Fractional Cholesterol Absorption Research Articles

Intestine-specific DGAT1 deficiency improves atherosclerosis in apolipoprotein E knockout mice by reducing systemic cholesterol burden

Background and aimsAcyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme catalyzing the final step of triglyceride synthesis by esterifying a diglyceride with a fatty acid. We have previously shown that apolipoprotein E-knockout (ApoE−/−) mice lacking Dgat1 have reduced intestinal cholesterol absorption and potentiated macrophage cholesterol efflux, and consequently, exhibit attenuated atherogenesis. However, hematopoietic Dgat1 deficiency lacked beneficial effects on atherosclerosis. Due to our recent results on the critical role of intestinal Dgat1 in murine cholesterol homeostasis, we delineated whether intestinal Dgat1 deficiency regulates atherogenesis in mice. MethodsWe generated intestine-specific Dgat1−/− mice on the ApoE−/− background (iDgat1−/−ApoE−/−) and determined cholesterol homeostasis and atherosclerosis development. ResultsWhen fed a Western-type diet, iDgat1−/−ApoE−/− mice exhibited a substantial decrease in fasting plasma cholesterol content in ApoB-containing lipoproteins. Although lipid absorption was delayed, iDgat1−/−ApoE−/− mice had reduced acute and fractional cholesterol absorption coupled with an elevated fecal caloric loss. In line, increased appearance of i.v. administered [³H]cholesterol in duodena and stool of iDgat1−/−ApoE−/− animals suggested potentiated cholesterol elimination. Atherosclerotic lesions were markedly smaller with beneficial alterations in plaque composition as evidenced by reduced macrophage infiltration and necrotic core size despite unaltered collagen content, indicating improved plaque stability. ConclusionsDisruption of Dgat1 activity solely in the small intestine of ApoE−/− mice strongly decreased plasma cholesterol levels by abrogating the assimilation of dietary cholesterol, partly by reduced absorption and increased excretion. Consequently, the reduced cholesterol burden significantly attenuated atherogenesis and improved the lesion phenotype in iDgat1−/−ApoE−/− mice.

Efficient reabsorption of transintestinally excreted cholesterol is a strong determinant for cholesterol disposal in mice

Transintestinal cholesterol excretion (TICE) is a major route for eliminating cholesterol from the body and a potential therapeutic target for hypercholesterolemia. The underlying mechanism, however, is largely unclear, and its contribution to cholesterol disposal from the body is obscured by the counteracting process of intestinal cholesterol reabsorption. To determine the quantity of TICE independent from its reabsorption, we studied two models of decreased intestinal cholesterol absorption. Cholesterol absorption was inhibited either by ezetimibe or, indirectly, by the genetic inactivation of the intestinal apical sodium-dependent bile acid transporter (ASBT; SLC10A2). Both ezetimibe treatment and Asbt inactivation virtually abrogated fractional cholesterol absorption (from 46% to 4% and 6%, respectively). In both models, fecal neutral sterol excretion and net intestinal cholesterol balance were considerably higher than in control mice (5- and 7-fold, respectively), suggesting that, under physiological conditions, TICE is largely reabsorbed. In addition, the net intestinal cholesterol balance was increased to a similar extent but was not further increased when the models were combined, suggesting that the effect on cholesterol reabsorption was already maximal under either condition alone. On the basis of these findings, we hypothesize that the inhibition of cholesterol (re)absorption combined with stimulating TICE will be most effective in increasing cholesterol disposal.

The emerging concept of "individualized cholesterol-lowering therapy": A change in paradigm.

High LDL-cholesterol concentrations constitute a risk for atherosclerotic cardiovascular disease. By consensus, cholesterol-lowering therapy is initiated with a statin that reduces endogenous cholesterol synthesis, upregulates hepatic LDL receptor activity, increases LDL clearance and lowers LDL-cholesterol concentrations in the bloodstream. The efficacy of statin treatment is dose dependent and achieves a risk reduction of up to 50%. However, a substantial body of evidence suggests that a quarter of statin-treated patients do not respond adequately as a result of low endogenous cholesterol synthesis. In humans fractional cholesterol absorption varies from 20% to 80%. High cholesterol absorbers, which are characterized by a low-to-normal cholesterol synthesis, exhibit poor responsiveness to statin treatment. On the other hand, the cholesterol absorption inhibitor ezetimibe effectively reduces serum cholesterol levels in these patients. On this background, we suggest to "get personal" and individualize cholesterol-lowering therapies, according to the individual's status of cholesterol synthesis and absorption.

Modifying Serum Plant Sterol Concentrations: Effects on Markers for Whole Body Cholesterol Metabolism in Children Receiving Parenteral Nutrition and Intravenous Lipids.

Background: Non-cholesterol sterols are validated markers for fractional intestinal cholesterol absorption (cholestanol) and endogenous cholesterol synthesis (lathosterol). This study’s objective was to evaluate markers for cholesterol synthesis and absorption in children exposed to two different intravenous lipid emulsions that rapidly change serum plant sterol concentrations as part of their parenteral nutrition (PN). Methods: Serum samples from two different studies were used: (1) nine PN-dependent children with intestinal failure associated liver disease (IFALD) whose soy-based, plant sterol-rich lipid (SO) was replaced with a fish-based, plant sterol-poor (FO) lipid; and (2) five neonates prescribed SO after birth. In the first study, samples were collected at baseline (prior to FO initiation) and after 3 and 6 months of FO. In study 2, samples were collected at 1 and 3 weeks of age. Results: In study 1, a 7-fold reduction in campesterol, a 12-fold reduction in sitosterol, and a 15-fold reduction in stigmasterol was observed 6 months after switching to FO. Serum cholesterol concentrations did not change, but cholesterol-standardized lathosterol increased (3-fold) and cholesterol-standardized cholestanol decreased (2-fold). In study 2, after 3 weeks of SO, sitosterol and campesterol concentrations increased 4-5 fold. At the same time, cholesterol-standardized lathosterol increased 69% and cholesterol-standardized cholestanol decreased by 29%. Conclusion: Based on these finding we conclude that changes in serum plant sterol concentrations might have direct effects on endogenous cholesterol synthesis, although this needs to be confirmed in future studies. Moreover, we speculate that this changed synthesis subsequently affects intestinal cholesterol absorption.

Small Heterodimer Partner and Fibroblast Growth Factor 19 Inhibit Expression of NPC1L1 in Mouse Intestine and Cholesterol Absorption

The nuclear receptor subfamily 0 group B member 2 (NR0B2, also called SHP) is expressed at high levels in the liver and intestine. Postprandial fibroblast growth factor 19 (human FGF19, mouse FGF15) signaling increases the transcriptional activity of SHP. We studied the functions of SHP and FGF19 in the intestines of mice, including their regulation of expression of the cholesterol transporter NPC1L1 )NPC1-like intracellular cholesterol transporter 1) and cholesterol absorption. We performed histologic and biochemical analyses of intestinal tissues from C57BL/6 and SHP-knockout mice and performed RNA-sequencing analyses to identify genes regulated by SHP.The effects of fasting and refeeding on intestinal expression of NPC1L1 were examined in C57BL/6, SHP-knockout, and FGF15-knockout mice. Mice were given FGF19 daily for 1 week; fractional cholesterol absorption, cholesterol and bileacid (BA) levels, and composition of BAswere measured. Intestinal organoids were generated from C57BL/6 and SHP-knockout mice, and cholesterol uptake was measured. Luciferase reporter assays were performed with HT29 cells. We found that the genes that regulate lipid and ion transport in intestine, including NPC1L1, were up-regulated and that cholesterol absorption was increased in SHP-knockout mice compared with C57BL/6 mice. Expression of NPC1L1 was reduced in C57BL/6 mice after refeeding after fasting but not in SHP-knockout or FGF15-knockout mice. SHP-knockout mice had altered BA composition compared with C57BL/6 mice. FGF19 injection reduced expression of NPC1L1, decreased cholesterol absorption, and increased levels of hydrophilic BAs, including tauro-α- and -β-muricholic acids; these changes were not observed in SHP-knockout mice. SREBF2 (sterol regulatory element binding transcription factor 2), which regulates cholesterol, activated transcription of NPC1L1. FGF19 signaling led to phosphorylation of SHP, which inhibited SREBF2 activity. Postprandial FGF19 and SHP inhibit SREBF2, which leads to repression of intestinal NPC1L1 expression and cholesterol absorption. Strategies to increase FGF19 signaling to activateSHP might be developed for treatment of hypercholesterolemia.

Intestinal Farnesoid X Receptor Controls Transintestinal Cholesterol Excretion in Mice

The role of the intestine in the maintenance of cholesterol homeostasis increasingly is recognized. Fecal excretion of cholesterol is the last step in the atheroprotective reverse cholesterol transport pathway, to which biliary and transintestinal cholesterol excretion (TICE) contribute. The mechanisms controlling the flux of cholesterol through the TICE pathway, however, are poorly understood. We aimed to identify mechanisms that regulate and stimulate TICE. We performed studies with C57Bl/6J mice, as well as with mice with intestine-specific knockout of the farnesoid X receptor (FXR), mice that express an FXR transgene specifically in the intestine, and ABCG8-knockout mice. Mice were fed a control diet or a diet supplemented with the FXR agonist PX20606, with or without the cholesterol absorption inhibitor ezetimibe. Some mice with intestine-specific knockout of FXR were given daily injections of fibroblast growth factor (FGF)19. To determine fractional cholesterol absorption, mice were given intravenous injections of cholesterol D5 and oral cholesterol D7. Mice were given 13C-acetate in drinking water for measurement of cholesterol synthesis. Bile cannulations were performed and biliary cholesterol secretion rates were assessed. In a separate set of experiments, bile ducts of male Wistar rats were exteriorized, allowing replacement of endogenous bile by a model bile. In mice, we found TICE to be regulated by intestinal FXR via induction of its target gene Fgf15 (FGF19 in rats and human beings). Stimulation of this pathway caused mice to excrete up to 60% of their total cholesterol content each day. PX20606 and FGF19 each increased the ratio of muricholate:cholate in bile, inducing a more hydrophilic bile salt pool. The altered bile salt pool stimulated robust secretion of cholesterol into the intestinal lumen via the sterol-exporting heterodimer adenosine triphosphate binding cassette subfamily G member 5/8 (ABCG5/G8). Of note, the increase in TICE induced by PX20606 was independent of changes in cholesterol absorption. Hydrophilicity of the bile salt pool, controlled by FXR and FGF15/19, is an important determinant of cholesterol removal via TICE. Strategies that alter bile salt pool composition might be developed for the prevention of cardiovascular disease. Transcript profiling: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=irsrayeohfcntqx&acc=GSE74101.

Measurement of Intestinal and Peripheral Cholesterol Fluxes by a Dual-Tracer Balance Method.

Long-term elevated plasma cholesterol levels put individuals at risk for developing atherosclerosis. Plasma cholesterol levels are determined by the balance between cholesterol input and output fluxes. Here we describe in detail the methodology to determine the different cholesterol fluxes in mice. The percentage of absorbed cholesterol is calculated from a stable isotope-based double-label method. Cholesterol synthesis is calculated from MIDA after 13 C-acetate enrichment. Cholesterol is removed from the body via the feces. The fecal excretion route is either biliary or non-biliary. The non-biliary route is dominated by trans-intestinal cholesterol efflux, or TICE. Biliary excretion of cholesterol is measured by collecting bile. Non-biliary excretion is calculated by computational modeling. In this article, we describe methods and procedures to measure and calculate dietary intake of cholesterol, fractional cholesterol absorption, fecal neutral sterol output, biliary cholesterol excretion, TICE, cholesterol synthesis, peripheral fluxes, and whole-body cholesterol balance. © 2016 by John Wiley & Sons, Inc.

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine.

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes.

Fractional cholesterol absorption measurements in humans: determinants of the blood-based dual stable isotope tracer technique.

The flux of absorbed cholesterol is a controlling element in the regulation of cholesterol biosynthesis and catabolism. A review of 5 published methods to measure cholesterol absorption is presented, including 2 dual stable isotope approaches. The continuous dual isotope feeding procedure is accurate, but only suitable for small-scale studies. The blood-based dual stable isotope technique is the least invasive and complex procedure, but leads to large variations in individual (<10%->90%) and mean population values (24%-70%) for healthy subjects. The results may be partly determined by the experimental and analytical procedures. Fifteen blood-based dual stable isotope studies published between 1993 and 2013 have been analyzed. The results were related to the methodologies applied and evidence was sought for accordance to the test principles. Seven different isotopic tracers, 3 cholesterol subcompartments in blood, and 6 mass spectrometry techniques were applied. The oral and intravenous test formulations were presented in only 1 study. Time points for blood sampling and methodologies for blood sample preparation and analysis were highly variable. No definite proofs were supplied for the fates of the oral and intravenous cholesterol tracers. Isotope enrichment measurements in free and total cholesterol in plasma and erythrocytes were never compared. Fractional cholesterol absorption rate values depend strongly on the mass spectrometry methodology. Dual-inlet isotope ratio mass spectrometry appears to be the method of choice. Dual stable isotope approaches require validation and standardization of administration and analysis procedures. A control group must always be included to correct for methodological differences.

Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice.

Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal(-/-) and matching lal(+/+) mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal(-/-) mice sequestered cholesterol at an average rate of 3.2 mg·day(-1)·animal(-1). The proportion of the body sterol pool contained in the liver of the lal(-/-) mice was 64 vs. 6.3% in their lal(+/+) controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal(-/-) mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal(-/-) mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal(+/+) controls. The rate of cholesterol synthesis in the lal(-/-) mice exceeded that in the lal(+/+) controls by 3.7 mg·day(-1)·animal(-1). Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal(-/-) mice, but their rate of neutral sterol excretion was 59% higher than in their lal(+/+) controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal(-/-) mice was 355 days.

Berberine decreases cholesterol levels in rats through multiple mechanisms, including inhibition of cholesterol absorption

ObjectiveThe objective was to determine the mechanisms of action of berberine (BBR) on cholesterol homeostasis using in vivo and in vitro models. MethodsMale Sprague–Dawley rats were fed the AIN-93G diet (normal control) or modified AIN-93G diet containing 28% fat, 2% cholesterol and 0.5% cholic acid with treatment of 0 (atherogenic control), 50, 100, and 150mg/kg·d of BBR, respectively by gavaging in water for 8weeks. Cholesterol absorption rate was measured with the dual stable isotope ratio method, and plasma lipids were determined using the enzymatic methods. Gene and protein expressions of Acyl-coenzyme A:cholesterol acyltransferase-2 were analyzed in vivo and in vitro. Cholesterol micellarization, uptake and permeability were determined in vitro. ResultsRats on the atherogenic diet showed significantly hypercholesterolemic characteristics compared to normal control rats. Treatment with BBR in rats on the atherogenic diet reduced plasma total cholesterol and nonHDL cholesterol levels by 29%–33% and 31%–41%, respectively, with no significant differences being observed among the three doses. The fractional dietary cholesterol absorption rate was decreased by 40%–51%. Rats fed the atherogenic diet showed lower plasma triacylglycerol levels, and no changes were observed after the BBR treatment. BBR interfered with cholesterol micellarization, decreased cholesterol uptake by Caco-2 cells and permeability through Caco-2 monolayer. BBR also inhibited the gene and protein expressions of acyl-coenzyme A cholesterol acyltransferease-2 in the small intestine and Caco-2 cells. ConclusionBBR lowered blood cholesterol levels at least in part through inhibiting the intestinal absorption and further by interfering with intraluminal cholesterol micellarization and decreasing enterocyte cholesterol uptake and secretion.

Cholesterol metabolism and serum non-cholesterol sterols: summary of 13 plant stanol ester interventions.

BackgroundThe efficacy and safety of plant stanols added to food products as serum cholesterol lowering agents have been demonstrated convincingly, but their effects on cholesterol metabolism and on serum non-cholesterol sterols is less evaluated. The aim of this study was to assess the validity of serum non-cholesterol sterols and squalene as bioindices of cholesterol synthesis and absorption, and to examine how the individual serum non-cholesterol sterols respond to consumption of plant stanols.MethodsWe collected all randomized, controlled plant stanol ester (STAEST) interventions in which serum cholestanol, plant sterols campesterol and sitosterol, and at least two serum cholesterol precursors had been analysed. According to these criteria, there was a total of 13 studies (total 868 subjects without lipid-lowering medication; plant stanol doses varied from 0.8 to 8.8 g/d added in esterified form; the duration of the studies varied from 4 to 52 weeks). Serum non-cholesterol sterols were assayed with gas–liquid chromatography, cholesterol synthesis with the sterol balance technique, and fractional cholesterol absorption with the dual continuous isotope feeding method.ResultsThe results demonstrated that during the control and the STAEST periods, the serum plant sterol/cholesterol- and the cholestanol/cholesterol-ratios reflected fractional cholesterol absorption, and the precursor sterol/cholesterol-ratios reflected cholesterol synthesis. Plant sterol levels were dose-dependently reduced by STAEST so that 2 g of plant stanols reduced serum campesterol/cholesterol-ratio on average by 32%. Serum cholestanol/cholesterol-ratio was reduced less frequently than those of the plant sterols by STAEST, and the cholesterol precursor sterol ratios did not change consistently in the individual studies emphasizing the importance of monitoring more than one surrogate serum marker.ConclusionsSerum non-cholesterol sterols are valid markers of cholesterol absorption and synthesis even during cholesterol absorption inhibition with STAEST. Serum plant sterol concentrations decrease dose-dependently in response to plant stanols suggesting that the higher the plant stanol dose, the more cholesterol absorption is inhibited and the greater the reduction in LDL cholesterol level is that can be achieved.Trial registrationClinical Trials Register # NCT00698256 [Eur J Nutr 2010, 49:111-117]

Sustained and selective suppression of intestinal cholesterol synthesis by Ro 48-8071, an inhibitor of 2,3-oxidosqualene:lanosterol cyclase, in the BALB/c mouse

The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterol cyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A.

Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BI(hApoCIII-ApoAIV-Tg)). SR-BI(hApoCIII-ApoAIV-Tg) mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BI(hApoCIII-ApoAIV-Tg) mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BI(hApoCIII-ApoAIV-Tg) mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

Plasma plant sterols serve as poor markers of cholesterol absorption in man

The validation of the use of plasma plant sterols as a marker of cholesterol absorption is frail. Nevertheless, plant sterol concentrations are routinely used to describe treatment-induced changes in cholesterol absorption. Their use has also been advocated as a clinical tool to tailor cholesterol-lowering therapy. Prior to wider implementation, however, the validity of plant sterols as absorption markers needs solid evaluation. Therefore, we compared plasma plant sterol concentrations to gold-standard stable isotope-determined cholesterol absorption. Plasma campesterol/TC concentrations (camp/TC) were measured in a population of 175 mildly hypercholesterolemic individuals (age: 59.7 ± 5.6 years; BMI: 25.5 ± 2.9 kg/m(2); LDL-C: 4.01 ± 0.56 mmol/l). We compared cholesterol absorption according to the plasma dual-isotope method in subjects with the highest camp/TC concentrations (N = 41, camp/TC: 2.14 ± 0.68 μg/mg) and the lowest camp/TC concentrations (N = 39, camp/TC: 0.97 ± 0.22 μg/mg). Fractional cholesterol absorption did not differ between the groups (24 ± 12% versus 25 ± 16%, P = 0.60), nor was it associated with plasma camp/TC concentrations in the total population of 80 individuals (β = 0.13; P = 0.30, adjusted for BMI and plasma triglycerides). Our findings do not support a relation between plasma plant sterol concentrations and true cholesterol absorption and, therefore, do not favor the use of these sterols as markers of cholesterol absorption. This bears direct consequences for the interpretation of earlier studies, as well as for future studies targeting intestinal regulation of cholesterol metabolism.

Phytosterols Reduce Cholesterol Absorption by Inhibition of 27-Hydroxycholesterol Generation, Liver X Receptor α Activation, and Expression of the Basolateral Sterol Exporter ATP-Binding Cassette A1 in Caco-2 Enterocytes1–3

Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 μmol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar 3H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXRα) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXRα-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXRα activation in human enterocytes. Exogenous LXRα agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXRα-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXRα-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXRα antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol.

Delineation of biochemical, molecular, and physiological changes accompanying bile acid pool size restoration in Cyp7a1(-/-) mice fed low levels of cholic acid.

Cholesterol 7α-hydroxylase (CYP7A1) is the initiating and rate-limiting enzyme in the neutral pathway that converts cholesterol to primary bile acids (BA). CYP7A1-deficient (Cyp7a1(-/-)) mice have a depleted BA pool, diminished intestinal cholesterol absorption, accelerated fecal sterol loss, and increased intestinal cholesterol synthesis. To determine the molecular and physiological effects of restoring the BA pool in this model, adult female Cyp7a1(-/-) mice and matching Cyp7a1(+/+) controls were fed diets containing cholic acid (CA) at modest levels [0.015, 0.030, and 0.060% (wt/wt)] for 15-18 days. A level of just 0.03% provided a CA intake of ~12 μmol (4.8 mg) per day per 100 g body wt and was sufficient in the Cyp7a1(-/-) mice to normalize BA pool size, fecal BA excretion, fractional cholesterol absorption, and fecal sterol excretion but caused a significant rise in the cholesterol concentration in the small intestine and liver, as well as a marked inhibition of cholesterol synthesis in these organs. In parallel with these metabolic changes, there were marked shifts in intestinal and hepatic expression levels for many target genes of the BA sensor farnesoid X receptor, as well as genes involved in cholesterol transport, especially ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG8. In Cyp7a1(+/+) mice, this level of CA supplementation did not significantly disrupt BA or cholesterol metabolism, except for an increase in fecal BA excretion and marginal changes in mRNA expression for some BA synthetic enzymes. These findings underscore the importance of using moderate dietary BA levels in studies with animal models.

The Role of Bile Acid Excretion in Atherosclerotic Coronary Artery Disease

The impact of cholesterol and different classes of lipoproteins on the development of coronary artery disease (CAD) has been investigated in extensively during the past 50 years. The cholesterol metabolism is dependent on numerous factors, including dietary fat, fractional absorption of dietary cholesterol, tissue stores of cholesterol, endogenous cholesterol synthesis, and fecal bile excretion. Several studies showed significantly lower amounts of bile acid secretion in adult patients with CAD compared to non-CAD patients. Could it be that the inability to efficiently excrete bile acids may lead to CAD development?

A Reappraisal of the Mechanism by Which Plant Sterols Promote Neutral Sterol Loss in Mice

Dietary plant sterols (PS) reduce serum total and LDL-cholesterol in hyperlipidemic animal models and in humans. This hypocholesterolemic effect is generally ascribed to inhibition of cholesterol absorption. However, whether this effect fully explains the reported strong induction of neutral sterol excretion upon plant sterol feeding is not known. Recent data demonstrate that the intestine directly mediates plasma cholesterol excretion into feces, i.e., without involvement of the hepato-biliary route.ObjectiveAim of this study was to determine whether stimulation of fecal neutral sterol loss during PS feeding is (partly) explained by increased intestinal cholesterol excretion and to assess the role of the cholesterol transporter Abcg5/Abcg8 herein.Methods and ResultsWild-type mice were fed a control diet or diets enriched with increasing amounts of PS (1%, 2%, 4% or 8%, wt/wt) for two weeks. In addition, Abcg5-/- mice were fed either control or 8% PS diet. PS feeding resulted in a dose-dependent decrease of fractional cholesterol absorption (∼2–7-fold reduction) in wild-type mice and ∼80% reduction in Abcg5-/- mice. Furthermore, PS feeding led to a strong, dose-independent induction of neutral sterol excretion (3.4-fold in wild-types and 2.7-fold in Abcg5-/- mice) without changes in biliary cholesterol secretion. It was calculated that PS feeding stimulated intestinal cholesterol excretion by ∼500% in wild-type mice and by ∼250% in Abcg5-/-.ConclusionsOur data indicate that in mice the cholesterol-lowering effects of PS are to a large extent attributable to stimulation of intestinal, non-bile derived, cholesterol excretion. The Abcg5/Abcg8 heterodimer is involved in facilitating this PS-induced flux of cholesterol.

Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis

We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1-tg mice) are protected against high fat diet-induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had two-fold higher Cyp7a1 activity and bile acid pool than did wild-type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild-type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild-type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild-type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate-binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis.