Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.

Evaluation of the MA-10 cell line as a model of insl3 regulation and Leydig cell function.

Cryptorchidism, the failure of one or both testes to descend to an extra-abdominal location during mammalian development, is a common reproductive malady that often requires surgical intervention to remedy. Leydig cells are responsible for producing insulin-like peptide 3 (INSL3), a peptide hormone that is essential for normal testicular descent. The insl3 promoter in Leydig cells can be activated by cAMP through the transcription factor Nur77, which also regulates the promoters of the steroidogenic enzymes, cyp17 and 3ß-hsd. While the mechanism of LH action on testosterone production is well characterized, the effect of LH on insl3 abundance has not been described. The MA-10 Leydig cell line was used to test the hypothesis that abundance of insl3 mRNA is increased by LH/CG via the cAMP pathway. Cells treated with hCG had a transient robust increase in abundance of nur77 mRNA, while insl3 mRNA abundance remained unchanged. Further, while cAMP addition increased nur77 mRNA abundance, it failed to affect insl3 mRNA. Inhibition of LH-receptor-linked signal transduction pathways in the presence of hCG implicated multiple signaling networks in the regulation of both the insl3 and nur77 genes. Treatment with hCG and cAMP also increased abundance of cyp17 mRNA but not 3ß-hsd. Abundance of insl3 mRNA was not affected by hCG, testosterone or the combination of hCG and testosterone. Collectively, these results provide support for the constitutive regulation of insl3 mRNA abundance in the MA-10 Leydig cell line rather than acute regulation by LH/CG and cAMP.

Thermodynamic effects of commercially available ice boots.

The limb of the equine athlete is subjected to all types of various stressors during exercise. To ensure the health of the horse and to prevent the possibility of lameness, it has been a common practice to apply cold therapy to the distal limb of the horse pre- and post-exercise. Commercially available boots are widely available for the application of cold therapy. To test the effectiveness of the boot, 6 healthy performance level sport type horses were exercised at a walk, trot, and canter in a round-pen and then subjected to the application of the ice boot for 20 minutes on a treatment leg, and no ice boot on an untreated leg. Thermal images were taken of the 3rd metacarpal region pre-exercise, post-exercise, post-ice boot, and every 2 minutes after until the difference between the temperatures of the control leg and the treatment leg became zero. The images were analyzed using an analysis software (FLIR Tools) to determine the average temperature of the 3rd metacarpal region at each time point. The measured temperatures between treatments were found to be significantly different due to the application of the ice boot, providing evidence that the boot sufficiently cools the leg (P<.01). Thereafter, a 95% confidence interval was created to depict the average time it took for the cooled leg to return to average temperature post-ice boot, suggesting that it takes about 14.67 minutes for the difference between the temperatures of the cooled leg versus the non-cooled leg to become zero. This finding is significant to horse owners, trainers, and veterinarians that use this commonly available tool. These findings lend evidence to support the common practice of using cold therapy in treatment of disease in the horse.

Sources of spermatozoa loss during collection and artificial insemination of horses.

During artificial insemination of horses, it is important to accurately estimate the number of spermatozoa in each insemination dose. However, little research exists regarding sources of spermatozoa loss during collection and artificial insemination. Therefore, spermatozoal losses were quantified in the dismount loss (187.6×10(6)±62.5×10(6)spermatozoa), gel fraction (179.8×10(6)±61.7×10(6)spermatozoa), and the collection receptacle (136.1×10(6)±26.9×10(6)spermatozoa). Spermatozoal losses were examined in the centrifuge tube (25.8×10(6)±2.1×10(6)spermatozoa), AI pipette during the air removal (90.9×10(6)±8.5×10(6)spermatozoa), and spermatozoa remaining in the AI pipette after insemination (342.9×10(6)±21.4×10(6)spermatozoa). The average cumulative loss was 14.2±2.9% of the total spermatozoa ejaculated with approximately half of the loss due to the process of semen collection and half due to the process of artificial insemination. Spermatozoa retained in the AI pipette, after insemination with extended semen, represented the greatest source of loss.