Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep (-/-) mice.

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2ß-, 6ß-, and 15ß-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Impact characteristics of female children running in adult versus youth shoes of the same size.

The purpose of this study was to determine if ground reaction forces were influenced by shoe design (adult vs. youth) for female children when running. Subjects (n = 10, 12.0 ± 1.1 years old; 154 ± 4.9 cm; 46.2 ± 14.3 kg; shoe size 3.5-7 youth) were fit with a shoe model available in youth and adult sizes. Subjects ran 10 trials per shoe condition across a force platform placed in the middle of a 9-m runway. Impact force, second maximum force, loading rate, stance time and average vertical ground reaction forces were recorded for each trial. Shoes underwent a mechanical impact test with peak force, peak acceleration, and percent energy returned recorded. Each variable was compared between shoe conditions. From the impact testing, it was determined that peak force, peak acceleration and percent energy return were 7.1%, 7.1%, and 18.9% greater, respectively, for the youth vs. adult shoe (p < .001). From the running tests, it was determined that loading rate was different (p = .009) between shoe conditions whereas impact force, second maximum force, average force and stance time were not different between shoes (p > .01). Young girls had a greater loading rate when running in youth vs. adult shoes even though the shoe size was the same.

Determination of muscle activity during running at reduced body weight.

The aim of this study was to investigate how lower extremity muscles are influenced by body weight support during running at different speeds. Nine participants (age 24 ± 2 years, height 1.75 ± 0.12 m, mass 73.5 ± 15.7 kg) ran at 100%, 115%, and 125% of preferred speed at 100%, 90%, 80%, 70%, and 60% of body weight on a treadmill that provided body weight support. Preferred speed was self-selected by each participant and represented a speed that he or she could sustain if going for a 30 min run. Electromyography (EMG) data were recorded (1000 Hz, 1 min) from the bicep femoris, rectus femoris, tibialis anterior, and gastrocnemius for each condition together with knee angle (electrogoniometer). Average and root mean square EMG were calculated across 30 s. Muscle patterns were determined by smoothing (low-pass filter, 4 Hz) and extracting patterns for 49 cycles defined by consecutive maximum knee flexion angles. Repeated-measures analyses of variance were used to compare average and root mean square across body weight and speeds. Correlations were computed between the 100% speed/100% body weight condition and all other conditions per muscle. There was no interaction between body weight and speed (P > 0.05). Average and root mean square decreased as body weight decreased for all muscles (P < 0.05) and increased across speeds for all muscles (P < 0.05). Correlations for all muscles between conditions were high (range: 0.921-0.999). Although a percent reduction in body weight did not lead to the same reduction in muscle activity, it was clear that reducing body weight leads to a reduction in muscle activity with no changes in muscle activity patterns.

Polymorphisms in multiple genes are associated with resting heart rate in a stepwise allele-dependent manner.

OBJECTIVE: The purpose of this study was to use a candidate gene approach to identify common polymorphisms that are associated with resting sinus heart rate in a population without overt cardiovascular disease. BACKGROUND: Increased resting heart rate is significantly associated with susceptibility to development of myocardial infarction, sudden cardiac death, and overall cardiac mortality. METHODS: A longitudinal cohort of 1468 individuals (active and retired middle-aged Canadian firefighters) who were enrolled in the Firefighters and Their Endothelium (FATE) study was evaluated. Resting heart rate was recorded from the electrocardiogram (ECG) obtained at enrollment. Candidate genes were selected for their known roles in sinus node automaticity and/or its regulation, and single nucleotide polymorphisms (SNPs) with a minor allele frequency of > or =0.20 were targeted. A total of 53 SNPs in 46 genes were selected and analyzed in a screening sample, and 33 SNPs in 29 genes were evaluated in the full population. RESULTS: Univariate analysis detected five putative associations between HR and SNPs. As expected, environmental covariates were identified. Three polymorphisms, ADRB1 G389R, SCN5a H558R, and CASQ1 intron 2, remained statistically significant and independent of covariates. Some alleles were associated with higher and some with lower heart rates. A stepwise increase in heart rate was observed that was dependent on the number of tachycardia-associated alleles with progressive increases in mean heart rate from 51 to 66 bpm. CONCLUSIONS: Common polymorphisms are associated with heart rate in a stepwise allele-dependent manner.

Severe cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein.

Intrahepatic cholestasis is often associated with impairment of biliary bile acid secretion, a process mediated by the sister of P-glycoprotein (Spgp or Abcb11) also known as the bile salt export pump (Bsep). In humans, mutations in the Spgp gene are associated with a fatal childhood disease, type 2 progressive familial intrahepatic cholestasis (PFIC2). However in mice, the "knockout" of Spgp only results in mild cholestasis. In this study, we fed spgp(-/-) knockout mice with a cholic acid (CA)-supplemented diet to determine whether a more pronounced PFIC2-like phenotype could be induced. Such mice developed severe cholestasis characterized by jaundice, weight loss, elevated plasma bile acid, elevated transaminase, cholangiopathy (proliferation of bile ductules and cholangitis), liver necrosis, high mortality, and wide-ranging changes in the mRNA expression of major liver genes (16/36 examined). A surprising observation was that the bile acid output and bile flow in CA-fed mutant mice was significantly higher than anticipated. This suggests that the spgp(-/-) mice are able to utilize an alternative bile salt transport system. However, unlike Spgp, this system is insufficient to protect the knockout mice from cholestasis despite its high capacity. In conclusion, the spgp(-/-) mice provide a unique model to investigate molecular pathways associated with cholestasis and related diseases.

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice.

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.