Cholesterol-to-Coprostanol Conversion by the Gut Microbiota: What We Know, Suspect, and Ignore

Every day, up to 1 g of cholesterol, composed of the unabsorbed dietary cholesterol, the biliary cholesterol secretion, and cholesterol of cells sloughed from the intestinal epithelium, enters the colon. All cholesterol arriving in the large intestine can be metabolized by the colonic bacteria. Cholesterol is mainly converted into coprostanol, a non-absorbable sterol that is excreted in the feces. Interestingly, cholesterol-to-coprostanol conversion in human populations is variable, with a majority of high converters and a minority of low or inefficient converters. Two major pathways have been proposed, one involving the direct stereospecific reduction of the Δ5 double bond direct while the indirect pathway involves the intermediate formation of 4-cholelesten-3-one and coprostanone. Despite the fact that intestinal cholesterol conversion was discovered more than a century ago, only a few cholesterol-to-coprostanol-converting bacterial strains have been isolated and characterized. Moreover, the responsible genes were mainly unknown until recently. Interestingly, cholesterol-to-coprostanol conversion is highly regulated by the diet. Finally, this gut bacterial metabolism has been linked to health and disease, and recent evidence suggests it could contribute to lower blood cholesterol and cardiovascular risks.

Simultaneous Determination of Biliary and Intestinal Cholesterol Secretion Reveals That CETP (Cholesteryl Ester Transfer Protein) Alters Elimination Route in Mice

Objective: Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol–fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Conclusions: Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway.

Abstract 390: The Absence of Abcg5 Abcg8 Reveals a Sexually Dimorphic Adaption to Impaired Biliary Cholesterol Secretion

Objective: The ABCG5 ABCG8 (G5G8) sterol transporter is the primary mechanism for biliary cholesterol secretion, but mice maintain fecal sterol excretion in its absence. The mechanism by which mice maintain sterol excretion in the absence of this pathway is not known. Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion. We investigated the impact of G5G8 deficiency on TICE in the absence of Sitosterolemia. Methods and Results: We compared both hepatobiliary and transintestinal cholesterol excretion rates in wild-type (WT) and G5G8 deficient mice of both sexes. WT and G5G8 were maintained on a plant-sterol free diet from the time of weaning to prevent the development of secondary phenotypes associated with Sitosterolemia. Biliary and intestinal cholesterol secretion rates were determined by biliary diversion with simultaneous perfusion of the proximal 10 cm of the small bowel. Among WT mice, biliary cholesterol secretion was greater in female mice compared to males. Conversely, male mice exhibited greater rates of TICE than females. As expected, WT mice had higher biliary cholesterol secretion rates than their G5G8 deficient littermates. However, the decline in biliary cholesterol secretion was far less in male mice compared to females in the absence of G5G8. In female mice, the absence of G5G8 resulted in a two-fold increase in TICE, whereas males were unaffected. Conclusion: Female mice are more dependent upon the biliary pathway for cholesterol excretion, whereas males are more dependent upon TICE. G5G8 independent pathways are present for both biliary and intestinal cholesterol secretion. Female and male mice differ in their adaptation to G5G8 deficiency in order to maintain fecal sterol excretion.

Abstract 439: Atherosclerosis Development is Reduced in Mice With Blunted Biliary Cholesterol Secretion

Excessive accumulation of cholesterol in the arteries drives atherosclerosis development. It is believed that biliary cholesterol secretion is crucial for eliminating excess cholesterol from the body via reverse cholesterol transport. In the current study, we wanted to determine the impact of reduced biliary cholesterol secretion on atherosclerosis development in mice. Decreased biliary cholesterol secretion was achieved by hepatic over-expression of human NPC1L1 (L1Tg mice) combined with knockdown of hepatic ABCG5/G8 function using an ABCG8 antisense oligonucleotide (ASO). LDLR-/- and LDLR-/-/L1Tg mice received either control or ABCG8 ASO and were fed a high fat (42% Kcal)/low cholesterol diet (0.015% wt/wt) for 20 weeks. As expected, L1Tg mice and mice with hepatic ABCG8 knockdown had an >70% reduction in biliary cholesterol. The dramatic decrease in biliary cholesterol did not increase plasma cholesterol, and in fact mice with hepatic ABCG8 knockdown had reduced VLDL cholesterol. Even more surprising, aortic atherosclerosis was significantly decreased in mice with compromised biliary cholesterol secretion. LDLR-/-/L1Tg treated with ABCG8 ASO had a >90% reduction in biliary cholesterol yet had ~70% less atherosclerosis compared to LDLR-/- controls. Moreover, reducing biliary cholesterol had no impact on macrophage reverse cholesterol transport, fecal excretion of neutral sterol, and hepatic expression of genes involved in cholesterol synthesis (HMG CoA reductase/synthase) and HDL metabolism (ABCA1 and SR-BI). These results indicate that atherosclerosis development can be decreased by shunting cholesterol away from biliary secretion and potentially towards trans-intestinal cholesterol excretion or bile acid synthesis.

Abstract 629: FGF15/19 Upregulates Hepatic ABCG5 and ABCG8 to Promote Biliary Cholesterol Secretion

Background: Elevated hepatic cholesterol is thought to contribute to the development of nonalcoholic fatty liver disease, a condition highly associated with cardiovascular risk factors. The ABCG5 and ABCG8 (G5G8) heterodimer is responsible for up to 90% of biliary cholesterol secretion and is a potential therapeutic target for promoting cholesterol elimination. We have previously demonstrated that ursodiol (UDCA), a pharmacologic bile acid, increases G5G8 protein expression and biliary cholesterol secretion. However, whole body cholesterol elimination is minimized likely due to simultaneous suppression of bile acid synthesis through upregulation of FGF15/19. The objectives of this study are to determine whether FGF15/19 regulates G5G8 and determine whether UDCA requires FGF15/19 signaling in order to upregulate G5G8. Methods: Mice were injected with two doses of FGF19 or carrier (PBS) 1μg/g body weight within an 8-hour treatment window. A separate group of wild type (WT) and G5G8 knockout (KO) mice were similarly injected with FGF19. In another experiment, WT mice were fed chow or UDCA-supplemented diet in the absence or presence of FGF15/19 signaling inhibition which was achieved by FGFR4 antisense oligonucleotide (ASO) supplied by Ionis Pharmaceuticals. In all experiments, body weight, liver weight, bile flow rate and plasma, hepatic and biliary lipids were measured. Immunoblotting of G5G8 and real-time PCR of genes involved in cholesterol metabolism were also conducted. Results: Mice injected with FGF19 had increased biliary lipids (PBS: 5.287±0.5720, FGF19: 8.098±0.6114, n=6), decreased Cyp7a1 (PBS: 1.021±0.1064 FGF19: 0.07787±0.01345 n=5-7) and Cyp8b1 (PBS: 1.018±0.09846, FGF19: 0.2647±0.05609, n=5-7) expression, and increased G5G8 protein expression compared to mice injected with PBS. In G5G8 KO mice injected with FGF19, there was only a small increase in plasma free cholesterol (WT: 51.96±2.098, KO: 62.24±2.562, n=4) and no other significant changes in cholesterol metabolism compared to wild type mice injected with FGF19. Conclusion: In conclusion, FGF15/19 suppresses bile acid synthesis and post-transcriptionally upregulates G5G8. However, in the absence of G5G8, FGF15/19 did not disrupt cholesterol metabolism.

Abstract 131: Restoration of ER Stress and Induction of FGF15/19 Independently Rescue ABCG5 ABCG8 Sterol Transporter

Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic ABCG5 ABCG8 (G5G8). Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanism responsible for G5G8 regulation remains unclear. In the current study, we conducted a series of experiments to address potential mechanisms. To determine if leptin signaling directly regulates hepatic G5G8 abundance, we acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8. Alternatively, the G5G8 protein complex may be decreased due to compromised ER stress. It has been previously reported that tauroursodeoxycholate (TUDCA) alleviates ER stress. It also increases G5G8 and biliary cholesterol in both lean and db/db mice. The ER chaperone protein, glucose regulated protein 78-kDa (GRP78) can restore ER function and reduce unfolded protein response (UPR) signaling. Therefore, we tested the hypothesis that expression of GRP78 could rescue G5G8 in db/db mice. Adenovirus encoding GRP78 was administered to db/db mice and the effect on hepatic G5G8 was determined. G5 and G8 proteins and biliary cholesterol were increased in the absence of changes in mRNAs encoding either protein. However, TUDCA has also been shown to induce FGF15. In several models of bile acid feeding, FGF15/19 is stimulated in ileum and activates its receptor in liver to repress bile acid synthesis. Simultaneously, G5G8 and biliary cholesterol secretion are elevated. To determine if FGF15/19 had a direct effect on hepatic G5G8, we injected C57BL/6 mice with recombinant FGF19. CYP7A1 and CYP8B1 mRNA expression were both strongly suppressed, whereas G5G8 increased at both mRNA and protein levels. In conclusion, G5G8 can be rescued in ob/ob and db/db mice through multiple mechanisms that include restoration of ER functions and FGF15/19 signaling. Counter regulation of CYP7A1, CYP8B1, and G5G8 by FGF15/19 allows for the maintenance of hepatic sterol elimination in the face of expanded bile acid pool.

Abstract 317: CETP Alters Routes of Total and HDL-derived Cholesterol Elimination in Mice

Objective: Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion for cholesterol elimination. The cholesterol donors contributing to this pathway in plasma remain unclear, but appear to include both ApoA and ApoB lipoproteins. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma. Our study was aimed at determining the impact of CETP on cholesterol elimination pathways in mice. Methods and Results: We compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (TG) mice at age of 12 weeks. WT and TG mice did not differ either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to high fat, high cholesterol Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to the bile and intestinal lumen. Unlike bulk cholesterol secretion, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP transgenic mice. To further explore the mechanism, we injected radiolabeled HDLs from both WT mice (WT-HDL) and TG mice (CETP-HDL) back into male WT mice. Although CETP did not alter HDL-CE delivery to bile, cholesterol from HDL isolated from CETP TG mice were secreted into the intestinal lumen at a greater rate than WT mice. Conclusion: The data suggest that CETP alters routes of total and HDL cholesterol elimination from the body in mice.