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

Toyoda, Yu; Takada, Tappei; Umezawa, Masakazu; Tomura, Fumiya; Yamanashi, Yoshihide; Takeda, Ken; Suzuki, Hiroshi

doi:10.1096/fba.2018-00044

Cited by 20 publications

(30 citation statements)

References 34 publications

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“…Regarding experimental results, all statistical analyses were performed by using EXCEL 2013 (Microsoft Corp., Redmond, WA, USA) with Statcel3 add-in software (OMS publishing Inc., Saitama, Japan) according to our previous study [38]. The specific statistical tests that were used for individual experiments are described in the figure legends.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

Toyoda

Mančíková

Krylov

et al. 2019

Cells

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ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein.

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

Toyoda

Mančíková

Krylov

et al. 2019

Cells

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“…Diets containing ezetimibe (16 μg/g diet) were made by mixing powdered HFD with ezetimibe before use. Of note, it was previously confirmed that the dose of ezetimibe used in the present study is enough for chemical inhibition of hepatic NPC1L1 in the liver of mice fed the HFD with ezetimibe [11]. At the indicated time points, blood specimens were taken immediately and serum specimens were prepared as described previously [11].…”

Section: Methodsmentioning

confidence: 57%

“…Transgenic mice expressing human NPC1L1 in hepatocytes (L1-Tg mice) [7] (B6;D2-Tg(APOE-NPC1L1)20Lqyu/J) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and backcrossed at least eight generations to C57BL/6 J mice (Japan SLC, Shizuoka, Japan) as described previously [11]. All experiments used hemizygous L1-Tg mice and WT littermate controls.…”

Section: Methodsmentioning

confidence: 99%

“…Nevertheless, due to the species differences in NPC1L1 tissue distribution, the pathophysiological impact of hepatic NPC1L1 on liver diseases has been overlooked in a lot of previous studies using murine models. Regarding this issue, using transgenic mice with hepatic expression of human NPC1L1 under a liver-specific ApoE promoter (L1-Tg mice) [7], a recent study identified hepatic NPC1L1 as an NAFLD risk factor amendable to therapeutic intervention [11]. Indeed, L1-Tg mice fed with a western diet exhibited steatosis characterized by the elevation of hepatic cholesterol and triglyceride (TG) levels within a few weeks, which was prevented and rescued by the administration of ezetimibe.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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BackgroundNon-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.MethodsTo 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.ResultsUnlike 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.ConclusionHepatic 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.

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“…However, due to differences in the tissue distribution of NPC1L1 between humans and rodents, a feasible pathophysiological involvement of hepatic NPC1L1 in atherosclerosis has been overlooked in many studies using mouse models. Indeed, although the importance of hepatic NPC1L1 in the physiology and pathophysiology of bile formation and liver diseases has been intensively studied so far (Temel et al, 2007;Toyoda et al, 2019), there is little information about its effect on atherosclerosis progression. Therefore, the purpose of our study was to explore the effect of hepatic NPC1L1 on atherosclerotic plaque formation in mouse models.…”

Section: Introductionmentioning

confidence: 99%

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

Yamamoto

Yamanashi

Takada

et al. 2019

Molecular Pharmacology

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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 STATEMENTNiemann-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.

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

Cited by 20 publications

References 34 publications

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

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

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

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