Cholesterol Excretion In Mice Research Articles

Supplementation of Weizmannia coagulans BC2000 and Ellagic Acid Inhibits High-Fat-Induced Hypercholesterolemia by Promoting Liver Primary Bile Acid Biosynthesis and Intestinal Cholesterol Excretion in Mice

The probiotic Weizmannia coagulans (W. coagulans) BC2000 can increase the abundance of intestinal transforming ellagic acid (EA) bacteria and inhibit metabolic disorders caused by hyperlipidemia by activating liver autophagy. This study aimed to investigate the inhibitory effects of W. coagulans BC2000 and EA on hyperlipidemia-induced cholesterol metabolism disorders. C57BL/6J mice (n = 10 in each group) were fed a low-fat diet, high-fat diet (HFD), HFD supplemented with EA, HFD supplemented with EA and W. coagulans BC77, HFD supplemented with EA, and W. coagulans BC2000. EA and W. coagulans BC2000 supplementation prevented HFD-induced hypercholesterolemia and promoted fecal cholesterol excretion. Transcriptome analysis showed that primary bile acid biosynthesis in the liver was significantly activated by EA and W. coagulans BC2000 treatments. EA and W. coagulans BC2000 treatment also significantly increased the intestinal Eggerthellaceae abundance and the liver EA metabolites, iso-urolithin A, Urolithin A, and Urolithin B. Therefore, W. coagulans BC2000 supplementation promoted the intestinal transformation of EA, which led to the upregulation of liver bile synthesis, thus preventing hypercholesterolemia.

FXR activation promotes intestinal cholesterol excretion and attenuates hyperlipidemia in SR-B1-deficient mice fed a high-fat and high-cholesterol diet.

Obeticholic acid (OCA) activates the farnesoid X receptor (FXR) to lower circulating total cholesterol (TC) and high density lipoprotein‐cholesterol (HDL‐C) concentrations and to stimulate fecal cholesterol excretion in mice by increasing hepatic SR‐B1 expression. Here we show that hepatic SR‐B1 depletion by an adenovirus expressing Sr‐b1 shRNA (Ad‐shSR‐B1) attenuated these beneficial effects of OCA in mice on a chow diet. The mRNA levels of ABC cholesterol transporter genes (Abca1, Abcg1, Abcg5, and Abcg8) were unchanged in the liver of hepatic SR‐B1‐depleted mice regardless of OCA treatment; however, a modest increase in Abca1, Abcg5, and Abcg8 mRNA levels was observed in the ileum of vehicle‐treated control mice and Abca1 and Abcg8 mRNA levels were increased more by OCA administration. OCA treatment of Sr‐b1 knock out (KO) mice (Sr‐b1‐/‐) fed a normal chow diet (NCD) displayed a similar lack of transhepatic cholesterol movement, as well as a modest increase in the levels of ileum cholesterol transporter expression. However, OCA treatment of Sr‐b1 KO mice fed a cholesterol‐enriched diet reduced circulating cholesterol and increased fecal cholesterol output to comparable degrees to that of wild‐type (WT) mice, and these effects were accompanied by substantial elevations of mRNA levels of Abca1, Abcg1, Abcg5, and Abcg8 in the ileum of Sr‐b1 KO mice. Our studies suggest that FXR activation stimulates intestinal cholesterol excretion and reduces diet‐induced hyperlipidemia through increased expression of ileal cholesterol transporters when hepatic SR‐B1‐mediated cholesterol movement is absent.

Wheat alkylresorcinols reduce micellar solubility of cholesterol in vitro and increase cholesterol excretion in mice

Epidemiological studies have shown that the consumption of whole grains can reduce risk for metabolic disorders. We recently showed that chronic supplementation with wheat alkylresorcinols (ARs) prevents glucose intolerance and insulin resistance with hepatic lipid accumulation induced in mice by a high-fat high-sucrose diet (HFHSD). This study examines the effects of ARs on the micellar solubility of cholesterol in vitro, as well as the effects of transient AR supplementation on faecal lipid excretion and plasma lipid levels in mice. We found that ARs formed bile micelles with taurocholate independently of phospholipids, and dose-dependently decreased the micellar solubility of cholesterol in a biliary micelle model. Transient AR supplementation with HFHSD increased faecal cholesterol and triglyceride contents and decreased plasma cholesterol concentrations. These suggest that one underlying mechanism through which ARs suppress diet-induced obesity is by interfering with the micellar cholesterol solubilisation in the digestive tract, which subsequently decreases cholesterol absorption.

Abstract 22: LDLR Promotes and PCSK9 Inhibits LDL-Derived Transintestinal Cholesterol Excretion

Direct transintestinal cholesterol excretion (TICE) is an alternative path to biliary secretion and accounts for 33% of fecal cholesterol excretion in mice. Objectives: We aimed at identifying i) the lipoproteins involved in TICE ii) the role of intestinal LDL receptor (LDLR) and of its natural inhibitor, circulating proprotein convertase subtilisin kexine type 9 (PCSK9) at the basolateral pole iii) the implication of ATP binding cassette transporter ABCB1ab, a cholesterol floppase localized at the enterocytes apical side. Methods: We labelled human lipoproteins with free 3H cholesterol (free3H) or with 3H-cholesteryl oleate (3HCO). We measured lipoprotein-derived 3H-cholesterol TICE ex vivo in intestinal explants mounted in Ussing chambers. Human explants were obtained from 4 patients undergoing bariatric surgery with their informed consent. In vivo, TICE was measured in mice i.v. injected with radiolabelled lipoproteins, by cannulation of the proximal intestine and concomitant surgical bile diversion, counting radioactivity in intestinal perfusates over 120 minutes. Results: For the first time, we showed direct evidence of TICE in human duodenal explants, from both LDL and HDL. Both lipoproteins (labelled with free3H or 3HCO ) contributed to TICE in mouse explants; TICE was highly responsive to changes in temperature and medium oxygenation. LDL-derived TICE was decreased by 58% (p<0.05) in explants from LDLR knockout mice (LDLR KO), compared with control C57Bl6J. In vivo free3H- or 3HCO-LDL-derived-TICE was conserved in LDLRKO mice, suggestive of a compensatory mechanism. LDL-derived TICE was increased by 62% (p<0.01) in PCSK9KO that present with ∼300% more intestinal LDLR. Acute depletion of intestinal LDLR with purified recombinant PCSK9 (i.v.) led to 40% (p<0.05) less TICE in PCSK9KO but had no effect in LDLRKO, confirming the implication of this receptor. Lovastatin (0.02% W/W 10d) increased TICE by 71% (p < 0.05) in C57Bl6J but not in LDLRKO. Interestingly, using 3H-cholesterol diluted in intralipid as a source, we showed that ABCB1 plays an important role in TICE. Indeed ABCB1ab -/- mice presented with 26% (p<0.05) less TICE than FVB controls in vivo and ABCB1 inhibitor PSC-833 (5mircoM) decreased TICE by 64% in an ABCB1 dependent fashion in explants. Collectively, these results provide the first molecular understanding of TICE.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARdelta activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARdelta agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion ( approximately 200%) and TICE ( approximately 100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARdelta activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARdelta stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.

Diosgenin stimulation of fecal cholesterol excretion in mice is not NPC1L1 dependent

Diosgenin exists in some food supplements and herbal medicines and lowers plasma cholesterol by increasing fecal cholesterol excretion. It is believed that diosgenin promotes fecal cholesterol excretion by stimulating biliary cholesterol secretion and decreasing intestinal cholesterol absorption. Niemann-Pick C1-like 1 (NPC1L1) was recently identified as an essential protein for intestinal cholesterol absorption. To determine the relative contribution of biliary secretion and intestinal absorption of cholesterol in diosgenin-stimulated fecal cholesterol excretion, wild-type (WT) and NPC1L1-knockout (L1KO) mice were fed a diet with or without 1% diosgenin. Fecal cholesterol excretion (mumol/day/100 g body weight) increased in diosgenin-fed WT and L1KO mice from 4.2 to 52 and from 63 to 140, respectively. Surprisingly, this increase in diosgenin-treated versus untreated L1KO mice (77) was even greater than that seen in diosgenin-treated versus untreated WT mice (47.8). Additionally, WT and L1KO mice fed the diosgenin diet had similar increases in biliary cholesterol concentration, despite unaltered hepatic expression of the hepatobiliary cholesterol transporter, ATP binding cassette transporters G5 and G8. Facilitated cholesterol excretion in diosgenin-treated WT and L1KO mice was associated with decreased hepatic and plasma cholesterol and increased liver expression of cholesterol synthetic genes. In contrast, diosgenin had no effect on the intestinal expression of NPC1L1 and cholesterol synthetic genes. In an in vitro assay, diosgenin was unable to block NPC1L1-dependent cholesterol uptake. In conclusion, diosgenin stimulation of fecal cholesterol excretion is independent of NPC1L1-mediated cholesterol absorption.

Regulation of direct transintestinal cholesterol excretion in mice

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.

Abcg5/8 Independent Biliary Cholesterol Excretion in Atp8b1-Deficient Mice

ATP8B1 is a phosphatidylserine flippase in the canalicular membrane; patients with mutations in ATP8B1 develop severe chronic (PFIC1) or periodic (BRIC1) cholestatic liver disease. We have observed that Atp8b1 deficiency leads to enhanced biliary cholesterol excretion. It has been established that biliary cholesterol excretion depends on transport by the heterodimer Abcg5/Abcg8. We hypothesized that the increased cholesterol output was due to enhanced extraction from the altered canalicular membrane rather than to higher Abcg5/Abcg8 activity. We therefore studied the relation between Abcg5/Abcg8 expression and biliary cholesterol excretion in mice lacking Atp8b1, Abcg8, or both (GF mice). Bile formation was studied in LXR agonist-fed wild-type mice as well as mice lacking Atp8b1 or Abcg8, or in GF mice upon infusion of taurocholate. Bile samples were analyzed for cholesterol, bile salt, phospholipids, and ectoenzyme content. LXR agonist increased Abcg5/8 expression, and this was accompanied by increased biliary cholesterol output in both wild-type and Atp8b1(G308V/G308V) mice. However, Atp8b1(G308V/G308V) mice maintained higher cholesterol output. Although in Abcg8(-/-) mice biliary cholesterol output was severely reduced, GF mice displayed high biliary cholesterol output, which was comparable with wild-type mice. Bile of both Atp8b1(G308V/G308V) and GF mice displayed elevated levels of phosphatidylserine and sphingomyelin, indicating membrane stress. Our data demonstrate that the increased biliary cholesterol excretion in Atp8b1-deficient mice is independent of Abcg5/8 activity. This implicates that Atp8b1 deficiency leads to a decrease in the detergent resistance and subsequent nonspecific extraction of cholesterol from the canalicular membrane by bile salts.

Direct Intestinal Cholesterol Secretion Contributes Significantly to Total Fecal Neutral Sterol Excretion in Mice

Hepatobiliary secretion is generally believed to be an integral step in the pathway of cholesterol excretion from the body. Here we have investigated the validity of this paradigm in mice. Cholesterol balance was assessed by measuring intake, excretion, and biliary output in different mouse models. Direct secretion of cholesterol from the luminal side of enterocytes was studied by perfusion of isolated segments of the small intestine in mice. Cholesterol input and output measurements in different mouse models revealed that fecal neutral sterol excretion was higher than the sum of dietary cholesterol intake and biliary cholesterol secretion indicating the existence of an alternative pathway. Here we show that substantial amounts of cholesterol can be secreted directly by enterocytes. Transintestinal cholesterol secretion is a specific process observed throughout the small intestine (proximal > medial > distal). Secretion depended on the presence of a cholesterol acceptor and was strongly stimulated by bile salts and phospholipids. The capacity of the pathway was sufficient to account for the missing cholesterol in the balance studies. The contribution of this pathway to cholesterol excretion in mice is approximately twice that of the biliary pathway. In mice, the intestine plays a significant role in removal of cholesterol from the body.

Abcg5/Abcg8-independent pathways contribute to hepatobiliary cholesterol secretion in mice

The ATP-binding cassette (ABC) half-transporters ABCG5 and ABCG8 heterodimerize into a functional complex that mediates the secretion of plant sterols and cholesterol by hepatocytes into bile and their apical efflux from enterocytes. We addressed the putative rate-controlling role of Abcg5/Abcg8 in hepatobiliary cholesterol excretion in mice during (maximal) stimulation of this process. Despite similar bile salt (BS) excretion rates, basal total sterol and phospholipid (PL) output rates were reduced by 82% and 35%, respectively, in chow-fed Abcg5(-/-) mice compared with wild-type mice. When mice were infused with the hydrophilic BS tauroursodeoxycholate, similar relative increases in bile flow, BS output, PL output, and total sterol output were observed in wild-type, Abcg5(+/-), and Abcg5(-/-) mice. Maximal cholesterol and PL output rates in Abcg5(-/-) mice were only 15% and 69%, respectively, of wild-type values. An infusion of increasing amounts of the hydrophobic BS taurodeoxycholate increased cholesterol excretion by 3.0- and 2.4-fold in wild-type and Abcg5(-/-) mice but rapidly induced cholestasis in Abcg5(-/-) mice. Treatment with the liver X receptor (LXR) agonist T0901317 increased the maximal sterol excretion capacity in wild-type mice (fourfold), concomitant with the induction of Abcg5/Abcg8 expression, but not in Abcg5(-/-) mice. In a separate study, mice were fed chow containing 1% (wt/wt) cholesterol. As expected, hepatic expression of Abcg5 and Abcg8 was strongly induced (fivefold and fourfold) in wild-type but not LXR-alpha-deficient (Lxra(-/-)) mice. Surprisingly, hepatobiliary cholesterol excretion was increased to the same extent, i.e., 2.2-fold in wild-type mice and 2.0-fold in Lxra(-/-) mice, upon cholesterol feeding. Our data confirm that Abcg5, as part of the Abcg5/Abcg8 heterodimer, strongly controls hepatobiliary cholesterol secretion in mice. However, our data demonstrate that Abcg5/Abcg8 heterodimer-independent, inducible routes exist that can significantly contribute to total hepatobiliary cholesterol output.