Long-term incubation of proteins with glucose leads to advanced glycation end products (AGE) with fluorescence and a brown color. We recently immunologically demonstrated the intracellular ASE-accumulation in macrophage and smooth muscle cell (SMC)-derived foam cells in the human advanced atherosclerotic lesions by using antibody against AGE. Immunoelectron microscopic observation also revealed the localization of AGE in lysosomal lipid vacuoles, or electron-dense granules of the foam cells. To understand the mechanism of AGE-accumulation in these foam cells, we characterized the interaction of AGE-proteins with macrophage and SMC.
In the present study, we examined whether macrophage scavenger receptor (MSR) could mediate the endocytic uptake of AGE-proteins by using Chinese hamster ovary cells overexpressing bovine type II MSR (CHO-SRII). ¹²⁵I-AGE-bovine serum albumin (¹²⁵I-AGE-BSA) as well as ¹²⁵I-acetylated low density lipoprotein (¹²⁵I-acetyl-LDL) underwent endocytic degradation by CHO-SRII cells, but not by control CHO cells. Endocytic degradation of ¹²⁵I-acetyl-LDL and ¹²⁵I-AGE-BSA by CHO-SRII cells was significantly inhibited by unlabeled AGE-BSA, as well as by acetyl-LDL. Moreover, the endocytic uptake of ¹²⁵I-AGE-BSA by peritoneal macrophages from MSR knock out mice was decreased 70-80% as compared with that of control. We recently demonstrated that the cell growth of starchinduced mouse peritoneal macrophages was induced by AGE-proteins. This AGE-induced macrophage growth was accompanied by a significant induction of granulocyte/monocyte colony stimulating factor (GM-CSF) at the mRNA level, and was effectively inhibited by an anti-GM-CSF antibody, suggesting that AGE-proteins may stimulate macrophages to secrete GM-CSF, which might stimulate cell growth in an autocrine or paracrine fashion.
In experiments by using rabbit SMC, ¹²⁵I-AGE-BSA showed a dose-dependent saturable binding with an apparent dissociation constant (Kd) of 4.0μg/ml at 4°C. In experiments at 37°C, AGE-BSA underwent receptormediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of ¹²⁵I-AGE-BSA was effectively inhibited by unlabeled AGE-proteins such as AGE-BSA and AGE-hemoglobin, but not by acetyl-LDL and oxidized LDL, well-known ligands for MSR. These results indicate that the endocytic uptake of AGE-proteins by SMC is mediated by an AGE receptor which is different from MSR. To determine the functional role of this AGE receptor, the effects of AGE-BSA on the cell migration of these SMC were examined. Incubation of SMC with 1-100μg/ml of AGE-BSA resulted in the significant cell migration.
Based on our findings, we will discuss the pathophysiological role of AGE in atherosclerosis.

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Diabetes mellitus and aging are known to accelerate atherosclerotic lesions, but the mechanisms of this acceleration are unclear. We attempted to elucidate the role of nonenzymatic glycation of extracellular matrix on the development of atherosclerotic lesions. Advanced glycation end products (AGEs) were found in the extracellular matrix of intima of fatty streaks and atherosclerotic plaques. Some foamy macrophages showed an accumulation of AGEs in their cytoplasms. The staining pattern of AGEs was similar to that obtained with antibody against apolipoprotein-B. Streptozotocininduced diabetic rats showed a significant increase in the percentages of pepsin-insoluble collagen to total collagen and AGE contents of collagen as compared to control after 28 weeks. Although AGEs had no effects on the DNA synthesis of cultured smooth muscle cells, they accelerated the fibronectin-production of smooth muscle cells. The glycated BSA was prepared by incubation with 1.67M glucose at 37°C for 2 (gB2) or 16 (gB16) weeks. Both gB2 and gB16 had a cytotoxic effect on the clutured smooth muscle cells and induced the extracellular superoxide generation in the presence of cupric ion. Although the early products of nonenzymatic glycation were present in both gB2 and gB16, AGEs were not formed in gB2, but in gB16. These results suggest that the nonenzymatic glycation of extracellualr matrices, especially collagen, may accelerate the atherosclerosis by the insolubility and deposition of collagen in the extracellular matrix and increased production of fibronectin of smooth muscle cells, in relation to diabetes mellitus and aging. The early products of nonenzymatic glycation may contribute to the oxidation of LDL and oxidative damage of smooth muscle cells which play a role in the development of atherosclerotic lesions.

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Using glycolaldehyde (two carbons, C2) and monosaccharides of the aldose D series, i. e., glyceraldehyde (C3), erythrose (C4), arabinose (C5), and glucose (C6), new modified low density lipoproteins (LDLs) were produced to determine if degradation of modified LDLs by the human monocyte-derived macrophage (Mφ) scavenger receptors is based on the molecular weight of the substance used to modify the LDL free lysine.
By using a trinitrobenzensulfonic acid (TNBS) assay, it was found that the smaller the molecular weight of the substances used for LDL modification, the more rapid and extensive is the decrease of free lysine, which plateaued after 24h. Based on findings of glycolaldehyde-LDL, glyceraldehyde-LDL and erythrose-LDL, double reciprocal plots were made. Results revealed that all three reactions occured through the same mechanism, although the Km values in the three modified LDLs differed. Further, the electrophoretic mobility of these modified LDLs correlated with a decrease in the percentage of TNBS reactivity.
Using Mφ, degradation studies of mildly modified LDLs revealed that, in contrast to native LDL, little degradation occured. However, after extensive modifications, less degradation was seen in glycolaldehyde-LDL, glyceraldehyde-LDL and erythrose-LDL than in the acetyl-LDL, but more degradation occured in these new modified LDLs than in native LDL. Further, degradation of 125-I-glyceraldehyde-LDL was remarkedly inhibited in unlabeled acetyl-LDL, 125-Iglycolaldehyde-LDL and fucoidin, whereas in unlabeled oxidized LDL and advanced glycoslation end products(AGE)-modified bovine serum albumin, degradation of 125-I-glyceraldehyde-LDL was extremely slight. In contrast, unlabeled native LDL produced no inhibitory effect on the degradation of 125-I-glyceraldehyde-LDL. These findings would seem to suggest that after extensive modification, receptor recognition of glyceraldehyde-LDL changes from the LDL receptor to the scavenger one.
Degradation of new modified LDLs appeared to depend on a decrease in the percentage of TNBS reactivity. Further, even though these modified LDLs showed the same ΔTNBS decrease, these degradation progressively increased in the following order: erythrose-LDL. glyceraldehyde-LDL, and glycolaldehyde-LDL, These findings suggest that the degree of LDL degradation is not only associated with an increase in the negative charge and decrease in the percentage of TNBS reactivity, but also with the molecular weight of the substance used to modify the LDLs.
Arabinose-LDL and glycosylated LDL showed less degradation than native LDL, although LDLs were incubated for 5 days with a high concentration of their respective modifying substance. Degradation decreased even further when NaBH₃CN was used for extensive LDL modification. These findings suggest that the particle size and/or the stereoscopic strucure of modified LDLs may play an important role in becoming the ligand of the scavenger receptor.
Glucose promotes oxidative LDL modification. Further, glycosylated LDL are easily oxidized by metalic ions, and oxidized LDL are extensively degraded by the scavenger receptors. However, our results have demonstrated that glycosylated LDL is not recognized by the scavenger receptors even after extensive modification. We thus have speculated that under hyperglycemic conditions, LDLs may promote the formation of foam cells as not only AGE but also oxidized and/or glycoxidized LDLs.

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Obesity is frequently accompanied by diabetes mellitus, hyperlipidemia and hypertension. We have clarified body fat distribution rather than body fat mass is a critical determinant for the development of obesityrelated disorders. Accumulation of intra-abdominal visceral fat was seen even in the subjects whose body mass indexes fall in the normal range and those subjects were accompanied by multiple coronary risk factors such as glucose intolerance, hyperlipidemia, and hypertension. We designated this feasible condition to atherosclerosis as “visceral fat syndrome”. In the current study, we investigated hepatic regulation of VLDL synthesis and the molecules secreted from adipose tissue in visceral fat accumulation.
Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of visceral fat syndrome characterized by visceral fat accumulation, hyperlipidemia and late onset insulin resistance. OLETF rats cause hypertriglyceridemia before the manifestation of insulin resistance. In 6 week-old OLETF rats, in which insulin resistance has not manifested, visceral fat weight was heavier and portal free fatty acids and VLDL-triglyceride levels were higher than in age-matched LETO rats. Hepatic acyl-CoA synthetase activity and mRNA levels, and microsomal triglyceride transfer protein mRNA levels werehigher in OLETF rats than in LETO rats. These results suggest that the enhanced expression of ACS and MTP genes may be one of the mechanisms for hyperlipidemia in visceral fat accumulation.
Recent studies on adipocyte-biology revealed that adipose tissues secrete various biologically active molecules. Type 1 plasminogen activator inhibitor (PAI-1) regulates fibrinolysis and plasma levels of PAI-1 are elevated in patients with thrombotic disorders. We found plasma levels of PAI-1 were closely correlated with visceral fat area in human subjects but not with subcutaneous fat area. Direct secretion of PAI-1 fromvisceral adipose tissue may be responsible for high levels of plasma PAI-1 and may have a role in the development of vascular disease in visceral fat accumulation. PAI-1 mRNA was detected in both the visceral and subcutaneous fat in obese rats but was only increased in visceral fat during the development of obesity. Elevated PAI-1 gene expression was induced in 3T3-L1 cells after differentiation into adipocytes, which strongly suggested that the adipocyte itself expressed the PAI-1 gene.
These data suggested that portal free fatty acids and adipocytokines secreted from accumulated visceral fat may play an important role on the development of multiple clinical features in visceral fat syndrome.

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Familial combined hyperlipidemia (FCHL) is a common genetic hyperlipidemia, and has been known to cause premature coronary heart disease (CHD). The impaired glucose tolerance with hyperinsulinemia also is a potent risk factor for CHD. Familial hyperlipidemia and hyperinsulinemia in diabetic patients, may be important elements linkaging diabetes and CHD. In the present study, insulin responses after 75g glucose administration in non-obese FCHL patients (BMI<25, FCHL group, n=20), were compared with those in familial hypercholesterolemic (FH group, n=49) or normolipidemic patients (Control group, n=41). The patients were divided to the normal, impaired and diabetic glucose tolerance groups according to the WHO criteria.
Seven FCHL patients (35%) showed an impaired glucose tolerance and 5 FCHL patients (25%) diabetic glucose tolerance. Fasting plasma glucose levels and area under the plasma glucose concentration-time curve showed no differences among FCHL, FH and control groups in each normal, impaired and diabetic glucose tolerance group. However, the FCHL patients with normal glucose tolerance had a significant higher level of plasma insulin 60 minute after glucose administration than the FH and control groups (p<0.05), and the FCHL patients with impaired glucose tolerance a significant higher level of plasma insulin 30 and 120 minute after glucose administration than FH and control groups (p<0.05). Area under the plasma insulin concentration-time curve (AUC-IRI) in the FCHL patients with normal and impaired glucose tolerances were 162±42μU/ml/h and 208±60μU/ml/h, respectively, which were significant higher than those in the control group (77±6μU/ml/h and 103±18μU/ml/h)(p<0.05). There were significant relationships between AUC-IRI and LDL-cholesterol levels in FCHL patients (p<0.05), but no relationships between serum triglyceride or HDL-cholesterol levels, and AUC-IRI.
In conclusion, FCHL patients showed hyperinsulinemia after glucose administration, and may contribute to the increased CHD in the patients with glucose intolerance, according to the similar manner to the insulin resistance syndrome. Therefore, FCHL should be considered in the pathogenesis of CHD in the diabetic patients with hyperlipidemia, hyperinsulinemia and CHD.

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