00 AM), with ad libitum access to water and mouse chow diet (Arie Blok, Woerden, The Netherlands). Animal experiments were performed in conformity with PHS policy and in accordance with the national selleck chemical laws. All protocols were approved by the responsible ethics committee of the University of Groningen. Induction of type 1 diabetes mellitus To induce experimental T1DM, wild-type C57BL/6J were injected intravenously with a single dose of alloxan (65 mg/kg body weight, Sigma, St. Louis, MO), while control mice received an equivalent volume of PBS. Blood glucose levels were assessed by tail bleeding using a Onetouch Ultra glucosemeter (LifeScan Benelux, Beerse, Belgium). Plasma insulin levels were determined using an ultrasensitive mouse insulin ELISA kit (Alpco Diagnostics, Salem, NH).

Plasma lipid and lipoprotein analysis Plasma total cholesterol, triglycerides, free fatty acids, and phospholipids were measured enzymatically using commercially available reagents (Roche Diagnostics, Basel, Switzerland and Wako Pure Chemical Industries, Neuss, Germany). Pooled plasma samples from mice of the same experimental group were subjected to fast protein liquid chromatography (FPLC) gel filtration using a Superose 6 column (GE Healthcare, Uppsala, Sweden) as described (14). Samples were chromatographed at a flow rate of 0.5 ml/min, and fractions of 500 ��l each were collected. Individual fractions were assayed for cholesterol concentrations as described above. Plasma plant sterol levels were measured by gas chromatography exactly as previously published (15).

Analysis of liver lipid composition To determine hepatic cholesterol, phospholipid, and triglyceride content, liver tissue was homogenized, and lipids were extracted following the general procedure of Bligh and Dyer as described (16). Triglycerides and cholesterol were measured using commercial kits as detailed above. Phospholipid content of the liver was determined essentially as published previously (16). Bile collection and assessment of biliary excretion of cholesterol, phospholipids, and BAs Continuous bile cannulation was performed on day 10 after injection with either alloxan or PBS. Bile was collected during 30 min under Hypnorm (fentanyl/fluanisone; 1 ml/kg) and diazepam (10 mg/kg) anesthesia using a humidified incubator to maintain body temperature. Bile production was determined gravimetrically.

Biliary bile salt, cholesterol, and phospholipid concentrations were determined, and the respective biliary excretion rates were calculated as previously described (17). Fecal BA and neutral sterol analysis Feces of individually housed mice were collected over a period of 24 h, starting on day 8. Fecal samples were dried, weighed, and thoroughly ground. Aliquots were Carfilzomib used for determination of BAs and neutral sterols by gas liquid chromatography as described (17).