Risk Factors for Vulnerable Plaque Detected Using Near-Infrared Spectroscopy in Patients Receiving Statin Therapy with No History of Coronary Artery Disease.

Residual risk of atherosclerosis remains high despite the use of lipid-lowering therapy with statins. Near-infrared spectroscopy intravascular ultrasound imaging (NIRS-IVUS) can identify vulnerable plaque via the detection of lipid-rich plaque. This study aimed to reveal the clinical characteristics of patients with vulnerable plaque despite statin therapy.NIRS-IVUS was used to determine the maximum 4 mm Lipid Core Burden Index (MaxLCBI4 mm) values of 38 de novo culprit lesions from 32 patients with acute coronary syndrome (53%) (mean age: 73.1 ± 13.1 years) who underwent percutaneous coronary intervention after a minimum 6 months of statin therapy for primary prevention. A patient with vulnerable plaque was defined as an individual presenting at least 1 target lesion with a vulnerable plaque (MaxLCBI4 mm > 400). Overall, the average low-density lipoprotein cholesterol (LDL-C) level was 95.5 ± 27.2 mg/dL. Patients in the vulnerable plaque group were younger and had higher LDL-C, triglycerides, and non-high-density lipoprotein cholesterol (HDL-C) levels than those in the non-vulnerable plaque group. The MaxLCBI4 mm was positively correlated with LDL-C (P = 0.0002), triglycerides (P = 0.0003), and non-HDL-C (P = 0.0001). In multivariate analysis, all 3 treatable lipid components failed to show an independent relationship with the patients with vulnerable plaque. Using receiver-operating characteristics curve analysis, the cutoff points for LDL-C, triglycerides, and non-HDL-C were determined to be 78 mg/dL, 108 mg/dL, and 111 mg/dL, respectively, at MaxLCBI4 mm > 400. In conclusion, this study supports a more comprehensive and aggressive lipid-lowering therapy for the primary prevention of coronary artery disease.

Apolipoprotein M/sphingosine 1-phosphate protects against diabetic nephropathy.

Diabetic nephropathy remains a common cause of end-stage renal failure and its associated mortality around the world. Sphingosine 1-phosphate (S1P) is a multifunctional lipid mediator and binds to HDL via apolipoprotein M (ApoM). Since HDL has been reported to be epidemiologically associated with kidney disease, we attempted to investigate the involvement of the ApoM/S1P axis in the pathogenesis/progression of diabetic nephropathy. In type 2 diabetic patients, the serum ApoM levels were inversely correlated with the clinical stage of diabetic nephropathy. The decline in the eGFR over a 5-year observation period proceeded more rapidly in subjects with lower serum ApoM levels. In a mouse model of streptozotocin-induced diabetes, deletion of ApoM deteriorated the phenotypes of diabetic nephropathy: the urinary albumin and plasma creatinine levels increased, the kidneys enlarged, and renal fibrosis and thickening of the basement membrane progressed. On the other hand, overexpression of ApoM ameliorated these phenotypes. These protective effects of ApoM were partially inhibited by treatment with VPC23019, an antagonist of S1P1 and S1P3, but not by treatment with JTE013, an antagonist of S1P2. ApoM/S1P axis attenuated activation of the Smad3 pathway, while augmented eNOS phosphorylation through the S1P1 pathway. Moreover, ApoM/S1P increased the SIRT1 protein levels and enhanced mitochondrial functions by increasing the S1P content of the cell membrane, which might cause selective activation of S1P1. ApoM might be a useful biomarker for predicting the progression of diabetic nephropathy, and the ApoM/S1P-S1P1 axis might serve as a novel therapeutic target for preventing the development/progression of diabetic nephropathy.

Apolipoprotein D modulates lipid mediators and osteopontin in an anti-inflammatory direction.

BACKGROUND: HDL has been proposed to possess anti-inflammatory properties; however, the detail mechanisms have not been fully elucidated. METHODS: We investigated the roles of Apolipoprotein D (ApoD) in the pathogenesis of inflammation in the mouse model of diet-induced obesity and that of lipopolysaccharide-induced sepsis and the in vitro experiments. Furthermore, we analyzed serum ApoD levels in human subjects. RESULTS: The overexpression of human ApoD decreased the plasma IL-6 and TNF-a levels in both mice models. Lipidomics analyses demonstrated association of ApoD with increase of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, as well as of their metabolites, and of the anti-inflammatory molecule sphingosine 1-phosphate, and decrease of proinflammatory lysophosphatidic acids and lysophosphatidylinositol. ApoD-containing lipoproteins might directly bind eicosapentaenoic acid and docosahexaenoic acid. The modulations of the lysophosphatidic acid and sphingosine 1-phosphate levels resulted from the suppression of autotaxin expression and elevation of apolipoprotein M (ApoM), respectively. Moreover, ApoD negatively regulated osteopontin, a proinflammatory adipokine. The activation of PPARg by ApoD might suppress autotaxin and osteopontin. Serum ApoD levels were negatively correlated with the serum osteopontin and autotaxin levels and, positively with serum ApoM levels. CONCLUSION: ApoD is an anti-inflammatory apolipoprotein, which modulates lipid mediators and osteopontin in an anti-inflammatory direction.

Isoform-Dependent Effects of Apolipoprotein E on Sphingolipid Metabolism in Neural Cells.

BACKGROUND: Sphingosine 1-phosphate (S1P) and ceramides have been implicated in the development of Alzheimer's disease. Apolipoprotein E (ApoE) isoforms are also involved in the development of Alzheimer's disease. OBJECTIVE: We aimed at elucidating the potential association of the ApoE isoforms with sphingolipid metabolism in the central nervous system. METHODS: We investigated the modulations of apolipoprotein M (apoM), a carrier of S1P, S1P, and ceramides in Apoeshl mice, which spontaneously lack apoE, and U251 cells and SH-SY5Y cells infected with adenovirus vectors encoding for apoE2, apoE3, and apoE4. RESULTS: In the brains of Apoeshl mice, the levels of apoM were lower, while those of ceramides were higher. In U251 cells, cellular apoM and S1P levels were the highest in the cells overexpressing apoE2 among the apoE isoforms. The cellular and medium contents of ceramides decreased in the order of the cells overexpressing apoE3 > apoE2 and increased in the cells overexpressing apoE4. In SH-SY5Y cells, apoM mRNA and medium S1P levels were also the highest in the cells overexpressing apoE2. The cellular contents of ceramides decreased in the order of the cells overexpressing apoE3 > apoE2 = apoE4 and those in medium decreased in the order of the cells overexpressing apoE3 > apoE2, while increased in the cells overexpressing apoE4. CONCLUSION: The modulation of apoM and S1P might partly explain the protective effects of apoE2 against Alzheimer's disease, and the modulation of ceramides might be one of the mechanisms explaining the association of apoE4 with the development of Alzheimer's disease.

Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins.

Malaria parasites cannot multiply in host erythrocytes without cholesterol because they lack complete sterol biosynthesis systems. This suggests parasitized red blood cells (pRBCs) need to capture host sterols, but its mechanism remains unknown. Here we identified a novel high-density lipoprotein (HDL)-delivery pathway operating in blood-stage Plasmodium. In parasitized mouse plasma, exosomes positive for scavenger receptor CD36 and platelet-specific CD41 increased. These CDs were detected in pRBCs and internal parasites. A low molecular antagonist for scavenger receptors, BLT-1, blocked HDL uptake to pRBCs and suppressed Plasmodium growth in vitro. Furthermore, platelet-derived exosomes were internalized in pRBCs. Thus, we presume CD36 is delivered to malaria parasites from platelets by exosomes, which enables parasites to steal HDL for cholesterol supply. Cholesterol needs to cross three membranes (RBC, parasitophorous vacuole and parasite's plasma membranes) to reach parasite, but our findings can explain the first step of sterol uptake by intracellular parasites.

Modulation of sphingosine 1-phosphate by hepatobiliary cholesterol handling.

Hepatobiliary cholesterol handling, mediated by Niemann-Pick C1-like 1 protein (NPC1L1) and ABCG5/8, is well-known to contribute to the homeostasis of cholesterol. We attempted to elucidate the impact of hepatobiliary cholesterol handling on the homeostasis of sphingolipids and lysophospholipids, especially sphingosine 1-phosphate (S1P). We induced the overexpression of NPC1L1 or ABCG5/8 in the mouse liver. Hepatic NPC1L1 overexpression increased the plasma and hepatic S1P levels, while it decreased the biliary S1P levels, and all of these changes were inhibited by ezetimibe. The ability of HDL to activate Akt in the endothelial cells was augmented by hepatic NPC1L1 overexpression. NPC1L1-mediated S1P transport was confirmed by both in vitro and in vivo studies conducted using C17 S1P, an exogenous S1P analog. Upregulation of apolipoprotein M (apoM) was involved in these modulations, although apoM was not necessary for these modulations. Moreover, the increase in the plasma S1P levels also observed in ABCG5/8-overexpressing mice was dependent on the elevation of the plasma apoM levels. In regard to other sphingolipids and lysophospholipids, ceramides were similarly modulated by NPC1L1 to S1P, while other lipids were differently influenced by NPC1L1 or ABCG5/8 from S1P. Hepatobiliary cholesterol handling might also regulate the functional lipids, such as S1P.

Fructo-oligosaccharides ameliorate steatohepatitis, visceral adiposity, and associated chronic inflammation via increased production of short-chain fatty acids in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Within the spectrum of NAFLD, non-alcoholic steatohepatitis (NASH) in combination with hepatic inflammation and fibrosis can lead to liver cirrhosis and hepatocellular carcinoma. Dysbiosis was reported to contribute to NASH pathogenesis. This study aimed to determine the effects of fructo-oligosaccharides (FOS) on steatohepatitis and visceral adiposity in an obese mouse model of NASH. METHODS: Twelve newborn C57BL/6 J male mice were subcutaneously injected with monosodium glutamate (MSG) to induce obesity on a conventional diet. Six mice were also administered 5% FOS via drinking water from 10 weeks of age. At 18 weeks, histological characteristics of the liver and epididymal fat were compared between the groups. Hepatic mRNA expression of lipid metabolism enzymes and SCFA in feces and sera were measured. RESULTS: Hepatic steatosis, inflammatory cell infiltration, and hepatocyte ballooning in the liver and increased hepatic mRNA expression of fatty acid synthase and glycerol-3-phosphate acyltransferase were observed in the MSG-treated mice. FOS treatment improved the liver pathology and blunted the increases in the mRNA expression levels of lipid metabolism enzymes. In addition, FOS inhibited adipocyte enlargement and formation of crown-like structures and reduced the M1 macrophage frequency in the epididymal fat of the MSG mice (39.4% ± 3.0% vs. 22.8% ± 0.7%; P = 0.001). FOS increased not only the fecal concentrations of n-butyric acid (0.04 ± 0.01 vs. 0.38 ± 0.14 mg/g, P = 0.02), propionic acid (0.09 ± 0.03 vs. 0.42 ± 0.16 mg/g, P = 0.02), and acetic acid (0.65 ± 0.16 vs. 1.48 ± 0.29 mg/g, P = 0.03) but also the serum concentration of propionic acid (3.9 ± 0.5 vs. 8.2 ± 0.5 µmol/L, P = 0.001). CONCLUSIONS: FOS ameliorates steatohepatitis, visceral adiposity, and chronic inflammation by increasing SCFA production.

Protection Against Insulin Resistance by Apolipoprotein M/Sphingosine-1-Phosphate.

Subjects with low serum HDL cholesterol levels are reported to be susceptible to diabetes, with insulin resistance believed to be the underlying pathological mechanism. Apolipoprotein M (apoM) is a carrier of sphingosine-1-phosphate (S1P), a multifunctional lipid mediator, on HDL, and the pleiotropic effects of HDL are believed to be mediated by S1P. In the current study, we attempted to investigate the potential association between apoM/S1P and insulin resistance. We observed that the serum levels of apoM were lower in patients with type 2 diabetes and that they were negatively correlated with BMI and the insulin resistance index. While deletion of apoM in mice was associated with worsening of insulin resistance, overexpression of apoM was associated with improvement of insulin resistance. Presumably, apoM/S1P exerts its protective effect against insulin resistance by activating insulin signaling pathways, such as the AKT and AMPK pathways, and also by improving the mitochondrial functions through upregulation of SIRT1 protein levels. These actions of apoM/S1P appear to be mediated via activation of S1P1 and/or S1P3. These results suggest that apoM/S1P exerts protective roles against the development of insulin resistance.

Regulation of plasma glycero-lysophospholipid levels by lipoprotein metabolism.

Glycero-lysophospholipids, such as lysophosphatidic acids and lysophosphatidylserine, are gathering attention, since specific receptors have been identified. Most of these compounds have been proposed to be bound to albumin, while their associations with lipoproteins have not been fully elucidated. Therefore, in this study, we aimed to investigate the contents of glycero-lysophospholipids (lysophosphatidic acids, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine) on lipoproteins and the modulation of their metabolism by lipoprotein metabolism. We observed that moderate amounts of glycero-lysophospholipids, with the exception of lysophosphatidylserine, were distributed on the LDL and HDL fractions, and glycero-lysophospholipids that had bound to albumin were observed in lipoprotein fractions when they were co-incubated. The overexpression of cholesteryl ester transfer protein decreased the plasma levels of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, and lysophosphatidylinositol and it increased their contents in apoB-containing lipoproteins, while it decreased their contents in HDL and lipoprotein-depleted fractions in mice. The overexpression of the LDL receptor (LDLr) decreased the plasma levels of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, and lysophosphatidylinositol and decreased the contents of these compounds in the LDL, HDL, and lipoprotein-depleted fractions, while the knockdown of the LDLr increased them. These results suggest the potential importance of glycero-lysophospholipids in the pleiotropic effects of lipoproteins as well as the importance of lipoprotein metabolism in the regulation of glycero-lysophospholipids.

Regulation of the metabolism of apolipoprotein M and sphingosine 1-phosphate by hepatic PPARγ activity.

Apolipoprotein M (apoM) is a carrier and a modulator of sphingosine 1-phosphate (S1P), an important multifunctional bioactive lipid. Since peroxisome proliferator-activated receptor γ (PPARγ) is reportedly associated with the function and metabolism of S1P, we investigated the modulation of apoM/S1P homeostasis by PPARγ. First, we investigated the modulation of apoM and S1P homeostasis by the overexpression or knockdown of PPARγ in HepG2 cells and found that both the overexpression and the knockdown of PPARγ decreased apoM expression and S1P synthesis. When we activated or suppressed the PPARγ more mildly with pioglitazone or GW9662, we found that pioglitazone suppressed apoM expression and S1P synthesis, while GW9662 increased them. Next, we overexpressed PPARγ in mouse liver through adenoviral gene transfer and observed that both the plasma and hepatic apoM levels and the plasma S1P levels decreased, while the hepatic S1P levels increased, in the presence of enhanced sphingosine kinase activity. Treatment with pioglitazone decreased both the plasma and hepatic apoM and S1P levels only in diet-induced obese mice. Moreover, the overexpression of apoM increased, while the knockdown of apoM suppressed PPARγ activities in HepG2 cells. These results suggested that PPARγ regulates the S1P levels by modulating apoM in a bell-shaped manner, with the greatest levels of apoM/S1P observed when PPARγ was mildly expressed and that hepatic apoM/PPARγ axis might maintain the homeostasis of S1P metabolism.

Sitosterol prevents obesity-related chronic inflammation.

The physiological roles of phytosterols in chronic inflammation, which are believed to be involved in the underlying mechanisms for metabolic diseases, have yet to be elucidated. Therefore, in the present study, we aimed to elucidate the physiological roles of phytosterols in both clinical studies and animal experiments. We observed the existence of rather specific negative correlations between the serum sitosterol level and the serum IL-6 and the TNF-α levels in both diabetic subjects (n=46) and non-diabetic subjects (n=178). Multiple regression analyses also revealed that the serum IL-6 and TNF-α levels exhibited strong negative correlations with the serum sitosterol levels. When ABCG5/8 KO mice with markedly elevated plasma sitosterol levels and ABCG5/8 hetero mice were fed a high-fat diet, we observed that the increase in body weight, the fatty liver changes, and the expansion of perigonadal adipose tissues were suppressed in ABCG5/8 KO mice without any modulation of food intake. We also observed that the plasma IL-6 and TNF-α levels, the expressions of TNF-α and PAI-1 in the liver and the expressions of the IL-6, TNF-α, and MCP-1 levels in the adipose tissue were lower in ABCG5/8 KO mice. These results suggest that sitosterol might suppress obesity-related chronic inflammation and might be applicable to the treatment of metabolic diseases.

Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis.

Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.

Involvement of CETP (Cholesteryl Ester Transfer Protein) in the Shift of Sphingosine-1-Phosphate Among Lipoproteins and in the Modulation of its Functions.

OBJECTIVE: Sphingosine-1-phosphate (S1P) is a vasoprotective lipid mediator. About two thirds of plasma S1P rides on high-density lipoprotein (HDL), and several pleiotropic properties of HDL have been ascribed to S1P. In human subjects, CETP (cholesteryl ester transfer protein) greatly influences HDL quantities. In this study, we attempted to elucidate the roles of CETP in the metabolism of S1P. APPROACH AND RESULTS: We overexpressed CETP in mice that lacked CETP and found that CETP overexpression decreased the HDL level but failed to modulate the levels of S1P and apolipoprotein M (apoM), a carrier of S1P, in the total plasma. We observed, however, that the distribution of S1P and apoM shifted from HDL to apoB-containing lipoproteins. When we administered C17S1P bound to apoM-containing lipoprotein, C17S1P and apoM were rapidly transferred to apoB-containing lipoproteins in CETP-overexpressing mice. When HDL containing C17S1P was mixed with low-density lipoprotein ex vivo, C17S1P shifted to the low-density lipoprotein fraction independent of the presence of CETP. Concordant with these results, apoM was distributed mainly to the same fraction as apo AI in a CETP-deficient subject, although apoM was also detected in apo AI-poor fractions in a corresponding hypercholesterolemia subject. About the bioactivities of S1P carried on each lipoprotein, S1P riding on apoB-containing lipoproteins induced the phosphorylation of Akt (AKT8 virus oncogene cellular homolog) and eNOS (endothelial nitric oxide synthase) in human umbilical vein endothelial cells, and CETP overexpression increased insulin secretion and sensitivity, which was inhibited by an S1P receptor 1 or 3 antagonist. CONCLUSIONS: CETP modulates the distribution of S1P among lipoproteins, which affects the bioactivities of S1P.

Genome-wide association study of serum lipids confirms previously reported associations as well as new associations of common SNPs within PCSK7 gene with triglyceride.

Previous reports including genome-wide association studies (GWASs) have described associations of serum lipids with genomic variations. In the present study, we examined the association of ∼2.5 million single-nucleotide polymorphisms (SNPs) from 3041 Japanese healthy volunteers obtained from the Japan Pharmacogenomics Data Science Consortium (JPDSC) database with serum lipids. We confirmed the previously reported associations of 14 SNPs in 5 regions for low-density lipoprotein (LDL) cholesterol, 23 SNPs in 12 regions for high-density lipoprotein (HDL) cholesterol, 16 SNPs in 6 regions for triglyceride and 5 SNPs in 1 region for phospholipid. Furthermore, we identified 16 possible novel candidate genes associated with LDL cholesterol, HDL cholesterol or triglycerides, where SNPs had P-values of <1 × 10(-5). Further replication analyses of these genes with Korean data revealed significant associations of SNPs located within the PCSK7 gene and triglyceride (Pmeta=7.98 × 10(-9) and 1.91 × 10(-8) for rs508487 and rs236911, respectively). These associations remained significant even by the conditional analysis adjusting for three neighboring variations associated with triglyceride. Our present data suggest that PCSK7 as well as PCSK9 may be associated with lipids, especially triglyceride, and may serve as a candidate for a new drug target to treat lipid abnormality syndromes.

Resveratrol exerts a biphasic effect on apolipoprotein M.

BACKGROUND AND PURPOSE: Resveratrol exerts a range of beneficial actions in several areas of pathophysiology, including vascular biology. Here, we have investigated the effects of resveratrol on apolipoprotein M (apoM), a carrier and modulator of sphingosine 1-phosphate (S1P), a vasoactive lipid mediator. EXPERIMENTAL APPROACH: We used a hepatoma cell line (HepG2), human primary hepatocytes and C57BL/6 mice. We measured apoM, S1P and related enzymes, LDL receptors and sirtuin1 activity, using Western blotting, RT-PCR and enzyme assays. We also used si-RNA to knock-down sirtuin1 in HepG2 cells. KEY RESULTS: In cultures of HepG2 cells, resveratrol (1-10 µM) increased intracellular apoM and S1P. High concentrations of resveratrol (100 µM) decreased extracellular (in the culture medium) apoM, whereas moderate concentrations of resveratrol (1-10 µM) increased extracellular apoM. High concentrations of resveratrol also increased LDL receptor expression, while all concentrations of resveratrol activated the histone deacetylase sirtuin1. In cultures of human primary hepatocytes, resveratrol, at all concentrations, increased both intra- and extracellular apoM. When wild-type mice were fed a resveratrol-containing chow (0.3% w/w) for 2 weeks, both the plasma and hepatic apoM and S1P levels were increased. However, the resveratrol diet did not affect hepatic LDL receptor levels in this in vivo study. CONCLUSIONS AND IMPLICATIONS: Resveratrol increased intra- and extracellular levels of apoM, along with intracellular S1P levels, while a high concentration of resveratrol reduced extracellular apoM. The present findings suggest that resveratrol has novel effects on the metabolic kinetics of S1P, a multi-functional bioactive phospholipid.

Accumulated myeloid-derived suppressor cells demonstrate distinct phenotypes and functions in two non-alcoholic steatohepatitis mouse models.

BACKGROUND: To examine the steady state of hepatic myeloid-derived suppressor cells (MDSCs) and the lipid accumulation and inflammation-related changes in these cells, we analyzed the presence and functions of hepatic MDSCs in the following two non-alcoholic steatohepatitis (NASH) mouse models. METHODS: Monosodium glutamate (MSG) model; MSG was subcutaneously injected into neonatal male C57BL/6J mice that were fed with normal diet up to 18 weeks of age. Methionine/choline-deficient diet (MCD) model; 16-week-old male C57BL/6J mice were fed with an MCD for 2 weeks. Those hepatic MDSCs were evaluated by flow cytometry and immunohistochemically. RESULTS: Both MSG and MCD mice exhibited greater numbers of hepatic lipid droplets than 18-week-old male control mice. Hepatocellular ballooning was obvious in MSG, whereas inflammatory cell infiltration were apparent in MCD mice. CD11b, CD115, and Gr-1-positive hepatic MDSCs were increased in both models but higher in MCD mice, and demonstrated higher expression of an M2 macrophage marker CD206 mean fluorescence intensity (MFI) in MSG compared to MCD mice. Degree of reactive oxygen species production was evaluated using the DCFDA MFI values, which were significantly elevated in hepatic MDSCs from MCD mice. MSG mouse livers demonstrated Gr-1 positive cell accumulation around lipid droplets, mimicking crown-like structures in adipose tissues. In contrast, hepatic Gr-1 positive cells were primarily located in inflammatory cell aggregates in MCD mice. CONCLUSIONS: These results suggest that hepatic fatty changes promote MDSC accumulation, and inflammatory changes induce phenotypic and functional alteration in hepatic MDSCs in NASH mouse models.

Hepatic NPC1L1 overexpression ameliorates glucose metabolism in diabetic mice via suppression of gluconeogenesis.

OBJECTIVE: Inhibition of intestinal NPC1L1 by ezetimibe has been demonstrated to improve glucose metabolism in rodent models; however, the role of hepatic NPC1L1 in glucose metabolism has not been elucidated. In this study, we analyzed the effects of hepatic NPC1L1 on glucose metabolism. MATERIAL AND METHODS: We overexpressed NPC1L1 in the livers of lean wild type mice, diet-induced obesity mice and db/db mice with adenoviral gene transfer. RESULTS: We found that in all three mouse models, hepatic NPC1L1 overexpression lowered fasting blood glucose levels as well as blood glucose levels on ad libitum; in db/db mice, hepatic NPC1L1 overexpression improved blood glucose levels to almost the same as those found in lean wild type mice. A pyruvate tolerance test revealed that gluconeogenesis was suppressed by hepatic NPC1L1 overexpression. Further analyses revealed that hepatic NPC1L1 overexpression decreased the expression of FoxO1, resulting in the reduced expression of G6Pase and PEPCK, key enzymes in gluconeogenesis. CONCLUSIONS: These results indicate that hepatic NPC1L1 might have distinct properties of suppressing gluconeogenesis via inhibition of FoxO1 pathways.