Inefficient chronic activation of parietal cells in Ae2a,b(-/-) mice.

In parietal cells, basolateral Ae2 Cl(-)/HCO(3)(-) exchanger (Slc4a2) appears to compensate for luminal H(+) pumping while providing Cl(-) for apical secretion. In mouse and rat, mRNA variants Ae2a, Ae2b1, Ae2b2, and Ae2c2 are all found in most tissues (although the latter at very low levels), whereas Ae2c1 is restricted to the stomach. We studied the acid secretory function of gastric mucosa in mice with targeted disruption of Ae2a, Ae2b1, and Ae2b2 (but not Ae2c) isoforms. In the oxyntic mucosa of Ae2(a,b)(-/-) mice, total Ae2 protein was nearly undetectable, indicating low gastric expression of the Ae2c isoforms. In Ae2(a,b)(-/-) mice basal acid secretion was normal, whereas carbachol/histamine-stimulated acid secretion was impaired by 70%. These animals showed increased serum gastrin levels and hyperplasia of G cells. Immunohistochemistry and electron microscopy revealed baseline activation of parietal cells with fusion of intracellular H(+)/K(+)-ATPase-containing vesicles with the apical membrane and degenerative changes (but not substantial apoptosis) in a subpopulation of these cells. Increased expression of proliferating cell nuclear antigen in the oxyntic glands suggested enhanced Ae2(a,b)(-/-) parietal cell turnover. These data reveal a critical role of Ae2a-Ae2b1-Ae2b2 isoforms in stimulated gastric acid secretion whereas residual Ae2c isoforms could account to a limited extent for basal acid secretion.

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.

Emerging pharmacological approaches to the treatment of obesity.

The obesity epidemic has been recognized by the World Health Organization (WHO) as one of the top 10 global health problems. Worldwide, more than one billion adults are overweight and over 300 million are obese. The majority of developed countries, including the United States, Canada and England are experiencing dramatic increases in obesity. Obesity is a condition associated with the accumulation of excessive body fat resulting from chronic imbalance of energy whereby the intake of energy exceeds expenditure. The excess body fat predisposes an obese individual to chronic diseases, such as coronary heart disease, type 2 diabetes and diseases of the gall bladder and cancer. The high incidence of obesity and the lack of safe pharmaceutical agents have fuelled an increase in anti-obesity drug-related research. Although a number of pharmacological approaches have been investigated in recent years, few safe, therapeutically effective products have been developed. This commentary focuses on emerging pharmacological approaches targeted for the treatment of obesity.

Disodium Ascorbyl Phytostanyl Phosphates (FM-VP4) reduces plasma cholesterol concentration, body weight and abdominal fat gain within a dietary-induced obese mouse model.

PURPOSE: The purpose of this study was to determine if Disodium Ascorbyl Phytostanol Phosphates (FM-VP4) alters animal body weight and plasma lipid levels in a dietary-induced obese mouse model. METHODS: Twenty-four C57BL6 mice (28 days old) were housed individually and fed a standard mouse diet for 2 weeks upon arrival. After 2 weeks the animals were weighed and divided in 4 groups of similar average weight, and the groups received a low fat (10% kcal from fat) and high fat (45% kcal from fat) diet with or without FM-VP4 (2% w/w) for 12 continuous weeks. Food, water and caloric intake and body weight were recorded on a daily basis throughout the duration of the study. Following the 12th week of the study all animals were humanely sacrificed and blood and abdominal fat pads were harvested for further analysis. Plasma cholesterol, triglyceride, AST/ALT and creatinine levels were measured using enzymatic kits. RESULTS: There is a significant difference in weight gain between the low-fat diet and the low-fat diet + 2% w/w FM-VP4 treatment groups (P<0.05), as well as between the high-fat diet and the high-fat diet + 2% w/w FM-VP4 treatment groups (P<0.05). However, the reduction of weight gain of the high-fat diet + 2% FM-VP4 treatment group compared to the high-fat group was 51%, while the reduction in weight gain between the low-fat diet + 2% w/w FM-VP4 treatment group and the low-fat diet group was 17% over the duration of the study. No significant differences in food and water intakes, serum creatinine and AST/ALT levels were observed between the four groups. No significant differences in caloric intake between the low-fat diet and the low-fat diet + 2% w/w FM-VP4. However, a significant difference in caloric intake between high-fat diet and the high-fat diet + 2% w/w FM-VP4 treatment groups was observed. In addition, significant reductions in plasma cholesterol levels and abdominal fat pad weight between diet alone and diet + FM-VP4 treatment groups were observed. CONCLUSIONS: These findings suggest that FM-VP4 may have potential weight-loss and cholesterol lowering activity in both High Fat and Low Fat Diets treated groups.

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

BACKGROUND: Mutations 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. METHODS: In 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. RESULTS: Mice 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. CONCLUSION: These 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.

A mouse genetic model for familial cholestasis caused by ATP8B1 mutations reveals perturbed bile salt homeostasis but no impairment in bile secretion.

Mutations in ATP8B1, a broadly expressed P-type ATPase, result, through unknown mechanisms, in disorders of bile secretion. These disorders vary in severity from mild and episodic to progressive with liver failure. We generated Atp8b1G308V/G308V mutant mice, which carry a mutation orthologous to that present in homozygous form in patients from the Amish index kindred for severe ATP8B1 disease. In contrast to human patients, Atp8b1(G308V/G308V) mice had unimpaired bile secretion and no liver damage, but showed mild abnormalities including depressed weight at weaning and elevated serum bile salt levels. We challenged the hepatobiliary metabolism of Atp8b1G308V/G308V mice by administering exogenous bile salts. Upon bile salt feeding, Atp8b1G308V/G308V mice, but not wild-types, demonstrated serum bile salt accumulation, hepatic injury and expansion of the systemic bile salt pool. Unexpectedly, this failure of bile salt homeostasis occurred in the absence of any defect in hepatic bile secretion. Upon infusion of a hydrophobic bile salt, wild-type mice developed cholestasis while Atp8b1G308V/G308V mice maintained high biliary output and more extensively rehydroxylated the infused bile salt. Increased bile salt hydroxylation, which reduces bile salt toxicity, may explain the milder phenotype in Atp8b1G308V/G308V mice compared with humans with the equivalent mutation. These results demonstrate the key role of Atp8b1 in bile salt homeostasis and highlight the importance of bile salt hydroxylation in the prevention of cholestasis. The mouse phenotype reveals that loss of Atp8b1 disrupts bile salt homeostasis without impairment of canalicular bile secretion; in humans this process is likely to be obscured by early onset of severe liver disease.

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 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.

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.