The fecal sterol analysis clearly showed that dietary

The fecal sterol analysis clearly showed that dietary piperine significantly increased fecal excretion of cholesterol and total neutral sterols in a dose-dependent pattern (Table 4). In humans, 1200–1300 mg cholesterol enters the small intestine lumen with 300–400 mg coming from the diet and the rest from bile duct each day (Grundy, 1983). Most dietary and biliary cholesterol are absorbed in the small intestine and the remainder is excreted out into feces (Grundy, 1983) Most cholesterol, when passing through the large intestine, is utilized by gut microbiota and fermented into its derivatives namely, coprostanol, coprostanone, and dihydrocholesterol. In this regard, neutral sterols in feces should be summed as a biomarker of total cholesterol MG-262 and excretion (Zhang et al., 2017). In the present study, PL and PH diets remarkably increased fecal total neutral sterols by 84% and 109%, respectively (Table 4), suggesting that piperine reduced plasma TC via a mechanism of inhibiting cholesterol absorption and stimulating fecal output of neutral sterols. The present study is the first time to investigate the effect of piperine on intestinal sterol transporters and enzymes in the small intestine. NPC1L1 is a sterol transporter responsible for taking up cholesterol from the lumen to enterocytes (Davis et al., 2004). ACAT2 is an enzyme, which esterifies cholesterol to cholesteryl ester (CE) in enterocytes. Subsequently, MTP assembles CE with apolipoprotein B into chylomicrons before entering the lymphatic system (Jamil et al., 1995). Results clearly showed piperine supplementation was associated with down-regulation on the mRNA of intestinal NPC1L1, ACAT2, and MTP in a dose-dependent manner and protein mass of of ACAT2 and MTP (Fig. 3). In fact, piperine has been shown to reduce the cholesterol uptake and inhibit the gene expression of NPC1L1 in Caco-2 monolayer (Duangjai, Ingkaninan, Praputbut, & Limpeanchob, 2013). It was therefore concluded that plasma cholesterol-lowering activity of piperine was mediated by inhibition on cholesterol absorption via down-regulation of NPC1L1, ACAT2 and MTP. Excess cholesterol in the liver is eliminated via the conversion of cholesterol to bile acids (Chen et al., 2008). In the present study, we found that piperine supplementation only caused a slight increase in the fecal exertion of total bile acids. In the liver, CYP7A1 is an enzyme, which catalyzes the synthesis of bile acids, while LXRα mediates the stimulation of gene expression of CYP7A1. Dietary piperine administration did not significantly alter the mRNA and protein abundances of LXRα and CYP7A1, suggesting that plasma cholesterol-lowering activity of piperine was unlikely mediated via enhancing the synthesis or elimination of bile acids. HMGCR is an enzyme regulating cholesterol synthesis, while LDLR mediates the cholesterol removal from circulation. SREBP2 regulates the transcription of HGGCR and LDLR. Results from RT-PCR and Western Blot analyses did not find dietary piperine had any significant effect on SREBP2, HMGCR and LDLR, indicating that plasma cholesterol-lowering activity was unlikely mediated by inhibition on hepatic cholesterol synthesis or removal.