ABCG5 and ABCG8 Are Involved in Vitamin K Transport.

ATP-binding cassette protein G5 (ABCG5)/ABCG8 heterodimer exports cholesterol from cells, while Niemann-Pick C1-like 1 (NPC1L1) imports cholesterol and vitamin K. We examined whether ABCG5/ABCG8 transports vitamin K similar to NPC1L1. Since high concentrations of vitamin K3 show cytotoxicity, the cytoprotective effects of ABCG5/ABCG8 were examined. BHK cells expressing ABCG5/ABCG8 were more resistant to vitamin K3 cytotoxicity than control cells, suggesting that ABCG5/ABCG8 transports vitamin K3 out of cells. The addition of vitamin K1 reversed the effects of ABCG5/ABCG8, suggesting that vitamin K1 competitively inhibits the transport of vitamin K3. To examine the transport of vitamin K1 by ABCG5/ABCG8, vitamin K1 levels in the medium and cells were measured. Vitamin K1 levels in cells expressing ABCG5/ABCG8 were lower than those in control cells, while vitamin K1 efflux increased in cells expressing ABCG5/ABCG8. Furthermore, the biliary vitamin K1 concentration in Abcg5/Abcg8-deficient mice was lower than that in wild-type mice, although serum vitamin K1 levels were not affected by the presence of Abcg5/Abcg8. These findings suggest that ABCG5 and ABCG8 are involved in the transport of sterols and vitamin K. ABCG5/ABCG8 and NPC1L1 might play important roles in the regulation of vitamin K absorption and excretion.

Hepatic Niemann-Pick C1-Like 1 exacerbates non-alcoholic fatty liver disease by re-absorbing specific biliary oxysterols.

BACKGROUND: Dietary oxysterols are believed to be associated with the progression of non-alcoholic fatty liver disease (NAFLD). However, the molecular basis of the association between dietary oxysterols and NAFLD is poorly understood. We hypothesized that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver, would regulate hepatic oxysterol levels and affects NAFLD progression. METHODS AND RESULTS: Considering the species differences in hepatic NPC1L1 expression, we used liver-specific NPC1L1 transgenic (NPC1L1Tg) mice as a human model and demonstrated that oxysterol-rich heated cholesterol exacerbated high-fat diet-induced steatosis, an early stage of NAFLD, in a hepatic NPC1L1-dependent manner. Analyses of hepatic and biliary oxysterol levels in NPC1L1Tg mice and in vitro oxysterol uptake assays with NPC1L1-overexpressing cells revealed that NPC1L1 can uptake some, but not all, oxysterols and suppress their biliary excretion. Furthermore, in vitro and in vivo analyses revealed that 22(R)-hydroxycholesterol (22R-OHC) and 25-hydroxycholesterol (25-OHC), which are NPC1L1 substrates, were primarily involved in steatosis progression, via the activation of liver X receptor α and retinoid-related orphan receptor γ, respectively. Consistent with these results, examination of clinical specimens revealed that among the 14 major oxysterols analyzed, plasma concentrations of 22R-OHC and 25-OHC were significantly positively correlated with hepatic fat accumulation in humans. CONCLUSIONS: Among the major dietary oxysterols, 22R-OHC and 25-OHC are particularly potent in promoting the progression of hepatic steatosis in a hepatic NPC1L1-dependent manner.

Increase of serum uric acid levels associated with APOE ε2 haplotype: a clinico-genetic investigation and in vivo approach.

Elevated serum uric acid (SUA)-hyperuricemia-is caused by overproduction of urate or by its decreased renal and/or intestinal excretion. This disease, which is increasing in prevalence worldwide, is associated with both gout and metabolic diseases. Several studies have reported relationships between apolipoprotein E (APOE) haplotypes and SUA levels in humans; however, their results remain inconsistent. This prompted us to investigate the relationship between APOE polymorphisms and SUA levels. Our subjects were 5,272 Japanese men, premenopausal women, and postmenopausal women. Multiple linear regression analyses revealed the ε2 haplotype of APOE to be independently associated with higher SUA in men (N = 1,726) and postmenopausal women (N = 1,753), but not in premenopausal women (N = 1,793). In contrast, the ε4 haplotype was little related to SUA levels in each group. Moreover, to examine the effect of Apoe deficiency on SUA levels, we conducted animal experiments using Apoe knockout mice, which mimics ε2/ε2 carriers. We found that SUA levels in Apoe knockout mice were significantly higher than those in wild-type mice, which is consistent with the SUA-raising effect of the ε2 haplotype observed in our clinico-genetic analyses. Further analyses suggested that renal rather than intestinal underexcretion of urate could be involved in Apoe deficiency-related SUA increase. In conclusion, we successfully demonstrated that the ε2 haplotype, but not the ε4 haplotype, increases SUA levels. These findings will improve our understanding of genetic factors affecting SUA levels.

NPC1L1 Facilitates Sphingomyelin Absorption and Regulates Diet-Induced Production of VLDL/LDL-associated S1P.

Niemann-Pick C1-Like 1 (NPC1L1) is a cholesterol importer and target of ezetimibe, a cholesterol absorption inhibitor used clinically for dyslipidemia. Recent studies demonstrated that NPC1L1 regulates the intestinal absorption of several fat-soluble nutrients, in addition to cholesterol. The study was conducted to reveal new physiological roles of NPC1L1 by identifying novel dietary substrate(s). Very low-density lipoprotein and low-density lipoprotein (VLDL/LDL) are increased in Western diet (WD)-fed mice in an NPC1L1-dependent manner, so we comprehensively analyzed the NPC1L1-dependent VLDL/LDL components. Apolipoprotein M (apoM), a binding protein of sphingosine-1-phosphate (S1P: a lipid mediator), and S1P were NPC1L1-dependently increased in VLDL/LDL by WD feeding. S1P is metabolized from sphingomyelin (SM) and SM is abundant in WD, so we focused on intestinal SM absorption. In vivo studies with Npc1l1 knockout mice and in vitro studies with NPC1L1-overexpressing cells revealed that SM is a physiological substrate of NPC1L1. These results suggest a scenario in which dietary SM is absorbed by NPC1L1 in the intestine, followed by SM conversion to S1P and, after several steps, S1P is exported into the blood as the apoM-bound form in VLDL/LDL. Our findings provide insight into the functions of NPC1L1 for a better understanding of sphingolipids and S1P homeostasis.

Pathophysiological importance of bile cholesterol reabsorption: hepatic NPC1L1-exacerbated steatosis and decreasing VLDL-TG secretion in mice fed a high-fat diet.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, although its pathogenesis remains to be elucidated. A recent study revealed that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver expressed on the bile canalicular membrane, is an exacerbation factor of NAFLD. Indeed, transgenic mice with hepatic expression of human NPC1L1 under a liver-specific promoter (L1-Tg mice) developed steatosis with a high-fat diet (HFD) containing cholesterol within a few weeks. However, the mechanism underlying diet-induced hepatic NPC1L1-mediated lipid accumulation is poorly defined. METHODS: To achieve a deeper understanding of steatosis development in L1-Tg mice, the biochemical features of hepatic NPC1L1-mediated steatosis were investigated. Hemizygous L1-Tg mice and wild-type littermate controls fed a HFD or control-fat diet were used. At the indicated time points, the livers were evaluated for cholesterol and triglyceride (TG) contents as well as mRNA levels of hepatic genes involved in the maintenance of lipid homeostasis. The hepatic ability to secrete very low-density lipoprotein (VLDL)-TG was also investigated. RESULTS: Unlike the livers of wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice displayed time-dependent changes that indicated steatosis formation. In steatosis, there were three different stages of development: mild accumulation of hepatic cholesterol and TG (early stage), acceleration of hepatic TG accumulation (middle stage), and further accumulation of hepatic cholesterol (late stage). In the early stage, between WT and L1-Tg mice fed a HFD for 2 weeks, there were no significant differences in the hepatic expression of Pparα, Acox1, Fat/Cd36, Srebf1, and Srebf2; however, the hepatic ability to secrete VLDL-TG decreased in L1-Tg mice (P < 0.05). Furthermore, this decrease was completely prevented by administration of ezetimibe, an NPC1L1-selective inhibitor. CONCLUSION: Hepatic NPC1L1 exacerbates diet-induced steatosis, which was accompanied by decreased hepatic ability of VLDL-TG secretion. The obtained results provide a deeper understanding of L1-Tg mice as a promising NAFLD animal model that is able to re-absorb biliary-secreted cholesterol similar to humans. Furthermore, this work supports further studies of the pathophysiological impact of re-absorbed biliary cholesterol on the regulation of hepatic lipid homeostasis.

[Translational Research Based on Understanding the Regulatory Mechanisms of in Vivo Behaviors of Fat-soluble Compounds].

Several fat-soluble compounds such as cholesterol and fat-soluble vitamins have important physiological activities in the body, and their excess and/or deficiency have been reported to be closely associated with the onset and progression of several conditions such as lifestyle-related diseases. It is important to clarify not only the physiological activities but also in vivo kinetics of fat-soluble compounds to understand their in vivo activity (toxicity). This review introduces our recent (reverse) translational research in a combination of basic and clinical studies to reveal the regulatory mechanisms of in vivo behaviors of fat-soluble compounds and effects of their disruption in humans.

Niemann-Pick C1-like 1 Promotes Intestinal Absorption of Siphonaxanthin.

Siphonaxanthin is a carotenoid found in certain green algae, and its promising beneficial properties, such as its anti-obesity effect, have recently been demonstrated. However, there is little information about the molecular mechanisms underlying intestinal absorption of siphonaxanthin. In this study, we aimed to elucidate how siphonaxanthin is transported across the intestinal epithelium using differentiated Caco-2 cells (dCaco-2 cells), recombinant proteins, and an animal model. Siphonaxanthin was taken up by dCaco-2 cells, a model of intestinal epithelial cells, and its uptake linearly increased up to at least 6 h. Pharmacological inhibition of Nieman-Pick C1-like 1 (NPC1L1), but not that of scavenger receptor class B type 1 (SR-B1), significantly suppressed siphonaxanthin uptake by dCaco-2 cells. Results from an in vitro binding assay suggested that the N-terminal domain of NPC1L1, which is an extracellular domain of NPC1L1, binds with siphonaxanthin. Moreover, pretreatment with ezetimibe, an inhibitor of NPC1L1, significantly decreased the plasma level of siphonaxanthin following oral administration in mice. Considered together, we concluded that NPC1L1 promotes siphonaxanthin transport across the intestinal epithelium.

Hepatic Expression of Niemann-Pick C1-Like 1, a Cholesterol Reabsorber from Bile, Exacerbates Western Diet-Induced Atherosclerosis in LDL Receptor Mutant Mice.

Westernization of dietary habits increases lipid intake and is responsible for increased numbers of patients with atherosclerotic diseases. Niemann-Pick C1-Like 1 (NPC1L1)-a cholesterol importer-plays a crucial role in dietary cholesterol absorption in the intestine and is closely associated with several lipid-related diseases, including atherosclerosis. NPC1L1 is highly expressed in the liver and intestine in humans, whereas NPC1L1 expression is low in the rodent liver. Due to species differences in the tissue distribution of NPC1L1, there are limited studies on the pathophysiological role of hepatic NPC1L1, a cholesterol reabsorber from bile. In the present study, to explore whether hepatic NPC1L1 is involved in the development/progression of atherosclerosis, we compared four kinds of atherosclerosis mouse models with different expression levels of NPC1L1 in the intestinal and liver tissues in a genetic background of dysfunctional low-density lipoprotein receptor mutation. Western diet (WD)-induced hyperlipidemia and atherosclerotic plaque formation were more severe in mice expressing NPC1L1 in both the liver and intestine (plasma cholesterol, 839.5 mg/dl; plaque area, 29.5% of total aorta), compared with mice expressing NPC1L1 only in the intestine (plasma cholesterol, 573.1 mg/dl; plaque area, 13.3% of total aorta). Such hepatic NPC1L1-mediated promotion of hyperlipidemia and atherosclerosis was not observed in mice not expressing intestinal NPC1L1 and mice treated with ezetimibe, an NPC1L1 inhibitor used clinically for dyslipidemia. These results suggested that hepatic NPC1L1 promotes WD-induced dyslipidemia and atherosclerosis in concert with intestinal NPC1L1. Our findings provide novel insights into the pathophysiological importance of hepatic NPC1L1 in development/progression of atherosclerosis. SIGNIFICANCE STATEMENT: Niemann-Pick C1-Like 1 (NPC1L1) protein, a cholesterol importer and a molecular target of ezetimibe clinically used for dyslipidemia, is highly expressed not only in the intestine, but also in the liver in humans, although the pathophysiological importance of hepatic NPC1L1 in atherosclerotic diseases remained unclear. By using novel mouse models to separately analyze the effects of hepatic and intestinal NPC1L1 on the development/progression of atherosclerosis, we first demonstrated that hepatic NPC1L1 accelerates Western diet-induced atherosclerotic plaque formation in an intestinal NPC1L1-dependent and an ezetimibe-sensitive manner.

Mechanisms of Niemann-Pick type C1 Like 1 protein degradation in intestinal epithelial cells.

Intestinal Niemann-Pick C1 Like 1 (NPC1L1) protein plays a key role in cholesterol absorption. A decrease in NPC1L1 expression has been implicated in lowering plasma cholesterol and mitigating the risk for coronary heart disease. Little is known about the mechanisms responsible for NPC1L1 protein degradation that upon activation may lead to a reduction in NPC1L1 protein levels in intestinal epithelial cells (IECs). In current studies, the human intestinal Caco-2 and HuTu-80 cell lines expressing NPC1L1-hemagglutinin fusion protein were used to investigate the mechanisms of NPC1L1 protein degradation. Incubation with the proteasome inhibitors MG-132 and lactacystin (10 µM, 24 h) significantly increased NPC1L1 protein levels in IECs. Also, the inhibition of the lysosomal pathway with bafilomycin A1 (80 nM, 24 h) resulted in a significant increase in NPC1L1 protein levels. Immunoprecipitation studies showed that NPC1L1 protein is both a poly- and monoubiquinated polypeptide and that the inhibition of the proteasomal pathway remarkably increased the level of the polyubiquinated NPC1L1. The surface expression of NPC1L1 was increased by the inhibition of both proteasomal and lysosomal pathways. Furthermore, the pharmacological inhibition of mitogen-activated protein kinase pathway (PD-98059, 15 µM, 24 h) and siRNA silencing of ERK1/2 resulted in a significant decrease in NPC1L1 protein levels in IECs. In conclusion, our results showed that basal level of intestinal cholesterol transporter NPC1L1 protein is modulated by both ubiquitin proteasome- and lysosome-dependent degradation as well as by ERK1/2-dependent pathway. The modulation of these pathways may provide novel clues for therapeutic intervention to inhibit cholesterol absorption and lower plasma cholesterol.

Identification of hepatic NPC1L1 as an NAFLD risk factor evidenced by ezetimibe-mediated steatosis prevention and recovery.

Non-alcoholic fatty liver disease (NAFLD) is a serious global public health concern. Nevertheless, there are no specific medications for treating the associated abnormal accumulation of hepatic lipids such as cholesterol and triglycerides. While seminal findings suggest a link between hepatic cholesterol accumulation and NAFLD progression, the molecular bases of these associations are not well understood. Here, we experimentally demonstrate that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver, can cause steatosis, an early stage of NAFLD using genetically engineered L1-Tg mice characterized by hepatic expression of NPC1L1 under the control of ApoE promoter. Contrary to wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice fed a high-fat diet became steatotic within only a few weeks. Moreover, hepatic NPC1L1-mediated steatosis was not only prevented, but completely rescued, by orally administered ezetimibe, a well-used lipid-lowering drug on the global market, even under high-fat diet feedings. These results indicate that hepatic NPC1L1 is an NAFLD-exacerbating factor amendable to therapeutic intervention and would extend our understanding of the vital role of cholesterol uptake from bile in the development of NAFLD. Furthermore, administration of a TLR4 inhibitor also prevented the hepatic NPC1L1-mediated steatosis formation, suggesting a latent link between physiological roles of hepatic NPC1L1 and regulation of innate immune system. Our results revealed that hepatic NPC1L1 is a novel NAFLD risk factor contributing to steatosis formation that is rescued by ezetimibe; additionally, our findings uncover feasible opportunities for repositioning drugs to treat NAFLD in the near future.

Clinical Importance of Drug-Drug Interaction Between Warfarin and Prednisolone and Its Potential Mechanism in Relation to the Niemann-Pick C1-Like 1-Mediated Pathway.

BACKGROUND: Warfarin is an anticoagulant drug used to prevent thromboembolic disorders, but its pharmacological effect is affected by co-administered drugs. Therefore, careful management of warfarin-related drug-drug interactions (DDIs) is necessary for its safety and effectiveness. Recently, intestinal vitamin K1absorption through the Niemann-Pick C1-like 1 (NPC1L1)-mediated pathway was found to affect the pharmacological effect of warfarin. This study aimed to identify high-frequency warfarin-related DDIs in a clinical setting and elucidate their mechanism(s) in terms of changes in NPC1L1 expression and/or activity. Methods and Results: Prednisolone was the most frequently suspected drug in retrospective surveys of medical records of patients who experienced warfarin-related DDIs. Prednisolone significantly increased the international normalized ratio of prothrombin time (PT-INR) values in warfarin-treated patients. To demonstrate the involvement of NPC1L1 in warfarin-prednisolone DDI, we conducted an in vitro vitamin K1uptake assay using NPC1L1-overexpressing cells and found that prednisolone inhibited NPC1L1-mediated vitamin K1uptake. Additionally, we found that prednisolone downregulates NPC1L1 in a glucocorticoid receptor α-dependent manner. CONCLUSIONS: Co-administration of warfarin and prednisolone frequently enhanced the anticoagulant effect of warfarin in a clinical setting. Prednisolone-mediated suppression of NPC1L1 expression and activity could be the mechanism of DDI between warfarin and prednisolone. To manage warfarin therapy, the potential of concomitant drugs to change its anticoagulant effect through NPC1L1-related mechanisms merits consideration.

Associations between Lifestyle-Related Diseases and Transporters Involved in Intestinal Absorption and Biliary Excretion of Cholesterol.

Westernization of dietary habits leads to an increase in lipid intake and is thought to be responsible for an increase in patients with dyslipidemia. It is a well-known fact that the impaired cholesterol homeostasis is closely related to the development of various lifestyle-related diseases such as fatty liver, diabetes, and gallstone as well as dyslipidemia leading to atherosclerosis and cardiovascular diseases such as heart attack and stroke. Therefore, appropriate management of cholesterol levels in the body is considered important in prevention and treatments of these lifestyle-related diseases and in addition, molecular mechanisms controlling plasma (and/or hepatic) cholesterol levels have been intensively studied. Due to its hydrophobicity, cholesterol was long believed to pass through cell membranes by passive diffusion. However, recent studies have identified a number of plasma membrane transporters that are responsible for the cellular uptake or efflux of cholesterol and involved in developments of lifestyle-related diseases. In this review, we focus on Niemann-Pick C1 Like 1 (NPC1L1) and a heterodimer of ATP-binding cassette transporter G5 and G8 (ABCG5/G8), both of which are responsible for intestinal cholesterol absorption and biliary cholesterol secretion, and discuss the relationship between these cholesterol transporters and lifestyle-related diseases. In addition, we also discuss the related uncertainties that need to be explored in future studies.

VLDL/LDL acts as a drug carrier and regulates the transport and metabolism of drugs in the body.

Only free drugs have been believed to be carried into tissues through active or passive transport. However, considering that lipoproteins function as carriers of serum lipids such as cholesterol and triglycerides, we hypothesized that lipoproteins can associate with certain drugs and mediate their transport into tissues in lipid-associated form. Here, in vitro and in vivo studies with low density lipoprotein receptor (LDLR)-overexpressing or -knockdown cells and wild-type or LDLR-mutant mice were used to show the association of various drugs with lipoproteins and the uptake of lipoprotein-associated drugs through a lipoprotein receptor-mediated process. In clinical studies, investigation of the effect of lipoprotein apheresis on serum drug concentrations in patients with familial hypercholesterolemia demonstrated that lipoprotein-mediated drug transport occurs in humans as well as in mice. These findings represent a new concept regarding the transport and metabolism of drugs in the body and suggest that the role of lipoprotein-mediated drug transport should be considered when developing effective and safe pharmacotherapies.

Transporters for the Intestinal Absorption of Cholesterol, Vitamin E, and Vitamin K.

Humans cannot synthesize fat-soluble vitamins such as vitamin E and vitamin K. For this reason, they must be obtained from the diet via intestinal absorption. As the deficiency or excess of these vitamins has been reported to cause several types of diseases and disorders in humans, the intestinal absorption of these nutrients must be properly regulated to ensure good health. However, the mechanism of their intestinal absorption remains poorly understood. Recent studies on cholesterol using genome-edited mice, genome-wide association approaches, gene mutation analyses, and the development of cholesterol absorption inhibitors have revealed that several membrane proteins play crucial roles in the intestinal absorption of cholesterol. Surprisingly, detailed analyses of these cholesterol transporters have revealed that they can also transport vitamin E and vitamin K, providing clues to uncover the molecular mechanisms underlying the intestinal absorption of these fat-soluble vitamins. In this review, we focus on the membrane proteins (Niemann-Pick C1 like 1, scavenger receptor class B type I, cluster of differentiation 36, and ATP-binding cassette transporter A1) that are (potentially) involved in the intestinal absorption of cholesterol, vitamin E, and vitamin K and discuss their physiological and pharmacological importance. We also discuss the related uncertainties that need to be explored in future studies.

NPC1L1 is a key regulator of intestinal vitamin K absorption and a modulator of warfarin therapy.

Vitamin K (VK) is a micronutrient that facilitates blood coagulation. VK antagonists, such as warfarin, are used in the clinic to prevent thromboembolism. Because VK is not synthesized in the body, its intestinal absorption is crucial for maintaining whole-body VK levels. However, the molecular mechanism of this absorption is unclear. We demonstrate that Niemann-Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter, plays a central role in intestinal VK uptake and modulates the anticoagulant effect of warfarin. In vitro studies using NPC1L1-overexpressing intestinal cells and in vivo studies with Npc1l1-knockout mice revealed that intestinal VK absorption is NPC1L1-dependent and inhibited by ezetimibe, an NPC1L1-selective inhibitor clinically used for dyslipidemia. In addition, in vivo pharmacological studies demonstrated that the coadministration of ezetimibe and warfarin caused a reduction in hepatic VK levels and enhanced the pharmacological effect of warfarin. Adverse events caused by the coadministration of ezetimibe and warfarin were rescued by oral VK supplementation, suggesting that the drug-drug interaction effects observed were the consequence of ezetimibe-mediated VK malabsorption. This mechanism was supported by a retrospective evaluation of clinical data showing that, in more than 85% of warfarin-treated patients, the anticoagulant activity was enhanced by cotreatment with ezetimibe. Our findings provide insight into the molecular mechanism of VK absorption. This new drug-drug interaction mechanism between ezetimibe (a cholesterol transport inhibitor) and warfarin (a VK antagonist and anticoagulant) could inform clinical care of patients on these medications, such as by altering the kinetics of essential, fat-soluble vitamins.

ABCG2 dysfunction increases serum uric acid by decreased intestinal urate excretion.

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.

Sindbis viral vectors transiently deliver tumor-associated antigens to lymph nodes and elicit diversified antitumor CD8+ T-cell immunity.

Tumors are theoretically capable of eliciting an antitumor immune response, but are often poorly immunogenic. Oncolytic viruses (OVs) have recently emerged as a promising strategy for the immunogenic delivery of tumor-associated antigens (TAAs) to cancer patients. However, safe and effective OV/TAA therapies have not yet been established. We have previously demonstrated that vectors based on Sindbis virus (SV) can inhibit tumor growth and activate the innate immune system in mice. Here, we demonstrate that SV vectors carrying a TAA generate a dramatically enhanced therapeutic effect in mice bearing subcutaneous, intraperitoneal, and lung cancers. Notably, SV/TAA efficacy was not dependent on tumor cell targeting, but was characterized by the transient expression of TAAs in lymph nodes draining the injection site. Early T-cell activation at this site was followed by a robust influx of NKG2D expressing antigen-specific cytotoxic CD8+ T cells into the tumor site, subsequently leading to the generation of long-lasting memory T cells which conferred protection against rechallenge with TAA-positive as well as TAA-negative tumor cells. By combining in vivo imaging, flow cytometry, cytotoxicity/cytokine assays, and tetramer analysis, we investigated the relationship between these events and propose a model for CD8+ T-cell activation during SV/TAA therapy.

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis.

STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10(-/-)) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10(-/-) mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10(-/-) mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10(-/-) mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10(-/-) mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10(-/-) mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10(-/-) mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.

Ursodeoxycholic acid stimulates the formation of the bile canalicular network.

Ursodeoxycholic acid (UDCA) is a hepatoprotective bile acid used in the treatment of chronic liver diseases. Although several pharmacological effects, including choleresis and inhibition of apoptosis, have been proposed, the impact of UDCA on hepatic structure is not well understood. Here, the influence of UDCA on bile canalicular (BC) morphology was evaluated in vitro in immortalized rat hepatocytes (McA-RH 7777 cells) and primary rat hepatocytes. Cells cultured for 3 days in the presence of UDCA, the BC lumen was enlarged and the bile canaliculi were surrounded by multiple cells (≥5) with a continuous canal-like structure, reminiscent of the in vivo BC network. The effects were dependent on p38MAPK and conventional PKC in McA-RH cells, and partially dependent on p38MAPK, MAPK/ERK kinase, and conventional PKC in primary rat hepatocytes. These findings were then studied in vivo in a rat model of dimethylnitrosamine-induced hepatic injury, in which the BC network is significantly disrupted. In accordance with the in vitro observations, administration of UDCA (40 mg/kg/day) to the injured rats for 18 days improved the BC network compared with the vehicle control. Serum hepatic markers were not altered by UDCA treatment, suggesting that the morphological effects were due to the direct actions of UDCA on network formation. Our data provide new evidence of the pharmacological potential of UDCA in accelerating or regenerating BC network formation in vitro, in hepatic cell culture models, and in vivo in a rat model of hepatic injury, and provide a basis for understanding its hepatoprotective effects.

Decreased extra-renal urate excretion is a common cause of hyperuricemia.

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate 'overproduction type' and/or 'underexcretion type' based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the 'overproduction type' in the current concept of hyperuricemia be renamed 'renal overload type', which consists of two subtypes-'extra-renal urate underexcretion' and genuine 'urate overproduction'-providing a new concept valuable for the treatment of hyperuricemia and gout.