Cryo-EM analysis reveals human SID-1 transmembrane family member 1 dynamics underlying lipid hydrolytic activity.

Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for extracellular dsRNA uptake or for release of incorporated dsRNA from lysosome to cytoplasm. SIDT1/2 is also suggested to be involved in cholesterol transport and lipid metabolism. Here, we determine the cryo-electron microscopy structures of human SIDT1, homodimer in a side-by-side arrangement, with two distinct conformations, the cholesterol-bound form and the unbound form. Our structures reveal that the membrane-spanning region of SIDT1 harbors conserved histidine and aspartate residues coordinating to putative zinc ion, in a structurally similar manner to alkaline ceramidases or adiponectin receptors that require zinc for ceramidase activity. We identify that SIDT1 has a ceramidase activity that is attenuated by cholesterol binding. Observations from two structures suggest that cholesterol molecules serve as allosteric regulator that binds the transmembrane region of SIDT1 and induces the conformation change and the reorientation of the catalytic residues. This study represents a contribution to the elucidation of the cholesterol-mediated mechanisms of lipid hydrolytic activity and RNA transport in the SID-1 family proteins.

Association of Serum Oxysterols with Cholesterol Metabolism Markers and Clinical Factors in Patients with Coronary Artery Disease: A Covariance Structure Analysis.

Oxysterols have been implicated in the pathogenesis of cardiovascular diseases. Serum levels of oxysterols could be positively correlated with cholesterol absorption and synthesis. However, physiological regulation of various serum oxysterols is largely unknown. The aim of this study was to investigate the relationship between clinical factors and cholesterol metabolism markers, and identify oxysterols associated with cholesterol absorption and synthesis in patients with coronary artery disease. Subjects (n = 207) who underwent coronary stenting between 2011 and 2013 were studied cross-sectionally. We measured lipid profiles including serum oxysterols. As for the serum biomarkers of cholesterol synthesis and absorption, oxysterol levels were positively correlated with campesterol and lathosterol. Covariance structure analysis revealed that dyslipidemia and statin usage had a positive correlation with "cholesterol absorption". Statin usage also had a positive correlation with "cholesterol synthesis". Several oxysterols associated with cholesterol absorption and/or synthesis. In conclusion, we elucidated the potential clinical factors that may affect cholesterol metabolism, and the associations between various oxysterols with cholesterol absorption and/or synthesis in patients with coronary artery disease.

Piceatannol Prevents Obesity and Fat Accumulation Caused by Estrogen Deficiency in Female Mice by Promoting Lipolysis.

Postmenopausal women have a higher susceptibility to obesity and chronic disease. Piceatannol (PIC), a natural analog of resveratrol, was reported to inhibit adipogenesis and to have an antiobesity effect. In this study, PIC's effect on postmenopausal obesity and the mechanism of its action were investigated. C57BL/6J female mice were divided into four groups and half of them were ovariectomized (OVX). Both OVX and sham-operated mice were fed a high-fat diet (HFD) with and without the addition of 0.25% of PIC for 12 weeks. The abdominal visceral fat volume was higher in the OVX mice than the sham-operated mice, and PIC significantly decreased the fat volume only in the OVX mice. Unexpectedly, expression levels of adipogenesis-related proteins in white adipose tissue (WAT) were suppressed in the OVX mice, and PIC did not affect lipogenesis in either the OVX or sham-operated mice. Regarding the expression of proteins associated with lipolysis, PIC activated the phosphorylation of hormone-sensitive lipase much more in the OVX mice, but it did not affect the expression of adipose triglyceride lipase. PIC also tended to induce the expression of uncoupled protein 1 in brown adipose tissue (BAT). These results suggest that by promoting lipolysis in WAT and deconjugation in BAT, PIC is a potential agent to inhibit fat accumulation caused by menopause.

Association between Serum Oxysterols and Coronary Plaque Regression during Lipid-Lowering Therapy with Statin and Ezetimibe: Insights from the CuVIC Trial.

AIM: Several clinical trials using intravascular ultrasound (IVUS) evaluation have demonstrated that intensive lipid-lowering therapy by statin or a combination therapy with statin and ezetimibe results in significant regression of coronary plaque volume. However, it remains unclear whether adding ezetimibe to statin therapy affects coronary plaque composition and the molecular mechanisms of plaque regression. We conducted this prospective IVUS analysis in a subgroup from the CuVIC trial. METHODS: The CuVIC trial was a prospective randomized, open, blinded-endpoint trial conducted among 11 cardiovascular centers, where 260 patients with coronary artery disease who received coronary stenting were randomly allocated into either the statin group (S) or the combined statin and ezetimibe group (S＋E). We enrolled 79 patients (S group, 39 patients; S＋E group, 40 patients) in this substudy, for whom serial IVUS images of nonculprit lesion were available at both baseline and after 6-8 months of follow-up. RESULTS: After the treatment period, the S＋E group had significantly lower level of low-density lipoprotein cholesterol (LDL-C; 80.9±3.7 vs. 67.7±3.8 mg/dL, p=0.0143). Campesterol, a marker of cholesterol absorption, and oxysterols (ß-epoxycholesterol, 4ß-hydroxycholesterol, and 27-hydroxycholesterol) were also lower in the S＋E group. IVUS analyses revealed greater plaque regression in the S＋E group than in the S group (-6.14% vs. -1.18% for each group, p=0.042). It was noteworthy that the lowering of campesterol and 27-hydroxycholesterol, but not LDL-C, had a significant positive correlation with plaque regression. CONCLUSIONS: Compared with statin monotherapy, ezetimibe in combination with statin achieved significantly lower LDL-C, campesterol, and 27-hydroxycholesterol, which resulted in greater coronary plaque regression.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Impact of the COVID-19 pandemic on the glycemic control, eating habits, and body compositions of people with diabetes mellitus: A retrospective longitudinal observational study.

AIMS/INTRODUCTION: To evaluate the impact of the COVID-19 pandemic on the glycemic control, eating habits, and body composition of people with diabetes mellitus; to identify the determinants of worsening glycemic control in people with diabetes mellitus. MATERIALS AND METHODS: This retrospective, longitudinal observational study was performed in outpatients with diabetes mellitus who visited our hospital between April 2019 and March 2020 (pre-COVID-19 period) and continued for follow up from April 2020 to March 2021 (COVID-19 period). We compared the glycemic control, nutritional intakes, and body composition of people with diabetes mellitus between the two periods. The changes in the HbA1c values (ΔHbA1c) and other study variables were compared between the two periods. Logistic regression analysis was performed to identify the factors associated with the increase of HbA1c levels. RESULTS: A significant increase of HbA1c was observed during the COVID-19 period. The percent fat mass (FM) also increased, while the percent skeletal muscle mass (SMM) decreased during the COVID-19 period. After adjustments for age and sex, the ΔBMI (OR:2.33), ΔFM (OR:1.45), and ΔSMM (OR:0.51) were identified as being associated with elevated levels of HbA1c. CONCLUSIONS: The COVID-19 pandemic had a negative impact on the glycemic control and body composition of people with diabetes mellitus. The increased body weight and FM and decreased SMM observed during the pandemic were associated with poor glycemic control in people with diabetes mellitus.

GPER/PKA-Dependent Enhancement of Hormone-Sensitive Lipase Phosphorylation in 3T3-L1 Adipocytes by Piceatannol.

We previously reported that piceatannol (PIC) had an anti-obesity effect only in ovariectomized (OVX) postmenopausal obesity mice. PIC was found to induce the phosphorylation of hormone-sensitive lipase (pHSL) in OVX mice. To elucidate the mechanism by which PIC activates HSL, we investigated the effect of PIC using 3T3-L1 adipocytes. PIC induced HSL phosphorylation at Ser563 in 3T3-L1 cells, as in vivo experiments showed. pHSL (Ser563) is believed to be activated through the ß-adrenergic receptor (ß-AR) and protein kinase A (PKA) pathways; however, the addition of a selective inhibitor of ß-AR did not inhibit the effect of PIC. The addition of a PKA inhibitor with PIC blocked pHSL (Ser563), suggesting that the effects are mediated by PKA in a different pathway than ß-AR. The addition of G15, a selective inhibitor of the G protein-coupled estrogen receptor (GPER), reduced the activation of HSL by PIC. Furthermore, PIC inhibited insulin signaling and did not induce pHSL (Ser565), which represents its inactive form. These results suggest that PIC acts as a phytoestrogen and phosphorylates HSL through a novel pathway that activates GPER and its downstream PKA, which may be one of the inhibitory actions of PIC on fat accumulation in estrogen deficiency.

A Dietary Oxysterol, 7-Ketocholesterol, Exacerbates Imiquimod-Induced Psoriasis-like Dermatitis in Steatohepatitic Mice.

Patients with psoriasis are at a higher risk of developing nonalcoholic fatty liver disease. We previously identified an oxidized derivative of cholesterol, 7-ketocholesterol (7KC), in diet-induced steatohepatitic mice. Here, we investigated whether 7KC exacerbates psoriasis-like dermatitis by accelerating steatohepatitis in mice. A high-fat/high-cholesterol/high-sucrose/bile salt diet (nonalcoholic steatohepatitis (NASH) diet) with or without 0.0125% 7KC was fed to C57BL/6 mice (7KC or control group) for three weeks to induce steatohepatitis. A 5% imiquimod cream was then applied to the ears and dorsal skin for four days to induce psoriasis-like dermatitis. Hepatic lipid accumulation and inflammatory cell infiltration were exacerbated in the 7KC group compared with the control group after three weeks. Serum tumor necrosis factor-α (TNF-α) levels were also elevated in the 7KC group (108.5 ± 9.8 vs. 83.1 ± 13.1 pg/mL, p < 0.005). Imiquimod cream increased the psoriasis area severity index (PASI) score in mice in the 7KC group (9.14 ± 0.75 vs. 5.17 ± 1.17, p < 0.0001). Additionally, Tnfa, Il23a, Il17a, and Il22 mRNA levels in the dorsal lesion were significantly upregulated. Finally, Th17 cell differentiation and the TNF signaling pathway were enhanced in the dorsal lesions and liver of mice in the 7KC group. These data suggest that steatohepatitis and psoriasis are linked by a potent, diet-related factor.

Dietary 7-ketocholesterol exacerbates myocardial ischemia-reperfusion injury in mice through monocyte/macrophage-mediated inflammation.

Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most abundant dietary oxysterols, causes inflammation and cardiovascular diseases. Here we show the deteriorating effects of dietary 7-KC on myocardial ischemia-reperfusion (IR) injury and detailed the molecular mechanisms. A high-fat high-cholesterol diet containing 7-KC (7KWD) for 3 weeks increased the plasma 7-KC level compared with high-fat high-cholesterol diet in mice. In wild-type mice but not in CCR2-/- mice, dietary 7-KC increased the myocardial infarct size after IR. Flow cytometry revealed that the ratio of Ly-6Chigh inflammatory monocytes to total monocytes was increased in the 7KWD group. Unbiased RNA sequencing using murine primary macrophages revealed that 7-KC regulated the expression of transcripts related to inflammation and cholesterol biosynthesis. We further validated that in vitro, 7-KC induced endoplasmic reticulum stress, mitochondrial reactive oxygen species production, and nuclear factor-kappa B activation, which are associated with increased mRNA levels of proinflammatory cytokines. Administration of N-acetyl-L-cysteine or siRNA-mediated knockdown of PKR-like endoplasmic reticulum kinase or endoplasmic reticulum oxidase 1α suppressed the levels of 7-KC-induced inflammation. Dietary 7-KC exacerbates myocardial IR injury through monocyte/macrophage-mediated inflammation. Endoplasmic reticulum stress and oxidative stress are involved in the 7-KC-induced proinflammatory response in macrophages.

Effect of tocotrienol on the primary progression of nonalcoholic steatohepatitis in a mouse model.

Tocotrienol (T3), a vitamin E (Vit E) isoform, is known to have both biological and antioxidant effects. Although alpha-tocopherol (α-Toc), another isoform of Vit E is suggested to be a useful treatment against nonalcoholic steatohepatitis (NASH), the effect of T3 on NASH is unclear. This study aimed to comparatively evaluate the effects of T3 and α-Toc on NASH in the early stage of NASH progression, using a recently established NASH mouse model induced by a choline-deficient l-amino acid-defined high-fat diet (CDAHFD). Six-week-old male mice were divided into four groups (n = 6 per group) and fed the CDAHFD for 1 week. The first group was given no other treatment (Pre). The other three groups continued the CDAHFD plus daily oral administration of Vit E-free corn oil (Control), corn oil containing α-Toc, or corn oil containing T3 for additional 2 weeks. Neither Vit E treatment changed the histologic features of NASH, but T3 significantly reduced the mRNA expression of several genes related to inflammation and fibrosis and α-Toc did not. These results suggested that oral T3 treatment was more effective than α-Toc at suppressing hepatic inflammation and fibrosis in the early stage of NASH progression in CDAHFD model mice.

Effects of Vitamin E and Dietary Conditions on the Differentiation and Maturation of Osteoclast.

α-Tocopherol is reported to activate the differentiation and fusion of osteoclast, however, it is not clear whether the excessive intake of vitamin E is a risk for osteoporosis. To investigate the effects of vitamin E and the dietary conditions on the osteoclastogenesis, osteoclast differentiation was evaluated using the bone marrow cells collected from mice fed various dietary conditions. Not only α-tocopherol but also γ-tocotrienol activated osteoclast differentiation in mice fed normal diet. Formation of large multinucleated cells was significantly increased by stimulation of nuclear factor-kappa B ligand (RANKL) in mice fed vitamin E deficient diet and was suppressed by the addition of α-tocopherol. Furthermore, there was no effect on bone density and no difference in osteoclast differentiation from the bone marrow cells collected from mice fed a high-fat diet with 0 or 1,000 mg/kg diet of α-tocopherol and tocotrienol, respectively. These results suggest that different type of diet affect the activation of osteoclast by α-tocopherol.

FADS2-dependent fatty acid desaturation dictates cellular sensitivity to ferroptosis and permissiveness for hepatitis C virus replication.

The metabolic oxidative degradation of cellular lipids severely restricts replication of hepatitis C virus (HCV), a leading cause of chronic liver disease, but little is known about the factors regulating this process in infected cells. Here we show that HCV is restricted by an iron-dependent mechanism resembling the one triggering ferroptosis, an iron-dependent form of non-apoptotic cell death, and mediated by the non-canonical desaturation of oleate to Mead acid and other highly unsaturated fatty acids by fatty acid desaturase 2 (FADS2). Genetic depletion and ectopic expression experiments show FADS2 is a key determinant of cellular sensitivity to ferroptosis. Inhibiting FADS2 markedly enhances HCV replication, whereas the ferroptosis-inducing compound erastin alters conformation of the HCV replicase and sensitizes it to direct-acting antiviral agents targeting the viral protease. Our results identify FADS2 as a rate-limiting factor in ferroptosis, and suggest the possibility of pharmacologically manipulating the ferroptosis pathway to attenuate viral replication.

Ablation of fatty acid desaturase 2 (FADS2) exacerbates hepatic triacylglycerol and cholesterol accumulation in polyunsaturated fatty acid-depleted mice.

Deficiency of polyunsaturated fatty acids (PUFAs) is known to induce hepatic steatosis. However, it is not clearly understood which type of PUFA is responsible for the worsening of steatosis. This study observed a marked accumulation of hepatic triacylglycerol and cholesterol in fatty acid desaturase 2 knockout (FADS2-/- ) mice lacking both C18 and ≥ C20 PUFAs that were fed a PUFA-depleted diet. Hepatic triacylglycerol accumulation was associated with enhanced sterol regulatory element-binding protein (SREBP)-1-dependent lipogenesis and decreased triacylglycerol secretion into the plasma via very-low-density lipoprotein (VLDL). Furthermore, upregulation of cholesterol synthesis contributed to increased hepatic cholesterol content in FADS2-/- mice. These results suggest that ≥ C20 PUFAs synthesized by FADS2 are important in regulating hepatic triacylglycerol and cholesterol accumulation during PUFA deficiency.

The effect of polyunsaturated fatty acid deficiency on allergic response in ovalbumin-immunized mice.

Polyunsaturated fatty acids (PUFAs) are present in biological membranes and influence membrane fluidity and immune responses. PUFAs such as 18:2n-6 and 18:3n-3 cannot be synthesized de novo in mammals and are thus called essential fatty acids (EFAs). In addition, PUFAs can be converted to very long-chain PUFAs (VLC-PUFAs), such as arachidonic acid and docosahexaenoic acid, in the body. Although avoiding allergens is an effective strategy for food-allergy patients, the dietary exclusion of several allergens reportedly induces deficiencies in essential nutrients such as PUFAs. In this study, we investigated whether an EFA-deficient (EFAD) diet influenced allergic symptoms in ovalbumin (OVA)-immunized mice. Unexpectedly, no exacerbation of immune responses after OVA-sensitization was observed in mice fed an EFAD diet, and no differences in serum PUFA levels between OVA-immunized and non-immunized mice fed the EFAD diet were detected. However, levels of VLC-PUFAs in the small intestine increased after OVA-sensitization and did not decrease during EFAD diet administration, showing that small intestinal VLC-PUFAs levels were strongly preserved in the food-allergy model mice. Further studies are required to elucidate the mechanisms by which small intestinal VLC-PUFAs are retained in food-allergy model mice.

Dietary Oxysterol, 7-Ketocholesterol Accelerates Hepatic Lipid Accumulation and Macrophage Infiltration in Obese Mice.

Non-alcoholic fatty liver disease is strongly associated with obese and type 2 diabetes. It has been reported that an oxidized cholesterol, 7-ketocholesterol (7KC), might cause inflammatory response in macrophages and plasma 7KC concentration were higher in patients with cardiovascular diseases or diabetes. Therefore, we have decided to test whether small amount of 7KC in diet might induce hepatic steatosis and inflammation in two types of obese models. We found that addition of 0.01% 7KC either in chow diet (CD, regular chow diet with 1% cholesterol) or western type diet (WD, high fat diet with 1% cholesterol) accelerated hepatic neutral lipid accumulation by Oil Red O staining. Importantly, by lipid extraction analysis, it has been recognized that triglyceride rather than cholesterol species was significantly accumulated in CD+7KC compared to CD as well as in WD+7KC compared to WD. Immunostaining revealed that macrophages infiltration was increased in CD+7KC compared to CD, and also in WD+7KC compared to WD. These phenotypes were accompanied by inducing inflammatory response and downregulating fatty acid oxidation. Furthermore, RNA sequence analysis demonstrated that 7KC reduced expression of genes which related to autophagy process. Levels of LC3-II protein were decreased in WD+7KC compared to WD. Similarly, we have confirmed the effect of 7KC on acceleration of steatohepatitis in db/db mice model. Collectively, our study has demonstrated that small amount of dietary 7KC contributed to accelerate hepatic steatosis and inflammation in obese mice models.

Lipid-Lowering Therapy With Ezetimibe Decreases Spontaneous Atherothrombotic Occlusions in a Rabbit Model of Plaque Erosion: A Role of Serum Oxysterols.

OBJECTIVE: Plaque erosion is increasing its importance as one of the mechanisms of acute coronary syndromes in this statin era. However, the clinical efficacy of currently used lipid-lowering agents in the prevention of thrombotic complications associated with plaque erosion has not been clarified. Therefore, we examined the therapeutic effects of ezetimibe or rosuvastatin monotherapy on spontaneous atherothrombotic occlusion. APPROACH AND RESULTS: Femoral arteries of Japanese white rabbits, fed a high-cholesterol diet, were injured by balloon catheter, and then angiotensin II was continuously administrated. In 94% of these arteries, spontaneous thrombotic occlusions were observed after 5 weeks (median) of balloon injury. Histochemical analyses indicated that the injured arteries had similar pathological features to human plaque erosions; (1) spontaneous thrombotic occlusion, (2) lack of endothelial cells, and (3) tissue factor expression in vascular smooth muscle cells. Ezetimibe (1.0 mg/kg per day), but not rosuvastatin (0.6 mg/kg per day), significantly decreased thrombotic occlusion of arteries accompanied with accelerated re-endothelialization and the decreases of serum oxysterols despite the comparable on-treatment serum cholesterol levels. The 7-ketocholesterol inhibited the migration of human umbilical vein endothelial cells. Both 7-ketocholesterol and 27-hydroxycholesterol increased tissue factor expression in cultured rat vascular smooth muscle cells. Tissue factor expression was also induced by serum from vehicle- or rosuvastatin-treated rabbits, but the induction was attenuated with serum from ezetimibe-treated rabbits. CONCLUSIONS: We have established a novel rabbit model of spontaneous atherothromobotic occlusion without plaque rupture that is feasible to test the therapeutic effects of various pharmacotherapies. Ezetimibe may decrease atherothrombotic complications after superficial plaque erosion by reducing serum oxysterols.

FADS2 inhibition in essential fatty acid deficiency induces hepatic lipid accumulation via impairment of very low-density lipoprotein (VLDL) secretion.

Fatty acid desaturase 2 (FADS2) is responsible for the first desaturation reaction in the synthesis of highly unsaturated fatty acids (HUFAs), such as arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), and is involved in Mead acid (20:3n-9) production during essential fatty acid deficiency (EFAD). In this study, an obvious hepatic lipid accumulation was observed in EFAD mice treated with a FADS2 inhibitor. FADS2 inhibition in the EFAD state reduced secretion of very low-density lipoprotein (VLDL) and markedly diminished Mead acid in phosphatidylcholine (PC) in the liver and plasma. As the results, the amount of C20 HUFAs in hepatic and plasma PC dramatically reduced in the EFAD mice treated with a FADS2 inhibitor, whereas the decrease of C20 HUFA levels of PC in EFAD mice was not observed because of the increased Mead acid in PC. These results supposed that Mead acid in PC is important as a component of VLDL. It is possible that Mead acid plays the role of a substitute of HUFAs in VLDL secretion during EFAD.

Oleuropein improves insulin resistance in skeletal muscle by promoting the translocation of GLUT4.

As the beneficial effects of the Mediterranean diet on human health are well established, the phenolic compounds in olive oil have been gaining interest. Oleuropein, a major phenolic compound in olives, is known to reduce the blood glucose levels in alloxan-induced diabetic rats and rabbits, however, its effect on type 2 diabetes caused by obesity is not clear. The purpose of this study is clarifying the effect of oleuropein on the glucose tolerance in skeletal muscle under the condition of lipotoxicity caused by type 2 diabetes. Oleuropein enhanced glucose uptake in C2C12 cells without insulin. Translocation of glucose transporter 4 (GLUT4) into the cell membrane was promoted by activation of adenosine monophosphate-activated protein kinase (AMPK) but not protein kinase B (Akt). Physiological concentration of oleuropein (10 µM) was sufficient to express beneficial effects on C2C12 cells. Oleuropein prevented palmitic acid-induced myocellular insulin resistance. Furthermore, in gastrocnemius muscles of mice fed a high fat diet, oleuropein also induced the GLUT4 localization into cell membrane. These results suggest the possibility of oleuropein to be effective for type 2 diabetes by reducing insulin resistance in skeletal muscles.

Excessive Vitamin E Intake Does Not Cause Bone Loss in Male or Ovariectomized Female Mice Fed Normal or High-Fat Diets.

Background: Animal studies on the effects of vitamin E on bone health have yielded conflicting and inconclusive results, and to our knowledge, no studies have addressed the effect of vitamin E on bone in animals consuming a high-fat diet (HFD).Objective: This study aimed to evaluate the effect of excessive vitamin E on bone metabolism in normal male mice and ovariectomized female mice fed a normal diet (ND) or HFD.Methods: In the first 2 experiments, 7-wk-old male mice were fed an ND (16% energy from fat) containing 75 (control), 0 (vitamin E-free), or 1000 (high vitamin E) mg vitamin E/kg (experiment 1) or an HFD (46% energy from fat) containing 0, 200, 500, or 1000 mg vitamin E/kg (experiment 2) for 18 wk. In the third experiment, 7-wk-old sham-operated or ovariectomized female mice were fed the ND (75 mg vitamin E/kg) or HFD containing 0 or 1000 mg vitamin E/kg for 8 wk. At the end of the feeding period, blood and femurs were collected to measure bone turnover markers and analyze histology and microcomputed tomography.Results: In experiments 1 and 2, vitamin E intake had no effect on plasma alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activity, or bone formation, resorption, or volume in femurs in mice fed the ND or HFDs. In experiment 3, bone volume was significantly reduced (85%) in ovariectomized mice compared with that in sham-operated mice (P < 0.05), but it did not differ among mice fed the 3 diets. Plasma ALP and TRAP activities and bone formation and resorption in femur were similar among ovariectomized mice fed the HFD containing 0 or 1000 mg vitamin E/kg.Conclusions: The results suggest that excess vitamin E intake does not cause bone loss in normal male mice or in ovariectomized or sham-operated female mice, regardless of dietary fat content.

Ezetimibe in Combination With Statins Ameliorates Endothelial Dysfunction in Coronary Arteries After Stenting: The CuVIC Trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction After Coronary Stenting), a Multicenter Randomized Controlled Trial.

OBJECTIVES: We sought to investigate whether treatment with ezetimibe in combination with statins improves coronary endothelial function in target vessels in coronary artery disease patients after coronary stenting. APPROACH AND RESULTS: We conducted a multicenter, prospective, randomized, open-label, blinded-end point trial among 11 cardiovascular treatment centers. From 2011 to 2013, 260 coronary artery disease patients who underwent coronary stenting were randomly allocated to 2 arms (statin monotherapy, S versus ezetimibe [10 mg/d]+statin combinational therapy, E+S). We defined target vessel dysfunction as the primary composite outcome, which comprised target vessel failure during treatment and at the 6- to 8-month follow-up coronary angiography and coronary endothelial dysfunction determined via intracoronary acetylcholine testing performed in cases without target vessel failure at the follow-up coronary angiography. Coadministration of ezetimibe with statins further lowered low-density lipoprotein cholesterol levels (83±23 mg/dL in S versus 67±23 mg/dL in E+S; P<0.0001), with significant decreases in oxidized low-density lipoprotein and oxysterol levels. Among patients without target vessel failure, 46 out of 89 patients (52%) in the S arm and 34 out of 96 patients (35%) in the E+S arm were found to have coronary endothelial dysfunction (P=0.0256), and the incidence of target vessel dysfunction at follow-up was significantly decreased in the E+S arm (69/112 (62%) in S versus 47/109 (43%) in E+S; P=0.0059). A post hoc analysis of post-treatment low-density lipoprotein cholesterol-matched subgroups revealed that the incidence of both target vessel dysfunction and coronary endothelial dysfunction significantly decreased in the E+S arm, with significant reductions in oxysterol levels. CONCLUSIONS: The CuVIC trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction after Coronary Stenting) has shown that ezetimibe with statins, compared with statin monotherapy, improves functional prognoses, ameliorating endothelial dysfunction in stented coronary arteries, and was associated with larger decreases in oxysterol levels.