Yukihiko Ueda

Inhibition of CCR2 Ameliorates Insulin Resistance and Hepatic Steatosis in db/db Mice

Objective—Recently, adipose tissue inflammation induced by macrophage infiltration through MCP-1/C-C chemokine receptor-2 (CCR2) pathway is considered to play a role in the development of visceral obesity and insulin resistance. In the present study, to further examine the role of CCR2 in the development of obesity and type 2 diabetes, we studied the effect of pharmacological inhibition of CCR2 from the early stage of obesity in db/db mice. Methods and Results—Db/+m (lean control) and db/db mice were fed with a standard diet with or without 0.005% propagermanium, as a CCR2 inhibitor for 12 weeks from 6 weeks of age. Propagermanium treatment decreased body weight gain, visceral fat accumulation, and the size of adipocytes only in db/db mice. Further, propagermanium suppressed macrophage accumulation and inflammation in adipose tissue. Propagermanium treatment also ameliorated glucose tolerance and insulin sensitivity, and decreased hepatic triglyceride contents in db/db mice. Conclusions—Propagermanium improved obesity and related metabolic disorders, such as insulin resistance and hepatic steatosis by suppressing inflammation in adipose tissue. Our data indicate that inhibition of CCR2 could improve obesity and type 2 diabetes by interfering adipose tissue inflammation, and that propagermanium may be a beneficial drug for the treatment of the metabolic syndrome.

Knockdown expression and hepatic deficiency reveal an atheroprotective role for SR-BI in liver and peripheral tissues

Scavenger receptor SR-BI has been implicated in HDL-dependent atheroprotective mechanisms. We report the generation of an SR-BI conditional knockout mouse model in which SR-BI gene targeting by loxP site insertion produced a hypomorphic allele (hypomSR-BI). Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-fold elevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BI gene inactivation of the hypomorphic SR-BI allele in hepatocytes (hypomSR-BI-KO(liver)) was associated with high plasma TC concentrations, increased plasma free cholesterol/TC (FC/TC) ratio, and a lipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TC levels were increased 2-fold in hypomSR-BI and control mice fed an atherogenic diet, whereas hypomSR-BI-KO(liver) and SR-BI-/- mice developed severe hypercholesterolemia due to accumulation of FC-rich, VLDL-sized particles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold) compared with that in controls, but to a much lower degree than in hypomSR-BI-KO(liver) (32-fold) and SR-BI-/- (48-fold) mice. The latter models did not differ in either plasma lipid levels or in the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading. However, reduced atherosclerosis in hypomSR-BI-KO(liver) mice was associated with decreased lesional macrophage content as compared with that in SR-BI-/- mice. These data imply that, in addition to its major atheroprotective role in liver, SR-BI may exert an antiatherogenic role in extrahepatic tissues.

Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit — long-term antiatherogenic effect and effects on established plaques

We performed two studies to investigate the effect of probucol on atherogenesis in vivo in the Watanabe heritable hyperlipidemic (WHHL) rabbit. In the first study (Study A), probucol was administered to 2-month-old WHHL rabbits, to evaluate its long-term effect. When killed at about 1.5 years of age, the percentage area of aorta covered with atherosclerotic plaque in probucol-treated rabbits was markedly less than that seen in non-treated rabbits (23.0 +/- 11.4% vs. 87.7 +/- 8.1%, M +/- S.D., P less than 0.001). In the second study (study B), administration of probucol was commenced with 8-month-old WHHL rabbits to investigate whether the drug was effective for limiting atherosclerosis in rabbits in which plaques had already developed. When killed after 6 months of treatment, the percentage area of aorta covered with plaque was 38.1 +/- 12.1% in treated rabbits and 82.7 +/- 22.6% in non-treated rabbits (P less than 0.02). Microscopic observations of lesions also supported the effect of probucol. Probucol treatment resulted in a change not only in the size but also the composition of lesions. Thus, probucol was effective in preventing atherosclerosis in long-term studies at both early and late stages.

Blockade of Platelet-Derived Growth Factor Receptor-β Pathway Induces Apoptosis of Vascular Endothelial Cells and Disrupts Glomerular Capillary Formation in Neonatal Mice

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymal-derived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-alpha and PDGFR-beta, have been identified and we previously analyzed the effects of blockade of PDGFR-alpha signal in neonatal mice. In the current study, we examined the role of PDGFR-beta in glomerular development by blocking PDGFR-beta signal in neonatal mice by administration of antagonistic anti-PDGFR-beta monoclonal antibody. Unlike the mice injected with anti-PDGFR-alpha antibody, the mice injected daily with anti-PDGFR-beta antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-beta is not expressed in the endothelial cells, the effects of the blockade of PDGFR-beta might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Mulberry leaf ameliorates the expression profile of adipocytokines by inhibiting oxidative stress in white adipose tissue in db/db mice

Previous study showed that mulberry (Morus Alba L.) leaf (ML) ameliorates atherosclerosis in apoE(-/-) mice. Although the adipocytokine dysregulation is an important risk factor for atherosclerotic cardiovascular disease, the effect of ML on metabolic disorders related to adipocytokine dysregulation and inflammation has not been studied. Therefore, we studied the effects of ML in metabolic disorders and examined the mechanisms by which ML ameliorates metabolic disorders in db/db mice. We treated db/db mice with ML, pioglitazone, or both for 12 weeks and found that ML decreased blood glucose and plasma triglyceride. Co-treatment with ML and pioglitazone showed additive effects compared with pioglitazone. Moreover, their co-treatment attenuated the body weight increase observed under the pioglitazone treatment. ML treatment also increased the expression of adiponectin, and decreased the expression of TNF-alpha, MCP-1, and macrophage markers in white adipose tissue (WAT). Furthermore, ML decreased lipid peroxides and the expression of NADPH oxidase subunits in WAT and liver. Their co-treatment enhanced these effects. Thus, ML ameliorates adipocytokine dysregulation at least in part through inhibiting oxidative stress in WAT of db/db mice, and that ML may be a basis for a pharmaceutical for the treatment of the metabolic syndrome as well as reducing adverse effects of pioglitazone.

Probucol enhances the expression of human hepatic scavenger receptor class B type I, possibly through a species-specific mechanism.

Objective—Scavenger receptor class B type I (SR-BI) is a major receptor for high-density lipoproteins (HDL) in the liver, which is the terminus of reverse cholesterol transport. Overexpression of SR-BI attenuated experimental atherosclerosis in murine models, concomitant with a reduction in plasma HDL-cholesterol levels. Probucol is known to be a potent hypolipidemic drug to regress xanthoma formation and carotid atherosclerosis in conjunction with a marked reduction in HDL-cholesterol levels. The aim of the present study was to know the effect of probucol on the expression of SR-BI and the underlying mechanism. Methods and Results—We found that probucol increased the expression of SR-BI proteins in in vitro human liver cells and an in vivo rabbit model, but not in wild-type C57Bl6 mice. The decay curve of SR-BI protein was markedly retarded in probucol-treated HepG2 cells in the presence of cycloheximide, indicating that probucol may stabilize human SR-BI protein. To determine the underlying mechanism for the observed species-specific effect, we conducted the following host-swap experiments, in which SR-BI was transfected or expressed in heterologous cells or hosts. Probucol did not increase human SR-BI protein in the liver of transgenic mice carrying the entire human SR-BI genome. Although probucol could stabilize even murine SR-BI, when transfected into a human cell line, HepG2, human SR-BI was not stabilized in a mouse hepatoma cell line, Hepa 1-6, treated with probucol. Conclusion—Probucol enhances hepatic SR-BI protein expression, possibly through species-specific stabilization of the protein.

Regulation of the human PDZK1 expression by peroxisome proliferator-activated receptor alpha.

Although PDZK1 is a well‐known adaptor protein, the mechanisms for its role in transcriptional regulation are largely unknown. The peroxisome proliferator‐activated receptor alpha (PPARα) is a ligand‐activated transcription factor that plays an important role in the regulation of lipid homeostasis. Previously, we established a tetracycline‐regulated human cell line that can be induced to express PPARα and identified candidate target genes, one of which was PDZK1. In this study, we cloned and characterized the promoter region of the human pdzk1 gene and determined the PPAR response element. Finally, we demonstrate that endogenous PPARα regulates PDZK1 expression.

Coexpression of CLA-1 and Human PDZK1 in Murine Liver Modulates HDL Cholesterol Metabolism

Objective—In rodents scavenger receptor class B type I (SR-BI) is a key molecule for selective uptake of cholesteryl ester from high-density lipoprotein (HDL). This study was aimed to clarify the role of the human SR-BI/CD36 and LIMP-II Analogues-1 (CLA-1) as a molecular target of selective uptake of cholesteryl ester from HDL in vivo. Methods and Results—To clarify the function and regulation of CLA-1 in vivo we produced CLA-1 BAC transgenic mice. In spite of abundant hepatic RNA expression of CLA-1, CLA-1 BAC transgenic mice had no significant effect on mouse HDL cholesterol. Although coexpression of a human scaffolding protein PDZK1 along with CLA-1 enhanced hepatic CLA-1 expression, it did not affect mouse HDL cholesterol levels, either. However, in the presence of human apoA-1, HDL cholesterol level and size were significantly reduced in CLA-1 transgenic mice, and its reduction was more pronounced in CLA-1/human PDZK1 double transgenic mouse. Conclusions—We established a mouse model to study human reverse cholesterol transport by expressing CLA-1, human PDZK1, and human apoA-I gene. Our results imply that enhancing CLA-1 expression by human PDZK1 in the liver can modulate HDL cholesterol metabolism and possibly enhance reverse cholesterol transport to prevent the progression of atherosclerosis in human.

Different expression of modified low density lipoprotein receptors in rabbit peritoneal macrophages and Kupffer cells

We have previously reported that mouse peritoneal macrophages have three types of modified low density lipoprotein (LDL) receptors. One is specific for acetylated LDL (Ac-LDL), the second is for oxidized LDL (Ox-LDL), and the third recognizes both (Arai, H. et al. (1989) Biochem. Biophys. Res. Commun. 159, 1375-1382). In the current study, the characteristics of modified LDL receptors in rabbit peritoneal macrophages and Kupffer cells from rabbits were investigated. Cross-competition studies of the degradation assay between Ox-LDL and Ac-LDL in rabbit peritoneal macrophages showed that the degradation of 125I-labeled Ox-LDL was almost completely inhibited by an excess amount of unlabeled Ac-LDL. On the other hand, an excess amount of unlabeled Ox-LDL suppressed 125I-labeled Ac-LDL degradation only partially. In contrast, in Kupffer cells an excess amount of unlabeled Ox-LDL inhibited the degradation of 125I-labeled Ac-LDL almost completely, whereas the degradation of 125I-labeled Ox-LDL was inhibited only partially by Ac-LDL. Scatchard analysis of binding assay showed that rabbit peritoneal macrophages have a single class of receptor for Ox-LDL, which binds maximally 0.31 microgram/mg cellular protein (Bmax) with an apparent dissociation constant (Kd) of 19.3 micrograms/ml, and two classes of receptors for Ac-LDL; one with high affinity (Bmax 0.025 microgram/mg cellular protein, Kd 0.040 micrograms/ml) and the other with low affinity (Bmax 0.08 microgram/mg cellular protein, Kd 11.31 micrograms/ml). On the other hand, Kupffer cells have two classes for Ox-LDL; one is a high affinity receptor (Bmax 0.53 microgram/mg cellular protein, Kd 0.99 microgram/ml) and the other is a low affinity receptor (Bmax 3.71 micrograms/mg cellular protein, Kd 16.2 micrograms/ml) and a single class for Ac-LDL (Bmax 0.60 microgram/mg cellular protein, Kd 7.24 micrograms/ml). These results indicate that rabbit peritoneal macrophages have two kinds of modified LDL receptors; one is specific for Ac-LDL, and the other recognizes both Ox-LDL and Ac-LDL.