Shogo Kurebayashi

Peroxisome Proliferator-Activated Receptor α Agonists Increase Nitric Oxide Synthase Expression in Vascular Endothelial Cells

Objective—There has been accumulating evidence demonstrating that activators for peroxisome proliferator-activated receptor &agr; (PPAR&agr;) have antiinflammatory, antiatherogenic, and vasodilatory effects. We hypothesized that PPAR&agr; activators can modulate endothelial nitric oxide synthase (eNOS) expression and its activity in cultured vascular endothelial cells. Methods and Results—Bovine aortic endothelial cells were treated with the PPAR&agr; activator fenofibrate. The amount of eNOS activity and the expression of eNOS protein and its mRNA were determined. Our data show that treatment with fenofibrate for 48 hours resulted in an increase in eNOS activity. Fenofibrate failed to increase eNOS activity within 1 hour. Fenofibrate also increased eNOS protein as well as its mRNA levels. RU486, which has been shown to antagonize PPAR&agr; action, inhibited the fenofibrate-induced upregulation of eNOS protein expression. WY14643 and bezafibrate also increased eNOS protein levels, whereas rosiglitazone did not. Transient transfection experiments using human eNOS promoter construct showed that fenofibrate failed to enhance eNOS promoter activity. Actinomycin D studies demonstrated that the half-life of eNOS mRNA increased with fenofibrate treatment. Conclusions—PPAR&agr; activators upregulate eNOS expression, mainly through mechanisms of stabilizing eNOS mRNA. This is a new observation to explain one of the mechanisms of PPAR&agr;-mediated cardiovascular protection.

Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor

Estrogens are the most effective agents available for preventing osteoporosis, and their principal role in bone metabolism is the inhibition of interleukin-6 (IL-6) production in osteoblasts and bone marrow stromal cells. We examined the mechanism of inhibitory effect of estrogens on the 190 bp proximal promoter of the IL-6 gene. Promoter activity induced by transfection of both NF-kappaB p65 subunit and NF-IL6 was decreased by 45% by estradiol (E2)-estrogen receptor (ER) complexes. The inhibitory effect of E2 was also observed on a mutant IL-6 promoter in which the NF-IL6 binding site was disrupted. E2 repressed the wild-type promoter activity induced by NF-kappaB p65 subunit alone, but had no effect on that induced by NF-IL6 alone. These findings suggested that estrogens inhibit IL-6 production by interfering with the function of NF-kappaB rather than that of NF-IL6. The ER mutant, HE19, which does not contain the A/B domain, repressed the induction by NF-kappaB to the same extent as wild-type ER HE0, whereas the effect of C-terminal deletion mutant, HE21, was only marginal. The antiestrogen, 4-hydroxytamoxifen (OHT), had no effect on IL-6 promoter activity, suggesting that E2-induced conformational change of the hormone binding domain plays an important role in protein-protein interaction between ER and NF-kappaB. E2 had no effect on the nuclear translocation of NF-kappaB, and electrophoretic mobility shift assay showed that the presence of E2-ER complexes did not affect the ability of NF-kappaB to bind to specific DNA sequences.

Thiazolidinediones Downregulate Stearoyl-CoA Desaturase 1 Gene Expression in 3T3-L1 Adipocytes

Thiazolidinediones (TZDs) are known to have potent increases of insulin sensitivity. Because peroxisome proliferator-activated receptor-γ (PPAR-γ), a receptor for TZDs, is mainly expressed in adipocytes, we tried to search the TZD-targeted genes in mouse 3T3-L1 adipocytes. By the mRNA differential display method, one band repressed by troglitazone was obtained, which corresponded to the partial sequences of the stearoyl-CoA desaturase 1 (SCD1) gene. Troglitazone dramatically decreased SCD1 mRNA levels in 3T3-L1 adipocytes in a dose-dependent manner. Pioglitazone also repressed the SCD1 mRNA expression, whereas WY-14,643 had no apparent effect. Both troglitazone and pioglitazone raised the composition (weight percentage) of myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0), but lowered the composition of the Δ9-cis desaturated fatty acids such as myristoleic acid (C14:l, Δ9), palmitoleic acid (C16:l, Δ9), oleic acid (C18:l, Δ9), and linoleic acid (C18:2, Δ9,12). These results indicate that TZDs repress SCD1 activity in 3T3-L1 adipocytes via downregulating SCD1 enzyme gene expression.

Increased Interleukin-6 Production in Mouse Osteoblastic MC3T3-E1 Cells Expressing Activating Mutant of the Stimulatory G Protein

The McCune–Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café‐au‐lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the α subunit of the Gs protein (Gsα) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin‐6 (IL‐6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL‐6 production by activating mutant Gsα in bone cells, we established mouse osteoblastic MC3T3‐E1 cells stably transfected with the activating mutant Gsα expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL‐6 than the cells transfected with control vector or wild‐type Gsα expression vector. Analysis of the IL‐6 promoter revealed that any of the AP‐1, nuclear factor (NF)‐IL6, and NF‐κB binding elements are important for the activating mutant Gsα‐induced gene expression. Electrophoretic mobility‐shift assays using nuclear extracts of the mutant Gsα‐expressing cells showed that phospho(Ser133)‐cAMP‐responsive element binding protein (CREB), AP‐1, NF‐IL6, and NF‐κB were increased, compared with the control cells or the wild‐type Gsα‐expressing cells. These results indicate that activating mutant Gsα increases the transcriptional factors binding to CRE, AP‐1, NF‐IL6, and NF‐κB elements to induce IL‐6 gene expression in the osteoblastic cells.

Strong association of C‐reactive protein with body mass index and 2‐h post‐challenge glucose in non‐diabetic, non‐smoker subjects without hypertension

Aims  Increases in C‐reactive protein (CRP) levels have been shown to be associated with cardiovascular diseases as well as asymptomatic atherosclerosis and to be closely related to traditional cardiovascular risk factors. The aim of this study was to determine the clinical and biochemical characteristics associated with CRP in non‐diabetic, non‐smoker subjects without hypertension.

Reduced IRS-2 and GLUT4 expression in PPARγ2-induced adipocytes derived from C/EBPβ and C/EBPδ-deficient mouse embryonic fibroblasts

In adipose tissue, the ability of cells to respond to insulin and to express genes such as those encoding fatty-acid-binding protein (422/aP2), lipoprotein lipase (LPL), adipsin and glucose transporter 4 (GLUT4) is acquired during their differentiation into mature adipocytes. It has been recognized that peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs) play critical roles in adipocyte differentiation. However, it remained uncertain whether PPARγ or which C/EBP is involved in the acquisition of these characteristics. We introduced PPARγ2 into C/EBPβ/δ-double deficient mouse embryonic fibroblasts (MEFs), followed by stimulation with its ligands, in order to define the roles of C/EBPβ and C/EBPδ in phenotypic acquisition during adipocyte differentiation. This procedure resulted in differentiation of these MEFs into mature adipocytes morphologically similar to wild-type MEFs. However, the adipocytes derived from the C/EBPβ/δ-deficient MEFs showed lower expression of GLUT4 and adipsin mRNA than those derived from wild-type MEFs, although aP2 and LPL mRNA levels were similar in both types. The C/EBPβ/δ-deficient adipocytes also expressed lower amounts of insulin receptor substrate 2 (IRS-2) than the adipocytes derived from wild-type MEFs, whereas the amounts of insulin receptor and IRS-1 were similar. Finally, insulin-responsive 2-deoxyglucose uptake was lower in the C/EBPβ/δ-deficient cells. It could thus be demonstrated that C/EBPβ and C/EBPδ are involved in the acquisition of IRS-2 and GLUT4 expression as well as in insulin-sensitive glucose uptake during adipocyte differentiation.

Risk factors for asymptomatic atherosclerosis in Japanese type 2 diabetic patients without diabetic microvascular complications.

Atherosclerotic vascular diseases are frequently associated with diabetes mellitus. There has been increasing evidence showing that the atherosclerotic diseases in diabetic patients are distinct from diabetic microvascular complications as to their pathophysiology and epidemiology. However, we have no information on the prevalence of asymptomatic atherosclerosis in diabetic patients before the onset of microvascular diseases. In the present investigation, we aimed to evaluate risk factors for the atherosclerosis in type 2 diabetic patients without the microvascular diseases. For this purpose, we evaluated atherosclerotic change of carotid arteries in 125 Japanese type 2 diabetic patients who had neither atherosclerotic vascular diseases nor diabetic microvascular complications. When atherosclerotic change was defined as the mean intima-media thickness (IMT) of >/= 1.1 mm and/or the presence of plaque lesion, 50% of patients had atherosclerosis of the carotid arteries. Risk factors for the carotid atherosclerosis were age, low-density lipoprotein (LDL)-cholesterol, hypertension, and diabetes treatment. Age and LDL-cholesterol were associated with mean IMT. Age, diabetes treatment, LDL-cholesterol, and hypertension were positively associated with plaque lesion, while high-density lipoprotein (HDL)-cholesterol was negatively associated with it. Fasting plasma glucose, glycosylated hemoglobin (HbA(1c)), and known diabetes duration remained unassociated with any parameters of asymptomatic atherosclerosis of the carotid arteries. These results indicate that glycemic control is unrelated with asymptomatic atherosclerosis in type 2 diabetic patients without diabetic microvascular complications. Conventional risk factors and diabetes treatment are independently associated with atherosclerosis of the carotid arteries in these patients.

Clinical and endocrinological characteristics of adrenal incidentaloma in Osaka region, Japan.

The aim of this study was to investigate the clinical and endocrinological characteristics of adrenal incidentalomas in Osaka region, Japan. The study was a multicenter retrospective analysis of 150 patients with adrenal incidentalomas who underwent radiographic and endocrine evaluations between 2005 and 2013. Most adrenal incidentalomas were discovered by computed tomography (77.0%) and the rest were identified by abdominal ultrasonography (14.6%), magnetic resonance imaging (4.2%), or positron emission tomography (4.2%). Adrenal incidentalomas were more frequently localized on the left side than on the right. The average diameter of tumors was 21 ± 11 mm. On endocrinological evaluation, 14 patients were diagnosed with primary aldosteronism (9.3%), 10 with subclinical Cushings syndrome (6.7%), 7 with pheochromocytoma (4.7%), 7 with Cushings syndrome (4.7%), 2 with both subclinical Cushings syndrome and primary aldosteronism (1.3%), and 110 with non-functioning tumors (73.3%). Patients with functioning tumors were significantly younger and had larger tumor diameters than those with non-functioning tumors. Except for hypertension, complications were comparable between patients with functioning and non-functioning tumors, including the presence of glucose intolerance, cardiovascular disease, and dyslipidemia. In conclusion, a higher prevalence of primary aldosteronism was observed compared with a previous report. Complications were comparable between patients with functioning and non-functioning tumors, including the frequencies of glucose intolerance, cardiovascular disease, and dyslipidemia. Long-term follow-up is required in patients with non-functioning tumors because the frequency of complications, such as glucose intolerance, cardiovascular disease, and dyslipidemia, was equal to that in patients with functioning tumors.

Estimation of HbA1c response to sitagliptin by change in glycated albumin level for 2 weeks

Aims/Introduction:  Since glycated albumin (GA) reflects shorter‐term (about 2 weeks) control of plasma glucose levels compared with HbA1c, GA is thought to be a useful glycemic control indicator for the early period following commencement of the treatment of diabetes. In this study, we attempted to estimate HbA1c using the change in GA level before and after the first 2 weeks (ΔGA2w) of administration of sitagliptin, a dipeptidyl peptidase‐4 (DPP‐4) inhibitor.

Sitagliptin Significantly Decreases the Ratio of Glycated Albumin to HbA1c in Patients with Type 2 Diabetes Mellitus

Background: Since glycated albumin (GA) is a glycemic control marker which reflects more postprandial plasma glucose (PPPG) and/or glycemic excursions than HbA1c, the GA/HbA1c ratio is a useful indicator for PPPG and/or glycemic excursions. In this study, we investigated the clinical significance of the GA/HbA1c ratio by administration of sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in patients with type 2 diabetes mellitus. Methods: Sitagliptin (50 mg/day) was administered for 24 weeks to 69 patients with type 2 diabetes mellitus with stable glycemic control. Results: With sitagliptin administration, both HbA1c and GA significantly decreased from baseline to the periods of week 4 and week 24. The GA/HbA1c ratio also significantly decreased (baseline 2.72 ± 0.42 vs. 24 weeks 2.60 ± 0.38, P<0.0001). The change in the GA/HbA1c ratio with the sitagliptin administration for 24 weeks was inversely correlated with baseline GA (R=-0.425, P<0.001) and baseline GA/HbA1c ratio (R=-0.354, P=0.003), but not with baseline HbA1c (R=-0.222, P=0.066). By tertile analysis based on the baseline GA/HbA1c ratio, the GA/HbA1c ratios were significantly associated with GA (P<0.0001), but not fasting plasma glucose (FPG) and HbA1c. Furthermore, changes in GA (P=0.010), but not FPG and HbA1c, were significantly correlated with the baseline GA/HbA1c ratio. Conclusions: Sitagliptin significantly decreased the GA/HbA1c ratio and this effect was more pronounced in patients with the higher baseline GA/HbA1c ratio. Our findings suggest that the effect of sitagliptin on the GA/HbA1c ratio might reflect improvement of PPPG levels and/or glycemic excursion.