Norbert Looije

Adenoviral overexpression of apolipoprotein A-V reduces serum levels of triglycerides and cholesterol in mice

Mice lacking ApoA-V, a novel HDL-associated apolipoprotein identified by our group and independently by Pennacchio et al. [Science 294 (2001) 169], were recently shown to be hypertriglyceridemic. To study the role of ApoA-V in triglyceride homeostasis, we compared lipid profiles in mice expressing normal and highly elevated levels of ApoA-V. For this purpose, adenoviral vectors expressing sense or antisense ApoA-V cDNA were constructed. Treatment of mice with sense adenoviral constructs resulted in circa 20-fold higher serum ApoA-V levels compared with mice injected with either PBS or antisense adenoviral constructs. ApoA-V overexpressing mice had markedly decreased (-70%) serum triglyceride levels caused primarily by lowered triglyceride content of the VLDL fraction. Furthermore, in these mice cholesterol levels were found to be lowered in all lipoprotein fractions with the largest mass decrease in the HDL fraction. This resulted in a 40% drop of serum cholesterol content. These findings suggest a role of ApoA-V in regulating levels of circulating triglycerides and cholesterol.

A mouse model of sitosterolemia: absence of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol

BackgroundMutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion.MethodsIn order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia.ResultsMice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice.ConclusionThese data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.

Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer

Bilirubin is glucuronidated by bilirubin UDP-glucuronyltransferase (UGT1A1) before biliary excretion. Because bilirubin is toxic, patients with Crigler–Najjar type I (CN), who have no UGT1A1 activity, suffer severe brain damage early in childhood. The Gunn rat is the model for CN type 1. Gunn rat fetuses were injected with 107 transducing units of UGT1A1 lentiviral vector at the end of the third trimester on embryonic day 19. Serum bilirubin of injected Gunn rats was lowered by 45% compared to untreated controls. This decrease was highly significant (P < 106) and was sustained for more than a year. In treated Gunn rats, bilirubin glucuronides were present in bile and UGT1A1 protein was detected in tissue. Liver, intestine, stomach, pancreas, and other organs were transduced and mostly contained 1% or less vector copies per genome. Tissue distribution was variable among experimental animals but high transduction levels were seen in pancreas and intestine in most animals. Immunohistochemistry of these organs revealed transduction of pancreatic acinar cells and intestinal epithelium. Injection of a lentiviral UGT1A1 vector into third-trimester Gunn rat fetuses corrects the metabolic deficiency and mediates a reduction of serum bilirubin levels that would be therapeutic in humans.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8+/+, Abcg8+/−, and Abcg8−/− mice with hydrophilic and hydrophobic bile salts. In Abcg8−/− mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8−/− mice at a much lower infusion rate compared with Abc8−/− and Abcg8+/− mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8−/− mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.

151 Long term correction of bilirubin UDP glucuronyltransferase deficiency in rats by in utero lentiviral gene transfer

Bilirubin is glucuronidated by bilirubin UDP-glucuronyltransferase (UGT1A1) before biliary excretion. Because bilirubin is toxic, patients with Crigler-Najjar type I (CN), who have no UGT1A1 activity, suffer severe brain damage early in childhood. The Gunn rat is the model for CN type 1. Gunn rat fetuses were injected with 10(7) transducing units of UGT1A1 lentiviral vector at the end of the third trimester on embryonic day 19. Serum bilirubin of injected Gunn rats was lowered by 45% compared to untreated controls. This decrease was highly significant (P < 10(6)) and was sustained for more than a year. In treated Gunn rats, bilirubin glucuronides were present in bile and UGT1A1 protein was detected in tissue. Liver, intestine, stomach, pancreas, and other organs were transduced and mostly contained 1% or less vector copies per genome. Tissue distribution was variable among experimental animals but high transduction levels were seen in pancreas and intestine in most animals. Immunohistochemistry of these organs revealed transduction of pancreatic acinar cells and intestinal epithelium. Injection of a lentiviral UGT1A1 vector into third-trimester Gunn rat fetuses corrects the metabolic deficiency and mediates a reduction of serum bilirubin levels that would be therapeutic in humans.