Mitsuyo Okazaki

Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca2+ in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5−/− islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5−/− islets. Furthermore, exposure of LRP5+/+ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that Wnt/LRP5 signaling contributes to the glucose-induced insulin secretion in the islets.

Identification of Unique Lipoprotein Subclasses for Visceral Obesity by Component Analysis of Cholesterol Profile in High-Performance Liquid Chromatography

Objective—The contribution of visceral fat accumulation to the development of coronary heart disease was previously reported, but the relation between visceral fat accumulation and serum lipoprotein subclasses was unknown. Methods and Results—We examined the relation of lipoprotein subclasses with visceral fat accumulation in 62 male subjects (aged 22 to 67 years) with visceral fat syndrome or obesity. Cholesterol levels in very low–density, low-density, and high-density lipoprotein subclasses (VLDL, LDL, and HDL) were determined by computer-assisted high-performance liquid chromatography. Subcutaneous fat area and visceral fat area were measured by computed tomographic scanning. There was no significant correlation between the subcutaneous fat area and the cholesterol levels in all lipoprotein subclasses. In contrast, the visceral fat area was correlated positively (P<0.002) with VLDL and LDL subclasses, except for large LDL, but negatively (P<0.001) with those in large and medium HDL subclasses. The observed positive correlations of small and very small LDL subclasses remained significant (P<0.005) after adjustment for serum cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, respectively, but a significant negative correlation (P<0.005) of large LDL was obtained after adjustment for LDL cholesterol. Conclusion—These findings indicate that this simple high-performance liquid chromatography method may be applied for easy detection and evaluation of abnormal distribution of lipoprotein subclasses.

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome

Non‐alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.

Angiopoietin-Like Protein3 Regulates Plasma HDL Cholesterol Through Suppression of Endothelial Lipase

Objectives—A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. Methods and Results—Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. Conclusions—Angptl3 acts as an inhibitor of EL and may be involved in the regulation of plasma HDL cholesterol and HDL-PL levels in humans and rodents.

Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E.

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had ∼60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were ∼3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced arthrosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.

Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity

Lipoprotein abnormalities were analyzed in 3 cases of marked hyperalphalipoproteinemia caused by complete deficiency of cholesteryl ester transfer activity. The probands were all men, aged 34, 43 and 48 years, respectively. The serum high density lipoprotein (HDL)-cholesterol levels of these patients were higher than 150 mg/dl (157-254 mg/dl), while serum total cholesterol levels ranged from 227 to 360 mg/dl. Sequential flotation-ultracentrifugation analysis disclosed that low density lipoprotein (LDL)-cholesterol was slightly decreased and that cholesteryl ester accumulated solely in the HDL2 fraction, which was also enriched with apolipoprotein E. Cholesteryl ester transfer activity was completely absent in all of these cases. High-performance liquid chromatography showed a decrease of LDL particle size in combination with a marked enlargement of HDL particle size. Analytical ultracentrifugation disclosed heterogeneity of LDL with the presence of small LDL subpopulations. We conclude that hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with cholesteryl ester and apolipoprotein E.

Dynamics of postprandial remnant-like lipoprotein particles in serum after loading of diacylglycerols.

BACKGROUND To compare the effects of diacylglycerol (DAG) on postprandial lipid metabolism with triacylglycerol (TAG), we examined the differences in the dynamics of remnant lipoproteins after loading of DAG or TAG of the same fatty acid composition. METHODS The subjects were comprised of 6 male volunteers who orally took creamed test meals prepared with either DAG or TAG at a dose of 30 g lipid/m(2) of body surface area in the early morning after fasting for at least 12 h. Blood was taken before and 2, 3, 4, 6 and 8 h after lipid loading. To quantify the amount of remnants, as the parameters, we used concentrations of cholesterol (RLP-C) and triacylglycerol (RLP-TG) in remnant-like lipoprotein particles (RLP) of serum. RESULTS The serum triacylglycerol concentration was markedly increased 2 h after loading of DAG and TAG. Serum triacylglycerol concentrations at 2, 3 and 8 h after loading of DAG were significantly lower than those after loading of TAG. The serum RLP-C concentration was significantly lower 2, 3 and 8 h after loading of DAG than TAG. There was a trend toward less increase in the RLP-TAG in the DAG group. The area under the curve (AUC) of serum RLP-TAG after DAG loading was significantly lower than that following TAG loading. CONCLUSIONS DAG might reduce the risk of coronary arteriosclerotic diseases by weakening the postprandial increase of RLP that is known to be closely correlated with atherosclerosis.

High-performance liquid chromatography of serum lipoproteins

Publisher Summary High-performance liquid chromatography (HPLC) has been widely used for the separation and analysis of many substances due to the short experimental time, small sample for analysis, high resolution, and excellent reproducibility. A simple and rapid method for lipoprotein analysis has been developed using HPLC with the gel permeation column. A direct quantitation method for cholesterol in each lipoprotein class using a very small amount of intact serum has been developed through a combination of the methods of (1) the separation by HPLC with gel permeation column, and (2) the selective detection of cholesterol in a post column effluent using an enzymatic reaction reagent. The HPLC technique of monitoring lipids in a post column effluent holds promise as a means of serum lipoprotein analysis from the following standpoints: simplicity of procedure, an analysis time of less than one hour, no need for sample pretreatment, small sample amount required (less than 20/μl of intact serum), high resolution, and good reproducibility.

Postprandial lipoprotein metabolism: VLDL vs chylomicrons.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

Deficiency of Interleukin-1 Receptor Antagonist Deteriorates Fatty Liver and Cholesterol Metabolism in Hypercholesterolemic Mice

Although the anti-inflammatory effect of interleukin-1 (IL-1) receptor antagonist (IL-1Ra) has been described, the contribution of this cytokine to cholesterol metabolism remains unclear. Our aim was to ascertain whether deficiency of IL-1Ra deteriorates cholesterol metabolism upon consumption of an atherogenic diet. IL-1Ra-deficient mice (IL-1Ra-/-) showed severe fatty liver and portal fibrosis containing many inflammatory cells following 20 weeks of an atherogenic diet when compared with wild type (WT) mice. Expectedly, the levels of total cholesterol in IL-1Ra-/- mice were significantly increased, and the start of lipid accumulation in liver was observed earlier when compared with WT mice. Real-time PCR analysis revealed that IL-1Ra-/- mice failed to induce mRNA expression of cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, with concurrent up-regulation of small heterodimer partner 1 mRNA expression. Indeed, IL-1Ra-/- mice showed markedly decreased bile acid excretion, which is elevated in WT mice to maintain cholesterol level under atherogenic diet feeding. Therefore, we conclude that the lack of IL-1Ra deteriorates cholesterol homeostasis under atherogenic diet-induced inflammation.