Takayuki Sasahara

International clinical harmonization of glycated hemoglobin in Japan: From Japan Diabetes Society to National Glycohemoglobin Standardization Program values

In 1999, the Japan Diabetes Society (JDS) launched the previous version of the diagnostic criteria of diabetes mellitus, in which JDS took initiative in adopting glycated hemoglobin (HbA1c) as an adjunct to the diagnosis of diabetes. In contrast, in 2009 the International Expert Committee composed of the members of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) manifested the recommendation regarding the use of HbA1c in diagnosing diabetes mellitus as an alternative to glucose measurements based on the updated evidence showing that HbA1c has several advantages as a marker of chronic hyperglycemia2–4. The JDS extensively evaluated the usefulness and feasibility of more extended use of HbA1c in the diagnosis of diabetes based on Japanese epidemiological data, and then the ‘Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus’ was published in the Journal of Diabetes Investigation5 and Diabetology International6. The new diagnostic criterion in Japan came into effect on 1 July 2010. According to the new version of the criteria, HbA1c (JDS) ≥6.1% is now considered to indicate a diabetic type, but the previous diagnosis criteria of high plasma glucose (PG) levels to diagnose diabetes mellitus also need to be confirmed. Those are as follows: (i) FPG ≥126 mg/dL (7.0 mmol/L); (ii) 2‐h PG ≥200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test; or (iii) casual PG ≥200 mg/dL (11.1 mmol/L). If both PG criteria and HbA1c in patients have met the diabetic type, those patients are immediately diagnosed to have diabetes mellitus5,6.

Inhibitory effects of tranilast on expression of transforming growth factor-β isoforms and receptors in injured arteries

Tranilast (N(3,4-dimethoxycinnamoyl)anthranilic acid), an agent which in cell culture inhibits transforming growth factor-beta (TGF-beta) secretion and antagonises the effects of TGF-beta and platelet-derived growth factor (PDGF) on cell migration and proliferation, has been reported to reduce the incidence of restenosis after angioplasty in angiographically validated human clinical trials. We investigated in a rat model of balloon angioplasty whether tranilasts effects in vivo could be attributed to inhibition of expression of TGF-beta and/or its receptor types. Using a standardised reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we examined the effects of three doses of tranilast (25, 50 and 100 mg/kg) on the expression of two TGF-beta isoforms, the types I and II TGF-beta receptors and two putative TGF-beta responses, induction of integrins alpha(v) and beta3 mRNA, 2 h after oral administration and 26 h after vessel injury. Tranilast attenuated in a dose-dependent and reversible manner the injury-induced increases in mRNA levels encoding TGF-beta1, TGF-beta3, two type I TGF-beta receptors ALK-5 and ALK-2, and the type II receptor TbetaRII. At the highest dose mRNA levels encoding TGF-beta1 and TbetaRII were attenuated to levels approaching or below those observed in uninjured vessels. Messenger RNAs encoding TGF-beta3, ALK-5 and ALK-2 were all attenuated by between 70 and 74% (all P < 0.05). Tranilast also attenuated in a reversible manner the elevations in mRNA levels for integrins alpha(v) and beta3 observed after vessel injury, by 90 and 72%, respectively. We also investigated, in cultured smooth muscle cells derived from injured carotid arteries, the extent to which tranilast (300 mg/l) attenuated any increases in expression of type I and type II receptors stimulated by PDGF-BB and TGF-beta1, growth factors implicated in smooth muscle cell migration and proliferation in injured vessels. Increases in mRNA levels of the type I receptors ALK-5 and ALK-2 induced by PDGF-BB and TGF-beta1 were almost completely prevented by tranilast. Tranilast also prevented the PDGF-BB induced increases in TbetaRII but only partially inhibited the TGF-beta1 induced upregulation of TbetaRII. We conclude that tranilast can inhibit transcriptional mechanisms associated with the upregulation of TGF-beta and its receptor types in balloon catheter injured vessels. It is possible that these mechanisms contribute to its ability to reduce the frequency of restenosis after angioplasty.

Glucocorticoid Inhibits Oxidized LDL-Induced Macrophage Growth by Suppressing the Expression of Granulocyte/Macrophage Colony-Stimulating Factor

Glucocorticoid, an anti-inflammatory agent, inhibits the development of atherosclerosis in various experimental animal models. This is partially explained by its ability to inhibit smooth muscle cell migration and proliferation in the intima and to reduce chemotaxis of circulating monocytes and leukocytes into the subendothelial spaces. We have recently demonstrated that oxidized LDL (Ox-LDL) has a mitogenic activity for macrophages in vitro in which Ox-LDL-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role. Proliferation of cellular components is one of the characteristic events in the development and progression of atherosclerotic lesions. In the present study, we investigated the effects of glucocorticoids on Ox-LDL-induced macrophage growth. Dexamethasone, prednisolone, and cortisol inhibited Ox-LDL-induced thymidine incorporation into macrophages by 85%, 70%, and 50%, respectively. Ox-LDL induced a significant production of GM-CSF by macrophages, which was effectively inhibited by dexamethasone, prednisolone, and cortisol by 80%, 65%, and 50%, respectively. Dexamethasone-mediated inhibition of Ox-LDL-induced GM-CSF mRNA expression and macrophage growth was significantly abrogated by RU-486, a glucocorticoid receptor antagonist. Our results suggest that the inhibitory effects of glucocorticoids on macrophage growth may be due to the inhibition of Ox-LDL-induced GM-CSF production through transactivation of the glucocorticoid receptor.

Genome-wide linkage analysis of type 2 diabetes mellitus reconfirms the susceptibility locus on 11p13–p12 in Japanese

AbstractType 2 diabetes mellitus is a heterogeneous disorder, and the development of type 2 diabetes mellitus is associated with both insulin secretion defect and insulin resistance. The primary metabolic defect leading to type 2 diabetes mellitus has been thought to be varied among populations, especially in Japanese and Caucasians. Here, we have done the genome-wide scan for type 2 diabetes mellitus using 102 affected Japanese sib-pairs to identify the genetic factors predisposing to type 2 diabetes mellitus. Nonparametric linkage analysis showed one suggestive evidence for linkage to 11p13-p12 [D11S905: two-point maximum LOD score (MLS) of 2.89 and multipoint MLS of 2.32] and one nominally significant evidence for linkage to 6q15-q16 (D6S462: two-point MLS of 2.02). Interestingly, the 11p13-p12 region was reported to be a susceptibility locus for Japanese type 2 diabetes mellitus with suggestive evidence of linkage, and D11S905 was within 5 cM to D11S935 with the highest MLS in the previous linkage analysis reported. The only overlapped susceptibility region with suggestive evidence of linkage for Japanese type 2 diabetes mellitus was D11S935-D11S905 among the three reports including this study. These results taken together suggest that a susceptibility gene for type 2 diabetes mellitus in Japanese will reside in 11p13-p12.

Acetyl-low density lipoprotein receptors on rat mesangial cells

To elucidate whether mesangial cells have any scavenger functions for modified lipoproteins, surface binding and cholesteryl ester (CE) formation by acetyl-low density lipoproteins (acetyl-LDL) have been studied in cultured rat renal mesangial cells. Specific binding kinetics for acetyl-LDL were observed with Kd = 28.3 micrograms/ml and Bmax = 1.1 ng/micrograms cell protein at 0 degrees C. The fluorescence microscopic finding demonstrated the enhanced uptake of DiI-acetyl-LDL in mesangial cells. Incorporation of [14C]oleate into CE was enhanced to 6-fold by loading 30 micrograms/ml of acetyl-LDL on 10 micrograms/ml of [14C]oleate-bovine serum albumin conjugate as compared with the control without lipoproteins (P < 0.05). The CE formation was completely inhibited by chloroquine. The light microscopic finding demonstrated the increased CE deposition by acetyl-LDL, resulting in foam cell formation. These results indicate biochemically and morphologically that the mesangial cells take up acetyl-LDL by receptor-mediated endocytosis, and that cholesterols in acetyl-LDL are converted to CE, resulting in an increased cellular cholesterol content. In conclusion, mesangial cells may have a scavenger function similar to macrophages.

Effects of probucol on cholesterol metabolism in mouse peritoneal macrophages: Inhibition of HDL-mediated cholesterol efflux

Macrophage-derived foam cells are known to play an essential role in the development and progression of atherosclerotic lesions. Probucol prevents oxidative modification of low-density lipoprotein (LDL) and lowers plasma contents of LDL and high-density lipoprotein (HDL). A recent report using apoE -/- mice demonstrated that probucol treatment enhanced atherosclerosis in apoE -/- mice more rapidly than that in untreated apoE -/- mice, and a reduction in plasma cholesterol by probucol was not the cause of enhancement of atherosclerotic lesions in probucol-treated apoE -/- mice. Moreover, probucol was reported to inhibit apoA-I mediated cholesterol efflux from mouse macrophages. These reports suggested that probucol might directly affect cholesterol metabolism in mouse macrophages. Thus, we investigated the effects of probucol on cholesterol metabolism in mouse resident peritoneal macrophages. Probucol did not affect degradation of acetylated LDL (Ac-LDL), degradation of LDL and endogenous cholesterol synthesis in mouse macrophages. However, it significantly inhibited HDL-mediated cholesterol efflux. Moreover, probucol partially (30%) inhibited the binding of HDL to mouse macrophages, and significantly activated acyl-coenzyme A:cholesterol acyltransferase (ACAT). Our results suggested that probucol inhibited HDL-mediated cholesterol efflux by inhibiting the binding of HDL to mouse macrophages and reducing HDL-accessible free cholesterol content by ACAT activation, thereby worsening atherosclerotic lesions in apoE -/- mice. However, it remains unclear whether probucol inhibits HDL-mediated cholesterol efflux from human macrophages.

β-Migrating very low density lipoproteins induce foam cell formation in mouse mesangial cells

To elucidate whether beta-migrating very low density lipoproteins (beta-VLDL) induce foam cell formation in mesangial cells or not, surface binding and foam cell formation with beta-VLDL were studied in mouse mesangial cells. Specific binding kinetics for beta-VLDL and low density lipoproteins (LDL) on the mesangial cells were observed with Kd = 3.8 and 13.7 micrograms/ml, and Bmax = 65.9 and 71.9 ng/ml cell protein at 4 degrees C, respectively. The binding of beta-VLDL was inhibited by excess amounts of LDL or beta-VLDL, but not by acetyl-low density lipoproteins. Ligand blotting using beta-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected the same apparent single protein (approx. 130 kDa). Incorporation of [14C]oleate into cholesteryl ester in mouse mesangial cells was enhanced by beta-VLDL to 3-fold higher than that by LDL, and it was inhibited by chloroquine or anti-human LDL receptor monoclonal antibody. The light microscopic findings also demonstrated that cholesteryl ester deposition increased in these cells incubated with beta-VLDL, but not with LDL. In conclusion, beta-VLDL was specifically taken up by receptor-mediated endocytosis in mouse mesangial cells through LDL receptors, resulting in foam cell formation.

The metabolic fate of apolipoprotein A-I-containing lipoproteins internalized into HepG2 cells: resecreted lipoproteins as a potent inducer for cholesterol efflux

In a chase study using double-radiolabeled apolipoprotein (apo) A-I-containing lipoproteins (14C-labeled cholesteryl ester and 125I-labeled apolipoprotein) with or without apo A-II (Lp A-I/A-II particle and Lp A-I particle), these lipoproteins internalized into HepG2 cells were demonstrated to be time-dependently released into the medium as trichloroacetic acid (TCA)-precipitable fraction. The molar ratio of 14C/125I-radioactivity of TCA-precipitable fraction in the medium was time-dependently decreased. In Sephacryl S-300 HR chromatography of both circulating mature and resecreted apo A-I-containing lipoproteins in the medium after the chase period, a single major protein peak corresponding to that of high density lipoproteins was detected by absorbance at 280 nm. The 14C-radioactivity in apo A-I-containing lipoproteins resecreted from HepG2 cells after 3-h chase was approximately one-fourth of that in circulating mature apo A-I-containing lipoproteins. Cholesterol mass in resecreted apo A-I-containing lipoproteins was three-tenths of that in circulating mature apo A-I-containing lipoproteins. In a cholesterol efflux experiment using macrophage foam cells labeled with [3H]cholesterol, apo A-I-containing lipoproteins resecreted significantly decreased cholesteryl ester radioactivity in macrophage foam cells, as compared with circulating mature apo A-I-containing lipoproteins. There were no remarkable differences in the metabolic fates and cholesterol efflux from macrophage foam cells between Lp A-I and Lp A-I/A-II particles. These results suggest that a part of apo A-I-containing lipoproteins internalized into HepG2 cells may be resecreted in the form of intact lipoproteins with lower cholesterol content, and apo A-I-containing lipoproteins resecreted may be a potent inducer for cholesterol efflux through the processes of reverse cholesterol transport.

Group-II phospholipase A2 enhances oxidized low density lipoprotein-induced macrophage growth through enhancement of GM-CSF release

Inflammatory process plays an important role in the development and progression of atherosclerotic lesions. Recently, group-II phospholipase A(2) (PLA(2)), an inflammatory mediator, was reported to exist in human atherosclerotic lesions and to enhance the development of murine atherosclerotic lesions. Oxidized low density lipoprotein (Ox-LDL) stimulates the growth of several types of macrophages in vitro. Since proliferation of macrophages occurs in atherosclerotic lesions, it is possible to assume that the Ox-LDL-induced macrophage proliferation might be involved in the progression of atherosclerosis. In this study, the role of group-II PLA(2) in the Ox-LDL-induced macrophage growth was investigated using thioglycollate-elicited mouse peritoneal macrophages. Thioglycollate-elicited macrophages significantly expressed group-II PLA(2) and released it into the culture medium. The Ox-LDL-induced thymidine incorporation into thioglycollate-elicited macrophages was three times higher than that into resident macrophages, whereas under the same conditions, granulocyte/macrophage colony-stimulating factor (GM-CSF) equally induced thymidine incorporation into both types of macrophages. Moreover, the Ox-LDL-induced GM-CSF release from thioglycollate-elicited macrophages was significantly higher than that from resident macrophages. In addition, the Ox-LDL-induced thymidine incorporation into macrophages obtained from human group-II PLA(2) transgenic mice and the GM-CSF release from these cells were significantly higher than those from their negative littermates, and the Ox-LDL-induced thymidine incorporation into human group-II PLA(2) transgenic macrophages was significantly inhibited by a polyclonal anti-human group-II PLA(2) antibody. These results suggest that the expression of group-II PLA(2) in thioglycollate-elicited macrophages may play an enhancing role in the Ox-LDL-induced macrophage growth through the enhancement of the GM-CSF release.