our findings indicate that these effects are mediated through induction of protein levels of hUGT1A1 in the intestine, but not in liver. We further demonstrate that in Ugt1a-deficient Gunn rats, UDCA but not OCA significantly decreases plasma bilirubin, indicating that at least some of the hypobilirubinemic effects of UDCA are independent of UGT1A1. Finally, using the synthetic, non-bile acid, FXR-agonist GW4064, we show that some of these effects are mediated through direct or indirect activation of FXR. Together, our study shows that therapeutic bile acids UDCA and OCA effectively reduce both plasma and brain bilirubin, highlighting their potential in the treatment of neonatal hyperbilirubinemia.

LED-phototherapy does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats.

BACKGROUND: Phototherapy (PT) is the standard treatment of neonatal unconjugated hyperbilirubinemia. Fluorescent tube (FT)-emitted PT light is known to induce oxidative DNA damage in neonates. Nowadays, however, FTs have largely been replaced by light-emitting diodes (LEDs) for delivering PT. Until now, it is unknown whether LED-PT causes oxidative DNA damage. We aim to determine whether LED-PT induces oxidative DNA damage in hyperbilirubinemic rats. METHODS: Adult Gunn rats, with genetically unconjugated hyperbilirubinemia, received LED-PT in the clinically relevant doses of 10 or 30 µW/cm2/nm. Urine was collected at 0, 24, and 48 h of PT. A group of young Gunn rats received intensive LED-PT of 100 µW/cm2/nm for 24 h. Urine was collected every 8 h and analyzed for the levels of oxidative DNA damage marker 8-hydroxy-2'deoxyguanosine (8-OHdG) and creatinine. DNA damage was evaluated by immunohistochemistry (γH2AX) of skin and spleen samples. RESULTS: LED-PT of 10 and 30 µW/cm2/nm did not affect urinary concentrations of 8-OHdG and creatinine or the 8-OHdG/creatinine ratio. Likewise, intensive LED-PT did not affect the 8-OHdG/creatinine ratio or the number of γH2AX-positive cells in the skin or spleen. CONCLUSIONS: Our results show that LED-PT does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats either at clinically relevant or intensive dosages.

Impaired Hepatic Phosphatidylcholine Synthesis Leads to Cholestasis in Mice Challenged With a High-Fat Diet.

Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic integral membrane protein localized to the endoplasmic reticulum (ER). PEMT catalyzes approximately 30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet (HFD) develop steatohepatitis. Interestingly, portions of the ER located close to the canaliculus are enriched in PEMT. Phospholipid balance and asymmetrical distribution by adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) on the canalicular membrane is required for membrane integrity and biliary processes. We hypothesized that PEMT is an important supplier of PC to the canaliculus and that PEMT activity is critical for the maintenance of canalicular membrane integrity and bile formation following HFD feeding when there is an increase in overall hepatic PC demand. Pemt+/+ and Pemt-/- mice were fed a chow diet, an HFD, or a choline-supplemented HFD. Plasma and hepatic indices of liver function and parameters of bile formation were determined. Pemt-/- mice developed cholestasis, i.e, elevated plasma bile acid (BA) concentrations and decreased biliary secretion rates of BAs and PC, during HFD feeding. The maximal BA secretory rate was reduced more than 70% in HFD-fed Pemt-/- mice. Hepatic ABCB11/bile salt export protein, responsible for BA secretion, was decreased in Pemt-/- mice and appeared to be retained intracellularly. Canalicular membranes of HFD-fed Pemt-/- mice contained fewer invaginations and displayed a smaller surface area than Pemt+/+ mice. Choline supplementation (CS) prevented and reversed the development of HFD-induced cholestasis. Conclusion: We propose that hepatic PC availability is critical for bile formation. Dietary CS might be a potential noninvasive therapy for a specific subset of patients with cholestasis.

Protection against the Metabolic Syndrome by Guar Gum-Derived Short-Chain Fatty Acids Depends on Peroxisome Proliferator-Activated Receptor γ and Glucagon-Like Peptide-1.

The dietary fiber guar gum has beneficial effects on obesity, hyperglycemia and hypercholesterolemia in both humans and rodents. The major products of colonic fermentation of dietary fiber, the short-chain fatty acids (SCFAs), have been suggested to play an important role. Recently, we showed that SCFAs protect against the metabolic syndrome via a signaling cascade that involves peroxisome proliferator-activated receptor (PPAR) γ repression and AMP-activated protein kinase (AMPK) activation. In this study we investigated the molecular mechanism via which the dietary fiber guar gum protects against the metabolic syndrome. C57Bl/6J mice were fed a high-fat diet supplemented with 0% or 10% of the fiber guar gum for 12 weeks and effects on lipid and glucose metabolism were studied. We demonstrate that, like SCFAs, also guar gum protects against high-fat diet-induced metabolic abnormalities by PPARγ repression, subsequently increasing mitochondrial uncoupling protein 2 expression and AMP/ATP ratio, leading to the activation of AMPK and culminating in enhanced oxidative metabolism in both liver and adipose tissue. Moreover, guar gum markedly increased peripheral glucose clearance, possibly mediated by the SCFA-induced colonic hormone glucagon-like peptide-1. Overall, this study provides novel molecular insights into the beneficial effects of guar gum on the metabolic syndrome and strengthens the potential role of guar gum as a dietary-fiber intervention.

Short-Chain Fatty Acids Protect Against High-Fat Diet-Induced Obesity via a PPARγ-Dependent Switch From Lipogenesis to Fat Oxidation.

Short-chain fatty acids (SCFAs) are the main products of dietary fiber fermentation and are believed to drive the fiber-related prevention of the metabolic syndrome. Here we show that dietary SCFAs induce a peroxisome proliferator-activated receptor-γ (PPARγ)-dependent switch from lipid synthesis to utilization. Dietary SCFA supplementation prevented and reversed high-fat diet-induced metabolic abnormalities in mice by decreasing PPARγ expression and activity. This increased the expression of mitochondrial uncoupling protein 2 and raised the AMP-to-ATP ratio, thereby stimulating oxidative metabolism in liver and adipose tissue via AMPK. The SCFA-induced reduction in body weight and stimulation of insulin sensitivity were absent in mice with adipose-specific disruption of PPARγ. Similarly, SCFA-induced reduction of hepatic steatosis was absent in mice lacking hepatic PPARγ. These results demonstrate that adipose and hepatic PPARγ are critical mediators of the beneficial effects of SCFAs on the metabolic syndrome, with clearly distinct and complementary roles. Our findings indicate that SCFAs may be used therapeutically as cheap and selective PPARγ modulators.

The short-chain fatty acid uptake fluxes by mice on a guar gum supplemented diet associate with amelioration of major biomarkers of the metabolic syndrome.

Studies with dietary supplementation of various types of fibers have shown beneficial effects on symptoms of the metabolic syndrome. Short-chain fatty acids (SCFAs), the main products of intestinal bacterial fermentation of dietary fiber, have been suggested to play a key role. Whether the concentration of SCFAs or their metabolism drives these beneficial effects is not yet clear. In this study we investigated the SCFA concentrations and in vivo host uptake fluxes in the absence or presence of the dietary fiber guar gum. C57Bl/6J mice were fed a high-fat diet supplemented with 0%, 5%, 7.5% or 10% of the fiber guar gum. To determine the effect on SCFA metabolism, 13C-labeled acetate, propionate or butyrate were infused into the cecum of mice for 6 h and the isotopic enrichment of cecal SCFAs was measured. The in vivo production, uptake and bacterial interconversion of acetate, propionate and butyrate were calculated by combining the data from the three infusion experiments in a single steady-state isotope model. Guar gum treatment decreased markers of the metabolic syndrome (body weight, adipose weight, triglycerides, glucose and insulin levels and HOMA-IR) in a dose-dependent manner. In addition, hepatic mRNA expression of genes involved in gluconeogenesis and fatty acid synthesis decreased dose-dependently by guar gum treatment. Cecal SCFA concentrations were increased compared to the control group, but no differences were observed between the different guar gum doses. Thus, no significant correlation was found between cecal SCFA concentrations and metabolic markers. In contrast, in vivo SCFA uptake fluxes by the host correlated linearly with metabolic markers. We argue that in vivo SCFA fluxes, and not concentrations, govern the protection from the metabolic syndrome by dietary fibers.

Optimizing exchange transfusion for severe unconjugated hyperbilirubinemia: studies in the Gunn rat.

BACKGROUND: Severe unconjugated hyperbilirubinemia carries the risk of neurotoxicity. Phototherapy (PT) and exchange transfusion (ET) are cornerstones in the treatment of unconjugated hyperbilirubinemia. Studies to improve ET efficacy have been hampered by the low application of ET in humans and by the lack of an in vivo model. The absence of an appropriate animal model has also prevented to determine the efficacy of adjunct or alternative treatment options such as albumin (Alb) administration. AIM: To establish an in vivo model for ET and to determine the most effective treatment (combination) of ET, PT and Alb administration. METHODS: Gunn rats received either PT, PT+Alb, ET, ET+PT, ET+PT+Alb or sham operation (each n = 7). ET was performed via the right jugular vein in ≈ 20 min. PT (18 µW/cm(2)/nm) was started after ET or at T0. Albumin i.p. injections (2.5 g/kg) were given after ET or before starting PT. Plasma unconjugated bilirubin (UCB), plasma free bilirubin (Bf), and brain bilirubin concentrations were determined. RESULTS: We performed ET in 21 Gunn rats with 100% survival. At T1, ET was profoundly more effective in decreasing both UCB -44%, p<0.01) and Bf -81%, p<0.05) than either PT or PT+Alb. After 48 h, the combination of ET+PT+Alb showed the strongest hypobilirubinemic effect (-54% compared to ET). CONCLUSIONS: We optimized ET for severe unconjugated hyperbilirubinemia in the Gunn rat model. Our data indicate that ET is the most effective treatment option, in the acute as well as the follow-up situation.

Acceleration of the gastrointestinal transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats.

BACKGROUND AND AIMS: Several conditions that delay gastrointestinal transit are associated with unconjugated hyperbilirubinaemia. We hypothesised that the gastrointestinal transit time is directly related to plasma unconjugated bilirubin (UCB) concentrations, and that this relationship can be used to develop a new therapeutic strategy for severe unconjugated hyperbilirubinaemia in the Gunn rat model. METHODS: Gunn rats received, for various time periods, oral polyethylene glycol (PEG) with or without conventional phototherapy treatment to accelerate, or oral loperamide to delay gastrointestinal transit. Gastrointestinal transit time and UCB concentrations in plasma, faeces, intestinal content and bile were determined. Results Within 36 h, PEG administration accelerated gastrointestinal transit by 45% and simultaneously decreased plasma UCB concentrations by 23% (each p<0.001). The decrease in plasma UCB coincided with an increased small intestinal UCB content (+340%, p<0.05) and an increased faecal UCB excretion (+153%, p<0.05). After 2 weeks, PEG decreased plasma UCB by 41% as single treatment, and by 62% if combined with phototherapy (each p<0.001). Loperamide delayed gastrointestinal transit by 57% and increased plasma UCB by 30% (each p<0.001). Dose-response experiments showed a strong, linear relation between the gastrointestinal transit time and plasma UCB concentrations (r=0.87, p<0.001). CONCLUSION: Gastrointestinal transit time and plasma UCB concentrations are linearly related in Gunn rats. This relationship can be exploited by pharmacologically accelerating the gastrointestinal transit, which increases transmucosal UCB diffusion and thereby effectively treats unconjugated hyperbilirubinaemia. Present results support the feasibility of PEG treatment, either solitary or combined with phototherapy, in patients with severe unconjugated hyperbilirubinaemia.

Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in Gunn rats.

BACKGROUND & AIMS: We tested the hypothesis that oral administration of bile salts, which are known to increase the biliary excretion of unconjugated bilirubin (UCB), decreases unconjugated hyperbilirubinemia in the Gunn rat model. METHODS: Adult Gunn rats were fed a standard diet or the same diet supplemented with 0.5 weight % ursodeoxycholic acid (UDCA) or cholic acid (CA) for 1 or 6 weeks. UCB and urobilinoids, a family of intestinal UCB breakdown products, were determined in plasma, feces, or both. After 6 weeks of treatment, tracer 3H-UCB was administered intravenously to determine steady-state UCB kinetics over the next 60 hours. RESULTS: One-week treatment with UDCA or CA decreased plasma UCB concentrations by 21% and 30%, respectively (each P < .01). During the first 4 days of treatment, both UDCA and CA increased the combined fecal excretion of UCB and urobilinoids (+52% and +32%, respectively; each P < .01). Prolongation of treatment to 6 weeks caused a persistent decrease in plasma UCB concentrations to approximately 40% below baseline (each bile salt P < .001). (3)H-UCB kinetic studies showed that UDCA and CA administration decreased UCB pool size (-33% and -32%, respectively; each P < .05) and increased UCB fractional turnover (+33% and +25%, respectively; each P < .05). CONCLUSIONS: Dietary bile salt administration induces a large, persistent decrease in plasma UCB concentrations in Gunn rats. Both UDCA and CA enhance UCB turnover by increasing its fecal disposal. These results support the application of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.

Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats.

Treatment with phototherapy or with the lipase inhibitor orlistat decreases plasma unconjugated bilirubin (UCB) concentrations in hyperbilirubinemic Gunn rats. We investigated the mechanism(s) underlying the effects of orlistat, phototherapy, and combined treatment, using steady-state 3H-UCB kinetics. After three weeks of treatment with orlistat (200 mg/kg chow), phototherapy (19 microW/cm2/nm) or combined treatment, tracer 3H-UCB was administered IV to treated and untreated (control) Gunn rats. Plasma samples and feces were collected every 12h for 60h, and bile for 30 min at 60h. The following results were obtained: 1) each treatment decreased plasma bilirubin levels compared with controls: orlistat- 19%, phototherapy-32%, combined treatment-53%; 2) plasma bilirubin concentrations were strongly, negatively correlated with fractional bilirubin turnover; 3) orlistat treatment induced net transmucosal excretion of UCB into the intestinal lumen, whereas phototherapy increased biliary UCB excretion rate; 4) all treatments profoundly increased the enterohepatic circulation of UCB derivatives, indicating enhanced metabolism by intestinal bacteria. In conclusion, orlistat and phototherapy lower plasma bilirubin concentrations in Gunn rats by increasing (net) intestinal influx of UCB, either by transmucosal excretion (orlistat), or increased biliary secretion (phototherapy). The mechanism of phototherapy and orlistat treatment involves increasing the availability of UCB in the intestinal lumen for fecal excretion and for metabolism by intestinal bacteria.

Effective oral treatment of unconjugated hyperbilirubinemia in Gunn rats.

We sought to develop an oral treatment for unconjugated hyperbilirubinemia. In the Gunn rat model of unconjugated hyperbilirubinemia, dietary supplementation with the lipase inhibitor orlistat (Orl) or with calcium phosphate (CaP) decreases plasma unconjugated bilirubin (UCB) levels. We determined whether Orl, CaP, or their combination is superior to phototherapy, the conventional treatment, and whether the effects of Orl and CaP are influenced by dietary fat content. Gunn rats were treated with Orl (200 mg/kg chow), CaP (20 g/kg chow), Orl + CaP, or continuous phototherapy (19 muW/cm(2)/nm) during a low-fat (LF) diet (13 energy%) or high-fat (HF) diet (35 energy%). Plasma UCB and fecal fat excretion were measured before, during, and/or at the end of treatment. Orl treatment for 2 weeks (HF diet) reduced plasma UCB concentrations similar to phototherapy (-34% and -28%, respectively); the combination of both was more effective than either treatment alone (-48%; P < .001). After 3 weeks of a HF diet, plasma UCB was 46% lower compared with the LF diet (P < .001). Plasma UCB concentrations were negatively correlated with fecal fat excretion (r = -0.96; P < .001). Irrespective of dietary fat content, 3 weeks of combined treatment (Orl + CaP) decreased plasma UCB by approximately 50% (P < .01) and was more effective than phototherapy (P < .05) at the intensity provided. In conclusion, plasma UCB concentrations in Gunn rats are negatively related to fecal fat excretion and dietary fat content. Orlistat is equally effective as phototherapy for the treatment of unconjugated hyperbilirubinemia in Gunn rats, and combined oral treatment with Orl + CaP is more effective than phototherapy. The present results support the feasibility of an efficient oral treatment of unconjugated hyperbilirubinemia.

Treatment of EFA deficiency with dietary triglycerides or phospholipids in a murine model of extrahepatic cholestasis.

Essential fatty acid (EFA) deficiency during cholestasis is mainly due to malabsorption of dietary EFA (23). Theoretically, dietary phospholipids (PL) may have a higher bioavailability than dietary triglycerides (TG) during cholestasis. We developed murine models for EFA deficiency (EFAD) with and without extrahepatic cholestasis and compared the efficacy of oral supplementation of EFA as PL or as TG. EFAD was induced in mice by feeding a high-fat EFAD diet. After 3 wk on this diet, bile duct ligation was performed in a subgroup of mice to establish extrahepatic cholestasis. Cholestatic and noncholestatic EFAD mice continued on the EFAD diet (controls) or were supplemented for 3 wk with EFA-rich TG or EFA-rich PL. Fatty acid composition was determined in plasma, erythrocytes, liver, and brain. After 4 wk of EFAD diet, induction of EFAD was confirmed by a sixfold increased triene-to-tetraene ratio (T/T ratio) in erythrocytes of noncholestatic and cholestatic mice (P < 0.001). EFA-rich TG and EFA-rich PL were equally effective in preventing further increase of the erythrocyte T/T ratio, which was observed in cholestatic and noncholestatic nonsupplemented mice (12- and 16-fold the initial value, respectively). In cholestatic mice, EFA-rich PL was superior to EFA-rich TG in decreasing T/T ratios of liver TG and PL (each P < 0.05) and in increasing brain PL concentrations of the long-chain polyunsaturated fatty acids (LCPUFA) docosahexaenoic acid and arachidonic acid (each P < 0.05). We conclude that oral EFA supplementation in the form of PL is more effective than in the form of TG in increasing LCPUFA concentrations in liver and brain of cholestatic EFAD mice.